TGFβ1-binding immunoglobulins and uses thereof

Abstract

Novel methods and compositions for modulating TGFβ signaling are provided that can be used to effectively and safely treat diseases and disorders in which TGFβ is involved. [Solution] The present invention provides methods and compositions for treating diseases associated with TGFβ1 signaling by administering to a subject an effective amount of a TGFβ1 isoform-specific or highly selective TGFβ1 inhibitor. In some embodiments, such a TGFβ1 isoform-selective or TGFβ1 isoform-specific inhibitor can be a small molecule drug or a biologic (e.g., an antibody). The use of any such inhibitor to reduce toxicity (e.g., adverse or side effects) associated with TGFβ inhibition in a subject is encompassed by the present invention.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 307,353, filed March 11, 2016, U.S. Provisional Application No. 62 / 443,615, filed January 6, 2017, and U.S. Provisional Application No. 62 / 452,866, filed January 31, 2017, the entire contents of each of which are expressly incorporated herein by reference in their entirety. [Background technology]

[0002] The transforming growth factor β (TGFβ) superfamily of growth factors participates in several signaling cascades that regulate diverse biological processes, including, but not limited to, cell proliferation inhibition, tissue homeostasis, extracellular matrix (ECM) remodeling, endothelial-mesenchymal transition (EMT), cell migration and invasion, and immune regulation / suppression, as well as mesenchymal-epithelial transition (MET). In the context of ECM remodeling, TGFβ signaling can increase fibroblast populations and ECM deposition (e.g., collagen). In the immune system, TGFβ ligands regulate regulatory T cell function and the 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, epithelial cells frequently lose their negative growth response to TGFβ. In this context, TGFβ can stimulate angiogenesis, alter the stromal environment, and induce local and systemic immunosuppression, potentially promoting tumorigenesis. For these and other reasons, TGFβ has become a therapeutic target for several clinical indications. Despite considerable efforts to date by several groups, clinical development of TGFβ therapeutics has been challenging.

[0003] Observations from preclinical studies, including in rats and dogs, have revealed certain toxicities associated with the inhibition of TGFβ in vivo. Furthermore, although several TGFβ inhibitors have been developed to date, most clinical programs targeting TGFβ have been discontinued due to side effects.

[0004] For example, Anderton et al. (Toxicology Pathology, Vol. 39:916-24, 2011) reported that a small molecule inhibitor of the TGFβ type I (ALK5) receptor induced cardiac valve lesions characterized by hemorrhage, inflammation, degeneration, and proliferation of valvular interstitial cells in a preclinical animal model. This toxicity was observed in all cardiac valves at all doses tested. Frazier et al. (Toxicology Pathology, Vol. 35:284-295, 2007) reported that administration of GW788388, a small molecule inhibitor of the TGFβ type I (ALK5) receptor, induced epiphyseal dysplasia in rats.

[0005] Stauber et al. (J. Clin. Practice, Vol. 4:3, 2014) reported that chronic administration (greater than 3 months) of LY2157299, an inhibitor of TGF-β receptor type I kinase that is being investigated for the treatment of certain cancers, caused multiple organ toxicity in rats and dogs, involving the cardiovascular, gastrointestinal, immune, bone / cartilage, reproductive, and renal systems.

[0006] Fresolimumab (GC1008), a "pan" TGFβ antibody capable of neutralizing all human isoforms of TGFβ, has been reported to induce epithelial hyperplasia of the gingiva, bladder, and nasal turbinate epithelium after multiple administration in cynomolgus monkeys (Lonning et al., Current Pharmaceutical Biotechnology, 12:2176-89, 2011). Similarly, various skin rashes / lesions, gingival bleeding, and fatigue have been reported in clinical trials after administration of multiple doses of the drug. The most notable adverse reactions to fresolimumab include the induction of cutaneous keratoacanthoma and / or squamous cell carcinoma in human cancer patients (see, e.g., Lacouture et al., 2015, Cancer Immunol Immunother, 64:437-46; Stevenson et al., 2013, OncoImmunology, 2:8, e26218; and Lonning et al., 2011). Additional evidence from clinical trials suggests that this antibody may accelerate tumor progression in some cases (Stevenson et al., 2013, OncoImmunology, 2:8, e26218).

[0007] Thus, there is a need for new methods and compositions for modulating TGFβ signaling that can be used to effectively and safely treat diseases and disorders in which TGFβ is involved, including, for example, cancer, fibrosis, and inflammation. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Lacouture et al., Cancer Immunol Immunother (2015) 64:437-46 [Non-patent document 2] Stevenson et al., OncoImmunology (2013) 2:8, e26218 Summary of the Invention [Means for solving the problem]

[0009] The present disclosure relates to selective modulation of a subset of TGFβ actions. The present invention is based, at least in part, on the concept that the lack of isoform specificity of currently known TGFβ antagonists may underlie the toxicity associated with TGFβ inhibition. Indeed, the inventors of the present disclosure have found that most TGFβ inhibitors described to date antagonize multiple or all TGFβ isoforms. Furthermore, a general trend described in the art is that inhibitors that antagonize multiple isoforms of TGFβ (e.g., anti-TGFβ antibodies) are advantageous because neutralization of multiple isoforms of TGFβ appears to be necessary or advantageous for achieving "maximal therapeutic efficacy" (see, e.g., Bedinger et al. (2016) MABS, Vol. 8(2): pp. 389-404).

[0010] Contrary to this general teaching, the present inventors instead sought to develop agents that would allow for isoform-specific inhibition of TGFβ1, as opposed to inhibition that also affects TGFβ2 and / or TGFβ3, with the goal of eliminating or significantly reducing the toxicity (e.g., adverse effects, side effects) observed in vivo with known TGFβ antagonists. This novel approach is based, at least in part, on the concept that the clinical usefulness of a TGFβ inhibitor may be based not only on its efficacy but also on its safety. It was determined that the ability to fine-tune the target with an unprecedented degree of specificity would allow for both efficacy and safety / tolerability in a clinical setting.

[0011] Therefore, the present invention encompasses the recognition that pharmaceutical agents that inhibit TGFβ signal transduction in an isoform-specific manner can provide an improved safety profile compared to agents that affect multiple TGFβ isoforms.In contrast, several known TGFβ antagonists in the art produce unacceptable levels of toxicity at doses that have been shown to be effective in vivo.In such cases, lower dosages can be used to avoid toxicity, but the reduced doses may no longer produce sufficient in vivo efficacy.Without wishing to be bound by any particular theory, it is expected that such toxicity is at least in part due to the lack of isoform specificity / selectivity of the agent.

[0012] Thus, in one aspect, the present invention provides a method for reducing toxicity (e.g., adverse effects, undesirable side effects) associated with TGFβ inhibition in a subject. According to the present invention, TGFβ1-specific inhibitors, such as those described herein, have a superior safety-efficacy profile compared to agents that elicit activity against a broader range of targets (e.g., more than one isoform of TGFβ). Thus, such TGFβ1-specific inhibitors can be administered to a subject in need thereof at therapeutically effective doses without causing adverse effects. Such an approach therefore broadens the range of dosages that can achieve both efficacy and safety / tolerability in patients. Thus, the present invention provides a method for treating a disease associated with TGFβ1 signaling by administering to a subject an effective amount of a TGFβ1 isoform-specific or highly selective TGFβ inhibitor. In some embodiments, such a TGFβ1 isoform-selective or TGFβ1 isoform-specific inhibitor can be a small molecule drug or a biologic (e.g., an antibody). The use of any such inhibitor to reduce toxicity (e.g., adverse or side effects) associated with TGFβ inhibition in a subject is encompassed by the present invention. According to the present invention, an effective amount is within a dosage range that allows for both i) efficacy (e.g., therapeutically beneficial effects); and ii) safety (e.g., adverse or side effects within an acceptable level). In some embodiments, adverse effects may include cardiovascular toxicity, gastrointestinal toxicity, immunotoxicity, bone / cartilage toxicity, reproductive toxicity, and nephrotoxicity. In some embodiments, cardiovascular toxicity includes, but is not limited to, cardiac valve lesions, such as bleeding, inflammation, degeneration, and proliferation of valvular interstitial cells. In some embodiments, adverse effects may include bleeding. In some embodiments, adverse effects may include skin lesions or tumors. In some embodiments, adverse effects may include tumor progression.

[0013] Thus, in some embodiments, the present invention provides isoform-specific TGFβ1 inhibitor antibodies or antigen-binding fragments thereof that selectively inhibit the in vivo TGFβ1 activation step but not the TGFβ2 and / or TGFβ3 activation steps. Such antibodies or fragments thereof can be administered to subjects who would benefit from TGFβ1 inhibition in an amount effective to achieve clinical efficacy without causing unacceptable or intolerable levels of adverse effects. Thus, the present invention teaches TGFβ1 isoform-specific inhibitors that are specifically selected to meet both efficacy and safety criteria for treating diseases or conditions associated with TGFβ signaling in human patients.

[0014] In a related aspect, the present invention provides methods for producing isoform-specific TGFβ modulators with improved safety profiles (e.g., reduced in vivo toxicity). Such methods require that candidate agents be tested and selected for isoform specificity. In some embodiments, candidate agents are selected for specific activity on TGFβ1 signaling but not on TGFβ2 and / or TGFβ3 signaling. In some embodiments, such agents are TGFβ1 isoform-specific inhibitors. In some embodiments, such agents are antibodies or antigen-binding fragments thereof that specifically bind to and block activation 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 factor that is not associated with the pro / latent complex.

[0015] In another aspect, the present invention provides compositions and related methods for achieving further fine-tuning of TGFβ signaling by modulating TGFβ activation in a context-dependent manner.

[0016] TGFβ is involved in the conferring of several cellular / tissue effects, each of which is mediated, in part, by interaction with so-called "presentation molecules." Because the expression of various presentation molecules is cell-type or tissue-specific, TGFβ is intended to confer cellular effects depending on its interaction with a particular presentation molecule (i.e., "context"). Accordingly, among other things, the present disclosure provides monoclonal antibodies that selectively bind to TGFβ present in a particular context (i.e., a complex comprising TGFβ and a presentation molecule). In some embodiments, such monoclonal antibodies specifically bind to at least one, at least two, or at least three of the following complexes: i) TGFβ1-GARP; ii) TGFβ1-LRRC33; iii) TGFβ1-LTBP1; and iv) TGFβ1-LTBP3. In some embodiments, such monoclonal antibodies specifically bind to one of the following complexes: i) TGFβ1-GARP; ii) TGFβ1-LRRC33; iii) TGFβ1-LTBP1; and iv) TGFβ1-LTBP3. In some embodiments, such monoclonal antibodies specifically bind to two of the following complexes: i) TGFβ1-GARP; ii) TGFβ1-LRRC33; iii) TGFβ1-LTBP1; and iv) TGFβ1-LTBP3. In some embodiments, such monoclonal antibodies specifically bind to three of the following complexes: i) TGFβ1-GARP; ii) TGFβ1-LRRC33; iii) TGFβ1-LTBP1; and iv) TGFβ1-LTBP3. In some embodiments, such monoclonal antibodies specifically bind to 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., not complexed with a presentation molecule).

[0017] The present disclosure includes monoclonal antibodies that bind to the small latent complex (eg, "C4S") of TGFβ1.

[0018] The present disclosure further provides monoclonal antibodies that selectively target and modulate TGFβ in certain contexts, hi some embodiments, such monoclonal antibodies inhibit or activate TGFβ in certain contexts.

[0019] Thus, the present invention provides compositions and methods for modulating (activating or inhibiting) a subset of TGFβ activity. Thus, the present invention includes monoclonal antibodies that can selectively modulate a subset of TGFβ-mediated signaling pathways without affecting other TGFβ-mediated signaling pathways. In some embodiments, the subset of TGFβ-mediated signaling pathways includes at least one, at least two, or at least three of: i) GARP-mediated TGFβ action, ii) LRRC33-mediated TGFβ action, iii) LTBP1-mediated TGFβ action, and iv) LTBP3-mediated TGFβ action. In some embodiments, such monoclonal antibodies specifically modulate one of the following TGFβ-mediated signaling pathways without modulating the other three: i) GARP-mediated TGFβ action, ii) LRRC33-mediated TGFβ action, iii) LTBP1-mediated TGFβ action, and iv) LTBP3-mediated TGFβ action. In some embodiments, such monoclonal antibodies specifically modulate two of the following TGFβ-mediated signaling pathways, without modulating the other two: i) GARP-mediated TGFβ action, ii) LRRC33-mediated TGFβ action, iii) LTBP1-mediated TGFβ action, and iv) LTBP3-mediated TGFβ action. In some embodiments, such monoclonal antibodies specifically modulate three of the following TGFβ-mediated signaling pathways, without modulating the other one: i) GARP-mediated TGFβ action, ii) LRRC33-mediated TGFβ action, iii) LTBP1-mediated TGFβ action, and iv) LTBP3-mediated TGFβ action. In some embodiments, such monoclonal antibodies specifically modulate all of the following TGFβ-mediated signaling pathways: i) GARP-mediated TGFβ action, ii) LRRC33-mediated TGFβ action, iii) LTBP1-mediated TGFβ action, and iv) LTBP3-mediated TGFβ action.In some embodiments, such monoclonal antibodies specifically regulate GARP-mediated TGFβ action. In some embodiments, such monoclonal antibodies specifically regulate LRRC33-mediated TGFβ action. In some embodiments, such monoclonal antibodies specifically regulate LTBP1-mediated TGFβ action. In some embodiments, such monoclonal antibodies specifically regulate LTBP3-mediated TGFβ action. Thus, the present invention provides methods for context-dependently selectively targeting TGFβ activity.

[0020] Aspects of the present invention include pharmaceutical compositions and methods for treating diseases or disorders in human subjects. In some embodiments, such diseases or disorders include conditions associated with immune dysregulation / dysregulation, conditions associated with T cell dysregulation / dysregulation, conditions associated with fibrotic features (fibrosis), and / or tumor-related conditions.

[0021] Because the antibodies or fragments thereof specifically targeting the TGFβ pro / latent complex described herein regulate the activation step (i.e., the release of free mature TGFβ growth factor from the inactive latent precomplex) as opposed to targeting the already released free mature growth factor, the mode of action of these regulators depends on the source of TGFβ growth factor in the tissue. Therefore, identifying the source of TGFβ involved in a disease state is intended to aid in the selection of agents that effectively regulate TGFβ in the appropriate context. For example, to treat a disease phenotype involving GARP-mediated TGFβ1 action, it is desirable to select an antibody or fragment thereof that specifically targets the GARP-proTGFβ1 complex. To treat a disease phenotype involving LRRC33-mediated TGFβ1 action, it is desirable to select an antibody or fragment thereof that specifically targets the LRRC33-proTGFβ1 complex. To treat a disease phenotype involving LTBP1-mediated TGFβ1 action, it is desirable to select an antibody or fragment thereof that specifically targets the LTBP1-proTGFβ1 complex. For treating disease phenotypes involving LTBP2-mediated TGFβ1 action, it is desirable to select an antibody or fragment thereof that specifically targets the LTBP2-proTGFβ1 complex. For treating disease phenotypes involving LTBP3-mediated TGFβ1 action, it is desirable to select an antibody or fragment thereof that specifically targets the LTBP3-proTGFβ1 complex. For treating disease phenotypes involving LTBP4-mediated TGFβ1 action, it is desirable to select an antibody or fragment thereof that specifically targets the LTBP4-proTGFβ1 complex. For treating disease phenotypes involving TGFβ1 action mediated by multiple (e.g., two or more) contexts, it is desirable to select an antibody or fragment thereof that can target the corresponding multiple TGFβ1-presenting contexts.

[0022] Certain diseases are associated with multiple biological roles of TGFβ signaling that are not limited to a single TGFβ functional context. In such situations, it may be beneficial to modulate TGFβ action across multiple contexts. Thus, in some embodiments, the present invention provides methods for targeting and modulating TGFβ1 isoform-specifically, rather than context-specifically. Such agents may be referred to as "isoform-specific, context-permissive" TGFβ1 modulators. In some embodiments, context-permissive TGFβ1 modulators target multiple contexts (e.g., multiple types of pro / latent TGFβ1 complexes). In some embodiments, context-permissive TGFβ1 modulators target all types of pro / latent TGFβ1 complexes (e.g., those associated with GARP, those associated with LRRC33, those associated with LTBP, etc.) to encompass all contexts.

[0023] While a context-permissive TGFβ1 modulator can target more than one type of pro / latent TGFβ1 complex (i.e., involving different presentation molecules), in some embodiments, such a modulator may prefer one or more contexts over others. Thus, in some embodiments, a context-permissive antibody that inhibits TGFβ1 activation preferentially inhibits TGFβ1 activation mediated by one presentation molecule over another, even if such an antibody can bind to both types of pro / latent complexes. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits the activation of TGFβ1 associated with LTBP, TGFβ1 associated with GARP, and TGFβ1 associated with LRRC33, but has preferential inhibitory activity against TGFβ1 associated with LTBP. In some embodiments, such antibodies are monoclonal antibodies that bind to and inhibit the activation of TGFβ1 associated with LTBP1, TGFβ1 associated with LTBP3, TGFβ1 associated with GARP, and TGFβ1 associated with LRRC33, but have preferential inhibitory activity against TGFβ1 associated with LTBP1 and LTBP-3. In some embodiments, such antibodies are monoclonal antibodies that bind to and inhibit the activation of TGFβ1 associated with LTBP1, TGFβ1 associated with LTBP3, TGFβ1 associated with GARP, and TGFβ1 associated with LRRC33, but have preferential inhibitory activity against TGFβ1 associated with GARP and TGFβ1 associated with LRRC33. In some embodiments, such antibodies are monoclonal antibodies that bind to and inhibit the activation of TGFβ1 associated with GARP and TGFβ1 associated with LRRC33, but have preferential inhibitory activity against TGFβ1 associated with GARP. In some embodiments, such antibodies are monoclonal antibodies that bind to and inhibit the activation of TGFβ1 associated with GARP and TGFβ1 associated with LRRC33, but have preferential inhibitory activity for TGFβ1 associated with LRRC33.

[0024] Thus, according to the present invention, varying degrees of selectivity can be generated to target subsets of TGFβ effects. Isoform-specific modulators of TGFβ (targeting a single isoform of TGFβ) provide greater selectivity than pan-TGFβ modulators (targeting multiple or all isoforms of TGFβ). Isoform-specific, context-permissive modulators of TGFβ (targeting multiple contexts of a single isoform of TGFβ) provide greater selectivity than isoform-specific modulators. Isoform-specific, context-permissive modulators of TGFβ (targeting a single context of a single isoform of TGFβ) provide much greater selectivity than isoform-specific, context-permissive modulators.

[0025] Thus, in some embodiments, the present invention includes methods for treating diseases associated with TGFβ signaling, comprising first identifying or confirming the source and / or context of TGFβ associated with the disease, and then selecting an agent that specifically targets a particular subpool of TGFβ within the tissue. It is thus contemplated that such an approach may allow for preferential modulation of disease-associated functions of TGFβ while preserving normal TGFβ function. In some embodiments, to identify the source / context of disease-associated TGFβ, the expression of TGFβ-presenting molecule(s) present in affected tissue can be assessed. For example, both LTBP-associated TGFβ1 latent complexes and GARP-associated TGFβ1 complexes may be present in diseased tissue, with only the latter of these two potentially manifesting as a disease phenotype. In this scenario, it is desirable to inhibit GARP-mediated TGFβ1 signaling while leaving LTBP-mediated TGFβ1 signaling intact. Determining the source / status of disease-associated TGFβ1 can be performed using antibodies that specifically bind to TGFβ1 latent complexes containing specific presentation molecules (e.g., GARP, LRRC33, LTBP, etc.) With regard to treating conditions associated with T cell regulation / dysregulation, some embodiments of the invention involve administering a composition comprising an effective amount of a monoclonal antibody that modulates GARP-mediated TGFβ action in a subject.

[0026] With respect to the treatment of conditions associated with immune regulation / dysregulation, some embodiments of the invention involve the administration of a composition comprising an effective amount of a monoclonal antibody that modulates GARP-mediated TGFβ action and / or LRRC33-mediated TGFβ action in a subject.

[0027] With respect to the treatment of conditions associated with fibrosis, some embodiments of the present invention involve the administration of a composition comprising an effective amount of a monoclonal antibody that modulates LTBP1-mediated TGFβ action and / or LTBP3-mediated TGFβ action in a subject.

[0028] With respect to the treatment of conditions associated with certain types of cancer, some embodiments of the present invention involve the administration of a composition comprising an effective amount of a monoclonal antibody that modulates GARP-mediated TGFβ action, LTBP1-mediated TGFβ action, and / or LTBP3-mediated TGFβ action in a subject.

[0029] Aspects of the present disclosure relate to immunoglobulins, such as antibodies or antigen-binding portions thereof, that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex. The antibodies or antigen-binding portions thereof described herein specifically bind to an epitope of TGFβ1 that is available for binding by the antibody or antigen-binding portion thereof when TGFβ1 is present in a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex.

[0030] In one aspect, provided herein is an isolated antibody, or antigen-binding portion thereof, that specifically binds to an epitope of TGFβ1, where 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 where the antibody does not bind to free, mature TGFβ1.

[0031] In some embodiments, the TGFβ1 is latent TGFβ1. In some embodiments, the TGFβ1 is pro-TGFβ1.

[0032] In some embodiments, the antibody or antigen-binding portion thereof does not bind to TGFβ2. In some embodiments, the antibody or antigen-binding portion thereof does not bind to TGFβ3. In some embodiments, the antibody or antigen-binding portion thereof does not interfere with the ability of TGFβ1 to bind to integrins.

[0033] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a 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 a 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 a 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] In some embodiments, the antibody or antigen-binding portion thereof inhibits TGFβ1 activation.

[0040] In some embodiments, the antibody or antigen-binding portion thereof inhibits release of mature TGFβ1 from a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, or an LRRC33-TGFβ1 complex.

[0041] In some embodiments, the antibody or antigen-binding portion thereof has a nucleotide sequence of at least about 10 -8M; at least about 10 -9 M; at least about 10 -10 M; at least about 10 -11 M; at least about 10 -12 M; and at least about 10 -13 M, a dissociation constant (K D )

[0042] In some embodiments, the antibody or antigen-binding portion thereof comprises an immunoglobulin heavy chain 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 an immunoglobulin heavy chain 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 an immunoglobulin heavy chain constant domain of a human IgG4 constant domain. In some embodiments, the antibody or antigen-binding portion thereof comprises an immunoglobulin heavy chain constant domain of a human IgG4 constant domain with a Ser to Pro backbone substitution that creates an IgG1-like hinge and allows interchain disulfide bond formation.

[0043] In some embodiments, the antibody or antigen-binding portion thereof further comprises an immunoglobulin light chain constant domain comprising a human Ig lambda constant domain or a human Ig kappa constant domain.

[0044] In some embodiments, the antibody is an IgG having four polypeptide chains: two heavy chains and two light chains.

[0045] 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.

[0046] In some embodiments, the antigen-binding portion is a Fab fragment, a F(ab')2 fragment, an scFab fragment, or an scFv fragment.

[0047] In one aspect, provided herein is an anti-TGFβ1 antibody, or antigen-binding portion thereof, that competes for binding with an antibody, or antigen-binding portion thereof, described herein.

[0048] In another aspect, provided herein is an anti-TGFβ1 antibody, or antigen-binding portion thereof, that binds to the same epitope as an antibody, or antigen-binding portion thereof, described herein.

[0049] In some embodiments, the antibody or antigen-binding portion thereof is conjugated to a drug or detectable moiety. In some embodiments, the antibody or antigen-binding portion thereof is conjugated to the drug or detectable moiety via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the detectable moiety is selected from the group consisting of a fluorescent agent, a luminescent agent, an enzymatic agent, and a radioactive agent.

[0050] In one aspect, provided herein is a pharmaceutical composition comprising an antibody, or antigen-binding portion thereof, described herein and a pharmaceutically acceptable carrier.

[0051] In another aspect, provided herein is a method for inhibiting TGFβ1 activation, the method comprising exposing a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, or an LRRC33-TGFβ1 complex to an antibody, antigen-binding portion thereof, or pharmaceutical composition described herein.

[0052] In some embodiments, the antibody or antigen-binding portion thereof inhibits release of mature TGFβ1 from a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, or an LRRC33-TGFβ1 complex.

[0053] In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo.

[0054] In some embodiments, the GARP-TGFβ1 complex or the LRRC33-TGFβ1 complex is present on the outer surface of the cell.

[0055] In some embodiments, the cell is a T cell, a fibroblast, a macrophage, a monocyte, a dendritic cell, an antigen-presenting cell, or a microglia.

[0056] In some embodiments, the LTBP1-TGFβ1 complex or the LTBP3-TGFβ1 complex is bound to an extracellular matrix. In some embodiments, the extracellular matrix comprises fibrillin. In some embodiments, the extracellular matrix comprises a protein comprising an RGD motif.

[0057] 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.

[0058] In some embodiments, the subject has or is at risk of having fibrosis. 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, renal fibrosis, or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis). In some embodiments, the subject has or is at risk of having cancer. In some embodiments, the subject has or is at risk of having dementia. In some embodiments, the subject has or is at risk of developing myelofibrosis.

[0059] In some embodiments, the subject further receives an additional therapy, in some embodiments, the additional therapy 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, or any combination thereof.

[0060] In some embodiments, the antibody or antigen-binding portion thereof reduces the suppressive activity of regulatory T cells.

[0061] 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 system toxicity, or nephrotoxicity.

[0062] In one aspect, provided herein is a method for treating cancer in a subject in need thereof, 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.

[0063] In another aspect, provided herein is a method of reducing tumor growth in a subject in need thereof, 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.

[0064] 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 a PD-1 antagonist, a PDL1 antagonist, a PD-L1 or PDL2 fusion protein, a CTLA4 antagonist, a GITR agonist, an anti-ICOS antibody, an anti-ICOSL antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-OX40 antibody, an anti-CD27 antibody, an anti-CD70 antibody, an anti-CD47 antibody, an anti-41BB antibody, an anti-PD-1 antibody, an oncolytic virus, and a PARP inhibitor. In some embodiments, the additional treatment is radiation, a chemotherapeutic agent, or a combination thereof. In some embodiments, the additional treatment is radiation. In some embodiments, the additional agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is taxol. In some embodiments, the additional agent is an anti-inflammatory agent. In some embodiments, the additional agent inhibits the process of monocyte / macrophage recruitment and / or tissue infiltration. In some embodiments, the additional agent is an inhibitor of hepatic stellate cell activation. In some embodiments, the additional agent is a chemokine receptor antagonist, e.g., a CCR2 antagonist and a CCR5 antagonist. In some embodiments, such a chemokine receptor antagonist is a bispecific antagonist, such as a CCR2 / CCR5 antagonist. In some embodiments, the additional agent administered as a combination therapy is or comprises a member of the TGFβ superfamily of growth factors or a regulator thereof. In some embodiments, such an agent is selected from modulators (e.g., inhibitors and activators) of GDF8 / myostatin and GDF11. In some embodiments, such an agent is an inhibitor of GDF8 / myostatin signaling. In some embodiments, such an agent is a monoclonal antibody that specifically binds to the pro / latent myostatin complex and blocks myostatin activation.In some embodiments, monoclonal antibodies that specifically bind to the pro / latent myostatin complex and block activation of myostatin do not bind to free mature myostatin.

[0065] In yet another aspect, provided herein is a method of treating renal injury in a subject in need thereof, comprising administering to the subject an effective amount of an antibody, antigen-binding portion thereof, or pharmaceutical composition described herein, thereby treating the renal injury in the subject.

[0066] In one aspect, provided herein is a nucleic acid encoding an antibody or antigen-binding portion thereof described herein. Also provided is a vector comprising the nucleic acid.

[0067] In another aspect, provided herein is a cell comprising a nucleic acid encoding an antibody, or antigen-binding portion thereof, described herein. Also provided is a cell comprising a vector comprising a nucleic acid encoding an antibody, or antigen-binding portion thereof, described herein.

[0068] In yet another aspect, provided herein is a kit comprising an antibody, antigen-binding portion thereof, or pharmaceutical composition described herein and instructions for its use.

[0069] In another aspect, provided herein are methods for treating muscle fiber injury, comprising administering to a subject with muscle fiber injury an agent that selectively inhibits TGFβ1 relative to TGFβ2 / 3, in an amount effective to: i) promote muscle fiber repair; ii) protect against contraction-induced damage; iii) reduce muscle inflammation; and / or iv) reduce muscle fibrosis. In some embodiments, the amount does not cause an unacceptable level of adverse effects in the subject.

[0070] In some embodiments, the muscle fiber injury is i) associated with muscular dystrophy; or ii) associated with acute muscle injury. In some embodiments, the agent blocks activation of TGFβ1 but not TGFβ2 or TGFβ3. In some embodiments, the agent is a monoclonal antibody. In some embodiments, the monoclonal antibody binds to a GARP-proTGFβ1 latent complex, a LRRC33-proTGFβ1 latent complex, a LTBP1-proTGFβ1 latent complex, a LTBP2-proTGFβ1 latent complex, a LTBP3-proTGFβ1 latent complex, and / or a LTBP4-proTGFβ1 latent complex. In some embodiments, the subject further receives a myostatin inhibitor.

[0071] In some embodiments, the method further comprises identifying a source or context of TGFβ1 that is associated with the disease.

[0072] In yet another aspect, provided herein is a method for producing a pharmaceutical composition that modulates TGFβ signaling, the method comprising the steps of providing one or more agents that modulate the signaling of at least one isoform of TGFβ; measuring the activity of the one or more agents against all isoforms of TGFβ; selecting an agent that is specific for a single isoform of TGFβ; and formulating into a pharmaceutical composition comprising an isoform-specific TGFβ modulator and a pharmaceutically acceptable excipient. Also provided is a pharmaceutical composition produced by this method.

[0073] In some embodiments, the isoform-specific TGFβ modulator is a TGFβ1-specific modulator. In some embodiments, the TGFβ1-specific modulator is an inhibitor of TGFβ1. In some embodiments, the isoform-specific TGFβ modulator is an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof specifically binds to the pro / latent complex of TGFβ1. In some embodiments, the antibody or fragment thereof does not bind to free mature TGFβ1 that is not present in the pro / latent complex. In some embodiments, the pro / latent complex comprises GARP, LRRC33, LTBP1, LTBP2, LTBP3, or LTBP4.

[0074] In another aspect, provided herein is a method for treating a disease associated with TGFβ signaling, comprising the step of administering to a subject in need thereof a pharmaceutical composition provided herein, in an amount effective to treat the disease, wherein the amount achieves statistically significant clinical efficacy and safety when administered to a patient population having the disease.

[0075] In yet another aspect, provided herein is a TGFβ inhibitor for use in reducing adverse effects in a subject, wherein the TGFβ inhibitor is an isoform-selective TGFβ inhibitor. In some embodiments, the TGFβ inhibitor is an antibody that specifically inhibits TGFβ1. In particular embodiments, for example, the following items are provided: (Item 1) 1. An isolated antibody or antigen-binding portion thereof that specifically binds to an epitope of TGFβ1, wherein the TGFβ1 is present in the following protein complex: GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex is available for binding by the antibody when present in two or more of The antibody does not bind to free mature TGFβ1. An antibody or antigen-binding portion thereof. (Item 2) 2. The antibody or antigen-binding portion thereof according to item 1, wherein the TGFβ1 is latent TGFβ1. (Item 3) 2. The antibody or antigen-binding portion thereof according to item 1, wherein the TGFβ1 is pro-TGFβ1. (Item 4) 4. The antibody or antigen-binding portion thereof of any one of items 1 to 3, wherein the antibody does not bind to TGFβ2. (Item 5) 5. The antibody or antigen-binding portion thereof of any one of items 1 to 4, which does not bind to TGFβ3. (Item 6) 6. The antibody or antigen-binding portion thereof of any one of items 1 to 5, which does not interfere with the ability of TGFβ1 to bind to integrins. (Item 7) 7. The antibody or antigen-binding portion thereof of any one of items 1 to 6, comprising a heavy chain variable region comprising a 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. (Item 8) 8. The antibody or antigen-binding portion thereof of any one of items 1 to 7, comprising a heavy chain variable region comprising a 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. (Item 9) 9. The antibody or antigen-binding portion thereof of any one of items 1 to 8, comprising a heavy chain variable region comprising a 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. (Item 10) 10. The antibody or antigen-binding portion thereof of any one of items 1 to 9, comprising 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. (Item 11) 11. The antibody or antigen-binding portion thereof of any one of items 1 to 10, comprising 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. (Item 12) 7. The antibody or antigen-binding portion thereof of any one of items 1 to 6, comprising 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. (Item 13) 13. The antibody or antigen-binding portion thereof of any one of items 1 to 6 and 12, comprising 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. (Item 14) 14. The antibody or antigen-binding portion thereof of any one of items 1 to 6, 12 and 13, comprising a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 8. (Item 15) 15. The antibody or antigen-binding portion thereof of any one of items 1 to 6 and 12 to 14, comprising 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. (Item 16) 16. The antibody or antigen-binding portion thereof of any one of items 1 to 6 and 12 to 15, comprising 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. (Item 17) 17. The antibody or antigen-binding portion thereof of any one of items 1 to 16, which inhibits TGFβ1 activation. (Item 18) 18. The antibody or antigen-binding portion thereof of any one of items 1 to 17, which inhibits the release of mature TGFβ1 from the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex. (Item 19) The dissociation constant (K D ) but at least about 10 -8 M; at least about 10 -9 M; at least about 10 -10 M; at least about 10 -11 M; at least about 10 -12 M; and at least about 10 -13 19. The antibody or antigen-binding portion thereof of any one of items 1 to 18, selected from the group consisting of: M (Item 20) 20. The antibody or antigen-binding portion thereof of any one of items 1 to 19, comprising an immunoglobulin heavy chain constant domain of a human IgG1 constant domain or a human IgG4 constant domain. (Item 21) 21. The antibody or antigen-binding portion thereof of any one of items 1 to 20, comprising an immunoglobulin heavy chain constant domain of a human IgG4 constant domain. (Item 22) 22. The antibody or antigen-binding portion thereof according to item 21, comprising an immunoglobulin heavy chain constant domain of a human IgG4 constant domain with a Ser to Pro backbone substitution that creates an IgG1-like hinge and allows interchain disulfide bond formation. (Item 23) 23. The antibody or antigen-binding portion thereof of any one of items 1 to 22, further comprising an immunoglobulin light chain constant domain comprising a human Ig lambda constant domain or a human Ig kappa constant domain. (Item 24) 24. The antibody of any one of items 1 to 23, which is an IgG having four polypeptide chains: two heavy chains and two light chains. (Item 25) 25. The antibody of any one of items 1 to 24, which is a humanized antibody, a diabody, or a chimeric antibody. (Item 26) 26. The antibody of any one of items 1 to 25, which is a humanized antibody. (Item 27) 27. The antibody of any one of items 1 to 26, which is a human antibody. (Item 28) 28. The antibody of any one of items 1 to 27, comprising a framework having a human germline amino acid sequence. (Item 29) 29. The antigen-binding portion of any one of items 1 to 28, which is a Fab fragment, a F(ab')2 fragment, an scFab fragment, or an scFv fragment. (Item 30) 30. An anti-TGFβ1 antibody or antigen-binding portion thereof that competes for binding with the antibody or antigen-binding portion thereof of any one of items 1 to 29. (Item 31) 30. An anti-TGFβ1 antibody or antigen-binding portion thereof that binds to the same epitope as the antibody or antigen-binding portion thereof of any one of items 1 to 29. (Item 32) 32. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of any one of items 1 to 31 and a pharmaceutically acceptable carrier. (Item 33) 32. A method for inhibiting TGFβ1 activation, comprising exposing a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, or an LRRC33-TGFβ1 complex to the antibody or antigen-binding portion thereof of any one of items 1 to 31 or the pharmaceutical composition of item 32. (Item 34) 34. The method of claim 33, wherein the antibody or antigen-binding portion thereof inhibits the release of mature TGFβ1 from the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex. (Item 35) 35. The method of item 33 or 34, which is carried out in vitro. (Item 36) 35. The method of item 33 or 34, which is carried out in vivo. (Item 37) 35. The method of any one of items 33 to 34, wherein the GARP-TGFβ1 complex or the LRRC33-TGFβ1 complex is present on the outer surface of a cell. (Item 38) 38. The method of claim 37, wherein the cell is a T cell, a fibroblast, a macrophage, a monocyte, a dendritic cell, an antigen-presenting cell, or a microglia. (Item 39) 37. The method of any one of items 33 to 36, wherein the LTBP1-TGFβ1 complex or the LTBP3-TGFβ1 complex is bound to an extracellular matrix. (Item 40) Item 39. The method of item 39, wherein the extracellular matrix comprises fibrillin. (Item 41) 41. The method of item 39 or 40, wherein the extracellular matrix comprises a protein containing an RGD motif. (Item 42) A method for reducing TGFβ1 activation in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding portion thereof described in any one of items 1 to 31 or the pharmaceutical composition described in item 32, thereby reducing TGFβ1 activation in the subject. (Item 43) 43. The method of claim 42, wherein the subject has or is at risk of having a condition selected from the group consisting of fibrosis, muscular dystrophy, cancer, dementia, and myelofibrosis. (Item 44) 44. The method of claim 43, wherein the subject has or is at risk of having liver fibrosis, renal fibrosis, or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis). (Item 45) 45. The method of item 44, wherein the subject further receives a treatment comprising 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, or any combination thereof. (Item 46) 46. ​​The method of any one of items 42 to 45, wherein the antibody or antigen-binding portion thereof reduces the suppressive activity of regulatory T cells. (Item 47) 47. The method of any one of items 33 to 46, wherein the antibody or antigen-binding portion thereof does not induce organ toxicity in the subject. (Item 48) 48. The method of claim 47, wherein the organ toxicity comprises cardiovascular toxicity, gastrointestinal toxicity, immunotoxicity, bone toxicity, cartilage toxicity, reproductive system toxicity, or nephrotoxicity. (Item 49) 32. A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen-binding portion thereof of any one of items 1 to 31 or the pharmaceutical composition of item 32, thereby treating the cancer in the subject. (Item 50) 50. The method of item 49, wherein the antibody or antigen-binding portion thereof is administered in combination with an additional agent or treatment. (Item 51) 50. The method of claim 49, wherein the additional agent is a checkpoint inhibitor. (Item 52) 50. The method of item 49, wherein the additional agent is selected from the group consisting of a PD-1 antagonist, a PDL1 antagonist, a PD-L1 or PDL2 fusion protein, a CTLA4 antagonist, a GITR agonist, an anti-ICOS antibody, an anti-ICOSL antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-OX40 antibody, an anti-CD27 antibody, an anti-CD70 antibody, an anti-CD47 antibody, an anti-41BB antibody, an anti-PD-1 antibody, an oncolytic virus, and a PARP inhibitor. (Item 53) 50. The method of claim 49, wherein the additional treatment is radiation, a chemotherapeutic agent, or a combination thereof. (Item 54) 32. A nucleic acid encoding the antibody or antigen-binding portion thereof of any one of items 1 to 31. (Item 55) A kit comprising the antibody or antigen-binding portion thereof according to any one of items 1 to 31 or the pharmaceutical composition according to item 32, and instructions for its use. (Item 56) A method for treating muscle fiber injury, comprising administering to a subject having muscle fiber injury an agent that selectively inhibits TGFβ1 relative to TGFβ2 / 3, i) promote muscle fiber repair; ii) protect against contraction-induced damage; iii) reducing muscle inflammation; and / or iv) reducing muscle fibrosis in an amount effective to (Item 57) 57. The method of claim 56, wherein the amount does not cause an unacceptable level of adverse effects in the subject. (Item 58) The muscle fiber injury is i) associated with muscular dystrophy; or ii) associated with acute muscle injury; Item 58. The method according to item 56 or 57. (Item 59) 59. The method of any one of items 56 to 58, wherein the agent blocks activation of TGFβ1 but does not block activation of TGFβ2 or TGFβ3. (Item 60) 60. The method of any one of items 56 to 59, wherein the agent is a monoclonal antibody. (Item 61) 61. The method of claim 60, wherein the monoclonal antibody binds to a GARP-proTGFβ1 latent complex, a LRRC33-proTGFβ1 latent complex, a LTBP1-proTGFβ1 latent complex, a LTBP2-proTGFβ1 latent complex, a LTBP3-proTGFβ1 latent complex, and / or a LTBP4-proTGFβ1 latent complex. (Item 62) 62. The method of any one of items 56 to 61, wherein the subject further receives a myostatin inhibitor. (Item 63) Identifying a source or condition of TGFβ1 associated with a disease 63. The method of any one of items 56 to 62, further comprising: (Item 64) 1. A method for producing a pharmaceutical composition that modulates TGFβ signaling, comprising: providing one or more agents that modulate signaling of at least one isoform of TGFβ; measuring the activity of said one or more agents against all isoforms of TGFβ; selecting an agent that is specific for a single isoform of TGFβ; formulating the isoform-specific TGFβ modulator into a pharmaceutical composition comprising the isoform-specific TGFβ modulator and a pharmaceutically acceptable excipient. A method comprising: (Item 65) 65. The method of claim 64, wherein the isoform-specific TGFβ modulator is a TGFβ1-specific modulator. (Item 66) 66. The method of item 65, wherein the TGFβ1-specific modulator is an inhibitor of TGFβ1. (Item 67) 65. The method of claim 64, wherein the isoform-specific TGFβ modulator is an antibody or a fragment thereof. (Item 68) 68. The method of claim 67, wherein the antibody or fragment thereof specifically binds to the pro / latent complex of TGFβ1. (Item 69) 69. The method of item 68, wherein the antibody or fragment thereof does not bind to free mature TGFβ1 that is not present in the pro / latent complex. (Item 70) 69. The method of item 68, wherein the pro / latent complex comprises GARP, LRRC33, LTBP1, LTBP2, LTBP3, or LTBP4. (Item 71) 65. A pharmaceutical composition produced by the method of item 64. (Item 72) 1. A method for treating a disease associated with TGFβ signaling, comprising: 72. The method of claim 71, wherein the pharmaceutical composition of claim 71 is administered to a subject in need thereof in an amount effective to treat the disease, wherein the amount achieves statistically significant clinical efficacy and safety when administered to a patient population having the disease. method. (Item 73) 1. A TGFβ inhibitor for use in reducing adverse effects in a subject, wherein the TGFβ inhibitor is an isoform-selective TGFβ inhibitor. (Item 74) 74. The use of item 73, wherein the TGFβ inhibitor is an antibody that specifically inhibits TGFβ1. [Brief explanation of the drawings]

[0076] [Figure 1] FIG. 1 is a schematic diagram showing TGFβ bound to latent complexes in the tissue microenvironment.

[0077] [Figure 2] Figures 2A and 2B are schematic diagrams showing niche regulation in the microenvironment. Figure 2A shows latent transforming growth factor beta-binding protein (LTBP) presenting TGFβ in fibrotic disease and wound healing niches. Figure 2B illustrates that glycoprotein-A repetitions predominant protein (GARP) regulates TGFβ activation in the inflammatory niche.

[0078] [Figure 3] Figure 3 illustrates the protein expression platform for generating GARP-TGFβ1 and LTBP-TGFβ1 complexes. A HEK293-based expression system uses NiNTA affinity purification and gel filtration to yield multimilligram quantities of purified protein. Schematics of wild-type proTGFβ1, LTPB1, sGARP, and proTGFβ1 C4S are shown.

[0079] [Figure 4A] Figures 4A and 4B show the purification of the sGARP-proTGFβ1 complex: Figure 4A is a chromatogram of the sample, and Figure 4B shows a blot of the product and a schematic illustrating the complex. [Figure 4B] Figures 4A and 4B show the purification of the sGARP-proTGFβ1 complex: Figure 4A is a chromatogram of the sample, and Figure 4B shows a blot of the product and a schematic illustrating the complex.

[0080] [Figure 5A] Figures 5A and 5B show the purification of the sGARP-TGFβ1 LAP complex: Figure 5A is a sample chromatogram, and Figure 5B shows a product blot and a schematic illustrating the complex. [Figure 5B] Figures 5A and 5B show the purification of the sGARP-TGFβ1 LAP complex: Figure 5A is a sample chromatogram, and Figure 5B shows a product blot and a schematic illustrating the complex.

[0081] [Figure 6A] Figures 6A and 6B show the purification of LTBP1 complexed with pro-TGFβ1. Figure 6A shows a sample chromatogram, and Figure 6B shows a product blot and a schematic diagram illustrating the complex. NR stands for non-reduced, and R stands for reduced. [Figure 6B] Figures 6A and 6B show the purification of LTBP1 complexed with pro-TGFβ1. Figure 6A shows a sample chromatogram, and Figure 6B shows a product blot and a schematic diagram illustrating the complex. NR stands for non-reduced, and R stands for reduced.

[0082] [Figure 7] FIG. 7 is a graph showing that Ab1 and Ab2 block activation of TGFβ1 activity.

[0083] [Figure 8] FIG. 8 shows the initial dose-response analysis of Ab1 in human cells.

[0084] [Figure 9] FIG. 9 shows a CAGA12 reporter cell assay illustrating TGFβ1 inhibition by Ab1 in human cells.

[0085] [Figure 10] FIG. 10 is a graph showing that inhibition of the GARP complex blocks the suppressive activity of regulatory T (Treg) cells in T cells isolated from the blood of healthy donors.

[0086] [Figure 11-1] Figures 11A-11C show the inhibition of integrin-mediated TGFβ1 release from fibroblasts. Figures 11A and 11B are graphs showing the inhibition of endogenous TGFβ1 in either normal human dermal fibroblasts (circles), normal human lung fibroblasts (squares), mouse C57BL / 6J lung fibroblasts (inverted triangles), or mouse DBA2 / J myofibroblasts (circles) using increasing concentrations of either Ab1 (Figure 11A) or Ab2 (Figure 11B). Figure 11C presents a schematic diagram of the coculture assay system. [Figure 11-2] Figures 11A-11C show the inhibition of integrin-mediated TGFβ1 release from fibroblasts. Figures 11A and 11B are graphs showing the inhibition of endogenous TGFβ1 in either normal human dermal fibroblasts (circles), normal human lung fibroblasts (squares), mouse C57BL / 6J lung fibroblasts (inverted triangles), or mouse DBA2 / J myofibroblasts (circles) using increasing concentrations of either Ab1 (Figure 11A) or Ab2 (Figure 11B). Figure 11C presents a schematic diagram of the coculture assay system.

[0087] [Figure 12] Figures 12A and 12B show the binding of either Ab1 (Figure 12A) or Ab2 (Figure 12B) to the LRRC33-proTGFβ1 complex.

[0088] [Figure 13A] Figures 13A and 13B are graphs showing inhibition of GARP-TGFβ1 complexes (Figure 13A) or LRRC33-TGFβ1 complexes (Figure 13B) in SW480 / β6 cell transfectants using increasing concentrations of either Ab1 ("Ab1"), Ab2 ("Ab2"), an isotype control IgG1 antibody ("Isotype Control"), or a vehicle control ("Vehicle"). [Figure 13B]Figures 13A and 13B are graphs showing inhibition of GARP-TGFβ1 complexes (Figure 13A) or LRRC33-TGFβ1 complexes (Figure 13B) in SW480 / β6 cell transfectants using increasing concentrations of either Ab1 ("Ab1"), Ab2 ("Ab2"), an isotype control IgG1 antibody ("Isotype Control"), or a vehicle control ("Vehicle").

[0089] [Figure 14] FIG. 14 is a bar graph showing hydroxyproline levels in kidney tissue from mice that underwent permanent right unilateral UUO surgery and received either PBS (control), 30 mg / kg mouse IgG1 control antibody, 3 mg / kg Ab2, or 30 mg / kg Ab2 intraperitoneally (ip) prior to surgical intervention (bars 2-5); or mice that received PBS and underwent laparotomy (sham control; bar 1).

[0090] [Figure 15-1] Figures 15A-15H show the plasminogen activity in kidney tissue from mice that underwent permanent right unilateral UUO surgery and received either PBS (control), 30 mg / kg mouse IgG1 control antibody, 3 mg / kg Ab2, or 30 mg / kg Ab2 intraperitoneally (ip) the day before the surgical intervention, and then 1 and 3 days after surgery (second to fifth bars in each graph); or mice that received PBS and underwent laparotomy (sham control; first bar in each graph). Figure 15A is a bar graph showing the relative mRNA levels of either activator inhibitor-1 (PAI-1; Figure 15A), connective tissue growth factor (CTGF; Figure 15B), transforming growth factor beta 1 (TGFβ1; Figure 15C), fibronectin-1 (Figure 15D), α-smooth muscle actin (α-SMA; Figure 15E), monocyte chemotactic protein 1 (MCP-1; Figure 15F), type I collagen alpha 1 (Col1a1; Figure 15G), or type III collagen alpha 1 chain (Col3a1; Figure 15H). Data are representative of multiple experiments. [Figure 15-2]Figures 15A-15H show the plasminogen activity in kidney tissue from mice that underwent permanent right unilateral UUO surgery and received either PBS (control), 30 mg / kg mouse IgG1 control antibody, 3 mg / kg Ab2, or 30 mg / kg Ab2 intraperitoneally (ip) the day before the surgical intervention, and then 1 and 3 days after surgery (second to fifth bars in each graph); or mice that received PBS and underwent laparotomy (sham control; first bar in each graph). Figure 15A is a bar graph showing the relative mRNA levels of either activator inhibitor-1 (PAI-1; Figure 15A), connective tissue growth factor (CTGF; Figure 15B), transforming growth factor beta 1 (TGFβ1; Figure 15C), fibronectin-1 (Figure 15D), α-smooth muscle actin (α-SMA; Figure 15E), monocyte chemotactic protein 1 (MCP-1; Figure 15F), type I collagen alpha 1 (Col1a1; Figure 15G), or type III collagen alpha 1 chain (Col3a1; Figure 15H). Data are representative of multiple experiments. [Figure 15-3] Figures 15A-15H show the plasminogen activity in kidney tissue from mice that underwent permanent right unilateral UUO surgery and received either PBS (control), 30 mg / kg mouse IgG1 control antibody, 3 mg / kg Ab2, or 30 mg / kg Ab2 intraperitoneally (ip) the day before the surgical intervention, and then 1 and 3 days after surgery (second to fifth bars in each graph); or mice that received PBS and underwent laparotomy (sham control; first bar in each graph). Figure 15A is a bar graph showing the relative mRNA levels of either activator inhibitor-1 (PAI-1; Figure 15A), connective tissue growth factor (CTGF; Figure 15B), transforming growth factor beta 1 (TGFβ1; Figure 15C), fibronectin-1 (Figure 15D), α-smooth muscle actin (α-SMA; Figure 15E), monocyte chemotactic protein 1 (MCP-1; Figure 15F), type I collagen alpha 1 (Col1a1; Figure 15G), or type III collagen alpha 1 chain (Col3a1; Figure 15H). Data are representative of multiple experiments.

[0091] [Figure 16]Figure 16 shows the composite cortical collagen volume fraction (CVF) of three serial sections of right kidneys collected from mice that underwent permanent right unilateral UUO surgery and were intraperitoneally (ip) administered PBS ("Veh"), 30 mg / kg mouse IgG1 control antibody ("IgG Ctrl"), 3 mg / kg Ab2 ("3 Ab2"), or 30 mg / kg Ab2 ("30 Ab2") prior to surgical intervention (second through fifth bars in each graph); or mice that received PBS and underwent laparotomy ("Sham"; first bar in each graph).

[0092] [Figure 17] Figure 17 shows the median tumor volume in C57 / BL / 6 mice, a syngeneic model of MC38 murine colon carcinoma, treated with a combination of a mouse IgG1 isotype control antibody and a rat IgG2a control antibody (group 1; control); a combination of Ab1 and a rat IgG2a control antibody (group 2); a combination of Ab2 and a rat IgG2a control antibody (group 3); a combination of a mouse IgG1 control antibody and an anti-PD-1 antibody (group 4); a combination of Ab1 and an anti-PD-1 antibody (group 5); or a combination of Ab2 and an anti-PD-1 antibody (group 6).

[0093] [Figure 18] Figures 18A-18C show the binding specificity of exemplary monoclonal antibodies. Figure 18A shows that Ab1 and Ab2 specifically bind to proTGFβ1, but not to proTGFβ2, proTGFβ3, or mature TGFβ1, as determined by ELISA. Figure 18B shows an example of purified LTBP-proTGFβ1 expressed and purified for use as an antigen to determine antibody binding specificity. Figure 18C shows an example of an antibody that specifically binds to the LTBP1-proTGFβ1 complex (as determined by ELISA).

[0094] [Figure 19]Figure 19 shows survival curves for rats treated with either vehicle control (PBS; "Control"); 200 mg / kg LY2109761; 300 mg / kg LY2109761; 100 mg / kg pan-TGFβ antibody ("pan-TGFβ Ab"); or 100 mg / kg Ab2.

[0095] [Figure 20] Figure 20 shows the body weights (mean / standard deviation) of rats treated with either vehicle control (PBS; "Control"); 200 mg / kg LY2109761; 300 mg / kg LY2109761; 100 mg / kg pan-TGFβ antibody ("pan-TGFβ Ab"); or 100 mg / kg Ab2.

[0096] [Figure 21] Figures 21A-21C show the body weights of individual rats treated with vehicle control (PBS; "Control"), 200 mg / kg LY2109761, or 300 mg / kg LY2109761 (Figure 21A); vehicle control (PBS; "Control"), or 100 mg / kg pan-TGFβ antibody ("pan-TGFβ Ab"); or vehicle control (PBS; "Control"), or 100 mg / kg Ab2.

[0097] [Figure 22] Figures 22A and 22B are graphs showing the inhibition of GARP-proTGFβ1 or LRRC33-proTGFβ1 complexes in SW480 / β6 cells transiently transfected with plasmids to express proTGFβ1 and the presentation molecule (i.e., GARP or LRRC33) using increasing concentrations of either Ab1 (Figure 22A) or Ab2 (Figure 22B). The IC50 (μg / mL) of Ab1 for the GARP-TGFβ1 complex was 0.445, and the IC50 (μg / mL) for the LRRC33-TGFβ1 complex was 1.325.

[0098] [Figure 23]Figure 23 shows microscopic images of hematoxylin-eosin-stained sections from heart valves from rats treated with either 200 mg / kg LY2109761 (top right panel); 100 mg / kg pan-TGFβ antibody ("pan-TGFβ Ab," bottom left panel); 100 mg / kg Ab2 (bottom right panel), or an untreated control (top left panel). Note: The bottom right panel (Ab2) is an oblique section with darker staining due to uneven thickness. DETAILED DESCRIPTION OF THE INVENTION

[0099] In mammals, the transforming growth factor-beta (TGFβ) superfamily consists of at least 33 gene products. These include bone morphogenetic proteins (BMPs), activins, growth differentiation factors (GDFs), and three isoforms of the TGFβ family: TGFβ1, TGFβ2, and TGFβ3. TGFβs are thought to play important roles in diverse processes, such as the inhibition of cell proliferation, extracellular matrix (ECM) remodeling, and immune homeostasis. The importance of TGFβ1 for T cell homeostasis is demonstrated by the finding that TGFβ1- / - mice survive only 3–4 weeks and die from multiple organ failure due to intense immune activation (Kulkarni, AB et al., Proc Natl Acad Sci USA, 1993, 90(2):770–4; Shull, MM et al., Nature, 1992, 359(6397):693–9). The roles of TGFβ2 and TGFβ3 are unclear. The three TGFβ isoforms have distinct temporal and spatial expression patterns while signaling 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, XH and R. Derynck, Annu Rev Cell Dev Biol 2005, 21:659-93; Massague, J., Annu Rev Biochem 1998, 67:753-91). Ligand-induced oligomerization of TGFβRI / II induces 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):2783-810).SMAD-independent TGFβ signaling pathways have also been described, for example, in cancers or aortic lesions in Marfan mice (Derynck, R. and Y. E. Zhang, Nature, 2003, 425(6958):577-84; Holm, T. M. et al., Science, 2011, 332(6027):358-61).

[0100] The biological importance of the TGF-β pathway in humans has been validated by genetic diseases. Kamurachi-Engelman disease results in bone dysplasia due to an autosomal dominant mutation in the TGFB1 gene, which leads to constitutive TGF-β1 signaling activation (Janssens, K. et al., J Med Genet, 2006, 43(1):1-11). Patients with Loeys / Dietz syndrome have autosomal dominant mutations in components of the TGF-β signaling pathway, which cause aortic aneurysms, sequestration, and cleft uvula (Van Laer, L., H. Dietz, and B. Loeys, Adv Exp Med Biol, 2014, 802:95-105). Because dysregulation of the TGF-β pathway has been implicated in numerous diseases, several drugs targeting the TGF-β pathway have been developed and tested in patients, but with limited success.

[0101] The inventors of the present disclosure have determined that although all three isoforms of TGFβ can signal through the same receptor and transduce downstream effectors in cells that express the receptor, each TGFβ isoform can produce distinct biological effects in vivo. Furthermore, the inventors have considered that, at least in some cases, the manner in which growth factor-receptor interactions are induced may further confer signal transduction specificity in vivo. With this recognition in mind, it is noted that TGFβ inhibitors described in the literature to date lack specificity, as briefly summarized below.

[0102] Fresolimumab, a humanized monoclonal antibody that binds to and inhibits all three isoforms of TGF-β, is being clinically tested in patients with focal segmental glomerulosclerosis, malignant melanoma, renal cell carcinoma, and systemic sclerosis (Rice, LM et al., J Clin Invest, 2015, 125(7):2795-807; Trachtman, H. et al., Kidney Int, 2011, 79(11):1236-43; Morris, JC et al., PLoS One, 2014, 9(3):e90353). Additional companies are developing monoclonal antibodies against TGF-β growth factors with varying degrees of selectivity for TGF-β isoforms. Residual activity against other TGF-β family members in addition to TGF-β1 may lead to in vivo toxicity for such agents. To the best of the inventors' knowledge, complete specificity to a single isoform has not been achieved by targeting mature growth factors due to the high degree of sequence identity between isoforms.

[0103] Other approaches to targeting the TGFβ pathway include ACE-1332, a soluble TGFβRII-Fc ligand trap from Acceleron (Yung, LM et al., Am J Respir Crit Care Med, 2016, 194(9):1140-1151), or small molecule inhibitors of ALK5 kinase, such as Lilly's galunisertib, which binds to TGFβ1 and TGFβ3 with equally high affinity (Yung, LM et al., Am J Respir Crit Care Med, 2016, 194(9):1140-1151). ALK5 inhibitors block the activity of all growth factors that signal through TGFR1. Substantial toxicity has been found in preclinical studies using ALK5 inhibitors (Anderton, MJ et al., Toxicol Pathol, 2011, 39(6):916-24; Stauber, A. et al., Clinical Toxicology, 2014, 4(3):1-10), and sophisticated clinical dosing schemes are needed to maintain efficacy while reducing adverse effects (Herbertz, S. et al., Drug Des Devel Ther, 2015, 9:4479-99). Indeed, questions regarding TGFβ signaling specificity and its potential impact on the toxicity observed with known TGFβ inhibitors have arisen for most, if not all, drug candidates attempting to block TGFβ. For example, the extent of toxicity resulting from inhibition of TGFβ1 versus TGFβ2 and / or TGFβ3 has not been addressed. Similarly, the mode of TGFβ activation has not been considered in the design or development of approaches to antagonize TGFβ signaling.

[0104] Recent structural insights into the activation mechanism of TGF-β1 have enabled us to take a novel, more specific approach to TGF-β inhibition (Shi, M. et al., Nature, 2011, Vol. 474 (No. 7351): 343-9). Unlike other cytokines, TGF-β superfamily members are not secreted as active growth factors but as dimeric proproteins consisting of an N-terminal prodomain and a C-terminal growth factor domain. Cleavage of pro-TGF-β1 by furin protease separates the homodimeric growth factor domain from the prodomain, also called the latent association peptide (LAP). However, the growth factor and LAP remain noncovalently associated, forming a latent complex that cannot bind to its receptor and induce signaling (Figure 1). During translation, latent TGF-β1, also called the small latent complex (SLC), is linked to a "presentation molecule" by disulfide bridges to form the large latent complex (LLC). These molecules allow pro-TGF-β1 to be presented in specific cell or tissue contexts. Two cysteines near the N-terminus of latent TGF-β1 link to appropriately positioned cysteines on the presentation molecule. The identity of the presentation molecule depends on the environment and the cell type producing latent TGF-β1. For example, fibroblasts secrete latent TGF-β1 tethered to latent TGF-β binding protein (LTBP), which then associates with proteins in the extracellular matrix (ECM) (i.e., fibronectin, fibrillin-1), linking latent TGF-β to the ECM (Robertson et al., Matrix Biol, 47:44-53 (2015) (Figure 2A)). On the surface of activated regulatory T cells, latent TGF-β1 is covalently bound to the transmembrane protein GARP (Figure 2B). Recently, a protein closely related to GARP, LRRC33, was identified as a presentation molecule for TGF-β1 on the surface of monocytes, macrophages, and microglia (Wang, R. et al., Mol Biol Cell, 2012, 23(6):1129-39 and TA Springer, Int. BMP Conference, 2016).

[0105] In mammals, there are four known LTBPs, LTBP1-4, each with multiple splice variants (Robertson, IB et al., Matrix Biol, 2015, vol. 47:44-53). LTBP2 is the only LTBP that does not associate with latent TGFβ (Saharinen, J. and J. Keski-Oja, Mol Biol Cell, 2000, vol. 11(8):2691-704). While the association of LTBP1 or LTBP3 with latent TGFβ1 has been well documented, the role of LTBP4 in TGFβ presentation remains unclear. The complex between LTBP4 and latent TGFβ1 appears to form much less efficiently, potentially due to the absence of several negatively charged residues in the TGFβ-binding domain of LTBP4 (Saharinen, J. and J. Keski-Oja, Mol Biol Cell, 2000, 11(8):2691-704; Chen, Y. et al., J Mol Biol, 2005, 345(1):175-86). Disruption of elastic fiber assembly occurs in both LTBP4S- / - mice and Urban-Rifkin-Davis syndrome patients with null mutations in LTBP4 (Urban, Z. et al., Am J Hum Genet, 2009, Vol. 85(5):593-605; Dabovic, B. et al., J Cell Physiol, 2015, Vol. 230(1):226-36). Furthermore, LTBP4S- / - mice have pulmonary septum formation and elastogenesis defects, whereas transgenic mice with LTBP4 that cannot form a complex with latent TGF-β1 have no obvious phenotype (Dabovic, B. et al., J Cell Physiol, 2015, Vol. 230(1):226-36). It is unclear whether LTBP4 is directly involved in latent TGFβ1 regulation by functioning as a presentation molecule; instead, LTBP4 may be required for the proper formation of elastic fibrils in the ECM, and its absence indirectly affects latent TGFβ1 activation through defects in the ECM.

[0106] Several studies have clarified the mechanism of TGF-β1 activation. Three integrins, αVβ6, αVβ8, and αVβ1, have been demonstrated to be key activators of latent TGF-β1 (Reed, NI et al., Sci Transl Med, 2015, 7(288):288ra79; Travis, MA and D. Sheppard, Annu Rev Immunol, 2014, 32(51-82); Munger, JS et al., Cell, 1999, 96(3):319-28). αV integrins bind with high affinity to the RGD sequence present in TGF-β1 and TGF-β1 LAP (Dong, X. et al., Nat Struct Mol Biol, 2014, 21(12):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):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):227-32). The key to integrin-dependent activation of latent TGFβ1 is its covalent tethering to a presentation molecule; disruption of the disulfide bond between GARP and TGFβ1 LAP by mutagenesis completely abolishes TGFβ1 activation by αVβ6, although it does not impair complex formation (Wang, R. et al., Mol Biol Cell, 2012, 23(6):1129-39). A recent structure of latent TGFβ1 elucidates how integrins enable active TGFβ1 to be released from the latent complex: the covalent link between latent TGFβ1 and its presentation molecule anchors it to the ECM via LTBP or to the cytoskeleton via GARP or LRRC33.Binding of integrins to the RGD sequence results in a force-dependent change in the conformation of LAP, which releases active TGF-β1 and allows it to bind to nearby receptors (Shi, M. et al., Nature, 2011, 474(7351):343-9). The importance of integrin-dependent TGF-β1 activation in disease has also been 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):288ra79), and antibody-based blockade of αVβ6 or lack of integrin β6 expression suppresses bleomycin-induced pulmonary fibrosis and radiation-induced fibrosis (Munger, JS et al., Cell, 1999, 96(3):319-28; Horan, GS et al., Am J Respir Crit Care Med, 2008, 177(1):56-65). In addition to integrins, other mechanisms of TGFβ1 activation have also been implicated, including activation by thrombospondin-1 and proteases such as matrix metalloproteinases (MMPs), cathepsin D, or kallikrein. However, the majority of these studies have been performed in vitro using purified proteins; evidence regarding the role of these molecules from in vivo studies is scarce. Knockout of thrombospondin-1 recapitulates some aspects of the TGFβ1- / - phenotype in some tissues but is not protective in bleomycin-induced pulmonary fibrosis, which is known to be TGFβ-dependent (Ezzie, ME et al., Am J Respir Cell Mol Biol, 2011, 44(4):556-61). Furthermore, knockout of candidate proteases did not result in the TGFβ1 phenotype (Worthington, JJ, JE Klementowicz, and MA Travis, Trends Biochem Sci, 2011, 36(1):47-54). This could be explained by redundancy or by the importance of these mechanisms in specific diseases rather than in development and homeostasis.

[0107] TGFβ has been implicated in several biological processes, including fibrosis, immunoregulation, and cancer progression. TGFβ1 was the first identified member of the TGFβ superfamily of proteins. TGFβ1 and its isoforms, TGFβ2 and TGFβ3, like other members of the TGFβ superfamily, are initially expressed as inactive precursor proprotein forms (referred to as pro-TGFβ). 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 "pro / latent TGFβ proteins." TGFβ1 can be presented in complexes with other molecules, including, for example, GARP (to form a GARP-TGFβ1 complex), LRRC33 (to form a LRRC33-TGFβ1 complex), LTBP1 (to form a LTBP1-TGFβ1 complex), and / or LTBP3 (to form a LTBP3-TGFβ1 complex). The TGFβ1 present in these complexes can be in its latent form (latent TGFβ1) or its precursor form (pro-TGFβ1).

[0108] The present invention relates to immunoglobulins, such as (1) antibodies or antigen-binding portions thereof that specifically bind to TGFβ proteins (e.g., pro / latent TGFβ1, pro / latent TGFβ2, and pro / latent TGFβ3) in complex with a GARP protein, (2) antibodies or antigen-binding portions thereof that specifically bind to TGFβ proteins in complex with an LTBP protein (e.g., LTBP1 or LTBP3), and / or (3) antibodies or antigen-binding portions thereof that specifically bind to TGFβ proteins in complex with an LRRC33 protein. In some embodiments, the antibodies or antigen-binding portions thereof disclosed herein bind to an epitope of TGFβ1 that is available for binding by the antibody or antigen-binding portion thereof when TGFβ1 is present in a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex. Without wishing to be bound by any particular theory, the ability of the antibodies and antigen-binding portions thereof disclosed herein to bind to TGFβ proteins (e.g., TGFβ1) that are present in a complex with either a GARP protein, an LTBP protein, and / or an LRRC33 protein allows for context-independent targeting of TGFβ proteins, making them particularly suitable for therapeutic applications.

[0109] definition To facilitate understanding of this disclosure, certain terms are first defined. These definitions should be read in light of the remainder of this disclosure and as would be understood by one of ordinary skill in the art. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

[0110] 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 binding to proteins in general. In some embodiments, an antibody or antigen-binding portion thereof binds to a target, e.g., TGFβ1, by binding to the target of the antibody. D is 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 In some embodiments, the terms "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, refer to binding to TGFβ1 and having a dissociation constant (K) determined by surface plasmon resonance. D ) is 1.0 × 10 -7 M or less. In one embodiment, the antibody or antigen-binding portion thereof can specifically bind to both a human ortholog and a non-human (e.g., murine) ortholog of TGFβ1.

[0111] In some embodiments, 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 is determined using an Octet assay. In some embodiments, an Octet assay is an assay that determines one or more kinetic parameters indicative of antibody-antigen binding. In some embodiments, 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 is determined using an Octet® system (ForteBio, Menlo Park, CA). For example, the binding affinity of an antibody can be determined using a forteBio Octet QK e 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 is determined using the protocol outlined in Table 6.

[0112] 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). In some embodiments, the proprotein or latent form of the TGFβ1 protein may be referred to as a "pro / latent TGFβ1 protein." In some embodiments, the GARP-TGFβ1 complex comprises GARP covalently linked to pro / latent TGFβ1 via one or more disulfide bonds. In other embodiments, the GARP-TGFβ1 complex comprises GARP non-covalently linked to pro / latent TGFβ1. In some embodiments, the GARP-TGFβ1 complex is a naturally occurring complex, e.g., an intracellular GARP-TGFβ1 complex. An exemplary GARP-TGFβ1 complex is shown in FIG. 3.

[0113] As used herein, the term "LTBP1-TGFβ1 complex" refers to a protein complex comprising the proprotein or latent form of transforming growth factor-β1 (TGFβ1) protein and latent TGF-beta binding protein 1 (LTBP1). In some embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 covalently linked to pro / latent TGFβ1 via one or more disulfide bonds. In other embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 non-covalently linked to pro / 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.

[0114] As used herein, the term "LTBP3-TGFβ1 complex" refers to a protein complex comprising the proprotein or latent form of transforming growth factor-β1 (TGFβ1) protein and latent TGF-beta binding protein 1 (LTBP3). In some embodiments, the LTBP3-TGFβ1 complex comprises LTBP3 covalently linked to pro / latent TGFβ1 via one or more disulfide bonds. In other embodiments, the LTBP3-TGFβ1 complex comprises LTBP1 non-covalently linked to pro / 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.

[0115] As used herein, the term "LRRC33-TGFβ1 complex" refers to a complex between the proprotein or latent form of the 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). In some embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 covalently linked to pro / latent TGFβ1 via one or more disulfide bonds. In other embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 non-covalently linked to pro / latent TGFβ1. In some embodiments, the LRRC33-TGFβ1 complex is a naturally occurring complex, e.g., an intracellular LRRC33-TGFβ1 complex.

[0116] The term "antibody" refers to an immunoglobulin molecule that specifically binds to a target antigen, including, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, although in some instances, such as antibodies that may contain only heavy chains naturally occurring in camelids, they may contain fewer chains. Antibodies may be derived from only a single source or may be "chimeric," i.e., different portions of the antibody are derived from two different antibodies. Antibodies or antigen-binding portions thereof may be produced in hybridomas, by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. As used herein, the term antibody 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.

[0117] 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" chain (in certain embodiments, approximately 25 kDa) and one full-length "heavy" chain (in certain embodiments, approximately 50-70 kDa). The amino-terminal portion of each chain typically contains a variable region of approximately 100-110 or more amino acids that is generally 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 mu, delta, gamma, alpha, or epsilon, which defines the antibody's isotype. Antibodies can be of any type (e.g., IgM, IgD, IgG, IgA, IgY, and IgE) and class (e.g., IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, and IgA2). In 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 or more amino acids (see, e.g., Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), incorporated by reference in its entirety). Typically, the variable regions of each light / heavy chain pair form the antigen-binding site.

[0118] Variable regions typically share the same general structure: three hypervariable regions, also called complementarity-determining regions or CDRs, joined to relatively conserved framework regions (FRs). Typically, the CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. Both light and heavy chain variable regions typically comprise, from N- to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain typically follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or the definitions of Chothia and Lesk (1987) J. Mol. Biol., 196:901-917; Chothia et al. (1989) Nature, 342:878-883. Light chain CDRs may also be referred to as CDR-L1, CDR-L2, and CDR-L3, and heavy chain CDRs may also be referred to as CDR-H1, CDR-H2, and CDR-H3. In some embodiments, an antibody may contain a small number of amino acids deleted from the carboxy terminus of the heavy chain(s). In some embodiments, an antibody comprises a heavy chain with a 1-5 amino acid deletion from the carboxy terminus of the heavy chain. In certain embodiments, the final delineation of the CDRs and identification of the residues comprising the antibody binding site are achieved by solving the structure of the antibody and / or the structure of an antibody-ligand complex. In certain embodiments, this may be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In some embodiments, various analytical methods can be used to identify or estimate 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.

[0119] A "functional antigen-binding site" of a binding protein is a site that can bind to a target, antigen, or ligand. The antigen-binding affinity of an antigen-binding site need not necessarily be as strong as that of the parent binding protein from which it is derived, but its ability to bind to an antigen must be measurable using any one of a variety of known methods for assessing binding of a binding protein to an antigen. Furthermore, the antigen-binding affinity of each of the antigen-binding sites of a multispecific binding protein herein need not be quantitatively the same.

[0120] The term "variable region" or "variable domain" typically refers to a portion of an antibody's light and / or heavy chain, comprising approximately the amino-terminal 120-130 amino acids of the heavy chain and approximately the amino-terminal 100-110 amino acids of the light chain. In certain embodiments, the variable regions of different antibodies vary extensively in amino acid sequence, even among antibodies of the same species. Typically, the variable regions of an antibody determine the specificity of a particular antibody for its target.

[0121] Immunoglobulin constant domain refers to a heavy chain constant domain or a light chain constant domain. The amino acid sequences of human IgG heavy and light chain constant domains are known in the art.

[0122] The term "compete," when used in reference to antigen-binding proteins (e.g., antibodies or antigen-binding portions thereof) that compete for the same epitope, refers to competition between antigen-binding proteins as determined by an assay 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). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, including solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays; solid-phase direct biotin-avidin EIAs; solid-phase direct label assays, and solid-phase direct label sandwich assays. Typically, when a competing antigen-binding protein is present in excess, the antigen-binding protein 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 examples, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0123] The term "antigen" refers to a molecular structure that provides an epitope, e.g., a molecule or portion of a molecule, or a complex of molecules or portions of molecules, to which a selective binding agent, such as an antigen-binding protein (including, e.g., an antibody), can bind. 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 is one that can be used in an animal to produce antibodies capable of binding to that antigen. An antigen can possess one or more epitopes that can interact with different antigen-binding proteins, e.g., antibodies.

[0124] As used herein, the term "CDR" refers to the complementarity determining region in antibody variable sequence.There are three CDRs in each of the variable regions of heavy and light chains, and these are called CDR1, CDR2 and CDR3 for each variable region.As used herein, the term "CDR set" refers to a group of three CDRs present in a single variable region that can bind to antigen.The exact boundaries of these CDRs are defined differently according to different systems. The system described in 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 the variable region of any antibody, but also provides the precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and 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 discovered that certain subportions within the CDRs adopt nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level. These subportions are designated L1, L2, and L3 or H1, H2, and H3, with "L" and "H" indicating the light chain and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, and have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the 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 adhere to one of the systems herein, but may still overlap with the Kabat CDRs, albeit shortened or lengthened in light of predictions or experimental findings that certain residues, groups of residues, or even entire CDRs do not significantly affect antigen binding. The methods used herein can utilize CDRs defined by any of these systems, although certain embodiments use CDRs defined by Kabat or Chothia.

[0125] The terms "crystal" and "crystallized," 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 crystal state. A crystal is composed of a regularly repeating three-dimensional array of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (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 repeats in a crystal is called the asymmetric unit. The repetition of the asymmetric unit in an arrangement that matches a given, well-defined crystallographic symmetry results in the "unit cell" of the crystal. The repetition of the unit cell by regular translation in all three dimensions results in a crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ed., pp. 201-16, Oxford University Press, New York, NY (1999).

[0126] 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 groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is a region of an antigen to which a binding protein binds. Thus, an epitope consists of amino acid residues in a region of an antigen (or fragment thereof) 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 in a complex mixture of proteins and / or macromolecules. For example, antibodies are said to "bind to the same epitope" if they cross-compete (one prevents the binding or modulatory action of the other). Furthermore, although structural definitions of epitopes (overlapping, similar, identical) are informative, functional definitions are often more relevant because they encompass structural (binding) and functional (regulatory, competitive) parameters.

[0127] The terms "treat" and "treatment" encompass therapeutic treatment, prophylactic treatment, and applications that reduce the risk of a subject developing a disorder or other risk factor. Treatment does not require a complete cure of a disorder, but encompasses embodiments that reduce symptoms or underlying risk factors.

[0128] Standard techniques for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection) can be used. Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can generally be performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for all purposes. Unless specific definitions are provided, the nomenclature utilized in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as laboratory procedures and techniques, are those well known and commonly used in the art. Standard techniques for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients can be used.

[0129] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody, as used herein, 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 derived, 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 can be obtained from a whole antibody molecule using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Non-limiting examples of antigen-binding portions include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is 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; and (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) dAb fragments (see, e.g., Ward et al. (1989) NATURE 341:544-546); and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs)). 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 the antibody domains and linker have one of the following orders from N-terminal to C-terminal: 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, and wherein the linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids.

[0130] An "isolated antibody," as used herein, refers to an antibody that is substantially free of other antibodies having different antigen specificities. In some embodiments, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0131] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more of its CDRs such that the antibody's affinity for the antigen is improved compared to a parent antibody that does not have these 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 describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues has been 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, and selective mutations at selective mutagenesis positions, contact or hypermutation positions with activity-enhancing amino acid residues are described in U.S. Pat. No. 6,914,128.

[0132] The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences derived from one species, but in which the sequences of one or more of the VH and / or VL CDR regions have been replaced with CDR sequences from another species, such as 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 with human CDR sequences.

[0133] 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, such as an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0134] 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 amino acid residues, for example in the CDRs, particularly CDR3, 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, does not include antibodies derived from the germline of another mammalian species, such as a mouse, in which CDR sequences have been grafted onto human framework sequences.

[0135] The term "humanized antibody" refers to an antibody containing heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which 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" also refers to an antibody, or variant, derivative, analog, or fragment thereof, that immunospecifically binds to an antigen of interest and comprises FR regions having substantially the amino acid sequence of a human antibody and CDR regions having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially," with respect to CDRs, refers to CDRs having amino acid sequences 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, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the FR regions correspond to those of a human immunoglobulin consensus sequence. In certain embodiments, a humanized antibody also typically comprises at least a portion of an immunoglobulin Fc region of a human immunoglobulin. In some embodiments, a humanized antibody contains a light chain and at least the variable domain of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain. In certain embodiments, a humanized antibody contains only a humanized variable domain of a light chain and / or a humanized heavy chain.

[0136] As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of a variable region minus the CDRs. The precise definition of a CDR sequence can be determined by different systems, and the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain and CDR-H1, CDR-H2, and CDR-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. Unless a specific subregion is designated as FR1, FR2, FR3, or FR4, the framework region, as referred to by others, represents the mixture of FRs in the variable region of a single naturally occurring immunoglobulin chain. As used herein, a FR refers to one of the four subregions that make up a framework region, and multiple FRs refer to two or more of the four subregions that make up a framework region.

[0137] 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 leads to gene rearrangement and mutation 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 is based on the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals within a species, and therefore are less likely to be recognized as being from a foreign source when used therapeutically in that species.

[0138] As used herein, the term "neutralizing" refers to the negation of the biological activity of an antigen when the binding protein specifically binds to the antigen. In certain embodiments, a neutralizing binding protein binds to an antigen / target, e.g., 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.

[0139] 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 diabodies.

[0140] The term "monoclonal antibody" or "mAb," when used in reference to a composition comprising same, can refer to an antibody preparation obtained from a population of substantially homogeneous antibodies, i.e., an antibody preparation in which 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, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.

[0141] The term "recombinant human antibody," as used herein, refers to any human antibody that is prepared, expressed, created, or isolated by recombinant means, e.g., an antibody expressed using a recombinant expression vector transfected into a host cell (described further in Section IIC, below), an antibody isolated from a recombinant combinatorial human antibody library (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, incorporated herein by reference), antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see Taylor, LD et 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 antibodies prepared, expressed, created, or isolated by any other means involving splicing other DNA sequences from human immunoglobulin gene 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 in vivo somatic mutagenesis when animals transgenic for human Ig sequences are used), and thus 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 occur within the human antibody germline repertoire in vivo.

[0142] As used herein, "dual variable domain immunoglobulins" or "DVD-IgTM" and the like include binding proteins comprising paired heavy and light chain DVD polypeptides, each paired heavy and light chain providing two antigen-binding sites. Each binding site comprises a total of six CDRs involved in antigen binding per antigen-binding site. DVD-IgTM typically have two arms connected to each other, at least in part, by dimerization of the CH3 domains, and each DVD arm is bispecific, resulting in an immunoglobulin with four binding sites. DVD-IgTM are described in U.S. Patent Application Publication Nos. 2010 / 0260668 and 2009 / 0304693, each of which is incorporated herein by reference, including its sequence listing.

[0143] As used herein, a "triple variable domain immunoglobulin" or "TVD-Ig" or the like refers to a binding protein that contains paired heavy and light chain TVD binding protein polypeptides, with each paired heavy and light chain providing three antigen-binding sites. Each binding site contains a total of six CDRs involved in antigen binding per antigen-binding site. The TVD binding protein may have two arms connected to each other, at least in part, by dimerization of the CH3 domains, and each arm of the TVD binding protein is trispecific, resulting in a binding protein with six binding sites.

[0144] As used herein, "receptor-antibody immunoglobulin" or "RAb-Ig" refers to a binding protein comprising a heavy chain RAb polypeptide and a light chain RAb polypeptide, which together form a total of three antigen-binding sites. The antibody heavy chain variable domain and light chain variable domain present in each of the heavy chain RAb polypeptide and the light chain RAb polypeptide form a single binding site with a total of six CDRs, resulting in a first antigen-binding site. The heavy chain RAb polypeptide and the light chain RAb polypeptide each contain receptor sequences that independently bind to ligands, resulting in second and third "antigen" binding sites. RAb-Ig typically has two arms connected to each other, at least in part, by dimerization of the CH3 domain, and each arm of RAb-Ig is trispecific, resulting in an immunoglobulin with six binding sites. RAb-Ig is described in U.S. Patent Application Publication No. 2002 / 0127231, the entire contents of which, including the sequence listing, are incorporated herein by reference.

[0145] The term "bispecific antibody", as used herein, and to distinguish it from "bispecific semi-Ig binding proteins" or "bispecific (semi-Ig) binding proteins", refers to antibodies that are synthesized by quadroma technology (see Milstein, C. and Cuello, A.C. (1983) Nature 305(5934):537-540), by chemical conjugation of two different monoclonal antibodies (see Staerz, U.D. et al. (1985) Nature 314(6012):628-631), or by introducing mutations in the knob-into-hole or Fc region that do not inhibit CH3-CH3 dimerization (see Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90(14):6444-6448), and refers to full-length antibodies produced by similar techniques, resulting in a large number of different immunoglobulin species, of which only one is a functional bispecific antibody. By molecular function, a bispecific antibody binds to one antigen (or epitope) on one of its two binding arms (one HC / LC pair) and to a different antigen (or epitope) on its second arm (a different HC / LC pair). By this definition, a bispecific antibody has two distinct antigen-binding arms (both in specificity and CDR sequence) and is monovalent for each antigen to which it binds.

[0146] The term "bispecific antibody," as used herein, and in distinction from bispecific semi-Ig binding proteins or bispecific binding proteins, refers to a full-length antibody that can bind to two different antigens (or epitopes) in each of its two binding arms (HC / LC pairs) (see PCT Publication No. WO 02 / 02773). Thus, a bispecific binding protein has two identical antigen-binding arms with the same specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.

[0147] The term "pan-TGFβ antibody" refers to any antibody that can bind to more than one isoform of TGFβ, for example, at least two of TGFβ1, TGFβ2, and TGFβ3. In some embodiments, the pan-TGFβ antibody binds to all three isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3. In some embodiments, the pan-TGFβ antibody binds to and neutralizes all three isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3.

[0148] The term “K on "K," as used herein, refers to the on rate constant for the association of a binding protein (e.g., an antibody) with an antigen to form, for example, an antibody / antigen complex, as known in the art. on " refers to the term "association rate constant" or "k" which are used interchangeably herein. a The value that indicates the rate of binding of an antibody to its target antigen or the rate of formation of an antibody-antigen complex is also given by the equation: antibody ("Ab") + antigen ("Ag") → Ab-Ag.

[0149] The term “K off "K," as used herein, is intended to refer to the off rate constant for dissociation of a binding protein (e.g., an antibody), e.g., from an antibody / antigen complex, as known in the art. off " refers to the term "dissociation rate constant" or "k" which are used interchangeably herein. d The rate of dissociation of an antibody from its target antigen, or the separation of an Ab-Ag complex into free antibody and antigen over time, is given by the equation: Ab + Ag ← Ab - Ag.

[0150] The terms "equilibrium dissociation constant" or "K" are used interchangeably herein. D" is the value obtained in a titration measurement at equilibrium, or the dissociation rate constant (k off ) as the binding rate constant (k on ) The association rate constant, dissociation rate constant, and equilibrium dissociation constant are used to describe the binding affinity of a binding protein, such as an antibody, to an antigen. Methods for determining association rate constants and dissociation rate constants are well known in the art. The use of fluorescence-based techniques offers high sensitivity and the ability to test samples in physiological buffer 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, a GE Healthcare company, Uppsala, Sweden). In addition, the KinExA® (Kinetic Exclusion Assay) assay available from Sapidyne Instruments (Boise, Idaho) can also be used.

[0151] The term "linker" is used to refer to a polypeptide that contains 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, e.g., 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); No. 69); ADAPTVSIFPP (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); GGGGSGGGSGGGGS (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).

[0152] The term "cancer," as used herein, refers to a physiological condition in multicellular eukaryotic organisms that is typically characterized by unregulated cell growth.

[0153] "Label" and "detectable label" or "detectable moiety" refer to a moiety attached to a specific binding partner, such as an antibody or an analyte, to render the reaction between members of a specific binding pair, such as, for example, an antibody and an analyte, detectable; a specific binding partner, such as, for example, an antibody or an analyte, so labeled is said to be "detectably labeled." Thus, the term "labeled binding protein," as used herein, refers to a protein incorporating a label that provides for identification of the binding protein. In certain embodiments, the label is a detectable marker that can produce a detectable signal by visual or instrumental means, for example, by incorporation of a radiolabeled amino acid or attachment of a biotinyl moiety to the polypeptide that can be detected by tagged 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., 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, and 153Sm); chromogens; fluorescent labels (e.g., FITC, rhodamine, and lanthanide phosphors); 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 type of detectable label.

[0154] The term "surface plasmon resonance" as used herein refers to an optical phenomenon that allows for the analysis of real-time bispecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Joensson, U. et al. (1993) Ann. Biol. Clin., 51:19-26; Joensson, U. et al. (1991) Biotechniques, 11:620-627; Johnson, B. et al. (1995) J. Mol. Recognit., 8:125-131; and Johnson, B. et al. (1991) Anal. Biochem., 198:268-277.

[0155] A "plasmid" or "vector" includes a nucleic acid construct designed for delivery into a host cell or transfer between different host cells. An "expression plasmid" or "expression vector" can be a plasmid capable of incorporating and expressing heterologous nucleic acid fragments in a cell. An expression plasmid can contain additional elements; for example, an expression vector can have two replication systems, thus allowing it to be maintained in two organisms. A nucleic acid incorporated into a plasmid can be operably linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of the polynucleotide sequence.

[0156] A "nucleic acid" or "nucleic acid sequence" may be any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. A nucleic acid may be either single-stranded or double-stranded. A single-stranded nucleic acid may be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, a single-stranded nucleic acid may be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one embodiment, a nucleic acid may be DNA. In another embodiment, a nucleic acid may be RNA. A suitable nucleic acid molecule is DNA, including genomic DNA or cDNA. Another suitable nucleic acid molecule is RNA, including mRNA.

[0157] Except in the working examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about," which when used in connection with percentages can mean ±1%.

[0158] While several embodiments of the present disclosure have been described and illustrated herein, various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein will readily occur to those skilled in the art, and each such variation and / or modification is considered to be within the scope of the present disclosure. More generally, all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and those skilled in the art will readily understand that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the present disclosure are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it should be understood that, within the scope of the appended claims and their equivalents, the present disclosure may be practiced other than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and / or methods is within the scope of the present disclosure, unless such features, systems, articles, materials, and / or methods are mutually inconsistent.

[0159] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0160] The phrase "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present conjunctively in some cases and disjunctively in other cases. Unless clearly indicated to the contrary, other elements, whether related or unrelated to the elements specifically identified, other than the elements specifically identified by the "and / or" clause may optionally be present. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so forth.

[0161] As used in this specification and claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the 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, nor excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the specifically identified elements. 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") can refer to, in one embodiment, at least one A, optionally including more than one, and no B (and optionally including elements other than B); in another embodiment, at least one B, optionally including more than one, and no A (and optionally including elements other than A); in yet another embodiment, at least one A, optionally including more than one, and at least one B (and optionally including other elements), etc.

[0162] The use of ordinal terms, such as "first," "second," "third," etc., to modify claim elements in the claims does not, by itself, indicate any priority, precedence, or order of one claim element relative to another claim element, nor does it indicate the chronological order in which actions of a method are performed, but is merely used as a marker to distinguish between claim elements having a particular name and another element having the same name (when no ordinal term is used).

[0163] It is understood that ranges provided herein are abbreviated to all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange 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.

[0164] Antibodies and antigen-binding portions thereof that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex The present invention is based, at least in part, on the discovery of antibodies and antigen-binding portions thereof that bind to TGFβ1 present in the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex. Accordingly, some aspects of the present invention relate to antibodies or antigen-binding portions thereof that specifically bind to an epitope of TGFβ1, wherein the epitope is available for binding by the antibody or antigen-binding portion thereof when TGFβ1 is present in the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex. In some embodiments, the epitope becomes available due to a change in conformation of TGFβ1 when it is in a complex with GARP, LTBP1, LTBP3, and / or LRRC33. In some embodiments, the epitope of 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 interfere with the binding of TGFβ1 to integrins. For example, in some embodiments, the antibody or antigen-binding portion thereof does not block 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 the release of mature TGFβ1 from the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex.

[0165] The antibodies or antigen-binding portions thereof provided herein specifically bind to an epitope of TGFβ1, wherein the epitope is available for binding by the antibody or antigen-binding portion thereof when TGFβ1 is present in a GARP-TGFβ1, LTBP1-TGFβ1 complex, LTBP2-TGFβ1 complex, and / or LRRC33-TGFβ1 complex. In some embodiments, TGFβ1 comprises a naturally occurring mammalian amino acid sequence. In some embodiments, TGFβ1 comprises a naturally occurring human amino acid sequence. In some embodiments, 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 either 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 amino acid sequence of TGFβ3 are set forth in SEQ ID NOs: 22 and 23, respectively. In some embodiments, an antibody or antigen-binding portion thereof described herein specifically binds to a TGFβ1 comprising a non-naturally occurring amino acid sequence (otherwise referred to herein as non-naturally occurring TGFβ1). For example, a non-naturally occurring TGFβ1 can include one or more recombinantly generated mutations compared to a 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 shown 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 shown in Table 2. [ka] [Table 1-1] Table 1-2 Table 1-3 Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5

[0166] In some embodiments, the antibodies or antigen-binding portions thereof described herein can bind to the LTBP1-TGFβ1 complex. In some embodiments, the antigenic protein complex (e.g., the LTBP-TGFβ1 complex) may comprise one or more LTBP proteins (e.g., LTBP1, LTBP2, LTBP3, and LTBP4). In some embodiments, the LTBP1 protein is a naturally occurring protein. In some embodiments, the LTBP1 protein is a non-naturally occurring protein. In some embodiments, the LTBP1 protein is a recombinant protein. Such recombinant LTBP1 proteins may comprise LTBP1, its alternatively spliced ​​variants, and / or fragments thereof. The recombinant LTBP1 protein may also 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 has been 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 NOs: 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.

[0167] In some embodiments, the antibodies or antigen-binding portions thereof described herein can bind to the LTBP3-TGFβ1 complex. In some embodiments, the LTBP3 protein is a naturally occurring protein. In some embodiments, the LTBP3 protein is a non-naturally occurring protein. In some embodiments, the LTBP3 protein is a recombinant protein. Such recombinant LTBP3 proteins may include LTBP3, alternatively spliced ​​variants thereof, and / or fragments thereof. In some embodiments, the LTBP3 protein includes a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP3 protein does not include a leader sequence (i.e., the leader sequence is processed or cleaved). The recombinant LTBP3 protein may also 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.

[0168] In some embodiments, the antibodies or antigen-binding portions thereof described herein can bind to the GARP-TGFβ1 complex. In some embodiments, the GARP protein is a naturally occurring protein. In some embodiments, the GARP protein is a non-naturally occurring protein. In some embodiments, the GARP protein is a recombinant protein. Such GARPs can be recombinant and are referred to herein as recombinant GARPs. Some recombinant GARPs can contain one or more modifications, truncations, and / or mutations compared to wild-type GARP. The recombinant GARP can be modified to be soluble. In some embodiments, the GARP protein contains a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the GARP protein does not contain a leader sequence (i.e., the leader sequence is processed or cleaved). In other embodiments, the recombinant GARP has been modified to contain one or more detectable labels. In further embodiments, such detectable labels can 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 the amino acid sequence set forth in SEQ ID NOs: 48-49 in Table 2. In some embodiments, the GARP protein comprises the amino acid sequence set forth in SEQ ID NOs: 52 and 53 in 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, such as 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 TGFβ1 is bound to any of the following presentation molecules: GARP, LTBP1, LTBP3, and / or LRCC33.

[0169] In some embodiments, the antibodies or antigen-binding portions thereof described herein can bind to the LRRC33-TGFβ1 complex. In some embodiments, the LRRC33 protein is a naturally occurring protein. In some embodiments, the LRRC33 protein is a non-naturally occurring protein. 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 contain 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 LRRC33 ectodomain can 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 is processed or cleaved). In other embodiments, the recombinant LRRC33 protein has been 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 85 in Table 4. [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2]

[0170] In some embodiments, antibodies or antigen-binding portions thereof of the invention that specifically bind to an epitope of TGFβ1 that is available for binding by the antibody when TGFβ1 is present in a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex, and nucleic acid molecules of the disclosure encoding the antibodies, comprise one or more of the CDR amino acid sequences shown in Table 5. [Table 5]

[0171] In some embodiments, antibodies of the invention that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex comprise any antibody or antigen-binding portion thereof that comprises a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, provided for any one of the antibodies shown in Table 5. 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 CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any one of the antibodies shown in Table 5. The present invention also provides any nucleic acid sequence encoding a molecule that comprises a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 provided for any one of the antibodies shown in Table 5. The antibody heavy and light chain CDR3 domains can play a particularly important role in the binding specificity / affinity of an antibody to an antigen. Thus, antibodies of the present disclosure that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex, or nucleic acid molecules encoding these antibodies or antigen-binding portions thereof, can comprise at least the heavy and / or light chain CDR3 of the antibodies shown in Table 5.

[0172] 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, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex and comprises six complementarity-determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.

[0173] In some embodiments, CDRH1 comprises the sequence set forth in any one of SEQ ID NOs: 1 and 2. In some embodiments, CDRH2 comprises the sequence set forth in any one of SEQ ID NOs: 3 and 4. In some embodiments, CDRH3 comprises the sequence set forth in any one of SEQ ID NOs: 5 and 6. CDRL1 comprises the sequence set forth in any one of SEQ ID NOs: 7 and 8. In some embodiments, CDRL2 comprises the sequence set forth in any one of SEQ ID NOs: 9 and 10. In some embodiments, CDRL3 comprises the sequence set forth in any one of SEQ ID NOs: 11 and 12.

[0174] In some embodiments (e.g., with respect to antibody Ab1 shown in Table 5), an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex comprises 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.

[0175] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a 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 a 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 a 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.

[0176] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable domain comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable domain comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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.

[0177] In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an 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, the 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.

[0178] In some embodiments (e.g., with respect to antibody Ab2 shown in Table 5), an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an 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.

[0179] 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.

[0180] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable domain comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable domain comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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.

[0181] In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an 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, the 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.

[0182] In some examples, antibodies of the present disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex include 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 comprise one or more CDR sequences shown in Table 5 (SEQ ID NOS: 1-12) that contain up to five, four, three, two, or one amino acid residue variations compared to the corresponding CDR region of any one of SEQ ID NOS: 1-12. The complete amino acid sequences of the heavy and light chain variable regions of the antibodies (e.g., Ab1 and Ab2) 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. [ka] [ka] [ka]

[0183] In some embodiments, antibodies of the present disclosure that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex include any antibodies comprising a heavy chain variable domain of SEQ ID NO: 13 or 17 or a light chain variable domain of SEQ ID NO: 14 or 18. In some embodiments, antibodies of the present disclosure that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex include any antibodies comprising the heavy chain variable and light chain variable pairs of SEQ ID NOs: 13 and 14; and 17 and 18.

[0184] Embodiments of the present disclosure provide antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the 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 or 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 or 17, or the light chain variable sequence of SEQ ID NO: 14 or 18. In some embodiments, the homologous heavy chain variable and / or light chain variable amino acid sequences do not vary among any of the CDR sequences 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 be present within the heavy chain variable and / or light chain variable amino acid sequences excluding any of the CDR sequences provided herein.

[0185] In some embodiments, antibodies of the present disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex include any antibody or antigen-binding portion thereof comprising 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 present disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex include any antibody comprising the heavy chain and light chain pair of SEQ ID NOs: 15 and 16; or 19 and 20.

[0186] Aspects of the present disclosure provide antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex, having heavy and / or light chain amino acid sequences homologous to any of those described herein. In some embodiments, the antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an 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 sequence of SEQ ID NO: 16 or 20. In some embodiments, 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, 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 exist within heavy and / or light chain amino acid sequences excluding any of the CDR sequences provided herein.

[0187] In some embodiments, antibodies of the present disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex include any antibody or antigen-binding portion thereof comprising 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 present disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex include any antibody comprising the heavy chain and light chain pair of SEQ ID NOs: 15 and 16; or 19 and 20.

[0188] Aspects of the present disclosure provide antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex, having heavy and / or light chain amino acid sequences homologous to any of those described herein. In some embodiments, antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex comprise a heavy chain 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 do not vary within any of the CDR sequences 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 exist within heavy and / or light chain amino acid sequences excluding any of the CDR sequences provided herein.

[0189] 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 as in 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 utilized 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.

[0190] In any of the antibodies or antigen-binding fragments described herein, one or more conservative mutations can be introduced into the CDR or framework sequences at residue positions unlikely to be involved in antibody-antigen interactions. In some embodiments, such conservative mutation(s) can be introduced into the CDR or framework sequences at residue positions unlikely to be involved in interactions with the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex, as determined based on crystal structures. In some embodiments, likely interfaces (e.g., residues involved in antigen-antibody interactions) can be predicted from known structural information about another antigen that shares structural similarity.

[0191] 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. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, such as those found in references summarizing such methods, for example, Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, edited by FM Ausubel et al., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions between amino acids in the following group: (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.

[0192] In some embodiments, the antibodies provided herein contain mutations that confer desirable properties to the antibody. For example, to avoid potential difficulties with Fab arm exchange, which is known to occur in natural IgG4 mAbs, the antibodies provided herein may contain a stabilizing "Adair" mutation in which serine 228 (EU numbering, residue 241 in Kabat numbering) is converted to proline to create an IgG1-like (CPPCP (SEQ ID NO: 54)) hinge sequence (Angal et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30:105-108; 1993). Thus, any of the antibodies may contain the stabilizing "Adair" mutation or the amino acid sequence CPPCP (SEQ ID NO: 54).

[0193] Antibodies of the present disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex may optionally comprise an antibody constant region or a portion thereof. L The domain may be attached at its C-terminus to a light chain constant domain such as Cκ or Cλ. H The domain or a portion thereof can be attached to all or part of a heavy chain, such as IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. The antibody can include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within this scope can include a V domain combined with any suitable constant region. H and V L domain, or an antigen-binding portion thereof.

[0194] In some embodiments, antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex may or may not comprise the framework regions of the antibodies of SEQ ID NOs: 13 to 20. In some embodiments, antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex are murine antibodies and comprise murine framework region sequences.

[0195] In some embodiments, antibodies of the present disclosure that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex have relatively high affinity for the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex, e.g., 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or less K D For example, an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex may bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an 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).

[0196] Antibodies that block TGFβ In one aspect, the present invention provides functional antibodies. As used herein, a "functional antibody" is one that confers one or more biological activities through its ability to bind to an antigen. Functional antibodies can include inhibitory antibodies (or inhibitory antibodies) and activating antibodies. Thus, the present disclosure includes TGFβ antibodies that can regulate (e.g., inhibit or activate) biological processes mediated by TGFβ signaling.

[0197] As used herein, the term "inhibitory antibody" refers to an antibody that inhibits the release of mature growth factors or reduces growth factor activity. Inhibitory antibodies include antibodies that target any epitope that reduces growth factor release or activity upon association with such an antibody. Such epitopes may be present on the prodomain of a TGFβ protein (e.g., TGFβ1), a growth factor, or other epitopes that, upon binding by the antibody, lead to a reduction in growth factor activity. Inhibitory antibodies of the present invention include, but are not limited to, TGFβ1-inhibitory antibodies.

[0198] Multiple embodiments of the present disclosure include methods of using inhibitory antibodies to alter growth factor signaling in solution, cell culture, and / or in a subject.

[0199] Polypeptides Some aspects of the present 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: 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: 15, and SEQ ID NO: 19.

[0200] Some aspects of the present 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: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:16, and SEQ ID NO:20.

[0201] Antibodies that compete with antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex Aspects of the present disclosure relate to antibodies that compete or cross-compete with any of the antibodies provided herein. The term "compete," as used herein with respect to antibodies, 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 a second antibody, such that the binding of the first antibody to its 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. Alternatively, the binding of the second antibody to its epitope may also be detectably reduced in the presence of the first antibody, but this need not be the case. That is, a first antibody may inhibit the binding of a 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, then the antibodies are said to "cross-compete" with each other for binding to 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 portion thereof), those skilled in the art will understand that such competing and / or cross-competing antibodies are encompassed by and may be useful in the methods and / or compositions provided herein.

[0202] Aspects of the present disclosure relate to antibodies that compete or cross-compete with any of the specific antibodies or antigen-binding portions thereof provided herein. In some embodiments, the antibody or antigen-binding portion thereof binds to or near the same epitope as any of the antibodies provided herein. In some embodiments, an antibody or antigen-binding portion thereof binds near an epitope if it binds within 15 or fewer amino acid residues from the epitope. In some embodiments, any of the antibodies or antigen-binding portions thereof provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues from the epitope to which any of the antibodies provided herein binds.

[0203] In another embodiment, the antibody is selected from the group consisting of a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and a LRRC33-TGFβ1 complex. -6 The equilibrium dissociation constant K between the antibody and the protein is less than M D Provided herein are antibodies or antigen-binding portions thereof that compete or cross-compete at 10 or more for binding to any of the antigens provided herein. -11 M to 10 -6 K to M D Provided herein are antibodies that compete or cross-compete for binding with an antibody, or antigen-binding portion thereof, described herein. In some embodiments, provided herein are anti-TGFβ1 antibodies, or antigen-binding portions thereof, that compete for binding with an antibody, or antigen-binding portion thereof, described herein. In some embodiments, provided herein are anti-TGFβ1 antibodies, or antigen-binding portions thereof, that bind to the same epitope as an antibody, or antigen-binding portion thereof, described herein.

[0204] Any antibody provided herein can be characterized using any suitable method. For example, one method is to identify the epitope to which the antigen binds, i.e., "epitope mapping." For example, as described in Chapter 11 of Harlow and Lane, "Using Antibodies," a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999, there are many suitable methods for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, and synthetic peptide-based assays. In another example, epitope mapping can be used to determine the sequence to which an antibody binds. The epitope may be a linear epitope, i.e., an epitope contained in a single stretch of amino acids, or a conformational epitope formed by the three-dimensional interaction of amino acids, which may not necessarily be contained in a single stretch (linear sequence of primary structure). In some embodiments, the epitope is a TGFβ1 epitope that becomes available for binding by an antibody or antigen-binding portion thereof described herein only when TGFβ1 is present in a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex. Peptides of various lengths (e.g., at least 4-6 amino acids in length) can be isolated or synthesized (e.g., recombinantly) and used in antibody-based binding assays. In another example, the epitope to which an antibody binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining antibody binding. In a gene fragment expression assay, the open reading frame encoding the target antigen is fragmented randomly or by specific gene construction, and the reactivity of the expressed antigen fragment with the antibody being tested is determined. Gene fragments are generated, for example, by PCR, and then transcribed and translated into protein in vitro in the presence of radioactive amino acids.The binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. A specific epitope can also be identified by using a large library of random peptide sequences (phage libraries) displayed on the surface of phage particles. Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. In a further example, mutagenesis of the antigen-binding domain, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen 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 with sequences from a closely related but antigenically distinct protein, such as another member of the TGFβ protein family (e.g., GDF11). By assessing the binding of the antibody 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 assessed.

[0205] Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as another antibody. Competition assays are well known to those skilled in the art.

[0206] Furthermore, the interaction of any of the antibodies 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 conventional techniques. For example, a crystal structure can be determined, and the distance between 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 distances, it can be determined whether specific residues in the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex interact with one or more residues in the antibody. Furthermore, suitable methods such as competition assays and targeted mutagenesis assays can be applied to determine the preferential binding of candidate antibodies.

[0207] Production of antibodies that bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex Numerous methods can be used to obtain the antibodies or antigen-binding fragments thereof of the present disclosure. For example, antibodies can be produced using recombinant DNA methods. Monoclonal antibodies can also be produced by generating hybridomas according to known methods (see, e.g., Kohler and Milstein (1975) Nature 256:495-499). The hybridomas thus formed are then 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 the designated antigen. Any form of the designated antigen can be used as an immunogen, for example, recombinant antigens, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptides thereof (e.g., any of the epitopes described herein within a scaffold as a linear epitope or as a conformational epitope). One typical method for generating antibodies involves screening a protein expression library, such as a phage or ribosome display library, which expresses antibodies or fragments thereof (eg, scFv). Phage display is described, for example, in Ladner et al., U.S. Pat. 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.

[0208] In addition to using a display library, a designated antigen (e.g., a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex) can be used to immunize a non-human animal, e.g., a rodent, e.g., a mouse, a hamster, or a rat. In one embodiment, the non-human animal is a mouse.

[0209] In another embodiment, monoclonal antibodies are obtained from non-human animals and then modified (e.g., chimeric) using suitable recombinant DNA techniques. Various approaches 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. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent Publication Nos. EP 171496, 0173494, and GB 2177096B.

[0210] For additional 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 source, method of production or other particular characteristics of the antibodies.

[0211] Some aspects of the present disclosure relate to host cells transformed with 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 host cell's genome or maintained extrachromosomally. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal, or human cell. In some embodiments, the fungal cell is, for example, of the genus Saccharomyces, specifically the species S. cerevisiae. The term "prokaryote" includes all bacteria that can be transformed or transfected with DNA or RNA molecules for expression of antibodies or corresponding immunoglobulin chains. Prokaryotic hosts may include gram-negative and gram-positive bacteria, such as E. coli, S. typhimurium, Serratia marcescens, and Bacillus subtilis. The term "eukaryote" includes yeast, higher plants, insects, and vertebrate cells, such as mammalian cells, such as NS0 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 also include an initial methionine amino acid residue.

[0212] 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, if desired, by recovery and purification of the immunoglobulin light chain, heavy chain, light / heavy chain dimer or intact antibody, antigen-binding fragment, or other immunoglobulin form; see Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY, (1979). The polynucleotide or vector is then introduced into a cell that produces the antibody or antigen-binding fragment. Furthermore, transgenic animals, preferably mammals, containing the aforementioned host cells may be used for large-scale production of antibodies or antibody fragments.

[0213] Transformed host cells may be grown in fermentors 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 by 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, for example, in microorganisms, may be by any conventional means, including, for example, preparative chromatographic separations and immunological separations, including, for example, those involving the use of monoclonal or polyclonal antibodies directed against the constant regions of the antibodies.

[0214] Aspects of the present disclosure relate to hybridomas that provide an indefinitely sustainable source of monoclonal antibodies. As an alternative to obtaining immunoglobulins directly from hybridoma cultures, immortalized hybridoma cells may be used as a source of rearranged heavy and light chain loci for subsequent expression and / or genetic manipulation. Rearranged antibody genes may be reverse transcribed from the appropriate mRNA to produce cDNA. In some embodiments, the heavy chain constant region may be replaced with one of a different isotype or removed entirely. Variable regions may be linked to encode a single-chain Fv region. Multiple Fv regions may be linked to confer binding ability to more than one target, or chimeric heavy and light chain combinations may be used. Any suitable method may be used for cloning antibody variable regions and generating recombinant antibodies.

[0215] In some embodiments, appropriate nucleic acids encoding the heavy and / or light chain variable regions are obtained and inserted into expression vectors that can be transfected into standard recombinant host cells. A variety of such host cells may be used. In some embodiments, mammalian host cells may 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 may be carried out by culturing the modified recombinant host under culture conditions appropriate for host cell growth and expression of the coding sequences. Antibodies or antigen-binding fragments may be recovered by isolating them from the culture. Expression systems may be designed to include a signal peptide so that the resulting antibody is secreted into the medium; however, intracellular production is also possible.

[0216] 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.

[0217] 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 comprising any of these polynucleotides, 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.

[0218] 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 translatable mRNA. Control elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may include a control sequence that promotes transcription initiation and, optionally, a polyA signal that promotes transcription termination and transcript stabilization. Additional control elements may include transcriptional and translational enhancers and / or naturally associated or heterologous promoter regions. Possible control elements that allow expression in prokaryotic host cells include, for example, the PL, Lac, Trp, or Tac promoters in E. coli. Examples of control elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast, or the CMV promoter, SV40 promoter, RSV promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells.

[0219] Such control elements, in addition to those involved in the initiation of transcription, may also include transcription termination signals downstream of the polynucleotide, such as the SV40 polyA site or the tk polyA site. Furthermore, depending on the expression system used, a leader sequence capable of directing the polypeptide into 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) is assembled in 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 portion thereof, for example, into the extracellular medium. Heterologous polynucleotide sequences encoding fusion proteins containing C- or N-terminal identification peptides that confer desirable characteristics, such as stabilization or simplified purification of the expressed recombinant product, may also optionally be used.

[0220] In some embodiments, the polynucleotides encoding at least the variable domains of the light and / or heavy chains may encode both immunoglobulin chains or only one variable domain. Similarly, the polynucleotides may be under the control of the same promoter or may be separately regulated for expression. Furthermore, some aspects relate to vectors conventionally used in genetic engineering, particularly plasmids, cosmids, viruses, and bacteriophages, that contain polynucleotides encoding the variable domains of an immunoglobulin chain of an antibody or antigen-binding fragment, optionally in combination with polynucleotides encoding the variable domains of other immunoglobulin chains of the antibody.

[0221] 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 to deliver polynucleotides or vectors to targeted cell populations (e.g., to engineer cells to express antibodies or antigen-binding fragments). Various suitable methods may be used to construct recombinant viral vectors. In some embodiments, polynucleotides and vectors may be reconstituted into liposomes for delivery to target cells. Vectors containing polynucleotides (e.g., immunoglobulin chain heavy and / or light chain variable domain(s) encoding sequences and expression control sequences) may be transferred into host cells by suitable methods, which vary depending on the type of cellular host.

[0222] qualification Antibodies or antigen-binding portions thereof of the present disclosure may be modified with a detectable label or moiety, 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. Detectable substances or moieties may be coupled or conjugated to the polypeptides of the present disclosure either directly or indirectly through an intermediate, such as a linker (e.g., a cleavable linker), using suitable techniques. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-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, for example, iodine ( 131 I, 125 I, 123 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115 mIn, 113 mIn, 112 In, 111 In), and technetium ( 99 Tc, 99 mTc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133Xe), fluorine ( 18 F), 153 Sm, Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 86 R, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, and tin ( 113 Sn, 117 Detectable substances include radioactive metal ions such as Sn), e.g., alpha emitters or other radioisotopes. Detectable substances may be coupled or conjugated to antibodies of the present disclosure that specifically bind to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex either directly or indirectly through an intermediate (e.g., a linker) using suitable techniques. Any of the antibodies provided herein that are conjugated to a detectable substance may be used in any suitable diagnostic assay, such as those described herein.

[0223] Additionally, antibodies or antigen-binding portions thereof of the present disclosure may be modified with drugs, which may be coupled or conjugated to the polypeptides of the present disclosure either directly or indirectly through an intermediate (e.g., a linker, such as a cleavable linker) using suitable techniques.

[0224] targeted drug In some embodiments, the methods of the present disclosure include the use of one or more targeting agents to target the antibodies or antigen-binding portions thereof disclosed herein to specific sites in a subject to regulate mature TGFβ release from the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex. For example, the LTBP1-TGFβ1 and LTBP3-TGFβ1 complexes are typically localized in the extracellular matrix. Thus, in some embodiments, the antibodies disclosed herein can be conjugated to an extracellular matrix targeting agent to localize the antibody to sites where the LTBP1-TGFβ1 and LTBP3-TGFβ1 complexes are present. In such embodiments, selective targeting of the antibody leads to selective regulation of the LTBP1-TGFβ1 and / or LTBP3-TGFβ1 complexes. In some embodiments, selective targeting of the antibody leads to selective inhibition of the LTBP1-TGFβ1 and / or LTBP3-TGFβ1 complex (e.g., to treat fibrosis). In some embodiments, the extracellular matrix targeting agent includes 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.

[0225] Similarly, the GARP-TGFβ1 complex typically binds to activated FOXP3 +It is localized on the surface of regulatory T cells (Treg). Thus, in some embodiments, the antibodies disclosed herein can be conjugated to an immune cell (e.g., Treg cell) binding agent to localize the antibody to the site where the GARP-TGFβ1 complex is present. In such embodiments, selective targeting of the antibody leads to selective modulation of the GARP-TGFβ1 complex. In some embodiments, selective targeting of the antibody leads to selective inhibition of the GARP-TGFβ1 complex (e.g., selective inhibition of release of mature TGFβ1 for immune modulation, e.g., in the treatment of cancer). In such embodiments, the Treg cell targeting agent can include, for example, CCL22 and CXCL12 proteins or fragments thereof.

[0226] In some embodiments, bispecific antibodies can be used that have 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).

[0227] Pharmaceutical Composition The present invention further provides pharmaceutical compositions for use as medicaments suitable for administration to human and non-human subjects. One or more 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 formulated or mixed with a pharmaceutically acceptable carrier (excipient), including, for example, a buffer, to form a pharmaceutical composition. Such formulations may be used to treat diseases or disorders involving TGFβ signaling. In some embodiments, such diseases or disorders associated with TGFβ signaling involve one or more conditions, i.e., TGFβ is associated with a specific type(s) of presentation molecule. In some embodiments, such conditions occur in a cell-type- and / or tissue-specific manner. In some embodiments, for example, such context-dependent effects of TGFβ signaling are mediated, in part, by GARP, LRRC33, LTBP1, and / or LTBP3.

[0228] In some embodiments, the antibodies of the present invention specifically bind to TGFβ in two or more contexts, thus binding to TGFβ in a complex with a presentation molecule selected from two or more of GARP, LRRC33, LTBP1, and LTBP3. Therefore, such pharmaceutical compositions can be administered to patients to alleviate TGFβ-related conditions (e.g., fibrosis, immune disorders, and / or cancer). By "acceptable," we mean that the carrier is compatible with the active ingredient of the composition (and preferably, can stabilize the active ingredient) and is not harmful to the subject being treated. Examples of pharmaceutically 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 Edition (2000), Lippincott Williams and Wilkins, ed., K.E. Hoover. In one example, the pharmaceutical compositions described herein contain 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.

[0229] The pharmaceutical composition used in the present method can contain a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or aqueous solution (Remington: The Science and Practice of Pharmacy, 20th Edition (2000), Lippincott Williams and Wilkins, edited by K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate, or other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as 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; and serum albumin. , gelatin, 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.

[0230] In some embodiments, the pharmaceutical compositions described herein include liposomes containing antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the 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 with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0231] Antibodies that specifically bind to GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex can also be entrapped in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, for example, in microcapsules prepared by coacervation technology or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively.Exemplary techniques have been previously described, see, for example, Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing (2000).

[0232] In other examples, the pharmaceutical compositions described herein may be formulated in a sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactide lactic acid (U.S. Pat. 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 composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0233] Pharmaceutical compositions to be 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.

[0234] 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 for administration by inhalation or insufflation.

[0235] To prepare solid compositions such as tablets, the primary active ingredient may be mixed with pharmaceutical carriers, such as conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, and other pharmaceutical diluents, such as water, or non-toxic pharmaceutically acceptable salts thereof, to form solid preformulation compositions containing a homogeneous mixture of the compounds of the present disclosure. When these preformulation compositions are referred to as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition, allowing the composition to be easily divided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformulation composition is then divided into unit dosage forms of the type described above containing from 0.1 mg to about 500 mg of the active ingredient of the present disclosure. Tablets or pills of the novel compositions may be coated or otherwise compounded to provide a dosage form offering the advantage of prolonged action. For example, the tablets or pills may comprise an inner dosage and an outer dosage component, the latter in the form of an envelope surrounding the former. The two components may 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.

[0236] Suitable surfactants include, in particular, non-ionic agents such as polyoxyethylene sorbitan (e.g., Tween™ 20, 40, 60, 80, or 85) and other sorbitan (e.g., Span™ 20, 40, 60, 80, or 85). Compositions that include a surfactant conveniently contain 0.05 to 5% surfactant, and may be 0.1 to 2.5%. It will be appreciated that other ingredients, such as mannitol or other pharmaceutically acceptable vehicles, may be added as required.

[0237] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™, and Lipiphysan™. The active ingredient can be dissolved in a premixed emulsion composition, or alternatively, in an emulsion formed by mixing 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) with 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, for example, 5 to 20%.

[0238] The emulsion composition may be prepared by mixing an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex with Intralipid™ and its components (soybean oil, egg phospholipids, glycerol, and water).

[0239] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0240] Compositions, preferably in sterile, pharmaceutically acceptable solvents, may be nebulized by the use of gases. Nebulized solutions may be breathed directly 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 from devices that deliver the formulation in an appropriate manner, preferably orally or nasally.

[0241] Uses of antibodies and antigen-binding portions thereof that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex In some 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. In some cases, such diseases, disorders, and / or conditions can be TGFβ-related indications. As used herein, the term "TGFβ-related indications" refers to any disease, disorder, and / or condition associated with the expression, activity, and / or metabolism of a TGFβ family member protein, or any disease, disorder, and / or condition in which modulation of the activity and / or levels of one or more TGFβ family member proteins can be beneficial. TGFβ-related indications include, but are not limited to, fibrosis, cancer (including but not limited to colon cancer, renal cancer, breast cancer, malignant melanoma, and glioblastoma), promoting rapid hematopoiesis after chemotherapy, bone healing, wound healing, dementia, myelofibrosis, kidney disease, unilateral ureteral obstruction (UUO), tooth loss and / or degeneration, endothelial proliferative syndrome, asthma and allergies, gastrointestinal disorders, age-related anemia, aortic aneurysm, rare indications (such as Marfan syndrome and Kamuracchi-Engelman disease), obesity, diabetes, arthritis, and multiple sclerosis. Additional indications may include muscular dystrophy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, metabolic syndrome, malnutrition, organ atrophy, chronic obstructive pulmonary disease (COPD), and anorexia. Additional indications may include any of the indications disclosed in U.S. Patent Application Publication No. 2013 / 0122007, U.S. Patent No. 8,415,459, or International Patent Application Publication No. WO2011 / 151432, the contents of each of which are incorporated herein by reference in their entirety.

[0242] fibrosis In some embodiments, the antibodies and / or compositions of the present disclosure may be useful for altering fibrosis. In some embodiments, such antibodies and / or compositions are antagonists of TGFβ (e.g., TGFβ1). TGFβ1 is recognized as a central orchestrator of the fibrotic response. In numerous preclinical models, antibodies targeting TGFβ1 reduce fibrosis. Examples of such antibodies and / or antibody-based compounds include LY2382770 (Eli Lilly, Indianapolis, IN). Also included are those described in U.S. Patent Nos. 6,492,497, 7,151,169, 7,723,486, and U.S. Patent Application Publication No. 2011 / 0008364, the entire contents of each of which are incorporated herein by reference.

[0243] 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 disorder (COPD), allergic asthma, acute lung injury, eosinophilic esophagitis, pulmonary arterial hypertension, and chemical gas-injury), renal indications (e.g., diabetic glomerulosclerosis, focal segmental glomerulosclerosis (FSGS), chronic kidney disease, fibrosis associated with renal 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, toxin-mediated fibrosis such as alcoholic fibrosis, nonalcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC), primary fibrosis, and fibrosis associated with pulmonary arterial hypertension, including pulmonary fibrosis, pulmonary arterial hypertension, and pulmonary arterial hypertension), and hepatocellular carcinoma (HCC). biliary cirrhosis, and sclerosing cholangitis), cardiovascular fibrosis (e.g., cardiomyopathy, hypertrophic cardiomyopathy, atherosclerosis and restenosis), systemic sclerosis, dermal fibrosis (e.g., dermal fibrosis in systemic sclerosis, diffuse cutaneous systemic sclerosis, scleroderma, pathological skin scars, keloids, postoperative scars, scar revision surgery, 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 associated with fibrosis that can be treated using the compounds and / or compositions of the present disclosure include, but are not limited to, Marfan syndrome, stiff skin syndrome, scleroderma, rheumatoid arthritis, myelofibrosis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, muscular dystrophies (e.g., DMD), Dupuytren's contracture, Kamraci-Engelmann disease, neural scarring, dementia, proliferative vitreoretinopathy, corneal injury, complications following glaucoma drainage surgery, and multiple sclerosis. Many such fibrotic indications are also accompanied by inflammation of the affected tissue(s), which indicates the involvement of an immune component.

[0244] The antibodies described herein can be used to treat fibrosis. In some embodiments, the TGFβ1 isoform-specific agent is administered to a 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, such an antibody is a context-specific antibody capable of blocking TGFβ1 activation mediated by TGFβ1 associated with ECM containing LTBP. In some embodiments, the LTBP is LTBP1 and / or LTBP3. In some embodiments, the antibody is a context-permissive antibody capable of blocking ECM-localized LTBP-mediated TGFβ1 activation and immune cell-localized GARP-mediated TGFβ1 activation. In some embodiments, the antibody is a context-permissive antibody capable of blocking ECM-localized LTBP-mediated TGFβ1 activation and monocyte / macrophage-localized LRRC33-mediated TGFβ1 activation. In some embodiments, the LTBP is LTBP1 and / or LTBP3. In some embodiments, it may be beneficial to target and inhibit TGFβ1 presented by LRRC33 on profibrotic M2-like macrophages in the fibrotic microenvironment.

[0245] Assays useful in determining the efficacy of antibodies and / or compositions of the present disclosure to alter fibrosis include, but are not limited to, histological assays for enumerating fibroblasts and basic immunohistochemical analyses known in the art.

[0246] cancer The antibodies and / or compositions of the present disclosure can be used to treat various cancers. As used herein, the term "cancer" refers to any of a variety of malignant neoplasms characterized by the proliferation of undifferentiated cells that tend to infiltrate surrounding tissues and metastasize to new body sites, and also refers to pathological conditions characterized by the proliferation of such malignant neoplasms. Cancer can be a tumor or a hematological malignancy, including, but 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, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, tailbone, testicles, thyroid gland and uterus.

[0247] In cancer, TGFβ (e.g., TGFβ1) can be either growth-promoting or growth-inhibitory. For example, in pancreatic cancer, SMAD4 wild-type tumors may undergo growth inhibition in response to TGFβ, but as the disease progresses, constitutively activated type II receptors typically exist. Additionally, SMAD4-null pancreatic cancers exist. In some embodiments, the antibodies, antigen-binding portions thereof, and / or compositions of the present 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 abnormal proliferation of white blood cells, or 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)], translocations between chromosomes 8 and 21 [t(8;21)], translocations between chromosomes 15 and 17 [t(15;17)], and inversions in chromosome 16 [inv(16)]); multilineage dysplasia, including patients who have previously had myelodysplastic syndrome (MDS) or myeloproliferative disorders that transform to AML). AML can be divided into four major classifications, including: AML with leukemia (myeloid leukemia, leukemia associated with leukemia-associated leukemia); AML and myelodysplastic syndrome (MDS), therapy-related; this category includes patients who have previously received chemotherapy and / or radiation and subsequently developed AML or MDS; d) AML not elsewhere categorized, which includes subtypes of AML that do not fall into the categories above; and e) acute leukemia of ambiguous lineage, which occurs 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).

[0248] Types of carcinoma include, but are not limited to, papilloma / carcinoma, choriocarcinoma, yolk sac tumor, teratoma, adenoma / adenocarcinoma, melanoma, fibroma, 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.

[0249] Types of sarcoma 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 schwannoma, osteosarcoma, and chondrosarcoma.

[0250] 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).

[0251] In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex described herein can be used in a method for treating cancer in a subject in need thereof, comprising administering the antibody or antigen-binding portion thereof to the subject such that the cancer is treated. In certain embodiments, the cancer is colon cancer.

[0252] In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex described herein can be used in methods for treating solid tumors. In some embodiments, the solid tumor can be a desmoplastic tumor, which is generally dense and difficult for therapeutic molecules to penetrate. By targeting components of the ECM of such tumors, such antibodies "loosen" and disrupt the dense tumor tissue, thereby facilitating the access of therapeutic agents to exert their anti-cancer effects. Therefore, additional therapeutic agents, such as any known anti-tumor agents, can be used in combination.

[0253] In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex described herein can be used in methods for inhibiting or reducing solid tumor growth in a subject having a solid tumor, comprising administering the antibody or antigen-binding portion thereof to the subject such that solid tumor growth is inhibited or reduced. In certain embodiments, the solid tumor is a colon cancer tumor. In some embodiments, antibodies or antigen-binding portions thereof useful for treating cancer are isoform-specific, context-permissive inhibitors of TGFβ1 activation. In some embodiments, such antibodies target the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex. In some embodiments, such antibodies target the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, and LTBP3-TGFβ1 complex. In some embodiments, such antibodies target the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex. In some embodiments, such antibodies target the GARP-TGFβ1 complex and the LRRC33-TGFβ1 complex.

[0254] In certain embodiments, an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex described herein is administered to a subject with cancer or a tumor either alone or in combination with an additional agent, such as an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist). Another combination therapy encompassed by the invention is administering an antibody or antigen-binding portion thereof described herein together with radiation or a chemotherapeutic agent. Exemplary additional agents include, but are not limited to, PD-1 antagonists, PDL1 antagonists, PD-L1 or PDL2 fusion proteins, CTLA4 antagonists, GITR agonists, anti-ICOS antibodies, anti-ICOSL antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-TIM3 antibodies, anti-LAG3 antibodies, anti-OX40 antibodies, anti-CD27 antibodies, anti-CD70 antibodies, anti-CD47 antibodies, anti-41BB antibodies, anti-PD-1 antibodies, oncolytic viruses, and PARP inhibitors.

[0255] The role of TGFβ in skeletal muscle condition In skeletal muscle, TGFβ plays various roles, including inhibiting proliferation and differentiation, inducing atrophy, and causing fibrosis. TGFβ reduces satellite cell proliferation and prevents differentiation (by inhibiting MyoD and myogenin) (Allen, RE and LK J Cell Physiol, 1987, 133(3):567-72; Brennan, TJ et al., Proc Natl Acad Sci USA, 1991, 88(9):3822-6; Massague, J. et al., Proc Natl Acad Sci USA, 1986, 83(21):8206-10; Olson, EN et al., J Cell Biol, 1986, 103(5):1799-805). These earlier papers did not identify the TGFβ isoform (i.e., TGFβ1, 2, or 3), but it is presumed to be TGFβ1. TGFβ also contributes to muscle fibrosis; direct injection of recombinant TGFβ1 induces skeletal muscle fibrosis, and pan-TGFβ inhibition reduces fibrosis in acutely and chronically injured muscle (Li, Y. et al., Am J Pathol, 2004, 164(3):1007-19; Mendias, CL et al., Muscle Nerve, 2012, 45(1):55-9; Nelson, CA et al., Am J Pathol, 2011, 178(6):2611-21).TGF-β1 is expressed by myofibers, macrophages, regulatory T cells, fibroblasts, and fibrocytes in skeletal muscle (Li, Y. et al., Am J Pathol, 2004, 164(3):1007-19; Lemos, DR et al., Nat Med, 2015, 21(7):786-94; Villalta, SA et al., Sci Transl Med, 2014, 6(258):258ra142; Wang, X. et al., J Immunol, 2016, 197(12):4750-4761), and expression is increased during injury and disease (Li, Y. et al., Am J Pathol, 2004, 164(3):1007-19; Nelson, CA et al., Am J Pathol, 2011, 178(6):2611-21; Bernasconi, P. et al., J Clin Invest, 1995, 96(2):1137-44; Ishitobi, M. et al., Neuroreport, 2000, 11(18):4033-5. TGF-β2 and TGF-β3 are also upregulated (at the mRNA level) in mdx muscle, although to a lesser extent than TGF-β1 (Nelson, CA et al., Am J Pathol, 2011, 178(6):2611-21; Zhou, L. et al., Neuromuscul Disord, 2006, 16(1):32-8). Pessina et al. recently used lineage tracing experiments to show that multiple cells of origin in dystrophic muscle adopt a fibrogenic fate via a TGFβ-dependent pathway (Pessina, P. et al., Stem Cell Reports, 2015, Vol. 4(6):1046-60).

[0256] TGF-β1 has been implicated in human muscular dystrophy. Duchenne muscular dystrophy (DMD) is a severe, progressive, and ultimately fatal disease caused by the absence of dystrophin (Bushby, K. et al., Lancet Neurol, 2010, 9(1):77-93). Dystrophin deficiency results in increased susceptibility to contraction-induced damage, leading to ongoing muscle degeneration (Petrof, BJ et al., Proc Natl Acad Sci USA, 1993, 90(8):3710-4; Dellorusso, C. et al., J Muscle Res Cell Motil, 2001, 22(5):467-75; Pratt, SJ et al., Cell Mol Life Sci, 2015, 72(1):153-64). Repeated rounds of repair contribute to chronic inflammation, fibrosis, depletion of satellite cell pools, and eventual loss of mobility and death (Bushby, K. et al., Lancet Neurol, 2010, 9(1):77-93; McDonald, C. M. et al., Muscle Nerve, 2013, 48(3):343-56). TGF-β1 expression is significantly increased in patients with DMD and correlates with the degree of fibrosis observed in these patients (Bernasconi, P. et al., J Clin Invest, 1995, 96(2):1137-44; Chen, Y. W. et al., Neurology, 2005, 65(6):826-34). Excessive ECM deposition can have detrimental effects on muscle contractile properties and limit nutrient availability as muscle fibers are isolated from the blood supply (Klingler, W. et al., Acta Myol, 2012, 31(3):184-95). Recently, additional data have further implicated TGFβ1 in muscular dystrophy. Variants of LTBP4 have been found to modulate disease severity in mice and humans.In mice, variants of LTBP4 are protective in mice lacking dystrophin or γ-sarcoglycan (Coley, W.D. et al., Hum Mol Genet, 2016, 25(1):130-45; Heydemann, A. et al., J Clin Invest, 2009, 119(12):3703-12). In humans, two groups independently identified variants of LTBP4 as protective in DMD, delaying the loss of locomotion by several years (Flanigan, K.M. et al., Ann Neurol, 2013, 73(4):481-8; van den Bergen, J.C. et al., J Neurol Neurosurg Psychiatry, 2015, 86(10):1060-5). Although the nature of the genetic variants differs in mice and humans, in both species protective variants result in reduced TGFβ signaling (Heydemann, A. et al., J Clin Invest, 2009, 119(12):3703-12; Ceco, E. et al., Sci Transl Med, 2014, 6(259):259ra144). Many of the functions of TGF-β1 in skeletal muscle biology have been inferred from experiments in which purified active growth factor was injected into animals or added to cells in culture (Massague, J. et al., Proc Natl Acad Sci USA, 1986, 83(21):8206-10; Li, Y. et al., Am J Pathol, 2004, 164(3):1007-19; Mendias, CL et al., Muscle Nerve, 2012, 45(1):55-9). Given the importance of cellular context for specific functions of TGF-β1 (see, e.g., Hinck et al., Cold Spring Harb. Perspect. Biol, 2016, 8(12)), some of the effects observed in these experiments may not reflect the cytokine's intrinsic role(s) in vivo.For example, treatment of human dermal fibroblasts with recombinant TGFβ1, myostatin, or GDF11 results in nearly identical gene expression changes in these cells, despite the fact that the roles of these proteins in vivo are quite different (Tanner, JW, Khalil, A., Hill, J., Franti, M., MacDonnell, SM, Growth Differentiation Factor 11 Potentiates Myofibroblast Activation, Fibrosis: From Basic Mechanisms to Targeted Therapies. 2016: Keystone, CO).

[0257] Many researchers have used inhibitors of TGFβ to clarify the role of growth factors in vivo. Treatment of mdx mice with the pan-TGFβ neutralizing antibody 1D11 clearly resulted in reduced fibrosis (by histology and hydroxyproline content), reduced muscle injury (reduced serum creatine kinase and greater muscle fiber density), and improved muscle function (increased plethysmography, isolated EDL muscle force production, and forelimb grip strength) (Nelson, CA et al., Am J Pathol, 2011, 178(6):2611-21; Andreetta, F. et al., J Neuroimmunol, 2006, 175(1-2):77-86; Gumucio, JP et al., J Appl Physiol (1985), 2013, 115(4):539-45). Furthermore, myofiber-specific expression of a dominant-negative TGF-β type II receptor protects against muscle injury after cardiotoxin injury and in δ-sarcoglycan − / − mice (Accornero, F. et al., Hum Mol Genet, 2014, 23(25):6903-15). The proteoglycan decorin, which is abundant in skeletal muscle and inhibits TGF-β activity, reduces muscle fibrosis in mdx mice and after laceration injury (Li, Y. et al., Mol Ther, 2007, 15(9):1616-22; Gosselin, L. E. et al., Muscle Nerve, 2004, 30(5):645-53). Other molecules with TGFβ inhibitory activity, such as suramin (an antitumor drug) and losartan (an angiotensin receptor blocker), have been effective in ameliorating muscle pathology and reducing fibrosis in mouse models of injury, Marfan syndrome, and muscular dystrophy (Spurney, CF et al., J Cardiovasc Pharmacol Ther, 2011, 16(1):87-95; Taniguti, AP et al., Muscle Nerve, 2011, 43(1):82-7; Bedair, HS et al., Am J Sports Med, 2008, 36(8):1548-54; Cohn, RD et al., Nat Med, 2007, 13(2):204-10).Although all of the above therapeutic agents inhibit TGFβ1 or its signal transduction, none are specific to the TGFβ1 isoform. For example, 1D11 binds to and inhibits TGFβ1, 2, and 3 isoforms (Dasch, JR et al., J Immunol, 1989, vol. 142(5): pp. 1536-41). Suramin inhibits the ability of many growth factors, including TGFβ1 as well as PDGF, FGF, and EGF, to bind to their receptors (Hosang, M., J Cell Biochem, 1985, vol. 29(3): pp. 265-73; Olivier, S. et al., Eur J Cancer, 1990, vol. 26(8): pp. 867-71; Scher, HI and WD Heston, Cancer Treat Res, 1992, vol. 59: pp. 131-51). Decorin also inhibits myostatin activity, both by direct binding and through upregulation of the myostatin inhibitor follistatin (Miura, T. et al., Biochem Biophys Res Commun, 2006, 340(2):675-80; Brandan, E., C. Cabello-Verrugio, and C. Vial, Matrix Biol, 2008, 27(8):700-8; Zhu, J. et al., J Biol Chem, 2007, 282(35):25852-63). Losartan affects additional signaling pathways through its effects on the renin-angiotensin-aldosterone system, including the IGF-1 / AKT / mTOR pathway (Burks, TN et al., Sci Transl Med, 2011, 3(82):82ra37; Sabharwal, R. and MW Chapleau, Exp Physiol, 2014, 99(4):627-31; McIntyre, M. et al., Pharmacol Ther, 1997, 74(2):181-94). Thus, all of these therapies inhibit additional molecules that may contribute to their therapeutic effects, as well as toxicity.

[0258] Given the postulated role of TGFβ in muscle homeostasis, repair, and regeneration, agents such as the monoclonal antibodies described herein that selectively modulate TGFβ1 signaling may be effective in treating muscle fiber injuries, such as those in chronic / genetic muscular dystrophies and acute muscle injury, without the toxicity associated with the more broadly acting TGFβ inhibitors developed to date.

[0259] Thus, the present invention provides methods for treating muscle fiber injury using agents that preferentially modulate a subset, but not all, of TGFβ actions in vivo. Such agents can selectively modulate TGFβ1 signaling ("isoform-specific modulation"). In some embodiments, such agents can also selectively modulate TGFβ1 in specific contexts ("context-specific modulation").

[0260] Muscle fiber repair in chronic muscle diseases The present invention encompasses methods for improving muscle quality and function in DMD patients by limiting fibrosis and contributing to the normalization of muscle morphology and function.Since TGFβ1 also inhibits myogenesis, TGFβ1 blockade may promote the regeneration of dystrophic muscle, which adds additional therapeutic benefits.TGFβ1 inhibitors can be used in combination with dystrophin upregulation therapy, such as Exondys 51 (Eteplirsen).Considering the potential therapeutic benefits of TGFβ1 inhibition in muscular dystrophy, it is important to (1) distinguish the role(s) of TGFβ1 from the role(s) of TGFβ2 and TGFβ3, and (2) clarify the molecular context(s) in which TGFβ1 inhibition is most beneficial. As mentioned above, significant toxicity is associated with pan-TGFβ inhibitors, limiting their clinical use (Anderton, MJ et al., Toxicol Pathol, 2011, 39(6):916-24; Stauber, A. et al., Clinical Toxicology, 2014, 4(3):1-10). It is unclear which TGFβ isoform(s) causes the toxicity. Some of the described toxicity may be due to TGFβ1 inhibition in the immune system. For example, 1D11 significantly reduces the level of fibrosis in the diaphragm, but this treatment also increases the number of CD4+ and CD8+ T cells in the muscle, suggesting an increased inflammatory response during pan-TGFβ inhibition, which may be detrimental with long-term treatment (Andreetta, F. et al., J Neuroimmunol, 2006, 175(1-2):77-86). Indeed, depletion of T cells from muscle ameliorates muscle pathology in mdx mice, suggesting that T cell-mediated inflammatory responses are detrimental to dystrophic muscle (Spencer, MJ et al., Clin Immunol, 2001, 98(2):235-43). The increase in T cell numbers upon 1D11 administration may be due to the effects of TGFβ1 on regulatory T (Treg) cells.Tregs present TGFβ1 on their cell surface via GARP, and release of TGFβ1 from this complex enhances Treg suppressive activity, thus limiting T cell-mediated inflammation (Wang, R. et al., Mol Biol Cell, 2012, 23(6):1129-39; Edwards, JP, AM Thornton, and EM Shevach, J Immunol, 2014, 193(6):2843-9; Nakamura, K. et al., J Immunol, 2004, 172(2):834-42; Nakamura, K., A. Kitani, and W. Strober, J Exp Med, 2001, 194(5):629-44). Indeed, depletion of Tregs using PC61 antibody increases inflammation and muscle injury in the diaphragm of mdx mice, and enhancing the number and activity of Tregs reduces muscle injury (Villalta, SA et al., Sci Transl Med, 2014, 6(258):258ra142). Interestingly, an additional population of immunosuppressive T cells, Tr1 cells, has recently been identified. These cells produce large amounts of TGFβ3, which is required for their suppressive activity (Gagliani, N. et al., Nat Med, 2013, 19(6):739-46; Okamura, T. et al., Proc Natl Acad Sci USA, 2009, 106(33):13974-9; Okamura, T. et al., Nat Commun, 2015, 6:6329). Although the role of Tr1 cells in skeletal muscle is unknown, inhibition of both TGFβ1 and TGFβ3 by 1D11 may have additive pro-inflammatory effects through inhibition of both Treg and Tr1 cells.

[0261] The above structural insights into TGFβ1 latency and activation enable novel approaches to discover drugs that specifically target TGFβ1 activation (Shi, M. et al., Nature, 2011, 474(7351):343-9). The high degree of sequence identity shared among the three mature TGFβ growth factors is not shared in the latent complex, enabling the discovery of antibodies highly specific for pro-TGFβ1. Using a proprietary approach for antibody discovery, we identified antibodies (Ab1 and Ab2) that specifically bind to pro-TGFβ1 (Figure 18A). Using an in vitro co-culture system, we demonstrated that these antibodies inhibit integrin-mediated release of TGFβ1. In this system, fibroblasts derived from human skin or mouse skeletal muscle are the source of latent TGFβ1, a cell line expressing αVβ6, which allows the release of active TGFβ1, which is then measured using a third cell line expressing a SMAD2 / 3-responsive luciferase reporter (Figure 11A-C). One of these antibodies, Ab1, has been tested in vivo and shown efficacy in the UUO (unilateral ureteral obstruction) mouse model of renal fibrosis. In this model, treating mice (n=10) with 9 mg / kg / week of Ab1 prevented the upregulation of TGFβ1-responsive genes (Figure 15) and reduced the extent of fibrosis after injury (by picrosirius red staining) (Figure 16). TGFβ1-specific therapy may have improved efficacy and safety profiles compared to pan-TGFβ inhibitors, which are critical aspects for therapeutics used long-term in the DMD population. TGFβ1 inhibitory antibodies can be used to determine whether specific TGFβ1 inhibition has potential as a therapeutic agent for DMD or other muscle diseases, and to clarify the role of TGFβ1 in skeletal muscle regeneration.

[0262] Chronic vs. acute myofiber injury and optimal treatment options In normal but regenerating muscle after acute injury (e.g., traumatic injury to otherwise healthy muscle or motor neurons), it is thought that initial infiltration of inflammatory macrophages is required to remove damaged tissue and secrete factors (e.g., cytokines) necessary for satellite cell activation. These cells then switch to an M2 phenotype to drive wound resolution.

[0263] In contrast, in chronic conditions, such as diseases including DMD, pro-inflammatory macrophages always predominate, and the switch to M2 never occurs (or at least not efficiently enough), and pro-inflammatory macrophages continue to drive inflammation and muscle injury. In DMD, the NFkB pathway is permanently active, resulting in constitutive inflammation. Therefore, in some embodiments, NFkB inhibitors can be administered to DMD patients to reduce chronic inflammation.

[0264] Therefore, in chronic conditions such as DMD, the therapeutic focus may be on muscle repair as opposed to muscle regeneration. This is because DMD muscle fibers are defective but not destroyed—they are injured by membrane tears, dysregulated calcium transients, and ROS damage from macrophages. In comparison, in the case of injury to healthy muscle, the therapeutic focus may be on regeneration. For example, in a cardiotoxin model, muscle fibers die and need to be regenerated, simulating the process following a traumatic injury such as a crush injury.

[0265] Evidence suggests that LRRC33 is expressed in thioglycollate-induced peritoneal macrophages, which have an M2-like phenotype (characterized by high expression of arginase, no expression of iNOS, and high expression of CD206).

[0266] In situations where LRRC33 is primarily expressed on M2 cells and where TGFβ1 presentation ("context") is important for the wound-healing-promoting effects of these cells, it may be beneficial to activate LRRC33-mediated TGFβ1 to promote repair and / or myogenesis. On the other hand, in situations where LRRC33 is also expressed on pro-inflammatory M1 cells, it may be beneficial to inhibit LRRC33-mediated TGFβ1, especially given that inflammation drives fibrosis in dystrophies such as DMD. Therefore, identifying the disease-relevant source / context of TGFβ1 may be an important step in selecting appropriate modulators of TGFβ signaling, informing what level of selectivity should be considered (e.g., isoform-specific, context-permissive, or context-specific TGFβ1 modulators; TGFβ1 inhibitors or activators, etc.).

[0267] A hallmark of DMD, besides chronic inflammation, is excessive and progressive fibrosis. In advanced disease, fibrosis can be so severe that individual muscle fibers may actually be isolated from their blood supply. Fibrosis also alters muscle contractile properties. In human patients, there is a strong correlation between the degree of TGFβ1 upregulation and fibrosis, and a strong association between the degree of fibrosis and negative mobility outcomes. Thus, in some embodiments, LTBP-proTGFβ1 inhibitors can be administered to dystrophic patients to selectively target ECM-associated TGFβ1 action in the disease to prevent and / or reduce fibrosis. In some embodiments, various isoform-selective and / or context-selective agents described herein can be used to achieve inhibitory signaling of TGFβ1 (e.g., through GARP or LRRC33) to prevent fibrosis and promote myogenesis while sparing undesirable effects on the immune system.

[0268] treatment To practice the methods disclosed herein, an effective amount of the pharmaceutical composition can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation, or topical. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, or lyophilized powders can be nebulized after reconstitution. Alternatively, antibodies or antigen-binding portions thereof that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex can be aerosolized using a fluorocarbon formulation and a metered-dose inhaler, or inhaled as a lyophilized and crushed powder.

[0269] The subject treated by the methods described herein can be a mammal, more preferably a human.Mammals include, but are not limited to, livestock, sport animals, pets, primates, horses, dogs, cats, mice and rats.The human subject in need of treatment can be a human patient who has, is at risk of, or is suspected of having TGFβ-related indications such as those described above.Subjects with TGFβ-related indications can be identified by routine medical examinations, such as clinical examinations, organ function tests, CT scans, or ultrasound.Subjects suspected of having any of these indications can show one or more symptoms of the indication.Subjects at risk of an indication can be subjects who have one or more risk factors for the indication.

[0270] As used herein, the terms "effective amount" and "effective dose" refer to any amount or dose of a compound or composition sufficient to achieve its intended purpose(s), i.e., a desired biological or medical response in a tissue or subject, at an acceptable benefit / risk ratio. For example, in certain embodiments of the present invention, the intended purpose may be to inhibit TGFβ-1 activation in vivo to achieve a clinically meaningful outcome associated with TGFβ-1 inhibition. As will be recognized by those skilled in the art, the effective amount will vary depending on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, the duration of treatment, the nature of concurrent treatments (if any), the particular route of administration, and similar factors within the knowledge and professional opinion of medical professionals. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. It is generally preferred that the maximum dose of the individual components or combinations thereof, i.e., the highest safe dose according to sound medical judgment, be used. However, it will be understood by those skilled in the art that a patient may insist on a lower or tolerable dose for medical reasons, psychological reasons, or virtually any other reason.

[0271] Empirical considerations such as half-life generally contribute to determining dosage. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, may be used to extend the half-life of the antibody and prevent it from being attacked by the host's immune system. The frequency of administration may be determined and adjusted over the course of treatment and is generally, but not necessarily, based on the treatment and / or suppression and / or amelioration and / or delay of TGFβ-related indications. Alternatively, sustained, sustained-release formulations of antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex may also be appropriate. Various formulations and devices for achieving sustained release will be apparent to those skilled in the art and are within the scope of the present disclosure.

[0272] In one example, the dosage of an antibody specifically binding to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex described herein can be empirically determined in an individual given one or more administrations of the antibody. The individual is given increasing dosages of the antagonist. To evaluate efficacy, indicators of TGFβ-related symptoms can be tracked. For example, methods for measuring muscle fiber damage, muscle fiber repair, muscle inflammation levels, and / or muscle fibrosis levels are well known to those skilled in the art.

[0273] The present invention encompasses the recognition that agents capable of isoform-specifically modulating the activation step of TGFβ offer improved safety profiles when used as pharmaceuticals. Accordingly, the present invention includes antibodies and antigen-binding fragments thereof that specifically bind to and inhibit the activation of TGFβ1, but do not bind to TGFβ2 or TGFβ3, thereby conferring specific inhibition of TGFβ1 signaling in vivo while minimizing undesirable side effects resulting from affected TGFβ2 and / or TGFβ3 signaling.

[0274] In some embodiments, the antibodies, or antigen-binding portions thereof, described herein are not toxic when administered to a subject. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein exhibit reduced toxicity when administered to a subject compared to an antibody that specifically binds both TGFβ1 and TGFβ2. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein exhibit reduced toxicity when administered to a subject compared to an antibody that specifically binds both TGFβ1 and TGFβ3. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein exhibit reduced toxicity when administered to a subject compared to an antibody that specifically binds TGFβ1, TGFβ2, and TGFβ3.

[0275] Generally, for administration of any of the antibodies described herein, the initial candidate dosage may be about 2 mg / kg. For purposes of this disclosure, a typical daily dosage may range anywhere from about 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg to 30 mg / kg to 100 mg / kg or more, depending on the factors discussed above. For repeated administration over several days or longer, treatment is sustained until desired symptomatic suppression occurs or until a sufficient therapeutic level is achieved to alleviate the TGFβ-related indication or its symptoms, depending on the condition. An exemplary dosing regimen involves an initial dose of about 2 mg / kg, followed by weekly maintenance doses of about 1 mg / kg of antibody, or followed by maintenance doses of about 1 mg / kg every other week. However, other dosing regimens may be useful depending on the pharmacokinetic decay pattern the clinician wishes to achieve. For example, dosing one to four times per week is contemplated. In some embodiments, doses ranging from about 3 μg / mg to about 2 mg / kg (e.g., about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 2 mg / kg, etc.) may be used. Pharmacokinetic studies have shown that serum concentrations of the antibodies disclosed herein (e.g., Ab2) remain stable for at least 7 days after administration to preclinical animal models (e.g., mouse models). While not wishing to be bound by any particular theory, this stability after administration can be advantageous because it allows the antibody to be administered less frequently while maintaining clinically effective serum concentrations in the subject (e.g., human subject) to whom the antibody is administered. In some embodiments, the administration frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks, or once every month, every 2 months, every 3 months, or longer. The progress of this therapy can be easily monitored by conventional techniques and assays. The dosage regimen (including the antibody used) can be varied over time.

[0276] In some embodiments, for a normal weight adult patient, a dose ranging from about 0.3 to 5.00 mg / kg may be administered. The particular dosing regimen, e.g., dose, timing, and repetition, will depend on the particular individual and their medical history and the characteristics of the individual drug (such as the drug's half-life and other relevant considerations).

[0277] For purposes of this disclosure, the appropriate dosage of an antibody that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex will depend on the specific antibody (or composition thereof) used, the type and severity of the indication, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the antagonist, and the judgment of the attending physician. In some embodiments, a clinician will administer an antibody that specifically binds to a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and / or a LRRC33-TGFβ1 complex until a dosage is reached that achieves the desired result. Administration of an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex, whether the purpose of administration is therapeutic or prophylactic, can be continuous or intermittent, depending, for example, on the physiological condition of the recipient and other factors known to a skilled physician. Administration of an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex can be essentially continuous over a preselected period of time, or in a series of spaced doses, e.g., either before, during, or after the onset of a TGFβ-related indication.

[0278] As used herein, the term "treat" refers to the application or administration of a composition containing one or more active agents to a subject having a TGFβ-related indication, symptoms of the indication, or predisposition to the indication, for the purpose of curing, healing, alleviating, mitigating, altering, remedying, ameliorating, improving, or affecting the indication, symptoms of the indication, or predisposition to the indication.

[0279] Alleviating a TGFβ-related indication using an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex includes delaying the onset or progression of the indication or reducing the severity of the indication. Alleviating an indication does not necessarily require a curative result. As used herein, "delaying" the onset of a TGFβ-related indication means deferring, hindering, slowing, retarding, stabilizing, and / or postponing the progression of the indication. This delay may be for a variety of lengths of time depending on the history of the indication and / or the individual being treated. A method of "delaying" or alleviating the onset of an indication, or delaying the onset of an indication, is a method that reduces the likelihood of onset of one or more symptoms of the indication in a given time frame and / or reduces the severity of the symptoms in a given time frame compared to not using the method. Such comparisons are typically based on clinical studies using a number of subjects sufficient to produce statistically significant results.

[0280] DBA2 / J mice have a 40-bp deletion in the LTBP4 allele. Dysregulation of the ECM associated with latent TGFb1 may expose the epitope to which Ab1 binds. Diseases in which the epitope to which Ab1 binds may be exposed may exist, and these diseases may represent therapeutic opportunities for Ab1 if TGFb1 inhibition is indicated.

[0281] Combination therapy The present disclosure encompasses pharmaceutical compositions and related methods used as combination therapies to treat subjects who may benefit from in vivo TGFβ inhibition. In any of these embodiments, such subjects can receive a combination therapy comprising a first composition containing at least one TGFβ inhibitor, such as an antibody or antigen-binding portion thereof described herein, in combination with a second composition containing at least one additional therapeutic agent intended to treat the same or an overlapping disease or clinical condition. The first and second compositions can act on the same or distinct cellular targets. In some embodiments, the first and second compositions can treat or alleviate the same or overlapping set of symptoms or aspects of a disease or clinical condition. In some embodiments, the first and second compositions can treat or alleviate separate sets of symptoms or aspects of a disease or clinical condition. For example, the first composition can treat a disease or condition associated with TGFβ signaling, while the second composition can treat inflammation or fibrosis associated with the same disease. Such combination therapies can be administered in conjunction with each other. The phrase "in conjunction with," in reference to combination therapy, means that the therapeutic effect of a first therapy overlaps temporally and / or spatially with the therapeutic effect of a second therapy in a subject receiving the combination therapy. Thus, the combination therapy may be formulated as a single formulation for simultaneous administration or as separate formulations for sequential administration of the therapies.

[0282] In a preferred embodiment, the combination therapy produces a synergistic effect in treating the disease. The term "synergistic" refers to an effect (e.g., greater efficacy) that is greater than the additive effect of each monotherapy taken together.

[0283] In some embodiments, a combination therapy comprising a pharmaceutical composition described herein produces overall efficacy comparable to that produced by another therapy (e.g., monotherapy with a second agent), but produces fewer undesirable adverse effects or less severe toxicity associated with the second agent compared to monotherapy with the second agent. In some embodiments, such a combination therapy allows for lower dosages of the second agent while maintaining overall efficacy. Such a combination therapy may be particularly suitable for patient populations where long-term treatment is warranted and / or involving pediatric patients.

[0284] Thus, the present invention provides pharmaceutical compositions and methods for use in combination therapy to reduce TGFβ1 protein activation and to treat or prevent diseases or conditions associated with TGFβ1 signaling as described herein. Thus, the methods or pharmaceutical compositions further include a second therapy. In some embodiments, the second therapy may be useful in treating or preventing diseases or conditions associated with TGFβ1 signaling. The second therapy may attenuate or treat at least one symptom(s) associated with the targeted disease. The first and second therapies may exert their biological effects through similar or unrelated mechanisms of action, or one or both of the first and second therapies may exert their biological effects through multiple mechanisms of action.

[0285] It should be understood that the pharmaceutical compositions described herein may have the first and second therapies for each of the described embodiments in the same pharmaceutically acceptable carrier or in different pharmaceutically acceptable carriers. It should further be understood that the first and second therapies may be administered simultaneously or sequentially within the described embodiments.

[0286] One or more anti-TGFβ antibodies, or antigen-binding portions thereof, of the present invention may be used in combination with one or more additional therapeutic agents. Examples of additional therapeutic agents that may be used with the anti-TGFβ antibodies of the present invention include, but are not limited to, myostatin inhibitors, VEGF agonists, IGF1 agonists, FXR agonists, CCR2 inhibitors, CCR5 inhibitors, dual CCR2 / CCR5 inhibitors, lysyl oxidase-like-2 inhibitors, ASK1 inhibitors, acetyl-CoA carboxylase (ACC) inhibitors, p38 kinase inhibitors, pirfenidone, nintedanib, GDF11 inhibitors, and the like.

[0287] In some embodiments, the additional agent is a checkpoint inhibitor. In some embodiments, the additional agent is selected from the group consisting of a PD-1 antagonist, a PDL1 antagonist, a PD-L1 or PDL2 fusion protein, a CTLA4 antagonist, a GITR agonist, an anti-ICOS antibody, an anti-ICOSL antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-OX40 antibody, an anti-CD27 antibody, an anti-CD70 antibody, an anti-CD47 antibody, an anti-41BB antibody, an anti-PD-1 antibody, an oncolytic virus, and a PARP inhibitor. In some embodiments, the additional treatment is radiation. In some embodiments, the additional agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is taxol. In some embodiments, the additional agent is an anti-inflammatory agent. In some embodiments, the additional agent inhibits the process of monocyte / macrophage recruitment and / or tissue infiltration. In some embodiments, the additional agent is an inhibitor of hepatic stellate cell activation. In some embodiments, the additional agent is a chemokine receptor antagonist, e.g., a CCR2 antagonist and a CCR5 antagonist. In some embodiments, such a chemokine receptor antagonist is a bispecific antagonist, such as a CCR2 / CCR5 antagonist. In some embodiments, the additional agent administered as a combination therapy is or includes a member of the TGFβ superfamily of growth factors or a regulator thereof. In some embodiments, such an agent is selected from modulators (e.g., inhibitors and activators) of GDF8 / myostatin and GDF11. In some embodiments, such an agent is an inhibitor of GDF8 / myostatin signaling. In some embodiments, such an agent is a monoclonal antibody that specifically binds to the pro / latent myostatin complex and blocks myostatin activation. In some embodiments, the monoclonal antibody that specifically binds to the pro / latent myostatin complex and blocks myostatin activation does not bind to free, mature myostatin.

[0288] Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

[0289] Regulation of TGFβ activity The methods of the present disclosure include methods of modulating growth factor activity in one or more biological systems. Such methods may include contacting one or more biological systems with an antibody and / or composition of the present disclosure. In some cases, these methods include modulating the level of free growth factor in the biological system (e.g., in a cellular niche or a subject). The antibody and / or composition from such methods may include, but is not limited to, a biological molecule, including, but not limited to, a recombinant protein, protein complex, and / or antibody or antigen-binding portion thereof described herein.

[0290] In some embodiments, the methods of the present disclosure can be used to reduce or eliminate growth factor activity, referred to herein as "inhibitory methods." Some such methods can involve retaining mature growth factors in TGFβ complexes (e.g., TGFβ1 complexed with GARP, LTBP1, LTBP3, and / or LRRC33) and / or promoting reassociation of growth factors into TGFβ complexes. In some cases, inhibitory methods can involve the use of antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex. According to some inhibitory methods, one or more inhibitory antibodies are provided.

[0291] In some embodiments, the antibodies, antigen-binding portions thereof, and compositions of the present disclosure can be used to inhibit TGFβ1 activation. In some embodiments, provided herein are methods for inhibiting TGFβ1 activation, comprising exposing a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex to an antibody, antigen-binding portion thereof, or pharmaceutical composition described herein. In some embodiments, the antibody, antigen-binding portion thereof, or pharmaceutical composition inhibits the release of mature TGFβ1 from a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed ex vivo.

[0292] In some embodiments, the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, or the LRRC33-TGFβ1 complex is present on the outer surface of a cell. In some embodiments, the cell is a T cell, a fibroblast, a macrophage, a monocyte, or a microglia.

[0293] In some embodiments, the LRRC33-TGFβ1 complex is present on the outer surface of profibrotic (M2-like) macrophages. In some embodiments, profibrotic (M2-like) macrophages are present in a fibrotic microenvironment. In some embodiments, targeting the LRRC33-TGFβ1 complex on the outer surface of profibrotic (M2-like) macrophages provides superior efficacy compared to simply targeting LTBP1-TGFβ1 and / or the LTBP1-TGFβ1 complex.

[0294] In some embodiments, the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex are bound to an extracellular matrix. In some embodiments, the extracellular matrix comprises fibrillin. In some embodiments, the extracellular matrix comprises a protein comprising an RGD motif.

[0295] In some embodiments, provided herein are methods for reducing TGFβ1 protein activation in a subject, comprising administering to the subject an antibody, antigen-binding portion thereof, or pharmaceutical composition described herein, thereby reducing TGFβ1 protein activation in the subject. In some embodiments, the subject has or is at risk of having fibrosis. In some embodiments, the subject has or is at risk of having cancer. In some embodiments, the subject has or is at risk of having dementia.

[0296] In some embodiments, the antibodies, or antigen-binding portions thereof, described herein reduce the suppressive activity of regulatory T cells (Tregs).

[0297] Kits for use in alleviating diseases / disorders associated with TGFβ-related indications The present disclosure also provides kits for use in alleviating a disease / disorder associated with a TGFβ-related indication. Such kits may include one or more containers containing an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex, such as any of those described herein.

[0298] In some embodiments, the kit may include instructions for use in any of the methods described herein. The included instructions may include instructions for administering an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex to treat, delay the onset of, or alleviate a target disease described herein. The kit may further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has the target disease. In yet other embodiments, the instructions include instructions for administering the antibody or antigen-binding portion thereof to an individual at risk for the target disease.

[0299] Instructions for use of an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex generally include information about the dosage, administration schedule, and route of administration for the intended treatment. Containers may be unit dose, bulk packages (e.g., multi-dose packages), or partial unit doses. Instructions provided in kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions written on a magnetic or optical storage disk) are also acceptable.

[0300] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or ameliorating a disease or disorder associated with a TGFβ-related indication. Instructions may be provided for practicing any of the methods described herein.

[0301] The kits of the present disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., atomizers), or injection devices such as minipumps, are also contemplated. The kits may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody or antigen-binding portion thereof that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex described herein.

[0302] Optionally, the kit can provide additional components such as buffers and interpretive information. Typically, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides an article of manufacture including the contents of the kit described above.

[0303] Assays for detecting GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex In some embodiments, the methods and compositions provided herein relate to methods for detecting a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex in a sample obtained from a subject. As used herein, "subject" refers to an individual organism, e.g., an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cow, poultry, cat, or dog. In some embodiments, the subject is a vertebrate, amphibian, reptile, fish, insect, fly, or nematode. In some embodiments, the subject is an experimental animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either gender and at any stage of development. In some embodiments, the subject is a patient or a healthy volunteer.

[0304] In some embodiments, a method for detecting a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex in a sample obtained from a subject includes: (a) contacting the sample with an antibody that specifically binds to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex under conditions suitable for binding of the antibody to the antigen, if the antigen is present in the sample, thereby forming a bound complex; and (b) determining the level of antibody bound to the antigen (e.g., determining the level of the bound complex).

[0305] In one embodiment, the screening assay utilizes a surface-immobilized biotinylated latent TGFβ1 complex, which provides a tether, thereby allowing integrin activation of latent TGFβ. Alternatively, non-integrin activators can be tested in the system. Readout can be via reporter cells or other TGFβ-dependent cellular responses.

[0306] Cell-based assays for measuring TGFβ activation TGFβ activation (and its inhibition by test TGFβ inhibitors, such as antibodies) can be measured by any suitable method known in the art. For example, integrin-mediated activation of TGFβ can be utilized in a cell-based assay, such as the "CAGA12" luciferase assay, described in more detail herein. An exemplary embodiment of such an assay is shown in FIG. 11C for illustrative purposes. As shown, such an assay system can include the following components: i) a source of TGFβ (recombinant, endogenous, or transfected); ii) a source of integrin (recombinant, endogenous, or transfected); and iii) a reporter system responsive to TGFβ activation, such as cells expressing a TGFβ receptor that can respond to TGFβ and convert a signal into a readable output (e.g., luciferase activity in CAGA12 cells or other reporter cell line). In some embodiments, the reporter cell line includes a reporter gene (e.g., a luciferase gene) under the control of a TGFβ-responsive promoter (e.g., the PAI-1 promoter). In some embodiments, certain promoter elements that confer sensitivity can be incorporated into the reporter system. In some embodiments, such a promoter element is a CAGA12 element. Reporter cell lines that can be used in the assay are described, for example, in Abe et al. (1994) Anal Biochem. 216(2):276-84, incorporated herein by reference. In some embodiments, each of the above-described assay components is provided from the same source (e.g., the same cells). In some embodiments, two of the above-described assay components are provided from the same source, and the third assay component is provided from a different source. In some embodiments, all three assay components are provided from different sources. For example, in some embodiments, the integrin and latent TGFβ complex (pro-TGFβ and presentation molecule) are provided for the assay from the same source (e.g., the same transfected cell line).In some embodiments, the integrin and TGF are provided for the assay from separate sources (e.g., two different cell lines, a combination of purified integrin and transfected cells). When cells are used as the source of one or more of the assay components, such assay components may be endogenous to the cells, stably expressed in the cells, transiently transfected, or any combination thereof. Results from a non-limiting exemplary embodiment of a cell-based assay for measuring TGFβ activation, in which inhibition of either the GARP-proTGFβ1 complex or the LRRC33-proTGFβ1 complex using antibodies Ab1 and Ab2 disclosed herein, are shown in Figures 22A and 22B, respectively. In this exemplary assay, the IC50 (μg / mL) of Ab1 for the GARP-TGFβ1 complex was 0.445, and the IC50 (μg / mL) of Ab1 for the LRRC33-TGFβ1 complex was 1.325.

[0307] Those skilled in the art can easily adapt such assays to various suitable configurations. For example, various sources of TGFβ can be considered. In some embodiments, the source of TGFβ is cells (e.g., primary cells, reproductive cells, immortalized cells, or cell lines) in which TGFβ is expressed and deposited. In some embodiments, the source of TGFβ is purified and / or recombinant TGFβ is immobilized in the assay system using suitable means. In some embodiments, the immobilized TGFβ in the assay system is present in an extracellular matrix (ECM) composition on the assay plate, with or without decellularization, to mimic TGFβ of fibroblast origin. In some embodiments, TGFβ is present on the cell surface of the cells used in the assay. Furthermore, selected presentation molecules can be included in the assay system to provide suitable latent TGFβ complexes. Those skilled in the art can easily determine which presentation molecule(s) may be present or expressed in a particular cell or cell type. Using such an assay system, the relative change in TGFβ activation in the presence or absence of a test agent (such as an antibody) can be readily measured to assess the effect of the test agent on TGFβ activation in vitro. Data from an exemplary cell-based assay is presented in the Examples section below.

[0308] Such cell-based assays can be modified or adjusted in several ways depending on the TGFβ isoform, type of latent complex (e.g., presentation molecule), etc. being tested. In some embodiments, cells known to express an integrin capable of activating TGFβ can be used as the source of integrin in the assay. Such cells include SW480 / β6 cells (e.g., clone 1E7). In some embodiments, integrin-expressing cells can be co-transfected with a plasmid encoding a presentation molecule of interest (e.g., GARP, LRRC33, LTBP (e.g., LTBP1 or LTBP3), etc.) and a plasmid encoding a pro-form of a TGFβ isoform of interest (e.g., pro-TGFβ1, etc.). After transfection, the cells are incubated for a sufficient time (e.g., about 24 hours) to allow expression of the transfected gene, washed, and incubated with serial dilutions of a test agent (e.g., an antibody). A reporter cell line (e.g., CAGA12 cells) is then added to the assay system, followed by incubation for an appropriate period of time to allow TGFβ signaling. After an incubation period (e.g., about 18-20 hours) following addition of the test agent, the signal / readout (e.g., luciferase activity) is detected using a suitable means (e.g., for reporter cell lines expressing luciferase, Bright-Glo reagent (Promega) can be used). In some embodiments, luciferase fluorescence can be detected using a BioTek (Synergy H1) plate reader with the autogain setting.

[0309] nucleic acid In some embodiments, the antibodies, antigen-binding portions thereof, and / or compositions of the present disclosure may be encoded by nucleic acid molecules. Such nucleic acid molecules include, without limitation, DNA molecules, RNA molecules, polynucleotides, oligonucleotides, mRNA molecules, vectors, plasmids, etc. In some embodiments, the present disclosure may include cells programmed or generated to express nucleic acid molecules encoding the compounds and / or compositions of the present disclosure. In some cases, the nucleic acids of the present disclosure include codon-optimized nucleic acids. Methods for generating codon-optimized nucleic acids are known in the art and may include, but are not limited to, those described in U.S. Patent Nos. 5,786,464 and 6,114,148, the contents of each of which are incorporated herein by reference in their entirety.

[0310] The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. The entire contents of all references, patents and published patent applications, and figures cited throughout this application are hereby incorporated by reference herein. [Example]

[0311] Example 1: Inhibition of TGFβ1 The TGFβ superfamily contains a propeptide complexed with the active growth factor (Figure 1). We developed a selection strategy to obtain antibodies that stabilize the complex, resulting in more selective and potent inhibition.

[0312] Using a HEK293-based expression system, NiNTA affinity and gel filtration were performed to obtain multimilligram quantities of purified protein, which were then used to generate TGFβ1 complexed with LTBP (LTBP-TGFβ1 complex) and TGFβ1 complexed with GARP (GARP-TGFβ1 complex) (Figure 3). The diversity of proteins produced allowed for species cross-reactivity testing and epitope mapping. Purification of the sGARP-proTGFβ complex (Figures 4A and 4B), sGARP-TGFβ LAP complex (Figure 5), and LTBP1-proTGFβ1 complex (Figure 6) is shown.

[0313] Candidate antibodies were tested using an in vitro luminescence assay (Figure 8). In the screen, antibodies that inhibited growth factor release "turned off" reporter cells in the face of normal activating stimuli. Ab1 and Ab2 were shown to be inhibitors of activation of the latent TGFβ1 complex (Figure 7) and to be cross-reactive in mouse.

[0314] An initial dose-response analysis curve of Ab1 in cells expressing human TGFβ1 demonstrated inhibition of TGFβ1 activity (Figure 8). Using the more sensitive CAGA12 reporter cell line, Ab1 demonstrated similar inhibition of human pro-TGFβ1 activity (Figure 9). Furthermore, inhibition of the GARP complex was shown to block the suppressive activity of regulatory T cells (Tregs), as measured by the percentage of dividing effector T cells (Teffs), in T cells isolated from the blood of healthy donors (Figure 10).

[0315] The affinity of the GARP-proTGFβ1 inhibitors was measured by an Octet assay on human GARP-proTGFβ1 cells, while the activity was measured by CAGA12 reporter cells testing human GARP-proTGFβ1 inhibition. The protocol used to measure the affinity of antibodies Ab1 and Ab2 to the conjugates provided herein is summarized in Table 6. The results are shown in Table 7. [Table 6-1] [Table 6-2] [Table 7]

[0316] Clones were further screened for binding selectivity (Table 8) and species cross-reactivity (Table 9). Ab1 and Ab2 did not bind to TGFβ1, TGFβ2, or TGFβ3, but did bind to the pro-TGFβ1 complex and also exhibited species cross-reactivity. [Table 8] [Table 9]

[0317] Example 2: Ab1 and Ab2 specifically bind to pro-TGFβ1 complexes from multiple species To determine whether Ab1 and Ab2 could specifically bind to proTGFβ1 complexes from multiple species, Octet binding assays were performed as described in Table 6. As shown in Table 10 (below), both antibodies (i.e., Ab1 and Ab2) specifically bound to human LTBP1-proTGFβ1 complexes, mouse LTBP1-proTGFβ1 complexes, human LTBP3-proTGFβ1 complexes, and human GARP-proTGFβ1 complexes. However, only Ab2 specifically bound to the rat LTBP1-proTGFβ1 complex. [Table 10]

[0318] Example 3: Ab1 and Ab2 inhibit endogenous TGFβ1 in human and mouse fibroblasts. To determine whether Ab1 and Ab2 could inhibit endogenous TGF-β1 secreted from cultured primary fibroblasts of different origins, we performed a quantitative in vitro assay in which the activity of secreted TGF-β1 was determined by measuring the luciferase levels produced by mink lung epithelial cells stably transfected with a nucleic acid containing a luciferase reporter gene fused to the CAGA12 synthetic promoter and cocultured with fibroblasts treated with either Ab1 or Ab2. As shown in Figures 11A and 11B, both Ab1 and Ab2 inhibited endogenous TGF-β1 secreted from normal human dermal fibroblasts, mouse C57BL.6J lung fibroblasts, and DBA2 / J myofibroblasts. The differences in maximal inhibition observed with each antibody were cell line-specific.

[0319] Example 4: Ab2 binds to LRRC33-proTGFβ1 To determine whether Ab1 and Ab2 bind to pro-TGFβ1 complexed with LRRC33, an Octet binding assay was performed. As shown in Figures 12A and 12B, both Ab1 and Ab2 can bind to the LRRC33-pro-TGFβ1 protein complex. However, Ab1 exhibits a slow on-rate for binding to the LRRC33-pro-TGFβ1 protein complex. The binding of Ab1 and Ab2 to the LRRC33-pro-TGFβ1 protein complex was further confirmed using ELISA.

[0320] Example 5: Ab1 and Ab2 inhibit the activity of both GARP-proTGFβ1 and LRRC33-proTGFβ1 To determine whether Ab1 and Ab2 inhibited GARP-proTGF-β1 and / or LRRC33-proTGF-β1 activity, an in vitro cell-based assay was performed. In this assay system, an engineered human colon cancer cell line (SW480 / β6 cells) stably transfected with β6 integrin was cotransfected with constructs for expressing proTGF-β1 and a presentation molecule (i.e., GARP or LRRC33). To express the presentation molecule, constructs encoding chimeric LRRC33-GARP (SEQ ID NO: 85) or GARP were used. Transfected cells were incubated to allow sufficient expression and deposition of the components (proTGF-β1 complexed with the integrin and the respective presentation molecule). Activation of TGF-β1 in the presence or absence of Ab1 or Ab2 was assayed using reporter cells (CAGA12 cells) expressing the TGF-β receptor coupled to downstream signal transduction pathways to measure the inhibitory activity of the antibodies. As shown in Figures 13A and 13B, Ab1 and Ab2 inhibited both GARP-proTGF-β1 and LRRC33-proTGF-β1.

[0321] Additional cell-based assays were performed to detect inhibition of either the GARP-proTGFβ1 complex or the LRRC33-proTGFβ1 complex using antibodies Ab1 and Ab2. As shown in Figures 22A and 22B, Ab1 and Ab2 inhibited both GARP-proTGF-β1 and LRRC33-proTGF-β1. In this assay, the IC50 (μg / mL) of Ab1 for the GARP-TGFβ1 complex was 0.445, and the IC50 (μg / mL) of Ab1 for the LRRC33-TGFβ1 complex was 1.325.

[0322] Example 6: Effects of Ab2 on renal biomarkers and fibrosis in a unilateral ureteral obstruction (UUO) mouse model The unilateral ureteral obstruction mouse model has been widely used to examine interstitial fibrosis, a common pathological process that can lead to end-stage renal disease (see Isaka et al. (2008) Contrib. Nephrol. 159:109-21 and Chevalier (1999) Pediatr. Nephrol. 13:612-9). UUO mice are characterized by renal myofibroblast activation, tubular atrophy, and interstitial fibrosis with minimal glomerular lesions (see Lian et al. (2011) Acta Pharmacol. Sin. 32:1513-21). Increased expression of TGFβ1 is thought to play a role in the phenotype observed in UUO mice. To evaluate the effect of Ab2 on the presentation of interstitial fibrosis in the UUO mouse model, the following experiment was performed.

[0323] Briefly, 7-8 week-old male CD-1 mice (Charles River Laboratories) were administered intraperitoneally (ip) to four groups of mice (n = 10) with either Ab2 (3 mg / kg or 30 mg / kg; dose volume 10 mL / kg), a mouse IgG1 control antibody (30 mg / kg; dose volume 10 mL / kg), or PBS as a vehicle control prior to surgical intervention. Treatments were administered 1 day before surgery (d-1), 1 day after surgery (d1), and 3 days after surgery (d3). On day 0 (d0), mice were anesthetized with isoflurane anesthesia using a nose cone, and a laparotomy was performed, followed by a permanent right unilateral UUO surgery. An additional control group of mice (n = 8) received PBS as described above, but underwent sham surgery (i.e., without ureteral obstruction). Immediately after the completion of the surgical procedure, all mice received a single subcutaneous injection of 0.001 mg / kg buprenorphine. Five days after surgery, the mice were sacrificed, and tissues were collected for analysis. After collection, both kidneys were placed in ice-cold 0.9% NaCl, deencapsulated, and weighed. Hydroxyproline levels were assessed to evaluate the collagen content of kidney tissue. As shown in Figure 14, kidney hydroxyproline levels, a marker of tissue fibrosis and collagen deposition, were significantly elevated in mice that underwent surgical intervention compared to mice that underwent sham surgery.

[0324] A midline transverse section of each right kidney was immersion fixed in 10% neutral-buffered formalin for 48 hours and then transferred to 70% ethanol for histological processing and analysis. Fixed kidney sections were paraffin-embedded, sectioned (three 5-µm serial sections taken 200–250 µm apart per kidney of an animal to allow for greater sampling and representation of kidney damage), stained with picrosirius red, and subjected to quantitative histological analysis using color spectral segmentation to determine the cortical collagen volume fraction (CVF). A single composite CVF score was calculated for each animal by determining the average CVF score for each of the three serial sections. Statistical analysis was performed using a one-tailed t-test. As shown in Figure 16, renal cortical fibrosis, as determined by CVF, was increased in UUO-obstructed kidneys compared to control sham-treated mice. Mice receiving 3 mg / kg or 30 mg / kg Ab2 showed a significant attenuation of the UUO-induced increase in CVF compared with mice receiving either the vehicle control (PBS) or the IgG control.

[0325] The relative mRNA expression levels of plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), TGFβ1, fibronectin-1, α-smooth muscle actin (α-SMA), monocyte chemoattractant protein 1 (MCP-1), type I collagen alpha 1 (Col1a1), and type III collagen alpha 1 chain (Col3a1) in the harvested kidney tissues were determined (Figures 15A-15H). The mRNA levels were normalized using the mRNA level of the housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT1). Furthermore, in mice receiving either 3 mg / kg or 30 mg / kg Ab2 before surgical intervention, the mRNA levels of PAI-1, CTGF, TGFβ1, fibronectin-1, Col1a1, and Col3a1 were significantly reduced compared with mice receiving the 30 mg / kg IgG1 control. Mice receiving 3 mg / kg Ab2 before surgical intervention had significantly reduced α-SMA mRNA levels compared with mice receiving 30 mg / kg IgG1 control.Furthermore, mice receiving 30 mg / kg Ab2 before surgical intervention had significantly reduced MCP-1 mRNA levels compared with mice receiving 30 mg / kg IgG1 control.

[0326] In summary, in the UUO mouse model, significant effects were observed in Ab2-treated mice, with the exception of hydroxyproline levels. As shown in Figures 15A–15H and 16, treatment with Ab2 significantly attenuated the UUO-induced increase in CVF and significantly reduced the gene expression of known fibrosis markers, such as PAI-1, CTGF, TGFβ1, fibronectin-1, Col1a1, and Col3a1. These data demonstrate that TGFβ1 is the major form of TGFβ that plays a role in renal disease and, surprisingly, that TGFβ2 and TGFβ3 may not be involved in pathogenesis.

[0327] Example 7: Effect of Ab1 and Ab2 alone or in combination with an anti-PD-1 antibody on tumor progression in the MC38 murine colon cancer syngeneic mouse model To evaluate the effect of Ab1 and Ab2, alone or in combination with anti-PD-1 antibody, on reducing colon cancer tumor progression, the MC38 murine colon carcinoma C57BL / 6 mouse syngeneic model was used.

[0328] Tumor cell culture MC38 mouse colon carcinoma cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 units / mL penicillin G sodium, 100 μg / mL streptomycin sulfate, 25 μg / mL gentamicin, and 2 mM glutamine. Cell cultures were maintained in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air.

[0329] In vivo implantation and tumor growth MC38 cells used for implantation were harvested during logarithmic phase growth and resuspended in phosphate-buffered saline (PBS). On the day of tumor implantation, 5 × 10 cells were implanted into the right flank of each test mouse. 5 10 cells (0.1 mL of cell suspension) were injected subcutaneously, and tumor growth was monitored until the tumor reached an average size of 80–120 mm. 3 After 11 days, designated Day 1 of the study, mice were monitored for tumor size calculations, each reaching an individual tumor volume of 63 mm. 3 from 196mm 3 The mean tumor volume of the group was 95-98 mm 3 Tumors were measured in two dimensions using calipers and calculated using the formula: [ka] The volume was calculated using the formula: where w = tumor width and l = tumor length in mm. Tumor weight was calculated as 1 mg = 1 mm of tumor volume. 3 can be estimated using the assumption that

[0330] treatment Briefly, on day 1, subcutaneous MC38 tumors (63–172 mm) were injected. 3Eight-week-old female C57BL / 6 mice (n=12) bearing tumor-specific IgG1 antibodies were intraperitoneally (ip) administered with Ab1, Ab2, or a mouse IgG1 control antibody (each at 30 mg / kg in a dose volume of 10 mL / kg) twice weekly for 4 weeks. 3 Once this was reached (day 6), mice were administered either a rat anti-mouse PD-1 antibody (RMP1-14) or a rat IgG2A control antibody ip twice weekly for 2 weeks (each antibody at 5 mg / kg in a dosing volume of 10 mL / kg).

[0331] Group 1 served as a tumor growth control and received a combination of a mouse IgG1 isotype control antibody and a rat IgG2a control antibody. Group 2 received a combination of Ab1 and a rat IgG2a control antibody. Group 3 received a combination of Ab2 and a rat IgG2a control antibody. Group 3 received a combination of a mouse IgG1 control antibody and an anti-PD-1 antibody. Group 4 received a combination of Ab1 and an anti-PD-1 antibody. Group 5 received a combination of Ab2 and an anti-PD-1 antibody. Group 6 (n=16) was untreated and served as a sample collection control group.

[0332] Endpoint and tumor growth delay (TGD) analysis Tumors were measured twice weekly using calipers, and for each animal, tumors were measured to reach an endpoint volume of 1,000 mm 3 Mice were euthanized when they reached a tumor volume endpoint or at the end of the study (day 60), whichever came first. Mice terminated due to tumor volume endpoint were recorded as euthanized due to tumor progression (TP) along with the date of euthanasia. For each mouse, the time to endpoint (TTE) for analysis was calculated using the following equation: [ka] (where TTE is expressed in days and endpoint volume is mm 3The TTE was calculated using the logarithmic transformation (TTE) function (where a is the intercept and m is the slope of the line obtained from linear regression of the log-transformed tumor growth dataset). The dataset consisted of the first observation that exceeded the endpoint volume used for analysis and the three consecutive observations immediately before this endpoint volume was achieved. The calculated TTE was usually less than the TP day, which is the day the animal was euthanized due to tumor size. Mice with tumors that did not reach the endpoint volume were assigned a TTE value equal to the last day of the study (day 60). In instances where the log-transformed calculated TTE preceded the day before the endpoint was reached or exceeded the day the tumor volume endpoint was reached, linear interpolation was performed to estimate the TTE. Mice classified as having died from non-treatment-related (NTR) causes were excluded from TTE calculations (and all further analyses). Animals classified as TR (treatment-related) deaths or NTRm (non-treatment-related deaths due to metastases) were assigned a TTE value equal to the day of death.

[0333] Treatment outcome was assessed in terms of tumor growth delay (TGD), defined as the increase in median time to endpoint (TTE) in the treated group compared to the control group: In days: TGD=TC, Or expressed as a percentage of the median TTE in the control group: [ka] (In the formula, T = median TTE for the treatment group, and C = median TTE for the specified control group).

[0334] Criteria for MTV and regression response The efficacy of treatment can be determined from the tumor volumes of the animals remaining in the study on the final day. MTV(n) was defined as the median tumor volume on the final day of the study for the number (n) of remaining animals whose tumors did not reach the endpoint volume.

[0335] Treatment efficacy can also be determined from the incidence and magnitude of regression responses observed during the study. Treatment can result in partial regression (PR) or complete regression (CR) of tumors in animals. A PR response is when tumor volume is 50% or less of its day 1 volume for three consecutive measurements during the course of the study and is 13.5 mm or less for one or more of those three measurements. 3 In CR responses, tumor volumes were equal to or greater than 13.5 mm for three consecutive measurements during the course of the study. 3 Animals with a CR response at the end of the study were further classified as tumor-free survivors (TFS). Animals were monitored for regression responses.

[0336] Tumor growth inhibition The tumor growth inhibition (TGI) assay evaluates the difference in median tumor volume (MTV) between treated and control mice. For this study, the endpoint for determining TGI was a mean tumor volume of 1500 mm in control mice. 3 The median tumor volume (MTV(n)) for n, the number of animals on the day of TGI analysis, was determined for each group. Percent tumor growth inhibition (%TGI) was defined as the difference between the MTV of the designated control group and the MTV of the drug-treated group, expressed as a percentage of the MTV of the control group: [ka]

[0337] The data set for the TGI analysis included all mice in the group except those that died from treatment-related (TR) or non-treatment-related (NTR) causes before the day of TGI analysis.

[0338] In this study, Ab1 and Ab2 were evaluated alone and in combination with anti-PD-1 in a C57BL / 6 mouse syngeneic model of MC38 murine colon carcinoma. Mice treated with Ab2 in combination with anti-PD-1 resulted in a significant 29-day TGI (P<0.05, Mann-Whitney U test), a survival benefit that was statistically significantly different from vehicle-treated controls using log-rank survival analysis (P<0.05, log-rank) (see Figure 17). Mice receiving Ab1 or Ab2 in combination with a rat IgG2a control antibody had regression responses of one CR and one PR, respectively. When combined with anti-PD-1, Ab1 and Ab2 had regression responses of one PR, one CR, and four CR, respectively. Combining Ab2 with anti-PD-1 produced significant short-term efficacy at day 29 and overall survival benefit in a 60-day TGD study in this MC38 murine colon carcinoma C57BL / 6 mouse syngeneic model.

[0339] Example 8: Role of TGFβ1 in muscular dystrophy TGFβ plays multiple roles in skeletal muscle function, including inhibiting myogenesis, regulating inflammation and muscle repair, and promoting fibrosis. TGFβ inhibition has attracted considerable interest as a treatment for a wide range of diseases, including muscular dystrophies; however, these treatments inhibit TGFβ1, TGFβ2, and TGFβ3, regardless of molecular context. The lack of specificity / selectivity of these inhibitors can result in unwanted side effects, leading to ineffective clinical doses. Pan-TGFβ inhibitory molecules have been reported to improve muscle function and reduce fibrosis in mdx mice, but whether these effects are due to inactivation of TGFβ1, β2, or β3 remains to be addressed.

[0340] To this end, an antibody was generated that specifically blocks integrin-mediated activation of latent TGFβ1 while sparing TGFβ2 and β3. To confirm the role of TGFβ1 specifically in muscle repair in dystrophic muscle, D2.mdx mice were treated with a pro-TGFβ1-specific antibody. The functional effects of TGFβ1 inhibition on protection from contraction-induced injury and on recovery from the same injury method were assessed. Histological evaluation included whether treatment affected muscle injury, fibrosis, and inflammation. Furthermore, potential toxicity could be assessed to determine whether the observed negative effects reported with pan-TGFβ inhibition in muscle (e.g., increased inflammation, long-term loss of muscle function) were due to inhibition of TGFβ1 or TGFβ2 / 3. To understand whether inhibition of TGFβ1 in a particular molecular context is more effective and / or has fewer negative (adverse) effects, the efficacy of LTBP-proTGFβ1 inhibitors in this model can be evaluated to deconvolute the role of TGFβ1 presented to immune cells from the role of TGFβ1 present within the extracellular matrix (ECM), potentially leading to safer and / or more effective antifibrotic treatments.

[0341] Dystrophic muscles are highly susceptible to contraction-induced injury. After injury, muscles from mdx mice exhibit significantly reduced force generation and increased Evans Blue dye uptake (an indicator of physical damage / injury to muscle fibers) compared to WT mice (Lovering, RM et al., Arch Phys Med Rehabil, 2007, 88(5):617-25). Therapeutic agents that reduce the extent of contraction-induced damage or improve recovery after injury would be of significant clinical benefit to patients with muscular dystrophy (Bushby, K. et al., Lancet Neurol, 2010, 9(1):77-93). Ab1 and Ab2 will be evaluated for their ability to i) prevent contraction-induced damage and ii) promote recovery from injury. In our experiments, the D2.mdx strain can be used, as opposed to the traditional mdx strain on the B10 background. These mice, generated by crossing mdx with a DBA2 / J background, have the non-protective variant of LTBP4 described above and therefore display disease pathology that is more severe, progressive, and more similar to human disease than the standard mdx strain (Coley, W.D. et al., Hum Mol Genet, 2016, 25(1):130-45). Because D2.mdx mice are used, DBA2 / J mice can serve as wild-type controls. Because DMD primarily affects males, studies can focus on male mice.

[0342] To investigate the ability of Ab1 and Ab2 to prevent / limit contraction-induced damage, 6-week-old male D2.mdx mice (n=10) were treated with either IgG control, Ab1, or Ab2 at 10 mg / kg / week for 6 weeks. To allow for comparison with published studies using pan-TGFβ inhibitors, a fourth group received 1D11 at 10 mg / kg / week. All antibodies were of the mIgG1 isotype, a dose previously shown to be effective in the UUO model (Figures 15 and 16). A WT group receiving the IgG control was also included. To allow for assessment of myofiber injury by fluorescence microscopy, mice were administered 1% Evans Blue Dye (EBD) in PBS (volume 1% of body weight) 24 h before sacrifice. At the end of treatment, mice were subjected to an in vivo eccentric contraction protocol. Stretch injury of the gastrocnemius muscle can be performed using the 305B muscle lever system (Aurora Scientific) as described (Khairallah, RJ et al., Sci Signal, 2012, Vol. 5(236):ra56). Briefly, 20 eccentric contractions with a 1-minute pause between them are performed, and a decrease in peak isometric muscle strength before the eccentric phase can be considered an indicator of muscle injury. The degree of muscle weakness and the percentage of EBD-positive fibers can be determined. DBA2 / J mice subjected to this protocol lose 30-40% of their initial muscle strength after 20 eccentric contractions. In contrast, as previously described, D2.mdx mice lose 80% of their initial muscle strength after the same protocol (Pratt, SJ et al., Cell Mol Life Sci, 2015, 72(1):153-64; Khairallah, RJ et al., Sci Signal, 2012, 5(236):ra56). The ability of Ab1 and Ab2 to reduce muscle loss after injury can be assessed. Mice are sacrificed at the end of the experiment, and both injured and uninjured gastrocnemius muscles can be harvested for histological analysis. EBD uptake can be assessed from both muscles. Myofiber cross-sectional area and the degree of fibrosis can be measured.To determine cross-sectional area, sections from the mid-abdomen of the muscle can be stained with wheat germ agglutinin conjugated with a fluorophore to visualize cell membranes. Sections can be digitized using a fluorescence microscope, and predictive software can be used to trace cell boundaries and determine cross-sectional area through unbiased automated measurements. To analyze fibrosis, sections can be stained with picrosirius red (PSR), and the PSR+ area per slide can be calculated by computer.

[0343] The ability of Ab1 and Ab2 to accelerate recovery from contraction-induced injury was assessed. 12-week-old DBA2 / J and D2.mdx mice were subjected to the same eccentric contraction protocol described above. After injury, mice were stratified into treatment groups (n = 10) and administered either IgG control (for WT and D2.mdx mice), 1D11, Ab1, or Ab2 (D2.mdx only). Antibodies were administered at 10 mg / kg / week for the duration of the experiment. Seven and 14 days after injury, maximal peak isometric muscle force, twitch-tetanic ratio, and force-frequency relationship were measured to assess the effect of treatment on recovery from injury. While Ab1 and Ab2 inhibit TGFβ1 release regardless of the presenting molecule, in DMD, selective release of TGFβ1 (i.e., presented by LTBP) from the extracellular matrix is ​​more beneficial because TGFβ1-driven Treg activity is preserved. To address this issue, specific LTBP-proTGFβ1 inhibitory antibodies can also be evaluated for their ability to both prevent contraction-induced injury and accelerate recovery from injury.

[0344] Example 9: Role of TGFβ1 in skeletal muscle regeneration after acute injury The role of TGFβ1 in muscle fiber regeneration, particularly after muscle injury, can be investigated. TGFβ1-specific antibodies can be used in cardiotoxin injury models to determine the role of TGFβ1 specifically during muscle fiber regeneration. Regeneration can be assessed histologically, and functional assessments of muscle strength and quality can be performed. Given the potential benefits of TGFβ1 inhibition on muscle regeneration, a therapy with beneficial effects without the toxicity observed with pan-TGFβ inhibition would be highly beneficial. This would allow for the investigation of the effects of TGFβ1-specific inhibition on satellite cell function, which could provide insights for satellite cell transplantation studies.

[0345] As noted above, TGFβ appears to have multiple effects on muscle biology, including inhibition of myoblast proliferation and differentiation and promotion of atrophy and fibrosis (Allen, RE and LK Boxhorn, J Cell Physiol, 1987, 133(3):567-72; Brennan, TJ et al., Proc Natl Acad Sci USA, 1991, 88(9):3822-6; Massague, J. et al., Proc Natl Acad Sci USA, 1986, 83(21):8206-10; Olson, EN et al., J Cell Biol, 1986, 103(5):1799-805; Li, Y. et al., Am J Pathol, 2004, 164(3):1007-19; Mendias, CL et al., Muscle Nerve, 2012, 45(1):55-9; Nelson, CA et al., Am J Pathol, 2011, 178(6):2611-21). However, these studies used recombinant TGF-β1 in culture or injected into mice, which may result in non-physiological results because the growth factor is removed from its molecular context. Alternatively, researchers have used TGF-β inhibitors that are not selective for TGF-β1.

[0346] To assess the isoform-specific effects of TGFβ1, multiple pro-TGFβ1 antibodies (e.g., Ab1 and Ab2) can be investigated for their ability to affect muscle regeneration after CTX-induced injury. These antibodies are "isoform-specific" and "context-permissive" inhibitors of TGFβ1 activation, and therefore specifically inhibit the release of TGFβ1 from any presenting molecule (as opposed to TGFβ2 or TGFβ3) and do not bind to the mature growth factor (Figure 18A).

[0347] Muscle regeneration can be induced in male DBA2 / J mice (n=10) by CTX injection into the right gastrocnemius muscle. The day before injury, mice can be administered 10 mg / kg of IgG control, 1D11, Ab1, or Ab2. Antibodies continue to be administered weekly until the end of the study. Seven and 14 days after injury, muscle strength measurements can be measured in vivo using a 305C muscle lever system (Aurora Scientific Inc., Aurora, CA). Briefly, plantarflexor muscle contractions...

Claims

1. a TGFβ1 isoform selective inhibitor that does not inhibit either TGFβ2 or TGFβ3; PD-1 or PD-L1 inhibitors that are PD-1 or PD-L1 antibodies or antigen-binding portions thereof a TGFβ1 isoform selective inhibitor for reducing the growth of cancer or solid tumors in a human subject, wherein the TGFβ1 isoform selective inhibitor reduces the growth of the cancer or tumor at a dose that does not cause cardiovascular toxicity; wherein the TGFβ1 isoform selective inhibitor is an anti-TGFβ1 antibody or an antigen-binding portion thereof; wherein the TGFβ1 isoform selective inhibitor binds to the TGFβ1 pro / latent complex and inhibits the release of TGFβ1 from the TGFβ1 pro / latent complex, thereby inhibiting the activation of TGFβ1; and Here, the combination is one in which the TGFβ1 isoform selective inhibitor does not bind to any of the TGFβ2 procomplex, the TGFβ3 procomplex, and free TGFβ1 that is not associated with the TGFβ1 pro / latent complex.

2. 2. The combination of claim 1, wherein the TGFβ1 isoform selective inhibitor and the PD-1 or PD-L1 inhibitor are formulated as a single formulation for simultaneous administration or as separate formulations for sequential administration.

3. The combination according to any one of claims 1 to 2, wherein the cardiovascular toxicity comprises valvular disease.

4. 4. The combination according to any one of claims 1 to 3, wherein the cancer or tumor is found in the colon, anus, bladder, bile duct, bone, brain, breast, cervix, rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovary, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, tailbone, testicles, thyroid gland and uterus.

5. The combination according to any one of claims 1 to 4, wherein the solid tumor is a desmoplastic tumor.

6. The combination according to any one of claims 1 to 5, wherein the antibody or antigen-binding portion thereof is a monoclonal antibody or antigen-binding fragment thereof.

7. The combination of any one of claims 1 to 6, wherein the anti-TGFβ1 antibody or antigen-binding portion thereof binds to a latent complex of TGFβ1, and wherein the latent complex of TGFβ1 comprises TGFβ1 having the sequence of SEQ ID NO: 21 or 25.

8. The combination according to any one of claims 1 to 7, wherein the antibody or antigen-binding portion thereof is of the human IgG1 or IgG4 subtype.

9. The combination according to any one of claims 1 to 8, wherein the antibody or antigen-binding portion thereof is human or humanized.

10. The combination according to any one of claims 1 to 9, wherein the dose that does not cause cardiovascular toxicity is up to 100 mg / kg.

11. The combination of any one of claims 1 to 10, further comprising an additional agent or treatment.

12. The combination of claim 11, wherein the additional treatment is radiation, a chemotherapy agent, or a combination thereof.

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