TGF-β Antibodies

Abstract

The present invention relates to antibodies capable of activating TGF-β1, in particular antibodies capable of binding to latent TGF-β1 and liberating mature TGF-β1 cytokine, and uses thereof.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to antibodies and antigen-binding fragments thereof that bind to latent TGF-β1 and activate TGF-β1. Because TGF-β1 is an important immunosuppressive cytokine, the antibodies and antigen-binding fragments described herein are useful in methods for suppressing pathological immune responses, for example, in the prevention and treatment of autoimmune and alloimmune diseases. The antibodies and antigen-binding fragments described herein are also useful in methods for preventing and treating tumor diseases. [Background technology]

[0002] TGF-β is a pleiotropic cytokine that regulates numerous biological processes, including cell growth, differentiation, tissue homeostasis, and extracellular matrix remodeling. It plays an important and complex role in regulating the function of the immune system (as reviewed in Travis and Sheppard, Annu. Rev. Immunol. 2014;32:51-82 and also Sanjabi et al., Cold Spring Harb Perspect Biol. 2017;9:a022236). TGF-β1-deficient mice die after birth but experience excessive inflammation, highlighting the importance of this cytokine in regulating the immune response (Shull et al., Nature, 1992;359:693-99).

[0003] Three isoforms of TGF-β have been found in mammals: TGF-β1; TGF-β2; and TGF-β3. The most abundant 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 (which targets TGF-β to the endoplasmic reticulum), a large N-terminal region called the latency-associated peptide (LAP), and a shorter C-terminal fragment that is the mature TGF-β cytokine. TGF-β is secreted from cells as a homodimer (containing three interchain disulfide bonds), and either before or after secretion, the LAP is separated from the mature cytokine via furin cleavage. The N-terminal LAP remains non-covalently associated with the mature cytokine, forming the latent TGF-β complex. In some cases, latent TGF-β binds to a family of regulatory proteins known as "latent TGF-β binding proteins" or "LTBPs." The complex of latent TGF-β and one or more LTBPs is sometimes referred to as the "large latent complex."

[0004] The crystal structure of latent TGF-β has been solved, revealing how TGF-β converts from its latent form to its active form (Shi et al., Nature 2011;474:343-49). The crystal structure revealed that in the latent form, the mature cytokine region is surrounded by LAP, which masks the site of the mature cytokine that can contact the TGF-β receptor complex. The mature cytokine is held in place by multiple contacts with a mobile loop structure in LAP called the "latency lasso." Release of LAP-mediated inhibition requires activation of TGF-β.

[0005] TGF-β is activated in vivo through a variety of mechanisms. An important mode of activation is via integrin binding to "RGD," a linear tripeptide motif within the LAP region of TGF-β. This binding of LAP to integrins exerts a deforming physical force on the tethered latent TGF-β complex, resulting in the release of the mature cytokine. Integrins of particular importance in the activation of latent TGF-β include αvβ6, which is normally expressed by epithelial cells, and αvβ8, which is normally expressed by dendritic cells. Additional mechanisms by which latent TGF-β is activated in vivo may include binding to thrombospondin-1 (TSP-1) or by enzymatic activation by proteases, such as serine and metalloproteases, that cleave the LAP region.

[0006] Upon activation, TGF-β mediates its effects through binding to the TGF-β receptor complex at 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 cytoplasmic regions with serine / threonine kinase activity, and binding of TGF-β to the receptor complex leads to phosphorylation of TGF-βR1 by TGF-βRII. Phosphorylation at the receptor level triggers an intracellular signaling 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, and this Smad complex translocates to the nucleus to activate or repress gene expression via binding to Smad-responsive regulatory regions in the promoter segments of TGF-β-regulated genes. 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 Rho GTPase family. In most cell types, including normal and premalignant or malignant epithelial cells, the signals induced by TGF-β1 are cytostatic. TGF-β1 thus exerts an inhibitory activity on epithelial cells in the early stages of cancer.

[0007] As mentioned above, TGF-β1 is also an important cytokine in the regulation of immune response.TGF-β1 is produced by various cells in the immune system and has effects on multiple cellular targets, especially at the level of T cells.In this regard, TGF-β1 plays an important role in regulating the differentiation of T cells into effector and regulatory subsets.

[0008] In its role 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 also of CD8+ T cell proliferation. In contrast, TGF-β1 actively promotes the generation of regulatory T cells (Tregs), both natural or thymic Tregs generated in the thymus at a young age, and also inducible or peripheral Tregs generated from naive CD4+ T cells in the periphery. In the thymus, TGF-β1 protects Tregs from apoptosis during the developmental selection process, and in the periphery, TGF-β1 promotes the conversion of naive CD4+ T cells into Tregs by upregulating the expression of the transcription factor Foxp3.

[0009] Activation of TGF-β1 on the surface of Tregs (and also certain other cell types) is regulated by binding to a transmembrane protein called "GARP" (Glycoprotein A repetitions predominant protein). Indeed, studies have revealed that GARP is required for surface expression of latent TGF-β on 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).

[0010] Considering the importance of TGF-β1 signaling in the regulatory aspects of immune responses, various attempts have been made to generate antibodies that bind to TGF-β1 and regulate its biological activity. International Publication Nos. WO2015 / 015003 and WO2016 / 125017 describe antibodies that can bind to the complex of GARP and latent TGF-β1 formed on the surface of Treg cells. These antibodies inhibit TGF-β1 activation, thereby weakening the immunosuppressive activity of Treg populations. These antibodies are therefore proposed for use as immunostimulatory antibodies in the treatment of conditions or diseases in which it is desirable to upregulate immune responses, for example for the immunotherapy of cancer.

[0011] However, there is still an unmet need to generate antibodies that bind and activate TGF-β1 (or promote TGF-β1 activation), thereby increasing the immunosuppressive activity of Treg populations.These antibodies, which are the goal of the present invention, are proposed for use as immunosuppressive antibodies in the treatment of conditions or diseases in which it is desirable to downregulate immune response, for example in the treatment of autoimmune and alloimmune diseases or conditions.They are also proposed for use in the prevention or treatment of conditions or diseases in which it is desirable to increase the tumor suppressive, cytostatic activity of TGF-β1, for example in premalignant, primary or secondary metastatic stage cancers. Summary of the Invention

[0012] The present invention provides antibodies capable of activating TGF-β1, i.e., antibodies capable of binding to latent TGF-β1 and relieving the mature TGF-β1 cytokine from the inhibition exerted by the LAP region of the full-length TGF-β1 protein.

[0013] 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.

[0014] In certain embodiments, the antibody or antigen-binding fragment binds to an epitope that includes one or more amino acids from a region of human or mouse latent TGF-β1 consisting of amino acids 87-222, where human latent TGF-β1 is represented by SEQ ID NO:1 and mouse latent TGF-β1 is represented by SEQ ID NO:5.

[0015] In certain embodiments, the antibody or antigen-binding fragment binds to an epitope that includes one or more amino acids from a region of human or mouse latent TGF-β1 consisting of amino acids 87-135, where human latent TGF-β1 is represented by SEQ ID NO:1 and mouse latent TGF-β1 is represented by SEQ ID NO:5.

[0016] 3. The antibody or antigen-binding fragment according to claim 1 or 2, which binds to an epitope containing one or more amino acids derived from a region of human or mouse latent TGF-β1 consisting of amino acids 136 to 200, wherein human latent TGF-β1 is represented by SEQ ID NO: 1 and mouse latent TGF-β1 is represented by SEQ ID NO: 5.

[0017] In certain embodiments, the antibody or antigen-binding fragment binds to an epitope that includes one or more amino acids from a region of human or mouse latent TGF-β1 consisting of amino acids 201-222, where human latent TGF-β1 is represented by SEQ ID NO:1 and mouse latent TGF-β1 is represented by SEQ ID NO:5.

[0018] In 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 including amino acid residues E119, K123, S138, R141, S209, R210, G212, E213, I214, and / or E215 of human latent TGF-β1.

[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 that includes amino acid residues G212 and E215 of human latent TGF-β1.

[0020] In embodiments in which the antibody or antigen-binding fragment binds to mouse latent TGF-β1, the antibody or antigen-binding fragment may bind to an epitope including amino acid residues S138, R141, P145, E146, S175, L208, N209 and / or D212 of mouse latent TGF-β1.

[0021] For embodiments in which the antibody or antigen-binding fragment binds to mouse latent TGF-β1, the antibody or antigen-binding fragment may bind to an epitope that includes amino acid residues L208 and D212 of mouse latent TGF-β1. For antibodies or antigen-binding fragments that bind to mouse latent TGF-β1 via an epitope that includes amino acid residues L208 and D212, the epitope may not include additional amino acid residues from within the region of mouse latent TGF-β1 consisting of amino acids 201-222.

[0022] In certain embodiments, the antibody or antigen-binding fragment activates TGF-β1 when latent TGF-β1 is complexed with GARP. In particular, the antibody or antigen-binding fragment can activate TGF-β1 when latent TGF-β1 is bound to the surface of one or more cell types selected from 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 is bound to the surface of regulatory T cells (Treg).

[0023] In certain embodiments, the antibody or antigen-binding fragment activates TGF-β1 when latent TGF-β1 is bound to one or more cell types selected from myeloid cells, lymphoid cells, hematopoietic cells, and pre-malignant stages of cancer.

[0024] In certain embodiments, the antibody or antigen-binding fragment is bivalent and is preferably an IgG antibody, more preferably an IgG1 antibody. In certain embodiments, the antibody or antigen-binding fragment comprises the CH1 domain, hinge region, CH2 domain and / or CH3 domain of human IgG, preferably human IgG1.

[0025] In certain embodiments, the antigen-binding fragment is selected from the group consisting of an antibody light chain variable domain (VL); an antibody heavy chain variable domain (VH or VHH); a single chain antibody (scFv); an (scFv)2 fragment, an F(ab')2 fragment; an Fab fragment; an Fd fragment; an Fv fragment; a one-armed (monovalent) antibody; a diabody; a triabody; a tetrabody; or any antigen-binding molecule formed by combining, assembling or conjugating such antigen-binding fragments.

[0026] In certain embodiments, the antibodies or antigen-binding fragments are defined with reference to specific CDR, VH and / or VL sequences described herein. For embodiments in which the domains of the antibody or antigen-binding fragment are defined by a specific percentage of sequence identity to a reference sequence, the VH and / or VL domains may retain identical CDR sequences as present in the reference sequence, such that changes are only present in the framework regions. The invention further 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.

[0027] Also provided are isolated nucleic acids encoding antibodies or antigen-binding fragments comprising polynucleotides encoding the VH, VHH and / or VL domains of the antibodies or antigen-binding fragments described herein. The invention further provides expression vectors comprising the aforementioned nucleic acids operably linked to regulatory sequences that allow for expression of the antibodies, antigen-binding fragments, variable heavy domains (VH or VHH) or variable light domains (VL) in a host cell or cell-free expression system. Also provided are host cells or cell-free expression systems comprising the aforementioned expression vectors.

[0028] The present invention also provides a pharmaceutical composition comprising an antibody or antigen-binding fragment according to the first or second aspect of the invention and at least one pharma- ceutically acceptable carrier or excipient.

[0029] Further provided is 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.

[0030] In a further aspect, the present invention provides a method of preventing or treating an autoimmune disease and / or an alloimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to the preceding aspect of the invention, or a pharmaceutical composition according to the invention. In a particular embodiment, the disease or condition is selected from the group consisting of inflammatory bowel disease (IBD), multiple sclerosis (MS), graft-versus-host disease (GVHD), allograft rejection, antibody-mediated allograft 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, autoimmune diseases of the adrenal gland, autoimmune encephalitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, immune thrombocytopenia (ITP), autoimmune pulmonary fibrosis (OPF ... or idiopathic thrombocytopenic purpura or idiopathic thrombocytopenic purpura or immune-mediated thrombocytopenia), autoimmune urticaria, Behçet's disease, bullous pemphigoid (BP), cardiomyopathy, Castleman syndrome, celiac sprue dermatitis dermatitis), chronic fatigue immune deficiency syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, COVID-19-mediated orthostatic tachycardia syndrome (POTS), Crohn's disease, delayed graft function after kidney transplantation, dilated cardiomyopathy, discoid lupus, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia and 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 polyneuropathy, juvenile arthritis, Kawasaki disease, lichen planusplanus), lichen sclerosus, lupus erythematosus, systemic lupus erythematosus (SLE), lupus nephritis, membranous neuropathy, membranous nephropathy, Meniere's disease, mixed connective tissue disease, mucous membrane pemphigoid, graft 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, polyfocal 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 arteritis) / giant cell arteritis, thrombotic thrombocytopenic purpura, thyroid ophthalmopathy, ulcerative colitis, uveitis, warm autoimmune hemolytic anemia (wAIHA), dermatitis herpetiformis, vasculitis, antineutrophil cytoplasmic antibody-associated vasculitis, leukoplakia, and Wegner's granulomatosis.

[0031] In a further aspect, the present invention provides a method for preventing or treating a tumor disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to the preceding aspect of the invention, or a pharmaceutical composition according to the invention.

[0032] The present invention also provides a kit comprising an antibody or antigen-binding fragment according to the first or second aspect of the invention, and optionally instructions for use. [Brief description of the drawings]

[0033] [Figure 1]14 monoclonal antibodies that activate latent TGF-β1. A. Schematic representation of results obtained by screening mAbs against GARP or latent TGF-β1 for TGF-β1 activating antibodies. B. Representative results of TGF-β1 reporter assay in HEK-293 cells. HEK-293T cells were transfected to express human (upper panel) or mouse (lower panel) GARP:TGF-β1 complexes as well as firefly luciferase under the control of a TGF-β1 responsive promoter (pCAGA-12), and Renilla luciferase under a constitutive promoter to control for transfection efficiency. Transfected cells were incubated with the indicated mAbs (20 μg / ml) or recombinant human TGF-β1 (rhTGF-β1) at the indicated concentrations. Bar graphs represent the ratio of firefly luciferase normalized to Renilla luciferase activity (mean + SD of three replicates). The shaded histograms represent antibodies that gave firefly / renilla ratios superior to those measured in the absence of rhTGF-β1 or mAb in at least three independent experiments. These mAbs were identified as TGF-β1 activating antibodies. C. EC50 concentrations for the indicated TGF-β1 activating antibodies. [Diagram 2]Bivalent monospecific antibodies and requirements for GARP expression. A. Antibodies activate mouse or human TGF-β1 when used as bivalent monospecific full-length mAbs or Fab'2 fragments, but not when used as monovalent Fab fragments. HEK-293T cells were transfected as shown in FIG. 1. Transfected cells were incubated with 20 μg / ml of the indicated full-length mAbs, Fab or Fab'2 fragments, or with the indicated concentrations of rhTGF-β1. Bar graphs represent the ratio of firefly luciferase normalized to Renilla luciferase activity (mean + SD of triplicates). Shaded histograms represent antibodies or antibody fragments that showed an increased signal compared to incubation in the absence of rhTGF-β1. B. TGF-β1-activating antibodies activate latent TGF-β1, regardless of whether latent TGF-β1 is presented by GARP on the cell surface or not. Cells were transfected and incubated with antibodies or rhTGF-β1 as indicated in A. C. Western blot analysis of TGF-β1 content in 25 μg of the indicated mAb preparations or in the indicated amounts of rhTGF-β1 using anti-mature TGF-β1 antibody for ECL revelation. [Diagram 3]The epitopes bound by the activating LMT and LHT mAbs are unique. A. Sequence alignment of human and mouse TGF-β1 proteins. Signal peptide: amino acids 1-29; LAP: amino acids 30-278; mature TGF-β1: amino acids 279-391. B. Binding of LMT and LHT mAbs to HEK-293T cells transfected to express GARP: latent TGF-β1 complexes containing the HA-tagged chimeric form of TGF-β1 diagrammed on the left. Values ​​on the right indicate % binding to a given chimera compared to binding to wild-type (WT) mouse TGF-β1 (100%). C. Alanine scanning analysis of antibody binding to alanine mutants in the region 201-222 of latent TGF-β1. Bar graphs show % binding to a given alanine mutant compared to binding to WT mouse TGF-β1 (100%). Binding less than 50% is indicated by white bars. Mean + SD of 2-3 experiments. D. Alanine scan analysis to identify residues in region 201-222 of latent TGF-β1 required for LHT-22 and LMT-12 to activate the cytokine. Bar graphs show % activation of corresponding single A mutants (alanine mutants) compared to WT TGF-β1 (normalized for expression levels of mutants vs. WT TGF-β1). Less than 50% binding is indicated by white bars. [Figure 4]LMT-10 and LMT-12 increased survival in a model of graft-versus-host disease. A. Schematic of the experimental design. B6D2 F1 mice were injected intraperitoneally with fresh splenocytes (70-90 million) from C57BL / 6 mice on day 0. 400 μg of mAb was injected intraperitoneally once a week starting on day -1. Mice were scored twice a week starting on day 0 and daily during the acute phase of the disease (days 13-20). Scores were determined based on weight loss (1 or 2 points), posture (1 or 2 points), mobility (1 or 2 points), and fur ruffled (1 or 2 points). Mice were euthanized when they reached a score of 6. B. Kaplan-Meier plots showing the proportion of surviving mice at the indicated days after splenocyte transplantation. P values ​​were calculated using the Gehan-Breslow-Wilcoxon test (GraphPad Prism). C. Pooled data from three independent experiments. C. Changes in GvHD scores in individual mice measured in three independent experiments. Ratios showing the proportion of mice that died (above the dotted line) or survived (below the dotted line) from GvHD 30–50 days after allogeneic splenocyte transplantation. [Diagram 5]LMT-12-activated TGF-β1 from a GARP-independent source in a model of graft-versus-host disease. A. Activation of latent TGF-β1 by activating mAbs is suppressed in the presence of blocking anti-GARP mAbs. HEK-293T cells were transfected as shown in FIG. 1 and incubated with antibodies or rhTGF-β1 at the indicated concentrations. Bar graphs represent the ratio of firefly luciferase normalized to Renilla luciferase activity (mean + SD of three replicates). Shaded histograms represent conditions where the firefly / renilla ratio was superior to that measured in the absence of rhTGF-β1 or mAb. B. LMT-12 exerted the same therapeutic activity in GvHD regardless of whether it was administered alone or in combination with the blocking anti-GARP latent TGF-β1 antibody 58A2. Induction and scoring of GvHD was performed as described in FIG. 4. Top panel - Kaplan-Meier plot showing the proportion of mice surviving the experiment, P values ​​were calculated using the Gehan-Breslow-Wilcoxon test (GraphPad Prism). Bottom panel - Changes in GvHD scores in individual mice. Ratios showing the proportion of mice that died (above the dotted line) or survived (below the dotted line) from GvHD 30-50 days after transplantation. [Figure 6] Specificity test of LMT-12 for mouse latent TGF-β1. The specificity of the binding of mAb LMT-12 was tested by flow cytometry as described in Example 9. mAb LMT-12 was found to specifically bind to mouse latent TGF-β1, but not to mouse latent TGF-β2 or mouse latent TGF-β3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of this invention.

[0035] "Transforming Growth Factor β" or "TGF-β" - As used herein, the term "TGF-β" refers to the cytokine "transforming growth factor β". As described elsewhere herein, TGF-β is a pleiotropic cytokine that regulates a multitude of effects, particularly within the immune system. As used herein, the term "TGF-β" is broad enough to encompass the human isoforms of this protein as well as any species homologue. There are three isoforms of TGF-β in humans: TGF-β1, TGF-β2, and TGF-β3. The amino acid sequences of full-length human TGF-β1 and full-length mouse TGF-β1 are shown in the table below as SEQ ID NOs: 1 and 5, respectively. These sequences correspond to the sequences deposited in the Uniprot database as human TGF-β1-P01137-1; and mouse TGF-β1-P04202-1.

[0036] The full-length TGF-β1 protein can be divided into (i) a signal peptide (amino acid residues 1-29); (ii) a latency 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, respectively, for human TGF-β1, and as SEQ ID NOs: 6, 7, and 8, respectively, for mouse TGF-β1. [Table 1] JPEG2025508073000002.jpg59159

[0037] "GARP" - As used herein, the term "GARP" refers to the protein "glycoprotein A repeat dominant", which is also known in the literature as "leucine-rich repeat protein 32" or "LRRC32". GARP is an 80 kDa type I transmembrane protein that functions as a receptor for latent TGF-β, particularly on the surface of regulatory T cells (Stockis et al., Eur J Immunol. 2009 Dec;39(12):3315-22).

[0038] 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. (SEQ ID NO:67)

[0039] "Antibody" or "Immunoglobulin" - As used herein, the term "immunoglobulin" includes a polypeptide having a combination of two heavy chains and two light chains, whether or not it has any associated specific immune reactivity. "Antibody" refers to such a construct that has significant known specific immune reactivity to an antigen of interest, herein latent TGF-β1. The term "latent TGF-β1 antibody" or "TGF-β1 antibody" as used herein refers to an antibody that exhibits immunological specificity for latent TGF-β1, including human latent TGF-β1 and, if specified, its homologous species. Antibodies and immunoglobulins include light and heavy chains, with or without interchain covalent bonds. The basic immunoglobulin structure of vertebrate systems is relatively well understood.

[0040] The collective term "immunoglobulin" includes five distinct, biochemically distinct classes of antibodies. All five classes of antibodies are within the scope of the present invention. The following discussion generally relates to the IgG class of immunoglobulin molecules. With respect to IgG, immunoglobulins contain two identical light chains of molecular weight approximately 23,000 daltons, and two identical heavy chains of molecular weight 53,000-70,000 daltons. The four chains are linked by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains, beginning at the mouth of the "Y" and continuing through the variable region.

[0041] The light chains of antibodies are classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with a kappa or lambda light chain. Usually, the light and heavy chains are covalently linked to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. In the heavy chain, the amino acid sequence runs from the N-terminus at the forked ends of the Y-shape to the C-terminus at the bottom of each chain. Those skilled in the art will understand that heavy chains are classified as gamma, mu, delta, or epsilon, (e.g., γ, μ, α, δ, or ε), with several subclasses within them (e.g., γ1-γ4). It is the nature of this chain that identifies the "class" of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and known to confer functional specialization. Variations of these classes and isotypes are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention.

[0042] As shown 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 present at the end of each arm of the Y. More specifically, the antigen-binding site is determined by three complementarity determining regions (CDRs) on each of the VH and VL chains. As used herein, the term "antibody" is also intended to encompass "VHH antibodies" or "heavy chain only antibodies".

[0043] "VHH antibody" - As used herein, the term "VHH antibody" or "heavy chain only antibody" refers to a type of antibody produced exclusively by Camelidae species, including 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 conventional heterotetrameric antibodies, i.e., the VH domains described above.

[0044] "Epitope" - As used herein, the term "epitope" refers to a region of a TGF-β1 protein to which an antibody binds. An antibody typically binds to its respective TGF-β1 epitope via a complementary binding site on the antibody. The epitope to which an antibody binds typically comprises multiple amino acids from the TGF-β1 protein. An epitope may comprise amino acids that are contiguous in the TGF-β1 protein, i.e., a linear epitope, or may comprise amino acids that are not contiguous in the TGF-β1 protein, i.e., a conformational epitope.

[0045] "Binding Site" - As used herein, the term "binding site" includes the region of an antibody or antigen-binding fragment that is involved in 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. Antibodies of the invention may comprise a single binding site or multiple (e.g., two, three, or four) binding sites.

[0046] "Variable region" or "variable domain" - the terms "variable region" and "variable domain" are used interchangeably herein and are intended to have equivalent meanings. The term "variable" refers to the fact that certain portions of the variable domains VH and VL vary widely in sequence between antibodies and are used in the binding and specificity of each particular antibody to its target antigen. However, the variability is not uniformly distributed throughout 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 a Vλ light chain domain are referred to herein as L1(λ), ​​L2(λ) and L3(λ), and in the VL domain may be defined as comprising residues 24-33 (9, 10 or 11 amino acid residues, L1(λ)), residues 49-53 (3 residues, L2(λ)), and residues 90-96 (5 residues, L3(λ)) (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 in the VL domain may be defined as comprising residues 25-33 (6, 7, 8, 11, 12 or 13 residues, L1(κ)), residues 49-53 (3 residues, L2(κ)) and residues 90-97 (6 residues, L3(κ)) (Morea et al., Methods 20:267-279 (2000)). The first, second and third hypervariable loops of a VH domain are referred to herein as H1, H2 and H3, and may be defined in the VH domain as comprising residues 25-33 (H1, consisting of 7, 8 or 9 residues), residues 52-56 (H2, consisting of 3 or 4 residues) and residues 91-105 (H3, highly variable in length) (Morea et al., Methods, 2000;20:267-279).

[0047] Unless otherwise specified, the terms L1, L2 and L3 refer to the first, second and third hypervariable loops, respectively, of the VL domain and include hypervariable loops from both Vκ and Vλ isotypes, and the terms H1, H2 and H3 refer to the first, second and third hypervariable loops, respectively, of the VH domain and include hypervariable loops from any known heavy chain isotype, including gamma, epsilon, delta, alpha or mu.

[0048] Each of the hypervariable loops L1, L2, L3, H1, H2 and H3 may comprise a portion of the "complementarity determining region" or "CDR" as defined below. The terms "hypervariable loop" and "complementarity determining region" are not strictly synonymous. This is because hypervariable loops (HV) are defined on the basis of structure, whereas complementarity determining regions (CDRs) are defined on the basis of 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 ends of HV and CDR may differ in some VH and VL domains.

[0049] The CDRs of the VL and VH domains are usually defined to include 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, HVs may be contained within the corresponding CDRs, and references herein to "hypervariable loops" of the VH and VL domains should also be construed to encompass the corresponding CDRs, and vice versa, unless otherwise specified.

[0050] The more highly conserved parts of the variable domains are called framework regions (FRs), as defined below. Each of the naturally occurring heavy and light chain variable domains contains four FRs (FR1, FR2, FR3 and FR4, respectively), which are mainly arranged in a β-sheet and connected by three hypervariable loops. The hypervariable loops in each chain are held together in close proximity by the FRs and, together with the hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of the antibody. Structural analysis of antibodies has revealed a relationship between the sequence and shape of the binding site formed by the complementarity determining regions (Chothia et al., J. Mol. Biol. 227:799-817 (1992)); Tramontano et al., J. Mol. Biol, 215:175-182 (1990)). Despite their high sequence variability, five of the six loops adopt only a few types of main-chain conformations, called "canonical structures". These conformations are determined firstly by the length of the loop and secondly by the presence of key residues at specific positions in the loop and in the framework regions that determine the conformation by their packing, hydrogen bonding or ability to adopt rare main-chain conformations.

[0051] "CDR" - As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions are described by Kabat et al., J.Biol.Chem.252,6609-6616(1977)and Kabat et al.,Sequences of proteins of immunological interest.(1991),and by Chothia et al., J.Mol.Biol.196:901-917(1987)and by MacCallum et al., J.Mol.Biol.262:732-745(1996), where the definitions include overlapping or subsets of amino acid residues when compared against each other. The amino acid residues encompassing the CDRs as defined by each of the above cited references are listed for comparison. Preferably, the term "CDR" refers to the CDRs defined by Kabat based on sequence comparison. [Table 2]

[0052] "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 CDRs (e.g., using the Kabat definition of CDRs). Thus, the variable region framework is about 100-120 amino acids long, but includes only amino acids other than the CDRs. For the specific example of a heavy chain variable domain, and for the CDRs defined by Kabat et al., framework region 1 corresponds to the domain of the variable region encompassing amino acids 1-30; framework region 2 corresponds to the domain of the variable region encompassing amino acids 36-49; framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94, and framework region 4 corresponds to the domain of the variable region from amino acid 103 to the amino acid at the end of the variable region. The framework regions for the light chain are similarly separated by each of the light chain variable region CDRs. Similarly, using the CDR definitions by Chothia et al., or McCallum et al., the framework region boundaries are separated by the respective CDR ends as described above. In a preferred embodiment, the CDRs are as defined by Kabat.

[0053] In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, non-contiguous sequences of amino acids that are specifically arranged to form an antigen-binding site when the antibody assumes its three-dimensional shape in an aqueous environment. The remainder of the variable domains of the heavy and light chains show less inter-molecular diversity in amino acid sequence and are called framework regions. The framework regions mainly adopt a β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, these framework regions act to form a scaffold for positioning the six CDRs in a correct orientation by inter-chain non-covalent interactions. The antigen-binding site formed by the arranged CDRs determines the surface complementarity to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. The location of the CDRs can be easily identified by those skilled in the art.

[0054] "Constant Region" - As used herein, the term "constant region" refers to the portion of an antibody molecule excluding the variable domain or regions. Immunoglobulin light chains have a single domain, the "constant region", usually called the "CL or CL1 domain". This domain is located C-terminal to the VL domain. Immunoglobulin heavy chains differ in their constant regions depending on the immunoglobulin class (gamma, mu, alpha, delta, epsilon). Heavy chains gamma, alpha, and delta have a constant region consisting of three immunoglobulin domains (called CH1, CH2, and CH3) with a flexible hinge region separating the CH1 and CH2 domains. Heavy chains mu and epsilon have a constant region consisting of four domains (CH1-CH4). The constant domain of the heavy chains is located C-terminal to the VH domain.

[0055] The numbering of the amino acid sequences in immunoglobulin heavy and light chains is done from the N-terminus at the forked end of the Y-shape to the C-terminus at the end of each chain. Different numbering schemes are used to define the constant domains of immunoglobulin heavy and light chains. According to the EU numbering scheme, the heavy chain constant domains of an 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 an IgG molecule are identified as follows: CH1-amino acid residues 114-223; CH2-amino acid residues 244-360; CH3-amino acid residues 361-477. The "Fc domain" or "Fc region" usually defines the portion of the constant region of the heavy chain that includes 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 to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three separate regions: the upper, middle, and lower hinge domains (Roux KHet al. J. Immunol. 161:4083-90 1998). Antibodies of the invention that contain a "fully human" hinge region may contain one of the hinge region sequences shown in Table 3 below. [Table 3]

[0056] "Antigen-binding fragment" - As used herein, the term "antigen-binding fragment" or "fragment" refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds an antigen or competes with an intact antibody (i.e., the intact antibody from which it is derived) for antigen binding (i.e., specific binding to TGF-β1). As used herein, the term "antigen-binding fragment" includes, for example, an antibody light chain variable domain (VL); an antibody heavy chain variable domain (VH); a single-chain antibody (scFv); an F(ab')2 fragment; an Fab fragment; an Fd fragment; an Fv fragment; a one-armed (monovalent) antibody; a diabody; a triabody; a tetrabody; or any antigen-binding molecule formed by such antigen-binding combinations, aggregations, or conjugations. As used herein, the term "antigen-binding fragment" is further intended to encompass an antibody fragment selected from the group consisting of a unibody, a domain antibody, and a nanobody. Fragments can be obtained, for example, by chemical or enzymatic treatment of an intact or complete antibody or antibody chain, or by recombinant means.

[0057] "Fab" - "Fab" or "Fab fragment" refers to a molecule composed of a heavy and a light chain, where the light chain consists of a VL domain and one constant domain (CL, Cκ or Cλ) and the heavy chain consists only of a VH domain and a CH1 domain. A Fab fragment is usually one arm of a conventional Y-shaped immunoglobulin molecule. Fab fragments can be generated from immunoglobulin molecules by the action of the enzyme papain. Papain cleaves the immunoglobulin molecule at the hinge region, thereby generating two Fab fragments and a separate Fc region.

[0058] "scFv" or "scFv fragment" - "scFv" or "scFv fragment" refers to a single chain variable fragment. An scFv is a fusion protein of the VH and VL domains of an antibody linked by a linker.

[0059] "Valency" - As used herein, the term "valency" refers to the number of possible target or antigen binding sites in an antibody. Each target or antigen binding site specifically binds one antigen or a particular site on a target antigen. If an antibody contains more than one target binding site, each target binding site may specifically bind the same or different molecules (e.g., may bind different ligands or different antigens, or different epitopes on the same antigen).

[0060] "Specificity" and "Multispecific Antibodies" - As used herein, the term "specificity" refers to the ability to bind (e.g., immunoreact with) a given target, e.g., TGF-β1. The antibodies described herein preferably "specifically bind" to their target antigen, and the term "specifically binds" refers to the ability of any antibody to selectively immunoreact with a given target. The antibodies described herein may be monospecific and contain one or more binding sites that specifically bind a particular target. The antibodies described herein may also be in the format of a "multispecific antibody," e.g., a bispecific antibody, which binds to more than one target antibody.

[0061] "Derived from" - As used herein, the term "derived from" a specified protein (e.g., a camelid antibody or 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 particular starting polypeptide is a CDR sequence or a sequence related thereto. In one embodiment, a polypeptide or amino acid sequence derived from a particular starting polypeptide is not contiguous. For example, in one embodiment, one, two, three, four, five, or six CDRs are derived from the starting antibody. In one embodiment, a polypeptide or amino acid sequence derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is substantially identical to the starting sequence or a portion thereof, which portion consists 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 which is otherwise identifiable by one of skill in the art as having its origin in the starting sequence. In one embodiment, one or more CDR sequences derived from a starting antibody are modified to produce variant CDR sequences, e.g., affinity variants, where the variant CDR sequences maintain target antigen binding activity.

[0062] "Camelid-derived" - In certain embodiments, the antibodies of the invention comprise framework and / or CDR amino acid sequences derived from camelid conventional or VHH antibodies produced by active immunization of camelids. However, antibodies of the invention comprising camelid-derived amino acid sequences may be engineered to include human amino acid sequences (i.e., human antibodies) or framework and / or constant regions derived from 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 a TGF-β1 antibody. In one embodiment, one or more non-camelid amino acids may be present in the framework regions of a "camelid-derived" antibody, for example, the camelid framework amino acid sequence may include one or more amino acid mutations, where a corresponding human or non-human primate amino acid residue is present. Furthermore, camelid-derived VH and VL domains, or humanized variants thereof, may be linked to constant domains of human antibodies, as described elsewhere herein, to produce chimeric molecules.

[0063] "Conservative Amino Acid Substitution" - A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include amino acids with 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), nonpolar 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 can be replaced with another amino acid residue from the same side chain family. In another embodiment, a series of amino acids can be replaced with structurally similar sequences that differ in the order and / or composition of side chain family members.

[0064] "Heavy chain portion" - As used herein, the term "heavy chain portion" includes amino acid sequences derived from a constant domain of an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. In one embodiment, an antibody or antigen-binding fragment of the invention may include an Fc portion of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In another embodiment, an antibody or antigen-binding fragment of the invention may lack at least a portion of a constant domain (e.g., all or a portion of a CH2 domain). In certain embodiments, at least one, and preferably all, of the constant domains are derived from a human immunoglobulin heavy chain. For example, in a preferred embodiment, the heavy chain portion includes a complete human hinge domain. In other preferred embodiments, the heavy chain portion includes a complete human Fc portion (e.g., hinge, CH2, and CH3 domain sequences from a human immunoglobulin).

[0065] In certain embodiments, the component constant domains of the heavy chain portion are derived from different immunoglobulin molecules. For example, the heavy chain portion of the polypeptide may comprise 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, the hinge may comprise a first portion derived from an IgG1 molecule and a second portion from an IgG3 or IgG4 molecule. As mentioned above, one skilled in the art will appreciate that the constant domains of the heavy chain portion may be modified such that they differ in amino acid sequence from the naturally occurring (wild type) immunoglobulin molecule. That is, the polypeptides of the invention disclosed herein may comprise changes or modifications to one or more heavy chain constant domains (CH1, hinge, CH2 or CH3) and / or to the light chain constant region domain (CL). Exemplary modifications include addition, deletion or substitution of one or more amino acids in one or more domains.

[0066] "Chimeric" - A "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence, which is not naturally linked to the second amino acid sequence in nature. The amino acid sequences may normally exist as separate proteins that are brought together in a fusion polypeptide, or they may normally exist in the same protein, but are arranged in a novel arrangement in the fusion polypeptide. Chimeric proteins may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. Exemplary chimeric antibodies of the invention include fusion proteins comprising VH and VL domains from a camelid, or humanized variants thereof, fused to the constant domains of a human antibody, e.g., human IgG1, IgG3 or IgG4.

[0067] "Synthetic" - As used herein, the term "synthetic" in reference to a polypeptide includes a polypeptide that includes an amino acid sequence that is not naturally occurring, e.g., a modified version of a naturally occurring polypeptide (e.g., containing mutations such as additions, substitutions or deletions), or a non-naturally occurring polypeptide that includes a first amino acid sequence (which may or may not be naturally occurring) linked to a second amino acid sequence (which may or may not be naturally occurring) to which it is not naturally linked in the linear sequence of amino acids.

[0068] "Engineered" - As used herein, the term "engineered" includes the manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, by in vitro peptide synthesis, enzymatic or chemical coupling of peptides or some combination of these techniques). Preferably, the antibodies of the invention have been engineered, and include, for example, humanized and / or chimeric antibodies, and antibodies that have been engineered to improve one or more properties, such as antigen binding, stability / half-life or effector function.

[0069] "Modified antibodies" - As used herein, the term "modified antibodies" includes synthetic forms of antibodies that are not of natural origin, e.g., antibodies that contain at least two heavy chain portions but not two complete heavy chains (e.g., domain deleted antibodies or minibodies); multispecific forms of antibodies that are altered to bind to two or more different antigens or to different epitopes on a single antigen (e.g., bispecific, trispecific, etc.); heavy chain molecules combined with scFv molecules; and the like. 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 antibodies" 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 antibodies of the invention are fusion proteins that contain at least one heavy chain portion that lacks a CH2 domain and that contain a binding domain of a polypeptide that contains the binding portion of one member of a receptor-ligand pair.

[0070] The term "modified antibody" may also be used herein to refer to amino acid sequence variants of the antibodies of the present invention as structurally defined herein. It will be understood by those skilled in the art that an antibody may be modified to produce a variant antibody having an altered amino acid sequence compared to the antibody from which it was derived. For example, nucleotide or amino acid substitutions resulting in conservative substitutions, or changes at "non-essential" amino acid residues may be made (e.g., in CDR and / or framework residues). Amino acid substitutions may include the replacement of one or more amino acids with naturally occurring or non-naturally occurring amino acids.

[0071] "Humanizing substitution" - As used herein, the term "humanizing substitution" refers to an amino acid substitution in which an amino acid residue present at a particular position in a VH or VL domain of an antibody (e.g., a camelid-derived TGF-β1 antibody) is replaced with an amino acid residue occurring at the equivalent position in a reference human VH or VL domain. The reference human VH or VL domain may be a human germline encoded VH or VL domain. Humanizing substitutions may be made in the framework regions and / or CDRs of an antibody as defined herein.

[0072] "Humanized variant" - As used herein, the term "humanized variant" refers to a variant antibody that contains one or more "humanizing substitutions" compared to a reference antibody, where a portion of the reference antibody (e.g., the VH domain and / or the VL domain or a portion thereof comprising at least one CDR) has amino acids derived from a non-human species, and the "humanizing substitutions" occur within the amino acid sequence derived from the non-human species.

[0073] "Germline variant" - As used herein, the term "germline variant" specifically refers to a "humanized variant" in which a "humanizing substitution" results in the replacement of one or more amino acid residues present at a particular position in a VH or VL domain of a non-human antibody with an amino acid residue that occurs at the equivalent position in a reference human VH or VL domain encoded by human germline. For any given "germline variant", the substituted amino acid residues substituted into the germline variant are obtained exclusively or primarily 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 production of a "humanized variant" of the camelid (llama)-derived VH or VL domain. When the substituted amino acid residues are derived primarily or exclusively 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.

[0074] "Affinity variant" - As used herein, the term "affinity variant" refers to a variant antibody that exhibits one or more changes in the amino acid sequence compared to a reference antibody, and the affinity variant exhibits an altered affinity for the target antigen compared to the reference antibody. For example, the affinity variant exhibits an altered affinity for TGF-β1 compared to a reference TGF-β antibody. Preferably, the affinity variant exhibits an improved affinity for the target antigen compared to the reference antibody. The affinity variant usually exhibits one or more changes in the amino acid sequence in the CDR compared to the reference antibody. Such substitutions may result in the replacement of an original amino acid present at a given position in the CDR with a different amino acid residue, which may be a naturally occurring amino acid residue or a non-naturally occurring amino acid residue. The amino acid substitutions may be conservative or non-conservative.

[0075] "Highly human homologous" - An antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) may be considered to have highly human homology if the VH and VL domains, taken together, exhibit at least 90% amino acid sequence identity with the closest matching human germline VH and VL sequences. Antibodies with highly human homology may include antibodies comprising the VH and VL domains of a conventional antibody, for example from a camelid, as well as engineered, particularly humanized or germline variants of such antibodies, and also antibodies comprising the VH and VL domains of a naturally occurring non-human antibody that exhibit a sufficiently high % sequence identity with human germline sequences, including "fully human" antibodies.

[0076] 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 even 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 compared to the original non-human VH domain sequence from which they are derived.

[0077] In one embodiment, the VH domain of an antibody with high human homology may contain one or more (e.g., 1 to 10) amino acid sequence mismatches across the framework regions FR1, FR2, FR3 and FR4 compared to the most closely matching human VH sequence.

[0078] In another embodiment, the VL domain of an antibody with high human homology may show 80% or more sequence identity or sequence homology with one or more human VL domains across the framework regions FR1, FR2, FR3 and FR4. In other embodiments, the amino acid sequence identity or sequence homology between the VL domain of the polypeptide of the invention and the closest matching human germline VL domain sequence may be 85% or more, 90% or more, 95% or more, 97% or more, or even up to 99% or even 100%. The VL domain variants with high human homology may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions compared to the original non-human VL domain sequence from which they are derived.

[0079] In one embodiment, the VL domain of an antibody with high human homology may contain one or more (e.g., 1 to 10) amino acid sequence mismatches across the framework regions FR1, FR2, FR3 and FR4 compared to the most closely matching human VL sequence.

[0080] B. TGF-β1 activating antibodies that bind latent TGF-β1 TGF-β1 activation In a first aspect, the invention provides antibodies, or antigen-binding fragments thereof, that bind to latent TGF-β1, and which antibodies or antigen-binding fragments thereof activate TGF-β1. The antibodies and antigen-binding fragments described herein are therefore referred to as either: "TGF-β1 activating antibodies"; "TGF-β1 activating antibodies"; "anti-latent TGF-β1 activating antibodies"; or "anti-latent TGF-β1 antibodies that activate the release of TGF-β1."

[0081] As described elsewhere herein, latent TGF-β1 consists of a TGF-β1 homodimer, with the two LAP domains of the TGF-β protein cleaved from the mature TGF-β1 cytokine domain at the C-terminus. The two N-terminal LAP polypeptides remain covalently linked to the C-terminal mature TGF-β1 cytokine, thereby preventing TGF-β1 from binding to its receptor complex and initiating downstream signaling cascades. The antibodies and antigen-binding fragments described herein can bind to the latent TGF-β1 complex and release the mature TGF-β1 cytokine from inhibition by the LAP domains. This can involve physical dissociation of the LAP polypeptides from the mature TGF-β1 cytokine. Alternatively, the antibodies or antigen-binding fragments described herein can bind to latent TGF-β1 and cause a deformation of the latent complex, thereby exposing the mature TGF-β1 cytokine, which can bind to the TGF-β receptor complex. The form of mature TGF-β1 that is free of inhibition from LAP and capable of binding to the TGF-β receptor complex is referred to herein as "active TGF-β1."

[0082] As used herein, "activation of TGF-β1" refers to the conversion of latent TGF-β1 to active TGF-β1. This activation can be measured by various methods, including, but not limited to, in vitro methods such as Western blotting, ELISA, TGF-β1 reporter assay, and TGF-β1 activity assay.

[0083] In certain embodiments, the activation of TGF-β1 is measured by exposing a cell expressing a 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 in addition, TGF-β1 activation can be measured by detecting an increase in phosphorylation of an R-Smad protein, such as Smad-2. The phosphorylation state of the TGF-β receptor complex and / or an R-Smad protein (e.g., Smad-2) can be measured using any suitable technique, including, but not limited to, Western blotting and ELISA. The level of phosphorylation observed when cells are exposed to a combination of latent TGF-β1 and an antibody or antigen-binding fragment of the present invention can be compared to one or more controls. For example, cells expressing the TGF-β receptor complex can be exposed to a range of concentrations of active TGF-β1, whereby the phosphorylation state of the TGF-β receptor complex and / or R-Smad proteins (e.g., Smad-2) can be determined in the presence of known concentrations of active TGF-β1. The level of phosphorylation observed when an antibody or antigen-binding fragment of the invention binds to latent TGF-β1 can then be compared to the activation observed in the presence of a standard concentration of active TGF-β1.

[0084] In certain embodiments, activation of TGF-β1 can be measured using an in vitro assay in which changes in gene expression serve as an indicator of TGF-β1 activation. Such assays include, but are not limited to, TGF-β1 reporter assays, such as the reporter assays described in the Examples presented herein below. TGF-β1 reporter assays typically involve the use of a TGF-β1-responsive reporter construct, in which a TGF-β1-responsive element (e.g., as 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 expression of the reporter gene. The TGF-β1-responsive element is a Smad complex binding element, such as (CAGA) 12(as described in Dennler et al., EMBO J. 1998;17:3091-3100).

[0085] A reporter gene can be any gene that codes for a protein whose expression can be measured and / or quantified.Preferred reporter genes include, but are not limited to, reporter genes that code for GFP and luciferase.The TGF-β1 reporter construct exemplified herein incorporates a luciferase reporter gene, so that the increase in luminescence can be used as a measure of TGF-β1 activation.

[0086] A complete TGF-β1 reporter assay may include (i) engineering cells to carry a TGF-β1 reporter 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 a reporter gene. As exemplified herein, cells engineered to carry a TGF-β1 reporter may also be engineered to express latent TGF-β1 protein. In certain embodiments, cells used in a TGF-β1 reporter assay are engineered to express latent TGF-β1 and GARP, whereby latent TGF-β1 is anchored to the cell surface. Controls may be incorporated into assays such as those described herein to control for gene transfer efficiency for the TGF-β1 reporter construct.

[0087] As described above with respect to assays based on measuring phosphorylation levels, the level of activity observed in the 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, thereby allowing the readout from the TGF-β1 reporter construct to be normalized to one or more known concentrations of active TGF-β1.The activation of the TGF-β1 reporter construct observed when the antibody or antigen-binding fragment of the present invention binds to latent TGF-β1 can then be compared to the activation observed in the presence of a standard concentration of active TGF-β1.

[0088] Alternatively or additionally, activation mediated by the antibodies and antigen-binding fragments described herein may be measured in comparison to activation mediated under standard or physiological conditions found in vitro or in vivo. As described elsewhere herein, latent TGF-β1 may be activated through various mechanisms, including binding of the "RGD" motif in LAP to integrins, binding to thrombospondin, and by protease-mediated cleavage. Given that binding of latent TGF-β1 to αVβ6 and αVβ8 is well established as the physiological mechanism by which latent TGF-β1 is activated in vivo, activation in the presence of the antibodies or antigen-binding fragments described herein may be measured in vitro in comparison to activation mediated by αVβ6 and / or αVβ8 integrins. A level of activation comparable to or higher than that seen with integrins may classify the antibody as a "TGF-β1 activating antibody."

[0089] The antibodies or antigen-binding fragments of the invention may activate TGF-β1 by binding to uncomplexed latent TGF-β1, i.e., when latent TGF-β1 is not bound by a regulatory protein such as LTBP or GARP. Alternatively or additionally, the antibodies or antigen-binding fragments of the invention may activate TGF-β1 by binding to latent TGF-β1 when latent TGF-β1 is complexed with one or more additional proteins. In certain embodiments, the antibodies or antigen-binding fragments of the 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 localized activation of TGF-β1 within the body, i.e., in the vicinity of cell types expressing GARP. In certain preferred embodiments, the antibodies or antigen-binding fragments described herein activate TGF-β1 when latent TGF-β1 is bound to the surface of Tregs. In certain embodiments, the antibodies or antigen-binding fragments described herein activate TGF-β1 when latent TGF-β1 is bound to the surface of megakaryocytes, platelets and / or endothelial cells. In certain embodiments, the antibodies or antigen-binding fragments described herein activate TGF-β1 when latent TGF-β1 is bound to the surface of B lymphocytes, fibroblasts, mesenchymal cells and / or hepatic stellate cells.

[0090] Latent TGF-β1 may be bound or anchored to the cell surface via a protein 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 bound to or anchored to the surface of one or more cell types selected from the group consisting of myeloid cells, lymphoid cells, hematopoietic cells, and pre-malignant stage cancer cells.

[0091] The TGF-β1 activating antibodies or antigen-binding fragments of the invention may bind to human latent TGF-β1 and / or to mouse latent TGF-β1. The sequence of human latent TGF-β1 is represented by SEQ ID NO:1, and the sequence of mouse latent TGF-β1 is represented by SEQ ID NO:5. The TGF-β1 activating antibodies and antigen-binding fragments described herein typically bind either human latent TGF-β1 or mouse latent TGF-β1, i.e., they are not cross-reactive between these two species. The TGF-β1 activating antibodies or antigen-binding fragments of the invention may exhibit binding specificity for TGF-β1, i.e., the antibodies or antigen-binding fragments bind to TGF-β1 but not to other isoforms, particularly TGF-β2 and TGF-β3.

[0092] Epitope The TGF-β1 activating antibodies or antigen-binding fragments of the present invention can be characterized by their unique binding epitopes within latent TGF-β1.

[0093] In some embodiments, the binding of an amino acid of a TGF-β1 antibody or antigen-binding fragment to an epitope within latent TGF-β1 refers to physical interactions well known in the art, including covalent bonds or hydrogen bonds.

[0094] In some embodiments, amino acid contacts of a TGF-β1 antibody or antigen-binding fragment to an epitope within latent TGF-β1 refer to non-physical interactions that contribute to the activation of latent TGF-β1.

[0095] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to an epitope that includes one or more amino acids from the region of TGF-β1 consisting of amino acids 87-222 (these amino acid positions are determined relative to the full-length sequences of the human and mouse TGF-β1 proteins, set forth in SEQ ID NOs: 1 and 5, respectively). In certain embodiments, the epitope includes two amino acids from within these regions.

[0096] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to an epitope that includes one or more amino acids from the region of TGF-β1 consisting of amino acids 87-135 (these amino acid positions are determined relative to the full-length sequences of the human and mouse TGF-β1 proteins, set forth in SEQ ID NOs: 1 and 5, respectively). In certain embodiments, the epitope includes two amino acids from within these regions.

[0097] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to an epitope that includes one or more amino acids from the region of TGF-β1 consisting of amino acids 136-200 (these amino acid positions are determined relative to the full-length sequences of the human and mouse TGF-β1 proteins, as set forth in SEQ ID NOs: 1 and 5, respectively). In certain embodiments, the epitope includes two amino acids from within these regions.

[0098] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to an epitope that includes one or more amino acids from the region of TGF-β1 consisting of amino acids 201-222 (these amino acid positions are determined relative to the full-length sequences of the human and mouse TGF-β1 proteins, as set forth in SEQ ID NOs: 1 and 5, respectively). In certain embodiments, the epitope includes two amino acids from within these regions.

[0099] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to a conformational epitope that requires one or more amino acids from the region of TGF-β1 consisting of amino acids 87-222 (these amino acid positions are determined relative to the full-length sequences of the human and mouse TGF-β1 proteins, set forth in SEQ ID NOs: 1 and 5, respectively). In certain embodiments, the conformational epitope requires two amino acids from within these regions.

[0100] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to a conformational epitope that requires one or more amino acids from the region of TGF-β1 consisting of amino acids 87-135 (these amino acid positions are determined relative to the full-length sequences of the human and mouse TGF-β1 proteins, set forth in SEQ ID NOs: 1 and 5, respectively). In certain embodiments, the conformational epitope requires two amino acids from within these regions.

[0101] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to a conformational epitope that requires one or more amino acids from the region of TGF-β1 consisting of amino acids 136-200 (these amino acid positions are determined relative to the full-length sequences of the human and mouse TGF-β1 proteins, set forth in SEQ ID NOs: 1 and 5, respectively). In certain embodiments, the conformational epitope requires two amino acids from within these regions.

[0102] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to a conformational epitope that requires one or more amino acids from the region of TGF-β1 consisting of amino acids 201-222 (these amino acid positions are determined relative to the full-length sequences of the human and mouse TGF-β1 proteins, set forth in SEQ ID NOs: 1 and 5, respectively). In certain embodiments, the conformational epitope requires two amino acids from within these regions.

[0103] The region of TGF-β1 spanning amino acids 87-222 differs between the human and mouse TGF-β1 proteins as follows: Human latent TGF-β1: (aa87~222) [ka] Mouse latent TGF-β1: (aa87~222) [ka]

[0104] The region of TGF-β1 spanning amino acids 87-135 differs between the human and mouse TGF-β1 proteins as follows: Human latent TGF-β1: (aa87~135) [ka] [ka]

[0105] The region of TGF-β1 spanning amino acids 136-200 differs between the human and mouse TGF-β1 proteins as follows: Human latent TGF-β1: (aa136~200) [ka] [ka]

[0106] 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 (as represented by SEQ ID NOs: 3 and 7 - human and mouse, respectively). This region is located close to the dimerization interface of the latent TGF-β1 protein (see Shi et al., Nature 2011;474:343-49). The "cryptic loop" or "latent lasso", i.e., the region of LAP that binds most closely to mature TGF-β1 in the latent complex (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 that includes amino acids from a region of LAP distal to the latent loop or latent lasso. These antibodies or antigen-binding fragments are nevertheless capable of activating TGF-β1 by relieving the inhibition mediated by LAP.

[0107] The region of TGF-β1 consisting of amino acids 201-222 differs between the human and mouse TGF-β1 proteins as follows: Human latent TGF-β1: (aa201-222) [ka] [ka]

[0108] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to human latent TGF-β1 binds to an epitope including 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, or a conformational epitope that requires these amino acid residues.

[0109] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to human latent TGF-β1 contacts an epitope including 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, or a conformational epitope requiring these amino acid residues.

[0110] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to human latent TGF-β1 binds to an epitope that includes amino acid residues E119 and / or K123 of the full-length TGF-β1 protein represented by SEQ ID NO: 1. In the case of an antibody or antigen-binding fragment that binds to an epitope that includes E119 and / or K123 of human latent TGF-β1, in certain embodiments, the epitope does not include additional amino acid residues from within the region of human latent TGF-β1 consisting of amino acids 87-135.

[0111] In certain embodiments, a TGF-β1 activating 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. In the case of an antibody or antigen-binding fragment that binds to a conformational epitope that requires E119 and / or K123 of human latent TGF-β1, in certain embodiments, the epitope does not require additional amino acid residues from within the region of human latent TGF-β1 consisting of amino acids 87-135.

[0112] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to human latent TGF-β1 contacts a region within amino acids 87-135 of human latent TGF-β1 that includes amino acid residues D122, F124 and / or F134 of the full-length TGF-β1 protein represented by SEQ ID NO:1, or a conformational epitope that requires these amino acid residues.

[0113] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to human latent TGF-β1 binds to an epitope that includes amino acid residues S138 and / or R141 of the full-length TGF-β1 protein represented by SEQ ID NO: 1. In the case of an antibody or antigen-binding fragment that binds to an epitope that includes S138 and / or R141 of human latent TGF-β1, in certain embodiments, the epitope does not include additional amino acid residues from within the region of human latent TGF-β1 consisting of amino acids 136-200.

[0114] In certain embodiments, a TGF-β1 activating 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. In the case of an antibody or antigen-binding fragment that binds to a conformational epitope that requires S138 and / or R141 of human latent TGF-β1, in certain embodiments, the epitope does not require additional amino acid residues from within the region of human latent TGF-β1 consisting of amino acids 136-200.

[0115] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to human latent TGF-β1 contacts a region within amino acids 136-200 of human latent TGF-β1 that includes 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, or a conformational epitope requiring these amino acid residues.

[0116] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to human latent TGF-β1 binds to an epitope that includes amino acid residues S209, R210, G212, E213, I214, and / or E215 of the full-length TGF-β1 protein represented by SEQ ID NO: 1. In the case of an antibody or antigen-binding fragment that binds to an epitope that includes S209, R210, G212, E213, I214, and / or E215 of human latent TGF-β1, in certain embodiments, the epitope does not include additional amino acid residues from within the region of human latent TGF-β1 consisting of amino acids 201-222. In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment does not bind to R205 and / or R210 and / or E213.

[0117] In certain embodiments, a TGF-β1 activating 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 additional amino acid residues from within the region of human latent TGF-β1 consisting of amino acids 201-222. In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment binds to a conformational epitope that does not require R205 and / or R210 and / or E213.

[0118] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to human latent TGF-β1 contacts a conformational epitope that includes or requires amino acid residues L208 and / or G211 of the full-length TGF-β1 protein represented by SEQ ID NO: 1 within a region of human latent TGF-β1 consisting of amino acids 201 to 222. In one embodiment, a conformational epitope that includes or requires amino acid residues L208 and / or G211 of the full-length TGF-β1 protein represented by SEQ ID NO: 1 is required for activity of the activating TGF-β1 antibody or antigen-binding fragment thereof.

[0119] In certain embodiments, the amino acid residues of human latent TGF-β1 represented by SEQ ID NO:1 required for activation of a TGF-β1 activating antibody or antigen-binding fragment include G212 and E215. In certain embodiments, the human latent TGF-β1 protein represented by SEQ ID NO:1 required for activation of a TGF-β1 activating antibody or antigen-binding fragment includes W207, L208, G211, G212, I214, E215, and F217.

[0120] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to mouse latent TGF-β1 binds to a conformational epitope that includes or requires 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.

[0121] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to mouse latent TGF-β1 binds to an epitope that includes amino acid residues S138, R141, P145, E146, and / or S175 of the full-length TGF-β1 protein represented by SEQ ID NO:5. For an antibody or antigen-binding fragment that binds to an epitope that includes S138, R141, P145, E146, and / or S175 of mouse latent TGF-β1, in certain embodiments, the epitope does not include additional amino acid residues from within the region of mouse latent TGF-β1 consisting of amino acids 136-200. In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment does not bind to G211 and / or F217.

[0122] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to mouse latent TGF-β1 binds to a conformational epitope that requires 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 conformational epitopes S138, R141, P145, E146, and / or S175 of mouse latent TGF-β1, in certain embodiments, the epitope does not require additional amino acid residues from within the region of mouse latent TGF-β1 consisting of amino acids 136-200. In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment binds to a conformational epitope that does not require G211 and / or F217.

[0123] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to mouse latent TGF-β1 contacts a region within amino acids 136-200 of mouse latent TGF-β1 that includes amino acid residues E142, P147, P148, L149, Y174, and / or N176 of the full-length TGF-β1 protein represented by SEQ ID NO:5, or a conformational epitope that requires these amino acid residues.

[0124] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to mouse latent TGF-β1 binds to an epitope that includes amino acid residues L208, N209, and D212 of the full-length TGF-β1 protein represented by SEQ ID NO:5. For an antibody or antigen-binding fragment that binds to an epitope that includes L208, N209, and D212 of mouse latent TGF-β1, in certain embodiments, the epitope does not include additional amino acid residues from within the region of mouse latent TGF-β1 consisting of amino acids 201-222. In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment does not bind to G211 and / or F217.

[0125] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to mouse 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 an antibody or antigen-binding fragment that binds to a conformational epitope that requires L208, N209, and D212 of mouse latent TGF-β1, in certain embodiments, the epitope does not require additional amino acid residues from within the region of mouse latent TGF-β1 consisting of amino acids 201-222. In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment binds to a conformational epitope that does not require G211 and / or F217.

[0126] In certain embodiments, a TGF-β1 activating antibody or antigen-binding fragment that binds to mouse latent TGF-β1 contacts a region within the region of mouse latent TGF-β1 consisting of amino acids 201-222 that includes amino acid residues L208 and G211 of the full-length TGF-β1 protein represented by SEQ ID NO:5, or a conformational epitope that requires these amino acid residues.

[0127] In certain embodiments, the amino acid residues of mouse latent TGF-β1 represented by SEQ ID NO:5 that are required for activation of a TGF-β1 activating antibody or antigen-binding fragment include L208 and / or D212. In certain embodiments, the amino acid residues of mouse latent TGF-β1 represented by SEQ ID NO:5 that are required for activation of a TGF-β1 activating antibody or antigen-binding fragment include L208 and / or D212.

[0128] Antibody and antigen-binding fragment formats The TGF-β1 antibodies and antigen-binding fragments of the present invention may take any suitable form. The term "antibody" is used in the broadest sense and includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), VHH antibodies, so long as they exhibit the appropriate immunological specificity for latent TGF-β1.

[0129] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Moreover, in contrast to conventional (polyclonal) antibody preparations, which usually contain different antibodies against different determinants (epitopes) on the antigen, each monoclonal antibody is directed against a single determinant or epitope on the antigen.

[0130] An "antibody fragment" or "antigen-binding fragment" comprises a portion of a full-length antibody, typically the antigen-binding or variable domain thereof. Antibody fragments are described elsewhere herein, and examples of antibody fragments include Fab, Fab', F(ab')2 bispecific Fab, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, single-chain variable fragments (ScFv); and multispecific antibodies formed from antibody fragments (see Holliger and Hudson, Nature Biotechnol. 23:1126-36 (2005), the contents of which are incorporated herein by reference).

[0131] 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, having two identical antigen-binding sites that immunoreact with the same epitope on latent 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.

[0132] The TGF-β1 antibodies and antigen-binding fragments described herein are intended for human therapeutic use and therefore are usually immunoglobulins of the IgA, IgD, IgE, IgG, IgM type, and often of the IgG type, in which case they belong to one of the four subclasses: IgG1, IgG2a and IgG2b, IgG3 or IgG4. In a preferred embodiment, the TGF-β1 antibodies are IgG antibodies. Particularly preferred are IgG1 antibodies. Monoclonal antibodies are highly specific and are therefore directed against a single antigenic site.

[0133] TGF-β1 antibodies and antigen-binding fragments may exhibit high human homology, as defined elsewhere herein. Such antibody molecules with high human homology may include antibodies that contain VH and VL domains of natural non-human antibodies that 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.

[0134] In a particular embodiment, the TGF-β1 antibody or antigen-binding fragment of the present invention is derived from a camelid. The camelid-derived antibody may be a heavy chain only antibody, i.e. a VHH antibody, or may be a conventional heterotetrameric antibody. In a preferred embodiment, the TGF-β1 and antigen-binding fragment are derived from a camelid heterotetrameric antibody. In a preferred embodiment, the TGF-β1 antibody is derived from a VHH antibody or a VHH antibody.

[0135] For example, TGF-β1 antibodies and antigen-binding fragments can be selected from an immune library obtained by a method comprising immunizing a camelid with a target of interest. The camelid can be immunized with a target protein or a polypeptide fragment thereof, or with an mRNA or cDNA molecule expressing the protein or a polypeptide fragment thereof. Methods for producing antibodies in camelid species and selecting antibodies against preferred targets from camelid immune libraries are described, for example, in International Publication No. WO2020 / 001251, which is incorporated herein by reference.

[0136] In certain embodiments, the TGF-β1 antibodies and antigen-binding fragments may be derived from a camelid in that they comprise at least one hypervariable (HV) loop or complementarity determining region derived from a VH or VL domain of a camelid species. In particular, the TGF-β1 antibodies and antigen-binding fragments may comprise a VH and / or VL domain, or CDRs thereof, derived by active immunization of an outbred camelid, such as a llama, with latent TGF-β1.

[0137] The term "derived from" in this context refers to a structural relatedness, in that the HV or CDRs of the antibody embody an amino acid sequence originally encoded by a camelid immunoglobulin gene (or a minor variant thereof), but this does not necessarily imply a particular relatedness in terms of the manufacturing process used to prepare the antibody or antigen-binding fragment thereof.

[0138] Camelid-derived antibodies or antigen-binding fragments thereof may be derived from any camelid species, including in particular llama, dromedary, alpaca, vicuna, guanaco or camel.

[0139] Antibody molecules comprising VH and VL domains, or CDRs thereof, from camelids are usually recombinantly expressed polypeptides and may be chimeric polypeptides. The term "chimeric polypeptide" refers to an artificial (non-naturally occurring) polypeptide created by juxtaposing two or more peptide fragments that are not otherwise contiguous. Included within this definition are "species" chimeric polypeptides created by juxtaposing peptide fragments encoded by two or more species, for example camelids and humans.

[0140] In certain embodiments, the complete VH domain and / or the complete VL domain may be obtained from a camelid species. The camelid-derived VH domain and / or the camelid-derived VL domain are then subjected to protein engineering, in which 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", in which one or more amino acid residues in a camelid-encoded VH or VL domain are replaced with the equivalent residue from a homologous human-encoded VH or VL domain.

[0141] Isolated camelid VH and VL domains obtained by active immunization of camelids (e.g. llamas) with latent TGF-β1 can be used as building blocks for engineering TGF-β1 antibodies and antigen-binding fragments according to the invention. Starting from intact camelid VH and VL domains, it is possible to engineer one or more amino acid substitutions, insertions or deletions which deviate from the starting camelid sequence. In certain embodiments, such substitutions, insertions or deletions may be present in the framework regions of the VH and / or VL domains.

[0142] In other embodiments, "chimeric" antibody molecules are provided that comprise VH and VL domains (or engineered variants thereof) from a camelid and one or more constant domains, such as human encoded constant domains (or engineered variants thereof). In such embodiments, both the VH and VL domains are preferably obtained from the same species of camelid, for example both the VH and VL may be from a llama, or both the VH and VL may be from an alpaca (prior to the introduction of engineered amino acid sequence changes). In such embodiments, both the VH and VL domains may be derived from a single animal, in particular a single animal that has been actively immunized with the antigen of interest.

[0143] Instead of engineered changes in the primary amino acid sequence of camelid VH and / or VL domains, individual camelid-derived hypervariable loops or CDRs, or a combination of these, are isolated from the camelid VH / VL domains and transferred by CDR grafting into another (i.e. non-camelid) framework, for example a human VH / VL framework.

[0144] In a non-limiting embodiment, the TGF-β1 antibody may comprise a CH1 domain and / or a CL domain (from the heavy and light chains, respectively), whose amino acid sequence is fully or substantially human. For antibody molecules intended for human therapeutic use, it is typical that the entire constant region of the antibody, or at least a portion thereof, has a fully 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 fully or substantially human in terms of their amino acid sequence. The CH1 domain, hinge region, CH2 domain, CH3 domain and / or CL domain (and CH4 domain, if present) may be derived from a human antibody, preferably from a human IgG antibody, more preferably from a human IgG1 antibody subtype IgG1, IgG2, IgG3 or IgG4.

[0145] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain, if present) may all have a fully or substantially human amino acid sequence. In the case of a constant region of a humanized or chimeric antibody or antibody fragment, the term "substantially human" refers to at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% amino acid sequence identity 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 "human" sequence that has been modified by one or more additions, deletions or substitutions, except in embodiments where the presence of a "fully human" hinge region is explicitly required with respect to the human sequence.

[0146] The TGF-β1 antibody may have one or more amino acid substitutions, insertions or deletions in the constant regions of the heavy and / or light chains, particularly in the constant regions in the Fc region. The amino acid substitutions may result in the replacement of the amino acid being replaced by a different naturally occurring amino acid, or by a non-natural or modified amino acid. Other structural modifications are also possible, such as, for example, changes in the glycosylation pattern (e.g., by adding or deleting N- or O-linked glycosylation sites).

[0147] The TGF-β1 antibody may be modified in the Fc region to increase binding affinity to the neonatal receptor FcRn. The increased binding affinity can be measured at an acidic pH (e.g., about pH 5.5 to about pH 6.0). The increased binding affinity can also be measured at a neutral pH (e.g., about pH 6.9 to about pH 7.4). "Increased binding affinity" refers to increased binding affinity to FcRn compared to an unmodified Fc region. Typically, the unmodified Fc region has the wild-type amino acid sequence of human IgG1, IgG2, IgG3 or IgG4. In such embodiments, the increased FcRn binding affinity of the antibody molecule having the modified Fc region is measured compared to the binding affinity to FcRn of wild-type IgG1, IgG2, IgG3 or IgG4.

[0148] In certain embodiments, one or more amino acid residues in the Fc region may be replaced with different amino acids to enhance binding to FcRn. Some Fc substitutions have been reported to enhance FcRn binding, thereby improving antibody pharmacokinetics. Such substitutions have been reported, for example, in 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 by reference in their entirety.

[0149] In certain embodiments, the TGF-β1 antibody comprises a modified human IgG Fc domain comprising or consisting of the amino acid substitutions H433K and N434F, where the Fc domain numbering is according to EU numbering. In further embodiments, the TGF-β1 antibody described herein comprises a modified human IgG Fc domain comprising or consisting of the amino acid substitutions M252Y, S254T, T256E, H433K and N434F, where the Fc domain numbering is according to EU numbering.

[0150] In certain embodiments, the TGF-β1 antibody comprises an altered 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 compared to the corresponding wild-type sequence.

[0151] In certain embodiments, the Fc region may be engineered to be absent of effector function. In certain embodiments, the antibodies of the invention may have an Fc region derived from a naturally occurring IgG isotype, such as IgG4, with reduced effector function. The IgG4-derived Fc region may be further modified to enhance therapeutic utility, for example, by introducing modifications that minimize arm exchange between IgG4 molecules in vivo. The IgG4-derived Fc region may be modified to incorporate an S228P substitution.

[0152] In certain embodiments, one, two or three 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 the 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.

[0153] 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. Such antibodies with altered, reduced or even eliminated effector functions are attractive in the context of the present invention.

[0154] In certain embodiments, the antibody or antigen-binding fragment is modified with respect to glycosylation. For example, an aglycosylated antibody (i.e., an antibody lacking glycosylation) can be generated. Glycosylation can be modified, for example, to increase the affinity of the antibody for a target antigen. Such carbohydrate modifications can be accomplished, 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 removal of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such deglycosylation can increase the affinity of the antibody for the antigen.

[0155] C. Exemplary TGF-β1 Activating Antibodies that Bind Latent TGF-β1 The present invention provides exemplary TGF-β1 antibodies and antigen-binding fragments thereof. Exemplary TGF-β1 antibodies and antigen-binding fragments of the present invention can be defined solely with respect to structural characteristics, as described below.

[0156] In some embodiments, the invention includes antibodies or antigen-binding fragments thereof, wherein the CDRs have amino acid sequences that share at least about 90%, or 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% identity to the SEQ ID NOs set forth herein.

[0157] In certain embodiments, an antibody or antigen-binding fragment thereof is provided that binds to human latent TGF-β1, the antibody or antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, the variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 11 [GGSIDLTYGMDY] or a sequence variant thereof; the variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 10 [AIRWNGVTTYYAESMKG] or a sequence variant thereof; the variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 9 [DYTMN] or a sequence variant thereof; the variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 26 [ALYMYTGSNNGRV] or a sequence variant thereof; the variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 25 [NTVARHS] or a sequence variant thereof; the variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 24 [GLSSGSVTRNNYPD] or a sequence variant thereof; and Sequence variants contain one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions, or deletions) in the listed sequence.

[0158] In certain embodiments, an antibody or antigen-binding fragment thereof is provided that binds to human latent TGF-β1, the antibody or antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, the variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 14 [SRYGVVITTTYFGGVDY] or a sequence variant thereof; the variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 13 [AINTGGGITYYTDSVKG] or a sequence variant thereof; the variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 12 [HYWMY] or a sequence variant thereof; the variable light chain CDR3 sequence comprises or consists of SEQ ID NO:29 [LSYESSGYAV] or a sequence variant thereof; the variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 28 [GNSNRPS] or a sequence variant thereof; the variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 27 [QGGNFGSYYAS] or a sequence variant thereof; and Sequence variants contain one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions, or deletions) in the listed sequence.

[0159] In certain embodiments, an antibody or antigen-binding fragment thereof is provided that binds to human latent TGF-β1, said antibody or antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, the variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO:57 [YITRAQLMGAWDY] or a sequence variant thereof; The variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 56 [RITNSGGTAFYTDSVEG] or a sequence variant thereof; the variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 55 [SYWMD] or a sequence variant thereof; the variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 60 [AQSTYYPYD] or a sequence variant thereof; the variable light chain CDR2 sequence comprises or consists of SEQ ID NO:59 [QVSNRAS] or a sequence variant thereof; the variable light chain CDR1 sequence comprises or consists of SEQ ID NO:58 [KASQSLVHTDGKTYLS] or a sequence variant thereof; and Sequence variants contain one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions, or deletions) in the listed sequence.

[0160] In certain embodiments, an antibody or antigen-binding fragment thereof is provided that binds to human latent TGF-β1, the antibody or antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, the variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 11 [GGSIDLTYGMDY] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 14 [SRYGVVITTTYFGGVDY] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 57 [YITRAQLMGAWDY] or a sequence variant thereof; The variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 10 [AIRWNGVTTYYAESMKG] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 13 [AINTGGGITYYTDSVKG] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 56 [RITNSGGTAFYTDSVEG] or a sequence variant thereof; the variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 9 [DYTMN] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 12 [HYWMY] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 55 [SYWMD] or a sequence variant thereof; the variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 26 [ALYMYTGSNNGRV] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 29 [LSYESSGYAV] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 60 [AQSTYYPYD] or a sequence variant thereof; the variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 25 [NTVARHS] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 28 [GNSNRPS] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 59 [QVSNRAS] or a sequence variant thereof; The variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 24 [GLSSGSVTRNNYPD] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 27 [QGGNFGSYYAS] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 58 [KASQSLVHTDGKTYLS] or a sequence variant thereof; and Sequence variants contain one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions, or deletions) in the listed sequence.

[0161] In certain embodiments, an antibody or antigen-binding fragment thereof is provided that binds to human latent TGF-β1, the antibody or antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, the variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 11 [GGSIDLTYGMDY] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 14 [SRYGVVITTTYFGGVDY] or a sequence variant thereof; the variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 10 [AIRWNGVTTYYAESMKG] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 13 [AINTGGGITYYTDSVKG] or a sequence variant thereof; the variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 9 [DYTMN] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 12 [HYWMY] or a sequence variant thereof; the variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 26 [ALYMYTGSNNGRV] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 29 [LSYESSGYAV] or a sequence variant thereof; the variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 25 [NTVARHS] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 28 [GNSNRPS] or a sequence variant thereof; the variable light chain CDR1 sequence comprises SEQ ID NO: 24 [GLSSGSVTRNNYPD] or a sequence variant thereof, or comprises or consists of SEQ ID NO: 27 [QGGNFGSYYAS] or a sequence variant thereof; and Sequence variants contain one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions, or deletions) in the listed sequence.

[0162] In certain embodiments, antibodies and antigen-binding fragments that bind to human latent TGF-β1 comprise or consist of a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises or consists of 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 the VL comprises or consists of the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, 99% identical thereto.

[0163] In certain embodiments, antibodies and antigen-binding fragments that bind to human latent TGF-β1 comprise or consist of a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises or consists of 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 the VL comprises or consists of the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, 99% identical thereto.

[0164] In certain embodiments, antibodies and antigen-binding fragments that bind to human latent TGF-β1 comprise or consist of a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises or consists of 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 the VL comprises or consists of the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, 99% identical thereto.

[0165] In certain embodiments, antibodies and antigen-binding fragments that bind human latent TGF-β1 comprise or consist of a variable heavy domain (VH) and a variable light domain (VL) selected from the group consisting of: (i) a VH comprising, or consisting of, 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, or consisting of, the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, 99% identical thereto; (ii) a VH comprising, or consisting of, 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, or consisting of, the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, 99% identical thereto; and (iii) a VH comprising or consisting of 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 or consisting of the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, 99% identical thereto.

[0166] In certain embodiments, antibodies and antigen-binding fragments that bind human latent TGF-β1 comprise or consist of a variable heavy domain (VH) and a variable light domain (VL) selected from the group consisting of: (i) a VH comprising, or consisting of, 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, or consisting of, the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, 99% identical thereto; and (ii) a VH comprising or consisting of 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 or consisting of the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, 99% identical thereto.

[0167] For embodiments in which antibody or antigen-binding fragment domains are defined by a particular percentage of sequence identity to a reference sequence, the VH and / or VL domain may retain identical CDR sequences as present in the reference sequence, such that changes are present only within the framework regions.

[0168] In certain embodiments, an antibody or antigen-binding fragment thereof is provided that binds to mouse latent TGF-β1, the antibody or antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, the variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 17 [DRIRSSYEYDS] or a sequence variant thereof; the variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 16 [HISAAGISRYYADAVKG] or a sequence variant thereof; the variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 15 [NYGVH] or a sequence variant thereof; the variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 32 [GCYDSSLSTGYV] or a sequence variant thereof; the variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 31 [NANNRAS] or a sequence variant thereof; the variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 30 [TGSSSNIGGGYYLS] or a sequence variant thereof; and Sequence variants contain one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions, or deletions) in the listed sequence.

[0169] In certain embodiments, an antibody or antigen-binding fragment thereof is provided that binds to mouse latent TGF-β1, the antibody or antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, the variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 20 [GLVSSGLNGMDY] or a sequence variant thereof; the variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 19 [AITPGGTATYYANSVKG] or a sequence variant thereof; the variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 18 [RSGMA] or a sequence variant thereof; the variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 34 [GCYDDSLNTYV] or a sequence variant thereof; the variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 33 [NTNNRAS] or a sequence variant thereof; the variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 30 [TGSSSNIGGGYYLS] or a sequence variant thereof; and Sequence variants contain one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions, or deletions) in the listed sequence.

[0170] In certain embodiments, an antibody or antigen-binding fragment thereof is provided that binds to mouse latent TGF-β1, the antibody or antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, the variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 23 [DLNSRSYKGMGD] or a sequence variant thereof; the variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 22 [GTSIGGGSTWYADSVKG] or a sequence variant thereof; the variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 21 [SYYMY] or a sequence variant thereof; the variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 36 [GCYDSSLSSVV] or a sequence variant thereof; the variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 31 [NANNRAS] or a sequence variant thereof; the variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 35 [TGSSSNIGGGYYLN] or a sequence variant thereof; and Sequence variants contain one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions, or deletions) in the listed sequence.

[0171] In certain embodiments, antibodies and antigen-binding fragments that bind human latent TGF-β1 comprise or consist of a variable heavy domain (VH) and a variable light domain (VL) selected from the group consisting of: (i) a VH comprising, or consisting of, 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, or consisting of, the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, 99% identical thereto; (ii) a VH comprising, or consisting of, 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, or consisting of, the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, 99% identical thereto; and (iii) a VH comprising or consisting of 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 or consisting of the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, 99% identical thereto.

[0172] For embodiments in which antibody or antigen-binding fragment domains are defined by a particular percentage of sequence identity to a reference sequence, the VH and / or VL domain may retain identical CDR sequences as present in the reference sequence, such that changes are present only within the framework regions.

[0173] The present invention also provides antibodies or antigen-binding fragments thereof that bind to the same epitope as the TGF-β1 antibodies exemplified herein.

[0174] 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 the mature TGF-β1 cytokine from LAP-mediated inhibition. The activating properties of the exemplary TGF-β1 antibodies and antigen-binding fragments can be measured by any of the methods described in Section B above. Additionally, the exemplary TGF-β1 antibodies and antigen-binding fragments described herein can take any of the antibody formats according to the embodiments described in Section B above.

[0175] In preferred embodiments, the exemplary TGF-β1 antibody molecules having the CDR sequences listed above exhibit high human homology, e.g., are humanized or germline variants of the antibody or antigen-binding fragment thereof from which the CDR sequences are derived.

[0176] In non-limiting embodiments, exemplary TGF-β1 antibodies and antigen-binding fragments thereof having the CDR, VH and / or VL sequences described herein may comprise a CH1 domain and / or a CL domain (from the heavy and light chains, respectively), whose amino acid sequences are fully or substantially human. For antibody molecules intended for human therapeutic use, it is typical that the entire constant region of the antibody, or at least a portion thereof, has a fully 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 fully or substantially human in terms of their amino acid sequences.

[0177] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain, if present) may all be fully 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 at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% amino acid sequence identity with a human constant region. In this context, the term "human amino acid sequence" 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 "human" sequence that has been modified with respect to the human sequence by one or more additions, deletions or substitutions, except in embodiments where the presence of a "fully human" hinge region is explicitly required. Any of the exemplary Fc region modifications described herein may be incorporated into a TGF-β1 antibody having the CDR and / or VH / VL domain sequences listed above. In certain embodiments, the TGF-β1 antibody having the CDRs and / or VH / VL domains listed above comprises a modified human IgG Fc domain comprising or consisting of the amino acid substitutions H433K and N434F, where the Fc domain numbering is according to EU numbering. In certain embodiments, the TGF-β1 antibody having the CDRs and / or VH / VL domains listed above comprises a modified human IgG Fc domain comprising or consisting of the amino acid substitutions M252Y, S254T, T256E, H433K and N434F.

[0178] Unless otherwise specified in this application, the % sequence identity between two amino acid sequences may be determined by comparing these two sequences aligned in an optimal manner and where the amino acid sequence being compared may contain additions or deletions relative to the reference sequence for optimal alignment of these two sequences. The percentage of identity is calculated by determining the number of identical positions where the amino acid residue is identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window to obtain the percentage of identity between these two sequences, and multiplying the result obtained by 100. For example, it is possible to use 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), available at the site http: / / www.ncbi.nlm.nih.gov / gorf / bl2.html, the parameters used are those given by default (in particular for the parameters “Open gap penalty”: 5, and “Extended gap penalty”: 2; the matrix selected 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.

[0179] For embodiments in which antibody or antigen-binding fragment domains are defined by a particular percentage of sequence identity to a reference sequence, the VH, VHH and / or VL domain may retain identical CDR sequences as present in the reference sequence, such that changes are present only within the framework regions.

[0180] D. Nucleic Acids Encoding TGF-β1 Activating Antibodies The present invention also provides polynucleotide molecules or nucleic acids encoding the TGF-β1 antibodies or antigen-binding fragments of the present invention. Polynucleotide molecules or nucleic acids encoding full-length antibodies are provided, along with polynucleotide molecules or nucleic acids encoding fragments or individual 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 comprising the above-described nucleotide sequences of the present invention operably linked to regulatory sequences enabling expression of the antibody or fragments thereof in a host cell or cell-free expression system, and host cells or cell-free expression systems comprising the expression vectors.

[0181] Polynucleotide molecules encoding the antibodies or antigen-binding fragments of the invention include, for example, recombinant DNA molecules. The terms "nucleic acid", "polynucleotide" or "nucleic acid molecule" are used interchangeably herein and refer to any DNA or RNA molecule, single-stranded or double-stranded, and, if single-stranded, to the molecule of its complementary sequence. In discussing nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule may be described herein according to the usual convention of giving the sequence in the 5' to 3' direction. In some embodiments of the invention, a nucleic acid or polynucleotide is "isolated". This term, as applied to a nucleic acid molecule, refers to a nucleic acid molecule that is separated from sequences that are immediately adjacent in the naturally occurring genome of the organism from which it originates. For example, an "isolated nucleic acid" may include a DNA molecule that is inserted into a vector, such as a plasmid or viral vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or a non-human host organism. As applied to RNA, the term "isolated polynucleotide" primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term can refer to an RNA molecule that has been purified / separated from other nucleic acids with which it is associated in its natural state (i.e., in cells or tissues). An isolated polynucleotide (either DNA or RNA) can also refer to a molecule that has been produced directly by biological or synthetic means and separated from other components during its production.

[0182] For recombinant production of an antibody according to the invention, a recombinant polynucleotide encoding it, or encoding different chains or domains, can be prepared (using standard molecular biology techniques) and inserted into a replicable vector for expression in a selected host cell or cell-free expression system. Suitable host cells can be prokaryotic, yeast, or higher eukaryotic cells, particularly mammalian cells. Examples of useful mammalian host cell lines include the SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth 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 cell cultures (CV1 ATCC CCL 70); African green monkey kidney cell cultures (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), as well as DSM's The PERC-6 cell line. Suitable expression vectors for use in each of these host cells are also generally known in the art.

[0183] It should be noted that the term "host cell" typically refers to a cultured cell line. Any human cell into which an expression vector encoding an antigen-binding polypeptide according to the invention has been introduced is expressly excluded from the definition of "host cell."

[0184] E. Antibody Production In a further aspect, the present invention also provides a method for producing an antibody or antigen-binding fragment of the present invention, comprising culturing a host cell (or a cell-free expression system) comprising a polynucleotide or nucleic acid (e.g., an expression vector) encoding the antibody or antigen-binding fragment under conditions allowing expression of the antibody or fragment, and recovering the expressed antibody. This recombinant expression method can be used for the mass production of antibodies, including activating TGF-β1 antibodies according to the present invention, including monoclonal antibodies for human therapeutic use. Suitable vectors, cell lines and production methods for large-scale production of recombinant antibodies suitable for in vivo therapeutic use are generally available in the art and known to the skilled artisan.

[0185] F. Pharmaceutical Compositions The scope of the present invention includes pharmaceutical compositions comprising one or a combination of the antibodies or antigen-binding fragments described herein, formulated with one or more pharma- ceutically acceptable carriers or excipients. Such compositions can comprise a combination of activating TGF-β1 antibodies (i.e., a combination of two or more different activating TGF-β1 antibodies). The techniques for formulating monoclonal antibodies for human therapeutic use are well known in the art, and are reviewed, for example, in Wang et al., Journal of Pharmaceutical Sciences 2007;96:1-26, the contents of which are incorporated herein in their entirety.

[0186] Pharmaceutically acceptable excipients that may be used to formulate the compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as 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 silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0187] In certain embodiments, the compositions are 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.

[0188] G. Treatment Methods As reported herein in the Examples, TGF-β1 activating antibodies according to the invention were able to improve the outcome in a mouse model of graft-versus-host disease (GvHD). Without intending to be bound by theory, this effect is likely due to the action of TGF-β1 as an immunosuppressive cytokine in regulating the immune response.

[0189] In view of the above, the activating TGF-β1 antibodies and antigen-binding fragments described herein can be used in a method of treatment.Thus, provided herein is a TGF-β1 activating antibody or antigen-binding fragment according to any aspect or embodiment described elsewhere herein for use as a drug.TGF-β1 antibodies and antigen-binding fragments for use as a drug are usually formulated as pharmaceutical compositions.Importantly, all of the above embodiments of the TGF-β1 activating antibodies and antigen-binding fragments are equally applicable to the methods described herein.

[0190] Provided herein is a method of suppressing a pathological immune response in a subject in need thereof, the method comprising 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 of preventing or treating an autoimmune disease in a subject in need thereof, the method comprising 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 of preventing or treating an alloimmune disease in a subject in need thereof, the method comprising 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.

[0191] As used herein, the term "therapeutically effective amount" is intended to mean an amount or dosage of TGF-β1 antibody or antigen-binding fragment sufficient to produce a therapeutic effect, such as the amount or dosage required to eradicate or at least reduce symptoms associated with a disease or condition. The appropriate amount or dosage can be determined by a physician as necessary. For example, dosage can be adjusted based on factors such as the size or weight of the subject being treated, the age of the subject being treated, the general health of the subject being treated, the condition being treated, and the route of administration.

[0192] The present invention also provides a TGF-β1 activating antibody or antigen-binding fragment thereof according to the invention for use in suppressing a pathological immune response in a subject in need thereof. Further provided is a TGF-β1 activating antibody or antigen-binding fragment thereof according to the invention for use in preventing or treating an autoimmune disease in a subject in need thereof. Further provided is a TGF-β1 activating antibody or antigen-binding fragment thereof according to the invention for use in preventing or treating an alloimmune disease in a subject in need thereof. The subject is typically a human subject.

[0193] Autoimmune and alloimmune diseases or conditions which may be prevented or treated according to the present invention include, but are not limited to, the group consisting of inflammatory bowel disease (IBD), multiple sclerosis (MS), graft versus host disease (GVHD), allograft rejection, antibody-mediated allograft 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, autoimmune diseases of the adrenal gland, autoimmune encephalitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, immune thrombocytopenia (ITP), and autoimmune pulmonary fibrosis (OPF). or idiopathic thrombocytopenic purpura or idiopathic thrombocytopenic purpura or immune-mediated thrombocytopenia), autoimmune urticaria, Behçet's disease, bullous pemphigoid (BP), cardiomyopathy, Castleman syndrome, celiac sprue dermatitis dermatitis), chronic fatigue immune deficiency syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, COVID-19-mediated orthostatic tachycardia syndrome (POTS), Crohn's disease, delayed graft function after kidney transplantation, dilated cardiomyopathy, discoid lupus, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia and 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 polyneuropathy, juvenile arthritis, Kawasaki disease, lichen planusplanus), lichen sclerosus, lupus erythematosus, systemic lupus erythematosus (SLE), lupus nephritis, membranous neuropathy, membranous nephropathy, Meniere's disease, mixed connective tissue disease, mucous membrane pemphigoid, graft 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, polyfocal 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 arteritis) / giant cell arteritis, thrombotic thrombocytopenic purpura, thyroid ophthalmopathy, ulcerative colitis, uveitis, warm autoimmune hemolytic anemia (wAIHA), dermatitis herpetiformis, vasculitis, antineutrophil cytoplasmic antibody-associated vasculitis, leukoplakia, and Wegner's granulomatosis.

[0194] In addition to its important role as an immunosuppressive cytokine in regulating immune responses, TGF-β1 plays a key role in cancer development and strategies targeting TGF-β1 have been employed in cancer therapy (Fonseca Teixeira et al., Front. Cell. Dev. Biol. 2020;8:605). In the case of cancer, TGF-β1 plays a complex dual role with both tumor-promoting and tumor-suppressing activities. Especially in the early stages of cancer development, TGF-β1 is thought to play an important immunosuppressive role by suppressing the proliferation of epithelial and precancerous cells. In the later stages of cancer progression, tumors can acquire resistance to the cytostatic effects mediated by TGF-β1.

[0195] In view of the important tumor suppressive function of TGF-β1, especially in the early stages of cancer development, provided herein is a method of preventing or treating a neoplastic disease in a subject in need thereof, comprising 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 invention for use in preventing or treating a neoplastic disease in a subject in need thereof, thereby effecting. When preventing or treating a neoplastic disease, the TGF-β1 activating antibody or antigen-binding fragment described herein may activate latent TGF-β1 bound to the surface of cells at a pre-malignant stage, e.g., cells of a benign neoplasm.

[0196] As used herein, a method of "preventing" a disease or condition means preventing the onset of the disease, preventing the worsening of symptoms, preventing the progression of the disease or condition, or reducing the risk that a subject will develop the disease or condition. As used herein, a method of "treating" a disease or condition means curing the disease or condition and / or reducing or eradicating the symptoms associated with the disease or condition such that the patient's suffering is reduced.

[0197] For clinical use, in certain embodiments, the TGF-β1 antibody is administered to the subject in one or more doses of about 0.1 mg / kg body weight to about 20 mg / kg body weight as described elsewhere herein. In certain embodiments, the TGF-β1 antibody is administered to the 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 the 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 the subject at a dose of about 1 mg / kg body weight to about 10 mg / kg body weight.

[0198] H.Kit Any of the TGF-β1 activating antibodies or antigen-binding fragments described herein or pharmaceutical compositions comprising them may be packaged as a kit, optionally including instructions for use.

[0199] In some embodiments, the kits are for carrying out the therapeutic methods described herein, hi some embodiments, the kits are for preventing or treating alloimmune and / or autoimmune diseases or conditions.

[0200] I.Other Further provided is 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 the manufacture of a medicament.

[0201] Provided herein is a method of activating TGF-β1 in a subject in need thereof, the method comprising 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.

[0202] Further provided is a method of increasing the activity of TGF-β1 in a subject in need thereof, the method comprising 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 the activity of TGF-β1" means increasing the activity of TGF-β1 by at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold compared to the activity of TGF-β1 in the absence of a therapeutically effective amount of a TGF-β1 activating antibody or antigen-binding fragment according to the invention. The activity of TGF-β1 can be assessed by any method known in the art.

[0203] Another object is a method of downregulating immunity in a subject in need thereof, comprising 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 "downregulating immunity" means 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 invention. The immunity of the subject can be assessed by any method known in the art.

[0204] Compared to prior art TGF-β1 antibodies, the antibodies of the invention may exhibit the following advantages: In some embodiments, the antibodies of the invention exhibit the capacity or ability to activate TGF-β1 or enhance the activity of TGF-β1. In some embodiments, the antibodies of the invention exhibit increased affinity for TGF-β1 compared to prior art TGF-β1 antibodies. In some embodiments, the antibodies of the invention exhibit increased binding to TGF-β1 compared to prior art TGF-β1 antibodies.

[0205] Working Example The invention will be further understood with reference to the following non-limiting examples. Example 1: 14 monoclonal antibodies that activate latent TGF-β1 We screened a total of 134 monoclonal antibodies (mAbs) against human or mouse GARP, GARP:(latent)TGF-β1 complexes, or latent TGF-β1 for their ability to activate TGF-β1 in a TGF-β reporter assay. 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 encoding firefly luciferase under the control of a TGF-β1-responsive promoter (pCAGA-12) and a control plasmid encoding Renilla luciferase under a constitutive promoter (pRL-TK) as a control for transfection efficiency. 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 after 24 h. The ratio of firefly luminescence to Renilla measured in the absence of rhTGF-β1 or mAb was taken as the reference negative signal. A firefly / renilla ratio higher than the mean + 2 SD minus sign was considered as a positive signal for TGF-β1 activity. Fourteen of the 134 mAbs tested activated latent TGF-β1 in these assays (see Figures 1A and 1B). The 14 TGF-β1 activating mAbs were either against human latent TGF-β1 (6 of the 24 mAbs called LHT-1 to LHT-24 against llama-derived anti-human TGF-β1) or against mouse latent TGF-β1 (8 of the 11 mAbs called LMT-1 to LMT-3 and LMT-5 to LMT-12 against llama-derived anti-mouse TGF-β1). None of the activating mAbs were directed against GARP, and there was no cross-reactivity against human and mouse latent TGF-β1 (Figure 1A). Results obtained with TGF-β1 activating mAbs and a few non-activating mAbs in the reporter assay are shown in Figure 1B (upper panel: LMT mAb; lower panel: LHT mAb). They are representative of at least three independent experiments.The hatched bars in Figure 1B highlight 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. Latent TGF-β1-activating mAbs evoked signals comparable to 40-320 pg / ml rhTGF-β1. Serial dilutions of several TGF-β1-activating mAbs were tested in the reporter assay to determine the EC50, which ranged from 0.2 to 2.1 nM (Figure 1C).

[0206] Example 2: Antibodies against murine or human latent TGF-β1 activate TGF-β1 when used as bivalent monospecific full-length mAbs or F(ab')2 fragments, but not when used as monovalent Fab fragments 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 assay described above (Figure 2A). The four LMT-activating antibodies activated TGF-β1 when used as bivalent full-length mAbs or F(ab')2 fragments, but did not activate TGF-β1 when used as monovalent Fab fragments (Figure 2A). We conclude that bivalent binding to latent TGF-β1 is required for monospecific antibodies to mediate TGF-β1 activation.

[0207] Example 3: TGF-β1-activating antibodies activate latent TGF-β1 regardless of whether latent TGF-β1 is presented by GARP on the cell surface 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. Indeed, during the production and processing of latent TGF-β1 in cells, GARP binds to the LAP portion of latent TGF-β1 via a disulfide bond, acts as a chaperone to increase latent TGF-β1 production, and finally presents and anchors latent TGF-β1 on the cell surface (Gauthy et al. PLoS One 2013;8(9):e76186; Lienart et al. Science 2018;362(6417):952-956). We tested whether TGF-β1-activating mAbs could also activate latent TGF-β1 in the absence of GARP in a reporter assay, and HEK-293 cells were transfected with a plasmid encoding latent TGF-β1 with or without a plasmid encoding GARP (Figure 2B). All three tested TGF-β1-activating mAbs LMT-7, -9, and -10 induced TGF-β1 activity well above background in reporter cells in the presence of GARP (Figure 2B, left panel). Two of the three mAbs, LMT-7 and -10, also induced TGF-β1 activity above background in the absence of GARP, but at a lower level in the presence of GARP (Figure 2B, middle panel). The reduced activity is likely due to lower levels of latent TGF-β1 tethered on the cell surface in the absence of GARP. None of the three mAbs induced TGF-β1 activity in reporter cells that were not transfected to express latent TGF-β1 (Fig. 2B, right panel), indicating that the TGF-β1-activating mAb preparations were not contaminated with active TGF-β1 that may have copurified with the mAbs. We confirmed by ELISA (data not shown) and immunoblotting (Fig. 2C) that active TGF-β1 was not contaminated into the mAb formulations.

[0208] Example 4: Unique epitopes bound by TGF-β1-activating LMT and LHT mAbs TGF-β1-activating mAbs bind either human or mouse TGF-β1, but not both (non-cross-reactive mAbs). We used 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 shows that most amino acid differences are located between residues 87-278, a region of LAP that is highly conserved and located outside the so-called "latent lasso" (Figure 3A). We constructed four plasmids encoding HA-tagged mouse-human-mouse chimeric TGF-β1. In each chimera, amino acids from one segment of mouse LAP were replaced by amino acids found in the corresponding segment of human LAP. The segments of mouse LAP replaced by the corresponding human LAP in the four plasmids included residues 87-135, 136-200, 201-222, or 223-278, respectively (Figure 3B). We cotransfected HEK-293T cells with plasmids encoding GARP and chimeric TGF-β1 to express chimeric latent TGF-β1 on the cell surface and analyzed the binding of TGF-β1-activating and nonactivating mAbs to the transfected cells by flow cytometry.

[0209] As expected, all LMT mAbs bound to cells expressing mouse TGF-β1 but not to cells expressing human TGF-β1. Loss of binding of any given LMT mAb to a particular mouse-human-mouse chimera compared to mouse TGF-β1 allows identification of the region of mouse TGF-β1 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 3B). We could refine this binding requirement more precisely to residues 158-163 within region 136-200 (data not shown). Three other activating LMT mAbs (LMT-2, -9 and -12) required region 201-222 for binding (Figure 3B). In contrast, nonactivated LMT-1 mAb required both (LMT-1) regions 136–200 and 201–222 for binding, whereas nonactivated LMT-8 did not require any of the four regions tested in the chimera ( Fig. 3B ).

[0210] Also as expected, all LHT mAbs bound to cells expressing human TGF-β1 but not to cells expressing mouse TGF-β1 (Figure 3B). Here, acquisition of binding of any given LHT mAb to a particular mouse-human-mouse chimera in comparison to mouse TGF-β1 identifies the region required for binding by that particular mAb. We observed that two activating LHT mAbs (LHT-18 and -22) required region 201-222 for binding (i.e., the same region required by activating LHT-2, -9, and -12). Non-activating LHT-8 mAb also required region 201-222 for binding, whereas activating LHT-15 mAb required region 87-135.

[0211] Taken together, we observed that region 201-222, but not the other regions, was required for binding by three activating LMT and two activating LHT mAbs, highlighting a common binding requirement for activating mAbs against mouse or human TGF-β1, respectively. Two non-activating mAbs (LHT-8, shown in Fig. 3C, and LHT-11, not shown) also required that region for binding, but not the other regions.

[0212] To further determine the amino acids required for binding by activating mAbs in region 201-222, we constructed a series of plasmids encoding HA-tagged mouse or human TGF-β1, each with a single amino acid substituted by alanine (A). A series of 20 alanine mutant plasmids contained 20 positions in region 201-222 in mouse TGF-β1, and another series of 20 plasmids contained 20 positions in human TGF-β1 (Figure 3C). To perform an "alanine scan" analysis, we cotransfected HEK-293T cells with plasmids encoding mouse or human GARP and single alanine mutants of mouse or human TGF-β1 and analyzed binding by TGF-β1 activating and nonactivating mAbs using flow cytometry (Figure 3C).

[0213] The mouse TGF-β1 activating mAbs LMT-2 and LMT-12 lost the ability to bind mouse TGF-β1 when the L at position 208 (L208) or the D at position 212 (D212) was mutated to A. Thus, activated LMT-2 and LMT-12 required L208 and D212, but not any other single residue, for binding to mouse TGF-β1. Other LMT or LHT mAbs that bind to region 201-222, whether activated or non-activated, did not require L208 and D212, but did not require any other amino acids, for binding. For example, non-activated LMT-1 mAb required L208, G211, D212, and F217, whereas non-activated LMT-8 mAb did not require any of the 20 residues in region 201-222. The requirement for L208 / D212 is unique to the TGF-β1 activating mAbs LMT-2 and LMT-12 and is not observed with non-activating mAbs that bind human or mouse TGF-β1.

[0214] The human TGF-β1 activating mAb LHT-22 lost its ability to bind human TGF-β1 when G at position 212 (G212) or E at position 215 (E215) was mutated to A. Thus, LHT-22 required G212 and E215 for binding to human TGF-β1, but no other single residues. Other LMT or LHT mAbs that bind to region 201-222, whether activated or non-activated, did not require G212 and E215 for binding, nor did they require any other amino acids. Non-activated LHT-8 mAb required E213 and E215, whereas non-activated LHT-11 required R205 and R210. The requirement for G212 / E215 is unique to the activating mAb LHT-22 and is not observed in non-activated mAbs that bind human or mouse TGF-β1.

[0215] Taken together, these observations indicated that the binding requirement for L208 / D212 in mouse TGF-β1 or G212 / E215 in human TGF-β1 is unique to TGF-β1 activating mAbs.

[0216] We also investigated whether LMT-12 and LHT-22 lost the ability to activate single A mutants in 293T reporter cells (Figure 3D). As expected, these mAbs lost the ability to activate mutants to which they do not bind (Figure 3C: L208A and D212A for LMT-12; G212A and E215A for LHT-22). In addition, however, LHT-22 also lost the ability to activate five single A mutants (W207A, L208A, G211A, I214A, and F217A) that bind similarly to WT TGF-β1. Thus, we identified five other TGF-β1 residues that are required for the activity of LHT-22.

[0217] Taken together, using the chimeras and single A mutants, we identified residues required for mAb binding and activity (Figure 3B, 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 epitopes of LHT-22 and LMT-12 are highly similar to each other and are not shared with non-activating mAbs. Interestingly, all TGF-β1 activating mAbs appear to bind to loops close to the LAP dimer interface (Figure 2D). This interface has been reported to be essential for TGF-β1 latency by holding LAP monomers together (Shi et al.).

[0218] Example 5: Treatment of mice with TGF-β1 activators LMT-10 and LMT-12 increased survival in a model of graft-versus-host disease Graft-versus-host disease (GvHD) is a life-threatening side effect in allogeneic hematopoietic stem cell transplantation due to the strong immune activity of donor-derived T cells against allogeneic recipient cells. Due to its strong immunomodulatory properties, TGF-β1 may reduce the activity of donor-derived T cells against allogeneic recipient antigens. We therefore tested the therapeutic efficacy of TGF-β1-activating mAbs to treat GvHD in mice. We used C57BL / 6 H-2 b / b Mouse-derived splenocytes were F1(C57BL / 6xDBA2)H-2 b / d We used a mouse model of GvHD in which allogeneic H-2 in F1 recipients was injected intraperitoneally (ip) into the recipient. d Donor T cell reactivity against MHC molecules leads to the development of GvHD, which can be scored by monitoring weight loss, hunched posture, loss of motor function and ruffled fur, and can lead to death 10-21 days after cell transfer. We have demonstrated that H-2 b / b Starting 1 day before splenocyte transfer, F1 H-2 b / d Recipients received TGF-β1-activating mAb or isotype control (400 μg / mouse by weekly intraperitoneal injection) ( Fig. 4A ).

[0219] Mice receiving TGF-β1-activated LMT-10 or LMT-12 had significantly improved survival compared to mice receiving isotype control antibody (Figure 4B). 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 showed longer survival (>30 days) without an estimate of median survival (Figure 4B). GvHD scores of individual mice treated with LMT-10 or LMT-12 showed delayed disease progression, or reduced disease severity, or even complete disappearance of GvHD compared to isotype control (Figure 4C).

[0220] Example 6: LMT-12 activated latent TGF-β1 from a GARP-independent source in GvHD As described in Example 4 and Figure 2, TGF-β1-activating mAbs can activate latent TGF-β1 regardless of whether latent TGF-β1 is presented by GARP on the cell surface. Despite this, when latent TGF-β1 is presented by GARP in HEK-293 cells, activation by TGF-β1-activating mAbs is completely suppressed in the presence of blocking anti-GARP mAbs. As shown in Figure 5A, blocking anti-human GARP mAb LHG-10 suppresses human TGF-β1 activation by LHT-4, -15, -18, -20, -22 and -25 (left panel), and blocking anti-mouse GARP: latent TGF-β1 complex mAb 58A2 suppresses mouse TGF-β1 activation by LMT-12 (right panel). This observation provides an opportunity to determine whether TGF-β1-activating mAbs can still exert therapeutic activity in murine GvHD in the presence of blocking anti-mouse GARP antibodies and indicates that TGF-β1-activating mAbs can activate latent TGF-β1 regardless of its presentation by GARP-expressing cells in vivo.

[0221] As mentioned above, we have F1 H-2 b / d Recipient mice were transfected with H-2 b / b Splenocytes were transplanted and recipient mice were treated with TGF-β1-activating LMT-12 alone 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, delaying disease progression, and reducing disease severity compared to an isotype control antibody. Interestingly, LMT-12 exerted the same therapeutic activity when it was administered alone or in combination with 58A2 (Figure 5B). We therefore conclude that TGF-β1-activating mAbs can exert their therapeutic activity by activating latent TGF-β1 produced by cells that do not express GARP in vivo. TGF-β1 activation by activating mAbs and the therapeutic activity of activating mAbs are context-independent and independent of GARP:TGF-β1 complexes.

[0222] Example 7: Binding activity of TGF-β1 antibodies The binding properties of activated anti-human and anti-mouse latent TGFβ1 antibodies binding to region 201-222 were tested by surface plasmon resonance (Biacore3000, GE Healthcare). CM5 chips were 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 spent HBS-EP buffer. The flow rate was set to 30 μl / min in "kinject" injection mode and a regenerative injection of 10 mM glycine pH 1.5. The on-rate (k a ), off rate (K d ) and affinity (K D ) was determined based on measurements of six concentrations per antibody: 100 nM, 50 nM, 25 nM, 12 nM, 6 nM and 3 nM. All tested clones were in a hIgG1 backbone except for LHT-18 which had a scFv-Fc hIgG1 backbone. All clones showed binding to immobilized latent TGFβ1, with K values ​​of 66 nM for LHT-18 and 2320 nM for LHT-22 for binding to human latent TGFβ1. D values, and the binding of the clones to mouse latent TGFβ1 ranged from 63 nM to 687 nM. D had value. [Table 4]

[0223] Example 8: Specificity determination assay The specificity of LHT-22 and LHT-18 for latent TGF-β1, compared with latent TGF-β2 and latent TGF-β3, was tested by ELISA experiments for human latent TGF-β and by flow cytometry for mouse latent TGF-β, both in a single experiment.

[0224] ELISA was performed to confirm that LHT-22 specifically binds to human latent TGF-β1 and 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-22 hIgG1 were then incubated and bound antibodies were detected with anti-hIgG-HRP antibody. Antibodies with OD values ​​<0.1 were defined as no binding. A negative control sample (Motavizumab) was also included. All concentrations tested for LHT-22 (4, 1 and 0.25 μg / mL) showed binding to human latent TGF-β1 and no binding to human latent TGF-β2 and latent TGF-β3 (Table 5).

[0225] Using flow cytometry, we confirmed that LMT-12 specifically binds to mouse latent TGF-β1 and not to mouse latent TGF-β2 and mouse latent TGF-β3. Briefly, 293T cells were transfected with plasmids encoding HA-tagged TGF-β1, β2, or β3, and a plasmid encoding GARP. They were then stained with the indicated concentrations of LMT-12 mIgG1 or anti-HA mIgG1 antibodies, followed by goat anti-mouse conjugated to FITC (GAM-FITC) as a secondary reagent. Using flow cytometry, histograms were gated on live cells identified by dead cell labeling reagent (Bioscience, Cat. No. 65-0865-14). All concentrations tested of LMT-12 showed binding to mouse latent TGF-β1 and no binding to mouse latent TGF-β2 and latent TGF-β3 (Figure 6). [Table 5] [Table 6] [Table 7] [Table 8] JPEG2025508073000018.jpg16159

[0226] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Moreover, all embodiments described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, if necessary.

[0227] Incorporation by Reference Various publications are cited within the foregoing description and throughout the examples which follow, each of which is incorporated herein by reference in its entirety.

Claims

1. An antibody or its antigen-binding fragment that binds to latent TGF-β1 and activates TGF-β1.

2. The antibody or antigen-binding fragment according to claim 1, which activates TGF-β1 by releasing mature TGF-β1 cytokines from suppression mediated by latent related peptides (LAPs).

3. The antibody or antigen-binding fragment according to claim 1, which binds to an epitope containing one or more amino acids derived from a region of human or mouse latent TGF-β1 consisting of 87 to 222 amino acids, 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, which binds to an epitope containing one or more amino acids derived from a region of human or mouse latent TGF-β1 consisting of 87 to 135 amino acids, 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, which binds to an epitope containing one or more amino acids derived from a region of human or mouse latent TGF-β1 consisting of 136 to 200 amino acids, 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, wherein it binds to an epitope containing one or more amino acids derived from a region of human or mouse latent TGF-β1 consisting of amino acids 201 to 222, the human latent TGF-β1 being represented by SEQ ID NO: 1 and the mouse latent TGF-β1 being represented by SEQ ID NO:

5.

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

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

1.

9. The antibody or antigen-binding fragment according to claim 3, which binds to mouse latent TGF-β1.

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

5.

11. The antibody or antigen-binding fragment according to claim 10, wherein the fragment binds to an epitope comprising the amino acid residues S138, R141, P145, E146, S175, L208, N209 and / or D212 of mouse latent TGF-β1, the epitope does not contain any other amino acid residues from within the region of mouse latent TGF-β1 consisting of amino acids 135 to 222, and mouse latent TGF-β1 is represented by Sequence ID No.

5.

12. The antibody or antigen-binding fragment according to claim 1, wherein latent TGF-β1 is activated when it is complexed with GARP.

13. The antibody or antigen-binding fragment according to claim 1, wherein latent TGF-β1 activates TGF-β1 when it is bound to the surface of one or more cell types selected from regulatory T cells (Treg); megakaryocytes; platelets; B lymphocytes; endothelial cells; fibroblasts; mesenchymal cells; and hepatic astrocytes.

14. The antibody or antigen-binding fragment according to claim 1, which activates latent TGF-β1 that binds to one or more cell types selected from bone marrow cells; lymphoid cells; hematopoietic cells; and pre-malignant stage cancers.

15. The antibody or antigen-binding fragment according to claim 1, which is bivalent.

16. The antibody according to claim 15, wherein it is IgG.

17. The antibody or antigen-binding fragment according to claim 15, comprising the CH1 domain, hinge region, CH2 domain and / or CH3 domain of human IgG.

18. An antigen-binding fragment according to claim 1, selected from the group consisting of an antibody light chain variable domain (VL); an antibody heavy chain variable domain (VH or VHH); a single-chain antibody (scFv); an (scFv)2 fragment; an F(ab')2 fragment; a Fab fragment; an Fd fragment; an Fv fragment; a one-armed (monovalent) antibody; a diabody; a triabody; and a tetrabody.

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

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

22. The antibody or antigen-binding fragment according to claim 21, comprising a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of the following: (i) VH containing 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 VL containing 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) VH containing 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 VL containing 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) VH containing 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 VL containing 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 that binds to latent TGF-β1, comprising a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains contain a CDR sequence selected from the group consisting of the following: (i) HCDR3 containing or consisting of SEQ ID NO: 11; HCDR2 containing or consisting of SEQ ID NO: 10; HCDR1 containing or consisting of SEQ ID NO: 9; LCDR3 containing or consisting of SEQ ID NO: 26; LCDR2 containing or consisting of SEQ ID NO: 25; LCDR1 containing or consisting of SEQ ID NO: 24; (ii) HCDR3 containing or consisting of SEQ ID NO: 14; HCDR2 containing or consisting of SEQ ID NO: 13; HCDR1 containing or consisting of SEQ ID NO: 12; LCDR3 containing or consisting of SEQ ID NO: 29; LCDR2 containing or consisting of SEQ ID NO: 28; LCDR1 containing or consisting of SEQ ID NO: 27; and (iii) HCDR3 containing or consisting of SEQ ID NO: 57; HCDR2 containing or consisting of SEQ ID NO: 56; HCDR1 containing or consisting of SEQ ID NO: 55; LCDR3 containing or consisting of SEQ ID NO: 60; LCDR2 containing or consisting of SEQ ID NO: 59; LCDR1 containing or consisting of SEQ ID NO: 58; Or any CDR having an amino acid sequence that shares at least approximately 90% identity with SEQ ID NOs: 9-14, 24-29, 55-60.

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

26. The antibody or antigen-binding fragment according to claim 25, comprising a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of the following: (i) VH containing 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 VL containing 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) VH containing 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 VL containing 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) VH containing 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 VL containing 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 its antigen-binding fragment that binds to the same epitope as the antibody described in claim 23.

28. An isolated nucleic acid encoding an antibody or antigen-binding fragment or its VH, VHH, or VL domain, as described in any one of claims 1 to 27.

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

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

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

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

33. An antibody or antigen-binding fragment according to any one of claims 1 to 27 for use as a drug.

34. An antibody or antigen-binding fragment according to any one of claims 1 to 27 for use in the treatment of autoimmune diseases and / or alloimmune diseases in subjects requiring it.

35. An antibody or antigen-binding fragment according to any one of claims 1 to 27 for use in the treatment of tumor diseases in subjects requiring it.

36. A kit comprising an antibody or antigen-binding fragment and instructions for use according to any one of claims 1 to 27.

37. The pharmaceutical composition according to claim 32 for use as a drug.

38. The pharmaceutical composition according to claim 32 for use in the treatment of autoimmune diseases and / or alloimmune diseases in subjects who require it.

39. The pharmaceutical composition according to claim 32 for use in the treatment of tumor diseases in subjects who require it.

40. A kit comprising the pharmaceutical composition and instructions for use described in Claim 32.

41. An antibody or its antigen-binding fragment that binds to the same epitope as the antibody described in Claim 24.

42. An antibody or its antigen-binding fragment that binds to the same epitope as the antibody described in Claim 25.

43. An antibody or its antigen-binding fragment that binds to the same epitope as the antibody described in Claim 26.

44. An isolated nucleic acid encoding an antibody or antigen-binding fragment or its VH, VHH, or VL domain, as described in any one of claims 41 to 43.

45. An expression vector comprising the nucleic acid according to claim 44, operably linked to a regulatory sequence, which enables the expression of the antibody, antigen-binding fragment, VH domain, VHH domain, or VL domain in host cells or a cell-free expression system.

46. A host cell or cell-free expression system comprising the expression vector according to claim 45.

47. A process for producing a recombinant antibody or antigen-binding fragment, comprising culturing the host cells or cell-free expression system described in claim 46 under conditions that enable the expression of an antibody or antigen-binding fragment, and recovering the expressed antibody or antigen-binding fragment.

48. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 41 to 43 and at least one pharmaceutically acceptable carrier or excipient.

49. The antibody or antigen-binding fragment according to any one of claims 41 to 43 for use as a drug.

50. The pharmaceutical composition according to claim 48 for use as a drug.

51. The antibody or antigen-binding fragment according to any one of claims 41 to 43 for use in the treatment of autoimmune diseases and / or alloimmune diseases in subjects requiring it.

52. The pharmaceutical composition according to claim 48 for use in the treatment of autoimmune diseases and / or alloimmune diseases in subjects who require it.

53. The antibody or antigen-binding fragment according to any one of claims 41 to 43 for use in the treatment of tumor diseases in subjects requiring it.

54. The pharmaceutical composition according to claim 48 for use in the treatment of tumor diseases in subjects who require it.

55. A kit comprising the antibody or antigen-binding fragment and instructions for use according to any one of claims 41 to 43.

56. A kit comprising the pharmaceutical composition and instructions for use described in Claim 48.

57. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 27, or the antibody or antigen-binding fragment, for use in the treatment of autoimmune diseases and / or alloimmune diseases in a subject requiring the same, wherein the disease is inflammatory bowel disease (IBD), multiple sclerosis (MS), graft-versus-host disease (GVHD), allograft rejection, antibody-mediated allograft 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, autoimmune diseases of the adrenal gland, autoimmune encephalitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, and immunothrombocytopenia (ITP) or idiopathic thrombocytopenic purpura or immune-mediated thrombocytopenia), autoimmune urticaria, Behçet's disease, bullous pemphigoid (BP), cardiomyopathy, Castleman syndrome, celiac sprue dermatitis dermatitis, chronic fatigue immunodeficiency syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, bullous pemphigoid, CREST syndrome, cold agglutinin disease, COVID-19 mediated orthostatic tachycardia syndrome (POTS), Crohn's disease, delayed graft function after kidney transplantation, dilated cardiomyopathy, lupus discoid, 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, fetal / neonatal hemolytic disease (HDFN), idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, IgA neuropathy, IgM polyneuropathy, juvenile arthritis, Kawasaki disease, lichen planusPlanus), lichen sclerosing, lupus erythematosus, systemic lupus erythematosus (SLE), lupus nephritis, membranous neuropathy, membranous nephropathy, Meniere's disease, mixed connective tissue disease, mucosal pemphigoid, graft-type 1 diabetes, multifocal motor neuropathy (MMN), myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), myasthenia gravis (MG), systemic myasthenia gravis (gMG), ocular myasthenia gravis (OMG), myositis, neuromyelitis optica (NMO), paraneoplastic bullous pemphigoid, pemphigoid of pregnancy, pemphigus vulgaris (PV), pemphigus foliaceus (PF), pernicious anemia, polyarteritis nodosa, polyarteritis Polychondritis, polysynthetic syndrome, polymyalgia rheumatica, polymyositis, dermatomyositis (DM), necrotizing autoimmune myopathy (NAM), antisynthetic enzyme 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, Stiffman syndrome, Takayasu's arteritis, toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS), temporal arteritis A pharmaceutical composition comprising an antibody or antigen-binding fragment selected from the group consisting of giant cell arteritis, thrombotic thrombocytopenic purpura, thyroid eye disease, ulcerative colitis, uveitis, warm autoimmune hemolytic anemia (wAIHA), herpetiform dermatitis, vasculitis, antineutrophil cytoplasmic antibody-associated vasculitis, vitiligo, and Wegner granulomatosis, or said antibody or antigen-binding fragment.

58. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to claim 23 or the antibody or antigen-binding fragment for use in the treatment of autoimmune diseases and / or alloimmune diseases in subjects requiring the use thereof, wherein the disease is inflammatory bowel disease (IBD), multiple sclerosis (MS), graft-versus-host disease (GVHD), allograft rejection, antibody-mediated allograft 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, autoimmune diseases of the adrenal gland, autoimmune encephalitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, and immunothrombocytopenia (ITP) or idiopathic thrombocytopenic purpura or immune-mediated thrombocytopenia), autoimmune urticaria, Behçet's disease, bullous pemphigoid (BP), cardiomyopathy, Castleman syndrome, celiac sprue dermatitis dermatitis, chronic fatigue immunodeficiency syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, bullous pemphigoid, CREST syndrome, cold agglutinin disease, COVID-19 mediated orthostatic tachycardia syndrome (POTS), Crohn's disease, delayed graft function after kidney transplantation, dilated cardiomyopathy, lupus discoid, 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, fetal / neonatal hemolytic disease (HDFN), idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, IgA neuropathy, IgM polyneuropathy, juvenile arthritis, Kawasaki disease, lichen planusPlanus), lichen sclerosing, lupus erythematosus, systemic lupus erythematosus (SLE), lupus nephritis, membranous neuropathy, membranous nephropathy, Meniere's disease, mixed connective tissue disease, mucosal pemphigoid, graft-type 1 diabetes, multifocal motor neuropathy (MMN), myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), myasthenia gravis (MG), systemic myasthenia gravis (gMG), ocular myasthenia gravis (OMG), myositis, neuromyelitis optica (NMO), paraneoplastic bullous pemphigoid, pemphigoid of pregnancy, pemphigus vulgaris (PV), pemphigus foliaceus (PF), pernicious anemia, polyarteritis nodosa, polyarteritis Polychondritis, polysynthetic syndrome, polymyalgia rheumatica, polymyositis, dermatomyositis (DM), necrotizing autoimmune myopathy (NAM), antisynthetic enzyme 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, Stiffman syndrome, Takayasu's arteritis, toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS), temporal arteritis A pharmaceutical composition comprising an antibody or antigen-binding fragment selected from the group consisting of giant cell arteritis, thrombotic thrombocytopenic purpura, thyroid eye disease, ulcerative colitis, uveitis, warm autoimmune hemolytic anemia (wAIHA), herpetiform dermatitis, vasculitis, antineutrophil cytoplasmic antibody-associated vasculitis, vitiligo, and Wegner granulomatosis, or said antibody or antigen-binding fragment.

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