TGFβ1-binding immunoglobulin and its use

Abstract

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

Description

[Technical Field]

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

[0002] The transforming growth factor β (TGFβ) superfamily of growth factors is involved in several signaling cascades that control diverse biological processes, including inhibition of cell proliferation, tissue homeostasis, extracellular matrix (ECM) remodeling, endothelial-mesenchymal transition (EMT), cell migration and invasion, and immunomodulation / suppression, as well as mesenchymal-epithelial transition, though not limited to these. In relation to ECM remodeling, TGFβ signaling may increase fibroblast populations and ECM deposition (e.g., collagen). In the immune system, TGFβ ligands regulate the function of regulatory T cells as well as the proliferation and homeostasis of immune progenitor cells. In normal epithelial cells, TGFβ is a potent growth inhibitor and cell differentiation promoter. However, as tumors develop and progress, epithelial cells frequently lose their negative growth response to TGFβ. In this context, TGFβ can become a promoter of tumorigenesis, as it can stimulate angiogenesis, alter the stromal environment, and induce local and systemic immunosuppression. For these and other reasons, TGFβ has been a therapeutic target for numerous clinical indications. Despite numerous efforts by various groups to date, the clinical development of TGFβ therapeutics remains challenging.

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

[0004] For example, Anderton et al. (Toxicology Pathology, Vol. 39: pp. 916-924, 2011) reported that small molecule inhibitors of the TGFβ1 (ALK5) receptor induce cardiac valve lesions characterized by bleeding, inflammation, degeneration, and proliferation of valve interstitial cells in preclinical animal models. This toxicity was observed in all cardiac valves at all doses tested. Frazier et al. (Toxicology Pathology, Vol. 35: pp. 284-295, 2007) reported that administration of GW788388, a small molecule inhibitor of the TGFβ1 (ALK5) receptor, induces epiphyseal chondrodysplasia in rats.

[0005] Stauber et al. (J. Clin. Practice, Vol. 4: No. 3, 2014) reported that long-term administration (more than 3 months) of LY2157299, a TGFβ receptor type I kinase inhibitor investigated in relation to certain cancer treatments, caused numerous organ toxicity involving the cardiovascular, gastrointestinal, immune, bone / cartilage, reproductive, and renal systems in rats and dogs.

[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 turbinates after multiple doses in studies using cynomolgus monkeys (Lonning et al., Current Pharmaceutical Biotechnology, Vol. 12: pp. 2176-2189, 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 fresolimmab include the induction of keratosinous acanthomas and / or squamous cell carcinomas of the skin in human cancer patients (see, e.g., Lacour et al., 2015, Cancer Immunol Immunother, Vol. 64: pp. 437-46; Stevenson et al., 2013, OncoImmunology, Vol. 2: No. 8, e. 26218; and London et al., 2011). Additional evidence from clinical trials suggests that in some cases this antibody may accelerate tumor progression (Stevenson et al., 2013, OncoImmunology, Vol. 2: No. 8, e. 26218).

[0007] Therefore, there is a need for novel methods and compositions for modulating TGFβ signaling that can be used to effectively and safely treat TGFβ-related diseases and disorders, 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), Vol. 64: pp. 437-446 [Non-Patent Document 2] Stevenson et al., OncoImmunology (2013), Vol. 2, No. 8, e26218. [Overview of the project] [Means for solving the problem]

[0009] This disclosure relates to the selective modulation of a subset of TGFβ activity. The invention is based, at least in part, on the concept that the lack of isoform specificity in currently known TGFβ antagonists may underlie the toxicity associated with TGFβ inhibition. Indeed, the inventors of this 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 antagonizing multiple TGFβ isoforms (e.g., anti-TGFβ antibodies) are advantageous because neutralization of multiple TGFβ isoforms appears necessary or advantageous to achieve “maximum therapeutic efficacy” (see, e.g., Bedinger et al. (2016), MABS, vol. 8(2): pp. 389-404).

[0010] Contrary to this general teaching, the inventors of this invention instead sought to develop a drug that enables isoform-specific inhibition of TGFβ1, as opposed to inhibition that also affects TGFβ2 and / or TGFβ3, with the aim of eliminating or significantly reducing the toxicity (e.g., adverse effects, side effects) observed with known TGFβ antagonists in vivo. This novel approach is based, at least in part, on the concept that the clinical utility of a TGFβ inhibitor may depend not only on its efficacy but also on its safety. They determined that by being able to fine-tune the target with an unprecedented degree of specificity, both efficacy and safety / tolerability in a clinical setting could be achieved.

[0011] Therefore, the present invention encompasses the recognition that pharmaceuticals that specifically inhibit TGFβ signaling isoforms may result in an improved safety profile compared to drugs 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 doses may be used to avoid toxicity, but these reduced doses may no longer produce sufficient in vivo efficacy. While we do not wish to be bound by any particular theory, such toxicity is expected to result, at least in part, from a lack of isoform specificity / selectivity of the drug.

[0012] Accordingly, in one embodiment, the present invention provides a method for reducing the 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 drugs that evoke activity against a broader range of targets (e.g., more than one isoform of TGFβ). Accordingly, such TGFβ1-specific inhibitors can be administered to subjects in need at therapeutically effective doses without causing adverse effects. Accordingly, such methods broaden the range of dosages in which both efficacy and safety / tolerability can be achieved in patients. Accordingly, the present invention provides a method for treating diseases related to TGFβ1 signaling by administering to a subject an effective dose of a TGFβ inhibitor that is specific to or highly selective for a TGFβ1 isoform. In some embodiments, such TGFβ1 isoform-selective or TGFβ1 isoform-specific inhibitors may be small molecule drugs or biologics (e.g., antibodies). The use of any such inhibitor to reduce the toxicity (e.g., adverse effects or side effects) associated with TGFβ inhibition in a subject is included in the present invention. According to the present invention, the effective dose is within the range of dosages that allows both i) efficacy (e.g., therapeutically beneficial effects); and ii) safety (e.g., adverse effects or side effects are within an acceptable range). In some embodiments, adverse effects may include cardiovascular toxicity, gastrointestinal toxicity, immunotoxicity, osteo / chondrosis toxicity, reproductive toxicity, and nephrotoxicity. In some embodiments, cardiovascular toxicity may include, but is not limited to, cardiac valve lesions, e.g., bleeding, inflammation, degeneration, and proliferation of valve 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] Accordingly, in some embodiments, the present invention provides isoform-specific TGFβ1 inhibitor antibodies or antigen-binding fragments thereof, characterized by selectively inhibiting the step of TGFβ1 activation in vivo without inhibiting the step of TGFβ2 and / or TGFβ3 activation. Such antibodies or fragments can be administered to subjects for whom TGFβ1 inhibition is beneficial in amounts effective enough to achieve clinical efficacy without causing unacceptable or tolerable levels of adverse effects. Accordingly, the present invention teaches TGFβ1 isoform-specific inhibitors that are specifically selected to satisfy both efficacy and safety criteria for treating diseases or conditions related to TGFβ signaling in human patients.

[0014] In related embodiments, the present invention provides a method for producing isoform-specific TGFβ modulators having an improved safety profile (e.g., reduced in vivo toxicity). Such a method requires testing and selecting candidate agents for isoform specificity. In some embodiments, candidate agents are selected for specific activity against TGFβ1 signaling rather than 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 TGFβ1 rather than TGFβ2 and / or TGFβ3 and block its activation. In some embodiments, such antibodies or antigen-binding fragments thereof do not bind to free mature TGFβ1 growth factor that is not associated with a pro / latent complex.

[0015] In another embodiment, the present invention provides compositions and related methods for achieving further fine-tuning of TGFβ signaling by context-dependently regulating TGFβ activation.

[0016] TGFβ is involved in the conferral of several cellular / tissue effects, each of which is mediated in part by interactions with so-called “presentation molecules.” Since the expression of various presentation molecules is cell-type specific or tissue-specific, TGFβ is intended to confer cellular effects depending on its interaction with its particular presentation molecule (i.e., “situation”). Accordingly, in particular, this disclosure provides monoclonal antibodies that selectively bind to TGFβ present in a specific situation (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 a monoclonal antibody specifically binds to one of the following complexes: i) TGFβ1-GARP; ii) TGFβ1-LRRC33; iii) TGFβ1-LTBP1; and iv) TGFβ1-LTBP3. In some embodiments, such a monoclonal antibody specifically binds to two of the following complexes: i) TGFβ1-GARP; ii) TGFβ1-LRRC33; iii) TGFβ1-LTBP1; and iv) TGFβ1-LTBP3. In some embodiments, such a monoclonal antibody specifically binds 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 presenting molecule).

[0017] This disclosure includes a monoclonal antibody that binds to a small latent complex of TGFβ1 (e.g., "C4S").

[0018] This disclosure further provides monoclonal antibodies that selectively target and modulate TGFβ in specific situations. In some embodiments, such monoclonal antibodies inhibit or activate TGFβ in specific situations.

[0019] Accordingly, the present invention provides compositions and methods for modulating (activating or inhibiting) a subset of TGFβ activity. Accordingly, 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 the following: i) GARP-mediated TGFβ activity, ii) LRRC33-mediated TGFβ activity, iii) LTBP1-mediated TGFβ activity, and iv) LTBP3-mediated TGFβ activity. In some embodiments, such a monoclonal antibody specifically modulates one of the following TGFβ-mediated signaling pathways without modulating the other three pathways: i) GARP-mediated TGFβ activity, ii) LRRC33-mediated TGFβ activity, iii) LTBP1-mediated TGFβ activity, and iv) LTBP3-mediated TGFβ activity. In some embodiments, such a monoclonal antibody specifically modulates 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 a monoclonal antibody specifically modulates 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 activity, ii) LRRC33-mediated TGFβ activity, iii) LTBP1-mediated TGFβ activity, and iv) LTBP3-mediated TGFβ activity.In some embodiments, such monoclonal antibodies specifically modulate GARP-mediated TGFβ activity. In some embodiments, such monoclonal antibodies specifically modulate LRRC33-mediated TGFβ activity. In some embodiments, such monoclonal antibodies specifically modulate LTBP1-mediated TGFβ activity. In some embodiments, such monoclonal antibodies specifically modulate LTBP3-mediated TGFβ activity. Thus, the present invention provides a method for context-dependent selective targeting of 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 related to immune dysregulation / dysregulation, conditions related to T cell dysregulation / dysregulation; conditions related to fibrotic features (fibrosis); and / or conditions related to tumors.

[0021] Antibodies or fragments that specifically target the pro / latent TGFβ complex described herein modulate the activation step (i.e., the release of free mature TGFβ growth factor from the inactive latent pre-form complex), as opposed to targeting already released free mature growth factor. Therefore, the mode of action of these modulants depends on the source of TGFβ growth factor in the tissue. Accordingly, identifying the TGFβ source involved in the disease situation is intended to help in selecting agents that effectively modulate TGFβ in the appropriate situation. For example, to treat disease phenotypes with GARP-mediated TGFβ1 activity, it is desirable to select antibodies or fragments that specifically target the GARP-proTGFβ1 complex. To treat disease phenotypes with LRRC33-mediated TGFβ1 activity, it is desirable to select antibodies or fragments that specifically target the LRRC33-proTGFβ1 complex. To treat disease phenotypes with LTBP1-mediated TGFβ1 activity, it is desirable to select antibodies or fragments that specifically target the LTBP1-proTGFβ1 complex. To treat disease phenotypes involving LTBP2-mediated TGFβ1 activity, it is desirable to select an antibody or fragment that specifically targets the LTBP2-pro-TGFβ1 complex. To treat disease phenotypes involving LTBP3-mediated TGFβ1 activity, it is desirable to select an antibody or fragment that specifically targets the LTBP3-pro-TGFβ1 complex. To treat disease phenotypes involving LTBP4-mediated TGFβ1 activity, it is desirable to select an antibody or fragment that specifically targets the LTBP4-pro-TGFβ1 complex. To treat disease phenotypes involving TGFβ1 activity mediated by multiple (e.g., two or more) situations, it is desirable to select an antibody or fragment capable of targeting the corresponding multiple TGFβ1 presentation situations.

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

[0023] While a context-tolerant TGFβ1 modulator can target more than one type of pro / latent TGFβ1 complex (i.e., with different presenting molecules), in some embodiments, such a modulator may prefer one or more contexts to others. Therefore, in some embodiments, a context-tolerant antibody that inhibits TGFβ1 activation will preferentially inhibit TGFβ1 activation mediated by one presenting 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 an antibody is a monoclonal antibody that binds to and inhibits the activation of TGFβ1 associated with LTBP1, TGFβ1 associated with LTBP3, TGFβ1 associated with GARP, and TGFβ1 associated with LRRC33, but has preferential inhibitory activity against TGFβ1 associated with LTBP1- and LTBP-3. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits the activation of TGFβ1 associated with LTBP1, TGFβ1 associated with LTBP3, TGFβ1 associated with GARP, and TGFβ1 associated with LRRC33, but has preferential inhibitory activity against TGFβ1 associated with GARP and TGFβ1 associated with LRRC33. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits the activation of TGFβ1 associated with GARP and TGFβ1 associated with LRRC33, but has 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 against TGFβ1 associated with LRRC33.

[0024] Therefore, according to the present invention, various degrees of selectivity can be produced to target a subset of TGFβ activity. A TGFβ isoform-specific modulator (targeting a single isoform of TGFβ) yields higher selectivity than a pan-TGFβ modulator (targeting multiple or all isoforms of TGFβ). A TGFβ isoform-specific, context-agreeable modulator (targeting multiple contexts of a single isoform of TGFβ) yields greater selectivity than an isoform-specific modulator. A TGFβ isoform-specific, context-agreeable modulator (targeting a single context of a single isoform of TGFβ) yields much higher selectivity than an isoform-specific, context-agreeable modulator.

[0025] Accordingly, in some embodiments, the present invention includes methods for treating diseases related to TGFβ signaling, comprising first identifying or confirming disease-related sources and / or circumstances of TGFβ, and then selecting agents that specifically target a particular subpool of TGFβ in the tissue. Thus, such methods are intended to preferentially modulate disease-related functions of TGFβ while preserving normal functions of TGFβ. In some embodiments, the expression of TGFβ-presenting molecules(s) present in the affected tissue can be evaluated to identify disease-related sources / circumstances of TGFβ. For example, both latent TGFβ1 complexes associated with LTBP and TGFβ1 complexes associated with GARP may be present in diseased tissue, and only the latter of these two may be expressed as the disease phenotype. In that scenario, it is desirable to inhibit GARP-mediated TGFβ1 signaling while maintaining intact LTBP-mediated TGFβ1 signaling. Determining the source / situation of disease-related TGFβ1 can be done using antibodies that specifically bind to latent TGFβ1 complexes containing specific presentation molecules (e.g., GARP, LRRC33, LTBP, etc.). With regard to the treatment of conditions related to T cell regulation / dysregulation, some embodiments of the present invention involve administering a composition containing an effective amount of a monoclonal antibody that modulates GARP-mediated TGFβ action in a subject.

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

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

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

[0029] Aspects of this disclosure relate to immunoglobulins, such as antibodies or their antigen-binding moieties, that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex. The antibodies or their antigen-binding moieties described herein specifically bind to TGFβ1 epitopes available for binding by the antibody or its antigen-binding moiety when TGFβ1 is present in the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex.

[0030] In one embodiment, an isolated antibody or its antigen-binding moiety that specifically binds to an epitope of TGFβ1 is provided herein, which is available for antibody binding when the epitope 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 the antibody does not bind to free mature TGFβ1.

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

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

[0033] In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region containing a complementarity-determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 5, and a light chain variable region containing a CDR3 having the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region containing a complementarity-determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region containing a CDR2 having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region containing a complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 1, and a light chain variable region containing a CDR1 having the amino acid sequence of SEQ ID NO: 7.

[0034] In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable domain containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 13, and a light chain variable domain containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 14.

[0035] In some embodiments, the antibody or its antigen-binding moiety includes a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 13 and a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 14.

[0036] In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region containing a CDR3 having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region containing a CDR3 having the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region containing a CDR2 having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region containing a CDR2 having the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region containing a CDR1 having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region containing a CDR1 having the amino acid sequence of SEQ ID NO: 8.

[0037] In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable domain containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 15, and a light chain variable domain containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 16.

[0038] In some embodiments, the antibody or its antigen-binding moiety includes a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 15 and a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 16.

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

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

[0041] In some embodiments, the antibody or its antigen-binding portion is 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 A dissociation constant (K) for the TGFβ1 epitope is selected from the group consisting of M. D ) has.

[0042] In some embodiments, the antibody or its antigen-binding moiety includes 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 its antigen-binding moiety includes an immunoglobulin heavy chain constant domain of a human IgG1 constant domain or a human IgG4 constant domain. In some embodiments, the antibody or its antigen-binding moiety includes an immunoglobulin heavy chain constant domain of a human IgG4 constant domain. In some embodiments, the antibody or its antigen-binding moiety includes an immunoglobulin heavy chain constant domain of a human IgG4 constant domain having a Ser-to-Pro scaffold substitution that generates an IgG1-like hinge and allows for the formation of interchain disulfide bonds.

[0043] In some embodiments, the antibody or its antigen-binding moiety further comprises an immunoglobulin light chain constant domain including 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, which consist of 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 moiety is a Fab fragment, an F(ab')2 fragment, an scFab fragment, or an scFv fragment.

[0047] In one embodiment, an anti-TGFβ1 antibody or its antigen-binding moiety that competes for binding to the antibody or its antigen-binding moiety described herein is provided herein.

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

[0049] In some embodiments, the antibody or its antigen-binding portion is conjugated with a drug or detectable portion. In some embodiments, the antibody or its antigen-binding portion is conjugated with a drug or detectable portion via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the detectable portion is selected from the group consisting of fluorescent agents, luminescent agents, enzyme agents, and radiopharmaceuticals.

[0050] In one embodiment, a pharmaceutical composition comprising an antibody or its antigen-binding moiety as described herein and a pharmaceutically acceptable carrier is provided herein.

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

[0052] In some embodiments, the antibody or its antigen-binding moiety inhibits the release of mature TGFβ1 from the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, or 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 located on the outer surface of the cell.

[0055] In some embodiments, the cells are T cells, fibroblasts, macrophages, monocytes, dendritic cells, antigen-presenting cells, or microglia.

[0056] In some embodiments, the LTBP1-TGFβ1 complex or the LTBP3-TGFβ1 complex is bound to the extracellular matrix. In some embodiments, the extracellular matrix contains fibrillin. In some embodiments, the extracellular matrix contains proteins containing the RGD motif.

[0057] In another embodiment, a method for reducing TGFβ1 activation in a subject is provided herein, comprising the step of administering to the subject an effective amount of an antibody, an antigen-binding moiety thereof, or a 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 hepatic 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 receives additional treatment. In some embodiments, the additional treatment is selected from the group consisting of 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, or any combination thereof.

[0060] In some embodiments, the antibody or its antigen-binding moiety reduces the inhibitory activity of regulatory T cells.

[0061] In some embodiments, the antibody or its antigen-binding moiety does not induce organ toxicity in the subject. In some embodiments, organ toxicity includes cardiovascular toxicity, gastrointestinal toxicity, immunotoxicity, osteotoxicity, chondrogenicity, reproductive system toxicity, or nephrotoxicity.

[0062] In one embodiment, a method for treating cancer in a subject requiring such treatment is provided herein, comprising the step of administering to the subject an effective amount of an antibody, an antigen-binding moiety thereof, or a pharmaceutical composition described herein, thereby treating the cancer in the subject.

[0063] In another embodiment, a method for reducing tumor growth in a subject requiring such reduction is provided herein, comprising the step of administering to the subject an effective amount of an antibody, an antigen-binding moiety thereof, or a pharmaceutical composition described herein, thereby reducing tumor growth in the subject.

[0064] In some embodiments, an antibody or its antigen-binding moiety is administered in combination with an additional agent or additional treatment. In some embodiments, the additional agent is a checkpoint inhibitor. In some embodiments, the additional agent is selected from the group consisting of PD-1 antagonists, PDL1 antagonists, PD-L1 or PDL2 fusion proteins, CTLA4 antagonists, 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. In some embodiments, the additional treatment is radiotherapy, chemotherapeutic agents, or a combination thereof. In some embodiments, the additional treatment is radiotherapy. 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 is one that 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 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, a monoclonal antibody that specifically binds to the pro / latent myostatin complex and blocks myostatin activation does not bind to free mature myostatin.

[0065] In yet another embodiment, a method for treating renal impairment in a subject requiring such treatment is provided herein, comprising the step of administering to the subject an effective amount of an antibody, an antigen-binding moiety thereof, or a pharmaceutical composition described herein, thereby treating the renal impairment in the subject.

[0066] In one embodiment, nucleic acids encoding an antibody or an antigen-binding portion thereof are provided herein. A vector containing said nucleic acid is also provided.

[0067] In another embodiment, cells comprising nucleic acids encoding the antibody or its antigen-binding moiety as described herein are provided herein. Cells comprising vectors comprising nucleic acids encoding the antibody or its antigen-binding moiety as described herein are also provided herein.

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

[0069] In another embodiment, a method for treating muscle fiber injury is provided herein, comprising the step of administering to a subject having muscle fiber injury an agent that selectively inhibits TGFβ1 compared to TGFβ2 / 3 in an amount effective to i) promote muscle fiber repair; ii) protect from contraction-induced injury; iii) reduce muscle inflammation; and / or iv) reduce muscle fibrosis. In some embodiments, the amount does not cause an unacceptable level of adverse effect in the subject.

[0070] In some embodiments, muscle fiber injury is associated with i) muscular dystrophy; or ii) acute muscle injury. In some embodiments, the agent blocks the 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 the GARP-proTGFβ1 latent complex, LRRC33-proTGFβ1 latent complex, LTBP1-proTGFβ1 latent complex, LTBP2-proTGFβ1 latent complex, LTBP3-proTGFβ1 latent complex, and / or LTBP4-proTGFβ1 latent complex. In some embodiments, the subject further receives a myostatin inhibitor.

[0071] In some embodiments, the method further includes the step of identifying disease-related sources or circumstances of TGFβ1.

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

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

[0074] In another embodiment, the present invention provides a method for treating a disease related to TGFβ signaling, comprising the step of administering to a subject in need of such treatment a pharmaceutical composition provided in the present invention in an amount effective for treating 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 embodiment, TGFβ inhibitors for use in reducing adverse effects in a subject are provided herein, which are isoform-selective. In some embodiments, the TGFβ inhibitor is an antibody that specifically inhibits TGFβ1. In certain embodiments, the following items are provided, for example: (Item 1) An isolated antibody or its antigen-binding moiety that specifically binds to an epitope of TGFβ1, wherein the epitope is a protein complex of the following: GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex If present in two or more of these, they are available for binding by the antibody, The aforementioned antibody does not bind to free mature TGFβ1. The antibody or its antigen-binding portion. (Item 2) The antibody or its antigen-binding portion as described in item 1, wherein the TGFβ1 is latent TGFβ1. (Item 3) The antibody or its antigen-binding portion as described in item 1, wherein the TGFβ1 is pro-TGFβ1. (Item 4) The antibody described in any one of items 1 to 3, wherein the antibody does not bind to TGFβ2, or the antigen-binding portion thereof. (Item 5) An antibody or its antigen-binding portion described in any one of items 1 to 4, which does not bind to TGFβ3. (Item 6) An antibody or its antigen-binding moiety as described in any one of items 1 to 5, which does not interfere with the ability of TGFβ1 to bind to integrin. (Item 7) An antibody or its antigen-binding moiety according to any one of items 1 to 6, comprising a heavy chain variable region containing complementarity-determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 5, and a light chain variable region containing CDR3 having the amino acid sequence of SEQ ID NO: 11. (Item 8) An antibody or its antigen-binding moiety according to any one of items 1 to 7, comprising a heavy chain variable region containing a complementarity-determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region containing a CDR2 having the amino acid sequence of SEQ ID NO: 9. (Item 9) An antibody or its antigen-binding moiety according to any one of items 1 to 8, comprising a heavy chain variable region containing complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 1, and a light chain variable region containing CDR1 having the amino acid sequence of SEQ ID NO: 7. (Item 10) An antibody or its antigen-binding moiety according to any one of items 1 to 9, comprising a heavy chain variable domain containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 13, and a light chain variable domain containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 14. (Item 11) An antibody or its antigen-binding moiety according to any one of items 1 to 10, comprising a heavy chain variable domain containing the amino acid sequence described in SEQ ID NO: 13 and a light chain variable domain containing the amino acid sequence described in SEQ ID NO: 14. (Item 12) An antibody or its antigen-binding moiety according to any one of items 1 to 6, comprising a heavy chain variable region containing a CDR3 having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region containing a CDR3 having the amino acid sequence of SEQ ID NO: 12. (Item 13) An antibody or its antigen-binding moiety according to any one of items 1 to 6 and 12, comprising a heavy chain variable region containing a CDR2 having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region containing a CDR2 having the amino acid sequence of SEQ ID NO: 10. (Item 14) An antibody or its antigen-binding moiety according to any one of items 1 to 6, 12, and 13, comprising a heavy chain variable region containing a CDR1 having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region containing a CDR1 having the amino acid sequence of SEQ ID NO: 8. (Item 15) An antibody or an antigen-binding portion thereof according to 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) An antibody or an antigen-binding portion thereof according to 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) An antibody or an antigen-binding portion thereof according to any one of items 1 to 16, which inhibits TGFβ1 activation. (Item 18) An antibody or an antigen-binding portion thereof according to 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 ) for the epitope of TGFβ1 is 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 M, and is selected from the group consisting of, an antibody or an antigen-binding portion thereof according to any one of items 1 to 18. (Item 20) An antibody or an antigen-binding portion thereof according to 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) An antibody or its antigen-binding moiety according to any one of items 1 to 20, comprising the immunoglobulin heavy chain constant domain of the human IgG4 constant domain. (Item 22) The antibody or its antigen-binding moiety as described in item 21, comprising a human IgG4 constant domain having a Ser-to-Pro scaffold substitution that generates an IgG1-like hinge and enables the formation of interchain disulfide bonds, the immunoglobulin heavy chain constant domain. (Item 23) An antibody or its antigen-binding moiety according to any one of items 1 to 22, further comprising an immunoglobulin light chain constant domain containing a human Ig lambda constant domain or a human Ig kappa constant domain. (Item 24) An antibody as described in any one of items 1 to 23, which is an IgG having four polypeptide chains, two heavy chains and two light chains. (Item 25) An antibody described in any one of items 1 through 24, which is a humanized antibody, a diabody, or a chimeric antibody. (Item 26) A humanized antibody, as described in any one of items 1 through 25. (Item 27) A human antibody, as described in any one of items 1 through 26. (Item 28) An antibody according to any one of items 1 to 27, comprising a framework having a human germline amino acid sequence. (Item 29) An antigen-binding moiety as described in any one of items 1 through 28, which is a Fab fragment, an F(ab')2 fragment, an scFab fragment, or an scFv fragment. (Item 30) An anti-TGFβ1 antibody or its antigen-binding moiety that competes for binding with the antibody or its antigen-binding moiety described in any one of items 1 to 29. (Item 31) An anti-TGFβ1 antibody or its antigen-binding moiety that binds to the same epitope as the antibody or its antigen-binding moiety described in any one of items 1 to 29. (Item 32) A pharmaceutical composition comprising an antibody or its antigen-binding moiety as described in any one of items 1 to 31 and a pharmaceutically acceptable carrier. (Item 33) A method for inhibiting TGFβ1 activation, comprising the step of exposing a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, or an LRRC33-TGFβ1 complex to an antibody or its antigen-binding moiety as described in any one of items 1 to 31, or a pharmaceutical composition as described in item 32. (Item 34) The method according to item 33, wherein the antibody or its antigen-binding moiety 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) The method described in item 33 or 34, performed in vitro. (Item 36) The method described in item 33 or 34, performed in vivo. (Item 37) The method according to any one of items 33 to 34, wherein the GARP-TGFβ1 complex or the LRRC33-TGFβ1 complex is present on the outer surface of the cell. (Item 38) The method according to item 37, wherein the cells are T cells, fibroblasts, macrophages, monocytes, dendritic cells, antigen-presenting cells, or microglia. (Item 39) The method according to any one of items 33 to 36, wherein the LTBP1-TGFβ1 complex or the LTBP3-TGFβ1 complex is bound to the extracellular matrix. (Item 40) The method according to item 39, wherein the extracellular matrix comprises fibrillin. (Item 41) The method according to 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 the step of administering to the subject an effective amount of an antibody or its antigen-binding moiety described in any one of items 1 to 31 or a pharmaceutical composition described in item 32, thereby reducing TGFβ1 activation in the subject. (Item 43) The method according to item 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) The method according to item 43, wherein the subject has or is at risk of having hepatic fibrosis, renal fibrosis, or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis). (Item 45) The method according to item 44, wherein the subject further receives 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) The method according to any one of items 42 to 45, wherein the antibody or its antigen-binding portion reduces the inhibitory activity of regulatory T cells. (Item 47) The method according to any one of items 33 to 46, wherein the antibody or its antigen-binding portion does not induce organ toxicity in the subject. (Item 48) The method according to item 47, wherein the organ toxicity includes cardiovascular toxicity, gastrointestinal toxicity, immunotoxicity, osteotoxicity, cartilage toxicity, reproductive system toxicity, or nephrotoxicity. (Item 49) A method for treating cancer in a subject requiring treatment for cancer, comprising the step of administering to the subject an effective amount of an antibody or antigen-binding portion thereof as described in any one of items 1 to 31 or a pharmaceutical composition as described in item 32, thereby treating the cancer in the subject. (Item 50) The method according to item 49, wherein the antibody or its antigen-binding portion is administered in combination with an additional drug or additional treatment. (Item 51) The method according to item 49, wherein the additional drug is a checkpoint inhibitor. (Item 52) The method according to item 49, wherein the additional agent is selected from the group consisting of 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. (Item 53) The method according to item 49, wherein the additional treatment is radiation, chemotherapy, or a combination thereof. (Item 54) A nucleic acid encoding an antibody or its antigen-binding portion as described in any one of items 1 through 31. (Item 55) A kit comprising an antibody or its antigen-binding moiety as described in any one of items 1 through 31, or a pharmaceutical composition as described in item 32, and instructions for its use. (Item 56) A method for treating muscle fiber injury, wherein a subject with muscle fiber injury is administered a drug that selectively inhibits TGFβ1 relative to TGFβ2 / 3. i) Promote muscle fiber repair; ii) Protect from contraction-induced injury; iii) Reduce muscle inflammation; and / or, iv) Reduce muscle fibrosis A method comprising the step of administering an effective amount. (Item 57) The method according to item 56, wherein the amount does not cause an unacceptable level of adverse effect in the subject. (Item 58) The aforementioned muscle fiber injury, i) related to muscular dystrophy; or ii) Related to acute muscle injury, The method described in item 56 or 57. (Item 59) The method according to any one of items 56 to 58, wherein the drug blocks the activation of TGFβ1 but does not block the activation of TGFβ2 or TGFβ3. (Item 60) The method according to any one of items 56 to 59, wherein the drug is a monoclonal antibody. (Item 61) The method according to item 60, wherein the monoclonal antibody binds to the GARP-proTGFβ1 latent complex, LRRC33-proTGFβ1 latent complex, LTBP1-proTGFβ1 latent complex, LTBP2-proTGFβ1 latent complex, LTBP3-proTGFβ1 latent complex, and / or LTBP4-proTGFβ1 latent complex. (Item 62) The method according to any one of items 56 to 61, wherein the subject further receives a myostatin inhibitor. (Item 63) Steps to identify disease-related sources or circumstances of TGFβ1 The method described in any one of items 56 to 62, further including the method described in any one of items 56 to 62. (Item 64) A method for producing a pharmaceutical composition that modulates TGFβ signaling, A step of providing one or more agents that modulate the signaling of at least one isoform of TGFβ; A step of measuring the activity of one or more agents against all isoforms of TGFβ; A step of selecting a drug that is specific to a single isoform of TGFβ; Step 1: Formulating a pharmaceutical composition comprising an isoform-specific TGFβ modulator and pharmaceutically acceptable excipients. Methods that include... (Item 65) The method according to item 64, wherein the isoform-specific TGFβ modulator is a TGFβ1-specific modulator. (Item 66) The method according to item 65, wherein the TGFβ1-specific modulator is a TGFβ1 inhibitor. (Item 67) The method according to item 64, wherein the isoform-specific TGFβ modulator is an antibody or a fragment thereof. (Item 68) The method according to item 67, wherein the antibody or fragment thereof specifically binds to the pro / latent complex of TGFβ1. (Item 69) The method according to 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) The method according to item 68, wherein the pro / latent complex comprises GARP, LRRC33, LTBP1, LTBP2, LTBP3, or LTBP4. (Item 71) A pharmaceutical composition produced by the method described in item 64. (Item 72) A method for treating diseases related to TGFβ signaling, The step of administering to a subject in need of it a pharmaceutical composition described in item 71 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) A TGFβ inhibitor for use in reducing adverse effects in a subject, wherein the TGFβ inhibitor is isoform selective. (Item 74) The use described in item 73, wherein the TGFβ inhibitor is an antibody that specifically inhibits TGFβ1. [Brief explanation of the drawing]

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

[0077] [Figure 2] Figures 2A and 2B are schematic diagrams illustrating niche regulation in the microenvironment. Figure 2A shows latent transforming growth factor beta-binding protein (LTBP) presenting TGFβ in the fibrous disease niche and wound healing niche. Figure 2B illustrates how TGFβ activation in the inflammatory niche is regulated by glycoprotein-A repetitions predominant protein (GARP).

[0078] [Figure 3] Figure 3 illustrates a protein expression platform for producing GARP-TGFβ1 and LTBP-TGFβ1 complexes. The HEK293-based expression system uses NiNTA affinity purification and gel filtration to obtain several milligrams of purified protein. Schematic diagrams of wild-type pro-TGFβ1, LTBP1, sGARP, and pro-TGFβ1 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 diagram 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 diagram illustrating the complex.

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

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

[0082] [Figure 7] Figure 7 is a graph showing that the activation of TGFβ1 activity is blocked by Ab1 and Ab2.

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

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

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

[0086] [Figure 11-1] Figures 11A-11C show inhibition of integrin-mediated TGFβ1 release from fibroblasts. Figures 11A and 11B are graphs showing inhibition of endogenous TGFβ1 in normal human dermal fibroblasts (circles), normal human lung fibroblasts (squares), mouse C57BL / 6J lung fibroblasts (inverted triangles), or mouse DBA2 / J myofibroblasts (circles) using either Ab1 (Figure 11A) or Ab2 (Figure 11B) at gradually increasing concentrations. Figure 11C presents a schematic diagram of the co-culture assay system. [Figure 11-2] Figures 11A-11C show inhibition of integrin-mediated TGFβ1 release from fibroblasts. Figures 11A and 11B are graphs showing inhibition of endogenous TGFβ1 in normal human dermal fibroblasts (circles), normal human lung fibroblasts (squares), mouse C57BL / 6J lung fibroblasts (inverted triangles), or mouse DBA2 / J myofibroblasts (circles) using either Ab1 (Figure 11A) or Ab2 (Figure 11B) at gradually increasing concentrations. Figure 11C presents a schematic diagram of the co-culture 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 the inhibition of the GARP-TGFβ1 complex (Figure 13A) or the LRRC33-TGFβ1 complex (Figure 13B) in SW480 / β6 cell transfectants using either Ab1 ("Ab1"), Ab2 ("Ab2"), isotype control IgG1 antibody ("isotype control"), or vehicle control ("vehicle") at increasing concentrations. [Figure 13B]Figures 13A and 13B are graphs showing the inhibition of the GARP-TGFβ1 complex (Figure 13A) or the LRRC33-TGFβ1 complex (Figure 13B) in SW480 / β6 cell transfectants using either Ab1 ("Ab1"), Ab2 ("Ab2"), isotype control IgG1 antibody ("isotype control"), or vehicle control ("vehicle") at increasing concentrations.

[0089] [Figure 14] Figure 14 is a bar graph showing the levels of hydroxyproline in kidney tissue from mice that underwent permanent right-sided UUO surgery and were administered intraperitoneally (ip) with either PBS (control), 30 mg / kg mouse IgG1 control antibody, 3 mg / kg Ab2, or 30 mg / kg Ab2 before surgical intervention (bars 2-5); or mice that were administered PBS and underwent laparotomy (false control; bar 1).

[0090] [Figure 15-1] Figures 15A-15H show the plasminogen activity in kidney tissue from mice that underwent permanent right-sided UUO surgery and were administered intraperitoneally (ip) with either PBS (control), 30 mg / kg mouse IgG1 control antibody, 3 mg / kg Ab2, or 30 mg / kg Ab2 (second to fifth bars in each graph) the day before the surgical intervention, and then one and three days after the surgery; or mice that were administered PBS and underwent laparotomy (false control; first bar in each graph). This bar graph shows the relative mRNA levels of one of the following: sex factor inhibitor-1 (PAI-1; Figure 15A), connective tissue growth factor (CTGF; Figure 15B), 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). This data is representative of multiple experiments. [Figure 15-2]Figures 15A-15H show the plasminogen activity in kidney tissue from mice that underwent permanent right-sided UUO surgery and were administered intraperitoneally (ip) with either PBS (control), 30 mg / kg mouse IgG1 control antibody, 3 mg / kg Ab2, or 30 mg / kg Ab2 (second to fifth bars in each graph) the day before the surgical intervention, and then one and three days after the surgery; or mice that were administered PBS and underwent laparotomy (false control; first bar in each graph). This bar graph shows the relative mRNA levels of one of the following: sex factor inhibitor-1 (PAI-1; Figure 15A), connective tissue growth factor (CTGF; Figure 15B), 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). This data is representative of multiple experiments. [Figure 15-3] Figures 15A-15H show the plasminogen activity in kidney tissue from mice that underwent permanent right-sided UUO surgery and were administered intraperitoneally (ip) with either PBS (control), 30 mg / kg mouse IgG1 control antibody, 3 mg / kg Ab2, or 30 mg / kg Ab2 (second to fifth bars in each graph) the day before the surgical intervention, and then one and three days after the surgery; or mice that were administered PBS and underwent laparotomy (false control; first bar in each graph). This bar graph shows the relative mRNA levels of one of the following: sex factor inhibitor-1 (PAI-1; Figure 15A), connective tissue growth factor (CTGF; Figure 15B), 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). This data is representative of multiple experiments.

[0091] [Figure 16]Figure 16 shows the percentage of composite cortical collagen volume (CVF) from three serial sections of the right kidney collected from mice, stained with picrosilius red and subjected to quantitative histological analysis using color spectral splitting. CVF is derived from mice that underwent permanent right unilateral UUO surgery and were intraperitoneally administered (ip) with 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 to fifth bars in each graph); or mice that were administered PBS and underwent laparotomy ("false"; first bar in each graph).

[0092] [Figure 17] Figure 17 shows the median tumor volume of MC38 mouse colon cancer syngeneic model C57 / BL / 6 mice administered one of the following combinations: mouse IgG1 isotype control antibody and rat IgG2a control antibody (Group 1; control); Ab1 and rat IgG2a control antibody (Group 2); Ab2 and rat IgG2a control antibody (Group 3); mouse IgG1 control antibody and anti-PD-1 antibody (Group 4); Ab1 and anti-PD-1 antibody (Group 5); or Ab2 and 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 pro-TGFβ1 as measured by ELISA, but do not bind to pro-TGFβ2, pro-TGFβ3, or mature TGFβ1. Figure 18B shows an example of purified LTBP-pro-TGFβ1 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-pro-TGFβ1 complex (as measured by ELISA).

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

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

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

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

[0098] [Figure 23]Figure 23 shows microscopic images of hematoxylin-eosin stained sections from heart valves of rats treated with either 200 mg / kg LY2109761 (upper right panel), 100 mg / kg pan-TGFβ antibody ("Pan-TGFβ Ab," lower left panel), or 100 mg / kg Ab2 (lower right panel), or untreated controls (upper left panel). Note: The lower right panel (Ab2) shows oblique sections that are stained more intensely due to uneven thickness. [Modes for carrying out 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 and differentiation factors (GDFs), and three isoforms of the TGFβ family: TGFβ1, TGFβ2, and TGFβ3. TGFβ is thought to play important roles in a variety of processes, including inhibition of cell proliferation, extracellular matrix (ECM) remodeling, and immune homeostasis. The importance of TGFβ1 to T cell homeostasis is demonstrated by the fact that TGFβ1- / - mice survive for only 3-4 weeks and die from multi-organ failure due to potent immune activation (Kulkarni, AB et al., Proc Natl Acad Sci USA, 1993, Vol. 90 (No. 2): pp. 770-774; Shull, MM et al., Nature, 1992, Vol. 359 (No. 6397): pp. 693-699). The roles of TGFβ2 and TGFβ3 are not clearly understood. The three TGFβ isoforms have distinct temporal and spatial expression patterns, while they signal through the same receptors, TGFβRI and TGFβRII. However, in some cases, such as with TGFβ2 signaling, type III receptors such as betaglycans are also required (Feng, XH and R. Derynck, Annu Rev Cell Dev Biol, 2005, Vol. 21: pp. 659-693; Massague, J., Annu Rev Biochem, 1998, Vol. 67: pp. 753-791). 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, Vol. 19 (No. 23): pp. 2783-810).SMAD-independent TGFβ signaling pathways have also been described, for example, in cancer or aortic lesions in Marfan mice (Derynck, R. and YE Zhang, Nature, 2003, Vol. 425 (No. 6958): pp. 577-584; Holm, TM et al., Science, 2011, Vol. 332 (No. 6027): pp. 358-361).

[0100] The biological importance of the TGFβ pathway in humans has been validated by genetic disorders. Kamrachi-Engelmann disease results in osteodysplasia due to an autosomal dominant mutation in the TGFB1 gene, which leads to constitutive TGFβ1 activation signaling (Janssens, K. et al., J Med Genet, 2006, Vol. 43 (No. 1): pp. 1-11). Patients with Loeys / Dietz syndrome have autosomal dominant mutations in components of the TGFβ signaling pathway, causing aortic aneurysm, schizophrenia, and cleft uvula (Van Laer, L., H. Dietz, and B. Loeys, Adv Exp Med Biol, 2014, Vol. 802: pp. 95-105). Since dysregulation of the TGFβ pathway is associated with numerous diseases, several drugs targeting the TGFβ pathway have been developed and tested in patients, but with limited success.

[0101] The inventors of this disclosure have determined that while all three isoforms of TGFβ signal through the same receptor and can transmit downstream effectors in cells expressing that receptor, each TGFβ isoform can produce distinct biological effects in vivo. Furthermore, the inventors consider that, at least in some cases, further signal transduction specificity in vivo may be provided by the mechanism by which growth factor-receptor interactions are induced. Based on this understanding, it should be noted that TGFβ inhibitors described in the literature to date lack specificity, as briefly summarized below.

[0102] Fresolimmab, a humanized monoclonal antibody that binds to and inhibits all three isoforms of TGFβ, has been clinically tested in patients with focal segmental glomerulosclerosis, malignant melanoma, renal cell carcinoma, and systemic sclerosis (Rice, LM et al., J Clin Invest, 2015, Vol. 125 (No. 7): pp. 2795-807; Trachtman, H. et al., Kidney Int, 2011, Vol. 79 (No. 11): pp. 1236-43; Morris, JC et al., PLoS One, 2014, Vol. 9 (No. 3): p. 90353). Further companies have developed monoclonal antibodies against TGFβ growth factor with varying degrees of selectivity for TGFβ isoforms. Such drugs may evoke in vivo toxicity due to residual activity against other TGFβ family members in addition to TGFβ1. To the best of the inventors' knowledge, targeting maturation growth factors has not achieved complete specificity for a single isoform, which is due to the high degree of sequence identity between isoforms.

[0103] Other methods for targeting the TGFβ pathway include ACE-1332 (Yung, LM et al.), a soluble TGFβRII-Fc ligand trap from Acceleron. This includes small molecule inhibitors of ALK5 kinase, such as Lilly's garnicertib (Am J Respir Crit Care Med, 2016, Vol. 194 (No. 9): pp. 1140-1151). On the other hand, ACE-1332 binds to TGFβ1 and TGFβ3 with equally high affinity (Yung, LM et al., Am J Respir Crit). Care Med, 2016, Vol. 194 (No. 9): pp. 1140-1151. ALK5 inhibitors block the activity of all growth factors that signal via TGFR1. Substantial toxicity has been observed in preclinical studies using ALK5 inhibitors (Anderton, MJ et al., Toxicol Pathol, 2011, Vol. 39 (No. 6): pp. 916-924; Stauber, A. et al., Clinical Toxicology, 2014, Vol. 4 (No. 3): pp. 1-10), and a sophisticated clinical drug administration scheme is needed to maintain efficacy while reducing adverse effects (Herbertz, S. et al., Drug Des Devel Ther, 2015, Vol. 9: pp. 4479-4499). Indeed, issues concerning TGFβ signaling specificity and its potential impact on toxicity observed with known TGFβ inhibitors arise in most, though not all, candidate drugs in which TGFβ blockade is being attempted. For example, the extent of toxicity resulting from TGFβ1 inhibition over TGFβ2 and / or TGFβ3 has not been addressed. Similarly, the mechanism of TGFβ activation has not been considered in the design or development of methods for antagonizing TGFβ signaling.

[0104] Recent structural insights into the activation mechanism of TGFβ1 have enabled the inventors to employ novel and more specific methods for TGFβ inhibition (Shi, M. et al., Nature, 2011, Vol. 474 (No. 7351): pp. 343-349). Unlike other cytokines, TGFβ 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-TGFβ1 by furin protease separates the homodimeric growth factor domain from the prodomain, also known as the latent associated peptide (LAP). However, the growth factor and LAP remain associated by non-covalent bonds, forming a latent complex that cannot bind to its receptor and induce signal transduction (Figure 1). During translation, the latent TGFβ1, also known as the small latent complex (SLC), links to the "presenting molecule" via disulfide crosslinking, forming the large latent complex (LLC). These molecules enable pro-TGFβ1 to be presented in specific cellular or tissue conditions. Two cysteine ​​molecules near the N-terminus of latent TGFβ1 bind to cysteine ​​molecules at the appropriate positions on the presenting molecule. The identity of the presenting molecule depends on the environment and the cell type producing latent TGFβ1. For example, fibroblasts secrete latent TGFβ1 anchored to latent TGFβ-binding protein (LTBP), which then associates with proteins in the extracellular matrix (ECM) (i.e., fibronectin, fibrillin-1), linking latent TGFβ to the ECM (Robertson et al., Matrix Biol, Vol. 47: pp. 44-53 (2015) (Figure 2A)). On the surface of activated regulatory T cells, latent TGFβ1 covalently binds to the transmembrane protein GARP (Figure 2B), and recently, the protein LRRC33, which is closely related to GARP, has been identified as a presentation molecule for TGFβ1 on the surfaces of monocytes, macrophages, and microglia (Wang, R. et al., Mol Biol). Cell, 2012, Vol. 23 (No. 6): pp. 1129-1139 and TA Springer, Int. BMP Conference, 2016).

[0105] In mammals, there are four known LTBPs, LTBP1-4, each possessing multiple splice variants (Robertson, IB et al., Matrix Biol, 2015, Vol. 47: pp. 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 (No. 8): pp. 2691-704). While the association of LTBP1 or LTBP3 with latent TGFβ1 has been well-established, the role of LTBP4 in TGFβ presentation remains unclear. The complex of LTBP4 and latent TGFβ1 is thought to be formed with much lower efficiency, 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, Vol. 11 (No. 8): pp. 2691-704; Chen, Y. et al., J Mol Biol, 2005, Vol. 345 (No. 1): pp. 175-86). Disruption of the elastic fiber assembly occurs in both LTBP4S- / - mice and patients with Urban-Rifkin-Davis syndrome who have a null mutation in LTBP4 (Urban, Z. et al., Am J Hum Genet, 2009, Vol. 85 (No. 5): pp. 593-605; Dabovic, B. et al., J Cell Physiol, 2015, Vol. 230 (No. 1): pp. 226-36). Furthermore, LTBP4S- / - mice have pulmonary septal formation and elastogenesis defects, and transgenic mice with LTBP4 that cannot form a complex with latent TGFβ1 do not exhibit a clear phenotype (Dabovic, B. et al., J Cell Physiol, 2015, Vol. 230 (No. 1): pp. 226-36). It is unclear whether LTBP4 is directly involved in latent TGFβ1 regulation by functioning as a presentation molecule; instead, LTBP4 may be necessary for proper formation of elastic fibrils in the extracellular matrix (ECM), and its absence may indirectly affect latent TGFβ1 activation through defects in the ECM.

[0106] Numerous studies have revealed the mechanism of TGFβ1 activation. Three integrins, αVβ6, αVβ8, and αVβ1, have been demonstrated to be important activators of latent TGFβ1 (Reed, NI et al., Sci Transl Med, 2015, Vol. 7 (No. 288): 288ra79; Travis, MA and D. Sheppard, Annu Rev Immunol, 2014, Vol. 32: pp. 51-82; Munger, JS et al., Cell, 1999, Vol. 96 (No. 3): pp. 319-328). αV integrins bind with high affinity to the RGD sequence present in TGFβ1 and TGFβ1 LAP (Dong, X. et al., Nat Struct Mol Biol, 2014, Vol. 21 (No. 12): pp. 1091-1096). Transgenic mice with mutations in the TGFβ1 RGD site that inhibit integrin binding but not secretion phenotypicly mimic TGFβ1- / - mice (Yang, Z. et al., J Cell Biol, 2007, Vol. 176 (No. 6): pp. 787-793). Mice lacking both β6 and β8 integrins reproduce all essential phenotypes of TGFβ1 and TGFβ3 knockout mice, including multi-organ inflammation and cleft palate, thus confirming the essential roles of these two integrins in TGFβ1 activation during development and homeostasis (Aluwihare, P. et al., J Cell Sci, 2009, Vol. 122 (Pt. 2): pp. 227-232). The key to integrin-dependent activation of latent TGFβ1 is covalent anchoring to the presenting molecule; disruption of the disulfide bond between GARP and TGFβ1 LAP by mutagenesis does not impair complex formation, but αVβ6-mediated TGFβ1 activation is completely eliminated (Wang, R. et al., Mol Biol Cell, 2012, Vol. 23 (No. 6): pp. 1129-1139). Recent structural studies of latent TGFβ1 elucidate how integrin-activated TGFβ1 can be released from the latent complex: covalent linkage between latent TGFβ1 and its presenting molecule allows latent TGFβ1 to be fixed to the ECM via LTBP, or to the cytoskeleton via GARP or LRRC33.The binding of integrins to the RGD sequence results in a force-dependent change in the structure of LAP, which in turn releases active TGFβ1, enabling it to bind to nearby receptors (Shi, M. et al., Nature, 2011, Vol. 474 (No. 7351): pp. 343-349). The importance of integrin-dependent TGFβ1 activation in disease has also been thoroughly investigated. Small molecule inhibitors of αVβ1 provide protection from bleomycin-induced pulmonary fibrosis and carbon tetrachloride-induced hepatic fibrosis (Reed, NI et al., Sci Transl Med, 2015, Vol. 7 (No. 288): 288ra79). Furthermore, bleomycin-induced pulmonary fibrosis and radiation-induced fibrosis are suppressed by antibody-based αVβ6 blockade or elimination of integrin β6 expression (Munger, JS et al., Cell, 1999, Vol. 96 (No. 3): pp. 319-328); Horan, GS et al., Am J Respir Crit Care Med, 2008, Vol. 177 (No. 1): pp. 56-65. In addition to integrins, other mechanisms of TGFβ1 activation, including activation by thrombospondin-1 and proteases such as matrix metalloproteinases (MMPs), cathepsin D, or kallikrein, have also been implicated. However, the vast majority of these studies were conducted in vitro using purified proteins; there is little evidence from in vivo studies regarding the role of these molecules. Knockout of thrombospondin-1 reproduces 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, Vol. 44 (No. 4): pp. 556-561). Furthermore, knockout of candidate proteases did not result in the TGFβ1 phenotype (Worthington, JJ, JE Klementowicz, and MA Travis, Trends Biochem Sci, 2011, Vol. 36 (No. 1): pp. 47-54). This could be explained by redundancy or by the fact that these mechanisms are important in specific diseases rather than development and homeostasis.

[0107] TGFβ is associated with several biological processes, including fibrosis, immunomodulation, and cancer progression. TGFβ1 is the first identified member of the TGFβ superfamily of proteins. TGFβ1, as well as its isoforms TGFβ2 and TGFβ3, like other members of the TGFβ superfamily, are initially expressed as an inactive precursor proprotein form (referred to as pro-TGFβ). TGFβ proteins (e.g., TGFβ1, TGFβ2, and TGFβ3) are cleaved by proteolysis by proprotein convertases (e.g., furin) to produce 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 to other molecules in complexes with multiple molecules, including, for example, GARP (to form the GARP-TGFβ1 complex), LRRC33 (to form the LRRC33-TGFβ1 complex), LTBP1 (to form the LTBP1-TGFβ1 complex), and / or LTBP3 (to form the LTBP3-TGFβ1 complex). The TGFβ1 present in these complexes may be in a latent form (latent TGFβ1) or a precursor form (pro-TGFβ1).

[0108] The present invention relates to immunoglobulins, for example, (1) an antibody or its antigen-binding moiety that specifically binds to TGFβ protein (e.g., pro / latent TGFβ1, pro / latent TGFβ2, and pro / latent TGFβ3) in a complex with a GARP protein, (2) an antibody or its antigen-binding moiety that specifically binds to TGFβ protein in a complex with an LTBP protein (e.g., LTBP1 or LTBP3), and / or (3) an antibody or its antigen-binding moiety that specifically binds to TGFβ protein in a complex with an LRRC33 protein. In some embodiments, the antibody or its antigen-binding moiety disclosed herein binds to an epitope of TGFβ1 that is available for binding by the antibody or its antigen-binding moiety when TGFβ1 is present in a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex. While we do not wish to be bound by any particular theory, the ability of the antibodies and their antigen-binding moieties disclosed herein to bind to TGFβ proteins (e.g., TGFβ1) present in complexes with any of the GARP protein, LTBP protein, and / or LRRC33 protein allows for context-independent targeting of TGFβ proteins and is particularly suitable for therapeutic applications.

[0109] definition To facilitate understanding of this disclosure, certain terms are defined first. These definitions should be read in light of the remainder of this disclosure and will be understood as those skilled in the art. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art. Additional definitions are provided throughout the detailed description.

[0110] As used herein, the terms “specific binding” or “specifically binding” mean that the interaction between an antibody or its antigen-binding moiety and an antigen depends on the presence of a specific structure (e.g., an antigenic determinant or epitope). For example, an antibody or its antigen-binding moiety binds to a specific protein rather than to proteins in general. In some embodiments, an antibody or its antigen-binding moiety binds to a target, such as TGFβ1, and the antibody binds to the target K D 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 If M is less than or equal to M, it binds specifically. In some embodiments, the terms “specific binding to the epitope of TGFβ1,” “specifically binding to the epitope of TGFβ1,” “specifically binding to TGFβ1,” or “specifically binding to TGFβ1” mean, as used herein, binding to TGFβ1 and dissociation constant (K) determined by surface plasmon resonance. D ) is 1.0 × 10 -7 This refers to an antibody or its antigen-binding moiety that is M or less. In one embodiment, the antibody or its antigen-binding moiety may specifically bind to both human and non-human (e.g., mouse) orthologs of TGFβ1.

[0111] In some embodiments, the binding affinity of an antibody or its antigen-binding moiety to the GARP-TGFβ1, LTBP1-TGFβ1, LTBP3-TGFβ1, and / or LRRC33-TGFβ1 complex is determined using an Octet assay. In some embodiments, the Octet assay is an assay that determines one or more kinetic parameters indicating antibody-antigen binding. In some embodiments, the Octet® system (ForteBio, Menlo Park, CA) is used to determine the binding affinity of an antibody or its antigen-binding moiety to the GARP-TGFβ1, LTBP1-TGFβ1, LTBP3-TGFβ1, and / or LRRC33-TGFβ1 complex. For example, the binding affinity of an antibody is determined using the forteBio Octet QK e This can be determined using readout label-free assay systems utilizing dip and biolayer interferometry. In some embodiments, the antigen is immobilized on a biosensor (e.g., a streptavidin-coated biosensor), and the antibody and complex (e.g., biotinylated GARP-TGFβ1 complex and biotinylated LTBP-TGFβ1 complex) are present in solution at high concentrations (50 μg / mL) to measure the binding interaction. In some embodiments, the binding affinity of the antibody or its antigen-binding moiety to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex is determined using the protocols outlined in Table 6.

[0112] As used herein, the term “GARP-TGFβ1 complex” refers to a protein complex comprising the proprotein or latent form of the transforming growth factor-β1 (TGFβ1) protein and glycoprotein A repeat dominant protein (GARP). In some embodiments, the proprotein or latent form of the TGFβ1 protein may be referred to as “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, such as the intracellular GARP-TGFβ1 complex. An exemplary GARP-TGFβ1 complex is shown in Figure 3.

[0113] As used herein, the term “LTBP1-TGFβ1 complex” refers to a protein complex comprising the proprotein or latent form of the 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 the pro / latent TGFβ1 via one or more disulfide bonds. In other embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 noncovalently linked to the pro / latent TGFβ1. In some embodiments, the LTBP1-TGFβ1 complex is a naturally occurring complex, such as the intracellular LTBP1-TGFβ1 complex. An exemplary LTBP1-TGFβ1 complex is shown in Figure 3.

[0114] As used herein, the term “LTBP3-TGFβ1 complex” refers to a protein complex comprising the proprotein or latent form of the 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 the pro / latent TGFβ1 via one or more disulfide bonds. In other embodiments, the LTBP3-TGFβ1 complex comprises LTBP1 noncovalently linked to the pro / latent TGFβ1. In some embodiments, the LTBP3-TGFβ1 complex is a naturally occurring complex, such as the intracellular LTBP3-TGFβ1 complex. An exemplary LTBP3-TGFβ1 complex is shown in Figure 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 a 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 the pro / latent TGFβ1 via one or more disulfide bonds. In other embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 noncovalently linked to the pro / latent TGFβ1. In some embodiments, the LRRC33-TGFβ1 complex is a naturally occurring complex, such as the intracellular LRRC33-TGFβ1 complex.

[0116] The term “antibody” refers to an immunoglobulin molecule that specifically binds to a target antigen, and includes, 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 some examples, such as antibodies that may contain only the heavy chain naturally present in camelid animals, may contain fewer chains. Antibodies may originate from only a single source, and may be “chimeric,” meaning that different parts of an antibody originate from two different antibodies. Antibodies or their antigen-binding portions 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, respectively, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimes”), chimeric antibodies, humanized antibodies, human antibodies, and antibody fusions (sometimes referred to herein as “antibody conjugates”). In some embodiments, this term also encompasses the term peptibody.

[0117] Naturally occurring antibody structural units typically consist of tetramers. Each such tetramer typically comprises a pair of two identical polypeptide chains, each pair having one full-length "light" chain (about 25 kDa in certain embodiments) and one full-length "heavy" chain (about 50–70 kDa in certain embodiments). The amino-terminal portion of each chain typically contains a variable region of about 100–110 or more amino acids, generally involved in antigen recognition. The carboxyl-terminal portion of each chain typically defines a constant region that may be involved in effector function. Human antibody light chains are typically classified into kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, which defines the antibody isotype. Antibodies may 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). Within the 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, and the heavy chain also contains a "D" region of about 10 or more amino acids (see, for example, Fundamental Immunology, Chapter 7 (Paul, W. ed., 2nd edition, Raven Press, NY (1989)) (the whole is incorporated by reference)). Typically, the variable region of each light / heavy chain pair forms the antigen-binding site.

[0118] The variable regions typically exhibit the same general structure, consisting of a relatively conserved framework region (FR) to which three hypervariable regions, also called complementarity-determining regions or CDRs, are attached. Typically, the CDRs derived from the two chains of each pair are aligned by the framework region, thereby enabling binding to specific epitopes. Typically, both the light-chain and heavy-chain variable regions contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. The amino acid assignments to each domain typically follow the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia and Lesk (1987) J. Mol. Biol., vol. 196: pp. 901-917; Chothia et al. (1989) Nature, vol. 342: pp. 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, the antibody may contain a few amino acid deletions from the carboxyl terminus of the heavy chain(s). In some embodiments, the antibody contains a heavy chain having 1 to 5 amino acid deletions from the carboxyl terminus of the heavy chain. In certain embodiments, the final demarcation of the CDRs and the identification of residues containing the antibody binding site are achieved by elucidating the structure of the antibody and / or the structure of the antibody-ligand complex. In certain embodiments, this can be achieved by any of the various techniques known to those skilled in the art, such as X-ray crystallography. In some embodiments, the CDR region can be identified or estimated using various analytical methods. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.

[0119] The "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 the antigen-binding site does not necessarily have to be as strong as that of the parent binding protein from which the antigen-binding site originates, but the ability to bind to the antigen must be measurable using one of the various known methods for evaluating the binding of a binding protein to an antigen. Furthermore, the antigen-binding affinities of each antigen-binding site of the multispecific binding proteins described herein do not need to 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, including approximately 120–130 amino acids at the amino terminus of the heavy chain and about 100–110 amino acids at the amino terminus of the light chain. In certain embodiments, the variable regions of different antibodies can vary extensively in amino acid sequence, even among antibodies of the same species. Typically, the variable region of an antibody determines the specificity of a particular antibody to its target.

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

[0122] The term "compete" means competition between antigen-binding proteins, as determined by assays, where 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) of the reference antigen-binding protein, when used in relation to antigen-binding proteins competing for the same epitope. Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, such as 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 labeling assays; and solid-phase direct labeling sandwich assays. Typically, when there is an excess of competing antigen-binding proteins, these proteins inhibit (e.g., reduce) the specific binding of the reference antigen-binding protein to the common antigen by at least 40–45%, 45–50%, 50–55%, 55–60%, 60–65%, 65–70%, 70–75%, or 75% or more. In some cases, binding is inhibited by at least 80–85%, 85–90%, 90–95%, 95–97%, or 97% or more.

[0123] The term "antigen" refers to a molecular structure that yields an epitope, such as a molecule or part of a molecule, or a complex of molecules or parts of molecules, to which a selective binding agent, such as an antigen-binding protein (e.g., an antibody), can bind. Therefore, a selective binding agent can specifically bind to an antigen formed by two or more components in the complex. In some embodiments, an antigen can be used in animals to produce antibodies capable of binding to that antigen. An antigen may harbor one or more epitopes that can interact with different antigen-binding proteins, such as antibodies.

[0124] As used herein, the term “CDR” refers to the complementarity-determining region within the antibody variable sequence. Three CDRs exist in each of the heavy and light chain variable regions, which are referred to as CDR1, CDR2, and CDR3 for each variable region. The term “CDR set” as used herein refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The precise boundaries of these CDRs are defined differently according to different systems. The system described by Kabat et al. (1987; 1991) in Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) provides not only a unique residue numbering system applicable to the variable region of any antibody, but also precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Biol., vol. 196: pp. 901-917; and Chothia et al. (1989) Nature, vol. 342: pp. 877-883) found that certain sub-regions within the Kabat CDR adopt nearly identical peptide skeletal conformations despite exhibiting significant diversity at the amino acid sequence level. These sub-regions are named L1, L2, and L3 or H1, H2, and H3, where "L" and "H" indicate the light chain and heavy chain regions, respectively. These regions can be referred to as the Chothia CDR and have boundaries that overlap with the Kabat CDR. Other boundaries defining CDRs that overlap with the Kabat CDR are described by Padlan (1995) FASEB J., vol. 9: pp. 133-139 and MacCallum (1996) J. Mol. Biol., vol. 262 (no. 5): pp. 732-745. Furthermore, while other CDR boundary definitions may not strictly adhere to one of the systems herein, they still overlap with Kabat CDRs, although they may be shortened or extended based on predictions or experimental findings that certain residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding. The methods used herein may utilize CDRs defined by any of these systems, but in certain embodiments, CDRs defined by Kabat or Chothia are used.

[0125] The terms “crystal” and “crystallized,” as used herein, refer to a binding protein (e.g., an antibody) or its antigen-binding portion existing in crystalline form. A crystal is one form of solid state of matter, distinct from other forms such as amorphous solid states or liquid crystal states. A crystal consists of a three-dimensional array of regular repeating 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 certain mathematical relationships well understood in the art. The repeating basic units or components in a crystal are called asymmetric units. The repeating of asymmetric units in an arrangement consistent with a given well-defined crystallographic symmetry results in the “unit cell” of the crystal. The repeating of the unit cell by regular translation in all three dimensions results in the crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd edition, pp. 201–20116, Oxford University Press, New York, New York (1999).

[0126] The term “epitope” includes any molecular determinant (e.g., polypeptide determinant) that can specifically bind to a binding agent, immunoglobulin, or T cell receptor. In certain embodiments, an epitope determinant includes a chemically active surface grouping of a molecule, such as an amino acid, sugar side chain, phosphoryl, or sulfonyl, and in certain embodiments, may have specific three-dimensional structural properties and / or specific charge properties. 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) that is known to bind to a complementary site on a specific binding partner. An antigen fragment may contain more than one epitope. In certain embodiments, an antibody is one that specifically binds to an antigen if it recognizes its target antigen in a complex mixture of proteins and / or macromolecules. For example, antibodies can be said to “bind to the same epitope” if they cross-compete (one interferes with the binding or regulatory action of the other). Furthermore, while structural definitions of epitopes (overlap, similarity, identity) are informative, functional definitions are often more relevant because they encompass both structural (coupling) and functional (regulating, competing) parameters.

[0127] The terms “treat” and “treatment” encompass therapeutic, preventive, and application that reduces the risk of injury or other risk factors occurring in a subject. Treatment does not require complete cure of injury and includes embodiments that reduce symptoms or underlying risk factors.

[0128] Standard techniques for recombinant DNA, oligonucleotide synthesis, and tissue cultures and transformations (e.g., electroporation, lipofection) may be used. Enzymatic reactions and purification techniques may be carried out according to the manufacturer's specifications, as commonly achieved in the art, or as described herein. The aforementioned techniques and procedures may generally be carried out according to conventional methods well known in the art, and as described in the various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for all purposes. Unless otherwise specified, the nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, as well as laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment may be used.

[0129] The terms “antigen-binding moiety” or “antigen-binding fragment” of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TGFβ1). Antigen-binding moieties include, but are not limited to, any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. In some embodiments, the antigen-binding moiety of an antibody may be obtained, for example, from the whole antibody molecule using any preferred standard technique such as protein digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding the antibody variable domain and optionally the constant domain. Non-limiting examples of antigen-binding moieties include: (i) Fab fragments, which are monovalent fragments consisting of a VL domain, VH domain, CL domain, and CH1 domain; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks in the hinge region; (iii) Fd fragments, which consist of a VH domain and a CH1 domain; (iv) Fv fragments, which consist of the VL domain and VH domain of a single arm of the antibody; and (v) single-chain Fv(scFv) molecules (e.g., Bird et al. (1988) SCIENCE, vol. 242: pp. 423-426; and Huston et al. (1988) PROC. NAT'L. ACAD. SCI. See USA, Vol. 85: pp. 5879-5883; (vi) dAb fragments (see, for example, Ward et al. (1989) NATURE, Vol. 341: pp. 544-546); and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementarity-determining regions (CDRs)). Other forms of single-chain antibodies, such as diabodies, are also included.The term antigen-binding portion of an antibody includes a "single-chain Fab fragment" also 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 domain and the linker have one of the following N-terminal to C-terminal sequences: 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 the linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids.

[0130] As used herein, "isolated antibody" refers to an antibody that substantially does not contain other antibodies having different antigen specificities. In some embodiments, the isolated antibody substantially does not contain other cellular material and / or chemical substances.

[0131] An "affinity-mature" antibody refers to an antibody that has one or more modifications to one or more CDRs, resulting in improved affinity for an antigen compared to a parent antibody without these modifications. Exemplary affinity-mature antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity-mature antibodies are prepared by procedures known in the art. Marks et al. (1992), Bio / Technology, Vol. 10: pp. 779-783, describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDRs and / or framework residues has been described by Barbas et al. (1994), Proc Nat. Acad. Sci. USA, Vol. 91: pp. 3809-3813; Schier et al. (1995), Gene, Vol. 169: pp. 147-155; Yelton et al. (1995), J. Immunol., Vol. 155: pp. 1994-2004; Jackson et al. (1995), J. Immunol., Vol. 154 (No. 7): pp. 3310-339; and Hawkins et al. (1992), J. Mol. Biol., Vol. 226: pp. 889-896. Selective mutations at mutagenesis sites, contact or hypermutation sites using amino acid residues that enhance activity are described in U.S. Patent 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 one or more sequences of the VH and / or VL CDR regions are replaced with CDR sequences from another species. For example, an antibody that has mouse heavy and light chain variable regions, in which one or more mouse CDRs (e.g., CDR3) are replaced with human CDR sequences.

[0133] The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences derived from one species as well as a constant region sequence derived from another species, such as an antibody having mouse heavy chain variable region and light chain variable region linked to a human constant region.

[0134] As used herein, the term "human antibody" includes antibodies having a variable region and a constant region derived from a human germline immunoglobulin sequence. The human antibodies of this disclosure may include, for example, CDRs, particularly CDR3, which contain amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by random mutagenesis or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, as used herein, the term "human antibody" does not include antibodies whose CDR sequence is derived from the germline of another mammalian species, such as a mouse, grafted onto a human framework sequence.

[0135] The term “humanized antibody” refers to an antibody that contains 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 has been modified 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 a human CDR sequence has been introduced into the non-human VH and VL sequences, replacing the corresponding non-human CDR sequence. A “humanized antibody” is also an antibody, or a variant, derivative, analog, or fragment thereof, that immunospecifically binds to an antigen of interest and also contains an FR region having substantially the amino acid sequence of a human antibody and a CDR region having substantially the amino acid sequence of a non-human antibody. As used herein, the term “substantially” means, with respect to the CDR, a CDR having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody contains at least one, typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin (i.e., donor antibody), and all or substantially all of the FR region corresponds to that of a human immunoglobulin consensus sequence. In some embodiments, the humanized antibody also typically contains at least a portion of the immunoglobulin Fc region of a human immunoglobulin. In some embodiments, the humanized antibody contains the light chain, as well as at least the variable domains of the heavy chain. The antibody may also contain the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only the humanized light chain. In some embodiments, the humanized antibody contains only the humanized heavy chain. In certain embodiments, the humanized antibody contains only the humanized variable domains of the light chain and / or the humanized heavy chain.

[0136] As used herein, the terms “framework” or “framework sequence” refer to the sequence remaining after removing the CDRs from the variable region. Since the precise definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 on the light chain and CDR-H1, CDR-H2, and CDR-H3 on the heavy chain) also divide the framework regions on the light and heavy chains into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, with CDR1 positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Unless a specific sub-region is explicitly identified as FR1, FR2, FR3, or FR4, the framework region represents a mixture of FRs within the variable region of a single naturally occurring immunoglobulin chain, as referred to by others. As used herein, one FR represents one of the four sub-regions that make up the framework area, and multiple FRs represent two or more of the four sub-regions that make up the framework area.

[0137] As used herein, the terms “germline antibody gene” or “gene fragment” refer to immunoglobulin sequences encoded by non-lymphoid cells that have not undergone the maturation process that leads to gene rearrangements and mutations relating to the expression of specific immunoglobulins (see, for example, Shapiro et al. (2002) Crit. Rev. Immunol., Vol. 22 (No. 3): pp. 183–200; Marchalonis et al. (2001) Adv. Exp. Med. Biol., Vol. 484: pp. 13–30). One of the advantages brought about by the various embodiments of this disclosure is the recognition that germline antibody genes are more likely to conserve essential amino acid sequence structures that are characteristic of individuals within a species than mature antibody genes, and therefore are less likely to be recognized as originating from an exogenous source when used therapeutically in that species.

[0138] As used herein, the term “neutralizing” refers to the ability of a binding protein to specifically bind to an antigen and neutralize the antigen’s biological activity. In some embodiments, a neutralizing binding protein binds to an antigen / target, such as a cytokine, kinase, growth factor, cell surface protein, soluble protein, phosphatase, or receptor ligand, and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.

[0139] The term “binding protein,” as used herein, includes, but is not limited to, antibodies or their antigen-binding moieties, DVD-Ig™, TVD-Ig, RAb-Ig, bispecific antibodies, and bispecific antibodies, as well as any polypeptide that specifically binds to an antigen (e.g., TGFβ1).

[0140] When used in relation to a composition containing it, the term “monoclonal antibody” or “mAb” may refer to a substantially homogeneous population of antibodies, i.e., an antibody preparation obtained from a population in which the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific and target a single antigen. Furthermore, in contrast to polyclonal antibody preparations, which typically contain different antibodies targeting different determinants (epitopes), each mAb targets a single determinant on an antigen. The modifier “monoclonal” is not interpreted as requiring the antibody to be produced by any particular method.

[0141] The term "recombinant human antibody," as used herein, refers to all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (as further described in Section II C below), and antibodies isolated from recombinant combinatorial human antibody libraries (Hoogenboom, HR (1997) TIB Tech., Vol. 15: pp. 62-70; Azzazy, H. and Highsmith, WE (2002) Clin. Biochem., Vol. 35: pp. 425-445; Gavilondo, JV and Larrick, JW (2002) BioTechniques, Vol. 29: pp. 128-145; Hoogenboom, H. and Chames, P. (2000) Immunol. This includes antibodies isolated from animals (e.g., mice) that are transgenic with respect to human immunoglobulin genes (see Taylor, LD et al. (1992) Nucl. Acids Res., vol. 20: pp. 6287-6295; Kellermann, SA. and Green, LL (2002) Cur. Opin. in Biotechnol., vol. 13: pp. 593-597; Little, M. et al. (2000) Immunol. Today, vol. 21: pp. 364-370), or antibodies prepared, expressed, created, or isolated by any other means involving splicing from human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or in vivo somatic mutagenesis if transgenic animals with respect to the human Ig sequence are used), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but which may not be naturally present within the human antibody germline repertoire in vivo.

[0142] As used herein, “Dual Variable Domain Immunoglobulin” or “DVD-Ig™” includes a binding protein comprising a paired heavy-chain DVD polypeptide and a light-chain DVD polypeptide, each paired heavy-chain and light-chain resulting in two antigen-binding sites. Each binding site contains a total of six CDRs involved in antigen binding per site. DVD-Ig™ typically has two arms linked to each other by dimerization of the CH3 domain in at least a portion, and each arm of the DVD is bispecific, resulting in an immunoglobulin with four binding sites. DVD-Ig™ are presented in U.S. Patent Applications Publications 2010 / 0260668 and 2009 / 0304693, respectively, which are incorporated herein by reference, including their sequence listings.

[0143] As used herein, "triply variable domain immunoglobulin" or "TVD-Ig," etc., is a binding protein comprising a paired heavy-chain TVD-binding protein polypeptide and a light-chain TVD-binding protein polypeptide, wherein each paired heavy-chain and light-chain yields three antigen-binding sites. Each binding site contains a total of six CDRs involved in antigen binding. The TVD-binding protein may have two arms linked to each other by dimerization of the CH3 domain in at least a portion of the protein, 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” is a binding protein comprising a heavy-chain RAb polypeptide and a light-chain RAb polypeptide that together form a total of three antigen-binding sites. One antigen-binding site is formed by the pairing of the antibody heavy-chain variable domain and the light-chain variable domain present in each of the heavy-chain and light-chain RAb polypeptides, forming a single binding site with a total of six CDRs, resulting in the first antigen-binding site. The heavy-chain and light-chain RAb polypeptides each contain receptor sequences that bind independently to a ligand, resulting in the second and third “antigen”-binding sites, respectively. RAb-Ig typically has two arms bound to each other by dimerization of the CH3 domain in at least part, and each arm of RAb-Ig is trispecific, resulting in an immunoglobulin with six binding sites. RAb-Ig is described in its entirety, including its sequence listing, in U.S. Patent Application Publication No. 2002 / 0127231, which is incorporated herein by reference.

[0145] The term "bispecific antibody," as used herein and to distinguish it from "bispecific semi-Ig-binding protein" or "bispecific (semi-Ig)-binding protein," refers to antibodies produced by quadroma technology (see Milstein, C. and Cuello, AC (1983), Nature, Vol. 305 (No. 5934): pp. 537-540), by chemical conjugation of two different monoclonal antibodies (see Staerz, UD et al. (1985), Nature, Vol. 314 (No. 6012): pp. 628-631), or by introducing a knob-into-hole or non-inhibitory mutation in the Fc region (see Holliger, P. et al. (1993), Proc. Natl. Acad. Sci). See USA, Vol. 90 (No. 14): pp. 6444-6448. This refers to full-length antibodies produced by a similar method, resulting in a number of different immunoglobulin species, each containing only one functionally 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). According to this definition, a bispecific antibody has two distinct antigen-binding arms (both in specificity and CDR sequence) and is monovalent for each antigen it binds to.

[0146] The term “bispecific antibody,” as used herein, and to distinguish it from bispecific semi-Ig binding proteins or bispecific binding proteins, refers to a full-length antibody capable of binding to two different antigens (or epitopes) in each of its two binding arms (HC / LC pairs) (see PCT Publication WO02 / 02773). Thus, a bispecific binding protein has two identical antigen-binding arms with identical 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 capable of binding to more than one isoform of TGFβ, such as 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 all three isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3, and neutralizes them.

[0148] The term “K on When used herein, "K" refers to the binding rate constant for the association of a binding protein (e.g., an antibody) and an antigen to form, for example, an antibody / antigen complex, as is known in the art. on In this specification, the term "association rate constant" or "k" is used interchangeably. a It is also known as "the rate at which an antibody binds to its target antigen, or the rate at which an antibody-antigen complex is formed, can also be expressed by the equation: antibody ("Ab") + antigen ("Ag") → Ab - Ag.

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

[0150] In this specification, the terms "equilibrium dissociation constant" or "K" are used interchangeably. D" is the value obtained in a titration measurement at equilibrium, or the dissociation rate constant (k off ) is the coupling rate constant (k on This refers to the value obtained by dividing by ). The binding rate constant, dissociation rate constant, and equilibrium dissociation constant are used to express the binding affinity of binding proteins, such as antibodies, to antigens. Methods for determining the binding rate constant and dissociation rate constant are well known in the art. Using fluorescence-based techniques provides high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental techniques and instruments can be used, such as the BIAcore® (Biomolecular Interaction Analysis) assay (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 containing two or more amino acid residues linked by a peptide bond and used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 6444–6448; Poljak, RJ et al. (1994) Structure, Vol. 2: pp. 1121–1123). Exemplary linkers include, but are not limited to, ASTKGPSVFPLAP (sequence number 55), ASTKGP (sequence number 56); TVAAPSVFIFPP (sequence number 57); TVAAP (sequence number 58); AKTTPKLEEGEFSEAR (sequence number 59); AKTTPKLEEGEFSEARV (sequence number 60); AKTTPKLGG (sequence number 61); SAKTTPKLGG (sequence number 62); SAKTTP (sequence number 63); RADAAP (sequence number 64); RADAAPTVS (sequence number 65); RADAAAAGGPGS (sequence number 66); RADAAAA(G4S)4 (sequence number 67); SAKTTPKLEEGEFSEARV (sequence number 68); ADAAP (array Includes number 69);ADAAPTVSIFPP(sequence number 70);QPKAAP(sequence number 71);QPKAAPSVTLFPP(sequence number 72);AKTTPP(sequence number 73);AKTTPPSVTPLAP(sequence number 74);AKTTAP(sequence number 75);AKTTAPSVYPLAP(sequence number 76);GGGGSGGGGSGGGGS(sequence number 77);GENKVEYAPALMALS(sequence number 78);GPAKELTPLKEAKVS(sequence number 79);GHEAAAVMQVQYPAS(sequence number 80);TVAAPSVFIFPPTVAAPSVFIFPP(sequence number 81); and ASTKGPSVFPLAPASTKGPSVFPLAP(sequence number 82).

[0152] As used herein, the term "cancer" typically refers to a physiological condition in multicellular eukaryotes characterized by uncontrolled cell proliferation.

[0153] "Label" and "detectable label" or "detectable moiety" mean a moiety attached to a specific binding partner, such as an antibody or a sample, in order to make the reaction between members of a specific binding pair, such as an antibody and a sample, detectable, and such a labeled specific binding partner, such as an antibody or a sample, is referred to as "detectable label." Thus, the term "labeled binding protein," as used herein, refers to a protein into which a label has been incorporated that results in the identification of the binding protein. In some embodiments, the label is a detectable marker that can produce a detectable signal by visual or instrumental means, such as the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to a polypeptide that can be detected by a marked avidin (e.g., a fluorescent marker or streptavidin containing enzymatic activity that can be detected by optical or colorimetric quantitative methods). Examples of labels on polypeptides include, but are not limited to, 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); pigments; fluorescent labels (e.g., FITC, rhodamine, and lanthanidrine luminescent agents); 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, secondary antibody binding sites, metal-binding domains, and epitope tags); and magnetic agents such as gadolinium chelates. Typical examples of labels commonly used in immunoassays include light-emitting moieties, e.g., acridinium compounds, and fluorescent moieties, e.g., fluorescein. Other labels are described herein. In this regard, a moiety itself may not be detectably labeled, but may become detectable upon reaction with 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 enables real-time analysis of 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 Jonesson, U. et al. (1993) Ann. Biol. Clin., Vol. 51: pp. 19-26; Jonesson, U. et al. (1991) Biotechniques, Vol. 11: pp. 620-627; Johnson, B. et al. (1995) J. Mol. Recognit., Vol. 8: pp. 125-131; and Johnson, B. et al. (1991) Anal. Biochem., Vol. 198: pp. 268-277.

[0155] A "plasmid" or "vector" contains a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. An "expression plasmid" or "expression vector" may be a plasmid that has the ability to incorporate and express a heterologous nucleic acid fragment into a cell. An expression plasmid may contain additional elements; for example, an expression vector may have two replication systems, thus enabling maintenance in two organisms. The nucleic acid incorporated into the plasmid may be operably ligated to an expression regulatory sequence if the expression regulatory sequence controls and regulates the transcription and translation of its polynucleotide sequence.

[0156] "Nucleic acid" or "nucleic acid sequence" may be any molecule, preferably a polymer molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or analogues. Nucleic acid may be single-stranded or double-stranded. Single-stranded nucleic acid may be one nucleic acid strand of denatured double-stranded DNA. Alternatively, single-stranded nucleic acid may be single-stranded nucleic acid not derived from any double-stranded DNA. In one embodiment, nucleic acid may be DNA. In another embodiment, nucleic acid may be RNA. A preferred nucleic acid molecule is DNA, including genomic DNA or cDNA. Another preferred nucleic acid molecule is RNA, including mRNA.

[0157] Unless otherwise indicated in the working examples or otherwise, all figures representing quantities of components or reaction conditions used herein should be understood to be modified in all cases by the term “approximately.” When used in relation to percentages, the term “approximately” may mean ±1%.

[0158] While several embodiments of the Disclosure have been described and explained herein, a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein will be readily conceivable to those skilled in the art, and each of such changes and / or modifications will be considered within the scope of the Disclosure. More generally, all parameters, dimensions, materials, and configurations described herein are illustrative, and those skilled in the art will readily understand that actual parameters, dimensions, materials, and / or configurations will depend on the particular application or the application in which the teachings of the Disclosure are used. Those skilled in the art can recognize or confirm many equivalents to specific embodiments of the Disclosure described herein by means of mere routine experimentation. Thus, the embodiments described herein are presented merely as examples, and it should be understood that within the scope of the appended claims and their equivalents, the Disclosure may be practiced in ways other than those specifically described and claimed. The Disclosure covers 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 included in the scope of this disclosure, provided that such features, systems, articles, materials, and / or methods are not inconsistent with each other.

[0159] When used herein and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless explicitly stated otherwise.

[0160] When used herein and in the claims, the phrase “and / or” should be understood to mean “either or both” of the elements thus connected, i.e., elements that are sometimes conjunctive and sometimes disjunctive. Unless explicitly stated otherwise, other elements besides those specifically identified by the “and / or” clause may exist, at their discretion, whether related to or unrelated to the specifically identified elements. Thus, as a non-restrictive example, when used in conjunction with open-ended words such as “comprising,” a reference to “A and / or B” may, in one embodiment, mean A without B (optionally including elements other than B); in another embodiment, B without A (optionally including elements other than A); and in yet another embodiment, both A and B (optionally including other elements), and so on.

[0161] As used herein and in the claims, the phrase “at least one” means, with respect to a list of one or more elements, at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and all elements specifically enumerated in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the presence of elements other than those specifically identified in the list of elements to which the phrase “at least one” refers, whether related to or unrelated to the specifically identified elements, at the discretion of the user. Therefore, as a non-restrictive 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”) could mean, in one embodiment, at least one A and no B (and optionally an element other than B); in another embodiment, at least one B and no A (and optionally an element other than A); in yet another embodiment, at least one A and at least one B (and optionally an element other than A), and so on.

[0162] The use of sequential terms such as "first," "second," and "third" to modify elements of a claim in the claims does not, by itself, indicate any priority, precedence, or order of one element of a claim over another, nor does it indicate a chronological order in which the actions of the method are performed, but is used simply as a distinguishing mark to differentiate elements of the claims from another element having the same name (if sequential terms are not used) as an element of one claim having a particular name.

[0163] It is understood that the ranges provided herein omit all values ​​within that range. For example, the range 1–50 is understood to encompass all numbers, combinations of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, such as 10–20, 1–10, 30–40, etc.

[0164] Antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, and their antigen-binding moieties. The present invention is, at least in part, based on the discovery of antibodies and their antigen-binding moieties that bind to TGFβ1 present in the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex. Accordingly, some aspects of the present invention relate to antibodies or their antigen-binding moieties that specifically bind to an epitope of TGFβ1, wherein the epitope is available for binding by the antibody or its antigen-binding moiety when TGFβ1 is present in the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex. In some embodiments, the epitope becomes available due to a conformational change of TGFβ1 when it is in a complex with GARP, LTBP1, LTBP3, and / or LRRC33. In some embodiments, the TGFβ1 epitope to which the antibody or its antigen-binding moiety binds is not available if TGFβ1 is not in a complex with GARP, LTBP1, LTBP3, and / or LRRC33. In some embodiments, the antibody or its antigen-binding moiety does not specifically bind to TGFβ2. In some embodiments, the antibody or its antigen-binding moiety does not specifically bind to TGFβ3. In some embodiments, the antibody or its antigen-binding moiety does not interfere with the binding of TGFβ1 to integrins. For example, in some embodiments, the antibody or its antigen-binding moiety does not shield the integrin-binding site of TGFβ1. In some embodiments, the antibody or its antigen-binding moiety inhibits the activation of TGFβ1. In some embodiments, the antibody or its antigen-binding moiety inhibits the release of mature TGFβ1 from the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex.

[0165] The antibodies or their antigen-binding moieties provided herein specifically bind to an epitope of TGFβ1, where the epitope is available for binding by the antibody or its antigen-binding moiety when TGFβ1 is present in the 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 their antigen-binding moieties described herein do not specifically bind to TGFβ2. In some embodiments, the antibodies or their antigen-binding moieties described herein do not specifically bind to TGFβ3. In some embodiments, the antibodies or their antigen-binding moieties described herein do not specifically bind to either TGFβ2 or TGFβ3. In some embodiments, the antibodies or their antigen-binding moieties described herein specifically bind to TGFβ1 comprising the amino acid sequence described in SEQ ID NO: 21. The amino acid sequences of TGFβ2 and TGFβ3 are described in SEQ ID NOs. 22 and 23, respectively. In some embodiments, the antibodies or antigen-binding moieties described herein specifically bind to TGFβ1 containing amino acid sequences not found in nature (otherwise referred to herein as non-naturally occurring TGFβ1). For example, non-naturally occurring TGFβ1 may contain one or more recombinant mutations compared to the naturally occurring TGFβ1 amino acid sequence. In some embodiments, the TGFβ1, TGFβ2, or TGFβ3 amino acid sequences include the amino acid sequences described in SEQ ID NOs. 24-35 shown in Table 1. In some embodiments, the TGFβ1, TGFβ2, or TGFβ3 amino acid sequences include the amino acid sequences described 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 moieties described herein can bind to an 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 protein that does not exist naturally. In some embodiments, the LTBP1 protein is a recombinant protein. Such a recombinant LTBP1 protein may comprise LTBP1, its alternatively spliced ​​variants, and / or fragments thereof. The recombinant LTBP1 protein may also be modified to include one or more detectable labels. In some embodiments, the LTBP1 protein comprises a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP1 protein does not comprise a leader sequence (i.e., the leader sequence is processed or cleaved). Examples of such detectable labels include, but are not limited to, biotin labels, polyhistidine tags, myc tags, HA tags, and / or fluorescent tags. 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 contains the amino acid sequences described in SEQ ID NOs. 46 and 47 of Table 2. In some embodiments, the LTBP1 protein contains the amino acid sequence described in SEQ ID NOs. 50 of Table 3.

[0167] In some embodiments, the antibodies or antigen-binding moieties 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 protein that does not exist naturally. In some embodiments, the LTBP3 protein is a recombinant protein. Such recombinant LTBP3 proteins may comprise LTBP3, its alternatively spliced ​​variants, and / or fragments thereof. In some embodiments, the LTBP3 protein comprises a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP3 protein does not comprise a leader sequence (i.e., the leader sequence is processed or cleaved). The recombinant LTBP3 protein may also be modified to include one or more detectable labels. Such detectable labels may include, but are not limited to, biotin labels, polyhistidine tags, myc tags, HA tags, and / or fluorescent tags. 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 contains the amino acid sequences described in SEQ ID NOs. 44 and 45 of Table 2. In some embodiments, the LTBP1 protein contains the amino acid sequence described in SEQ ID NO. 51 of Table 3.

[0168] In some embodiments, the antibody or its antigen-binding moiety 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 protein that does not exist naturally. In some embodiments, the GARP protein is a recombinant protein. Such GARP may be recombinant and is referred to herein as recombinant GARP. Some recombinant GARP may contain one or more modifications, shortenings and / or mutations compared to wild-type GARP. Recombinant GARP may be modified to be soluble. In some embodiments, the GARP protein includes a leader sequence (e.g., native or non-native leader sequence). In some embodiments, the GARP protein does not include a leader sequence (i.e., the leader sequence is processed or cleaved). In other embodiments, the recombinant GARP is modified to include one or more detectable labels. In further embodiments, such detectable labels may include, but are not limited to, biotin labels, polyhistidine tags, flag tags, myc tags, HA tags and / or fluorescent tags. In some embodiments, the GARP protein is a mammalian GARP protein. In some embodiments, the GARP protein is human, monkey, mouse, or rat GARP protein. In some embodiments, the GARP protein includes the amino acid sequences described in SEQ ID NOs. 48-49 of Table 2. In some embodiments, the GARP protein includes the amino acid sequences described in SEQ ID NOs. 52 and 53 of Table 4. In some embodiments, the antibody or its antigen-binding moiety described herein does not bind to TGFβ1 in a context-dependent manner; for example, binding to TGFβ1 occurs only when the TGFβ1 molecule is complexed with a specific presenting molecule such as GARP. Instead, the antibody and its antigen-binding moiety bind to TGFβ1 in a context-independent manner.In other words, the antibody or its antigen-binding moiety binds to TGFβ1 if TGFβ1 is bound to any of the presenting molecules: GARP, LTBP1, LTBP3, and / or LRCC33.

[0169] In some embodiments, the antibody or its antigen-binding moiety 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 protein that does not exist naturally. In some embodiments, the LRRC33 protein is a recombinant protein. Such LRRC33 may be recombinant and will be referred to herein as recombinant LRRC33. Some recombinant LRRC33 proteins may contain one or more modifications, shortenings and / or mutations compared to wild-type LRRC33. Recombinant LRRC33 proteins may be modified to be soluble. For example, in some embodiments, the external domain of LRRC33 can be expressed with a C-terminal His tag in order to express a soluble LRRC33 protein (sLRRC33; see, for example, 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 is modified to include one or more detectable labels. In further embodiments, such detectable labels may include, but are not limited to, biotin labels, polyhistidine tags, flag tags, myc tags, HA tags, and / or fluorescent tags. 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 contains the amino acid sequences described in SEQ ID NOs. 83, 84, and 85 of Table 4. [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2]

[0170] In some embodiments, the antibody of the present invention or its antigen-binding moiety, which specifically binds to an epitope of TGFβ1 available for antibody binding when TGFβ1 is present in the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, as well as the nucleic acid molecule of the present disclosure encoding the antibody, comprises one or more of the CDR amino acid sequences shown in Table 5. [Table 5]

[0171] In some embodiments, the antibodies of the present invention that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex include any antibody or its antigen-binding moiety, which includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, provided for any one of the antibodies shown in Table 5. In some embodiments, the antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex include CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, which include any one of the antibodies shown in Table 5. The present invention also provides any nucleic acid sequences encoding molecules containing CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, which are 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 antibody's binding specificity / affinity to the antigen. Therefore, antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex of this disclosure, or nucleic acid molecules encoding these antibodies or their antigen-binding moieties, may include at least the heavy chain and / or light chain CDR3 of the antibodies shown in Table 5.

[0172] Aspects of the present invention relate to a monoclonal antibody or its antigen-binding moiety that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, and also comprises six complementarity-determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.

[0173] In some embodiments, CDRH1 includes the sequence described in either sequence number 1 or 2. In some embodiments, CDRH2 includes the sequence described in either sequence number 3 or 4. In some embodiments, CDRH3 includes the sequence described in either sequence number 5 or 6. CDRL1 includes the sequence described in either sequence number 7 or 8. In some embodiments, CDRL2 includes the sequence described in either sequence number 9 or 10. In some embodiments, CDRL3 includes the sequence described in either sequence number 11 or 12.

[0174] In some embodiments (for example, with respect to antibody Ab1 shown in Table 5), the antibody or its antigen-binding moiety that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex includes CDRH1 containing the amino acid sequence described in SEQ ID NO: 1, CDRH2 containing the amino acid sequence described in SEQ ID NO: 3, CDRH3 containing the amino acid sequence described in SEQ ID NO: 5, CDRL1 containing the amino acid sequence described in SEQ ID NO: 7, CDRL2 containing the amino acid sequence described in SEQ ID NO: 9, and CDRL3 containing the amino acid sequence described in SEQ ID NO: 11.

[0175] In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region containing a complementarity-determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 5, and a light chain variable region containing a CDR3 having the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region containing a complementarity-determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region containing a CDR2 having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region containing a complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 1, and a light chain variable region containing 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 having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable domain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0177] In some embodiments, the 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 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 (for example, with respect to antibody Ab2 shown in Table 5), the antibody or its antigen-binding moiety that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex includes CDRH1 containing the amino acid sequence described in SEQ ID NO: 2, CDRH2 containing the amino acid sequence described in SEQ ID NO: 3, CDRH3 containing the amino acid sequence described in SEQ ID NO: 6, CDRL1 containing the amino acid sequence described in SEQ ID NO: 8, CDRL2 containing the amino acid sequence described in SEQ ID NO: 10, and CDRL3 containing the amino acid sequence described in SEQ ID NO: 12.

[0179] In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region containing a CDR3 having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region containing a CDR3 having the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region containing a CDR2 having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region containing a CDR2 having the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region containing a CDR1 having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region containing a CDR1 having the amino acid sequence of SEQ ID NO: 8.

[0180] In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable domain containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 15, and a light chain variable domain containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 16.

[0181] In some embodiments, an antibody or its antigen-binding moiety that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex includes 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 with the nucleic acid sequence described 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 with the nucleic acid sequence described in SEQ ID NO: 94.

[0182] In some embodiments, any antibody of this disclosure that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex comprises any antibody (including its antigen-binding moiety) having one or more CDR (e.g., CDRH or CDRL) sequences substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3. For example, the antibody may contain one or more CDR sequences (SEQ ID NOs: 1-12) shown in Table 5, which contain variations of up to five, four, three, two, or one amino acid residue compared to the corresponding CDR region of any one of SEQ ID NOs: 1-12. The complete amino acid sequences of the heavy chain variable regions and light chain variable regions of the antibodies listed in Table 5 (e.g., Ab1 and Ab2), as well as the nucleic acid sequences encoding the heavy chain variable regions and light chain variable regions of the antibodies, are presented below. [ka] [ka] [ka]

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

[0184] Aspects of this disclosure provide antibodies that specifically bind to GARP-TGFβ1 complexes, LTBP1-TGFβ1 complexes, LTBP3-TGFβ1 complexes, and LRRC33-TGFβ1 complexes, 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, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex include a heavy-chain variable amino acid sequence or a light-chain variable sequence that is at least 75% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the heavy-chain variable amino acid sequence of SEQ ID NO: 13 or 17, or the light-chain variable sequence of SEQ ID NO: 14 or 18. In some embodiments, homologous heavy-chain variable and / or light-chain variable amino acid sequences are not varied among any of the CDR sequences provided herein. For example, in some embodiments, the 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 in heavy-chain variable and / or light-chain variable amino acid sequences, excluding any of the CDR sequences provided herein.

[0185] In some embodiments, the antibodies of this disclosure that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex include any antibody or its antigen-binding moiety comprising the heavy chain of SEQ ID NO: 15 or 19 or the light chain of SEQ ID NO: 16 or 20.

[0186] Aspects of this disclosure provide antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, having heavy chain and / or light chain amino acid sequences homologous to any of those described herein. In some embodiments, antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex include a heavy chain sequence or a 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 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 among any of the CDR sequences provided herein. For example, in some embodiments, the 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 in heavy and / or light chain amino acid sequences other than any of the CDR sequences provided herein.

[0187] In some embodiments, the antibodies of this disclosure that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex include any antibody or its antigen-binding moiety comprising the heavy chain of SEQ ID NO: 15 or 19 or the light chain of SEQ ID NO: 16 or 20.

[0188] Aspects of this disclosure provide antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, having heavy chain and / or light chain amino acid sequences homologous to any of those described herein. In some embodiments, antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex include a heavy chain sequence or a 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 among any of the CDR sequences provided herein. For example, in some embodiments, the 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 in heavy and / or light chain amino acid sequences other than 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, Vol. 87: pp. 2264-2268, 1990, modified as described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 5873-5877, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. Vol. 215: pp. 403-4010, 1990. BLAST protein search can be performed using the XBLAST program, score=50, word length=3, to obtain homologous amino acid sequences to the target protein molecule. If a gap exists between two sequences, gap BLAST can be used as described by Altschul et al., Nucleic Acids Res. Vol. 25 (No. 17): pp. 3389-3402, 1997. When using BLAST and gap BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) may be used.

[0190] In any of the antibody or antigen-binding fragments described herein, one or more conserved mutations can be introduced into the CDR or framework sequence at the positions of residues unlikely to be involved in antibody-antigen interactions. In some embodiments, such conserved mutations can be introduced into the CDR or framework sequence at the positions of residues unlikely to be involved in interactions with the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex, as determined based on the crystal structure. In some embodiments, likely interfaces (e.g., residues involved in antigen-antibody interactions) can be inferred from known structural information relating to another antigen that shares structural similarities.

[0191] As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein being substituted. Modified versions are references that summarize such methods, e.g., Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd edition, Cold Spring Harbor Laboratory Press, Cold The preparations may be made according to methods for modifying polypeptide sequences that are known to those skilled in the art, such as those found in 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 made between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0192] In some embodiments, the antibodies provided herein include mutations that confer desirable properties to the antibody. For example, to avoid the difficult situations that can occur due to Fab arm exchange, which are known to occur in natural IgG4 mAbs, the antibodies provided herein may include a stabilizing "Adair" mutation in which serine 228 (EU numbered, Kabat numbered residue 241) is converted to proline, resulting in 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 Vol. 30, pp. 105-108; 1993). Thus, any of the antibodies may include a stabilizing "Adair" mutation or the amino acid sequence CPPP (SEQ ID NO: 54).

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

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

[0195] In some embodiments, the antibodies of this disclosure that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex have relatively high affinity for the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, for example, 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 the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex may bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex with an affinity between 5 pM and 500 nM, for example between 50 pM and 100 nM, or for example between 500 pM and 50 nM. This disclosure also includes antibodies or antigen-binding fragments that compete with any of the antibodies described herein for binding to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, and have an affinity of 50 nM or less (e.g., less than 20 nM, less than 10 nM, less than 500 pM, less than 50 pM, or less than 5 pM). The affinity and binding kinetics of antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex may be tested using any suitable method, including biosensor technologies (e.g., OCTET or BIACORE), but are not limited to these.

[0196] Antibodies that inhibit TGFβ In one embodiment, the present invention provides a functional antibody. As used herein, “functional antibody” means one that confers one or more biological activities by its ability to bind to an antigen. Functional antibodies may include inhibitory antibodies (or inhibitory antibodies) and activating antibodies. Accordingly, this disclosure includes TGFβ antibodies that can modulate (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, upon association with such an antibody, reduces the release or activity of growth factors. Such epitopes may be located on the prodomain of the TGFβ protein (e.g., TGFβ1), growth factors, or other epitopes, which, upon antibody binding, 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 this disclosure include methods for using inhibitory antibodies to modify growth factor signaling in solutions, cell cultures, and / or subjects.

[0199] Polypeptide Some aspects of this disclosure relate to polypeptides having sequences 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 described in SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 15, and SEQ ID NO: 19.

[0200] Some aspects of this disclosure relate to polypeptides having sequences 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 described 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, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex. Aspects of this disclosure relate to antibodies that compete with or cross-compete with any of the antibodies provided herein. The term “compete” as used herein with respect to antibodies means that the first antibody binds to an epitope (e.g., the epitopes of the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex) in a manner sufficiently similar to that of the second antibody, and that the binding of the first antibody to its epitope is, in the presence of the second antibody, detectably reduced compared to the binding of the first antibody in the absence of the second antibody. There may be alternatives where the binding of the second antibody to its epitope is similarly detected in the presence of the first antibody, but this is not required. That is, the first antibody may inhibit the binding of the second antibody to its epitope, but this may not be accompanied by the inhibition of the first antibody's binding to its respective epitope by the second antibody. However, if each antibody detectably inhibits the binding of other antibodies to their respective epitopes or ligands, then these antibodies can be said to “cross-compete” with each other for binding to their respective epitopes, whether to the same, greater, or lesser degree. Both competing antibodies and cross-competing antibodies are within the scope of this disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, or binding to a common epitope or part thereof), it will be understood by those skilled in the art that such competing and / or cross-competing antibodies are included in and may be useful in the methods and / or compositions provided herein.

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

[0203] In another embodiment, regarding binding to any of the antigens provided herein (e.g., GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex), 10 -6 Equilibrium dissociation constant K between antibodies and proteins less than M D Antibodies or their antigen-binding portions that compete or cross-compete are provided herein. In other embodiments, 10 for binding to any of the antigens provided herein. -11 M to 10 -6 K up to M D Antibodies that compete or cross-compete with the antibodies described herein are provided herein. In some embodiments, anti-TGFβ1 antibodies or their antigen-binding moieties that compete for binding with the antibodies or their antigen-binding moieties described herein are provided herein. In some embodiments, anti-TGFβ1 antibodies or their antigen-binding moieties that bind to the same epitope as the antibodies or their antigen-binding moieties described herein are provided herein.

[0204] Any antibody provided herein can be characterized using any preferred method. For example, one method is to identify the epitope to which the antigen binds, i.e., “epitope mapping”. There are many preferred methods for mapping and characterizing the location of epitopes on proteins, including elucidation of the crystalline structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, and assays based on synthetic peptides, as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In an additional 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 it may be a conformational epitope formed by three-dimensional interactions of amino acids, which do not necessarily have to be contained in a single stretch (linear primary structure sequence). In some embodiments, the epitope is a TGFβ1 epitope that becomes available for binding by the antibodies or their antigen-binding moieties described herein only when TGFβ1 is present in the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex. Peptides of various lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., by recombination) and used in antibody-based binding assays. In another example, the epitope to which the antibody binds can be determined in a systematic screening by using duplicate peptides derived from the target antigen sequence and determining antibody binding. According to gene fragment expression assays, an open reading frame encoding the target antigen is fragmented randomly or by specific gene construction, and the reactivity of the expressed antigen fragments with the antibody under test is determined. The gene fragments are generated, for example, by PCR, and then transcribed and translated into proteins in vitro in the presence of radioactive amino acids.Next, the binding of the antibody to the radiolabeled antigen fragment is determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using a large library of random peptide sequences displayed on the surface of phage particles (a phage library). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. In additional examples, antigen-binding domain mutagenesis, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify the sufficient and / or required residues for epitope binding. For example, domain swapping experiments may be performed using variants of target antigens in which various fragments of the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex have been replaced (swapped) with sequences derived from closely related but antigenically distinct proteins, such as another member of the TGFβ protein family (e.g., GDF11). By evaluating the binding of antibodies to variants of the GARP-TGFβ1, LTBP1-TGFβ1, LTBP3-TGFβ1, and / or LRRC33-TGFβ1 complexes, the importance of specific antigenic fragments to antibody binding can be assessed.

[0205] Alternatively, a competitive assay can be performed using other antibodies known to bind to the same antigen to determine whether the antibody binds to the same epitope as the other antibodies. Competitive assays are well known to those skilled in the art.

[0206] Furthermore, the interaction between any of the antibodies provided herein and one or more residues within the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex can be determined by conventional techniques. For example, the crystal structure can be determined, and accordingly, the distance between residues within the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex and one or more residues in the antibody can be determined. Based on such distances, it can be determined whether a particular residue within the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex interacts with one or more residues in the antibody. Furthermore, preferred methods such as competitive 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, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex. Numerous methods can be used to obtain the antibodies or antigen-binding fragments thereof of this disclosure. For example, antibodies may be produced using recombinant DNA methods. Monoclonal antibodies may also be produced by hybridoma generation according to known methods (see, e.g., Kohler and Milstein (1975) Nature, Vol. 256: pp. 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 a specified antigen. Any form of the specified antigen, e.g., recombinant antigen, naturally occurring form, any variant or fragment thereof, and its antigenic peptide (e.g., any of the epitopes described herein in the scaffold, either as a linear epitope or a conformational epitope) can be used as immunogens. One typical method of antibody production involves screening protein expression libraries, such as phage or ribosome display libraries, that express antibodies or fragments of antibodies (e.g., scFv). Phage displays are described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985), Science, Vol. 228, pp. 1315-1317; Clackson et al., (1991), Nature, Vol. 352, pp. 624-628; Marks et al., (1991), J. Mol. Biol., Vol. 222, pp. 581-597; WO92 / 18619; WO91 / 17271; WO92 / 20791; WO92 / 15679; WO93 / 01288; WO92 / 01047; WO92 / 09690; and WO90 / 02809.

[0208] In addition to using display libraries, non-human animals, such as rodents, such as mice, hamsters, or rats, can be immunized using specified antigens (e.g., GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex). 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 using preferred recombinant DNA techniques (e.g., chimeric). Various approaches for producing chimeric antibodies have been described. See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA vol. 81:p. 6851, 1985; Takeda et al., Nature vol. 314:p. 452, 1985; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication No. EP171496, European Patent Publication No. 0173494, and UK Patent No. GB2177096B.

[0210] For additional antibody production techniques, see Antibodies: A Laboratory Manual, edited by Harlow et al., Cold Spring Harbor Laboratory, 1988. This disclosure is not necessarily limited to any specific source, method of production, or other special features of any antibody.

[0211] Some aspects of this disclosure relate to host cells transformed with polynucleotides or vectors. The host cells may be prokaryotic or eukaryotic cells. The polynucleotides or vectors present in the host cells may be integrated into the host cell's genome or maintained extrachromosomally. The host cells may be any prokaryotic or eukaryotic cells, such as bacterial, insect, fungal, plant, animal, or human cells. In some embodiments, fungal cells are, for example, those 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 the expression of antibodies or corresponding immunoglobulin chains. Hosts of prokaryotes 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 NSO and CHO cells. Depending on the host used in the recombinant production procedure, the antibody or immunoglobulin chain encoded by the polynucleotide may or may not be glycosylated. The antibody or corresponding immunoglobulin chain may also contain an initiating methionine amino acid residue.

[0212] In some embodiments, once the vector is incorporated into a suitable host, the host may be maintained under conditions favorable for high-level expression of the nucleotide sequence, and optionally, recovery and purification of immunoglobulin light chains, heavy chains, light / heavy chain dimers or intact antibodies, antigen-binding fragments or other immunoglobulin forms may follow; see Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY, (1979). Thus, the polynucleotide or vector is then introduced into cells that produce antibodies or antigen-binding fragments. Furthermore, transgenic animals, preferably mammals, including the aforementioned host cells, may be used for large-scale production of antibodies or antibody fragments.

[0213] Transformed host cells may be grown in a fermenter and cultured using any suitable technique to achieve optimal cell growth. Once expressed, the 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, and gel electrophoresis; 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. For example, the isolation and purification of antibodies or antigen-binding fragments expressed in microorganisms may be by any conventional means, such as chromatographic separation for preparation and immunological separation, such as using monoclonal or polyclonal antibodies directed against the constant region of the antibody.

[0214] Aspects of this disclosure relate to hybridomas that provide an indefinitely extended source of monoclonal antibodies. As an alternative to obtaining immunoglobulins directly from hybridoma cultures, immortalized hybridoma cells may be used as a source of rearranged heavy and light chain loci for subsequent expression and / or genetic manipulation. The rearranged antibody gene may be reverse transcribed from 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. The variable region may be ligated to encode a single-stranded Fv region. Multiple Fv regions may be ligated to confer the ability to bind to more than one target, or chimeric heavy and light chain combinations may be used. Any suitable method may be used for cloning the antibody variable region and generating recombinant antibodies.

[0215] In some embodiments, suitable nucleic acids encoding the variable regions of the heavy and / or light chains are obtained and inserted into an expression vector that can be transfected into a standard recombinant host cell. Various such host cells may be used. In some embodiments, mammalian host cells may be advantageous for efficient processing and production. Typical 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 suitable for host cell proliferation and expression of the coding sequence. Antibodies or antigen-binding fragments can be recovered by isolating them from the culture. The expression system may be designed to include a signal peptide so that the resulting antibody is secreted into the culture medium; intracellular production is also possible.

[0216] This disclosure also includes polynucleotides encoding at least the variable region of the immunoglobulin chain of the antibodies described herein. In some embodiments, the variable region encoded by the polynucleotide includes at least one complementarity-determining region (CDR) of the VH and / or VL of the variable region of the antibody 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 a recombinantly produced chimeric nucleic acid molecule containing any of these polynucleotides, either alone or in combination, synthetically produced DNA or RNA. In some embodiments, the polynucleotide is part of the vector. Such a vector may contain further genes, such as marker genes, that enable vector selection in a suitable host cell and under suitable conditions.

[0218] In some embodiments, polynucleotides are operably ligated to expression regulatory sequences that enable expression in prokaryotic or eukaryotic cells. Polynucleotide expression involves transcription of the polynucleotide into translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may include regulatory sequences that promote transcription initiation and, optionally, polyA signals that promote transcription termination and transcript stabilization. Additional regulatory elements may include transcription and translation enhancers, and / or naturally occurring related or heterologous promoter regions. Regulatory elements that may enable expression in prokaryotic host cells include, for example, the PL, Lac, Trp, or Tac promoters in E. coli, while examples of regulatory elements that enable expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV promoter, SV40 promoter, RSV promoter (Roussarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells.

[0219] Such regulatory elements other than those involved in transcription initiation 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 culture medium may be added to the polynucleotide coding sequence, as previously described. The leader sequence(s) are assembled with translation, start, and stop sequences in appropriate phases, and preferably the leader sequence can lead to the secretion of the translated protein or a portion thereof into, for example, an extracellular culture medium. Heterogeneous polynucleotide sequences encoding a fusion protein containing a C or N-terminal discriminant peptide that provides desirable features, such as stabilization of the recombinant product being expressed or simplification of purification, may be optionally used.

[0220] In some embodiments, the polynucleotide encoding at least one variable domain of the light chain and / or heavy chain may encode both immunoglobulin chains or just one variable domain. Similarly, the polynucleotides may be under the regulation of the same promoter or may be regulated separately for expression. Further embodiments relate to vectors conventionally used in genetic engineering, particularly plasmids, cosmids, viruses and bacteriophages, comprising a polynucleotide encoding a variable domain of an immunoglobulin chain of an antibody or antigen-binding fragment in combination with a polynucleotide optionally encoding a variable domain of another immunoglobulin chain of the antibody.

[0221] In some embodiments, the regulatory expression sequence is provided as a eukaryotic promoter system in a vector that can transform or transfect eukaryotic host cells, although regulatory sequences for prokaryotic hosts may also be used. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, or bovine papillomavirus may be used to deliver polynucleotides or vectors to a targeted cell population (e.g., to manipulate 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., heavy and / or light chain variable domains of immunoglobulin chains encoding sequences and regulatory expression sequences) may be transferred to host cells by a preferred method that varies depending on the type of cell host.

[0222] qualification The antibodies or their antigen-binding moieties of the Disclosure may be modified with detectable labels or moieties, including but not limited to enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, non-radioactive paramagnetic metal ions, and affinity labels, for the detection and isolation of GARP-TGFβ1 complexes, LTBP1-TGFβ1 complexes, LTBP3-TGFβ1 complexes, and / or LRRC33-TGFβ1 complexes. The detectable substances or moieties may be coupled or conjugated either directly to the polypeptides of the Disclosure or indirectly through intermediates (e.g., linkers (e.g., cleavable linkers)) using preferred techniques. Non-limiting examples of preferred enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or acetylcholinesterase; non-limiting examples of preferred 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; examples of luminescent materials include luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include, 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 Sn), etc., radioactive metal ions, such as alpha emitters or other radioisotopes. The detectable substance may be coupled or conjugated either directly or, using suitable techniques, indirectly through an intermediate (such as a linker, etc.) to an antibody of the present disclosure that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex. Any of the antibodies provided herein conjugated to a detectable substance can be used in any suitable diagnostic assay such as those described herein.

[0223] Furthermore, the antibody or antigen-binding portion thereof of the present disclosure may be modified with a drug. The drug may be coupled or conjugated either directly to the polypeptide of the present disclosure or, using suitable techniques, indirectly through an intermediate (such as a linker (such as a cleavable linker), etc.).

[0224] Targeting agent In some embodiments, the methods of the present disclosure involve the use of one or more targeted agents to modulate mature TGFβ release from GARP-TGFβ1 complexes, LTBP1-TGFβ1 complexes, LTBP3-TGFβ1 complexes, and / or LRRC33-TGFβ1 complexes by targeting the antibodies disclosed herein or their antigen-binding moieties to specific sites in a subject. For example, LTBP1-TGFβ1 and LTBP3-TGFβ1 complexes are typically localized in the extracellular matrix. Therefore, in some embodiments, the antibodies disclosed herein may be conjugated with extracellular matrix-targeting agents to localize the antibodies to the sites where LTBP1-TGFβ1 and LTBP3-TGFβ1 complexes are present. In such embodiments, selective targeting of the antibody leads to selective modulation of the LTBP1-TGFβ1 and / or LTBP3-TGFβ1 complexes. In some embodiments, selective targeting of antibodies leads to selective inhibition of the LTBP1-TGFβ1 and / or LTBP3-TGFβ1 complex (for example, to treat fibrosis). In some embodiments, extracellular matrix-targeted agents include heparin-binding agents, matrix metalloproteinase-binding agents, lysyl oxidase-binding domains, fibrillin-binding agents, hyaluronic acid-binding agents, and others.

[0225] Similarly, the GARP-TGFβ1 complex typically interacts with cells, such as activated FOXP3 +It is localized on the surface of regulatory T cells (Tregs). Therefore, in some embodiments, the antibodies disclosed herein may be conjugated with immune cell (e.g., Treg cell) binding agents to localize the antibody to the site where the GARP-TGFβ1 complex is located. 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 the release of mature TGFβ1 for immunomodulation in cancer treatment). In such embodiments, the Treg cell targeting agent may include, for example, CCL22 and CXCL12 proteins or fragments thereof.

[0226] In some embodiments, a bispecific antibody can be used that has a first portion that selectively binds to the GARP-TGFβ1 complex and the LTBP-TGFβ1 complex, and a second portion that selectively binds to components of a target site, such as components of the ECM (e.g., fibrillin) or components of Treg cells (e.g., CTLA-4).

[0227] Pharmaceutical composition The present invention further provides pharmaceutical compositions for use as pharmaceuticals suitable for administration to human and non-human subjects. One or more antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex may be formulated or mixed with a pharmaceutically acceptable carrier (excipient), 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 related to TGFβ signaling involve one or more conditions, i.e., TGFβ associates with a particular type(s) of presenting molecule. In some embodiments, such conditions occur in a cell-type specific and / or tissue-specific manner. In some embodiments, for example, the condition-dependent action of such TGFβ signaling is 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 situations, and thus the antibodies bind to TGFβ in a complex with a presenting molecule selected from two or more of GARP, LRRC33, LTBP1, and LTBP3. Thus, such pharmaceutical compositions can be administered to patients to alleviate TGFβ-related indications (e.g., fibrosis, immunodeficiency, and / or cancer). "Acceptable" means that the carrier is compatible with (and preferably able to stabilize) the active ingredient of the composition 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, edited by K.E. Hoover. For example, the pharmaceutical compositions described herein contain one or more antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, wherein these antibodies recognize different epitopes / residues of the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex.

[0229] The pharmaceutical composition used in this method may contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized preparations or aqueous solutions (Remington: The Science and Practice of Pharmacy, 20th edition (2000), edited by Lippincott Williams and Wilkins, and KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphates, citrates, 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; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than approximately 10 residues) polypeptides; and serum albumin. The excipients may include proteins such as 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 nonionic 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 GARP-TGFβ1 complexes, LTBP1-TGFβ1 complexes, LTBP3-TGFβ1 complexes, and / or LRRC33-TGFβ1 complexes, which can be prepared by any suitable method, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, Vol. 82:p. 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, Vol. 77:p. 4030 (1980); and U.S. Patents No. 4,485,045 and No. 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 of lipid compositions containing phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a specified pore size to obtain liposomes with the desired diameter.

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

[0232] In other examples, the pharmaceutical compositions described herein may be formulated in a sustained-release form. A preferred example of a sustained-release preparation comprises a semipermeable matrix of a solid hydrophobic polymer containing an antibody, the matrix being in the form of a molded article, e.g., a film 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. Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable glycolic acid copolymers such as LUPRON DEPOT (an injectable microsphere composed of glycolic acid lactic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0233] Pharmaceutical compositions used for in vivo administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane. Therapeutic antibody compositions are generally placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be pierced by a subcutaneous needle.

[0234] The pharmaceutical compositions described herein may be in the form of tablets, pills, capsules, powders, granules, solutions or suspensions or suppositories for oral, parenteral, rectal, or inhalation or inhalation administration.

[0235] To prepare solid compositions such as tablets, the main active ingredient may be mixed with other pharmaceutical diluents, such as water or non-toxic, pharmaceutically acceptable salts thereof, to form a solid pre-formulation composition containing a conventional tableting component, such as a pharmaceutical carrier, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gum, and a homogeneous mixture of the compound of the Disclosure. When these pre-formulation compositions are referred to as homogeneous, it means that the active ingredient is homogeneously dispersed throughout the composition, thereby allowing the composition to be easily divided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid pre-formulation composition is then divided into the above-described unit dosage forms containing 0.1 mg to about 500 mg of the active ingredient of the Disclosure. Tablets or pills of the novel compositions may be coated or otherwise formulated to provide a dosage form that offers the benefit of long-term action. For example, a tablet or pill may contain an inner dosage and an outer dosage component, the latter in the form of an envelope covering the former. The two components may be separated by an enteric layer, which helps to resist disintegration in the stomach and allows the internal components to pass through the duodenum intact or delay their release. Various materials may be used for such an enteric layer or coating, and such materials include several polymer acids as well as mixtures of polymer acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0236] Suitable surfactants specifically include nonionic agents such as polyoxyethylene sorbitan (e.g., Tween® 20, 40, 60, 80, or 85) and other sorbitans (e.g., Span® 20, 40, 60, 80, or 85). The composition containing the surfactant may conveniently contain 0.05 to 5% surfactant, and may be 0.1 to 2.5%. It is understood that other components, such as mannitol or other pharmaceutically acceptable vehicles, may be added as needed.

[0237] Suitable emulsions may be prepared using commercially available lipid emulsions such as Intralipid®, Liposyn®, Infonutrol®, Lipofandin®, and Lipiphysan®. The active ingredient may be dissolved in a pre-mixed 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 phospholipid, soybean phospholipid, or soybean lecithin) with water. It is understood that other components, such as glycerol or glucose, may 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 the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex with Intralipid® or its components (soybean oil, egg phospholipid, glycerol, and water).

[0239] Pharmaceutical compositions for inhalation or inhalation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. Liquid or solid compositions may contain the preferred pharmaceutically acceptable excipients described above. In some embodiments, compositions are administered orally or via nasal respiratory routes for topical or systemic effects.

[0240] Preferably, the composition in a sterile, pharmaceutically acceptable solvent may be sprayed using a gas. The sprayed solution may be inhaled directly from a spraying device, or the spraying device may be attached to a face mask, tent, or intermittent positive airway pressure (PAP) respirator. The solution, suspension, or powder composition may be administered preferably orally or nasally from a device that delivers the formulation in an appropriate manner.

[0241] Use of antibodies that specifically bind to GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, and their antigen-binding moieties. In some embodiments, the antibodies, antigen-binding moieties, and compositions of this disclosure may be used to treat a wide variety of diseases, disorders, and / or conditions. In some cases, such diseases, disorders, and / or conditions may be TGFβ-related indications. As used herein, the term “TGFβ-related indication” means any disease, disorder, and / or condition relating to the expression, activity, and / or metabolism of TGFβ family member proteins, or any disease, disorder, and / or condition in which modulation of the activity and / or levels of one or more TGFβ family member proteins may be beneficial. TGFβ-related indications include, but are not limited to, fibrosis, cancer (but are not limited to, colon cancer, renal cancer, breast cancer, malignant melanoma and glioblastoma), rapid promotion of hematopoiesis after chemotherapy, bone healing, wound healing, dementia, myelofibrosis, kidney disease, unilateral ureteral obstruction (UUO), tooth loss and / or degeneration, endothelial hyperplasia, asthma and allergies, gastrointestinal disorders, age-related anemia, aortic aneurysm, rare indications (e.g., Marfan syndrome and Kamrachi-Engelmann disease), obesity, diabetes, arthritis, and multiple sclerosis. This may include conditions such as muscular dystrophy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, metabolic syndrome, malnutrition, organ atrophy, chronic obstructive pulmonary disease (COPD), and loss of appetite. Additional indications may include any indications disclosed in U.S. Patent Application Publication 2013 / 0122007, U.S. Patent No. 8,415,459, or International Patent Application Publication WO2011 / 151432, the entirety of which is incorporated herein by reference.

[0242] Fibrosis In some embodiments, the antibodies and / or compositions of this 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. Such antibodies and / or antibody-based compounds include LY2382770 (Eli Lilly, Indianapolis, IN). Also included are those described in U.S. Patents 6,492,497, 7,151,169, 7,723,486 and U.S. Patent Application Publication 2011 / 0008364, the entire contents of which are incorporated herein by reference.

[0243] Fibrotic indications for which the antibodies and / or compositions of this disclosure can be used therapeutically include, but are not limited to, pulmonary indications (e.g., idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), allergic asthma, acute lung injury, eosinophilic esophagitis, pulmonary hypertension, and chemical gas injury), renal indications (e.g., diabetic glomerulosclerosis, focal segmental glomerulosclerosis (FSGS), chronic kidney disease, fibrosis associated with kidney transplantation and chronic rejection, IgA nephropathy, and hemolytic uremic syndrome), hepatic fibrosis (e.g., non-alcoholic steatohepatitis (NASH), chronic viral hepatitis, parasitemia, congenital metabolic disorders, toxin-mediated fibrosis such as alcoholic fibrosis, non-alcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC), primary This includes biliary cirrhosis and sclerosing cholangitis), cardiovascular fibrosis (e.g., cardiomyopathy, hypertrophic cardiomyopathy, atherosclerosis and restenosis), systemic sclerosis, dermatofibrosis (e.g., dermatofibrosis in systemic sclerosis, diffuse cutaneous systemic sclerosis, scleroderma, pathological skin scars, keloids, postoperative scars, scar revision, 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 fibrosis-related diseases, disorders, or conditions that can be treated with the compounds and / or compositions of this 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 dystrophy (e.g., DMD), Dupuytren's contracture, Kamrachi-Engelmann disease, neural scarring, dementia, proliferative vitreoretinopathy, corneal injury, complications after glaucoma drainage, and multiple sclerosis. Many of these fibrotic indications also involve inflammation of the affected tissue(s) in which immune components are involved.

[0244] The antibodies described herein may be used to treat fibrosis. In some embodiments, a TGFβ1 isoform-specific agent is administered to a subject in an effective amount to treat fibrosis. The 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 containing LTBP that can block TGFβ1 activation mediated by TGFβ1 associated with the ECM. In some embodiments, the LTBP is LTBP1 and / or LTBP3. In some embodiments, the antibody is a context-tolerant antibody that can block TGFβ1 activation mediated by LTBP localized to the ECM and TGFβ1 activation mediated by GARP localized to immune cells. In some embodiments, the antibody is a context-tolerant antibody that can block TGFβ1 activation mediated by LTBP localized to the ECM and TGFβ1 activation mediated by LRRC33 localized to monocytes / macrophages. 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 pro-fibrotic M2-like macrophages in the fibrotic microenvironment.

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

[0246] cancer Various cancers can be treated with the antibodies and / or compositions of this disclosure. As used herein, the term “cancer” refers to any of the various malignant neoplasms characterized by the proliferation of undifferentiated cells that tend to invade surrounding tissues and metastasize to new sites of the body, and also refers to the pathological condition characterized by the proliferation of such malignant neoplasms. Cancer can be, but is not limited to, a tumor or hematological malignancy, including all types of lymphoma / leukemia, carcinomas and sarcomas, such as cancers or tumors found in the anus, bladder, bile ducts, bones, 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, coccyx, testes, thyroid and uterus.

[0247] In cancer, TGFβ (e.g., TGFβ1) can be either pro-growth or anti-growth. For example, in pancreatic cancer, SMAD4 wild-type tumors may undergo growth inhibition in response to TGFβ, but as the disease progresses, typically constitutively activated type II receptors are present. Furthermore, SMAD4 null pancreatic cancer exists. In some embodiments, the antibodies, their antigen-binding moieties, and / or compositions of this 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 the abnormal proliferation of white blood cells, i.e., leukocytes, includes acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia or acute myeloid leukemia (AML) (AML with translocations between chromosomes 10 and 11 [t(10,11)], between chromosomes 8 and 21 [t(8;21)], between chromosomes 15 and 17 [t(15;17)], and inversion on chromosome 16 [inv(16)]; and multilineage dysplasia, including in patients with a history of myelodysplastic syndrome (MDS) or myeloproliferative disorder that previously progressed to AML. AML can be divided into four main classifications, including d) AML with dysplasia; 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 that does not fall into the above categories, including subtypes of AML that do not fall into the above categories; and e) acute leukemia of unclear lineage, which occurs when leukemia cells cannot be classified as either myeloid or lymphoid cells, or when both types of cells are present; as well as chronic myeloid leukemia (CML).

[0248] Carcinoma types include, but are not limited to, papillomas / carcinomas, choriocarcinomas, yolk sac tumors, teratomas, adenomas / adenocarcinomas, melanomas, fibromas, lipomas, leiomyomas, rhabdomyomas, mesotheliomas, hemangiomas, osteomas, chondromos, gliomas, lymphomas / leukemias, squamous cell carcinomas, small cell carcinomas, anaplastic large cell carcinomas, basal cell carcinomas, and anaplastic sinus carcinomas.

[0249] Types of sarcomas include, but are not limited to, soft tissue sarcomas such as hydatidiform soft tissue sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumors, fibroplastic small round cell tumors, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangioectocytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (undifferentiated neuroectodermal tumor), malignant hemangioendothelioma, malignant Schwann cell tumor, osteosarcoma, and chondrosarcoma.

[0250] In some embodiments, the antibodies and methods of this disclosure may be used to treat one or more types of cancer or cancer-related conditions, including, but 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, antibodies or their antigen-binding moieties that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex described herein may be used in a method for treating cancer in a subject requiring such treatment, the method comprising the step of administering the antibody or its antigen-binding moiety to the subject so that the cancer is treated. In a particular embodiment, the cancer is colon cancer.

[0252] In some embodiments, antibodies or their antigen-binding moieties that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex described herein may be used in methods for treating solid tumors. In some embodiments, solid tumors may be fibrous tumors, which are generally dense and difficult for therapeutic molecules to penetrate. By targeting components of the ECM of such tumors, such antibodies "loosen" and disintegrate the dense tumor tissue, thereby facilitating access for therapeutic agents and exerting their anticancer effects. Thus, they can be used in combination with additional therapeutic agents, such as any known antitumor agents.

[0253] In some embodiments, antibodies or their antigen-binding moieties that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex described herein may be used in a method for inhibiting or reducing solid tumor growth in a subject having a solid tumor, the method comprising the step of administering the antibody or its antigen-binding moiety 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, the antibody or its antigen-binding moiety useful for treating cancer is an isoform-specific, context-tolerant inhibitor 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, antibodies or their antigen-binding moieties that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex described herein are administered to subjects with cancer or tumors either alone or in combination with additional agents, such as anti-PD-1 antibodies (e.g., anti-PD-1 antagonists). Other combination therapies encompassed in the present invention involve administering the antibodies or their antigen-binding moieties described herein together with radiotherapy or chemotherapeutic agents. 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 the state of skeletal muscle In skeletal muscle, TGFβ plays various roles, including inhibition of proliferation and differentiation, induction of atrophy, and development of fibrosis. TGFβ reduces satellite cell proliferation and hinders differentiation (by inhibition of MyoD and myogenin) (Allen, RE and LK, J Cell Physiol, 1987, Vol. 133 (No. 3): pp. 567-567; Brennan, TJ et al., Proc Natl Acad Sci USA, 1991, Vol. 88 (No. 9): pp. 3822-3826; Massague, J. et al., Proc Natl Acad Sci USA, 1986, Vol. 83 (No. 21): pp. 8206-8210; Olson, EN et al., J Cell Biol, 1986, Vol. 103 (No. 5): pp. 1799-805). These early papers do not identify the isoform of TGFβ (i.e., TGFβ1, 2, or 3), but it is presumed to be TGFβ1. TGFβ also contributes to myofascial pain; direct injection of recombinant TGFβ1 induces skeletal myofascial pain, and pan-TGFβ inhibition reduces fibrosis in acutely and chronically damaged muscles (Li, Y. et al., Am J Pathol, 2004, Vol. 164 (No. 3): pp. 1007-19; Mendias, CL et al., Muscle Nerve, 2012, Vol. 45 (No. 1): pp. 55-59; Nelson, CA et al., Am J Pathol, 2011, Vol. 178 (No. 6): pp. 2611-21).TGFβ1 is expressed by muscle fibers, macrophages, regulatory T cells, fibroblasts, and fibroblasts in skeletal muscle (Li, Y. et al., Am J Pathol, 2004, Vol. 164 (No. 3): pp. 1007-19; Lemos, DR et al., Nat Med, 2015, Vol. 21 (No. 7): pp. 786-794; Villalta, SA et al., Sci Transl Med, 2014, Vol. 6 (No. 258): 258ra142; Wang, X. et al., J Immunol, 2016, Vol. 197 (No. 12): pp. 4750-4761), and its expression increases during injury and disease (Li, Y. et al., Am J Pathol, 2004, Vol. 164 (No. 3): pp. 1007-19; Nelson, CA et al., Am J Pathol, 2011, Vol. 178 (No. 6): pp. 2611-2621; Bernasconi, P. et al., J Clin Invest, 1995, Vol. 96 (No. 2): pp. 1137-1144; Ishitobi, M. et al., Neuroreport, 2000, Vol. 11 (No. 18): pp. 4033-4035). In mdx muscle, TGFβ2 and TGFβ3 are also upregulated, to a lesser extent than TGFβ1 (at the mRNA level) (Nelson, CA et al., Am J Pathol, 2011, Vol. 178 (No. 6): pp. 2611-2621; Zhou, L. et al., Neuromuscul Disord, 2006, Vol. 16 (No. 1): pp. 32-38). Pessina et al. recently used lineage tracking experiments to show that cells of multiple origins within the muscle of dystrophy follow a fibrillating fate via a TGFβ-dependent pathway (Pessina, P. et al., Stem Cell Reports, 2015, Vol. 4 (No. 6): pp. 1046-1060).

[0256] TGFβ1 has been linked to 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, Vol. 9 (No. 1): pp. 77-93). Dystrophin deficiency results in increased susceptibility to contraction-induced injury, which leads to continuous muscle degeneration (Petrof, BJ et al., Proc Natl Acad Sci USA, 1993, Vol. 90 (No. 8): pp. 3710-374; Dellorusso, C. et al., J Muscle Res Cell Motil, 2001, Vol. 22 (No. 5): pp. 467-475; Pratt, SJ et al., Cell Mol Life Sci, 2015, Vol. 72 (No. 1): pp. 153-154). Repeated repair rounds contribute to chronic inflammation, fibrosis, depletion of the satellite cell pool, eventual loss of mobility, and death (Bushby, K. et al., Lancet Neurol, 2010, Vol. 9 (No. 1): pp. 77-93; McDonald, CM et al., Muscle Nerve, 2013, Vol. 48 (No. 3): pp. 343-56). TGFβ1 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, Vol. 96 (No. 2): pp. 1137-1144; Chen, YW et al., Neurology, 2005, Vol. 65 (No. 6): pp. 826-834. Excessive ECM deposition has detrimental effects on muscle contractile properties and can restrict nutrient utilization as muscle fibers are isolated from the blood supply (Klingler, W. et al., Acta Myol, 2012, Vol. 31 (No. 3): pp. 184-185). Recent additional data have further linked TGFβ1 to muscular dystrophy. Modified LTBP4 have been found to modulate disease severity in mice and humans. In mice, LTBP4 variants are protective in mice lacking dystrophin or γ-sarcoglycan (Coley, WD et al., Hum Mol Genet, 2016, Vol. 25 (No. 1): pp. 130-145; Heydemann, A. et al., J Clin Invest, 2009, Vol. 119 (No. 12): pp. 3703-3712). In humans, two groups independently identified that LTBP4 variants are protective in DMD and delay the loss of walking movement by several years (Flanigan, KM et al., Ann Neurol, 2013, Vol. 73 (No. 4): pp. 481-488; van den Bergen, JC et al., J Neurol Neurosurg Psychiatry, 2015, Vol. 86 (No. 10): pp. 1060-1065). Although the properties of genetically modified organisms differ between mice and humans, protective modified organisms in both species result in a decrease in TGFβ signaling (Heydemann, A. et al., J Clin Invest, 2009, Vol. 119 (No. 12): pp. 3703-3712; Ceco, E. et al., Sci Transl Med, 2014, Vol. 6 (No. 259): 259ra144).Much of the function of TGFβ1 in skeletal muscle biology has been inferred from experiments in which purified active growth factor is injected into animals or added to cells in culture (Massague, J. et al., Proc Natl Acad Sci USA, 1986, Vol. 83 (No. 21): pp. 8206-8210; Li, Y. et al., Am J Pathol, 2004, Vol. 164 (No. 3): pp. 1007-1019; Mendias, CL et al., Muscle Nerve, 2012, Vol. 45 (No. 1): pp. 55-59). Given the importance of the cellular context for the specific function of TGFβ1 (see, for example, Hinck et al., Cold Spring Harb. Perspect. Biol, 2016, Vol. 8 (No. 12)), some of the effects observed in these experiments may not reflect the intrinsic role of cytokines in vivo. For example, treating human dermal fibroblasts with recombinant TGFβ1, myostatin, or GDF11 results in nearly identical gene expression changes in these cells, despite the considerably different roles of these proteins in vivo (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] Numerous researchers have used TGFβ inhibitors to elucidate 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 (plethysmography, force generation in isolated EDL muscles, and increased forelimb grip strength) (Nelson, CA et al., Am J Pathol, 2011, Vol. 178 (No. 6): pp. 2611-21; Andreetta, F. et al., J Neuroimmunol, 2006, Vol. 175 (No. 1-2): pp. 77-86; Gumucio, JP et al., J Appl Physiol (1985), 2013, Vol. 115 (No. 4): pp. 539-45). Furthermore, muscle fiber-specific expression of dominant-negative TGFβ type II receptors provides protection from muscle injury after cardiotoxicity and in δ-sarcoglycan- / - mice (Accornero, F. et al., Hum Mol Genet, 2014, Vol. 23 (No. 25): pp. 6903-6915). Proteoglycan decorin, which is abundant in skeletal muscle and inhibits TGFβ activity, reduces myofibrosis in mdx mice and after laceration injury (Li, Y. et al., Mol Ther, 2007, Vol. 15 (No. 9): pp. 1616-1622; Gosselin, LE et al., Muscle Nerve, 2004, Vol. 30 (No. 5): pp. 645-6453). Other molecules with TGFβ inhibitory activity, such as suramin (an antitumor drug) and losartan (an angiotensin receptor blocker), were effective in improving muscle pathology and reducing fibrosis in mouse models of injury, Marfan syndrome, and muscular dystrophy (Spurney, CF et al., J Cardiovasc Pharmacol Ther, 2011, Vol. 16 (No. 1): pp. 87-95; Taniguti, AP et al., Muscle Nerve, 2011, Vol. 43 (No. 1): pp. 82-87; Bedair, HS et al., Am J Sports Med, 2008, Vol. 36 (No. 8): pp. 1548-1554; Cohn, RD et al., Nat Med, 2007, Vol. 13 (No. 2): pp. 204-2010. All of the above therapeutic agents inhibit TGFβ1 or its signaling pathway, but none are specific to TGFβ1 isoforms. For example, 1D11 binds to and inhibits TGFβ1, 2, and 3 isoforms (Dasch, JR et al., J Immunol, 1989, Vol. 142 (No. 5): pp. 1536-1541). Suramin inhibits the ability of numerous growth factors, including PDGF, FGF, and EGF, to bind to these receptors in addition to TGFβ1 (Hosang, M., J Cell Biochem, 1985, Vol. 29 (No. 3): pp. 265-263; Olivier, S. et al., Eur J Cancer, 1990, Vol. 26 (No. 8): pp. 867-861; Scher, HI and WD Heston, Cancer Treat Res, 1992, Vol. 59: pp. 131-161). Decorin also inhibits myostatin activity, both through direct binding and by upregulating follistatin, a myostatin inhibitor (Miura, T. et al., Biochem Biophys Res Commun, 2006, Vol. 340 (No. 2): pp. 675-670; Brandan, E., C. Cabello-Verrugio, and C. Vial, Matrix Biol, 2008, Vol. 27 (No. 8): pp. 700-778; Zhu, J. et al., J Biol Chem, 2007, Vol. 282 (No. 35): pp. 25852-25863). 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, Vol. 3 (No. 82): 82ra37; Sabharwal, R. and MW Chapleau, Exp Physiol, 2014, Vol. 99 (No. 4): pp. 627-631; McIntyre, M. et al., Pharmacol Ther, 1997, Vol. 74 (No. 2): pp. 181-194). Therefore, all of these therapies inhibit additional molecules that may contribute to their therapeutic effects and toxicity.

[0258] Given the hypothesized 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 injury, such as in chronic / genetic muscular dystrophy and acute muscle injury, without the toxicity associated with the more broadly acting TGFβ inhibitors developed to date.

[0259] Accordingly, the present invention provides a method for treating muscle fiber injury using agents that preferentially modulate a subset, rather than all, of TGFβ activity in vivo. Such agents can selectively modulate TGFβ1 signaling ("isoform-specific modulation"). In some embodiments, such agents can further selectively modulate TGFβ1 in specific situations ("situation-specific modulation").

[0260] Muscle fiber repair in chronic muscle diseases This invention encompasses methods for improving muscle quality and function in DMD patients by limiting fibrosis and contributing to the standardization of muscle morphology and function. Since TGFβ1 also inhibits myogenesis, TGFβ1 blockade may promote muscle regeneration in dystrophy, thereby adding further therapeutic benefits. TGFβ1 inhibitors may be used in combination with dystrophin upregulation therapies such as Exondys 51 (Eteplirsen). Given the potential therapeutic benefits of TGFβ1 inhibition in muscular dystrophy, it is important to (1) distinguish the role of TGFβ1(or more) from the roles of TGFβ2(or more) and TGFβ3(or more), and (2) clarify the molecular(or more) context in which TGFβ1 inhibition is most beneficial. As described above, pan-TGFβ inhibitors are associated with significant toxicity, which limits the clinical use of these compounds (Anderton, MJ et al., Toxicol Pathol, 2011, Vol. 39 (No. 6): pp. 916-924; Stauber, A. et al., Clinical Toxicology, 2014, Vol. 4 (No. 3): pp. 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 harmful with long-term treatment (Andreetta, F. et al., J Neuroimmunol, 2006, Vol. 175 (Vols. 1-2): pp. 77-86). In fact, depletion of T cells from the muscles improved the muscle pathology in mdx mice, suggesting that T cell-mediated inflammatory responses are harmful to the muscles of dystrophy (Spencer, MJ et al., Clin Immunol, 2001, Vol. 98 (No. 2): pp. 235-2343). The increase in T cell count upon 1D11 administration may be due to the effect of TGFβ1 on regulatory T (Treg) cells.Treg cells present TGFβ1 on their surface via GARP, and the release of TGFβ1 from this complex enhances Treg inhibitory activity, thus limiting T cell-mediated inflammation (Wang, R. et al., Mol Biol Cell, 2012, Vol. 23 (No. 6): pp. 1129-1139; Edwards, JP, AM Thornton, and EM Shevach, J Immunol, 2014, Vol. 193 (No. 6): pp. 2843-2849; Nakamura, K. et al., J Immunol, 2004, Vol. 172 (No. 2): pp. 834-842; Nakamura, K., A. Kitani, and W. Strober, J Exp Med, 2001, Vol. 194 (No. 5): pp. 629-644). Indeed, depleting Tregs using the PC61 antibody increased inflammation and muscle injury in the diaphragm of mdx mice, and muscle injury was reduced by enhancing the number and activity of Tregs (Villalta, SA et al., Sci Transl Med, 2014, Vol. 6 (No. 258): 258ra142). Interestingly, an additional population of immunosuppressive T cells, Tr1 cells, has recently been identified. These cells produce large amounts of TGFβ3 necessary for their suppressive activity (Gagliani, N. et al., Nat Med, 2013, Vol. 19 (No. 6): pp. 739-746; Okamura, T. et al., Proc Natl Acad Sci USA, 2009, Vol. 106 (No. 33): pp. 13974-79; Okamura, T. et al., Nat Commun, 2015, Vol. 6: p. 6329). Although the role of Tr1 cells in skeletal muscle is unknown, inhibition of both TGFβ1 and TGFβ3 by 1D11 may have an additional pro-inflammatory effect due to the inhibition of both Treg and Tr1 cells.

[0261] The above structural insights regarding TGFβ1 latent and activated states enable a novel approach to discovering drugs that specifically target TGFβ1 activation (Shi, M. et al., Nature, 2011, Vol. 474 (No. 7351): pp. 343-349). The high degree of sequence identity shared among the three mature TGFβ growth factors is not shared in the latent complex, which enables the discovery of antibodies highly specific to pro-TGFβ1. Using proprietary antibody discovery techniques, the inventors identified antibodies (Ab1 and Ab2) that specifically bind to pro-TGFβ1 (Figure 18A). Using an in vitro co-culture system, these antibodies were demonstrated to inhibit integrin-mediated release of TGFβ1. In this system, fibroblasts derived from human skin or mouse skeletal muscle are the source of latent TGFβ1, which are cell lines expressing αVβ6 that enable the release of active TGFβ1, and active TGFβ1 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 has demonstrated efficacy in a UUO (unilateral ureteral obstruction) mouse model of renal fibrosis. In this model, treatment of mice (n=10) with 9 mg / kg / week of Ab1 prevented upregulation of TGFβ1-responsive genes (Figure 15) and reduced the degree of post-injury fibrosis (by picrosilius red staining) (Figure 16). TGFβ1-specific therapy may have an improved efficacy and safety profile compared to pan-TGFβ inhibitors, which is a significant aspect for therapeutics to be used long-term in the DMD population. TGFβ1 inhibitory antibodies may be used to determine whether specific TGFβ1 inhibition has potential as a therapeutic agent for DMD or other muscle diseases, and to elucidate the role of TGFβ1 in skeletal muscle regeneration.

[0262] Chronic muscle fiber injury vs. acute muscle fiber injury and selection of the optimal treatment 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 by inflammatory macrophages is necessary to remove damaged tissue and to stimulate the secretion of factors (e.g., cytokines) required for satellite cell activation. Subsequently, these cells switch to the M2 phenotype to drive wound dissipation.

[0263] In contrast, in chronic conditions such as diseases including DMD, pro-inflammatory macrophages are always dominant, and switching to M2 macrophages does not occur at all (or at least not efficiently), with inflammation and muscle injury continuing to be driven by pro-inflammatory macrophages. In DMD, the NFκB pathway is permanently activated, resulting in constitutive inflammation. Therefore, in some embodiments, NFκB inhibitors can be administered to DMD patients to reduce chronic inflammation.

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

[0265] The evidence suggests that LRRC33 is expressed in thioglycolate-induced peritoneal macrophages that have an M2-like phenotype (characterized by high levels of arginase expression, absence of iNOS expression, and high levels of CD206 expression).

[0266] In situations where LRRC33 is primarily expressed on M2 cells, and where TGFβ1 presentation ("situation") is crucial for the wound-healing effects of these cells, activating LRRC33-mediated TGFβ1 to promote repair and / or myogenesis may be beneficial. On the other hand, in situations where LRRC33 is also expressed on pro-inflammatory M1 cells, particularly in dystrophy cases such as DMD, considering that inflammation drives fibrosis, inhibiting LRRC33-mediated TGFβ1 may be beneficial. Therefore, identifying disease-related TGFβ1 sources / situations can be a crucial step in selecting appropriate modulators of TGFβ signaling, indicating what level of selectivity should be considered (e.g., isoform-specific, situation-tolerant TGFβ1 modulators, or situation-specific TGFβ1 modulators; TGFβ1 inhibitors or activators, etc.).

[0267] Aside from chronic inflammation, a defining characteristic of DMD is excessive and progressive fibrosis. In progressive disease, fibrosis is so severe that individual muscle fibers may actually become isolated from their blood supply. Fibrosis also alters the contractile properties of muscles. In human patients, there is a strong correlation between the degree of TGFβ1 upregulation and fibrosis, and also a strong correlation between the degree of fibrosis and negative mobility outcomes. Therefore, in some embodiments, LTBP-pro-TGFβ1 inhibitors can be administered to dystrophy patients to selectively target TGFβ1 action associated with the ECM in the disease for the prevention and / or reduction of fibrosis. In some embodiments, the various isoform-selective and / or context-selective agents described herein may be used (e.g., via GARP or LRRC33) to achieve inhibitory signaling of TGFβ1 in such a way that it prevents fibrosis and promotes myogenesis, but without having undesirable effects on the immune system.

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

[0269] Subjects treated by the methods described herein may be mammals, more preferably humans. Mammals include, but are not limited to, livestock, sports animals, companion animals, primates, horses, dogs, cats, mice, and rats. Human subjects requiring treatment may be human patients who have, are at risk of, or are suspected of having TGFβ-related indications, such as those described above. Subjects with TGFβ-related indications can be identified by routine medical examinations, e.g., clinical examinations, organ function tests, CT scans, or ultrasound. Subjects suspected of having any of such indications may exhibit one or more symptoms of the indication. Subjects at risk of an indication may be subjects who have one or more risk factors for that indication.

[0270] As used herein, the terms “effective dose” and “effective amount” refer to any amount or dose of a compound or composition sufficient to satisfy its intended purpose, i.e., a desired biological or medical response in a tissue or subject with 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 dose varies depending on the specific 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 any concurrent treatments, the specific route of administration, and similar factors within the knowledge and expertise of healthcare professionals. These factors are well known to those skilled in the art and can be addressed to the extent of routine experimentation. It is generally preferable to use the maximum dose of an individual component or combination thereof, i.e., the highest safe dose by reasonable medical judgment. However, it will be understood by those skilled in the art that a patient may insist on a lower dose or an acceptable dose for medical reasons, psychological reasons, or virtually any other reason.

[0271] Empirical considerations such as half-life generally contribute to the determination of dosage. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, may be used to extend the antibody half-life and prevent the antibody from being attacked by the host 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-release formulations of antibodies that specifically bind to GARP-TGFβ1, LTBP1-TGFβ1, LTBP3-TGFβ1, and / or LRRC33-TGFβ1 complexes may also be appropriate. Various formulations and devices for achieving sustained release are apparent to those skilled in the art and are within the scope of this disclosure.

[0272] For example, the dosage of antibodies specifically binding to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex described herein may be empirically determined in an individual given one or more doses of the antibody. The individual is given an antagonist in gradually increasing doses. To evaluate efficacy, indicators of TGFβ-related indications may be tracked. For example, methods for measuring muscle fiber injury, 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 result in an improved safety profile when used as pharmaceuticals. Accordingly, the present invention includes an antibody and its antigen-binding fragment that specifically binds to TGFβ1 and inhibits its activation, but does not bind to TGFβ2 or TGFβ3, thereby conferring specific inhibition of TGFβ1 signaling in vivo while minimizing undesirable side effects resulting from the influence of TGFβ2 and / or TGFβ3 signaling.

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

[0275] In general, for any administration of the antibodies described herein, the initial candidate dose may be approximately 2 mg / kg. For the purposes of this disclosure, a typical daily dose may be in the range of approximately 0.1 μg / kg, 3 μg / kg, 30 μg / kg, 300 μg / kg, 3 mg / kg, 30 mg / kg, 100 mg / kg, or more, depending on the factors described above. For repeated administration over several days or longer, treatment should be continued, depending on the condition, until the desired symptom suppression occurs or until a sufficient therapeutic level is achieved to alleviate the TGFβ-related indication or its symptoms. An exemplary dosing regimen may include an initial dose of approximately 2 mg / kg, followed by weekly maintenance doses of approximately 1 mg / kg of antibody, or followed by every-weekly maintenance doses of approximately 1 mg / kg. However, other dosing regimens may be useful depending on the pharmacokinetic decay pattern that the clinician wishes to achieve. For example, administration 1 to 4 times per week is intended. In some embodiments, dosages ranging from approximately 3 μg / mg to approximately 2 mg / kg (e.g., approximately 3 μg / mg, approximately 10 μg / mg, approximately 30 μg / mg, approximately 100 μg / mg, approximately 300 μg / mg, approximately 1 mg / kg, and approximately 2 mg / kg) 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 we do not wish to be bound by any particular theory, this post-administration stability may be advantageous because it allows for the administration of antibodies at a lower frequency while maintaining clinically effective serum concentrations in the subjects receiving the antibodies (e.g., human subjects). In some embodiments, the administration frequency is once weekly, every two weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, or every ten weeks, or once monthly, every two months, every three months, or at longer intervals. The progression of this treatment can be easily monitored using conventional techniques and assays. The administration regimen (including the antibodies used) may vary over time.

[0276] In some embodiments, doses ranging from approximately 0.3 to 5.00 mg / kg may be administered to adult patients of normal weight. Specific dosing regimens, such as dose, timing, and repetition, depend on the specific individual, their medical history, and the characteristics of the individual drug (e.g., drug half-life and other relevant considerations).

[0277] For the purposes of this disclosure, the appropriate dosage of an antibody that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex depends on the specific antibody (or its composition) 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 antagonists, and the judgment of the attending physician. In some embodiments, the clinician administers an antibody that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex until a dosage is reached that achieves the desired outcome. Administration of antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex may be continuous or intermittent, depending on, for example, the recipient's physiological condition and other factors known to a skilled physician, regardless of whether the purpose of administration is therapeutic or prophylactic. Administration of antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex may be essentially continuous over a pre-selected period, or it may be a series of intervald doses, for example, before, during, or after the onset of a TGFβ-related indication.

[0278] As used herein, the term “treat” means the application or administration of a composition comprising one or more activators to a subject having a TGFβ-related indication, symptoms of an indication, or predisposition to an indication, with the aim of curing, healing, alleviating, reducing, altering, remedying, improving, or influencing such indication, symptoms of an indication, or predisposition to an indication.

[0279] Mitigating TGFβ-related indications with antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex includes delaying the onset or progression of the indication, or reducing the severity of the indication. Mitigating an indication does not necessarily require a curative outcome. 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 retarding may be of varying lengths depending on the history of the indication and / or the individual being treated. Methods that “delay” or mitigate the onset of an indication, or delay the onset of an indication, are methods that reduce the likelihood of developing one or more symptoms of the indication and / or reduce the severity of symptoms within a given time frame compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to yield statistically significant results.

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

[0281] Combination therapy This disclosure encompasses pharmaceutical compositions and related methods used as combination therapies for treating subjects in which in vivo TGFβ inhibition may be beneficial. In any of these embodiments, such a subject may receive combination therapy comprising a first composition comprising at least one TGFβ inhibitor, e.g., an antibody or antigen-binding moiety described herein, and a second composition comprising at least one further therapeutic agent intended for the treatment of the same or overlapping disease or clinical condition. The first and second compositions may both act on the same cellular target, or they may both act on distinct cellular targets. In some embodiments, the first and second compositions may treat or alleviate the same or overlapping set of symptoms or appearances of the disease or clinical condition. In some embodiments, the first and second compositions may treat or alleviate distinct sets of symptoms or appearances of the disease or clinical condition. For example, the first composition may treat a disease or condition related to TGFβ signaling, and the second composition may treat inflammation or fibrosis associated with the same disease, and so on. Such combination therapies may be administered in combination with each other. The phrase "in conjunction with" means, with respect to combination therapy, that the therapeutic effect of the first therapy temporarily and / or spatially overlaps with the therapeutic effect of the second therapy in a subject receiving combination therapy. Therefore, 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, combination therapy produces a synergistic effect in treating the disease. The term "synergistic" refers to an effect greater than the sum of the individual therapies (e.g., greater efficacy).

[0283] In some embodiments, combination therapy comprising the pharmaceutical compositions described herein produces an overall efficacy equivalent to that produced by another therapy (e.g., monotherapy of the second drug), but with fewer undesirable adverse effects or a lower severity of toxicity compared to monotherapy of the second drug. In some embodiments, such combination therapy makes it possible to use a lower dose of the second drug while maintaining the overall efficacy. Such combination therapy may be particularly suitable for patient populations where long-term treatment is guaranteed and / or involving pediatric patients.

[0284] Accordingly, 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 described herein. Accordingly, the methods or pharmaceutical compositions further include a second treatment. In some embodiments, the second treatment may be useful in treating or preventing diseases or conditions associated with TGFβ1 signaling. The second treatment can attenuate or treat at least one symptom(s) associated with the targeted disease. The first and second treatments may exert their biological effects by similar or unrelated mechanisms of action, or one or both of the first and second treatments may exert their biological effects by multiple mechanisms of action.

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

[0286] One or more anti-TGFβ antibodies or their antigen-binding moieties 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 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. 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 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 allow for the advantageous use of administered therapeutic agents in smaller doses, thus avoiding potential toxicity or complications associated with various monotherapies.

[0289] Regulation of TGFβ activity The methods of this disclosure include methods for modulating growth factor activity in one or more biological systems. Such methods may include contacting one or more biological systems with the antibodies and / or compositions of this disclosure. In some cases, these methods include a step of modulating the level of free growth factor in the biological system (e.g., in a cellular niche or in a subject). The antibodies and / or compositions of such methods may include, but are not limited to, recombinant proteins, protein complexes and / or biomolecules including antibodies or their antigen-binding moieties as described herein.

[0290] In some embodiments, the methods of this disclosure, referred herein as “inhibition methods,” may be used to reduce or eliminate growth factor activity. Some such methods may include the retention of mature growth factors in the TGFβ complex (e.g., TGFβ1 complexed with GARP, LTBP1, LTBP3, and / or LRRC33) and / or the promotion of the reassociation of growth factors into the TGFβ complex. In some cases, the inhibition method may include the use of antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex. According to some inhibition methods, one or more inhibitory antibodies are provided.

[0291] In some embodiments, the antibodies, antigen-binding moieties, and compositions of this disclosure may be used to inhibit TGFβ1 activation. In some embodiments, methods for inhibiting TGFβ1 activation are provided herein, comprising the step of exposing a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex to the antibodies, antigen-binding moieties, or pharmaceutical compositions described herein. In some embodiments, the antibodies, antigen-binding moieties, or pharmaceutical compositions inhibit the release of mature TGFβ1 from the GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex. In some embodiments, the method is carried out in vitro. In some embodiments, the method is carried out in vivo. In some embodiments, the method is carried out ex vivo.

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

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

[0294] In some embodiments, the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex are bound to the extracellular matrix. In some embodiments, the extracellular matrix contains fibrillin. In some embodiments, the extracellular matrix contains proteins containing the RGD motif.

[0295] In some embodiments, methods for reducing TGFβ1 protein activation in a subject are provided herein, comprising administering an antibody, an antigen-binding moiety thereof, or a pharmaceutical composition described herein to the subject, 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 described herein, or their antigen-binding moieties, reduce the inhibitory activity of regulatory T cells (Tregs).

[0297] A kit for use in alleviating diseases / disorders related to TGFβ-associated indications. This disclosure also provides kits for use in alleviating diseases / disorders associated with TGFβ-related indications. Such kits may comprise one or more containers containing an antibody or its antigen-binding moiety that specifically binds to a GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, for example, any of those described herein.

[0298] In some embodiments, the kit may include instructions for use by any of the methods described herein. The included instructions may include instructions for administering an antibody or its antigen-binding moiety that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex to treat, delay, or mitigate the target diseases described herein. The kit may further include instructions for selecting individuals suitable for treatment based on identifying whether the individuals have the target disease. In yet other embodiments, the instructions may include instructions for administering an antibody or its antigen-binding moiety to individuals at risk of the target disease.

[0299] Instructions for the use of antibodies or their antigen-binding moieties that specifically bind to GARP-TGFβ1 complexes, LTBP1-TGFβ1 complexes, LTBP3-TGFβ1 complexes, and / or LRRC33-TGFβ1 complexes generally include information on dosage, administration schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages) or partial unit doses. Instructions supplied in the kits of this disclosure are typically written instructions on labels or package inserts (e.g., paper sheets included in the kit), but machine-readable instructions (e.g., instructions written on magnetic or optical storage disks) are also acceptable.

[0300] Labels or package inserts indicate that the composition is used to treat, delay the onset of, and / or alleviate a disease or disorder associated with a TGFβ-related indication. Instructions may be provided for carrying out any of the methods described herein.

[0301] The kits of this disclosure are in preferred packaging. Preferred packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging (e.g., sealed Mylar or plastic bags). Packaging for use in combination with specific devices such as inhalers, nasal administration devices (e.g., atomizers) or infusion devices such as minipumps may also be considered. The kits may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a subcutaneous needle). The containers may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a subcutaneous needle). At least one activator in the composition is an antibody or its antigen-binding moiety that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex described herein.

[0302] The kit may optionally provide additional components such as buffers and interpretation information. Typically, the kit includes a container and a label or packaging insert(s) on or accompanying the container. In some embodiments, this disclosure provides a manufactured product containing the contents of the kit described above.

[0303] Assay 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 the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex in samples obtained from subjects. 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 any sex and at any developmental stage. In some embodiments, the subject is a patient or a healthy volunteer.

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

[0305] In one embodiment, a screening assay is performed using a surface-immobilized biotinylated latent TGFβ1 complex that enables integrin-mediated activation of latent TGFβ by providing anchoring. Alternatively, non-integrin activators can also be tested in the system. Readout may be via a reporter cell or other TGFβ-dependent cellular response.

[0306] Cell-based assay for measuring TGFβ activation TGFβ activation (and its inhibition by TGFβ test 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 cell-based assays such as the "CAGA12" luciferase assay described in more detail herein. An exemplary embodiment of such an assay is shown for illustrative purposes in Figure 11C. As shown, such an assay system may 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 the signal into a readable output (e.g., luciferase activity in CAGA12 cells or other reporter cell lines). 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., a PAI-1 promoter). In some embodiments, a specific promoter element that imparts sensitivity can be incorporated into the reporter system. In some embodiments, such a promoter element is the CAGA12 element. Reporter cell lines that can be used in the assay are described, for example, Abe et al. (1994) Anal Biochem., vol. 216(2): pp. 276-2784, which are incorporated herein by reference. In some embodiments, each of the components of the assay described above is supplied from the same source (e.g., the same cells). In some embodiments, two of the components of the assay described above are supplied from the same source, and the components of a third assay are supplied from a different source. In some embodiments, the components of all three assays are supplied from different sources. For example, in some embodiments, the integrin and latent TGFβ complex (pro-TGFβ and presenting molecule) are supplied for the assay from the same source (e.g., the same transfected cell line).In some embodiments, integrins and TGF are supplied for the assay from separate sources (e.g., two different cell lines, a combination of purified integrins and transfected cells). When cells are used as one or more sources of assay components, such assay components may be endogenous to the cells, stably expressed in the cells, transiently transfected, or any combination thereof. Results from non-limiting exemplary embodiments of cell-based assays for measuring TGFβ activation demonstrated by inhibition of either the GARP-pro-TGFβ1 complex or the LRRC33-pro-TGFβ1 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 against the GARP-TGFβ1 complex was 0.445, and the IC50 (μg / mL) of Ab1 against the LRRC33-TGFβ1 complex was 1.325.

[0307] Those skilled in the art can easily adapt such assays to a variety of suitable configurations. For example, various sources of TGFβ can be considered. In some embodiments, the source of TGFβ is cells (e.g., primary cells, breeding cells, immortalized cells, or cell lines) on which TGFβ is expressed and deposited. In some embodiments, the source of TGFβ is purified and / or recombinant TGFβ is immobilized in an assay system using preferred means. In some embodiments, the TGFβ immobilized in the assay system is present in the extracellular matrix (ECM) composition on the assay plate with or without decellularization, mimicking fibroblast-derived TGFβ. In some embodiments, the 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 a suitable latent TGFβ complex. Those skilled in the art can easily determine which presentation molecules may be present or expressed in a particular cell or cell type. Using such assay systems, the relative changes in TGFβ activation in the presence or absence of a test drug (e.g., an antibody) can be easily measured to evaluate the effect of the test drug on TGFβ activation in vitro. Data from exemplary cell-based assays are presented in the Examples section below.

[0308] Assays based on such cells can be modified or adapted in several ways depending on the TGFβ isoform being tested, the type of latent complex (e.g., the presenting molecule), etc. In some embodiments, cells known to express integrins capable of activating TGFβ can be used as the integrin source 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 the presenting molecule of interest (e.g., GARP, LRRC33, LTBP (e.g., LTBP1 or LTBP3)) and a plasmid encoding the pro-form of the TGFβ isoform of interest (e.g., pro-TGFβ1). After transfection, the cells are incubated for a sufficient amount of time (e.g., about 24 hours) to allow expression of the transfected gene, washed, and incubated with serial dilutions of the test drug (e.g., antibody). A reporter cell line (e.g., CAGA12 cells) is then added to the assay system and incubated for an appropriate time to allow TGFβ signaling. After an incubation period following the addition of the test drug (e.g., approximately 18-20 hours), the signal / readout (e.g., luciferase activity) is detected using a suitable method (for example, 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 auto-gain settings.

[0309] nucleic acid In some embodiments, the antibodies of the Disclosure, their antigen-binding portions, and / or compositions may be encoded by nucleic acid molecules. Such nucleic acid molecules include, but are not limited to, DNA molecules, RNA molecules, polynucleotides, oligonucleotides, mRNA molecules, vectors, plasmids, and the like. In some embodiments, the Disclosure may include cells programmed or generated to express nucleic acid molecules encoding the compounds and / or compositions of the Disclosure. In some cases, the nucleic acids of the 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. Patents 5,786,464 and 6,114,148, the entire contents of which are incorporated herein by reference.

[0310] The present invention is further illustrated by the following embodiments, which are not limiting in any way. All references, patent and published patent applications, and drawings cited throughout this application are thus incorporated herein by reference. [Examples]

[0311] (Example 1: Inhibition of TGFβ1) The TGFβ superfamily includes propeptides that form complexes with active growth factors (Figure 1). We obtained antibodies that stabilize these complexes and developed a selection strategy to achieve more selective and potent inhibition.

[0312] Using an expression system based on HEK293, several milligrams of purified protein were obtained by NiNTA affinity testing and gel filtration, and these were 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), the sGARP-TGFβ LAP complex (Figure 5), and the LTBP1-proTGFβ1 complex (Figure 6) is shown.

[0313] Candidate antibodies were tested using an in vitro luminescence assay (Figure 8). In screening, antibodies that inhibited growth factor release "off" reporter cells when faced with normal activation stimuli. Ab1 and Ab2 were inhibitors of latent TGFβ1 complex activation (Figure 7) and were shown to cross-react in mice.

[0314] Initial dose-response analysis curves of Ab1 in cells expressing human TGFβ1 demonstrated inhibition of TGFβ1 activity (Figure 8). Using the more sensitive CAGA12 reporter cell line, Ab1 showed similar inhibition of human pro-TGFβ1 activity (Figure 9). Furthermore, inhibition of the GARP complex was shown to block the inhibitory activity of regulatory T cells (Treg), as measured by the percentage of proliferating effector T cells (Teff) in T cells isolated from the blood of healthy donors (Figure 10).

[0315] The affinity of GARP-pro-TGFβ1 inhibitors was measured by the Octet assay against human GARP-pro-TGFβ1 cells, while their activity was measured using CAGA12 reporter cells to test for human GARP-pro-TGFβ1 inhibition. The protocols used to measure the affinity of antibodies Ab1 and Ab2 to the conjugates provided herein are summarized in Table 6. The results are shown in Table 7. [Table 6-1] [Table 6-2] [Table 7]

[0316] The 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 they 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 derived from multiple species.) To determine whether Ab1 and Ab2 can specifically bind to pro-TGFβ1 complexes derived 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-pro-TGFβ1 complexes, mouse LTBP1-pro-TGFβ1 complexes, human LTBP3-pro-TGFβ1 complexes, and human GARP-pro-TGFβ1 complexes. However, only Ab2 specifically bound to rat LTBP1-pro-TGFβ1 complexes. [Table 10]

[0318] (Example 3: Ab1 and Ab2 inhibit endogenous TGFβ1 in human fibroblasts and mouse fibroblasts.) To determine whether Ab1 and Ab2 can inhibit endogenous TGF-β1 secreted from cultured primary fibroblasts of different origins, a quantitative in vitro assay was performed in which the activity of secreted TGF-β1 was stably transfected with nucleic acids containing a luciferase reporter gene fused with the CAGA12 synthesis promoter, and luciferase levels were determined from mink lung epithelial cells co-cultured with fibroblasts treated with either Ab1 or Ab2. As shown in Figures 11A and 11B, endogenous TGF-β1 secreted from normal human dermal fibroblasts, mouse C57BL.6J lung fibroblasts, and DBA2 / J myofibroblasts was inhibited by both Ab1 and Ab2. The differences in maximum 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 association with respect 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-pro-TGFβ1 and LRRC33-pro-TGFβ1) To determine whether GARP-pro-TGF-β1 and / or LRRC33-pro-TGF-β1 activity is inhibited by Ab1 and Ab2, in vitro cell-based assays were performed. In this assay system, engineered human colon cancer cell lines (SW480 / β6 cells), stably transfected with β6 integrin, were co-transfected with constructs for expressing pro-TGF-β1 and constructs for expressing the presentation molecule (i.e., GARP or LRRC33). Chimeric LRRC33-GARP (SEQ ID NO: 85) or a construct encoding GARP was used to express the presentation molecule. Transfected cells were incubated to allow sufficient expression and deposition of the components (integrin and pro-TGFβ1 complexed with their respective presentation molecules). The activation of TGFβ1 in the presence or absence of Ab1 or Ab2 was assayed using reporter cells (CAGA12 cells) expressing the TGFβ receptor coupled to a downstream signal transduction pathway, and the inhibitory activity of the antibodies was measured. As shown in Figures 13A and 13B, both GARP-pro-TGF-β1 and LRRC33-pro-TGF-β1 were inhibited by Ab1 and Ab2.

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

[0322] (Example 6: Effects of Ab2 on renal biomarkers and fibrosis in a mouse model of unilateral ureteral obstruction (UUO)) The unilateral ureteral obstruction mouse model is widely used to test interstitial fibrosis, a common pathological process that can lead to end-stage renal disease (see Isaka et al. (2008) Contrib. Nephrol., vol. 159: pp. 109-21, and Chevalier (1999) Pediatr. Nephrol., vol. 13: pp. 612-619). 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., vol. 32: pp. 1513-21). Increased TGFβ1 expression 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 experiments were performed.

[0323] In short, male CD-1 mice (Charles River Laboratories), aged 7-8 weeks, were divided into four groups (n=10). They were administered either Ab2 (3 mg / kg or 30 mg / kg; 10 mL / kg dose), mouse IgG1 control antibody (30 mg / kg; 10 mL / kg dose), or PBS as a vehicle control intraperitoneally (ip), followed by surgical intervention. The procedure was performed one day before surgery (d-1), one day after surgery (d1), and three days after surgery (d3). On day 0 (d0), the mice were anesthetized with isoflurane using a nose cone, underwent laparotomy, and then a permanent right-sided unilateral ureteral ostomy (UUO) was performed. An additional control group of mice (n=8) were administered PBS as described above, but only sham surgery (i.e., without ureteral obstruction) was performed. Immediately after the completion of the surgical procedure, all mice received a single subcutaneous injection of 0.001 mg / kg of buprenorphine. The mice were sacrificed 5 days after surgery, and tissue was 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 the renal tissue. As shown in Figure 14, renal hydroxyproline levels, a marker of histofibrosis and collagen deposition, were significantly elevated in surgically intervened mice compared to sham-surgery-treated mice.

[0324] The central transverse section of each right kidney was fixed by immersion in 10% neutral buffered formalin for 48 hours, and then transferred to 70% ethanol for histological processing and analysis. The fixed kidney sections were paraffin-embedded, sectioned (three 5 μm serial sections obtained 200–250 μm apart per animal kidney to allow for larger sample sizes and representation of kidney damage), stained with picrosilius red, and subjected to quantitative histological analysis using color spectral splitting to determine the percentage of cortical collagen volume (CVF). A single composite CVF score was calculated for each animal by determining the mean of the CVF scores of each of the three serial sections. Statistical analysis was performed using a one-sided 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 of Ab2 showed a significant reduction in the UUO-inducible increase of CVF compared to mice receiving either a vehicle control (PBS) or an 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 chemotactic protein 1 (MCP-1), type I collagen alpha 1 (Col1a1), and type III collagen alpha 1 chain (Col3a1) were determined in recovered kidney tissue (Figure 15A-15H). mRNA levels were normalized using housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT1) mRNA levels. Furthermore, in mice that received either 3 mg / kg or 30 mg / kg of Ab2 prior to surgical intervention, mRNA levels of PAI-1, CTGF, TGFβ1, fibronectin 1, Col1a1, and Col3a1 were significantly reduced compared to mice that received 30 mg / kg of IgG1 control. Mice that received 3 mg / kg of Ab2 before surgical intervention showed significantly lower α-SMA mRNA levels compared to mice that received 30 mg / kg of IgG1 control. Furthermore, mice that received 30 mg / kg of Ab2 before surgical intervention showed significantly lower MCP-1 mRNA levels compared to mice that received 30 mg / kg of IgG1 control.

[0326] In summary, in the UUO mouse model, significant effects were observed in mice treated with Ab2, with the exception of hydroxyproline levels. As shown in Figures 15A–15H and 16, treatment with Ab2 significantly attenuated the UUO-inducible 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 the pathogenesis.

[0327] (Example 7: Effects of Ab1 and Ab2 alone or in combination with an anti-PD-1 antibody on tumor progression in an MC38 mouse syngeneic model of colon cancer) To evaluate the effects of Ab1 and Ab2, either alone or in combination with anti-PD-1 antibodies, on reducing colon cancer tumor progression, we used the MC38 mouse colon cancer C57BL / 6 mouse syngeneic model.

[0328] Tumor cell culture MC38 mouse colon cancer cells were grown in Dulbecco's Modified Eagle 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. The cell cultures were maintained in tissue culture flasks in a humidified incubator at 37°C, under an atmosphere of 5% CO2 and 95% air.

[0329] In vivo implantation and tumor growth MC38 cells for implantation were harvested during logarithmic growth and resuspended in phosphate-buffered saline (PBS). On the day of tumor implantation, 5 × 10¹⁶ cells were placed in the right flank of each test mouse. 5 Individual cells (0.1 mL of cell suspension) are subcutaneously injected to stimulate tumor growth, with an average size of 80-120 mm. 3 We monitored the mice as they approached the target range. On day 1 of the experiment, designated 11 days later, each mouse was measured according to its calculated tumor size, with an individual tumor volume of 63 mm². 3 From 196mm 3 Up to that point, the group average tumor volume was 95-98 mm 3 The animals were selected into a group of 12. Tumors were measured in two dimensions using a calyx, and the formula was: [ka] The volume was calculated using the formula (where w = tumor width and l = tumor length, in mm). Tumor weight was given as 1 mg per 1 mm of tumor volume. 3 It can be estimated using the assumption that it is equal to .

[0330] treatment In short, on day 1, a subcutaneous MC38 tumor (63-172 mm) was found. 3Eight-week-old female C57BL / 6 mice (n=12) with the following characteristics were administered intraperitoneally (ip) twice a week for four weeks with either Ab1, Ab2, or a mouse IgG1 control antibody (30 mg / kg in 10 mL / kg of administration volume). The tumor size in the control group was 150 mm. 3 Once the target was reached (day 6), mice were administered either rat anti-mouse PD-1 antibody (RMP1-14) or rat IgG2A control antibody twice a week for two weeks (each antibody at a dose of 5 mg / kg in 10 mL / kg).

[0331] Group 1 served as a tumor growth control and received a combination of mouse IgG1 isotype control antibody and rat IgG2a control antibody. Group 2 received a combination of Ab1 and rat IgG2a control antibody. Group 3 received a combination of Ab2 and rat IgG2a control antibody. Group 3 received a combination of mouse IgG1 control antibody and anti-PD-1 antibody. Group 4 received a combination of Ab1 and anti-PD-1 antibody. Group 5 received a combination of Ab2 and 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 a week using Calipas, and for each animal, the tumor was measured when the endpoint volume reached 1,000 mm³. 3 Mice were euthanized at the earlier of either reaching the tumor volume endpoint or at the end of the study (day 60). Mice that terminated the study due to tumor volume were recorded as having been euthanized due to tumor progression (TP), along with the date of euthanasia. For each mouse, the time to the endpoint for analysis (TTE) was calculated using the following equation: [ka] (In the formula, TTE is expressed in days, and endpoint volume is mm 3The TTE was calculated using (represented as b = √m, where b is the intercept and m is the slope of the line obtained from the linear regression of the log-transformed tumor growth dataset). The dataset consisted of the first observation exceeding the endpoint volume used for analysis and three consecutive observations immediately preceding this endpoint volume. The calculated TTE is typically smaller than the TP day, which is the day on which 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 cases where the log-transformed calculated TTE preceded a day before reaching the endpoint or exceeded the day on which the tumor volume endpoint was reached, linear interpolation was performed to estimate the TTE. Mice classified as dying from causes unrelated to treatment (NTR) were excluded from TTE calculation (and all further analysis). Animals classified as TR (treatment-related) death or NTRm (treatment-related death due to metastasis) were assigned a TTE value equal to the day of death.

[0333] The outcome of the treatment was evaluated based on tumor growth delay (TGD), and defined as an increase in median time to the endpoint (TTE) in the treatment group compared to the control group. Expressed in days: TGD=TC, Alternatively, expressed as a percentage of the median TTE of 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 the treatment can be determined from the tumor volume 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 remaining number (n) of animals whose tumors did not reach the endpoint volume.

[0335] The efficacy of the treatment can also be determined from the number and magnitude of regression responses observed during the study. The treatment may induce partial regression (PR) or complete regression (CR) of the tumor in animals. In a PR response, the tumor volume is 50% or less of its volume on day 1 for three consecutive measurements during the study, and 13.5 mm for one or more of these three measurements. 3 It was equal to or greater than . In the CR response, the tumor volume was 13.5 mm with respect to three consecutive measurements during the course of the test. 3 It was smaller than that. Animals that showed a complete response (CR) at the end of the study were further classified as tumor-free survivors (TFS). The animals were monitored for regression response.

[0336] Tumor growth inhibition Tumor Growth Inhibition (TGI) analysis evaluates the difference in median tumor volume (MTV) between treated mice and control mice. For this study, the endpoint for determining TGI is a mean tumor volume of 1500 mm³ in control mice. 3 It was the 29th day, the day when the TGI was reached. The number of animals on the TGI analysis day and the median tumor volume (MTV(n)) for n were determined for each group. Percentage 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 dataset for TGI analysis included all mice within the group except those that died before the TGI analysis date from treatment-related (TR) or treatment-related (NTR) causes.

[0338] In this study, Ab1 and Ab2 were evaluated alone and in combination with anti-PD-1 in a syngeneic C57BL / 6 mouse model of MC38 mouse colon cancer. Mice treated with Ab2 in combination with anti-PD-1 showed a significant day 29 TGI (P<0.05, Mann-Whitney U test), and a statistically significant survival benefit compared to vehicle-treated controls was observed using log-rank survival analysis (P<0.05, log-rank) (see Figure 17). Mice treated with Ab1 or Ab2 in combination with rat IgG2a control antibody showed one complete response (CR) and one partial response (PR), respectively. When combined with anti-PD-1, the regression responses for Ab1 and Ab2 were one PR and one CR, respectively, and four CRs. Combining Ab2 with anti-PD-1 resulted in a significant short-term efficacy on day 29, and also demonstrated overall survival benefit in a 60-day TGD trial in this MC38 mouse colon cancer C57BL / 6 mouse syngeneic model.

[0339] (Example 8: The role of TGFβ1 in muscular dystrophy) TGFβ plays numerous roles in skeletal muscle function, including inhibiting myogenesis, regulating inflammation and muscle repair, and promoting fibrosis. While TGFβ inhibition has garnered considerable interest as a treatment for a wide range of diseases, including muscular dystrophy, these treatments inhibit TGFβ1, TGFβ2, and TGFβ3 regardless of the molecular context. The lack of specificity / selectivity of these inhibitors can lead to undesirable side effects, resulting in ineffective clinical doses. While pan-TGFβ inhibitory molecules have been reported to improve muscle function and reduce fibrosis in mdx mice, it remains unclear whether these effects are due to the inactivation of TGFβ1, β2, or β3.

[0340] To address this, antibodies were generated that specifically block integrin-mediated activation of latent TGFβ1 while preserving TGFβ2 and β3. To confirm the specific role of TGFβ1 in muscle repair in dystrophy muscles, D2.mdx mice were treated with a pro-TGFβ1 specific antibody. The functional effects of TGFβ1 inhibition on protection from contraction-induced injury, as well as on recovery from the same type of injury, were evaluated. Histological evaluation included whether the 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 muscles (e.g., increased inflammation, long-term loss of muscle function) are attributable to TGFβ1 inhibition or to TGFβ2 / 3 inhibition. To understand whether inhibiting TGFβ1 in a specific molecular context is more effective and / or has fewer negative effects (adverse effects), the efficacy of LTBP-pro-TGFβ1 inhibitors in this model can be evaluated to deconvolute the role of TGFβ1 presented to immune cells from the role of TGFβ1 present in the extracellular matrix (ECM), potentially leading to safer and / or more effective anti-fibrotic therapies.

[0341] Muscles in dystrophy patients are highly sensitive to contraction-induced injury. After injury, muscles derived from mdx mice show a significant reduction in force generation and increased uptake of Evans blue dye (an indicator of physical damage / injury to muscle fibers) compared to WT mice (Lovering, RM et al., Arch Phys Med Rehabil, 2007, Vol. 88 (No. 5): pp. 617-625). Therapeutics that reduce the degree of contraction-induced injury or improve post-injury recovery have significant clinical utility in patients with muscular dystrophy (Bushby, K. et al., Lancet Neurol, 2010, Vol. 9 (No. 1): pp. 77-93). Ab1 and Ab2 are evaluated for i) their ability to prevent contraction-induced injury and ii) their ability to promote recovery from injury. In our experiments, the D2.mdx strain can be used in contrast to the conventional mdx strain with a B10 background. These mice, produced by crossing mdx with a DBA2 / J background, possess the unprotective variant of LTBP4 described above and therefore exhibit a more severe, progressive, and human-like disease pathology than the standard mdx line (Coley, WD et al., Hum Mol Genet, 2016, Vol. 25 (No. 1): pp. 130-145). Since D2.mdx mice are used, DBA2 / J mice can function as wild-type controls. As DMD primarily affects males, the study can focus on male mice.

[0342] To investigate the ability of Ab1 and Ab2 to prevent / limit contraction-induced injury, 6-week-old male D2.mdx mice (n=10) were treated for 6 weeks with either an IgG control, Ab1, or Ab2 at 10 mg / kg / week. A fourth group was administered 1D11 at 10 mg / kg / week to allow comparison with published studies using pan-TGFβ inhibitors. All antibodies were mIgG1 isotypes, and this dose has been previously shown to be effective in the UUO model (Figures 15 and 16). A WT group administered with an IgG control was also included. To allow evaluation of muscle fiber damage by fluorescence microscopy, mice were administered 1% Evans blue dye (EBD) in PBS (1% of body weight by volume) 24 hours before sacrifice. At the end of treatment, mice were subjected to an in vivo eccentric contraction protocol. Eccentric 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 (No. 236): ra56). Briefly, 20 eccentric contractions are performed with a 1-minute rest in between, and the 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 lost 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, Vol. 72 (No. 1): pp. 153-154; Khairallah, RJ et al., Sci Signal, 2012, Vol. 5 (No. 236): ra56). The ability of Ab1 and Ab2 to reduce post-injury muscle weakness can be evaluated. Mice can be sacrificed at the end of the experiment, and both injured and uninjured gastrocnemius muscles can be collected for histological analysis. EBD uptake can be evaluated from both muscles. Muscle fiber cross-sectional area and the degree of fibrosis can be measured.To determine the cross-sectional area, sections derived from the mid-belly of muscle can be stained with wheat germ agglutinin conjugated with fluorophores to visualize the cell membrane. Using a fluorescence microscope, the sections can be digitized, cell boundaries can be traced using predictive software, and the cross-sectional area can be determined by unbiased automated measurement. For fibrosis analysis, sections can be stained with picrosilius 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 is evaluated. 12-week-old DBA2 / J mice and D2.mdx mice can be subjected to the same eccentric contraction protocol described above. After injury, mice are divided into treatment groups (n=10) and administered either IgG control (for WT and D2.mdx mice), 1D11, Ab1, or Ab2 (D2.mdx only). Antibodies are administered at 10 mg / kg / week for the duration of the experiment. At 7 and 14 days after injury, the effect of treatment on recovery from injury can be evaluated by measuring peak isometric muscle strength, unicontraction-tetanic ratio, and force-frequency relationship. While Ab1 and Ab2 inhibit TGFβ1 release regardless of the presenting molecule, selective release of TGFβ1 from the extracellular matrix (i.e., presented by LTBP) is greater and more beneficial in DMD because Treg activity driven by TGFβ1 is conserved. To address this problem, specific LTBP-pro-TGFβ1 inhibitory antibodies can also be evaluated for both their ability to prevent contraction-induced injury and their ability to accelerate recovery from injury.

[0344] (Example 9: The role of TGFβ1 in skeletal muscle regeneration after acute injury) In particular, the role of TGFβ1 in muscle fiber regeneration after muscle injury can be investigated. TGFβ1-specific antibodies can be used in a cardiotoxicity injury model to determine the role of TGFβ1, especially during muscle fiber regeneration. Regeneration can be histologically evaluated, and functional assessments of muscle strength and quality can be performed. Considering the potential benefits of TGFβ1 inhibition for muscle regeneration, the benefits of a therapy that has beneficial effects without...

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[Claim 1] The invention as shown in the drawings.

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