Isoform-specific, context-permissive TGFβ1 inhibitors and use thereof

Abstract

To provide compositions for inhibiting TGFβ signaling that can be used to effectively and safely treat diseases and disorders involving TGFβ1, including proliferative disorders (e.g., cancer), fibrosis and inflammation.SOLUTION: A composition comprises an isoform-specific inhibitor of TGFβ1 and a pharmaceutically acceptable excipient, wherein the inhibitor targets both ECM-associated TGFβ1 and immune cell-associated TGFβ1 but does not target TGFβ2 or TGFβ3 in vivo, wherein optionally the inhibitor inhibits the activation step of TGFβ1.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This international application is a joint venture under 35 U.S.C. §119(e) with the following applications: U.S. Provisional Application No. 62 / 443,615, filed January 6, 2017; U.S. Provisional Application No. 62 / 452,866, filed January 31, 2017; U.S. Provisional Application No. 62 / 514,417, filed June 2, 2017; U.S. Provisional Application No. 62 / 529,616, filed July 7, 2017; U.S. Provisional Application No. 62 / 549,767, filed August 24, 2017; This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 558,311, filed September 13, 2017; U.S. Provisional Application No. 62 / 585,227, filed November 13, 2017; U.S. Provisional Application No. 62 / 587,964, filed November 17, 2017; and U.S. Provisional Application No. 62 / 588,626, filed November 20, 2017, the contents of each of which are expressly incorporated by reference herein in their entirety.

[0002] Array List This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. Said ASCII copy, created on January 5, 2018, is named 127036-02020_ST25.txt and is 221,821 bytes in size. [Background technology]

[0003] The transforming growth factor beta (TGFβ) superfamily of growth factors is involved in many signaling cascades that regulate a variety of biological processes, including, but not limited to: inhibition of cell proliferation, tissue homeostasis, extracellular matrix (ECM) remodeling, endothelial-mesenchymal transition (EMT), cell migration and invasion, and immune regulation / suppression, as well as mesenchymal-epithelial transition. With respect to ECM remodeling, TGFβ signaling can increase fibroblast population and ECM deposition (e.g., collagen). In the immune system, TGFβ ligands regulate the function of regulatory T cells and the maintenance of proliferation and homeostasis of immune progenitor cells. In normal epithelial cells, TGFβ is a potent growth inhibitor and promoter of cell differentiation. However, as tumors develop and progress, they frequently lose their negative growth response to TGFβ. In this context, TGFβ can be a promoter of tumor development due to its ability to stimulate angiogenesis, modify the stromal environment, and induce local and systemic immune suppression. With regard to these and other responses, TGFβ has become a therapeutic target for many clinical indications. Despite the many efforts made to date by many groups, successful clinical development of TGFβ therapy remains challenging.

[0004] Preclinical observations, including those in rats and dogs, have revealed certain toxicities associated with TGFβ inhibition in vivo. Furthermore, although several TGFβ inhibitors have been developed to date, most clinical programs targeting TGFβ have been discontinued due to side effects (e.g., summarized in WO2017 / 156500). Thus, despite the lines of direct and indirect evidence showing the involvement of TGFβ signaling in the progression of diseases such as cancer and fibrosis, there are no safe and efficacious TGFβ treatments available on the market.

[0005] Among proliferative diseases, dysregulation of TGFβ has also been implicated in myelofibrosis, a bone marrow disorder characterized by clonal myeloid proliferation, abnormal cytokine production, extramedullary hematopoiesis, and myelofibrosis. Somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of the disease, but ruxolitinib (Jakafi), a JAK1 / JAK2 inhibitor approved by the FDA for the treatment of myelofibrosis, has not shown efficacy in remitting documented myelofibrosis in patients. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need for improved methods and compositions for inhibiting TGFβ signaling that can be used to effectively and safely treat diseases and disorders involving TGFβ1, including, for example, proliferative diseases (e.g., cancer), fibrosis and inflammation. [Means for solving the problem]

[0007] The present invention encompasses the recognition that inhibition of TGFβ activation in multiple sources can provide greater clinical efficacy in the treatment of many diseases involving both ECM and immune aspects of TGFβ dysregulation.Therefore, the improved method for the treatment of such diseases with TGFβ1 inhibitors is provided herein, which is superior to conventional TGFβ antagonists in terms of their isoform selectivity, breadth of molecular targets within disease niches, durability of efficacy and safety.

[0008] Evidence supports the view that many diseases exhibit complex disruptions of TGFβ signaling, thought to involve the involvement of heterogeneous cell types that provide distinct effects of TGFβ function, which are mediated by their interaction with so-called presentation molecules. At least four such presentation molecules have been identified that can "present" TGFβ in various extracellular niches, allowing its activation in response to local stimuli. In one category, TGFβ is deposited within the ECM in association with ECM-associated presentation molecules such as LTBP1 and LTBP3 to mediate ECM-associated TGFβ activity. In another category, TGFβ is tethered on the surface of immune cells via presentation molecules such as GARP and LRRC33 to mediate specific immune functions. These presentation molecules show differential expression, localization and / or function in various tissues and cell types, indicating that the outcomes and triggering events of TGFβ activation differ depending on the microenvironment. Based on the view that many TGFβ effects can interact with and contribute to disease progression, therapeutic agents that can antagonize multiple facets of TGFβ function may provide greater efficacy.

[0009] Previously, the inventors have recognized that isoform-specific inhibition of TGFβ (as opposed to pan-inhibition) may provide an improved safety profile of antagonizing TGFβ in vivo (see WO2017 / 156500). In view of this, the inventors seek to develop TGFβ1 inhibitors that are i) isoform-specific; and ii) capable of broadly targeting multiple TGFβ1 signaling complexes associated with different presentation molecules as therapeutic agents for conditions driven by pleiotropic TGFβ1 effects and their dysregulation.

[0010] Thus, the present disclosure provides isoform-specific inhibitors that can target both ECM-associated and immune cell-associated TGFβ1, thereby blocking multiple origins of TGFβ1 presented in multiple contexts. Such inhibitors are herein referred to as "isoform-specific, context-tolerant" inhibitors of TGFβ1. The present invention also provides the use of these agents as therapeutics in the treatment of conditions characterized by dysregulation of TGFβ1 signaling associated with multiple aspects of TGFβ1 function. Such inhibitors can function as multifunctional agents to antagonize multiple TGFβ1 activities (e.g., TGFβ1 from multiple origins or contexts) to enhance clinical efficacy in the context of fibrosis, myelofibrosis, cancer, and other conditions.

[0011] The rationale for the advantageous use of context-tolerant (e.g., context-independent) inhibitors of TGFβ1 over context-specific inhibitors of TGFβ1 as therapeutics for treating certain diseases (described further herein) includes the following:

[0012] Involvement of heterogeneous TGFβ1 complexes in disease environments: First, various diseases involve heterogeneous populations of cells as TGFβ1 of multiple origins, which collectively contribute to the pathogenesis and / or progression of the disease. More than one type of TGFβ1-containing complex ("context") may coexist within the same disease microenvironment. In particular, such diseases may involve both the ECM component of TGFβ1 signaling and the immune component of TGFβ1 signaling. In such situations, selective targeting of a single TGFβ1 context (e.g., TGFβ1 associated with one type of presentation molecule) may provide limited relief. In contrast, context-tolerant inhibitors of TGFβ1 advantageously aim to target inactive (precursor / latent) TGFβ1 complexes more broadly, preventing activation of growth factors in multiple origins before mature TGFβ1 can be released for receptor binding to trigger downstream signaling, while maintaining isoform selectivity to minimize toxicity.

[0013] Common mechanisms underlying various diseases: Second, striking similarities in tissue / cell characteristics are observed between tumor stroma and fibrotic tissue. Many pathologies show crosstalk between i) TGFβ1-dependent profibrotic phenotype; ii) TGFβ1-dependent tumor-promoting phenotype; and iii) TGFβ-dependent immunosuppressive phenotype. Thus, the use of context-tolerance inhibitors that act broadly against many of these components may provide optimal therapeutic effects across various types of disease states. For example, clinical manifestations of primary myelofibrosis include fibrosis in bone marrow and abnormal proliferation of certain cell populations.

[0014] Combating drug resistance: Third, many studies have reported cancers / tumors that are resistant to anti-cancer treatments such as immune checkpoint inhibitors. In some cases, such resistance appears intrinsic to a particular cancer / tumor type in relation to the patient's background (typically referred to as congenital resistance, primary resistance, intrinsic resistance, or innate resistance; these terms are used interchangeably herein). Such resistance may be shown in patient subjects that respond poorly to cancer treatments such as immune checkpoint inhibitors, and in some cases reflects an immune exclusion environment. This is believed to be mediated at least in part by TGFβ1-dependent pathways. Thus, the isoform-selective inhibitors described herein may make resistant cancers more responsive to such treatments.

[0015] Alternatively, resistance may progress over time such that patients who show material clinical responsiveness to treatment become less responsive (i.e., adaptive or acquired resistance). For example, it has been reported that PD-1 treatment can result in adaptive resistance that correlates with upregulation of other T cell antigens (e.g., TCR components), suggesting that cancer cells evolve to evade PD-1 blockade via other mechanisms. A second checkpoint inhibitor targeting a different T cell receptor component, such as TIM3, can subsequently restore responsiveness to immunotherapy. These observations suggest that blocking multiple pathways to counteract the adaptive response of cancer cells may reduce the likelihood of cancer cells' ability to evade host immunity. A context-tolerance inhibitor of TGFβ1 that can target multiple TGFβ1 contexts may advantageously circumvent acquired drug resistance by providing blockade at multiple points of TGFβ1 function.

[0016] Tolerate expression adaptability: Finally, based on the notion that expression of various presentation molecules can change over time, for example in response to local cues (e.g., cytokines, chemokines, the ECM environment, etc.) and / or with changes in the disease microenvironment, it stands to reason that a context-tolerant inhibitor of TGFβ1, such as those described herein, could be used to tolerate such adaptability and to provide a broad and durable inhibitory effect even in the event of aberrant changes in presentation molecule expression.

[0017] In any of these scenarios, a context tolerance inhibitor of TGFβ1 is advantageously targeted to target precursor / latent forms of TGFβ1 associated with various presentation molecules, all or different combinations of which are present within the disease microenvironment(s). More specifically, in one modality, the inhibitor targets ECM-associated TGFβ1 (LTBP1 / 3-TGFβ1 complex). In another modality, the inhibitor targets immune cell-associated TGFβ1. This includes TGFβ1 presented by GARP, such as the GARP-TGFβ1 complex expressed on Treg cells, and the LRRC33-TGFβ1 complex expressed on macrophages and other myeloid / lymphoid cells, as well as certain cancer cells.

[0018] Such antibodies include isoform-specific inhibitors of TGFβ1 that bind to and block activation (or release) of mature TGFβ1 growth factors from precursor / latent TGFβ1 complexes in a context-tolerant (or context-independent) manner, and thus the antibodies can inhibit activation (or release) of TGFβ1 associated with multiple types of presentation molecules. In particular, the present invention provides antibodies capable of blocking at least one context of ECM-associated TGFβ1 (presented by LTBP and / or presented by LTBP3) and at least one context of cell-associated TGFβ1 (presented by GARP and / or presented by LRRC33).

[0019] Various disease states have been suggested to involve dysregulation of TGFβ signaling as a contributing factor. Indeed, the pathogenesis and / or progression of certain human conditions are believed to be predominantly driven by or dependent on TGFβ1 activity. In particular, many such diseases and disorders are believed to involve both ECM and immune components of TGFβ1 function, suggesting that TGFβ1 activation in multiple contexts (e.g., mediated by more than one type of presentation molecule) is involved. Furthermore, crosstalk between TGFβ1-responsive cells is believed to exist. In some cases, the interplay between the pleiotropic activities of the TGFβ1 axis can result in disease progression, exacerbation, and / or suppression of the host's ability to fight disease. For example, certain disease microenvironments, such as the tumor microenvironment (TME), can be associated with TGFβ1 presented by a number of different presentation molecules, such as LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, LRRC33-proTGFβ1, and any combination thereof. It is possible that TGFβ1 activity in one context may in turn regulate or affect TGFβ1 activity in another context, which, if dysregulated, may result in exacerbation of disease states. It is therefore desirable to broadly inhibit TGFβ1 across multiple modes of function (i.e., multiple contexts) while selectively restricting such inhibitory effects to TGFβ1 isoforms. The aim is not to disrupt homeostatic TGFβ signaling mediated by other isoforms, including TGFβ3, which plays an important role in wound healing.

[0020] To address this, the inventors of the present disclosure sought to produce isoform-specific, context-tolerant inhibitors of TGFβ1 that may be particularly advantageous for therapeutic use in the treatment of diseases driven by or dependent on TGFβ1 signaling or its dysregulation. The approach taken to meet the criteria for such inhibitors is: i) the ability to inhibit TGFβ1 signaling in an isoform-specific manner (without interfering with TGFβ2 and / or TGFβ3 activity); and, ii) the ability to inhibit both ECM-associated and immune cell-associated TGFβ1 signaling. The rationale for this approach is to balance the efficacy (and therefore clinical efficacy) of TGFβ1 inhibition against potential toxicity. More specifically, achieving selectivity toward TGFβ1 at therapeutic doses over other isoforms aims to reduce or minimize possible toxicities (e.g., unwanted side effects and adverse events) associated with pan-inhibition of TGFβ in vivo, some of which may be required for normal biological functions (e.g., wound healing). On the other hand, the inclusion of multiple contexts of TGFβ1 as a therapeutic target aims to ensure or optimize clinical efficacy in diseases involving dysregulation of multiple aspects of TGFβ1 signaling. Various embodiments of clinical applications and treatment regimens are encompassed by the present invention.

[0021] Thus, in one embodiment, an isoform-specific, context-tolerant inhibitor of TGFβ1 is provided herein, characterized in that such inhibitor has the ability to inhibit both ECM-associated TGFβ1 signaling and immune cell-associated TGFβ1 signaling.In particular, such inhibitor can block TGFβ1 presented in multiple contexts (i.e., TGFβ1 activity mediated by two or more types of presentation molecules) while keeping TGFβ2 and TGFβ3 activity intact.Thus, the TGFβ1 activity that can be inhibited by such inhibitor includes two or more of the following: i) TGFβ1 signaling associated with TGFβ1 presented by GARP; ii) TGFβ1 signaling associated with TGFβ1 presented by LRRC33; iii) TGFβ1 signaling associated with TGFβ1 presented by LTBP1; and iv) TGFβ1 signaling associated with TGFβ1 presented by LTBP3. In some embodiments, such inhibitors target at least two or at least three proprotein forms of the following complexes: i) TGFβ1-GARP; ii) TGFβ1-LRRC33; iii) TGFβ1-LTBP1; and iv) TGFβ1-LTBP3. In some embodiments, such inhibitors are monoclonal antibodies that specifically bind and inhibit i) TGFβ1-GARP; iii) TGFβ1-LTBP1; and iv) TGFβ1-LTBP3. In some embodiments, such monoclonal antibodies specifically bind and inhibit ii) TGFβ1-LRRC33; iii) TGFβ1-LTBP1; and iv) TGFβ1-LTBP3. In some embodiments, such monoclonal antibodies specifically bind and inhibit i) TGFβ1-GARP; ii) TGFβ1-LRRC33; and iii) TGFβ1-LTBP1. In some embodiments, such monoclonal antibodies specifically bind to and inhibit: i) TGFβ1-GARP; ii) TGFβ1-LRRC33; and iv) TGFβ1-LTBP3.In some embodiments, such monoclonal antibodies specifically inhibit all of the following complexes: i) TGFβ1-GARP; ii) TGFβ1-LRRC33; iii) TGFβ1-LTBP1; and iv) TGFβ1-LTBP3. In some embodiments, such monoclonal antibodies do not bind to mature TGFβ1, which is free TGFβ1 (e.g., growth factor released from or not complexed with a presentation molecule). Aspects of the invention include compositions comprising such inhibitors, including, for example, pharmaceutical compositions suitable for administration in human and non-human subjects to be treated. Such pharmaceutical compositions are typically sterile. In some embodiments, such pharmaceutical compositions may include at least one pharma- ceutically acceptable excipient, such as a buffer and a surfactant (e.g., polysorbate). Kits comprising such pharmaceutical compositions are also encompassed by the invention.

[0022] The isoform-specific, context-tolerant inhibitors described herein are suitable for use in the treatment of diseases or disorders involving multiple biological functions of TGFβ1 and their dysregulation. In particular, such diseases or disorders involve both the ECM component of TGFβ1 function and the immune component of TGFβ1 function. The administration of such inhibitors can thus inhibit each axis of the TGFβ1 signaling pathway in vivo, e.g., multiple TGFβ1 targets associated with the disease or disorder, to enhance therapeutic effects. Thus, in another aspect, the present invention includes the therapeutic use of such inhibitors in a method for treating a subject suffering from a disease associated with TGFβ1 dysregulation. The isoform-specific, context-tolerant or context-independent inhibitors of TGFβ1 signaling are particularly suitable for treating diseases driven by or dependent on multiple functions of TGFβ1 (e.g., both ECM component and immune component). Typically, such diseases involve multiple cell types or cell conditions in which TGFβ1 is presented by multiple types of presentation molecules (e.g., multiple contexts).

[0023] In related aspects, the present invention provides screening, production and manufacturing methods for isoform-specific, context-tolerant TGFβ1 inhibitors with improved safety profiles (e.g., reduced in vivo toxicity). Such methods require that candidate agents be tested and selected for TGFβ1 isoform specificity, e.g., candidate agents are selected for inhibitory activity against TGFβ1 signaling but not against TGFβ2 and / or TGFβ3 signaling. In accordance with the present invention, such isoform-specific inhibitors of TGFβ1 activity are capable of inhibiting multiple contexts of TGFβ1 function (see below).

[0024] In some embodiments, such agents are antibodies or antigen-binding fragments thereof that specifically bind and block the activity of TGFβ1, but not TGFβ2 and / or TGFβ3. In some embodiments, such antibodies or antigen-binding fragments thereof do not bind to free mature TGFβ1 growth factors that are not associated with precursor / latent complexes. Thus, relevant production methods may include a screening step in which candidate agents (e.g., candidate antibodies or fragments thereof) are evaluated for their ability to inhibit TGFβ1 associated with specific presentation molecules, e.g., GARP, LRRC33, LTBP1, and / or LTBP3. In some embodiments, inactive (e.g., latent) precursor complexes, e.g., GARP-proTGFβ1, LRRC33-proTGFβ1, LTBP1-proTGFβ1, and LTBP3-proTGFβ1, may be used to assay for activation of mature active TGFβ1 growth factors. TGFβ1 activation in the presence or absence of a test agent (i.e., a candidate inhibitor) can be measured by any suitable means, including, but not limited to, in vitro and cell-based assays. Similar screening steps can be used to test isoform specificity by using TGFβ2 and / or TGFβ3 counterparts. Such screening steps can be performed to identify candidate agents (e.g., candidate antibodies or fragments thereof) for their ability to inhibit TGFβ1 signaling in an i) isoform-specific manner; and ii) context-tolerant or context-independent manner.

[0025] Certain diseases are associated with dysregulation of multiple biological roles of TGFβ signaling that are not restricted to a single context of TGFβ function. In such situations, it may be beneficial to modulate TGFβ effects across multiple contexts involved in the process of development and / or disease progression. Thus, in some embodiments, the present invention provides a method for targeting and broadly inhibiting multiple TGFβ1 contexts, albeit in an isoform-specific manner. Such agents are referred to herein as "isoform-specific, context-tolerant" TGFβ1 inhibitors. Thus, context-tolerant TGFβ1 inhibitors target multiple contexts (e.g., multiple types of precursor / latent-TGFβ1 complexes). Preferably, such inhibitors target at least one type (or "context") of ECM-associated TGFβ1 pre-activation complexes (i.e., precursor / latent TGFβ1 complexes presented by ECM-associated presentation molecules), and at least one type (or "context") of cell surface-tethered TGFβ1 pre-activation complexes (i.e., precursor / latent TGFβ1 complexes presented by cell- or membrane-associated presentation molecules). In some embodiments, context-tolerant TGFβ1 modulators target all types of precursor / latent TGFβ1 complexes (e.g., GARP-associated, LRRC33-associated, LTBP-associated, etc.) to encompass all contexts regardless of the particular presentation molecule(s).

[0026] Although a context-tolerant TGFβ1 inhibitor can target more than one type of precursor / latent-TGFβ1 complex (i.e., with different presentation molecules), in some embodiments, such inhibitors may prefer (or show bias toward) one or more contexts over the other(s). Thus, in some embodiments, a context-tolerant antibody that inhibits TGFβ1 activation may preferentially inhibit TGFβ1 activation mediated by one presentation molecule over another presentation molecule (even if such an antibody is capable of binding to both types of precursor / latent complexes). In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits activation of LTBP1 / 3-associated TGFβ1, GARP-associated TGFβ1, and LRRC33-associated TGFβ1, but has selective inhibitory activity toward LTBP1 / 3-associated TGFβ1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits activation of LTBP1-associated TGFβ1, LTBP3-associated TGFβ1, GARP-associated TGFβ1, and LRRC33-associated TGFβ1, but has selective inhibitory activity toward LTBP1 and LTBP-3-associated TGFβ1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits activation of LTBP1-associated TGFβ1, LTBP3-associated TGFβ1, GARP-associated TGFβ1, and LRRC33-associated TGFβ1, but has selective inhibitory activity toward GARP-associated TGFβ1 and LRRC33-associated TGFβ1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits activation of GARP-associated TGFβ1 and LRRC33-associated TGFβ1, but has selective inhibitory activity toward GARP-associated TGFβ1. In some embodiments, such antibodies are monoclonal antibodies that bind to and inhibit activation of GARP-associated TGFβ1 and LRRC33-associated TGFβ1, but have selective inhibitory activity towards LRRC33-associated TGFβ1.

[0027] Thus, according to the invention, different degrees of selectivity can be produced to target subsets of TGFβ effects: isoform-specific inhibitors of TGFβ (which target a single isoform of TGFβ) offer greater selectivity than so-called pan-TGFβ inhibitors (which target many or all isoforms of TGFβ).

[0028] The present invention includes the use of such TGFβ1 inhibitors in the method for treating diseases associated with TGFβ1 dysregulation.The use of such inhibitors is particularly advantageous in conditions where TGFβ1 isoforms play a predominant role (rather than TGFβ2 / 3) in driving the disease, and where the disease involves both the ECM component and the immune component of TGFβ1 signaling.This approach aims to preserve normal or homeostatic TGFβ function while preferentially targeting disease-related TGFβ function.

[0029] Such inhibitors are preferably TGFβ1 activation inhibitors (i.e., inhibitors of the TGFβ1 activation step). In a preferred embodiment, such inhibitors can target inactive forms of TGFβ1 (e.g., precursor / latent-TGFβ1 complexes) prior to activation, resulting in more durable inhibition compared to targeting transient, already activated, soluble / free forms of growth factor released from latent complexes. Determination of the origin / context of disease-associated TGFβ1 can be performed by the use of antibodies that specifically bind to TGFβ1 latent complexes containing a particular presentation molecule of interest (e.g., GARP, LRRC33, LTBP1, LTBP3, etc.).

[0030] An embodiment of the present disclosure relates to an immunoglobulin, e.g., an antibody, or an antigen-binding portion thereof, that specifically binds to at least three of the following complexes: GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex. According to the present invention, such an immunoglobulin specifically binds to at least one type of ECM-associated (e.g., ECM-tethered) TGFβ1 complex (e.g., LTBP1 and / or LTBP3-associated TGFβ1 complex) and at least one type of cell-associated (e.g., cell surface-tethered) TGFβ1 complex (e.g., GARP and / or LRRC33-associated TGFβ1 complex), providing a broad inhibitory effect across multiple contexts. The antibodies, or antigen-binding portions thereof, described herein specifically bind to a component or components of a protein complex that includes TGFβ1 (e.g., LAP) or an epitope of TGFβ1 (e.g., LAP) that is available for binding by the antibody, or antigen-binding portion thereof, when TGFβ1 is present in a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex and / or a LRRC33-TGFβ1 complex.

[0031] In some embodiments, the epitope is available for binding by the antibody when TGFβ1 is present in two or more of the following protein complexes: GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex; and wherein the antibody does not bind to free mature TGFβ1 growth factor that is not associated with a precursor / latent complex.

[0032] In some embodiments, the TGFβ1 is precursor TGFβ1 and / or latent TGFβ1 (e.g., precursor / latent TGFβ1). In some embodiments, the TGFβ1 is latent TGFβ1. In some embodiments, the TGFβ1 is precursor TGFβ1.

[0033] Isoform-specific TGFβ1 inhibitors of the present invention do not bind to bound TGFβ2. Isoform-specific TGFβ1 inhibitors of the present invention do not bind to TGFβ3. In some embodiments, such inhibitors do not bind to precursor / latent TGFβ2. In some embodiments, such inhibitors do not bind to precursor / latent TGFβ3. In some embodiments, the antibody, or antigen-binding portion thereof, does not interfere with the ability of TGFβ1 to bind to integrins.

[0034] In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO: 87 and a light chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO: 90. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO: 86 and a light chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO: 89. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 85 and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 88.

[0035] In some embodiments, the antibody comprises a heavy chain polypeptide sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 99. In some embodiments, the antibody comprises a light chain polypeptide sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 100. In some embodiments, the antibody comprises a heavy chain polypeptide sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 99 and a light chain polypeptide sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 100. In some embodiments, such an antibody comprises the CDRs set forth in SEQ ID NOs: 85-90. In some embodiments, the antibody consists of two polypeptides of SEQ ID NO: 99 and two polypeptides of SEQ ID NO: 100.

[0036] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:95 and a light chain variable domain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:97.

[0037] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:95 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:97.

[0038] In some embodiments, the antibody, or antigen binding portion thereof, inhibits TGFβ1 activation, but does not inhibit TGFβ2 activation or TGFβ3 activation.

[0039] In some embodiments, the antibody, or antigen binding portion thereof, inhibits release of mature TGFβ1 from the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex.

[0040] In one aspect, provided herein is a pharmaceutical composition comprising an antibody, or an antigen-binding portion thereof, described herein and a pharma- ceutically acceptable carrier. Such pharmaceutical compositions are typically sterile and suitable for administration to human subjects. In some embodiments, such pharmaceutical compositions can be provided as kits encompassed by the present invention.

[0041] In another aspect, provided herein is a method of inhibiting TGFβ1 activation, the method comprising exposing a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, or a LRRC33-TGFβ1 complex to an antibody, antigen-binding portion thereof, or pharmaceutical composition described herein.

[0042] In some embodiments, the antibody, or antigen binding portion thereof, inhibits release of mature TGFβ1 from a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, or a LRRC33-TGFβ1 complex.

[0043] In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo.

[0044] Thus, the present invention includes a method for treating a disease associated with dysregulation of TGFβ1 signaling in a human subject, comprising administering to a human subject in need thereof a pharmaceutical composition provided herein in an amount effective to treat the disease, which amount achieves statistically significant clinical efficacy and safety when administered to a patient population having the disease.

[0045] In yet another aspect, provided herein is a TGFβ inhibitor for use in reducing side effects in a subject, wherein the TGFβ inhibitor is isoform selective. In some embodiments, the TGFβ inhibitor is an antibody that specifically inhibits TGFβ1 while broadly targeting multiple contexts.

[0046] In some embodiments, the cell expressing the GARP-TGFβ1 complex or the LRRC33-TGFβ1 complex is a T cell, a fibroblast, a myofibroblast, a macrophage, a monocyte, a dendritic cell, an antigen-presenting cell, a neutrophil, a myeloid-derived suppressor cell (MDSC), a lymphocyte, a mast cell, a megakaryocyte, a natural killer (NK) cell, a microglia, or a precursor of any one of such cells. In some embodiments, the cell expressing the GARP-TGFβ1 complex or the LRRC33-TGFβ1 complex is a hematopoietic stem cell. In some embodiments, the cell expressing the GARP-TGFβ1 complex or the LRRC33-TGFβ1 complex is a neural crest-derived cell. The T cell may be a regulatory T cell (e.g., an immunosuppressive T cell). The T cell may be a CD4 positive (CD4+) T cell and / or a CD8 positive (CD8+) T cell. The neuprophil may be an activated neutrophil. The macrophages may be polarized macrophages, including profibrotic and / or tumor-associated macrophages (TAMs), such as M2c and M2d subtype macrophages. The macrophages may be activated by one or more soluble factors, such as growth factors, cytokines, chemokines, and / or other molecules present in a particular disease microenvironment (e.g., TME), and may act in an autocrine, paracrine, and / or endocrine manner. In some embodiments, the macrophages are activated by M-CSF, such as M-CSF secreted by solid tumors. In some embodiments, the macrophages are activated by TGFβ1.

[0047] In some embodiments, the cells expressing GARP-TGFβ1 complex or LRRC33-TGFβ1 complex are cancer cells, e.g., circulating cancer cells and tumor cells. In some embodiments, the cells expressing GARP-TGFβ1 complex or LRRC33-TGFβ1 complex are recruited to disease sites such as TME (e.g., tumor infiltration). In some embodiments, the expression of GARP-TGFβ1 complex or LRRC33-TGFβ1 complex is induced by disease microenvironment (e.g., TME). In some embodiments, solid tumors include enhanced leukocyte infiltration, e.g., CD45+. Tumor-associated CD45+ cells are believed to include GARP-expressing and / or LRRC33-expressing cells.

[0048] In some embodiments, the LTBP1-TGFβ1 complex or the LTBP3-TGFβ1 complex is bound to an extracellular matrix (i.e., a component of the ECM). In some embodiments, the extracellular matrix comprises fibrin and / or fibronectin. In some embodiments, the extracellular matrix comprises a protein comprising an RGD motif. In some embodiments, the cells that produce and deposit the LTBP1-TGFβ1 complex or the LTBP3-TGFβ1 complex are present in solid tumors, such as cancer cells and stromal cells. In some embodiments, the cells that produce and deposit the LTBP1-TGFβ1 complex or the LTBP3-TGFβ1 complex are present in fibrous tissue. In some embodiments, the cells that produce and deposit the LTBP1-TGFβ1 complex or the LTBP3-TGFβ1 complex are present in bone marrow. In some embodiments, the cells that produce and deposit the LTBP1-TGFβ1 complex or the LTBP3-TGFβ1 complex are myofibroblasts or myofibroblast-like cells, including, for example, cancer-associated fibroblasts (CAFs).

[0049] In another aspect, provided herein is a method for reducing TGFβ1 activation in a subject, the method comprising administering to the subject an effective amount of an antibody, antigen-binding portion thereof, or pharmaceutical composition described herein, thereby reducing TGFβ1 activation in the subject.

[0050] In some embodiments, the subject has or is at risk of having a fibrotic disorder. In some embodiments, the fibrotic disorder comprises chronic inflammation of the affected tissue / organ. In some embodiments, the subject has muscular dystrophy. In some embodiments, the subject has Duchenne muscular dystrophy (DMD). In some embodiments, the subject has or is at risk of having liver fibrosis, kidney fibrosis, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), endometriosis or uterine fibrosis. In some embodiments, the subject has or is at risk of having cancer (e.g., solid tumors, blood cancers, and myelofibrosis). In some embodiments, the subject has or is at risk of having dementia.

[0051] In some embodiments, the subject further receives an additional treatment. In some embodiments, the additional treatment is selected from the group consisting of a myostatin inhibitor, a VEGF agonist, an IGF1 agonist, an FXR agonist, a CCR2 inhibitor, a CCR5 inhibitor, a dual CCR2 / CCR5 inhibitor, a lysyl oxidase-like 2 inhibitor, an ASK1 inhibitor, an acetyl-CoA carboxylase (ACC) inhibitor, a p38 kinase inhibitor, pirfenidone, nintedanib, a GDF11 inhibitor, a JAK inhibitor (e.g., a JAK2 inhibitor), or any combination thereof.

[0052] In some embodiments, the antibody, or antigen-binding portion thereof, reduces the suppressive activity of regulatory T cells (Tregs).

[0053] In some embodiments, the antibody, or antigen-binding portion thereof, does not induce organ toxicity in a subject, hi some embodiments, organ toxicity includes cardiovascular toxicity, gastrointestinal toxicity, immunotoxicity, bone toxicity, cartilage toxicity, reproductive toxicity, or nephrotoxicity.

[0054] In one aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody, antigen-binding portion thereof, or pharmaceutical composition described herein, thereby treating cancer in the subject.

[0055] In another aspect, provided herein is a method of reducing tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody, antigen-binding portion thereof, or pharmaceutical composition described herein, thereby reducing tumor growth in the subject.

[0056] In some embodiments, the antibody, or antigen-binding portion thereof, is administered in combination with an additional agent or treatment. In some embodiments, the additional agent is a checkpoint inhibitor. In some embodiments, the additional agent is selected from the group consisting of PD-1 antagonists, PDL1 antagonists, PD-L1 or PDL2 fusion proteins, CTLA4 antagonists, and the like. Such combination therapy may advantageously utilize lower doses of administered therapeutic agents, thus avoiding possible toxicities or complications associated with various monotherapies or conventional combination therapies that do not have the degree of selectivity / specificity achieved by the present invention.

[0057] In some embodiments, the method further comprises determining (e.g., testing or confirming) the involvement of TGFβ1 in the disease compared to TGFβ2 and TGFβ3. In some embodiments, the method further comprises identifying the origin (or context) of disease-associated TGFβ1. In some embodiments, the origin / context is assessed by determining the expression of TGFβ-presenting molecules, such as LTBP1, LTBP3, GARP and LRRC33, in clinical samples obtained from the patient.

[0058] In yet another aspect, methods are provided herein for making (e.g., producing, manufacturing) pharmaceutical compositions for inhibiting TGFβ signaling, comprising the steps of providing one or more agents that inhibit the signaling of at least one isoform of TGFβ; measuring the activity of one or more agents toward all isoforms of TGFβ; selecting an agent that is selective for TGFβ1; and formulating into a pharmaceutical composition comprising an isoform-specific TGFβ1 inhibitor and a pharma- ceutically acceptable excipient, such as a suitable buffer. Pharmaceutical compositions produced by such methods are also provided. In some embodiments, the methods further comprise determining (e.g., measuring, evaluating) the context-dependent inhibitory activity of one or more agents.

[0059] The subject matter of this disclosure is also related to PCT / US2013 / 068613, filed November 6, 2013; PCT / US2014 / 036933, filed May 6, 2014; and PCT / US2017 / 021972, filed March 10, 2017, the entire contents of each of which are incorporated herein by reference. [Brief description of the drawings]

[0060] [Figure 1] 1 provides a schematic showing TGFβ1 within a latent complex in the tissue microenvironment. [Figure 2A-2C] We show multiple contexts of TGFβ1 function: TGFβ1 presented by GARP is expressed on regulatory T cells and is involved in immune regulation (Figure 2A); TGFβ1 presented by LTBP1 / 3 is deposited within the ECM by fibroblasts and other cells (Figure 2B); TGFβ1 presented by LRRC33 is expressed on myeloid cells including macrophages (Figure 2C). [Diagram 3]Protein expression platforms for generating GARP-TGFβ1 and LTBP-TGFβ1 complexes are shown. The HEK293-based expression system yields multi-milligram quantities of purified protein using Ni-NTA affinity purification and gel filtration. An overview of wild-type proTGFβ1, LTPB1, sGARP, and proTGFβ1 C4S is shown. [Figure 4] Figure 4A shows the specific binding of Ab3 to latent TGFβ1. Figure 4B shows the binding specificity of exemplary monoclonal antibodies. Figure 4B shows that Ab1 and Ab2 specifically bind to proTGFβ1, but not proTGFβ2, proTGFβ3, or mature TGFβ1, as measured by ELISA. Figure 4C shows an example of an antibody that specifically binds to the LTBP1-proTGFβ1 complex (measured by ELISA). [Diagram 5] A panel of prior art antibodies raised against the mature TGFβ growth factor and their respective binding profiles for all three isoforms are provided. [Figure 6A-6B] Binding profiles of Ab1, Ab2 and Ab3, measured by Octet, are provided, which are isoform-specific, context-tolerant / independent TGFβ1 inhibitors. [Figures 7A-7H] A cell-based inhibition assay is provided. [Figure 8] 1 shows the inhibitory effect of Ab3 on kallikrein-induced activation of TGF-β1 in vitro. [Figure 9A-9B] 1 shows the inhibitory effects of Ab1 and Ab3 on regulatory T cell-dependent suppression of effector T cell proliferation. [Figure 10A-10C] 1 shows upregulation of cell surface LRRC33 expression in polarized macrophages. [Figure 11] Results from a T cell cotransfer colitis model are provided. [Figures 12A-12K] 1 shows the inhibitory effect of Ab2 on a TGFb1-dependent mechanistic disease model of UUO. [Figures 13A-13C]1 shows the inhibitory effect of Ab3 on a TGFb1-dependent mechanistic disease model of UUO. [Figure 14] 1 shows the inhibitory effect of Ab3 on a carbon tetrachloride-induced fibrosis model. [Figure 15] 1 provides the inhibitory effect of Ab3 on a translational model of fibrosis in Alport mice. [Figure 16] 1 shows the inhibitory effect of Ab2 on tumor growth in MC38 carcinoma. [Figure 17] 1 provides the effect of Ab3 in combination with a PD-1 antagonist on survival in an EMT-6 tumor model. [Fig. 18A-18F] Toxicology / tolerability data are provided showing the improved safety profile of Ab2 in rats. [Figure 19A-19B] Toxicology / tolerability data are provided showing the improved safety profile of Ab3 in rats. [Figure 20] 1 provides data showing in vivo isoform selectivity of Ab3 in homeostatic rat BAL cells. [Figures 21A-21D] Relative expression of TGFβ isoforms is provided. Figure 21A shows TGFβ isoform expression relative to normal comparator (by cancer type). Figure 21B shows the frequency of TGFβ isoform expression by human cancer type. Figure 21C shows TGFβ isoform expression in individual tumor samples by cancer type. Figure 21D shows TGFβ isoform expression in mouse syngeneic cancer cell model lines. [Figure 22] 1 shows microscopic cardiac findings from a pan-TGFβ antibody from a 1-week study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] In mammals, the transforming growth factor beta (TGFβ) superfamily consists of at least 33 gene products. These include bone morphogenetic proteins (BMPs), activins, growth and differentiation factors (GDFs), and three isoforms of the TGFβ family: TGFβ1, TGFβ2, and TGFβ3. TGFβ is thought to play a key role in various processes such as inhibition of cell proliferation, extracellular matrix (ECM) remodeling, and immune homeostasis. The importance of TGFβ1 for T cell homeostasis is evidenced by the observation that TGFβ1- / - mice survive only 3-4 weeks and die of multiple organ failure due to extensive immune activation (Kulkarni, AB, et al., Proc Natl Acad Sci USA, 1993.90(2):p.770-4; Shull, MM, et al., Nature, 1992.359(6397):p.693-9). The role of TGFβ2 and TGFβ3 is less clear. Although the three TGFβ isoforms have distinct temporal and spatial expression patterns, they signal through the same receptors, TGFβRI and TGFβRII, although in some cases, for example for TGFβ2 signaling, type III receptors such as betaglycan are also required (Feng, XHand R.Derynck, Annu Rev Cell Dev Biol, 2005.21:p.659-93; Massague, J., Annu Rev Biochem, 1998.67:p.753-91). Ligand-induced oligomerization of TGFβRI / II triggers phosphorylation of SMAD transcription factors, resulting in the transcription of target genes such as Col1a1, Col3a1, ACTA2, and SERPINE1 (Massague, J., J. Seoane, and D. Wotton, Genes Dev, 2005.19(23):p.2783-810).SMAD-independent TGFβ signaling pathways have also been described, for example, in cancers or aortic lesions in Marfan mice (Derynck, R. and YE Zhang, Nature, 2003. 425(6958): p. 577-84; Holm, TM, et al., Science, 2011. 332(6027): p. 358-61).

[0062] The biological importance of the TGFβ pathway in humans is confirmed by genetic diseases. Kamurati-Engelman disease results from an autosomal dominant mutation in the TGFB1 gene, resulting in dysplastic bone formation, leading to constitutive activation of TGFβ1 signaling (Janssens, K., et al., J Med Genet, 2006. 43(1): p. 1-11). Patients with Loeys / Dietz syndrome carry autosomal dominant mutations within components of the TGFβ signaling pathway, leading to aortic aneurysms, sequestration, and bifid uvula (Van Laer, L., H. Dietz, and B. Loeys, Adv Exp Med Biol, 2014. 802: p. 95-105). As TGFβ pathway dysregulation is involved in numerous diseases, several drugs targeting the TGFβ pathway have been developed and tested in patients, with limited success.

[0063] Dysregulation of TGFβ signaling is associated with a wide range of human diseases. Indeed, in many disease states, such dysregulation may involve multiple aspects of TGFβ function. Diseased tissues, such as fibrotic and / or inflammatory tissues and tumors, may create a local environment in which TGFβ activation may cause disease exacerbation or progression, which may be mediated at least in part by interactions between numerous TGFβ-responsive cells activated in an autocrine and / or paracrine manner, along with many other cytokines, chemokines and growth factors that play a role in certain disease situations. For example, the tumor microenvironment (TME) contains, in addition to cancer (i.e., malignant) cells, numerous cell types expressing TGFβ1, such as activated myofibroblast-like fibroblasts, stromal cells, infiltrating macrophages, MDSCs and other immune cells. Thus, the TME represents a heterogeneous population of cells that express and / or are responsive to TGFβ1 in the niche, but are associated with more than one type of presentation molecule, such as LTBP1, LTBP3, LRRC33 and GARP.

[0064] In order to effectively inhibit dysregulated or disease-driven TGFβ1 activity involving multiple cell types and signaling "contexts", the inventors of the present disclosure have sought to develop a class of agents capable of inhibiting multiple TGFβ1 functions, albeit in an isoform-specific manner. Such agents are referred to as "isoform-specific, context-tolerant" inhibitors of TGFβ1, as defined herein. In some embodiments, such inhibitors are isoform-specific, context-independent inhibitors of TGFβ1. It is believed that the use of isoform-specific, context-tolerant or context-independent inhibitors of TGFβ1 can exert their inhibitory effects on TGFβ1 function in multiple models of diseases involving the interaction of various cell types that express and / or respond to TGFβ1 signaling, thereby enhancing therapeutic efficacy by targeting multiple types of TGFβ1 precursor complexes. Thus, the therapeutic target of such inhibitors includes at least three of the following complexes: i) proTGFβ1 presented by GARP; ii) proTGFβ1 presented by LRRC33; iii) proTGFβ1 presented by LTBP1; and iv) proTGFβ1 presented by LTBP3. Typically, the above complexes (i) and (ii) are presented on the cell surface, since both GARP and LRRC33 are transmembrane proteins capable of presenting TGFβ1 on the extracellular surface, while complexes (iii) and (iv) are components of the extracellular matrix. Many studies have elucidated the mechanism of TGFβ1 activation. Three integrins, αVβ6, αVβ8, and αVβ1, have been shown to be major activators of latent TGFβ1 (Reed, NI, et al., Sci Transl Med, 2015.7(288):p.288ra79; Travis, MAand D. Sheppard, Annu Rev Immunol, 2014.32:p.51-82; Munger, JS, et al., Cell, 1999.96(3):p.319-28).αV integrins bind with high affinity to the RGD sequence present in TGFβ1 and TGFβ1 LAP (Dong, X., et al., Nat Struct Mol Biol, 2014.21(12):p.1091-6). Transgenic mice with a mutation in the TGFβ1 RGD site that prevents integrin binding but not secretion phenocopies TGFβ1- / - mice (Yang, Z., et al., J Cell Biol, 2007.176(6):p.787-93). Mice lacking both β6 and β8 integrins recapitulate all essential phenotypes of TGFβ1 and TGFβ3 knockout mice, including multiorgan inflammation and cleft palate, confirming the essential role of these two integrins for TGFβ1 activation in development and homeostasis (Aluwihare, P., et al., J Cell Sci, 2009.122(Pt2):p.227-32). Crucial for integrin-dependent activation of latent TGFβ1 is the covalent tethering to the presentation molecule; disruption of the disulfide bond between GARP and TGFβ1 LAP by mutagenesis does not impair complex formation but completely abolishes TGFβ1 activation by αVβ6 (Wang, R., et al., Mol Biol Cell, 2012.23(6):p.1129-39). Recent structures of latent TGFβ1 have elucidated how integrins enable the release of active TGFβ1 from the latent complex: covalent binding of latent TGFβ1 to its presentation molecules anchors it to the ECM via LTBP or to the cytoskeleton through GARP or LRRC33. Integrin binding to the RGD sequence leads to a force-dependent change in the structure of LAP, allowing active TGFβ1 to be released and bind to nearby receptors (Shi, M., et al., Nature, 2011.474(7351):p.343-9). The importance of integrin-dependent TGFβ1 activation in disease is also well documented.Small molecule inhibitors of αVβ1 protect against bleomycin-induced pulmonary fibrosis and carbon tetrachloride-induced liver fibrosis (Reed, NI, et al., Sci Transl Med, 2015.7(288):p.288ra79), and antibody-mediated αVβ6 blockade or loss of integrin β6 expression suppresses bleomycin-induced pulmonary fibrosis and radiation-induced fibrosis (Munger, JS, et al., Cell, 1999.96(3):p.319-28; Horan, GS, et al., Am J Respir Crit Care Med, 2008.177(1):p.56-65). In addition to integrins, other mechanisms of TGFβ1 activation have been implicated, including activation by thrombospondin-1 and proteases such as matrix metalloproteinases (MMPs), cathepsin D or kallikrein. However, most of these studies were performed in vitro with purified proteins; there is little evidence for the role of these molecules from in vivo studies. Knockout of thrombospondin-1 recapitulates some aspects of the TGFβ1- / - phenotype in some tissues, but does not protect in bleomycin-induced pulmonary fibrosis, known to be TGFβ-dependent (Ezzie, ME, et al., Am J Respir Cell Mol Biol, 2011. 44(4): p. 556-61). In addition, knockout of candidate proteases did not result in a TGFβ1 phenotype (Worthington, JJ, JE Klementowicz, and MA Travis, Trends Biochem Sci, 2011. 36(1): p. 47-54). This could be explained by multiplicity or by these mechanisms being important in specific diseases rather than development and homeostasis.

[0065] Thus, the isoform-specific, context-tolerant inhibitors of TGFβ1 described herein include inhibitors that act by interfering with a step of TGFβ1 activation. In some embodiments, such inhibitors can inhibit integrin-dependent (e.g., mechanical or force-driven) activation of TGFβ1 (see FIG. 2). In some embodiments, such inhibitors can inhibit protease-dependent or protease-induced activation of TGFβ1. The latter includes inhibitors that inhibit TGFβ1 activation steps in an integrin-independent manner. In some embodiments, such inhibitors can inhibit TGFβ1 activation regardless of the mode of activation, e.g., inhibit both integrin-dependent and protease-dependent activation of TGFβ1. Non-limiting examples of proteases that can activate TGFβ1 include serine proteases such as kallikrein, chemotrypsin, trypsin, elastase, plasmin, and zinc metalloproteases (MMP family), such as MMP-2, MMP-9, and MMP-13. Kallikreins include plasma kallikrein and tissue kallikrein, such as KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14, and KLK15. Figure 8 provides an example of an isoform-specific, context-independent inhibitor of TGFβ1 that can inhibit kallikrein-dependent activation of TGFβ1 in vitro. In some embodiments, inhibitors of the invention prevent the release or dissociation of active (mature) TGFβ1 growth factor from the latent complex, hi some embodiments, such inhibitors may act by stabilizing an inactive (e.g., latent) conformation of the complex.

[0066] TGFβ is involved in many biological processes, including fibrosis, immune regulation, and cancer progression. TGFβ1 was the first identified member of the TGFβ superfamily of proteins. Like other members of the TGFβ superfamily, TGFβ1 and isoforms TGFβ2 and TGFβ3 are initially expressed as inactive precursor proprotein forms (referred to as proTGFβ). TGFβ proteins (e.g., TGFβ1, TGFβ2, and TGFβ3) are proteolytically cleaved by proprotein convertases (e.g., furin) to generate latent forms (referred to as latent TGFβ). In some embodiments, the proprotein or latent forms of TGFβ proteins (e.g., TGFβ1, TGFβ2, and TGFβ3) may be referred to as "precursor / latent TGFβ proteins." TGFβ1 can be presented in complexes with a number of molecules, including, for example, GARP (which forms the GARP-TGFβ1 complex), LRRC33 (which forms the LRRC33-TGFβ1 complex), LTBP1 (which forms the LTBP1-TGFβ1 complex), and / or LTBP3 (which forms the LTBP3-TGFβ1 complex). The TGFβ1 present in these complexes can be in either a latent form (latent TGFβ1) or a precursor form (proTGFβ1).

[0067] The present invention is particularly useful for therapeutic use for certain diseases associated with multiple biological roles of TGFβ1 signaling that are not limited to a single context of TGFβ1 function. In such situations, it may be beneficial to inhibit TGFβ1 effects across multiple contexts. Thus, in some embodiments, the present invention provides methods for targeting and inhibiting TGFβ1 in an isoform-specific manner rather than a context-specific manner. Such agents may be referred to as "isoform-specific, context-tolerant" TGFβ1 modulators. In some embodiments, context-tolerant TGFβ1 modulators target multiple contexts (e.g., multiple types of precursor / latent-TGFβ1 complexes). In some embodiments, context-tolerant TGFβ1 modulators target all types of precursor / latent TGFβ1 complexes (e.g., GARP-related, LRRC33-related, LTBP-related, etc.) to encompass all contexts.

[0068] Although a context-tolerant TGFβ1 inhibitor can target more than one type of precursor / latent-TGFβ1 complex (i.e., with different presentation molecules), in some embodiments, such inhibitors may prefer one or more contexts over others. Thus, in some embodiments, a context-tolerant antibody that inhibits TGFβ1 activation may preferentially inhibit TGFβ1 activation mediated by one presentation molecule over another, even if such an antibody is capable of binding to both types of precursor / latent complexes. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits activation of LTBP-associated TGFβ1, GARP-associated TGFβ1, and LRRC33-associated TGFβ1, but has selective inhibitory activity toward LTBP-associated TGFβ1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits activation of LTBP1-associated TGFβ1, LTBP3-associated TGFβ1, GARP-associated TGFβ1, and LRRC33-associated TGFβ1, but has selective inhibitory activity toward LTBP1 and LTBP-3-associated TGFβ1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits activation of LTBP1-associated TGFβ1, LTBP3-associated TGFβ1, GARP-associated TGFβ1, and LRRC33-associated TGFβ1, but has selective inhibitory activity toward GARP-associated TGFβ1 and LRRC33-associated TGFβ1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits activation of GARP-associated TGFβ1 and LRRC33-associated TGFβ1, but has selective inhibitory activity toward GARP-associated TGFβ1. In some embodiments, such antibodies are monoclonal antibodies that bind to and inhibit activation of GARP-associated TGFβ1 and LRRC33-associated TGFβ1, but have selective inhibitory activity towards LRRC33-associated TGFβ1.

[0069] Thus, according to the present invention, different degrees of selectivity can be produced to target a subset of TGFβ effects. Isoform-specific inhibitors of TGFβ1 (targeting a single isoform of TGFβ, e.g., TGFβ1) provide better selectivity than pan-TGFβ inhibitors (targeting multiple or all isoforms of TGFβ). Isoform-specific, context-tolerant inhibitors of TGFβ1 (targeting multiple contexts of a single isoform of TGFβ1) provide better selectivity than isoform-specific inhibitors. Isoform-specific, context-independent inhibitors of TGFβ1 (targeting and inhibiting TGFβ1 function regardless of which presentation molecule is associated) provide isoform specificity while allowing broader coverage of inhibitory effects across multiple activities of TGFβ1.

[0070] [Definition] In order to make this disclosure easier to understand, certain terms are first defined.These definitions should be read in light of the remainder of this disclosure and are as understood by those skilled in the art.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.Further definitions are provided throughout the detailed description.

[0071] antibody: The term "antibody" encompasses any naturally occurring, recombinant, modified or engineered immunoglobulin or immunoglobulin-like structure or antigen-binding fragment or portion thereof, or derivatives thereof, as further described elsewhere herein. Thus, the term refers to an immunoglobulin molecule that specifically binds to a target antigen, including, for example, chimeric, humanized, fully human, and bispecific antibodies. An intact antibody generally contains at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, such as naturally occurring antibodies in camelids, which may contain only heavy chains. An antibody may be derived only from a single source, or may be "chimeric", i.e., different portions of the antibody may be derived from two different antibodies. An antibody, or an antigen-binding portion thereof, may be produced in a hybridoma, by recombinant DNA technology, or by enzymatic or chemical cleavage of an intact antibody. The term "antibody" as used herein includes monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, and antibody fusions (sometimes referred to herein as "antibody conjugates"). In some embodiments, the term also encompasses peptibodies.

[0072] antigen The term "antigen" refers to a molecular structure that provides an epitope, e.g., a molecule or molecular moiety, or a complex of molecules or molecular moieties, capable of being bound by a selective binding agent, such as an antigen-binding protein (including, e.g., an antibody). Thus, a selective binding agent can specifically bind to an antigen formed by two or more components in a complex. In some embodiments, an antigen can be used in an animal to produce antibodies capable of binding to the antigen. An antigen can bear one or more epitopes capable of interacting with different antigen-binding proteins, e.g., antibodies. Antigen-binding portions / fragmentsAs used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TGFβ1). Antigen-binding portions include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. In some embodiments, an antigen-binding portion of an antibody may be obtained from an intact antibody molecule using any suitable standard technique, such as proteolytic or recombinant genetic engineering techniques, including, for example, the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Non-limiting examples of antigen-binding portions include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a single-chain Fv (scFv) molecule (see, e.g., Bird et al. (1988) SCIENCE 242:423-426; and Huston et al. (1988) PROC. NAT'L. ACAD. SCI. USA 85:5879-5883); (vi) a dAb fragment (see, e.g., Ward et al. (1989) NATURE 341:544-546); and (vii) a minimal recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)). Other forms of single chain antibodies, such as diabodies, are also encompassed.The term antigen-binding portion of an antibody includes a "single-chain Fab fragment" (otherwise known as "scFab"), which comprises an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; wherein said linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids.

[0073] cancer The term "cancer" as used herein refers to a physiological condition in multicellular eukaryotic organisms that is typically characterized by unregulated cell proliferation and malignant tumors.The term therefore broadly encompasses solid tumors, blood cancers (e.g., leukemia), as well as myelofibrosis and multiple myeloma.

[0074] Cell-associated TGFβ1 The term refers to membrane-bound (e.g., tethered to the cell surface) TGFβ1 or its signaling complex (e.g., precursor / latent TGFβ1). Typically, such cells are immune cells. TGFβ1 presented by GARP or LRRC33 is cell-associated TGFβ1.

[0075] Checkpoint inhibitors: In the context of this disclosure, checkpoint inhibitor refers to immune checkpoint inhibitor and has the meaning understood in the art. Typically, the target is a receptor molecule on T cells or NK cells, or a corresponding cell surface ligand on antigen presenting cells (APCs) or tumor cells. Immune checkpoints are activated in immune cells to avoid inflammatory immune development against "self". Therefore, altering the balance of the immune system through checkpoint inhibition should allow it to be fully activated to detect and eliminate cancer. The most known inhibitory receptors involved in the control of immune response are cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM3), lymphocyte activation gene 3 (LAG3), killer cell immunoglobulin-like receptor (KIR), glucocorticoid-induced tumor necrosis factor receptor (GITR) and suppressor of T cell activation containing V-domain immunoglobulin (Ig) (VISTA). Non-limiting examples of checkpoint inhibitors include: nivolumab, pembrolizumab, BMS-936559, atezolizumab, avelumab, durvalumab, ipilimumab, tremelimumab, IMP-321, BMS-986016, and lirilumab.

[0076] clinical benefit The term "clinical benefit" as used herein is intended to include both efficacy and safety of treatment. Thus, a therapeutic treatment that achieves a desired clinical benefit is both effective and safe (e.g., tolerable or acceptable with respect to toxicity or adverse events).

[0077] Combination therapy: "Combination therapy" refers to a therapeutic regimen for a clinical indication that includes two or more therapeutic agents. Thus, the term refers to a therapeutic regimen in which a first treatment that includes a first composition (e.g., an active ingredient) is administered to a patient together with a second treatment that includes a second composition (active ingredient), intended to treat the same or overlapping disease or clinical condition. Both the first and second compositions may act on the same cellular target, or on separate cellular targets. The phrase "together with" in the context of combination therapy means that the therapeutic effect of the first treatment overlaps temporally and / or spatially with the therapeutic effect of the second treatment in the subject receiving the combination therapy. Thus, combination therapy may be formulated as a single formulation for simultaneous administration, or as separate formulations for sequential administration of the treatments.

[0078] Combined or Combinatorial Epitopes In some embodiments, the inhibitory antibodies of the present invention can bind to epitopes formed by two or more components (e.g., parts or segments) of the precursor / latent TGFβ1 complex. Such epitopes are called combined or combinatorial epitopes. Thus, a combined epitope may include an amino acid residue(s) from a first component of the complex, an amino acid residue(s) from a second component of the complex, and so on. Each component may consist of a single protein or two or more proteins of the antigenic complex. The binding of an antibody to a combined epitope does not depend solely on the primary amino acid sequence of the antigen. Rather, a combined epitope is formed by structural contributions from two or more components (e.g., parts or segments such as amino acid residues) of the antigen or antigen complex.

[0079] Conflict or cross-conflictThe term "competition", when used in the context of competing antigen-binding proteins (e.g., antibodies or antigen-binding portions thereof) for the same epitope, refers to competition between antigen-binding proteins in which the antigen-binding protein being tested prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein to a common antigen (e.g., TGFβ1 or a fragment thereof) as determined by the assay. Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another (e.g., solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay; solid-phase direct biotin-avidin EIA; solid-phase direct target assay, and solid-phase direct target sandwich assay). Typically, when a competing antigen-binding protein is present in excess, it inhibits (e.g., reduces) specific binding of a reference antigen-binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more. In some embodiments, the first antibody or antigen-binding portion thereof and the second antibody or antigen-binding portion thereof cross-block each other with respect to the same antigen, as analyzed, for example, by Biacor or Octet, using standard test conditions (e.g., binding is analyzed at room temperature, about 20-25° C.), e.g., according to the manufacturer's instructions. In some embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have the same epitope. In other embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have non-identical but overlapping epitopes. In further embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have distinct (different) epitopes that are in close proximity in three-dimensional space such that antibody binding is cross-blocked through steric hindrance."Cross-blocking" means that the binding of a first antibody to an antigen prevents the binding of a second antibody to the same antigen, and similarly, the binding of the second antibody to an antigen prevents the binding of the first antibody to the same antigen.

[0080] Complementarity determining regionAs used herein, the term "CDR" refers to the complementarity determining region in an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2 and CDR3 for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs that give rise to a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are defined differently by different systems. The system described by Kabat (Kabat et al. (1987; 1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; and Chothia et al. (1989) Nature 342:877-883) have used the Kabat CDRs to It has been found that certain sub-portions within the CDRs adopt nearly identical peptide backbone conformations, despite being highly diverse at the amino acid sequence level. These sub-portions are designated L1, L2 and L3 or H1, H2 and H3 (where "L" and "H" designate the light and heavy chain regions, respectively). These regions may be called Chothia CDRs, and have overlapping boundaries with Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat CDRs are described by Padlan (1995) FASEB J.9:133-139 and MacCallum (1996) J.Mol.Biol.262(5):732-45. Still other CDR boundary definitions may not strictly follow one of the systems herein, but may still overlap with Kabat CDRs, but may be shorter or longer in light of predictions or experimental findings that certain residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding.The methods used herein may utilize CDRs defined according to any of these systems, although particular embodiments use the CDRs defined by Kabat or Chothia.

[0081] Conformational epitopes In some embodiments, the inhibitory antibodies of the present invention may bind to conformation-specific epitopes. Such epitopes are referred to as conformational, conformation-specific, conformation-dependent, or conformation-sensitive epitopes. The corresponding antibodies or fragments thereof that specifically bind to such epitopes may be referred to as conformation-specific, conformation-selective, or conformation-dependent antibodies. The binding of an antigen to a conformational epitope depends on the three-dimensional structure (conformation) of the antigen or antigen complex.

[0082] Constant region : Immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy and light chain constant domain amino acid sequences are known in the art.

[0083] Context-tolerant; context-independent : "context-tolerant" and "context-independent" TGFβ inhibitors are broad-spectrum context inhibitors that can act on more than one mode of TGFβ function. A "context-tolerant inhibitor" of TGFβ is an agent capable of inhibiting TGFβ activity associated with multiple contexts of TGFβ function, for example, at least two of the following: GARP (also called LRRC32), LRRC33, LTBP1, and LTBP3. Among context-tolerant inhibitors, if an agent is capable of inhibiting TGFβ activity regardless of the specific presentation molecule, such an inhibitor is called a "context-independent" inhibitor. Thus, a context-independent inhibitor of TGFβ can inhibit TGFβ activity associated with all of the following: GARP, LRRC33, LTBP1, and LTBP3. In some embodiments, context-tolerant and context-independent inhibitors can exert selective or biased inhibitory activity toward one or more contexts over others.

[0084] ECM-associated TGFβ1 The term refers to TGFβ1 or its signaling complexes (e.g., precursor / latent TGFβ1) that are components of (e.g., deposited in) the extracellular matrix. TGFβ1 presented by LTBP1 or LTBP3 is ECM-associated TGFβ1.

[0085] Effective dose An "effective amount" (or therapeutically effective amount) is a dose or dosing regimen that achieves a statistically significant clinical benefit in a patient population.

[0086] Epitope : The term "epitope" includes any molecular determinant (e.g., polypeptide determinant) capable of specific binding to a binding agent, immunoglobulin, or T-cell receptor. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and in certain embodiments may have specific three-dimensional structural characteristics, and / or specific charge characteristics. An epitope is a region of an antigen that is bound by a binding protein. An epitope therefore consists of amino acid residues in a region of an antigen (or a fragment thereof) that is known to bind to a complementary site on a specific binding partner. An antigenic fragment may contain more than one epitope. In certain embodiments, an antibody specifically binds to an antigen if it recognizes its target antigen within a complex mixture of proteins and / or macromolecules. For example, antibodies are said to "bind to the same epitope" if the antibodies cross-compete (one prevents the binding or modulating effect of the other). In addition, while the structural definition of an epitope (overlapping, similar, identical) is informative, the functional definition is often more relevant because it encompasses both structural (binding) and functional (regulatory, competitive) parameters.

[0087] Fibrosis The term "fibrosis" or "fibrotic condition / disorder" refers to a process or indication characterized by the pathological accumulation of extracellular matrix (ECM) components, such as collagen, in a tissue or organ.

[0088] GARP-TGFβ1 complex As used herein, the term "GARP-TGFβ1 complex" refers to a protein complex comprising a proprotein or latent form of transforming growth factor β1 (TGFβ1) protein and a glycoprotein A repeat dominant protein (GARP) or a fragment or variant thereof. In some embodiments, the proprotein or latent form of the TGFβ1 protein may be referred to as a "precursor / latent TGFβ1 protein". In some embodiments, the GARP-TGFβ1 complex comprises a GARP covalently linked to a precursor / latent TGFβ1 via one or more disulfide bonds. In other embodiments, the GARP-TGFβ1 complex comprises a GARP non-covalently linked to a precursor / latent TGFβ1. In some embodiments, the GARP-TGFβ1 complex is a naturally occurring complex, e.g., a GARP-TGFβ1 complex in a cell. An exemplary GARP-TGFβ1 complex is shown in FIG. 3.

[0089] Human antibodies The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include, for example, within the CDRs and particularly CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted into human framework sequences.

[0090] Humanized antibodiesThe term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse), but at least a portion of the VH and / or VL sequences have been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. A "humanized antibody" is also an antibody, or a variant, derivative, analog, or fragment thereof, that immunospecifically binds to an antigen of interest, and comprises a FR region having substantially the amino acid sequence of a human antibody and a CDR region having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR that has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises substantially all of at least one, typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), with all or substantially all of the CDR regions and all or substantially all of the FR regions corresponding to those of a non-human immunoglobulin (i.e., donor antibody) being of a human immunoglobulin consensus sequence. In one embodiment, the humanized antibody also comprises at least a portion of an immunoglobulin Fc region (typically that of a human immunoglobulin). In some embodiments, the humanized antibody comprises a light chain and at least the variable domain of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only a humanized light chain. In some embodiments, the humanized antibody comprises only a humanized heavy chain. In certain embodiments, the humanized antibody comprises only a humanized variable domain of the light chain and / or a humanized heavy chain.

[0091] Isoform specificThe term "isoform specificity" refers to the ability of an agent to discriminate one isoform over other structurally related isoforms (i.e., selectivity). An isoform-specific TGFβ inhibitor will direct its inhibitory activity toward one isoform of TGFβ at a given concentration, but not other isoforms of TGFβ. For example, an isoform-specific TGFβ1 antibody will selectively bind to TGFβ1. A TGFβ1 specific inhibitor (antibody) will preferentially target (bind and thereby inhibit) the TGFβ1 isoform over TGFβ2 or TGFβ3 with substantially greater affinity. For example, selectivity in this context can refer to at least a 500-1000-fold difference in the respective affinities as measured by in vitro binding assays such as Octet and Biacor. In some embodiments, the selectivity is such that the inhibitor will not inhibit TGFβ2 and TGFβ3 when used at a dose effective to inhibit TGFβ1 in vivo. For example, an antibody may preferentially bind to TGFβ1 with an affinity of about 1 pM, while the same antibody may bind to TGFβ2 and / or TGFβ3 with about 0.5-50 nM. For such inhibitors to be useful as therapeutics, the dose to achieve the desired effect (e.g., a therapeutically effective amount) must fall within a window in which the inhibitor can effectively inhibit the TGFβ1 isoform without inhibiting TGFβ2 or TGFβ3.

[0092] To be isolated As used herein, an "isolated" antibody refers to an antibody that is substantially free from other antibodies having different antigenic specificities. In some embodiments, an isolated antibody is substantially free from other unintended cellular material and / or chemicals.

[0093] LocalizedThe term "localized" (e.g., "localized tumor") in the context of this disclosure refers to an anatomically isolated or isolatable abnormality, e.g., a solid malignant tumor, as opposed to a systemic disease. Certain leukemias, etc., may have both a localized (e.g., bone marrow) and a systemic (e.g., circulating blood cells) component to the disease.

[0094] LRRC33-TGFβ1 complex As used herein, the term "LRRC33-TGFβ1 complex" refers to a complex between the proprotein or latent form of transforming growth factor β1 (TGFβ1) protein and leucine-rich repeat-containing protein 33 (LRRC33; also known as negative regulator of reactive oxygen species or NRROS) or a fragment or variant thereof. In some embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 covalently bound to precursor / latent TGFβ1 via one or more disulfide bonds. In other embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 non-covalently bound to precursor / latent TGFβ1. In some embodiments, the LRRC33-TGFβ1 complex is a naturally occurring complex, for example, an intracellular LRRC33-TGFβ1 complex.

[0095] LTBP1-TGFβ1 complex As used herein, the term "LTBP1-TGFβ1 complex" refers to a protein complex comprising a proprotein or latent form of transforming growth factor β1 (TGFβ1) protein and latent TGF-β binding protein 1 (LTBP1) or a fragment or variant thereof. In some embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 covalently bound to precursor / latent TGFβ1 via one or more disulfide bonds. In other embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 non-covalently bound to precursor / latent TGFβ1. In some embodiments, the LTBP1-TGFβ1 complex is a naturally occurring complex, e.g., an intracellular LTBP1-TGFβ1 complex. An exemplary LTBP1-TGFβ1 complex is shown in FIG. 3.

[0096] LTBP3-TGFβ1 complex As used herein, the term "LTBP3-TGFβ1 complex" refers to a protein complex comprising a proprotein or latent form of transforming growth factor β1 (TGFβ1) protein and latent TGF-β binding protein 3 (LTBP3) or a fragment or variant thereof. In some embodiments, the LTBP3-TGFβ1 complex comprises LTBP3 covalently bound to precursor / latent TGFβ1 via one or more disulfide bonds. In other embodiments, the LTBP3-TGFβ1 complex comprises LTBP1 non-covalently bound to precursor / latent TGFβ1. In some embodiments, the LTBP3-TGFβ1 complex is a naturally occurring complex, e.g., an intracellular LTBP3-TGFβ1 complex. An exemplary LTBP3-TGFβ1 complex is shown in FIG. 3.

[0097] Myelofibrosis : "Myelofibrosis", also known as bone myelofibrosis, is a relatively rare myeloproliferative disease (e.g., cancer) that belongs to a group of diseases called myeloproliferative disorders. Myelofibrosis is classified in the Philadelphia chromosome negative (-) branch of myeloproliferative neoplasms. Myelofibrosis is characterized by abnormal clonal proliferation of hematopoietic stem cells in the bone marrow and other sites, resulting in fibrosis, or replacement of the marrow by scar tissue. The term myelofibrosis refers to primary myelofibrosis (PMF) unless otherwise specified. It may also be called chronic idiopathic myelofibrosis (cIMF) (the terms idiopathic and primary imply that in these cases the disease is of unknown or spontaneous origin). This is in contrast to myelofibrosis that occurs secondary to polycythemia vera or essential thrombocythemia. Myelofibrosis is a form of myelogenous dysplasia, which refers to changes in cell types within the blood-forming tissue of the bone marrow, and the two terms are often used interchangeably. The terms idiopathic myelodysplasia and myelofibrosis with myelodysplasia (MMM) are also used to refer to primary myelofibrosis.

[0098] Pan-TGFβ inhibitorsThe term "pan-TGFβ inhibitor" refers to any agent capable of inhibiting or antagonizing multiple isoforms of TGFβ. Such inhibitors may be small molecule inhibitors of TGFβ isoforms. The term includes pan-TGFβ antibodies, which refer to any agent capable of binding to more than one isoform of TGFβ, for example, at least two of TGFβ1, TGFβ2, and TGFβ3. In some embodiments, pan-TGFβ antibodies bind to all three isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3. In some embodiments, pan-TGFβ antibodies bind to and neutralize all three isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3.

[0099] presentation molecule The term "presenting molecule" or "presentation molecule" of TGFβ refers to a protein entity capable of binding / linking to the inactive form(s) of TGFβ, thereby "presenting" the proprotein in the extracellular domain. Four TGFβ presenting molecules have been identified to date: latent TGFβ binding protein-1 (LTBP1) and LTBP3 are deposited in the extracellular matrix (i.e., components of the ECM), while glycoprotein A repeat dominant (GARP / LRRC32) and leucine-rich repeat containing protein 33 (LRRC33) contain transmembrane domains and present latent TGFβ1 on the surface of certain cells, such as immune cells. The TGFβ1 isoform alone is involved in many biological processes, both in normal and disease states. These include, but are not limited to, the maintenance of tissue homeostasis, inflammatory responses, ECM recognition, e.g., wound healing, and regulation of immune responses, as well as organ fibrosis, cancer, and autoimmunity.

[0100] ProTGFβ1The term "proTGFβ1" as used herein is intended to encompass the precursor form of the inactive TGFβ1 complex that contains the prodomain sequence of TGFβ1 within the complex. Thus, the term may include precursor-, as well as latent-forms of TGFβ1. The expressions "precursor / latent TGFβ1" may be used interchangeably. The "precursor" form of TGFβ1 exists prior to proteolytic cleavage at the furin site. Once cleaved, the resulting form is referred to as the "latent" form of TGFβ1. The "latent" complex remains associated until further activation triggers, such as an integrin-driven activation event. As shown in FIG. 3, the proTGFβ1 complex consists of dimeric TGFβ1 proprotein polypeptides linked by disulfide bonds. It should be noted that the adjective "latent" may be commonly used to describe the "inactive" state of TGFβ1 prior to integrin-mediated or other activation events.

[0101] regulatory T cells : "Regulatory T cells" or Tregs are characterized by the expression of biomarkers CD4, FOXP3, and CD25. Tregs are sometimes called suppressive T cells and represent a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T (Teff) cells. Tregs can arise in the thymus (so-called CD4+Foxp3+ "intrinsic" Tregs) or differentiate from naive CD4+ T cells in the periphery, for example after exposure to TGFβ or retinoic acid.

[0102] Solid tumors The term "solid tumor" refers to a proliferative disorder that results in an abnormal growth or mass of tissue that does not usually contain cysts or liquid areas. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Solid tumors can be composed of cancerous (malignant) cells, stromal cells including CAFs, and infiltrating white blood cells such as macrophages and lymphocytes.

[0103] Specific binding As used herein, the terms "specific binding" or "specifically binds" mean that the interaction of an antibody, or antigen-binding portion thereof, with an antigen is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope). For example, an antibody, or antigen-binding portion thereof, binds to a specific protein rather than to proteins in general. In some embodiments, an antibody, or antigen-binding portion thereof, is characterized in that the antibody binds to at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or less. In some embodiments, the term "specific binding to an epitope of TGFβ1," "specifically binds to an epitope of TGFβ1," "specific binding to TGFβ1," or "specifically binds to TGFβ1" as used herein means binding to TGFβ1 and having a KD of 1.0×10 as determined by surface plasmon resonance. -7 It refers to an antibody, or antigen-binding portion thereof, that has a dissociation constant (KD) of less than or equal to M. In one embodiment, the antibody, or antigen-binding portion thereof, can specifically bind to both human and non-human (e.g., mouse) orthologs of TGFβ1.

[0104] subjectThe term "subject" in the context of therapeutic applications refers to an individual who receives clinical care or intervention, such as treatment, diagnosis, etc. Suitable subjects include vertebrates, including but not limited to mammals (e.g., human and non-human mammals). When the subject is a human subject, the term "patient" may be used interchangeably. In a clinical context, the term "patient population" or "patient subpopulation" is used to refer to a group of individuals that fall into a set of criteria, such as clinical criteria (e.g., disease presentation, disease stage, susceptibility to a particular condition, responsiveness to treatment, etc.), medical history, health status, sex, age group, genetic criteria (e.g., carriers of a particular mutation, polymorphism, gene duplication, DNA sequence repeat, etc.) and lifestyle factors (e.g., smoking, alcohol consumption, exercise, etc.).

[0105] TGF-β1-Related Disorders By "TGFβ1-associated disorder" is meant any disease or disorder whose etiology and / or progression can be attributed, at least in part, to TGFβ1 signaling or dysregulation thereof.

[0106] TGFβ inhibitors The term "TGFβ inhibitor" refers to any agent capable of antagonizing the biological activity or function of a TGFβ growth factor (e.g., TGFβ1, TGFβ2 and / or TGFβ3). The term is not intended to be limited by its mechanism of action and includes, for example, neutralizing inhibitors, receptor antagonists, soluble ligand traps, and activation inhibitors of TGFβ.

[0107] The "TGFβ family" is a class within the TGFβ superfamily that includes three structurally similar isoforms: TGFβ1, TGFβ2, and TGFβ3.

[0108] toxicity As used herein, the term "toxicity" refers to unwanted in vivo effects in a patient associated with a treatment administered to the patient, such as unwanted side effects and adverse events. "Tolerability" refers to the level of toxicity associated with a treatment or therapeutic regimen that can be reasonably tolerated by a patient without discontinuing treatment due to toxicity.

[0109] Treat / treatment The term "treat" or "treatment" includes therapeutic treatment, prophylactic treatment, and applications that reduce the risk of a subject developing a disorder or other risk factor. Thus, the term is intended to broadly mean causing a therapeutic benefit in a patient, for example, by enhancing or boosting the body's immunity; by reducing or reversing immunosuppression; by reducing, removing, or eradicating harmful cells or substances from the body; by reducing disease burden (e.g., tumor burden); by preventing recurrence or relapse; by extending refractory periods, and / or otherwise improving survival. The term includes therapeutic treatment, prophylactic treatment, and applications that reduce the risk of a subject developing a disorder or other risk factor. Treatment does not require complete treatment of the disorder, but encompasses embodiments that reduce symptoms or underlying risk factors. In the context of combination therapy, the term may refer to i) the ability of the second therapeutic to reduce the effective dose of the first therapeutic, reducing side effects and increasing tolerability; ii) the ability of the second therapeutic to make the patient more responsive to the first therapeutic; and / or iii) the ability to effect additive or synergistic clinical benefit.

[0110] Tumor-associated macrophages : "Tumor-associated macrophages (TAM)" are polarized / activated macrophages with a tumor-promoting phenotype. TAMs can be tissue-resident macrophages derived from erythro-myeloid progenitors or medulla-derived monocytes / macrophages recruited to tumor sites. The differentiation of monocytes / macrophages into TAMs is influenced by many factors, including local chemical signals, such as cytokines, chemokines, growth factors, and other molecules acting as ligands, as well as cell-cell interactions between monocytes / macrophages present within the niche (tumor microenvironment). In general, monocytes / macrophages can be polarized into so-called "M1" or "M2" subtypes, the latter being associated with a more tumor-promoting phenotype. In solid tumors, up to 50% of the tumor mass can correspond to macrophages that are preferentially M2 polarized.

[0111] Tumor microenvironment The term "tumor microenvironment (TME)" refers to the local disease niche in which a tumor (e.g., a solid tumor) resides in vivo.

[0112] Variable region The term "variable region" or "variable domain" refers to portions of the light and / or heavy chains of an antibody, typically comprising approximately the amino terminal 120-130 amino acids in the heavy chain and about the amino terminal 100-110 amino acids in the light chain. In certain embodiments, the variable regions of different antibodies differ extensively in amino acid sequence, even among antibodies of the same species. The variable regions of an antibody typically determine the specificity of a particular antibody for its target.

[0113] Other than in the operating examples, or unless otherwise indicated, all numbers expressing reaction conditions or amounts of ingredients used herein should be understood to be modified in all instances by the term "about," which when used in reference to percentages may mean ±1%.

[0114] As used herein, in the specification and the claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless clearly dictated to the contrary.

[0115] In the specification and claims, the term "and / or" as used herein should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements, whether related or unrelated to those elements specifically identified, other than the elements expressly identified by the "and / or" clause may optionally be present, unless expressly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and / or B," when used with open-ended language such as "comprising," may refer in one embodiment to A without B (which may optionally include elements other than B); in another embodiment to B without A (which may optionally include elements other than A); in yet another embodiment to both A and B (which may optionally include other elements); and so forth.

[0116] As used herein in the specification and claims, the phrase "at least one" in a reference to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements other than those specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present (whether related or unrelated to those elements specifically identified). Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") may refer, in one embodiment, to at least one (optionally, may include more than one) A with no B (optionally, may include elements other than B); in another embodiment, to at least one (optionally, may include more than one) B with no A (optionally, may include elements other than A); in yet another embodiment, to at least one (optionally, may include more than one) A and at least one (optionally, may include more than one) B (optionally, may include other elements); etc.

[0117] The use of ordinal terms in the claims, such as "first," "second," "third," etc., to modify claim elements does not, in and of itself, imply any priority, precedence, or order, or chronological order, in which method actions are performed of one claim element over another, but is merely used as a label to distinguish one claim element having a particular name from another element having the same name (if no ordinal term is used).

[0118] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1-50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, such as 10-20, 1-10, 30-40, etc.

[0119] [TGFβ1 isoform-selective, context-tolerant / context-independent antibodies] The present invention provides antibodies, and antigen-binding portions thereof, that bind to two or more of the following complexes that contain precursor / latent-TGFβ1: GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex. Accordingly, some aspects of the present invention relate to antibodies, or antigen-binding portions thereof, that specifically bind to an epitope within such TGFβ1 complexes, where the epitope is available for binding by the antibody, or antigen-binding portion thereof, when TGFβ1 is present within the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex. In some embodiments, the epitope is available due to a conformational change in TGFβ1 when in complex with GARP, LTBP1, LTBP3, and / or LRRC33. In some embodiments, the epitope in TGFβ1 to which the antibody, or antigen binding portion thereof, binds is not available when TGFβ1 is not in a complex with GARP, LTBP1, LTBP3, and / or LRRC33. In some embodiments, the antibody, or antigen binding portion thereof, does not specifically bind to TGFβ2. In some embodiments, the antibody, or antigen binding portion thereof, does not specifically bind to TGFβ3. In some embodiments, the antibody, or antigen binding portion thereof, does not prevent TGFβ1 from binding to an integrin. For example, in some embodiments, the antibody, or antigen binding portion thereof, does not mask the integrin binding site of TGFβ1. In some embodiments, the antibody, or antigen binding portion thereof, inhibits activation of TGFβ1. In some embodiments, the antibody, or antigen binding portion thereof, inhibits release of mature TGFβ1 from the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex.

[0120] The antibodies, or antigen-binding portions thereof, provided herein specifically bind to epitopes of multiple (i.e., two or more) TGFβ1 complexes, and the epitopes are available for binding by the antibodies, or antigen-binding portions thereof, when TGFβ1 is present in a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP2-TGFβ1 complex, a LTBP3-TGFβ1 complex, a LTBP4-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex. In some embodiments, the TGFβ1 comprises a naturally occurring mammalian amino acid sequence. In some embodiments, the TGFβ1 comprises a naturally occurring human amino acid sequence. In some embodiments, the TGFβ1 comprises a human, monkey, rat, or mouse amino acid sequence. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein do not specifically bind to TGFβ2. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein do not specifically bind to TGFβ3. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein do not specifically bind to TGFβ2 or TGFβ3. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein specifically bind to TGFβ1 comprising the amino acid sequence set forth in SEQ ID NO:21. The amino acid sequence of TGFβ2 and the TGFβ3 amino acid sequence are set forth in SEQ ID NOs:22 and 23, respectively. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein specifically bind to TGFβ1 comprising a non-naturally occurring amino acid sequence (otherwise referred to herein as non-naturally occurring TGFβ1). For example, non-naturally occurring TGFβ1 may include one or more recombinantly produced mutations to the naturally occurring TGFβ1 amino acid sequence. In some embodiments, the TGFβ1, TGFβ2, or TGFβ3 amino acid sequence comprises the amino acid sequence set forth in SEQ ID NOs:24-35 as set forth in Table 1. In some embodiments, the TGFβ1, TGFβ2, or TGFβ3 amino acid sequence comprises the amino acid sequence set forth in SEQ ID NOs:36-43 as set forth in Table 2.

[0121] TGFβ1 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 21)

[0122] TGFβ2 SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS (SEQ ID NO: 22)

[0123] TGFβ3 SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEAASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS (SEQ ID NO: 23) JPEG2025000891000001.jpg124166 JPEG2025000891000002.jpg225166 JPEG2025000891000003.jpg225166 JPEG2025000891000004.jpg114166 JPEG2025000891000005.jpg102166 JPEG2025000891000006.jpg227166 JPEG2025000891000007.jpg229166 JPEG2025000891000008.jpg226166 JPEG2025000891000009.jpg228166 JPEG2025000891000010.jpg229166 JPEG2025000891000011.jpg225166 JPEG2025000891000012.jpg23166

[0124] In some embodiments, an antigenic protein complex (e.g., LTBP-TGFβ1 complex) may comprise one or more LTBP proteins (e.g., LTBP1, LTBP2, LTBP3, and LTBP4) or fragment(s) thereof. In some embodiments, an antibody described herein, or an antigen-binding portion thereof, is capable of binding to a LTBP1-TGFβ1 complex. In some embodiments, the LTBP1 protein is a naturally occurring protein or a fragment thereof. In some embodiments, the LTBP1 protein is a non-naturally occurring protein or a fragment thereof. In some embodiments, the LTBP1 protein is a recombinant protein. Such a recombinant LTBP1 protein may comprise LTBP1, or a splice variant thereof and / or a fragment thereof. The recombinant LTBP1 protein may be modified to comprise one or more detectable labels. In some embodiments, the LTBP1 protein comprises a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP1 protein does not comprise a leader sequence (i.e., the leader sequence is processed or cleaved). Such detectable labels may include, but are not limited to, a biotin label, a polyhistidine tag, a myc tag, an HA tag, and / or a fluorescent tag. In some embodiments, the LTBP1 protein is a mammalian LTBP1 protein. In some embodiments, the LTBP1 protein is a human, monkey, mouse, or rat LTBP1 protein. In some embodiments, the LTBP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 46 and 47 in Table 2. In some embodiments, the LTBP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 50 in Table 3.

[0125] In some embodiments, the antibodies, or antigen-binding portions thereof, described herein are capable of binding to the LTBP3-TGFβ1 complex. In some embodiments, the LTBP3 protein is a naturally occurring protein or a fragment thereof. In some embodiments, the LTBP3 protein is a non-naturally occurring protein or a fragment thereof. In some embodiments, the LTBP3 protein is a recombinant protein. Such recombinant LTBP3 proteins may comprise LTBP3, or a splice variant and / or a fragment thereof. In some embodiments, the LTBP3 protein comprises a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP3 protein does not comprise a leader sequence (i.e., the leader sequence is processed or cleaved). The recombinant LTBP3 protein may be modified to include one or more detectable labels. Such detectable labels may include, but are not limited to, a biotin label, a polyhistidine tag, a myc tag, an HA tag, and / or a fluorescent tag. In some embodiments, the LTBP3 protein is a mammalian LTBP3 protein. In some embodiments, the LTBP3 protein is a human, monkey, mouse, or rat LTBP3 protein. In some embodiments, the LTBP3 protein comprises the amino acid sequence set forth in SEQ ID NOs: 44 and 45 in Table 2. In some embodiments, the LTBP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 51 in Table 3.

[0126] In some embodiments, the antibodies, or antigen-binding portions thereof, described herein are capable of binding to the GARP-TGFβ1 complex. In some embodiments, the GARP protein is a naturally occurring protein or a fragment thereof. In some embodiments, the GARP protein is a non-naturally occurring protein or a fragment thereof. In some embodiments, the GARP protein is a recombinant protein. Such GARPs may be recombinant and are referred to herein as recombinant GARPs. Some recombinant GARPs may contain one or more modifications, truncations and / or mutations compared to wild-type GARP. Recombinant GARPs may be modified to be soluble. In some embodiments, the GARP protein includes a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the GARP protein does not include a leader sequence (i.e., the leader sequence is processed or cleaved). In other embodiments, the recombinant GARP is modified to include one or more detectable labels. In further embodiments, such detectable labels may include, but are not limited to, a biotin label, a polyhistidine tag, a flag tag, a myc tag, an HA tag, and / or a fluorescent tag. In some embodiments, the GARP protein is a mammalian GARP protein. In some embodiments, the GARP protein is a human, monkey, mouse, or rat GARP protein. In some embodiments, the GARP protein comprises an amino acid sequence set forth in SEQ ID NOs: 48-49 of Table 2. In some embodiments, the GARP protein comprises an amino acid sequence set forth in SEQ ID NOs: 52 and 53 of Table 4. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein do not bind to TGFβ1 in a context-dependent manner (e.g., binding to TGFβ1 occurs only when the TGFβ1 molecule is complexed with a specific presentation molecule, e.g., GARP). Instead, the antibodies, or antigen-binding portions thereof, bind to TGFβ1 in a context-independent manner. In other words, the antibody, or antigen-binding portion thereof, binds to TGFβ1 when bound to any of the presentation molecules: GARP, LTBP1, LTBP3, and / or LRCC33.

[0127] In some embodiments, the antibodies, or antigen-binding portions thereof, described herein are capable of binding to the LRRC33-TGFβ1 complex. In some embodiments, the LRRC33 protein is a naturally occurring protein or a fragment thereof. In some embodiments, the LRRC33 protein is a non-naturally occurring protein or a fragment thereof. In some embodiments, the LRRC33 protein is a recombinant protein. Such LRRC33 may be recombinant, and is referred to herein as recombinant LRRC33. Some recombinant LRRC33 proteins may include one or more modifications, truncations, and / or mutations compared to wild-type LRRC33. The recombinant LRRC33 protein may be modified to be soluble. For example, in some embodiments, the ectodomain of LRRC33 may be expressed with a C-terminal His tag to express a soluble LRRC33 protein (sLRRC33; see, e.g., SEQ ID NO:84). In some embodiments, the LRRC33 protein includes a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LRRC33 protein does not include a leader sequence (i.e., the leader sequence has been processed or cleaved). In other embodiments, the recombinant LRRC33 protein is modified to include one or more detectable labels. In further embodiments, such detectable labels may include, but are not limited to, a biotin label, a polyhistidine tag, a flag tag, a myc tag, an HA tag, and / or a fluorescent tag. In some embodiments, the LRRC33 protein is a mammalian LRRC33 protein. In some embodiments, the LRRC33 protein is a human, monkey, mouse, or rat LRRC33 protein. In some embodiments, the LRRC33 protein comprises the amino acid sequence set forth in SEQ ID NOs: 83, 84, and 101 of Table 4. JPEG2025000891000013.jpg229166 JPEG2025000891000014.jpg201166 JPEG2025000891000015.jpg229166 JPEG2025000891000016.jpg234166 JPEG2025000891000017.jpg124166

[0128] [TGFβ1 antagonist] To carry out the method of the present invention, any suitable inhibitor of TGFβ1 may be used, provided that such agent inhibits or antagonizes TGFβ1 across multiple biological effects (e.g., TGFβ1 from multiple cell origins) with sufficient selectivity with respect to the TGFβ1 isoform. Preferably, such inhibitor of TGFβ1 has no measurable inhibitory activity toward TGFβ2 and TGFβ3 at a dose that provides clinical benefit (e.g., therapeutic efficacy and acceptable toxicity profile) when administered to a human subject. Suitable inhibitors include small molecules, nucleic acid-based agents, biologics (e.g., polypeptide-based agents, such as antibodies and other finding-agents), and any combination thereof. In some embodiments, such agents are antibodies or fragments thereof, as further described below. These include neutralizing antibodies that bind to TGFβ1 growth factor and thereby neutralize its action.

[0129] [Functional antibody selectively inhibiting TGFβ1]

[0130] The present invention, in one aspect, encompasses the use of functional antibodies. As used herein, a "functional antibody" confers one or more biological activities by virtue of its ability to bind to an antigen. Functional antibodies thus include those capable of modulating the activity / function of a target molecule (i.e., an antigen). Such modulating antibodies include inhibitory (or inhibitory) and activating antibodies. The present disclosure includes TGFβ antibodies capable of inhibiting biological processes mediated by TGFβ1 signaling associated with multiple contexts of TGFβ1. It is intended that the inhibitors used to carry out the invention, such as the antibodies described herein, are TGFβ1 selective and do not target or interfere with TGFβ2 and TGFβ3 when administered at therapeutically effective doses (doses at which sufficient efficacy is achieved within acceptable toxicity levels).

[0131] Based on earlier recognition by the applicants of the present disclosure that the lack of isoform specificity of conventional TGFβ antagonists may underlie the origin of toxicity associated with TGFβ inhibition (see PCT / US2017 / 021972), the inventors sought to further achieve broad-spectrum TGFβ1 inhibition to treat a variety of diseases exhibiting pleiotropic TGFβ1 dysregulation, while maintaining the safety / tolerability aspects of isoform-selective inhibitors.

[0132] In a broad sense, the term "inhibitory antibody" refers to an antibody that antagonizes or neutralizes a target function, e.g., growth factor activity. Advantageously, preferred inhibitory antibodies of the present disclosure are capable of inhibiting the release of mature growth factors from latent complexes, thereby reducing growth factor signaling. Inhibitory antibodies include antibodies that target any epitope that, when associated with such an antibody, reduces the release or activity of a growth factor. Such epitopes may be present on the pro-domain of a TGFβ protein (e.g., TGFβ1), a growth factor, or other epitopes that, when bound by the antibody, result in a reduction in growth factor activity. Inhibitory antibodies of the present invention include, but are not limited to, TGFβ1 inhibitory antibodies. In some embodiments, inhibitory antibodies of the present disclosure specifically bind combinatorial epitopes, i.e., epitopes formed by two or more components / portions of an antigen or antigen complex. For example, combinatorial epitopes may be formed by contributions from multiple portions of a single protein, i.e., amino acid residues from more than one non-contiguous segment of the same protein. Alternatively, combinatorial epitopes may be formed by contributions from multiple protein components of an antigen complex. In some embodiments, the inhibitory antibodies of the present disclosure specifically bind to a conformational epitope (or a conformation-specific epitope), e.g., an epitope that is sensitive to the tertiary structure (i.e., conformation) of an antigen or antigen complex.

[0133] Traditional approaches to antagonize TGFβ signaling have been to i) directly neutralize the mature growth factor after it has already become active, so as to exhaust the free ligand available for receptor binding (e.g., released from its latent precursor complex); ii) utilize soluble receptor fragments capable of sequestering the free ligand (e.g., so-called ligand traps); or iii) target its cell surface receptor(s) to block the ligand-receptor interaction. Each of these traditional approaches requires the antagonist to compete for the endogenous counterpart. Moreover, the first two approaches above (i and ii) target active ligands, which are transient species. Thus, such antagonists must be able to kinetically outcompete the endogenous receptor during a short time window. The third approach may provide a durable effect in comparison, but inadvertently results in unwanted inhibitory effects (and therefore possible toxicity) since many growth factors (e.g., up to about 20) signal through the same receptor(s).

[0134] To provide a solution to these shortcomings and to further enable better selectivity and localized action, the preferred mechanism of action underlying inhibitory antibodies such as those described herein acts upstream of TGFβ1 activation and ligand-receptor interaction. Therefore, it is considered that an isoform-specific, context-tolerant inhibitor of TGFβ1 suitable for carrying out the present invention should preferably target inactive (e.g., latent) precursor TGFβ1 complexes (e.g., complexes containing precursor / latent TGFβ1) prior to their activation in order to block the activation step at their origin (e.g., within the disease microenvironment). According to a preferred embodiment of the present invention, such inhibitors target ECM-associated and / or cell surface-tethered precursor / latent TGFβ1 complexes, rather than free ligands that are temporarily available for receptor binding.

[0135] Thus, some embodiments of the present invention use agents that specifically bind to TGFβ1-containing complexes, thereby inhibiting the function of TGFβ1 in an isoform-selective manner. Such agents are preferably antibodies that bind to epitopes within protein complexes that contain precursor / latent TGFβ1 (e.g., inactive TGFβ1 precursor). In some embodiments, the epitopes are available for binding by the antibodies when TGFβ1 is present in two or more of the following: GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex. In some embodiments, such antibodies bind to two or more of the TGFβ1-containing complexes provided above (e.g., "context-tolerant"), while in other embodiments, such antibodies bind to all four of the TGFβ1-containing complexes provided above (e.g., "context-independent"). In some embodiments, any such antibody may exhibit differential species selectivity. The epitope may be within the pro-domain of the TGFβ1 complex. The epitope may be a combination epitope, such that the epitope is formed by two or more parts / segments (e.g., amino acid residues) of one or more components of the complex. The epitope may be a conformational epitope, such that the epitope is sensitive to a specific three-dimensional structure of the antigen (e.g., the TGFβ1 complex). An antibody or fragment thereof that specifically binds to a conformational epitope is called a conformational antibody or a conformation-specific antibody.

[0136] Embodiments of the present disclosure include methods of using inhibitory antibodies in solution, in cell culture and / or in a subject to modify growth factor signaling, including for the purpose of providing clinical benefit to patients.

[0137] Exemplary antibodies, and corresponding nucleic acid sequences encoding the antibodies, useful in practicing the present invention contain one or more of the CDR amino acid sequences shown in Table 5. JPEG2025000891000018.jpg104166

[0138] In some embodiments, an antibody of the invention that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex comprises any antibody, or antigen-binding portion thereof, comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, as provided for any one of the antibodies shown in Table 5. In some embodiments, an antibody that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any one of the antibodies shown in Table 5. The invention also provides any nucleic acid sequence encoding a molecule comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 as provided for any one of the antibodies shown in Table 5. Antibody heavy and light chain CDR3 domains may play a particularly important role in the binding specificity / affinity of an antibody for an antigen. Thus, antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex of the present disclosure, or nucleic acid molecules encoding these antibodies or antigen-binding portions thereof, may comprise at least the heavy and / or light chain CDR3 of the antibodies shown in Table 5.

[0139] An embodiment of the present invention relates to a monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex and comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.

[0140] In some embodiments, CDRH1 comprises the sequence set forth in any one of SEQ ID NOs: 1, 2, and 85. In some embodiments, CDRH2 comprises the sequence set forth in any one of SEQ ID NOs: 3, 4, and 86. In some embodiments, CDRH3 comprises the sequence set forth in any one of SEQ ID NOs: 5, 6, and 87. CDRL1 comprises the sequence set forth in any one of SEQ ID NOs: 7, 8, and 88. In some embodiments, CDRL2 comprises the sequence set forth in any one of SEQ ID NOs: 9, 10, and 89. In some embodiments, CDRL3 comprises the sequence set forth in any one of SEQ ID NOs: 11, 12, and 90.

[0141] In some embodiments (e.g., for antibody Ab1 shown in Table 5), an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex includes: a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO:1, a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO:3, a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO:5, a CDRL1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO:9, and a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO:11.

[0142] In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO:5, and a light chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO:11. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO:3, and a light chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO:9. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:1, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO:7.

[0143] In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable domain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:14.

[0144] In some embodiments, an antibody or antigen binding portion thereof that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex comprises a heavy chain variable domain amino acid sequence encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 91, and a light chain variable domain amino acid sequence encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 92. In some embodiments, an antibody or antigen binding portion thereof comprises a heavy chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 91, and a light chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 92.

[0145] In some embodiments (e.g., for antibody Ab2 shown in Table 5), an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex comprises a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO:2, a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO:3, a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO:6, a CDRL1 comprising the amino acid sequence set forth in SEQ ID NO:8, a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO:10, and a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO:12.

[0146] In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO:6 and a light chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO:12. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO:4 and a light chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO:10. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO:2 and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO:8.

[0147] In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable domain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:16.

[0148] In some embodiments, an antibody or antigen binding portion thereof that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex comprises a heavy chain variable domain amino acid sequence encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 93, and a light chain variable domain amino acid sequence encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 94. In some embodiments, an antibody or antigen binding portion thereof comprises a heavy chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 93, and a light chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 94.

[0149] In some embodiments (e.g., for antibody Ab3 shown in Table 5), an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex comprises a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 85, a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 86, a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 87, a CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 88, a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 89, and a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 90.

[0150] In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO: 87 and a light chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO: 90. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO: 86 and a light chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO: 89. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 85 and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 88.

[0151] In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 95, and a light chain variable domain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 95, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 97.

[0152] In some embodiments, an antibody or antigen binding portion thereof that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex comprises a heavy chain variable domain amino acid sequence encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 96, and a light chain variable domain amino acid sequence encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 98. In some embodiments, an antibody or antigen binding portion thereof comprises a heavy chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 96, and a light chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 98.

[0153] In some examples, any antibody of the disclosure that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex includes any antibody (including an antigen-binding portion thereof) having one or more CDR (e.g., CDRH or CDRL) sequences substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3. For example, an antibody may include one or more CDR sequences set forth in Table 5 (SEQ ID NOs: 1-12 and 85-90) that contain up to 5, 4, 3, 2, or 1 amino acid residue variations compared to the corresponding CDR regions in any one of SEQ ID NOs: 1-12 and 85-90. The complete amino acid sequences for the heavy and light chain variable regions of the antibodies (e.g., Ab1, Ab2, and Ab3) listed in Table 5, as well as the nucleic acid sequences encoding the heavy and light chain variable regions of the antibodies, are provided below:

[0154] Ab1-heavy chain variable region amino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDIRPYGDYSAAFDIWGQGTLVTVSS (SEQ ID NO: 13)

[0155] Ab1-heavy chain variable region nucleic acid sequence GAGGTGCAACTCGTGGAGTCAGGCGGTGGACTTGTTCAGCCTGGGCGAAGTCTGAGACTCTCATGTGCAGCAAGTGGATTCACTTTCTCCAGTTACGGCATGCACTGGGTGAGACAGGCGCCTGGAAAGGGTTTGGAATGGGTCGCTGTGATCTCTTACGACGGGTCAAACAAATATTACGCGGATTC AGTGAAAGGGCGGTTCACTATTTCACGGGATAACTCCAAGAACACCCTGTATCTGCAGATGAATAGCCTGAGGGCAGAGGACACCGCTGTGTACTATTGTGCCCGGCATAAGGCCTTACGGCGATTACAGCGCCGCATTTGATATTTGGGGACAAGGCACCCTTGTGACAGTATCTTCT (SEQ ID NO: 91)

[0156] Ab1-Light chain variable region amino acid sequence NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSAPSIVIFEDNQRPSGAPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNHGGVFGGGTQLTVL (SEQ ID NO: 14)

[0157] Ab1-Light Chain Variable Region Nucleic Acid Sequence AATTTTATGCTTACCCAACCACATAGTGTGAGTGAGTCTCCCGGCAAGACTGTAACAATTTCATGTACCGGCAGCAGTGGCTCCATCGCTAGCAATTATGTGCAATGGTACCAACAGCGCCCCGGGAGCGCACCTTCAAATAGTGATATTCGAGGATAACCAACGGCCTAGTGG GGCTCCCGATAGATTTAGTGGGAGTATAGATAGCTCCTCCAACTCTGCCTCTCTCACCATTAGCGGGCTGAAAACAGAGGATGAAGCCGACTATTACTGCCAAAGCTATGATTCTAGCAACCACGGCGGAGTGTTTGGCGGAGGAACACAGCTGACAGTCCTAGG (SEQ ID NO: 92)

[0158] Ab1-heavy chain amino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDIRPYGDYSAAFDIWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 15)

[0159] Ab1-light chain amino acid sequence NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSAPSIVIFEDNQRPSGAPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNHGGVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 16)

[0160] Ab2-heavy chain variable region amino acid sequence EVQLVQSGAEMKKPGESLKISCKGSGYNFASDWIGWVRQTPGKGLEWMGVIYPGDSDTRYSASFQGQVTISADKSINTAYLQWSSLKASDTAMYYCASAAGIAAAGHVTAFDIWGQGTMVTVSS (SEQ ID NO: 17)

[0161] Ab2-heavy chain variable region nucleic acid sequence GAGGTGCAACTGGTGCAATCCGGAGCCGAGATGAAAAAGCCAGGGGAGAGCCTGAAGATCTCTTGTAAGGGCTCTGGCTATAACTTCGCTAGTGATTGGATCGGATGGGTGAGGCAAACCCCCGAAAGGGCCTCGAGTGGATGGGCGTGATCTACCCCGGCGACTCCGACACACGCTATAGCGCCTCAT TCCAGGGCCAGGTCACCATAAGTGCTGATAAATCAATAAATACAGCCTACTTGCAATGGTCAAGTCTGAAAGCCTCAGATACTGCCATGTACTATTGTGCCTCTGCCGCCGGCATTGCCGCGGCCGGTCACGTCACCGCCTTCGACATTTGGGGTCAGGGCACTATGGTCACTGTAAGCTCC (SEQ ID NO: 93)

[0162] Ab2-Light chain variable region amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPVTFGQGTKLEIK (SEQ ID NO: 18)

[0163] Ab2-Light Chain Variable Region Nucleic Acid Sequence GACATAGTCATGACCCAGTCACCTGACTCTTTGGCCGTGTCTCTGGGGGAGAGAGCCACAATAAATTGCAAGTCATCACAGAGCGTCCTGTACTCCTCCAATAATAAAAATTACCTGGCCTGGTACCAGCAAAAGCCCGGGCAACCCCCCAAATTGTTGATTTACTGGGCTAG TACAAGGGAATCTGGAGTGCCAGACCGGTTTTCTGGTTCTGGATCTGGTACTGACTTCACCCTGACAATCAGCTCCCTGCAGGCCGAAGACGTGGCTGTGTACTATTGTCAGCAGTACTATAGTACACCAGTTACTTTCGGCCAAGGCACTAAACTCGAAATCAAG (SEQ ID NO: 94)

[0164] Ab2-heavy chain amino acid sequence EVQLVQSGAEMKKPGESLKISCKGSGYNFASDWIGWVRQTPGKGLEWMGVIYPGDSDTRYSASFQGQVTISADKSINTAYLQWSSLKASDTAMYYCASAAGIAAAGHVTAFDIW GQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 19)

[0165] Ab2-light chain amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPVTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 20)

[0166] Ab3-heavy chain variable region amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVSSISGSGGATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSSGHWDFDYWGQGTLVTVSS (SEQ ID NO: 95)

[0167] Ab3-heavy chain variable region nucleic acid sequence GAGGTTCAGCTTCTGGAGAGCGGCGGTGGTCTTGTACAACCTGGAGGATCACTCAGGTTGTCATGTGCCGCAAGCGGGTTTACATTCAGGAACTATGCAATGAGCTGGGTCAGACAGGCTCCCGGCAAGGGACTTGAGTGGGTATCTTCCATCAGCGGATCTGGAGGAGCAACATATTATGCAGATAGTGTCAAAGGCAGGTTCACAATAAGCCGCGACAATTCTAAAAATACTCTTTATCTTCAAATGAATAGCCTTAGGGCTGAGGATACGGCGGTGTATTATTGTGCCCGCGTCTCAAGCGGGCATTGGGACTTCGATTATTGGGGGCAGGGTACTCTGGTTACTGTTTCCTCC (SEQ ID NO: 96)

[0168] Ab3 - Light chain variable region amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPFTFGQGTKVEIK (SEQ ID NO: 97)

[0169] Ab3 - Light chain variable region nucleic acid sequence GACATCCAAATGACACAGAGCCCGTCTTCCCTCTCAGCTTCAGTCGGTGATCGAGTGACGATTACGTGCCGCGCCAGCCAAAGCATCTCCTCCTATCTTAACTGGTATCAGCAGAAACCCGGAAAGGCCCCAAAGTTGCTTATTTACGACGCATCCTCCCTTCAATCTGGTGTGCCCAGCAGGTTCTCAGGCAGCGGTTCAGGAACGGATTTTACTCTTACCATTTCTAGTCTTCAACCTGAGGATTTTGCGACGTATTACTGTCAACAGAGCTACAGTGCGCCGTTCACCTTTGGGCAGGGTACTAAGGTTGAGATAAAGC (SEQ ID NO: 98)

[0170] Ab3-heavy chain amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFR NYAMS WVRQAPGKGLEWVS SISGSGGATYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARVSSGHWDFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 99)

[0171] Ab3-light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIYDA SSLQS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQSYSAPFT FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 100)

[0172] In some embodiments, antibodies of the disclosure that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex include any antibody that comprises a heavy chain variable domain of SEQ ID NO: 13, 17, or 95, or a light chain variable domain of SEQ ID NO: 14, 18, or 97. In some embodiments, antibodies of the disclosure that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex include any antibody that comprises a heavy chain variable and light chain variable pair of SEQ ID NO: 13 and 14; 17 and 18; and 95 and 97.

[0173] Embodiments of the disclosure provide antibodies that specifically bind to two or more of the following complexes: GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex having heavy chain variable and / or light chain variable amino acid sequences homologous to any of those described herein. In some embodiments, antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex comprise a heavy chain variable sequence or a light chain variable sequence that is at least 75% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the heavy chain variable amino acid sequence of SEQ ID NO: 13, 17, or 95, or the light chain variable sequence of SEQ ID NO: 14, 18, or 97. In some embodiments, the homologous heavy chain variable and / or light chain variable amino acid sequence is unchanged within any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variability (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) may occur within the heavy chain variable and / or light chain variable amino acid sequences excluding any CDR sequences provided herein.

[0174] In some embodiments, antibodies of the disclosure that specifically bind to two or more of the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex include any antibody, or antigen-binding portion thereof, that comprises a heavy chain of SEQ ID NO: 15 or 19 or a light chain of SEQ ID NO: 16 or 20. In some embodiments, antibodies of the disclosure that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex include any antibody that comprises the heavy and light chain pairs of SEQ ID NOs: 15 and 16; or 19 and 20.

[0175] Embodiments of the disclosure provide antibodies that specifically bind to two or more of the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex having heavy and / or light chain amino acid sequences homologous to any of those described herein. In some embodiments, an antibody that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex comprises a heavy or light chain sequence that is at least 75% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the heavy chain sequence of SEQ ID NO: 15, or 19, or the light chain amino acid sequence of SEQ ID NO: 16, or 20. In some embodiments, the homologous heavy and / or light chain amino acid sequences do not vary within any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variability (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) may occur within the heavy and / or light chain amino acid sequences excluding any CDR sequences provided herein.

[0176] In some embodiments, antibodies of the disclosure that specifically bind to two or more of the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex include any antibody, or antigen-binding portion thereof, that comprises a heavy chain of SEQ ID NO: 15 or 19 or a light chain of SEQ ID NO: 16 or 20. In some embodiments, antibodies of the disclosure that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex include any antibody that comprises the heavy and light chain pairs of SEQ ID NOs: 15 and 16; or 19 and 20.

[0177] Aspects of the disclosure provide antibodies that specifically bind to two or more of the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex that have heavy and / or light chain amino acid sequences homologous to any of those described herein. In some embodiments, antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex comprise a heavy or light chain sequence that is at least 75% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the heavy chain sequence of SEQ ID NO: 15 or 19 or the light chain amino acid sequence of SEQ ID NO: 16 or 20. In some embodiments, homologous heavy and / or light chain amino acid sequences are not varied within any CDR sequence provided herein.For example, in some embodiments, a degree of sequence variation (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) may occur within heavy and / or light chain amino acid sequences, excluding any CDR sequence provided herein.

[0178] In some embodiments, the "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990 (modified by Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0179] In any antibody or antigen-binding fragment described herein, one or more conservative mutations may be introduced into CDR or framework sequences at positions where the residues are not likely to participate in antibody-antigen interactions.In some embodiments, such conservative mutation(s) may be introduced into CDR or framework sequences at positions where the residues are not likely to participate in interactions with GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex, as determined based on crystal structure.In some embodiments, similar interfaces (e.g., residues involved in antigen-antibody interactions) may be predicted from known structural information on another antigen that shares structural similarity.

[0180] As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Mutations can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, such as those found in references that collect such methods (e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York). Conservative amino acid substitutions include those made between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0181] In some embodiments, the antibodies provided herein contain mutations that confer desirable properties to the antibody. For example, to avoid potential complications due to Fab arm exchange (known to occur in native IgG4 mAbs), the antibodies provided herein may contain a stabilizing "Adair" mutation (Angal et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline to provide an IgG1-like (CPPCP (SEQ ID NO: 54)) hinge sequence. Thus, any antibody may contain a stabilizing "Adair" mutation or the amino acid sequence CPPCP (SEQ ID NO: 54).

[0182] The isoform-specific, context-tolerant inhibitors (including context-independent inhibitors) of TGFβ1 of the present disclosure, such as antibodies that specifically bind to two or more of the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex, may optionally include an antibody constant region or a portion thereof. For example, a VL domain may be attached at its C-terminus to a light chain constant domain such as Cκ or Cλ. Similarly, a VH domain or a portion thereof may be attached to all or a portion of a heavy chain such as IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. The antibody may include a suitable constant region (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of this disclosure may comprise VH and VL domains, or antigen-binding portions thereof, in combination with any appropriate constant region.

[0183] Additionally or alternatively, such antibodies may or may not include the framework regions of the antibodies of SEQ ID NOs: 13 to 20. In some embodiments, the antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex are murine antibodies and include murine framework region sequences.

[0184] In some embodiments, such antibodies have relatively high affinity (e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11The antibody binds to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex with an affinity of between 5 pM and 500 nM, e.g., between 50 pM and 100 nM, e.g., between 500 pM and 50 nM. The present disclosure also includes antibodies or antigen-binding fragments that compete with any of the antibodies described herein for binding to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex and have an affinity of 50 nM or less (e.g., 20 nM or less, 10 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding kinetics of antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex can be tested using any suitable method, including, but not limited to, biosensor technology (e.g., OCTET or BIACORE).

[0185] In some embodiments, inhibitors of cell-associated TGFβ1 (e.g., TGFβ1 presented by GARP and TGFβ1 presented by LRRC33) according to the present invention comprise antibodies or fragments thereof that specifically bind to such complexes (e.g., GARP-precursor / latent TGFβ1 and LRRC33-precursor / latent TGFβ1) and trigger internalization of the complexes. This mode of action results in the removal or depletion of inactive TGFβ1 complexes from the cell surface (e.g., Tregs, macrophages, etc.), thus reducing TGFβ1 available for activation. In some embodiments, such antibodies or fragments thereof bind to the target complex in a pH-dependent manner, such that binding occurs at neutral or physiological pH, but the antibody dissociates from its antigen at acidic pH. Such antibodies or fragments thereof can function as regenerating antibodies.

[0186] [Polypeptide] Some aspects of the disclosure relate to a polypeptide having a sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:95, SEQ ID NO:15, and SEQ ID NO:19. In some embodiments, the polypeptide is a variable heavy chain domain or a heavy chain domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to any one of the amino acid sequences set forth in SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:95, SEQ ID NO:15, and SEQ ID NO:19.

[0187] Some aspects of the disclosure relate to a polypeptide having a sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:97, SEQ ID NO:16, and SEQ ID NO:20. In some embodiments, the polypeptide is a variable light chain domain or a light chain domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to any one of the amino acid sequences set forth in SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:97, SEQ ID NO:16, and SEQ ID NO:20.

[0188] [TGF-β1 isoform-specific, context-tolerant, and competitive blocking antibodies] An embodiment of the present disclosure relates to an antibody that competes or cross-competes with any of the antibodies provided herein. The term "competes" as used herein with respect to an antibody means that a first antibody binds to an epitope (e.g., an epitope of the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex) in a manner sufficiently similar to that of the second antibody, such that the result of the binding of the first antibody to that epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative may be similar, but not necessarily, that the binding of the second antibody to that epitope is also detectably reduced in the presence of the first antibody. That is, the first antibody may inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its epitope or ligand, whether to the same, greater or lesser extent, the antibodies are said to "cross-compete" with each other for the binding of their respective epitope(s). Both competing and cross-competing antibodies are within the scope of the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or part thereof), those skilled in the art will understand that such competing and / or cross-competing antibodies are included and can be useful in the methods and / or compositions provided herein.

[0189] Aspects of the present disclosure relate to antibodies that compete or cross-compete with any specific antibody provided herein, or antigen-binding portion thereof. In some embodiments, an antibody, or antigen-binding portion thereof, binds at or near the same epitope as any antibody provided herein. In some embodiments, an antibody, or antigen-binding portion thereof, binds near an epitope if it binds within 15 amino acid residues or less of the epitope. In some embodiments, any antibody provided herein, or antigen-binding portion thereof, binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues of the epitope bound by any antibody provided herein.

[0190] In another embodiment, 10 -6 Provided herein are antibodies, or antigen-binding portions thereof, that compete or cross-compete for binding to any of the antigens provided herein (e.g., GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex) with an equilibrium dissociation constant (KD) between the antibody and the protein of less than M. In other embodiments, the antibody is -11 M~10 -6 Competes or cross-competes for binding to any of the antigens provided herein with a KD within the range of M. In some embodiments, provided herein are anti-TGFβ1 antibodies, or antigen-binding portions thereof, that compete for binding with the antibodies, or antigen-binding portions thereof, described herein. In some embodiments, provided herein are anti-TGFβ1 antibodies, or antigen-binding portions thereof, that bind to the same epitope as the antibodies, or antigen-binding portions thereof, described herein.

[0191] Any antibody provided herein can be characterized using any suitable method. For example, one method is to identify the epitope that an antigen binds, or "epitope mapping". There are many suitable methods for mapping and characterizing the location of an epitope on a protein, including crystal structure analysis of antibody-antigen complex, competitive assay, gene fragment expression assay, and synthetic peptide-based assay (e.g., as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999). In a further example, epitope mapping can be used to determine the sequence that an antibody binds to. An epitope can be a linear epitope, i.e., contained within a single stretch of amino acids, or a conformational epitope formed by the three-dimensional interaction of amino acids, which does not necessarily have to be contained within a single stretch (linear sequence of primary structure). In some embodiments, when TGFβ1 is within a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex, the epitope is a TGFβ1 epitope that is only available for binding by an antibody, or antigen-binding portion thereof, described herein. Peptides of different lengths (e.g., at least 4-6 amino acids in length) may be isolated or synthesized (e.g., recombinantly) and used in antibody binding assays. In another example, the epitope to which an antibody binds may be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to a gene fragment expression assay, an open reading frame encoding a target antigen is fragmented, either randomly or by specific gene construction, and the degree of reactivity of the expressed fragments of the antigen with the antibody to be tested is determined.Gene fragments may be produced, for example, by PCR, and then transcribed and translated in vitro into proteins in the presence of radioactive amino acids. Binding of antibodies to the radiolabeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes may be identified by using large libraries of random peptide sequences (phage libraries) displayed on the surface of phage particles. Alternatively, defined libraries of overlapping peptide fragments may be tested for binding to a test antibody in a simple binding assay. In a further example, mutagenesis of antigen-binding domains, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues necessary, sufficient, and / or essential for epitope binding. For example, domain swapping experiments can be performed using mutants of target antigens in which various fragments of the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex are replaced (swapped) with sequences from a closely related but antigenically distinct protein, such as another member of the TGFβ protein family (e.g., GDF11). By assessing antibody binding to mutants of the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex, the importance of specific antigen fragments to antibody binding can be evaluated.

[0192] Alternatively, competition assays can be performed with other antibodies known to bind to the same antigen to determine if an antibody binds to the same epitope as the other antibody. Competition assays are well known to those of skill in the art.

[0193] Furthermore, the interaction of any antibody provided herein with one or more residues in the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex can be determined by routine techniques. For example, a crystal structure can be determined, and the distance between the residues in the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex and one or more residues in the antibody can be determined accordingly. Based on such distance, it can be determined whether a specific residue in the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex interacts with one or more residues in the antibody. Furthermore, the selective binding of a candidate antibody can be determined using suitable methods such as competition assays and targeted mutagenesis assays.

[0194] [Various modifications and variations of antibodies] Non-limiting variations, modifications, and properties of any of the antibodies or antigen-binding fragments thereof encompassed by this disclosure are briefly described below. Embodiments of related analytical methods are also provided.

[0195] Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer typically consists of two identical pairs of polypeptide chains, each pair having one full-length "light" (in certain embodiments, about 25 kDa) and one full-length "heavy" chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100-110 or more amino acids that is typically responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that may be responsible for effector function. Human antibody light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, which defines the antibody's isotype. Antibodies may be of any type (e.g., IgM, IgD, IgG, IgA, IgY, and IgE) and class (e.g., IgG1 , IgG 2 , IgG 3 , IgG 4 , IgM 1 , IgM 2 , IgA 1 , and IgA 2 Within full-length light and heavy chains, the variable and constant regions are typically joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids (see, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)) (incorporated by reference in its entirety)). The variable regions of each light / heavy chain pair typically form the antigen-binding site.

[0196] Variable regions typically exhibit the same general structure of relatively conserved framework regions (FRs) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by framework regions, which can enable them to bind to a specific epitope. From the N-terminus to the C-terminus, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The amino acid assignment for each domain typically follows the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia&Lesk (1987) J.Mol.Biol.196:901-917; Chothia et al. (1989) Nature 342:878-883. The CDRs of the light chain may be referred to as CDR-L1, CDR-L2, and CDR-L3, and the CDRs of the heavy chain may be referred to as CDR-H1, CDR-H2, and CDR-H3. In some embodiments, the antibody may comprise a small number of amino acid deletions from the carboxy terminus of the heavy chain(s). In some embodiments, the antibody comprises a heavy chain having a 1-5 amino acid deletion within the carboxy terminus of the heavy chain. In certain embodiments, definitive delineation of the CDRs and identification of the residues that comprise the antibody's binding site is achieved by analyzing the structure of the antibody and / or by analyzing the structure of the antibody-ligand complex. In certain embodiments, it may be achieved by any of a variety of techniques known to those of skill in the art, for example, X-ray crystallography. In some embodiments, various methods of analysis may be used to identify or estimate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.

[0197] An "affinity matured" antibody is an antibody that has one or more alterations in one or more of its CDRs that result in improved affinity of the antibody for the antigen compared to a parent antibody that does not possess these alterations or alterations. Exemplary affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. (1992) Bio / Technology 10:779-783 describes affinity maturation by shuffling VH and VL domains. Random mutagenesis of CDR and / or framework residues is described by Barbas, et al. (1994) Proc Nat. Acad. Sci. USA 91:3809-3813; Schier et al. (1995) Gene 169:147-155; Yelton et al., (1995) J. Immunol. 155:1994-2004; Jackson et al. (1995) J. Immunol. 154(7):3310-9; and Hawkins et al. (1992) J. Mol. Biol. 226:889-896; selective mutagenesis at contact or hypermutation positions with activity enhancing amino acid residues is described in U.S. Patent No. 6,914,128.

[0198] The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species, but in which the sequences of one or more CDR regions of the VH and / or VL have been replaced by CDR sequences from another species, e.g., an antibody having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) have been replaced by human CDR sequences.

[0199] The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, e.g., an antibody having murine heavy and light chain variable regions joined to human constant regions.

[0200] The term "framework" or "framework sequence" as used herein refers to the remaining sequences of the variable region minus the CDRs. The exact definition of the CDR sequence can be determined by different systems, so the meaning of the framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of the light chain and CDR-H1, -H2, and -H3 of the heavy chain) also divide the framework regions on the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, with CDR1 located between FR1 and FR2, CDR2 located between FR2 and FR3, and CDR3 located between FR3 and FR4. Without specifying a particular subregion as FR1, FR2, FR3, or FR4, the framework region, as referred to by others, refers to the combined FR's in the variable region of a single, naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FRs refers to two or more of the four subregions that make up a framework region.

[0201] In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain immunoglobulin constant domain of a human IgM constant domain, a human IgG constant domain, a human IgG1 constant domain, a human IgG2 constant domain, a human IgG2A constant domain, a human IgG2B constant domain, a human IgG2 constant domain, a human IgG3 constant domain, a human IgG3 constant domain, a human IgG4 constant domain, a human IgA constant domain, a human IgA1 constant domain, a human IgA2 constant domain, a human IgD constant domain, or a human IgE constant domain. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain immunoglobulin constant domain of a human IgG1 constant domain or a human IgG4 constant domain. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain immunoglobulin constant domain of a human IgG4 constant domain. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain immunoglobulin constant domain of a human IgG4 constant domain with a backbone substitution of Ser to Pro creating an IgG1-like hinge allowing interchain disulfide bond formation.

[0202] In some embodiments, the antibody or antigen binding portion thereof further comprises a light chain immunoglobulin constant domain comprising a human Igλ constant domain or a human Igκ constant domain.

[0203] In some embodiments, the antibody is an IgG having four polypeptide chains: two heavy chains and two light chains.

[0204] In some embodiments, the antibody is a humanized antibody, a diabody, or a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody comprises a framework having a human germline amino acid sequence.

[0205] In some embodiments, the antigen-binding portion is a Fab fragment, a F(ab')2 fragment, an scFab fragment, or an scFv fragment.

[0206] As used herein, the term "germline antibody gene" or "gene fragment" refers to an immunoglobulin sequence encoded by a non-lymphoid cell that has not undergone the maturation process that results in mutations and gene rearrangements for expression of a specific immunoglobulin (see, e.g., Shapiro et al. (2002) Crit. Rev. Immunol. 22(3):183-200; Marchalonis et al. (2001) Adv. Exp. Med. Biol. 484:13-30). One advantage provided by various embodiments of the present disclosure stems from the recognition that germline antibody genes are more likely to preserve the essential amino acid sequence structural characteristics of an individual in a species than mature antibody genes, and therefore are less likely to be recognized as derived from a foreign source when used therapeutically in that species.

[0207] The term "neutralizing" as used herein refers to opposing the biological activity of an antigen when the binding protein specifically binds to the antigen. In one embodiment, the neutralizing binding protein binds to an antigen / target, such as a cytokine, kinase, growth factor, cell surface protein, soluble protein, phosphatase, or receptor ligand, and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%.96%, 97%.98%, 99% or more.

[0208] The term "binding protein" as used herein includes any polypeptide that specifically binds to an antigen (e.g., TGFβ1), including, but not limited to, antibodies, or antigen-binding portions thereof, DVD-IgTM, TVD-Ig, RAb-Ig, bispecific antibodies, and dual specific antibodies.

[0209] The term "monoclonal antibody" or "mAb", when used in the context of a composition comprising it, may refer to an antibody preparation obtained from a population of substantially homogenous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method.

[0210] As used herein, the term "recombinant human antibody" refers to any human antibody that is prepared, expressed, generated or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section IIC below), antibodies isolated from recombinant, combinatorial human antibody libraries (Hoogenboom, H R (1997) TIB Tech. 15:62-70; Azzazy, H. and Highsmith, W E (2002) Clin. Biochem. 35:425-445; Gavilondo, J V and Larrick, J W (2002) BioTechniques 29:128-145; Hoogenboom, H. and Chames, P. (2000) Immunol. Today 21:371-378, which are incorporated herein by reference), antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (Taylor, L D et al., 2003). al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann, SA. and Green, LL (2002) Cur. Opin. in Biotechnol. 13:593-597; Little, M. et al. (2000) Immunol. Today 21:364-370), or any other means including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, where animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0211] As used herein, "dual variable domain immunoglobulin" or "DVD-IgTM" and the like includes a binding protein that includes a pair of heavy chain DVD polypeptides and a light chain DVD polypeptide, each pair of heavy chain and light chain providing two antigen binding sites. Each binding site includes a total of six CDRs involved in antigen binding per antigen binding site. DVD-IgTM typically has two arms that are at least partially connected to each other by dimerization of CH3 domains, and each arm of the DVD is bispecific, providing four binding sites to the immunoglobulin. DVD-IgTM is provided in U.S. Patent Publication Nos. 2010 / 0260668 and 2009 / 0304693, each of which is incorporated herein by reference, including the sequence listing.

[0212] As used herein, a "triple variable domain immunoglobulin" or "TVD-Ig" or the like is a binding protein that includes a pair of heavy and light chain TVD binding protein polypeptides, where each pair of heavy and light chains provides three antigen binding sites. Each binding site includes a total of six CDRs involved in antigen binding per antigen binding site. The TVD binding protein may have two arms that are at least partially linked to each other by dimerization of the CH3 domains, and each arm of the TVD binding protein is trispecific, providing the binding protein with six binding sites.

[0213] As used herein, a "receptor-antibody immunoglobulin" or "RAb-Ig" or the like is a binding protein that includes a heavy chain RAb polypeptide and a light chain RAb polypeptide, which together form a total of three antigen binding sites. One antigen binding site is formed by a pair of heavy and light antibody variable domains present in each of the heavy chain RAb polypeptide and the light chain RAb polypeptide to form a single binding site with a total of six CDRs that provide a first antigen binding site. Each of the heavy chain RAb polypeptide and the light chain RAb polypeptide contains a receptor sequence that binds independently to a ligand, providing a second and a third "antigen" binding site. RAb-Ig typically has two arms that are at least partially connected to each other by dimerization of the CH3 domain, and each arm of RAb-Ig is trispecific, providing six binding sites for the immunoglobulin. RAb-Igs are described in U.S. Patent Application Publication No. 2002 / 0127231, the entire contents of which, including the sequence listing, are incorporated herein by reference.

[0214] The term "bispecific antibody" as used herein, as distinguished from "bispecific half-Ig binding protein" or "bispecific (half-Ig) binding protein", refers to full-length antibodies produced by chemical conjugation of two different monoclonal antibodies (see Staerz, UD et al. (1985) Nature 314(6012):628-631), or by knob-into-hole or similar techniques (introducing mutations in the Fc region that do not inhibit CH3-CH3 dimerization) (see Holliger, P. et al. (1993) Proc. Natl. Acad. Sci USA 90(14):6444-6448), by quadroma technology (see Milstein, C. and Cuello, AC (1983) Nature 305(5934):p.537-540), resulting in a large number of different immunoglobulin species, only one of which is a functional bispecific antibody. By molecular function, a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC / LC) and binds a different antigen (or epitope) on its second arm (a different pair of HC / LC). By this definition, a bispecific antibody has two separate antigen-binding arms (both in specificity and CDR sequences) and is monovalent with respect to each antigen it binds.

[0215] The term "dual-specific antibody" as used herein is distinct from a bispecific half-Ig binding protein or a bispecific binding protein and refers to a full-length antibody that can bind two different antigens (or epitopes) in each of its two binding arms (HC / LC pairs) (see PCT Publication No. WO02 / 02773). Thus, a dual-specific binding protein has two identical antigen-binding arms, has the same specificity and identical CDR sequences, and is bivalent with respect to each antigen to which it binds.

[0216] The term "Kon" as used herein is intended to refer to the on rate constant for the association of a binding protein (e.g., an antibody) to an antigen to form an antibody / antigen complex, e.g., as known in the art. "Kon" is also known by the term "association rate constant" or "ka", as used interchangeably herein. This value, which indicates the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and an antigen, is also represented by the equation: antibody ("Ab") + antigen ("Ag") → Ab-Ag.

[0217] The term "Koff" as used herein is intended to refer to the off rate constant for dissociation of a binding protein (e.g., an antibody) from an antibody / antigen complex, e.g., as known in the art. "Koff" is also known by the term "dissociation rate constant" or "kd", as used interchangeably herein. This value indicates the dissociation rate of an antibody from its target antigen or the separation of the Ab-Ag complex into free antibody and antigen over time, as indicated by the equation: Ab+Ag←Ab-Ag.

[0218] The term "equilibrium dissociation constant" or "KD" used interchangeably herein refers to the value obtained in a titration measurement at equilibrium or the value obtained by dividing the dissociation rate constant (koff) by the association rate constant (kon). The association rate constant, dissociation rate constant, and equilibrium dissociation constant are used to express the binding affinity of a binding protein (e.g., an antibody to an antigen). Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to test samples in physiological buffers at equilibrium. Other experimental techniques and instruments, such as the BIAcore® (Biomolecular Interaction Analysis) assay, can be used (e.g., instruments available from BIAcore International AB, GE Healthcare, Uppsala, Sweden). In addition, the KinExA® (Kinetic Exclusion Assay) assay available from Sapidyne Instruments (Boise, Idaho) may be used.

[0219] The terms "crystal" and "crystallization" as used herein refer to a binding protein (e.g., an antibody), or an antigen-binding portion thereof, that exists in the form of a crystal. A crystal is one form of the solid state of matter, distinct from other forms such as the amorphous solid state or the liquid crystalline state. A crystal consists of a regular repeating three-dimensional array of atoms, ions, molecules (e.g., proteins such as antibodies), or collections of molecules (e.g., antigen / antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well understood in the art. The basic unit, or building block, that is repeated within a crystal is called the asymmetric unit. The repetition of the asymmetric unit in an arrangement according to a given, well-defined crystallographic symmetry provides the "unit cell" of the crystal. The repetition of the unit cell by regular translation in all three dimensions provides a crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 201-16, Oxford University Press, New York, New York, (1999). The term "linker" is used to refer to a polypeptide comprising two or more amino acid residues joined by peptide bonds, and is used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2:1121-1123).Exemplary linkers include, but are not limited to, ASTKGPSVFPLAP (SEQ ID NO:55), ASTKGP (SEQ ID NO:56); TVAAPSVFIFPP (SEQ ID NO:57); TVAAP (SEQ ID NO:58); AKTTPKLEEGEFSEAR (SEQ ID NO:59); AKTTPKLEEGEFSEARV (SEQ ID NO:60); AKTTPKLGG (SEQ ID NO:61); SAKTTPKLGG (SEQ ID NO:62); SAKTTP (SEQ ID NO:63); RADAAP (SEQ ID NO:64); RADAAPTVS (SEQ ID NO:65); RADAAAAAGGPGS (SEQ ID NO:66); RADAAAA(G4S)4 (SEQ ID NO:67); SAKTTPKLEEGEFSEARV (SEQ ID NO:68); ADAAP (SEQ ID NO: 69);ADAAPTVSIFPP (SEQ ID NO: 70);QPKAAP (SEQ ID NO: 71);QPKAAPSVTLFPP (SEQ ID NO: 72);AKTTPP (SEQ ID NO: 73);AKTTPPSVTPLAP (SEQ ID NO: 74);AKTTAP (SEQ ID NO: 75);AKTTAPSVYPLAP (SEQ ID NO: 76);GGGGSGGGGSGGGGS (SEQ ID NO: 77);GENKVEYAPALMALS (SEQ ID NO: 78);GPAKELTPLKEAKVS (SEQ ID NO: 79);GHEAAAVMQVQYPAS (SEQ ID NO: 80);TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO: 81); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 82).

[0220] "Label" and "detectable label" or "detectable moiety" refer to a moiety attached to a specific binding partner (e.g., antibody or analyte) to render, for example, a reaction between members of a specific binding pair (e.g., antibody and analyte) detectable; a specific binding partner (e.g., antibody or analyte) so labeled is said to be "detectably labeled." Thus, as used herein, the term "labeled binding protein" refers to a protein having an incorporated label that provides identification of the binding protein. In one embodiment, the label is a detectable marker capable of producing a signal detectable by visual or instrumental means (e.g., incorporation of a radiolabeled amino acid or attachment of a biotinyl moiety to a polypeptide that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods)). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm); chromogens; fluorescent labels (e.g., FITC, rhodamine, and lanthanide fluorophores); enzyme labels (e.g., horseradish peroxidase, luciferase, and alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, and epitope tags); and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-producing moieties such as acridinium compounds, and fluorescence-producing moieties such as fluorescein. Other labels are described herein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with yet another moiety; the use of "detectably labeled" is intended to encompass the latter types of detectable labels.

[0221] In some embodiments, the binding affinity of an antibody or antigen-binding portion thereof to an antigen (e.g., a protein complex), such as a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex, is determined using an Octet assay. In some embodiments, an Octet assay is an assay that determines one or more kinetic parameters indicative of binding between an antibody and an antigen. In some embodiments, an Octet® system (ForteBio, Menlo Park, CA) is used to determine the binding affinity of an antibody or antigen-binding portion thereof to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex. For example, the binding affinity of an antibody can be determined using a forteBio Octet QKe dip and read label-free assay system that utilizes biolayer interferometry. In some embodiments, the antigen is immobilized on a biosensor (e.g., a streptavidin-coated biosensor), and the antibody and complex (e.g., biotinylated GARP-TGFβ1 complex and biotinylated LTBP-TGFβ1 complex) are in solution at high concentration (50 μg / mL) to measure binding interactions. In some embodiments, the binding affinity of the antibody, or antigen-binding portion thereof, to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex is determined using the protocol outlined in Table 6. The term "surface plasmon resonance" as used herein refers to an optical phenomenon that allows the analysis of bispecific interactions in real time by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ).For further explanation, see Jonsson, U. et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U. et al. (1991) Biotechniques 11:620-627; Johnsson, B. et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B. et al. (1991) Anal. Biochem. 198:268-277.

[0222] [Identification and production / manufacturing of TGF-β1 isoform-specific, context-specific inhibitors of tolerance] The present invention encompasses methods of screening for, methods of producing, and processes for manufacturing antibodies or fragments thereof that bind to two or more of the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex, as well as pharmaceutical compositions and related kits containing same.

[0223] Numerous methods can be used to obtain the antibody of the present disclosure, or an antigen-binding fragment thereof. For example, the antibody can be produced using recombinant DNA methods. Monoclonal antibodies can be produced by hybridoma generation according to known methods (see, for example, Kohler and Milstein (1975) Nature, 256:495-499). Hybridomas formed in this manner can then be screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (e.g., OCTET or BIACORE) analysis, to identify one or more hybridomas that produce antibodies that specifically bind to a particular antigen. Any form of a particular antigen, such as a recombinant antigen, a naturally occurring form, any variant or fragment thereof, as well as antigenic peptides thereof (e.g., any epitope described herein within a scaffold as a conformational epitope or as a linear epitope) can be used as an immunogen. One exemplary method for producing antibodies includes screening protein expression libraries, such as phage or ribosome display libraries, that express antibodies or fragments thereof (e.g., scFv). Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597; WO92 / 18619; WO91 / 17271; WO92 / 20791; WO92 / 15679; WO93 / 01288; WO92 / 01047; WO92 / 09690; and WO90 / 02809.

[0224] In addition to the use of display libraries, specific antigens (e.g., GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex) can be used to immunize non-human hosts, e.g., non-mammalian hosts such as rabbits, guinea pigs, rats, mice, hamsters, sheep, goats, chickens, camels, and sharks. In one embodiment, the non-human animal is a mouse.

[0225] In another embodiment, monoclonal antibodies are obtained from non-human animals and then modified using appropriate recombinant DNA techniques (e.g., chimeric).Various techniques for producing chimeric antibodies have been described.See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication EP 171496; European Patent Publication 0173494, British Patent GB 2177096B.

[0226] For further antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. The present disclosure is not necessarily limited to any particular origin, method of production, or other particular characteristics of the antibodies.

[0227] Some aspects of the present disclosure relate to a host cell transformed by a polynucleotide or vector. The host cell may be a prokaryotic or eukaryotic cell. The polynucleotide or vector present in the host cell may be integrated into the genome of the host cell or may be maintained extrachromosomally. The host cell may be any prokaryotic or eukaryotic cell, for example, a bacterial, insect, fungal, plant, animal or human cell. In some embodiments, the fungal cell is, for example, of the genus Saccharomyces, particularly of the species S. cerevisiae. The term "prokaryote" includes all bacteria that can be transformed or transfected with DNA or RNA molecules for the expression of an antibody or the corresponding immunoglobulin chain. Prokaryotic hosts may include gram-negative and gram-positive bacteria, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryote" includes yeast, higher plants, insects and vertebrate cells, for example, mammalian cells such as NSO and CHO cells. Depending on the host employed in a recombinant production procedure, the antibody or immunoglobulin chain encoded by the polynucleotide may be glycosylated or may be non-glycosylated. The antibody or the corresponding immunoglobulin chain may include an initial methionine amino acid residue.

[0228] In some embodiments, once the vector has been incorporated into a suitable host, the host may be maintained under conditions suitable for high level expression of the nucleotide sequence, followed by collection and purification of the immunoglobulin light chain, heavy chain, light / heavy chain dimer or intact antibody, antigen-binding fragment or other immunoglobulin form as desired; see Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY, (1979). Thus, the polynucleotide or vector is introduced into a cell, which in turn produces the antibody or antigen-binding fragment. Additionally, transgenic animals, preferably mammals, containing the host cells described above may be used for large-scale production of antibodies or antibody fragments.

[0229] The transformed host cells may be grown in fermenters and cultured using any suitable technique to achieve optimal cell growth. Once expressed, whole antibodies, their dimers, individual light and heavy chains, other immunoglobulin forms, or antigen-binding fragments may be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like; see Scopes, "Protein Purification", Springer Verlag, NY (1982). The antibodies or antigen-binding fragments may then be isolated from the growth medium, cell lysates, or cell membrane fractions. Isolation and purification of antibodies or antigen-binding fragments expressed by, for example, microorganisms may be by any conventional means, such as immunological separation, such as those involving the use of monoclonal or polyclonal antibodies directed against the constant regions of the antibodies, and preparative chromatographic separation.

[0230] Aspects of the present disclosure relate to hybridomas that provide an indefinitely extended source of monoclonal antibodies. As an alternative to obtaining immunoglobulins directly from hybridoma cultures, immortalized hybridoma cells can be used as a source of rearranged heavy and light chain loci for subsequent expression and / or genetic engineering. Rearranged antibody genes can be reverse transcribed from the appropriate mRNA to produce cDNA. In some embodiments, the heavy chain constant region can be exchanged for that of a different isotype or removed entirely. Variable regions can be combined to encode a single chain Fv region. Multiple Fv regions can be combined to confer binding capacity for more than one target, or chimeric heavy and light chain combinations can be used. Any suitable method can be used for cloning antibody variable regions and producing recombinant antibodies.

[0231] In some embodiments, suitable nucleic acids encoding the variable regions of the heavy and / or light chains are obtained and inserted into expression vectors that can be transfected into standard recombinant host cells. A variety of such host cells can be used. In some embodiments, mammalian host cells can be advantageous for efficient processing and production. Exemplary mammalian cell lines useful for this purpose include CHO cells, 293 cells, or NSO cells. Production of antibodies or antigen-binding fragments can be carried out by culturing the modified recombinant host under culture conditions suitable for host cell growth and expression of the coding sequence. Antibodies or antigen-binding fragments can be recovered by isolating them from the culture. Expression systems can be designed to include a signal peptide so that the resulting antibody is secreted into the medium; however, intracellular production is also possible.

[0232] The present disclosure also includes polynucleotides encoding at least the variable regions of the immunoglobulin chains of the antibodies described herein. In some embodiments, the variable regions encoded by the polynucleotides comprise at least one complementarity determining region (CDR) of the VH and / or VL variable regions of the antibodies produced by any one of the hybridomas described above.

[0233] The polynucleotide encoding the antibody or antigen-binding fragment may be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule, including any of these polynucleotides, either alone or in combination. In some embodiments, the polynucleotide is part of a vector. Such vectors may contain additional genes, such as marker genes, that allow for the selection of the vector in a suitable host cell and under suitable conditions.

[0234] In some embodiments, the polynucleotide is operably linked to an expression control sequence that allows expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide includes transcription of the polynucleotide into a translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may include regulatory sequences that facilitate transcription initiation and optionally poly-A signals that facilitate transcription termination and transcription stabilization. Further regulatory elements may include transcriptional and translational enhancers and / or naturally associated or heterologous promoter regions. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, PL, Lac, Trp or Tac promoters in E. coli, and examples of regulatory elements that allow expression in eukaryotic host cells are AOX1 or GAL1 promoters in yeast, or CMV promoter, SV40 promoter, RSV promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer or globin intron in mammalian and other animal cells.

[0235] Besides the elements responsible for transcription initiation, such regulatory elements may also include transcription termination signals downstream of the polynucleotide, such as the SV40-poly-A site or the tk-poly-A site. Furthermore, depending on the expression system used, leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide, as previously described. The leader sequence(s) are assembled in the appropriate phase with the translation, initiation and termination sequences, and preferably the leader sequence is capable of directing the secretion of the translated protein, or a part thereof, for example, into the extracellular medium. Optionally, heterologous polynucleotide sequences may be used that code for fusion proteins containing C- or N-terminal identification peptides that confer desired properties, for example simplified purification or stabilization of the expressed recombinant product.

[0236] In some embodiments, the polynucleotides encoding at least the variable domains of the light and / or heavy chains may encode the variable domains of both or only one of the immunoglobulin chains. Similarly, the polynucleotides may be under the control of the same promoter and may be separately controlled for expression. Furthermore, some aspects relate to vectors, particularly plasmids, cosmids, viruses and bacteriophages, conventionally used in genetic engineering, that contain a polynucleotide encoding the variable domain of an immunoglobulin chain of an antibody or antigen-binding fragment; optionally in combination with a polynucleotide encoding the variable domain of the other immunoglobulin chain of the antibody.

[0237] In some embodiments, the expression control sequences are provided as eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, although control sequences for prokaryotic hosts may also be used. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses may be used for delivery of polynucleotides or vectors to targeted cell populations (e.g., to engineer cells to express antibodies or antigen-binding fragments). Recombinant viral vectors may be constructed using a variety of suitable methods. In some embodiments, polynucleotides and vectors may be reconstituted into liposomes for delivery to target cells. Vectors containing polynucleotides (e.g., sequences encoding immunoglobulin chains and heavy and / or light variable domain(s) of expression control sequences) may be transferred into host cells by suitable methods that vary depending on the type of cellular host.

[0238] The screening method may include evaluating or confirming a desired activity of the antibody or fragment thereof. In some embodiments, the step includes selecting for an ability to inhibit a target function, e.g., inhibiting release of mature TGFβ1 from a latent complex. In some embodiments, the step includes selecting for an antibody or fragment thereof that promotes internalization and subsequent removal of the antibody-antigen complex from the cell surface. In some embodiments, the step includes selecting for an antibody or fragment thereof that induces ADCC. In some embodiments, the step includes selecting for an antibody or fragment thereof that accumulates in a desired site(s) in vivo (e.g., cell type, tissue, or organ). In some embodiments, the step includes selecting for an antibody or fragment thereof that has the ability to close the blood-brain barrier. The method may optionally include optimizing one or more antibodies or fragments thereof to provide mutant counterparts that possess a desired profile as determined by criteria such as stability, binding affinity, functionality (e.g., inhibitory activity, Fc function, etc.), immunogenicity, pH sensitivity, and developability (e.g., high solubility, low self-association, etc.). Such steps may include affinity maturation of the antibody or its fragment. The resulting optimized antibody is preferably a fully human or humanized antibody suitable for human administration. The manufacturing process for pharmaceutical compositions containing such antibodies or their fragments may include steps such as purification, formulation, sterile filtration, packaging, etc. Certain steps, such as sterile filtration, are performed according to the guidelines set forth by relevant regulatory agencies, such as the FDA. Such compositions may be made available in the form of single-use containers, such as pre-filled syringes, or multi-dose containers, such as vials.

[0239] [Modification] The antibodies of the disclosure, or antigen-binding portions thereof, may be modified with detectable labels or moieties, including, but not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, non-radioactive paramagnetic metal ions, and affinity labels for detection and isolation of GARP-TGFβ1 complexes, LTBP1-TGFβ1 complexes, LTBP3-TGFβ1 complexes, and / or LRRC33-TGFβ1 complexes. The detectable substance or moiety may be linked or conjugated directly to the polypeptides of the disclosure or indirectly through an intermediate, such as a linker (e.g., a cleavable linker), using appropriate techniques. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, L-galactosidase, glucose oxidase, or acetylcholinesterase; non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; non-limiting examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin; an example of a suitable radioactive material includes a radioactive metal ion, e.g., an α-emitter or other Radioisotopes, such as iodine (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In), and technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), and molybdenum (99Mo) , xenon (133Xe), fluorine (18F), 153Sm, Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 86R, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, and tin (113Sn, 117Sn).The detectable substance may be linked or conjugated directly to an antibody of the present disclosure that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex, or indirectly through an intermediate (such as a linker) using a suitable technique. Any antibody provided herein that is conjugated to a detectable substance may be used in any suitable diagnostic assay, such as those described herein.

[0240] In addition, the antibodies of the present disclosure, or antigen-binding portions thereof, may be modified with a drug. The drug may be linked or conjugated directly to the polypeptides of the present disclosure, or indirectly through an intermediate, such as a linker (e.g., a cleavable linker), using appropriate techniques.

[0241] [Targeting Agents] In some embodiments, the disclosed method includes the use of one or more targeting agents to target the antibody disclosed herein, or an antigen-binding portion thereof, to a specific site in a subject for the purpose of regulating mature TGFβ release from GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex. For example, LTBP1-TGFβ1 and LTBP3-TGFβ1 complexes are typically localized in the extracellular matrix. Thus, in some embodiments, the antibody disclosed herein can be conjugated to an extracellular matrix targeting agent for the purpose of localizing the antibody to the site where LTBP1-TGFβ1 and LTBP3-TGFβ1 complexes are present. In such an embodiment, the selective targeting of the antibody leads to the selective regulation of LTBP1-TGFβ1 and / or LTBP3-TGFβ1 complexes. In some embodiments, the selective targeting of the antibody leads to selective inhibition of the LTBP1-TGFβ1 and / or LTBP3-TGFβ1 complex (e.g., for purposes of treating fibrosis). In some embodiments, the extracellular matrix targeting agent comprises a heparin binding agent, a matrix metalloproteinase binding agent, a lysyl oxidase binding domain, a fibrillin binding agent, a hyaluronic acid binding agent, and others.

[0242] Similarly, GARP-TGFβ1 complexes are typically localized on the surface of cells such as activated FOXP3+ regulatory T cells (Treg). Thus, in some embodiments, the antibodies disclosed herein can be conjugated to immune cell (e.g., Treg cell) binding agents for the purpose of localizing the antibody to the site where the GARP-TGFβ1 complex is present. In such embodiments, the selective targeting of the antibody leads to selective modulation of the GARP-TGFβ1 complex. In some embodiments, the selective targeting of the antibody leads to selective inhibition of the GARP-TGFβ1 complex (such as selective inhibition of the release of mature TGFβ1 for the purpose of immune regulation in cancer treatment). In such embodiments, the Treg cell targeting agent can include, for example, CCL22 and CXCL12 proteins or fragments thereof.

[0243] In some embodiments, bispecific antibodies may be used having a first portion that selectively binds to GARP-TGFβ1 complexes and LTBP-TGFβ1 complexes, and a second portion that selectively binds to a component of a target site, such as a component of the ECM (e.g., fibrillin) or a component of Treg cells (e.g., CTLA-4).

[0244] [Pharmaceutical composition] The present invention further provides pharmaceutical compositions for use as medicines suitable for administration in human and non-human subjects. One or more antibodies specifically binding to GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex can be formulated or mixed with a pharma- ceutically acceptable carrier (excipient), including, for example, a buffer, to form a pharmaceutical composition. Such formulations can be used for the treatment of diseases or disorders involving TGFβ signaling. In some embodiments, such diseases or disorders associated with TGFβ signaling include one or more contexts, i.e., TGFβ is associated with a particular type(s) of presentation molecule. In some embodiments, such contexts occur in a cell type-specific and / or tissue-specific manner. In some embodiments, for example, such context-dependent effects of TGFβ signaling are mediated in part through GARP, LRRC33, LTBP1, and / or LTBP3.

[0245] In some embodiments, the antibody of the present invention specifically binds to two or more contexts of TGFβ, such that the antibody binds to TGFβ in a complex with a presentation molecule selected from two or more of GARP, LRRC33, LTBP1 and LTBP3. Thus, such pharmaceutical compositions can be administered to patients to alleviate TGFβ-related symptoms (e.g., fibrosis, immune disease, and / or cancer). "Acceptable" means that the carrier is compatible with (and preferably capable of stabilizing) the active ingredient of the composition and is not harmful to the subject being treated. Examples of pharma-ceutically acceptable excipients (carriers), including buffers, will be apparent to those skilled in the art and have been previously described. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover. In one example, a pharmaceutical composition described herein comprises more than one antibody that specifically binds to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex, wherein the antibodies recognize different epitopes / residues of the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex.

[0246] The pharmaceutical compositions used in this method may contain a pharma- ceutically acceptable carrier, excipient, or stabilizer (in the form of a lyophilized formulation or an aqueous solution) (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, etc. The excipients may include proteins such as saccharides, peptides, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0247] In some examples, the pharmaceutical compositions described herein include liposomes that contain antibodies that specifically bind to GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, which can be prepared by any suitable method, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al. Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition that includes phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0248] In some embodiments, the pharmaceutical composition of the present invention may comprise or be used together with an adjuvant. It is believed that certain adjuvants can boost the subject's immune response to, for example, tumor antigens, and can promote Teffector function, DC differentiation from monocytes, high antigen uptake, and presentation by APC, etc. Suitable adjuvants include, but are not limited to, retinoic acid-based adjuvants and their derivatives, oil-in-water emulsion-based adjuvants, such as MF59 and other squalene-containing adjuvants, Toll-like receptor (TRL) ligands, α-tocopherol (vitamin E) and their derivatives.

[0249] Antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex may be entrapped in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, e.g., in microcapsules prepared by coacervation techniques or interfacial polymerization, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively. Exemplary techniques have been previously described, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0250] In other examples, the pharmaceutical compositions described herein can be formulated in sustained release form.Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies, the matrices being in the form of shaped articles, such as films, or microcapsules.Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres made of lactic acid-glycolic acid copolymers and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0251] Pharmaceutical compositions used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0252] The pharmaceutical compositions described herein may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0253] To prepare solid compositions such as tablets, the main active ingredient may be mixed with pharmaceutical carriers such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums such as conventional tableting ingredients, and other pharmaceutical diluents (e.g., water) to form solid preformulation compositions, including homogenous mixtures of the compounds of the present disclosure, or non-toxic pharmaceutically acceptable salts thereof. When these preformulation compositions are referred to as homogenous, it is meant that the active ingredient is evenly distributed throughout the composition, so that the composition can be easily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above, containing 0.1 mg to about 500 mg of the active ingredient of the present disclosure. The tablets or pills of the novel composition may be coated or otherwise compounded to provide a dosage form that provides the benefit of extended action. For example, the tablet or pill may include an inner dose and an outer dose component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0254] Suitable surfactants include, inter alia, non-ionic agents such as polyoxyethylene sorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions having a surfactant suitably contain 0.05-5% surfactant, and may be 0.1-2.5%. It will be appreciated that other ingredients, such as mannitol or other pharma- ceutically acceptable vehicles, may be added if necessary.

[0255] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be dissolved in a premixed emulsion composition or alternatively in an emulsion formed upon mixing of oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and phospholipids (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water. It is understood that other ingredients such as glycerol or glucose can be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to 20% oil (e.g. 5-20%).

[0256] The emulsion composition may be prepared by mixing an antibody that specifically binds to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water).

[0257] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0258] Compositions in preferably sterile pharma- ceutically acceptable solvents may be nebulized by using gas. Nebulized solutions may be directly inhaled from the nebulizing device, or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from a device that delivers the formulation in an appropriate manner.

[0259] [Therapeutic indications and / or selection of subjects likely to respond to treatments that include TGFβ1-selective broad-spectrum inhibitors] With regard to identifying / selecting appropriate indications and / or patient populations in which an isoform-specific, context-tolerant inhibitor of TGFβ1 such as those described herein may have beneficial effects, two questions can be asked: i) whether the disease is driven by or dependent on a TGFβ1 isoform predominately over other isoforms in humans; and ii) whether the disease involves dysregulation of multiple aspects of TGFβ1 function.

[0260] Differential expression of three known TGFβ isoforms, namely TGFβ1, TGFβ2, and TGFβ3, has been observed in various tissues under normal (healthy; homeostatic) as well as diseased conditions. Nevertheless, the concept of isoform selectivity has not been fully utilized or achieved by conventional approaches that favor pan-inhibition of TGFβ across multiple isoforms. Furthermore, the expression patterns of isoforms may be differentially regulated not only in normal (homeostatic) versus abnormal (pathological) conditions, but also in different subpopulations of patients. Since most preclinical studies are performed in a limited number of animal models, the data obtained by using such models may be biased, leading to misinterpretation of data or confusing conclusions regarding applicability to human conditions.

[0261] Thus, the present invention includes the recognition that differential expression of TGFβ isoforms must be considered in predicting the efficacy of a particular inhibitor, as well as in interpreting preclinical data regarding translatability to human conditions. As illustrated in FIG. 21, TGFβ1 and TGFβ3 are co-dominant in certain murine isogenic cancer models (e.g., EMT-6 and 4T1) that are widely used in preclinical studies. In contrast, numerous other cancer models (e.g., S91, B16 and MBT-2) almost exclusively express TGFβ1, similar to what is observed in many human tumors, where TGFβ1 is believed to be the predominant isoform more frequently than TGFβ2 / 3. Furthermore, the TGFβ isoform(s) that are predominantly expressed under homeostatic conditions may not be disease-associated isoform(s). For example, in normal lung tissue in healthy rats, tonic TGFβ signaling is believed to be mediated primarily by TGFβ3. However, TGFβ1 is thought to be significantly upregulated in disease states such as pulmonary fibrosis. Taken together, it is beneficial to test or confirm the relative expression of TGFβ isoforms in clinical samples to select appropriate treatments to which patients are likely to respond.

[0262] As described herein, isoform-selective TGFβ1 inhibitors are particularly advantageous for treating diseases in which the TGFβ1 isoform is predominantly expressed compared to other isoforms. As an example, FIG. 21D provides a non-limiting list of human cancer clinical samples with relative expression levels of TGF1 (left), TGFβ2 (middle) and TGFβ3 (right). Each horizontal lime across the three isoforms represents a single patient. As can be seen, overall TGFβ1 expression is significantly higher in the majority of these human tumors than the other two isoforms across many tumor types, suggesting that TGFβ1 selective inhibition may be beneficial. However, certain exceptions should be noted. First, such trends are not always applicable in a particular individual patient. That is, even in cancer types that show almost uniform TGFβ1 dominance over TGFβ2 / 3, there are some individuals that do not follow this general rule. Patients that fall into minority subpopulations may therefore not respond to isoform-specific inhibitor therapy in a way that works for the majority of patients. Secondly, there are some cancer types in which TGFβ1 is co-dominant with another isoform, or TGFβ2 and / or TGFβ3 expression is significantly greater than TGFβ1. In these situations, TGFβ1 selective inhibitors such as those described herein may not be efficatious. Therefore, it is beneficial to test or confirm the relative expression levels of the three TGFβ isoforms (i.e., TGFβ1, TGFβ2 and TGFβ3) in clinical samples collected from individual patients. Such information can provide better predictions about the effectiveness of a particular treatment in individual patients, and can help ensure the selection of appropriate treatment (e.g., individualized treatment) to increase the likelihood of clinical response.

[0263] Thus, the present invention includes a method for selecting a patient population or subject that may respond to a treatment comprising an isoform-specific, context-tolerant TGFβ1 inhibitor. Such a method includes the steps of providing a biological sample (e.g., a clinical sample) collected from a subject, determining (e.g., measuring or analyzing) the relative levels of TGFβ1, TGFβ2 and TGFβ3 in the sample, and administering a composition comprising an isoform-specific, context-tolerant TGFβ1 inhibitor to the subject if TGFβ1 is the predominant isoform over TGFβ2 and TGFβ3; and / or TGFβ1 is significantly overexpressed or upregulated compared to a control. The relative levels of isoforms can be determined by RNA-based assays and / or protein-based assays, which are well known in the art. In some embodiments, the administration step may include another treatment, such as an immune checkpoint inhibitor, or other agent provided elsewhere herein. Such methods may optionally include a step of evaluating therapeutic response by monitoring changes in the relative levels of TGFβ1, TGFβ2 and TGFβ3 at two or more time points. In some embodiments, clinical samples (e.g., biopsies) are collected both before and after administration. In some embodiments, clinical samples (e.g., biopsies) are collected multiple times following treatment to evaluate in vivo effects over time.

[0264] In addition to the first question, which is attracted by the aspect of isoform selectivity, the second question asks the breadth of TGFβ1 function involved in a particular disease. This can be indicated by the number of TGFβ1 contexts (i.e., which presented molecule(s) mediate disease-related TGFβ1 function). TGFβ1-specific, broad-context inhibitors, such as context-tolerant and context-independent inhibitors, are advantageous for treating diseases that involve both immune and ECM components of TGFβ1 function. Such diseases may be associated with dysregulation in the ECM and disruptions in immune response or immune cell function. Thus, the TGFβ1 inhibitors described herein are capable of targeting ECM-associated TGFβ1 (e.g., presented by LTBP1 or LTBP3) as well as immune cell-associated TGFβ1 (e.g., presented by GARP or LRRC33). In some embodiments, such inhibitors target at least three of the following therapeutic targets (e.g., "context-tolerant" inhibitors): GARP-associated precursor / latent TGFβ1; LRRC33-associated precursor / latent TGFβ1; LTBP1-associated precursor / latent TGFβ1; and LTBP3-associated precursor / latent TGFβ1. In some embodiments, such inhibitors inhibit all four of the therapeutic targets (e.g., "context-independent" inhibitors): GARP-associated precursor / latent TGFβ1; LRRC33-associated precursor / latent TGFβ1; LTBP1-associated precursor / latent TGFβ1; and LTBP3-associated precursor / latent TGFβ1, so as to broadly inhibit TGFβ1 function in these contexts.

[0265] Whether a particular condition of a patient involves or is driven by multiple aspects of TGFβ1 function can be evaluated by evaluating the expression profile of the presenting molecules in clinical samples collected from the patient. Various assays are known in the art, including RNA-based and protein-based assays, and can be performed to obtain an expression profile. The relative expression levels (and / or changes / alterations) of LTBP1, LTBP3, GARP, and LRRC33 in a sample(s) can indicate the origin and / or context of TGFβ1 activity associated with the condition. For example, a biopsy sample obtained from a solid tumor can show high expression of all four presenting molecules. For example, LTBP1 and LTBP3 can be highly expressed in CAFs within the tumor stroma, while GARP and LRRC33 can be highly expressed by tumor-associated immune cells such as Tregs and leukocyte infiltrates, respectively.

[0266] Thus, the present invention includes methods for determining (e.g., testing or confirming) the involvement of TGFβ1 in disease compared to TGFβ2 and TGFβ3. In some embodiments, the method further comprises identifying the origin (or context) of disease-associated TGFβ1. In some embodiments, the origin / context is assessed by determining the expression of TGFβ-presenting molecules, such as LTBP1, LTBP3, GARP and LRRC33, in clinical samples obtained from patients.

[0267] Isoform-selective TGFβ1 inhibitors such as those described herein can be used to treat a wide variety of diseases, disorders and / or conditions associated with TGFβ1 dysregulation (i.e., TGFβ1-related indications) in human subjects (subhects), and as used herein, "disease (disorder or condition) associated with TGFβ1 dysregulation" or "TGFβ1-related indication" means any disease, disorder and / or condition associated with expression, activity and / or metabolism of TGFβ1 or any disease, disorder and / or condition that may benefit from inhibition of activity and / or levels of TGFβ1.

[0268] Thus, the present invention includes the use of isoform-specific, context-tolerant TGFβ1 inhibitors in methods for treating diseases associated with TGFβ1 dysregulation in human subjects.Such inhibitors are typically formulated into pharmaceutical compositions further comprising pharma-ceutical acceptable excipients.Advantageously, the inhibitor targets both ECM-associated TGFβ1 and immune cell-associated TGFβ1 in vivo, but does not target TGFβ2 or TGFβ3.In some embodiments, the inhibitor inhibits the activation step of TGFβ1. The disease is characterized by dysregulation or dysfunction in at least two of the following attributes: a) regulatory T cells (Treg); b) effector T cell (Teff) proliferation or function; c) myeloid cell proliferation or differentiation; d) monocyte recruitment or differentiation; e) macrophage function; f) epithelial-mesenchymal transition (EMT) and / or endothelial-mesenchymal transition (EndMT); g) gene expression in one or more marker genes selected from the group consisting of PAI-1, ACTA2, CCL2, Col1a1, Col3a1, FN-1, CTGF, and TGFβ1; h) ECM components or functions; and i) fibroblast differentiation. A therapeutically effective amount of such an inhibitor is administered to a subject suffering from or diagnosed with the disease.

[0269] In some embodiments, diseases involving dysregulation or dysfunction of an ECM component or function include those exhibiting increased collagen I deposition.

[0270] In some embodiments, the dysregulated or impaired differentiation of fibroblasts comprises an expansion of myofibroblasts or myofibroblast-like cells. In some embodiments, the myofibroblasts or myofibroblast-like cells are cancer-associated fibroblasts (CAFs). In some embodiments, CAFs are associated with the tumor stroma and can produce CCL2 / MCP-1 and / or CXCL12 / SDF-1.

[0271] In some embodiments, dysregulation or dysfunction of regulatory T cells comprises increased Treg activity.

[0272] In some embodiments, dysregulation or impairment of effector T cell (Teff) proliferation or function comprises suppression of CD4+ / CD8+ cell proliferation.

[0273] In some embodiments, the dysregulated or impaired myeloid cell proliferation or differentiation comprises increased proliferation of myeloid progenitor cells. Increased proliferation of myeloid cells can occur within the bone marrow.

[0274] In some embodiments, dysregulated or impaired monocyte differentiation comprises increased differentiation of bone marrow-derived and / or tissue-resident monocytes into macrophages in disease sites such as fibrotic tissues and / or solid tumors.

[0275] In some embodiments, dysregulated or impaired monocyte recruitment includes increased bone marrow-derived monocyte recruitment to disease sites, such as the TME, leading to increased macrophage differentiation and M2 polarization, followed by an expansion of TAMs.

[0276] In some embodiments, the dysregulated or impaired macrophage function comprises increased polarization of macrophages towards the M2 phenotype.

[0277] In some embodiments, the dysregulated or impaired myeloid cell proliferation or differentiation comprises an increase in the numbers of Tregs, MDSCs and / or TANs.

[0278] TGFβ-related indications may include conditions involving immune-deprived disease microenvironments, such as tumors or cancerous tissues, that suppress the body's normal defense mechanisms / immunity in part by depriving effector immune cells (e.g., CD4+ and / or CD8+ T cells). In some embodiments, such immune-deprived conditions are associated with poor responsiveness to treatment. Without intending to be bound by a particular theory, it is believed that TGFβ inhibitors, such as those described herein, may help counter the ability of tumors to deprive anti-cancer immunity by restoring T cell access.

[0279] Non-limiting examples of TGFβ-associated indications include: fibrosis, including organ fibrosis (e.g., kidney fibrosis, liver fibrosis, heart / cardiovascular fibrosis and pulmonary fibrosis), scleroderma, Alport syndrome, cancer (including but not limited to: blood cancers such as leukemia, myelofibrosis, multiple myeloma, colon cancer, renal cancer, breast cancer, malignant melanoma, glioblastoma), fibrosis associated with solid tumors (e.g., cancer fibrosis, e.g., desmoplastic melanoma, pancreatic cancer-associated fibrosis and breast carcinoma fibrosis), interstitial fibrosis (e.g., interstitial fibrosis of the breast), radiation-induced fibrosis (e.g., radiation fibrosis syndrome), promotion of rapid hematopoiesis after chemotherapy, bone healing, wound healing, dementia, bone marrow fibrosis, and osteoporosis. fibrosis, myelodysplastic syndromes (e.g., myelodysplastic syndromes or MDS), renal disease (e.g., end stage renal disease or ESRD), unilateral ureteral obstruction (UUO), tooth loss and / or degeneration, endothelial proliferation syndrome, asthma and allergies, gastrointestinal disorders, anemia of aging, aortic aneurysms, orphan indications (e.g., Marfan syndrome and Kamuracchi-Engelman disease), obesity, diabetes, arthritis, multiple sclerosis, muscular dystrophies, amyotrophic lateral sclerosis (ALS), Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, metabolic syndrome, nutritional diseases, organ atrophy, chronic obstructive pulmonary disease (COPD), and anorexia. Additional indications may include any of those disclosed in U.S. Publication No. 2013 / 0122007, U.S. Patent No. 8,415,459, or International Publication No. WO2011 / 151432, the contents of each of which are incorporated by reference herein in their entirety.

[0280] In preferred embodiments, the antibodies, antigen-binding portions thereof, and compositions of the present disclosure can be used to treat a wide variety of diseases, disorders, and / or conditions associated with TGFβ1 signaling. In some embodiments, the target tissue / cell preferentially expresses the TGFβ1 isoform over other isoforms. Thus, the present invention includes methods for treating such conditions associated with TGFβ1 expression (e.g., dysregulation of TGFβ1 signaling and / or upregulation of TGFβ1 expression) using pharmaceutical compositions comprising the antibodies or antigen-binding portions thereof described herein.

[0281] In some embodiments, the disease involves TGFβ1 associated with (e.g., presented on or deposited by) multiple cell origins, hi some embodiments, such disease involves both immune and ECM components of TGFβ1 function. In some embodiments, such diseases include: i) dysregulation of the ECM (e.g., overproduction / deposition of ECM components such as collagens and proteases; altered stiffness of the ECM matrix; abnormal or pathological activation or differentiation of fibroblasts, such as myofibroblasts and CAFs); ii) immune suppression due to expansion of Tregs and / or suppression of effector T cells (Teff), e.g., an elevated Treg / Teff ratio; increased leukocyte infiltration (e.g., macrophages and MDSCs) causing suppression of CD4 and / or CD8 T cells; and / or iii) abnormal or pathological activation, differentiation, and / or recruitment of myeloid cells, such as macrophages (e.g., bone marrow-derived monocytic / macrophages and tissue-resident macrophages), monocytes, myeloid-derived suppressor cells (MDSCs), neutrophils, dendritic cells, and NK cells.

[0282] In some embodiments, the condition comprises TGFβ1 presented by more than one type of presentation molecule (e.g., two or more of GAPR, LRRC33, LTBP1 and / or LTBP3). In some embodiments, the affected tissue / organ / cell comprises TGFβ1 from multiple cellular origins. To provide but one example, solid tumors (which may include proliferative disorders involving bone marrow, e.g., myelofibrosis and multiple myeloma) may comprise TGFβ1 from multiple origins, e.g., cancer cells, stromal cells, surrounding healthy cells, and / or infiltrating immune cells (e.g., CD45+ leukocytes), and engage different types of presentation molecules. Relevant immune cells include, but are not limited to, myeloid and lymphoid cells, e.g., neutrophils, eosinophils, basophils, lymphocytes (e.g., B cells, T cells, and NK cells), and monocytes. Context-independent or context-tolerant inhibitors of TGFβ1 may be useful in treating such conditions.

[0283] Non-limiting examples of conditions or disorders that can be treated by an isoform-specific, context-tolerance inhibitor of TGFβ1, such as an antibody or fragment thereof described herein, are provided below.

[0284] Disorders with abnormal gene expression: Abnormal activation of the TGFβ1 signaling pathway in various disease states has been observed to be associated with altered gene expression of many markers. These gene expression markers (e.g., measured by mRNA) include, but are not limited to: Serpine1 (encoding PAI-1), MCP-1 (also known as CCL2), Col1a1, Col3a1, FN1, TGFβ1, CTGF, and ACTA2 (encoding α-SMA). Interestingly, many of these genes are implicated as playing a role in a diverse set of disease states, including various types of organ fibrosis, as well as in many cancers, including myelofibrosis. Indeed, a pathophysiological relationship has been suggested between fibrotic conditions and abnormal cell proliferation, tumor formation, and metastasis. See, for example, Cox and Erler (2014) Clinical Cancer Research 20(14):3637-43 "Molecular pathways: connecting fibrosis and solid tumor metastasis"; Shiga et al. (2015) Cancers 7:2443-2458 "Cancer-associated fibroblasts: their characteristics and their roles in tumor growth"; Wynn and Barron (2010) Semin. Liver Dis. 30(3):245-257 "Macrophages: master regulators of inflammation and fibrosis", the contents of which are incorporated herein by reference. Without wishing to be bound by any particular theory, the inventors of the present disclosure contemplate that the TGFβ1 signaling pathway may indeed be an important link between these broad pathologies.

[0285] For example, MCP-1 / CCL2 is thought to play a role in both fibrosis and cancer. MCP-1 / CCL2 is characterized as a profibrotic chemokine and is a monocyte chemoattractant, and evidence suggests that it may be involved in both the initiation and progression of cancer. In fibrosis, MCP-1 / CCL2 has been shown to play an important role in the inflammatory phase of fibrosis. For example, neutralization of MCP-1 resulted in a dramatic reduction in glomerular crescent formation and deposition of type I collagen.

[0286] The ability of MCP-1 / CCL2 to recruit monocytes / macrophages has crucial consequences in cancer progression. Tumor-derived MCP-1 / CCL2 can promote a "cancer-promoting" phenotype in macrophages. For example, in lung cancer, MCP-1 / CCL2 has been shown to be produced by stromal cells and promote metastasis. In human pancreatic cancer, tumors secrete CCL2 and immunosuppressive CCR2-positive macrophages infiltrate these tumors. Patients with tumors that show high CCL2 expression / low CD8 T cell infiltration have significantly reduced survival.

[0287] Similarly, the involvement of PAI-1 / Serpine1 has been implicated in various cancers, angiogenesis, inflammation, and neurodegenerative diseases (e.g., Alzheimer's disease). Elevated expression of PAI-1 in tumors and / or serum correlates with poor prognosis (e.g., shorter survival, increased metastasis) in various cancers such as breast and bladder cancers (e.g., transitional cell carcinoma) and in myelofibrosis. In the context of fibrotic conditions, PAI-1 has been recognized as an important downstream effector of TGFβ1-induced fibrosis, and increased PAI-1 expression has been observed in various forms of tissue fibrosis, including pulmonary fibrosis (e.g., IPF), renal fibrosis, liver fibrosis, and scleroderma.

[0288] In some embodiments, the in vivo effect of TGFβ1 inhibitor therapy can be evaluated by measuring changes in genetic markers. Suitable markers include TGFβ (e.g., TGFβ1, TGFβ2, and TGFβ3). In some embodiments, suitable markers include mesenchymal transition genes (e.g., AXL, ROR2, WNT5A, LOXL2, TWIST2, TAGLN, and / or FAP), immunosuppressive genes (e.g., IL10, VEGFA, VEGFC), monocyte and macrophage chemotaxis genes (e.g., CCL2, CCL7, CCL8, and CCL13), and / or various fibrotic markers discussed herein. Preferred markers are plasma markers.

[0289] As shown in the Examples herein, the isoform-specific, context-independent inhibitors of TGFβ1 described herein can reduce the expression levels of many of these markers in mechanistic animal models such as UUO, which have been shown to be TGFβ1 dependent. Thus, such inhibitors can be used to treat diseases or disorders characterized by aberrant expression (e.g., overexpression / upregulation or underexpression / downregulation) of one or more gene expression markers.

[0290] Thus, in some embodiments, the isoform-specific, context-tolerant or context-independent inhibitors of TGFβ1 are used in the treatment of diseases associated with overexpression of one or more of the following: PAI-1 (also known as Serpine1), MCP-1 (also known as CCL2), Col1a1, Col3a1, FN1, TGFβ1, CTGF, α-SMA, ITGA11, and ACTA2, the treatment comprising administering to a subject suffering from the disease an amount of the inhibitor effective to treat the disease. In some embodiments, the inhibitors are used to treat diseases associated with overexpression of PAI-1, MCP-1 / CCL2, CTGF, and / or α-SMA. In some embodiments, the disease is myelofibrosis. In some embodiments, the disease is cancer, e.g., the cancer comprises a solid tumor. In some embodiments, the disease is organ fibrosis, e.g., fibrosis of liver, kidney, lung, and / or heart or cardiovascular tissue.

[0291] Protease-related disorders: Activation of TGFβ from its latent complex can be triggered by integrins in a force-dependent manner and / or by proteases. Evidence suggests that certain classes of proteases, including but not limited to Ser / Thr proteases such as kallikrein, chemotrypsin, elastase, plasmin, and zinc metalloproteases of the MMP family such as MMP-2, MMP-9, and MMP-13, may be involved in the process. MMP-2 degrades the most abundant component of the basement membrane, collagen IV, raising the possibility that it may play a role in ECM-associated TGFβ1 regulation. MMP-9 has been implicated as playing a central role in tumor progression, angiogenesis, stromal remodeling, and metastasis. Thus, protease-dependent activation of TGFβ1 in the ECM may be important for the treatment of cancer.

[0292] Kallikreins (KLKs) are trypsin- or chymotrypsin-like serine proteases, including plasma kallikrein and tissue kallikrein. ECM plays a role in tissue homeostasis, acting as a structural and signaling scaffold and a barrier to inhibit malignant growths. KLKs may play a role in the degradation of ECM proteins and other components that may promote tumor expansion and invasion. For example, KLK1 is highly upregulated in certain breast cancers and can activate pro-MMP-2 and pro-MMP-9. KLK2 activates latent TGFβ1 to make prostate cancer adjacent to fibroblasts permissive to cancer growth. KLK3 has been widely studied as a diagnostic marker for prostate cancer (PSA). KLK3 can directly activate TGFβ1 by processing plasminogen to plasmin, which proteolytically cleaves LAP. KLK6 may be a potential marker for Alzheimer's disease.

[0293] Moreover, data provided in Example 8 indicates that such a protease may be kallikrein. Thus, the present invention encompasses the use of isoform-specific, context-independent or permissive inhibitors of TGFβ in methods for treating diseases associated with kallikrein or kallikrein-like proteases.

[0294] All known activators of TGFβ1, such as plasmin, TSP-1 and αVβ6 integrin, directly interact with LAP. It is hypothesized that proteolytic cleavage of LAP may destabilize LAP-TGFβ interaction, thereby releasing active TGFβ1. It has been suggested that the region containing 54-LSKLRL-59 is important for maintaining TGFβ1 latency. Thus, agents (e.g., antibodies) that block proteolytic cleavage of LAP or stabilize the interaction may prevent TGFβ activation.

[0295] Diseases involving epithelial-mesenchymal transition (EMT): EMT (epithelial-mesenchymal transition) is a process in which epithelial cells with tight junctions switch to mesenchymal characteristics (phenotypes), such as loose cell-cell contacts. The process is observed in many normal biological processes as well as pathological situations, including embryogenesis, wound healing, cancer metastasis and fibrosis (reviewed, for example, in Shiga et al. (2015) "Cancer-Associated Fibroblasts: Their Characteristics and Their Roles in Tumor Growth" Cancers, 7:2443-2458). In general, EMT signals are thought to be primarily induced by TGFβ. Many types of cancer, for example, are thought to involve the transdifferentiation of cells toward a mesenchymal phenotype (e.g., CAFs), which correlates with poor prognosis. Therefore, isoform-specific, context-tolerant inhibitors of TGFβ1, such as those described herein, can be used to treat diseases initiated or driven by EMT. Indeed, the data illustrated herein (e.g., Figures 12 and 13) show that such inhibitors have the ability to suppress the expression of CAF markers such as α-SMA, Col1 (type I collagen), and FN (fibronectin) in vivo.

[0296] Diseases involving endothelial-mesenchymal transition (EndMT): Similarly, TGFβ is also a key regulator of endothelial-mesenchymal transition (EndMT) observed in normal development, such as cardiogenesis. However, the same or similar phenomenon is also seen in many diseases, such as cancer stroma. In some disease processes, endothelial markers, such as CD31, become downregulated upon TGFβ1 exposure, and instead, the expression of mesenchymal markers, such as FSP-1, α-SMA and fibronectin, becomes induced. In fact, stromal CAFs can be derived from vascular endothelial cells. Therefore, isoform-specific, context-tolerant inhibitors of TGFβ1, such as those described herein, can be used to treat diseases initiated or driven by EndMT.

[0297] Diseases involving matrix stiffening and remodeling: The progression of fibrotic conditions involves an increase in the levels of matrix components deposited on the ECM and / or the maintenance / remodeling of the ECM. TGFβ1 at least partially contributes to this process. This is supported by the observation that the mechanical properties of the ECM (e.g., matrix / substrate stiffness) can be modified by increasing the deposition of ECM components such as collagen, a phenomenon associated with TGFβ1 signaling. To confirm this view, we have evaluated the role of matrix stiffness in the effect of integrin-dependent activation of TGFβ in primary fibroblasts transfected with proTGFβ and LTBP1 and grown on silicon-based substrates with defined stiffness (e.g., 5 kPa, 15 kPa or 100 kPa). As summarized in the Examples section below, matrices with greater stiffness enhance TGFβ1 activation, which can be suppressed by isoform-specific, context-tolerant inhibitors of TGFβ1 such as those described herein. This observation suggests that TGFβ1 affects ECM properties (e.g., stiffness), which in turn can further induce TGFβ1 activation (reflecting disease progression). Thus, isoform-specific, context-tolerant inhibitors of TGFβ1, such as those described herein, can be used to block this process, combating disease progression involving ECM alterations, such as fibrosis, tumor growth, invasion, metastasis, and fibrosis. The LTBP arm of such inhibitors can directly block ECM-associated precursor / latent TGFβ complexes presented by LTBP1 and / or LTBP3, thereby preventing growth factor release / activation from the complexes within the disease niche. In some embodiments, isoform-specific, context-tolerant TGFβ1 inhibitors, such as those described herein, can normalize ECM stiffness to treat diseases involving integrin-dependent signaling. In some embodiments, the integrin includes α11 chain, β1 chain, or both.

[0298] Fibrosis: According to the present invention, isoform-specific, context-tolerant inhibitors of TGFβ1, such as those described herein, are used in the treatment of fibrosis (e.g., fibrotic symptoms, fibrotic conditions) in a subject. Suitable inhibitors for carrying out the present invention include antibodies and / or compositions according to the present disclosure that may be useful for modifying or ameliorating fibrosis. More specifically, such antibodies and / or compositions are selective antagonists of TGFβ1 that can target TGFβ1 presented by various types of presentation molecules. TGFβ1 is recognized as a central orchestrator of fibrotic responses. Antibodies targeting TGFβ reduce fibrosis in numerous preclinical models. Such antibodies and / or antibody-based compounds include LY2382770 (Eli Lilly, Indianapolis, IN). including those described in U.S. Patent Nos. US 6,492,497, US 7,151,169, US 7,723,486, and U.S. Application Publication No. 2011 / 0008364, the contents of each of which are incorporated by reference in their entirety. Prior art TGFβ antagonists include, for example, agents that target and block integrin-dependent activation of TGFβ.

[0299] However, evidence suggests that such prior art agents may not mediate isoform-specific inhibition and may cause unwanted effects by inadvertently blocking the normal function of TGFβ2 and / or TGFβ3.Indeed, the data provided herein supports this view.Normal (non-diseased) lung tissue contains relatively low but measurable levels of TGFβ2 and TGFβ3, but especially less TGFβ1.In comparison, in certain disease states, such as fibrosis, TGFβ1 is preferentially upregulated compared to other isoforms.Preferably, TGFβ antagonists for use in treating such conditions will only direct their inhibitory activity toward disease-inducing or disease-associated isoforms, while preventing the function of other isoforms that are normally expressed and mediate tonic signaling in tissues. Advantageously, as shown in Example 20 below, the isoform-specific, context-tolerant TGFβ1 inhibitors encompassed by the present disclosure show little effect in bronchoalveolar lavage (BAL) of healthy rats, supporting the notion that tonic TGFβ signaling (e.g., TGFβ2 and / or TGFβ3) is not disrupted. In contrast, both prior art inhibitors (LY2109761, a small molecule TGFβ receptor antagonist, and a monoclonal antibody targeting αVβ6 integrin) were shown to inhibit tonic signaling downstream of TGFβ in non-diseased rat BAL, raising the possibility that these inhibitors may cause unwanted side effects. Alternatively or additionally, agents that target and block integrin-dependent activation of TGFβ may be able to block only a subset of integrins responsible for disease-associated TGFβ1 activation, among the numerous integrin types expressed by various cell types and playing a role in pathogenesis. Furthermore, even if such antagonists could selectively block integrin-mediated activation of TGFβ1 isoforms, they might not be effective in blocking TGFβ1 activation triggered by other modes, such as protease-dependent activation.In contrast, isoform-specific, context-tolerant inhibitors of TGFβ1, such as those described herein, aim to prevent the activation step of TGFβ1 regardless of the specific mode of activation, while maintaining isoform selectivity.

[0300] Fibrotic indications for which the antibodies and / or compositions of the disclosure may be used therapeutically include, but are not limited to, pulmonary indications (e.g., idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), allergic asthma, acute lung injury, eosinophilic esophagitis, pulmonary arterial hypertension, and chemical gas disorders), renal indications (e.g., diabetic glomerulosclerosis, focal segmental glomerulosclerosis (FSGS), chronic kidney disease (CKD), fibrosis associated with kidney transplantation and chronic rejection, IgA nephropathy, and hemolytic uremic syndrome), hepatic fibrosis (e.g., nonalcoholic steatohepatitis (NASH), chronic viral hepatitis, parasitemia, inborn errors of metabolism, toxins, and the like). Intervening fibrosis, such as alcoholic fibrosis, non-alcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC), primary biliary cirrhosis, and sclerosing cholangitis), cardiovascular fibrosis (e.g., cardiomyopathies, hypertrophic cardiomyopathy, atherosclerosis and restenosis), systemic sclerosis, dermal fibrosis (e.g., in systemic sclerosis, diffuse cutaneous systemic sclerosis, scleroderma, pathological skin scars, keloids, post-operative scars, scar revision surgical procedures, radiation-induced scars and chronic wounds), and cancer or secondary fibrosis (e.g., myelofibrosis, head and neck cancer, M7 acute megakaryoblastic leukemia, and mucositis). Other diseases, disorders or conditions involving fibrosis (including degenerative disorders) that may be treated using the compounds and / or compositions of the present disclosure include, but are not limited to, adenomyosis, endometriosis, Marfan syndrome, scleroderma syndrome, scleroderma, rheumatoid arthritis, myelofibrosis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, muscular dystrophies (e.g., DMD), Parkinson's disease, ALS, Dupuytren's contracture, Kamurati-Engelman disease, neural scarring, dementia, proliferative vitreoretinopathy, corneal injury, complications following glaucoma drainage surgery, and multiple sclerosis. Many such fibrotic indications are also associated with inflammation in the affected tissue(s), indicating the involvement of an immune component.

[0301] In some embodiments, fibrotic indications that may be treated by the compositions and / or methods described herein include organ fibrosis, such as pulmonary fibrosis (e.g., IPF), renal fibrosis (e.g., fibrosis associated with CKD), liver fibrosis, cardiac or cardiac tissue fibrosis, dermal fibrosis (e.g., scleroderma), uterine (e.g., endometrial, myometrial) fibrosis, and bone marrow fibrosis. In some embodiments, such treatment may reduce or delay the need for organ transplantation in patients. In some embodiments, such treatment may prolong the survival of patients.

[0302] For treating IPF, patients who may benefit from treatment include those with familial IPF and those with sporadic IPF. Administration of a therapeutically effective amount of an isoform-specific, context-tolerant inhibitor of TGFβ1 may reduce myofibroblast accumulation in lung tissue, reduce collagen deposition, reduce IPF symptoms, improve or maintain lung function, and prolong survival. In some embodiments, the inhibitor blocks the activation of ECM-associated TGFβ1 (e.g., precursor / latent TGFβ1 presented by LTBP1 / 3) in the fibrotic environment of IPF. The inhibitor may optionally further block macrophage-associated TGFβ1 (e.g., precursor / latent TGFβ1 presented by LRRC33), such as the activation of alveolar macrophages. As a result, the inhibitor may suppress fibronectin release and other fibrosis-related factors.

[0303] Isoform-specific, context-tolerant TGFβ1 inhibitors such as those provided herein can be used to treat fibrotic conditions of the liver, such as fatty liver (e.g., NASH). Fatty liver may or may not be inflamed. Liver inflammation caused by fatty liver (i.e., steatohepatitis) can develop into scarring (fibrosis), which often progresses to cirrhosis (scarring that distorts the liver's structure and impairs its function). Inhibitors can therefore be used to treat such conditions. In some embodiments, inhibitors block the activation of ECM-associated TGFβ1 (e.g., precursor / latent TGFβ1 presented by LTBP1 / 3) within the fibrotic environment of the liver. Inhibitors may optionally further block the activation of macrophage-associated TGFβ1 (e.g., precursor / latent TGFβ1 presented by LRRC33), such as Kupffer cells (also known as stellate macrophages) and infiltrating monocyte-derived macrophages and MDSCs. As a result, the inhibitor can suppress fibrosis-related factors. Administration of the inhibitor in a subject with such a condition can reduce one or more symptoms, prevent or hinder disease progression, reduce or stabilize fat accumulation in the liver, reduce disease-related biomarkers (e.g., serum collagen fragments), reduce liver scarring, reduce liver stiffness, and / or otherwise produce clinically meaningful outcomes in a patient population treated with the inhibitor compared to a control population not treated with the inhibitor. In some embodiments, an effective amount of the inhibitor can achieve both a reduction in liver fat and a reduction in fibrosis (e.g., scarring) in NASH patients. In some embodiments, an effective amount of the inhibitor can achieve at least one stage of fibrosis improvement in NASH patients without worsening steatohepatitis. In some embodiments, an effective amount of the inhibitor can reduce the incidence of liver failure and / or liver cancer in NASH patients. In some embodiments, an effective amount of an inhibitor may normalize the levels of a number of inflammatory or fibrotic serum biomarkers compared to a control, as assessed, for example, 12-36 weeks after initiation of treatment.In some embodiments in NASH patients, an isoform-specific, context-tolerant TGFβ1 inhibitor may be administered in patients receiving one or more additional therapies (including but not limited to myostatin inhibitors) to generally enhance metabolic control in patients with clinical signs of metabolic syndrome, including NASH.

[0304] Isoform-specific, context-tolerant TGFβ1 inhibitors such as those described herein can be used to treat kidney fibrotic conditions, such as diseases characterized by extracellular matrix accumulation (IgA nephropathy, focal and segmental glomerulosclerosis, crescentic glomerulonephritis, lupus nephritis and diabetic nephropathy), in which significantly increased expression of TGFβ in glomeruli and tubulointerstitium has been observed. Glomerular and tubulointerstitial deposition of two matrix components induced by TGFβ, fibronectin EDA+ and PAI-1, was significantly elevated in all diseases with matrix accumulation, but correlation analysis reveals a close relationship mainly with TGFβ1 isoform. Therefore, isoform-specific, context-tolerant TGFβ1 inhibitors are useful as treatments for various human glomerular disorders in which TGFβ is associated with pathological accumulation of extracellular matrix.

[0305] In some embodiments, the renal fibrotic condition is associated with chronic kidney disease (CKD). CKD is mainly caused by hypertension or diabetes and claims more than one million lives each year. CKD patients require lifelong medical care ranging from strict diet and medication to dialysis and transplantation. In some embodiments, TGFβ1 inhibitor therapy as described herein may reduce or delay the need for dialysis and / or transplantation. In some embodiments, such treatment may reduce the need (e.g., dose, frequency) of other treatments. In some embodiments, isoform-specific, context-tolerant TGFβ1 inhibitors may be administered in patients undergoing one or more additional treatments, including but not limited to myostatin inhibitors, which may generally enhance metabolic regulation in patients with CKD.

[0306] Organ fibrosis that can be treated by the methods provided herein includes cardiac (e.g., cardiovascular) fibrosis. In some embodiments, cardiac fibrosis is associated with heart failure, such as chronic heart failure (CHF). In some embodiments, heart failure can be associated with myocardial disease and / or metabolic disease. In some embodiments, an isoform-specific, context-tolerant TGFβ1 inhibitor can be administered in patients who undergo one or more additional therapies (including, but not limited to, myostatin inhibitors in patients with cardiac dysfunction, including cardiac fibrosis and metabolic disorders).

[0307] In some embodiments, fibrotic conditions that can be treated by the compositions and / or methods described herein include fibrosis. Fibrosis can occur around neoplasms, resulting in dense fibrosis (e.g., desmoplastic stroma) around tumors or scar tissue in the abdomen after abdominal surgery. In some embodiments, fibrosis is associated with malignant tumors. Due to the dense formation surrounding malignant tumors, conventional anti-cancer treatments (e.g., chemotherapy) cannot effectively penetrate to reach cancerous cells for clinical efficacy. Isoform-specific, context-tolerant inhibitors of TGFβ1, such as those described herein, can be used to disrupt fibrosis so that fibrosis formation can be relaxed to aid the efficacy of anti-cancer treatment. In some embodiments, isoform-specific, context-tolerant inhibitors of TGFβ1 can be used as monotherapy (further below).

[0308] To treat a patient with a fibrotic condition, a TGFβ1 isoform-specific, context-tolerant inhibitor is administered to the subject in an amount effective to treat fibrosis. An effective amount of such an antibody is an amount effective to achieve both therapeutic efficacy and clinical safety in the subject. In some embodiments, the inhibitor is a context-tolerant antibody capable of blocking activation of LTBP-mediated TGFβ1 localized (e.g., tethered) in the ECM and GARP-mediated TGFβ1 localized (e.g., tethered) in immune cells. In some embodiments, the antibody is a context-tolerant antibody capable of blocking activation of LTBP-mediated TGFβ1 localized in the ECM and LRRC33-mediated TGFβ1 localized (e.g., tethered) in monocytes / macrophages. In some embodiments, the LTBP is LTBP1 and / or LTBP3. In some embodiments, targeting and inhibiting TGFβ1, which is presented by LRRC33 on pro-fibrotic, M2-like macrophages in a fibrotic microenvironment, may be beneficial.

[0309] Assays useful for determining the effectiveness of an antibody and / or composition of the disclosure with respect to altering fibrosis include, but are not limited to, histological assays for enumerating fibroblasts and basic immunohistochemical analyses known in the art.

[0310] Myelofibrosis: Myelofibrosis, also known as bone marrow fibrosis, is a relatively rare myeloproliferative disease (cancer) that belongs to a group of diseases called myeloproliferative disorders. Myelofibrosis is classified as the Philadelphia chromosome negative (-) branch of myeloproliferative neoplasms. Myelofibrosis is characterized by clonal myeloid proliferation, abnormal cytokine production, extramedullary hematopoiesis, and myelofibrosis. Abnormal clonal proliferation of hematopoietic stem cells in the bone marrow and other sites leads to fibrosis, or replacement of the marrow by scar tissue. The term myelofibrosis refers to primary myelofibrosis (PMF) unless otherwise specified. It may also be called chronic idiopathic myelofibrosis (cIMF) (the terms idiopathic and primary in these cases mean that the disease is of unknown or spontaneous origin). This is in contrast to myelofibrosis that occurs secondary to polycythemia vera or essential thrombocythemia. Myelofibrosis is a form of myelodysplasia, which refers to changes in cell types within the blood-forming tissue of bone marrow, and the two terms are often used interchangeably. The terms idiopathic myelodysplasia and myelofibrosis with myelodysplasia (MMM) are also used to refer to primary myelofibrosis. In some embodiments, hematological proliferative disorders that can be treated according to the present invention include myeloproliferative disorders such as myelofibrosis. The so-called "classical group" of BCR-ABL (Ph) negative chronic myeloproliferative disorders includes essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF).

[0311] Myelofibrosis disrupts the body's normal production of blood cells. The result is extensive scarring within the bone marrow, leading to severe anemia, weakness, fatigue, and often an enlarged spleen. Production of cytokines such as fibroblast growth factor by abnormal hematopoietic cell clones (especially by megakaryocytes) leads to replacement of hematopoietic tissue in the bone marrow by connective tissue via collagen fibrosis. Reduction of hematopoietic tissue impairs the patient's ability to produce new blood cells, leading to progressive pancytopenia (deficiency of all blood cell types). However, fibroblast proliferation and collagen deposition are thought to be secondary phenomena, and fibroblasts themselves may not be part of the abnormal cell clone.

[0312] Myelofibrosis can be caused by abnormal blood stem cells in the bone marrow that rapidly proliferate and produce mature and less differentiated cells that take over the bone marrow, leading to both fibrosis (scar tissue formation) and chronic inflammation.

[0313] Primary myelofibrosis is associated with mutations in Janus kinase 2 (JAK2), thrombopoietin receptor (MPL) and calreticulin (CALR), which may result in constitutive activation of the JAK-STAT pathway, progressive scarring, or fibrosis of the resulting bone marrow. As hematopoietic cells are forced to migrate to other areas (especially the liver and spleen), patients may develop extramedullary hematopoiesis, i.e., blood cell formation occurring in sites other than the bone marrow. This results in enlargement of these organs. In the liver, the abnormal size is called hepatomegaly. Enlargement of the spleen is called splenomegaly and also contributes to causing pancytopenia, particularly thrombocytopenia and anemia. Another complication of extramedullary hematopoiesis is poikilocytosis, or the presence of abnormally shaped red blood cells.

[0314] The main site of extramedullary hematopoiesis in myelofibrosis is the spleen, which is usually significantly enlarged in patients with myelofibrosis. As a result of the large enlargement of the spleen, multiple subcapsular infarctions often occur in the spleen, meaning that partial or complete tissue death occurs due to the interrupted oxygen supply to the spleen. At the cellular level, the spleen contains erythroid precursors, granulocyte precursors and megakaryocytes, with megakaryocytes being prominent in their numbers and their abnormal shape. Megakaryocytes may be involved in causing the secondary fibrosis seen in this condition.

[0315] It has been suggested that TGFβ may be involved in the fibrotic aspects of the pathogenesis of myelofibrosis (see, e.g., Agarwal et al., "Bone marrow fibrosis in primary myelofibrosis: pathogenic mechanisms and the role of TGFβ" (2016) Stem Cell Investig 3:5). Bone marrow pathology in primary myelofibrosis is characterized by fibrosis, neoangeogenesis and osteosclerosis, with fibrosis associated with increased production of collagen that is deposited within the ECM.

[0316] Many biomarkers have been described, the alterations of which indicate or correlate with disease. In some embodiments, the biomarker is a cellular biomarker. Such disease-related biomarkers are useful for diagnosing and / or monitoring disease progression and treatment effectiveness (e.g., patient responsiveness to treatment). These biomarkers include many fibrotic markers, as well as cellular markers. In lung cancer, for example, TGFβ1 concentration in bronchoalveolar lavage (BAL) fluid has been reported to be significantly higher (about 2+-fold increase) in patients with lung cancer compared to patients with benign disease, and may also serve as a biomarker for diagnosing and / or monitoring lung cancer progression or treatment effectiveness.

[0317] Since primary myelofibrosis is associated with abnormal megakaryocyte development, specific cell markers of megakaryocytes and their progenitors of stem cell lineage may serve as markers for diagnosing and / or monitoring disease progression and efficacy of treatment. In some embodiments, useful markers include, but are not limited to: cell markers of differentiated megakaryocytes (e.g., CD41, CD42, and TpoR), cell markers of megakaryocyte-erythroid progenitors (e.g., CD34, CD38, and CD45RA-), cell markers of general myeloid progenitors (e.g., IL-3α / CD127, CD34, SCFR / c-kit, and Flt-3 / Flk-2), and cell markers of hematopoietic stem cells (e.g., CD34, CD38-, Flt-3 / Flk-2). In some embodiments, useful biomarkers include fibrotic markers. These include, but are not limited to: TGFβ1, PAI-1 (also known as Serpine1), MCP-1 (also known as CCL2), Col1a1, Col3a1, FN1, CTGF, α-SMA, ACTA2, Timp1, Mmp8, and Mmp9. In some embodiments, useful biomarkers are serum markers (e.g., proteins or fragments found and detected in serum samples).

[0318] Based on the knowledge that TGFβ is a component of the leukemic bone marrow niche, targeting the bone marrow microenvironment with TGFβ inhibitors may be a promising approach to reduce leukemic cells expressing presentation molecules that regulate local TGFβ availability in affected tissues.

[0319] Indeed, due to the multifaceted nature of the pathology, which shows TGFβ-dependent dysregulation in both myeloproliferative and fibrotic aspects (as the term "myelofibrosis" itself suggests), TGFβ1 isoform-specific, context-tolerant inhibitors such as those described herein may provide particularly advantageous therapeutic effects to patients suffering from myelofibrosis. It is believed that the LTBP arm of such inhibitors can target ECM-associated TGFβ1 complexes in the bone marrow, while the LRRC33 arm of inhibitors can block myeloid cell-associated TGFβ1. In addition, the abnormal megakaryocyte biology associated with myelofibrosis may involve both GARP- and LTBP-mediated TGFβ1 activity. TGFβ1 isoform-specific, context-tolerant inhibitors can target such complexes and thus inhibit the release of active TGFβ1 in the niche.

[0320] Such TGFβ1 inhibitors are therefore useful for treating patients with polycythemia vera who have responded inadequately to or are intolerant to other (or standard of care) therapies, such as hydroxyurea and JAK inhibitors. Such inhibitors are also useful for treating patients with intermediate or high risk myelofibrosis (MF), including primary MF, post-polycythemia vera MF, and post-essential thrombocythemia MF.

[0321] Thus, one aspect of the present invention relates to a method for treating primary myelofibrosis, comprising administering to a patient suffering from primary myelofibrosis a therapeutically effective amount of a composition comprising a TGFβ inhibitor that causes a decrease in TGFβ availability. In some embodiments, an isoform-specific, context-tolerant monoclonal antibody inhibitor of TGFβ1 activation is administered to a patient with myelofibrosis. Such antibodies may be administered at a dose ranging from 0.1 to 100 mg / kg, e.g., 1 to 30 mg, e.g., 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, etc. The preferred route of administration of pharmaceutical compositions comprising antibodies is intravenous or subcutaneous. If the composition is administered intravenously, the patient may be given treatment for an appropriate duration (e.g., approximately 60 minutes per treatment) and then repeated every few weeks (e.g., 3, 4, 6 weeks, etc.) for a total of several cycles (e.g., 4, 6, 8, 10, 12 cycles, etc.). In some embodiments, the patient is treated with a composition comprising an inhibitory antibody at a dosage level of 1-10 mg / kg (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg) via intravenous administration for 6 or 12 cycles every 28 days (4 weeks). In some embodiments, such treatment is administered as a chronic (long-term) treatment (e.g., continued indefinitely as long as deemed beneficial) instead of being discontinued after a set number of cycles of administration.

[0322] In some embodiments, the TGFβ inhibitor is an antibody or antigen-binding portion thereof that binds to inactive (e.g., latent) proTGFβ complexes, thereby preventing the release of active or mature TGFβ from the complexes, effectively inhibiting the activation step. In some embodiments, such an antibody or antigen-binding portion specifically binds to proTGFβ complexes associated with LRRC33, GARP, LTBP1, LTBP3, or any combination thereof. In some embodiments, such an antibody or antigen-binding portion specifically binds to proTGFβ complexes tethered by cells. In some embodiments, the antibody or portion thereof selectively binds to proTGFβ complexes associated with LRRC33 and / or GARP (but not associated with LTBP1 or LTBP3). In some embodiments, the antibody or portion thereof specifically binds to proTGFβ complexes associated with LRRC33. In some embodiments, the antibody or portion thereof specifically binds to proTGFβ complexes associated with GARP. In some embodiments, the antibody or portion thereof specifically binds to proTGFβ complexes associated with LRRC33 as well as proTGFβ complexes associated with GARP.

[0323] Alternatively or in addition to the above embodiments, the TGFβ inhibitor is an antibody or antigen binding thereof that binds LRRC33 and / or GARP and includes a domain for further effector function. In some embodiments, the domain for further effector function may be an Fc or Fc-like domain that mediates ADCC in target cells. Preferably, the antibody that induces ADCC does not trigger or promote internalization sufficiently to allow ADCC-mediated target cell death.

[0324] Alternatively or additionally to the above-mentioned embodiments, the antibody or antigen-binding portion thereof may comprise an additional moiety to carry a "payload" of interest (e.g., an antibody-drug conjugate, or ADC). Examples of suitable payloads include, but are not limited to: therapeutic agents / drugs, toxins, markers, and detection / imaging labels, etc. Such payloads may be chemicals, small molecules, polypeptides, nucleic acids, radioisotopes, etc. Preferably, an antibody suitable for an ADC-mediated mechanism of action is capable of triggering effective internalization of the antigen-antibody complex upon binding to a cell surface target to deliver the payload into the cell.

[0325] Since myelofibrosis is a progressive disease that shows many facets of pathology in multiple affected tissues or organs, therapeutic approaches may vary according to disease progression. For example, at the primary site of disease (bone marrow), appropriate treatment would include LRRC33 inhibitors as described herein that can target hematopoietic cells expressing LRRC33. This can be achieved by administration of a composition comprising an antibody that binds to the proTGFβ complex presented by LRRC33 and inhibits activation of TGFβ in the patient. It can also be achieved by administration of a composition comprising an antibody that binds to LRRC33 and induces death of target cells in the patient. Alternatively, these approaches may be combined, using an antibody that is a TGFβ activation inhibitor and also comprises an additional moiety to mediate cellular cytotoxicity. For example, the additional moiety may be a toxin conjugated to the antibody as a payload or an Fc or Fc-like domain to induce ADCC (e.g., antibody-drug conjugate, or ADC).

[0326] Myelofibrosis may be considered a type of leukemia, but is characterized by signs of fibrosis. TGFβ is known to regulate aspects of ECM homeostasis, and its dysregulation may result in tissue fibrosis, and in some embodiments, it may be desirable to inhibit TGFβ activity associated with ECM. Thus, antibodies or fragments thereof that bind to and inhibit proTGFβ presented by LTBPs (e.g., LTBP1 and LTBP3) are encompassed by the present invention. In some embodiments, antibodies or fragments thereof suitable for treating myelofibrosis are "context tolerant" in that they can bind to multiple contexts of proTGFβ complexes, such as those associated with LRRC33, GARP, LTBP1, LTBP3, or any combination thereof. In some embodiments, such antibodies are context-independent inhibitors of TGFβ activation, characterized in that the antibodies can bind to and inhibit any of the following latent complexes: LTBP1-proTGFβ, LTBP3-proTGFβ, GARP-proTGFβ, and LRRC33-proTGFβ. In some embodiments, such antibodies are isoform-specific antibodies that bind to and inhibit such latent complexes containing one of the TGFβs (but not other isoforms). These include, for example, LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. In some embodiments, such antibodies are isoform-selective antibodies that preferentially bind to and inhibit one or more isoforms of TGFβ. Antibodies that can inhibit TGFβ1 activation in a context-tolerant or context-independent manner would be advantageous for use in the treatment of myelofibrosis.

[0327] Suitable patient populations with myeloproliferative neoplasms that may be treated by the compositions and methods described herein may include, but are not limited to: a) a patient population that is Philadelphia(+); b) a patient population that is Philadelphia(-); c) a patient population classified as "classical" (PV, ET and PMF); d) a patient population that harbors the mutation JAK2V617F(+); e) a patient population that harbors JAK2V617F(-); f) a patient population that has JAK2 exon 12(+); g) a patient population that has MPL(+); and h) a patient population that has CALR(+).

[0328] In some embodiments, the patient population includes patients with intermediate-2 or high-risk myelofibrosis. In some embodiments, the patient population includes patients with myelofibrosis who are refractory to, or are not candidates for, available treatments. In some embodiments, subjects receive 100-200×10 9 / L. In some embodiments, the subject has a platelet count of >200×10 9 / L platelet count.

[0329] In some embodiments, subjects who receive (and may benefit from) isoform-specific, context-tolerant TGFβ1 inhibitor therapy are diagnosed with intermediate-1 or higher primary myelofibrosis (PMF), or late-stage (post-) polycythemia vera / essential thrombocythemia myelofibrosis (late-stage (post-)PV / ETMF). In some embodiments, the subject has documented myelofibrosis prior to treatment. In some embodiments, the subject has grade MF-2 or higher as assessed by the European Concordance Grade Score, and grade 3 or higher by the modified Bauermeister scale, prior to treatment. In some embodiments, the subject has an ECOG performance status of 1 prior to treatment. In some embodiments, the subject has a white blood cell count in the range of 5-120 (10 9 / L). In some embodiments, the subject has a JAK2V617F allele burden in the range of 10-100%.

[0330] In some embodiments, subjects receiving (and who may benefit from) isoform-specific, context-tolerant TGFβ1 inhibitor therapy are transfusion-dependent (prior to treatment), characterized in that the subject has a history of at least 2 units of red blood cell transfusions in the last month due to hemoglobin levels below 8.5 g / dL that are not associated with clinically overt bleeding.

[0331] In some embodiments, the subject receiving (and may benefit from) isoform-specific, context-tolerant TGFβ1 inhibitor therapy has previously received a therapy to treat myelofibrosis. In some embodiments, the subject has been treated with one or more therapies, including but not limited to: AZD1480, panobinostat, EPO, IFNα, hydroxyurea, pegylated interferon, thalidomide, prednisone, and JAK2 inhibitors (e.g., lestaurtinib, CEP-701).

[0332] In some embodiments, the patient has extramedullary hematopoiesis. In some embodiments, the extramedullary hematopoiesis is in the liver, lungs, spleen, and / or lymph nodes. In some embodiments, the pharmaceutical composition of the present invention is administered locally to one or more localized sites of disease manifestations.

[0333] The isoform-specific, context-tolerant TGFβ1 inhibitor is administered to a patient in an amount effective to treat myelofibrosis. A therapeutically effective amount is an amount sufficient to alleviate one or more symptoms and / or complications of myelofibrosis in the patient, including but not limited to: excessive deposition of ECM in the bone marrow stroma, angiogenesis, osteosclerosis, splenomegaly, hematomegaly, anemia, bleeding, bone pain and other bone-related morbidity, extramedullary hematopoiesis, thrombocytosis, leukopenia, cachexia, infection, thrombosis and death.

[0334] In some embodiments, the amount is effective to reduce TGFβ1 expression and / or secretion in the patient (e.g., in megakaryocytic cells). Such inhibitors may thus reduce TGFβ1 mRNA levels in the treated patient. In some embodiments, such inhibitors reduce TGFβ1 mRNA in bone marrow, e.g., in mononuclear cells. PMF patients typically exhibit elevated plasma TGFβ1 levels above about 2,500 pg / mL (as opposed to the normal range of about 600-2,000 pg / mL as measured by ELISA), e.g., above 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 pg / mL (see, e.g., Mascaremhas et al. (Leukemia & Lymphoma, 2014, 55(2):450-452)). Zingariello (Blood, 2013, 121(17):3345-3363) quantified bioactive and total TGFβ1 content in plasma of PMF patients and control individuals. According to this reference, the median bioactive TGFβ1 in PMF patients was 43 ng / mL (range 4-218 ng / mL) and the total TGFβ1 was 153 ng / mL (range 32-1000 ng / mL), while in the control counterparts, the values ​​were 18 (0.05-144) and 52 (8-860), respectively. Thus, based on these reports, plasma TGFβ1 content in PMF patients is elevated several fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, etc.) compared to control or healthy plasma samples. Treatment with an inhibitor as described herein, e.g., following administration of 4 to 12 cycles (e.g., 2, 4, 6, 8, 10, 12 cycles) or chronic or long-term treatment (e.g., every 4 weeks at a dose of 0.1 to 100 mg / kg, e.g., 1 to 30 mg / kg of monoclonal antibody), may reduce plasma TGFβ1 levels by at least 10%, e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50%, compared to the corresponding baseline (pre-treatment).

[0335] Some therapeutic effects may be observed relatively quickly after the initiation of treatment, for example, after 1, 2, 3, 4, 5 or 6 weeks. For example, the inhibitor may effectively increase the number of stem and / or progenitor cells in the bone marrow of a patient treated with the inhibitor within 1-8 weeks. These include hematopoietic stem cells and blood progenitor cells. Bone marrow biopsies may be performed to assess changes in the frequency / number of marrow cells. Correspondingly, patients may show improvement in symptoms such as bone pain and fatigue.

[0336] One of the morphological halomarks of myelofibrosis is fibrosis within the bone marrow (e.g., medullary stroma), characterized in part by abnormal ECM. In some embodiments, the amount is effective to reduce excess collagen deposition, e.g., by mesenchymal stromal cells. In some embodiments, the inhibitor is effective to reduce the number of CD41 positive cells (e.g., megakaryocytes) in treated subjects compared to untreated control subjects. In some embodiments, the baseline frequency of megakaryocytes in PMF bone marrow is measured as the number of cells per square millimeter (mm ) in PMF spleens, as determined in randomly selected cross sections. 2 ) and 200-700 cells per square millimeter (mm 2 In contrast, megakaryocyte frequency in bone marrow and spleen of normal donors is less than 140 and less than 10, respectively. Treatment with an inhibitor can reduce the number (e.g., frequency) of megakaryocytes in bone marrow and / or spleen. In some embodiments, treatment with an inhibitor can cause a decrease in the level of downstream effector signaling, such as phosphorylation of SMAD2 / 3.

[0337] Patients with myelofibrosis may suffer from an enlarged spleen. Thus, the clinical efficacy of treatment may be evaluated by monitoring changes in spleen size. Spleen size may be tested by known techniques, such as assessing spleen length by palpation and / or assessing spleen volume by ultrasound. In some embodiments, subjects treated with TGFβ1 isoform-specific, context tolerance inhibitors have a baseline spleen length (pre-treatment) of 5 cm or more, e.g., in the range of 5-30 cm, as assessed by palpation. In some embodiments, subjects treated with TGFβ1 isoform-specific, context tolerance inhibitors have a baseline spleen volume (pre-treatment) of 300 mL or more, e.g., in the range of 300-1500 mL, as assessed by ultrasound. Treatment with the inhibitor, e.g., after 4-12 cycles (e.g., 2, 4, 6, 8, 10, 12 cycles) of administration at a dose of, e.g., 0.1-30 mg / kg monoclonal antibody every 4 weeks, as described herein, may reduce spleen size in a subject. In some embodiments, an effective amount of the inhibitor is sufficient to reduce spleen size by at least 10%, 20%, 30%, 35%, 40%, 50%, and 60% in a patient population receiving the inhibitor treatment, compared to the corresponding baseline value. For example, the treatment is effective to achieve a ≥ 35% reduction in spleen volume from baseline at 12-24 weeks as measured by MRI or CT scan, compared to a placebo control. In some embodiments, the treatment is effective to achieve a ≥ 35% reduction in spleen volume from baseline at 24-48 weeks as measured by MRI or CT scan, compared to the best available treatment control. The best available treatments may include hydroxyurea, glucocorticoids, as well as the non-pharmaceuticals, anagrelide, epoetin alfa, thalidomide, lenalidomide, mercaptopurine, thioguanine, danazol, peginterferon alfa-2a, interferon-alpha, melphalan, acetylsalicylic acid, cytarabine, and colchicine.

[0338] In some embodiments, patient populations treated with isoform-specific, context-tolerant TGFβ1 inhibitors such as those described herein exhibit a statistically improved treatment response as measured, for example, by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) criteria, a degree of change in myelofibrosis grade as measured by the modified Bauermeister scale and the European Concordance Grading System after treatment (e.g., 4, 6, 8, or 12 cycles), and a symptomatic response using the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF).

[0339] In some embodiments, treatment with an isoform-specific, context-tolerant TGFβ1 inhibitor such as those described herein achieves a statistically improved therapeutic response, as assessed, for example, by a modified Myelofibrosis Symptom Assessment Form (MFSAF), where symptoms are measured by the MFSAF tool (e.g., v2.0), a daukt diary that captures debilitating symptoms of myelofibrosis (abdominal discomfort, early satiety, pain under the left rib cage, pruritus, night sweats, and bone / muscle pain) using a scale of 0 to 10, where 0 is absent and 10 is the worst imaginable. In some embodiments, treatment is effective to achieve a ≧50% reduction in total MFSAF score from baseline, for example, within 12 to 24 weeks. In some embodiments, a significant fraction of patients receiving treatment achieves a ≧50% improvement in Total Symptom Score compared to patients receiving placebo. For example, the fraction of the patient pool achieving a ≧50% improvement may be greater than 40%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.

[0340] In some embodiments, a therapeutically effective amount of an inhibitor is an amount sufficient to effect clinical improvement as assessed by anemia response, for example, an improved anemia response may include a longer duration of transfusion independence (e.g., 8 weeks or more) after 4-12 cycles (e.g., 6 cycles) of treatment.

[0341] In some embodiments, a therapeutically effective amount of an inhibitor is sufficient to maintain persistent disease for a duration such as 6 weeks, 8 weeks, 12 weeks, 6 months, etc. In some embodiments, disease progression may be assessed by changes in overall bone marrow cellularity, the extent of reticulin or collagen fibrosis, and / or changes in JAK2V617F allele burden.

[0342] In some embodiments, a patient population treated with an isoform-specific, context-tolerant TGFβ1 inhibitor such as those described herein shows a statistically improved survival compared to a control population that does not receive treatment. For example, in the control group, the median survival of PMF patients is approximately 6 years (approximately 16 months for high-risk patients), and less than 20% of patients are expected to survive more than 10 years after diagnosis. Treatment with an isoform-specific, context-tolerant TGFβ1 inhibitor such as those described herein can extend survival time by at least 6 months, 12 months, 18 months, 24 months, 30 months, 36 months, or 48 months. In some embodiments, treatment is effective to achieve improved overall survival at 26 weeks, 52 weeks, 78 weeks, 104 weeks, 130 weeks, 144 weeks, or 156 weeks compared to patients receiving a placebo.

[0343] The clinical benefit of treatments such as those exemplified above can be seen in patients with or without new-onset anemia.

[0344] One of the advantageous features of isoform-specific, context-tolerant TGFβ1 inhibitors is that they maintain the improved safety profile that is enabled by isoform selectivity, compared with conventional TGFβ antagonists that lack selectivity.Therefore, it is expected that treatment with isoform-specific, context-tolerant inhibitors such as those described herein can reduce adverse events in a patient population, in terms of the frequency and / or severity of such events, compared with a comparable patient population treated with conventional TGFβ antagonists.Therefore, isoform-specific, context-tolerant TGFβ1 inhibitors can provide a larger therapeutic window in terms of the dose and / or duration of treatment.

[0345] Adverse events may be graded by any suitable art-recognized method, such as the Common Terminology Criteria for Adverse Events (CTCAE) version 4. Adverse events previously reported in human patients receiving TGFβ antagonists such as GC1008 include: leukocytosis (grade 3), fatigue (grade 3), hypoxia (grade 3), cardiac arrest (grade 5), leukopenia (grade 1), recurrent, transient, tender erythematous, nodular skin lesions, suppurative dermatitis, and herpes zoster.

[0346] Isoform-specific, context-tolerant TGFβ1 inhibitor therapy may result in less frequent and / or less severe adverse events (side effects) than JAK inhibitor therapy in myelofibrosis patients, e.g., with respect to anemia, thrombocytopenia, neutropenia, hypercholesterolemia, elevated alanine transaminase (ALT), elevated aspartate transaminase (AST), bruising, dizziness, and headaches, and thus provide a safer treatment option.

[0347] It is believed that the inhibitor of TGFβ1 signaling may be used as a combination therapy with one or more therapies for the treatment of myelofibrosis. In some embodiments, the inhibitor of TGFβ1 activation described herein is administered to a patient suffering from myelofibrosis who is receiving a JAK1 inhibitor, a JAK2 inhibitor, or a JAK1 / JAK2 inhibitor. In some embodiments, such a patient is responsive to JAK1 inhibitor, a JAK2 inhibitor, or a JAK1 / JAK2 inhibitor therapy, while in other embodiments, such a patient is poorly responsive or non-responsive to JAK1 inhibitor, a JAK2 inhibitor, or a JAK1 / JAK2 inhibitor therapy. In some embodiments, the use of the isoform-specific inhibitor of TGFβ1 described herein may make a poorly responsive or non-responsive person to a JAK1 inhibitor, a JAK2 inhibitor, or a JAK1 / JAK2 inhibitor therapy more responsive. In some embodiments, the use of the isoform-specific inhibitor of TGFβ1 described herein may allow for a reduced dose of JAK1 inhibitor, JAK2 inhibitor or JAK1 / JAK2 inhibitor, which still results in less or less drug-related toxicity or adverse events (such as those listed above) but comparable clinical efficacy in patients. In some embodiments, treatment with the TGFβ1 activation inhibitor described herein used with JAK1 inhibitor, JAK2 inhibitor or JAK1 / JAK2 inhibitor therapy may produce synergistic or additive therapeutic effects in patients. In some embodiments, treatment with the TGFβ1 activation inhibitor described herein may boost the benefit of JAK1 inhibitor, JAK2 inhibitor or JAK1 / JAK2 inhibitor or other treatment given to treat myelofibrosis. In some embodiments, the patient may additionally receive treatment to address anemia associated with myelofibrosis.

[0348] cancer: A variety of cancers involve TGFβ1 activity and may be treated by the antibodies and / or compositions of the present disclosure. The term "cancer" as used herein refers to any of a variety of malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissues and metastasize to new body sites, as well as disease states characterized by such malignant neoplastic growth. Cancer may be localized (e.g., solid tumors) or systemic. The term "localized" (e.g., "localized tumor") in the context of the present disclosure refers to an anatomically isolated or isolatable abnormality (e.g., solid malignant tumors) as opposed to a systemic disease. Certain cancers, such as certain leukemias (e.g., myelofibrosis) and multiple myeloma, may have both a local (e.g., bone marrow) and a systemic (e.g., circulating blood cells) component to the disease. In some embodiments, the cancer may be systemic (e.g., hematological malignancies). Cancers that can be treated according to the present disclosure include, but are not limited to, all types of lymphoma / leukemia, carcinoma and sarcoma, such as cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, neck, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (thorax), mouth, ovary, pancreas, penis, prostate, skin, small intestine, stomach, spinal medulla, tailbone, testicle, thyroid and uterus. In cancer, TGFβ (e.g., TGFβ1) can be either growth promoting or growth inhibiting. As an example, in pancreatic cancer, SMAD4 wild-type tumors can experience growth inhibition in response to TGFβ, but as the disease progresses, constitutively activated type II receptors are typically present. In addition, SMAD4-null pancreatic cancers exist. In some embodiments, the antibodies, antigen-binding portions thereof, and / or compositions of the disclosure are designed to selectively target components of the TGFβ signaling pathway that function uniquely in one or more forms of cancer.Leukemia, or cancer of the blood or bone marrow characterized by an abnormal proliferation of white blood cells (i.e., leukocytes), includes acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia or acute myeloid leukemia (AML) (AML with translocations between chromosomes 10 and 11 [t(10,11)], chromosomes 8 and 21 [t(8;21)], chromosomes 15 and 17 [t(15;17)], and an inversion within chromosome 16 [inv(16)]); AML with multilineage dysplasia (AML with myelodysplastic syndrome (MDS) or myeloproliferative disorder prior to transformation to AML); AML can be divided into four main classifications, including: AML and myelodysplastic syndromes (MDS) (which are therapy-related and the category includes patients who have previously had chemotherapy and / or radiation and subsequently develop AML or MDS); d) AML not otherwise classified (which includes subtypes of AML that do not fall into the categories above); and e) acute leukemia of ambiguous lineage (which arises when the leukemia cells cannot be classified as either myeloid or lymphoid cells or when both types of cells are present); and chronic myeloid leukemia (CML).

[0349] Isoform-specific, context-tolerance inhibitors of TGFβ1, such as those described herein, can be used to treat multiple myeloma. Multiple myeloma is a cancer of B lymphocytes (e.g., plasma cells, plasmablasts, memory B cells), which develops and expands in bone marrow, causing destructive bone lesions (i.e., osteolytic lesions). Typically, the disease shows high osteoclast bone resorption, suppressed osteoblast differentiation (e.g., differentiation block), and impaired bone formation, characterized in part by osteolytic lesions, osteopenia, osteoporosis, hypercalcemia, as well as plasmacytoma, thrombocytopenia, neutropenia, and neuropathy. TGFβ1-selective, context-tolerance inhibitor therapy as described herein can be effective in ameliorating one or more of such clinical signs or symptoms in patients. TGFβ1 inhibitors can be administered to patients undergoing additional treatment or treatment for treating multiple myeloma, including those listed elsewhere herein. In some embodiments, multiple myeloma may be treated with a TGFβ1 inhibitor (e.g., an isoform-specific context-tolerance inhibitor) in combination with a myostatin inhibitor or an IL-6 inhibitor. In some embodiments, the TGFβ1 inhibitor may be used with traditional multiple myeloma treatments, such as bortezomib, lenalidomide, carfilzomib, pomalidomide, thalidomide, doxorubicin, corticosteroids (e.g., dexamethasone and prednisone), chemotherapy (e.g., melphalan), radiation therapy, stem cell transplantation, plitidepsin, elotuzumab, ixazomib, masitinib, and / or panobinostat.

[0350] The types of carcinomas that can be treated by the methods of the present invention include, but are not limited to, papilloma / carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma / adenocarcinoma, melanoma, fibroadenoma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, hemangioma, osteoma, chondroma, glioma, lymphoma / leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, basal cell carcinoma, and sinonasal undifferentiated carcinoma.

[0351] Types of sarcomas include, but are not limited to, soft tissue sarcomas, such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant neurilemmoma, osteosarcoma, and chondrosarcoma.

[0352] Isoform-selective, context-tolerant / independent inhibitors of TGFβ1 activation, such as those described herein, may be suitable for treating malignancies involving cells of neural crest origin. Cancers of neural crest lineage (i.e., tumors of neural crest origin) include, but are not limited to: melanoma (cancer of melanocytes), neuroblastoma (cancer of sympathetic adrenal precursors), ganglioneuroma (cancer of peripheral nervous system ganglia), medullary thyroid carcinoma (cancer of thyroid C cells), pheochromocytoma (cancer of chromaffin cells of the adrenal medulla), and MPNST (cancer of Schwann cells). In some embodiments, the antibodies and methods of the present disclosure may be used to treat one or more types of cancer or cancer-related conditions, which may include, but are not limited to, colon cancer, renal cancer, breast cancer, malignant melanoma, and glioblastoma (Schlingensiepen et al., 2008; Ouhtit et al., 2013).

[0353] A growing line of evidence suggests a role for macrophages in tumor / cancer progression. The present invention embraces the notion that this is partially mediated by TGFβ1 activation in disease environments such as the TME. Bone marrow-derived monocytes (e.g., CD11b+) are recruited to tumor sites in response to tumor-derived cytokines / chemokines, where they undergo differentiation and polarization to acquire a cancer-promoting phenotype (e.g., M2-biased, TAM or TAM-like cells). As shown in the examples provided in this disclosure, monocytes isolated from human PBMCs can be induced to polarize into different subtypes of macrophages, such as M1 (profibrotic, anticancer) and M2 (cancer-promoting). The majority of TAMs in many tumors are M2-biased. Among M2-like macrophages, M2c and M2d subtypes, but not M1, have been found to express enhanced LRRC33 on the cell surface. Furthermore, macrophages may be further skewed or activated by M-CSF exposure, resulting in a significant increase in LRRC33 expression, consistent with TGFβ1 expression. Increased circulating M-CSF (i.e., serum M-CSF concentration) has also been observed in patients with myeloproliferative disorders (e.g., myelofibrosis). In general, tumors with high macrophages (TAMs) and / or MDSC infiltration are associated with poor prognosis. Similarly, elevated levels of M-CSF also indicate poor prognosis.

[0354] As described above, the context-tolerant / independent inhibitors of TGFβ1 activation can be used to treat melanoma. Types of melanoma that can be treated by such inhibitors include, but are not limited to: lentigo maligna; lentigo maligna melanoma; superficial spreading melanoma; acral lentigo melanoma; mucosal melanoma; nodular melanoma; polypoid melanoma and desmoplastic melanoma. In some embodiments, the melanoma is metastatic melanoma.

[0355] Recently, immune checkpoint inhibitors have been used to effectively treat patients with advanced melanoma. In particular, anti-programmed death (PD)-1 antibodies (e.g., nivolumab and pembrolizumab) have now become the standard of care for certain types of cancer (e.g., advanced melanoma), demonstrating significant activity and durable responses with a manageable toxicity profile. However, effective clinical application of PD-1 antagonist...

Claims

1. 1. A composition for use in a method for treating a disease associated with TGFβ1 dysregulation in a human subject, comprising: The composition comprises an isoform-specific inhibitor of TGFβ1 and a pharma- ceutically acceptable excipient; said inhibitor targets both ECM-associated TGFβ1 and immune cell-associated TGFβ1 in vivo, but not TGFβ2 or TGFβ3, optionally said inhibitor may inhibit the activation step of TGFβ1; and The disease has the following attributes: a) regulatory T cells (Tregs); b) effector T cell (Teff) proliferation or function; c) myeloid cell proliferation or differentiation; d) monocyte recruitment or differentiation; e) macrophage function; f) epithelial-mesenchymal transition (EMT) and / or endothelial-mesenchymal transition (EndMT); g) gene expression in one or more marker genes selected from the group consisting of PAI-1, ACTA2, CCL2, Col1a1, Col3a1, FN-1, CTGF, and TGFβ1; h) ECM components or functions; i) fibroblast differentiation; characterized by dysregulation or dysfunction of at least two of The method includes administering to a human subject diagnosed with the disease a therapeutically effective amount of the composition. composition.

2. A composition for use according to claim 1, comprising The ECM-associated TGFβ1 is TGFβ1 presented by LTBP1 and / or TGFβ1 presented by LTBP3; the immune cell-associated TGFβ1 is TGFβ1 presented by GARP and / or TGFβ1 presented by LRRC33. composition.

3. A composition for use according to claim 1 or 2, comprising The human subject suffers from a disease involving a proliferative and / or fibrotic component. composition.

4. A composition for use according to claim 3, comprising The disease is cancer, and optionally, the cancer may be a metastatic cancer; composition.

5. A composition for use according to claim 4, comprising the cancer comprises a solid tumor that is TGFβ1 positive, and optionally the solid tumor may be a desmoplastic tumor; composition.

6. A composition for use according to claim 4, comprising the cancer is a myeloproliferative disorder, optionally the myeloproliferative disorder may be essential thrombocythemia (ET), polycythemia vera (PV) or primary myelofibrosis (PMF); composition.

7. A composition for use according to claim 4, comprising The cancer is associated with an increase in the number of Tregs, TAMs, TANs, MDSCs, CAFs, or any combination thereof; composition.

8. A composition for use according to claim 4, comprising the cancer has responded poorly to a cancer treatment selected from the group consisting of radiation therapy, chemotherapy, and checkpoint inhibitor therapy; The checkpoint inhibitor therapy may optionally include a PD-1 antagonist, a PD-L1 antagonist, or a CTLA-4 antagonist; Further optionally, the poor response may be due to intrinsic or acquired resistance. composition.

9. A composition for use according to claim 8, comprising The subject is having a cancer resistant to immune checkpoint inhibitors selected from the group consisting of myelofibrosis, melanoma, renal cell carcinoma, bladder cancer, colon cancer, hematological malignancies, non-small cell carcinoma, non-small cell lung cancer (NSCLC), lymphoma (classical Hodgkin and non-Hodgkin), head and neck cancer, urothelial carcinoma, microsatellite instability high cancer, mismatch repair deficient cancer, gastric cancer, renal cancer, and hepatocellular carcinoma; composition.

10. A composition for use according to claim 8, comprising the clinical sample from the human subject exhibits expression of GARP and / or LRRC33; and / or TGFβ1 expression in the clinical sample is greater than TGFβ2 or TGFβ3 expression, optionally wherein said expression may be determined by RNA level and / or protein level; composition.

11. A composition for use according to any one of claims 4 to 10, comprising The therapeutically effective amount is intended to produce the following clinical effects: a) reduction in tumor growth; b) reduction in metastasis; c) reduced tumor invasion; d) reduced angiogenesis and angiogenesis / vascularity; e) reduced monocyte recruitment to the tumor site; f) reduced TAM infiltration of tumors; g) reduced macrophage activation; h) an increase in the ratio of M1 to M2 (TAM-like) macrophage populations at the tumor site; i) a reduction in the number of CAFs at the tumor site; j) decrease in immunosuppression; k) enhanced responsiveness to cancer treatment; l) prolonged survival; m) prolonged refractory period; n) an increase in the rate of complete remission or complete response; o) a reduction in the ratio of Treg / Teff cells at the tumor site; p) an increase in the number of Teff cells at the tumor site; q) a reduction in the number of Treg cells at the tumor site; r) a reduction in the number of MDSCs and / or TANs in said subject; and the clinical benefit(s) is achieved with an acceptable level of toxicity in the subject; composition.

12. A composition for use according to claims 4, 6, 8 to 10, comprising The therapeutically effective amount is intended to provide one or more of the following clinical benefits: a) reduction of fibrosis in bone marrow; b) enhancing hematopoiesis of differentiated blood cells in the bone marrow; c) a reduction in proliferation of abnormal stem cells in the bone marrow, optionally said abnormal stem cells being CD133 positive; d) a decrease in megakaryocytes in the bone marrow and / or spleen; e) reducing the occurrence and / or extent of extramedullary hematopoiesis in said subject, optionally wherein said extramedullary hematopoiesis is within the spleen; f) reduced need for bone marrow transplants; g) prolonged survival; h) normalization of the levels of one or more expression markers, wherein said expression markers may optionally be selected from the group consisting of BMP1, BMP6, BMP7, and BMP receptor 2, PLOD2, TGFβ1, bFGF, platelet derived growth factor (PDGF), Col1, metalloproteinases, FN1, CXCL12, VEGF, CXCR4, IL-2, IL-3, IL-9, CXCL1, IL-5, IL-12, TNFα, Bmp2, Bmp5, Acvrl1, Tgfblil, Igf1, Cdkn1a, Ltbp1, Gdf2, Lefty1 and Nodal; and i) Reduction of chronic inflammation in bone marrow In an amount effective to achieve at least two of the following: composition.

13. A composition for use according to claim 3, comprising The human subject has organ fibrosis, and optionally the organ fibrosis may be liver fibrosis, pulmonary fibrosis, kidney fibrosis, skin fibrosis and / or cardiac fibrosis; composition.

14. A composition for use according to claim 13, comprising The subject has organ fibrosis and is not a candidate for organ transplantation. composition.

15. A composition for use according to any one of claims 3, 13 and 14, comprising The subject has a fibrotic disorder associated with chronic inflammation. composition.

16. A composition for use according to claim 15, comprising the fibrotic disorder is a muscular dystrophy; Optionally, the muscular dystrophy may be DMD. composition.

17. A composition for use according to any one of claims 1 to 16, comprising The isoform selective inhibitors inhibit the following TGFβ1 activities: a) GARP-mediated TGFβ1 activity; b) LRRC33-mediated TGFβ1 activity; c) LTBP1-mediated TGFβ1 activity, and d) LTBP3-mediated TGFβ1 activity; Inhibit three or more of the following: Optionally, the inhibitor may inhibit all of the TGFβ1 activities (a)-(d); composition.

18. 18. The composition according to any one of claims 1 to 17, The inhibitor is a monoclonal antibody or a fragment thereof. composition.

19. A composition for use according to claim 18, comprising The monoclonal antibody or fragment thereof binds to a protein complex containing precursor / latent TGFβ1, Optionally, the protein complex may further comprise a presentation molecule selected from the group consisting of LTBP1, LTBP3, GARP and LRRC33; composition.

20. 20. The composition of claim 18 or 19, The antibody or fragment thereof specifically binds to an epitope in precursor / latent TGFβ1, Optionally, the epitope may be within the prodomain of the precursor / latent TGFβ1; composition.

21. 21. The composition of claims 18 to 20, The antibody or fragment thereof specifically binds to a combinatorial and / or conformational epitope, composition.

22. 22. The composition of claims 18 to 21, The monoclonal antibody inhibits the release of mature TGFβ1 growth factor from a latent protein complex containing precursor / latent TGFβ1. composition.

23. A composition for use according to claims 18 to 22, comprising The antibody or fragment thereof is a fully human or humanized antibody. composition.

24. A composition for use according to claims 18 to 23, comprising The antibody is a human IgG ４ and optionally said human IgG ４ The antibody may comprise backbone substitutions. composition.

25. A composition for use according to claims 18 to 24, comprising The antibody has the following CDR sequences: CDR-H1: NYAMS (SEQ ID NO:85); CDR-H2: SISGSGGATYYADSVKG (SEQ ID NO:86); CDR-H3: ARVSSGHWDFDY (SEQ ID NO:87); CDR-L1: RASQSISSYLN (SEQ ID NO:88); CDR-L2: SSLQS (SEQ ID NO:89); and CDR-L3: QQSYSAPFT (SEQ ID NO: 90) having Optionally, it may have up to three substitutions; composition.

26. an antibody comprising a heavy chain variable region polypeptide that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:95, and a light chain variable region polypeptide that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:97; Pharmaceutical compositions.

27. 1. A pharmaceutical composition comprising an antibody having the following CDR sequences: CDR-H1: NYAMS (SEQ ID NO:85); CDR-H2: SISGSGGATYYADSVKG (SEQ ID NO:86); CDR-H3: ARVSSGHWDFDY (SEQ ID NO:87); CDR-L1: RASQSISSYLN (SEQ ID NO:88); CDR-L2: SSLQS (SEQ ID NO:89); and CDR-L3: QQSYSAPFT (SEQ ID NO: 90) Optionally, it may have up to three substitutions. Pharmaceutical compositions.

28. 28. The pharmaceutical composition of claim 27, The antibody has CDR sequences without substitutions. Pharmaceutical compositions.

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