BR122024003721A2 - TGFbeta1-BINDING IMMUNOGLOBULINS AND USES OF THESE - Google Patents

Abstract

Disclosed in this descriptive report are immunoglobulins, such as antibodies, and antigen-binding portions thereof, which specifically bind to the GARP-TGFβ1, LTBP1-TGFβ1, LTBP3-TGFβ1 and/or LRRC33-TGFβ1 complexes. The application also provides methods of using these immunoglobulins for, for example, inhibiting TGFβ1 activity and treating individuals suffering from TGFβ1-related disorders, such as cancer and fibrosis.

Description

RELATED REQUESTS

[001] This application claims priority to U.S. Provisional Application No. 62/307,353, filed on March 11, 2016; U.S. Provisional Application No. 62/443,615, filed January 6, 2017; and U.S. Provisional Application No. 62/452,866, filed on January 31, 2017; the entire contents of each of which are expressly incorporated into this descriptive report by reference in their entirety.

FUNDAMENTALS

[002] The transforming growth factor β (TGFβ) superfamily of growth factors is involved in diverse signaling cascades that regulate diverse biological processes that include, without limitation: cell growth inhibition, tissue homeostasis, extracellular matrix remodeling ( ECM), endothelial to mesenchymal transition (EMT), cell migration and invasion, and immune modulation/suppression, as well as mesenchymal to epithelial transition. In relation to ECM remodeling, TGFβ signaling can increase fibroblast populations and ECM (e.g. collagen) deposition. In the immune system, TGFβ ligand modulates regulatory T cell function and the maintenance of immune precursor cell growth and homeostasis. In normal epithelial cells, TGFβ is a potent growth inhibitor and promoter of cell differentiation. However, as tumors develop and progress, they often lose their negative growth response to TGFβ. In this context, TGFβ can become a promoter of tumor development due to its ability to stimulate angiogenesis, alter the stromal environment and induce local and systemic immunosuppression. For these and other reasons, TGFβ has been a therapeutic target for several clinical indications. Despite much effort made to date by several groups, the clinical development of a TGFβ therapeutic agent has been challenging.

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

[004] For example, Anderton et al. (Toxicology Pathology, 39: 916-24, 2011) reported that small molecule inhibitors of TGFβ type I receptor (ALK5) induced heart valve injuries characterized by hemorrhage, inflammation, degeneration, and proliferation of valvular interstitial cells in a preoperative animal model. -clinical. Toxicity was observed in all heart valves at all doses tested. Frazier et al. (Toxicology Pathology, 35: 284-295, 2007) reported that administration of the TGFβ type I receptor (ALK5) small molecule inhibitor GW788388 induced physeal dysplasia in rats.

[005] Stauber et al. (J. Clin. Practice 4:3, 2014) reported that chronic administration (>3 months) of the TGFβ receptor I kinase inhibitor, LY2157299, which is being investigated for certain cancer treatments, caused multiple organ toxicities, which involve the cardiovascular, gastrointestinal, immune, bone/cartilage, reproductive and renal systems in rats and dogs.

[006] Fresolimumab (GC1008), a “pan” antibody against TGFβ capable of neutralizing all human isoforms of TGFβ, has been reported to induce epithelial hyperplasia of the gingiva, bladder and nasal turbinate epithelium after multiple administrations in studies with Cynomolgus monkeys ( Lonning et al., Current Pharmaceutical Biotechnology 12: 2,176-89, 2011). Similarly, several cases of skin rashes/lesions, gingival bleeding, and fatigue have been reported in clinical trials following administration of multiple doses of the drug. The most notable adverse reaction to fresolimumab includes the induction of cutaneous keratoacanthomas and/or squamous cell carcinomas in human cancer patients (see, for example: Lacouture et al., 2015, Cancer Immunol. Immunother., 64:437-46; Stevenson et al., 2013, Oncolmmunology, 2:8, e26218; and Lonning et al., 2011). Additional evidence from a clinical trial suggests that in some cases this antibody may accelerate tumor progression (Stevenson et al., 2013, Oncolmmunology, 2:8, e26218).

[007] Therefore, new methods and compositions are needed for modulating TGFβ signaling that can be used to effectively and safely treat diseases and disorders involving TGFβ, including, for example, cancer, fibrosis and inflammation.

SUMMARY OF THE INVENTION

[008] The present disclosure is related to the selective modulation of a subset of TGFβ effects. The invention is based, at least in part, on the realization that the lack of isoform specificity of TGFβ antagonists known to date may be responsible for the source of toxicities associated with TGFβ inhibition. Indeed, the inventors of the present disclosure have discovered that most of the TGFβ inhibitors described to date antagonize several or all TGFβ isoforms. Furthermore, the general trend described in the art has been that inhibitors (e.g., anti-TGFβ antibodies) that antagonize multiple TGFβ isoforms are favored, with the rationale being that neutralization of multiple TGFβ isoforms would be necessary or advantageous to obtain “ maximum therapeutic efficacy” (see, for example, Bedinger et al. (2016) MABS 8 (2): 389-404).

[009] Contrary to this general teaching, the inventors of the present invention, instead, sought to develop agents that allow inhibition of specific TGFβ1- isoform, as opposed to inhibition that also affects TGFβ2 and/or TGFβ3, with the aim of eliminating or markedly reduce toxicities (e.g., adverse events, side effects) observed with known TGFβ antagonists in vivo. This new approach is based, at least in part, on the realization that the clinical utility of a TGFβ inhibitor may not only be based on its efficacy but also on its safety. It was reasoned that the ability to fine-tune the target with an unprecedented degree of specificity can achieve both efficacy and safety/tolerability in a clinical setting.

[0010] Consequently, the invention encompasses the recognition that pharmaceutical agents that inhibit TGFβ signaling in an isoform-specific manner can provide improved safety profiles over agents that affect multiple TGFβ isoforms. By contrast, several TGFβ antagonists known in the art produce unacceptable levels of toxicity at a dose proven to be effective in vivo. In these cases, dosage reduction may be employed to avoid toxicity, but this may no longer produce sufficient in vivo efficacy at the reduced dose. Without being tied to a particular theory, it is contemplated that this toxicity, at least in part, is due to the lack of isoform specificity/selectivity of the agent.

[0011] Thus, in one aspect, the invention provides methods for reducing toxicities (e.g., adverse events, unwanted side effects) associated with TGFβ inhibition in an individual. According to the invention, TGFβl-specific inhibitors, such as those described in this specification, have a superior safety-efficacy profile, compared to agents that evoke activity against a broader target (for example, more than one TGFβ isoform). . These TGFβl-specific inhibitors can therefore be administered to individuals in need at a therapeutically effective dose without causing adverse effects. This approach would therefore expand the dosage range at which both efficacy and safety/tolerability can be achieved in patients. Thus, the invention provides methods for treating a disease associated with TGFβ1 signaling, by administering to an individual an effective amount of a TGFβ inhibitor that is specific or highly selective for the TGFβ1 isoform. In some embodiments, these TGFβ1 isoform-selective or TGFe1 isoform-specific inhibitors may be small molecule agents or biologics (e.g., antibodies). The use of any of these inhibitors to reduce toxicities (e.g., adverse events or side effects) associated with the inhibition of TGFβ in an individual is encompassed by the present invention. According to the invention, the effective amount is within a dosage range that allows for: i) efficacy (e.g., therapeutically beneficial effects); and, ii) safety (e.g., within acceptable levels of adverse effects or side effects). In some embodiments, adverse effects may include cardiovascular toxicities, gastrointestinal toxicities, immune toxicities, bone/cartilage toxicities, reproductive toxicities, and renal toxicities. In some embodiments, cardiovascular toxicities include, without limitation: heart valve injuries, e.g., hemorrhage, 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.

[0012] Consequently, in some embodiments, the invention provides TGFβ1-specific isoform inhibitory antibodies or antigen-binding fragments thereof, characterized by the fact that they selectively inhibit the activation step of TGFβ1 in vivo, but do not inhibit the activation step of TGFβ2 and/or TGFβ3. These antibodies or fragments thereof can be administered to individuals who benefit from TGFβ1 inhibition, in an amount effective to achieve clinical efficacy without causing unacceptable or intolerable levels of adverse effects. Thus, the present invention teaches that TGFβl-specific isoform inhibitors are specifically selected to meet both efficacy and safety criteria for the treatment of a disease or condition associated with TGFβ signaling in human patients.

[0013] In a related aspect, the invention provides production methods for specific TGFβ- isoform modulators with a superior safety profile (e.g., reduced in vivo toxicity). These methods require that candidate agents be tested and screened for isoform specificity. In some embodiments, candidate agents are selected for specific activities against TGFβ1 signaling, and not TGFβ2 and/or TGFβ3 signaling. In some embodiments, these agents are TGFβl isoform-specific inhibitors. In some embodiments, these agents are antibodies or antigen-binding fragments thereof that specifically bind and block the activation of TGFβ1, but not TGFβ2 and/or TGFβ3. In some embodiments, these antibodies or antigen-binding fragments thereof do not bind free mature TGFβ1 growth factor that is not associated with a pro/latent complex.

[0014] In another aspect, the present invention provides related compositions and methods for obtaining additional fine-tuning of TGFβ signaling by modulating TGFβ activation in a context-dependent manner.

[0015] TGFβ is involved in the conferral of several cellular/tissue effects, and each of these effects is mediated, in part, by its interaction with so-called “presentation molecules”. Because the expression of various presentation molecules is cell-type- or tissue-specific, it is contemplated that TGFβ confers a cellular effect depending on its interaction with a particular presentation molecule (i.e., “context”). Thus, among other things, the present disclosure provides monoclonal antibodies that selectively bind to TGFβ present in a particular context (i.e., a complex comprising TGFβ and a presentation molecule). In some embodiments, these 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, these monoclonal antibodies specifically bind to one of the following complexes: i) TGFβ1-GARP; ii) TGFβ1-LRRC33; iii) TGFβ1-LTBP1; and, iv) TGFβ1-LTBP3. In some embodiments, these monoclonal antibodies specifically bind to two of the following complexes: i) TGFβ1-GARP; ii) TGFβ1-LRRC33; iii) TGFβ1-LTBP1; and, iv) TGFβ1-LTBP3. In some embodiments, these monoclonal antibodies specifically bind to three of the following complexes: i) TGFβ1-GARP; ii) TGFβ1-LRRC33; iii) TGFβ1-LTBP1; and, iv) TGFβ1- LTBP3. In some embodiments, these 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, these monoclonal antibodies do not bind to mature TGFβ1 that is free TGFβ1 (e.g., not complexed with a presentation molecule).

[0016] The present disclosure includes monoclonal antibodies that bind to the small latent complex (e.g., “C4S”) of TGFβ1.

[0017] The disclosure further provides monoclonal antibodies that selectively target and modulate TGFβ in a particular context. In some embodiments, these monoclonal antibodies inhibit or activate TGFβ in a particular context.

[0018] Consequently, the invention provides compositions and methods for modulating (activating or inhibiting) a subset of TGFβ activities. Thus, the invention includes monoclonal antibodies that can selectively modulate a subset of TGFβ-mediated signaling pathways without affecting other TGFβ-mediated signaling pathways. In some embodiments, the subset of TGFβ-mediated signaling pathways includes at least one, at least two, or at least three of: i) GARP-mediated TGFβ effects, ii) LRRC33-mediated TGFβ effects, iii) TGFβ effects mediated by LTBP1, and iv) effects of TGFβ mediated by LTBP3. In some embodiments, these monoclonal antibodies specifically modulate one of the following TGFβ-mediated signaling pathways, without modulating the other three pathways: i) GARP-mediated TGFβ effects, ii) LRRC33-mediated TGFβ effects, iii) TGFβ-mediated effects by LTBP1, and iv) LTBP3-mediated TGFβ effects. In some embodiments, these monoclonal antibodies specifically modulate two of the following TGFβ-mediated signaling pathways, without modulating the other two pathways: i) GARP-mediated TGFβ effects, ii) LRRC33-mediated TGFβ effects, iii) TGFβ-mediated effects by LTBP1, and iv) LTBP3-mediated TGFβ effects. In some embodiments, these monoclonal antibodies specifically modulate three of the following TGFβ-mediated signaling pathways, without modulating the other pathway: i) GARP-mediated TGFβ effects, ii) LRRC33-mediated TGFβ effects, iii) TGFβ-mediated effects LTBP1, and iv) LTBP3-mediated TGFβ effects. In some embodiments, these monoclonal antibodies specifically modulate all of the following TGFβ-mediated signaling pathways: i) GARP-mediated TGFβ effects, ii) LRRC33-mediated TGFβ effects, iii) LTBP1-mediated TGFβ effects, and iv) LTBP1-mediated TGFβ effects of TGFβ mediated by LTBP3. In some embodiments, these monoclonal antibodies specifically modulate GARP-mediated effects of TGFβ. In some embodiments, these monoclonal antibodies specifically modulate LRRC33-mediated TGFβ effects. In some embodiments, these monoclonal antibodies specifically modulate LTBP1-mediated effects of TGFβ. In some embodiments, these monoclonal antibodies specifically modulate LTBP3-mediated effects of TGFβ. Thus, the present invention provides methods for selectively targeting TGFβ activities in a context-dependent manner.

[0019] Aspects of the present invention include pharmaceutical compositions and methods for treating a disease or disorder in a human subject. In some embodiments, this disease or disorder includes conditions associated with immune regulation/dysregulation, conditions associated with T cell regulation/dysregulation; conditions associated with fibrotic characteristics (fibrosis); and/or tumor-associated conditions.

[0020] How antibodies or fragments thereof that specifically target the pro/latent TGFβ complex described in this specification modulate the activation step (i.e., release of a free, mature TGFβ growth factor from the latent preform complex, inactive), as opposed to targeting mature, free, already released growth factor, the mode of action of these modulating agents depends on the source of the TGFβ growth factor in the tissue. It is therefore contemplated that identifying the source of TGFβ involved in a disease context may help select an agent that can effectively modulate TGFβ in the correct context. For example, to treat a disease phenotype that involves GARP-mediated effects of TGFβ1, it is desirable to select antibodies or fragments thereof that specifically target a GARP-pro-TGFβ1 complex. To treat a disease phenotype that involves LRRC33-mediated effects of TGFβ1, it is desirable to select antibodies or fragments thereof that specifically target a LRRC33-pro-TGFβ1 complex. To treat a disease phenotype that involves LTBP1-mediated effects of TGFβ1, it is desirable to select antibodies or fragments thereof that specifically target an LTBP1-pro-TGFβ1 complex. To treat a disease phenotype that involves LTBP2-mediated effects of TGFβ1, it is desirable to select antibodies or fragments thereof that specifically target an LTBP2-pro-TGFβ1 complex. To treat a disease phenotype that involves LTBP3-mediated effects of TGFβ1, it is desirable to select antibodies or fragments thereof that specifically target an LTBP3-pro-TGFβ1 complex. To treat a disease phenotype that involves LTBP4-mediated effects of TGFβ1, it is desirable to select antibodies or fragments thereof that specifically target an LTBP4-pro-TGFβ1 complex.

[0021] To treat a disease phenotype that involves TGFβ1 effects mediated by multiple (e.g., two or more) contexts, it is desirable to select antibodies or fragments thereof that can target the corresponding multiple TGFβ1 presentation contexts.

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

[0023] Although context-permissive TGFβ1 modulators are capable of targeting more than one type of pro/latent TGFβ1 complexes (i.e., with different presenting molecules), in some embodiments, these modulators may favor one or more contexts over to others. Thus, in some embodiments, a context-permissive antibody that inhibits TGFβ1 activation may preferentially inhibit TGFβ1 activation mediated by one presenting molecule over another presenting molecule, even if one antibody is capable of binding to both. the types of pro/latent complexes. In some embodiments, this antibody is a monoclonal antibody that binds to and inhibits the activation of LTBP-associated TGFβ1, GARP-associated TGFβ1, and LRRC33-associated TGFβ1, but with preferential inhibitory activities against LTBP-associated TGFβ1. In some embodiments, this antibody is a monoclonal antibody that binds to and inhibits the activation of LTBP1-associated TGFβ1, LTBP3-associated TGFβ1, GARP-associated TGFβ1, and LRRC33-associated TGFβ1, but with preferential inhibitory activities against LTBP1-associated TGFβ1 and LTBP-3. In some embodiments, this antibody is a monoclonal antibody that binds to and inhibits the activation of LTBP1-associated TGFβ1, LTBP3-associated TGFβ1, GARP-associated TGFβ1, and LRRC33-associated TGFβ1, but with preferential inhibitory activities against GARP-associated TGFβ1 and TGFβ1. associated with LRRC33. In some embodiments, this antibody is a monoclonal antibody that binds to and inhibits the activation of GARP-associated TGFβ1 and LRRC33-associated TGFβ1, but with preferential inhibitory activities against GARP-associated TGFβ1. In some embodiments, this antibody is a monoclonal antibody that binds to and inhibits the activation of GARP-associated TGFβ1 and LRRC33-associated TGFβ1, but with preferential inhibitory activities against LRRC33-associated TGFβ1.

[0024] In this way, according to the invention, varying degrees of selectivity can be generated in order to target a subset of TGFβ effects. Isoform-specific TGFβ modulators (which target a single TGFβ isoform) provide greater selectivity than pan-TGFβ modulators (which target multiple or all TGFβ isoforms). Context-permissive, isoform-specific TGFβ modulators (which target multiple contexts of a single TGFβ isoform) provide greater selectivity than isoform-specific modulators. Context-specific, isoform-specific TGFβ modulators (which target single context of a single TGFβ isoform) provide even greater selectivity than context-permissive, isoform-specific modulators.

[0025] Thus, in some embodiments, the invention includes methods for treating a disease associated with TGFβ signaling that comprise first identifying or confirming the source and/or context of TGFβ associated with the disease, and then selecting a agent that specifically targets the particular subset of TGFβ in the tissue. Thus, it is contemplated that this approach may preserve normal TGFβ function while preferentially modulating the disease-associated function of TGFβ. In some embodiments, to identify the source/context of disease-associated TGFβ, the expression of the TGFβ presentation molecule (or molecules) present in the diseased tissue can be assessed. To give just one example, within a diseased tissue there can be both a latent TGFβ1 complex associated with LTBP and a TGFβ1 complex associated with GARP, and only the latter of the two can be expressed as a disease phenotype. In that scenario, it is desirable to inhibit GARP-mediated TGFβ1 signaling while keeping LTBP-mediated TGFβ1 signaling intact. Determining the source/context of disease-associated TGFβ1 can be accomplished with the use of antibodies that specifically bind to the latent TGFβ1 complex that includes a particular presenting molecule (e.g., GARP, LRRC33, LTBPs, etc.). For the treatment of conditions associated with T cell regulation/dysregulation, some embodiments of the invention comprise administering a composition comprising an effective amount of a monoclonal antibody that modulates the GARP-mediated effects of TGFβ in the individual.

[0026] For the treatment of conditions associated with immune regulation/dysregulation, some embodiments of the invention comprise administering a composition comprising an effective amount of a monoclonal antibody that modulates the GARP-mediated effects of TGFβ and/or the effects of TGFβ mediated by LRRC33 in the individual.

[0027] For the treatment of conditions associated with fibrosis, some embodiments of the invention comprise administering a composition comprising an effective amount of a monoclonal antibody that modulates the effects of TGFβ mediated by LTBP1 and/or the effects of TGFβ mediated by LTBP3 in the individual.

[0028] For the treatment of conditions associated with certain types of cancer, some embodiments of the invention comprise administering a composition comprising an effective amount of a monoclonal antibody that modulates the effects of TGFβ mediated by GARP, the effects of TGFβ mediated by LTBP1 and/or LTBP3-mediated effects of TGFβ in the individual.

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

[0030] In one aspect, provided in this specification is an isolated antibody, or antigen-binding portion thereof, that specifically binds to an epitope of TGFβ1, wherein the epitope is available for binding by the antibody when TGFβ1 is present in two or more of the following protein complexes: a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and a LRRC33-TGFβ1 complex; and wherein the antibody does not bind to free mature TGFβ1.

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

[0032] In some embodiments, the antibody, or antigen-binding portion thereof, does not bind to TGFβ2. In some embodiments, the antibody, or antigen-binding portion thereof, does not bind to TGFβ3. In some embodiments, the antibody, or antigen-binding portion thereof, does not prevent the ability of TGFβl to bind to the integrin.

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

[0034] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity for the amino acid sequence shown in the ID. OF SEQ. No.: 13 and a light chain variable domain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of identity to the amino acid sequence presented in the ID. OF SEQ. No.: 14.

[0035] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain that comprises an amino acid sequence shown in the ID. OF SEQ. No.: 13 and a light chain variable domain comprising an amino acid sequence shown in the ID. OF SEQ. No.: 14.

[0036] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region that comprises a CDR3 that has the amino acid sequence of the ID. OF SEQ. No.: 6 and a light chain variable region comprising a CDR3 having the amino acid sequence of the ID. OF SEQ. No.: 12. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a CDR2 having the ID amino acid sequence. OF SEQ. No.: 4 and a light chain variable region comprising a CDR2 having the amino acid sequence of the ID. OF SEQ. No.: 10. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a CDR1 having the ID amino acid sequence. OF SEQ. No.: 2 and a light chain variable region comprising a CDR1 having the amino acid sequence of the ID. OF SEQ. No.: 8.

[0037] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in the ID. OF SEQ. No.: 15 and a light chain variable domain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of identity to the amino acid sequence presented in the ID. OF SEQ. No.: 16.

[0038] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain that comprises an amino acid sequence shown in the ID. OF SEQ. No.: 15 and a light chain variable domain comprising an amino acid sequence shown in the ID. OF SEQ. No.: 16.

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

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

[0041] In some embodiments, the antibody, or antigen-binding portion thereof, has a dissociation constant (KD) for the TGFβ1 epitope selected from the group consisting of: 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.

[0042] In some embodiments, the antibody, or antigen-binding portion thereof, comprises an immunoglobulin heavy chain constant domain, 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, a human IgA1 constant domain, a human IgA2 constant domain, a human IgD constant domain, or a human IgE constant domain. In some embodiments, the antibody, or antigen-binding portion thereof, comprises an immunoglobulin heavy chain constant domain of a human IgG1 constant domain or a human IgG4 constant domain. In some embodiments, the antibody, or antigen-binding portion thereof, comprises an immunoglobulin heavy chain constant domain of a human IgG4 constant domain. In some embodiments, the antibody, or antigen-binding portion thereof, comprises an immunoglobulin heavy chain constant domain of a human IgG4 constant domain that has a Ser to Pro scaffold substitution that produces an IgG1-like hinge and allows the formation of interchain disulfide bonds.

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

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

[0045] In some embodiments, wherein 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 that has a human germline amino acid sequence.

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

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

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

[0049] In some embodiments, the antibody, or antigen-binding portion thereof, is conjugated to a drug or a detectable portion. In some embodiments, the antibody, or antigen-binding portion thereof, is conjugated to the 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 a fluorescent agent, a luminescent agent, an enzymatic agent, and a radioactive agent.

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

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

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

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

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

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

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

[0057] In another aspect, there is provided in this specification a method for reducing the activation of TGFβ1 in an individual, the method comprising administering to the individual an effective amount of an antibody, an antigen-binding portion thereof, or a pharmaceutical composition, as described in this specification, thus reducing the activation of TGFβ1 in the individual.

[0058] In some embodiments, the individual has or is at risk of having fibrosis. In some embodiments, the individual has muscular dystrophy. In some embodiments, the individual has Duchenne muscular dystrophy (DMD). In some embodiments, the individual has or is at risk for hepatic fibrosis, renal fibrosis, or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis). In some embodiments, the individual has or is at risk of having cancer. In some embodiments, the individual has or is at risk of having dementia. In some embodiments, the individual has or is at risk for developing myelofibrosis.

[0059] In some modalities, the individual still receives additional therapy. In some embodiments, the additional therapy is selected from the group consisting of a myostatin inhibitor, a VEGF agonist, an IGF1 agonist, an FXR agonist, a CCR2 inhibitor, a CCR5 inhibitor, a dual CCR2/CCR5 inhibitor. , a lysyl oxidase-like-2 inhibitor, an ASK1 inhibitor, an Acetyl-CoA Carboxylase (ACC) inhibitor, a p38 kinase inhibitor, Pirfenidone, Nintedanib, a GDF11 inhibitor, or any combination thereof.

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

[0061] In some embodiments, the antibody, or the antigen-binding portion thereof, does not induce organic toxicity in the individual. In some embodiments, organic toxicity comprises cardiovascular toxicity, gastrointestinal toxicity, immunotoxicity, bone toxicity, cartilage toxicity, reproductive system toxicity, or renal toxicity.

[0062] In one aspect, there is provided in this specification a method for treating cancer in an individual in need, the method comprising administering to the individual an effective amount of an antibody, an antigen-binding portion thereof, or an pharmaceutical composition, as described in this specification, thus treating cancer in the individual.

[0063] In another aspect, there is provided in this specification a method of reducing tumor growth in an individual in need, the method comprising administering to the individual an effective amount of an antibody, an antigen-binding portion thereof, or a pharmaceutical composition, as described in this specification, thus reducing tumor growth in the individual.

[0064] In some embodiments, the antibody, or antigen-binding portion thereof, is administered in combination with an additional agent or an additional therapy. In some embodiments, the additional agent is a checkpoint inhibitor. In some embodiments, the additional agent is selected from the group consisting of a PD-1 antagonist, a PDL1 antagonist, a PD-L1 or PDL2 fusion protein, a CTLA4 antagonist, a GITR agonist, an anti-ICOS antibody. , an anti-ICOSL antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-0X40 antibody, an anti-CD27 antibody, an anti-CD70 antibody, an anti-CD47 antibody, an anti-41BB antibody, an anti-PD-1 antibody, an oncolytic virus, and a PARP inhibitor. In some embodiments, the additional therapy is radiation, a chemotherapy drug, or a combination thereof. In some embodiments, additional therapy 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., CCR2 antagonists and CCR5 antagonists. In some embodiments, this chemokine receptor antagonist is a dual specific antagonist, e.g., a CCR2/CCR5 antagonist. In some embodiments, the additional agent to be administered as combination therapy is or comprises a member of the TGFβ superfamily of growth factors or regulators thereof. In some embodiments, this agent is selected from modulators (e.g., inhibitors and activators) of GDF8/myostatin and GDF11. In some embodiments, this agent is an inhibitor of GDF8/myostatin signaling. In some embodiments, this agent is a monoclonal antibody that specifically binds to a pro/latent myostatin complex and blocks myostatin activation. In some embodiments, the monoclonal antibody that specifically binds to a pro/latent myostatin complex and blocks myostatin activation does not bind to free, mature myostatin.

[0065] In yet another aspect, there is provided in this specification a method of treating a renal disorder in an individual in need, the method comprising administering to the individual an effective amount of an antibody, an antigen-binding portion thereof or of a pharmaceutical composition, as described in this specification, thus treating the renal disorder in the individual.

[0066] In one aspect, there is provided in this specification a nucleic acid encoding an antibody, or an antigen-binding portion thereof, as described in this specification. Vectors comprising nucleic acid are also provided.

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

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

[0069] In another aspect, there is provided in this specification a method for treating a myofiber damage, the method comprising the step of: administering to an individual who has a myofiber damage an agent that selectively inhibits TGFβ1 with respect to TGFβ2/3 in an amount effective to i) promote myofiber repair; ii) protect from contraction-induced injury; iii) reduce inflammation in the muscle; and/or, iv) reduce fibrosis in the muscle. In some embodiments, the amount does not cause an unacceptable level of adverse effects on the individual.

[0070] In some embodiments, myofiber damage is i) associated with muscular dystrophy; or, ii) associated with an 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 a latent GARP-pr0-TGFβ1 complex, a latent LRRC33-pr0-TGFβ1 complex, a latent LTBP1-pr0-TGFβ1 complex, a latent LTBP2-pr0-TGFβ1 complex, an LTBP3- latent pr0-TGFβ1, and/or a latent LTBP4-pr0-TGFβ1 complex. In some embodiments, the individual further receives a myostatin inhibitor.

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

[0072] In yet another aspect, provided in this specification is a method for producing a pharmaceutical composition that modulates TGFβ signaling, the method comprising the steps of: providing one or more agents that modulate the signaling of at least one TGFβ isoform; measuring the activities of (one or more) agents against all TGFβ isoforms; selection of an agent that is specific for a single TGFβ isoform; formulation in a pharmaceutical composition comprising a TGFβ-specific isoform modulator and a pharmaceutically acceptable excipient. A pharmaceutical composition produced by this method is also provided.

[0073] In some embodiments, the TGFβ isoform-specific modulator is a TGFβl-specific modulator. In some embodiments, the TGFe1-specific modulator is a TGFβ1 inhibitor. In some embodiments, the specific TGFβ- isoform modulator is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof specifically binds to a pro/latent TGFβ1 complex. In some embodiments, the antibody or fragment thereof does not bind to free mature TGFβ1 that is not in the pro/latent complex. In some embodiments, the pro/latent complex comprises GARP, LRRC33, LTBP1, LTBP2, LTBP3, or LTBP4.

[0074] In another aspect, there is provided in this specification a method for treating a disease associated with TGFβ signaling, the method comprising a step of: administering to an individual in need of a pharmaceutical composition provided in this specification, in an amount effective to treat the disease, in which the amount achieves statistically significant clinical efficacy and safety when administered to a population of patients who have the disease.

[0075] In yet another aspect, there is provided in this specification a TGFβ inhibitor for use in reducing adverse effects in an individual, wherein the TGFβ inhibitor is isoform-selective. In some embodiments, the TGFβ inhibitor is an antibody that specifically inhibits TGFβ1. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 is a schematic representation that depicts TGFβ bound in a latent complex in the tissue microenvironment.

[0077] Figures 2A and 2B are schematic representations that depict niche modulation in the microenvironment. Figure 2A shows latent transforming growth factor-beta binding proteins (LTBPs) that present TGFβ in a fibrotic disease niche and a wound healing niche. Figure 2B illustrates that glycoprotein-A repeat predominant protein (GARP) modulates TGFβ activation in the inflammatory niche.

[0078] Figure 3 illustrates a protein expression platform for the production of a GARP-TGFβ1 complex and an LTBP-TGFβ1 complex. The HEK293-based expression system uses NiNTA affinity purification and gel filtration to obtain multimilligram amounts of purified protein. Schematic representations of wild-type pr0-TGFβ1, LTPB1, sGARP, and pr0-TGFβ1 C4S are shown.

[0079] Figures 4A and 4B show the purification of the sGARP-pro-TGFβ1 complex. Figure 4A is a chromatogram of the sample and Figure 4B shows a blot of the products and a schematic representation illustrating the complex.

[0080] Figures 5A and 5B show the purification of the sGARP-TGFβl LAP complex. Figure 5A is a chromatogram of the sample and Figure 5B shows a blot of the products and a schematic representation illustrating the complex.

[0081] Figures 6A and 6B show the purification of LTBP1 complexed with pro-TGFβ1.

[0082] Figure 6A is a chromatogram of the sample and Figure 6B shows a blot of the products and a schematic representation that illustrates the complex. NR means reducer and R means reducer.

[0083] Figure 7 is a graph showing that Ab1 and Ab2 block the activation of TGFβ1 activity.

[0084] Figure 8 shows an initial dose-response analysis of Ab1 in human cells.

[0085] Figure 9 shows CAGA12 reporter cell assays illustrating the inhibition of TGFβ1 by Ab1 in human cells.

[0086] Figure 10 is a graph showing that inhibition of the GARP complex blocks the suppressive activity of regulatory T cells (Treg) in T cells isolated from healthy donor blood.

[0087] Figures 11A-11C show the inhibition of integrin-mediated TGFβ1 release by fibroblasts. Figures 11A and 11B are graphs depicting inhibition of endogenous TGFβ1 in normal human dermal fibroblasts (circles), normal human lung fibroblasts (squares), C57BL/6J murine lung fibroblasts (inverted triangles), or DBA2/J murine muscle fibroblasts (circles). ) using increasing concentrations of Ab1 (Figure 11A) or Ab2 (Figure 11B). Figure 11C provides a schematic representation of the coculture assay system.

[0088] Figures 12A and 12B depict the binding of Ab1 (Figure 12A) or Ab2 (Figure 12B) to the LRRC33-pro-TGFβ1 complex.

[0089] Figure 13A and 13B are graphs depicting the inhibition of the GARP-TGFβ1 complex (Figure 13A) or the LRRC33-TGFβ1 complex (Figure 13B) in SW480/06 cell transfectants using increasing concentrations of Ab1 (“Ab1”) , Ab2 (“Ab2”), IgG1 isotype control antibody (“Isotype Control”) or vehicle control (“Vehicle”).

[0090] Figure 14 is a bar graph depicting hydroxyproline levels in kidney tissue of mice that underwent permanent right unilateral UUO surgery and received administration of PBS (control), 30 mg/kg IgG1 control antibody murine, 3 mg/kg Ab2, or 30 mg/kg Ab2 intraperitoneally (i.p.) before surgical intervention (second to fifth bars); or mice that received PBS administration and underwent a laparotomy (sham control; first bar).

[0091] Figures 15A-15H are bar graphs depicting the relative mRNA levels of plasminogen activator inhibitor-1 (PAI-1; Figure 15A), connective tissue growth factor (CTGF; Figure 15B), TGFβ1 (Figure 15C), fibronectin-1 (Figure 15D), smooth muscle actin-α (SMA-α; Figure 15E), monocyte chemoattractant protein 1 (MCP-1; Figure 15F), type I collagen alpha 1 chain ( Col1a1; Figure 15G), or collagen type III alpha 1 chain (Col3a1; Figure 15H) in kidney tissue from mice that underwent permanent right unilateral UUO surgery and received administration of PBS (control), 30 mg/kg of antibody. murine IgG1 control, 3 mg/kg Ab2 or 30 mg/kg Ab2 intraperitoneally (i.p.) on the day before surgical intervention, and then 1 and 3 days after surgery (second to fifth bars of each graph); or mice that received PBS administration and underwent a laparotomy (sham control; first bar of each graph). These data are representative of multiple experiments.

[0092] Figure 16 depicts the composite cortical collagen volume fraction (CVF) of three serial sections of the right kidney collected from mice that were stained with Picrosirius-Red and subjected to quantitative histological analyzes using color spectrum segmentation. CVF of mice that underwent permanent right unilateral UUO surgery and received administration of PBS (“Veh”), 30 mg/kg murine IgG1 control antibody (“IgG Ctrl”), 3 mg/kg Ab2 (“3 Ab2”), or 30 mg/kg of Ab2 (“30 Ab2”) intraperitoneally (i.p.) before surgical intervention (second to fifth bars of each graph); or mice that received PBS administration and underwent a laparotomy (“Sham”; first bar of each graph).

[0093] Figure 17 depicts the median tumor volume of C57/BL/6 mice from the MC38 syngeneic murine colon carcinoma model that received administration of murine IgG1 isotype control antibody in combination with rat IgG2a control antibody (group 1; control); Ab1 in combination with rat IgG2a control antibody (group 2); Ab2 in combination with rat IgG2a control antibody (group 3); murine IgG1 control antibody in combination with anti-PD-1 antibody (group 4); Ab1 in combination with anti-PD-1 antibody (group 5); Ab2 in combination with anti-PD-1 antibody (group 6).

[0094] Figures 18A-18C show the binding specificity of exemplary monoclonal antibodies. Figure 18A depicts that Ab1 and Ab2 specifically bind pro-TGFβ1, as measured by ELISA, but not pr0-TGFβ2, pr0-TGFβ3, or mature TGFβ1. Figure 18B depicts an example of purified LTBP-pro-TGFe1 expressed and purified for use as antigens to determine antibody binding specificity. Figure 18C depicts an example of an antibody that binds (as measured by ELISA) specifically to the LTBP1-prδ-TGFβ1 complex.

[0095] Figure 19 depicts a survival curve of rats treated with vehicle control (PBS; “Control”); 200 mg/kg of LY2109761; 300 mg/kg of LY2109761; 100 mg/kg of a pan-TGFβ antibody (“Pan-TGFβ Ab”); or 100 mg/kg Ab2.

[0096] Figure 20 depicts the body weight (mean/standard deviation) of rats treated with vehicle control (PBS; “Control”); 200 mg/kg of LY2109761; 300 mg/kg of LY2109761; 100 mg/kg of a pan-TGFβ antibody (“Pan-TGFβ Ab”); or 100 mg/kg Ab2.

[0097] Figures 21A-21C depict the body weight of individual rats treated with vehicle control (PBS; “Control”), 200 mg/kg LY2109761, or 300 mg/kg LY2109761 (Figure 21A); vehicle control (PBS; “Control”), or 100 mg/kg of a pan-TGFβ antibody (“Pan-TGFβ Ab”); or vehicle control (PBS; “Control”), or 100 mg/kg Ab2.

[0098] Figures 22A and 22B are graphs depicting the inhibition of the GARP-pr0-TGFβ1 complex or the LRRC33-pr0-TGFβ1 complex in SW480/β6 cells transiently transfected with plasmids to express pr0-TGFβ1 and a presentation molecule (or i.e., GARP or LRRC33) using increasing concentrations of Ab1 (Figure 22A) or Ab2 (Figure 22B). The IC50 (μ m/ml) of Ab1 for the GARP-TGFβ1 complex was 0.445, and the IC50 (μ m/ml) of Ab1 for the LRRC33-TGFβ1 complex was 1.325.

[0099] Figure 23 depicts microscope images of hematoxylin and eosin-stained sections of heart valves from rats treated with 200 mg/kg of LY2109761 (upper right panel); 100 mg/kg of a pan-TGFβ antibody (“Pan-TGFβ Ab”, lower left panel); 100 mg/kg Ab2 (lower right panel), or untreated control (upper left panel). Note: The lower right panel (Ab2) is an oblique cut that stained darker due to the uneven thickness.

DETAILED DESCRIPTION

[00100] In mammals, the transforming growth factor-beta (TGFβ) superfamily is composed of at least 33 gene products. These include bone morphogenetic proteins (BMPs), activins, growth and differentiation factors (GDFs), and the three isoforms of the TGFβ family: TGFβ1, TGFβ2, and TGFβ3. TGFβs are believed to have crucial roles in several processes, e.g., inhibition of cell proliferation, extracellular matrix (ECM) remodeling, and immune homeostasis. The importance of TGFβ1 for T cell homeostasis is demonstrated by the observation that TGFβ1-/- mice survive only 3 to 4 weeks, succumbing to multiorgan failure as a consequence of massive immune activation (Kulkarni, A.B., et al., Proc. Natl. Acad. Sci. U.S.A., 1993. 90 (2): pages 770-4; Shull, M.M., et al., Nature, 1992. 359 (6397): pages 693-9). The roles of TGFβ2 and TGFβ3 are less clear. Although the three TGFβ isoforms have distinct temporal and spatial expression patterns, they signal through the same receptors, TGFβRI and TGFβRII although, in some cases, for example, for TGFβ2 signaling, type III receptors such as beta- glycan, are also necessary (Feng, ). Ligand-induced oligomerization of TGFβRI/II triggers phosphorylation of SMAD transcription factors, resulting in the transcription of target genes, e.g., Col1a1, Col3a1, ACTA2, and SERPINE1 (Massague, J., J. Seoane, and D. Wotton , Genes Dev., 2005. 19 (23): pages 2,783-810). SMAD-independent TGFβ signaling pathways have also been described, for example, in cancer or aortic lesions of Marfan mice (Derynck, R. and Y.E. Zhang, Nature, 2003. 425 (6,958): pages 577-84; Holm, T.M., et al., Science, 2011. 332 (6027): pages 358-61).

[00101] The biological importance of the TGFβ pathway in humans has been validated by genetic diseases. Camurati-Engelman disease results in bone dysplasia as a consequence of an autosomal dominant mutation in the TGFB1 gene, leading to constitutive activation of TGFβ1 signaling (Janssens, K., et al., J. Med. Genet., 2006. 43 ( 1): pages 1-11). Patients with Loeys/Dietz syndrome carry autosomal dominant mutations in components of the TGFβ signaling pathway, which cause aortic aneurysm, hypertelorism, and bifid uvula (Van Laer, L., H. Dietz, and B. Loeys, Adv. Exp. Med Biol., 2014. 802: pages 95-105). Dysregulation of the TGFβ pathway has been implicated in several diseases, several drugs that target the TGFβ pathway have been developed and tested in patients, but with limited success.

[00102] The inventors of the present disclosure have considered that, although the three TGFβ isoforms are capable of signaling through the same receptors and transduction of downstream effectors in cells that express the receptors, each TGFβ isoform can generate distinct biological effects in vivo . Furthermore, the inventors contemplate that, at least in some circumstances, the manner in which the growth factor-receptor interaction is triggered may further provide signaling specificity in vivo. In light of this recognition, it is observed that TGFβ inhibitors described in the literature to date do not have specificity, as briefly summarized below.

[00103] Fresolimumab, a humanized monoclonal antibody that binds 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, L.M., et al., J . Clin. Invest., 2015. 125 (7): pages 2,795-807; , PLoS One, 2014. 9 (3): page e90353). Additional companies have developed monoclonal antibodies against the TGFβ growth factors with varying degrees of selectivity for TGFβ isoforms. These agents likely evoke toxicities in vivo through residual activity against TGFβ family members other than TGFβ1. To the knowledge of the inventors of the present disclosure, complete specificity for a single isoform has not been obtained by targeting the mature growth factor, due to the high degree of sequence identity between isoforms.

[00104] Other approaches to targeting the TGFβ pathway include ACE-1332, a soluble TGFβRII-Fc ligand trap from Acceleron (Yung, L.M., et al., A. Am. J. Respir. Crit. Care Med., 2016. 194 (9): pages 1,140-1,151), or small molecule inhibitors of ALK5 kinase, for example, Lilly's galunisertib. Although ACE-1332 binds to TGFβ1 and TGFβ3 with equally high affinity (Yung, L.M., et al., Am. J. Respir. Crit. Care Med., 2016. 194 (9): pages 1140-1151). ALK5 inhibitors block the activity of all growth factors that signal through TGFR1. Substantial toxicities have been found in preclinical studies with the use of ALK5 inhibitors (Anderton, M.J., et al., Toxicol. Pathol., 2011. 39 (6): pages 916-24; Stauber, A., et al. , Clinical Toxicology, 2014. 4 (3): pages 1-10), and sophisticated clinical dosing schedules are needed to maintain efficacy while reducing adverse events (Herbertz, S., et al., Drug Des. Devel . Ther., 2015. 9: pages 4,479-99). In fact, the question of the specificity of TGFβ signaling and its possible effect on toxicity observed with known TGFβ inhibitors has not yet been raised in most, if not all, candidate drugs that have attempted to block TGFβ. For example, how much of the toxicities are caused by inhibition of TGFβ1 versus TGFβ2 and/or TGFβ3 remains to be answered. Similarly, modes of TGFβ activation have not yet been taken into account in design or development ways to antagonize TGFβ signaling.

[00105] Recent structural knowledge on the mechanism of TGFβ1 activation has allowed the present inventors to take new, more specific approaches to TGFβ inhibition (Shi, M., et al., Nature, 2011. 474 (7351): pages 343 -9). Unlike other cytokines, members of the TGFβ superfamily 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 pr0-TGFβ1 by furin proteases separates the homodimeric growth factor domain from its prodomain, also called latency-associated peptide (LAP). However, the growth factor and LAP remain non-covalently associated, forming a latent complex that is unable to bind to its receptors and induce signaling (Figure 1). During translation, latent TGFβ1, also called the small latent complex (SLC), is linked to “presentation molecules” through disulfide bonds, forming the large latent complex (LLC). These molecules allow pr0-TGFβ1 to be presented in specific cellular or tissue contexts. Two cysteines near the N-terminus of latent TGFβ1 bind to appropriately positioned cysteines 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 tethered to latent TGFβ-binding proteins (LTBPs), which then associates with proteins in the extracellular matrix (ECM) (i.e., fibronectin, fibrillin-1) to bind latent TGFβ to the ECM (Robertson and cols. Matrix Biol. 47: 44-53 (2015) (Figure 2A). , was identified as a presentation molecule for TGFβ1 on the surface of monocytes, macrophages and microglia (Wang, R., et al., Mol. Biol. Cell., 2012. 23 (6): pages 1,129-39 and T.A. Springer, Int. BMP Conference 2016).

[00106] In mammals there are four known LTBPs, LTBP1-4, each with several splice variants (Robertson, I.B., et al., Matrix Biol., 2015. 47: pages 44-53). LTBP2 is the only LTBP that does not associate with latent TGFβ (Saharinen, J. and J. Keski-Oja, Mol. Biol. Cell., 2000. 11 (8): pages 2691-704). Although the association between LTBP1 or LTBP3 and latent TGFβ1 has been well validated, the role of LTBP4 in TGFβ presentation is less clear. The complex with LTBP4 and latent TGFβ1 appears to form much less efficiently, potentially due to the absence of several negatively charged residues in the TGFβ-binding domain of LTBP4 (Saharinen, J. and J. Keski-Oja, Mol. Biol. Cell. , 2000. 11 (8): pages 2,691-704; Chen, Y., et al., J. Mol. Biol., 2005. 345 (1): pages 175-86). Both LTBP4S-/- mice and patients with Urban-Rifkin-Davis syndrome, which have null mutations in LTBP4, suffer from disrupted elastic fiber assembly (Urban, Z., et al., Am. J. Hum. Genet., 2009. 85 (5): pages 593-605; Dabovic, B., et al., J. Physiol., 2015. 230 (1): pages 226-36). Additionally, although LTBP4S-/- mice have lung septation and a defect in elastogenesis, transgenic mice with an LTBP4 that cannot form a complex with latent TGFβ1 have no obvious phenotype (Dabovic, B., et al., J. Cell. Physiol ., 2015. 230 (1): pages 226-36). Whether LTBP4 is directly involved in the regulation of latent TGFβ1 by functioning as a presentation molecule is uncertain; LTBP4 may instead be required for the proper formation of elastic fibrils in the ECM and its loss indirectly affects the activation of latent TGFβ1 through defects in the ECM.

[00107] Several studies have sought to clarify the mechanisms of TGFβ1 activation. Three integrins, αVβ6, αVβ8, and αVβ1, have been shown to be crucial activators of latent TGFβ1 (Reed, N.I., et al., Sci. Transl. Med., 2015. 7 (288): page 288ra79; Travis, M.A., and D. Sheppard, Annu. Rev. Immunol., 2014. 32: pages 51-82; Munger, J.S., et al., Cell, 1999. 96 (3): pages 319-28). αV integrins bind to the RGD sequence present in TGFβ1 and TGFβ1 LAPs with high affinity (Dong, X., et al., Nat. Struct. Mol. Biol., 2014. 21 (12): pages 1,091-6). Transgenic mice with a mutation in the RGD site of TGFβ1 that prevents integrin binding, but not secretion, phenocopies the TGFβ1-/- mouse (Yang, Z., et al., J. Cell. Biol., 2007. 176 ( 6): pages 787-93). Mice lacking β6 and β8 integrins recapitulate the essential phenotypes of TGFβ1 and TGFβ3 knockout mice, including multiorgan inflammation and cleft palate, confirming the essential role of these two integrins for TGFβ1 activation in development and homeostasis (Aluwihare, P., and cols., J. Cell. Sci., 2009. 122 (Part 2): pages 227-32). Crucial for integrin-dependent activation of latent TGFβ1 is covalent adhesion to presenting molecules; disruption of the disulfide bonds between GARP and TGFβ1 LAP by mutagenesis does not impair complex formation, but completely abolishes TGFβ1 activation by αVβ6 (Wang, R., et al., Mol. Biol. Cell., 2012. 23 (6 ): pages 1,129-39). The recent structure of latent TGFβ1 illuminates how integrins enable the release of active TGFβ1 from the latent complex: covalent binding of latent TGFβ1 to its presentation molecule anchors latent TGFβ1 to the ECM via LTBPs, or to the cytoskeleton via GARP or LRRC33 . Integrin binding to the RGD sequence results in a force-dependent change in the structure of LAP, allowing active TGFβ1 to be released and bind to nearby receptors (Shi, M., et al., Nature, 2011. 474 (7351): pages 343-9). The importance of TGFβ1 integrin-dependent activation in disease has also been well validated. A small molecular inhibitor of αVβ1 protects against bleomycin-induced pulmonary fibrosis and carbon tetrachloride-induced liver fibrosis (Reed, N.I., et al., Sci. Transl. Med., 2015. 7(288): page 288ra79), and the blocking αVβ6 with an antibody or loss of β6 integrin expression suppresses bleomycin-induced pulmonary fibrosis and radiation-induced fibrosis (Munger, J.S., et al., Cell, 1999. 96 (3): pages 319-28); Horan, G.S., et al., Am. J. Respir. Crit. Care Med., 2008. 177 (1): page 5,665). In addition to integrins, other mechanisms of TGFβ1 activation have been implicated, including thrombospondin-1 and activation by proteases such as matrix metalloproteinases (MMPs), cathepsin D or kallikrein. However, most of these studies were performed in vitro using purified proteins; There is less evidence for the role of these molecules from in vivo studies. Thrombospondin-1 knockout recapitulates some aspects of the TGFβ1-/- phenotype in some tissues, but is not protective in bleomycin-induced pulmonary fibrosis, known to be TGFβ-dependent (Ezzie, M.E., et al., Am. J. Respir. Cell . Mol. Biol., 2011. 44 (4): pages 556-61). Additionally, knockout of candidate proteases does not result in a TGFβ1 phenotype (Worthington, J.J., J.E. Klementowicz and M.A. Travis, Trends Biochem. Sci., 2011. 36 (1): pages 47-54). This could be explained by redundancies or by the fact that these mechanisms are crucial in specific diseases rather than development and homeostasis.

[00108] TGFβ has been implicated in several biological processes, including fibrosis, immune modulation and cancer progression. TGFβ1 was the first identified member of the TGFβ superfamily of proteins. Like other members of the TGFβ superfamily, TGFβ1 and the isoforms TGFβ2 and TGFβ3 are initially expressed as inactive proprotein precursor forms (termed pr0-TGFβ). TGFβ proteins (e.g., TGFβ1, TGFβ2, and TGFβ3) are proteolytically cleaved by proprotein convertases (e.g., furin) to generate the latent form (termed latent TGFβ). In some embodiments, a proprotein form or latent form of a TGFβ protein (e.g., TGFβ1, TGFβ2, and TGFβ3) may be referred to as a “pro/latent TGFβ protein.” TGFβ1 can be presented to other molecules in complex with various molecules including, for example, GARP (to form a GARP-TGFβ1 complex), LRRC33 (to form a LRRC33-TGFβ1 complex), LTBP1 (to form an LTBP1-TGFβ1 complex), and/or LTBP3 (to form an LTBP3-TGFβ1 complex). The TGFβ1 present in these complexes can be in latent form (latent TGFβl) or in precursor form (pr0-TGFβ1).

[00109] The present invention is directed to immunoglobulins, for example, antibodies or antigen-binding portions thereof that specifically bind to (1) a TGFβ protein (e.g., pro/latent TGFβ1, pro/latent TGFβ2 and pro/latent TGFβ3 latent) in complex with a GARP protein, (2) a TGFβ protein in complex with an LTBP protein (e.g., LTBP1 or LTBP3), and/or (3) a TGFβ protein in complex with a LRRC33 protein. In some embodiments, the antibodies, or antigen-binding portions thereof, disclosed in this specification bind to an epitope of TGFβ1 that is available for binding by the antibody, or antigen-binding portions thereof, when TGFβ1 is present in a complex GARP-TGFβ1, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and/or a LRRC33-TGFβ1 complex. Without being bound by a particular theory, the ability of the antibodies, and antigen-binding portions thereof, disclosed in this specification to bind to the TGFβ protein (e.g., TGFβ1) that is in complex with a GARP protein, an LTBP protein, and /or an LRRC33 protein allows targeting of the TGFβ protein in a context-independent manner, and may be particularly suitable for therapeutic applications.

Definitions

[00110] In order for the revelation to be more easily understood, first certain terms will be defined. These definitions should be read in light of the remainder of the disclosure and as understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings commonly understood by those skilled in the art. Additional definitions are presented throughout the detailed description.

[00111] As used in this specification, the term “specific binding” or “specifically binds” means that the interaction of the antibody, or antigen-binding portion thereof, with an antigen is dependent on the presence of a particular structure (e.g. , an antigenic determinant or epitope). For example, the antibody, or antigen-binding portion thereof, binds to a specific protein rather than to proteins in general. In some embodiments, an antibody, or antigen-binding portion thereof, specifically binds to a target, e.g., TGFβ1, if the antibody has a KD for the target of at least about 10-4 M, 10-5 M , 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, 10-13M, or less. In some embodiments, the term “specifically binds to a TGFβ1 epitope”, “specifically binds to a TGFβ1 epitope”, “specifically binds to TGFβ1”, or “specifically binds to TGFβ1”, as used in this specification, if refers to an antibody, or antigen-binding portion thereof, that binds to TGFβ1 and has a dissociation constant (KD) of 1.0 x 10-7 M or less, as determined by surface plasmon resonance. In one embodiment, an antibody, or antigen-binding portion thereof, can specifically bind to both human and non-human (e.g., mouse) orthologs of TGFβ1.

[00112] In some embodiments, the binding affinity of an antibody, or antigen-binding portion thereof, to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex is determined using an Octet assay. In some embodiments, an Octet assay is an assay that determines one or more kinetic parameters indicative of binding between an antibody and antigen. In some embodiments, an Octet® system (ForteBio, Menlo Park, CA) is used to determine the binding affinity of an antibody, or antigen-binding portion thereof, to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex. For example, antibody binding affinities can be determined using the “forteBio Octet QKe Dip and Read Label Free” assay system that utilizes biolayer interferometry. In some embodiments, antigens are immobilized to biosensors (e.g., streptavidin-coated biosensors) and antibodies and complexes (e.g., biotinylated GARP-TGFβ1 complexes and biotinylated LTBP-TGFβ1 complexes) are presented in solution at high concentration (50 μg /ml) to mediate binding interactions. In some embodiments, the binding affinity of an antibody, or antigen-binding portion thereof, to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and/or a LRRC33-TGFβ1 complex is determined using the protocol described in Table 6.

[00113] As used in this specification, the term “GARP-TGFβ1 complex” refers to a protein complex comprising a proprotein form or latent form of a transforming growth factor-β1 (TGFβ1) protein and a glycoprotein-A repeat predominant protein (GARP). In some embodiments, a proprotein form or latent form of TGFβ1 protein may be referred to as “pro/latent TGFβ1 protein”. In some embodiments, a GARP-TGFβ1 complex comprises GARP covalently linked to pro/latent TGFβ1 through one or more disulfide bonds. In other embodiments, a GARP-TGFβ1 complex comprises GARP non-covalently linked to pro/latent TGFβ1. In some embodiments, a GARP-TGFβ1 complex is a naturally occurring complex, e.g., a GARP-TGFβ1 complex in a cell. An exemplary GARP-TGFβ1 complex is shown in Figure 3.

[00114] As used in this specification, the term “LTBP1-TGFβ1 complex” refers to a protein complex comprising a proprotein form or latent form of transforming growth factor-β1 (TGFβ1) protein and a latent TGF-beta binding protein 1 (LTBP1). In some embodiments, an LTBP1-TGFβ1 complex comprises LTBP1 covalently linked to pro/latent TGFβ1 via one or more disulfide bonds. In other embodiments, an LTBP1-TGFβ1 complex comprises LTBP1 non-covalently bound to pro/latent TGFβ1. In some embodiments, an LTBP1-TGFβ1 complex is a naturally occurring complex, e.g., an LTBP1-TGFβ1 complex in a cell. An exemplary LTBP1-TGFβ1 complex is shown in Figure 3.

[00115] As used in this specification, the term “LTBP3-TGFβ1 complex” refers to a protein complex comprising a proprotein form or latent form of transforming growth factor-β1 (TGFβ1) protein and a latent TGF-beta binding protein 1 (LTBP3). In some embodiments, an LTBP3-TGFβ1 complex comprises LTBP3 covalently linked to pro/latent TGFβ1 via one or more disulfide bonds. In other embodiments, an LTBP3-TGFβ1 complex comprises LTBP1 non-covalently bound to pro/latent TGFβ1. In some embodiments, an LTBP3-TGFβ1 complex is a naturally occurring complex, e.g., an LTBP3-TGFβ1 complex in a cell. An exemplary LTBP3-TGFβ1 complex is shown in Figure 3.

[00116] As used in this specification, the term “LRRC33-TGFβ1 complex” refers to a complex between a proprotein form or latent form of transforming growth factor-β1 protein (TGFβ1) and the Repeat Containing Protein Rich in Leucine 33 (LRRC33; also known as Negative Regulator of Reactive Oxygen Species or NRROS). In some embodiments, a LRRC33-TGFβ1 complex comprises LRRC33 covalently linked to pro/latent TGFβ1 via one or more disulfide bonds. In other embodiments, a LRRC33-TGFβ1 complex comprises LRRC33 non-covalently bound to pro/latent TGFβ1. In some embodiments, a LRRC33-TGFβ1 complex is a naturally occurring complex, e.g., a LRRC33-TGFβ1 complex in a cell.

[00117] The term “antibody” refers to an immunoglobulin molecule that specifically binds to a target antigen, and includes, for example, chimeric, humanized, fully human and bispecific antibodies. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains, for example, naturally occurring antibodies in camelids may comprise only heavy chains. Antibodies can be derived exclusively from a single source, or they can be “chimeric”, that is, different portions of the antibody can be derived from two different antibodies. Antibodies, or antigen-binding portions thereof, can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. The term “antibodies” as used in this specification includes monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to in this specification as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies , antibody fusions (sometimes referred to in this specification as “antibody conjugates”), respectively. In some embodiments, the term also encompasses peptibodies.

[00118] Structural units of a naturally occurring antibody typically comprise a tetramer. The tetramer typically is composed of two identical pairs of polypeptide chains, each pair having a full-length “light” chain (in certain embodiments, about 25 kDa) and a full-length “heavy” chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids that is typically responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that may be responsible for effector function. Human antibody light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha or epsilon, and define the isotype of the antibody. An antibody can be of any type (e.g., IgM, IgD, IgG, IgA, IgY, and IgE) and class (e.g., IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, and IgA2). Within the full-length light and heavy chains, typically the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 or more amino acids. amino acids (see, for example, “Fundamental Immunology”, Chapter 7 (Paul, W., ed., 2nd Edition, Raven Press, N.Y. (1989)) (incorporated by reference in its entirety)). The variable regions of each light/heavy chain pair typically form the antigen-binding site.

[00119] Variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs of the two chains of each pair are typically aligned by framework regions, which may allow binding to a specific epitope. From the N-terminus to the C-terminus, the light chain and heavy chain variable regions typically comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. The allocation of amino acids to each domain is typically in accordance with the definitions of “Kabat Sequences of Proteins of Immunological Interest” (“National Institutes of Health”, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. The CDRs of a light chain can also be called CDR-L1, CDR-L2, and CDR-L3, and the CDRs of a heavy chain can also be called CDR-H1, CDR-H2, and CDR-H3. In some embodiments, an antibody may comprise a small number of amino acid deletions from the carboxy end of the heavy chain(s). In some embodiments, an antibody comprises a heavy chain that has 1-5 amino acid deletions at the carboxy end of the heavy chain. In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody are obtained by solving the structure of the antibody and/or solving the structure of the antibody-ligand complex. In certain embodiments, this may be achieved by any of several methodologies known to those skilled in the art, for example, X-ray crystallography. In some embodiments, various analysis methods can be employed to identify or approximate CDR regions. Examples of these methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.

[00120] A “functional antigen binding site” of a binding protein is one that can bind to a target, antigen or ligand. The antigen-binding affinity of the antigen-binding site is not necessarily as strong as that of the parental binding protein from which the antigen-binding site is derived, but the ability to bind antigen should be measurable using any of several known methods for evaluating the binding of the binding protein to an antigen. Furthermore, the antigen-binding affinity of each of the antigen-binding sites of a multispecific binding protein of this specification need not be quantitatively the same.

[00121] The term “variable region” or “variable domain” refers to a portion of the light and/or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids from the amino terminus of the light chain. In certain embodiments, the variable regions of different antibodies differ greatly in terms of amino acid sequence, even between antibodies of the same species. The variable region of an antibody typically determines the specificity of a particular antibody for its target.

[00122] An immunoglobulin constant domain refers to a heavy or light chain constant domain. The amino acid sequences of the constant domain of the human IgG heavy chain and light chain are known in the art.

[00123] The term “compete”, when used in the context of antigen-binding proteins (e.g., an antibody or antigen-binding portion thereof) that compete for the same epitope, means competition between antigen-binding proteins, as determined by an assay in which the antigen-binding protein being tested prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein to a common antigen (e.g., TGFβ1 or a fragment thereof) . Several types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, for example: direct or indirect solid-phase radioimmunoassay (RIA), direct or indirect solid-phase enzyme immunoassay (EIA), sandwich competition; Direct solid-phase biotin-avidin EIA; solid phase direct marked test and solid phase direct marked sandwich test. Typically, when a competing antigen-binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of a reference antigen-binding protein to a common antigen by at least 40-45%, 45 50 %, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[00124] The term “antigen” refers to a molecular structure that provides an epitope, for example, a molecule or a portion of a molecule or a complex of molecules or portions of molecules, capable of being bound by a selective binding agent , for example, an antigen-binding protein (including, for example, an antibody). In this way, a selective binding agent can specifically bind to an antigen that is formed by two or more components in a complex. In some embodiments, the antigen is capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen may possess one or more epitopes that are capable of interacting with different antigen-binding proteins, e.g., antibodies.

[00125] As used in this specification, the term “CDR” refers to the complementarity-determining region within variable antibody sequences. There are three CDRs in each of the heavy chain and light chain variable regions, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term “set of CDRs”, as used in this specification, refers to a group of three CDRs that occur in a single variable region that can bind antigen. The exact limits of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al. (1987; 1991) “Sequences of Proteins of Immunological Interest” (“National Institutes of Health”, Bethesda, Md.) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. and Chothia et al (1989) Nature 342: 877-883) found that certain subportions within the Kabat CDRs adopt nearly identical peptide framework conformations despite having great diversity at the amino acid sequence level. , L2 and L3 or H1, H2 and H3, where the “L” and “H” designate the light chain and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, which have boundaries that overlap. overlap with Kabat CDRs. Other boundaries defining CDRs overlap with Kabat CDRs have been described by Padlan (1995) FASEB J. 9: 133-139 and MacCallum (1996) J. Mol. Biol. 262 (5): 732-45. Still other definitions of CDR limits may not strictly follow one of the systems presented in this specification, but nevertheless overlap with Kabat's CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used in this specification may utilize CDRs defined in accordance with any of these systems, although certain embodiments use CDRs defined by Kabat or Chothia.

[00126] The terms “crystal” and “crystallized”, as used in this specification, refer to a binding protein (for example, an antibody), or antigen-binding portion thereof, which exists in the form of a crystal. Crystals are a form of the solid state of matter, which is distinct from other forms, such as the amorphous solid state or the liquid crystalline state. Crystals are composed of regular, repetitive, three-dimensional arrangements of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen/antibody complexes). These three-dimensional arrangements are arranged according to specific mathematical relationships that are well understood in the field. The fundamental unit, or modular block, that is repeated in a crystal is called the asymmetric unit. The repetition of the asymmetric unit in an arrangement that conforms to a certain well-defined crystallographic symmetry provides the “unit cell” of the crystal. Repetition of the unit cell by regular translations in all three dimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett, “Crystallization of Nucleic Acids and Proteins, a Practical Approach”, 2nd Edition, pages 201-16, Oxford University Press, New York, New York, (1999).

[00127] 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, epitope determinants include chemically active groupings of molecules on the surface, for example, amino acids, sugar, phosphoryl or sulfonyl side chains and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by a binding protein. Thus, an epitope consists of amino acid residues from a region of an antigen (or fragment thereof) that are known to bind to the complementary site on the specific binding partner. An antigenic fragment may contain more than one epitope. In certain embodiments, an antibody is said to specifically bind to an antigen when it recognizes its target antigen in a complex mixture of proteins and/or macromolecules. For example, antibodies are said to “bind to the same epitope” if the antibodies cross-compete (one prevents binding or modulates the effect of the other). Furthermore, structural definitions of epitopes (overlapping, similar, identical) are informative, but functional definitions are often more relevant, as they encompass both structural (binding) and functional (modulation, competition) parameters.

[00128] The term “treat” and “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which the risk that an individual will develop a disorder or other risk factor is reduced. Treatment does not require the complete cure of a disorder and encompasses modalities in which underlying symptoms or risk factors are reduced.

[00129] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be carried out in accordance with the manufacturer's specifications or as commonly obtained in the art or as described in this specification. The techniques and procedures cited above can generally be carried out in accordance with conventional methods well known in the art and as described in several general and more specific references that are cited and discussed throughout this specification. See, for example, Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated into this specification by reference for any purpose. Unless specific definitions are provided, the nomenclatures used in connection with, and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry described in this specification are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and release, and patient treatment.

[00130] The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody, as used in this specification, refer to one or more fragments of an antibody that retain the ability to specifically bind to a antigen (e.g. TGFβ1). Antigen-binding moieties include, without limitation, any naturally occurring, enzymatically obtainable, synthetic or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. In some embodiments, an antigen-binding portion of an antibody can be derived, for example, from whole antibody molecules using any suitable standard techniques such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression. of DNA encoding variable domains and, optionally, antibody constant domains. Non-limiting examples of antigen-binding moieties include: (i) Fab fragments, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments, which consist of the VH and CH1 domains; (iv) Fv fragments, which consist of the VL and VH domains of a single arm of an antibody; (v) single-chain Fv (scFv) molecules (see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Nat'l. Acad. Sci. USA 85: 5879-5883); (vi) dAb fragments (see, for example, Ward et al. (1989) Nature 341: 544-546); and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR). Other forms of single-chain antibodies, e.g., diabodies, are also encompassed. The term “antigen-binding portion” of an antibody includes a “single-chain Fab fragment” also known as a “scFab”, which comprises an antibody heavy chain variable domain (VH), a constant domain 1 (CH1) antibody, an antibody light chain (VL) variable domain, an antibody light chain (CL) constant domain, and a linker, wherein said antibody domains and said linker have one of the following orders in the terminal direction -N to C-terminal: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1- linker-VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids.

[00131] An “isolated antibody”, as used in this specification, refers to an antibody that is substantially free of other antibodies that have different antigenic specificities. In some embodiments, an isolated antibody is substantially free of other cellular material and/or chemicals.

[00132] An “affinity matured” antibody is an antibody with one or more changes in one or more of its CDRs, which result in an increase in the affinity of the antibody for the antigen, compared to a parental antibody, which does not have those changes. Exemplary affinity-matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. (1992) Bio/Technology 10: 779-783 describes affinity maturation by shuffling of the VH and VL domains. Random mutagenesis of CDR and/or framework residues is described by Barbas, et al. (1994) Proc. Nat. Acad. Sci. USA 91: 3809-3813; Schier et al. (1995) Gene 169: 147155; Yelton et al., (1995) J. Immunol. 155: 1994-2004; Jackson et al. (1995) J. Immunol. 154(7): 3310-9; and Hawkins et al. (1992) J. Mol. Biol. 226: 889-896; and selective mutation at selective mutagenesis positions, contact positions or hypermutation with an activity-enhancing amino acid residue is described in U.S. Patent No. 6,914,128.

[00133] The term “CDR-grafted antibody” refers to antibodies that comprise heavy and light chain variable region sequences of a species, but in which the sequences of one or more of the CDR regions of VH and/or VL are substituted with CDR sequences from another species, for example, antibodies that have murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.

[00134] The term “chimeric antibody” refers to antibodies that comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, for example, antibodies that have murine heavy and light chain variable regions. linked to human constant regions.

[00135] The term “human antibody”, as used in this specification, is intended to include antibodies that have variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs and , in particular, CDR3. However, the term “human antibody”, as used in this specification, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, for example, a mouse, have been grafted onto human framework sequences.

[00136] The term “humanized antibody” refers to antibodies that comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse), but in which at least a portion of the VH sequence and/or or VL was altered to be more “human-like”, that is, more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Furthermore, a “humanized antibody” is an antibody, or a variant, derivative, analogue, or fragment thereof, that immunologically specifically binds to an antigen of interest and that comprises an FR region that substantially has the amino acid sequence of a human antibody. and a CDR region that substantially has the amino acid sequence of a non-human antibody. As used in this specification, the term “substantially,” in the context of a CDR, refers to a CDR that has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. (i.e., donor antibody) and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. In one embodiment, a humanized antibody also comprises at least a portion of an immunoglobulin Fc region, typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains the light chain as well as at least the variable domain of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain. In specific embodiments, a humanized antibody contains only a humanized variable domain of a humanized light chain and/or heavy chain.

[00137] As used in this specification, the term “framework” or “framework sequence” refers to the remaining sequences of a variable region, minus the CDRs. As the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to consequently different interpretations. The six CDRs (light chain CDR-L1, L2 and -L3 and heavy chain CDR-H1, -H2 and -H3) also divide the framework regions in the light chain and heavy chain into four subregions (FR1, FR2, FR3 and FR4) on each chain, where CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying particular subregions such as FR1, FR2, FR3 or FR4, a framework region, as cited by others, represents the combined FRs within the variable region of a single naturally occurring immunoglobulin chain. As used in this specification, a “FR” represents one of the four subregions, and FRs represent two or more of the four subregions that constitute a framework region.

[00138] As used in this specification, the term “germline antibody gene” or “gene fragment” refers to an immunoglobulin sequence encoded by nonlymphoid cells that has not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin (see, for example, Shapiro et al. (2002) Crit. Rev. Immunol. 22 (3): 183200; Marchalonis et al. (2001) Adv. Exp. Med. Biol. 484: 13 -30). One of the advantages provided by various embodiments of the present disclosure is due to the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species and, thus, a greater probability to be recognized as from a foreign source, when used therapeutically in that species.

[00139] As used in this specification, the term “neutralizing” refers to the action contrary to the biological activity of an antigen when a binding protein specifically binds to the antigen. In one embodiment, the neutralizing binding protein binds to the antigen/target, e.g., cytokine, kinase, growth factor, cell surface protein, soluble protein, phosphatase, or receptor ligand, and reduces its biological activity by at least about of 20%, 40%, 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.

[00140] The term “binding protein”, as used in this specification, includes any polypeptide that specifically binds to an antigen (e.g., TGFβ1) including, without limitation, an antibody, or antigen-binding portions thereof, an DVD-IgTM, a TVD-Ig, a RAb-Ig, a bispecific antibody and a dual specific antibody.

[00141] The term “monoclonal antibody” or “mAb”, when used in the context of a composition comprising the same, may refer to an antibody preparation obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies that comprise the population are identical, except for possible naturally occurring mutations that may be present in small quantities. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The “monoclonal” modifier should not be considered as requiring the production of the antibody by any particular method.

[00142] The term “recombinant human antibody”, as used in this specification, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, for example, antibodies expressed using a recombinant expression vector transfected into a host cell (described in more detail in Section II C, below), antibodies isolated from a recombinant human antibody combinatorial library (Hoogenboom, H.R. (1997) TIB Tech. 15: 62-70; Azzazy, H. and Highsmith, W.E. (2002) Clin. Biochem. 35: 425-445; , incorporated into this specification by reference), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, Taylor, L. D. et al. (1992) Nucl. Acids Res. 20: 6.287- 6.295; Kellermann, S. A. and Green, L. L. (2002) Cur. 13: 593-597; Little, M. et al. (2000) Immunol. Today 21: 364-370) or antibodies prepared, expressed, created or isolated by other means involving splicing of human immunoglobulin gene sequences with other DNA sequences. These recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, these recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thereby the amino acid sequences of the VH and VL of recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[00143] As used in this specification, the term “Dual Variable Domain Immunoglobulin” or “DVD-IgTM” and the like include binding proteins comprising a heavy chain DVD polypeptide and a light chain DVD polypeptide paired, with each paired heavy and light chain providing two antigen binding sites. Each binding site includes a total of 6 CDRs involved in antigen binding per antigen binding site. A DVD-IgTM typically has two arms linked together, at least in part, by dimerization of the CH3 domains, with each arm of the DVD being bispecific, providing an immunoglobulin with four binding sites. DVD-IgTM are provided in U.S. Patent Publications Nos. 2010/0260668 and 2009/0304693, each of which is incorporated into this specification by reference, including sequence listings.

[00144] As used in this specification, the term “Triple Variable Domain Immunoglobulin” or “TVD-Ig” and the like are binding proteins comprising a heavy chain TVD-binding protein polypeptide and a TVD-binding protein polypeptide. of the paired light chain with each paired heavy and light chain providing three antigen binding sites. Each binding site includes a total of 6 CDRs involved in antigen binding per antigen binding site. A TVD-binding protein can have two arms linked together, at least in part, by dimerization of the CH3 domains, with each arm of the TVD-binding protein being trispecific, providing a binding protein with six binding sites.

[00145] As used in this specification, the term “Receptor-Antibody Immunoglobulin” or “RAb-Ig” and the like are binding proteins comprising a heavy chain RAb polypeptide and a light chain RAb polypeptide, which together form three antigen binding sites in total. An antigen binding site is formed by pairing the heavy and light antibody variable domains present on each of the heavy chain RAb polypeptide and the light chain RAb polypeptide to form a single binding site with a total of 6 CDRs, which provide a first antigen binding site. The heavy chain RAb polypeptide and light chain RAb polypeptide each includes a receptor sequence that independently binds a ligand providing second and third “antigen” binding sites. A RAb-Ig typically has two arms linked together, at least in part, by dimerization of the CH3 domains, with each arm of the RAb-Ig being trispecific, providing an immunoglobulin with six binding sites. RAb-Igs are described in U.S. Patent Application Publication No. 2002/0127231, the entire contents of which, including sequence listings, are incorporated into this specification by reference.

[00146] The term “bispecific antibody”, as used in this specification, and as distinguished from a “half-Ig bispecific binding protein” or “bispecific binding protein (half-Ig)”, refers to full-length antibodies which are generated by quadroma technology (see Milstein, C. and Cuello, A.C. (1983) Nature 305 (5934): pages 537-540), by chemical conjugation of two different monoclonal antibodies (see Staerz, U.D. et al. (1985 ) Nature 314 (6012): 628-631), or by knob-into-hole or similar approaches, which introduce mutations in the Fc region that do not inhibit CH3-CH3 dimerization (see Holliger, P. et al. (1993) Proc . Natl. Acad. Sci USA 90 (14): 6,444-6,448), resulting in multiple different immunoglobulin species, only one of which is the functional bispecific antibody. By molecular function, a bispecific antibody binds to an antigen (or epitope) on one of its two binding arms (an HC/LC pair), and binds to a different antigen (or epitope) on its second arm (a different HC/LC pair). By this definition, a bispecific antibody has two distinct antigen-binding arms (in terms of specificity and CDR sequences), and is monovalent for each antigen to which it binds.

[00147] The term “dual specific antibody”, as used in this specification, and as distinguished from a half-Ig bispecific binding protein or a bispecific binding protein, refers to full-length antibodies that can bind to two antigens different (or epitopes) on each of its two binding arms (a HC/LC pair) (see PCT Publication No. WO 02/02773). Consequently, a dual-specific binding protein has two identical antigen-binding arms, with identical specificity and identical CDR sequences, and is bivalent to each antigen to which it binds.

[00148] The term “pan-TGFβ antibody” refers to any antibody that is capable of binding to more than one isoform of TGFβ, for example, at least two of TGFβ1, TGFβ2 and TGFβ3. In some embodiments, a pan-TGFβ antibody binds to all three isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3. In some embodiments, a pan-TGFβ antibody binds and neutralizes all three isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3.

[00149] The term “Kon”, as used in this specification, is intended to refer to the rate constant for association of a binding protein (e.g., an antibody) with the antigen to form, for example, the antigen/ antibody, as is known in the art. “Kon” is also known by the terms “membership fee constant”, or “ka”, as used interchangeably in this specification. This value that indicates the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and antigen is also shown by the equation: Antibody (“Ab”) + Antigen (“Ag”) ^ Ab- Ag.

[00150] The term “Koff”, as used in this specification, is intended to refer to the off rate constant for dissociation of a binding protein (for example, an antibody), for example, from the antigen/antibody complex, as is known in the art. “Koff” is also known by the terms “dissociation rate constant” or “kd”, as used interchangeably in this specification. This value indicates the rate of dissociation of an antibody from its target antigen or separation of the Ag-Ab complex over time into free antibody and antigen, as shown by the equation: Ab + Ag ^ Ab-Ag.

[00151] The terms “equilibrium dissociation constant” or “KD”, as used interchangeably in this specification, refer to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (koff ) by the association rate constant (kon). The association rate constant, dissociation rate constant, and equilibrium dissociation constant are used to represent the binding affinity of a binding protein, e.g., antibody, to an antigen. Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments, for example, a BIAcore® assay (biomolecular interaction analysis), can be used (e.g., instrument available from BIAcore International AB, a GE Healthcare Company, Uppsala, Sweden). Additionally, a KinExA® assay (Kinetic Exclusion Assay), available from Sapidyne Instruments (Boise, Idaho), can also be used.

[00152] The term “linker” is used to designate polypeptides that comprise two or more amino acid residues joined by peptide bonds and are used to link one or more binding moieties to the antigen. Such linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 6,444-6,448; Poljak, R.J. et al. (1994) Structure 2 : 1,121-1,123). Exemplary linkers include, without limitation, ASTKGPSVFPLAP (SEQ ID NO: 55), ASTKGP (SEQ ID NO: 56); TVAAPSVFIFPP (SEQ ID NO: 57); TVAAP (SEQ ID NO: 58); AKTTPKLEEGEFSEAR (SEQ ID NO: 59); AKTTPKLEEGEFSEARV (SEQ ID NO: 60); AKTTPKLGG (SEQ ID NO: 61); SAKTTPKLGG (SEQ ID NO: 62); SAKTTP (SEQ ID NO: 63); RADAAP (SEQ ID NO: 64); RADAAPTVS (SEQ ID NO: 65); RADAAAAGGPGS (SEQ ID NO: 66); RADAAAA(G4S)4 (SEQ ID NO: 67); SAKTTPKLEEGEFSEARV (SEQ ID NO: 68); ADAAP (SEQ ID NO: 69); ADAAPTVSIFPP (SEQ ID NO: 70); QPKAAP (SEQ ID NO: 71); QPKAAPSVTLFPP (SEQ ID NO: 72); AKTTPP (SEQ ID NO: 73); AKTTPPSVTPLAP (SEQ ID NO: 74); AKTTAP (SEQ ID NO: 75); AKTTAPSVYPLAP (SEQ ID NO: 76); GGGGSGGGGSGGGGS (SEQ ID NO: 77); GENKVEYAPALMALS (SEQ ID NO: 78); GPAKELTPLKEAKVS (SEQ ID NO: 79); GHEAAAVMQVQYPAS (SEQ ID NO: 80); TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO: 81); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 82).

[00153] The term “cancer”, as used in this specification, refers to the physiological condition in multicellular eukaryotes that is typically characterized by unregulated cell proliferation.

[00154] The terms “tag” and “detectable tag” or “detectable moiety” mean a moiety attached to a specific binding partner, e.g., an antibody or an analyte, e.g., to make the reaction between members of a pair specific binding partner, e.g., an antibody and an analyte, is detectable, and the specific binding partner, e.g., antibody or analyte, so labeled is called “detectably labeled.” Thus, the term “labeled binding protein”, as used in this specification, refers to a protein with an incorporated marker that allows identification of the binding protein. In one embodiment, the label is a detectable label that can produce a signal that is detectable by visual or instrumental means, e.g., incorporation of a radiolabeled amino acid or adhesion to a polypeptide of biotinyl moieties that can be detected by labeled avidin (e.g. example, streptavidin containing a fluorescent label or an enzymatic activity that can be detected by optical or colorimetric methods). Examples of tags for polypeptides include, without limitation, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm); chromogens; fluorescent markers (e.g. FITC, rhodamine and lanthanide phosphors); enzyme markers (e.g., horseradish peroxidase, luciferase, and alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, and epitope tags); and magnetic agents, for example, gadolinium chelates. Representative examples of labels commonly employed for immunoassays include light-producing moieties, e.g., acridinium compounds, and fluorescence-producing moieties, e.g., fluorescein. Other markers are described in this specification. In this regard, the portion itself may not be detectably labeled, but may become detectable upon reaction with yet another portion. The use of the term “detectably marked” is intended to encompass the latter type of detectable marking.

[00155] The term “surface plasmon resonance”, as used in this specification, refers to an optical phenomenon that allows the analysis of bispecific interactions in real time by detecting changes in protein concentrations within a biosensor matrix, e.g. example, using the BIAcore® system (BIAcore International AB, a GE Healthcare Company, Uppsala, Sweden and Piscataway, NJ). For additional descriptions, see Jonsson, U. et al. (1993) Ann. Biol. Clinic. 51: 19-26; Jonsson, U. et al. (1991) Biotechniques 11: 620-627; Johnsson, B. et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnson, B. et al. (1991) Anal. Biochem. 198: 268-277.

[00156] A “plasmid” or “vector” includes a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. An “expression plasmid” or “expression vector” may be a plasmid that has the ability to incorporate and express heterologous nucleic acid fragments in a cell. An expression plasmid may comprise additional elements, for example, the expression vector may have two replication systems, thereby allowing it to be maintained in two organisms. The nucleic acid incorporated into the plasmid may be operatively linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence.

[00157] A “nucleic acid” or “sequence of nucleic acids” can be any molecule, preferably a polymeric molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid or an analogue thereof. Nucleic acid can be single-stranded or double-stranded. A single-stranded nucleic acid can be a nucleic acid strand of denatured double-stranded DNA. Alternatively, it may be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid may be DNA. In another aspect, the nucleic acid may be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.

[00158] Except in operational examples, or when otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used in this specification should be understood as modified in all instances by the term “about”. The term “about”, when used in connection with percentages, can mean ± 1%.

[00159] Although various embodiments of the present disclosure have been described and illustrated in this specification, those skilled in the art will readily foresee various other means and/or structures for carrying out the functions and/or for obtaining the results and/or one or more of the advantages described in this specification, and each of these variations and/or modifications is considered included within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials and configurations described in this specification are intended to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend on the specific application or applications for which the teachings of the present revelation are used. Those skilled in the art will recognize, or be able to verify using at most routine experimentation, many equivalents to the specific embodiments of disclosure described in this specification. It must, therefore, be understood that the modalities described above are presented by way of example only and that, within the scope of the attached claims and their equivalents, the disclosure may be practiced in another way in relation to that specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material and/or method described in this specification. Furthermore, any combination of two or more such features, systems, articles, materials and/or methods, if such features, systems, articles, materials and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[00160] The indefinite articles “a” and “an”, as used herein and in the specification and claims, unless clearly indicated otherwise, should be understood as meaning “at least one”.

[00161] The phrase “and/or”, as used herein and in the specification and in the claims, should be understood as meaning “one or both” of the elements so related, that is, elements that are present jointly in some cases and separately present in other cases. Other elements may optionally be present, in addition to the elements specifically identified by the “and/or” clause, whether or not related to those specifically identified, unless clearly indicated otherwise. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “that understands”, may refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, B without A (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other elements); etc.

[00162] As used herein and in the specification and claims, the phrase “at least one”, in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present in addition to the specifically identified elements within the list of elements to which the phrase “at least one” refers, whether or not they are related to those specifically identified elements. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B” or, equivalently “at least one of A and/or B”) can refer , in one embodiment, at least one, optionally including more than one, A, without B present (and optionally including elements other than B); in another embodiment, at least one, optionally including more than one, B, without A present (and optionally including elements other than A); in yet another embodiment, at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[00163] The use of ordinal terms such as, for example, “first”, “second”, “third”, etc., in the claims to modify an element of the claim does not, in itself, mean any priority, precedence or order of an element of the claim in relation to another or the temporal order in which actions of a method are performed, but are used simply as markers to distinguish one element of the claim that has a certain name from another element that has the same name (but for use of the term ordinal) to distinguish the elements of the claim.

[00164] It is understood that ranges provided in this specification are a shorthand for all values within the range. For example, a range from 1 to 50 is understood to include any number, combination of numbers, or subrange of 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, for example, 10-20, 1-10, 30-40 etc.

Antibodies and antigen-binding portions thereof that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and/or a LRRC33-TGFβ1 complex

[00165] The present invention is based, at least in part, on the discovery of antibodies, and antigen-binding portions thereof, that bind to TGFβ1 which is present in a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 and/or a LRRC33-TGFβ1 complex. Accordingly, some aspects of the invention relate to antibodies or antigen-binding portions thereof that specifically bind to an epitope of TGFβ1, wherein the epitope is available for binding by the antibody, or antigen-binding portions thereof, where TGFβ1 is available. present in a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex. In some embodiments, the epitope is available as a result of a conformational change in TGFβ1 when in complex with GARP, LTBP1, LTBP3 and/or LRRC33. In some embodiments, the epitope on TGFβ1 to which antibodies, or antigen-binding portions thereof, bind is not available when TGFβ1 is not in complex with GARP, LTBP1, LTBP3 and/or LRRC33. In some embodiments, the antibodies, or antigen-binding portions thereof, do not specifically bind to TGFβ2. In some embodiments, the antibodies, or antigen-binding portions thereof, do not specifically bind to TGFβ3. In some embodiments, the antibodies, or antigen-binding portions thereof, do not prevent TGFβ1 from binding to the integrin. For example, in some embodiments, the antibodies, or antigen-binding portions thereof, do not mask the TGFβ1 integrin binding site. In some embodiments, the antibodies, or antigen-binding portions thereof, inhibit the activation of TGFβ1. In some embodiments, the antibodies, or antigen-binding portions thereof, inhibit the release of mature TGFβ1 from a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex.

[00166] Antibodies, or antigen-binding portions thereof, provided in this specification specifically bind to an epitope of TGFβ1, where the epitope is available for binding by the antibody, or antigen-binding portions thereof, when TGFβ1 is present in a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP2-TGFβ1 complex and/or a 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, an antibody, or antigen-binding portion thereof, described in this specification does not specifically bind to TGFβ2. In some embodiments, an antibody, or antigen-binding portion thereof, described in this specification does not specifically bind to TGFβ3. In some embodiments, an antibody, or antigen-binding portion thereof, described in this specification does not specifically bind to TGFβ2 or TGFβ3. In some embodiments, an antibody, or antigen-binding portion thereof, described in this specification specifically binds to a TGFβ1 that comprises the amino acid sequence shown in the ID. OF SEQ. No.: 21. The amino acid sequences of TGFβ2 and the amino acid sequence of TGFβ3 are described in the IDS. OF SEQ. Nos: 22 and 23, respectively. In some embodiments, an antibody, or antigen-binding portion thereof, described in this specification specifically binds to a TGFβ1 that comprises a non-naturally occurring amino acid sequence (also referred to in this specification as a non-naturally occurring TGFβ1). For example, a non-naturally occurring TGFβ1 may comprise one or more recombinantly generated mutations relative to a naturally occurring TGFβ1 amino acid sequence. In some embodiments, an amino acid sequence of TGFβ1, TGFβ2, or TGFβ3 comprises the amino acid sequence as set forth in the IDS. OF SEQ. Nos.: 24-35, as shown in Table 1. In some embodiments, an amino acid sequence of TGFβ1, TGFβ2, or TGFβ3 comprises the amino acid sequence as set forth in the IDS. OF SEQ. Nos: 36-43, as shown in Table 2. VRQ WLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPL ERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLG PCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMI VRSCKCS (SEQ ID NO.: 21) TGFβ2 SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLL LHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDG TSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQ DNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPE ASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS (SEQ ID NO: 22) TGFβ3 SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTREL LEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSS VEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTA (ID. OF SEQ. No.: 23) Table 1. Amino acid sequences of exemplary TGFβ1, TGFβ2 and TGFβ3.

[00167] In some embodiments, an antibody, or antigen-binding portion thereof, as described in this specification, is capable of binding to an LTBP1-TGFβ1 complex. In some embodiments, antigenic protein complexes (e.g., an LTBP-TGFβ1 complex) may comprise one or more LTBP proteins (e.g., LTBP1, LTBP2, LTBP3, and LTBP4). In some embodiments, the LTBP1 protein is a naturally occurring protein. In some embodiments, the LTBP1 protein is a non-naturally occurring protein. In some embodiments, the LTBP1 protein is a recombinant protein. Such recombinant LTBP1 protein may comprise LTBP1, alternatively splice variants thereof and/or fragments thereof. Recombinant LTBP1 proteins can also be modified to comprise one or more detectable markers. In some embodiments, the LTBP1 protein comprises a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP1 protein does not comprise a leader sequence (i.e., the leader sequence has been processed or cleaved). Such detectable tags may include, without limitation, biotin tags, 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 comprises an amino acid sequence as set forth in the IDS. OF SEQ. Nos: 46 and 47 in Table 2. In some embodiments, the LTBP1 protein comprises an amino acid sequence as shown in the ID. OF SEQ. No.: 50 in Table 3.

[00168] In some embodiments, an antibody, or antigen-binding portion thereof, as described in this specification, is capable of binding to an LTBP3-TGFβ1 complex. In some embodiments, the LTBP3 protein is a naturally occurring protein. In some embodiments, the LTBP3 protein is a non-naturally occurring protein. In some embodiments, the LTBP3 protein is a recombinant protein. Such recombinant LTBP3 protein may comprise LTBP3, alternatively splice variants thereof 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 has been processed or cleaved). Recombinant LTBP3 proteins can also be modified to comprise one or more detectable markers. Such detectable tags may include, without limitation, biotin tags, 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 comprises an amino acid sequence as set forth in the IDS. OF SEQ. Nos: 44 and 45 in Table 2. In some embodiments, the LTBP1 protein comprises an amino acid sequence as shown in the ID. OF SEQ. No.: 51 in Table 3.

[00169] In some embodiments, an antibody, or antigen-binding portion thereof, as described in this specification, is capable of binding to a GARP-TGFβ1 complex. In some embodiments, the GARP protein is a naturally occurring protein. In some embodiments, the GARP protein is a non-naturally occurring protein. In some embodiments, the GARP protein is a recombinant protein. This GARP protein can be recombinant, referred to in this specification as recombinant GARP. Some recombinant GARPs may comprise one or more modifications, truncations and/or mutations, compared to wild-type GARP. Recombinant GARPs can be modified to be soluble. In some embodiments, the GARP protein comprises a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the GARP protein does not comprise a leader sequence (i.e., the leader sequence has been processed or cleaved). In other embodiments, recombinant GARPs are modified to comprise one or more detectable markers. In additional embodiments, such detectable tags may include, without limitation, biotin tags, 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 a human, monkey, mouse, or rat GARP protein. In some embodiments, the GARP protein comprises an amino acid sequence as set forth in the IDS. OF SEQ. Nos: 48-49 in Table 2. In some embodiments, the GARP protein comprises an amino acid sequence as set forth in the IDS. OF SEQ. Nos: 52 and 53 in Table 4. In some embodiments, the antibodies, or antigen-binding portions thereof, described in this specification do not bind to TGFβ1 in a context-dependent manner, for example, binding to TGFβ1 would only occur when the TGFβ1 molecule was complexed with a specific presentation molecule, for example, GARP. Instead, antibodies, and antigen-binding portions thereof, bind to TGFβ1 in a context-independent manner. In other words, antibodies, or antigen-binding portions thereof, bind to TGFβ1 when linked to any presentation molecule: GARP, LTBP1, LTBP3 and/or LRCC33.

[00170] In some embodiments, an antibody, or antigen-binding portion thereof, as described in this specification, is capable of binding to a LRRC33-TGFβ1 complex. In some embodiments, the LRRC33 protein is a naturally occurring protein. In some embodiments, the LRRC33 protein is a non-naturally occurring protein. In some embodiments, the LRRC33 protein is a recombinant protein. This LRRC33 may be recombinant, referred to in this specification as recombinant LRRC33. Some recombinant LRRC33 proteins may comprise one or more modifications, truncations and/or mutations, compared to wild-type LRRC33. Recombinant LRRC33 proteins can be modified to be soluble. For example, in some embodiments, the LRRC33 ectodomain can be expressed with a C-terminal His-tag in order to express soluble LRRC33 protein (sLRRC33; see, for example, SEQ ID NO: 84). In some embodiments, the LRRC33 protein comprises a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LRRC33 protein does not comprise a leader sequence (i.e., the leader sequence has been processed or cleaved). In other embodiments, recombinant LRRC33 proteins are modified to comprise one or more detectable markers. In additional embodiments, such detectable tags may include, without limitation, biotin tags, 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 comprises an amino acid sequence as set forth in the IDS. OF SEQ. Nos: 83, 84 and 85 in Table 4. Table 3. Exemplary LTBP amino acid sequences.

[00171] In some embodiments, the antibodies of the present invention, or antigen-binding portions thereof, which specifically bind to an epitope of TGFβ1 that is available for binding by the antibody when TGFβ1 is present in a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex and the nucleic acid molecules of the present disclosure that encode antibodies include one or more of the CDR amino acid sequences shown in Table 5. Table 5. Regions Heavy chain complementarity determinants (CDRHs) and light chain complementarity determinants (CDRLs), as determined using the Kabat numbering scheme, are shown for antibodies Ab1 and Ab2.

[00172] In some embodiments, antibodies of the present invention that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex include any antibody, or binding moiety to the antigen thereof, which comprises a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided for any of the antibodies shown in Table 5. In some embodiments, antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex include the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of any of the antibodies shown in Table 5. The present invention also provides any sequence of nucleic acids encoding a molecule comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 or CDRL3, as provided for any of the antibodies shown in Table 5. Antibody heavy and light chain CDR3 domains may play a particularly important role in binding specificity/affinity of an antibody to an antigen. Accordingly, antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex of the disclosure, or the nucleic acid molecules encoding such antibodies, or portions of antigen binding of these, may include at least the CDR3s of the heavy and/or light chain of the antibodies as shown in Table 5.

[00173] Aspects of the invention relate to a monoclonal antibody, or antigen-binding portion thereof, that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-complex. TGFβ1, and which comprises six complementarity-determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3.

[00174] In some embodiments, CDRH1 comprises a sequence as shown in any of the IDS. OF SEQ. Nos: 1 and 2. In some embodiments, CDRH2 comprises a sequence as set forth in any of the IDS. OF SEQ. Nos: 3 and 4. In some embodiments, CDRH3 comprises a sequence as set forth in any of the IDS. OF SEQ. Nos: 5 and 6. CDRL1 comprises a sequence as shown in any of the IDS. OF SEQ. Nos: 7 and 8. In some embodiments, CDRL2 comprises a sequence as set forth in any of the IDS. OF SEQ. Nos: 9 and 10. In some embodiments, CDRL3 comprises a sequence as set forth in any of the IDS. OF SEQ. Nos: 11 and 12.

[00175] In some embodiments (e.g., as for the Ab1 antibody, shown in Table 5), the antibody or antigen-binding portion thereof, which specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex comprises: a CDRH1 comprising an amino acid sequence as shown in the ID. OF SEQ. No.: 1, a CDRH2 comprising an amino acid sequence as shown in ID. OF SEQ. No.: 3, a CDRH3 comprising an amino acid sequence as shown in ID. OF SEQ. No.: 5, a CDRL1 comprising an amino acid sequence as shown in ID. OF SEQ. No.: 7, a CDRL2 comprising an amino acid sequence as shown in ID. OF SEQ. No.: 9, and a CDRL3 comprising an amino acid sequence as shown in the ID. OF SEQ. No.: 11.

[00176] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region that comprises a complementarity determining region 3 (CDR3) that has the amino acid sequence of the ID. OF SEQ. No.: 5 and a light chain variable region comprising a CDR3 having the amino acid sequence of the ID. OF SEQ. No.: 11. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 2 (CDR2) having the ID amino acid sequence. OF SEQ. No.: 3 and a light chain variable region comprising a CDR2 having the amino acid sequence of the ID. OF SEQ. No.: 9. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 1 (CDR1) having the amino acid sequence of the ID. OF SEQ. No.: 1 and a light chain variable region comprising a CDR1 having the amino acid sequence of the ID. OF SEQ. No.: 7.

[00177] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity for the amino acid sequence shown in the ID. OF SEQ. No.: 13 and a light chain variable domain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of identity to the amino acid sequence presented in the ID. OF SEQ. No.: 14. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence shown in the ID. OF SEQ. No.: 13 and a light chain variable domain comprising an amino acid sequence shown in the ID. OF SEQ. No.: 14.

[00178] In some embodiments, the antibody or antigen-binding portion thereof, which specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex comprises a sequence of amino acids of the variable domain of the heavy chain encoded by a nucleic acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity for the nucleic acid sequence shown in the ID. OF SEQ. No.: 91, and an amino acid sequence of the light chain variable domain encoded by a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98% or 99% identity to the nucleic acid sequence presented in the ID. OF SEQ. No.: 92. In some embodiments, the antibody or antigen-binding portion thereof comprises an amino acid sequence of the heavy chain variable domain encoded by the nucleic acid sequence shown in the ID. OF SEQ. No.: 91, and an amino acid sequence of the light chain variable domain encoded by the nucleic acid sequence shown in the ID. OF SEQ. No.: 92.

[00179] In some embodiments (e.g., as for the Ab2 antibody, shown in Table 5), the antibody or antigen-binding portion thereof, which specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex comprises a CDRH1 comprising an amino acid sequence as shown in the ID. OF SEQ. No.: 2, a CDRH2 comprising an amino acid sequence as shown in ID. OF SEQ. No.: 3, a CDRH3 comprising an amino acid sequence as shown in ID. OF SEQ. No.: 6, a CDRL1 comprising an amino acid sequence as shown in ID. OF SEQ. No.: 8, a CDRL2 comprising an amino acid sequence as shown in ID. OF SEQ. No.: 10, and a CDRL3 comprising an amino acid sequence as shown in the ID. OF SEQ. No.: 12.

[00180] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region that comprises a CDR3 that has the amino acid sequence of the ID. OF SEQ. No.: 6 and a light chain variable region comprising a CDR3 having the amino acid sequence of the ID. OF SEQ. No.: 12. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a CDR2 having the ID amino acid sequence. OF SEQ. No.: 4 and a light chain variable region comprising a CDR2 having the amino acid sequence of the ID. OF SEQ. No.: 10. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a CDR1 having the ID amino acid sequence. OF SEQ. No.: 2 and a light chain variable region comprising a CDR1 having the amino acid sequence of the ID. OF SEQ. No.: 8.

[00181] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity for the amino acid sequence shown in the ID. OF SEQ. No.: 15 and a light chain variable domain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of identity to the amino acid sequence presented in the ID. OF SEQ. No.: 16. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence shown in the ID. OF SEQ. No.: 15 and a light chain variable domain comprising an amino acid sequence shown in the ID. OF SEQ. No.: 16.

[00182] In some embodiments, the antibody or antigen-binding portion thereof, which specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex comprises a sequence of amino acids of the variable domain of the heavy chain encoded by a nucleic acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity for the nucleic acid sequence shown in the ID. OF SEQ. No.: 93, and an amino acid sequence of the light chain variable domain encoded by a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98% or 99% identity to the nucleic acid sequence presented in the ID. OF SEQ. No.: 94. In some embodiments, the antibody or antigen-binding portion thereof comprises an amino acid sequence of the heavy chain variable domain encoded by the nucleic acid sequence shown in the ID. OF SEQ. No.: 93, and an amino acid sequence of the light chain variable domain encoded by the nucleic acid sequence shown in the ID. OF SEQ. No.: 94.

[00183] In some examples, any of the antibodies of the disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex include any antibody (including portions of antigen binding thereof) that has one or more CDR sequences (e.g., CDRH or CDRL) substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3. For example, the antibodies may include one or more CDR sequences as shown in Table 5 (SEQ ID. Nos: 1-12) containing up to 5, 4, 3, 2 or 1 amino acid residue variations, compared to the corresponding CDR region in any of the IDS. OF SEQ. Nos: 1 12. The complete amino acid sequences for the heavy chain variable region and light chain variable region of the antibodies listed in Table 5 (e.g., Ab1 and Ab2), as well as the nucleic acid sequences encoding the variable region of the heavy chain and light chain variable region of antibodies are given below. Ab1 - Amino acid sequence of the heavy chain variable region TGCAACTCGTGGAGTCAGGCGGTGGACTTGTTCAGCCTGGGCGAAGTCTGAGACT CTCATGTGCAGCAAGTGGATTCACTTTCTCCAGTTACGGCATGCACTGGGTGAGACAGG CGCCTGGAAAGGGTTTGGAATGGGTCGCTGTGATCTCTTACGACGGGTCAAACAAATAT TACGCGGATTCAGTGAAAGGGCGGTTCACTATTTCACGGGATAACTCCAAGAACACCCT GTATCTGCAGATGAATAGCCTGAGGG CAGAGGACACCGCTGTGTACTATTGTGCCCGGG ACATAAGGCCTTACGGCGATTACAGCGCCGCATTTGATATTTGGGGACAAGGCACCCTT GTGACAGTATCTTCT (SEQ ID. No.: 91) Ab1 - Amino acid sequence of the light chain variable region TTATGCTTACCCAACCACATAGTGTGAGTGAGTCTCCCGGCAAGACTGTAACAAT TTCATGTACCGGCAGCAGTGGCTCCATCGCTAGCAATTATGTGCAATGGTACCAACAGC GCCCCGGGAGCGCACCTTCAATAGTGATATTCGAGGATAACCAACGGCCTAGTGGGGCT CCCGATAGATTTAGTGGGAGTATAGATAGCTCCTCCAACTCTGCCTCTCTCACCATTAG CGGGCTGAAAACAGAGGATGA AGCCGACTATTACTGCCAAAGCTATGATTCTAGCAACC ACGGCGGAGTGTTTGGCGGAGGAACACAGCTGACAGTCCTAGG (ID. OF SEQ. No.: 92) Ab1 - Heavy chain amino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDIRPYGDYSAAFDIWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVL QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 15) Ab1 - Light chain amino acid sequence NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSAPSIVIFEDNQRPSGA PDRFSGSIDSSSNSASLTISGL KTEDEADYYCQSYDSSNHGGVFGGGTQLTVLGQPKAA PSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNK YAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID. No.: 16) Ab2 - Amino acid sequence of the variable region of the heavy chain EVQLVQSGAEMKKPGESLKISCKGSGYNFASDWIGWVRQTPGKGLEWMGVIYPGDSDTR YSASFQGQVTISADKSINTAYLQWSSLKASDTAMYYCASAAGIAAAGHVTAFDIWGQGT MVTVSS (ID. OF SEQ. No.: 17) Ab2 - Nucleic acid sequence of the variable region of the heavy chain GAGGTGCAACTGGTGCAATCCGGAGCCGAGATGAAAAAGCCAGGGGAGAGCCTGAAGAT CTCTTGTAAGGGCTCTGGCTATAACTTCGCTAGTGATTGGATCGGATGGGTGAGGCAAA CCCCCGGAAAGGGCCTCGAGTGGATGGGCGTGATCTACCCCGGCGACTCCGACACACGC TATAGCGCCTCATTCCAGGGCCA GGTCACCATAAGTGCTGATAAATCAATAAATACAGC CTACTTGCAATGGTCAAGTCTGAAAGCCTCAGATACTGCCATGTACTATTGTGCCTCTG CCGCCGGCATTGCCGCGGCCGGTCACGTCACCGCCTTCGACATTTGGGGTCAGGGCACT ATGGTCACTGTAAGCTCC (SEQ ID NO: 93) Ab2 - Amino acid sequence of the light chain variable region DIVMTQSPDSLAVSLGERATINCK SSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPVTFGQGTKLEIK (ID . GGAGTGCCAGACCGGTTTTCTGGTTCTGGATCTGGTACTGACTTCACCCT GACAATCAGCTCCCTGCAGGCCGAAGACGTGGCTGTGTACTATTGTCAGCAGTACTATA GTACACCAGTTACTTTCGGCCAAGGCACTAAACTCGAAATCAAG (ID. OF SEQ. No.: 94) Ab2 - Heavy chain amino acid sequence EVQLVQSGAEMKKPGESLKISCKGSGYNFASDWIGWVRQTPGKGLEWMGVIYPGDSDTR YSASFQGQVTISADKSINTAYLQWSSLKASDTAMYYCASAAGIAAAGHVTAFDIWGQGT MVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTF PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 19) Ab2 - Light chain amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVP DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPVTFGQGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID. No.: 20)

[00184] In some embodiments, antibodies of the disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and a LRRC33-TGFβ1 complex include any antibody that includes a variable domain of the heavy chain of ID. OF SEQ. No.: 13 or 17, or a light chain variable domain of the ID. OF SEQ. No.: 14 or 18. In some embodiments, antibodies of the disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and a LRRC33-TGFβ1 complex include any antibody that includes chain pairs variable heavy chain and variable light chain of IDS. OF SEQ. Nos: 13 and 14; and 17 and 18.

[00185] Aspects of the disclosure provide antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and a LRRC33-TGFβ1 complex that have a variable heavy chain amino acid sequence and/or variable light chain homologous to any of those described in this specification. In some embodiments, the antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and a LRRC33-TGFβ1 complex comprises a heavy chain variable sequence or a light chain variable sequence that is at least 75% (e.g. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the ID heavy chain variable amino acid sequence. OF SEQ. No.: 13 or 17, or a variable sequence of the ID light chain. OF SEQ. No.: 14 or 18. In some embodiments, the homologous variable heavy chain and/or variable light chain amino acid sequences do not vary within any of the CDR sequences provided in this specification. 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 occur within a variable heavy chain and/or variable light chain amino acid sequence, excluding either of the CDR sequences provided in this specification.

[00186] In some embodiments, antibodies of the disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex include any antibody, or antigen-binding moiety of this, which includes a heavy chain of the ID. OF SEQ. No.: 15 or 19, or a light chain ID. OF SEQ. No.: 16 or 20. In some embodiments, antibodies of the disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex include any antibody that includes the heavy chain and light chain pairs of the IDS. OF SEQ. Nos: 15 and 16; or 19 and 20.

[00187] Aspects of the disclosure provide antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex that have a heavy chain amino acid sequence and/or a light chain amino acid sequence homologous to any of those described in this specification. In some embodiments, the antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and/or a LRRC33-TGFβ1 complex comprises 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 ID heavy chain sequence. OF SEQ. No.: 15, or 19, or an amino acid sequence from the light chain of the ID. OF SEQ. No.: 16, or 20. In some embodiments, the heavy chain and/or light chain homologous amino acid sequences do not vary within any of the CDR sequences provided in this specification. 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 occur within a heavy chain amino acid sequence and/or a light chain amino acid sequence, excluding any of the CDR sequences provided in this specification.

[00188] In some embodiments, antibodies of the disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex include any antibody, or antigen-binding moiety of this, which includes a heavy chain of the ID. OF SEQ. No.: 15 or 19, or a light chain ID. OF SEQ. No.: 16 or 20. In some embodiments, antibodies of the disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex include any antibody that includes the heavy chain and light chain pairs of the IDS. OF SEQ. Nos: 15 and 16; or 19 and 20.

[00189] Aspects of the disclosure provide antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex that have a heavy chain amino acid sequence and/or a light chain amino acid sequence homologous to any of those described in this specification. In some embodiments, the antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and/or a LRRC33-TGFβ1 complex comprises 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 ID heavy chain sequence. OF SEQ. No.: 15 or 19, or a light chain amino acid sequence of the ID. OF SEQ. No.: 16 or 20. In some embodiments, the heavy chain and/or light chain homologous amino acid sequences do not vary within any of the CDR sequences provided in this specification. 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 occur within a heavy chain amino acid sequence and/or a light chain amino acid sequence, excluding any of the CDR sequences provided in this specification.

[00190] In some embodiments, the “percent identity” of two amino acid sequences is determined using the Karlin and Altschul Proc algorithm. Natl. Academic. Sci. U.S.A. 87:226468, 1990, modified as in Karlin and Altschul Proc. Natl. Academic. Sci. U.S.A. 90: 5873-77, 1993. An algorithm of this type is incorporated into the NBLAST and XBLAST (version 2.0) programs by Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the program XBLAST, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of interest. When gaps exist between two sequences, the Gapped BLAST program can be used as described in Altschul et al., Nucleic Acids Res. 25 (17): 3,389-3,402, 1997. When the BLAST and Gapped BLAST programs are used, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[00191] In any of the antibodies or antigen-binding fragments described in this specification, one or more conservative mutations may be introduced into the CDR or framework sequences at positions where the residues are not likely to be involved in an antibody-antigen interaction. . In some embodiments, these conservative mutations can be introduced into CDR or framework sequences at positions where the residues are not likely to be involved in interaction with a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and a LRRC33-TGFβ1, as determined based on crystal structure. In some embodiments, the likely interface (e.g., residues involved in an antigen-antibody interaction) can be deduced from known structural information in another antigen that shares structural similarities.

[00192] As used in this specification, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering the polypeptide sequence known to those skilled in the art, such as are found in references that compile these methods, for example, “Molecular Cloning: A Laboratory Manual”, J. Sambrook, et al. , eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or “Current Protocols in Molecular Biology”, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made between amino acids within 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.

[00193] In some embodiments, the antibodies provided in this specification comprise mutations that confer desirable properties to the antibodies. For example, to avoid potential complications resulting from Fab-arm switching, which are known to occur with native IgG4 mAbs, the antibodies provided in this specification may comprise an 'Adair' stabilizing mutation (Angal et al., “A Single Amino Acid Substitution Abolishes the Heterogeneity of Chimeric Mouse/Human (IgG4) Antibody”, Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 in Kabat numbering) is converted to proline, resulting in a sequence of IgG1-like hinge (CPPCP (SEQ ID NO: 54)). Consequently, any of the antibodies may include a stabilizing 'Adair' mutation or the amino acid sequence CPPCP (SEQ ID NO: 54).

[00194] Antibodies of this disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex may optionally comprise antibody constant regions or parts thereof. For example, a VL domain may be attached at its C-terminal end to a light chain constant domain such as CK or CÀ. Similarly, a VH domain or portion thereof may be attached to all or part of a heavy chain such as IgA, IgD, IgE, IgG and IgM, and any isotype subclass. Antibodies may include suitable constant regions (see, for example, Kabat et al., “Sequences of Proteins of Immunological Interest”, No. 91 3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Therefore, antibodies within the scope of this disclosure may include VH and VL domains, or an antigen-binding portion thereof, combined with any suitable constant regions.

[00195] In some embodiments, antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex may or may not include the framework region of the IDS antibodies. OF SEQ. Nos: 13-20. In some embodiments, antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex are murine antibodies and include sequences from the murine framework region.

[00196] In some embodiments, antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex of the disclosure can bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex with relatively high affinity, for example, with a KD of less than 10-6 M, 10-7 M, 10-8 M, 10- 9M, 10-10M, 10-11M or smaller. For example, antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and/or a LRRC33-TGFβ1 complex can bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex with an affinity between 5 pM and 500 nM, for example between 50 pM and 100 nM, for example between 500 pM and 50 nM. The disclosure also includes antibodies or antigen-binding fragments that compete with any of the antibodies described in this specification for binding to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex. and which have an affinity of 50 nM or less (e.g., 20 nM or less, 10 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding kinetics of antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex may be tested using any suitable method including, without limitation , biosensor technology (e.g. OCTET or BIACORE).

Antibodies that inhibit TGFβ

[00197] The present invention, in one aspect, provides functional antibodies. As used in this specification, the term “a functional antibody” confers one or more biological activities by virtue of its ability to bind to an antigen. Functional antibodies may include inhibitory antibodies (or inhibitory antibodies) and activating antibodies. Thus, the present disclosure includes antibodies against TGFβ that can modulate (e.g., inhibit or activate) a biological process mediated by TGFβ signaling.

[00198] As used in this specification, the term “inhibiting antibody” refers to an antibody that inhibits the release of mature growth factor or reduces the activity of the growth factor. Inhibitory antibodies include antibodies that target any epitope that reduces growth factor release or activity when associated with these antibodies. These epitopes may lie on prodomains of TGFβ proteins (e.g., TGFβ1), growth factors, or other epitopes that lead to reduced growth factor activity when bound by antibody. Inhibitory antibodies of the present invention include, without limitation, TGFβ1 inhibitory antibodies.

[00199] Embodiments of the present disclosure include methods of using inhibitory antibodies in solution, in cell culture and/or in individuals to modify growth factor signaling.

Polypeptides

[00200] Some aspects of the disclosure relate to a polypeptide that has a sequence selected from the group consisting of the ID. OF SEQ. No.: 13, ID. OF SEQ. No.: 17, ID. OF SEQ. No.: 15 and ID. OF SEQ. No.: 19. In some embodiments, the polypeptide is a heavy chain variable 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 of the amino acid sequences shown in the ID. OF SEQ. No.: 13, ID. OF SEQ. No.: 17, ID. OF SEQ. No.: 15 and ID. OF SEQ. No.: 19.

[00201] Some aspects of the disclosure relate to a polypeptide that has a sequence selected from the group consisting of the ID. OF SEQ. No.: 14, ID. OF SEQ. No.: 18, ID. OF SEQ. No.: 16 and ID. OF SEQ. No.: 20. In some embodiments, the polypeptide is a light chain variable 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 of the amino acid sequences shown in the ID. OF SEQ. No.: 14, ID. OF SEQ. No.: 18, ID. OF SEQ. No.: 16 and ID. OF SEQ. No.: 20.

Antibodies that compete with antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex

[00202] Aspects of the disclosure relate to antibodies that compete or cross-compete with any of the antibodies provided in this specification. The term “compete,” as used in this specification with respect to an antibody, means that a first antibody binds to an epitope (e.g., an epitope of a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3- TGFβ1 and/or a LRRC33-TGFβ1 complex) in a manner sufficiently similar to binding of a second antibody, such that the result of binding of the first antibody to its epitope is detectably diminished in the presence of the second antibody, compared to binding of the first antibody in the absence of the second antibody. The alternative, when the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, may, but need not, be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, when each antibody detectably inhibits the binding of the other antibody to its epitope or ligand, whether to an equal, greater or lesser degree, the antibodies are said to “cross-compete” with each other for the binding of their respective( s) epitope(s). Competitive and cross-competitive antibodies are within the scope of this disclosure. Regardless of the mechanism by which the competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), those skilled in the art would observe that these competing and cross-competing antibodies are encompassed and can be useful for the methods and/or compositions provided in this specification.

[00203] Aspects of the disclosure relate to antibodies that compete or cross-compete with any of the specific antibodies, or antigen-binding portions thereof, as provided in this specification. In some embodiments, an antibody, or antigen-binding portion thereof, binds to the same epitope or near the same epitope as any of the antibodies provided in this specification. In some embodiments, an antibody, or antigen-binding portion thereof, binds near an epitope if it binds within 15 or fewer amino acid residues of the epitope. In some embodiments, any of the antibodies, or antigen-binding portion thereof, as provided in this specification, binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14 or 15 amino acid residues of an epitope that is bound by any of the antibodies provided in this specification.

[00204] In another embodiment, there is provided in this specification an antibody, or antigen-binding portion thereof, that competes or cross-competes for binding to any of the antigens provided in this specification (for example, a GARP- TGFβ1, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and/or a LRRC33-TGFβ1 complex) with an equilibrium dissociation constant, KD, between the antibody and protein of less than 10-6 M. In other embodiments , an antibody competes or cross-competes for binding to any of the antigens provided in this specification with a KD in a range of 10-11 M to 10-6 M. In some embodiments, an anti-antibody antibody is provided in this specification. TGFβ1, or antigen-binding portion thereof, which competes for binding with an antibody, or antigen-binding portion thereof, described in this specification. In some embodiments, there is provided in this specification an anti-TGFβ1 antibody, or antigen-binding portion thereof, that binds to the same epitope as an antibody, or antigen-binding portion thereof, described in this specification.

[00205] Any of the antibodies provided in this specification can be characterized using any suitable methods. For example, one method is to identify the epitope to which the antigen binds, or “epitope mapping”. There are many methods suitable for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described by For example, in Chapter 11 of Harlow and Lane, “Using Antibodies, a Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In a further example, epitope mapping can be used to determine the sequence which an antibody binds. The epitope can be a linear epitope, that is, contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch (linear sequence of the primary structure). In some embodiments, the epitope is an epitope of TGFβ1 that is only available for binding by the antibody, or antigen-binding portion thereof, described in this specification, when TGFβ1 is in a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex. Peptides of variable lengths (e.g., at least 4-6 amino acids in length) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with an antibody. In another example, the epitope to which the antibody binds can be determined in a systematic evaluation by using overlapping peptides derived from the sequence of the target antigen and determining binding by the antibody. According to gene fragment expression assays, the open reading frame encoding the target antigen is fragmented randomly or by specific genetic constructs and the reactivity of the expressed antigen fragments with the antibody to be tested is determined. Gene fragments can be produced, for example, by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. Antibody binding to radioactive antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In a further example, mutagenesis of an antigen-binding domain, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or required for epitope binding. For example, domain swapping experiments can be performed using a mutant of a target antigen in which several fragments of the GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex have been replaced. (swapped) with sequences from a closely related but antigenically distinct protein, e.g., another member of the TGFβ protein family (e.g., GDF11). By assessing antibody binding to the mutant of a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex, the importance of the particular antigen fragment for antibody binding can be assessed. .

[00206] Alternatively, competition assays can be performed using other antibodies that are known to bind to the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition trials are well known to those skilled in the art.

[00207] Furthermore, the interaction of any of the antibodies provided in this specification with one or more residues in a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex may be determined by routine technology. For example, a crystal structure can be determined, and the distances between residues in a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex and one or more residues in the antibody can be determined. be determined accordingly. Based on this distance, it can be determined whether a specific residue in a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex interacts with one or more residues in the antibody. Furthermore, suitable methods, for example, competition assays and target mutagenesis assays, can be applied to determine the preferential binding of a candidate antibody.

Production of antibodies that bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex

[00208] Various methods can be used to obtain disclosure antibodies, or antigen-binding fragments thereof. For example, antibodies can be produced using recombinant DNA methods. Monoclonal antibodies can also be produced by generating hybridomas (see, for example, Kohler and Milstein (1975) Nature, 256: 495-499) according to known methods. Hybridomas formed in this way are then evaluated using standard methods, e.g., enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance analysis (e.g., OCTET or BIACORE), to identify one or more hybridomas that produce an antibody. that specifically binds to a specified antigen. Any form of the specified antigen may be used as the immunogen, e.g., recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof (e.g., any of the epitopes described in this specification as a linear epitope or within a scaffold as a conformational epitope). An exemplary method of producing antibodies includes evaluating protein expression libraries that express antibodies or fragments thereof (e.g., scFv), e.g., phage or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228: 1315-1317; Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809.

[00209] In addition to the use of display libraries, the specified antigen (e.g., a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex) can be used to immunize a non-human animal, e.g. a rodent, e.g. a mouse, hamster or rat. In one embodiment, the non-human animal is a mouse.

[00210] In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, for example, chimeric, using suitable recombinant DNA techniques. Several approaches for producing chimeric antibodies have been described. See, for example, Morrison et al., Proc. Natl. Academic. Sci. U.S.A. 81: 6851, 1985; Takeda et al., Nature 314: 452, 1985, Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494, UK Patent GB 2177096B.

[00211] For additional antibody production techniques, see “Antibodies: A Laboratory Manual”, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. The present disclosure is not necessarily limited to any particular source, production method or other special characteristics of an antibody.

[00212] Some aspects of the present disclosure relate to host cells transformed with a polynucleotide or vector. Host cells can be a prokaryotic or eukaryotic cell. The polynucleotide or vector that is present in the host cell can be integrated into the host cell genome or can be maintained extrachromosomally. The host cell can be any prokaryotic or eukaryotic cell, for example, a bacterial, insect, fungal, plant, animal or human cell. In some embodiments, fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. The term “prokaryotic” includes all bacteria that can be transformed or transfected with a DNA or RNA molecule for the expression of an antibody or corresponding immunoglobulin chains. Prokaryotic hosts can include gram-negative as well as gram-positive bacteria, e.g., E. coli, S. typhimurium, Serratia marcescens, and Bacillus subtilis. The term “eukaryotic” includes cells from yeasts, higher plants, insects and vertebrates, for example, from mammalian cells, for example, NSO and CHO cells. Depending on the host employed in a recombinant production procedure, the antibodies or immunoglobulin chains encoded by the polynucleotide may be glycosylated or may be non-glycosylated. Antibodies or corresponding immunoglobulin chains may also include an initial methionine amino acid residue.

[00213] In some embodiments, after a vector has been incorporated into an appropriate host, the host can be maintained under suitable conditions for high-level expression of the nucleotide sequences and, as desired, the collection and purification of light chains, heavy chains , immunoglobulin light/heavy chain dimers or intact antibodies, antigen-binding fragments, or other forms of immunoglobulin may occur; see, Beychok, “Cells of Immunoglobulin Synthesis”, Academic Press, N.Y., (1979). In this way, polynucleotides or vectors are introduced into cells which, in turn, produce the antibody or antigen-binding fragments. Furthermore, transgenic animals, preferably mammals, comprising the aforementioned host cells can be used for large-scale production of the antibody or antibody fragments.

[00214] Transformed host cells can be grown in fermentors and cultivated using any suitable techniques to obtain optimal cell growth. Once expressed, entire antibodies, their dimers, individual light and heavy chains, other forms of immunoglobulin, or antigen-binding fragments, can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns. , column chromatography, gel electrophoresis and the like; see, Scopes, “Protein Purification”, Springer Verlag, N.Y. (1982). The antibody or antigen-binding fragments can then be isolated from growth medium, cell lysates, or cell membrane fractions. Isolation and purification, for example, of microbially expressed antibodies, or antigen-binding fragments, may be by any conventional means, such as preparative chromatographic separations and immunological separations, such as those involving the use of monoclonal antibodies. or polyclonals directed, for example, against the constant region of the antibody.

[00215] Aspects of the disclosure are related to a hybridoma, which provides an indefinitely prolonged source of monoclonal antibodies. As an alternative to obtaining immunoglobulins directly from hybridoma culture, immortalized hybridoma cells can be used as a source of rearranged heavy chain and light chain loci for expression and/or subsequent genetic manipulation. Rearranged antibody genes can be reverse transcribed from appropriate mRNAs to produce cDNA. In some embodiments, the constant region of the heavy chain may be exchanged for that of a different isotype or eliminated entirely. The variable regions can be linked to encode single-chain Fv regions. Multiple Fv regions can be ligated to confer binding ability to more than one target or chimeric heavy and light chain combinations can be employed. Any appropriate method can be used for cloning antibody variable regions and generating recombinant antibodies.

[00216] In some embodiments, an appropriate nucleic acid encoding variable regions of a heavy and/or light chain is obtained and inserted into an expression vector that can be transfected into standardized recombinant host cells. Several of these host cells can 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 the antibody or antigen-binding fragment can be carried out by culturing a modified recombinant host under culture conditions suitable for the growth of the host cells and the expression of the coding sequences. Antibodies or antigen-binding fragments can be recovered by isolating them from the culture. Expression systems can be designed to include signal peptides so that the resulting antibodies are secreted into the medium; however, intracellular production is also possible.

[00217] The disclosure also includes a polynucleotide that encodes at least one variable region of an immunoglobulin chain of the antibodies described in this specification. In some embodiments, the variable region encoded by the polynucleotide comprises at least one complementarity determining region (CDR) of the VH and/or VL of the variable region of the antibody produced by any of the hybridomas described above.

[00218] Polynucleotides encoding antibody or antigen-binding fragments may be, for example, DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides alone or in combination. In some embodiments, a polynucleotide is part of a vector. These vectors may comprise additional genes, such as marker genes, which allow selection of the vector in a suitable host cell and under suitable conditions.

[00219] In some embodiments, a polynucleotide is operatively linked to expression control sequences that allow expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide comprises the transcription of the polynucleotide into a translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may include regulatory sequences that facilitate transcription initiation and, optionally, poly-A signals that facilitate transcription termination and transcript stabilization. Additional regulatory elements may include transcription enhancers, as well as translation enhancers, and/or naturally associated or heterologous promoter regions. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, the PL, Lac, Trp, or Tac promoter in E. coli, and examples of regulatory elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV promoter, SV40 promoter, RSV (Rous sarcoma virus) promoter, CMV enhancer, SV40 enhancer or a globin intron in mammalian cells and other animal cells.

[00220] In addition to elements that are responsible for initiating transcription, these regulatory elements may also include transcription termination signals, for example, the SV40-poly-A site or the tk-poly-A site, below the polynucleotide. Furthermore, depending on the expression system used, leader sequences capable of directing the polypeptide to a cellular compartment or its secretion into the medium can be added to the polynucleotide coding sequence and have been previously described. The leader sequence (or sequences) is assembled in an appropriate phase with translation initiation and termination sequences and, preferably, a leader sequence capable of directing the secretion of the translated protein, or a portion thereof, for example, into the extracellular environment. Optionally, a heterologous polynucleotide sequence encoding a fusion protein may be used that includes a C- or -N-terminal identification peptide that imparts desired characteristics, e.g., stabilization or simplified purification of the expressed recombinant product.

[00221] In some embodiments, polynucleotides that encode at least the variable domain of the light and/or heavy chain may encode the variable domains of both immunoglobulin chains or just one. Likewise, polynucleotides may be under the control of the same promoter or may be separately controlled for expression. Furthermore, some aspects are related to vectors, particularly plasmids, cosmids, viruses and bacteriophages, conventionally used in genetic engineering that comprise a polynucleotide encoding a variable domain of an immunoglobulin chain of an antibody or antigen-binding fragment; optionally in combination with a polynucleotide encoding the variable domain of the other immunoglobulin chain of the antibody.

[00222] In some embodiments, expression control sequences are provided as eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts can also be used. Expression vectors derived from viruses, such as retroviruses, vaccinia virus, adeno-associated virus, herpes virus, or bovine papillomavirus, can be used to deliver the polynucleotides or vector into the targeted cell population (e.g., to genetically modify a cell to express an antibody or antigen-binding fragment). Several appropriate methods can be used to construct recombinant viral vectors. In some embodiments, polynucleotides and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing the polynucleotides (e.g., the heavy and/or light variable domain (or domains) of sequences encoding immunoglobulin chains and expression control sequences) can be transferred into the host cell by suitable methods, which vary depending on the type of cellular host.

Modifications

[00223] Disclosure antibodies, or antigen-binding portions thereof, may be modified with a detectable marker or detectable portion including, without limitation, an enzyme, prosthetic group, fluorescent material, luminescent material, bioluminescent material, radioactive material, emitting metal positron, non-radioactive paramagnetic metal ion and affinity tag for detection and isolation of a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex. The detectable substance or moiety may be coupled or conjugated directly to the polypeptides of the disclosure or indirectly, through an intermediate (such as, for example, a linker (e.g., a cleavable linker)) using suitable techniques. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or acetylcholinesterase; Non-limiting examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; Non-limiting examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; Non-limiting examples of bioluminescent materials include luciferase, luciferin and aquorin; and examples of suitable radioactive material include a radioactive metal ion, e.g. alpha emitters or other radioisotopes, e.g. iodine (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H) , indium (115mIn, 113mIn, 112In, 111In) and technetium (99Tc, 99mTc), thallium (201T1), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F) 153Sm, Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 86R, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, b, 51Cr, 54Mn, 75Se and tin ( 113Sn, 117Sn). The detectable substance can be coupled or conjugated directly to antibodies of the disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex, or indirectly, through an intermediary (such as a linker) using appropriate techniques. Any of the antibodies provided in this specification that are conjugated to a detectable substance can be used for any suitable diagnostic assays, such as those described in this specification.

[00224] Furthermore, disclosure antibodies, or antigen-binding portions thereof, can also be modified with a drug. The drug may be coupled or conjugated directly to the polypeptides of the disclosure, or indirectly, through an intermediate (such as, for example, a linker (e.g., a cleavable linker)) using suitable techniques.

Targeting agents

[00225] In some embodiments, the methods of the present disclosure comprise using one or more targeting agents to direct an antibody, or antigen-binding portion thereof, as disclosed in this specification, to a particular location on an individual for purposes of modulation of mature TGFβ release by a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex. For example, LTBP1-TGFβ1 and LTBP3-TGFβ1 complexes are typically localized to the extracellular matrix. Thus, in some embodiments, the antibodies disclosed in this specification can be conjugated to extracellular matrix targeting agents for the purpose of localizing the antibodies to the sites where the LTBP1-TGFβ1 and LTBP3-TGFβ1 complexes reside. In these embodiments, selective antibody targeting leads to selective modulation of LTBP1-TGFβ1 and/or LTBP3-TGFβ1 complexes. In some embodiments, selective antibody targeting leads to selective inhibition of LTBP1-TGFβ1 and/or LTBP3-TGFβ1 complexes (e.g., for purposes of treating fibrosis). In some embodiments, extracellular matrix targeting agents include heparin binding agents, matrix metalloproteinase binding agents, lysyl oxidase binding domains, fibrillin binding agents, hyaluronic acid binding agents, and others.

[00226] Similarly, GARP-TGFβ1 complexes are typically located on the surface of cells, for example, activated FOXP3+ regulatory T cells (Tregs). Thus, in some embodiments, the antibodies disclosed in this specification can be conjugated to immune cell binding agents (e.g., Treg cell) for the purpose of localizing the antibodies to the sites where the GARP-TGFβ1 complexes reside. In these embodiments, selective antibody targeting leads to selective modulation of GARP-TGFβ1 complexes. In some embodiments, selective antibody targeting leads to selective inhibition of GARP-TGFβ1 complexes (e.g., selective inhibition of mature TGFβ1 release for immune modulation purposes, e.g., in cancer treatment). In these embodiments, Treg cell targeting agents may include, for example, CCL22 and CXCL12 proteins or fragments thereof.

[00227] In some embodiments, bispecific antibodies may be used that have a first portion that selectively binds to a GARP-TGFβ1 complex and an LTBP-TGFβ1 complex and a second portion that selectively binds to a component of a target site , for example, a component of the ECM (e.g., fibrillin) or a component of a Treg cell (e.g., CTLA-4).

Pharmaceutical compositions

[00228] The invention further provides pharmaceutical compositions used as a medicine suitable for administration to human and non-human subjects. One or more antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex can be formulated or mixed with a pharmaceutically acceptable carrier (excipient), including, for example, a buffer, to form a pharmaceutical composition. These formulations can be used to treat a disease or disorder that involves TGFβ signaling. In some embodiments, this disease or disorder associated with TGFβ signaling involves one or more contexts, that is, TGFβ is associated with a particular type or types of presentation molecules. In some embodiments, this context occurs in a cell-specific and/or tissue-specific manner. In some embodiments, for example, this context-dependent action of TGFβ signaling is mediated, in part, through GARP, LRRC33, LTBP1 and/or LTBP3.

[00229] In some embodiments, the antibody of the present invention specifically binds to two or more TGFβ contexts, such that the antibody binds to TGFβ in a complex with presentation molecules selected from two or more of: GARP, LRRC33, LTBP1 and LTBP3. Thus, these pharmaceutical compositions can be administered to patients for the relief of a TGFβ-related indication (e.g., fibrosis, immune disorders and/or cancer). “Acceptable” means that the carrier is compatible with the active ingredient in the composition (and, preferably, capable of stabilizing the active ingredient) and not harmful to the individual being treated. Examples of pharmaceutically acceptable excipients (carriers), including buffers, would be apparent to those skilled in the art and have been described previously. See, for example, “Remington: The Science and Practice of Pharmacy,” 20th Edition (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. In one example, a pharmaceutical composition described in this specification contains more than one antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex, wherein the antibodies recognize different epitopes/residues of a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex.

[00230] The pharmaceutical compositions to be used in the present methods may comprise pharmaceutically acceptable carriers, excipients or stabilizers in the form of lyophilized formulations or aqueous solutions (“Remington: The Science and Practice of Pharmacy”, 20th Edition (2000) Lippincott Williams and Wilkins , Ed. K. E. Hoover). Acceptable carriers, excipients or stabilizers are non-toxic to receptors at the dosages and concentrations used, and may comprise buffers such as, for example, phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol ; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins, for example serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as, for example, polyvinylpyrrolidone; amino acids such as, for example, glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as, for example, TWEENTM, PLURONICSTM or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described in this specification.

[00231] In some examples, the pharmaceutical composition described in this specification comprises liposomes containing an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex, which may be prepared by any suitable method, for example, as described in Epstein et al., Proc. Natl. Academic. Sci. U.S.A. 82: 3688 (1985); Hwang et al. Proc. Natl. Academic. Sci. U.S.A. 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to generate liposomes of the desired diameter.

[00232] Antibodies that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex can also be captured in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions . Exemplary techniques have been described previously; see, for example, “Remington, The Science and Practice of Pharmacy,” 20th Edition, Mack Publishing (2000).

[00233] In other examples, the pharmaceutical composition described in this specification can be formulated in a sustained release format. Suitable examples of sustained release preparations include semipermeable matrices of hydrophobic solid polymers containing the antibody, which matrices are in the form of shaped articles, for example, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and 7 -ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as, for example, LUPRON DEPOTTM (injectable microspheres formed by lactic acid-glycolic acid copolymer and leuprolide acetate), isobutyrate acetate sucrose and poly-D-(-)-3-hydroxybutyric acid.

[00234] Pharmaceutical compositions to be used for in vivo administration must be sterile. This is easily achieved, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed in a container that has a sterile access port, for example, an intravenous solution bag or vial that has a cap pierceable by a hypodermic injection needle.

[00235] The pharmaceutical compositions described in this specification may be in unit dosage forms such as, for example, tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[00236] For the preparation of solid compositions such as, for example, tablets, the main active ingredient can be mixed with a pharmaceutical carrier, for example, conventional ingredients in tablet production such as, for example, corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, for example, water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present disclosure, or a pharmaceutical salt acceptable non-toxic of this. When such pre-formulation compositions are said to be homogeneous, it means that the active ingredient is dispersed equally throughout the composition so that the composition can be easily subdivided into equally effective unit dosage forms such as tablets, pills. and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 mg to about 500 mg of the active ingredient of the present disclosure. Tablets or pills of the new composition may be coated or otherwise formed to provide a dosage form that provides the advantage of prolonged action. For example, the tablet or pill may comprise an internal dosage component and an external dosage component, the latter being in the form of an envelope over the former. The two components may be separated by an enteric layer that serves to resist disintegration in the stomach and allows the internal component to pass intact into the duodenum or to have its release delayed. Various materials can be used for these enteric layers or coatings, for example, materials including various polymeric acids and mixtures of polymeric acids with materials such as, for example, shellac, cetyl alcohol and cellulose acetate. Suitable surfactants include, in particular, nonionic agents, for example polyoxyethylene sorbitans (e.g. TweenTM 20, 40, 60, 80 or 85) and other sorbitans (e.g. SpanTM 20, 40, 60, 80 or 85). Compositions with a surfactant will conveniently comprise between 0.05 and 5% surfactant, and may be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable carriers, if necessary.

[00237] Suitable emulsions can be prepared using commercially available fat emulsions, for example, IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM. The active ingredient may be dissolved in a premixed emulsion composition or, alternatively, may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed by mixing with a phospholipid (for example, egg phospholipids, soy phospholipids or soy lecithin) and water. It will be appreciated that other ingredients may be added, for example, glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example between 5 and 20%.

[00238] Emulsion compositions can be those prepared by mixing an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex with IntralipidTM or the components of these (soybean oil, egg phospholipids, glycerol and water).

[00239] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered orally or nasally for local or systemic effect.

[00240] Compositions in preferably sterile pharmaceutically acceptable solvents can be nebulized using gases. Nebulized solutions can be breathed directly from the nebulizing device or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure respirator. Solution, suspension or powder compositions may be administered, preferably orally or nasally, by devices that release the formulation in an appropriate manner.

The use of antibodies and antigen-binding portions thereof, which specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex

[00241] In some embodiments, antibodies, antigen-binding portions thereof, and disclosure compositions can be used to treat a wide variety of diseases, disorders and/or conditions. In some cases, these diseases, disorders and/or conditions may be indications related to TGFβ. As used in this specification, the term “TGFβ-related indication” refers to any disease, disorder and/or condition related to the expression, activity and/or metabolism of a TGFβ family member protein or any disease, disorder and/or condition that may benefit from modulating the activity and/or levels of one or more proteins that are members of the TGFβ family. TGFβ-related indications may include, without limitation, fibrosis, cancer (including, without limitation, colon cancer, renal cancer, breast cancer, malignant melanoma and glioblastoma, facilitation of rapid hematopoiesis following chemotherapy, bone healing, wound healing, dementia , myelofibrosis, a kidney disease, unilateral ureteral obstruction (UUO), tooth loss and/or degeneration, endothelial proliferation syndromes, asthma and allergy, gastrointestinal disorders, anemia of aging, aortic aneurysm, orphan indications (e.g., Marfan syndrome and Camurati-Engelmann disease), obesity, diabetes, arthritis, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, metabolic syndromes, nutritional disorders, organ atrophy, chronic obstructive pulmonary disease (COPD) and anorexia may include any of those disclosed in U.S. Publication No. 2013/0122007, U.S. Patent No. 8,415,459 or International Publication No. WO 2011/151432, the contents of each of which are incorporated in this report. descriptive by reference in its entirety.

Fibrosis

[00242] In some embodiments, antibodies and/or compositions of the present disclosure may be useful for altering fibrosis. In some embodiments, these antibodies and/or compositions are TGFβ antagonists (e.g., TGFβ1). TGFβ1 is recognized as the central orchestrator of the fibrotic response. Antibodies targeting TGFβ1 decrease fibrosis in several preclinical models. Such antibodies and/or antibody-based compounds include LY2382770 (Eli Lilly, Indianapolis, IN). Also included are those described in U.S. Patent Numbers US 6,492,497, US 7,151,169, US 7,723,486 and U.S. Application Publication No. 2011/0008364, the contents of each of which are incorporated into this specification by reference in their entirety. .

[00243] Fibrotic indications for which the antibodies and/or compositions of the present disclosure can be used therapeutically include, without limitation, pulmonary indications (e.g., idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disorder (COPD), allergic asthma, acute lung injury, eosinophilic esophagitis, pulmonary arterial hypertension, and chemical gas injury), renal indications (e.g., diabetic glomerulosclerosis, focal segmental glomerulosclerosis (FSGS), chronic kidney disease, fibrosis associated with kidney transplantation and chronic rejection, IgA nephropathy, and hemolytic uremic syndrome), hepatic fibrosis (e.g., non-alcoholic steatohepatitis (NASH), chronic viral hepatitis, parasitemia, inborn errors of metabolism, toxin-mediated fibrosis, e.g., alcoholic fibrosis, non-alcoholic steatohepatitis-hepatocellular carcinoma (NASH- HCC), primary biliary cirrhosis and sclerosing cholangitis), cardiovascular fibrosis (e.g., cardiomyopathy, hypertrophic cardiomyopathy, atherosclerosis and restenosis), systemic sclerosis, cutaneous fibrosis (e.g., cutaneous fibrosis in systemic sclerosis, diffuse cutaneous systemic sclerosis, scleroderma, scarring cutaneous pathology, keloid, post-surgical scarring, scar revision surgery, radiation-induced scarring, and chronic wounds) and cancers or secondary fibrosis (e.g., myelofibrosis, head and neck cancer, M7 acute megakaryoblastic leukemia, and mucositis). Other diseases, disorders or conditions related to fibrosis that can be treated using compounds and/or compositions of the present disclosure include, without limitation, Marfan syndrome, tight skin syndrome, scleroderma, rheumatoid arthritis, bone marrow fibrosis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, muscular dystrophy, (e.g., DMD), Dupuytren's contracture, Camurati-Engelmann disease, neural scarring, dementia, proliferative vitreoretinopathy, corneal injury, complications after glaucoma drainage surgery, and multiple sclerosis. Many of these fibrotic indications are also associated with inflammation of the affected tissue (or tissues), indicating involvement of an immune component.

[00244] The antibodies described in this specification can be used to treat fibrosis. In some embodiments, TGFβ1 isoform-specific agents are administered to an individual in an amount effective to treat fibrosis. The effective amount of an antibody is an effective amount to achieve both therapeutic efficacy and clinical safety in the individual. In some embodiments, this antibody is a context-specific antibody that can block TGFβ1 activation that is mediated by an ECM-associated TGFβ1 containing LTBP. In some embodiments, the LTBP is LTBP1 and/or LTBP3. In some embodiments, the antibody is a context-permissive antibody that can block the activation of an LTBP-mediated TGFβ1 located in the ECM and GARP-mediated TGFβ1 located in immune cells. In some embodiments, the antibody is a context-permissive antibody that can block the activation of an LTBP-mediated TGFβ1 located in the ECM and LRRC33-mediated TGFβ1 located in monocytes/macrophages. In some embodiments, the LTBP is LTBP1 and/or LTBP3. In some embodiments, targeting and inhibition of TGFβ1 presented by LRRC33 in pro-fibrotic, M2-like macrophages in the fibrotic microenvironment may be beneficial.

[00245] Assays useful in determining the effectiveness of the antibodies and/or compositions of the present disclosure for altering fibrosis include, without limitation, histological assays for counting fibroblasts and basic immunohistochemical analyzes known in the art.

Cancer

[00246] Various cancers can be treated with antibodies and/or compositions of the present disclosure. As used in this specification, the term “cancer” refers to any of a variety of malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new sites in the body, and also refers to the pathological condition characterized by these malignant neoplastic growths. Cancers may be tumors or hematologic malignancies and include, without limitation, all types of lymphomas/leukemias, carcinomas, and sarcomas, such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/ rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, coccyx, testicles, thyroid and uterus.

[00247] In cancer, TGFβ (e.g., TGFβ1) can be growth promoting or growth inhibitory. As an example, in pancreatic cancers, wild-type SMAD4 tumors may show inhibited growth in response to TGFβ, but as the disease progresses, constitutively activated type II receptor is typically present. Additionally, there are SMAD4-null pancreatic cancers. In some embodiments, antibodies, antigen-binding portions thereof, and/or compositions of the present disclosure are designed to selectively target components of the TGFβ signaling pathways that function uniquely in one or more forms of cancer. Leukemias, or cancers of the blood or bone marrow that are characterized by an abnormal proliferation of white blood cells, i.e. leukocytes, can be divided into four main classifications that include acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia or acute myeloid leukemia (AML) (AML with translocations between chromosome 10 and 11 [t(10, 11)], chromosome 8 and 21 [t(8;21)], chromosome 15 and 17 [t(15 ;17)], and inversions on chromosome 16 [inv(16)]; AML with multilineage dysplasia, which includes patients who have had a myelodysplastic syndrome (MDS) or previous myeloproliferative disease that transforms into AML and myelodysplastic syndrome (MDS) , therapy-related, which category includes patients who have had prior chemotherapy and/or radiation and subsequently develop AML or MDS; d) AML not otherwise categorized, which includes subtypes of AML that do not fall into the above categories; and e) acute leukemias of ambiguous lineage, which occur when leukemic cells cannot be classified as myeloid or lymphoid cells, or when both types of cells are present); and chronic myelogenous leukemia (CML).

[00248] Types of carcinomas include, without limitation, papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma /leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma and undifferentiated sinonasal carcinoma.

[00249] Types of sarcomas include, without limitation, soft tissue sarcoma such as, for example, alveolar soft tissue sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma , hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and chondrosarcoma.

[00250] In some embodiments, the antibodies and methods of the disclosure may be used to treat one or more types of cancer or cancer-related conditions which may include, without limitation, colon cancer, kidney cancer, breast cancer, malignant melanoma and glioblastomas (Schlingensiepen et al., 2008; Ouhtit et al., 2013).

[00251] In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex, as described in this specification, may be used in methods for treating cancer in an individual in need, said method comprising administering the antibody, or antigen-binding portion thereof, to the individual, such that the cancer is treated. In certain embodiments, the cancer is colon cancer.

[00252] In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex, as described in this descriptive report, can be used in methods for the treatment of solid tumors. In some embodiments, solid tumors may be desmoplastic tumors, which are typically dense and hard for therapeutic molecules to penetrate. By targeting the ECM component of these tumors, these antibodies can “loosen” dense tumor tissue to disintegrate, facilitating therapeutic access to exert their anticancer effects. In this way, additional therapies, for example, any known antitumor drugs, can be used in combination.

[00253] In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex, as described in this specification report, may be used in methods for inhibiting or decreasing the growth of solid tumors in an individual having a solid tumor, said method comprising administering the antibody, or antigen-binding portion thereof, to the individual in such a manner that solid tumor growth is inhibited or slowed. In certain embodiments, the solid tumor is a colon carcinoma tumor. In some embodiments, the antibodies, or antigen-binding portions thereof, useful for treating a cancer is an isoform-specific, context-permissive inhibitor of TGFβ1 activation. In some embodiments, these antibodies target a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and a LRRC33-TGFβ1 complex. In some embodiments, these antibodies target a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, and an LTBP3-TGFβ1 complex. In some embodiments, these antibodies target an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and a LRRC33-TGFβ1 complex. In some embodiments, these antibodies target a GARP-TGFβ1 complex and a LRRC33-TGFβ1 complex.

[00254] In certain embodiments, antibodies or antigen-binding portions thereof that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex, as described in this specification, are administered to an individual who has cancer or a tumor, alone or in combination with an additional agent, for example, an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist). Other combined therapies that are included in the invention are the administration of an antibody, or antigen-binding portion thereof, described in this specification, with radiation, or a chemotherapeutic agent. Exemplary additional agents include, without limitation, a PD-1 antagonist, a PDL1 antagonist, a PD-L1 or PDL2 fusion protein, a CTLA4 antagonist, a GITR agonist, an anti-ICOS antibody, an anti-ICOSL antibody. , an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-OX40 antibody, an anti-CD27 antibody, an anti-CD70 antibody, an anti-CD47 antibody, an anti-41BB antibody, an anti-PD-1 antibody, an oncolytic virus and a PARP inhibitor.

Role of TGFβ in musculoskeletal conditions

[00255] In skeletal muscle, TGFβ plays several roles, including inhibition of proliferation and differentiation, induction of atrophy and development of fibrosis. TGFβ reduces satellite cell proliferation and prevents differentiation (through inhibition of MyoD and myogenin) (Allen, R.E. and L.K. J. Cell. Physiol., 1987. 133 (3): pages 567-72; Brennan, T.J., et al. , Proc. Natl. Sci. U.S.A., 1991. 88 (9): pages 3.822-6; 8,206-10; Olson, E.N., et al., J. Biol., 1986. 103 (5): pages 1,799-805). The isoform of TGFβ (i.e., TGFβ1, 2, or 3) is not specified in these early works, but is assumed to be TGFβ1. TGFβ also contributes to muscle fibrosis; direct injection of recombinant TGFβ1 results in fibrosis of skeletal muscle, and inhibition of pan-TGFβ decreases fibrosis in acutely and chronically injured muscle (Li, Y., et al., Am. J. Pathol., 2004. 164 ( 3): pages 1,007-19; Mendias, C.L., et al., Muscle Nerve, 2012. 45 (1): pages 55-9; Nelson, C.A., et al., Am. J. Pathol., 2011. 178 ( 6): pages 2,611-21). TGFβ1 is expressed by myofibers, macrophages, regulatory T cells, fibroblasts, and fibrocytes within skeletal muscle (Li, Y., et al., Am. J. Pathol., 2004. 164 (3): pages 1007-19; Lemos, D.R., et al., Nat. Med., 2015. 21 (7): pages 786-94; Villalta, S.A., et al., Sci. Transl. X., et al., J. Immunol., 2016. 197 (12): pages 4750-4761); and expression is increased upon injury and in disease (Li, Y., et al., Am. J. Pathol., 2004. 164 (3): pages 1007-19; Nelson, C.A., et al., Am. J . Pathol., 2011. 178 (6): pages 2,611-21; Bernasconi, P., et al., J. Clin. Invest., 1995. 96 (2): pages 1,137-44; cols., Neuroreport, 2000. 11 (18): pages 4,033-5). TGFβ2 and TGFβ3 are also upregulated (at the mRNA level) in mdx muscle, although to a lesser extent than TGFβ1 (Nelson, C.A., et al., Am. J. Pathol., 2011. 178 (6): pages 2,611-21; Zhou, L., et al., Neuromuscul., 2006. 16 (1): pages 32-8). Pessina, et al, recently used lineage tracing experiments to show that cells of multiple origins within dystrophic muscle adopt a fibrogenic fate via a TGFβ-dependent pathway (Pessina, P., et al, Stem Cell Reports, 2015. 4 (6): pages 1,046-60).

[00256] TGFβ1 has been implicated in human muscular dystrophies. Duchenne muscular dystrophy (DMD) is a serious, progressive and ultimately fatal disease caused by the absence of dystrophin (Bushby, K., et al., Lancet Neurol., 2010. 9 (1): pages 77-93). The absence of dystrophin results in increased susceptibility to contraction-induced injury, leading to continued muscle degeneration (Petrof, B.J., et al., Proc. Natl. Acad. Sci. U.S.A., 1993. 90 (8): pages 3,710-4; Dellorusso, C., et al., J. Muscle Res. Cell. Motil., 2001. 22 (5): pages 467-75; Pratt, S.J., et al., Cell. (1): pages 153-64). Repeated rounds of repair contribute to chronic inflammation, fibrosis, exhaustion of the satellite cell pool, eventual loss of mobility and death (Bushby, K., et al., Lancet Neurol., 2010. 9 (1): pages 77-93 ; McDonald, C.M., et al., Muscle Nerve, 2013. 48 (3): pages 343-56). TGFβ1 expression is significantly increased in patients with DMD and correlates with the extent of fibrosis observed in these patients (Bernasconi, P., et al., J. Clin. Invest., 1995. 96 (2): pages 1,137-44 ; Chen, Y.W., et al., Neurology, 2005. 65 (6): pages 826-34). Excessive deposition of ECM has detrimental effects on the contractile properties of the muscle and can limit access to nutrition, as myofibers are isolated from their blood supply (Klingler, W., et al., Acta Myol., 2012. 31 (3): pages 184-95). Recently, additional data have further implicated TGFβ1 in muscular dystrophies. Variants in LTBP4 have been found to modify disease severity in mice and humans. In mice, a variant of LTBP4 is protective in mice that lack dystrophin or γ-sarcoglycan (Coley, W.D., et al., Hum. Mol. Genet., 2016. 25 (1): pages 130-45; Heydemann, A ., et al., J. Clin. Invest., 2009. 119 (12): pages 3,703-12). In humans, two groups independently identified an LTBP4 variant as protective in DMD, delaying loss of ambulation for several years (Flanigan, K.M., et al., Ann. Neurol., 2013. 73 (4): pages 481-8; van den Bergen, J.C., et al., J. Neurol. Psychiatry, 2015. 86 (10): pages 1060-5). Although the nature of the genetic variants in mice and humans differs, in both species the protective variant results in diminished TGFβ signaling (Heydemann, A., et al., J. Clin. Invest., 2009. 119 (12): pages 3,703-12); Ceco, E., et al., Sci. Transl. Med., 2014. 6 (259): page 259ra144). Many of the functions of TGFβ1 in skeletal muscle biology have been deduced from experiments in which purified active growth factor is injected into animals or added to cultured cells (Massague, J., et al., Proc. Natl. Acad. Sci. U.S.A., 1986. 83 (21): pages 8,206-10; Li, Y., et al., Am. J. Pathol., 2004. 164 (3): pages 1007-19; Muscle Nerve, 2012. 45 (1): pages 55-9). Considering the importance of cellular context for specific functions of TGFβ1 (see, e.g., Hinck et al., Cold Spring Harb. Perspect. Biol., 2016. 8(12)) it is possible that some of the effects observed in these experiments do not reflect the endogenous role (or roles) of the cytokine in vivo. For example, treatment of human dermal fibroblasts with recombinant TGFβ1, myostatin, or GDF11 results in nearly identical changes in gene expression in these cells, although the in vivo roles of these proteins are quite different (Tanner, J.W., Khalil, A., Hill, J. ., Franti, M., MacDonnell, S.M., “Growth Differentiation Factor 11 Potentiates Myofibroblast Activation, in Fibrosis: From Basic Mechanisms to Targeted Therapies 2016: Keystone, CO).

[00257] Several investigators have used TGFβ inhibitors to clarify the role of the growth factor in vivo. Treatment of mdx mice with the pan-TGFβ neutralizing antibody 1D11 clearly results in reduced fibrosis (by histology and hydroxyproline content), reduced muscle damage (reduced serum creatine kinase and increased myofiber density), and improved muscle function (by plethysmography, force generation by isolated EDL muscles, and increased forepaw grip strength) (Nelson, C.A., et al., Am. J. Pathol., 2011. 178 (6): pages 2,611-21; Andreetta, F. , et al., J. Neuroimmunol., 2006. 175 (1-2): pages 77-86; Gumucio, J.P., et al., J. Appl. 539-45). Furthermore, myofiber-specific expression of a dominant-negative type II TGFβ receptor protects against muscle damage after cardiotoxin injury and in δ-sarcoglycan-/- mice (Accornero, F., et al., Hum. Mol. Genet ., 2014. 23 (25): pages 6,903-15). The proteoglycan decorin, which is abundant in skeletal muscle and inhibits TGFβ activity, decreases muscle fibrosis in mdx mice and after laceration injury (Li, Y., et al., Mol. Ther., 2007. 15 (9) : pages 1,616-22; Gosselin, L.E., et al., Muscle Nerve, 2004. 30 (5): pages 645-53). Other molecules with TGFβγ inhibitory activity, for example, suramin (an antineoplastic agent) and losartan (an angiotensin receptor blocker) have been effective in ameliorating muscle pathology and reducing fibrosis in mouse models of injury, Marfan syndrome, and muscular dystrophy (Spurney, C.F., et al., J. Cardiovasc. Pharmacol. Ther., 2011. 16 (1): pages 87-95; Taniguti, A.P., et al., Muscle Nerve, 2011. 43 (1): pages 82-7; Bedair, H.S., et al., Am. J. Sports Med., 2008. 36 (8): pages 1548-54; 2): pages 204-10). Although all of the therapeutic agents described above actually inhibit TGFβ1 or its signaling, none of them are specific for the TGFβ1 isoform. For example, 1D11 binds and inhibits TGFβ1, isoforms 2 and 3 (Dasch, J.R., et al., J. Immunol., 1989. 142 (5): pages 1536-41). Suramin inhibits the ability of several growth factors to bind to their receptors, including PDGF, FGF and EGF, in addition to TGFβ1 (Hosang, M., J. Cell. Biochem., 1985. 29 (3): pages 265-73 ; Olivier, S., et al., Eur. J. Cancer, 1990. 26 (8): pages 867-71; . Decorin also inhibits myostatin activity, both by direct binding and through upregulation of follistatin, a myostatin inhibitor (Miura, T., et al., Biochem. Biophys. Res. Commun., 2006. 340 (2 ): pages 675-80; Brandan, E., C. Cabello-Verrugio, and C. Vial, Matrix Biol., 2008. 27 (8): pages 700-8; Biol. Chem., 2007. 282 (35): pages 25,852-63). Losartan affects additional signaling pathways through its effects on the renin-angiotensin-aldosterone system, including the IGF-1/AKT/mTOR pathway (Burks, T.N., et al., Sci. Transl. Med., 2011. 3 ( 82): page 82ra37; Sabharwal, R. and M.W. Chapleau, Exp. Physiol., 2014. 99 (4): pages 627-31; ): pages 181-94). Therefore, all of these therapies inhibit additional molecules that may contribute to their therapeutic effects as well as toxicities.

[00258] Considering the postulated role of TGFβ in muscle homeostasis, repair and regeneration, agents, for example, monoclonal antibodies described in this specification, that selectively modulate TGFβ1 signaling may be effective for treating damaged muscle fibers, e.g. in chronic/genetic muscular dystrophies and acute muscle injuries, without the toxicities associated with the broadest-acting TGFβ inhibitors developed to date.

[00259] Consequently, the present invention provides methods for treating damaged muscle fibers with the use of an agent that preferentially modulates a subset, but not all, of the effects of TGFβ in vivo. These agents can selectively modulate TGFβ1 signaling (“isoform-specific modulation”). In some embodiments, these agents can also selectively modulate TGFβ1 in a particular context (“context-specific modulation”).

Muscle Fiber Repair in Chronic Muscle Disease

[00260] The invention encompasses methods to increase muscle quality and function in patients with DMD, by limiting fibrosis and contributing to the normalization of muscle morphology and function. As TGFβ1 also inhibits myogenesis, blockade of TGFβ1 may promote regeneration in dystrophic muscle, adding further therapeutic benefit. TGFβ1 inhibitors can be used in combination with dystrophin upregulating therapies, e.g., Exondys 51 (Eteplirsen). Considering the potential therapeutic benefits of TGFβ1 inhibition in muscular dystrophy, it is crucial (1) to differentiate the role (or roles) of TGFβ1 from that of TGFβ2 and TGFβ3, and (2) to clarify in which molecular context (or contexts) TGFβ1 inhibition would be the most beneficial. As mentioned above, pan-TGFβ inhibitors have been associated with significant toxicities, which limit the clinical use of these compounds (Anderton, M.J., et al., Toxicol Pathol., 2011. 39 (6): pages 916-24; Stauber, A. , et al., Clinical Toxicology, 2014. 4 (3): pages 1-10). It is uncertain which of the TGFβ isoforms cause these toxicities. Some of the toxicities described may be caused by inhibition of TGFβ1 in the immune system. For example, although 1D11 significantly reduced fibrosis levels in the diaphragm, treatment also increased the numbers of CD4+ and CD8+ T cells in the muscle, suggesting an enhanced inflammatory response upon pan-TGFβ inhibition that would be detrimental with long-term treatment (Andreetta , F., et al., J Neuroimmunol., 2006. 175 (1-2): pages 77-86). Indeed, depletion of T cells from muscle improves muscle pathology in mdx mice, suggesting that T cell-mediated inflammatory responses are detrimental to dystrophic muscle (Spencer, M.J., et al., Clin. Immunol., 2001. 98 (2 ): pages 235-43). Increases in T cell numbers following 1D11 administration are likely caused by the effects of TGFβ1 on regulatory T cells (Treg). Tregs present TGFβ1 on their cell surface via GARP, and release of TGFβ1 by this complex increases Treg suppressive activity, thereby limiting T cell-mediated inflammation (Wang, R., et al., Mol. Biol . Cell., 2012. 23 (6): pages 1,129-39; Edwards, J.P., A.M. Thornton and E.M. Shevach, J. Immunol., 2014. 193 (6): pages 2,843-9; ., J. Immunol., 2004. 172 (2): pages 834-42; Nakamura, K., A. Kitani and W. Strober, J. Exp. Med., 2001. 194 (5): pages 629-44 ). In fact, depletion of Tregs using the PC61 antibody resulted in increased inflammation and muscle damage in the diaphragm of mdx mice, while increasing Treg numbers and activity reduced muscle damage (Villalta, S.A., et al., Sci. Transl. Med., 2014. 6 (258): page 258ra142). Interestingly, an additional population of immunosuppressive T cells, Tr1 cells, has recently been identified. These cells produce greater amounts of TGFβ3, which is necessary for its suppressive activity (Gagliani, N., et al., Nat. Med., 2013. 19 (6): pages 739-46; Okamura, T., et al ., Proc. Natl. Acad. Sci. U.S.A., 2009. 106 (33): pages 13,974-9; Although the role of Tr1 cells in skeletal muscle is unknown, there is a possibility that inhibition of TGFβ1 and TGFβ3 by 1D11 could have additive pro-inflammatory effects by inhibiting Tregs and Tr1 cells.

[00261] The structural clarifications described above regarding TGFβ1 latency and activation allow for new approaches to drug discovery that specifically target TGFβ1 activation (Shi, M., et al., Nature, 2011. 474 (7,351): pages 343-9). The high degree of sequence identity shared between the three mature TGFβ growth factors is not shared by the latent complexes, allowing the discovery of antibodies that are perfectly specific for prδ-TGFβl. Using proprietary approaches to antibody discovery, the present inventors have identified antibodies (Abl and Ab2) that specifically bind to pro-TGFe1 (Figure 18A). Using an in vitro coculture system, these antibodies were shown to inhibit integrin-mediated TGFβ1 release. In this system, fibroblasts derived from human skin or mouse skeletal muscle are the source of latent TGFβ1, a cell line expressing αVβ6 allows the release of active TGFβ1, which is then measured using a third cell line expressing a SMAD2/3-responsive luciferase reporter (Figures 11A-C). One of these antibodies, Ab1, was tested in vivo and demonstrated efficacy in the 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 the upregulation of TGFβ1-responsive genes (Figure 15) and reduced the extent of fibrosis after injury (by staining with Picrosirius-Red ) (Figure 16). TGFe1-specific therapies may have improved efficacy and safety profiles compared with pan-TGFβ inhibitors, a crucial aspect for a therapy that would be used long-term as in the DMD population. TGFβ1 inhibitory antibodies can be used to determine whether specific inhibition of TGFβ1 has potential as a therapeutic for DMD or other muscle diseases, and to clarify the role of TGFβ1 in skeletal muscle regeneration.

Chronic vs. chronic injuries acute myofibers and selection of optimal therapies

[00262] In normal but regenerating muscle following an acute injury (e.g., traumatic injury to otherwise healthy muscles or motor neurons), initial infiltration of inflammatory macrophages is believed to be necessary to clear the damaged tissue and secrete factors (e.g., cytokines) necessary for satellite cell activation. Subsequently, these cells switch to the M2 phenotype to drive wound resolution.

[00263] By contrast, in chronic conditions, for example, diseases including DMD, pro-inflammatory macrophages predominated at all times, and that switch to M2 does not occur (or at least not efficiently enough), and pro-inflammatory macrophages -inflammatories continue to drive inflammation and muscle damage. In DMD, the NFkB pathway is perpetually active, resulting in constitutive inflammation. In some embodiments, therefore, an NFkB inhibitor can be administered to patients with DMD in order to reduce chronic inflammation.

[00264] Therefore, in chronic conditions such as, for example, DMD, the therapeutic focus may be on muscle repair, as opposed to muscle regeneration. This is because the muscle fibers in DMD are damaged but not destroyed - they are damaged by membrane tears, dysregulation of calcium transport and ROS damage by macrophages. In comparison, in cases of injury to healthy muscles, the therapeutic focus may be on regeneration. For example, in cardiotoxin models, muscle fibers are killed and must be regenerated. This simulates the recovery process after a traumatic injury, for example crush injury.

[00265] Evidence suggests that LRRC33 is expressed in thioglycolate-induced peritoneal macrophages, which have an M2-like phenotype (characterized by the fact that they express high levels of Arginase, in iNOS, and high levels of CD206).

[00266] In situations in which LRRC33 is expressed primarily in M2 cells and in which its presentation of TGFβ1 (“context”) is important for the pro-wound healing effects of these cells, it may be beneficial to activate LRRC33-mediated TGFβ1 to promote repair and/or myogenesis. On the other hand, in situations in which LRRC33 is also expressed on pro-inflammatory M1 cells, then it may be beneficial to inhibit LRRC33-mediated TGFβ1, considering that inflammation drives fibrosis, especially in a dystrophic setting, e.g., DMD.

[00267] Thus, identifying the source/context of disease-associated TGFβ1 may be an important step in selecting the correct modulator of TGFβ signaling, which will inform what level of selectivity should be considered (e.g., context-specific TGFβ1 modulators). permissive, isoform-specific, or context-specific TGFβ1 modulators; TGFβ1 inhibitors or activators, etc.).

[00268] In addition to chronic inflammation, the hallmark of DMD is excessive and progressive fibrosis. In advanced disease, fibrosis is so severe that it can actually isolate individual muscle fibers from their blood supply. It also changes the contractile properties of the muscle. In human patients, there is a strong correlation between the extent of TGFβ1 upregulation and fibrosis, and a strong link between the extent of fibrosis and negative mobility outcomes. Therefore, in some embodiments, LTBP-pr0-TGFβ1 inhibitors can be administered to dystrophic patients for the prevention and/or reduction of fibrosis to selectively target the effects of ECM-associated TGFβ1 on the disease. In some embodiments, various isoform- and/or context-selective agents described in this specification can be employed to achieve inhibition of TGFβ1 signaling to prevent fibrosis and promote myogenesis, but without having undesirable effects on the immune system (e.g. , through GARP or LRRC33).

Treatment

[00269] To practice the method disclosed in this specification, an effective amount of the pharmaceutical composition described above can be administered to an individual (e.g., a human) in need of treatment via a suitable route, e.g., intravenous administration. , for example, as a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intraarticularly, intrasynovically, intrathecally, orally, by inhalation or topically . Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be nebulized directly and lyophilized powder can be nebulized after reconstitution.

[00270] Alternatively, antibodies or antigen-binding portions thereof that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex can be aerosolized using a formulation of fluorocarbon and a metered dose inhaler, or inhaled as a freeze-dried and crushed powder.

[00271] The individual to be treated by the methods described in this specification may be a mammal, more preferably a human. Mammals include, without limitation, farm animals, sporting animals, pets, primates, horses, dogs, cats, mice and rats. A human subject in need of treatment may be a human patient who has, is at risk for, or is suspected of having a TGFβ-related indication, such as those noted above. An individual who has a TGFβ-related indication can be identified by routine medical examination, for example, laboratory tests, organ functional tests, CT scans, or ultrasounds. An individual suspected of having any such indication may exhibit one or more symptoms of the indication. An individual at risk for the indication may be an individual who has one or more of the risk factors for that indication.

[00272] As used in this specification, the terms “effective amount” and “effective dose” refer to any amount or dose of a compound or composition that is sufficient to meet its desired objective (or objectives), that is, a response desired biological or medicinal product in a tissue or individual in an acceptable risk/benefit ratio. For example, in certain embodiments of the present invention, the desired objective may be to inhibit TGFβ1 activation in vivo, to obtain a clinically significant end result associated with TGFβ1 inhibition. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the patient's individual parameters including age, physical condition, size, sex and weight, the duration of treatment, the nature of the current therapy (if any), the specific route of administration, and similar factors within the knowledge and experience of the healthcare professional. These factors are well known to those skilled in the art and can be addressed with, at most, routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, i.e., the highest safe dose according to sound medical judgment. It will be understood by those skilled in the art, however, that a patient may insist on a lower dose or a tolerable dose for medical reasons, physiological reasons or for practically any other reasons.

[00273] Empirical considerations, for example, half-life, will generally contribute to determining the dosage. For example, antibodies that are compatible with the human immune system, e.g., humanized antibodies or fully human antibodies, can be used to prolong the half-life of the antibody and to prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted throughout the therapy cycle, and is generally, but not necessarily, based on treatment and/or suppression and/or improvement and/or delay of a TGFβ-related indication. Alternatively, continuous sustained release formulations of an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex may be suitable. Various formulations and devices for achieving sustained release would be apparent to those skilled in the art and are within the scope of this disclosure.

[00274] In one example, dosages for an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex as described in this specification can be determined empirically in individuals who received one or more administrations of the antibody. Subjects receive incremental dosages of the antagonist. To assess efficacy, an indicator of the TGFβ-related indication can be monitored. For example, methods for measuring myofiber damage, myofiber repair, levels of inflammation in muscle and/or levels of fibrosis in muscle are well known to those skilled in the art.

[00275] The present invention encompasses the recognition that agents capable of modulating the activation step of TGFβs in an isoform-specific manner can provide improved safety profiles when used as a medicine. Accordingly, the invention includes antibodies and antigen-binding fragments thereof that specifically bind and inhibit the activation of TGFβ1, but not TGFβ2 or TGFβ3, thereby conferring specific inhibition of TGFβ1 signaling in vivo while minimizing the unwanted side effects resulting from alterations in TGFβ2 and/or TGFβ3 signaling.

[00276] In some embodiments, antibodies, or antigen-binding portions thereof, as described in this specification, are non-toxic when administered to an individual. In some embodiments, the antibodies, or antigen-binding portions thereof, as described in this specification, exhibit reduced toxicity when administered to an individual, compared to an antibody that specifically binds to both TGFβ1 and TGFβ2. In some embodiments, the antibodies, or antigen-binding portions thereof, as described in this specification, exhibit reduced toxicity when administered to an individual, compared to an antibody that specifically binds to both TGFβ1 and TGFβ3. In some embodiments, antibodies, or antigen-binding portions thereof, as described in this specification, exhibit reduced toxicity when administered to an individual, compared to an antibody that specifically binds to TGFβ1, TGFβ2, and TGFβ3.

[00277] Generally, for administration of any of the antibodies described in this specification, an initial candidate dosage may be about 2 mg/kg. For purposes of the present disclosure, a typical daily dosage may range from about any one 0.1 μg/kg to 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg to 30 mg/kg up to 100 mg/kg or more depending on the factors mentioned above. For repeated administrations over several days or more, depending on the condition, treatment is continued until a desired suppression of symptoms occurs or until sufficient therapeutic levels are obtained to alleviate a TGFβ-related indication, or a symptom thereof. An exemplary dosage regimen comprises administering an initial dose of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of the antibody, or followed by a maintenance dose of about 1 mg/kg. kg every other week. However, other dosage regimens may be useful depending on the pattern of pharmacokinetic decline the practitioner wishes to achieve. For example, a dosage of one to four times per week is contemplated. In some embodiments, a dosage ranging from about 3 μg/mg to about 2 mg/kg (e.g., about 3 μg/mg, about 10 μg/mg, about 30 μg/mg, about 100 μg/mg, about 300 μg/mg, about 1 mg/kg, and about 2 mg/kg) can be used. Pharmacokinetic experiments have demonstrated that the serum concentration of an antibody disclosed in this specification (e.g., Ab2) remains stable for at least 7 days after administration in a preclinical animal model (e.g., a mouse model). Without being bound by a particular theory, such post-administration stability may be advantageous in that the antibody can be administered less frequently while maintaining a clinically effective serum concentration in the individual to whom the antibody is administered (e.g. , a human individual). In some embodiments, the dosage frequency is once per week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once a month, every 2 months, or every 3 months, or more. The progress of this therapy is easily monitored by conventional techniques and assays. The dosage regimen (including the antibody used) may vary over time.

[00278] In some embodiments, for a normal-weight adult patient, doses ranging from about 0.3 to 5.00 mg/kg can be administered. The particular dosage regimen, e.g., dose, timing, and repetition, will depend on the particular individual and that individual's medical history, as well as the properties of the individual agents (e.g., the half-life of the agent, and other relevant considerations). ).

[00279] For the purposes of the present disclosure, the appropriate dosage of an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex will depend on the specific antibody (or compositions thereof) employed, the type and severity of the indication, whether the antibody is administered for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the antagonist, and at the discretion of the attending physician. In some embodiments, a physician will administer an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and/or a LRRC33-TGFβ1 complex, until a dosage is reached that obtains the desired result. Administration of an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex can be essentially continuous over a preselected period of time or can be in a series of spaced doses, for example, before, during or after the development of a TGFβ-related indication.

[00280] As used in this specification, the term “treatment” refers to the application or administration of a composition that includes one or more active agents to an individual, who has a TGFβ-related indication, a symptom of the indication, or a predisposition for the indication, with the aim of curing, scarring, alleviating, altering, remedying, improving, enhancing or affecting the indication, the symptom of the indication, or the predisposition to the indication.

[00281] Relieving a TGFβ-related indication with an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex includes delaying development or progression of the indication, or reducing the severity of the indication. Indication relief does not necessarily require curative results. As used in this specification, “delaying” the development of an indication associated with a TGFβ-related indication means postponing, preventing, slowing down, delaying, stabilizing and/or postponing the progression of the indication. This delay may be of varying lengths of time depending on the history of the indication and/or the individuals being treated. A method that “delays” or alleviates the development of an indication, or delays the onset of the indication, is a method that reduces the probability of development of one or more symptoms of the indication within a certain time interval and/or reduces the extent of the symptoms in a certain period of time, when compared to not using the method. These comparisons are typically based on clinical studies, using a sufficient number of subjects to generate a statistically significant result.

[00282] DBA2/J mice have a 40 bp deletion in the LTBP4 allele. Dysregulation of the ECM with which latent TGFb1 is associated may expose the epitope to which Ab1 binds. There may be diseases in which the epitope to which Ab1 binds is exposed, and those diseases may be therapeutic opportunities for Ab1 if inhibition of TGFb1 is indicated.

Combination therapies

[00283] The disclosure further encompasses pharmaceutical compositions and related methods used as combined therapies for treating individuals who may benefit from TGFβ inhibition in vivo. In any of these embodiments, such individuals may receive combination therapies that include a first composition comprising at least one TGFβ inhibitor, e.g., antibody or antigen-binding portion thereof, described in this specification, together with a second composition that comprises at least one additional therapy intended to treat the same disease or medical condition or an overlapping disease or medical condition. The first and second compositions can both act on the same cellular target, or on different cellular targets. In some embodiments, the first and second compositions may treat or alleviate the same set of symptoms or aspects of a disease or clinical condition or overlapping set of symptoms or aspects of a disease or clinical condition. In some embodiments, the first and second compositions can treat or alleviate a separate set of symptoms or aspects of a disease or clinical condition. To give just one example, the first composition can treat a disease or condition associated with TGFβ signaling, while the second composition can treat inflammation or fibrosis associated with the same disease, etc. These combined therapies can be administered together. The phrase “in conjunction with” in the context of combination therapies means that the therapeutic effects of a first therapy may overlap temporally and/or spatially with the therapeutic effects of a second therapy in the individual receiving the combination therapy. Thus, combined therapies can be formulated as a single formulation for concomitant administration, or as separate formulations for sequential administration of the therapies.

[00284] In preferred embodiments, combined therapies produce synergistic effects in the treatment of a disease. The term “synergistic” refers to effects that are greater than the additive effects (e.g., increased efficacy) of each individual monotherapy.

[00285] In some embodiments, combination therapies comprising a pharmaceutical composition described in this specification produce efficacy that is generally equivalent to that produced by another therapy (e.g., monotherapy of a second agent), but are associated with fewer unwanted adverse effects or less severe toxicity associated with the second agent compared with monotherapy of the second agent. In some embodiments, these combination therapies allow for a lower dosage of the second agent but maintain overall efficacy. These combination therapies may be particularly suitable for patient populations in which long-term treatment is recommended and/or involving pediatric patients.

[00286] Consequently, the invention provides pharmaceutical compositions and methods for use in combined therapies for reducing TGFβ1 protein activation and in the treatment or prevention of diseases or conditions associated with TGFβ1 signaling, as described in this specification. Accordingly, the pharmaceutical methods or compositions further comprise a second therapy. In some embodiments, the second therapy may be useful in treating or preventing diseases or conditions associated with TGFβ1 signaling. The second therapy may lessen or treat at least one symptom (or symptoms) associated with the targeted disease. The first and second therapies may exert their biological effects through similar or unrelated mechanisms of action; or one or both of the first and second therapies may exert their biological effects through different mechanisms of action.

[00287] It should be understood that the pharmaceutical compositions described in this specification may have the first and second therapies in the same pharmaceutically acceptable carrier or in a different pharmaceutically acceptable carrier for each described modality. It should further be understood that the first and second therapies may be administered simultaneously or sequentially within the described embodiments.

[00288] The (one or more) anti-TGFβ antibodies of the invention, or antigen-binding portions thereof, can be used in combination with one or more additional therapeutic agents. Examples of additional therapeutic agents that can be used with an anti-TGFβ antibody of the invention include, without limitation, 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, and the like.

[00289] In some embodiments, the additional agent is a checkpoint inhibitor. In some embodiments, the additional agent is selected from the group consisting of a PD-1 antagonist, a PDL1 antagonist, a PD-L1 or PDL2 fusion protein, a CTLA4 antagonist, a GITR agonist, an anti-ICOS antibody. , an anti-ICOSL antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-0X40 antibody, an anti-CD27 antibody, an anti-CD70 antibody, an anti-CD47 antibody, an anti-41BB antibody, an anti-PD-1 antibody, an oncolytic virus, and a PARP inhibitor. In some embodiments, additional therapy 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., CCR2 antagonists and CCR5 antagonists. In some embodiments, this chemokine receptor antagonist is a dual specific antagonist, e.g., a CCR2/CCR5 antagonist. In some embodiments, the additional agent to be administered as combination therapy is or comprises a member of the TGFβ superfamily of growth factors or regulators thereof. In some embodiments, this agent is selected from modulators (e.g., inhibitors and activators) of GDF8/myostatin and GDF11. In some embodiments, this agent is an inhibitor of GDF8/myostatin signaling. In some embodiments, this agent is a monoclonal antibody that specifically binds to a pro/latent myostatin complex and blocks myostatin activation. In some embodiments, the monoclonal antibody that specifically binds to a pro/latent myostatin complex and blocks myostatin activation does not bind to free, mature myostatin.

[00290] These combined therapies can advantageously use lower dosages of the therapeutic agents administered, thus avoiding possible toxicities or complications associated with the various monotherapies.

Modulation of TGFβ activity

[00291] The methods of the present disclosure include methods of modulating growth factor activity in one or more biological systems. These methods may include contacting one or more biological systems with an antibody and/or disclosure composition. In some cases, these methods include modifying the level of free growth factor in a biological system (e.g., in a cell niche or individual). Antibodies and/or compositions according to these methods may include, without limitation, biomolecules including, without limitation, recombinant proteins, protein complexes and/or antibodies, or antigen-binding portions thereof, described in this specification.

[00292] In some embodiments, the methods of the present disclosure can be used to reduce or eliminate growth factor activity, referred to as “inhibition methods” in this specification. Some of these methods may comprise retaining mature growth factor in a TGFβ complex (e.g., a TGFβ1 complexed with GARP, LTBP1, LTBP3 and/or LRRC33) and/or promoting reassociation of growth factor into a TGFβ complex. . In some cases, inhibition methods may comprise the use of an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex. According to some inhibition methods, one or more inhibition antibodies are provided.

[00293] In some embodiments, antibodies, antigen-binding portions thereof, and disclosure compositions can be used to inhibit TGFβ1 activation. In some embodiments, there is provided in this specification a method for inhibiting TGFβ1 activation that comprises exposing a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex to an antibody. , an antigen-binding portion thereof, or a pharmaceutical composition described in this specification. In some embodiments, the antibody, antigen-binding portion thereof, or pharmaceutical composition, inhibits the release of mature TGFβ1 by the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and/or the LRRC33-TGFβ1 complex. . In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed ex vivo.

[00294] In some embodiments, the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, or the LRRC33-TGFβ1 complex is present on the outer surface of a cell. In some embodiments, the cell is a T cell, a fibroblast, a macrophage, a monocyte, or a microglia.

[00295] In some embodiments, the LRRC33-TGFβ1 complex is present on the external surface of pro-fibrotic (M2-like) macrophages. In some embodiments, pro-fibrotic (M2-like) macrophages are present in the fibrotic microenvironment. In some embodiments, targeting the LRRC33-TGFβ1 complex on the outer surface of pro-fibrotic (M2-like) macrophages provides a superior effect compared exclusively to targeting LTBP1-TGFβ1 complexes and/or LTBP1-TGFβ1 complexes.

[00296] In some embodiments, the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex and/or the LRRC33-TGFβ1 complex is linked to an extracellular matrix. In some embodiments, the extracellular matrix comprises fibrillin. In some embodiments, the extracellular matrix comprises a protein comprising an RGD motif.

[00297] In some embodiments, there is provided in this specification a method for reducing TGFβ1 protein activation in an individual that comprises administering an antibody, an antigen-binding portion thereof, or a pharmaceutical composition described in this specification to the individual. thus reducing the activation of TGFβ1 protein in the individual. In some embodiments, the individual has or is at risk for fibrosis. In some embodiments, the individual has or is at risk of having cancer. In some embodiments, the individual has or is at risk of having dementia.

[00298] In some embodiments, antibodies, or antigen-binding portions thereof, as described in this specification, reduce the suppressive activity of regulatory T cells (Tregs).

Kits for use in alleviating diseases/disorders associated with a TGFβ-related indication

[00299] The present disclosure also provides kits for use in alleviating diseases/disorders associated with a TGFβ-related indication. Such kits may include one or more containers comprising an antibody, or antigen-binding portion thereof, that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33- complex. TGFβ1, for example, any of those described in this specification.

[00300] In some embodiments, the kit may comprise instructions for use in accordance with any of the methods described in this specification. The included instructions may comprise a description of the administration of the antibody, or antigen-binding portion thereof, that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex. to treat, delay the onset of or alleviate a target disease, such as those described in this specification. The kit may further comprise a description of the selection of a suitable individual for treatment based on identifying whether that individual has the target disease. In still other embodiments, the instructions comprise a description of administering an antibody, or antigen-binding portion thereof, to an individual at risk for the target disease.

[00301] Instructions relating to the use of antibodies, or antigen-binding portions thereof, that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex generally include information regarding dosage, dosage schedule and route of administration for the desired treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages) or subunit doses. The instructions provided in developing kits are typically instructions written on a label or leaflet (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions on a magnetic or optical storage disk) are also acceptable. The label or leaflet indicates that the composition is used for the treatment, delaying the onset and/or alleviation of a disease or disorder associated with a TGFβ-related indication. Instructions may be provided for practicing any of the methods described in this specification.

[00302] The kits for this disclosure are in suitable packaging. Suitable packaging includes, without limitation, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic pouches), and the like. Also contemplated are packages for use in combination with a specific device, for example, an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as, for example, a minipump. A kit may have a sterile access port (for example, the container may be a bag of intravenous solution or a vial that has a cap pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example, the container may be an intravenous solution bag or a vial that has a cap pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody, or antigen-binding portion thereof, that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex, such as those described in this specification.

[00303] Kits may optionally provide additional components such as buffers and interpretive information. Typically, the kit comprises a container and a label or leaflet(s) on or associated with the container. In some embodiments, the disclosure provides manufactured articles comprising the contents of the kits described above.

Assays for detection of a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex

[00304] In some embodiments, the methods and compositions provided in this specification relate to a method for detecting a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex in a sample obtained from an individual. As used in this specification, an “individual” refers to an individual organism, for example, an individual mammal. In some embodiments, the individual is a human. In some embodiments, the individual is a non-human mammal. In some embodiments, the individual is a non-human primate. In some embodiments, the individual is a rodent. In some embodiments, the individual is a sheep, a goat, cattle, poultry, a cat, or a dog. In some embodiments, the individual is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the individual is a research animal. In some embodiments, the individual is genetically modified, e.g., a genetically modified non-human individual. The individual can be of any sex and at any stage of development. In some embodiments, the individual is a patient or healthy volunteer.

[00305] In some embodiments, a method for detecting a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and/or a LRRC33-TGFβ1 complex in a sample obtained from an individual involves (a) contacting of the sample with an antibody that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex and/or a LRRC33-TGFβ1 complex under conditions suitable for binding of the antibody to the antigen, if the antigen is present in the sample, thus forming binding complexes; and (b) determining the level of antibody bound to the antigen (e.g., determining the level of binding complexes).

[00306] In one embodiment, a screening assay that uses biotinylated latent TGFβ1 complexes immobilized on a surface, which allows activation of latent TGFβ by integrins by providing a tether. Other, non-integrin activators could also be tested in that system. The reading can be through reporter cells or other TGFβ-dependent cellular responses.

Cell-based assays for measuring TGFβ activation

[00307] Activation of TGFβ (and its inhibition by a TGFβ test inhibitor, e.g., an antibody) can be measured by any suitable method known in the art. For example, integrin-mediated activation of TGFβ can be used in a cell-based assay, e.g., the “CAGA12” luciferase assay, described in more detail in this specification. An exemplary embodiment of such an assay is depicted in Figure 11C for illustrative purposes. As shown, such an assay system may comprise the following components: i) a source of TGFβ (recombinant, endogenous or transfected); ii) an integrin source (recombinant, endogenous or transfected); and iii) a reporter system that responds to TGFβ activation, e.g., cells expressing TGFβ receptors capable of responding to TGFβ and translating the signal into a readable output (e.g., luciferase activity in CAGA12 cells or other cell lines). reporter cells). In some embodiments, the reporter cell line comprises 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, certain sensitivity-conferring promoter elements may be incorporated into the reporter system. In some embodiments, this promoter element is the CAGA12 element. Reporter cell lines that can be used in the assay have been described, for example, in Abe et al. (1994) Anal Biochem. 216 (2): 276 84, incorporated into this specification by reference. In some embodiments, each of the aforementioned assay components is provided from the same source (e.g., the same cell). In some embodiments, two of the aforementioned assay components are provided from the same source, and a third assay component is provided from a different source. In some embodiments, all three components of the assay are provided from different sources. For example, in some embodiments, the integrin and the latent TGFβ complex (pr0-TGFβ and a presentation molecule) are provided for the assay from the same source (e.g., the same transfected cell line). In some embodiments, the integrin and TGF are provided for the assay from separate sources (e.g., two different cell lines, a purified integrin combination, and a transfected cell). When cells are used as the source of one or more of the assay components, those assay components may be endogenous to the cell, stably expressed in the cell, transiently transfected, or any combination thereof. The results of an exemplary non-limiting embodiment of a cell-based assay for measuring TGFβ activation demonstrating inhibition of GARP-pr0-TGFβ1 complex or LRRC33-pr0-TGFβ1 complex using Ab1 and Ab2 antibodies, disclosed in this descriptive report, are shown in Figures 22A and Figure 22B, respectively. In this exemplary assay, the IC50 (μ m/ml) of Ab1 for the GARP-TGFβ1 complex was 0.445, and the IC50 (μ m/ml) of Ab1 for the LRRC33-TGFβ1 complex was 1.325.

[00308] Those skilled in the art could easily adapt these assays to various suitable configurations. For example, several sources of TGFβ can be considered. In some embodiments, the source of TGFβ is a cell that expresses and deposits TGFβ (e.g., a primary cell, a propagated cell, an immortalized cell or cell line, etc.). In some embodiments, the source of TGFβ is purified and/or recombinant TGFβ immobilized in the assay system using suitable media. In some embodiments, TGFβ immobilized in the assay system is presented within an extracellular matrix (ECM) composition on the assay plate, with or without decellularization, that simulates fibroblast-originated TGFβ. In some embodiments, TGFβ is presented on the cell surface of a cell used in the assay. Additionally, a presentation molecule of choice can be included in the assay system to provide suitable TGFβ-latent complex. Those skilled in the art can readily determine which presentation molecule (or molecules) may be present or expressed in certain cells or cell types. Using these assay systems, relative changes in TGFβ activation in the presence or absence of a test agent (e.g., an antibody) can be easily measured to evaluate the effects of the test agent on TGFβ activation in vitro. Exemplary cell-based assay data is provided in the Example section below.

[00309] These cell-based assays can be modified or adjusted in various ways, depending on the TGFβ isoform being studied, the type of latent complex (e.g., presentation molecule), and the like. In some embodiments, a cell known to express an integrin capable of activating TGFβ can be used as the source of integrin in the assay. These cells include SW480/06 cells (e.g., clone 1E7). In some embodiments, integrin-expressing cells can be cotransfected with a plasmid encoding a display molecule of interest (e.g., GARP, LRRC33, LTBP (e.g., LTBP1 or LTBP3), etc.) and a plasmid encoding a pro- form of the TGFβ isoform of interest (e.g., pr0-TGFβ1). After transfection, the cells are incubated long enough to allow expression of the transfected genes (e.g., about 24 hours), the cells are washed, and incubated with serial dilutions of a test agent (e.g., an antibody). Next, the reporter cell line (e.g., CAGA12 cells) is added to the assay system, followed by an appropriate incubation time to allow for TGFβ signaling. After an incubation period (e.g., about 18-20 hours) after addition of the test agent, the signal readout (e.g., luciferase activity) is detected using suitable media (e.g., for cell lines- reporter expressing luciferase, Bright-Glo reagent (Promega) can be used). In some embodiments, Luciferase fluorescence can be detected using a BioTek plate reader (Synergy H1), with autogain settings. Nucleic acids

[00310] In some embodiments, antibodies, antigen-binding portions thereof and/or compositions of the present disclosure may be encoded by nucleic acid molecules. These nucleic acid molecules include, without limitation, DNA molecules, RNA molecules, polynucleotides, oligonucleotides, mRNA molecules, vectors, plasmids and the like. In some embodiments, the present disclosure may comprise cells programmed or generated to express nucleic acid molecules that encode compounds and/or compositions of the present disclosure. In some cases, the nucleic acids of the disclosure include codon-optimized nucleic acids. Methods of generating codon-optimized nucleic acids are known in the art and may include, without limitation, those described in U.S. Patent Nos. 5,786,464 and 6,114,148, the contents of each of which are incorporated into this specification by reference in their entirety. .

[00311] The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. The entire contents of all references, patents and published patent applications cited throughout this application, as well as in the Figures, are incorporated herein into this specification by reference.

EXAMPLES Example 1: Inhibition of TGFβ1

[00312] The TGFβ superfamily includes pro-peptides complexed with active growth factors (Figure 1). Selection strategies to obtain antibodies that stabilize the complex, resulting in more selective and potent inhibition, have been developed.

[00313] Using a HEK293-based expression system, NiNTA affinity and gel filtration were performed to obtain multimilligram amounts of purified protein, which were used to generate TGFβ1 complexed with LTBP (LTBP-TGFβ1 complex) and TGFβ1 complexed to GARP (GARP-TGFβ1 complex) (Figure 3). The diversity of proteins manufactured allowed cross-species cross-reactivity testing and epitope mapping. Purification of a sGARP-pr0-TGFβ complex (Figure 4A and 4B), a sGARP-TGFβ LAP complex (Figure 5), and an LTBP1-pr0-TGFβ1 complex (Figure 6) is shown.

[00314] Candidate antibodies were tested using in vitro luminescence assays (Figure 8). In the evaluation, antibodies that inhibited the release of growth factor “turned off” the reporter cells when in contact with a stimulus for normal activation. Ab1 and Ab2 were shown to be inhibitors of the activation of latent TGFβ1 complexes (Figure 7) and cross-reacted with mouse.

[00315] Initial dose-response analysis curves of Ab1 in cells expressing human TGFβ1 exhibited TGFβ1 inhibition activity (Figure 8). Using a more sensitive CAGA12 reporter cell line, Ab1 showed similar inhibition of human pr0-TGFβ1 activity (Figure 9). Furthermore, inhibition of a GARP complex has been shown to block the suppressive activity of regulatory T cells (Tregs), as measured by the percentage of dividing effector T cells (Teff) in T cells isolated from healthy donor blood (Figure 10 ).

[00316] The affinity of GARP-pr0-TGFβ1 inhibitors was measured by the Octet assay in human GARP-pr0-TGFβ1 cells, while activity was measured by CAGA12 reporter cells that test inhibition of human GARP-pro-TGFe1. The protocol used to mediate the affinity of Ab1 and Ab2 antibodies to the complexes provided in this specification is summarized in Table 6. The results are shown in Table 7. Table 6: Protocol for performing the Octet binding assay. Materials:

[00317] The clones were further evaluated for binding selectivity (Table 8) and cross-species reactivity (Table 9). Ab1 and Ab2 did not bind to TGFβ1, TGFβ2, or TGFβ3, but they did bind to pro-TGFe1 complexes and showed interspecies cross-reactivity. Table 8: Selectivity of GARP-pr0-TGFβ1 inhibitors.

Example 2: Ab1 and Ab2 specifically bind to pr0-TGFβ1 complexes from various species.

[00318] To determine whether Ab1 and Ab2 are capable of specifically binding pr0-TGFβ1 complexes from various 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 and murine LTBP1-pr0-TGFβ1 complexes, human LTBP3-pr0-TGFβ1 complexes, and human GARP-pr0-TGFβ1 complexes. However, only Ab2 specifically bound to rat LTBP1-pro-TGFe1 complexes. Table 10. Affinity of Ab1 and Ab2 for pr0-TGFβ1 complexes from various species.

Example 3: Ab1 and Ab2 inhibit endogenous TGFβ1 in human and murine fibroblasts

[00319] To determine whether Ab1 and Ab2 were capable of inhibiting endogenous TGFβ1 secreted by cultured primary fibroblasts of different origins, a quantitative in vitro assay was performed in which the activity of secreted TGFβ1 was determined by measuring the levels of luciferase produced by epithelial cells mink lung cells that were stably transfected with a nucleic acid comprising a luciferase reporter gene fused to a synthetic CAGA12 promoter, and co-cultured with fibroblasts treated with Ab1 or Ab2. As shown in Figures 11A and 11B, both Ab1 and Ab2 inhibited endogenous TGFβ1 secreted by normal human dermal fibroblasts, C57BL.6J murine lung fibroblasts, and DBA2/J muscle fibroblasts. Differences in maximum inhibition observed with each antibody were cell line-specific.

Example 4: Ab2 binds to LRRC33-pr0-TGFβ1

[00320] To determine whether Ab1 and Ab2 bind to pro-TGFβ1 that is complexed with LRRC33, Octet binding assays were performed. As shown in Figures 12A and 12B, both Ab1 and Ab2 are capable of binding to the LRRC33-pr0-TGFβ1 protein complex. However, Ab1 shows a slow on-rate for binding to the LRRC33-pr0-TGFβ1 protein complex. The binding of Ab1 and Ab2 to the LRRC33-pro-TGFβ1 protein complex was further confirmed using ELISA.

Example 5: Ab1 and Ab2 inhibit the activity of both GARP-pr0-TGFβ1 and LRRC33-pr0-TGFβ1

[00321] To determine whether Ab1 and Ab2 inhibit the activity of GARP-pro-TGF-P1 and/or LRRC33-pro-TGFβ1, an in vitro cell-based assay was performed. In this assay system, a genetically modified human colon cancer cell line (SW480/06 cells) stably transfected with β6 integrin was cotransfected with a construct to express pro-TGF-P1 and a construct to express a presentation molecule (or i.e. GARP or LRRC33). To express the display molecules, constructs encoding chimeric LRRC33-GARP (SEQ ID NO: 85) or GARP were employed. Transfected cells were incubated to allow sufficient expression and deposition of the components (integrins and pr0-TGFβ1 complexed with a respective presentation molecule). Activation of TGFβ1 in the presence or absence of Ab1 or Ab2 was tested using reporter cells (CAGA12 cells) that express TGFβ receptors coupled to its downstream signal transduction pathway, to mediate the inhibitory activity of the antibody. As shown in Figures 13A and 13B, Ab1 and Ab2 inhibited both GARP-pro-TGF-β1 and LRRC33-pro—TGF—β1.

[00322] An additional cell-based assay was performed to detect inhibition of the GARP-pro-TGFβ1 complex or the LRRC33-pr0-TGFβ1 complex using the Ab1 and Ab2 antibodies. As shown in Figures 22A and Figure 22B, Ab1 and Ab2 inhibited both GARP-pr0-TGF-β1 and LRRC33-pro-TGF-β1. In this assay, the IC50 (μ m/ml) of Ab1 for the GARP-TGFβ1 complex was 0.445, and the IC50 (μ m/ml) of Ab1 for the LRRC33-TGFβ1 complex was 1.325.

Example 6: Effects of Ab2 on biomarkers and renal fibrosis in a mouse model of Unilateral Ureteral Occlusion (UUO)

[00323] The mouse model of unilateral ureteral occlusion has been widely used to study interstitial fibrosis, a common pathological process that can lead to end-stage renal disease (see Isaka et al. (2008) Contrib. Nephrol. 159: 109- 21, and Chevalier (1999) Pediatr. Mice with UUO are characterized by renal myofibroblast activation, tubular atrophy, and interstitial fibrosis, with minimal glomerular lesions (see Lian et al. (2011) Acta Pharmacol. Sin. 32: 1513-21). Increased expression of TGFβ1 is thought to play a role in the phenotype observed in mice with UUO. To evaluate the effect of Ab2 on the presentation of interstitial fibrosis in the mouse model of UUO, the following experiment was performed.

[00324] Briefly, 78-week-old male CD-1 mice (Charles River Laboratories) were divided into 4 groups of mice (n = 10) that received administration of Ab2 (3 mg/kg or 30 mg/kg ; dosing volume of 10 ml/kg), murine IgG1 control antibody (30 mg/kg; dosing volume of 10 ml/kg) or PBS, as vehicle control, intraperitoneally (i.p.) before surgical intervention. Treatments were administered one day before surgery (d-1), one day after surgery (d1) and 3 days after surgery (d3). On day 0 (d0), mice were anesthetized with isoflurane anesthesia in a nasal cone, and a laparotomy performed, followed by permanent right unilateral UUO surgery. An additional group of control mice (n = 8) received PBS administration as described above, but underwent exclusively sham surgery (i.e., laparotomy without ureteral occlusion). Immediately after completion of the surgical procedure, all mice received a subcutaneous injection of 0.001 mg/kg buprenorphine. Mice were sacrificed five days after surgery and tissues were collected for analysis. After collection, both kidneys were placed in ice-cold 0.9% NaCl, decapsulated and weighed. Hydroxyproline levels were evaluated to assess the collagen content of renal tissue. As shown in Figure 14, kidney levels of hydroxyproline, a marker of fibrosis and tissue collagen deposition, were significantly increased in mice receiving surgical intervention compared to mice receiving sham surgery.

[00325] A mid-section of each right kidney was fixed by immersion in 10% neutral buffered formalin for 48 hours, which was then transferred to 70% ethanol for histological processing and analysis. Fixed kidney sections were embedded in paraffin, cut (three serial 5 μm sections acquired at 200-250 μm spacing per animal kidney to allow for greater sampling and representation of renal injury), stained with Picrosirius-Red, and submitted to quantitative histological analyzes using color spectrum segmentation to determine cortical collagen volume fraction (CVF). A composite FVC score was calculated for each animal by determining the mean FVC score for each of the three serial sections. Statistical analyzes were performed using the unpaired t-test. As shown in Figure 16, renal cortical fibrosis, as determined by FVC, was increased in UUO-obstructed kidneys compared to sham-treated control mice. Mice receiving 3 mg/kg or 30 mg/kg Ab2 showed a significant attenuation in UUO-induced increases in FVC compared to mice receiving vehicle control (PBS) or IgG control.

[00326] The relative levels of mRNA expression of plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), TGFβ1, fibronectin-1, smooth muscle actin-α (SMA-α ), monocyte chemotactic protein 1 (MCP-1), collagen type I alpha 1 (Col1a1), and collagen type III alpha 1 chain (Col3a1) in the collected kidney tissue were determined (Figure 15A-15H). mRNA levels were normalized using hypoxanthine phosphoribosyltransferase 1 (HPRT1) housekeeping gene mRNA levels. Furthermore, in mice receiving 3 mg/kg or 30 mg/kg of Ab2 before surgical intervention, the mRNA levels of PAI-1, CTGF, TGFβ1, fibronectin 1, Col1a1, and Col3a1 were significantly decreased, compared with mice that received 3 mg/kg or 30 mg/kg of Ab2 before surgical intervention. receive 30 mg/kg IgG1 control. In mice receiving 3 mg/kg of Ab2 before surgical intervention, SMA-α mRNA levels were significantly decreased compared to mice receiving 30 mg/kg of IgG1 control. Furthermore, in mice receiving 30 mg/kg of Ab2 before surgical intervention, MCP-1 mRNA levels were significantly decreased compared to mice receiving 30 mg/kg of IgG1 control.

[00327] In summary, significant effects were observed in mice treated with Ab2 in the UUO mouse model, with the exception of hydroxyproline levels. As shown in Figures 15A-15H and 16, Ab2 treatment significantly attenuated the UUO-induced increases in FVC, and significantly decreased gene expression of known fibrosis markers, e.g., PAI-1, CTGF, TGFβ1, fibronectin 1, Col1a1. and Col3a1. These data demonstrate that TGFβ1 is the major form of TGFβ, playing a role in kidney disease and that, surprisingly, TGFβ2 and TGFβ3 are unlikely to be involved in pathogenesis.

Example 7. Effects of Ab1 and Ab2 alone or in combination with anti-PD-1 antibody on tumor progression in the MC38 murine colon carcinoma syngeneic mouse model

[00328] To evaluate the effects of Ab1 and Ab2, alone or in combination with an anti-PD-1 antibody to decrease colon carcinoma tumor progression, the C57BL/6 syngeneic MC38 murine colon carcinoma mouse model was used.

Tumor cell culture

[00329] MC38 murine colon carcinoma cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 25 μg/ml ml of gentamicin and 2 mM glutamine. Cell cultures were maintained in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air.

IN VIVO tumor implantation and growth

[00330] MC38 cells used for implantation were collected during log phase growth and resuspended in phosphate buffered saline (PBS). On the day of tumor implantation, each test mouse was injected subcutaneously into the right flank with 5 x 105 cells (0.1 ml cell suspension), and tumor growth was monitored as the average size approached the range -target from 80 to 120 mm3. Eleven days later, designated as Day 1 of the study, mice were separated into groups according to calculated tumor size, each consisting of twelve animals with individual tumor volumes ranging from 63 to 196 mm3 and group mean tumor volumes. 95 to 98 mm3. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula: where w = width and l = length, in mm, of the tumor. Tumor weight can be estimated by assuming that 1 mg is equivalent to 1 mm3 of tumor volume.

Treatment

[00331] Briefly, eight-week-old female C57BL/6 mice (n=12) harboring subcutaneous MC38 tumors (63-172 mm3) on Day 1 were administered intraperitoneally (i.p.) twice weekly for four weeks of Ab1, Ab2, murine IgG1 control antibody (each at 30 mg/kg in a dosing volume of 10 ml/kg). When tumors reached 150 mm3 (Day 6) in the control groups, mice were administered rat anti-mouse PD-1 antibody (RMP1-14) or rat IgG2A control antibody i.p. twice weekly for two weeks (each antibody at 5 mg/kg in a dosing volume of 10 ml/kg).

[00332] Group 1 served as tumor growth controls, and received murine IgG1 isotype control antibody in combination with rat IgG2a control antibody. Group 2 received Ab1 in combination with rat IgG2a control antibody. Group 3 received Ab2 in combination with rat IgG2a control antibody. Group 3 received murine IgG1 control antibody in combination with anti-PD-1 antibody. Group 4 received Ab1 in combination with anti-PD-1 antibody. Group 5 received Ab2 in combination with anti-PD-1 antibody. Group 6 (n=16) was not treated and served as a sampling control group.

End point and tumor growth retardation (TGD) analysis

[00333] Tumors were measured using calipers twice a week, and each animal was sacrificed when its tumor reached the endpoint volume of 1,000 mm3 or at the end of the study (Day 60), whichever came first. Mice that exited the study by the tumor volume endpoint were documented as euthanized for tumor progression (TP), with the date of sacrifice. The time to endpoint (TTE) for analysis was calculated for each mouse using the following equation:x where TTE is expressed in days, endpoint volume is expressed in mm3, b is the intercept, m is the slope of the line obtained by linear regression of a log-transformed tumor growth data set. The data set consisted of the first observation that exceeded the endpoint volume used in the analysis and the three consecutive observations that immediately preceded obtaining that endpoint volume. The calculated TTE is typically less than the PT date, 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 in which the calculated log-transformed TTE preceded the day before reaching the tumor volume endpoint or exceeded the day of reaching the tumor volume endpoint, a linear interpolation was performed to approximate the TTE. Mice classified as having died from causes unrelated to non-treatment (NTR) were excluded from TTE calculations (and all subsequent analyses). Animals classified as TR (treatment-related) or NTRm (non-treatment-related death due to metastasis) deaths received a TTE value equal to the day of death.

[00334] The final result of treatment was evaluated from tumor growth retardation (TGD), defined as the increase in the median time to end point (TTE) in a treatment group, compared to the control group: expressed in days, or as a percentage of the median TTE of the control group: where: T = median TTE for a treatment group, and C = median TTE for the designated control group.

MTV and criteria for regression responses

[00335] The effectiveness of the treatment can be determined by the tumor volumes of animals that remain in the study on the last day. The MTV (n) was defined as the median tumor volume on the last day of the study in the number of remaining animals (n) whose tumors did not reach the endpoint volume.

[00336] The effectiveness of the treatment can also be determined from the incidence and magnitude of regression responses observed during the study. Treatment can cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume was 50% or less than its volume on Day 1 for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm3 for one or more of these three measurements. In a CR response, the tumor volume was less than 13.5 mm3 for three consecutive measurements during the course of the study. An animal with a CR response at the termination of a study was further classified as a tumor-free survivor (TFS). Animals were monitored for regression responses.

Inhibition of tumor growth

[00337] Tumor growth inhibition (TGI) analysis evaluates the difference in median tumor volumes (MTVs) of treated and control mice. For this study, the end point for determining TGI was Day 29, which was the day that control mice reached a mean tumor volume of 1,500 mm3. MTV (n), the median tumor volume for the number of animals, n, on the day of TGI analysis, was determined for each group. Percent tumor growth inhibition (%TGI) was defined as the difference between the MTV of the designated control group and the MTV of the drug-treated group, expressed as a percentage of the MTV of the control group:

[00338] The data set for the TGI analysis included all mice in one group, except those that died due to treatment-related (TR) or non-treatment-related (NTR) causes before the day of the TGI analysis.

[00339] In the present study, Ab1 and Ab2 were evaluated alone and in combination with anti-PD-1 in the C57BL/6 syngeneic mouse model of MC38 murine colon carcinoma. Mice administered Ab2 in combination with anti-PD-1 resulted in significant GIT on Day 29 (P < 0.05, Mann-Whitney U test), producing a survival benefit that was statistically significantly different from controls treated with vehicle using Log-rank survival analyzes (P < 0.05, Log-rank) (see Figure 17). Mice receiving Ab1 or Ab2 in combination with rat IgG2a control antibody had regression responses of 1 CR and 1 PR, respectively. In combination with anti-PD-1, Ab1 and Ab2 regression responses were 1 PR and 1 CR, and 4 CRs, respectively. Ab2 in combination with anti-PD-1 produced significant short-term efficacy at Day 29 and produced an overall survival benefit in this 60-day TGD study in the C57BL/6 syngeneic mouse model of MC38 murine colon carcinoma.

Example 8: Role of TGFβ1 in muscular dystrophy

[00340] TGFβ plays several roles in skeletal muscle function, including inhibiting myogenesis, regulating inflammation and muscle repair, and promoting fibrosis. Although there is considerable interest in TGFβ inhibition as a therapy for a wide range of diseases, including muscular dystrophies, these therapies inhibit TGFβ1, TGFβ2, and TGFβ3 regardless of molecular context. The lack of specificity/selectivity of these inhibitors can result in unwanted side effects that lead to clinical doses with insufficient efficacy. Although pan-TGFβ inhibitory molecules have been reported to improve muscle function and reduce fibrosis in the mdx mouse, it remains to be clarified whether those effects are caused by inactivation of TGFβ1, β2, or β3.

[00341] For this purpose, antibodies have been generated that specifically block integrin-mediated activation of latent TGFβ1, which spares TGFβ2 and β3. D2.mdx mice are treated with antibodies specific for pro-TGFβ1, in order to verify the role of TGFβ1 specifically in muscle repair in dystrophic muscle. The functional effects of TGFβ1 inhibition on protection from contraction-induced injury are evaluated, as well as on recovery from the same method of injury. Histological evaluation includes whether treatment affects muscle damage, fibrosis, and inflammation. Additionally, potential toxicities can be evaluated to determine whether the observed negative effects reported with pan-TGFβ inhibition in muscle (e.g., increased inflammation, long-term deficits in muscle function) are due to TGFβ1 or TGFβ2/3 inhibition. . To understand whether TGFβ1 inhibition in specific molecular contexts is more effective and/or has fewer negative effects (adverse effects), the efficacy of LTBP-pr0-TGFβ1 inhibitors in this model can be evaluated in order to clarify the role of TGFβ1 presented. by the immune cell from that presented in the extracellular matrix (ECM), potentially leading to safer and/or more effective antifibrotic therapies.

[00342] Dystrophic muscle is highly susceptible to contraction-induced injury. After injury, muscle from mdx mice shows a significant reduction in force generation and increased Evan's Blue dye uptake, indicative of injury/physical damage to the muscle fiber, compared to WT (Lovering, R.M., et al., Arch. Phys. Med. Rehabil., 2007. 88 (5): pages 617-25). Therapeutic agents that reduce the extent of contraction-induced injury, or improve recovery after injury, would be of significant clinical benefit for patients with muscular dystrophy (Bushby, K., et al., Lancet Neurol., 2010. 9(1): pages 77-93). Ab1 and Ab2 will be evaluated for their ability to i) prevent contraction-induced injury, as well as ii) promote injury recovery. The D2.mdx strain can be used for our experiments, unlike the traditional mdx strain in the B10 background level. These mice, generated by crossing mdx onto a DBA2/J background, possess the non-protective LTBP4 variant described above and therefore exhibit disease pathology that is more severe, progressive, and similar to human disease than the mdx strain. standardized (Coley, W.D., et al., Hum. Mol. Genet., 2016. 25 (1): pages 130-45). To the extent that D2.mdx mice are being used, DBA2/J mice can serve as wild-type controls. Because DMD primarily affects males, studies can focus on male mice.

[00343] To examine the ability of Ab1 and Ab2 to prevent/limit contraction-induced injury, 6-week-old male D2.mdx mice (n=10) are treated with 10 mg/kg/week of IgG control, Ab1 or Ab2 for 6 weeks. To allow comparison with published work using a pan-TGFβ inhibitor, a fourth group is dosed with 10 mg/kg/week of 1D11. All antibodies are of the mIgG1 isotype and it has previously been demonstrated that this dose is effective in the UUO model (Figures 15 and 16). A WT group dosed with the IgG control is also included. Twenty-four hours before sacrifice, mice are administered 1% Evan's Blue Dye (EBD) in PBS (1% volume of body weight) to allow assessment of myofiber damage by fluorescence microscope. At the end of treatment, the mice are subjected to an in vivo eccentric contraction protocol. The eccentric lesion of the gastrocnemius muscle will be performed with a 305B muscle lever system (Aurora Scientific), as described (Khairallah, R.J., et al., Sci. Signal., 2012. 5 (236): page ra56). Briefly, 20 eccentric contractions are performed with 1-minute pauses in between, and a decrease in peak isometric strength before the eccentric phase can be considered as an indication of muscle damage. The extent of strength loss and the percentage of EBD-positive fibers can be determined. DBA2/J mice subjected to this protocol lose 30-40% of initial strength after 20 eccentric contractions. In contrast, D2.mdx mice lose 80% of initial strength following the same protocol as previously described (Pratt, S.J., et al., Cell. Mol. Life Sci., 2015. 72 (1): pages 153-64; Khairallah, R.J., et al., Sci. Signal., 2012. 5 (236): page ra56). The ability of Ab1 and Ab2 to reduce strength loss after injury can be assessed. The mice are sacrificed at the end of the experiment and both injured and uninjured gastrocnemius muscles can be collected for histological analysis. EBD uptake can be assessed from both muscles. The cross-sectional area of the myofiber and the extent of fibrosis can be measured. To determine the cross-sectional area, sections from the middle part of the muscle can be stained with wheat germ agglutinin conjugated to a fluorophore to visualize cell membranes. Sections can be digitized using a fluorescent microscope, cell boundaries traced using predictive software, and cross-sectional area determined using balanced automated measurements. For fibrosis analysis, sections can be stained with Picrosirius-Red (PSR) and the PSR+ area per slide computed.

[00344] The ability of Ab1 and Ab2 to accelerate recovery from contraction-induced injury is evaluated. 12-week-old DBA2/J 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 an IgG control (for WT and D2.mdx mice), 1D11, Ab1, or Ab2 (D2.mdx only). Antibodies can be dosed at 10 mg/kg/week for the duration of the experiment. Seven and 14 days post-injury, maximum peak isometric strength, twitch-to-tetanic ratio, and force-frequency relationship can be measured to assess the effect of treatment on injury recovery. Although Ab1 and Ab2 inhibit TGFβ1 release independently of the presenting molecule, selective release of TGFβ1 by the extracellular matrix (i.e., presented by LTBP) could have a greater benefit in DMD as a consequence of preservation of TGFβ1-driven Treg activity. To address this question, LTBP-pr0-TGFβ1-specific inhibitory antibodies can also be evaluated for both the ability to prevent contraction-induced injury and to accelerate recovery from injury.

Example 9: Role of TGFβ1 in skeletal muscle regeneration after acute injury.

[00345] The role of TGFβ1 specifically in myofiber regeneration after muscle injury can be investigated. TGFβl-specific antibodies can be employed in the cardiotoxin injury model to determine the role of TGFβ1 specifically during myofiber regeneration. Regeneration can be evaluated histologically and functional assessments of muscle strength and quality can be performed. Considering the potential benefits of TGFβ1 inhibition for muscle regeneration, therapies that have beneficial effects without the toxicities seen with pan-TGFβ inhibition would be highly beneficial. This allows for an investigation of the effects of TGFe1-specific inhibition on satellite cell function and may provide data on satellite cell transplantation studies.

[00346] As described above, TGFβ appears to have multiple effects on muscle biology, including inhibition of myoblast proliferation and differentiation, as well as the promotion of atrophy and fibrosis (Allen, R.E. and L.K. Boxhorn, J. Cell. Physiol., 1987 . 133 (3): pages 567-72; Brennan, T.J., et al., Proc. Proc. Natl. Sci. U.S.A., 1986. 83 (21): pages 8,206-10; , Y., et al., Am. J. Pathol., 2004. 164 (3): pages 1,007-19; Mendias, C.L., et al., Muscle Nerve, 2012. 45 (1): pages 55-9; Nelson, C.A., et al., Am. J. Pathol., 2011. 178 (6): pages 2,611-21). However, these studies used recombinant TGFβ1 in culture or injected into mice and may have non-physiological results, as the growth factor is removed from its molecular context. Alternatively, investigators have used TGFβ inhibitors that are not selective for TGFβ1.

[00347] To evaluate the isoform-specific effects of TGFβ1, multiple antibodies against pr0-TGFβ1 (e.g., Ab1 and Ab2) can be examined for their ability to affect muscle regeneration after CTX-induced injury. These antibodies are “isoform-specific” and “context-permissive” inhibitors of TGFβ1 activation, such that they specifically inhibit the release of TGFβ1 (as opposed to TGFβ2 or TGFβ3) by any presenting molecule and do not bind to TGFβ1 factors. mature growth (Figure l8A).

[00348] Muscle regeneration can be induced in male DBA2/J mice (n=10) by injecting CTX into the right gastrocnemius muscle. One day before injury, mice can be administered 10 mg/kg of control IgG, lDll, Abl, or Ab2. Antibodies are continued until measured weekly until the end of the study. At 7 and 14 days post-injury, muscle strength measurements can be made in vivo with a 305C muscle lever system (Aurora Scientific Inc., Aurora, CAN). Briefly, for the dorsal flexor muscle group, contractions are evoked by percutaneous electrical stimulation of the sciatic nerve in anesthetized mice, and a series of stimuli is then performed at increasing stimulus frequency (0.2 ms pulse, train duration 500 ms): 1, 10, 20, 40, 60, 80, 100, 150 Hz, followed by a final stimulus at 1 Hz. Maximum peak isometric force, twitch-to-tetanic ratio and force-frequency relationship will be determined. After strength measurements, the injured gastrocnemius and soleus muscles are collected and prepared for histology. The myofiber cross-sectional area and %PSR+ area can be determined as described in Example 8 above.

[00349] Treatment with Ab1 and Ab2 can result in reduced fibrosis and improved muscle function. However, considering the role of TGFβ1 in regulating immune activation, it is possible that we may observe increased inflammation with antibodies, as has been reported with 1D11 treatment (Andreetta, F., et al., J. Neuroimmunol., 2006. 175 (1-2): pages 77-86). If increased inflammation may limit the therapeutic effects of TGFβ1 inhibition, context-specific antibodies can subsequently be evaluated to provide an additional degree of specificity, which may limit toxicity. For example, antibodies that inhibit the release of TGFβ1 by LTBPs can only be used using the readouts and methods described above. These antibodies can limit the release of TGFβ1 only by the ECM, without affecting the release by Tregs or macrophages.

Example 10: Selection of suitable TGFβ1 inhibitory agents.

[00350] Analysis of the expression of pr0-TGFβ1 and its presenting molecules in healthy, regenerating and diseased muscle can provide useful information to aid in the selection of an optimal therapeutic approach. Considering the potential benefits of TGFβ1 inhibition in muscle regeneration and repair, understanding the context of pr0-TGFβ1 presentation (e.g., in ECM or immune cells) in skeletal muscle under different conditions (healthy, acutely injured, and chronically injured) may help inform the therapeutic utility of antibodies and ultimately provide insight into the degree of specificity/selectivity required to achieve both clinical efficacy and safety. The nature of TGFβ1 presentation may vary depending on the health status of the muscle and over the course of the disease, which could have implications for any therapies targeting TGFβ1. Understanding the expression profiles of these molecules will also help in selecting appropriate dosing timing for potential therapeutic molecules. With the use of western blot, immunohistochemistry and immunoprecipitation, the expression of pr0-TGFβ1 and its presentation molecules can be evaluated in normal, acutely injured (cardiotoxin injury) and chronically regenerating muscle (D2.mdx mouse). The expression of these molecules can be specifically investigated in crucial cell types or subset of cell types (e.g., satellite cells, macrophages, fibro-adipogenic progenitors, etc.) under the different conditions described above.

[00351] Although the expression of TGFβ isoforms has been examined in muscles of mdx mice, previous work has focused on the expression of mature growth factors (Nelson, C.A., et al., Am. J. Pathol., 2011. 178 ( 6): pages 2,611-21; Zhou, L., et al., Neuromuscul., 2006. 16 (1): pages 32-8). Considering the target specificity of the antibodies against TGFβ1 described in this specification, it is essential that expression patterns are examined not only for mature TGFβ1 and prδ-TGFβ1, but also those of the presenting molecules, which should provide information as to the source and /or context of a TGFβ1 pool of interest. Ideally, it is desirable to gain understanding of the expression patterns of latent complexes, not simply of each component.

[00352] Antibodies are evaluated by western blot and IHC for targets of interest. Antibodies against mouse TGFβl-LAP, LTBP1, LTBP3 and LTBP4 are commercially available. The antibody against TGFβl-LAP (clone TW7-16B4) has been extensively characterized and is effective in both flow cytometry and western blot (Oida, T. and H.L. Weiner, PLoS One, 20l0. 5 (ll): page el5523). Antibodies against LTBPl (ProteinTech #22065-l-AP) and LTBP3 (Millipore #ABT3l6) were validated in-house using SW480 cells transfected with LTBP1-pro-TGFe1 or LTBP3-pro-TGFe1 and demonstrated that they are specific for their targets. The usefulness of these antibodies for IHC can be determined. Muscles from healthy and D2.mdx mice are cut and antibodies tested on frozen and FFPE sections. Antibodies can be validated by including conditions with 100x excess target protein or purified complex (made in-house, see Figures 18B, for example) to ensure that the observed signal is specific.

[00353] Previous work identified antibodies that specifically bind to a certain latent complex, but do not have inhibitory activity. Antigen binding by these antibodies was confirmed by ELISA (Figures 18B and 18C) and can also be evaluated for their usefulness in IHC (considering the three-dimensional structure of these epitopes, these antibodies are unlikely to be effective as western blot reagents). . The presence of latent TGFβ1 complexes from bulk tissue can also be assessed by western blot or immunoprecipitation. Latent complexes can be identified by western blot by processing the same sample under reducing and nonreducing conditions. Under reducing conditions, TGFβ1, LAP, and the presenting molecule separate, and all three molecules can be identified on the same blot but using two-color western blot methods. Under nonreducing conditions, the LAP:presentation molecule complex remains associated, while TGFβ1 is released; the complex migrates more slowly than the empty presentation molecule and migrates together with TGFβ1-LAP (Figure 18B). Various antibodies are also evaluated for their ability to immunoprecipitate latent muscle complexes to demonstrate direct TGFβ1 binding to specific presenting molecules.

[00354] After appropriate antibodies have been identified, expression in healthy, regenerating and dystrophic muscle is evaluated, by western and/or IHC, depending on the antibodies available. Tibialis anterior (TA) muscle and diaphragm can be harvested from DBA2/J and D2.mdx mice at 4, 8, and 12 weeks of age. For regenerating muscle, cardiotoxin can be injected into the TA of 12-week-old DBA2/J mice, and the muscles harvested at 3, 7, and 14 days post-injury. Tissues from at least 4 mice can be used for each condition/time point. Co-staining experiments can also be performed to identify cell populations that express the various molecules (e.g. CD11b for macrophages, FoxP3 for Tregs, MyoD for myogenic cells).

Example 11: Ab2 exhibits reduced toxicity compared to the ALK5 kinase inhibitor LY2109761 and a pan-TGFβ antibody

[00355] To evaluate the toxicity of Ab2, compared with the small molecule TGF-β receptor type I (ALK5) kinase inhibitor LY2109761 and with a pan-TGFβ antibody (hIgG4), toxicity studies were performed in rats. Briefly, female F344/NHsd mice were administered Ab2 at 3 mg/kg (1 group, n=5), 30 mg/kg (1 group, n=5) or 100 mg/kg (1 group, n=5); a pan-TGFβ antibody at 3 mg/kg (1 group, n=5), at 30 mg/kg (1 group, n=5) or at 100 mg/kg (1 group, n=5); LY2109761 at 200 mg/kg (1 group, n=5) or 300 mg/kg (1 group, n=5); or PBS vehicle control (pH 7.4) (1 group, n=5). Animals receiving Ab2, the pan-TGFβ antibody, or vehicle control were dosed once intravenously (on day 1), and mice receiving LY2109761 were dosed by oral gavage once daily for 7 days (7 doses). . Animal body weight was determined on days 1, 3 and 7 of the dosing phase. The animals were sacrificed on day 8 and necropsies were performed.

[00356] As shown in the survival data shown in Figure 19, Ab2 exhibited reduced toxicity compared to the other treatment groups. All animals that received administration of 300 mg/kg of the ALK5 kinase inhibitor LY2109761 were euthanized in a moribund condition or were found dead on days 3, 6, or 7 of the study. Two of the animals administered 200 mg/kg LY2109761 were found dead on day 7 of the study. One animal administered 100 mg/kg of the pan-TGFβ antibody was found dead on day 6 of the study. All animals administered up to 100 mg/kg of Ab 2 survived until terminal sacrifice.

[00357] Furthermore, the toxicity of the treatments was assessed by monitoring the body weights of the animals during the dosing phase. As shown in Figures 20 and 21A-21C, animals receiving LY2109761 at 200 mg/kg or 300 mg/kg exhibited decreased body weight throughout the course of the study.

[00358] The weight of the animal's organs was also evaluated post-mortem. As shown in Table 11, increased heart weights were observed in animals administered >200 mg/kg LY2109761. Increased heart weights were also observed in animals administered >30 mg/kg of the pan-TGFβ antibody. No effect on organ weight was observed in animals administered up to 100 mg/kg Ab2. Table 11. Changes in organ weights across treatment groups. NE = not evaluated due to early mortality. Note: Values for absolute weight and proportion of organ weights (in relation to body or brain) for each treatment group expressed as mean value of the percentage control. a Vehicle control = phosphate buffered saline (PBS), pH 7.4.

[00359] Although no macroscopic findings were observed in animals administered up to 100 mg/kg of Ab2 or the pan-TGFβ antibody, an abnormally shaped sternum was observed in four animals from each treatment group that received 200 mg/kg or 300 mg/kg of LY2109761. 2.5 ml of clear fluid was observed in the thoracic cavity and an enlarged thymus due to excess fluid (i.e., edema) was observed in an animal administered 300 mg/kg LY2109761 that was found dead on Day 3 of the study.

[00360] As shown in Table 12, at the microscopic level, animals administered > 200 mg/kg of LY2109761 exhibited heart valve findings (i.e., valvulopathy). Valvulopathy was characterized by thickening of the heart valve as a result of hemorrhage, endothelial hyperplasia, mixed inflammatory cell infiltrates, and/or stromal hyperplasia (see Figure 23, upper right panel). Most animals had multiple affected valves. Additionally, findings in the atrium were noted, including minimal to discrete mixed inflammatory cell infiltrates, minimal hemorrhage, and/or minimal hyperplasia of the endothelium (endocardium) resulting in increased basophilic staining of the atrium in hematoxylin and eosin-stained sections. Myocardial findings were also observed primarily at the base of the heart and consisted of minimal to mild degeneration/necrosis, slight hemorrhage, and/or mild mixed inflammatory cell infiltrates. One animal administered 300 mg/kg LY2109761 had mild necrosis with inflammation of a coronary artery. Additionally, two animals administered 200 mg/kg LY2109761 had minimal mixed inflammatory cell infiltrates or aortic root hemorrhage. Table 12. Microscopic findings in the heart in animals receiving LY2109761.

[00361] As shown in Table 13, animals administered > 3 mg/kg of the pan-TGFβ antibody exhibited heart valve findings (i.e., valvulopathy) similar to those described in animals administered LY2109761, as described above (see also Figure 23, lower left panel). Animals administered >30 mg/kg of pan-TGFβ antibody exhibited atrium findings similar to those described in animals administered LY2109761. Animals administered 100 mg/kg of pan-TGFβ antibody exhibited myocardial findings similar to those described in animals administered LY2109761, and animals administered 30 mg/kg of pan-TGFβ antibody had myocardial hemorrhage. One animal administered 100 mg/kg of the pan-TGFβ antibody had moderate intramural necrosis with hemorrhage in a coronary artery, which was associated with mild perivascular infiltrates of mixed inflammatory cells. Table 13. Microscopic findings in the heart in animals receiving the pan-TGFβ antibody. Pan-TGFβ antibody

[00362] In contrast, a single animal in the treatment group administered with 100 mg/kg Ab2 had minimal mixed inflammatory cell infiltrates in a single heart valve leaflet of the left atrioventricular valve (see Figure 23, lower right panel), which was consistent with a background finding due to the single incidence and absence of concomitant valvular findings. Thus, Ab2 treatment surprisingly resulted in reduced mortality and reduced cardiotoxicity compared with treatment with the ALK5 kinase inhibitor LY2109761 or with treatment with the pan-TGFβ antibody.

Claims (25)

1. Method for producing a pharmaceutical composition comprising a specific inhibitor of the TGFβ1 isoform, the method characterized by comprising the steps of: i) selecting a specific inhibitor of the TGFβ1 isoform; ii) formulate into a pharmaceutical composition comprising the specific inhibitor of the TGFβ1 isoform and a pharmaceutically acceptable excipient. 2. Pharmaceutical composition characterized by being produced by the method as defined in claim 1. 3. Method for producing a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof which is a specific inhibitor of the TGFβ1 isoform, the method characterized by comprising the steps of: i) providing one or more antibodies or fragments of antigen binding thereof that inhibit signaling of at least one TGFβ isoform selected from the group consisting of TGFβ1, TGFβ2 and TGFβ3; ii) measuring activities of one or more antibodies or antigen-binding fragments thereof in relation to all TGFβ isoforms; iii) selecting an antibody or antigen-binding fragment thereof for specific activity against activation of the TGFβ1 isoform, wherein the selected antibody or antigen-binding fragment thereof specifically inhibits TGFβ1, but not TGFβ2 or TGFβ3 ; iv) formulate into a pharmaceutical composition the specific inhibitor of the TGFβ1 isoform selected in step iii) and a pharmaceutically acceptable excipient. 4. Method, according to claim 3, characterized by further comprising the step of selecting a specific inhibitor of the TGFβ1 isoform that is effective in vivo at a dose that does not cause cardiovascular toxicity in a preclinical model. 5. Method according to claim 4, characterized by the fact that the specific inhibitor of the TGFβ1 isoform is effective for reducing tumor growth when used in combination with a checkpoint inhibitor in a tumor model of mouse. 6. Method according to claim 3, characterized in that the antibody or antigen-binding fragment thereof specifically binds to a pro/latent TGFβ1 complex comprising GARP, LRRC33, LTBP1 or LTBP3. 7. Method according to claim 6, characterized by the fact that the antibody or antigen-binding fragment thereof specifically binds to the pro/latent complex of TGFβ1, but does not bind to free mature TGFβ1 that is not associated with a pro/latent complex. 8. Method according to claim 7, characterized by the fact that the pro/latent complex comprises GARP, LRRC33, LTBP1 or LTBP3. 9. Method according to claim 6, characterized in that the antibody or antigen-binding fragment thereof inhibits the release of mature TGFβ1 from the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex and/or LRRC33-TGFβ1 complex. 10. Method according to claim 1 or 3, characterized in that the antibody or antigen-binding fragment thereof is a neutralizing antibody or antigen-binding fragment thereof. 11. Method according to claim 1 or 3, characterized by the fact that the specific inhibitor of the TGFβ1 isoform cross-competes with an antibody or an antigen-binding fragment thereof, comprising six complementarity determining regions (CDRs ): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, wherein CDRH1 comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, wherein CDRH2 comprises an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4, wherein CDRH3 comprises an amino acid sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 6, wherein CDRL1 comprises an amino acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 8, wherein CDRL2 comprises an amino acid sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 10, wherein CDRL3 comprises an amino acid sequence as set forth in SEQ ID NO: 11 or SEQ ID NO: 12. 12. Method according to claim 1 or 3, characterized in that it further comprises a step of: selecting an antibody or an antigen-binding fragment thereof, which cross-competes with an antibody or an antigen-binding fragment thereof. same, comprising six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, wherein CDRH1 comprises an amino acid sequence as set forth in SEQ ID NO: 2, wherein CDRH2 comprises an amino acid sequence as set forth in SEQ ID NO: 2, wherein CDRH2 comprises an amino acid sequence as set forth in SEQ ID NO: 4, wherein CDRH3 comprises an amino acid sequence as set forth in SEQ ID NO: 6, wherein CDRL1 comprises an amino acid sequence as set forth in SEQ ID NO: 8, wherein CDRL2 comprises an amino acid sequence as set forth in SEQ ID NO: 10, wherein CDRL3 comprises an amino acid sequence as set forth in SEQ ID NO: 12. 13. Method according to claim 1 or 3, characterized in that the specific inhibitor of the TGFβ1 isoform achieves reduced expression of at least one gene selected from the group consisting of: PAI-1, CTGF, MCP-1, TGFB1, α-SMA, fibronectin-1, Col1a1 and Col3a1. 14. Pharmaceutical composition characterized by being produced by the method as defined in any one of claims 1 or 3 to 13. 15. Pharmaceutical composition according to claim 2 or 14, characterized by the fact that the specific inhibitor of the TGFβ1 isoform is in a dose of about 0.1 μg/kg to about 30 mg/kg. 16. Pharmaceutical composition according to claim 2 or 14, characterized in that it further comprises an additional agent or therapy. 17. Pharmaceutical composition according to claim 16, characterized by the fact that the additional agent is a checkpoint inhibitor. 18. Pharmaceutical composition according to claim 17, characterized in that the additional agent is selected from the group consisting of an anti-PD1 antibody, a PD-1 antagonist, a PDL1 antagonist or a PD fusion protein -L1 or PDL2, a CTLA4 antagonist, a GITR agonist, an anti-ICOS antibody, an anti-ICOSL antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody , an anti-OX40 antibody, an anti-CD27 antibody, an anti-CD70 antibody, an anti-CD47 antibody, an anti-41BB antibody, an oncolytic virus and a PARP inhibitor. 19. An isolated antibody or antigen-binding portion thereof characterized by specifically binding to an epitope of TGFβ1, wherein the epitope is available for binding by the antibody when TGFβ1 is present in two or more of the following protein complexes: a complex GARP-TGFβ1, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and a LRRC33-TGFβ1 complex; and wherein the antibody does not bind to free mature TGFβ1. 20. Use of the antibody or antigen-binding portion thereof, as defined in claim 19, or of the pharmaceutical composition, as defined in any one of claims 2 or 14 to 18, characterized in that it is in the manufacture of a medicament to inhibit activation of TGFβ1, comprising exposing a GARP-TGFβ1 Complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex or a LRRC33-TGFβ1 complex to the antibody, or to the antigen-binding portion thereof, or to the pharmaceutical composition. 21. Use of the antibody or antigen-binding portion thereof, as defined in claim 19, or of the pharmaceutical composition, as defined in any one of claims 2 or 14 to 18, characterized in that it is in the manufacture of a medicament for the treatment of a disease associated with TGFβ signaling. 22. Use according to claim 21, characterized by the fact that the disease is myofiber damage, cancer or myelofibrosis. 23. Use of a TGFβ1 isoform-specific inhibitor in the manufacture of a medicament for treating cancer or myelofibrosis, wherein the isoform-specific inhibitor is: (a) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a CDRH1, CDRH2 and CDRH3 and a light chain variable region comprising a CDRL1, CDRL2 and CDRL3, wherein: CDRH1 comprises a SEQ ID NO: 2; CDRH2 comprises a SEQ ID NO: 4; CDRH3 comprises a SEQ ID NO: 6; CDRL1 comprises a SEQ ID NO: 8; CDRL2 comprises a SEQ ID NO: 10; and CDRL3 comprises a sequence as set out in the sequence as set out in the sequence as set out in the sequence as set out in the sequence as set out in SEQ ID NO: 12; (b) an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region comprising a CDRH1, CDRH2 and CDRH3 and a light chain variable region comprising a CDRL1, CDRL2 and CDRL3, wherein: CDRH1 comprises a sequence as set forth in SEQ ID NO: 1; CDRH2 comprises a sequence as set forth in SEQ ID NO: 3; CDRH3 comprises a sequence as set forth in SEQ ID NO: 5; CDRL1 comprises a sequence as set forth in SEQ ID NO: 7; CDRL2 comprises a sequence as set forth in SEQ ID NO: 9; and CDRL3 comprises a sequence as set forth in SEQ ID NO: 11; or or (c) an antibody or antigen-binding fragment thereof that competes or cross-competes with the antibody defined in parts (a) or (b) by binding to TGFβ1; wherein antibodies or antigen-binding fragments thereof specifically bind to a pro/latent TGFβ1 complex, comprising LTBP1, LTBP3, GARP or LRRC33; wherein antibodies or antigen-binding fragments of the same isoform-specific inhibitor do not specifically bind to TGFβ2 or TGFβ3. 24. Nucleic acid characterized by encoding the antibody or an antigen-binding portion thereof, as defined in claim 19, or the pharmaceutical composition, as defined in any one of claims 2 or 14 to 18. 25. Kit characterized by comprising the antibody or an antigen-binding portion thereof, as defined in claim 19, or the pharmaceutical composition, as defined in any one of claims 2 or 14 to 18.