EA045239B1 - ISOFORM-SPECIFIC, CONTEXT-PERMISSIVE TGFβ1 INHIBITORS AND THEIR APPLICATIONS - Google Patents

Description

This application claims benefit and priority under 35 U.S.C. § 119(e) of the following applications: US Provisional Application No. 62/443615, filed January 6, 2017; US Provisional Application No. 62/452866, filed January 31, 2017; US Provisional Application No. 62/514417, filed June 2, 2017; US Provisional Application No. 62/529616, filed July 7, 2017; US Provisional Application No. 62/549767, filed August 24, 2017; US Provisional Application No. 62/558311, filed September 13, 2017; US Provisional Application No. 62/582527, filed November 13, 2017; US Provisional Application No. 62/587964, filed November 17, 2017, and US Provisional Application No. 62/588626, filed November 20, 2017, the contents of each of which are incorporated herein by reference in their entirety.

List of sequences

This application contains a sequence listing that was submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The specified ASCII copy, created on January 5, 2018, is named 127036-02020_ST25.txt and is 221821 bytes.

BACKGROUND OF THE INVENTION

The transforming growth factor β (TGFe) superfamily of growth factors is involved in a number of signaling cascades that regulate diverse biological processes, including but not limited to: cell growth inhibition, tissue homeostasis, extracellular matrix (ECM) remodeling, endothelial-mesenchymal transition (EMT) , cell migration and invasion and immune modulation/suppression, and mesenchymal-epithelial transition. In relation to ECM remodeling, TGFe signaling may increase fibroblast populations and ECM (e.g., collagens) deposition. In the immune system, TGFe ligand modulates T-regulatory cell function and maintains immune cell progenitor growth and homeostasis. In normal epithelial cells, TGFe is a potent growth inhibitor and promoter of cell differentiation. However, as tumors develop and progress, they often lose their negative growth response to TGFe. In this situation, TGFe may 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, TGFe has been a therapeutic target for a number of clinical indications. Despite significant efforts to date by a number of groups, successful clinical development of therapeutic TGFe has proven challenging.

Observations from preclinical studies, including rats and dogs, have identified certain toxic effects associated with TGFe inhibition in vivo. Moreover, although several TGFe inhibitors have been developed to date, most clinical programs targeting TGFe have been discontinued due to side effects (summarized, for example, in WO 2017/156500). Thus, despite a number of direct and indirect evidence implicating TGFe signaling in the progression of diseases such as cancer and fibrosis, there are no TGFe therapeutics available on the market that are safe and effective.

Among proliferative disorders, TGFe dysregulation is also involved in myelofibrosis, which is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in disease pathogenesis, ruxolitinib (Jakafi), which is a JAK1/JAK2 inhibitor approved by the FDA for the treatment of myelofibrosis, has not demonstrated efficacy in improving established bone marrow fibrosis in patients.

Thus, there is a need for improved methods and compositions for inhibiting TGFe signaling that can be used to effectively and safely treat TGFe1-related diseases and disorders, including, for example, proliferative disorders (eg, cancer), fibrosis, and inflammation.

Brief Disclosure of the Invention

The present invention embraces the recognition that blocking TGFe activation at multiple sources may provide greater clinical benefits in the treatment of a number of diseases involving both the ECM aspect and the immune aspect of TGFe dysregulation. Accordingly, this document provides improved methods for treating such diseases with TGFe1 inhibitors that are superior to traditional TGFe antagonists with respect to their isoform selectivity, breadth of molecular targets in the disease niche, duration of effects and safety.

There is a body of evidence that many diseases exhibit complex disturbances in TGFe signaling that are likely to involve heterogeneous cell types that produce differential effects of TGFe function that are mediated by its interactions with so-called presentation molecules. At least four such presenting molecules have been identified that can “present” TGFe in various extracellular niches, which

- 1 045239 would ensure its activation in response to local stimuli. In one category, TGFe is deposited in the ECM in association with ECM-associated presentation molecules such as LTBP1 and LTBP3, which mediate ECM-associated TGFe activities. In another category, TGFe binds to the surface of immune cells through presentation molecules such as GARP and LRRC33, which mediate specific immune function. These presentation molecules exhibit differential expression, localization, and/or function in different tissues and cell types, indicating that the triggering events and outcome of TGFe activation will vary depending on the microenvironment. Based on the notion that multiple effects of TGFe may interact to contribute to disease progression, therapeutics that can counteract multiple aspects of TGFe function may provide greater efficacy.

We have previously recognized that isoform-specific inhibition (as opposed to pan-inhibition) of TGFe may result in improved safety profiles of anti-TGFe in vivo (see WO 2017/156500). Taking this into account, we sought to develop TGFe1 inhibitors that are both i) isoform specific and ii) capable of broadly targeting multiple TGFe1 signaling complexes that are associated with different presentation molecules, as therapeutics for conditions caused by multifaceted effects of TGFe1 and their dysregulation.

Accordingly, the present disclosure provides isoform-specific inhibitory agents capable of targeting both ECM-associated TGFe1 and immune cell-associated TGFe1, thereby blocking multiple sources of TGFe1 presented in multiple contexts. Such inhibitory agents are referred to herein as isoform-specific, context-permissive TGFe1 inhibitors. The present invention also provides for the use of these agents as a therapeutic agent in the treatment of conditions that are characterized by dysregulation of TGFe1 signaling associated with multiple aspects of TGFe1 function. Such inhibitors may function as multifunctional agents to counteract multiple activities of TGFe1 (e.g., TGFe1 from multiple sources or contexts) to enhance clinical effects in the context of fibrosis, myelofibrosis, cancer and other conditions.

The rationale for the preferential use of context-permissive (such as context-independent) TGFe1 inhibitors over context-specific TGFe1 inhibitors as therapeutic agents for the treatment of certain diseases (as described in more detail herein) includes the following:

Involvement of heterogeneous TGFe1 complexes in the disease environment. First, various diseases involve heterogeneous cell populations as multiple sources of TGFe1 that collectively contribute to pathogenesis and/or disease progression. More than one type of TGFe1-containing complexes (contexts) are likely to coexist in the same disease microenvironment. In particular, such diseases may involve both an ECM component of TGFe1 signaling and an immune component of TGFe1 signaling. In such situations, selectively targeting one TGFe1 context (e.g., TGFe1 associated with one type of presentation molecule) may provide limited relief. In contrast, context-permissive TGFe1 inhibitors are primarily designed to more broadly target inactive (pro/latent) TGFe1 complexes and prevent growth factor activation at multiple sources before mature TGFe1 can be released to bind to the receptor to initiate onward transmission signals while maintaining isoform selectivity to minimize toxicity.

Common mechanisms underlying various diseases. Second, there are marked similarities in tissue/cellular characteristics between tumor stroma and fibrotic tissues. Indicating crosstalk between and among: i) TGFe1-dependent profibrotic phenotypes; ii) TGFe1-dependent pro-tumor phenotypes and iii) TGFe-dependent immunosuppressive phenotypes observed in a number of pathological conditions. Thus, the use of context-permissive inhibitors that broadly target many of these components may provide optimal therapeutic effects across different types of pathological conditions. For example, the clinical manifestations of primary myelofibrosis include abnormal proliferation of certain cell populations and fibrosis in the bone marrow.

Countering drug resistance. Third, a number of studies have reported cancers/tumors that are resistant to anticancer therapies such as immune checkpoint inhibitors. In some cases, such resistance appears to be intrinsic to a particular type of cancer/tumor relative to the patient's clinical background (usually referred to as true resistance, primary resistance, intrinsic resistance, or true resistance; these

- 2 045239 terms are used interchangeably in this document). Such resistance may be present in a population of patients who respond poorly to cancer treatments such as immune checkpoint inhibitors, and perhaps reflects an immune-prejudicial environment. This is likely mediated, at least in part, by a TGFβ1-dependent pathway. Thus, the isoform-selective inhibitor described herein may render resistant cancers more susceptible to such treatments.

Alternatively, resistance may develop over time such that patients who exhibit a significant clinical response to treatment become poor responders (ie, adaptive or acquired resistance). For example, it has been reported that PD-1 therapy can lead to adaptive resistance that correlates with the activation of other T cell antigens (eg, TCR components), suggesting that cancer cells evolve to evade PD-1 blockade through a different mechanism. Subsequently, a second checkpoint inhibitor targeting a different T cell receptor component, such as TIM3, may restore sensitivity to immunotherapy. These observations suggest that blocking multiple pathways to counteract the adaptive responses of cancer cells may reduce the likelihood that cancer cells can evade host immunity. Context-permissive TGFe1 inhibitors, which are capable of targeting multiple TGFe1 contexts, may preferentially circumvent acquired drug resistance by providing blockade at multiple points of TGFe1 function.

Tolerate expression plasticity: and finally, based on the notion that the expression of various presentation molecules can change over time, for example in response to local signals (e.g. cytokines, chemokines, ECM environment, etc.) and/or with changes in the disease microenvironment, it is argued that context-permissive TGFe1 inhibitors, such as those described herein, can be used to counter such plasticity and provide broad, long-lasting inhibitory effects even when abnormal changes in the expression of presenting molecules occur.

In either of these scenarios, context-permissive TGFe1 inhibitors preferentially target pro/latent forms of TGFe1 in combination with various presentation molecules, all of which or various combinations of which are present in the disease microenvironment(s). More specifically, in one method, the inhibitor targets ECM-associated TGFe1 (LTBP1/3-TGFe1 complexes). In another method, the inhibitor targets immune cell-associated TGFe1. This includes GARP-presented TGFe1, such as the GARPTGFe1 complexes expressed on Treg cells and the LRRC33-TGFe1 complexes expressed on macrophages and other myeloid/lymphoid cells, as well as some cancer cells.

Such antibodies include isoform-specific TGFe1 inhibitors that bind and prevent the activation (or release) of the mature growth factor TGFe1 from the TGFe1 pro/latent complex in a context-permissive (or context-independent) manner such that the antibodies can inhibit activation ( or release) TGFe1 associated with several types of presentation molecules. In particular, the present invention provides antibodies capable of blocking at least one context of ECM-associated TGFe1 (presented by LTBP and/or presented by LTBP3), and at least one cell-associated TGFe1 (presented by GARP and/or presented by LRRC33).

Various diseases have been proposed to involve dysregulation of TGFe signaling as a contributing factor. Indeed, the pathogenesis and/or progression of certain human conditions appears to be predominantly driven by or dependent on TGFe1 activity. In particular, many such diseases and disorders appear to involve both an ECM component and an immune component of TGFe1 function, suggesting that activation of TGFe1 in multiple contexts (e.g., mediated by more than one type of presentation molecule) is involved. In addition, cross-talk is suggested to exist between TGFe1-responsive cells. In some cases, interactions between the multifaceted activities of the TGFe1 axis may lead to disease progression, exacerbation and/or suppression of the host's ability to fight disease. For example, a specific disease microenvironment, such as the tumor microenvironment (TME), may be associated with TGFe1 presented by a variety of different presentation molecules, for example, LTBPl-pro-TGFβ1, LTBP3-pro-TGFβ1, GARP-proTGFe1, LRRC33-pro-TGFβ1 and any combinations thereof. The TGFe1 activities of one context may in turn regulate or influence the TGFe1 activities of another context, raising the possibility that when dysregulated it may lead to exacerbation of pathological conditions. Therefore, it is desirable to inhibit TGFe1 function broadly in multiple modes (i.e., in multiple contexts), while selectively limiting such inhibitory effects to the TGFe1 isoform. The goal is not to disrupt homeostatic TGFe signaling mediated by other isoforms, including TGFe3, which plays an important role in wound healing.

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To address this problem, we have attempted to create isoform-specific, context-permissive TGFe1 inhibitors that may be particularly useful for therapeutic use in the treatment of diseases that are caused by or dependent on TGFe1 signaling or dysregulation. The approach taken to meet the criteria for such inhibitors is the following: i) the ability to inhibit TGFe1 signaling in an isoform-specific manner (without interfering with the activities of TGFe2 and/or TGFe3); and, ii) the ability to inhibit both ECM-associated and immune cell-associated TGFe1 signaling. The rationale for this approach is to balance the efficacy (and therefore clinical effectiveness) of TGFe1 inhibition against potential toxicity. More specifically, achieving selectivity for TGFe1 at a therapeutic dosage over other isoforms is aimed at reducing or minimizing possible toxic effects (eg, unwanted side effects and adverse events) associated with pan-inhibition of TGFe in vivo, some of which may be required for normal biological function (such as wound healing). On the other hand, the inclusion of multiple TGFe1 contexts as a therapeutic target aims to provide or optimize clinical efficacy in a disease that involves dysregulation of multiple aspects of TGFe1 signaling. Various clinical uses and treatment regimens are covered by the present invention.

Accordingly, in one aspect, provided herein are isoform-specific, context-permissive TGFe1 inhibitors, characterized in that such inhibitors have the ability to inhibit both ECM-associated TGFp1 signaling and immune cell-associated TGFe1 signaling. In particular, such inhibitors can block TGFe1 presented in multiple contexts, e.g. TGFe1 activities mediated by two or more types of presentation molecules, while maintaining the activities of TGFe2 and TGFe3 unchanged. Thus, TGFe1 activities that may be inhibited by such inhibitors include two or more of the following: i) TGFe1 signaling associated with GARP-presented TGFe1; ii) TGFe1 signaling associated with LRRC33-presented TGFe1; iii) TGFe1 signaling associated with LTBP1-presented TGFe1; and iv) TGFe1 signaling associated with LTBP3-presented TGFe1. In some embodiments, such inhibitors target at least two or at least three proprotein forms of the following complexes: i) TGFe1-GARP; ii) TGFe1-LRRC33; iii) TGFe1-LTBP1 and iv) TGFe1-LTBP3. In some embodiments, such inhibitors are monoclonal antibodies that specifically bind and inhibit i) TGFe1GARP; iii) TGFe1-LTBP1 and iv) TGFe1-LTBP3. In some embodiments, such monoclonal antibodies specifically bind and inhibit ii) TGFe1-LRRC33; iii) TGFe1-LTBP1 and iv) TGFe1-LTBP3. In some embodiments, such monoclonal antibodies specifically bind and inhibit i) TGFe1-GARP; ii) TGFe1-LRRC33 and iii) TGFe1-LTBP1. In some embodiments, such monoclonal antibodies specifically bind and inhibit i) TGFe1-GARP; ii) TGFe1-LRRC33 and iv) TGFe1-LTBP3. In some embodiments, such monoclonal antibodies specifically inhibit all of the following complexes: i) TGFe1-GARP; ii) TGFe1-LRRC33; iii) TGFe1-LTBP1 and iv) TGFe1-LTBP3. In some embodiments, such monoclonal antibodies do not bind mature TGFe1, which is free TGFe1 (eg, a growth factor that is released or does not form a complex with the presentation molecule). An aspect of the present invention includes compositions containing such an inhibitor, including, for example, pharmaceutical compositions that are suitable for administration to human and non-human subjects to be treated. Such pharmaceutical compositions are generally sterile. In some embodiments, such pharmaceutical compositions may also contain at least one pharmaceutically acceptable excipient, such as a buffer and a surfactant (eg, polysorbates). Kits containing such a pharmaceutical composition are also covered by the present invention.

The isoform-specific, context-permissive inhibitors described herein are suitable for use in the treatment of a disease or disorder involving multiple biological functions of TGFe1 and its dysregulation. In particular, such a disease or disorder includes both an ECM component of TGFe1 function and an immune component of TGFe1 function. Therefore, administration of such an inhibitor can inhibit each axis of the TGFe1 signaling pathway in vivo, for example, multiple TGFe1 targets associated with a disease or disorder, enhancing therapeutic effects. Accordingly, in another aspect, the present invention includes the therapeutic use of such inhibitors in a method of treating a subject who suffers from a disease associated with TGFe1 dysregulation.

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Isoform-specific, context-permissive, or context-independent inhibitors of TGFe1 signaling are particularly suitable for treating disease that is caused by or depends on multiple functions (eg, both the ECM component and the immune component) of TGFe1. Typically, such diseases involve multiple cell types or cell statuses in which TGFe1 is represented by multiple types of presentation molecules (eg, in multiple contexts).

In a related aspect, the present invention provides methods for screening, preparing and manufacturing isoform-specific, context-permissive TGFe1 inhibitors with an improved safety profile (eg, reduced in vivo toxicity). Such methods require that candidate agents be screened and selected for TGFe1 isoform specificity, eg, candidate agents are selected for inhibitory activities on TGFe1 signaling rather than TGFe2 and/or TGFe3 signaling. In accordance with the present invention, such isoform-specific inhibitors of TGFe1 activities can inhibit multiple contexts of TGFe1 function (see below).

In some embodiments, such agents are antibodies or antigen-binding fragments thereof that specifically bind and block activation of TGFe1, but not TGFe2 and/or TGFe3. In some embodiments, such antibodies or antigen-binding fragments thereof do not bind free mature growth factor TGFe1 that is not associated with the pro/latent complex. Thus, suitable production methods may include a screening step that evaluates potential agents (such as potential antibodies or fragments thereof) for their ability to inhibit TGFe1, which is associated with specific presentation molecules, for example, GARP, LRRC33, LTBP1 and/or LTBP3. In some embodiments, an inactive (e.g., latent) precursor complex, such as GARP-pro-TGFβ1, LRRC33-pro-TGFβ1, LTBP1-pro-TGFβ1, and LTBP3-pro-TGFβ1, can be used in an assay to activate mature active growth factor TGFe1. Activation of TGFe1 in the presence or absence of a test agent (ie, a potential inhibitor) can be measured by any suitable means, including, without limitation, in vitro assays and cellular assays. A similar screening step can be used to examine isoform specificity using TGFe2 and/or TGFe3 analogues. Such a screening step may be conducted to identify potential agents (such as candidate antibodies or fragments thereof) for their ability to inhibit TGFe 1 signaling: i) in an isoform-specific manner and ii) in a context-permissive or context-independent manner.

Some diseases are associated with dysregulation of multiple biological roles of TGFe signaling that are not limited to one context of TGFe function. In such situations, it may be useful to modulate the effects of TGFe in multiple contexts involved in the onset and/or progression of the disease. Thus, in some embodiments, the present invention provides methods for targeting and broadly inhibiting multiple contexts of TGFp1, but in an isoform-specific manner. Such agents are referred to herein as isoform-specific, context-permissive TGFe1 inhibitors. Thus, context-permissive TGFe1 inhibitors target multiple contexts (e.g., multiple types of pro/latent-TGFe1 complexes). Preferably, such inhibitors target at least one type (or context) of the TGFe1 preactivation complex that is ECM-associated (i.e., the pro/latent TGFe1 complex presented by an ECM-associated presenting molecule), and additionally at least one the type (or context) of the TGFe1 preactivation complex associated with the cell surface (i.e., the pro/latent TGFe1 complex presented by a cell- or membrane-associated presenting molecule). In some embodiments, context-permissive TGFe1 modulators target all types of TGFe1 pro/latent complexes (e.g., GARP-associated, LRRC33-associated, LTBP-associated, etc.) to cover all contexts regardless of the specific presenting molecule (molecules).

Although context-permissive TGFe1 inhibitors are capable of targeting more than one type of pro/latent-TGFe1 complexes (i.e., with different presentation molecules), in some embodiments such inhibitors may have a preference for (or exhibit a bias towards) one or multiple contexts over other(s). Thus, in some embodiments, a context-permissive antibody that inhibits TGFe1 activation may preferentially inhibit TGFe1 activation mediated by one presentation molecule over another presentation molecule, even though such antibody is capable of binding to both types of prolatent complexes. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits the activation of LTBP1/3-associated TGFe1, GARP-associated

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TGFei and LRRC33-associated TGFei, but with preferential inhibitory activity against LTBPl/3-associated TGFe1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits the activation of LTBP1-associated TGFe1, LTBP3-associated TGFe1, GARP-associated TGFe, and LRRC33-associated TGFe1, but with preferential inhibitory activity against LTBP1- and LTBP-3-associated TGFe1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits activation of LTBP1-associated TGFe1, LTBP3-associated TGFe1, GARP-associated TGFe1, and LRRC33-associated TGFe1, but with preferential inhibitory activity against GARP-associated TGFe1 and LRRC33-associated TGFe1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits the activation of GARP-associated TGFe1 and LRRC33-associated TGFe1, but with preferential inhibitory activity against GARP-associated TGFe1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits the activation of GARP-associated TGFe1 and LRRC33-associated TGFe1, but with preferential inhibitory activity against LRRC33-associated TGFe1.

Thus, in accordance with the present invention, different degrees of selectivity can be created to target a subset of the effects of TGFe. Isoform-specific TGFe inhibitors (which target a single TGFe isoform) provide higher selectivity than so-called pan-TGFe inhibitors (which target multiple or all TGFe isoforms).

The present invention includes the use of such TGFe1 inhibitors in methods of treating a disease associated with TGFe1 dysregulation. The use of such inhibitors is particularly beneficial in settings where the TGFe1 isoform plays a dominant role (relative to TGFe2/3) in disease progression, and where the disease involves both an ECM component and an immune component of TGFe1 signaling. This approach aims to preserve normal or homeostatic TGFe function while preferentially targeting disease-associated TGFe function.

Such an inhibitor is preferably a TGFe1 activation inhibitor (ie, a TGFe1 activation step inhibitor). In preferred embodiments, such an inhibitor is capable of targeting inactive forms of TGFe1 (e.g., pro/latent-TGFe1 complexes) prior to activation to provide longer-lasting inhibition compared to targeting a transient, already activated, soluble/free form of growth factor that has been released from the latent complex. Determining the source/context of disease-associated TGFe1 can be accomplished using antibodies that specifically bind to the latent TGFp1 complex that includes the presentation molecule of interest (eg, GARP, LRRC33, LTBP1, LTBP3, etc.).

Aspects of the present invention relate to immunoglobulins, such as antibodies or antigen-binding portions thereof, that specifically bind at least three of the following complexes: the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and the LRRC33TGFe1 complex. In accordance with the present invention, such immunoglobulins specifically bind at least one type of ECM-associated (e.g., ECM-associated) TGFe1 complexes (e.g., LTBP1- and/or LTBP3-associated TGFe1 complexes) and at least one type of cell-associated (e.g., cell surface-associated) TGFe1 complexes, (eg, GARP-associated and/or LRRC33-associated TGFe1 complexes) to exert broad inhibitory effects in multiple contexts. Antibodies or antigen-binding portions thereof described herein specifically bind to an epitope of TGFe1 (e.g., LAP) or component(s) of a protein complex containing TGFe1 (e.g., LAP) that is accessible to binding by the antibodies or antigen-binding portions thereof when TGFe1 is present in the complex GARPTGFp1, LTBP1-TGFp1 complex, LTBP3-TGFe1 complex and/or LRRC33-TGFp1.

In some embodiments, the epitope is available for antibody binding when TGFe1 is present in two or more of the following protein complexes: GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and LRRC33-TGFe1 complex; and wherein the antibody does not bind free mature growth factor TGFe1, which is not associated with the pro/latent complex. In some embodiments, TGFe1 is pro-TGFe 1 and/or latent TGFe1 (eg, pro/latent TGFe1). In some embodiments, TGFe1 is latent TGFe1. In some embodiments, TGFe1 is pro-TGFe 1.

The isoform-specific TGFe1 inhibitors of the present invention do not bind to

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TGFe2. The isoform-specific TGFei inhibitors of the present invention do not bind to TGFe3. In some embodiments, such inhibitors do not bind to pro/latent TGFe2. In some embodiments, such inhibitors do not bind to pro/latent TGFe3. In some embodiments, the antibody or antigen-binding portion thereof does not interfere with the ability of TGFe1 to bind to integrin.

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

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

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

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

In some embodiments, the antibody or antigen-binding portion thereof inhibits TGFe1 activation, but not TGFe2 activation or TGFe3 activation.

In some embodiments, the antibody or antigen-binding portion thereof inhibits the release of mature TGFe1 from the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFp1 complex.

In one aspect, provided herein is a pharmaceutical composition comprising an antibody or antigen binding portion thereof described herein and a pharmaceutically acceptable carrier. Such pharmaceutical compositions are generally sterile and suitable for administration to human subjects. In some embodiments, such pharmaceutical compositions may be provided in kit form, which are covered by the present invention.

In another aspect, provided herein is a method of inhibiting TGFe1 activation comprising exposing the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, or the LRRC33-TGFe1 complex to an antibody, an antigen binding moiety thereof, or a pharmaceutical composition described herein.

In some embodiments, the antibody or antigen-binding portion thereof inhibits the release of mature TGFe1 from the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, or the LRRC33-TGFp1 complex.

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

Thus, the present invention includes a method of treating a disease associated with dysregulation of TGFe1 signaling in a human subject. Such a method comprises the step of: administering to a human subject in need thereof a pharmaceutical composition provided herein in an amount effective to treat a disease, the amount achieving statistically significant clinical efficacy and safety when administered to a patient population having the disease.

In yet another aspect, provided herein is a TGFe inhibitor for use in reducing adverse events in a subject, wherein the TGFe inhibitor is isoform selective. In some embodiments, a TGFe inhibitor is an antibody that specifically inhibits TGFe1 while broadly targeting multiple contexts.

In some embodiments, the cell expressing the GARP-TGFe1 complex or the LRRC33-TGFe1 complex is a T cell, fibroblast, myofibroblast, macrophage, monocyte, dendritic cell, antigen presenting cell, neutrophil, myeloid-derived suppressor cell (MDSC), lymphocyte, a mast cell, a megakaryocyte, a natural killer (NK) cell, a microglia, or a progenitor cell of any such cell. In some embodiments, the cell expressing the GARP-TGFe1 complex or the LRRC33-TGFe1 complex is a hematopoietic stem cell. In some embodiments, the cell expressing the GARP-TGFe1 complex or the LRRC33-TGFe1 complex is a neural crest-derived cell. The T cell may be a regulatory T cell (eg, an immunosuppressive T cell). The T cell may be a CD4 positive (CD4+) T cell and/or a CD8 positive (CD8+) T cell. The neutrophil may be an activated neutrophil. The macrophage may be a polarized macrophage, including profibrotic and/or tumor-associated macrophages (TAMs), for example, M2c subtype and M2d subtype macrophages. A macrophage may be activated by one or more soluble factors, such as growth factors, cytokines, chemokines, and/or other molecules that are present in the specific disease microenvironment (eg, TME), which may function in an autocrine, paracrine, and/or endocrine manner. In some embodiments, the macrophage is activated by M-CSF, such as solid tumor-secreted M-CSF. In some embodiments, the macrophage is activated by TGFe1.

In some embodiments, the cell expressing the GARP-TGFe1 complex or the LRRC33-TGFe1 complex is a cancer cell, such as circulating cancer cells and tumor cells. In some embodiments, a cell expressing the GARP-TGFe1 complex or the LRRC33-TGFe1 complex is recruited to a site of disease, such as the TME (eg, tumor infiltrate). In some embodiments, expression of the GARP-TGFe1 complex or LRRC33-TGFe1 complex is induced by the disease microenvironment (eg, TME). In some embodiments, the solid tumor contains increased infiltrates of leukocytes, such as CD45+. Tumor-associated CD45+ cells are predicted to include GARP-expressing and/or NRRC33-expressing cells.

In some embodiments, the LTBP1-TGFe1 complex or the LTBP3TGFe1 complex is associated with the extracellular matrix (ie, ECM components).

In some embodiments, the extracellular matrix comprises fibrillin and/or fibronectin. In some embodiments, the extracellular matrix comprises a protein containing an RGD motif. In some embodiments, cells that produce and deposit the LTBP1-TGFe1 complex or the LTBP3-TGFe1 complex are present in a solid tumor, such as malignant cells and stromal cells. In some embodiments, cells that produce and deposit the LTBP1-TGFe1 complex or the LTBP3-TGFe1 complex are present in fibrous tissue. In some embodiments, the cells that produce and deposit the LTBP1-TGFe1 complex or the LTBP3-TGFe1 complex are present in the bone marrow. In some embodiments, the cells that produce and deposit the LTBP1-TGFe1 complex or the LTBP3-TGFe1 complex are myofibroblasts or myofibroblast-like cells, including, for example, cancer-associated fibroblasts (CAFs).

In another aspect, provided herein is a method of reducing TGFe1 activation in a subject, comprising administering to the subject an effective amount of an antibody, antigen binding portion thereof, or pharmaceutical composition as described herein, thereby reducing TGFe1 activation in the subject.

In some embodiments, the subject has or is at risk of developing a fibrotic disorder. In some embodiments, a fibrotic disorder includes chronic inflammation of the affected tissue/organ. In some embodiments, the subject is characterized by having muscular dystrophy. In some embodiments, the subject has Duchenne muscular dystrophy (DMD). In some embodiments, the subject has or is at risk of developing liver fibrosis, renal fibrosis, pulmonary fibrosis (eg, idiopathic pulmonary fibrosis), endometriosis, or

- 8 045239 fibrosis of the uterus. In some embodiments, the subject has or is at risk of developing cancer (eg, solid tumor, blood cancer, and myelofibrosis). In some embodiments, the subject has or is at risk of developing dementia.

In some embodiments, the subject additionally receives additional therapy. In some embodiments, the adjunctive 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 inhibitor 2, an ASK1 inhibitor, an acetyl-CoA carboxylase inhibitor (ACC), a p38 kinase inhibitor, pirfenidone, nintedanib, a GDF11 inhibitor, a JAK inhibitor (eg, a JAK2 inhibitor), or any combination thereof.

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

In some embodiments, the antibody or antigen-binding portion thereof does not cause organ toxicity in the subject. In some embodiments, organ toxicity includes cardiovascular toxicity, gastrointestinal toxicity, immunotoxicity, bone toxicity, cartilage toxicity, reproductive toxicity, or kidney toxicity.

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

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

In some embodiments, the antibody or antigen-binding portion thereof is administered in combination with an additional agent or additional treatment. In some embodiments, the additional agent is a checkpoint inhibitor. In some embodiments, the additional agent is selected from the group consisting of a PD-1 antagonist, a PDL1 antagonist, a PD-L1 or PDL2 fusion protein, a CTLA4 antagonist, etc. Such combination therapies may advantageously use lower doses of therapeutic agents administered, thereby avoiding the potential toxicities or complications associated with various monotherapies or traditional combination therapies that lack the degree of selectivity/specificity achieved by the present invention.

In some embodiments, the method further comprises determining (eg, testing or confirming) the involvement of TGFe1 in the disease as compared to TGFe2 and TGFe3. In some embodiments, the method further comprises the step of identifying the source (or context) of disease-associated TGFe1. In some embodiments, the source/context is assessed by determining the expression of TGF presenting molecules, such as LTBP1, LTBP3, GARP, and LRRC33, in a clinical sample collected from patients.

In yet another aspect, provided herein is a method of producing (eg, producing, manufacturing) a pharmaceutical composition for inhibiting TGFe signaling, the method comprising the steps of: providing one or more agents that inhibit signaling of at least one TGFe isoform; measuring the activity of one or more agents against all TGFe isoforms; selection of an agent selective for TGFe 1; formulating the composition into a pharmaceutical composition containing an isoform-specific TGFe1 inhibitor and a pharmaceutically acceptable excipient, such as a suitable buffer. A pharmaceutical composition obtained by this method is also presented. In some embodiments, the method further includes the step of determining (eg, measuring, analyzing) the context-dependent inhibitory activities of the one or more agents.

The subject matter of this disclosure also relates to document PCT/US2013/068613, filed November 6, 2013; PCT/US2014/036933 filed May 6, 2014; and PCT/US2017/021972, filed March 10, 2017, the entire contents of each of which are incorporated herein by reference.

Brief description of graphic materials

In fig. Figure 1 is a schematic depicting TGFe1 in a latent complex in the tissue microenvironment.

In fig. Figures 2A-2C show multiple contexts of TGFe1 function: GARP-presented TGFe1 is expressed on regulatory T cells that are involved in immune regulation (Figure 2A); LTBP1/3-presented TGFe1 is deposited by fibroblasts and other cells in the ECM (Fig. 2B) and LRRC33-presented TGFe1 is expressed on myeloid cells, including macrophages (Fig. 2C).

In fig. 3 shows the protein expression platform for producing the GARP-TGFei complex and the LTBP-TGFei complex. The HEK293-based expression system uses Ni-NTA affinity purification and gel filtration to produce multi-milligram amounts of purified protein. Diagrams of wild-type pro-TGFe 1, LTPB1, sGARP, and pro-TGFβ1 C4S are shown.

In fig. Figure 4A shows specific binding of Ab3 to latent TGFe1. In fig. 4B shows the binding specificity of exemplary monoclonal antibodies. In fig. 4B shows that Ab1 and Ab2 bind specifically to pro-TGFe 1, as measured by ELISA, but not to pro-TGFβ2, pro-TGFβ3, or mature TGFe1. In fig. 4C shows an example of an antibody that specifically binds (as measured by ELISA) to the LTBP1-pro-TGFe1 complex.

In fig. 5 shows a panel of prior art antibodies generated against the mature growth factor TGFe and their corresponding binding profiles for all three isoforms.

In fig. 6A, 6B show binding profiles measured by Octet for Ab1, Ab2 and Ab3, which are isoform-specific, context-permissive/context-independent inhibitors of TGFe1.

In fig. 7A-7H show cell-based inhibition assays.

In fig. Figure 8 shows the inhibitory effect of Ab3 on kallikrein-induced TGFe1 activation in vitro.

In fig. 9A, 9B show the inhibitory effects of Ab1 and Ab3 on regulatory T cell-dependent suppression of effector T cell proliferation.

In fig. 10A-10C show upregulation of cell surface expression of LRRC33 in polarized macrophages.

In fig. Figure 11 shows the results of the T-cell co-transfer colitis model.

In fig. 12A-12K show the inhibitory effects of Ab2 in a mechanistic model of TGFb1-dependent UUO disease.

In fig. 13A-13C show the inhibitory effects of Ab3 in a mechanistic model of TGFb1-dependent UUO disease.

In fig. 14 shows the inhibitory effects of Ab3 in a model of carbon tetrachloride-induced fibrosis.

In fig. Figure 15 shows the inhibitory effects of Ab3 in a translational model of fibrosis in mice with Alport syndrome.

In fig. 16 shows the inhibitory effects of Ab2 on tumor growth in MC38 carcinoma.

In fig. 17 shows the effects of Ab3 in combination with a PD-1 antagonist on survival in the EMT-6 tumor model.

In fig. 18A-18F provide toxicology/tolerability data showing the improved safety profiles of Ab2 in rats.

In fig. 19A, 19B provide toxicology/tolerability data showing improved safety profiles of Ab3 in rats.

In fig. 20 presents data showing in vivo selectivity for Ab3 isoforms in homeostatic rat BAL cells.

In fig. 21A-21D show the relative expression of TGFe isoforms. In fig. 21A shows the expression of the TGFe isoform compared to a normal comparator (by cancer type). In fig. 21B shows the frequency of expression of the TGFe isoform by human cancer type. In fig. 21C shows TGFe isoform expression in individual tumor samples by cancer type. In fig. 21D shows expression of the TGFe isoform in mouse syngeneic cancer cell lines.

In fig. Figure 22 shows cardiac microscopic findings obtained from the pan-TGFe antibody from the 1-week study.

Detailed Description of Some Embodiments

In mammals, the transforming growth factor-beta (TGFe) superfamily consists of at least 33 gene products. These include bone morphogenic proteins (BMPs), activins, growth and differentiation factors (GDFs), and three isoforms of the TGFe family: TGFe1, TGFe2, and TGFe3. TGFe are thought to play key roles in diverse processes such as inhibition of cell proliferation, extracellular matrix (ECM) remodeling, and immune homeostasis. The importance of TGFe1 for T cell homeostasis is demonstrated by the observation that TGFe1-/- mice survive only 3-4 weeks, succumbing to multiple organ failure due to massive immune activation (Kulkarni, A.B., et al., Proc Natl Acad Sci USA, 1993 90(2): p.770-4; Shull, M.M., et al., Nature, 1992. 359(6397): p.693-9). The roles of TGFe2 and TGFe3 are less clear. Although the three TGFe isoforms have different temporal and spatial expression profiles, they signal through the same receptors, TGFeRI and TGFeRII, although in some cases, such as TGFe2 signaling, type III receptors such as betaglycan are also required (Feng, H. N. and R. Derynck, Annu Rev Cell Dev Biol, 2005.

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21:p. 659-93; Massague, 1, Annu Rev Biochem, 1998. 67: p. 753-91). Ligand-induced oligomerization of TGFeRI/II triggers phosphorylation of SMAD transcription factors, leading to transcription of target genes such as Colla1, Col3a1, ACTA2, and SERPINE1 (Massague, J., J. Seoane, and D. Wotton, Genes Dev, 2005. 19 (23): p. 2783-810). SMAD-independent TGFe signaling pathways have also been described, for example, in aortic cancer or lesions in Marfan mice (Derynck, R. and Y.E. Zhang, Nature, 2003. 425(6958): p. 577-84; Holm, T.M., et al ., Science, 2011. 332(6027): p. 358-61).

The biological significance of the TGFe pathway in humans has been documented in genetic diseases. Camurati-Engelman disease results in bone dysplasia due to an autosomal dominant mutation in the TGFB1 gene, leading to constitutive activation of TGFe1 signaling (Janssens, K., et al., J Med Genet, 2006. 43(1): p. 1-11 ). Patients with Loeys/Dietz syndrome carry autosomal dominant mutations in components of the TGFe signaling pathway, which cause aortic aneurysm, hypertelorism, and cleft uvula (Van Laer, L., H. Dietz, and B. Loeys, Adv Exp Med Biol, 2014. 802: p. 95-105). Because dysregulation of the TGFe pathway has been implicated in multiple diseases, several drugs that target the TGFe pathway have been developed and tested in patients, with limited success.

Dysregulation of TGFe signaling has been associated with a wide range of human diseases. Indeed, in a range of pathological conditions, such dysregulation may involve multiple aspects of TGFe function. Affected tissue, such as fibrous and/or inflamed tissues and tumors, may create a local environment in which TGFe activation can cause exacerbation or disease progression, which may be at least partially mediated by interactions between a variety of TGFe-responsive cells that are activated by autocrine and /or in a paracrine manner together with a number of other cytokines, chemokines and growth factors that play a role in certain disease conditions. For example, the tumor microenvironment (TME) contains many cell types expressing TGFe1, such as activated myofibroblast-like fibroblasts, stromal cells, infiltrating macrophages, MDSCs and other immune cells, in addition to cancerous (ie, malignant) cells. Thus, the TME is a heterogeneous population of cells expressing and/or responsive to TGFe1, but in association with more than one type of presentation molecules, such as LTBP1, LTBP3, LRRC33, and GARP, in a niche.

To effectively inhibit dysregulated or disease-causing TGFe1 activity involving multiple cell types and signaling contexts, we sought to develop a class of agents that have the ability to inhibit multiple TGFe1 functions in an isoform-specific manner. Such agents are referred to herein as isoform-specific, context-permissive TGFp1 inhibitors. In some embodiments, such inhibitors are isoform-specific, context-independent TGFe1 inhibitors. It is hypothesized that the use of an isoform-specific, context-permissive, or context-independent TGFe1 inhibitor may have an inhibitory effect on multiple modes of TGFe1 function in disease, which involves the interaction of different cell types that express and/or respond to TGFe1 signaling , thereby enhancing therapeutic effects by targeting multiple types of TGFe1 precursor complexes. Accordingly, therapeutic targets of such an inhibitor include at least three of the following complexes: i) pro-TGFe1 presented by GARP; ii) pro-TGFe1 presented by LRRC33; iii) pro-TGFe1 presented by LTBP1, and iv) pro-TGFe1 presented by LTBP3. Typically, the above complexes (i) and (ii) are present on the cell surface, since both GARP and LRRC33 are transmembrane proteins capable of presenting TGFe 1 on the extracellular surface, while complexes (iii) and (iv) are components extracellular matrix. A number of studies have shed light on the mechanisms of TGFe1 activation. Three integrins, aVe6, aVe8 and aVe1, have been shown to be key activators of latent TGFe1 (Reed, N.I., et al., Sci Transl Med, 2015. 7(288): p. 288ra79; Travis, M.A. and D. Sheppard, Annu Rev Immunol, 2014. 32: p. 51-82; Munger, J.S., et al., Cell, 1999. 96(3): p. 319-28). aV integrins bind the RGD sequence present in the TGFe1 and TGFp1 LAPs with high affinity (Dong, X., et al., Nat Struct Mol Biol, 2014. 21(12): p. 1091-6). Transgenic mice with a mutation in the RGD site of TGFe1, which prevents integrin binding but not secretion, phenocopy the TGFe1-/- mouse (Yang, Z., et al., J Cell Biol, 2007. 176(6): p. 787-93 ). Mice lacking e6 and e8 integrins recapitulate all of the significant phenotypes of TGFe1 and TGFe3 knockout mice, including multiorgan inflammation and cleft palate, suggesting an important role for these two integrins in activating TGFe1 in development and homeostasis (Aluwihare, P., et al., J Cell Sci, 2009. 122(Pt 2): p. 227-32). The key to integrin-dependent activation of latent TGFe1 is covalent bonding to presentation molecules; destruction of disulfide bonds between GARP and TGFe1 LAP by mutagenesis does not disrupt complex formation, but completely eliminates activation

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TGFei via ανβ6 (Wang, R., et al., Mol Biol Cell, 2012. 23(6): p. 1129-39). A recent structure of latent TGFe1 reveals how integrins mediate the release of active TGFe1 from the latent complex: covalent association of latent TGFe1 with its presentation molecule tethers latent TGFe1 either to the ECM via LTBP or to the cytoskeleton via GARP or LRRC33. Integrin binding to the RGD sequence results in a force-dependent change in LAP structure, allowing the release of active TGFe1 and binding of nearby receptors (Shi, M., et al., Nature, 2011. 474(7351): p. 343-9). The importance of integrin-dependent activation of TGFe1 in disease is also well documented. A small molecule inhibitor of aVe1 protects against bleomycin-induced pulmonary fibrosis and carbon tetrachloride-induced liver fibrosis (Reed, N.I., et al., Sci Transl Med, 2015. 7(288): p. 288ra79), and blockade of aVe6 with antibody or loss of expression β6 integrin suppresses bleomycin-induced pulmonary fibrosis and radiation-induced fibrosis (Munger, J.S., et al., Cell, 1999. 96(3): p. 319-28); Horan, G.S., et al., Am J Respir Crit Care Med, 2008. 177(1): p. 56-65). In addition to integrins, other mechanisms of TGFe1 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; the role of these molecules is less well established in in vivo studies. Thrombospondin-1 knockout recapitulates some aspects of the TGFe1-/- phenotype in some tissues but does not protect in bleomycin-induced pulmonary fibrosis, which is known to be TGFe dependent (Ezzie, M.E., et al., Am J Respir Cell Mol Biol, 2011. 44(4): pp. 556-61). In addition, knockout of potential proteases did not result in a TGFe1 phenotype (Worthington, J. J., J. E. Klementowicz, and M. A. Travis, Trends Biochem Sci, 2011. 36(1): p. 47-54). This may be explained by the redundancy or criticality of these mechanisms in specific diseases, rather than by development and homeostasis.

Thus, the isoform-specific, context-permissive TGFe1 inhibitors described herein include inhibitors that work by preventing the activation step of TGFe1. In some embodiments, such inhibitors may inhibit integrin-dependent (eg, mechanical or forced) activation of TGFe1 (see FIG. 2). In some embodiments, such inhibitors can inhibit protease-dependent or protease-induced activation of TGFe1. The latter includes inhibitors that inhibit the TGFe1 activation step independently of integrin. In some embodiments, such inhibitors can inhibit TGFe1 activation regardless of the mode of activation, for example, inhibit both integrin-dependent activation and protease-dependent activation of TGFe1. Non-limiting examples of proteases that can activate TGFe1 include serine proteases such as kallikreins, chemotrypsin, trypsin, elastases, plasmin, as well as zinc metalloproteases (MMP family) such as MMP-2, MMP-9 and MMP-13. Kallikreins include plasma kallikreins and tissue kallikreins such as KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14 and KLK14. In fig. 8 provides one example of an isoform-specific, context-independent TGFe1 inhibitor that can inhibit kallikrein-dependent activation of TGFe1 in vitro. In some embodiments, the inhibitors of the present invention prevent the release or dissociation of the active (mature) growth factor TGFe1 from the latent complex. In some embodiments, such inhibitors may work by stabilizing an inactive (eg, latent) conformation of the complex.

TGFe is involved in a number of biological processes, including fibrosis, immunomodulation and cancer progression. TGFe1 was the first identified member of the TGFe superfamily of proteins. Like other members of the TGFe superfamily, TGFe1 and the TGFe2 and TGFe3 isoforms are initially expressed as inactive precursor proprotein forms (termed proTGFe). TGFe proteins (eg, TGFe1, TGFe2, and TGFe3) are proteolytically cleaved by proprotein convertases (eg, furin) to produce a latent form (called latent TGFe). In some embodiments, the proprotein form or latent form of the TGFe protein (eg, TGFe1, TGFe2, and TGFe3) may be referred to as the pro/latent TGFe protein. TGFe1 can be presented to other molecules in a complex with several molecules, including, for example, GARP (to form the GARP-TGFe1 complex), LRRC33 (to form the LRRC33TGFp1 complex), LTBP1 (to form the LTBP1-TGFe1 complex) and/or LTBP3 ( to form the LTBP3-TGFe1 complex). TGFp1 present in these complexes can be in latent form (latent TGFe1) or precursor form (pro-TGFe1).

The present invention is particularly useful for therapeutic use in certain diseases that are associated with multiple biological roles of TGFe1 signaling that are not limited to one context of TGFe1 function. In such situations, it may be useful to inhibit the effects of TGFe1 in different contexts. Thus, in some embodiments,

- 12 045239 The present invention relates to methods of targeting and inhibiting TGFei in an isoform-specific manner rather than a context-specific manner. Such agents may be referred to as isoform-specific, context-permissive TGFe1 modulators. In some embodiments, context-permissive TGFe1 modulators target multiple contexts (eg, multiple types of pro/latentTGFe1 complexes). In some embodiments, context-permissive TGFe1 modulators target all types of TGFe1 pro/latent complexes (eg, GARP-associated, LRRC33-associated, LTBP-associated, etc.) to cover all contexts.

Although context-permissive TGFe1 inhibitors are capable of targeting more than one type of pro/latent-TGFe1 complexes (i.e., with different presentation molecules), in some embodiments such inhibitors may favor one or more contexts over with others. Thus, in some embodiments, a context-permissive antibody that inhibits TGFe1 activation may preferentially inhibit TGFe1 activation mediated by one presentation molecule over another presentation molecule, even though such antibody is capable of binding to both types of prolatent complexes. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits the activation of LTBP-associated TGFe1, GARP-associated TGFe1, and LRRC33-associated TGFe1, but with preferential inhibitory activity against LTBP-associated TGFe1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits the activation of LTBP1-associated TGFe1, LTBP3-associated TGFe1, GARP-associated TGFe1, and LRRC33-associated TGFe1, but with preferential inhibitory activity against LTBP1- and LTBP3-associated TGFe1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits the activation of LTBP1-associated TGFe1, LTBP3-associated TGFe1, GARP-associated TGFe1, and LRRC33-associated TGFe1, but with preferential inhibitory activity against GARP-associated TGFe1 and LRRC33-associated TGFe1 . In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits the activation of GARP-associated TGFe1 and LRRC33-associated TGFe1, but with preferential inhibitory activity against GARP-associated TGFe1. In some embodiments, such an antibody is a monoclonal antibody that binds to and inhibits the activation of GARP-associated TGFe1 and LRRC33-associated TGFe1, but with preferential inhibitory activity against LRRC33-associated TGFe1.

Thus, in accordance with the present invention, different degrees of selectivity can be created to target a subset of the effects of TGFe. Isoform-specific TGFe1 inhibitors (which target a single TGFe isoform, e.g., TGFe1) provide higher selectivity than pan-TGFe inhibitors (which target multiple or all TGFe isoforms). Isoform-specific, context-permissive TGFe1 inhibitors (which target multiple contexts of a single TGFe1 isoform) provide higher selectivity than isoform-specific inhibitors. Isoform-specific, context-independent TGFe1 inhibitors (which target and inhibit TGFe1 functions regardless of which presenting molecule is associated) provide isoform specificity while providing broader coverage of inhibitory effects across multiple TGFe1 activities.

Definitions.

In order that the present disclosure may be clearer, certain terms are first defined. These definitions should be read in light of the rest of this disclosure and will be understood by one skilled in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Additional definitions are provided in the detailed description.

Antibody. The term antibody encompasses any naturally occurring recombinant, modified or engineered immunoglobulin or immunoglobulin-like structure or antigen-binding fragment or portion thereof, or derivative thereof, as further described elsewhere herein. Thus, the term 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 typically include at least two full-length heavy chains and two full-length light chains

- 13 045239 chains, but in some cases may include a smaller number of chains, such as antibodies found naturally in camels, which may contain only heavy chains. Antibodies may come exclusively from a single source or may be chimeric, i.e. different parts of an antibody can come from two different antibodies. Antibodies or antigen-binding portions thereof can be produced in hybridomas by recombinant DNA methods or by enzymatic or chemical digestion of intact antibodies. As used herein, the term antibodies includes monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as antibody mimetics), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as antibody conjugates), respectively. In some embodiments, the term also includes peptide antibodies.

Antigen. The term antigen refers to a molecular structure that provides an epitope, for example a molecule or part of a molecule or a complex of molecules or parts of molecules capable of being bound by a selective binding agent such as an antigen binding protein (including, for example, an antibody). Thus, the selective binding agent can specifically bind to an antigen that is formed by two or more components in a complex. In some embodiments, an antigen can be used in an animal to produce antibodies capable of binding to the antigen. An antigen may have one or more epitopes that are capable of interacting with various antigen-binding proteins, such as antibodies. Antigen-binding moiety/fragment: As used herein, the terms antigen-binding moiety or antigen-binding fragment of an antibody refer to one or more antibody fragments that retain the ability to specifically bind an antigen (eg, TGFe1). Antigen-binding moieties include, without limitation, any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. In some embodiments, the antigen-binding portion of an antibody can be produced, for example, from whole antibody molecules using any suitable standard methods, such as proteolytic digestion or recombinant genetic engineering methods involving the manipulation and expression of DNA encoding variable and optionally constant domains of the antibody. Non-limiting examples of antigen binding moieties include: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of one arm of the antibody; (v) single chain Fv (scFv) molecules (Bird et al. (1988) SCIENCE 242:423426 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 the antibody (for example, a dedicated complementarity determining region ( CDR)). Other forms of single chain antibodies, such as diabodies, are also included. The term antigen binding portion of an antibody includes a single chain Fab fragment, otherwise known as a scFab, containing an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL ) and a linker, wherein said antibody domains and said linker are in one of the following orders in the direction from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1 , c) VH-CL-linker-VL-CH1 or d) VL-CH1-linkerVH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably from 32 to 50 amino acids.

Cancer. As used herein, the term cancer refers to a physiological condition of multicellular eukaryotes that is typically characterized by unregulated cell proliferation and malignancy. Thus, the term broadly covers solid tumors, blood cancers (eg, leukemia), as well as myelofibrosis and multiple myeloma.

Cell-associated TGFe1: The term refers to TGFe1 or its signaling complex (eg, pro/latent TGFe1) that is membrane associated (eg, tethered to the cell surface). Typically, such a cell is an immune cell. GARP- or LRRC33-presented TGFe1 is cell-associated TGFe1.

Checkpoint inhibitor. In the context of the present disclosure, checkpoint inhibitors refer to immune checkpoint inhibitors and have a meaning understood in the art. Typically, the target is a receptor molecule on T cells or NK cells or a corresponding cell surface ligand on antigen presenting cells (APCs) or tumor cells. Immune checkpoints are activated in immune cells to prevent the development of inflammatory immunity against themselves. Therefore, changing the balance of the immune system through checkpoint inhibition should allow it to be fully

- 14 045239 can be activated to detect and eliminate cancer. The best known inhibitory receptors involved in the control of the immune response are cytotoxic T lymphocyte antigen4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM3), activation gene 3 lymphocyte receptor (LAG3), killer immunoglobulin-like receptor (KIR), glucocorticoid-induced tumor necrosis factor receptor (GITR), and V-domain-containing immunoglobulin (Ig) suppressor of T-cell activation (VISTA). Non-limiting examples of checkpoint inhibitors include: nivolumab, pembrolizumab, BMS-936559, atezolizumab, avelumab, durvalumab, ipilimumab, tremelimumab, IMP-321, BMS-986016 and lirilumab.

Clinical benefit. As used herein, the term clinical benefit refers to both the effectiveness and safety of a therapy. Thus, a therapeutic treatment that provides the desired clinical benefit is both effective and safe (eg, with tolerable or acceptable toxicities or adverse events).

Combination Therapy: Combination therapy refers to clinically indicated treatment regimens that contain two or more therapeutic agents. Thus, the term refers to a therapeutic regimen in which a first therapy containing a first composition (eg, an active ingredient) is administered in combination with a second therapy containing a second composition (active ingredient) to a patient intended to treat the same or overlapping disease or clinical condition. The first and second compositions may act on a single cellular target or on separate cellular targets. The phrase in combination with, in the context of combination therapy, means that the therapeutic effects of the first therapy overlap in time and/or space with the therapeutic effects of the second therapy in a subject receiving the combination therapy. Thus, the combination therapy may be formulated as a single formulation for simultaneous administration or as separate formulations for sequential administration of the therapies.

Combination or combinatorial epitope. In some embodiments, inhibitory antibodies of the present invention can bind an epitope formed by two or more components (eg, portions or segments) of the TGFe1 pro/latent complex. Such an epitope is referred to as a combination or combinatorial epitope. Thus, the combination epitope may comprise amino acid residue(s) from the first complex component and amino acid residue(s) from the second complex component, and so on. Each component may consist of a single protein or two or more proteins of an antigenic complex. The binding of an antibody to a combination epitope depends not only on the primary amino acid sequence of the antigen. Rather, a combination epitope is formed with structural contributions from two or more components (eg, parts or segments, such as amino acid residues) of an antigen or antigenic complex.

Compete or cross-compete: The term compete, when used in the context of antigen-binding proteins (e.g., an antibody or an 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 of interest prevents or inhibits (eg, reduces) specific binding of a reference antigen binding protein to a common antigen (eg, TGFe1 or a fragment thereof). To determine whether one antigen-binding protein competes with another, many types of competitive binding assays can be used, for example: direct or indirect radioimmunoassay (RIA), direct or indirect enzyme-linked immunosorbent assay (EIA), sandwich competition assay; solid phase direct biotin-avidin EIA; solid phase direct labeling assay and solid phase sandwich direct labeling assay. Typically, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of the reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60% , 60-65%, 65-70%, 70-75% or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more. In some embodiments, the first antibody or antigen-binding portion thereof and the second antibody or antigen-binding portion thereof cross-block each other to the same antigen, e.g., as assayed by Biacor or Octet, using standard assay conditions, e.g., according to with the manufacturer's instructions (eg, binding is analyzed at room temperature, ~20-25°C). In some embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have the same epitope. In other embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have non-identical but overlapping epitopes. In yet other embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have separate (different) epitopes that are in close proximity in three-dimensional space such that binding of the antibody is cross-blocked by steric hindrance. Cross-blocking means that binding of a first antibody to an antigen prevents a second antibody from binding to the same antigen, and similarly, binding of a second antibody to an antigen prevents a first antibody from binding to the same antigen.

Complementarity-determining region. As used herein, the term CDR refers to the complementarity determining region in antibody variable 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. As used herein, the term CDR set refers to a group of three CDRs that occur in a single variable region that can bind antigen. The exact boundaries of these CDRs have been defined differently depending on different systems. The system described by Kabat et al. (1987; 1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) not only provides a unique residue numbering system applicable to any antibody variable region, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as the Kabat CDRs. Chothia and co-workers (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917 and Chothia et al. (1989) Nature 342: 877 -883) found that some subunits in the Kabat CDR adopt almost identical peptide backbone conformations despite great diversity at the amino acid sequence level.These subunits were designated L1, L2 and L3 or H1, H2 and H3, where L and H denote the regions light chain and heavy chain, respectively. These regions may be referred to as the Chothia CDRs, which have boundaries that overlap with the Kabat CDR. Other boundaries defining the overlap of the CDR with the Kabat CDR have been described by Padlan (1995), FASEB J. 9: 133-139 and MacCallum (1996) J. Mol. Biol. 262(5): 732-45. However, other CDR boundary definitions may not strictly follow one of the systems described herein but will nonetheless overlap with the Kabat CDR, although they may be shortened or lengthened in light of the prediction or experimental results that particular residues or groups of residues or even entire CDRs do not have a significant effect on antigen binding. The methods used herein may use CDRs defined in accordance with any of these systems, although some embodiments use CDRs defined by Kabat or Chothia.

Conformational epitope. In some embodiments, the inhibitory antibodies of the present invention can bind an epitope that is conformation specific. Such an epitope is referred to as a conformational epitope, conformation-specific epitope, conformation-dependent epitope, or conformation-sensitive epitope. A corresponding antibody or fragment thereof that specifically binds such an epitope may be referred to as a conformation-specific antibody, a conformation-selective antibody, or a conformation-dependent antibody. The binding of an antigen to a conformational epitope depends on the three-dimensional structure (conformation) of the antigen or antigenic complex.

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

Permissive in relation to the context; Context-independent: Context-permissive and context-independent TGFe inhibitors are broad context inhibitors that can target more than one mode of TGFe function. A context-permissive TGFe inhibitor is an agent capable of inhibiting multiple contexts of TGFe function, for example, TGFe activities associated with at least two of the following: GARP (also called LRRC32), LRRC33, LTBP1 and LTBP3. Among context-permissive inhibitors, where the agent is capable of inhibiting TGFe activities independently of specific presenting molecules, such an inhibitor is called a context-independent inhibitor. Thus, a context-independent TGFe inhibitor can inhibit TGFe activities associated with all of the following: GARP, LRRC33, LTBP1, and LTBP3. In some embodiments, context-permissive and context-independent inhibitors may exhibit preferential or biased inhibitory activity against one or more contexts over others.

ECM-associated TGFe1. The term refers to TGFe1 or its signaling complex (eg, pro/latent TGFe1), which is a component of (eg, deposited in) the extracellular matrix. TGFe1, which is presented by LTBP1 or LTBP3, is an ECM-associated TGFe1.

Effective amount. An effective amount (or therapeutically effective amount) is a dosage or dosage schedule that provides statistically significant clinical benefits in a patient population.

Epitope. The term epitope includes any molecular determinant (eg, polypeptide determinant) that can specifically bind to a binding agent, immunoglobulin, or T cell receptor. According to some embodiments of the determinant

- 16045239 epitopes include reactive surface moieties of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and in some 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 of a region of an antigen (or fragment thereof) that is known to bind to the complementary site of a specific binding partner. An antigenic fragment may contain more than one epitope. In some embodiments, an antibody specifically binds to an antigen when it recognizes a target antigen in a complex mixture of proteins and/or macromolecules. For example, antibodies are considered to bind to the same epitope if the antibodies cross-compete (one prevents the binding or modulating effect of the other). Additionally, structural definitions of epitopes (overlapping, similar, identical) are informative, but functional definitions are often more relevant because they cover structural (binding) and functional (modulation, competition) parameters.

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

GARP-TGFe1 complex. As used herein, the term GARP-TGFe1 complex refers to a protein complex containing the proprotein form or latent form of transforming growth factor-β1 (TGFe1) protein and glycoprotein-A repeat predominant protein (GARP) or a fragment or variant thereof. In some embodiments, the pro-protein form or latent form of the TGFe1 protein may be referred to as the pro/latent TGFe1 protein. In some embodiments, the GARP-TGFe1 complex comprises GARP covalently linked to pro/latent TGFe1 through one or more disulfide bonds. In other embodiments, the GARP-TGFe1 complex comprises GARP non-covalently associated with pro/latent TGFe1. In some embodiments, the GARP-TGFe1 complex is a naturally occurring complex, such as the GARP-TGFe1 complex in a cell. An exemplary GARP-TGFe1 complex is shown in FIG. 3.

Human antibody. As used herein, the term human antibody includes antibodies containing variable and constant regions derived from human germline immunoglobulin sequences. 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 somatic mutation in vivo), for example, in the CDR and, in particular, CDR3. However, as used herein, the term human antibody is not intended to include antibodies in which CDR sequences derived from the germ line of other mammalian species, such as mouse, have been grafted onto human framework sequences.

Humanized antibody. The term humanized antibody refers to antibodies that contain heavy and light chain variable region sequences from a non-human species (eg, mouse), but in which at least a portion of the VH and/or VL sequence has been altered to be more human-like, i.e. .e. more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Also, a humanized antibody is an antibody or a variant, derivative, analog, or fragment thereof that immunospecifically binds to an antigen of interest and that contains an FR region having essentially the amino acid sequence of a human antibody and a CDR region having essentially the amino acid sequence of a non-human antibody . As used herein, the term substantially in the context of a CDR refers to a CDR comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the CDR of a non-human antibody. A humanized antibody contains 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 non-human immunoglobulin (ie, donor antibody) and all or substantially all of the FR regions are those of the human immunoglobulin consensus sequence. In one embodiment, the humanized antibody also comprises at least a portion of the Fc region of an immunoglobulin, typically that of a human immunoglobulin. In some embodiments, the humanized antibody comprises a light chain as well as at least a heavy chain variable domain. The antibody may also include the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only a humanized light chain. In some embodiments, the humanized antibody comprises only a humanized heavy chain. In certain embodiments, the humanized antibody comprises only a humanized light chain and/or a humanized heavy chain variable domain.

Isoform specific. The term isoform specificity refers to the ability of an agent to distinguish one isoform from other structurally related isoforms (ie, selectivity). An isoform-specific TGFe inhibitor exhibits its inhibitory activity against one TGFe isoform but not other TGFe isoforms at a given concentration. For example, a TGFe1 isoform-specific antibody selectively binds TGFe1. A TGFe1-specific inhibitor (antibody) preferentially targets (binds, thereby inhibiting) the TGFe1 isoform over TGFe2 or TGFe3 with significantly higher affinity. For example, selectivity in this context may refer to at least a 500- to 1000-fold difference in the corresponding affinity values measured by in vitro binding assays such as Octet and Biacor. In some embodiments, the selectivity is such that the inhibitor, when used at a dosage effective to inhibit TGFe1 in vivo, does not inhibit TGFe2 and TGFe3. For example, an antibody may preferentially bind to TGFe1 with an affinity of ~1 pM, whereas the same antibody may bind to TGFe2 and/or TGFe3 at ~0.5-50 nM. For such an inhibitor to be useful as a therapeutic agent, the dosage to achieve the desired effects (eg, therapeutically effective amounts) must be within the window within which the inhibitor can effectively inhibit the TGFe1 isoform without inhibiting TGFe2 or TGFe3.

Dedicated. As used herein, the term isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities. In some embodiments, the isolated antibody is substantially free of other unwanted cellular material and/or chemicals.

Localized: As used herein, the term localized (as in a localized tumor) refers to an anatomically isolated or separable disorder such as a solid malignancy, as opposed to a systemic disease. For example, a certain leukemia may have both a localized component (eg, bone marrow) and a systemic component (eg, circulating blood cells) to the disease.

LRRC33-TGFe1 complex. As used herein, the term LRRC33TGFe1 complex refers to the complex between the proprotein form or latent form of transforming growth factor-b1 (TGFe 1) protein and leucine-rich repeat-containing protein 33 (LRRC33; also known as negative regulator of reactive oxygen species or NRROS) or a fragment thereof or option. In some embodiments, the LRRC33-TGFe1 complex contains LRRC33 covalently linked to pro/latent TGFe1 through one or more disulfide bonds. In other embodiments, the LRRC33-TGFe1 complex contains LRRC33 non-covalently associated with pro/latent TGFe1. In some embodiments, the LRRC33-TGFe1 complex is a naturally occurring complex, such as the LRRC33TGFe1 complex in a cell.

LTBP1-TGFe1 complex. As used herein, the term LTBP1-TGFe1 complex refers to a protein complex containing the proprotein form or latent form of transforming growth factor-β1 (TGFe1) protein and latent TGF-beta binding protein 1 (LTBP1) or a fragment or variant thereof. In some embodiments, the LTBP1-TGFe1 complex comprises LTBP1 covalently linked to pro/latent TGFe1 through one or more disulfide bonds. In other embodiments, the LTBP1-TGFe1 complex contains LTBP1 non-covalently associated with pro/latent TGFe1. In some embodiments, the LTBP1-TGFe1 complex is a naturally occurring complex, such as the LTBP1TGFe1 complex in a cell. An exemplary LTBP1-TGFe1 complex is shown in FIG. 3.

LTBP3-TGFe1 complex. As used herein, the term LTBP3-TGFe1 complex refers to a protein complex containing the proprotein form or latent form of transforming growth factor-β1 (TGFe1) protein and latent TGF-beta binding protein 3 (LTBP3) or a fragment or variant thereof. In some embodiments, the LTBP3-TGFe1 complex comprises LTBP3 covalently linked to pro/latent TGFe1 through one or more disulfide bonds. In other embodiments, the LTBP3-TGFe1 complex contains LTBP1 non-covalently associated with pro/latent TGFe1. In some embodiments, the LTBP3-TGFe1 complex is a naturally occurring complex, such as the LTBP3TGFe1 complex in a cell. An exemplary LTBP3-TGFe1 complex is shown in FIG. 3.

Myelofibrosis. Myelofibrosis, also known as osteomyelofibrosis, is a relatively rare proliferative disorder of the bone marrow (eg, cancer) that belongs to a group of diseases called myeloproliferative disorders. Myelofibrosis is classified into the Philadelphia chromosome-negative (-) branch of myeloproliferative neoplasms. Myelofibrosis is characterized by proliferation of an abnormal clone of hematopoietic stem cells in the bone marrow and other sites, leading to fibrosis, or replacement of the bone marrow by scar tissue. The term myelofibrosis, unless otherwise noted, refers to primary myelofibrosis (PMF). It may also be referred to as chronic idiopathic myelofibrosis (cIMF) (the terms idiopathic and primary mean that in these cases the disease is of unknown or spontaneous origin). This contrasts with myelofibrosis, which occurs secondary to polycythemia or essential thrombocythemia. Myelofibrosis is a form of myeloid metaplasia that refers to a change in the cell type in the hematopoietic tissue of the bone marrow, and often the two terms are used interchangeably. The terms agnogenic myeloid metaplasia and myelofibrosis with myeloid metaplasia (MMM) are also used to refer to primary myelofibrosis.

Pan-TGFe inhibitor. The term pan-TGFe inhibitor refers to any agent that is capable of inhibiting or antagonistizing multiple TGFe isoforms. Such an inhibitor may be a small molecule inhibitor of TGFe isoforms. The term includes a pan-TGFe antibody, which refers to any agent that is capable of binding to more than one TGFe isoform, such as at least two of TGFe1, TGFe2 and TGFe3. In some embodiments, the pan-TGFe antibody binds all three isoforms, i.e. TGFe1, TGFe2 and TGFe3. In some embodiments, a pan-TGFe antibody binds and neutralizes all three isoforms, i.e. TGFp1, TGFe2 and TGFe3.

Presenting molecule. The term presenting molecule or TGFe presentation molecule is a protein entity that is capable of binding to the inactive form(s) of TGFe, thereby presenting a protein in the extracellular domain. To date, four TGFe presenting molecules have been identified: latent TGFe binding protein-1 (LTBP1) and LTBP3 are deposited in the extracellular matrix (i.e., ECM components), while the predominant glycoprotein-A repeat (GARP/LRRC32) and containing leucine repeat-rich protein 33 (LRRC33) contains a transmembrane domain and presents latent TGFe1 on the surface of certain cells, such as immune cells. One isoform, TGFe1, is involved in a number of biological processes in both normal and pathological conditions. These include, but are not limited to, maintenance of tissue homeostasis, inflammatory response, ECM reorganization such as wound healing, and regulation of immune responses, as well as organ fibrosis, cancer, and autoimmunity.

pro-TGFe1. As used herein, the term pro-TGFe1 is intended to encompass the precursors of the inactive TGFe1 complex, which includes the TGFe1 prodomain sequence within the complex. Thus, the term may include both pro- and latent forms of TGFp1. The expression pro/latent TGFe1 can be used interchangeably. The pro TGFe1 form exists prior to proteolytic cleavage at the furin site. Once cleaved, the resulting form is said to be the latent form of TGFe1. The latent complex remains associated until the next activation trigger, such as an integrin-driven activation event. As shown in FIG. 3, the pro-TGFe1 complex consists of dimeric polypeptides of the TGFe1 protein linked by disulfide bonds. It should be noted that the adjective latent can generally be used to describe the inactive state of TGFe1 prior to integrin-mediated or other activation events.

Regulatory T cells. Regulatory T cells or Tregs are characterized by the expression of the biomarkers CD4, FOXP3 and CD25. Tregs are sometimes called suppressor T cells and are a subset of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs are immunosuppressive and typically suppress or suppress the induction and proliferation of effector T cells (Teff). Tregs can develop in the thymus (so-called CD4+ Foxp3+ natural Tregs) or differentiate from naive CD4+ T cells in the periphery, for example, after exposure to TGFe or retinoic acid.

Solid tumor. The term solid tumor refers to a proliferative disorder resulting in abnormal growth or mass of tissue that typically does not contain cysts or fluid areas. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Solid tumors may be composed of cancer cells, stromal cells including CAFs, and infiltrating leukocytes such as macrophages and lymphocytes.

Specific binding. As used herein, the term specific binding or specifically binds means that the interaction of an antibody or antigen-binding portion thereof with an antigen depends on the presence of a specific structure (eg, an antigenic determinant or epitope). For example, an antibody or antigen-binding portion binds to a specific protein rather than to proteins in general. In some embodiments, the antibody or antigen binding agents thereof

- 19 045239 the moiety specifically binds to a target, for example, TGFei, if the antibody has a KD for the target of at least about 10 ^-4 M, 10 ^-5 M, 10 ^-6 M, 10 ^-7 M, 10 ^-8 M , 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M, 10 ^-13 M or less. In some embodiments, as used herein, the term TGFe1 epitope-specific binding, TGFe1 epitope-specific binding, TGFe1-specific binding, or TGFe1-specific binding refers to an antibody or antigen-binding portion thereof that binds to TGFe1 and has a dissociation constant (KD) of 1 ,0x10' ⁷ M or less, which is determined by surface plasmon resonance. In one embodiment, the antibody or antigen-binding portion thereof can specifically bind to both human and non-human (eg, murine) TGFe1 orthologues.

Subject. The term subject in the context of therapeutic applications refers to an individual who is receiving clinical care or intervention, such as treatment, diagnosis, etc. Suitable subjects include vertebrates, including, but not limited to, mammals (eg, human and non-human mammals). When the subject is a human, the term patient can be used interchangeably. In a clinical context, the term patient population or patient subpopulation is used to refer to a group of individuals who fall under a set of criteria, such as clinical criteria (eg, disease manifestations, stages of disease, susceptibility to certain conditions, responsiveness to therapy, etc.), history diseases, health status, gender, age group, genetic criteria (for example, carrier of a certain mutation, polymorphism, gene duplications, DNA sequence repeats, etc.) and lifestyle factors (for example, smoking, alcohol consumption, exercise, etc.). d.).

TGFβ1-associated disorder. TGFβ1-associated disorder means any disease or disorder in which at least part of the pathogenesis and/or progression is due to TGFe1 signaling or dysregulation.

TGFe inhibitor. The term TGFe inhibitor refers to any agent capable of antagonizing the biological activity or function of the growth factor TGFe (eg, TGFe1, TGFe2 and/or TGFe3). The term is not intended to limit its mechanism of action and includes, for example, neutralizing inhibitors, receptor antagonists, soluble ligand decoys, and TGFe activation inhibitors.

The TGFe family is a class within the TGFe superfamily and contains three isoforms: TGFp1, TGFe2 and TGFe3, which are structurally similar.

Toxicity. As used herein, the term toxicity or toxicity refers to undesirable effects in vivo in patients associated with therapy administered to patients, such as unwanted side effects and adverse events. Tolerability refers to the level of toxicity associated with a therapy or treatment regimen that a patient can reasonably tolerate without discontinuing therapy due to toxicity.

Treat/cure. The term treat or treatment includes therapeutic treatment, prophylactic treatment, and applications that reduce the risk of a subject developing a disorder or other risk factor. Thus, the term is intended in a broad sense: to produce a therapeutic benefit in a patient, for example by enhancing or enhancing the body's immunity; reducing or changing immunosuppression; reducing, removing or destroying harmful cells or substances from the body; reducing disease burden (eg, tumor burden); preventing relapse or return of the disease; prolongation of the refractory period and/or other improvement in survival. The term includes therapeutic treatment, prophylactic treatment, and applications in which the risk of a subject developing a disorder or other risk factor is reduced. Treatment does not require complete cure of the disorder and covers embodiments in which symptoms or underlying risk factors can be reduced. In the context of combination therapy, the term may also refer to: i) the ability of a second therapeutic agent to reduce the effective dosage of the first therapeutic agent to reduce side effects and increase tolerability; ii) the ability of the second therapy to make the patient more responsive to the first therapy and/or iii) the ability to produce additional or synergistic clinical benefits.

Tumor-associated macrophages. Tumor-associated macrophages (TAMs) are polarized/activated macrophages with pro-tumor phenotypes. TAMs can be either bone marrow-derived monocytes/macrophages recruited to the tumor site or sedentary tissue macrophages derived from erythromyeloid progenitors. Monocyte/macrophage differentiation into TAMs depends on a number of factors, including local chemical signals such as cytokines, chemokines, growth factors and other molecules that act as ligands, as well as cell-cell interactions between monocytes/macrophages present in the niche (tumor microenvironment) . Typically, monocytes/macrophages can be polarized into so-called M1 or M2 subtypes, with the latter being associated with more pro-tumor

- 20 045239 phenotype. In a solid tumor, up to 50% of the tumor mass may correspond to macrophages, which are preferentially M2-polarized.

Tumor microenvironment. The term tumor microenvironment (TME) refers to the local disease niche in which a tumor (eg, solid tumor) resides in vivo.

Variable region. The term variable region or variable domain refers to a portion of the light and/or heavy chain of an antibody, typically comprising about 120 to 130 amino-terminal amino acids in the heavy chain and about 100 to 110 amino-terminal amino acids in the light chain. In some embodiments, the variable regions of different antibodies vary greatly in amino acid sequence, even among antibodies of the same species. The variable region of an antibody typically determines the specificity of a particular antibody for its target.

Except in the worked examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all cases by the term approximately. The term approximately when used in connection with percentages can mean ±1%.

As used herein in the specification and claims, the singular number, unless expressly indicated otherwise, is to be understood to mean at least one.

The phrase and/or, as used herein in the description and claims, is to be understood to mean one or both of the elements so connected, i.e. elements which are in some cases present together and in other cases present separately. If desired, other elements may be present other than those specifically identified in the proposal and/or whether or not related to those elements specifically identified, unless explicitly stated otherwise. Thus, as a non-limiting example, a reference to A and/or B, when used in conjunction with open language such as containing, may refer, according to one embodiment, to A without B (optionally including elements other than B) ; according to another embodiment, on B without A (optionally including elements other than A); according to yet another embodiment, A and B (optionally including other elements); and so on.

As used herein in the specification and claims, the term at least one, with respect to a list of one or more elements, is to be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically identified in the list of elements, and without excluding any combination of elements in the list of elements. This definition also allows that, if desired, elements other than the elements specifically defined in the list of elements to which the phrase at least one refers may be present, whether or not they are related to those elements specifically identified. Thus, as a non-limiting example, at least one of A and B (or, equivalently, at least one of A or B, or, equivalently, at least one of A and/or B) may be treated according to one embodiment implementation to at least one, optionally including more than one A, without the presence of B (and optionally including elements other than B); according to another embodiment, to at least one, optionally including more than one B, without the presence of A (and optionally including elements other than A); according to 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), and the like.

The use of ordinal numeral terms, such as first, second, third, etc., in a claim to modify an element of the claim does not of itself imply any priority, precedence, or order of one claimed element over another, or temporal order, in which performs the steps of the method, but is used merely as labels to distinguish one claim element having a specific name from another claim element having the same name (but to use an ordinal term) to distinguish the claim elements.

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

Isoform-selective, context-permissive/context-independent TGFe1 antibodies.

The present invention relates to antibodies and antigen-binding portions thereof that bind to two or more of the following pro/latent-TGFβ1-containing complexes: the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and the LRRC33-TGFe1 complex.

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Accordingly, some aspects of the present invention relate to antibodies or antigen-binding portions thereof that specifically bind to an epitope in such a TGFe1 complex, wherein the epitope is available for binding by the antibody or antigen-binding portions thereof when the TGFe1s are present in the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 and/or the LRRC33-TGFe1 complex. In some embodiments, the epitope is accessible due to a conformational change of TGFe1 in complex with GARP, LTBP1, LTBP3, and/or LRRC33. In some embodiments, the epitope on TGFe1 to which antibodies or antigen-binding portions thereof bind is not accessible when TGFe1 is not in a complex with GARP, LTBP1, LTBP3, and/or LRRC33. In some embodiments, the antibodies or antigen-binding portions thereof do not specifically bind TGFe2. In some embodiments, the antibodies or antigen-binding portions thereof do not specifically bind TGFe3. In some embodiments, the antibodies or antigen-binding portions thereof do not interfere with TGFe1 binding to integrin. For example, in some embodiments, the antibodies or antigen-binding portions thereof do not mask the TGFe1 integrin-binding site. In some embodiments, the antibodies or antigen-binding portions thereof inhibit TGFe1 activation. In some embodiments, the antibodies or antigen-binding portions thereof inhibit the release of mature TGFe1 from the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex.

The antibodies or antigen-binding portions thereof provided herein specifically bind to an epitope of multiple (i.e., two or more) TGFe1 complexes, wherein the epitope is available for binding by the antibody or antigen-binding portions thereof when TGFe1 is present in the GARP-TGFe1 complex, LTBP1 complex -TGFe1, LTBP2-TGFe1 complex, LTBP3-TGFe1 complex, LTBP4-TGFe1 complex and/or LRRC33-TGFe1 complex. In some embodiments, TGFe1 comprises a naturally occurring mammalian amino acid sequence. In some embodiments, TGFe1 comprises a naturally occurring human amino acid sequence. In some embodiments, TGFe1 comprises a human, monkey, rat, or mouse amino acid sequence. In some embodiments, an antibody or antigen-binding portion thereof described herein does not specifically bind TGFe2. In some embodiments, an antibody or antigen-binding portion thereof described herein does not specifically bind TGFe3. In some embodiments, an antibody or antigen-binding portion thereof described herein does not specifically bind TGFe2 or TGFe3. In some embodiments, an antibody or antigen-binding portion thereof described herein specifically binds to TGFe1 comprising the amino acid sequence set forth in SEQ ID NO: 21. The amino acid sequences of TGFe2 and the amino acid sequence of TGFe3 are set forth in SEQ ID NOs: 22 and 23, respectively. In some embodiments, an antibody or antigen-binding portion thereof described herein specifically binds to a TGFe1 containing a non-naturally occurring amino acid sequence (otherwise referred to herein as non-naturally occurring TGFe1). For example, non-naturally occurring TGFe1 may contain one or more recombinantly generated mutations relative to the naturally occurring amino acid sequence of TGFe1. In some embodiments, the amino acid sequence of TGFe1, TGFe2, or TGFe3 comprises the amino acid sequence set forth in SEQ ID NO: 24-35, as shown in Table. 1. In some embodiments, the amino acid sequence of TGFe1, TGFe2, or TGFe3 comprises the amino acid sequence as set forth in SEO ID NO: 36-43, as shown in Table. 2.

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TGF 1

LSTCKTmMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAG ESAEPEPPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLL SRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWL SRGGEIEGFRLSAHCSCDSRD NTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERA QHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPC PYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVR SCKCS (SEQ ID NO: 21)

TGFp2

SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPPEVISIYNSTRDLLQEK ASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNA SNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSF DVTDAVHEWLHHK DRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTS GDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCL RPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASP CCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCK CS (SEQ ID NO: 22)

TGFp3

SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRELLE EMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVE KNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWL SFDVTDTVREWLLRRESNLGLE ( SEQ Ш NO: 23)

- 23 045239

Table 1

Exemplary amino acid sequences of TGFei, TGFe2 and TGFe3

Protein Subsequence SEQ ID NO προΤΘΡβΙ LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEV PPGPLPEAVLALYNSTRDRVAGESAEPEPPEADYYA KEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELRE AVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNS WRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIE GFRLSAHCSCD SRDNTLQVDINGFTTGRRGDLATIHG MNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSST EI<NCCVRQLYIDFRI<DLGWKWIHEPI<GYHANFCLGP CPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALE PLPIVYYVGRKPKVEQLSNMIVRSCKC S 24 npoTGFpl C4S LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEV PPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYA KEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELRE AVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNS WRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIE GFRLSAHCS CDSRDNTLQVDINGFTTGRRGDLATIHG MNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSST EI<NCCVRQLYIDFRI<DLGWKWIHEPI<GYHANFCLGP CPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALE PLPIVYYVGRKPKVEQLSNMIVRSCKC S 25 npoTGFpl D2G LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEV PPGPLPEAVLALYNSTRDRVAGESAEPEPPEADYYA KEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELRE AVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNS WRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIE GFRLSAHCSCD SRDNTLQVDINGFTTTGRRGDLATIHG MNRPFLLLMATPLERAQHLQS SRHGALDTNYCF S STE KNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPC PYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEP LPIVYYVGRKPKVEQLSNMIVRSCKC S 26 npoTGFpl C4S D2G LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEV PPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYA KEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELRE AVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNS WRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIE GFRLSAHCS CDSRDNTLQVDINGFTTTGRRGDLATIHG MNRPFLLLMATPLERAQHLQS SRHGALDTNYCF S STE KNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPC PYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEP LPIVYYVGRKPKVEQLSNMIVRSCKC S 27 npoTGFp2 SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYP EPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEE YYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSA MEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILK SKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHE 28

- 24 045239

WLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELE ARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLM LLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLR PLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSS DTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIG KTPKIE QLSNMIVKSCKCS npoTGFp2 C5S SLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYP EPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEE YYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSA MEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILK SKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHE WLHH KDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELE ARFAGIDGTSTYTSGDQKTIKSTRKNSGKTPHLLLM LLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLR PLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSS DTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIG KTPKIEQLSNMIVKS CKCS 29 npoTGFp2 C5S D2G SLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYP EPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEE YYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSA MEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILK SKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHE WLHH KDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELE ARFAGIDGTSTYTSGDQKTIKSTRKNSGKTPHLLLM LLPSYRLESQQTNRRKGALDAAYCFRNVQDNCCLRPL YIDFKRDLGWK W IHEPKG YN ANFC AG ACP YLWS SDT QHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKT PKIEQLSNM IVKSCKCS thirty npoTGFp2 D2G WLHH KDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELE ARFAGTOGTSTYTSGDQKTIKSTRKNSGKTPHLLLM LLPSYRLESQQTNRRKGALDAAYCFRNVQDNCCLRPL YIDFKRDLGWK W IHEPKG YN ANFC AG ACP YLWS SDT QHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKT PKIEQLSNM IVKSCKCS 31 npoTGF33 SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPT VMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENT ESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRF NVS S VEK NRTN LFR A EFR VLR VPNP S SKRNEQRIELFQ ILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVRE WLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIK FKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPP HRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLY IDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTT HSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRT PKVEQ LSNMVVKSCKCS 32 npoTGFp3 C7S SLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPT VMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENT 33

- 25 045239

ESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRF NVS S VEKNRTNLFRAEFRVLRVPNP S SKRNEQRIELFQ ILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDDVTDTVRE WLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIK FKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMI PP HRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLY IDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTT HSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRT PKVEQLSNMVVKSCKCS npoTGFp3 C7S D2G SLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPT VMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENT ESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRF NVS S VEKNRTNLFRAEFRVLRVPNP S SKRNEQRIELFQ ILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVRE WL LRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIK FKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPP HRLDNPGQGGQRKGALDTNYCFRNLEENCCVRPLYI DFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTH STVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTP KVEQLSNMVV KSCKC S 34 npoTGFp3 D2G SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPT VMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENT ESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRF NVS S VEKNRTNLFRAEFRVLRVPNP S SKRNEQRIELFQ ILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVRE WL LRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIK FKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPP HRLDNPGQGGQRKGALDTNYCFRNLEENCCVRPLYI DFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTH STVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTP KVEQLSNMVV KSCKC S 35

Table 2 Non-human amino acid sequences

Protein View Subsequence SEQ ID NO npoTGFpl Mouse LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQG EVPPGPLPEAVLALYNSTRDRVAGESADPEPEPEAD YYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNT SDIREAVPEPPLLSRAELRLQRLKS S VEQHVELYQK YSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQW LNQGDGIQGFRF S ANS SC DSKDNKLHVEINGISPKR RGDLGTIHDMNRPFLLLM ATPLERAQHLHS SRHRR ALDTNYCFS STEKNCC VRQLYIDFRKDLGWK WIHE PKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNP GASASPCCVPQALEPLPIVYYVGRKPKVEQLSNMIV RSCKCS 36 npoTGFpl Cynomolgus macaque LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQG EVPPGPLPEAVLALYNSTRDRVAGESAEPEPPEPEAD YYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNT 37

- 26 045239

SELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQK YSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQW LSRGGEIEGFRLSAHCSCDSKDNTLQVDINGFTTGR RGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRR ALDTNYCFSSTEI<NCCVRQLYIDFRI<DLGWI<WIHE PKGYHANFCLGPCPYIWSLD TQYSKVLALYNQHNP GASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMI VRSCKCS TGFpl LAP C4S Mouse LSTSKTroMELVKRKRIEAIRGQILSKLRLASPPSQG EVPPGPLPEAVLALYNSTRDRVAGESADPEPEPEAD YYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNT SDIREAVPEPPLLSRAELRLQRLKS S VEQHVELYQK YSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQW LNQGDGIQGFRF SAHC SC DSKDNKLHVEINGISPKR RGDLGTIHDMNRPFLLLM ATPLERAQHLHS SRHRR 38 TGFpl LAP C4S Cynomolgus macaque LSTSI<TIDMELVI<RI<RIEAIRGQILSKLRLASPPSQG EVPPGPLPEAVLALYNSTRDRVAGESAEPEPPEPEAD YYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNT SELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQK YSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQW LSRGGEIEGFRLSAHCSCDS KDNTLQVDINGFTTGR RGDLATIHGMNRPFLLLMATPLERAQHLQS SRHRR 39 npoTGFpl C4S D2G Mouse LSTSI<TIDMELVI<RI<RIEAIRGQILSKLRLASPPSQG EVPPGPLPEAVLALYNSTRDRVAGESADPEPEPEAD YYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNT SDIREAVPEPPLLSRAELRLQRLKS S VEQHVELYQK YSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQW LNQGDGIQGFRF SAHC SC DSKDNKLHVEINGISPKR RGDLGTIHDMNRPFLLLM ATPLERAQHLHS SRHGA LDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPG ASASPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVR SCKCS 40 npoTGFpl C4S Mouse LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQG EVPPGPLPEAVLALYNSTRDRVAGESADPEPEPEAD YYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNT SDIREAVPEPPLLSRAELRLQRLKS S VEQHVELYQK YSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQW LNQGDGIQGFRF SAHC SC DSKDNKLHVEINGISPKR RGDLGTIHDMNRPFLLLM ATPLERAQHLHS SRHRR ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHE PKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNP GASASPCCVPQALEPLPIVYYVGRKPKVEQLSNMIV RSCKCS 41 npoTGFpl C4S Cynomolgus macaque LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQG EVPPGPLPEAVLALYNSTRDRVAGESAEPEPPEPEAD YYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNT SELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQK YSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQW LSRGGEIEGFRLSAHCSCDS KDNTLQVDINGFTTGR RGDLATIHGMNRPFLLLMATPLERAQHLQS SRHRR ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHE 42

- 27 045239

PKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNP GASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMI VRSCKCS npoTGFpl C4S D2G Cynomolgus macaque LSTSKTmMELVKRKRIEAIRGQILSKLRLASPPSQG EVPPGPLPEAVLALYNSTRDRVAGESAEPEPPEPEAD YYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNT SELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQK YSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQW LSRGGEIEGFRLSAHCSCDS KDNTLQVDINGFTTGR RGDLATIHGMNRPFLLLMATPLERAQHLQSSRHGA LDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPG ASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVR SCKCS 43 LTBP3 Cynomolgus macaque GPAGERGAGGGGGALARERFKVVFAPVICKRTCLKG QCRDSCQQGSNMTLIGENGHSTDTLTGSGFRVVVC PLPCMNGGQC S SRNQCLCPPDFTGRFCQVP AGGAG GGTGGSGPGLSRAGALSTGALPPLAPEGDSVASKH AIYAVQVIADPPGPGEGPPAQHAAFLVPLGPGQISA E VQ APPP VVNVRVHHPPEAS VQ VHRIES SNAEGAA PSQHLLPHPKPSHPRPPTQKPLGRCFQDTLPKQPCG SNPLPGLTKQEDCCGSIGTAWGQSKCHKCPQLQYT GVQKPGPVRGEVGADCPQGYKRLNSTHCQDINEC AMPGVCRHGDCLNNPGSYRCVCPPGHSLGPSRTQC IADKPEEKSLCFRLVSPEHQCQHPLTTRLTRQLCCC SV GKAWGARCQRCPADGTAAFKEICPAGKGYHILT SHQTLTIQGESDFSLFLHPDGPPKPQQLPESPSQAPP PEDTEEERGVTTDSPVSEERSVQQSHPTATTSPARP YPELISRPSPPTMRWFLPDLPPSRSAVEIAPTQVTET DECRLNQNICGHGECVPGPPDYSCHCNPGYRSHPQ HRYCVDVNECEAEPCGPGRGICMNTGG SYNCHCN RGYRLHVGAGGRSCVDLNECAKPHLCGDGGFCINF PGHYKCNCYPGYRLKASRPPVCEDIDECRDPSSCPD GKCENKPGSFKCIACQPGYRSQGGGACRDVNECAE GSPCSPGWCENLPGSFRCTCAQGYAPAPDGRSCVD VDECEAGDVCDNGICTNTPGSFQCQCLSGYHLSRD RSHCEDIDECDFPAACIGGDCINTNG SYRCLCPQGH RLVGGRKCQDIDECTQDPGLCLPHGACKNLQGSYV CVCDEGFTPTQDQHGCEEVEQPHHKKECYLNFDDT VFCDSVLATNVTQQECCCSLGAGWGDHCEIYPCPV YS S AEFHSLCPDGKGYTQDNNIVNYGIP AHRDIDEC MLFGAEICKEGKCVNTQPGYECYCKQGFYYDGNL LECVDVDECLDESNCRNGVCENTRGGYRCACTPPA EYSPAQRQCLSPEEMDVDECQDPAACRPGRCVNLP GSYRCECRPPWVPGPSGRDCQLPESPAERAPERRD VCWSQRGEDGMCAGPQAGPALTFDDCCCRQGRG WGAQCRPCPPRGAGSQCPTSQSESNSFWDTSPLLL GKPRRDEDSSEEDSDECRCVSGRCVPRPGGAVCEC PGGF QLDASRARCVDIDECRELNQRGLLCKSERCV NTSGSFRCVCKAGFARSRPHGACVPQRRR 44 LTBP3 Mouse GPAGERGTGGGGALARERFKVVFAPVICKRTCLKG QCRDSCQQGSNMTLIGENGHSTDTLTGSAFRVVVC 45

- 28 045239

PLPCMNGGQCSSRNQCLCPPDFTGRFCQVPAAGTG AGTGSSGPGLARTGAMSTGPLPPLAPEGESVASKH AIYAVQVIADPPGPGEGPPAQHAAFLVPLGPGQISA EVQAPPPVVNVRVHHPPEASVQVHRIEGPNAEGPA SSQHLLPHPKPPHPRPPTQKPLGRCFQDTLPKQPCG SNPLPGLTKQEDCCGSIGTAWGQSK LED TEEERGVTMDPPVSEERSVQQSHPTTTTSPPRP YPELISRPSPPTFHRFLPDLPPSRSAVEIAPTQVTED ECRLNQNICGHGQCVPGPSDYSCHCNAGYRSHPQH RYCVDVNECEAEPCGPGKGICMNTGGSYNCHCNR GYRLHVGAGGRSCVDLNECAKPHLCGDGGFCINFP GHYKCNCYPGYRLKASRPPICEDIDECRDPSTCPD G KCENKPGSFKCIACQPGYRSQGGGACRDVNECSEG TPCSPGWCENLPGSYRCTCAQYEPAQDGLSCIDVD ECEAGKVCQDGICTNTPGSFQCQCLSGYHLSRDRS RCEDIDECDFPAACIGGDCINTNGSYRCLCPLGHRL VGGRKCKKDIDECSQDPGLCLPHACENLQGSYVCV CDEGFTLTQDQHGCEEVEQPHHKKECY LNFDDTVF CDSVLATNVTQQECCCSLGAGWGDHCEIYPCPVYS SAEFHSLVPDGKRLHSGQQHCELCIPAHRDIDECILF GAEICKEGKCVNTQPGYECYCKQGFYYDGNLLEC VDVDECLDESNCRNGVCENTRGGYRCACTPPAEYS PAQAQCLIPERWSTPQRDVKCAGASEERTACVWGP WAGPALTFDDCCCRQPRL GTQCRPCPPRGTGSQCP TSQSESNSFWDTSPLLLGKSPRDEDSSEEDSDECRC VSGRCVPRPGGAVCECPGGFQLDASRARCVDIDEC RELNQRGLLCKSERCVNTSGSFRCVCKAGFTRSRP HGPACLSAAADDAAIAHTSVIDHRGYFH LTBP1S Cynomolgus macaque NHTGRIKVVFTPSICKVTCTKGSCQNSCEKGNTTTL ISENGHAADTLT ATNFRVVLCHLPCMNGGQC S SRD KCQCPPNFTGKLCQIPVHGASVPKLYQHSQQPGKA LGTHVIHSTHTLPLTVTSQQGVKVKFPPNIVNIHVK HPPEASVQIHQVSRIDGPTGQKTKEAQPGQSQVSYQ GLPV QKTQTIHSTYSHQQVIPHVYPVAAKTQLGRCF QETIGSQCGKALPGLSKQEDCCGTVGTSWGFNKCQ KCPKKPSYHGYNQMMECLPGYKRVNNTFCQDINE CQLQGVCPNGECLNTMGSYRCTCKIGFGPDPTFSSC VPDPPVISEEKGPCYRLVSSGRQCMHPLSVHLTKQL CCCSVGKAWG PHCEKCPLPGTAAFKEICPGGGMGYT VSGVHRRRPIHHHVGKGPVFVKPKNTQPVAKSTHP PPLPAKEEPVEALTFSREHGPGVAEPEVATAPPEKEI PSLDQEKTKLEPGQPQLSPGISTIHLHPQFPVVIEKTS PPVPVEVAPEASTSSASQVIAPTQVTEINECTVNPDI CGAGHCINLPVRYTCICYEGYK FSEQQRKCVDIDEC TQVQHLCSQGRCENTEGSFLCICPAGFMASEEGTNC IDVDECLRPVCGEGHCVNTVGAFRCEYCDSGYR MTQRGRCEDIDECLNPSTCPDEQCVNSPGSYQCVP 46

- 29 045239

CTEGFRGWNGQCLD VDECLEPNVCTNGDC SNLEG SYMCSCHKGYTRTPDHKHCKDIDECQQGNLCVNG QCKNTEGSFRCTCGQGYQLSAAKDQCEDIDECQHH HLCAHGQCRNTEGSFQCVCDQGYRASGLGDHCEDI NECLEDKSVCQRGDCINTAGSYDCTCPDGFQLDDN KTCQDINECEHPGLCGPQGECLNTEG SFHCVCQQG FSISADGRTCEDIDECVNNTVCDSHGFCDNTAGSFR CLCYQGFQAPQDGQGCVDVNECELLSGVCGEAFC ENVEGSFLCVCADENQEYSPMTGQCRSRTSTDLDV EQPKEEKKECYNLNDASLCDNVLAPNVTKQECC CTSGAGWGDNCEIFPCPVLGTAEFTEMCPKGKGFV P AGES S SEAGGENYKD ADECLLFGQEICKNGFCLNT RPGYECYCKQGTYYDPVKLQCFDMDECQDPSSCID GQCVNTEGSYNCFCTHPMVLDASEKRCIRPAESNE QIEETDVYQDLCWEHLSDEYVCSRPLVGKQTTYTE CCCLYGEAWGMQCALCPMKDSDDYAQLCNIPVTG RRQP YGRDALVDF SEQ Y APE ADP YFIQDRFLNSFEE LQAEECGILNGCENGRCVRVQEGYTCDCFDGYHLD TAKMTCVDVNECDELNNRMSLCKNAKCINTEGSY KCLCLPGYVPSDKPNYCTPLNTALNLEKDSDLE LTBP1S Mouse NHTGRIKVVFTPSICKVTCTKGNCQNSCQKGNTTTL ISENGHAADTLT ATNFRVVICHLPCMNGGQC S SRD KCQCPPNFTGKLCQIPVLGASMPKLYQHAQQQGKA LGSHVIHSTHTLPLTMTSQQGVKVKFPPNIVNIHVK HPPEASVQIHQVSRIDSPGGQKVKEAQPGQSQVSYQ GLPVQKTQTVHSTYSHQQLIPHVYPVAAKTQLGRC FQETIGSQCGKALPGLSKQEDCCGTVGTSWGFNKC QKCPKKQSYHGYTQMMECLQGYKRVNNTFCQDIN ECQLQGVCPNGECLNTMGSYRCSCKMGFGPDPTFS SCVPDPPVISEEKGPCYRLVSPGRHCMHPLSVHLTK QICCCSVGK AWGPHCEKCPLPGTAAFKEICPGGGMG YTVSGVHRRRPIHQHIGKEAVYVKPKNTQPVAKST HPPPLPAKEEPVEALTSSWEHGPRGAEPEVVTAPPE KEIPSLDQEKTRLEPGQPQLSPGVSTIHLHPQFPVVV EKTSPPVPVEVAPEASTSSASQVIAPTQVTEINECTV NPDICGAGHCINLPVRYTCICYEGYKF SEQLRKCVD IDECAQVRHLCSQGRCENTEGSFLCVCPAGFMASE EGTNCIDVDECLRPDMCRDGRCINTAGAFRCEYCD SGYRMSRRGYCEDIDECLKPSTCPEEQCVNTPGSYQ CVPCTEGFRGWNGQCLDVDECLQPKVCTNGSCTN LEGSYMCSCHRGYSPTPDHRHCQDIDECQQGNLCM NGQCRNTDGSFRCTCGQGYQ LSAAKDQCEDIDECE HHHLC SHGQCRNTEGSFQC VCNQGYRAS VLGDHC EDINECLEDSSVCQGGDCINTAGSYDCTCPDGFQLN DNKGCQDINECAQPGLCGSHGECLNTQGSFHCVCE QGFSISADGRTCEDIDECVNNTVCDSHGFCDNTAGS FRCLCYQGFQAPQDGQGCVDVNECELLSGVCGEAF CENVEGSFLCVCADEN QEYSPMTGQCRSRVTEDSG VDRQPREEKKECYYNLNDASLCDNVLAPNVTKQE CCCTSGAGWGDNCEIFPCPVQGTAEFTEMCPRGKG LVP AGES S YDTGGENYKD ADECLLFGEEICKNGYC LNTQPGYECYCKQGTYYDPVKLQCFDMDECQDPN 47

- 30 045239

SCroGQCVNTEGSYNCFCTHPMVLDASEKRCVQPT ESNEQIEETD VYQDLCWEHLSEEYVC SRPLVGKQT TYTECCCLYGEAWGMQCALCPMKDSDDYAQLCNI R VTGRRRP YGRD ALVDF SEQ YGPETDP YFIQDRFLN SFEELQAEECGILNGCENGRCVRVQEGYTCDCFDG YH LDM A K MTS VD VN EU SELNNRMSLCKNAKCINT EGSYKCLCLPGYIPSDKPNYCTPLNSALNLDKESDL E GARP Mouse ISQRREQVPCRTVNKEALCHGLGLLQVPSVLSLDIQ ALYLSGNQLQSILVSPLGFYTALRHLDLSDNQISFLQ AGVFQALPYLEHLNLAHNRLATGMALNSGGLGRL PLLVSLDLSGNSLHGNLVERLLGETPRLRTLSLAEN SLTRLARHTFWGMPAVEQLDLHSNVLMDIEDGAFE ALPHLTHLNLSRNSLTCISDFSLQQ LQVLDLSCNSIE AFQTAPEPQAQFQLAWLDLRENKLLHFPDLAVFPR LIYLNVSNNLIQLPAGLPRGSEDLHAPSEGWSASPLS NPSRNASTHPLSQLLNLDLSYNEIELVPASFLEHLTS LRFLNLSRNCLRSFEARQVDSLPCLVLLDLSHNVLE ALELGTKVLGSLQTLLLQDNALQELPPYTFASLASL QRLNLQGNQV SPCGGPAEPGPPGCVDFSGIPTLHVL NMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVA TGALVGLEASLEVLELQGNGLTVLRVDLPCFLRLK RLNLAENQLSHLP AWTRAVSLEVLDLRNNSF SLLP GNAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLH QGRVDVDATQDLICRFGSQEELSLSLVRPEDCEKG GLKNV NLILLLSFTLVSAIVLTTLATICFLRRQKLSQ QYKA 48 sGARP Mouse ISQRREQVPCRTVNKEALCHGLGLLQVPSVLSLDIQ ALYLSGNQLQSILVSPLGFYTALRHLDLSDNQISFLQ AGVFQALPYLEHLNLAHNRLATGMALNSGGLGRL PLLVSLDLSGNSLHGNLVERLLGETPRLRTLSLAEN SLTRLARHTFWGMPAVEQLDLHSNVLMDIEDGAFE ALPHLTHLNLSRNSLTCISDFSLQQ LQVLDLSCNSIE AFQTAPEPQAQFQLAWLDLRENKLLHFPDLAVFPR LIYLNVSNNLIQLPAGLPRGSEDLHAPSEGWSASPLS NPSRNASTHPLSQLLNLDLSYNEIELVPASFLEHLTS LRFLNLSRNCLRSFEARQVDSLPCLVLLDLSHNVLE ALELGTKVLGSLQTLLLQDNALQELPPYTFASLASL QRLNLQGNQV SPCGGPAEPGPPGCVDFSGIPTLHVL NMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVA TGALVGLEASLEVLELQGNGLTVLRVDLPCFLRLK RLNLAENQLSHLP AWTRAVSLEVLDLRNNSF SLLP GNAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLH QGRVDVDATQDLICRFGSQEELSLSLVRPEDCEKG GLKNV N 49

In some embodiments, antigenic protein complexes (eg, LTBP-TGFpi complex) may comprise one or more LTBP proteins (eg, LTBP1, LTBP2, LTBP3, and LTBP4) or fragment(s) thereof. In some embodiments, an antibody or antigen-binding portion thereof described herein is capable of binding to the LTBP1-TGFp1 complex. In some embodiments, the LTBP1 protein is a naturally occurring protein or fragment thereof. In some embodiments, the LTBP1 protein is a non-naturally occurring protein or fragment thereof. In some embodiments, the LTBP1 protein is a recombinant protein. Such a recombinant LTBP1 protein may contain LTBP1, alternatively spliced variants thereof, and/or fragments thereof. Recombinant LTBP1 proteins can also be modified to include one or more detectable tags. In some embodiments, the LTBP1 protein comprises a leader sequence (eg, a native or non-native leader sequence). In some embodiments, the LTBP1 protein does not contain a leader sequence (ie, the leader sequence has been processed or cleaved). Such detectable tags may include, but are not limited to, 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 the amino acid sequence set forth in SEQ ID NOs: 46 and 47 in Table. 2. In some embodiments, the LTBP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 50 in Table. 3.

In some embodiments, an antibody or antigen-binding portion thereof described herein is capable of binding to the LTBP3-TGFP1 complex. In some embodiments, the LTBP3 protein is a naturally occurring protein or fragment thereof. In some embodiments, the LTBP3 protein is a non-meeting

- 31 045239 protein or its fragment occurs in nature. In some embodiments, the LTBP3 protein is a recombinant protein. Such a recombinant LTBP3 protein may contain LTBP3, alternatively spliced variants thereof, and/or fragments thereof. In some embodiments, the LTBP3 protein comprises a leader sequence (eg, a native or non-native leader sequence). In some embodiments, the LTBP3 protein does not contain a leader sequence (ie, the leader sequence has been processed or cleaved). Recombinant LTBP3 proteins can also be modified to include one or more detectable tags. Such detectable tags may include, but are not limited to, 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 the amino acid sequence set forth in SEQ ID NOs: 44 and 45 in Table. 2. In some embodiments, the LTBP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 51 in Table. 3.

In some embodiments, an antibody or antigen-binding portion thereof described herein is capable of binding to the GARP-TGFe1 complex. In some embodiments, the GARP protein is a naturally occurring protein or fragment thereof. In some embodiments, the GARP protein is a non-naturally occurring protein or fragment thereof. In some embodiments, the GARP protein is a recombinant protein. Such GARP may be recombinant, referred to herein as recombinant GARP. Some recombinant GARPs may contain 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 (eg, a native or non-native leader sequence). In some embodiments, the GARP protein does not contain a leader sequence (ie, the leader sequence has been processed or cleaved off). In other embodiments, the recombinant GARPs are modified to include one or more detectable tags. In other embodiments, such detectable labels may include, but are not limited to, 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 the amino acid sequence set forth in SEQ ID NO: 48-49 in Table. 2. In some embodiments, the GARP protein comprises the amino acid sequence set forth in SEQ ID NOs: 52 and 53 in Table. 4. In some embodiments, the antibodies or antigen-binding portions thereof described herein do not bind to TGFe1 in a context-dependent manner, for example, binding to TGFe1 will only occur when the TGFe1 molecule forms a complex with a specific presentation molecule, such as GARP. Instead, antibodies and their antigen-binding moieties bind to TGFe1 in a context-independent manner. In other words, antibodies or antigen-binding portions thereof bind to TGFe1 upon binding to any of the presenting molecule: GARP, LTBP1, LTBP3 and/or LRCC33.

In some embodiments, an antibody or antigen-binding portion thereof described herein is capable of binding to the LRRC33-TGFe1 complex. In some embodiments, the LRRC33 protein is a naturally occurring protein or fragment thereof. In some embodiments, the LRRC33 protein is a non-naturally occurring protein or fragment thereof. In some embodiments, the LRRC33 protein is a recombinant protein. Such LRRC33 may be recombinant, referred to herein as recombinant LRRC33. Some recombinant LRRC33 proteins may contain one or more modifications, truncations and/or mutations compared to wild-type LRRC33. 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 to express soluble LRRC33 protein (sLRRC33; see, for example, SEQ ID NO: 84). In some embodiments, the LRRC33 protein comprises a leader sequence (eg, a native or non-native leader sequence). In some embodiments, the LRRC33 protein does not contain a leader sequence (ie, the leader sequence has been processed or cleaved). In other embodiments, the recombinant LRRC33 proteins are modified to include one or more detectable tags. In other embodiments, such detectable tags may include, but are not limited to, biotin tags, polyhistidine tags, flag tags, myc tags,

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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 the amino acid sequence set forth in SEQ ID NO: 83, 84, and 101 in Table 4.

Table 3 Exemplary LTBP amino acid sequences

Protein Subsequence SEQ ID NO LTBP1S V K AWGPHCEKCPLPGTAAFKEICPGGMGYTVSGVHR RRPIHHHVGKGPVFVKPKNTQPVAKSTHPPPLPAKE EPVEALTFSREHGPGVAEPEVATAPPEKEIPSLDQEK TKLEPGQPQLSPGISTIHLHPQFPVVIEKTSPPVPVEV APEASTSSASQVIAPTQVTEINECTVNPDICGAGHCIN LPVRYTCICYEGY RFSEQQRKCVDIDECTQVQHLCS QGRCENTEGSFLCICPAGFMASEEGTNCIDVDECLRP DVCGEGHCVNTVGAFRCEYCDSGYRMTQRGRCEDI DECLNPSTCPDEQCVNSPGSYQCVPCTEGFRGWNG QCLDVDECLEPNVCANGDCSNLEGSYMCSCHKGYT RTPDHKHCRDIDECQQGNLCVNGQCKNTEGSFRCT CGQGYQLS AAKDQCEDIDECQHRHLCAHGQCRNTE GSFQCVCDQGYRASGLGDHCEDINECLEDKSVCQR GDCINTAGSYDCTCPDGFQLDDNKTCQDINECEHPG LCGPQGECLNTEGSFHCVCQQGFSISADGRTCEDIDE CVNNTVCDSHGFCDNTAGSFRCLCYQGFQAPQDGQ GCVDVNECELLSGVCGEAFCENVEGSFLCVCADEN QEYSP MTGQCRSRTSTDLDVDVDQPKEEKKECYYN LNDASLCDNVLAPNVTKQECCCTSGVGWGDNCEIF PCPVLGTAEFTEMCPKGKGFVP AGES S SEAGGENYK DADECLLFGQEICKNGFCLNTRPGYECYCKQGTYY DPVKLQCFDMDECQDPSSCIDGQCVNTEGSYNCFCT HPMVLDASEKRCIRPAESNE QIEETDVYQDLCWEHL SDEYVCSRPLVGKQTTYTECCCLYGEAWGMQCALC PLKDSDDYAQLCNIPVTGRRQPYGRDALVDFSEQYT PEADPYFIQDRFLNSFEELQAEECGILNGCENGRCVR VQEGYTCDCFDGYHLDTAKMTCVDVNECDELNNR MSLCKNAKCINTDGSYKCLCLPGYVPSDKPNYCTPL NTAL NLEKDSDLE 50 LTBP3 GPAGERGAGGGGGALARERFKVVFAPVICKRTCLKG QCRDSCQQGSNMTLIGENGHSTDTLTGSGFRVVVCP LPCMNGGQCSSRNQCLCPPDFTGRFCQVPAGGAGG GTGGSGPGLSRTGALSTGALPPLAPEGDSVASKHAI 51

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YAVQVIADPPGPGEGPPAQHAAFLVPLGPGQISAEV QAPPPVVNVRVHHPPEASVQVHRIESSNAESAAPSQ HLLPHPKPSHPRPPTQKPLGRCFQDTLPKQPCGSNPL PGLTKQEDCCGSIGTAWGQSKCHKCPQLQYTGVQK PGPVRGEVGADCPQGYKRLNSTHCQDINECAMPGV CRHGDCLNNPG SYRCVCPPGHSLGPSRTQCIADKPE EKSLCFRLVSPEHQCQHPLTTRLTRQLCCCSVGKAW GARCQRCPTDGTAAFKEICPAGKGYHILTSHQTLTIQ GESDFSLFLHPDGPPKPQQLPESPSQAPPPEDTEEERG VTTDSPVSEERSVQQSHPTATTTPARPYPELISRPSPP TMRWFLPDLPPSRSAVEIAPTQVTETDECRL NQNICG HGECVPPGPDYSCHCNPGYRSHPQHRYCVDVNECE AEPCGPGRGICMNTGGSYNCHCNRGYRLHVGAGGR SCVDLNECAKPHLCGDGGFCINFPGHYKCNCYPGY RLKASRPPVCEDIDECRDPSSCPDGKCENKPGSFKCI ACQPGYRSQGGGACRDVNECAEGSPCSPGWCENLP GSFRCTCAQGYAPAPDGRSCLDV DECEAGDVCDNG ICSNTPGSFQCQCLSGYHLSRDRSHCEDIDECDFPAA CIGGDCINIWSYRCLCPQGHRLVGGRKCQDIDECS QDPSLCLPHGACKNLQGSYVCVCDEGFTPTQDQHG CEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQE CCCSLGAGWGDHCEIYPCPVYSSAEFHSLCPDGKGY TQDNNIV NYGIPAHRDIDECMLFGSEICKEGKCVNT QPGYECYCKQGFYYDGNLLECVDVDECLDESNCRN GVCENTRGGYRCACTPPAEYSPAQRQCLSPEEMDV DECQDPAACRPGRCVNLPGSYRCECRPPWVPGPSGR DCQLPESPAERAPERRDVCWSQRGEDGMCAGPLAG PALTFDDCCCRQGRGWGAQCRPCPPRGAGSHCPTS QSESNSFWD TSPLLLGKPPRDEDSSEEDSDECRCVSG RCVPRPGGAVCECPGGFQLDASRARCVE)IDECRELN QRGLLCKSERCVNTSGSFRCVCKAGFARSRPHGACV PQRRR

Table 4

Exemplary amino acid sequences of GARP and LRRC33

Protein Subsequence SEQ ID NO GARP AQHQDKVPCKMVDKKVSCQVLGLLQVPSVLPPDTET LDLSGNQLRSILASPLGFYTALRHLDLSTNEISFLQPGA FQALTHLEHLSLAHNRLAMATALSAGGLGPLPRVTSL DLSGNSLYSGLLERLLGEAPSLHTLSLAENSLTRLTRH TFRDMPALEQLDLHSNVLMDIEDGAFEGLPRLTHLNL SRNSLTCISDFSLQQLRVLD LSCNSIEAFQTASQPQAEF QLTWLDLRENKLLHFPDLAALPRLIYLNLSNNLIRLPT GPPQDSKGIHAPSEGWSALPLSAPSGNASGRPLSQLLN LDLSYNEIELIPDSFLEHLTSLCFLNLSRNCLRTFEARR LGSLPCLMLLDLSHNALETLELGARALGSLRTLLLQG NALRDLPPYTFANLASLQRLNLQGNRVSPCGGPDEPG PSGCV AFSGITSLRSLSLVDNEIELLRAGAFLHTPLTEL 52

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DLSSNPGLEVATGALGGLEASLEVLALQGNGLMVLQ VDLPCFICLKRLNLAENRLLSHLPAWTQAVSLEVLDLR NNSFSLLPGSAMGGLETSLRRLYLQGNPLSCCGNGWL AAQLHQGRVDATQDLICRFSSQEEVSLSHVRPEDC EKGGLKNINLIIILTFILVSAILLTTLAACCCVRRQKFNQ QYKA sGARP AQHQDKVPCKMVDKKVSCQVLGLLQVPSVLPPDTET LDLSGNQLRSILASPLGFYTALRHLDLSTNEISFLQPGA FQALTHLEHLSLAHNRLAMATALSAGGLGPLPRVTSL DLSGNSLYSGLLERLLGEAPSLHTLSLAENSLTRLTRH TFRDMPALEQLDLHSNVLMDIEDGAFEGLPRLTHLNL SRNSLTCISDFSLQQLRVLD LSCNSIEAFQTASQPQAEF QLTWLDLRENKLLHFPDLAALPRLIYLNLSNNLIRLPT GPPQDSKGIHAPSEGWSALPLSAPSGNASGRPLSQLLN LDLSYNEIELIPDSFLEHLTSLCFLNLSRNCLRTFEARR LGSLPCLMLLDLSHNALETLELGARALGSLRTLLLQG NALRDLPPYTFANLASLQRLNLQGNRVSPCGGPDEPG PSGCV AFSGITSLRSLSLVDNEIELLRAGAFLHTPLTEL DLSSNPGLEVATGALGGLEASLEVLALQGNGLMVLQ VDLPCFICLKRLNLAENRLLSHLPAWTQAVSLEVLDLR NNSFSLLPGSAMGGLETSLRRLYLQGNPLSCCGNGWL AAQLHQGRVDATQDLICRFSSQEEVSLSHVRPEDC EKGGLKNIN 53 LRRC33 (also known as NRROS; Accession no. Uniprot Q86YC3) MELLPLWLCLGFHFLTVGWRNRSGTATAASQGVC KLVGGAADCRGQSLASVPSSLPPHARMLTLDANPLKT LWNHSLQPYPLLESLSLHSCHLERISRGAFQEQGHLRS LVLGDNCLSENYEETAAALHALPGLRRLDLSGNALTE DMAALMLQNLSSLRSVSLAGNTIMRLDDSVFEGLERL RELDLQRNYIFEIEGGAFDGLAELR HLNLAFNNLPCIV DFGLTRLRVLNVSYNVLEWFLATGGEAAFELETLDLS HNQLLFFPLLPQYSKLRTLLLRDNNMGFYRDLYNTSS PREMVAQFLLVDGNVTNITT VSLWEEF S S SDL ADLRF LDMSQNQFQYLPDGFLRKMPSLSHLNLHQNCLMTLHI REHEPPGALTELDLSHNQLSELHLAPGLASCLGSLRLF NLSSN QLLGVPPGLFANARNITTLDMSHNQISLCPLPA ASDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPF QGTSLTYLDLSSNWGVLNGSLAPLQDVAPMLQVLSL RNMGLHS SFMALDF SGFGNLRDLDLSGNCLTTFPRFG GSLALETLDLRRNSLTALPQKAVSEQLSRGLRTIYLSQ NPYDCCGVDGWGALQHGQTVAD WAMVTCNLSSKII RVTELPGGVPRDCKWERLDLGLLYLVLILPSCLTLLV ACTVIVLTFKKPLLQVIKSRCHWSSVY *Native signal peptide shown in bold 83 Soluble LRRC33 (SLRRC33) MDMRVPAQLLGLLLLWFSGVLGWRNRSGTATAAS QGVCKLVGGAADCRGQSLASVPSSLPPHARMLTLDA NPLKTLWNHSLQPYPLLESLSLHSCHLERISRGAFQEQ GHLRSLVLGDNCLSENYEETAAALHALPGLRRLDLSG NALTEDMAALMLQNLSSLRSVSLAGNTIMRLDDSVFE GLERLRELDLQRNYIFEIEGGAFDGLA ELRHLNLAFNN LPCIVDFGLTRLRVLNVSYNVLEWFLATGGEAAFELE 84

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TLDLSHNQLLFFPLLPQYSKLRTLLLRDNNMGFYRDL YNT S SPREMVAQFLLVDGNVTNITT VSLWEEF S S SDL ADLRFLDMSQNQFQYLPDGFLRKMPSLSHLNLHQNC LMTLHIREHEPPGALTELDLSHNQLSELHLAPGLASCL GSLRLFNLSSNQLLGVPPGLFANARNITTLDMSHNQIS LCPLPAAS DRVGPPSCVDFRNMASLRSLSLEGCGLGA LPDCPFQGTSLTYLDLSSNWGVLNGSLAPLQDVAPML QVLSLRNMGLHS SFMALDF SGFGNLRDLDLSGNCLTT FPRFGGSLALETLDLRRNSLTALPQKAVSEQLSRGLRT IYLSQNPYDCCGVDGWGALQHGQTVADWAMVTCNL S SKIIRVTELPGVPRDCKWERLDL GLHHHHHH *Modified human kappa light chain signal peptide is shown in bold. **Histidine tag is underlined. Chimera LRRC33- Human GARP MDMRVPAQLLGLLLLWFSGVLGWRNRSGTATAAS OGVCKLVGGAADCRGQSLASVPSSLPPHARMLTLDA 101 NPLKTLWNHSLOPYPLLESLSLHSCHLERISRGAFOEO GHLRSLVLGDNCLSENYEETAAALHALPGLRRLDLSG NALTEDMAALMLQNLSSLRSVSLAGNTIMRLDDSVFE GLERLRELDLQRNYIFEIEGGAFDGLAELRHLNLAFNN LPCIVDFGLTRLRVLNVSYNVLEWFLATGGEAAFELE TLDLSHNQLLFFPLLPQYSKLRTLLLRDNNMGFYRDL YNT S SPREMVAQFLLVDGNVTNITT VSLWEEF S S SDL ADLRFLDMSONQFQYLPDGFLRKMPSLSHLNLHONC LMTLHIREHEPPGALTELDLSHNOLSELHLAPGLASCL GSLRLFNLSSNOLLGVPPGLFANARNITTLDMSHNOIS LCPLPAASDRVGPPSCVDFRNMASLRSLSLEGCGLGA LPDCPFOGTSLTYLDLSSNWGVLNGSLAPLODVAPML 0 VLSLRNMGLHS SFMALDF SGFGNLRDLDLSGNCLTT FPRFGGSLALETLDLRRNSLTALPQKAVSEQLSRGLRT IYLSONPYDCCGVDGWGALOHGOTVADWAMVTCNL S S K HRVT ELPGGVPRDC K W ERLDLGL/.////. TFIL VSAIL TmA/lCCCVRROKFNOOYKA *Modified human kappa light chain signal peptide is shown in bold. **The ectodomain of LRRC33 is underlined. ^The transmembrane domain of GARP is in italics. ##The intracellular tail of GARP is underlined by a double line.

TGFei antagonists.

Any suitable TGFP1 inhibitory agent may be used to carry out the methods of the present invention, provided that such agent inhibits or antagonizes TGF31 in multiple biological effects (eg, TGF31 from multiple cellular sources) with sufficient selectivity for the TGF31 isoform. Preferably, such TGFp1 inhibitory agents do not have measurable TGFe2 and TGF33 inhibitory activities at a dosage that provides clinical benefits (eg, therapeutic efficacy and acceptable toxicity profiles) when administered to human subjects. Suitable inhibitory agents include small molecules, nucleic acid-based agents, biological agents (eg, polypeptide-based agents such as antibodies and other detection agents), and any combination thereof. In some embodiments, such agents are antibodies or fragments thereof, as further described below. These include neutralizing antibodies, which bind to the growth factor TGFe1, thereby neutralizing its action.

Functional Antibodies that Selectively Inhibit TGFe1 The present invention, in one aspect, covers the use of functional antibodies. As used herein, the term functional antibody confers one or more biological activities due to its ability to bind an antigen. Therefore, functional antibodies include antibodies capable of modulating the activity/function of target molecules (ie, antigen). Such modulating antibodies include inhibitory antibodies (or inhibitory antibodies) and activating antibodies. The present disclosure includes anti-TGFe antibodies that can inhibit a biological process mediated by TGFp1 signaling associated with multiple TGF31 contexts. Inhibitory agents used to practice the present invention, such as the antibodies described herein, are intended to be selective for TGF31 and do not target or affect TGFe2 and TGFe3 when administered at a therapeutically effective dose (a dose that achieves sufficient effectiveness within acceptable toxicity levels).

Based on Applicant's earlier recognition of the present disclosure (see PCT/US2017/021972) that lack of isoform specificity of traditional TGF3 antagonists may underlie the source of toxicity associated with TGFe inhibition, the present inventors sought to further achieve broad-spectrum inhibition. TGFe1 spectrum for the treatment of various diseases that exhibit multifaceted TGFe1 dysregulation while maintaining the safety/tolerability aspect of isoform-selective inhibitors.

In a broad sense, the term inhibitory antibody refers to an antibody that antagonizes or neutralizes a target function, such as growth factor activity. Advantageously, preferred inhibitory antibodies of the present invention are capable of inhibiting the release of mature growth factor from the latent complex, thereby reducing growth factor signaling. Inhibitory antibodies include antibodies targeting any epitope that reduces growth factor release or activity when bound by such antibodies. Such epitopes may lie on the prodomains of TGFe proteins (eg, TGFe1), growth factors, or other epitopes that result in decreased activity of the growth factor upon binding to the antibody. The inhibitory antibodies of the present invention include, but are not limited to, TGFe1 inhibitory antibodies. In some embodiments, the inhibitory antibodies of the present invention specifically bind a combined epitope, i.e. an epitope formed by two or more components/parts of an antigen or complex of antigens. For example, a combinatorial epitope can be formed by contributions from several parts of a single protein, i.e. amino acid residues from more than one non-contiguous segment of the same protein. Alternatively, a combinatorial epitope can be formed by contributions from multiple protein components of the antigenic complex. In some embodiments, the inhibitory antibodies of the present invention specifically bind a conformational epitope (or conformation-specific epitope), for example, an epitope that is sensitive to the three-dimensional structure (ie, conformation) of an antigen or complex of antigens.

Traditional approaches to antagonizing TGFe signaling have been to: i) directly neutralize the mature growth factor after it has already become active, so as to deplete free ligands (e.g., released from its latent precursor complex) that are available for binding to receptor; ii) use soluble receptor fragments capable of sequestering free ligands (eg, so-called ligand decoys); or w) target its receptor(s) on the cell surface to block ligand-receptor interactions. Each of these traditional approaches requires the antagonist to compete with endogenous counterparts. Additionally, the first two approaches (i and ii) above target the active ligand, which is a transient form. Therefore, such an antagonist should be able to kinetically outcompete the endogenous receptor within a short time window. The third approach may provide a longer lasting effect when compared, but inadvertently leads to undesirable inhibitory effects (hence possible toxicity) since many growth factors (e.g., up to ~20) signal through the same receptor(s).

To address these drawbacks and further provide greater selectivity and localized action, the preferred mechanism of action underlying inhibitory antibodies such as those described herein acts prior to TGFe1 activation and ligand-receptor interaction. Thus, it is contemplated that isoform-specific, context-permissive TGFe1 inhibitors suitable for practicing the present invention should preferentially target an inactive (e.g., latent) TGFe1 precursor complex (e.g., a pro/latent TGFe1-containing complex) prior to its activation to block the activation step at its source (for example, in the disease microenvironment). In preferred embodiments of the present invention, such inhibitors target ECM-associated and/or cell surface-associated TGFe1 pro/latent complexes rather than free ligands that are transiently available for receptor binding.

Accordingly, some embodiments of the present invention utilize agents that specifically bind to TGFe1-containing complexes, thereby inhibiting TGFe1 function in an isoform-selective manner. Such agents are preferably antibodies that bind an epitope within a protein complex containing pro/latent TGFe1 (eg, an inactive TGFe1 precursor). In some embodiments, the epitope is available for antibody binding when TGFe1 is present in two or more of the following components: the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and the LRRC33-TGFe1 complex. In some embodiments, such antibodies bind two or more of the TGFe1-containing complexes presented above (e.g., context-permissive), while in other embodiments, such antibodies bind all four of the TGFe1-containing complexes presented above (e.g., context-independent). context). In some embodiments, any of such antibodies may exhibit differential form selectivity. The epitope may be located in the pro-domain of the TGFe1 complex. The epitope may be a combined epitope such that the epitope is formed by two or more

- 37 045239 parts/segments (for example, amino acid residues) of one or more components of the complex. The epitope may be a conformational epitope such that the epitope is sensitive to a particular three-dimensional structure of the antigen (eg, the TGFe1 complex). An antibody or fragment thereof that specifically binds to a conformational epitope is called a conformational antibody or conformation-specific antibody.

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

Exemplary antibodies and corresponding nucleic acid sequences that encode antibodies useful for practicing the present invention include one or more CDR amino acid sequences shown in table. 5.

Table 5

The complementarity determining regions of the heavy chain (CDRH) and light chain (CDRL), defined using the Kabat numbering scheme, are shown for antibodies Ab 1 Ab2 and Ab3

Antibody AY AB2 ABZ CDRH1 SYGMH (SEQ Ш NO: 1) SDWIG (SEQ GO NO: 2) NYAMS (SEQ ID NO: 85) CDRH2 VISYDGSNKYYADSV KG (SEQ Ш NO: 3) VIYPGDSDTRYSAS FQG (SEQ GO NO: 4) SISGSGGATYYADSVK G (SEQ ID NO: 86) CDRH3 DIRPYGDYSAAFDI (SEQ GO NO: 5) AAGIAAAGHVTAF D.I. (SEQ GO NO: 6) ARVSSGHWDFDY (SEQ ID NO: 87) CDRL1 TGSSGSIASNYVQ (SEQ GO NO: 7) KSSQSVLYSSNNNKN YLA (SEQ GO NO: 8) RASQSISSYLN (SEQ ID NO: 88) CDRL2 EDNQRPS (SEQ GO NO: 9) WASTRES (SEQIDNO: 10) SSLQS (SEQ ID NO: 89) CDRL3 QSYDSSNHGGV (SEQIDNO: AND) QQYYSTPVT (SEQ GO NO: 12) QQSYSAPFT (SEQ ID NO: 90)

In some embodiments, antibodies of the present invention that specifically bind to the GARP-TGF31 complex, the LTBP1-TGFp1 complex, the LTBP3TGFp1 complex, and/or the LRRC33-TGFp1 complex include any antibody or antigen binding portion thereof comprising 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 the GARP-TGF31 complex, the LTBP1TGFp1 complex, the LTBP3-TGFp1 complex, and/or the LRRC33-TGFp1 complex include CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any of the antibodies shown in table. 5. The present invention also provides any nucleic acid sequence that encodes a molecule containing CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 or CDRL3, as provided for any of the antibodies shown in table. 5. The CDR3 domains of an antibody's heavy and light chain may play a particularly important role in the binding specificity/affinity of the antibody for an antigen. Accordingly, antibodies that specifically bind to the GARP-TGFP1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex and/or LRRC33-TGF31 complex of the present disclosure, or nucleic acid molecules encoding these antibodies or antigen-binding portions thereof, may include at least CDR3 of the heavy and/or light chain of the antibodies, as shown in table. 5.

Aspects of the present invention relate to a monoclonal antibody or antigen binding portion thereof that specifically binds to the GARP-TGFP1 complex, the LTBP1TGF31 complex, the LTBP3-TGFe1 complex and/or the LRRC33-TGF31 complex, and which contains six complementarity determining regions (CDRs): CDRH1, CDRH2 , CDRH3, CDRL1, CDRL2 and CDRL3.

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

In some embodiments (eg, with respect to the Ab1 antibody shown in Table 5), the antibody or antigen-binding portion thereof that specifically binds to the GARP-TGFp1 complex, the LTBP1-TGFp1 complex, the LTBP3-TGFp1 complex, and/or the LRRC33TGFp1 complex comprises : CDRH1 containing the amino acid sequence presented in SEQ

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ID NO: 1, CDRH2, containing the amino acid sequence presented in SEQ ID NO: 3,

CDRH3 containing the amino acid sequence set forth in SEQ ID NO: 5, CDRL1 containing the amino acid sequence set forth in SEQ ID NO: 7, CDRL2 containing the amino acid sequence set forth in SEQ ID NO: 9, and CDRL3 containing the amino acid sequence set forth in in SEQ ID NO: 11.

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

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

In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex comprises a heavy chain variable domain amino acid sequence encoded by a nucleic acid sequence characterized by an identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to the nucleic acid sequence set forth in SEQ ID NO: 91, and a light chain variable domain amino acid sequence encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to relative to the nucleic acid sequence set forth in SEQ ID NO: 92. In some embodiments, the antibody or antigen binding portion thereof comprises a heavy chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 91 and a light chain variable domain amino acid sequence chain encoded by the nucleic acid sequence shown in SEQ ID NO: 92.

In some embodiments (e.g., with respect to the Ab2 antibody shown in Table 5), the antibody or antigen-binding portion thereof that specifically binds to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33TGFe1 complex comprises CDRH1, containing the amino acid sequence set forth in SEQ ID NO: 2, CDRH2 containing the amino acid sequence set forth in SEQ ID NO: 3, CDRH3 containing the amino acid sequence set forth in SEQ ID NO: 6, CDRL1 containing the amino acid sequence set forth in SEQ ID NO: 8, CDRL2 containing the amino acid sequence presented in SEQ ID NO: 10, and CDRL3 containing the amino acid acid sequence presented in SEQ ID NO: 12.

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

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

In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex comprises a heavy chain variable domain amino acid sequence encoded by a nucleic acid sequence characterized by identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with respect to the nucleic acid sequence presented in SEQ ID NO: 93, and the amino acid sequence of a light chain variable domain encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to relative to the nucleic acid sequence set forth in SEQ ID NO: 94. In some embodiments, the antibody or antigen binding portion thereof comprises a heavy chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 93 and a light chain variable domain amino acid sequence chain encoded by the nucleic acid sequence shown in SEQ ID NO: 94.

In some embodiments (eg, with respect to the Ab3 antibody shown in Table 5), the antibody or antigen-binding portion thereof that specifically binds to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33TGFe1 complex comprises CDRH1 containing the amino acid sequence presented in SEQ ID NO: 85, CDRH2 containing the amino acid sequence presented in SEQ ID NO: 86, CDRH3 containing the amino acid sequence presented in SEQ ID NO: 87, CDRL1 containing the amino acid sequence presented in SEQ ID NO: 88, CDRL2 containing the amino acid sequence presented in SEQ ID NO: 89, and CDRL3 containing the amino acid sequence presented in SEQ ID NO: 90.

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

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

In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33-TGFe1 complex contains the amino acid sequence ba

- 40 045239 riable heavy chain domain encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity across relative to the nucleic acid sequence set forth in SEQ ID NO: 96, and the amino acid sequence of the light chain variable domain encoded by a nucleic acid sequence having an identity of at least 70%, 75%, 80%, 85%, 90%, 95 %, 96%, 97%, 98%, or 99% with respect to the nucleic acid sequence set forth in SEQ ID NO: 98. In some embodiments, the antibody or antigen binding portion thereof comprises a heavy chain variable domain amino acid sequence encoded by the nucleic acid sequence, presented in SEQ ID NO: 96, and the amino acid sequence of the light chain variable domain encoded by the nucleic acid sequence presented in SEQ ID NO: 98.

In some examples, any of the antibodies of the present disclosure that specifically bind to the GARP-TGFe1 complex, the LTBP1-TGFp1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex includes any antibody (including their antigen-binding portions) containing one or more CDR sequences (eg, CDRH or CDRL) substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3. For example, antibodies may include one or more CDR sequences, as shown in table. 5 (SEQ ID NOs: 1-12 and 85-90) containing up to 5, 4, 3, 2 or 1 amino acid residue variation compared to the corresponding CDR region in any of SEQ ID NOs: 1-12 and 85-90. The complete amino acid sequences for the heavy chain variable region and light chain variable region of the antibodies listed in table. 5 (eg, Ab1, Ab2 and Ab3), as well as the nucleic acid sequences encoding the heavy chain variable region and the light chain variable region of the antibodies are given below.

Ab1-Amino acid sequence of the heavy chain variable region

EVQLVESGGGL VQPGRSLRLSC AASGFTF SS YGMHWVRQAPGKGLEW VAVIS YDGSNK

YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDIRPYGDYSAAFDIWGQG

TLVTVSS (SEQIDNO: 13)

Ab1-Heavy chain variable region nucleic acid sequence GAGGTGCAACTCGTGGAGTCAGGCGGTGGACTTGTTCAGCCTGGGCGAAGTCTGAG

ACTCTCATGTGCAGCAAGTGGATTCACTTTCTCCAGTTACGGCATGCACTGGGTGAG ACAGGCGCCTGGAAAGGGTTTGGAATGGGTCGCTGTGATCTCTTACGACGGGTCAA ACAAATATTACGCGGATTCAGTGAAAGGGCGGTTCACTATTTCACGGGATAACTCCA AGAACACCCTGTATCTGCAGATGAATAGCCTGAGGGCAGAGGACACCGCTGTGTAC TATTGTGCCCGGGAC ATAAGGCCTTACGGCGATTACAGCGCCGCATTTGATATTTGG GGACAAGGCACCCTTGTGACAGTATCTTCT (SEQ IDNO: 91)

Ab1-Amino acid sequence of the light chain variable region

NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSAPSIVIFEDNQRPSGAPD RFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNHGGVFGGGTQLTVL (SEQ Ш NO: 14)

Ab1-Light chain variable region nucleic acid sequence AATTTTATGCTTACCCAACCACATAGTGTGAGTGAGTCTCCCGGCAAGACTGTAACA

ATTTCATGTACCGGCAGCAGTGGCTCCATCGCTAGCAATTATGTGCAATGGTACCAA

CAGCGCCCCGGGAGCGCACCTTCAATAGTGATATTCGAGGATAACCAACGGCCTAG

TGGGGCTCCCGATAGATTTAGTGGGAGTATAGATAGCTCCTCCAACTCTGCCTCTCT

CACCATTAGCGGGCTGAAAACAGAGGATGAAGCCGACTATTACTGCCAAAGCTATG ATTCTAGCAACCACGGCGGAGTGTTTGGCGGAGGAACACAGCTGACAGTCCTAGG (SEQ Ш NO: 92)

Ab1-Heavy chain amino acid sequence

EVQLVESGGGLVQPGRSLRLSC AASGFTF S S YGMHWVRQAPGKGLEW VAVIS YDGSNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDIRPYGDYSAAFDIWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF P AVLQ S SGL YSLS S V VTVP S S SLGTKT YTCNVDHKP SNTKVDKRVESK YGPPCPPCP APE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQIDNO: 15)

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Abl-Amino acid sequence of light chain

NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSAPSIVIFEDNQRPSGAPD RFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNHGGVFGGGTQLTVLGQPKAAPSV TLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAA SSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS (SEQ Ш NO: 16)

AB2-Amino acid sequence of the heavy chain variable region

EVQLVQSGAEMKKPGESLKISCKGSGYNFASDWIGWVRQTPGKGLEWMGVIYPGDSDT RYSASFQGQVTISADKSINTAYLQWSSLKASDTAMYYCASAAGIAAAGHVTAFDIWGQ GTMVTVSS (SEQIDNO: 17)

Ab2-Heavy chain variable region nucleic acid sequence

GAGGTGCAACTGGTGCAATCCGGAGCCGAGATGAAAAAGCCAGGGGAGAGCCTGA

AGATCTCTTGTAAGGGCTCTGGCTATAACTTCGCTAGTGATTGGATCGGATGGGTGA GGCAAACCCCCGAAAGGGCCTCGAGTGGATGGGCGTGATCTACCCCGGCGACTCC GACACACGCTATAGCGCCTCATTCCAGGGCCAGGTCACCCATAAGTGCTGATAAATC AATAAATACAGCCTACTTGCAATGGTCAAGTCTGAAAGCCTCAGATACTGCCATGTA CTATTGTGCCTCTG CCGCCGGCATTGCCGCGGCCGGTCACGTCACCCGCCTTCGACAT TTGGGGTCAGGGCACTATGGTCACTGTAAGCTCC (SEQ ID NO: 93)

Ab2-Amino acid sequence of the light chain variable region

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPVTFGQGTKLEIK (SEQ Ш NO: 18)

Ab2-Light chain variable region nucleic acid sequence

GACATAGTCATGACCCAGTCACCTGACTCTTTGGCCGTGTCTCTGGGGGAGAGAGCC

ACAATAAATTGCAAGTCATCACAGAGCGTCCTGTACTCCTCCAATAATAAAAATTAC CTGGCCTGGTACCAGCAAAAGCCCGGGCAACCCCCCAAATTGTTGATTTACTGGGCT AGTACAAGGGAATCTGGAGTGCCAGACCGGTTTTCTGGTTCTGGATCTGGTACTGAC TTCACCCTGACAATCAGCTCCCTGCAGGCCGAAGACGTGGCTGTGTACTATTGTCAG CAGTACT ATAGTACACCAGTTACTTTCGGCCAAGGCACTAAACTCGAAATCAAG (SEQ Ш NO: 94)

Ab2-Heavy chain amino acid sequence

EVQLVQSGAEMKKPGESLKISCKGSGYNFASDWIGWVRQTPGKGLEWMGVIYPGDSDT RYS ASFQGQ VTIS ADKSINT AYLQW S SLKASDTAMYYC AS AAGIAAAGHVTAFDIWGQ GTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQIDNO: 19)

Ab2-Amino acid sequence of light chain

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPVTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ Ш NO: 20)

Ab3-Amino acid sequence of the heavy chain variable region

EVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVSSISGSGGAT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSSGHWDFDYWGQGTLV TVSS (SEQ Ш NO: 95)

Ab3-Heavy chain variable region nucleic acid sequence

GAGGTTCAGCTTCTGGAGAGCGGCGGTGGTCTTGTACAACCTGGAGGATCACTCAG

GTTGTCATGTGCCGCAAGCGGGTTTACATTCAGGAACTATGCAATGAGCTGGGTCAG ACAGGCTCCCGGCAAGGGACTTGAGTGGGTATCTTCCATCAGCGGATCTGGAGGAG CAACATATTATGCAGATAGTGTCAAAGGCAGGTTCACAATAAGCCGCGACAATTCT AAAAATACTCTTTATCTTCAAATGAATAGCCTTAGGCTGAGGATACGGCGGTGTAT TATTGTGCCCGCGTCTCAA GCGGGCATTGGGACTTCGATTATTGGGGGCAGGGTACT CTGGTTACTGTTTCCTCC (SEQ ID NO: 96)

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AL3-Amino acid sequence of the light chain variable region

DIQMTQSPSSLSSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASSLQSGVPSR

FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPFTFGQGTKVEIK (SEQ Ш NO: 97)

AL3-Nucleic acid sequence of the light chain variable region GACATCCAAATGACACAGAGCCCGTCTTCCCTCTCAGCTTCAGTCGGTGATCGAGTG

ACGATTACGTGCCGCGCCAGCCAAAGCATCTCCTCCTATCTTAACTGGTATCAGCAG AAACCCGGAAAGGCCCCAAAGTTGCTTATTTACGACGCATCCTCCCTTCAATCTGGT GTGCCCAGCAGGTTCTCAGGCAGCGGTTCAGGAACGGATTTTACTCTTACCATTTCT AGTCTTCAACCTGAGGATTTTGCGACGTATTACTGTCAACAGAGCTACAGTGCGCCG TTCACCTTT GGGCAGGGTACTAAGGTTGAGATAAAGC (SEQ Ш NO: 98)

AB3-Amino acid sequence of the heavy chain

EVOLLESGGGLVOPGGSLRLSCAASGFTFRNYAMSWVROAPGKGLEWVSSISGSGGAT

YYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARVSSGHWDFDYWGOGTLV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCV VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLG (SEQ Ш NO: 99)

AB3-Amino acid sequence of light chain

DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYDASSLOSGVPSR

FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPFTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ Ш NO: 100)

In some embodiments, antibodies of the present disclosure that specifically bind to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3TGFe1 complex, and the LRRC33-TGFp1 complex include any antibody that includes a heavy chain variable domain SEQ ID NO: 13 , 17 or 95 or a light chain variable domain SEQ ID NO: 14, 18 or 97. In some embodiments, antibodies of the present disclosure that specifically bind to the GARP-TGFp1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGF31 complex, and the LRRC33- complex TGFP1 include any antibody that includes the heavy chain and light chain variable pairs of SEQ ID NOs: 13 and 14; 17 and 18 and 95 and 97.

Aspects of the present disclosure provide antibodies that specifically bind to two or more of the following complexes: the GARP-TGFpt complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and the LRRC33-TGFe1 complex, comprising the amino acid sequence of a heavy chain and/or light chain variable region, homologous to any of those described herein. In some embodiments, an antibody that specifically binds to the GARP-TGFp1 complex, the LTBP1-TGFP1 complex, the LTBP3-TGFp1 complex, and the LRRC33-TGFe1 complex comprises a heavy chain variable region sequence or a light chain variable region 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 amino acid sequence of the heavy chain variable region of SEQ ID NO: 13, 17, or 95 or the sequence of the light chain variable region of SEQ ID NO: 14, 18, or 97. In some embodiments, homologous the amino acid sequences of the heavy chain variable region and/or light chain do not vary within any of the CDR sequences. 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 in the heavy chain variable region amino acid sequence and/or light chain variable region amino acid sequence, excluding any of the CDR sequences presented herein .

In some embodiments, antibodies of the present disclosure that specifically bind to two or more of: the GARP-TGFp1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFp1 complex, and the LRRC33-TGF31 complex include any antibody or antigen-binding portion thereof that includes comprising a heavy chain of SEQ ID NO: 15 or 19 or a light chain of SEQ ID NO: 16 or 20. In some embodiments, antibodies of the present disclosure that specifically bind to the GARP-TGF31 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFP1 complex, and /or the LRRC33-TGFP1 complex include any antibody that includes the heavy chain and light chain pairs of SEQ ID NOs: 15 and 16 or 19 and 20.

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Aspects of the present disclosure provide antibodies that specifically bind to two or more of: the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and the LRRC33-TGFe1 complex, characterized by a heavy chain and/or light chain amino acid sequence homologous to any of those described in this document. In some embodiments, an antibody that specifically binds to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex contains a heavy chain sequence or a light chain sequence that is at least 75% (e.g. , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 99%) identical to the heavy chain sequence of SEQ ID NO: 15 or 19 or the light chain amino acid sequence of SEQ ID NO: 16 or 20. In some embodiments, homologous heavy chain and/or light chain amino acid sequences are not vary within any of the CDR sequences presented herein. For example, in some embodiments, the degree of sequence variation (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99%) may occur in the heavy chain and/or light chain amino acid sequence excluding any of the CDR sequences provided herein.

In some embodiments, antibodies of the present disclosure that specifically bind to two or more of: the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex include any antibody or antigen-binding portion thereof, which includes a heavy chain of SEQ ID NO: 15 or 19 or a light chain of SEQ ID NO: 16 or 20. In some embodiments, antibodies of the present disclosure that specifically bind to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3- TGFe1 and/or the LRRC33-TGFe1 complex include any antibody that includes the heavy chain and light chain pairs of SEQ ID NOs: 15 and 16 or 19 and 20.

Aspects of the present disclosure provide antibodies that specifically bind to two or more of: the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex, characterized by a heavy chain and/or light chain amino acid sequence homologous to any of those described in this document. In some embodiments, an antibody that specifically binds to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex contains a heavy chain sequence or a light chain sequence that is at least 75% (e.g. , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 99%) identical to the heavy chain sequence of SEQ ID NO: 15 or 19 or the light chain amino acid sequence of SEQ ID NO: 16 or 20. In some embodiments, homologous heavy chain and/or light chain amino acid sequences are not vary within any of the CDR sequences presented herein. For example, in some embodiments, the degree of sequence variation (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99%) may occur in the heavy chain and/or light chain amino acid sequence excluding any of the CDR sequences provided herein.

In some embodiments, the percentage identity of two amino acid sequences is determined using an algorithm from Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:587377, 1993. This algorithm is included in the NBLAST and XBLAST programs (version 2.0) Altschul, et al. J. Mol. Biol. 215: 403-10, 1990. A protein BLAST search can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (for example, XBLAST and NBLAST) may be used.

In any of the antibodies or antigen binding fragments described herein, one or more conservative mutations may be introduced into the CDRs or framework sequences at positions where residues are unlikely to be involved in antibody-antigen interaction. In some embodiments, such conservative mutation(s) may be introduced into CDR or framework sequences at position(s) where residues are unlikely to be involved in interaction with the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and LRRC33 complex -TGFe1, as determined based on crystal structure. In some embodiments, a plausible interface (eg, residues involved in an antigen-antibody interaction) can be inferred from known structural information about another antigen having structural similarities.

As used herein, the term conservative amino acid substitution refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which

- 44 045239 amino acid substitution is performed. Variants can be prepared in accordance with methods for altering the polypeptide sequence known to one skilled in the art, such as those found in the references that constitute such 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 among amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f)Q,N and (g)E,D.

In some embodiments, antibodies provided herein contain mutations that impart desirable properties to the antibodies. For example, to avoid potential complications due to Fab arm exchange, which is known to occur with native IgG4 monoclonal antibodies, the antibodies presented herein may contain 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), where serine 228 (EU numbering; Kabat numbering of residue 241) is converted to proline, resulting in an IgG1-like (CPPCP (SEQ ID ) No.: 54)) hinge sequence. Accordingly, any of the antibodies may include an Adair stabilizing mutation or the amino acid sequence CPPCP (SEQ ID NO: 54).

Isoform-specific, context-permissive inhibitors (which include context-independent inhibitors) of TGFe1 of the present disclosure, for example, antibodies that specifically bind to two or more of: the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3 complex -TGFe1 and the LRRC33-TGFe1 complex may optionally contain antibody constant regions or portions thereof. For example, the VL domain may be attached at its C terminus to a light chain constant domain like C or Cλ. Likewise, the VH domain or a portion thereof may be attached to all or part of a heavy chain such as IgA, IgD, IgE, IgG and IgM and to 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 the present disclosure may include VH and VL domains or an antigen binding portion thereof in combination with any suitable constant regions.

Additionally or alternatively, such antibodies may or may not include the framework region of antibodies SEQ ID NO: 13-20. In some embodiments, the antibodies that specifically bind to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex are murine antibodies and include murine framework sequences.

In some embodiments, such antibodies bind to the GARPTGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33-TGFe1 complex with relatively high affinity, for example, with a KD of less than 10 ^-6 M, 10 ^-7 M, 10 ^{- 8} M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M or lower. For example, such antibodies may bind to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex with an affinity of 5 pM to 500 nM, e.g., 50 pM to 100 nM, e.g., 500 pM up to 50 nM. The present disclosure also includes antibodies or antigen binding fragments that compete with any of the antibodies described herein for binding to the GARP-TGFe1 complex, the LTBP1TGFe1 complex, the LTBP3-TGFe1 complex and/or the LRRC33-TGFe1 complex and that have an affinity of 50 nM or lower (eg, 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pM or lower). The affinity and binding kinetics of antibodies that specifically bind to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex can be studied using any suitable method, including, but not limited to, biosensor technology ( e.g. OCTET or BIACORE).

In some embodiments, inhibitors of cell-associated TGFe1 (e.g., GARP-presented TGFe1 and LRRC33-presented TGFe1) of the present invention include antibodies or fragments thereof that specifically bind to such a complex (e.g., GARP-pro/latent TGFe1 and LRRC33-pro /latent TGFe1) and trigger the internalization of the complex. This mode of action causes the removal or depletion of inactive TGFe1 complexes from the cell surface (eg Tregs, macrophages, etc.), hence reducing the TGFe1 available for activation. In some embodiments, such antibodies or fragments thereof bind the target complex in a pH-dependent manner such that binding occurs at neutral or physiological pH, but the antibody dissociates from its antigen at acidic pH. Such antibodies or fragments thereof may function as recycling antibodies.

Polypeptides.

Certain aspects of the present disclosure relate to a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 95,

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SEQ ID NO: 15 and SEQ ID 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 set forth in SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 95, SEQ ID NO: 15 and SEQ ID NO: 19.

Certain aspects of the present disclosure relate to a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 97, SEQ ID NO: 16, and SEQ ID NO: 20. In some embodiments In embodiment, the polypeptide is a light chain variable domain or 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 set forth in SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 97, SEQ ID NO: 16 and SEQ ID NO: 20.

Antibodies Competing with Isoform-Specific, Context-Permissive Inhibitory Antibodies to TGFe1

Aspects of the present disclosure relate to antibodies that compete or cross-compete with any of the antibodies provided herein. As used herein, the term compete with an antibody means that the first antibody binds to an epitope (e.g., an epitope of the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3TGFe1 complex, and/or LRRC33-TGFe1 complex) in a manner sufficiently similar to the binding of the second antibody, so that the binding effect of the first antibody to its epitope is markedly reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Alternatively, binding of the second antibody to its epitope may also be markedly reduced in the presence of the first antibody, but may not necessarily occur. That is, the 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 corresponding epitope. However, in cases where each antibody detectably inhibits the binding of another antibody to its epitope or ligand, to the same, greater or lesser extent, the antibodies are considered to cross-compete with each other for binding of their respective epitope(s). Both competitive and cross-competitive antibodies are included within the scope of the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one of ordinary skill in the art will appreciate that such competing and/or cross-competing antibodies are covered and may be applicable to the methods and/or compositions presented herein.

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

In another embodiment, provided herein is an antibody or antigen-binding portion thereof that competes or cross-competes for binding to any of the antigens provided herein (e.g., GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33 complex -TGFe1) with an equilibrium dissociation constant, KD, between the antibody and the protein of less than 10 ^-6 M. In other embodiments, the antibody competes or cross-competes for binding to any of the antigens provided herein with a KD ranging from 10 ^-11 M to 10 ^-6 M. In some embodiments, provided herein is an anti-TGFe1 antibody or antigen-binding portion thereof that competes for binding with an antibody or antigen-binding portion thereof described herein. In some embodiments, provided herein is an anti-TGFe1 antibody or antigen-binding portion thereof that binds to the same epitope as an antibody or antigen-binding portion thereof described herein.

Any of the antibodies presented herein 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 suitable methods for mapping and characterizing the location of epitopes on proteins, including identifying

- 46 045239 crystal structure of the antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, 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 an additional example, epitope mapping can be used to determine the sequence to which an antibody binds. The epitope may be a linear epitope, i.e. contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids, which may not necessarily be contained in a single stretch (linear sequence of primary structure). In some embodiments, the epitope is a TGFe1 epitope that is only accessible to binding by an antibody described herein or an antigen-binding portion thereof when TGFe1 is in the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33TGFe1 complex. Peptides of various lengths (eg, at least 4-6 amino acids in length) can be isolated or synthesized (eg, recombinantly) and used for antibody binding assays. In another example, the epitope to which an antibody binds can be determined in a systematic screen using overlapping peptides derived from the target antigen sequence and determining antibody binding. In gene fragment expression assays, the open reading frame encoding the target antigen is fragmented either randomly or using specific genetic constructs, and the reactivity of the expressed antigen fragments with the antibody of interest is determined. Gene fragments can, for example, be produced by PNR and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. The binding of the antibody to radiolabeled antigen fragments is then determined using immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified 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 screened for binding to the antibody of interest in simple binding assays. In a further example, antigen binding domain mutagenesis, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or essential for epitope binding. For example, domain shuffling experiments can be performed using a mutant of a target antigen in which various fragments of the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex have been replaced (swapped) with sequences from a closely related , but an antigenically distinct protein, such as another member of the TGF family of proteins (eg, GDF11). By assessing antibody binding to a mutant of the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33-TGFe1 complex, the importance of a particular antigen fragment for antibody binding can be assessed.

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

In addition, the interaction of any of the antibodies presented herein with one or more residues in the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex and/or LRRC33-TGFe1 complex can be determined using routine technology. For example, the crystal structure can be determined and the distances between residues in the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex and/or LRRC33TGFe1 complex and one or more residues in the antibody, respectively, can be determined. Based on this distance, it can be determined whether a particular residue in the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33-TGFe1 complex interacts with one or more residues in the antibody. In addition, suitable methods, such as competition assays and targeted mutagenesis assays, can be used to determine the preferential binding of a candidate antibody.

Various modifications and variations of antibodies.

Non-limiting variations, modifications and features of any of the antibodies or antigen binding fragments thereof covered by the present disclosure are briefly discussed below. Embodiments of appropriate analytical methods are also provided.

The structural units of a naturally occurring antibody typically include a tetramer. Each such tetramer typically consists of two identical pairs of polypeptide chains, each pair containing one full-length light chain (in some embodiments, approximately 25 kDa) and one full-length heavy chain (in some embodiments, approximately 50-70 kDa). The amino terminal portion of each chain typically includes a variable region of approximately 100 to 110 or more amino acids, which typically responds

- 47 045239 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 generally classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon and determine the isotype of the antibody. The antibody can be of any type (eg, IgM, IgD, IgG, IgA, IgY and IgE) and class (eg, IgG1, IgG2, IgG3, IgG ₄ , IgMi, IgM ₂ , IgA1 and IgA2). Within the full-length light and heavy chains, typically the variable and constant regions are connected by a J region of about 12 or more amino acids, with the heavy chain also containing a D region of about 10 more amino acids (see, for example, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989) (incorporated by reference in its entirety)). The variable regions of each light/heavy chain pair typically form the antigen binding site.

Variable regions are typically characterized by the same overall structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs of the two strands of each pair are typically aligned to framework regions that can mediate binding to a specific epitope. From N-terminus to C-terminus, the variable regions of both the light and heavy chains typically contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. The distribution of amino acids within each domain generally follows 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 light chain CDRs may also be referred to as CDR-L1, CDR-L2 and CDR-L3, and the heavy chain CDRs may also be referred to as CDR-H1, CDR-H2 and CDR-H3. In some embodiments, the antibody may contain a small number of amino acid deletions from the carboxy terminus of the heavy chain(s). In some embodiments, the antibody comprises a heavy chain having 1-5 amino acid deletions at the carboxy terminus of the heavy chain. In some embodiments, specific delineation of CDRs and identification of residues containing the antibody binding site is achieved by determining the structure of the antibody and/or determining the structure of the antibody-ligand complex. In certain embodiments, this may be accomplished by any of a variety of methods known to those skilled in the art, such as x-ray crystallography. In some embodiments, various analysis techniques may be used to identify or approximate CDR regions. Examples of such methods include, but are not limited to, Kabat determination, Chothia determination, AbM determination, and contact determination.

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

The term grafted CDR antibody refers to antibodies that contain heavy and light chain variable region sequences from one species, but in which the sequences of one or more CDR regions in VH and/or VL are replaced by CDR sequences of another species, such as antibodies having murine variable regions heavy and light chains in which one or more murine CDRs (eg, CDR3) have been replaced by human CDR sequences.

The term chimeric antibody refers to antibodies that contain heavy and light chain variable region sequences of one kind and constant region sequences of another, such as antibodies having mouse heavy and light chain variable regions linked to human constant regions.

As used herein, the term framework or framework sequence refers to the remaining variable region sequences minus the CDRs. Since the precise definition of the CDR sequence may be determined by different systems, the meaning of the framework sequence is subject to correspondingly different interpretations. Six CDRs (light chain CDR-L1, -L2 and -L3 and heavy chain CDR-H1, -H2 and -H3) also divide the light chain and heavy chain framework regions into four subregions (FR1, FR2, FR3 and FR4) each a chain in which CDR1 is located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying specific subregions such as FR1, FR2, FR3 or FR4, the framework region referred to by others is the combined FRs in the variable region of a single naturally occurring immunoglobulin chain.

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As used herein, FR represents one of four sub-regions, and FR represents two or more of the four sub-regions constituting a framework region.

In some embodiments, the antibody or antigen-binding portion thereof comprises an immunoglobulin heavy chain constant domain of a human IgM constant domain, a human IgG constant domain, a human IgG1 constant domain, a human IgG2 constant domain, a human IgG2A constant domain, a human IgG2B constant domain, a human IgG2 constant domain, human IgG3 constant domain, human IgG3 constant domain, human IgG4 constant domain, human IgA constant domain, human IgA1 constant domain, human IgA2 constant domain, human IgD constant domain or 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 a human IgG4 immunoglobulin heavy chain constant domain. In some embodiments, the antibody or antigen-binding portion thereof comprises a human IgG4 immunoglobulin heavy chain constant domain comprising a Ser to Pro backbone substitution that forms an IgG1-like hinge and allows for the formation of interchain disulfide bonds.

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

In some embodiments, the antibody is an IgG containing four polypeptide chains, which are two heavy chains and two light chains.

In some embodiments, the antibody is a humanized antibody, diabody, or 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 characterized by a human germline amino acid sequence.

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

As used herein, the term germline antibody gene or gene fragment refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that results in genetic rearrangement and mutation to express a particular immunoglobulin (see, for example, Shapiro et al. (2002) Crit Rev Immunol 22(3): 183-200; Marchalonis et al (2001) Adv Exp Med Biol 484: 13-30). One of the advantages provided by various embodiments of the present disclosure relates to the recognition that germline antibody genes are more likely than mature antibody genes to retain the structures of essential amino acid sequences characteristic of individuals of a given species, and therefore are less likely to be recognized as foreign source for therapeutic use in this species.

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

As used herein, the term binding protein includes any polypeptide that specifically binds an antigen (e.g., TGFe1), including, without limitation, an antibody or antigen-binding portions thereof, DVD-IgTM, TVD-Ig, RAb-Ig, bispecific antibody and double specific antibody.

The term monoclonal antibody or mAb, when used in the context of a composition containing it, can refer to an antibody preparation derived from a population of essentially homogeneous antibodies, i.e. the individual antibodies that make up the population are identical, except for possible naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific and directed against a single antigen. Additionally, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. The monoclonal modifier should not be construed as requiring the antibody to be produced by any particular method.

As used herein, the term recombinant human antibody is intended to include all human antibodies that are produced, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in section

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IIC 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: 425445; Gavilondo, J.V. and Larrick, J.W. (2002) BioTechniques 29: 128-145; Hoogenboom, H. and Chames, P. (2000) Immunol. Today 21: 371-378, incorporated herein by reference), antibodies isolated from an animal ( e.g., mouse) that is transgenic for human immunoglobulin genes (see Taylor, L. D. et al. (1992) Nucl. Acids Res. 20: 6287-6295; Kellermann, S. A. and Green, L. L. (2002) Cur. Opin. in Biotechnol. 13: 593-597; Little, M. et al. (2000) Immunol. Today 21: 364-370) or antibodies produced, expressed, created or isolated by any other method that involves splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are subject to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, to in vivo somatic mutagenesis) and, therefore, the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although and are derived from and related to human germline VH and VL sequences, would not naturally exist in the human antibody germline repertoire in vivo.

As used herein, the term dual variable domain immunoglobulin or DVD-IgTM or the like is used. comprises binding proteins comprising a paired heavy chain DVD polypeptide and a light chain DVD polypeptide, 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. DVD-IgTM typically contains two arms linked to each other, at least in part, by dimerization of CH3 domains, with each DVD arm being bispecific, providing an immunoglobulin with four binding sites. DVD-IgTM are disclosed in US Patent Publications No. 2010/0260668 and 2009/0304693, each of which is incorporated herein by reference, including its sequence listings.

As used herein, the term triple variable domain immunoglobulin or TVD-Ig or the like is used. are binding proteins comprising a paired heavy chain TVD binding protein polypeptide and a light chain TVD binding protein polypeptide, 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. The TVD binding protein may have two arms linked to each other at least in part by dimerization of CH3 domains, with each TVD binding protein arm being trispecific, providing a binding protein with six binding sites.

As used herein, the term immunoglobulin receptor-antibody or RAb-Ig and the like. are binding proteins containing a heavy chain RAb polypeptide and a light chain RAb polypeptide, which together form three antigen binding sites. A single antigen binding site is formed by pairing of the antibody heavy and light chain variable domains present in each of a heavy chain RAb polypeptide and a light chain RAb polypeptide to form a single binding site with a total of 6 CDRs providing the first antigen binding site. Each heavy chain RAb polypeptide and light chain RAb polypeptide includes a receptor sequence that independently binds a ligand providing a second and third antigen binding site. RAb-Ig typically contains two arms linked to each other, at least in part, by dimerization of CH3 domains, with each RAb-Ig arm being trispecific, providing an immunoglobulin with six binding sites. RAb-Igs are described in US Patent Application Publication No. 2002/0127231, the entire contents of which, including sequence listings, are incorporated herein by reference.

The term bispecific antibody as used herein, distinguished from bispecific half-Ig binding protein or bispecific binding protein (half Ig), refers to full-length antibodies that are produced by quadhybridoma technology (see Milstein, C. and Cuello, A.C. (1983) Nature 305(5934): p. 537-540), by chemical conjugation of two different monoclonal antibodies (see Staerz, U.D. et al. (1985) Nature 314(6012): 628-631) or by the projection-to-convex method or similar approaches that 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): 6444-6448), resulting in many different types of immunoglobulins, of which only one is a functional bispecific antibody. In molecular function, a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one HC/LC pair) and binds another antigen (or epitope) on its second arm (the other HC/LC pair). According to this definition, a bispecific antibody contains two different antigen-binding arms (both specificity and CDR sequences) and is monovalent for each antigen to which it binds.

The term dual-specific antibody as used herein, distinguished from half-Ig bispecific protein or bispecific binding protein, refers to full-length antibodies that can bind two different antigens (or epitopes) in each of its two binding arms (HC/pair). LC) (see PCT Publication No. WO 02/02773). Accordingly, a dual specificity binding protein contains two identical antigen binding arms with the same specificity and identical CDR sequences and is bivalent for each antigen to which it binds.

As used herein, the term Kon is intended to denote the rate constant for the association of a binding protein (eg, antibody) with an antigen to form, for example, an antibody/antigen complex, as is known in the art. The term Kon is also known by the terms association rate constant or ka, which are used interchangeably herein. This value indicates the rate of binding of an antibody to its target antigen, or the rate of complex formation between the antibody and the antigen, also represented by the equation: antibody (Ab) + antigen (Ag)^ Ab-Ag.

As used herein, the term Koff is intended to denote the rate constant for the dissociation of a binding protein (eg, antibody), for example, from an antibody/antigen complex, as is known in the art. Koff” is also known by the terms dissociation rate constant or kd, which are used interchangeably herein. This value indicates the rate of dissociation of an antibody from its target antigen or separation of the Ab-Ag complex over time into free antibody and antigen, as shown by the equation: Ab + Ag ^ AbAg.

The terms equilibrium dissociation constant or KD, used interchangeably herein, refer to the value obtained by measuring the titration at steady state, 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, such as an antibody, to an antigen. Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based methods provides high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and tools can be used, such as the BIAcore® (Biomolecular Interaction Assay) assay (eg, instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). In addition, the KinExA® (Kinetic Exclusion Assay), available from Sapidyne Instruments (Boise, Idaho), can also be used.

As used herein, the terms crystalline and crystallized refer to a binding protein (eg, antibody) or antigen-binding portion thereof that exists in the form of a crystal. Crystals are one form of solid state of matter that is distinct from other forms such as the amorphous solid state or liquid crystalline state. Crystals are composed of regular, repeating three-dimensional arrays of atoms, ions, molecules (eg, proteins such as antibodies), or molecular assemblies (eg, antigen/antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well understood in the art. The basic unit, or building block, that is repeated in a crystal is called an asymmetric unit. The repetition of an asymmetric unit in an arrangement that corresponds to a given, well-defined crystallographic symmetry provides the elementary unit of the crystal. Repeating an elementary link with regular translations in all three dimensions produces a crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 201-16, Oxford University Press, New York, New York, (1999). The term linker is used to refer to polypeptides containing two or more amino acid residues joined by peptide bonds and is used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J. et al. (1994) Structure 2 :1121-1123). Exemplary linkers include, but are not limited to, ASTKGPSVFPLAP (SEQ ID NO: 55), ASTKGP (SEQ ID NO: 56); TVAAAPSVFLFPP (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); ADAAPTVSLFPP (SEQ ID NO: 70); QPKAAP (SEQ ID NO: 71); QPKAAPSVTLFPP (SEQ ID NO: 72); AKTTPP (SEQ ID NO: 73); AKTTPPPSVTPLAP (SEQ ID NO: 74); AKTTAP (SEQ ID NO: 75); AKTTAPSVYPLAP(SEQ ID NO: 76); GGGGSGGGGSGGGGS (SEQ ID NO:

- 51 045239

77); GENKVEYAPALMALS (SEQ ID NO: 78); GPAKELTPLKEAKVS (SEQ ID NO: 79); GHEAAAVMQVQYPAS (SEQ ID NO: 80); TVAAPSVFLFPPTVAAPSVFLFPP (SEQ ID NO: 81) and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 82).

Label and detectable label or detectable fragment means a fragment attached to a specific binding partner, such as an antibody or analyte, for example, to make the reaction between members of a particular binding pair, such as antibody and analyte, detectable, and the specific binding partner, for example , an antibody or analyte so labeled is designated as detectably labeled. Thus, as used herein, the term tagged binding protein refers to a protein with a tag included that allows identification of the binding protein. In one embodiment, the label is a detectable marker that can produce a signal that can be detected visually or instrumentally, for example, by incorporating a radiolabeled amino acid or by attaching biotinyl moieties to the polypeptide that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (eg, 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, I66Ho and 153Sm); chromogens; fluorescent tags (for example, FITC, rhodamine and lanthanide complex phosphors); enzymatic tags (eg, horseradish peroxidase, luciferase and alkaline phosphatase); chemiluminescent markers; biotinyl groups; predefined polypeptide epitopes recognized by a secondary reporter (eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, and epitope tags) and magnetic agents such as gadolinium chelates. Representative examples of labels typically used for immunoassays include moieties that produce light, such as acridinium compounds, and moieties that produce fluorescence, such as fluorescein. Other labels are described in this document. Therefore, the fragment itself may not be detectably labeled, but may become detectable when reacted with another fragment. The use of detectable marking is intended to cover the latter type of detectable marking.

In some embodiments, the binding affinity of an antibody or antigen-binding portion thereof to an antigen (e.g., a protein complex), such as the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex is determined using an Octet assay. In some embodiments, an Octet assay is an assay that determines one or more kinetic parameters indicative of binding between an antibody and an antigen. In some embodiments, the Octet® system (ForteBio, Menlo Park, CA) is used to determine the binding affinity of an antibody or antigen binding portion thereof to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex. For example, antibody binding affinity can be determined using the forteBio Octet QKe label-free test strip assay system using biolayer interferometry. In some embodiments, antigens are immobilized in biosensors (eg, streptavidin-coated biosensors) and antibodies and complexes (eg, biotinylated GARP-TGFe1 complexes and biotinylated LTBP-TGFe1 complexes) are presented in solution at a high concentration (50 μg/ml) to measure binding interactions. In some embodiments, the binding affinity of an antibody or antigen-binding portion thereof to the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33-TGFe1 complex is determined using the set forth in Table. 6 protocols. As used herein, the term surface plasmon resonance refers to an optical phenomenon that allows the analysis of bispecific interactions in real time by detecting changes in protein concentrations in a biosensor matrix, for example, using the BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, New Jersey). For additional descriptions see Jonsson, U. et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U. et al. (1991) Biotechniques 11: 620-627; Johnsson, B. et al. (1995) J. Mol. Recognize 8: 125-131 and Johnson, B. et al. (1991) Anal. Biochem. 198: 268-277.

Identification and preparation/production of isoform-specific, context-permissive TGFe1 inhibitors

The present invention covers screening methods, production methods and processes for producing antibodies or fragments thereof that bind two or more of: the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex and/or the LRRC33-TGFe1 complex, and pharmaceutical compositions containing their related sets.

Numerous methods can be used to produce the antibodies or antigen-binding fragments thereof of the present invention. For example, antibodies can be produced using recombinant DNA methods. Monoclonal antibodies can also be produced by

- 52 045239 produced by hybridomas (see, for example, Kohler and Milstein (1975) Nature, 256: 495-499) according to known methods. The hybridomas thus formed are then screened using standard techniques such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance assays (eg, 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 can be used as an immunogen, for example, a recombinant antigen, naturally occurring forms, any variants or fragments thereof, and an antigenic peptide thereof (for example, any of the epitopes described herein as a linear epitope or within a framework as conformational epitope). One exemplary method for producing antibodies involves screening protein expression libraries that express antibodies or fragments thereof (eg, scFv), such as phage or ribosome display libraries. Phage display is described, for example, in Ladner et al., US patent No. 5223409; 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.

In addition to using display libraries, the antigen (e.g., GARPTGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33-TGFe1 complex) can be used to immunize a non-human host, e.g., rabbit, guinea pig, rat, mice, hamsters, sheep, goats, chickens, camels, and non-mammalian hosts such as sharks. In one embodiment, the non-human animal is a mouse.

In another embodiment, the monoclonal antibody is obtained from a non-human animal and is then modified, for example, in a chimeric manner using suitable recombinant DNA techniques. Various approaches have been described for the production of chimeric antibodies. See, for example, Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81:6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., US Patent No. 4816567; Boss et al., US patent. No. 4816397; Tanaguchi et al., European Patent Publication 171496; European patent publication 0173494, UK patent GB 2177096B.

For additional methods of producing antibodies, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. The present disclosure is not necessarily limited to any particular source, method of preparation, or other special characteristics of the antibody.

Certain aspects of the present disclosure relate to host cells transformed with a polynucleotide or vector. Host cells may be prokaryotic or eukaryotic cells. The polynucleotide or vector that is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. In some embodiments, the fungal cells are, for example, cells of the genus Saccharomyces, in particular cells of the species S. cerevisiae. The term prokaryotic includes all bacteria that can be transformed or transfected with DNA or RNA molecules to express an antibody or corresponding immunoglobulin chains. Prokaryotic hosts may include gram-negative as well as gram-positive bacteria, such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term eukaryotic includes yeast, higher plants, insect and vertebrate cells, for example, mammalian cells such as NSO and CHO cells. Depending on the host used in the 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 the parent amino acid residue methionine.

In some embodiments, once the vector has been incorporated into a suitable host, the host may be maintained under conditions suitable for high levels of expression of nucleotide sequences and, if desired, collection and purification of light chains, heavy chains, light chain/heavy chain immunoglobulin dimers may follow. or intact antibodies, antigen-binding fragments or other forms of immunoglobulin; see Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N.Y., (1979). Thus, polynucleotides or vectors are introduced into cells, which in turn produce antibodies or antigen-binding fragments. In addition, transgenic animals, preferably mammals, containing the above host cells can be used for large-scale production of antibody or antibody fragments.

Transformed host cells can be grown in fermenters and cultured using any suitable methods to achieve optimal cell growth. Once expressed, whole antibodies, their dimers, individual light and heavy chains, other forms of immunoglobulin or antigen binding fragments can be purified according to standard procedures

- 53 045239 of the present art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc., see Scopes, Protein Purification, Springer Verlag, N.Y. (1982). The antibody or antigen binding fragments can then be isolated from the culture medium, cell lysates or cell membrane fractions. Isolation and purification of, for example, microbially expressed antibodies or antigen-binding fragments can be accomplished by any conventional methods, such as, for example, preparative chromatographic separation and immunological separation, for example, involving the use of monoclonal or polyclonal antibodies directed, for example, against the constant region antibodies.

Aspects of the present disclosure relate to a hybridoma that provides an indefinitely long-lasting 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 subsequent expression and/or genetic manipulation. Rearranged antibody genes can be reverse transcribed from the corresponding mRNAs to produce cDNAs. In some embodiments, the heavy chain constant region may be replaced with a region of a different isotype or eliminated entirely. Variable regions can be linked to encode single-stranded Fv regions. Multiple Fv regions can be linked to impart binding ability to more than one target, or chimeric combinations of heavy and light chains can be used. Any suitable method can be used to clone antibody variable regions and produce recombinant antibodies.

In some embodiments, a suitable nucleic acid that encodes heavy and/or light chain variable regions is prepared and inserted into expression vectors that can be transfected into standard recombinant host cells. A variety of such host cells can be used. In some embodiments, mammalian host cells may be useful for efficient processing and production. Typical mammalian cell lines useful for this purpose include CHO cells, 293 cells or NSO cells. Production of an antibody or antigen binding fragment can be accomplished by culturing the modified recombinant host under culture conditions suitable for host cell growth and expression of coding sequences. Antibodies or antigen-binding fragments can be isolated by isolating them from a 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.

The present disclosure also includes a polynucleotide encoding at least a variable region of an immunoglobulin chain of the antibodies described herein. In some embodiments, the variable region encoded by the polynucleotide comprises at least one VH and/or VL complementarity determining region (CDR) of an antibody produced by any of the above-described hybridomas.

The polynucleotides encoding the 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 containing any of these polynucleotides, either alone or in combination. In some embodiments, the polynucleotide is part of a vector. Such vectors may contain additional genes, such as marker genes, which allow selection of the vector in a suitable host cell and under suitable conditions.

In some embodiments, the polynucleotide is operably linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells. Expression of a polynucleotide involves the transcription of the polynucleotide into translated mRNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. These 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 transcriptional as well as translational enhancers and/or naturally occurring or heterologous promoter regions. Possible regulatory elements for expression in prokaryotic host cells include, for example, the PL, Lac, Trp, or Tac promoter in E. coli, and examples of regulatory elements for expression in eukaryotic host cells include the AOX1 or GAL1 promoter in yeast or CMV promoter, SV40 promoter, RSV promoter (Rouse sarcoma virus), CMV enhancer, SV40 enhancer or globin intron in mammalian and other animal cells.

In addition to elements that are responsible for the initiation of transcription, such regulatory elements may also include transcription termination signals, such as the SV40 polyA site or the tkpoly A site, downstream of the polynucleotide. In addition, depending on the expression system used, leader sequences capable of directing

- 54 045239 polypeptide into the cellular compartment or secrete it into the environment, to the coding sequence of the polynucleotide, and they have been described previously. The leader sequence(s) are assembled in appropriate phase with the translation, initiation and termination sequences and, preferably, with a leader sequence capable of directing secretion of the translated protein or part thereof, for example, into the extracellular environment. Optionally, a heterologous polynucleotide sequence may be used that encodes a fusion protein including a C- or N-terminal identification peptide that imparts desired characteristics, such as stabilization or simplified purification of the expressed recombinant product.

In some embodiments, polynucleotides encoding at least a light and/or heavy chain variable domain may encode variable domains of both immunoglobulin chains or just one. Likewise, polynucleotides may be under the control of the same promoter or may be controlled separately for expression. In addition, some aspects relate to vectors, in particular plasmids, cosmids, viruses and bacteriophages, typically used in genetic engineering, which contain a polynucleotide encoding the variable domain of an immunoglobulin chain of an antibody or antigen binding fragment; optionally in combination with a polynucleotide that encodes the variable domain of another immunoglobulin chain of the antibody.

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 viruses, or bovine papillomavirus, can be used to deliver polynucleotides or a vector to a target population of cells (for example, to engineer a cell to express an antibody or antigen-binding fragment ). A variety of suitable methods can be used to construct recombinant viral vectors. In some embodiments, polynucleotides and vectors can be formulated into liposomes for delivery to target cells. Vectors containing polynucleotides (eg, immunoglobulin heavy and/or light chain variable domain(s), coding sequences and expression control sequences) can be transferred into a host cell by suitable methods that vary depending on the type of cellular host.

Screening methods may include the step of assessing or confirming the desired activity of an antibody or fragment thereof. In some embodiments, the step involves selecting the ability to inhibit a target function, for example, inhibiting the release of mature TGFe1 from a latent complex. In some embodiments, this step involves selecting antibodies or fragments thereof that facilitate the internalization and subsequent removal of antibody-antigen complexes from the cell surface. In some embodiments, this step involves selecting antibodies or fragments thereof that induce ADCC. In some embodiments, this step involves selecting antibodies or fragments thereof that accumulate at the desired site(s) in vivo (eg, cell type, tissue, or organ). In some embodiments, this step involves selecting antibodies or fragments thereof that are capable of crossing the blood-brain barrier. The methods may optionally include the step of optimizing one or more antibodies or fragments thereof to produce analogue variants that have desired profiles, as determined by criteria such as stability, binding affinity, functionality (e.g., inhibitory activity, Fc function, etc.), immunogenicity , pH sensitivity and developmental ability (e.g. high solubility, low self-association, etc.). Such a step may involve affinity maturation of the antibody or fragment thereof. The resulting optimized antibody is preferably a fully human antibody or a humanized antibody suitable for administration to humans. The process for producing a pharmaceutical composition containing such an antibody or fragment thereof may involve steps of purification, formulation, sterile filtration, packaging, etc. Some steps, such as sterile filtration, for example, are carried out in accordance with the guidelines laid down by relevant regulatory authorities such as the FDA. Such compositions may be made available in the form of single-use containers, such as prefilled syringes, or multiple-dose containers, such as vials.

Modifications.

The antibodies or antigen-binding portions thereof of the present invention may be modified with a detectable label or detectable moiety, including, but not limited to, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, a radioactive material, a positron-emitting metal, a non-radioactive ion

- 55 045239 paramagnetic metal and affinity tag for the detection and isolation of the GARP-TGFei complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex and/or the LRRC33-TGFe1 complex. The detectable substance or fragment can be linked or conjugated either directly to the polypeptides of the present invention, or indirectly, through an intermediate (such as, for example, a linker (eg, 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 the luminescent material includes luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin and aequorin and examples of suitable radioactive material include a radioactive metal ion, for example, alpha emitters or other radioisotopes such as, for example, iodine (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In) and technetium (99Ts, 99mTc), thallium (201Ti), 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, 169Yb, 51Cr, 54Mn and tin (113Sn, 117Sn). The detectable substance may be bound or conjugated either directly to antibodies of the present invention that specifically bind to the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3TGFe1 complex and/or LRRC33-TGFe1 complex, or indirectly, through an intermediate (such as, for example , linker) using suitable methods. Any of the antibodies provided herein that are conjugated to a detectable substance can be used for any suitable diagnostic assays, such as those described herein.

In addition, the antibodies or antigen-binding portions thereof of the present disclosure may also be modified with a drug. A drug can be linked or conjugated either directly to the polypeptides of the present disclosure or indirectly through an intermediate (such as, for example, a linker (eg, a cleavable linker)) using suitable techniques.

Targeting agents.

In some embodiments, the methods of the present invention include the use of one or more targeting agents to target an antibody or antigen binding portion thereof to a specific site in a subject to modulate the release of mature TGFe from the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3 complex -TGFe1 and/or the LRRC33-TGFe1 complex. For example, the LTBP1-TGFe1 and LTBP3TGFp1 complexes are typically localized in the extracellular matrix. Thus, in some embodiments, antibodies disclosed herein can be conjugated to extracellular matrix targeting agents to localize the antibodies to sites where the LTBP1-TGFe1 and LTBP3-TGFe1 complexes reside. In such embodiments, selective targeting of antibodies results in selective modulation of the LTBP1-TGFe1 and/or LTBP3-TGFe1 complexes. In some embodiments, selective targeting of antibodies results in selective inhibition of LTBP1-TGFe1 and/or LTBP3-TGFe1 complexes (eg, for the treatment of fibrosis). In some embodiments, extracellular matrix-targeting agents include heparin binding agents, matrix metalloproteinase binding agents, lysyl oxidase domain binding agents, fibrillin binding agents, hyaluronic acid binding agents, and others.

Likewise, GARP-TGFe1 complexes are typically localized to the surface of cells, such as activated FOXP3+ regulatory T cells (Tregs). Thus, in some embodiments, antibodies disclosed herein can be conjugated to immune cell binding agents (eg, Treg cells) to localize the antibodies to sites where GARP-TGFe1 complexes are located. In such embodiments, selective targeting of antibodies results in selective modulation of GARP-TGFe1 complexes. In some embodiments, selective targeting of antibodies results in selective inhibition of GARP-TGFe1 complexes (e.g., selective inhibition of mature TGFe1 release for immunomodulatory purposes, such as in the treatment of cancer). In such embodiments, Treg cell-targeting agents may include, for example, the CCL22 and CXCL12 proteins or fragments thereof.

In some embodiments, bispecific antibodies may be used comprising a first portion that selectively binds to the GARP-TGFe1 complex and the LTBP-TGFe1 complex, and a second portion that selectively binds to a target site component,

- 56 045239 for example, an ECM component (eg fibrillin) or a Treg cell component (eg CTLA4).

Pharmaceutical compositions.

The present invention further provides pharmaceutical compositions useful as a medicament suitable for administration to human and non-human subjects. One or more antibodies that specifically bind to the GARPTGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex and/or LRRC33-TGFe1 complex can be formulated or mixed with a pharmaceutically acceptable carrier (excipient), including, for example, a buffer , for the formation of a pharmaceutical composition. Such formulations may be used to treat a disease or disorder that involves TGFe signaling. In some embodiments, such a disease or disorder associated with TGFe signaling includes one or more contexts, i.e. TGFe is associated with a specific type or types of presenting molecules. In some embodiments, such context occurs depending on the cell and/or tissue type. In some embodiments, for example, such context-dependent effects of TGFe signaling are mediated in part through GARP, LRRC33, LTBP1, and/or LTBP3.

In some embodiments, an antibody of the present invention specifically binds to two or more TGFe contexts such that the antibody binds to the TGFe in complex with presentation molecules selected from two or more of: GARP, LRRC33, LTBP1, and LTBP3. Thus, such pharmaceutical compositions can be administered to patients to alleviate TGF-related indications (eg, fibrosis, immune disorders and/or cancer). Acceptable means that the carrier is compatible with the active ingredient of the composition (and preferably is capable of stabilizing the active ingredient) and is not harmful to the subject being treated. Examples of pharmaceutically acceptable excipients (carriers), including buffers, will be apparent to one skilled in the art and have been previously described. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover. In one example, a pharmaceutical composition described herein contains more than one antibody that specifically binds to the GARP-TGFe1 complex, the LTBP1TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex, where the antibodies recognize different epitopes/residues of the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex and/or LRRC33-TGFe 1 complex.

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

In some examples, the pharmaceutical composition described herein contains liposomes containing an antibody that specifically binds to the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex and/or LRRC33-TGFe1 complex, which can be prepared by any suitable method such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al. Proc. Natl. Acad. Sci. USA 77:4030 (1980) and US Patent Nos. 4,485,045 and 4,544,545. Extended circulation time liposomes are described in US Pat. No. 5,013,556. Particularly useful liposomes can be prepared by a reverse phase evaporation process with a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with specific pore sizes to obtain liposomes with the desired diameter.

In some embodiments, the pharmaceutical compositions of the present invention may contain or may be used in combination with an adjuvant drug

- 57 045239 means. It is believed that a certain adjuvant drug may enhance a subject's immune responses, for example, to tumor antigens, and facilitate Teffector function, DC differentiation from monocytes, enhanced antigen uptake and APC presentation, etc. Suitable excipients include, but are not limited to, retinoic acid-based excipients and derivatives thereof, oil-in-water emulsion excipients such as MF59 and other squalene-containing excipients, Toll-like ligands receptor (TRL), α-tocopherol (vitamin E) and their derivatives.

Antibodies that specifically bind to the GARP-TGFe1 complex, the LTBP1TGFe1 complex, the LTBP3-TGFe1 complex and/or the LRRC33-TGFe1 complex can also be enclosed in microcapsules prepared, for example, by coacervation or interfacial polymerization methods, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacylate) microcapsules, respectively, in colloidal drug delivery systems (eg liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Exemplary methods have been described previously, see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein may be formulated in a sustained release form. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, these matrices being in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOTTM (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate and poly-D-(-)-3-hydroxy-butyric acid .

Pharmaceutical compositions used for in vivo administration must be sterile. This is easily achieved, for example, by filtration through sterile filter membranes. Therapeutic antibody compositions are typically placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper pierced by a hypodermic needle.

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

For the preparation of solid compositions such as tablets, the main active ingredient can be mixed with a pharmaceutical carrier, for example, conventional tabletting ingredients such as 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 composition before forming it into a dosage form containing a homogeneous mixture of a compound of the present disclosure or a non-toxic pharmaceutically acceptable salt thereof. When these compositions are referred to as homogeneous before being formulated into a dosage form, this means that the active ingredient is uniformly distributed throughout the composition so that the composition can be easily divided into equally effective unit dosage forms such as tablets, pills and capsules. This solid composition, before being formulated into a dosage form, is then divided into unit dosage forms of the type described above, containing from 0.1 mg to about 500 mg of the active ingredient of the present disclosure. Tablets or pills of the novel composition may be coated or otherwise formulated to provide a dosage form that provides the benefit of extended release. For example, a tablet or pill may comprise an inner dosage component and an outer dosage component, the latter being in the form of a shell over the former. The two components may be separated by an enteric layer, which serves to resist breakdown in the stomach and allows the internal component to pass unchanged into the duodenum or be retained upon excretion. A variety of materials can be used for such enteric layers or coatings, such materials include a number of polymer acids and mixtures of polymer acids with materials such as shellac, cetyl alcohol and cellulose acetate.

Suitable surfactants include, in particular, non-ionic agents such as polyoxyethylene sorbitans (eg TweenTM 20, 40, 60, 80 or 85) and other sorbitans (eg SpanTM 20, 40, 60, 80 or 85). Surfactant compositions will typically contain from 0.05 to 5% surfactant and may range from 0.1 to 2.5%. It will be appreciated that other ingredients, such as mannitol or other pharmaceutically acceptable excipients, may be added if desired.

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Suitable emulsions can be prepared using commercially available fat emulsions such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM. The active ingredient can either be dissolved in a pre-mixed emulsion composition or, alternatively, it can be dissolved in an oil (for example, soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed after mixing with a phospholipid (eg egg phospholipids, soy phospholipids or soy lecithin) and water. It is understood that other ingredients, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to 20% oil, for example 5 to 20%.

Emulsion compositions can be those prepared by mixing an antibody that specifically binds to the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3TGFe1 complex and/or LRRC33-TGFe1 complex with IntralipidTM or its components (soybean oil, egg phospholipids, glycerol and water) .

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

The compositions in preferably sterile pharmaceutically acceptable solvents can be nebulized using gases. Nebulized solutions can be inhaled directly from the nebulizing device, or the nebulizing device can be attached to a face mask, swab, or intermittent positive pressure breathing apparatus. The compositions, in the form of a solution, suspension or powder, can be administered, preferably orally or nasally, from devices that deliver the composition in an appropriate manner.

Selection of therapeutic indications and/or subjects likely to respond to therapy containing a broad-spectrum TGFe1-selective inhibitory agent.

Two queries can be made regarding the identification/selection of appropriate indications and/or patient populations for which isoform-specific, context-permissive TGFe1 inhibitors, such as those described herein, are likely to have beneficial effects: i) when disease-specific or depends predominantly on the TGFe1 isoform over other isoforms in humans; and ii) whether the disease involves dysregulation of multiple aspects of TGFe1 function.

Differential expression of the three known TGFe isoforms, namely TGFe1, TGFe2 and TGFe3, has been observed under normal (healthy; homeostatic) as well as pathological conditions in various tissues. However, the concept of isoform selectivity has not been fully exploited and has not been achieved using traditional approaches that promote pan-inhibition of TGFe through multiple isoforms. Moreover, isoform expression profiles may be differentially regulated not only in normal (homeostatic) versus abnormal (pathological) states, but also in different patient subgroups. Because most preclinical studies are conducted in a limited number of animal models, data obtained using such models may be biased, leading to misinterpretation of the data or misleading conclusions regarding applicability to human conditions.

Accordingly, the present invention includes the recognition that differential expression of TGFe isoforms should be taken into account when predicting the effectiveness of specific inhibitors, as well as when interpreting preclinical data regarding translational capacity in human conditions. As shown in FIG. 21 TGFe1 and TGFe3 are dominant in several murine syngeneic cancer models (eg, EMT-6 and 4T1), which are widely used in preclinical studies. In contrast, many other cancer models (eg, S91, B16, and MBT-2) express almost exclusively TGFe1, similar to what is observed in many human tumors in which TGFe1 appears to be the more commonly dominant isoform over TGFe2/3. In addition, the TGFe isoform(s) predominantly expressed under homeostatic conditions may not represent the disease-associated isoform(s). For example, in normal lung tissues from healthy rats, tonic TGFe signaling appears to be mediated primarily by TGFe3. However, TGFe1 appears to be markedly upregulated in diseases such as pulmonary fibrosis. Taken together, it is useful to examine or confirm the relative expression of TGFe isoforms in clinical samples to select appropriate therapeutics to which a patient is likely to respond.

As described herein, isoform-selective TGFe1 inhibitors are particularly useful for treating diseases in which the TGFe1 isoform is predominantly expressed.

- 59 045239 compares with other isoforms. As an example, in FIG. 21D shows a non-limiting list of human cancer clinical samples with relative expression levels of TGF1 (left), TGFe2 (center), and TGFe3 (right). Each horizontal line on the three isoforms represents one patient. As can be seen, overall TGFe1 expression is significantly higher in most of these human tumors than in the other two isoforms for many tumor types, suggesting that selective inhibition of TGFe1 may be useful. However, some exceptions should be noted. First, this trend does not always apply to individual patients. Thus, even in a cancer type that shows almost uniform dominance of TGFe1 over TGFe2/3, there are a few individuals that do not follow this general rule. Thus, patients who fall into the minority subpopulation may not respond to isoform-specific therapy in the same way that it works for the majority of patients. Second, there are several types of cancer in which TGFe1 is co-dominant with another isoform or in which the expression of TGFe2 and/or TGFe3 is significantly higher than TGFe1. In these situations, TGFe1-selective inhibitors, such as those described herein, are unlikely to be effective. Therefore, it is useful to examine or confirm the relative expression levels of the three TGFe isoforms (i.e., TGFe1, TGFe2, and TGFe3) in clinical samples collected from individual patients. Such information may provide a better prediction of the effectiveness of a particular therapy in individual patients, which may help ensure the selection of appropriate treatment (eg, individualized treatment) to increase the likelihood of clinical response.

Accordingly, the present invention includes a method for selecting a patient population or subject that is likely to respond to therapy containing an isoform-specific, context-permissive TGFe1 inhibitor.

Such a method involves the following steps: providing a biological sample (eg, a clinical sample) taken from a subject, determining (eg, measuring or analyzing) the relative levels of TGFe1, TGFe2, and TGFe3 in the sample, and administering to the subject a composition comprising an isoform-specific, permissive to the context of a TGFe1 inhibitor if TGFe1 is the dominant isoform over TGFe2 and TGFe3; and/or if TGFe1 is significantly overexpressed or activated compared to controls. Relative levels of isoforms can be determined using RNA-based assays and/or protein-based assays, which are well known in the art. In some embodiments, the administration step may also include other therapy, such as immune checkpoint inhibitors or other agents provided elsewhere herein. Such methods may optionally include the step of assessing therapeutic response by monitoring changes in the relative levels of TGFe1, TGFe2 and TGFe3 at two or more time points. In some embodiments, clinical samples (such as biopsies) are collected both before and after administration. In some embodiments, clinical samples (such as biopsies) are collected multiple times after exposure to assess in vivo effects over time.

In addition to the first study addressing the isoform selectivity aspect, the second study explores the breadth of TGFe1 function involved in a specific disease. This can be represented by the number of TGFe1 contexts, namely which presentation molecule(s) mediate the disease-associated function of TGFe1. TGFe1-specific broad context inhibitors, such as context-permissive and context-independent inhibitors, are preferred for treating diseases that involve both an ECM component and an immune component of TGFe1 function. Such disease may be associated with dysregulation in the ECM, as well as with impaired immune cell function or immune response. Thus, the TGFe1 inhibitors described herein are capable of targeting ECM-associated TGFe1 (eg, presented by LTBP1 or LTBP3), as well as immune cell-associated TGFe1 (eg, presented by GARP or LRRC33). In some embodiments, such inhibitors target at least three of the following therapeutic targets (eg, context-permissive inhibitors): GARP-associated pro/latent TGFe1; LRRC33-associated pro/latent TGFe1; LTBP1-associated pro/latent TGFe1 and LTBP3-associated pro/latent TGFe1. In some embodiments, such inhibitors inhibit all four therapeutic targets (eg, context-independent inhibitors): GARP-associated pro/latent TGFe 1; LRRC33-associated pro/latent TGFe1; LTBP1-associated pro/latent TGFe1 and LTBP3-associated pro/latent TGFe1 to broadly inhibit TGFe1 function in these contexts.

Whether a particular patient's condition is associated or driven by multiple aspects of TGFe1 function can be assessed by assessing the expression profiles of presenting molecules in a clinical sample collected from the patient. Various assays are known in the art,

- 60 045239 including RNA-based assays and protein-based assays that can be performed to obtain expression profiles. The relative expression levels (and/or changes therein) of LTBP1, LTBP3, GARP, and LRRC33 in the sample(s) may indicate the source and/or context of condition-associated TGFe1 activities. For example, a biopsy sample taken from a solid tumor may exhibit high expression of all four presenting molecules. For example, LTBP1 and LTBP3 may be highly expressed in CAFs in the tumor stroma, whereas GARP and LRRC33 may be highly expressed by tumor-associated immune cells such as Tregs and leukocyte infiltrate, respectively.

Accordingly, the present invention includes a method for determining (eg, testing or confirming) the involvement of TGFe1 in a disease compared to TGFe2 and TGFe3. In some embodiments, the method further comprises the step of: identifying the source (or context) of disease-associated TGFe1. In some embodiments, the source/context is assessed by determining the expression of TGF presenting molecules, such as LTBP1, LTBP3, GARP, and LRRC33, in a clinical sample collected from patients.

Isoform-selective TGFe1 inhibitors, such as those described herein, can be used to treat a wide range of diseases, disorders and/or conditions that are associated with TGFe1 dysregulation (ie, TGFe1-related indications) in human subjects. As used herein, the term disease (disorder or condition) associated with TGFe1 dysregulation or indication associated with TGFe1 means any disease, disorder and/or condition associated with the expression, activity and/or metabolism of TGFe1, or any disease, disorder and/or condition associated with the expression, activity and/or metabolism of TGFe1. /or a condition that may benefit from inhibition of TGFe1 activity and/or levels.

Accordingly, the present invention includes the use of an isoform-specific, context-permissive TGFe1 inhibitor in a method of treating a disease associated with TGFe1 dysregulation in a human. Such an inhibitor is typically formulated into a pharmaceutical composition which further contains a pharmaceutically acceptable excipient. Advantageously, the inhibitor targets both ECM-associated TGFe1 and immune cell-associated TGFe1, but does not target TGFe2 or TGFe3 in vivo. In some embodiments, the inhibitor inhibits the activation step of TGFe1. The disease is characterized by dysregulation or impairment of at least two of the following: a) regulatory T cells (Treg); b) proliferation or function of effector T cells (Teff); c) proliferation or differentiation of myeloid cells; d) recruitment or differentiation of monocytes; f) functions of macrophages; f) epithelial-mesenchymal transition (EMT) and/or endothelial-mesenchymal transition (EndMT); g) gene expression in one or more marker genes selected from the group consisting of: PAI-1, ACTA2, CCL2, Collal, Col3a1, FN-1, CTGF and TGFe1; h) ECM components or function; i) fibroblast differentiation. A therapeutically effective amount of such an inhibitor is administered to a subject suffering from or diagnosed with a disease.

In some embodiments, the disease includes dysregulation or disruption of ECM components, or the function involves increased collagen I deposition.

In some embodiments, dysregulation or impairment of fibroblast differentiation involves an increase in the number of myofibroblasts or myofibroblast-like cells. In some embodiments, the myofibroblasts or myofibroblast-like cells are cancer-associated fibroblasts (CAFs). In some embodiments, CAFs are associated with tumor stroma and can produce CCL2/MCP-1 and/or CXCL12/SDF-1.

In some embodiments, dysregulation or disruption of regulatory T cells involves increased Treg activity.

In some embodiments, dysregulation or impairment of effector T cell proliferation or function (Teff) involves suppressed proliferation of CD4+/CD8+ cells.

In some embodiments, dysregulation or impairment of myeloid cell proliferation or differentiation involves increased proliferation of myeloid progenitor cells. Increased proliferation of myeloid cells may occur in the bone marrow,

In some embodiments, dysregulated or impaired monocyte differentiation involves increased differentiation of bone marrow-derived and/or tissue-resident monocytes into macrophages at a disease site, such as fibrous tissue and/or solid tumor.

In some embodiments, dysregulated or impaired monocyte recruitment involves increased recruitment of bone marrow-derived monocytes to a disease site, such as the TME, resulting in increased macrophage differentiation and M2 polarization with subsequent increase in TAM.

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In some embodiments, the dysregulation or dysfunction of macrophages involves increased polarization of macrophages into M2 phenotypes.

In some embodiments, the dysregulation or impairment of myeloid cell proliferation or differentiation involves increased numbers of Tregs, MDSCs, and/or

TAN.

TGF-related indications may include conditions containing an immune-prejudicing disease microenvironment, such as tumor or malignant tissue, that partially suppresses normal defense/immunity to the exclusion of effector immune cells (eg, CD4+ and/or CD8+ T cells) . In some embodiments, such immune-preventing conditions are associated with poor response to treatment. Without wishing to be limited by theory, it is believed that TGF inhibitors, such as those described herein, may help counteract the tumor's ability to exclude anticancer immunity by restoring access to T cells.

Non-limiting examples of TGF-related indications include: fibrosis, including organ fibrosis (eg, renal fibrosis, liver fibrosis, cardiac/cardiovascular fibrosis, and pulmonary fibrosis), scleroderma, Alport syndrome, cancer (including, without limitation: blood cancers such as leukemia, myelofibrosis, multiple myeloma, colon cancer, kidney cancer, breast cancer, malignant melanoma, glioblastoma), fibrosis associated with solid tumors (eg, malignant desmoplasia such as desmoplastic melanoma, desmoplasia , associated with pancreatic cancer, and desmoplasia in breast carcinoma), stromal fibrosis (eg, breast stromal fibrosis), radiation-induced fibrosis (eg, radiation fibrosis syndrome), promoting rapid hematopoiesis after chemotherapy, bone healing, wound healing, dementia, myelofibrosis, myelodysplasia (eg, myelodysplastic syndromes or MDS), kidney disease (eg, end-stage renal disease or ESRD), unilateral ureteral obstruction (UUO), tooth loss and/or degeneration, endothelial proliferation syndromes, asthma and allergies, gastrointestinal disorders, anemia with aging, aortic aneurysm, rare indications (such as Marfan syndrome and Camurati-Engelman disease), obesity, diabetes mellitus, arthritis, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, Parkinson's disease, osteoporosis, osteoarthritis , osteopenia, metabolic syndromes, nutritional disorders, organ atrophy, chronic obstructive pulmonary disease (COPD) and anorexia. Additional indications may include any of those disclosed in US Publication No. 2013/0122007, US Publication No. 8415459, or International Publication No. WO 2011/151432, the contents of each of which are incorporated herein by reference in their entirety.

In preferred embodiments, the antibodies, antigen binding portions thereof, and compositions of the present invention can be used to treat a wide range of diseases, disorders, and/or conditions associated with TGFe1 signaling. In some embodiments, target tissues/cells preferentially express the TGFe1 isoform over other isoforms. Thus, the present invention includes methods of treating such a condition associated with TGFe1 expression (eg, dysregulation of TGFe1 signaling and/or activation of TGFe1 expression) using a pharmaceutical composition that contains an antibody or an antigen-binding portion thereof described herein.

In some embodiments, the disease includes TGFe1 associated with (eg, presented to or deposited from) multiple cellular sources. In some embodiments, such a disease includes both an immune component and an ECM component of TGFe1 function. In some embodiments, such disease includes: i) ECM dysregulation (eg, overproduction/deposition of ECM components such as collagens and proteases; altered ECM substrate stiffness; abnormal or abnormal activation or differentiation of fibroblasts such as myofibroblasts and CAFs); ii) immune suppression due to increased Tregs and/or suppressed effector T cells (Teff), for example, increased Treg/Teff ratios; increased leukocyte infiltrate (eg, macrophages and MDSCs), which causes suppression of CD4 and/or CD8 T cells; and/or iii) abnormal or pathological activation, differentiation and/or recruitment of myeloid cells, such as macrophages (eg, bone marrow-derived monocytes/macrophages and tissue resident macrophages), monocytes, myeloid cell-derived suppressor cells (MDSCs), neutrophils, dendritic cells and NK cells.

In some embodiments, the condition includes TGFe1 presented by more than one type of presentation molecule (eg, two or more of: GAPR, LRRC33, LTBP1, and/or LTBP3). In some embodiments, tissues/organs/cells are affected that include TGFe1 from a variety of cellular sources. To give just one example, a solid tumor (which may also include proliferative diseases involving the bone marrow, such as myelofibrosis and multiple myeloma) may include TGFe1 from several sources, such as cancer cells, stromal cells surrounding healthy cells and/or infiltrating immune cells (eg, CD45+ leukocytes), including various types of presentation molecules. Suitable immune cells include, but are not limited to, myeloid cells and lymphoid cells, such as neutrophils, eosinophils, basophils, lymphocytes (eg, B cells, T cells and NK cells) and monocytes. Context-independent or context-permissive TGFe1 inhibitors may be useful for treating such conditions.

Non-limiting examples of conditions or disorders that can be treated with isoform-specific, context-permissive TGFe1 inhibitors, such as the antibodies or fragments thereof described herein, are provided below.

Diseases with aberrant gene expression.

Abnormal activation of the TGFe1 signal transduction pathway in various pathological conditions has been observed to be associated with altered gene expression of a number of markers. These gene expression markers (e.g., measured by mRNA) include, but are not limited to: Serpinel (encoding PAI-1), MCP-1 (also known as CCL2), Col1a1, Col3a1, FN1, TGFe1, CTGF, and ACTA2 (encoding α-SMA). Interestingly, many of these genes are involved in a variety of diseases, including various types of organ fibrosis, as well as many cancers, which include myelofibrosis. Indeed, a pathophysiological link has been proposed between fibrotic conditions and abnormal cell proliferation, tumorigenesis and metastasis. See, for example, Cox and Erler (2014) Clinical Cancer Research 20(14): 3637-43 Molecular pathways: connecting fibrosis and solid tumor metastasis; Shiga et al. (2015) Cancers 7:2443–2458 Cancer-associated fibroblasts: their characteristics and their roles in tumor growth; Wynn and Barron (2010) Semin. Live Dis. 30(3): 245-257 Macrophages: master regulators of inflammation and fibrosis, the contents of which are incorporated herein by reference. Without wishing to be bound by theory, the present disclosure suggests that the TGFe1 signaling pathway may in fact be a key link between these broad pathologies.

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

The ability of MCP-1/CCL2 to recruit monocytes/macrophages is critical for cancer progression. Tumor-derived MCP-1/CCL2 can promote malignant phenotypes in macrophages. For example, in lung cancer, MCP-1/CCL2 has been shown to be produced by stromal cells and promote metastasis. In human pancreatic cancer, tumors secrete CCL2, and immunosuppressive CCL2-positive macrophages infiltrate these tumors. Patients with tumors that exhibit high CCL2 expression/low CD8 T-cell infiltrate had significantly reduced survival.

Similarly, PAI-1/Serpinel has been implicated in various cancers, angiogenesis, inflammation, and neurodegenerative diseases (e.g., Alzheimer's disease). Increased expression of PAI-1 in tumor and/or serum correlates with poor prognosis (eg, shorter survival, increased metastasis) in various cancers, such as breast cancer and bladder cancer (eg, transitional cell carcinoma), as well as myelofibrosis. In the context of fibrotic conditions, PAI-1 has been recognized as an important downstream effector of TGFe1-induced fibrosis, and increased expression of PAI-1 has been observed in various forms of tissue fibrosis, including pulmonary fibrosis (such as IPF), renal fibrosis, liver fibrosis, and scleroderma.

In some embodiments, the in vivo effects of TGFe1 inhibitor therapy can be assessed by measuring changes in gene markers. Suitable markers include TGFe (eg TGFe1, TGFe2 and TGFe3). In some embodiments, suitable markers include mesenchymal transition genes (e.g., AXL, ROR2, WNT5A, LOXL2, TWIST2, TAGLN, and/or FAP), immunosuppressive genes (e.g., IL10, VEGFA, VEGFC), monocyte and macrophage chemotactic genes ( for example, CCL2, CCL7, CCL8 and CCL13) and/or various fibrotic markers discussed herein. The preferred markers are plasma markers.

As shown in the example provided herein, the isoform-specific, context-independent TGFe1 inhibitors described herein can reduce the expression levels of many of these markers in a mechanistic animal model such as UUO, which has been shown to be TGFβ1-dependent. Therefore, such inhibitors can be used to treat a disease or disorder characterized by abnormal expression (eg, overexpression/upregulation or underexpression/downregulation) of one or more gene expression markers.

Thus, in some embodiments, an isoform-specific, context-permissive, or context-independent TGFe1 inhibitor is used in the treatment of a disease associated with overexpression of one or more of the following: PAI-1 (also known as Serpinel), MCP-1 ( also known as CCL2), Col1a1, Col3a1, FN1, TGFe1, CTGF, α-SMA, ITGA11 and ACTA2, the treatment comprising administering the inhibitor to a subject suffering from the disease in an amount effective to treat the disease. In some embodiments, the inhibitor is used to treat a disease associated with overexpression of PAI-1, MCP-1/CCL2, CTGF, and/or α-SMA. In some embodiments, the disease is myelofibrosis. In some embodiments, the disease is a cancer, such as a cancer comprising a solid tumor. In some embodiments, the disease is organ fibrosis, such as fibrosis of the liver, kidney, lung, and/or cardiac or cardiovascular tissue.

Diseases involving proteases.

Activation of TGFe from its latent complex can be triggered by integrin in a force-dependent manner and/or by proteases. Evidence suggests that certain classes of proteases may be involved in this process, including, but not limited to, Ser/Thr proteases such as kallikreins, chemotrypsin, elastases, plasmin, and MMP family zinc metalloproteases such as MMP-2 , MMP-9 and MMP-13. MMP-2 degrades the most abundant basement membrane component, collagen IV, raising the possibility that it may play a role in the regulation of ECM-associated TGFe1. MMP-9 plays a central role in tumor development, angiogenesis, stromal remodeling and metastasis. Thus, protease-dependent activation of TGFe1 in the ECM may be important for cancer treatment.

Kallikreins (KLKs) are trypsin- or chymotrypsin-like serine proteases that include plasma kallikreins and tissue kallikreins. The ECM plays a role in tissue homeostasis, serving as a structural and signaling scaffold and barrier to the suppression of malignant growth. KLKs may play a role in the degradation of ECM proteins and other components that may promote tumor growth and invasion. For example, KLK1 is highly elevated in certain types of breast cancer and can activate pro-MMP-2 and pro-MMP-9. KLK2 activates latent TGFp1, making prostate cancer adjacent to fibroblasts permissive to cancer growth. KLK3 has been widely studied as a diagnostic marker for prostate cancer (PSA). KLK3 can directly activate TGFe1 by converting plasminogen to plasmin, which proteolytically cleaves LAP. KLK6 may be a potential marker for Alzheimer's disease.

In addition, the data presented in Example 8 indicate that such proteases may be kallikrein. Thus, the present invention encompasses the use of an isoform-specific, context-independent or context-permissive TGFe inhibitor in a method of treating a disease associated with kallikrein or kallikrein-like protease.

Known TGFe1 activators such as plasmin, TSP-1, and aVe6 integrin interact directly with LAP. It is postulated that proteolytic cleavage of LAP may destabilize the LAP-TGFe interaction, thereby releasing active TGFe1. The region containing 54-LSKLRL-59 is predicted to be important for maintaining TGFe1 latency. Thus, agents (eg, antibodies) that stabilize the interaction or block proteolytic cleavage of LAP may prevent TGFe activation.

Diseases Involving Epithelial-Mesenchymal Transition (EMT): EMT (epithelial mesenchymal transition) is the process by which epithelial cells with rigid junctions switch to mesenchymal properties (phenotypes), such as loose intercellular junctions. The process is observed in a number of normal biological processes as well as in pathological situations, including embryogenesis, wound healing, cancer metastasis and fibrosis (see, for example, Shiga et al. (2015) Cancer-Associated Fibroblasts: Their Characteristics and Their Roles in Tumor Growth. Cancers, 7: 2443-2458). EMT signals are generally thought to be induced primarily by TGFe. For example, many cancers appear to involve cell transdifferentiation toward a mesenchymal phenotype (such as CAF), which is correlated with a worse prognosis. Thus, isoform-specific, context-permissive TGFe1 inhibitors, such as those described herein, can be used to treat disease that is initiated or caused by EMT. Indeed, exemplary data (eg, Figs. 12 and 13) show that such inhibitors have the ability to suppress the expression of CAF markers in vivo, such as α-SMA, Coll (type I collagen) and FN (fibronectin). Diseases involving endothelial-mesenchymal transition (EndMT): Similarly, TGFe also represents a key regulator of endothelial-mesenchymal transition (EndMT) observed

- 64 045239 during normal development, such as heart formation. However, the same or similar phenomenon is also observed in many diseases, such as malignant tumor stroma. In some pathological processes, endothelial markers such as CD31 are reduced by TGFe1, and the expression of mesenchymal markers such as FSP-1, α-SMA and fibronectin is instead induced. Indeed, stromal CAFs can be derived from vascular endothelial cells. Thus, isoform-specific, context-permissive TGFe1 inhibitors, such as those described herein, can be used to treat disease that is initiated or caused by EndMT.

Diseases Involved in Matrix Compaction and Remodeling: Progression of fibrotic conditions involves increased levels of matrix components deposited in the ECM and/or maintenance/remodeling of the ECM. TGFe1 at least partially contributes to this process. This is supported, for example, by the observation that increased deposition of ECM components such as collagens can alter the mechanophysical properties of the ECM (e.g., matrix/substrate stiffness), and this phenomenon is associated with TGFe1 signaling. To validate this notion, we assessed the role of matrix stiffness in influencing integrin-dependent activation of TGFe in primary fibroblasts transfected with pro-TGFe and LTBP1 and grown on silica-based substrates with defined stiffness (e.g., 5 kPa, 15 kPa, or 100 kPa). kPa). As summarized in the Examples section below, matrices with greater stiffness enhance TGFp1 activation, and this can be suppressed by isoform-specific, context-permissive TGFe1 inhibitors, such as those described herein. These observations suggest that TGFe1 influences ECM properties (such as stiffness), which in turn may further cause TGFe1 activation reflecting disease progression. Thus, isoform-specific, context-permissive TGFe1 inhibitors, such as those described herein, can be used to block this process to counter disease progression involving ECM changes such as fibrosis, tumor growth, invasion, metastasis and desmoplasia. The LTBP group of such inhibitors can directly block ECM-associated pro/latent TGFe complexes, which are represented by LTBP1 and/or LTBP3, thereby preventing growth factor activation/release from the complex in the disease niche. In some embodiments, isoform-specific, context-permissive TGFe1 inhibitors, such as those described herein, can normalize ECM stiffness to treat a disease that involves integrin-dependent signaling. In some embodiments, the integrin comprises an α1 chain, a β1 chain, or both.

Fibrosis.

According to the present invention, isoform-specific, context-permissive TGFe1 inhibitors, such as those described herein, are used in the treatment of fibrosis (eg, fibrotic indications, fibrotic conditions) in a subject. Suitable inhibitors for practicing the present invention include antibodies and/or compositions according to the present disclosure that may be useful for modifying or attenuating fibrosis. More specifically, such antibodies and/or compositions are selective TGFe1 antagonists that are capable of targeting TGFe1 presented by various types of presentation molecules. TGFe1 is recognized as a central orchestrator of the fibrotic response. Antibodies targeting TGF reduce fibrosis in numerous preclinical models. Such antibodies and/or antibody-based compounds include LY2382770 (Eli Lilly, Indianapolis, IN). Also included are those described in US Patent No. US 6,492,497, US 7,151,169, US 7,723,486 and US Patent Application Publication No. 2011/0008364, the contents of each of which are incorporated herein by reference in their entirety. Prior art TGFe antagonists include, for example, agents that target and block integrin-dependent activation of TGFe.

However, evidence suggests that such prior art agents may not mediate isoform-specific inhibition and may cause undesirable effects by inadvertently blocking the normal function of TGFe2 and/or TGFe3. Indeed, the data presented herein support this notion. Normal (non-diseased) lung tissue contains relatively low but measurable levels of TGFe2 and TGFe3, but markedly less TGFe1. In comparison, under certain pathological conditions such as fibrosis, TGFe1 becomes preferentially activated compared to other isoforms. Preferably, TGFe antagonists for use in the treatment of such conditions exhibit their inhibitory activity only on the disease-induced or disease-associated isoform, while retaining the function of other isoforms that are normally expressed to mediate tonic signaling in tissue. Advantageously, as illustrated in Example 20 below, the isoform-specific, context-permissive TGFe1 inhibitor encompassed by this isoform

- 65 045239 by breeching demonstrates little effect in bronchoalveolar lavage (BAL) fluid from healthy rats, supporting the view that tonic TGFe (eg, TGFe2 and/or TGFe3) signaling is unperturbed. In contrast, prior art inhibitors (LY2109761, a small molecule TGFe receptor antagonist, and a monoclonal antibody that targets integrin aVe6) have both been shown to inhibit subsequent tonic TGFe signaling in disease-intact rat BAL, raising the possibility that these inhibitors may cause unwanted side effects. Alternatively or additionally, agents that target and block integrin-dependent TGFe activation may be able to block only the subset of integrins responsible for disease-associated TGFe1 activation, among the numerous integrin types that are expressed by different cell types and play a role in pathogenesis. Moreover, even though such antagonists can selectively block integrin-mediated activation of the TGFp1 isoform, it may not be effective in blocking TGFe1 activation triggered by other pathways, such as protease-dependent activation. In contrast, isoform-specific, context-permissive TGFe1 inhibitors, such as those described herein, aim to prevent the TGFe1 activation step, regardless of the specific mode of activation, while maintaining isoform selectivity.

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

In some embodiments, fibrotic indications that can be treated with the compositions and/or methods described herein include organ fibrosis, such as pulmonary fibrosis (e.g., IPF), renal fibrosis (e.g., CKD-associated fibrosis) , fibrosis of the liver, fibrosis of the heart or cardiac tissue, fibrosis of the skin (eg, scleroderma), fibrosis of the uterus (eg, endometrium, myometrium), and bone marrow fibrosis. In some embodiments, such therapy can reduce or delay the need for organ transplantation in patients. In some embodiments, such therapy may prolong survival of patients.

For the treatment of IPF, patients who may benefit from treatment include patients with familial LPF and patients with sporadic IPF. Administration of a therapeutically effective amount of an isoform-specific, context-permissive TGFe1 inhibitor can reduce the accumulation of myofibroblasts in lung tissue, reduce collagen deposition, reduce symptoms of IPF, improve or maintain lung function, and prolong survival. In some embodiments, the inhibitor blocks activation of ECM-associated TGFe1 (eg, pro/latent TGFe1 presented by LTBP1/3) in the fibrotic environment of the LPF. The inhibitor may optionally further block activation of macrophage-associated TGFe1 (eg, pro/latent TGFe1 presented by LRRC33), for example, by alveolar macrophages. As a result, the inhibitor may suppress the release of fibronectin and other factors associated with fibrosis.

Isoform-specific, context-permissive TGFe1 inhibitors such as

- 66 045239 those presented herein can be used to treat fibrotic liver conditions such as fatty liver disease (eg, NASH). Fatty liver disease may or may not be inflamed. Inflammation of the liver due to fatty liver disease (ie, steatohepatitis) can develop into scarring (fibrosis), which then often progresses to cirrhosis (scarring that distorts the structure of the liver and impairs its function). Therefore, the inhibitor can be used to treat such conditions. In some embodiments, the inhibitor blocks activation of ECM-associated TGFe1 (eg, pro/latent TGFe1 presented by LTBP1/3) in the fibrotic environment of the liver. The inhibitor may optionally further block activation of macrophage-associated TGFe1 (eg, pro/latent TGFe1 presented by LRRC33), such as Kupffer cells (also known as stellate macrophages), as well as infiltrating monocyte-derived macrophages and MDSCs. As a result, the inhibitor may suppress factors associated with fibrosis. Administration of an inhibitor to a subject with such conditions may reduce one or more symptoms, prevent or slow disease progression, reduce or stabilize fat accumulation in the liver, reduce disease-related biomarkers (such as serum collagen fragments), reduce liver scarring, reduce liver stiffness and/or otherwise lead to clinically significant results in a patient population treated with an inhibitor compared to a control group not treated with an inhibitor. In some embodiments, an effective amount of an inhibitor can result in both a reduction in liver fat content and a reduction in fibrosis (eg, scarring) in patients with NASH. In some embodiments, an effective amount of an inhibitor can lead to improvement in at least one stage of fibrosis without exacerbation of steatohepatitis in patients with NASH. In some embodiments, an effective amount of an inhibitor can reduce the incidence of liver failure and/or liver cancer in patients with NASH. In some embodiments, an effective amount of an inhibitor can normalize, relative to control, levels of multiple inflammatory or fibrotic serum biomarkers assessed after initiation of therapy, for example, 12-36 weeks. In some embodiments, in patients with NASH, isoform-specific, context-permissive TGFe1 inhibitors may be administered to patients who are receiving one or more additional therapies, including, but not limited to, myostatin inhibitors, which generally may enhance metabolic regulation in patients with clinical manifestations of metabolic syndrome, including NASH.

Isoform-specific, context-permissive TGFe1 inhibitors, such as those presented herein, can be used to treat fibrotic conditions of the kidney, such as diseases characterized by extracellular matrix accumulation (IgAnephropathy, focal and segmental glomerulosclerosis, crescentic glomerulonephritis, lupus nephritis and diabetic nephropathy), in which there was a significant increase in TGFe expression in the glomeruli and tubulointerstitium. While glomerular and tubulointerstitial deposition of two TGFe-induced matrix components, fibronectin EDA+ and PAI-1, were significantly increased in all matrix storage diseases, correlation analysis revealed a strong association primarily with the TGFe1 isoform. Accordingly, isoform-specific, context-permissive TGFe1 inhibitors are useful as therapeutics for a spectrum of human glomerular disorders in which TGFe is associated with pathological extracellular matrix accumulation.

In some embodiments, the fibrotic condition of the kidney is associated with chronic kidney disease (CKD). CKD is caused primarily by high blood pressure or diabetes and claims more than one million lives every year. Patients with CKD require lifelong medical care, which ranges from strict diets and medications to dialysis and transplantation. In some embodiments, TGFe1 inhibitor therapy described herein may reduce or delay the need for dialysis and/or transplantation. In some embodiments, such therapy may reduce the need (eg, dosage, frequency) of other treatments. In some embodiments, isoform-specific, context-permissive TGFe1 inhibitors can be administered to patients who are receiving one or more additional therapies, including, but not limited to, myostatin inhibitors, which generally may enhance metabolic regulation in patients with CKD .

Organ fibrosis that can be treated using the methods presented herein includes cardiac fibrosis (eg, cardiovascular). In some embodiments, cardiac fibrosis is associated with heart failure, such as chronic heart failure (CFLF). In some embodiments, heart failure may be associated with myocardial diseases and/or metabolic diseases. In some embodiments, isoform-specific, context-permissive TGFei inhibitors can be administered to patients who are receiving one or more additional therapies, including, but not limited to, myostatin inhibitors in patients with cardiac dysfunction that includes cardiac fibrosis and metabolic disorder.

In some embodiments, fibrotic conditions that can be treated with the compositions and/or methods described herein include desmoplasia. Desmoplasia can occur around a neoplasm, causing dense fibrosis around the tumor (eg, desmoplastic stroma) or scar tissue in the abdomen after abdominal surgery. In some embodiments, the desmoplasia is associated with a malignant tumor. Due to its dense formation surrounding the malignant tumor, traditional anticancer therapeutics (such as chemotherapy) may not effectively penetrate cancer cells for clinical effects. Isoform-specific, context-permissive TGFe1 inhibitors, such as those described herein, can be used to disrupt desmoplasia so that fibrous formation can be attenuated to promote the effects of anticancer therapy. In some embodiments, isoform-specific, context-permissive TGFe1 inhibitors can be used as monotherapy (more detail below).

To treat patients with fibrotic conditions, isoform-specific, context-permissive TGFe1 inhibitors are administered to the subject in an amount effective to treat the fibrosis. An effective amount of such an antibody is an amount effective to achieve both therapeutic efficacy and clinical safety in a subject. In some embodiments, the inhibitor is a context-permissive antibody that can block the activation of LTBP-mediated TGFe1 localized (eg, bound) to the ECM and GARP-mediated TGFe1 localized (eg, bound) to immune cells. In some embodiments, the antibody is a context-permissive antibody that can block activation of LTBP-mediated TGFe1 located in the ECM and LRRC33-mediated TGFe1 localized to (eg, bound to) monocytes/macrophages. In some embodiments, the LTBP is LTBP1 and/or LTBP3. In some embodiments, targeting and inhibiting TGFe1 presented by LRRC33 on profibrotic, M2-like macrophages in a fibrotic microenvironment may be beneficial.

Assays useful for determining the effectiveness of the antibodies and/or compositions of the present invention in reversing fibrosis include, but are not limited to, histological fibroblast counting assays and basic immunohistochemical assays known in the art.

Myelofibrosis.

Myelofibrosis, also known as osteomyelofibrosis, is a relatively rare proliferative bone marrow disease (cancer) that belongs to a group of diseases called myeloproliferative disorders. Myelofibrosis is classified into the Philadelphia chromosome-negative (-) branch of myeloproliferative neoplasms. Myelofibrosis is characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Proliferation of an abnormal clone of hematopoietic stem cells in the bone marrow and other sites leads to fibrosis, or replacement of the bone marrow by scar tissue. The term myelofibrosis, unless otherwise noted, refers to primary myelofibrosis (PMF). It may also be referred to as chronic idiopathic myelofibrosis (CLMF) (the terms idiopathic and primary mean that in these cases the disease is of unknown or spontaneous origin). This contrasts with myelofibrosis, which occurs secondary to polycythemia vera or essential thrombocythemia. Myelofibrosis is a form of myeloid metaplasia that refers to a change in the cell type in the hematopoietic tissue of the bone marrow, and often the two terms are used interchangeably. The terms agnogenic myeloid metaplasia and myelofibrosis with myeloid metaplasia (MMM) are also used to refer to primary myelofibrosis. In some embodiments, hematologic proliferative disorders that can be treated in accordance with the present invention include myeloproliferative disorders such as myelofibrosis. The so-called classical BCR-ABL (Ph) group of negative chronic myeloproliferative disorders includes essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF).

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

Myelofibrosis can be caused by abnormal blood stem cells in the bone marrow. The abnormal stem cells produce mature and poorly differentiated cells that grow rapidly and invade the bone marrow, causing both fibrosis (scar tissue formation) and chronic inflammation.

Primary myelofibrosis is associated with mutations in Janus kinase 2 (JAK2), thrombopoietin receptor (MPL), and calreticulin (CALR), which can lead to constitutive activation of the JAK-STAT pathway and progressive bone marrow scarring or fibrosis. Patients may develop extramedullary hematopoiesis, i.e. the formation of blood cells that occurs in sites other than the bone marrow as hematopoietic cells are forced to migrate to other areas, particularly the liver and spleen. This causes these organs to enlarge. In the liver, the abnormal size is called hepatomegaly. An enlarged spleen is called splenomegaly, which also contributes to pancytopenia, particularly thrombocytopenia and anemia. Another complication of extramedullary hematopoiesis is poikilocytosis, or the presence of abnormally formed red blood cells.

The main site of extramedullary hematopoiesis in myelofibrosis is the spleen, which, as a rule, is noticeably enlarged in patients suffering from myelofibrosis. As a result of massive enlargement of the spleen, multiple subcapsular infarcts often occur in the spleen, meaning that partial or complete tissue death occurs due to the interruption of oxygen supply to the spleen. At the cellular level, the spleen contains erythrocyte precursors, granulocyte precursors, and megakaryocytes, with megakaryocytes predominant in number and abnormal shape. Megakaryocytes may be involved in the secondary fibrosis observed in this condition.

It has been suggested that TGFe may be involved in the fibrotic aspect of the pathogenesis of myelofibrosis (see, for example, Agarwal et al., Bone marrow fibrosis in primary myelofibrosis: pathogenic mechanisms and the role of TGFe (2016) Stem Cell Investig 3:5). Bone marrow pathology in primary myelofibrosis is characterized by fibrosis, neoangeogenesis and osteosclerosis, and fibrosis is associated with an increase in the production of collagens deposited in the ECM.

A number of biomarkers have been described, the alternation of which indicates or correlates with disease. In some embodiments, the biomarkers are cellular markers. Such disease-related biomarkers are useful for diagnosing and/or monitoring disease progression, as well as the effectiveness of therapy (eg, patient response to therapy). These biomarkers include a number of fibrotic markers as well as cellular markers. For example, in lung cancer, TGFei concentrations in bronchoalveolar lavage fluid (BAL) have been reported to be significantly higher in patients with lung cancer compared with patients with benign disease (approximately 2-fold increase), which may also serve as a biomarker for diagnosis and /or monitoring the progression or treatment of consequences of lung cancer.

Because primary myelofibrosis is associated with abnormal megakaryocyte development, certain cellular markers of megakaryocytes, as well as their stem cell lineage precursors, may serve as markers for diagnosis and/or monitoring of disease progression, as well as the effectiveness of therapy. In some embodiments, useful markers include, but are not limited to: cellular markers of differentiated megakaryocytes (e.g., CD41, CD42, and Tro R), cellular markers of megakaryocyte-erythroid progenitor cells (e.g., CD34, CD38, and CD45RA-), cellular markers of general myeloid progenitor cells (eg, IL3a/CD127, CD34, SCF R/c-kit and Flt-3/Flk-2) and hematopoietic stem cell markers (eg, CD34, CD38-, Fit-3/Flk-2) . In some embodiments, useful biomarkers include fibrotic markers. These include, but are not limited to: TGFei, PAI-1 (also known as Serpinel), MCP-1 (also known as CCL2), Collai, Col3a1, FN1, CTGF, α-SMA, ACTA2, Timpi, Mmp8 and Mmp9. In some embodiments, the useful biomarkers are serum markers (eg, proteins or fragments found and detected in serum samples).

Based on the fact that TGFe is a component of the leukemic bone marrow niche, it is suggested that targeting the bone marrow microenvironment with TGFe inhibitors may be a promising approach to reduce leukemia cells expressing presenting molecules that regulate the local availability of TGFe in affected tissue.

Indeed, due to the multifaceted nature of the pathology, which exhibits TGFe-dependent dysregulation in both myeloproliferative and fibrotic aspects (as the term myelofibrosis itself suggests), isoform-specific, context-permissive TGFei inhibitors such as those described herein, may provide particularly applicable

- 69 045239 therapeutic effects for patients suffering from myelofibrosis. It is hypothesized that the LTBP group of such an inhibitor may target the ECM-associated TGFe1 complex in the bone marrow, whereas the LRRC33 group of the inhibitor may block myeloid cell-associated TGFe1. Additionally, abnormal megakaryocyte biology associated with myelofibrosis may involve GARP- and LTBP-mediated TGFe1 activity. An isoform-specific, context-permissive TGFe1 inhibitor is able to target such complexes, thereby inhibiting the release of active TGFe1 in the niche.

Thus, such TGFe1 inhibitors are useful for treating patients with polycythemia vera who have had an inadequate response to or intolerance to other (or standard) treatments such as hydroxyurea and JAK inhibitors. Such inhibitors are also useful for the treatment of patients with intermediate or high-risk myelofibrosis (MF), including primary MF, MF following polycythemia vera, and MF following thrombocythemia.

Accordingly, one aspect of the present invention relates to methods for treating primary myelofibrosis. The method involves administering to a patient suffering from primary myelofibrosis a therapeutically effective amount of a composition containing a TGFe inhibitor that causes a decrease in the availability of TGFe. In some embodiments, patients with myelofibrosis are administered an isoform-specific, context-permissive inhibitor of a TGFe1 activation monoclonal antibody. Such an antibody can be administered in doses ranging from 0.1 to 100 mg/kg, for example 1 to 30 mg, for example 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg /kg. 20 mg/kg, etc. Preferred routes of administration of a pharmaceutical composition containing an antibody are intravenous or subcutaneous administration. When the composition is administered intravenously, the patient may be given the therapeutic agent for a suitable period of time, for example about 60 minutes, for treatment and then repeated every few weeks, for example, 3 weeks, 4 weeks, 6 weeks, etc. for a total of several cycles, such as 4 cycles, 6 cycles, 8 cycles, 10 cycles, 12 cycles, etc. In some embodiments, patients are treated with a composition comprising an inhibitory antibody at a dose level of 1-10 mg/kg (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg) by intravenous administration every 28 days (4 weeks) for 6 cycles or 12 cycles. In some embodiments, such treatment is administered as chronic (long-term) therapy (eg, to be continued for an indefinite period of time as long as is considered beneficial) rather than discontinued after a set number of administration cycles.

In some embodiments, a TGFe inhibitor is an antibody or antigen-binding portion thereof that binds to an inactive (eg, latent) pro-TGFe complex, thereby preventing the release of active or mature TGFe from the complex, effectively inhibiting the activation step. In some embodiments, such an antibody or antigen-binding portion thereof specifically binds to a pro-TGF complex that is associated with LRRC33, GARP, LTBP1, LTBP3, or any combination thereof. In some embodiments, such an antibody or antigen-binding portion thereof specifically binds to a cell-associated pro-TGFe complex. In some embodiments, the antibody or portion thereof selectively binds to a pro-TGF complex that is associated with either LRRC33 and/or GARP (but not LTBP1 or LTBP3). In some embodiments, the antibody or portion thereof specifically binds to the pro-TGFe complex that is associated with LRRC33. In some embodiments, the antibody or portion thereof specifically binds to the pro-TGFe complex that is associated with GARP. In some embodiments, the antibody or portion thereof specifically binds to the pro-TGFe complex that is associated with LRRC33, as well as the pro-TGFe complex that is associated with GARP.

Alternatively or in addition to the embodiments discussed above, a TGFe inhibitor is an antibody or antigen-binding portion thereof that binds to LRRC33 and/or GARP and contains a domain for additional effector functions. In some embodiments, the domain for additional effector function may be an Fc or Fc-like domain for mediating ADCC in target cells. Preferably, the ADCC-inducing antibody does not trigger or facilitate internalization to sufficiently allow ADCC-mediated killing of target cells.

Alternatively or in addition to the embodiments discussed above, the antibody or antigen-binding portion thereof may include an additional moiety to carry the molecular cargo of interest (eg, antibody-drug conjugates or ADCs). Examples of suitable molecular cargo include, but are not limited to: therapeutics/drugs, toxins, detection/imaging markers and tags, etc. Such molecular cargo may be chemical entities, small molecules, polypeptides, nucleic acids, radioisotopes, etc. Preferably, antibodies that are suitable for the ADC-mediated mechanism of action can, upon binding to the target cell surface, trigger efficient internalization of the antigen-antibody complex to deliver molecular

- 70 045239 cular cargo in a cage.

Because myelofibrosis is a progressive disease that manifests many aspects of pathology in many affected tissues or organs, the therapeutic approach may vary depending on the progression of the disease. For example, at the primary site of the disease (bone marrow), suitable therapy is contemplated to include an LRRC33 inhibitor described herein that can target hematopoietic cells expressing LRRC33. This can be achieved by administering a composition containing an antibody that binds to the LRRC33 presenting pro-TGFe complex and inhibits TGFe activation in the patient. This can also be achieved by administering a composition containing an antibody that binds to LRRC33 and induces the destruction of target cells in the patient. Alternatively, these approaches can be combined to use an antibody that is an inhibitor of TGFe activation and also contains an additional moiety that mediates cellular cytotoxicity. For example, the additional fragment may be an Fc or Fc-like domain for inducing ADCC or a toxin conjugated to an antibody as a molecular cargo (eg, antibody-drug conjugates or ADCs).

Although myelofibrosis can be considered a type of leukemia, it is characterized by the appearance of fibrosis. Since TGFe is known to regulate aspects of ECM homeostasis, the dysregulation of which can lead to tissue fibrosis, it is contemplated that in some embodiments it is desirable to inhibit ECM-related TGFe activity. Accordingly, antibodies or fragments thereof that bind and inhibit pro-TGFe presented by LTBP (such as LTBP1 and LTBP3) are covered by the present invention. In some embodiments, antibodies or fragments thereof useful for treating myelofibrosis are context permissive in the sense that they can bind to multiple pro-TGFe complex contexts, such as those associated with LRRC33, GARP, LTBP1, LTBP3 or any combination thereof. In some embodiments, such an antibody is a context-independent inhibitor of TGFe activation, characterized in that the antibody can bind and inhibit any of the following latent complexes: LTBP1-pro-TGFe, LTBP3-pro-TGFe, GARP-pro-TGFe, and LRRC33-proTGFe . In some embodiments, such an antibody is an isoform-specific antibody that binds and inhibits latent complexes that contain one but not other TGFe isoforms. These include, for example, LTBP1-pro-TGFe1, LTBP3-pro-TGFe1, GARP-pro-TGFe1 and LRRC33-pro-TGFe1. In some embodiments, such an antibody is an isoform-selective antibody that substantially binds and inhibits one or more TGFe isoforms. It is believed that antibodies that can inhibit TGFe1 activation in a context-permissive or context-independent manner are preferred for use in the treatment of myelofibrosis.

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

In some embodiments, the patient population includes patients at intermediate level 2 or high risk for myelofibrosis. In some embodiments, the patient population comprises subjects with myelofibrosis who are refractory to treatment or are not candidates for available therapy. In some embodiments, the subject's platelet count is between 100-200 x 10 ⁹ /L. In some embodiments, the subject's platelet count is >200 x 10 ⁹ /L before receiving treatment.

In some embodiments, a subject who is to receive (and who may benefit from receiving) isoform-specific, context-permissive TGFp1 inhibitor therapy is diagnosed with primary myelofibrosis (PMF) at intermediate 1 or higher, or post-polycythemia vera myelofibrosis. essential thrombocythemia (post-PV/ET MF). In some embodiments, the subject has documented bone marrow fibrosis prior to treatment. In some embodiments, the subject is MF-2 or higher as assessed by the European Global Assessment Score and grade 3 or higher on the Modified Bauermeister Scale prior to treatment. In some embodiments, the subject has an ECOG performance status of 1 before treatment. In some embodiments, the subject's white blood cell count (10 ⁹ /L) ranges from 5 to 120 before treatment. In some embodiments, the subject has a JAK2V617F allelic load that is in the range of 10-100%.

In some embodiments, a subject who is receiving (and who may benefit from receiving) isoform-specific, context-permissive TGFei inhibitor therapy is transfusion dependent (pre-treatment), characterized in that the subject has a history of have had at least two units of red blood cell transfusion in the past month with a hemoglobin level of less than 8.5 g/dL that is not associated with clinically overt bleeding.

In some embodiments, the subject who is to receive (and who may benefit from receiving) isoform-specific, context-permissive TGFe1 inhibitor therapy has previously received therapy to treat myelofibrosis. In some embodiments, the subject has been treated with one or more therapies, including, but not limited to: AZD1480, panobinostat, EPO, IFNa, hydroxyurea, pegylated interferon, thalidomide, prednisone, and a JAK2 inhibitor (eg, lestaurtinib, CEP-701).

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

An isoform-specific, context-permissive TGFe1 inhibitor is administered to patients in an amount effective to treat myelofibrosis. A therapeutically effective amount is an amount sufficient to relieve one or more symptoms and/or complications of myelofibrosis in patients, including, but not limited to: excessive deposition of ECM in the bone marrow stroma, neoangiogenesis, osteosclerosis, splenomegaly, hematomegaly, anemia, bleeding, pain in bones and other bone-related diseases, extramedullary hematopoiesis, thrombocytosis, leukopenia, cachexia, infections, thrombosis and death.

In some embodiments, the amount is effective to reduce TGFe1 expression and/or secretion (such as megakaryocyte cells) in patients. Therefore, such an inhibitor may reduce TGFe1 mRNA levels in treated patients. In some embodiments, such an inhibitor reduces TGFe1 mRNA levels in the bone marrow, such as in mononuclear cells. Patients with PMF typically have elevated plasma TGFe1 levels greater than ~2500 pg/mL, e.g., greater than 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, and 10,000 pg/mL (in contrast from normal ranges of ~600-2000 pg/ml by ELISA (see, for example, Mascaremhas et al. (Leukemia & Lymphoma, 2014, 55(2): 450-452)) Zingariello (Blood, 2013, 121(17) : 3345-3363) quantified the bioactive and total TGFe1 content in the plasma of patients with PMF and control individuals. According to this reference, the median bioactive TGFe1 in patients with PMF was 43 ng/ml (range 4-218 ng/ml), and total TGFe1 was 153 ng/ml (32-1000 ng/ml), while the control counterparts had values of 18 (0.05-144) and 52 (8860), respectively.Thus, based on these reports, plasma TGFe1 levels in patients with PMF increases several times, for example, 2, 3, 4, 5 times, etc. compared to control or healthy plasma samples.Treatment with an inhibitor, for example, after 4-12 cycles of administration (for example, 2, 4 6, 8, 10, 12 cycles) or chronic or long-term treatment, e.g., every 4 weeks, at a dose of 0.1-100 mg/kg (e.g., 1-30 mg/kg monoclonal antibody) described herein, may reduce plasma TGFe1 levels by at least 10% relative to the corresponding baseline (pretreatment), for example, by at least 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50% .

Some therapeutic effects may be observed relatively quickly after initiation of treatment, for example, after 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. For example, the inhibitor can effectively increase the number of stem cells and/or progenitor cells in the bone marrow of patients treated with the inhibitor within 1-8 weeks. These include hematopoietic stem cells and blood progenitor cells. A bone marrow biopsy may be performed to evaluate changes in the frequency/number of bone marrow cells. Accordingly, the patient may experience improved symptoms such as bone pain and fatigue.

One of the morphological hallmarks of myelofibrosis is fibrosis in the bone marrow (eg, bone marrow stroma), which is characterized in part by aberrant ECM. In some embodiments, the amount is effective to reduce excessive collagen deposition, for example, by mesenchymal stromal cells. In some embodiments, the inhibitor is effective in reducing the number of CD41-positive cells, such as megakaryocytes, in treated subjects compared to control subjects who do not receive treatment. In some embodiments, baseline frequencies of megakaryocytes in bone marrow PMF may range from 200 to 700 cells per square millimeter (mm ² ) and from 40 to 300 megakaryocytes per square millimeter (mm ² ) in spleen PMF, as determined using randomly selected slices. In contrast, the frequency of megakaryocytes in the bone marrow and spleen of normal donors is less than 140 and less than 10, respectively. Exposure to an inhibitor may reduce the number (eg, frequency) of megakaryocytes in the bone marrow and/or

- 72 045239 spleen. In some embodiments, exposure to an inhibitor may cause decreased levels of downstream effector signaling, such as phosphorylation

SMAD2/3.

Patients with myelofibrosis may suffer from an enlarged spleen. Thus, the clinical effects of a therapeutic agent can be assessed by monitoring changes in spleen size. The size of the spleen can be examined by known methods, such as assessing the length of the spleen by palpation and/or assessing the volume of the spleen using ultrasound. In some embodiments, a subject being treated with an isoform-specific, context-permissive TGFe1 inhibitor has a baseline (pre-treatment) spleen length of 5 cm or more, e.g., in the range of 5 to 30 cm, as assessed by palpation . In some embodiments, a subject to be treated with an isoform-specific, context-permissive TGFe1 inhibitor has a baseline (pre-treatment) spleen volume of 300 mL or greater, for example, in the range of 300-1500 mL, as assessed by ultrasound. Treatment with an inhibitor described herein, for example, for 4-12 cycles of administration (for example, 2, 4, 6, 8, 10, 12 cycles), for example, every 4 weeks, at a dose of 0.1-30 mg/kg monoclonal antibodies) may reduce the size of the subject's spleen. In some embodiments, the effective amount of the inhibitor is sufficient to reduce the size of the spleen in a population of patients receiving treatment with the inhibitor by at least 10%, 20%, 30%, 35%, 40%, 50%, and 60%, relative to the corresponding baseline. values. For example, treatment is effective in achieving a reduction in spleen volume of >35% from baseline after 12 to 24 weeks, as measured by MRI or CT scan, compared with a placebo control. In some embodiments, the treatment is effective in achieving a 35% reduction in spleen volume from baseline after 24 to 48 weeks, as measured by MRI or CT scan, compared with the best available theoretical control. Best available therapy may include hydroxyurea, glucocorticoids, and medications, anagrelide, epoetin alfa, thalidomide, lenalidomide, mercaptopurine, thioguanine, danazol, peginterferon alfa-2a, interferon-α, melphalan, acetylsalicylic acid, and acetylsalicylic acid.

In some embodiments, a population of patients treated with an isoform-specific, context-permissive TGFe1 inhibitor, such as those described herein, demonstrates a statistically improved response to treatment, as assessed, for example, by the International Working Group on Myelofibrosis Research and Treatment (IWG) criteria -MRT), degree of change in the degree of bone marrow fibrosis measured by the modified Bauermeister score and the global European scoring system after treatment (eg, 4, 6, 8 or 12 cycles), symptom response using the Myeloproliferative Neoplasm Symptom Assessment Form (MPN- SAF).

In some embodiments, treatment with an isoform-specific, context-permissive TGFe1 inhibitor, such as that described herein, achieves a statistically improved response to treatment, as assessed, for example, by a modified Myelofibrosis Symptom Assessment Form (MFSAF), in which symptoms measured using the MFSAF tool (eg v2.0), a diary documenting debilitating symptoms of myelofibrosis (abdominal discomfort, early satiety, pain under left ribs, itching, night sweats and bone/muscle pain) using a 0 to 10 scale , where 0 is absent and 10 is the worst possible value. In some embodiments, treatment is effective in achieving a 50% reduction in total MFSAF score compared to baseline, for example, after 12-24 weeks. In some embodiments, a significant proportion of patients receiving therapy achieve >50% of their total symptom score compared to patients receiving placebo. For example, the proportion of the patient pool to achieve >50% improvement may be greater than 40%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%.

In some embodiments, a therapeutically effective amount of an inhibitor is an amount sufficient to achieve clinical improvement as measured by anemia response. For example, improved anemia response may include longer periods of transfusion independence, for example, 8 weeks or more, after treatment with 4-12 cycles, for example, 6 cycles.

In some embodiments, a therapeutically effective amount of an inhibitor is an amount sufficient to maintain stable disease for a period of time, for example, 6 weeks, 8 weeks, 12 weeks, six months, etc. In some embodiments, disease progression can be assessed by changes in overall bone marrow cellularity, degree of reticulin or collagen fibrosis, and/or changes in JAK2V617F allele load.

In some embodiments, a patient population that is treated with an isoform-specific, context-permissive TGFe1 inhibitor, such as those described herein, exhibits statistically improved survival by

- 73 045239 compared with a control group that does not receive treatment. For example, in control groups, the median survival of patients with PMF is approximately six years (approximately 16 months in high-risk patients), and less than 20% of patients are expected to survive 10 years or longer after diagnosis. Treatment with an isoform-specific, context-permissive TGFe1 inhibitor, such as those described herein, can prolong survival time by at least 6 months, 12 months, 18 months, 24 months, 30 months, 36 months, or 48 months. In some embodiments, the treatment is effective in achieving improved overall survival at 26 weeks, 52 weeks, 78 weeks, 104 weeks, 130 weeks, 144 weeks, or 156 weeks compared to patients who receive placebo.

Clinical benefits of therapy, such as those exemplified above, may be seen in patients with or without new-onset anemia.

One of the advantageous features of isoform-specific, context-permissive TGFe1 inhibitors is that they maintain improved safety profiles due to isoform selectivity compared to conventional TGFe antagonists that are not selective. It is therefore expected that treatment with an isoform-specific, context-permissive inhibitor, such as those described herein, may reduce adverse events in patient populations compared to equivalent patient populations treated with conventional TGFe antagonists, in terms of incidence and/or or the severity of such events. Thus, isoform-specific, context-permissive TGFe1 inhibitors may provide a wider therapeutic window in terms of dosage and/or duration of treatment.

Adverse events can be assessed using appropriate methods recognized in the art, such as the Common Terminology Criteria for Adverse Events (CTCAE), version 4. Previously reported adverse events in people treated with TGFe antagonists, such as GC1008, include: leukocytosis (grade 3), fatigue (grade 3), hypoxia (grade 3), asystole (grade 5), leukopenia (grade 1), recurrent, transient, painful erythematous, nodular skin lesions, purulent dermatitis and herpes zoster.

Isoform-specific, context-permissive TGFe1 inhibitor therapy may cause less frequent and/or less severe adverse events (side effects) compared to JAK inhibitor therapy in patients with myelofibrosis, for example, anemia, thrombocytopenia, neutropenia, hypercholesterolemia, elevated alanine transaminase (ALT), elevated aspartate transaminase (AST), bruising, dizziness and headache, thus offering a safer treatment option.

It is proposed that inhibitors of TGFe1 signaling may be used in combination with one or more therapeutic agents for the treatment of myelofibrosis as a combination therapy. In some embodiments, a TGFe1 activation inhibitor described herein is administered to patients suffering from myelofibrosis who have received a JAK1 inhibitor, a JAK2 inhibitor, or a JAK1/JAK2 inhibitor. In some embodiments, such patients respond to therapy with a JAK1 inhibitor, JAK2 inhibitor, or JAK1/JAK2 inhibitor, while in other embodiments, such patients respond poorly or do not respond to therapy with a JAK1 inhibitor, JAK2 inhibitor, or JAK1/JAK2 inhibitor. In some embodiments, use of an isoform-specific TGFe1 inhibitor described herein may render those who respond poorly or do not respond to JAK1 inhibitor therapy, JAK2 inhibitor therapy, or JAK1/JAK2 inhibitor therapy to greater sensitivity. In some embodiments, use of an isoform-specific TGFe1 inhibitor described herein may allow a dosage reduction of a JAK1 inhibitor, a JAK2 inhibitor, or a JAK1/JAK2 inhibitor that still produces equivalent clinical efficacy in patients but exhibits less drug-related toxicity. remedy, or adverse events (such as those listed above). In some embodiments, treatment with a TGFe1 activation inhibitor described herein, used in combination with JAK1 inhibitor, JAK2 inhibitor, or JAK1/JAK2 inhibitor therapy, can produce synergistic or additive therapeutic effects in patients. In some embodiments, treatment with a TGFe1 activation inhibitor described herein may enhance the benefits of a JAK1 inhibitor, a JAK2 inhibitor, or a JAK1/JAK2 inhibitor or other therapy used to treat myelofibrosis. In some embodiments, patients may additionally receive a therapeutic agent to treat anemia associated with myelofibrosis.

Cancer.

Various types of cancer involve TGFe1 activity and may be treated with the antibodies and/or compositions of the present invention. As used herein, the term cancer refers to any of various malignant neoplasms characterized by

- 74 045239 fermentation of anaplastic cells, which tend to penetrate the surrounding tissue and metastasize to new areas of the body, and also refers to the pathological condition characterized by such malignant neoplasms. Cancer can be localized (eg, solid tumors) or systemic. As used herein, the term localized (as in a localized tumor) refers to an anatomically isolated or discrete lesion such as a solid malignancy, as opposed to a systemic disease. Some cancers, such as leukemia (eg, myelofibrosis) and multiple myeloma, for example, may have both a localized component (eg, bone marrow) and a systemic component (eg, circulating blood cells) to the disease. In some embodiments, the cancer may be systemic, such as hematologic malignancies. Cancers that can be treated in accordance with the present invention include, without limitation, all types of lymphomas/leukemias, carcinomas and sarcomas, such as cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast gland, cervix, colon/rectum, endometrium, esophagus, eye, gall bladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, tailbone, testicles, thyroid gland and uterus. In cancer, TGFe (eg, TGFe1) can be either growth promoting or growth inhibiting. For example, in pancreatic cancer, wild-type SMAD4 tumors may experience inhibited growth in response to TGF, but as the disease progresses, a constitutively activated type II receptor is typically present. In addition, there are SMAD4-null pancreatic cancers. In some embodiments, the antibodies, antigen binding portions thereof, and/or compositions of the present invention are designed to selectively target components of the TGF signaling pathways that uniquely function in one or more forms of cancer. Leukemia or cancer of the blood or bone marrow, which is characterized by abnormal proliferation of white blood cells, i.e. white blood cells can be divided into four main categories, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia, or acute myeloid leukemia (AML) (AML with translocations between chromosomes 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 with preexisting myelodysplastic syndrome (MDS) or a myeloproliferative disorder that transforms into AML; therapy-related AML and myelodysplastic syndrome (MDS), defined as patients who have previously received chemotherapy and/or radiation and subsequently develop AML or MDS d) AML not elsewhere classified, which includes subtypes of AML that do not fall into the above categories; and f) acute leukemias of ambiguous origin, which occur when leukemic cells cannot be classified as myeloid or lymphoid cells or when both cell types are present); and chronic myelogenous leukemia (CML).

Isoform-specific, context-permissive TGFe1 inhibitors, such as those described herein, can be used to treat multiple myeloma. Multiple myeloma is a cancer of B lymphocytes (eg, plasma cells, plasmablasts, memory B cells) that develops and expands in the bone marrow, causing destructive bone lesions (ie, osteolytic injury). Typically, the disease exhibits increased osteoclastic bone resorption, suppressed osteoblastic differentiation (eg, delayed differentiation), and impaired bone formation characterized by partially osteolytic lesions, osteopenia, osteoporosis, hypercalcemia, as well as plasmacytoma, thrombocytopenia, neutropenia, and neuropathy. The TGFe1-selective, context-permissive inhibitor therapy described herein may be effective in reducing one or more of these clinical manifestations or symptoms in patients. The TGFe1 inhibitor can be administered to patients who are receiving additional therapy or therapy for the treatment of multiple myeloma, including those listed elsewhere herein. In some embodiments, multiple myeloma can be treated with a TGFe1 inhibitor (such as an isoform-specific, context-permissive inhibitor) in combination with a myostatin inhibitor or an IL-6 inhibitor. In some embodiments, a TGFe1 inhibitor may be used in combination with conventional treatments for multiple myeloma, such as bortezomib, lenalidomide, carfilzomib, pomalidomide, thalidomide, doxorubicin, corticosteroids (e.g., dexamethasone and prednisone), chemotherapy (e.g., melphalan), radiation therapy, stem cell transplantation, plidepsin, elotuzumab, ixazomib, masitinib and/or panobinostat.

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

Types of sarcoma include, but are not limited to, soft tissue sarcoma such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosaroma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyoma sarcoma, 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.

Isoform-selective, context-permissive/context-independent inhibitors of TGFe1 activation, such as those described herein, may be useful for the treatment of malignancies involving cells characterized by neural crest origin. Neural crest cell line cancers (i.e., tumors derived from the neural crest) include, but are not limited to: melanoma (cancer of the melanocytes), neuroblastoma (cancer of the sympathoadrenal system precursors), ganglioneuroma (cancer of the ganglia of the peripheral nervous system), medullary thyroid carcinoma glands (thyroid cell cancer), pheochromocytoma (adrenal medulla chromaffin cell cancer), and MPNST (Schwann cell cancer). In some embodiments, the antibodies and methods of the present invention can be used to treat one or more types of cancer or cancer-related conditions, which may include, but are not limited to, colon cancer, kidney cancer, breast cancer, malignant melanoma, and glioblastomas. (Schlingensiepen et al., 2008; Ouhtit et al., 2013).

Increasing evidence points to the role of macrophages in tumor/cancer progression. The present invention embraces the idea that this is mediated in part by activation of TGFe1 in a disease environment such as the TME. Bone marrow-derived monocytes (eg, CD11b+) are recruited to tumor sites in response to tumor-derived cytokines/chemokines, where the monocytes undergo differentiation and polarization to acquire a pro-tumor phenotype (eg, M2-biased, TAM, or TAM-like cells ). As shown in the examples presented herein, monocytes isolated from human PBMCs can be induced to polarize into various macrophage subtypes, for example, M1 (profibrotic, anticancer) and M2 (procancer). The majority of TAMs in many tumors are M2-biased. Among M2-like macrophages, the M2c and M2d subtypes, but not M1, were found to express increased LRRC33 on the cell surface. In addition, macrophages may be further distorted or activated by M-CSF exposure, resulting in a marked increase in LRRC33 expression, which coincides with TGFe1 expression. An increase in circulating M-CSF (ie, serum MCSF concentration) has also been observed in patients with a myeloproliferative disorder (eg, myelofibrosis). In general, tumors with high macrophage content (TAM) and/or MDSC are associated with poor prognosis. Likewise, elevated M-CSF levels also indicate poor prognosis.

As stated above, context-permissive/context-independent inhibitors of TGFe1 activation can be used in the treatment of melanoma. Types of melanoma that can be treated with such inhibitors include, but are not limited to: lentigo maligna; melanoma type lentigo maligna; superficial spreading melanoma; acral lentiginous melanoma; mucosal melanoma; nodular melanoma; polypoid melanoma and desmoplastic melanoma. In some embodiments, the melanoma is metastatic melanoma.

Recently, immune checkpoint inhibitors have been used to effectively treat patients with advanced melanoma. In particular, antibodies against programmed death protein (PD)-1 (eg, nivolumab and pembrolizumab) have now become standard of care for certain cancers, such as advanced melanoma, which have demonstrated significant activity and durable responses with a manageable toxicity profile. However, effective clinical use of PD-1 antagonists is hampered by high levels of innate resistance (~60-70%) (see Hugo et al. (2016) Cell 165: 35-44), illustrating that ongoing challenges continue to include patient selection issues and response and resistance rates, and optimization of combination strategies (Perrot et al. (2013) Ann Dermatol 25(2): 135-144). Moreover, studies have shown that approximately 25% of melanoma patients who initially respond to anti-PD therapy eventually develop acquired resistance (Ribas et al. (2016) JAMA 315: 1600-9).

The number of tumor infiltrating CD8+ T cells expressing PD-1 and/or CTLA-4 appears to be a key indicator of success with checkpoint inhibition, and blockade of both PD-1 and CTLA-4 can increase infiltrating T cells. However, in patients with higher macrophage infiltration, the anticancer effects of CD8 cells may be suppressed.

It is proposed that LRRC33-expressing cells, such as myeloid cells, including

- 76 045239 themselves myeloid progenitors, MDSC and TAM, can create or maintain an immunosuppressive environment (such as TME and myelofibrotic bone marrow) by inhibiting T cells (eg, T cell depletion), such as CD4 and/or CD8 T cells , which may, at least in part, underscore the observed anti-PD-1 resistance in certain patient populations. Indeed, evidence suggests that resistance to anti-PD-1 monotherapy was marked by failure to accumulate cytotoxic CD8+ T cells and a reduced Teff/Treg ratio. Notably, the present inventors recognized that there is a certain bifurcation among some cancer patients, such as the melanoma patient population, with respect to LRRC33 expression levels: one group exhibits high expression of LRRC33 (LRRC33 ^high ), while another group exhibits relatively low expression LRRC33 (LRRC33 ^low ). Thus, the present invention includes the concept that the LRRC33 ^high patient population may represent those who respond poorly to immune therapy or are resistant to checkpoint inhibitor therapy. Accordingly, agents that inhibit LRRC33, such as those described herein, may be particularly useful for the treatment of cancers such as melanoma, lymphoma and myeloproliferative disorders that are refractory to checkpoint inhibitor therapy (eg, anti-PD-1).

In some embodiments, the cancer/tumor is inherently resistant or unresponsive to an immune checkpoint inhibitor. To give just one example, some lymphomas appear to respond poorly to immune checkpoint inhibition such as anti-PD-I therapy. Likewise, a subset of melanoma patients are known to exhibit resistance to immune checkpoint inhibitors. Without intending to be limited by theory, the present disclosure suggests that this may be, at least in part, due to activation of TGFe1 signaling pathways, which may create an immunosuppressive microenvironment in which checkpoint inhibitors are unable to exert their effects. Inhibiting TGFe1 may make such cancers more sensitive to checkpoint inhibitor therapy. Non-limiting examples of cancer types that may benefit from the combination of an immune checkpoint inhibitor and a TGFe1 inhibitor include: myelofibrosis, melanoma, renal cell cancer, bladder cancer, colon cancer, hematologic malignancies, non-small cell carcinoma, non-small cell lung cancer ( NSCLC), lymphoma (classical Hodgkin and non-Hodgkin), head and neck cancer, urothelial cancer, cancer with high microsatellite instability, cancer with mismatch repair deficiency, gastric cancer, kidney cancer and hepatocellular cancer. However, any cancer (eg, patients with such cancer) in which TGFe1 is overexpressed or is a dominant isoform compared to TGFe2/3, as determined, for example, by biopsy, can be treated with an isoform-selective TGFe1 inhibitor in accordance with the present disclosure.

In some embodiments, the cancer/tumor becomes resistant over time. This phenomenon is called acquired resistance or adaptive resistance. Like intrinsic resistance, in some embodiments acquired resistance is at least partially mediated by TGFβ1-dependent pathways. The isoform-specific TGFe1 inhibitors described herein may be effective in restoring anticancer immunity in these cases.

In some embodiments, a combination therapy comprising an immune checkpoint inhibitor and an LRRC33 inhibitor (such as those described herein) may be effective for treating such cancer. Additionally, a high LRRC33-positive cellular infiltrate in tumors or other sites/tissues with abnormal cell proliferation may serve as a biomarker for host immunosuppression and immune checkpoint resistance. Likewise, effector T cells may be excluded from the immunosuppressive niche that limits the body's ability to fight cancer. Moreover, as shown in the Examples section below, Tregs that express TGFe1 presented by GARP suppress the proliferation of effector T cells. Taken together, TGFe1 is likely a key factor in the production and maintenance of an immunoinhibitory disease microenvironment (such as the TME), and multiple contexts of TGFe1 presentation are important in tumors. In some embodiments, combination therapy can achieve more favorable Teff/Treg ratios.

In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33-TGFe1 complex, as described herein, can be used in methods of treating cancer in a subject in need thereof, the method comprising administering the antibody or an antigen-binding portion thereof to the subject such that the cancer is treated. In certain embodiments, the cancer is colon cancer.

In some embodiments, antibodies or antigen-binding portions thereof that

- 77 045239 These specifically bind to the GARP-TGFei complex, LTBPl-TGFei complex, LTBP3-TGFei complex and/or LRRC33-TGFei complex, as described herein, can be used in methods of treating solid tumors. In some embodiments, solid tumors may be desmoplastic tumors, which are typically dense and difficult for therapeutic molecules to penetrate. By targeting the ECM component of such tumors, such antibodies can weaken dense tumor tissue for disintegration, facilitating therapeutic access to exert its anticancer effects. Thus, additional therapeutic agents, such as any known anticancer drugs, can be used in combination.

Additionally or alternatively, isoform-specific, context-permissive antibodies or fragments thereof that are capable of inhibiting TGFe1 activation, such as those described herein, can be used in combination with chimeric antigen receptor T cell (CAR-T) technology ) as cellular immunotherapy, such as cancer immunotherapy to fight cancer.

In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33-TGFe1 complex, as described herein, can be used in methods of inhibiting or reducing growth a solid tumor in a subject characterized by the presence of a solid tumor, the method comprising administering an antibody or an antigen-binding portion thereof to the subject such that the growth of the solid tumor is inhibited or reduced. In certain embodiments, the solid tumor is colon carcinoma. In some embodiments, the antibodies or antigen-binding portions thereof used to treat cancer are an isoform-specific, context-permissive inhibitor of TGFe1 activation. In some embodiments, such antibodies target the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and the LRRC33-TGFe1 complex. In some embodiments, such antibodies target the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, and the LTBP3-TGFe1 complex. In some embodiments, such antibodies target the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and the LRRC33-TGFe1 complex. In some embodiments, such antibodies target the GARP-TGFe1 complex and the LRRC33-TGFe1 complex.

The present invention includes the use of context-permissive (context-independent) isoform-specific TGFe1 inhibitors in the treatment of cancer containing a solid tumor in a subject. In some embodiments, such a context-permissive (context-independent), isoform-specific inhibitor can inhibit TGFe1 activation. In preferred embodiments, such an activation inhibitor is an antibody or an antigen-binding portion thereof that binds to the pro-TGFβ1 complex. Binding can occur when the complex associates with any of the presentation molecules, such as LTBP1, LTBP3, GARP or LRRC33, thereby inhibiting the release of the mature growth factor TGFe1 from the complex. In some embodiments, a solid tumor is characterized by having stroma enriched in CD8+ T cells that come into direct contact with CAFs and collagen fibers. Such a tumor may create an immunosuppressive environment that prevents antitumor immune cells (eg, effector T cells) from effectively infiltrating the tumor, limiting the body's ability to fight cancer. Instead, such cells may accumulate within or near the tumor stroma. These features may make such tumors poorly responsive to immune checkpoint inhibitor therapy. As discussed in more detail below, TGFe1 inhibitors disclosed herein can unblock suppression to allow effector cells to reach and kill cancer cells, for example, when used in combination with an immune checkpoint inhibitor.

TGFe1 is proposed to play multifaceted roles in the tumor microenvironment, including tumor growth, host immune suppression, malignant cell proliferation, vascularization, angiogenesis, migration, invasion, metastasis, and chemotherapy resistance. Therefore, each environmental context of TGFe1 presentation may be involved in the regulation (or dysregulation) of disease progression. For example, the GARP axis is particularly important in the Treg response, which regulates effector T cell responses to mediate the host immune response to fight cancer cells. The LTBP1/3 axis may regulate the ECM, including the stroma, where cancer-associated fibroblasts (CAFs) play a role in cancer pathogenesis and progression. The LRRC33 axis may play a critical role in the recruitment of circulating monocytes to the tumor microenvironment, subsequent differentiation into tumor-associated macrophages (TAMs), infiltration into tumor tissue, and disease exacerbation.

In some embodiments, TGFe1-expressing cells infiltrate the tumor

- 78 045239 hol, creating an immunosuppressive local environment. The extent to which such infiltration occurs may correlate with a worse prognosis. In some embodiments, higher infiltration indicates a poorer response to treatment with other cancer therapies, such as immune checkpoint inhibitors. In some embodiments, TGFe1-expressing cells in the tumor microenvironment contain Tregs and/or myeloid cells. In some embodiments, myeloid cells include, but are not limited to: macrophages, monocytes (tissue resident or bone marrow derived), and MDSC.

In some embodiments, the LRRC33-expressing cells in the TME are myeloid-derived suppressor cells (MDSCs). MDSC infiltration (eg, solid tumor infiltration) may highlight at least one mechanism of immune evasion by creating an immunosuppressive niche from which host antitumor immune cells are excluded. Evidence suggests that MDSCs are mobilized by inflammation-related signals such as tumor-associated inflammatory factors, Orop mobilization, MDSCs may influence immunosuppressive effects by damaging disease-fighting cells such as CD8+ T cells and NK cells. In addition, MDSCs can induce Treg differentiation by secreting TGFe and IL-10. Thus, an isoform-specific, context-permissive TGFe1 inhibitor, such as those described herein, can be administered to patients with immune evasion (eg, impaired immunosurveillance) to restore or enhance the body's ability to fight a disease (such as tumor). As described in more detail herein, this may further enhance (eg, restore or activate) the body's sensitivity or sensitivity to other therapy, such as cancer therapy.

In some embodiments, increased frequency (e.g., number) of circulating MDSCs in patients is predictive of poor response to checkpoint blockade therapy, such as PD-1 antagonists and PD-L1 antagonists. For example, biomarker studies showed that pre-treatment circulating HLA-DR lo/CD14+/CD11b+ myeloid-derived suppressor cells (MDSCs) were associated with progression and worsening OS (p = 0.0001 and 0.0009). Additionally, resistance to PD-1 checkpoint blockade in inflammatory head and neck carcinoma (HNC) is associated with the expression of GM-CSF and myeloid-derived suppressor cell (MDSC) markers. This observation suggested that MDSC depletion strategies such as chemotherapy should be considered in combination or sequentially with anti-PD-1. LRRC33 or LRRC33-TGFe complexes represent a novel target for cancer immunotherapy due to selective expression on immunosuppressive myeloid cells. Therefore, without intending to be limited by any particular theory, targeting this complex may improve the effectiveness of standard care checkpoint inhibitor therapy in patient populations.

Thus, the present invention provides the use of an isoform-specific, context-permissive or context-independent TGFe1 inhibitor described herein for the treatment of cancer that contains a solid tumor. Such treatment involves administering an isoform-specific, context-permissive, or context-independent TGFe1 inhibitor to a subject diagnosed with cancer that includes at least one localized tumor (solid tumor) in an amount effective to treat the cancer.

Evidence suggests that cancer progression (eg, tumor proliferation/growth, invasion, angiogenesis, and metastasis) may be, at least in part, driven by tumor-stromal interactions. In particular, CAFs may contribute to this process by secreting various cytokines and growth factors and remodeling the ECM. Factors involved in the process include, but are not limited to, stromal cell-derived factor 1 (SCD-1), MMP2, MMP9, MMP3, MMP-13, TNF-α, TGFe1, VEGF, IL-6, M-CSF . In addition, CAFs can recruit TAMs by secreting factors such as CCL2/MCP-1 and SDF-1/CXCL12 at tumor sites; subsequently, a pro-TAM niche is created (e.g., hyaluronan-enriched stromal regions) where TAMs preferentially attach. Since TGFe1 has been proposed to promote activation of normal fibroblasts into myofibroblast-like CAFs, administration of an isoform-specific, context-permissive, or context-independent TGFe1 inhibitor, such as those described herein, may be effective in counteracting cancer-promoting CAF activation. Indeed, the data presented herein suggest that an isoform-specific, context-independent antibody that blocks TGFe1 activation can inhibit UUO-induced activation of producer genes such as CCL2/MCP-1, α-SMA, FN1, and Col1, which are also involved in many types of cancer.

In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33-TGFe1 complex, as described herein, are administered to a subject

- 79 045239 suffering from cancer or tumor, alone or in combination with an additional agent, for example, an antibody to PD-1 (for example, an antagonist to PD-1). Other combination therapies that are included in the present invention are the administration of an antibody or antigen-binding portion thereof as described herein with the help of radiation or a chemotherapeutic agent. Exemplary additional agents include, but are not limited to, 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 TIM3, anti-LAG3, anti-OX40, anti-CD27, anti-CD70, anti-CD47, anti-41BB, anti-PD-1, anti-CD20, oncolytic virus and PARP inhibitor.

In some embodiments, determining or selecting a therapeutic approach for combination therapy that is appropriate for certain cancer types or patient populations may include the following: a) considerations regarding cancer types for which standard therapy is available (eg, approved immunotherapy indications); b) considerations regarding treatment-resistant subpopulations; and c) considerations regarding cancers/tumors that are active in the TGFe1 pathway or otherwise at least partially dependent on TGFe1 (eg, sensitive to TGFe1 inhibition). For example, many cancer samples show that TGFe1 is the predominant isoform, such as TCGA RNAseq analysis. In some embodiments, more than 50% (eg, more than 50%, 60%, 70%, 80%, and 90%) of the samples from each tumor type are positive for TGFe1 isoform expression. In some embodiments, cancers/tumors that are active in the TGFe1 pathway or otherwise at least partially dependent on TGFe1 (e.g., sensitive to TGFe1 inhibition) contain at least one Ras mutation, such as mutations in K -ras, N-ras and/or H-ras. In some embodiments, the cancer/tumor contains at least one K-ras mutation.

In some embodiments, an isoform-specific, context-permissive TGFe1 inhibitor is administered in combination with a checkpoint inhibitor therapy to patients diagnosed with cancer for which one or more checkpoint inhibitor therapies are approved. These include, but are not limited to: urothelial cancer of the bladder, squamous cell carcinoma (such as head and neck), clear cell carcinoma of the kidney, papillary cell carcinoma of the kidney, hepatocellular carcinoma of the liver, adenocarcinoma of the lung, cutaneous melanoma and adenocarcinoma of the stomach. In preferred embodiments, such patients respond poorly or do not respond to checkpoint inhibitor therapy.

The role of TGFe in musculoskeletal conditions.

In the musculoskeletal system, which consists of skeletal bones, muscles, cartilage, tendons, ligaments, joints and other connective tissues that support and bind tissues and organs together, TGFe plays various roles, including inhibition of proliferation and differentiation, induction of atrophy and the development of fibrosis. TGFe reduces satellite cell proliferation and prevents differentiation (via inhibition of MyoD and myogenin) (Allen, R.E. and L.K. J Cell Physiol, 1987. 133(3): p. 567-72; Brennan, T.J., et al., Proc Natl Acad Sci USA, 1991. 88(9): p. 3822-6; Massague, J., et al., Proc Natl Acad Sci USA, 1986. 83(21): p. 8206-10; Olson, E. N., et al. , J Cell Biol, 1986. 103(5): p. 1799805). The TGFe isoform (i.e., TGFe1, 2, or 3) was not specified in these early papers but is assumed to be TGFe1. TGFe also promotes muscle fibrosis; direct injection of recombinant TGFe1 results in skeletal muscle fibrosis, and inhibition of pan-TGFe reduces fibrosis in acute and chronic muscle injury (Li, Y., et al., Am J Pathol, 2004. 164(3): p. 1007-19; Mendias, C.L., et al., Muscle Nerve, 2012. 45(1): p. 55-9; Nelson, C. A., et al., Am J Pathol, 2011. 178(6): p. 261121). TGFe1 is expressed by muscle fibers, macrophages, regulatory T cells, fibroblasts and fibrocytes in skeletal muscle (Li, Y., et al., Am J Pathol, 2004. 164(3): p. 1007-19; Lemos, D.R., et al., Nat Med, 2015. 21(7): p. 786-94; Villalta, S. A., et al., Sci Transl Med, 2014. 6(258): p. 258ral42; Wang, X., et al. , J Immunol, 2016. 197(12): p. 4750-4761); and expression increases with injury and disease (Li, Y., et al., Am J Pathol, 2004. 164(3): p. 1007-19; Nelson, C.A., et al., Am J Pathol, 2011. 178( 6): p. 2611-21; Bernasconi, P., et al., J Clin Invest, 1995. 96(2): p. 1137-44; Ishitobi, M., et al., Neuroreport, 2000. 11( 18): p. 4033-5). TGFe2 and TGFe3 are also upregulated (at the mRNA level) in mdx muscle, although to a lesser extent than TGFe1 (Nelson, C.A., et al., Am J Pathol, 2011. 178(6): p. 2611-21; Zhou, L., et al., Neuromuscul Disord, 2006. 16(1): pp. 32-8). Pessina et al. recently used cell lineage tracing experiments to show that cells of multiple origins in dystrophic muscle adopt a fibrogenic fate through a TGFe-dependent pathway (Pessina, P., et al., Stem Cell Reports, 2015. 4(6): p. 1046 -60).

Bone is the largest storage site for TGFe in the body. Indeed, the TGFe pathway is thought to play an important role in bone homeostasis and remodeling, at least in part by regulating osteoblast differentiation and/or osteoclast resorption. This process is involved in both

- 80 045239 normal, as well as in abnormal situations which, if unregulated, can cause or aggravate disease, such as bone-related conditions and cancer. Thus, TGFe1-selective inhibitors, such as those described herein, can be used to treat such conditions. In some embodiments, administration of such inhibitors is effective in restoring or normalizing bone formation-resorption balance. In some embodiments, the TGFe1 inhibitor is administered to subjects in combination with another therapy, such as a myostatin inhibitor and/or bone enhancing agents as a combination therapy.

Bone conditions (eg, skeletal diseases) include osteoporosis, dysplasia, and bone cancer. In addition to primary bone cancer, which originates in the bone, many malignancies are known to metastasize to the bone; these include, but are not limited to, breast cancer, lung cancer (eg, squamous cell carcinoma), thyroid cancer, testicular cancer, renal cell cancer, prostate cancer, and multiple myeloma.

In some embodiments, such conditions are associated with muscle weakness.

TGFe1 may play a role in fibrotic conditions that accompany chronic inflammation of diseased tissue, such as human muscular dystrophies. Duchenne muscular dystrophy (DMD) is a severe, progressive and ultimately fatal disease caused by a lack of dystrophin (Bushby, K., et al., Lancet Neurol, 2010. 9(1): p. 77-93). Dystrophin deficiency results in increased susceptibility to contraction-induced injury, resulting in permanent muscle degeneration (Petrof, B.J., et al., Proc Natl Acad Sci USA, 1993. 90(8): p. 3710-4; Dellorusso, C, et al., J Muscle Res Cell Motil, 2001. 22(5): p. 467-75; Pratt, S. J., et al., Cell Mol Life Sci, 2015. 72(1): p. 153-64). Repeated cycles of repair contribute to chronic inflammation, fibrosis, depletion of the satellite cell pool, eventual loss of motility and death (Bushby, K., et al., Lancet Neurol, 2010. 9(1): p. 77-93; McDonald, S.M. ., et al., Muscle Nerve, 2013. 48(3): pp. 343-56). TGFe1 expression is significantly increased in patients with DMD and correlates with the degree of fibrosis observed in these patients (Bernasconi, P., et al., J Clin Invest, 1995. 96(2): p. 1137-44; Chen, Y.W., et al., Neurology, 2005. 65(6): p. 826-34). Excessive ECM deposition has a detrimental effect on muscle contractile properties and can limit access to nutrition as myofibers are isolated from their blood supply (Klingler, W., et al., Acta Myol, 2012. 31(3): p. 184-95). Recently, additional evidence further implicates the presence of TGFe1 in muscular dystrophy. Variants in LTBP4 have been found to modify disease severity in mice and humans. In mice, the LTBP4 variant is protective in those mice lacking dystrophin or γ-sarcoglycan (Coley, W.D., et al., Hum Mol Genet, 2016. 25(1): p. 130-45; Heydemann, A., et al. al., J Clin Invest, 2009. 119(12): p. 3703-12). In humans, two groups independently identified the LTBP4 variant as protective in DMD, delaying loss of ambulation for several years (Flanigan, K.M., et al., Ann Neurol, 2013. 73(4): p. 481-8; van den Bergen, J.C., et al., J Neurol Neurosurg Psychiatry, 2015. 86(10): p. 1060-5). Although the nature of the genetic variants differs between mice and humans, in both species a protective variant results in decreased TGFe signaling (Heydemann, A., et al., J Clin Invest, 2009. 119(12): p. 3703-12); Ceco, E., et al., Sci Transl Med, 2014. 6(259): p. 259ra144). Many functions of TGFe1 in skeletal muscle biology have been inferred from experiments in which purified active growth factor was administered to animals or added to cells in culture (Massague, J., et al., Proc Natl Acad Sci USA, 1986. 83(21): p 8206-10; Li, Y., et al., Am J Pathol, 2004. 164(3): p. 1007-19; Mendias, C. L., et al., Muscle Nerve, 2012. 45(1): p 55-9). Given the importance of cellular context for specific TGFe1 functions (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 an endogenous role ( i) cytokine in vivo. For example, exposure of human dermal fibroblasts to recombinant TGFe1, myostatin, or GDF11 results in almost identical changes in gene expression in these cells, although the roles of these proteins in vivo 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).

Numerous investigators have used TGF inhibitors to elucidate the role of the growth factor in vivo. Treatment of mdx mice with the pan-TGFe neutralizing antibody 1D11 clearly results in decreased fibrosis (via histology and hydroxyproline content), reduced muscle damage (decreased serum creatine kinase and greater myofibril density), and improved muscle function (via plethysmography, force generation of isolated EDL muscles and increased forelimb grip strength) (Nelson, S.A., et al., Am J Pathol, 2011. 178(6): p. 2611-21; Andreetta, F., et al., J Neuroimmunol, 2006. 175(1-2): pp. 77-86; Gumucio, J.P., et al., J Appl Physiol (1985), 2013. 115(4): pp. 539-45). Additionally, muscle fiber-specific expression of dominant negative TGFe receptor type II protects against muscle damage following cardiotoxin injury and in 5sarcoglycan-/- mice (Accornero, F., et al., Hum Mol Genet, 2014. 23(25) : p. 6903-15). The proteoglycan decorin, which is abundant in skeletal muscle and inhibits TGFe activity, reduces

- 81 045239 cervical fibrosis in mdx mice and after laceration injury (Li, Y., et al., Mol Ther, 2007. 15(9): p. 1616-22; Gosselin, L.E., et al., Muscle Nerve, 2004. 30(5): pp. 645-53). Other molecules with TGFe inhibitory activity, such as suramin (an antineoplastic agent) and losartan (an angiotensin receptor blocker), were effective in improving muscle pathology and reducing fibrosis in a mouse model of injury, Marfan syndrome and muscular dystrophy (Spurney, C.F., et al., J Cardiovasc Pharmacol Ther, 2011. 16(1): p. 87-95; Taniguti, A.P., et al., Muscle Nerve, 2011. 43(1): p. 82-7; Bedair, H.S., et al., Am J Sports Med, 2008. 36(8): p. 1548-54; Cohn, R. D., et al., Nat Med, 2007. 13(2): p. 20410). Although all of the therapeutic agents described above inhibit TGFe1 or its signaling, none are TGFe1 isoform specific. For example, 1D11 binds and inhibits TGFp1, 2 and 3 isoforms (Dasch, J.R., et al., J Immunol, 1989. 142(5): p. 1536-41). Suramin inhibits the ability of multiple growth factors to bind to their receptors, including PDGF, FGF and EGF, in addition to TGFe1 (Hosang, M., J Cell Biochem, 1985. 29(3): p. 265-73; Olivier, S ., et al., Eur J Cancer, 1990. 26(8): p. 867-71; Scher, H. I. and W. D. Heston, Cancer Treat Res, 1992. 59: p. 131-51). Decorin also inhibits myostatin activity, both through direct binding and through activation of follistatin, a myostatin inhibitor (Miura, T., et al., Biochem Biophys Res Commun, 2006. 340(2): p. 675-80; Brandan, E. , C Cabello-Verrugio, and C Vial, Matrix Biol, 2008. 27(8): p. 700-8; Zhu, J., et al., J Biol Chem, 2007. 282(35): p. 25852- 63). Losartan affects additional signaling pathways by affecting the renin-angiotensin-aldosterone system, including the IGF-1/AKT/mTOR pathway (Burks, T.N., et al., Sci Transl Med, 2011. 3(82): p. 82ra37 ; Sabharwal, R. and M.W. Chapleau, Exp Physiol, 2014. 99(4): p. 627-31; McIntyre, M., et al., Pharmacol Ther, 1997. 74(2): p. 181-94) . Therefore, all of these treatments inhibit additional molecules that may contribute to their therapeutic effect as well as toxicity.

Given the putative role of TGFe in muscle homeostasis, repair and regeneration, agents such as the monoclonal antibodies described herein that selectively modulate TGFe1 signaling may be effective in treating damaged muscle fibers such as in chronic/genetic muscular dystrophies and acute muscular dystrophies. lesions, without the toxicity associated with the more broadly acting TGFe inhibitors developed to date.

Accordingly, the present invention provides methods for treating damaged muscle fibers using an agent that advantageously modulates a subset, but not all, of the effects of TGFe in vivo. Such agents can selectively modulate TGFe1 signaling (isoform-specific modulation).

Muscle Fiber Restoration in Chronic Muscle Diseases: The present invention provides methods for improving muscle quality and function in patients with DMD by limiting fibrosis and promoting normalization of muscle morphology and function. Since TGFe1 also inhibits myogenesis, blockade of TGFe1 may promote regeneration in dystrophic muscles, adding an additional therapeutic benefit. TGFe1 inhibitors can be used in combination with dystrophin-regulating therapy such as Exondys 51 (Eteplirsen). Given the potential therapeutic benefits of TGFe1 inhibition in muscular dystrophy, it is critical to (1) differentiate the role(s) of TGFe1 from those of TGFe2 and TGFe3 and (2) clarify in which molecular context(s) TGFe1 inhibition would be most beneficial. As stated above, pan-TGFe inhibitors have been associated with significant toxicity, limiting the clinical use of these compounds (Anderton, M.J., et al., Toxicol Pathol, 2011. 39(6): p. 916-24; Stauber, A., et al., Clinical Toxicology, 2014. 4(3):

p. 1-10). It is unclear which TGFe isoform causes these toxicities. Some of the reported toxicities may be due to inhibition of TGFe1 in the immune system. For example, while 1D11 significantly reduced fibrosis levels in the diaphragm, treatment also increased the number of CD4+ and CD8+ T cells in muscle, suggesting an increased inflammatory response with pan-TGFe inhibition that may be detrimental with long-term treatment (Andreetta, F ., et al., J Neuroimmunol, 2006. 175(1-2): pp. 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): p 235-43). The increase in T cell numbers with 1D11 administration is likely due to the effect of TGFe1 on regulatory T cells (Tregs). Tregs present TGFe1 on their cell surface via GARP, and release of TGFe1 from this complex enhances Treg suppressive activity, thereby limiting T cell-mediated inflammation (Wang, R., et al., Mol Biol Cell, 2012. 23(6) ): p. 1129-39; Edwards, J. P., A. M. Thornton, and E. M. Shevach, J Immunol, 2014. 193(6): p. 2843-9; Nakamura, K., et al., J Immunol, 2004. 172 (2): p. 834-42; Nakamura, K., A. Kitani, and W. Strober, J Exp Med, 2001. 194(5): p. 629-44). Indeed, depletion of Treg using the PC61 antibody resulted in increased inflammation and muscle damage in the diaphragm of mdx mice, whereas increasing Treg number and activity reduced muscle damage.

- 82 045239 muscle movement (Villalta, S.A., et al., Sci Transl Med, 2014. 6(258): p. 258ra142). Interestingly, an additional population of immunosuppressive T cells, Tr1 cells, has recently been identified. These cells produce large amounts of TGFe3, which is necessary for their suppressive activity (Gagliani, N., et al., Nat Med, 2013. 19(6): p. 739-46; Okamura, T., et al., Proc Natl Acad Sci USA, 2009. 106(33): p. 13974-9; Okamura, T., et al., Nat Commun, 2015. 6: p. 6329). Although the role of Tr1 cells in skeletal muscle is unknown, there is a possibility that inhibition of both TGFe1 and TGFe3 by 1D11 may have additive proinflammatory effects by inhibiting Treg and Tr1 cells.

The structural insights described above regarding TGFe1 latency and activation enable new approaches to drug discovery that specifically target TGFe1 activation (Shi, M., et al., Nature, 2011. 474(7351): p. 343-9 ). The high degree of sequence identity shared by the three mature TGFe growth factors is not shared by the latent complexes, allowing the detection of antibodies that are exclusively specific to pro-TGFβ1. Using proprietary antibody discovery approaches, we have identified antibodies (Ab1, Ab2 and Ab3) that specifically bind to pro-TGFβ1 (see, for example, Fig. 4B). Using an in vitro coculture system, these antibodies were demonstrated to inhibit integrin-mediated release of TGF β1. In this system, fibroblasts derived from human skin or mouse skeletal muscle are the source of latent TGFe1, a cell line expressing ανβ6 allows the release of active TGFp1, which is then measured using a third cell line expressing a SMAD2/3-responsive luciferase reporter (Fig. 7G-7H). One of these antibodies, Ab1, was tested in vivo and showed efficacy in a mouse model of UUO (unilateral ureteral obstruction) renal fibrosis. In this model, treatment of mice (n=10) with 9 mg/kg/week Ab1 prevented the activation of TGFe1-responsive genes (Fig. 12A-12J) and reduced the degree of fibrosis after injury (by picrosirius red staining) (Fig. 12K). TGFe1-specific therapies may have improved efficacy and safety profiles compared to pan-TGFe inhibitors, a critical aspect for a therapeutic that will be used long-term as in the DMD population. TGFe1 inhibitory antibodies can be used to determine whether specific inhibition of TGFe1 has potential for the treatment of DMD or other muscle diseases and to elucidate the role of TGFe1 in skeletal muscle regeneration.

Chronic and acute muscle fiber injuries and selection of optimal therapeutic agents.

In normal but regenerating muscle following acute injury (such as traumatic injury to healthy muscle or motor neurons), the initial infiltration of inflammatory macrophages is thought to be necessary to clear the damaged tissue and release factors (eg, cytokines) necessary for satellite cell activation. These cells subsequently switch to the M2 phenotype to drive wound healing.

In contrast, in chronic conditions such as diseases including DMD, pro-inflammatory macrophages have been predominant all along, and this switch to M2 does not occur (or at least is not effective enough), and pro-inflammatory macrophages continue to drive inflammation and muscle damage. In DMD, the NFkB pathway is continuously active, leading to constitutive inflammation. Therefore, in some embodiments, an NFkB inhibitor can be administered to patients with DMD to reduce chronic inflammation.

Thus, in chronic conditions such as DMD, the therapeutic focus may be on muscle repair rather than muscle regeneration. This is because DMD muscle fibers are defective, but they are not destroyed, they are damaged by breaks in the membrane, dysregulated calcium transients, and ROS damage from macrophages. In comparison, in the case of injury to healthy muscles, the therapeutic emphasis may be on regeneration. For example, in cardiotoxin models, muscle fibers are killed and must be repaired. This models the recovery process from a traumatic injury such as a crush injury.

Data suggest that LRRC33 is expressed in thioglycollate-induced peritoneal macrophages, which are characterized by an M2-like phenotype (characterized by them expressing high levels of arginase, no iNOS, and high levels of CD206).

In situations where LRRC33 is expressed primarily on M2 cells and where its presentation to TGFe1 (context) is important for the wound healing effects of these cells, it may be useful to activate LRRC33-mediated TGFe1 to promote repair and/or myogenesis. On the other hand, in situations where LRRC33 is also expressed on pro-inflammatory Ml cells, then it may be beneficial to inhibit LRRC33-mediated TGFe1, given that inflammation promotes fibrosis, especially in dystrophic conditions such as DMD. Thus, identifying the source/context of disease-associated TGFe1 may be an important step in selecting the correct modulator of TGFe signaling that will inform what level of selectivity to consider (eg, isoform-specific, context-permissive, or context-specific TGFe1 modulators). TGFe 1 modulators, TGFe1 inhibitors or activators, etc.).

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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 muscles. In human patients, there is a strong correlation between the degree of TGFe1 upregulation and fibrosis, as well as a strong association between the degree of fibrosis and negative mobility outcomes. Therefore, in some embodiments, LTBP-proTGFe1 inhibitors can be administered to patients with dystrophy to prevent and/or reduce fibrosis to selectively target the ECM-related effects of TGFe1 in the disease. In some embodiments, various isoform- and/or context-selective agents described herein can be used to achieve inhibition of TGFe1 signaling to prevent fibrosis and promote myogenesis, but without adversely affecting the immune system (eg, through GARP or LRRC33).

Treatment, introduction.

To carry out the method disclosed herein, an effective amount of the pharmaceutical composition described above can be administered to a subject in need of treatment (e.g., a human) by a suitable route, such as intravenous administration, for example, as a bolus or continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhalation or topical route. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are suitable for administration. Liquid formulations can be sprayed directly, and lyophilized powder can be sprayed after reconstitution. Alternatively, antibodies or antigen-binding portions thereof that specifically bind to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex can be aerosolized using a fluorocarbon formulation and a metered dose inhaler or inhaled as a lyophilized and crushed powder.

The subject to be treated by the methods described herein may be a mammal, more preferably a human.

Mammals include, but are not limited to, farm animals, sporting animals, pets, primates, horses, dogs, cats, mice and rats. The subject in need of treatment may be a human patient at risk for or suspected of having a TGFe-related indication, such as those noted above. A subject having a TGFe-related indication may be identified by routine medical examination, such as laboratory tests, organ function tests, CT scan, or ultrasound. A subject suspected of having any of these indications may exhibit one or more symptoms of the indication. A subject at risk for developing an indication may be a subject having one or more risk factors for developing that indication.

As used herein, the terms effective amount and effective dose refer to any amount or dose of a compound or composition that is sufficient to achieve the intended purpose(s), i.e. a desired biological or medical response in a tissue or subject with an acceptable benefit/risk ratio. For example, in some embodiments of the present invention, the intended purpose may be to inhibit TGFe-1 activation in vivo to achieve a clinically significant result associated with TGFe-1 inhibition. Effective amounts will vary, as will be recognized by those skilled in the art, depending on the specific condition being treated, the severity of the condition, individual patient characteristics including age, physical condition, size, sex and weight, duration of treatment, nature of concurrent therapy (if there is one), the specific route of administration and similar factors within the knowledge and experience of the doctor. These factors are well known to those skilled in the art and can be eliminated through routine experimentation. In general, it is preferable to use the maximum dose of individual components or combinations thereof, i.e. the highest safe dose according to reasonable medical judgment. Those skilled in the art will appreciate, however, that a patient may insist on a lower dose or an acceptable dose for medical reasons, psychological reasons, or virtually any other reason.

Empirical considerations such as half-life will generally guide dosage determination. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to prolong the half-life of the antibody and prevent the antibody from being attacked by the host immune system. The frequency of administration can be determined and adjusted during the course of therapy and is generally, but not necessarily, based on treatment and/or suppression and/or attenuation and/or delay of TGF-related indications. Alternatively, sustained sustained release formulations of an antibody that specifically binds to the GARP-TGFei complex, the LTBP1TGFei complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex may be suitable. Various formulations and devices for achieving sustained release will be apparent to one skilled in the art and are within the scope of the present disclosure.

In one example, dosages for an antibody that specifically binds to the GARPTGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex, as described herein, can be determined empirically in individuals who have received one or more administrations antibodies. Individuals receive additional doses of the antagonist. A metric related to TGFe can be used to evaluate performance. For example, methods for measuring muscle fiber damage, muscle fiber recovery, muscle inflammation levels, and/or muscle fibrosis levels are well known to one of ordinary skill in the art.

The present invention embraces the recognition that agents capable of modulating the activation step of TGFe in an isoform-specific manner may provide improved safety profiles when used as a drug. Accordingly, the present invention includes antibodies and antigen-binding fragments thereof that specifically bind and inhibit activation of TGFe1, but not TGFe2 or TGFe3, thereby conferring specific inhibition of TGFe1 signaling in vivo while minimizing undesirable side effects from interfering with TGFe2 signaling and/or TGFe3.

In some embodiments, the antibodies or antigen-binding portions thereof described herein are non-toxic when administered to a subject. In some embodiments, antibodies or antigen-binding portions thereof described herein exhibit reduced toxicity when administered to a subject compared to an antibody that specifically binds to both TGFe1 and TGFe2. In some embodiments, antibodies or antigen-binding portions thereof described herein exhibit reduced toxicity when administered to a subject compared to an antibody that specifically binds to both TGFe1 and TGFe3. In some embodiments, antibodies or antigen-binding portions thereof described herein exhibit reduced toxicity when administered to a subject compared to an antibody that specifically binds TGFe1, TGFe2, and TGFe3.

Typically, for administration of any of the antibodies described herein, the initial potential dose may be approximately 2 mg/kg. For purposes of this disclosure, a typical daily dose may range from about 0.1 mcg/kg to 3 mcg/kg, up to 30 mcg/kg, up to 300 mcg/kg, up to 3 mg/kg, up to 30 mg/kg, up to 100 mg. /kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment is continued until the desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate the TGFe-related indication or symptom thereof. An exemplary dosage regimen involves administering an initial dose of approximately 2 mg/kg followed by a weekly maintenance dose of approximately 1 mg/kg of antibody or followed by a maintenance dose of approximately 1 mg/kg every other week. However, other dosage regimens may be appropriate depending on the nature of the pharmacokinetic breakdown that the practitioner wishes to achieve. For example, dosing is suggested between one and four times per week. In some embodiments, the dosage is 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 , approximately 1 mg/kg and approximately 2 mg/kg). Pharmacokinetic experiments have shown that serum concentrations of antibodies disclosed herein (eg, Ab2) remain stable for at least 7 days after administration in a preclinical animal model (eg, mouse model). Without wishing to be bound by any particular theory, this post-administration stability may be advantageous because the antibody can be administered less frequently while maintaining a clinically effective serum concentration in the subject to whom the antibody is administered (eg, a human subject). In some embodiments, the frequency of administration is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once a month, every 2 months or every 3 months or longer. The progress of this therapy is easily monitored using traditional methods and tests. The dosage regimen (including the antibody used) may change over time.

In some embodiments, dosages in the range of about 0.3 to 5.00 mg/kg may be administered to an adult patient of normal weight. The specific dosage regimen, such as dosage, timing, and repetition, will depend on the individual and the individual's medical history, as well as the properties of the individual agents (such as the half-life of the agent and other relevant considerations).

For purposes of the present disclosure, the appropriate dosage of an antibody that specifically binds to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFei complex will depend on the specific antibody (or composition thereof) used. the type and severity of the indication, whether the antibody is being administered for prophylactic or therapeutic purposes, prior therapy, the patient's medical history and response to the antagonist, and the discretion of the treating physician. In some embodiments, the physician will administer an antibody that specifically binds to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3TGFe1 complex, and/or the LRRC33-TGFe1 complex until a dosage is reached that achieves the desired result. Administration of an antibody that specifically binds to the GARPTGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex and/or LRRC33-TGFe1 complex may be continuous or intermittent, depending, for example, on the physiological state of the recipient, whether the purpose of administration is therapeutic or prophylactic , and other factors known to qualified specialists. Administration of an antibody that specifically binds to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex may be substantially continuous over a preselected period of time or may occur in a series of spaced doses, e.g. before, during, or after the development of a TGF-related indication.

As used herein, the term treatment refers to the use or administration of a composition comprising one or more active agents to a subject who has a TGF-related indication, a symptom of an indication, or a predisposition to an indication, for the purpose of treating, curing, alleviating, ameliorating, altering , correcting, eliminating, improving, or influencing an indication, a symptom of an indication, or a predisposition to an indication.

Alleviation of a TGFe-related indication with an antibody that specifically binds to the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex and/or LRRC33TGFp1 complex includes delaying the development or progression of the indication or reducing the severity of the indication. Relief of an indication does not necessarily require therapeutic results. As used herein, delaying the development of a TGF-related indication means delaying, preventing, slowing, inhibiting, stabilizing, and/or delaying the progression of the indication. This delay may vary in duration depending on the history of the indication and/or the individuals being treated. A method that delays or facilitates the development of an indication, or delays the onset of an indication, is a method that reduces the likelihood of developing one or more symptoms of an indication in a given period of time and/or reduces the severity of symptoms in a given period of time, compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to obtain a statistically significant result.

DBA2/J mice contain a 40 bp deletion. in the LTBP4 allele. Dysregulation of the ECM, to which latent TGFb1 is bound, may expose the epitope to which Ab1 binds. There may be diseases that target the epitope to which Ab1 binds, and these diseases may represent therapeutic opportunities for Ab1 if inhibition of TGFb1 is indicated.

Combination therapy.

The present disclosure further covers pharmaceutical compositions and related methods used as combination therapy to treat subjects who may benefit from TGFe inhibition in vivo. In any of these embodiments, such subjects may receive a combination therapy that includes a first composition containing at least one TGFe inhibitor, such as an antibody or antigen-binding moiety described herein, in combination with a second composition containing at least one additional therapeutic agent intended to treat the same or overlapping disease or clinical condition. The first and second compositions may act on a single cellular target or on separate cellular targets. In some embodiments, the first and second compositions may treat or alleviate the same or overlapping set of symptoms or aspects of a disease or clinical condition. In some embodiments, the first and second compositions may treat or alleviate a distinct set of symptoms or aspects of a disease or clinical condition. To give just one example, a first composition may treat a disease or condition associated with TGF signaling, while a second composition may treat inflammation or fibrosis associated with the same disease, etc. Such combination therapies can be administered in combination with each other. The phrase in combination with, in the context of combination therapy, means that the therapeutic effects of the first therapy overlap temporally and/or spatially with the therapeutic effects of the second therapy in a subject receiving the combination therapy. Thus, the combination therapy may be formulated as a single composition for simultaneous administration or as separate compositions for sequential administration of the therapy.

In preferred embodiments, the combination therapy produces a synergistic effect in treating the disease. The term synergistic refers to effects that are greater than the additive effects (eg, greater effectiveness) of each monotherapy combined.

In some embodiments, a combination therapy comprising a pharmaceutical composition described herein provides efficacy that is generally equivalent to that obtained with another therapy (such as monotherapy with a second agent) but is associated with less unwanted side effect or less severe toxicity associated with second agent, compared with monotherapy of the second agent. In some embodiments, such combination therapies reduce the dosage of the second agent while maintaining overall effectiveness. Such combination therapy may be particularly suitable for patient populations requiring long-term treatment and/or pediatric patients.

Accordingly, the present invention provides pharmaceutical compositions and methods for use in combination therapy to reduce TGFe1 protein activation and treat or prevent diseases or conditions associated with TGFe1 signaling, as described herein. Accordingly, the methods or pharmaceutical compositions further comprise a second therapy. In some embodiments, the second therapy may be useful in the treatment or prevention of diseases or conditions associated with TGFe1 signaling. The second therapy may reduce or treat at least one symptom(s) associated with the target disease. The first and second types of therapy may exert their biological effects through similar or unrelated mechanisms of action; or one or both of the first and second treatments may exert their biological effects through multiple mechanisms of action.

It should be understood that the pharmaceutical compositions described herein may contain the first and second therapy in the same pharmaceutically acceptable carrier or in a different pharmaceutically acceptable carrier for each embodiment described. In addition, it should be understood that the first and second therapies may be administered simultaneously or sequentially within the described embodiments.

One or more anti-TGFe antibodies or antigen-binding portions thereof of the present invention may be used in combination with one or more additional therapeutic agents. Examples of additional therapeutic agents that can be used with the anti-TGFe antibody of the present invention include, but are not limited to: a modulator of a member of the TGFe superfamily, such as a myostatin inhibitor and a GDF11 inhibitor; VEGF agonist; IGF1 agonist; FXR agonist; CCR2 inhibitor; CCR5 inhibitor; dual CCR2/CCR5 inhibitor; lysyl oxidase-2 inhibitor; ASK1 inhibitor; acetyl-CoA carboxylase (ACC) inhibitor; p38 kinase inhibitor; pirfenidone; nintedanib; M-CSF inhibitor (eg, M-CSF receptor antagonist and M-CSF neutralizing agents); a MAPK inhibitor (eg, Erk inhibitor), immune checkpoint agonist or antagonist; IL-11 antagonist and IL-6 antagonist and the like. Other examples of additional therapeutic agents that can be used with TGFe inhibitors include, but are not limited to, an indoleamine 2,3-dioxygenase (IDO) inhibitor, a tyrosine kinase inhibitor, a Ser/Thr kinase inhibitor, a dual-specific kinase inhibitor. In some embodiments, such an agent may be a PI3K inhibitor, a PKC inhibitor, or a JAK inhibitor.

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, anti-TIM3, anti-LAG3, anti-OX40, anti-CD27, anti-CD70, anti-CD47, anti-41BB, anti-PD-1, oncolytic virus and PARP inhibitor.

In some embodiments, the additional agent binds a T cell costimulation molecule, such as inhibitory costimulation molecules and activating costimulation molecules. In some embodiments, the additional agent is selected from the group consisting of an anti-CD40 antibody, an anti-CD38 antibody, an anti-KIR antibody, an anti-CD33 antibody, an anti-CD137 antibody, and an anti-CD74 antibody.

In some embodiments, the 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 monocyte/macrophage recruitment and/or tissue infiltration. In some embodiments, the additional agent is a hepatic stellate cell activation inhibitor. In some embodiments, the additional agent is a chemokine receptor antagonist, for example, CCR2 antagonists and CCR5 antagonists. In some embodiments, such a chemokine receptor antagonist is a dual specific antagonist, such as a CCR2/CCR5 antagonist. In some embodiments, the additional agent to be administered as a combination therapy is or includes a member of the TGFe superfamily of growth factors or their regulators. In some embodiments, such an agent is selected from the modulators (eg, inhibitors and activators) of GDF8/myostatin and GDF11. In some embodiments, such an agent is an inhibitor of GDF8/myostatin signaling. In some embodiments, such an agent is a monoclonal antibody that specifically binds to the myostatin pro-/latent complex and blocks myostatin activation. In some embodiments, a monoclonal antibody that specifically binds to the myostatin pro/latent complex and blocks myostatin activation does not bind to free mature myostatin.

In some embodiments, the adjunctive therapy comprises a CAR-T therapy.

Such combination therapy may advantageously use lower doses of therapeutic agents administered, thereby avoiding possible toxic effects or complications associated with different monotherapies. In some embodiments, use of an isoform-specific TGFe1 inhibitor described herein may render those who respond poorly or do not respond to therapy (eg, standard of care) to greater sensitivity. In some embodiments, use of an isoform-specific TGFe1 inhibitor described herein may allow for a reduced dosage of therapy (e.g., standard of care) that still provides equivalent clinical efficacy in patients but less toxicity or drug-related side effects.

Inhibition of TGFe1 activity.

Methods of the present disclosure include methods of inhibiting the activity of the growth factor TGFe1 in one or more biological systems. Such methods may include contacting one or more biological systems with an antibody and/or composition of the present disclosure. In some cases, these methods include changing the level of free growth factor in a biological system (eg, in a niche of a cell or subject). Antibodies and/or compositions in accordance with such methods may include, without limitation, biomolecules, including, without limitation, recombinant proteins, protein complexes and/or antibodies or antigenic portions thereof described herein.

In some embodiments, the methods of the present disclosure can be used to reduce or eliminate the activity of a growth factor, referred to herein as inhibition methods. Some such methods may involve retaining the mature growth factor in a TGFe complex (eg, TGFe1 complexed with GARP, LTBP1, LTBP3, and/or LRRC33) and/or promoting reassociation of the growth factor into the TGFe complex. In some cases, the inhibition methods may involve the use of an antibody that specifically binds to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex. Some inhibition methods provide one or more inhibitory antibodies.

In some embodiments, antibodies, antigen binding portions thereof, and compositions of the present disclosure can be used to inhibit TGFe1 activation. In some embodiments, provided herein is a method of inhibiting TGFe1 activation comprising exposing the GARP-TGFe1 complex, the LTBP1TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex to an antibody, an antigen-binding portion thereof, or a pharmaceutical composition described herein. In some embodiments, the antibody, antigen binding moiety, or pharmaceutical composition inhibits the release of mature TGFe1 from the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 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.

In some embodiments, the GARP-TGFe1 complex or the LRRC33TGFe1 complex is present on the outer surface of the cell.

In some embodiments, the cell expressing the GARP-TGFe1 complex or the LRRC33-TGFe1 complex is a T cell, fibroblast, myofibroblast, macrophage, monocyte, dendritic cell, antigen presenting cell, neutrophil, myeloid-derived suppressor cell (MDSC), lymphocyte, mast cell or microglia. The T cell may be a regulatory T cell (eg, an immunosuppressive T cell). The neutrophil may be an activated neutrophil. A macrophage may be an activated (eg, polarized) macrophage, including profibrotic and/or tumor-associated

- 88 045239 macrophages (TAM), for example, macrophages of the M2c subtype and M2d subtype. In some embodiments, macrophages are exposed to tumor-derived factors (eg, cytokines, growth factors, etc.) that can further induce pro-cancer phenotypes in the macrophages. In some embodiments, such tumor factor is CSF1/M-CSF.

In some embodiments, the cell expressing the GARP-TGFe1 complex or the LRRC33-TGFe1 complex is a cancer cell, such as circulating cancer cells and tumor cells.

In some embodiments, the LTBP1-TGFe1 complex or the LTBP3TGFe1 complex is associated with the extracellular matrix (ie, ECM components). In some embodiments, the extracellular matrix comprises fibrillin and/or fibronectin. In some embodiments, the extracellular matrix comprises a protein containing an RGD motif.

LRRC33 is expressed in selective cell types, particularly myeloid lineages, including monocytes and macrophages. Monocytes originate from precursors in the bone marrow and circulate in the bloodstream and reach peripheral tissues. Circulating monocytes can then migrate into tissues, where they become exposed to the local environment (eg, tissue-specific, disease-associated, etc.), which includes a panel of various factors, such as cytokines and chemokines, that trigger the differentiation of monocytes into macrophages, dendritic cells, etc. These include, for example, alveolar macrophages in the lung, osteoclasts in the bone marrow, microglia in the central nervous system, histiocytes in connective tissue, Kupffer cells in the liver, and brown adipose tissue macrophages in brown adipose tissues. In a solid tumor, the infiltrated macrophages may be tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), and myeloid-derived suppressor cells (MDSCs), etc. Such macrophages can activate and/or associate with activated fibroblasts, such as carcinoma (or cancer)-associated fibroblasts and/or stroma. Thus, the TGFe1 activation inhibitors described herein, which inhibit the release of mature TGFe1 from LRRC33-containing complexes, can target any of these LRRC33-pro-TGFβ1-expressing cells at the cell surface.

In some embodiments, the LRRC33-TGFe1 complex is present on the outer surface of profibrotic (M2-like) macrophages. In some embodiments, profibrotic (M2-like) macrophages are present in a fibrotic microenvironment. In some embodiments, targeting the LRRC33-TGFe1 complex to the outer surface of profibrotic (M2-like) macrophages provides a superior effect compared to solely targeting the LTBP1-TGFe1 and/or LTBP1-TGFe1 complexes. In some embodiments, M2-like macrophages are further distributed into multiple subtypes with different phenotypes, such as M2c and M2d TAM-like macrophages. In some embodiments, macrophages can be activated by various factors (e.g., growth factors, chemokines, cytokines, and ECM remodeling molecules) present in the tumor microenvironment, including, but not limited to, TGFe1, CCL2 (MCP-1), CCL22, SDF-1 /CXCL12, M-CSF (CSF-1), IL-6, IL-8, IL-10, IL-11, CXCR4, VEGF, PDGF, prostaglandin-regulating agents such as arachidonic acid and cyclooxygenase-2 (COX-2 ), parathyroid hormone-related protein (PTHrP), RUNX2, HIF1a and metalloproteinases. Exposure to one or more of these factors may further drive monocytes/macrophages toward protumor phenotypes. In turn, these activated tumor-associated cells may also promote the accumulation and/or differentiation of other cells into protumor cells, such as CAFs, TANs, MDSCs, etc. Stromal cells may also respond to macrophage activation and influence ECM remodeling and ultimately vascularization, invasion, and metastasis.

In some embodiments, the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex are associated with the extracellular matrix. In some embodiments, the extracellular matrix comprises fibrillin. In some embodiments, the extracellular matrix comprises a protein containing an RGD motif.

In some embodiments, provided herein is a method of reducing activation of TGFe1 protein in a subject, comprising administering to the subject an antibody, an antigen binding portion thereof, or a pharmaceutical composition described herein, thereby reducing activation of TGFe1 protein in the subject. In some embodiments, the subject has or is at risk of developing fibrosis. In some embodiments, the subject has or is at risk of developing cancer. In some embodiments, the subject has or is at risk of developing dementia.

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

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Kits for use in the relief of diseases/disorders associated with a TGFe indication.

The present disclosure also provides kits for use in alleviating diseases/disorders associated with TGFe indications. Such kits may include one or more containers containing an antibody or antigen-binding portion thereof that specifically binds to the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex and/or LRRC33-TGFe1 complex, such as any of those described in this document.

In some embodiments, the kit may contain instructions for use in accordance with any of the methods described herein. Included instructions may describe administration of an antibody or antigen-binding portion thereof that specifically binds to the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33-TGFe1 complex to treat, delay the onset of, or alleviate the target disease, as described in this document. The kit may further comprise a description of selecting an individual suitable for treatment based on determining whether the individual has the target disease. In other embodiments, the instructions describe administering the antibody or antigen-binding portion thereof to an individual at risk of the target disease.

Instructions for the use of antibodies or antigen-binding portions thereof that specifically bind to the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33-TGFe1 complex generally contain information regarding dosage, dosing schedule, and route of administration. for the intended treatment. Containers may be single dose, bulk pack (eg, multi-dose packs), or sub-unit dose. The instructions provided in the kits of the present disclosure are typically written instructions on a label or package insert (e.g., a piece of paper included in the kit), but machine-readable instructions (e.g., instructions stored on a magnetic or optical disk for storage) also acceptable.

The label or package insert indicates that the composition is used to treat, delay the onset of, and/or alleviate a disease or disorder associated with a TGFe-related indication. Instructions may be provided for performing any of the methods described herein.

The kits of the present invention are provided in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, a nasal administration device (eg, a nebulizer), or an infusion device, such as a minipump. The kit may have a sterile access port (eg, the container may be an IV bag or a vial with a stopper pierced by a hypodermic needle). The container may also have a sterile access port (eg, the container may be an intravenous solution bag or a vial with a stopper pierced by a hypodermic needle). At least one active agent in the composition is an antibody or an antigen-binding portion thereof that specifically binds to the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3TGFe1 complex, and/or the LRRC33-TGFe1 complex, as described herein.

Sets may optionally provide additional components such as buffers and interpretive information. Typically, the kit contains a container and a label or package insert(s) on or associated with the container. In some embodiments, the present disclosure provides articles containing the contents of the kits described above.

Assays to detect the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3TGFe1 complex, and/or the LRRC33-TGFe1 complex.

In some embodiments, the methods and compositions provided herein relate to a method for detecting the GARP-TGFe1 complex, the LTBP1-TGFe1 complex, the LTBP3-TGFe1 complex, and/or the LRRC33-TGFe1 complex in a sample obtained from a subject. As used herein, the term subject refers to a single organism, such as a single mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cattle, bird, cat, or dog. In some embodiments, the subject is a vertebrate, amphibian, reptile, fish, insect, fly, or nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically modified, e.g.

- 90 045239 modified non-human subject. The subject can be of any gender and at any stage of development. In some embodiments, the subject is a patient or healthy volunteer.

In some embodiments, a method for detecting the GARP-TGFe1 complex, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex, and/or LRRC33-TGFe1 complex in a sample obtained from a subject comprises (a) contacting the sample with an antibody that specifically binds to the GARP-TGFe1 complex. TGFe1, LTBP1-TGFe1 complex, LTBP3-TGFe1 complex and/or LRRC33-TGFe1 complex under conditions suitable for the antibody to bind to the antigen if antigen is present in the sample, thereby forming binding complexes; and (b) determining the level of antibody bound to the antigen (eg, determining the level of binding complexes).

In one embodiment, a screening assay that uses biotinylated latent TGFe1 complexes immobilized on a surface allows for activation of latent TGFe by integrins by providing binding. Other, non-integrin activators can also be investigated in this system. Readout may be through reporter cells or other TGFe-dependent cellular responses.

Cell studies to measure TGFe activation.

TGFe activation (and its inhibition by a TGFe inhibitor test such as an antibody) can be measured by any suitable method known in the art. For example, integrin-mediated activation of TGFe can be used in a cell assay, such as the CAGA12 luciferase assay described in more detail herein. As shown, such an assay system may contain the following components: i) a source of TGF (recombinant, endogenous or transfected); ii) a source of activator such as an integrin (recombinant, endogenous or transfected) and iii) a reporter system that responds to TGFe activation, such as cells expressing TGFe receptors capable of responding to TGFe and translating the signal into a readable output (e.g. luciferase activity in CAGA12 cells or other reporter cell lines). In some embodiments, the reporter cell line comprises a reporter gene (eg, a luciferase gene) under the control of a TGFe-responsive promoter (eg, the PAI-1 promoter). In some embodiments, certain promoter elements that confer sensitivity may be included in a reporter system. In some embodiments, such a promoter element is the CAGA12 element. Reporter cell lines that can be used in the assay are described, for example, in Abe et al. (1994) Anal Biochem. 216(2): 276-84, incorporated herein by reference. In some embodiments, each of the above assay components is provided from the same source (eg, the same cell). In some embodiments, two of the above analysis components are provided from the same source, and the third analysis component is provided from a different source. In some embodiments, all three analysis components are provided from different sources. For example, in some embodiments, the integrin and latent TGFe complex (proTGFe and presentation molecule) are provided for analysis from the same source (eg, the same transfected cell line). In some embodiments, the integrin and TGF are provided for analysis from separate sources (eg, two different cell lines, a combination of purified integrin and a transfected cell). When cells are used as the source of one or more assay components, such assay components may be endogenous to the cell, stably expressed in the cell, transiently transfected, or any combination thereof. The results of a non-limiting exemplary embodiment of a cell study to measure TGFe activation demonstrating inhibition of either the GARP-pro-TGFe1 complex or the LRRC33-pro-TGFe1 complex using antibodies Ab1 and Ab2 are disclosed herein. In this example assay, the Ab1 IC50 (μg/mL) for the GARPTGFe1 complex was 0.445 and the Ab1 IC50 (μg/mL) for the LRRC33-TGFe1 complex was 1.325.

One skilled in the art can easily adapt such assays to various suitable configurations. For example, different sources of TGFe may be considered. In some embodiments, the source of TGFe is a cell that expresses and deposits TGFe (eg, a primary cell, an expanded cell, an immortalized cell or cell line, etc.). In some embodiments, the TGFe source is purified and/or recombinant TGFe immobilized in the assay system using suitable means. In some embodiments, the TGFe immobilized in the assay system is presented in an extracellular matrix (ECM) on the assay plate with or without decellularization that mimics fibroblast-derived TGFe. In some embodiments, TGFe is presented on the cell surface of a cell used in the assay. Additionally, the selected presentation molecule can be included in the assay system to provide a suitable latent-TGFe complex. One skilled in the art can readily determine which presentation molecule may be present or expressed in certain cells or cell types. Using such assay systems, the relative changes in TGFe activation in the presence or absence of a test agent (such as an antibody) can be readily measured to assess the effect of the test agent on TGFe activation in vitro. Data from illustrative cell studies are presented in the Examples section below.

Such cell studies can be modified or adapted in various ways depending on the TGF isoform being studied, the type of latent complex (eg, presentation molecule), etc. In some embodiments, a cell known to express an integrin capable of activating TGF can be used as a source of integrin in the assay. Such cells include SW480/e6 cells (eg, clone 1E7). In some embodiments, integrin-expressing cells can be co-transfected with a plasmid encoding the molecule of interest (such as GARP, LRRC33, LTBP (e.g., LTBP1 or LTBP3), etc.) and a plasmid encoding the proform of the isoform of interest TGFe (such as pro-TGFe 1). After transfection, the cells are incubated for a time sufficient to express the transfected genes (eg, approximately 24 hours), the cells are washed and incubated with serial dilutions of the test agent (eg, antibody). A reporter cell line (eg, CAGA12 cells) is then added to the assay system, followed by appropriate incubation times to allow for TGFe signaling. After an incubation period (e.g., approximately 18-20 hours) after addition of the test agent, the signal/readout (e.g., luciferase activity) is detected using suitable means (e.g., for luciferase-expressing reporter cell lines, Bright-Glo reagent (Promega) can be used) . In some embodiments, luciferase fluorescence can be detected using a BioTek plate reader (Synergy H1) with auto gain settings.

Representative results from studies on TGFe cells are shown in FIG. 7 in this document. The data demonstrate that exemplary antibodies of the present invention are capable of selectively inhibiting TGFe1 activation regardless of context.

Nucleic acids.

In some embodiments, the antibodies, antigen binding portions thereof, and/or compositions of the present invention may be encoded by nucleic acid molecules. Such nucleic acid molecules include, but are not limited to, DNA molecules, RNA molecules, polynucleotides, oligonucleotides, mRNA molecules, vectors, plasmids, and the like. In some embodiments, the present disclosure may comprise cells programmed or engineered to express nucleic acid molecules encoding the compounds and/or compositions of the present disclosure. In some cases, the nucleic acids of the present disclosure include codon-optimized nucleic acids. Methods for producing codon-optimized nucleic acids are known in the art and may include, without limitation, those described in US Pat. Nos. 5,786,464 and 6,141,448, the contents of each of which are incorporated herein by reference in their entirety.

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 in this application, as well as figures, are incorporated herein by reference.

The present invention is further illustrated by the following examples, which should not be construed as limiting.

Examples

Example 1: Inhibition of TGFe1.

The TGFe superfamily includes propeptides that form a complex with active growth factors (Fig. 1). Selection strategies have been developed to produce antibodies that stabilize the complex, resulting in more selective and potent inhibition.

Using a HEK293-based expression system, NiNTA affinity and gel filtration were performed to obtain milligram quantities of purified protein, which was used to produce TGFp1 complexed with LTBP (LTBP-TGFe1 complex) and TGFe1 complexed with GARP (GARP-TGFe1 complex ) (Fig. 3). The diversity of proteins produced has allowed for cross-reactivity studies of forms and epitope mapping.

Potential antibodies were screened using in vitro luminescence assays. In the screen, antibodies that inhibited the release of growth factors turned off reporter cells when they encountered a stimulus for normal activation. Ab1 and Ab2 have been shown to be inhibitors of activation of latent TGFe1 complexes and are cross-reactive to murine ones.

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Initial dose response curves for Ab1 in cells expressing human TGFei showed inhibition of TGF31 activity. Using the more sensitive reporter cell line CAGA12, Ab1 demonstrated similar inhibition of human pro-TGFe 1 activity. In addition, inhibition of the GARP complex was shown to block the suppressive activity of T regulatory cells (Tregs), as measured by the percentage of dividing T effector cells (Teff) in T cells isolated from the blood of a healthy donor (Fig. 9A).

Similar results were observed for Ab3. Dose response curves of Ab3 in human liver stellate cells and human skin fibroblasts showed inhibition of TGFe1 activity (Figure 7F), and Ab3 was also shown to inhibit Treg suppressive activity (Figure 9B).

The affinity of GARP-pro-TGFβ1 inhibitors was measured using the Octet assay on human GARP-pro-TGFβ1 cells, while activity was measured using CAGA12 reporter cells examining human GARP-pro-TGFβ1 inhibition. The protocol used to measure the affinity of Ab1 and Ab2 antibodies for the complexes presented herein is shown in table. 6. The results are shown in table. 7.

Table 6

Protocol for performing Octet binding assay

Materials:

- 96-well black polypropylene plates

- Streptavidin coated tips for Octet

- 10x kinetic buffer (diluted 1:10 in PBS)__________________________

1. Soak the required number of streptavidin tips in 1x kinetic buffer; place in the equilibration apparatus_______________________ 2. Load sample plates:

- 200 µl of buffer or antibody dilution per well

A. Column 1 - initial level (buffer)

b. Column 2 - biotinylated protein (for example, sGARP-npoTGFpi or LTBP1-

InpoTGFpi); diluted to 5 µg/ml

With. Column 3 - original level 2 (buffer)

d. Column 4 - antibody association for Abi

e. Column 5 - association of antibodies for AB2 £ Column 6 - dissociation of AB (buffer)

g. Column 7 - dissociation of AB2 (buffer)_____________________________________________

3. Make dilutions in a 96-well plate:

A. Dilute both antibodies to 50 µg/ml in 300 µl 1x buffer in row A.

b. Add 200 µl buffer to the rest of each column

With. Transfer 100 µl down the column to make 3-fold dilutions________

4, Place the sample plate in the instrument next to the tip plate_____

5. Set up the software

A. Specify buffer, loading, sample (one assay per antibody of interest)

b. Specify protocol stages (baseline, loading, association, dissociation) for a given amount of time:

• Baseline: 60 seconds • Loading: 300 seconds • Baseline 2: 60 seconds • Association: 300 seconds * Dissociation: 600 seconds______________________________________________________________

6. Data analysis

A. Subtract the original level from the reference well

b. Set normalization to the last five seconds of baseline _____s. Even out dissociation_________________________________________________

d. Association and dissociation assay (1:1 binding model, curve fitting)

e. Determine the best values of R ² ; include concentrations with best R ² values

f. Select global fit

g. Set sample colors by sensor type

h. Analyze _____i. Save table and export__________________________________________

Table 7

Affinity and activity of GARP-pro-TGFβ1 inhibitors

Clone Affinity for GARPnpoTGFpi (nM ± SEM) Inhibition (IC50) GARP-npoTGFpi (nM; 95% CI) Maximum effect (% inhibition) AY 0.046 ± 0.043 3.4 (2.1-5.4) 75% AB2 0.561 ±0.014 3.9(1.5-10.3) 50%

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The clones were then screened for binding selectivity (Table 8) and form cross-reactivity (Table 9). AL and AB2 did not bind to ΤΟΡβΙ, ΤΘΡβ2 or ΤΟΡβ3, but bound to προ-ΤΘΡβΙ complexes and exhibited cross-reactivity of forms.

Table 8

Selectivity of ΟΑΚΡ-προ-ΤϋΡβΙ inhibitors

Clone GARP-npoTGFpi LTBPl-npoTGFpi LTBP3-npoTGFpi AY +++ +++ +++ AB2 +++ +++ +++

Table 9

Cross-reactivity of ΘΑΚΡ-προ-ΤΘΡβΙ inhibitor forms

Clone huGARP-npoTGFpi muGARP-npoTGFpi cyGARP-npoTGFpi AY +++ ++ +++ AB2 +++ +++ +++

+++ KD < 1 nM, ++KD 1 - 10 nM, + KD 10-100 nM. - No binding.

Binding specificity for Al3 was further examined using the Octet binding assay. As shown in FIG. 4A, Al3 binds specifically to latent ΤΘΡβΙ, but not to latent ΤΘΡβ2 or latent ΤΘΡβ3, whereas antibodies to naH-TGF-beta are not isoform specific (Fig. 5). These data demonstrate that Al3 binds to TΤΘΡβ in an isoform-specific manner.

Example 2. AL, AL2 and AL3 bind specifically to προ-ΤΘΡβΙ complexes from several species.

To determine whether Al, Al2, and Al3 were able to specifically bind to proΤΟΡβΙ complexes from several species, Octet binding assays were performed as described in Table 1. 6. As shown in table. 10 (below), all three antibodies (i.e., AL, AL2, and AL3) specifically bound to human and mouse ΕΤΒΡΙ-προ-ΤΘΡβΙ complexes, human ΕΤΒΡ3-προ-ΤΰΡβ1 complexes, and human ΟΑΚΡ-προ-Τ complexes. ΟΡβΙ. However, only AB2 and AL3 specifically bound to rat ΕΤΒΡΙ-προ-ΤΘΡβΙ complexes.

Table 10

Affinity of AL, AL2 and AL3 to the προ-ΤΟΡβ complexes! from several types

Аы (K _D ) AB2 (K _D ) ABZ (K _D ) Human LTBPl-npoTGFpl 16+ 1.3 5.8 + 0.6 1.1 + 0.07 Human LTBP3-npoTGFpl 85 + 5.0 122 + 3.9 0.12 + 0.04 LTBPl-npoTGFpi mice 203 ± 13 61+4.0 0.68 ± 0.06 LTBPl-npoTGFpi rats Linking is not discovered 38 + 6.8 0.93 ± 0.03 Human GARP-npoTGFpl 293 ± 22 58 + 6.2 4.9 + 0.11

Example 3. AB2 and AL3 bind to ΕΚΚΟ33-προ-ΤΟΡβ1.

To determine whether Al, Al2, and Al3 bind to προ-ΤΘΡβΙ, which is complexed with LRRC33, Octet binding assays were performed. As shown in FIG. 12C, Al, Al2 and Al3 are able to bind to the protein complex ΕΚΚΟ33-προ-ΤΟΡβ1. However, AI demonstrates a slow rate of binding of the ΕΚΚΟ33-προ-ΤΟΡβ1 protein complex. The binding of Al, Al2 and Al3 to the protein complex ΕΚΚΟ33-προ-ΤΟΡβ1 was further confirmed using ELISA.

Example 4. AL, AL2 and AL3 inhibit the activity of both ΘΑΚΡ-προ-ΤΟΡβΙ and LRRC33-npoΤΟΡβΕ

To determine whether Al, Al2 and Al3 inhibit the activity of GARP-npo-TGF-βΙ and/or LRRC33npo-TGF-βΙ, an in vitro cell study was performed. In this assay system, an engineered human colon cancer cell line (SW480/β6 cells) stably transfected with β6 integrin was cotransfected with an npo-TGF-βΙ expression construct and a presentation molecule expression construct (i.e., GARP or LRRC33). Constructs encoding chimeric LRRC33-GARP (SEQ ID NO: 101) or GARP were used to express presentation molecules. Transfected cells were incubated to ensure sufficient expression and sedimentation

-94045239 components (integrins and pro-TGFe 1 in complex with the corresponding presenting molecule).

Activation of TGFe1 in the presence or absence of Ab1 or Ab2 or Ab3 was analyzed using reporter cells (CAGA12 cells) expressing TGFe receptors coupled to their downstream signal transduction pathway to measure the inhibitory activity of the antibody. As shown in FIG. 7A and 7B, Ab1, Ab2, and Ab3 inhibited both GARP-pro-TGF-e1 and LRRC33-pro-TGF-e1.

Additionally, a cell study was performed to detect inhibition of either the GARP-pro-TGFe1 complex or the LRRC33-pro-TGFe1 complex using antibodies Ab1 and Ab2. Ab1 and Ab2 inhibited both GARP-pro-TGF-e1 and LRRC33-pro-TGF-e1. In this assay, the IC50 (μg/mL) of Ab1 for the GARP-TGFe1 complex was 0.445 and the IC50 (μg/mL) of Ab1 for the LRRC33-TGFe1 complex was 1.325.

Example 5 Assays to detect LTBP-TGFe1-specific activation.

In some embodiments, the methods and compositions provided herein provide a method for detecting an LTBP-TGFe1 complex, such as an LTBP1 or LTBP3-TGFe1 complex, in a sample.

A. Activation of latent TGFe1 deposited in the ECM.

In this assay, presentation molecules were cotransfected with pro-TGFe 1 in integrin-expressing cells. Transiently transfected cells are plated into assay dishes in the presence of inhibitors. The latent LTBP-pro-TGFe1 complex is inserted into the ECM. TGFe reporter cells are then added to the system; free growth factor (released by integrin) signals and is detected by a luciferase assay.

The following protocol is one example for measuring extracellular matrix activation (presented by LTBP) by integrin cells. Materials include: MvLu1-CAGA12 cells (clone 4A4); SW480/e6 cells (clone 1E7) (aV subunit is endogenously expressed at high levels; β6 subunit is consistently overexpressed); LN229 cell line (high level of endogenous V8 integrin); Costar treated white wall TC 96 well assay plate #3903; Greiner Bio-One High Binding 96-well Assay Plate #655094; human fibronectin (Corning #354008); multichannel pipette P200; pipettes P20, P200 and P1000 with sterile filter tips for each; sterile microcentrifuge tubes and rack; sterile containers with reagents; 0.4% trypan blue; sterile pipettes with a volume of 2, 5, 10 and 25 ml; tissue culture-treated 100 mm or 150 mm plates; 70% ethanol; Opti-MEM reduced serum (Life Tech #31985-070); Lipofectamine 3000 (Life Tech No. L3000015); Bright-Glo Luciferase Assay Reagent (Promega #E2620); Trispin at a concentration of 0.25% + EDTA at a concentration of 0.53 mM; human pro-TGFb1 expression plasmid (SR005); human LTBP1S expression plasmid (SR044); human LTBP3 expression plasmid (SR117); human LRRC32 expression plasmid (GARP) (SR116) and human LRRC33 expression plasmid (SR386). Equipment used includes: BioTek Synergy H1 tablet reader; TS funnel; tabletop centrifuge; CO ₂ incubator with a temperature of 37°C and 5% CO ₂ ; water/ball bath at 37°C; shaker with platform; microscope and hemocytometer/analyzer.

CAGA12 4A4 cells are derivatives of MvLu1 cells (mink lung epithelial cells) stably transfected with a synthetic CAGA12 promoter driving luciferase gene expression. DMEM-0.1% BSA is the assay medium; the base medium is DMEM (Gibco Cat. No. 11995-065), the medium also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin and 4 mM glutamine. D10 refers to DMEM 10% FBS, P/S, 4 mM glutamine, 1% NEAA, 1x GlutaMAX (Gibco Cat# 35050061). SW480/e6 medium refers to D10 + 1000 µg/ml G-418. CAGA12 medium (4A4) refers to D10 + 0.75 μg/ml puromycin.

On day 0, cells are plated for transfection. SW480/e6 cells (clone 1E7) were detached with trypsin and pelleted (rotation 5 min at 200xg). The cell pellet is resuspended in D10 medium and the number of viable cells per ml is counted. Cells are seeded in an amount of 5.0e6 cells/12 ml/100 mm TC dish. For CAGA12 cells, cells are passaged at a density of 1.0 ppm onto a T75 flask for use in the day 3 assay. Cultures are incubated at 37°C and 5% CO2.

On day 1, integrin-expressing cells are transfected. Follow the manufacturer's protocol for transfection with Lipofectamine 3000 reagent. Briefly, the following are diluted in OptiMEM I for 125 µl per well: 7.5 µg DNA (presenting molecule) + 7.5 µg DNA (pro-TGFe 1), 30 µl P3000 and up to 125 µl with OptiMEM I. The well is mixed by pipetting the DNA together, then OptiMEM is added. Add P3000 and mix everything well with a pipette. Prepare a master mixture of Lipofectamine 3000 for addition to DNA mixtures: for LTBP1 analysis: 15 μl Lipofectamine 3000, up to 125 μl in OptiMEM I per well; for LTBP3 assay: 45 µl Lipofectamine 3000, up to 125 µl in OptiMEM I per well. Diluted Lipofectamine 3000 is added to the DNA, mixed well with a pipette and incubated at room temperature for 15 minutes. After incubation, the solution is stirred several times

- 95 045239 pipette and then add 250 µl DNA: Lipofectamine 3000 (2x125 µl) dropwise per dish. Each plate is gently shaken to mix, and the plate is returned to the tissue culture incubator for ~24 h.

Equivalent amounts of each plasmid are generally optimal for co-transfection. However, co-transfection can be optimized by changing the ratio of plasmid DNA for the presenting molecule and pro-TGFe 1.

On days 1-2, assay plates were coated with human fibronectin. Specifically, lyophilized fibronectin is diluted to 1 mg/ml in ultrapure distilled water (sterile). 1 mg/ml stock solution is diluted to 19.2 μg/ml in PBS (sterile). 50 µl/well is added to the assay plate (high binding) and incubated O/N in a tissue culture incubator (temperature 37°C and 5% CO ₂ ). The final concentration is 3.0 μg/cm ² .

On day 2, transfected cells were plated for analysis and inhibitor addition. First, the fibronectin coating is washed by adding 200 μl/well of PBS to the fibronectin solution already in the assay plate. The wash is removed manually using a multichannel pipette. The washing is repeated twice. The plate is allowed to dry at room temperature with the lid closed before adding cells. Cells are then seeded by trypsin detachment and pelleted (rotate for 5 min at 200xg). The pellet is resuspended in assay medium and the number of viable cells is counted per ml. For the LTBP1 assay, cells are diluted to 0.10e6 cells/ml and plated at 50 μl per well (5000 cells per well). For the LTBP3 assay, cells are diluted to 0.05e6 cells/mL and seeded at 50 μL per well (2500 cells per well). To prepare functional dilutions of antibodies, antibodies are pre-diluted to a constant working concentration in the vehicle. Stock antibodies are serially diluted in vehicle (PBS is optimal; sodium citrate buffer should be avoided). Each serial dilution point is diluted in assay medium to 4 times the final antibody concentration. 25 μl per well of 4x antibody was added and the cultures were incubated at 37°C and 5% CO ₂ for ~24 h.

On day 3, TGF reporter cells are added. CAGA12 cells (clone 4A4) for analysis were separated with trypsin and pelleted (rotation 5 min at 200xg). The pellet is resuspended in assay medium and the number of viable cells per ml is counted. Cells are diluted to 0.46 cells/ml and seeded at 50 µl per well (20,000 cells per well). The cells are returned to the incubator.

On day 4, the assay is read (16-20 hours after the addition of antibodies and/or reporter cells). The reagent and Bright-Glo plate of interest are allowed to warm to room temperature before reading. Readout settings in BioTek Synergy H1 are set using the TMLC_std protocol - this method has an automatic gain setting. Positive control wells are selected for autoscaling (high level). Add 100 µl of Bright-Glo reagent to each well. Incubate for 2 minutes with shaking at room temperature; protecting the tablet from light. The plate is read on a BioTek Synergy H1.

Data obtained from this assay reflect LTBP1-TGFe1 and/or LTBP3-TGFe1 binding activity in cell supernatants.

B. Activation of latent TGFe1 present on the cell surface.

To detect activation of latent TGFe1 presented on the cell surface, presenting molecules were cotransfected with pro-TGFe1 in integrin-expressing cells. Latent TGFe1 is expressed on the cell surface by GARP or LRRC33. Reporter cells and TGFe inhibitors are then added to the system; free growth factor (released by integrin) signals and is detected by a luciferase assay. This assay or direct transfection protocol is optimal for cell surface integrin activation of TGFe1 (GARP or LRRC33 presenter).

Materials used included: MvLu1-CAGA12 cells (clone 4A4); SW480/e6 cells (clone 1E7) (aV subunit is endogenously expressed at high levels; β6 subunit is consistently overexpressed); LN229 cell line (high level of endogenous V8 integrin); Costar treated white wall TC 96 well assay plate #3903; Greiner Bio-One High Binding 96-well Assay Plate #655094; human fibronectin (Coining #354008); multichannel pipette P200; pipettes P20, P200 and P1000 with sterile filter tips for each; sterile microcentrifuge tubes and rack; sterile containers with reagents; 0.4% trypan blue; sterile pipettes with volumes of 2 ml, 5 ml, 10 ml and 25 ml; tissue culture-treated 100 mm or 150 mm plates; 70% ethanol; Opti-MEM reduced serum (Life Tech #31985-070); Lipofectamine 3000 (Life Tech No. L3000015); Bright-Glo Luciferase Assay Reagent (Promega #E2620); Trispin at a concentration of 0.25% + EDTA at a concentration of 0.53 mM; human pro-TGFb1 expression plasmid (SR005); human LTBP1S expression plasmid (SR044); human LTBP3 expression plasmid (SR117); human LRRC32 expression plasmid (GARP) (SR116) and human LRRC33 expression plasmid (SR386).

Equipment used includes: BioTek Synergy H1 tablet reader; TS funnel; 96 045239 benchtop centrifuge; CO ₂ -incubator with a temperature of 37°C and 5% CO2; water/ball bath at 37°C; shaker with platform; microscope and hemocytometer/analyzer.

The term CAGA12 4A4 cells refers to a derivative of MvLu1 cells (mink lung epithelial cells) stably transfected with a synthetic CAGA12 promoter driving luciferase gene expression. DMEM-0.1% BSA refers to assay media; the base medium is DMEM (Gibco Cat. No. 11995-065), the medium also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin and 4 mM glutamine. D10 refers to DMEM 10% FBS, P/S, 4 mM glutamine, 1% NEAA, 1x GlutaMAX (Gibco catalog 35050061). SW480/e6 medium refers to D10 + 1000 µg/ml G-418. CAGA12 medium (4A4) refers to D10 + 0.75 μg/ml puromycin.

On day 0, integrin-expressing cells are plated for transfection. Cells are separated with trypsin and pelleted (rotation 5 min at 200xg). The cell pellet is resuspended in D10 medium and the number of viable cells per ml is counted. Cells are diluted to 0.1e5 cells/ml and seeded at 100 µl per well (10,000 cells per well) into the assay plate. For CAGA12 cells, cells are passaged at a density of 1.5 ppm per T75 flask to be used for analysis on day 2. Cultures are incubated at 37°C and 5% CO ₂ .

On day 1, cells are transfected. The manufacturer's protocol for transfection with Lipofectamine 3000 reagent is followed. Briefly, the following are diluted in OptiMEM I at 5 μl per well: 0.1 μg DNA (presenting molecule) + 0.1 μg DNA (pro-TGFβ1), 0.4 μl P3000 and up to 5 µl with OptiMEM I. Mix the well by pipetting the DNA together. OptiMEM is then added. Add P3000 and mix everything well with a pipette. A master solution is made with Lipofectamine 3000, for adding DNA to mixtures: 0.2 µl Lipofectamine 3000, up to 5 µl in OptiMEM I, per well. Diluted Lipofectamine 3000 is added to the DNA, mixed well with a pipette and incubated at room temperature for 15 minutes. After incubation, the solution is mixed several times with a pipette, and then 10 µl per well of DNA:lipofectamine 3000 (2x5 µl) is added. The cell plate is returned to the tissue culture incubator for ~24 hours.

On day 2, antibody and TGF reporter cells are added. To prepare functional antibody dilutions, the original antibody in vehicle (PBS is optimal) is serially diluted. Each spot is then diluted in assay medium to 2 times the final antibody concentration. After antibody production, the cell plate is washed twice with assay medium by aspiration (vacuum aspirator) followed by the addition of 100 μl per well of assay media. After the second wash, the assay medium is replaced with 50 μl per well of 2× antibody. The plate with cells is returned to the incubator for ~15-20 minutes.

To prepare CAGA12 cells (clone 4A4) for analysis, cells were detached with trypsin and pelleted (rotation 5 min at 200xg). The pellet is resuspended in assay medium and the number of viable cells per ml is counted. Cells are diluted to 0.36 cells/ml and seeded at 50 µl per well (15,000 cells per well). The cells are returned to the incubator.

On Day 3, the assay is read 16-20 hours after addition of the antibody and/or reporter cell. Allow the reagent and Bright-Glo test plate to reach room temperature before reading. Readout settings in BioTek Synergy H1 are configured to use the TMLCstd protocol - this method has an automatic gain setting. Positive control wells are set to autoscale (high). Add 100 µl of Bright-Glo reagent to each well. Incubate for 2 minutes with shaking at room temperature; protecting the tablet from light. The plate is read on a BioTek Synergy H1.

Data obtained from this assay reflect TGFe1 activity in cell supernatants. Raw data units are relative light units (RLU). Samples with high RLU values contain high amounts of free TGFe1, samples with low RLU values contain low levels of TGFe1.

Example 6 Ab1 and Ab2 inhibit endogenous TGFe1 in human and mouse fibroblasts.

To determine whether Ab1 and Ab2 are able to inhibit endogenous TGF-βΠ secreted by primary cultured fibroblasts of various origins, an in vitro quantitative assay was performed in which the activity of secreted TGF-e1 was determined by measuring the levels of luciferase produced by mink lung epithelial cells that were stably transfected with nucleic acid. acid containing a luciferase reporter gene fused to the synthetic CAGA12 promoter and cocultured with fibroblasts treated with either Ab1 or Ab2. As shown in FIG. 7G and 7H, both Ab1 and Ab2 inhibited endogenous TGF-βΠ secreted by normal human dermal fibroblasts, C57BL.6J murine lung fibroblasts, and DBA2/J muscle fibroblasts. The differences in maximum inhibition observed with each antibody were cell line specific.

Example 7. The role of matrix stiffness and the influence of TGFe1-specific, context-independent antibodies on integrin-induced TGFe1 activation in vitro.

To investigate whether substrates with different degrees of stiffness can modulate TGFei activation, silica-based substrates with controlled stiffness (5 kPa, 15 kPa, and 100 kPa) were used to measure integrin-dependent TGFe1 activation in primary fibroblasts coated with them. . Briefly, SW480 cells were cotransfected with pro-TGFe 1 and LTBP1 to promote extracellular presentation of the latent TGFe1 complex. Cells overexpressing ave6 integrin were added to the assay system to trigger TGFe1 activation. TGFe1 activation was determined by measuring the activation of a TGFe-responsive reporter gene. In this case, ave6 integrin caused an approximately twofold increase in LTBP1-mediated TGFe1 activation in cells plated on high-stiffness (100 kPa) silica substrates compared to cells cultured on lower (5 or 15 kPa) stiffness silica substrates, all else being equal. conditions. We have found that isoform-specific, context-permissive inhibitors of TGFe1 activation, such as those described herein, can suppress this effect, reducing TGFe1 activation to approximately half the level compared to no antibody control at all stringencies tested.

Example 8. Effect of TGFe1-specific, context-independent antibodies on protease-induced TGFe1 activation in vitro.

To examine integrin-independent, protease-dependent activation of TGFe1 in vitro, purified recombinant LTBP3-pro-TGFe1 complex was incubated with kallikrein (KLK), and TGFe1 activation was measured using a reporter cell system as described. TGFe1 was released from the latent complex after incubation with KLK, but not with vehicle alone, indicating that the ECM-associated activity of TGFe1 may be triggered in a protease-dependent manner.

To further examine the ability of an isoform-specific, context-independent inhibitory antibody to inhibit an alternative mode (eg, integrin-independent) of TGFe1 activation, an in vitro assay was performed to assess kallikrein activation of TGFe1.

Briefly, CAGA reporter cells were seeded 24 h before the assay. pro-TGFe-C4S was titrated onto CAGA cells. Plasma KLK protease was added at a fixed concentration of 1 microgram per ml or 500 nanograms per ml. The assay mixture was incubated for approximately 18 hours. TGFe activation was measured by luciferase assay. The data is shown in Fig. 8. In the presence of KLK, pro-TGFe 1 is activated (positive control). This activation of TGFe was effectively inhibited by the addition of Ab3, indicating that in addition to integrin-dependent activation of TGFe1, an isoform-specific, context-independent inhibitory antibody can also block KLK-dependent activation of TGFe1 in vitro. Similarly, inhibition of KLK-activated TGFe1 was also observed with the addition of Ab1 (data not shown).

Example 9 Expression of LRRC33 in polarized and activated macrophages.

TGFe signaling has previously been described to be involved in macrophage maturation, differentiation, and eventual phenotypes. Monocyte-derived macrophages are predicted to express LRRC33. Additional studies of polarized macrophages showed that not all polarized macrophages express LRRC33. The present inventors have found that so-called classical M1 macrophages exhibit low expression of LRRC33, while M2 macrophages exhibit increased expression of LRRC33. Surprisingly, among the M2 macrophage subtypes, we observed LRRC33 expression only in M2c and M2d TAM-like macrophages. The first is the so-called profibrotic macrophages, and the second is a TAM-like or mimicking tumor-associated phenotype. These results indicate that LRRC33 expression is restricted to a selective subset of polarized macrophages.

Evidence suggests that tumor cells and/or surrounding tumor stromal cells secrete a range of cytokines, growth factors, and chemokines that can influence the phenotypes (eg, activation, differentiation) of various cells in the TME. For example, macrophage colony-stimulating factor (M-CSF, also called CSF-1) is a known tumor factor that may regulate TAM activation and phenotype.

Fluorescence-activated cell sorting (FACS) assays were performed to examine the effect of M-CSF exposure on LRRC33 expression in macrophages. Briefly, human PBMCs were collected from healthy donors. Primary cells were cultured for one week in medium containing 10% human serum plus GM-CSF or M-CSF. To induce different phenotypes of M2 macrophages, cells were cultured for an additional 2-3 days in the presence of IL-10 and TGFe for the M2c subtype and IL-6 for the M2d subtype. Antibodies against cell surface markers as shown in the figure were used in FACS analyses. CD14+ immunomagnetic selection indicates monocytes.

Surprisingly, the results showed that cell surface LRRC33 activation on macrophages was significantly increased by exposure to M-CSF (also known as CSF-1). In fig. 10A shows that M-CSF-treated macrophages uniformly correspond to M2-polarized macrophages. In addition, exposure to M-CSF caused macrophages to uniformly express LRRC33 on the cell surface (see Fig. 10B). As shown in FIG. 10C, stable expression was observed

LRRC33 on M-CSF-activated macrophages. These results suggest that tumor-derived factors such as M-CSF may induce activation of local macrophages to support tumor growth.

Example 10. Effect of Ab3 on the activity of regulatory T cells (Treg) in vivo.

GARP has been shown to be expressed on regulatory T cells. The effect of Ab3 on T cell regulatory activity in vivo was assessed using the T cell transfer colitis model (Powrie et al., 1993 International Immunology, 5(11): 1464-1474; Powrie et al., 1994 Immunity, 1: 553 -562; Powrie et al., 1996 J. Exp. Med., 186: 2669-2674). Co-transfer of CD45Rbhi T cells into severe combined immunodeficiency (SCID) mice is known to cause colitis, and co-transfer of CD45Rblo CD25+ regulatory T cells (Tregs) inhibits the development of colitis and is protective in mice. As shown in FIG. 11, mice treated with 30 mg/kg Ab3 abolished the protective effect demonstrated by co-transfer of CD45Rblo CD25+ Tregs. Specifically, mice treated with 30 mg/kg Ab3 showed a significant increase in proximal colon inflammation score and colon weight-to-length ratio, as well as a significant decrease in body weight gain compared to the IgG control. These data demonstrate that Ab3 is capable of suppressing T cell regulatory activity in vivo.

Example 11. Effect of Ab1 and Ab2 alone or in combination with an anti-PD-1 antibody on tumor progression in the MC38 syngeneic mouse model of colon carcinoma.

To evaluate the effect of Ab1 and Ab2, alone or in combination with anti-PD-1 antibody, in reducing colon carcinoma tumor progression, a syngeneic C38BL/6 mouse model of MC38 mouse colon carcinoma was used.

Tumor cell culture.

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

In vivo implantation and tumor growth.

MC38 cells used for implantation were harvested during the logarithmic growth phase and resuspended in phosphate-buffered saline (PBS). On the day of tumor implantation, each study mouse was injected subcutaneously into the right flank with 5 x 105 cells (0.1 ml cell suspension), and tumor growth was monitored when the average size approached the target range of 80 to 120 mm ³ . Eleven days later, designated as day 1 of the study, mice were sorted according to the calculated tumor size into groups, each of which consisted of twelve animals with individual tumor volumes ranging from 63 to 196 mm ³ and group average tumor volumes from 95 to 98 mm ³ . Tumors were measured in two dimensions using a caliper and the volume was calculated using the formula:

Tumor volume (mm ³ ) = w ² x / where w = width and l = length in mm of the tumor. Tumor weight can be estimated with the assumption that 1 mg is equivalent to 1 mm ³ tumor volume.

Treatment.

Briefly, eight-week-old female C57BL/6 mice (n=12) bearing subcutaneous MC38 tumors (63-172 mm ³ ) on day 1 were treated intraperitoneally (ip) twice weekly for four weeks with either Ab1, Ab2, mouse IgG1 control antibody ( each at a dose of 30 mg/kg in a dosage volume of 10 ml/kg). When tumors reached 150 mm ³ (day 6) in the control groups, mice were treated with either rat anti-mouse PD-1 antibody (RMP1-14) or rat IgG2A control antibody, i.e. twice a week for two weeks (each antibody at a dose of 5 mg/kg in a dosage volume of 10 ml/kg).

Group 1 served as a tumor growth control and received a mouse IgG1 isotype control antibody in combination with a rat IgG2a control antibody. Group 2 received Ab1 in combination with a control rat IgG2a antibody. Group 3 received Ab2 in combination with a control rat IgG2a antibody. Group 3 received a mouse IgG1 control antibody in combination with an 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 control group.

Endpoint and tumor growth delay (TGD) analysis.

Tumors were measured using calipers twice a week, and each animal was euthanized when its tumor reached an endpoint volume of 1000 mm ³ or at the end of the study (day 60), whichever occurred first. Mice that exited the study at the tumor volume endpoint were recorded as sacrificed at the time of tumor progression (TP) with a date of euthanasia. Time to end point (TTE) for the assay was calculated for each mouse according to the methods described in US Provisional Application No. 62/558311, filed September 13, 2017.

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MTV and regression response criteria.

The effectiveness of treatment can be determined by the volume of tumors in animals remaining in the study on the last day. MTV (n) was defined as the average tumor volume on the last day of the study from the remaining animals (n) whose tumors did not reach the final volume.

The effectiveness of treatment can also be determined by the frequency and magnitude of regression responses observed during the study. Treatment can cause partial regression (PR) or complete regression (CR) of the tumor in the animal. In the PR response, tumor volume was 50% or less of its volume on day 1 for three consecutive measurements during the study and equal to or greater than 13.5 mm ³ for one or more of these three measurements. In the CR response, tumor volume was less than 13.5 mm ³ for three consecutive measurements during the study. An animal with a CR response at the end of the study was further classified as a tumor-free survivor (TFS). Animals were monitored by regression reactions.

Inhibition of tumor growth.

The tumor growth inhibition (TGI) assay assesses the difference in mean tumor volumes (MTV) between treated and control mice. For this study, the end point for determining TGI was day 29, when control mice reached a mean tumor volume of 1500 mm ³ . MTV(n), the average 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 determined as the difference between the MTV of the indicated control group and the MTV of the drug-treated group, expressed as a percentage of the MTV of the control group.

The TGI analysis data set included all mice in the group except those that died due to treatment-related (TR) or non-treatment-related (NTR) causes prior to the day of TGI analysis.

In the present study, Ab1 and Ab2 were evaluated alone and in combination with anti-PD-1 in a syngeneic mouse model of C57BL/6 MC38 colon carcinoma. Mice treated with Ab2 in combination with anti-PD-1 had significant TGI values at day 29 (P < 0.05, Mann-Whitney U test), which had a positive effect on survival that was statistically significantly different from controls exposed to vehicle using the log-rank test in a survival analysis (P < 0.05, log-rank test) (see FIG. 16). Mice treated with Ab1 or Ab2 in combination with a rat IgG2a control antibody exhibited 1 CR and 1 PR regression responses, respectively. In combination with anti-PD-1, the regression responses of AB1 and Ab2 were 1 PR and 1 CR and 4 CR, respectively. Ab2 in combination with anti-PD-1 produced significant short-term efficacy at day 29 and an overall survival benefit in this 60-day TGD study in the C38BL/6 syngeneic mouse model of MC38 mouse colon carcinoma.

Example 12: In vivo survival effects of Ab3 in combination with a PD-1 inhibitor in the TGFe1/3 model.

EMT-6 is an orthotopic mouse tumor model in which immune checkpoint inhibitor treatment alone has shown limited effects on tumor growth and survival. The present inventors have discovered that several syngeneic tumor models express multiple TGFe isoforms, as assessed by RNAseq. Both TGFe1 and TGFe3 are dominant in EMT-6 (see Fig. 21), which are expressed in almost equal amounts. The present inventors therefore conclude that in this particular model, a pan-TGFe isoform inhibitor may provide broader in vivo efficacy compared to an isoform-selective inhibitor.

Study design.

To test this hypothesis, female Balb/c mice aged 8–12 weeks were injected subcutaneously into the flank with 0.1 ml containing 5x106 EMT6 breast cancer cells in 0% Matrigel. Animals were monitored every two weeks throughout the study to measure tumor weight and size. When the tumors reached a volume of 30-80 mm ³ , the animals were randomized into 6 groups and dosing was started as follows: group 1: HuNeg-rIgG1/HuNeg-mIgG1; group 2: anti-PD1-rIgG1/HuNeg-mIgG1; group 3: anti-PD1-rIgG1/pan-TGFe Ab-mIgG1; group 4: anti-PD1-r[gG1/Ab3-mIgG1; group 5: HuNeg-rIgG1/pan-TGFe Ab-mIgG1 and group 6: HuNeg-rIgG1/Ab3-mIgG1. The anti-PD1 clone was RMP1-14 (BioXCell), which was administered at a dose of 5 mg/kg twice weekly. HuNeg-rIgG1 was used as an isotype control and administered similarly. Ab3-mIgG1 was administered at a dose of 30 mg/kg once a week, and HuNeg-mIgG1 was administered in a similar manner. Pan-TGFe Ab-mIgG1 was dosed at 5 mg/kg twice a week. All doses were administered intraperitoneally at 10 ml/kg. When tumors exceeded 2000 mm ³ , animals were sacrificed, serum was collected, and the tumor was removed and snap frozen for later analysis. No animals were sacrificed due to significant weight loss, and one animal in group 2 was found dead (not determined to be related to treatment).

Results.

EMT6 is a rapidly progressing syngeneic tumor model. Animals in Group 1 and Group 6 had a median survival of 18 days, which is typical of the treatment effect in this model. Anti-PD1 is known to have limited effect in this model and, as such, increased median survival to 19.5 days when administered alone (arm 2). Group 5 also had a slight increase in median survival to 21 days. Group 4 saw a modest increase in survival to 25 days with two animals still alive at 34 days. In group 3 there were only 3 deaths by day 34, indicating a significant effect on survival of this combination. Inhibition of TGFe1 by administration of Ab3-mIgG1 alone had no effect on tumor volume growth, however, when combined with anti-PD1, 5 animals showed slower tumor growth and one animal showed a complete response. Pan-TGFe antibody alone slowed tumor growth in 3 animals, but when combined with anti-PD1, 4 animals had significantly slower tumor growth, and 5 animals had a complete response. These results are consistent with publicly available information, such as the tumor-wide RNAseq database (Crown Bioscience MuBase) showing that EMT6 tumors exhibit almost equal levels of TGFe1 and TGFe3 expression.

Example 13. Effect of Ab2 and Ab3 on renal biomarkers and fibrosis in a unilateral ureteral occlusion (UUO) model.

The mouse unilateral ureteral occlusion model 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. Nephrol. 13: 612-9). UUO mice are characterized by renal myofibroblast activation, tubular atrophy and interstitial fibrosis with minimal glomerular involvement (see Lian et al. (2011) Acta Pharmacol. Sin. 32: 1513-21). Increased expression of TGFe1 is thought to play a role in the phenotype observed in UUO mice. To evaluate the effect of Ab2 on the presentation of interstitial fibrosis in a mouse model of UUO, the following experiment was performed.

Briefly, 7-8 week old male CD-1 mice (Charles River Laboratories) in 4 groups of mice (n=10) were treated with either Ab2 (3 mg/kg or 30 mg/kg; dosage volume 10 ml/kg), mouse control IgG1 antibody (30 mg/kg; dosage volume 10 ml/kg) or PBS, as vehicle control, intraperitoneal (i.p.) before surgery. Treatment was carried out 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 nasal cone anesthesia and laparotomy was performed followed by permanent right unilateral UUO surgery. An additional control group of mice (n=8) were treated with PBS as described above but only underwent sham surgery (ie, laparotomy without ureteral occlusion). Immediately after completion of surgery, all mice received a single 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 content was assessed to assess the collagen content of renal tissue. Kidney hydroxyproline content, a marker of tissue fibrosis and collagen deposition, was significantly increased in surgically treated mice compared with sham-surgery-treated mice.

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, sectioned (three consecutive 5 µm sections taken at a distance of 200-250 µm per animal kidney to allow better sampling and representation of kidney lesions), stained with picrosirius red and subjected to quantitative histological analysis using color segmentation spectrum to determine the volume fraction of cortical collagen (CVF). One composite CVF score was calculated for each animal by determining the average CVF score for each of the three serial sections. Statistical analysis was performed using an unpaired t test. As shown in FIG. 12K, renal cortical fibrosis, as determined by CVF, was increased in UUO-obstructed kidneys compared to control sham-surged mice. Mice receiving either 3 mg/kg or 30 mg/kg Ab2 exhibited a significant attenuation of the UUO-induced increase in CVF compared to mice receiving either vehicle control (PBS) or IgG control.

The relative levels of mRNA expression of plasminogen activator inhibitor-1 (PAI1), connective tissue growth factor (CTGF), TGFe1, fibronectin-1, α-smooth muscle actin (αSMA), monocyte chemotactic protein 1 (MCP-1), alpha 1 collagen were determined type I (Col1a1) and type III collagen alpha 1 chain (Col3a1) in collected kidney tissue (Fig. 12A-12H). The mRNA levels were normalized using the housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT1) mRNA levels. Additionally, in mice treated with 3 mg/kg or 30 mg/kg Ab2 prior to surgery, PAI-1, CTGF, TGFe1, fibronectin 1, Col1a1, and Col3a1 mRNA levels were significantly reduced compared to mice treated with 30 mg/kg IgG1 control. In mice treated with 3 mg/kg Ab2 before surgery, α-SMA mRNA levels were significantly reduced compared with mice treated with 30 mg/kg IgG1 control. In addition, in mice treated with 30 mg/kg Ab2 prior to surgery, MCP-1 mRNA levels were significantly reduced compared to mice treated with 30 mg/kg IgG1 control.

The effect of Ab3 on mRNA expression levels of known fibrosis markers was also assessed. As shown in FIG. 12I and 12J, in mice treated with 3 mg/kg or 30 mg/kg Ab3 before surgery, PAI-1 and Col1a1 mRNA levels were significantly reduced compared with mice treated with IgG1 control.

In summary, significant effects were observed in Ab2- or Ab3-treated mice in the UUO mouse model, with the exception of hydroxyproline levels. As shown in FIG. 12A-12H and 12K, Ab2 treatment significantly attenuated the UUO-induced increase in CVF and significantly reduced gene expression of known fibrosis markers such as PAI-1, CTGF, TGFe1, fibronectin 1, Col1a1 and Col3a1. Likewise, as shown in FIG. 12I-12J, Ab3 exposure significantly reduced gene expression of known fibrosis markers such as PAI-1 and Col1a1. These data demonstrate that TGFe1 is the major form of TGFe playing a role in kidney disease and that, surprisingly, TGFe2 and TGFe3 are not likely to be involved in pathogenesis.

Example 14. Effect of a TGFe1-specific, context-independent antibody on a mouse model of Alport syndrome renal fibrosis.

The Col4a3-/- mouse model is an established genetic model of autosomal recessive Alport syndrome. Mice with Alport syndrome lack functional collagen 4 A3 (Col4a3-/-) and therefore cannot form type IV collagen, which requires the α3, α4 and α5 chains. Col4a3-/- mice develop fibrosis in the kidneys consistent with human renal fibrosis, including glomerulosclerosis, interstitial fibrosis, and tubular atrophy, and all Col4a3-/- mice develop end-stage renal disease (ESRD) between 10 and 10 years of age. 30 weeks, depending on the genetic background of the mouse. The structural and functional manifestations of renal pathology in Col4a3-/- mice, coupled with the progression of ESRD, make Col4a3-/ mice an ideal model for understanding renal fibrosis. Previous reports indicate the importance of the TGFe signaling pathway in this process, and treatment with either integrin aVe6, a known TGFe activator, or a TGFe ligand decoy was reported to prevent renal fibrosis and inflammation in mice with Alport syndrome (Hahm et al. (2007) The American Journal of Pathology, 170(1): 110-125).

Ab3, which is an isoform-specific, context-independent inhibitor of TGFe1 activation, was tested for its ability to inhibit or mitigate renal fibrosis in Alport syndrome mice as follows.

F1 progeny from a 129:B16 heterozygous cross (moderate progression model) were used for the study. Antibody dosing for Ab3 began six weeks after birth, at a dose of 5 mg/kg, twice weekly (ie, 10 mg/kg/week) for the study period of six weeks. A pan-TGFe neutralizing antibody was used as a positive control (dosage 5 mg/kg twice a week), while IgG was used as a negative control. All antibodies were administered intraperitoneally. After six weeks of antibody treatment (12 weeks after birth), animals were sacrificed and kidneys were collected for analyses.

It is well documented that activation of the TGFe receptor leads to a subsequent signaling cascade of intracellular events, including phosphorylation of Smad2/3. Therefore, the effects of Ab3 antibody treatment were assessed in kidney lysate samples by measuring relative levels of Smad2/3 phosphorylation as analyzed by Cell Signaling ELISA according to the manufacturer's instructions. In fig. 15 is a graph showing the relative ratios of phosphorylated and total (phosphorylated and unphosphorylated) Smad2/3. Whole kidney lysates obtained from Ab3-treated animal samples showed a significant decrease in relative Smad2/3 phosphorylation compared with the negative control. The mean ratios were equivalent to those from the heterozygous control.

The 12-week-old F1 mice with Alport syndrome described above had early signs of renal fibrosis at the end of the study, measured by both collagen deposition (quantified by picrosirius red) and blood urea nitrogen (BUN) accumulation, each of which indicates fibrosis. Consistent with the inhibitory activity of Ab3 observed on downstream TGFe receptor signaling, tissues treated with Ab3 showed decreased signs of fibrosis. For example, the mean BUN level for control animals with Alport syndrome that did not receive Ab3 treatment was greater than 50 mg/dL, while the mean BUN level for Ab3-treated animals was reduced to less than 30 mg/dL, which suggests that Ab3 is capable of improving fibrosis.

Example 15: Effect of a TGFe1-specific, context-independent antibody on carbon tetrachloride-induced liver fibrosis.

TGFe activity appears to play a role in the pathology of organ fibrosis such as liver fibrosis. A soluble TGFBRII agent was previously reported to prevent liver fibrosis in a carbon tetrachloride (CCl4) model of liver fibrosis (Yata et al., Hepatology, 2002).

- 102 045239

Similarly, antisense inhibition of TGFei (via adenovirus delivery) attenuates liver fibrosis due to bile duct ligation (Arias et al., BMC Gastroenterology, 2003). Additionally, 1D11, a pan-TGFe antibody that neutralizes all TGFe isoforms, has been shown to reduce liver fibrosis and cholangiocarcinoma in TAA-treated rats (Ling et al., PLoS ONE, 2013).

Herein, a carbon tetrachloride (CCl4)-induced liver fibrosis mouse model was used to evaluate the effect of a context-independent activation inhibitor TGFe1 on fibrosis in vivo. Liver fibrosis was induced in male BALB/c mice by CCl4 given twice weekly for six weeks i.p. After the first two weeks of CCl4 exposure, animals were treated with a weekly therapeutic dose of Ab3 (30 mg/kg). Therapeutic antibody dosing began after two weeks and continued for four weeks.

Animals were randomized based on blood chemistry data. During the four-week Ab3 dosing period, blood samples were collected for serum AST/ALT and total bilirubin analysis. Animals were weighed twice a week to monitor body weight during the study. After the six-week study, the liver and spleen were collected and weighed to determine the liver-to-spleen weight ratio. Liver pathology was assessed histologically on picrosirius red-stained liver sections. The degree of liver fibrosis was assessed according to Masson or Picrosirius red stained sections and viewed under a 10x or 20x magnification lens throughout the entire section using the criteria listed below.

Table 14

Fibrosis assessment criteria

Grade Central vein thickening (CLV) Intersinusoidal vein (PS) Portal vein (rt) Fibrosis Areas of involvement (NS) Fiber layers (W S) 0 Normal Absent Absent Absent 1 Slightly thickened Focal Average quantity < 6 layers racing and unrelated 2 Moderately thickened Moderate amount Moderate amount < 6 layers racing and unrelated Indistinguishable Vast quantity Cirrhosis Nodule formation Thick fibrous tissue 4 — — >2/3 cut

Fibrous scores were then calculated using the formula SSS=CLV+PS+PT+2x(NSxWS), which takes into account thickening of the central vein, intersinusoidal vein, portal vein, and affected areas and tissue layers.

As shown in FIG. 14, four weekly doses of Ab3 treatment significantly reduced CCl4-induced liver fibrosis.

Similarly, the antifibrotic effects of Ab2 and Ab3 at multiple doses (3, 10, and 30 mg/kg) were examined by histological quantification (% area) of picrosirius red staining in formalin-fixed, paraffin-embedded sections of one lobe of the liver. Quantification was performed by a pathologist in a blinded manner. Consistent with the observation presented above, liver sections from antibody-treated animals showed a significant reduction in CCl4-induced fibrosis as measured by picrosirius red staining, which corresponds to the relative amounts of tissue collagen. The results showed that Ab2 and Ab3 were each effective in reducing liver fibrosis even at the lowest dose tested (3 mg/kg). More specifically, CCl4-treated animals that received Ab2 treatment at 3 mg/kg reduced collagen volume fraction (% area) to 2.03% compared to IgG control (3.356%) (p < 0.0005). Similarly, CCl4-treated animals that received Ab3 treatment at 3 mg/kg reduced collagen volume fraction (% area) to 1.92% compared to IgG control (3.356%) (p < 0.0005). Double negative control animals that received no CCl4 exposure exhibited a background collagen volume fraction of 1.14%.

Additionally, preliminary data indicate that Ab3 exposure caused a significant decrease in phosphorylated SMAD2/3 levels, as measured by ELISA as the ratio of phospho-SMAD2/3 to total SMAD2/3, indicating that the TGFe downstream signaling pathway was suppressed by administration of a context-independent TGFe1 inhibitor in vivo.

Example 16. Role of TGFe1 in muscular dystrophy.

TGFe plays multiple roles in skeletal muscle function, including inhibition of myogenesis, regulation of inflammation and muscle repair, and promotion of fibrosis. Although there is significant interest in TGFe inhibition as a therapy for a wide range of diseases,

- 103 045239 including muscular dystrophies, these treatments inhibit TGFei, TGFe2 and TGFe3 regardless of the molecular context. The lack of specificity/selectivity of these inhibitors may result in undesirable side effects resulting in clinical dosages with insufficient efficacy. Although pan-TGFe inhibitory molecules have been reported to improve muscle function and reduce fibrosis in mdx mice, whether these effects are caused by inactivation of TGFe1, β2, or β3 has not yet been resolved.

To this end, antibodies have been created that specifically block integrin-mediated activation of latent TGFe1 while sparing TGFe2 and P3. D2.mdx mice were treated with pro-TGFβ1-specific antibodies to determine the role of TGFe1 specifically in dystrophic muscle repair. The functional impact of TGFe1 inhibition on protection from contraction-induced injury as well as recovery from the same mode of injury is assessed. Histological assessment includes whether the treatment affects muscle damage, fibrosis and inflammation. In addition, potential toxicity can be assessed to determine whether the observed negative effects reported with pan-TGFe inhibition in muscle (eg, increased inflammation, long-term deficits in muscle function) are a result of TGFe1 or TGFe2/3 inhibition. To understand whether inhibition of TGFe1 in certain molecular contexts is more effective and/or has fewer negative effects (adverse events), the effectiveness of LTBP-pro-TGFe1 inhibitors in this model can be assessed to understand the role of immune cell-presented TGFe1 among those presented in extracellular matrix (ECM), potentially leading to safer and/or more effective antifibrotic treatments.

Dystrophic muscle is very susceptible to contraction-induced damage. Following injury, muscle from mdx mice exhibits a significant reduction in force generation and increased uptake of Evan's blue dye, indicating physical damage/disruption of the muscle fiber, compared to WT (Lovering, R.M., et al., Arch Phys Med Rehabil, 2007. 88(5) ): p. 61725). Therapeutics that reduce the extent of contraction-induced damage or improve recovery from injury will have significant clinical benefit for patients with muscular dystrophy (Bushby, K., et al., Lancet Neurol, 2010. 9(1): p. 77-93 ). Investigational inhibitors such as Ab1, Ab2 and Ab3 can be evaluated for their ability to: i) prevent contraction-induced damage, and ii) promote recovery from damage. The D2.mdx strain can be used for the experiments of the authors of the present invention, in contrast to the traditional mdx strain on the B10 background. These mice, generated by crossing mdx into a DBA2/J background, contain the non-protective LTBP4 variant described above and therefore exhibit disease pathology that is more severe, progressive, and similar to human disease than the standard mdx strain (Coley, W.D., et al., Hum Mol Genet, 2016. 25(1): pp. 130-45). Because D2.mdx mice are used, DBA2/J mice can serve as wild-type controls. Because DMD predominantly affects males, studies may focus on male mice.

To examine the ability of Ab1 and Ab2 to prevent/limit contraction-induced injury, 6-week-old male D2.mdx mice (n=10) were treated with 10 mg/kg/week of either IgG control, Ab1, or Ab2 for 6 weeks. For comparison with published work using a pan-TGFe inhibitor, a fourth group was dosed at 10 mg/kg/week 1D11. All antibodies are of the mIgG1 isotype, and this dose has previously been shown to be effective in the UUO model (FIGS. 12A-12K). The WT group receiving the IgG control was also included. 24 h before sacrifice, mice were injected with 1% Evans blue dye (EBD) in PBS (1% body weight volume) to assess muscle fiber damage by fluorescence microscopy. At the end of treatment, mice are subjected to an in vivo eccentric contraction protocol. Eccentric injury to the gastrocnemius muscle can be performed using the 305B muscle lever system (Aurora Scientific) as described (Khairallah, R.J., et al., Sci Signal, 2012. 5 (236): p. Ra56). Briefly, 20 eccentric contractions are performed with 1-minute rests in between, and the reduction in peak isometric strength to the eccentric phase can be taken as an indicator of muscle damage. The degree 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 described previously (Pratt, S.J., et al., Cell Mol Life Sci, 2015. 72(1): p. 153-64; Khairallah, R.J. , et al., Sci Signal, 2012. 5(236): p. ra56). The ability of Ab1 and Ab2 to reduce strength loss after injury can be assessed. Mice are sacrificed at the end of the experiment, and injured and uninjured gastrocnemius muscles can be collected for histological analysis. EBD absorption can be assessed in both muscles. The cross-sectional area of the muscle fiber and the degree of fibrosis can be measured. To determine cross-sectional area, sections of mid-abdominal muscle can be stained with wheat germ agglutinin conjugated to a fluorophore to visualize cell membranes. Sections can be digitized using fluorescence

- 104 045239 microscopy, cell boundaries are traced using predictive software, and cross-sectional area is determined using unbiased automatic measurements. For fibrosis analysis, sections can be stained with picrosirius red (PSR) and the PSR+ area per slide can be calculated.

The ability of Ab1, Ab2 or Ab3 to accelerate recovery from contraction-induced injury is assessed. DBA2/J and D2.mdx mice at 12 weeks of age can be subjected to the same eccentric contraction protocol described above. After injury, mice were divided into treatment groups (n=10) and treated with either IgG control (for WT and D2.mdx mice), 1D11, Ab1, Ab2, or Ab3 (D2.mdx only). Antibodies can be dosed at 10 mg/kg/week throughout the experiment. Peak isometric force, contraction-tetany ratio, and force-frequency ratio can be measured at 7 and 14 days post-injury to assess the effect of treatment on injury recovery. While Ab1, Ab2, and Ab3 inhibit TGFe1 release independent of the presenting molecule, selective release of TGFe1 from the extracellular matrix (i.e., presented by LTBP) may have a greater benefit in DMD by preserving TGFβ1-driven Treg activity. To answer this question, specific antibodies that inhibit LTBP-pro-TGFβ1 can also be evaluated for both their ability to prevent contraction-induced injury and to accelerate recovery from injury.

Example 17: Role of TGFe1 in skeletal muscle after acute injury.

The role of TGFe1, particularly in the regeneration of muscle fibers after muscle injury, could be explored. TGFe1-specific antibodies can be used in a cardiotoxin injury model to determine the role of TGFe1 specifically during muscle fiber regeneration. Regeneration can be assessed histologically, and functional assessments of muscle strength and quality can be made. Considering the potential benefits of TGFe1 inhibition for muscle regeneration, a therapy that produces beneficial effects without the toxicity observed with pan-TGFe inhibition would be highly beneficial. This allows investigation of the effect of specific TGFe1 inhibition on satellite cell function and may provide insight into satellite cell transplantation studies.

As described above, TGFe appears to have multiple effects on muscle biology, including inhibition of myoblast proliferation and differentiation, as well as promotion of atrophy and fibrosis (Allen, R.E. and L.K. Boxhorn, J Cell Physiol, 1987. 133(3) : pp. 567-72; Brennan, T.J., et al., ProcNatl Acad Sci USA, 1991. 88(9): p. 3822-6; Massague, J., et al., Proc Natl Acad Sci USA, 1986. 83(21): p. 8206-10; Olson, E.N., et al., J Cell Biol, 1986. 103(5): p. 1799-805; Li, Y., et al., Am J Pathol, 2004 164(3): pp. 1007-19; Mendias, C. L., et al., Muscle Nerve, 2012. 45(1): pp. 55-9; Nelson, C. A., et al., Am J Pathol, 2011. 178(6): pp. 2611-21). However, these studies either used recombinant TGFe1 in culture or injected it into mice, which may have nonphysiological effects because the growth factor is removed from its molecular context. Alternatively, researchers have used TGFe inhibitors that are not selective for TGFe1.

To evaluate the isoform-specific, context-permissive effects of TGFe1, multiple anti-TGFe1 antibodies (e.g., Ab3) can be tested for their ability to influence muscle regeneration after CTX-induced injury. These antibodies are isoform-specific and context-permissive inhibitors of TGFe1 activation such that they specifically inhibit the release of TGFe1 (as opposed to TGFe2 or TGFe3) from any presentation molecule and do not bind to mature growth factors (Fig. 4B).

Muscle regeneration could be induced in male DBA2/J mice (n=10) by injection of CTX into the right gastrocnemius muscle. One day before injury, mice can be treated with 10 mg/kg control IgG, 1D11, Ab1, or Ab2. Antibodies continue to be administered weekly until the end of the study. At 7 and 14 days after injury, muscle strength measurements can be taken in vivo using a 305C muscle lever system (Aurora Scientific Inc., Aurora, CAN). Briefly, for the plantar flexor muscle group, contractions are induced by transcutaneous electrical stimulation of the sciatic nerve in anesthetized mice, and then a series of stimulations are performed at increasing stimulation rates (pulse 0.2 ms, transmission duration 500 ms): 1.10, 20, 40, 60 , 80, 100, 150 Hz followed by final stimulation at 1 Hz. Maximum peak isometric force, the relationship between contraction and tetany, and the force-frequency relationship will be determined. After strength measurements, the injured gastrocnemius and plantaris muscles are harvested and prepared for histology. The muscle fiber cross-sectional area and %PSR+ area can be determined as described in Example 8 above.

Treatment with Ab3 may result in decreased fibrosis and improved muscle function. However, given the role of TGFe1 in regulating immune activation, it is possible that increased inflammation with antibodies could be observed, as reported with 1D11 treatment (Andreetta, F., et al., J Neuroimmunol, 2006. 175(12): p. 77-86 ). In the event that increased inflammation may limit the therapeutic effects of TGFei inhibition, context-specific antibodies can subsequently be evaluated to provide an additional degree of specificity that may limit toxicity. For example, antibodies that inhibit the release of TGFe1 only from LTBP can be used using the indications and methods described above. These antibodies may limit the release of TGFe1 only from

ECM without affecting release from Tregs or macrophages.

Example 18: Selection of appropriate TGFe1 inhibitors for muscle disorders.

Analysis of the expression of pro-TGFe 1 and its presenting molecules in healthy, regenerating and diseased muscle may provide useful information to assist in choosing the optimal therapeutic approach. Given the potential benefits of TGFe1 inhibition in muscle regeneration and repair, understanding the context of pro-TGFe 1 presentation (e.g., in the ECM or on immune cells) in skeletal muscle under different conditions (healthy, acutely injured, and chronically injured) may help inform the therapeutic utility of antibodies and ultimately provides insight into the degree of specificity/selectivity required to achieve both clinical efficacy and safety. The pattern of TGFe1 presentation may vary depending on the state of muscle health and over the course of disease, which may have implications for any therapy targeting TGFe1. Understanding the expression profiles of these molecules will also help in selecting appropriate dosing timing for potential therapeutic molecules. Using Western blotting, immunohistochemistry, and immunoprecipitation, the expression of pro-TGFe 1 and its presenting molecules can be assessed in normal, acutely injured (cardiotoxin injury), and chronically regenerating (D2.mdx mouse) muscle. The expression of these molecules can be examined specifically in key cell types or a subset of cell types (eg, satellite cells, macrophages, fibroadipogenic progenitors, etc.) under the various conditions described above.

Although the expression of TGFe isoforms has been examined in muscle from mdx mice, previous work has focused on the expression of mature growth factors (Nelson, S.A., et al., Am J Pathol, 2011. 178(6): p. 2611-21; Zhou , L., et al., Neuromuscul Disord, 2006. 16(1): pp. 32-8). Given the target specificity of the anti-TGFe1 antibodies described herein, it is important that expression profiles be examined not only for mature and pro-TGFe1, but also for presentation molecules, which should provide information regarding the source and/or context of the TGFe1 pool of interest. Ideally, it would be desirable to gain insight into the expression profiles of latent complexes, rather than just each component.

Antibodies are screened by Western blotting and IHC for the target of interest. Antibodies to mouse TGFe1-LAP, LTBP1, LTBP3 and LTBP4 are commercially available. Antibody to TGFe1-LAP (clone TW7-16B4) has been extensively characterized and is effective for both flow cytometry and Western blotting (Oida, T. and H.L. Weiner, PLoS One, 2010. 5(11): p. e15523). Antibodies to LTBP1 (ProteinTech #22065-1-AP) and LTBP3 (Millipore #ABT316) were tested in-house using SW480 cells transfected with LTBP1-pro-TGFe1 or LTBP3-pro-TGFe1 and were shown to be specific for their targets. The utility of these antibodies for IHC can be determined. Muscle from healthy and D2.mdx mice was sectioned, and antibodies were tested on frozen and FFPE sections. Antibodies can be validated by running conditions with a 100-fold excess of purified target protein or complex (made in-house) to ensure that the observed signal is specific.

Previous work identified antibodies that specifically bind to this latent complex but do not have inhibitory activity. Antigen binding by these antibodies was confirmed by ELISA (Fig. 4C) and can also be assessed for their utility in IHC (given the three-dimensional structure of these epitopes, these antibodies are unlikely to be effective as Western blotting reagents). The presence of latent TGFe1 complexes from bulk tissue can also be assessed by Western blotting or immunoprecipitation. Latent complexes can be identified by Western blotting using the same sample under reducing and non-reducing conditions. Under reducing conditions, TGFe1, LAP, and the presenting molecule are separated, and the three molecules can be identified from the same blot, but using two-color Western blot techniques. Under non-reducing conditions, the LAP:presenting molecule complex remains bound while TGFe 1 is released; the complex migrates more slowly than the empty presentation molecule and migrates together with TGFe1-LAP. Various antibodies are also assessed for their ability to immunoprecipitate latent complexes from muscle to demonstrate direct binding of TGFe1 to specific presentation molecules.

Once suitable antibodies are identified, expression in healthy, regenerating and dystrophic muscles is assessed using Western blotting and/or IHC, depending on the antibodies available. Tibialis anterior (TA) and diaphragm muscles can be harvested from DBA2/J and D2.mdx mice at 4, 8, and 12 weeks of age. For regenerating muscles, cardiotoxin can be injected into the TA

- 106 045239 DBA2/J mice at 12 weeks of age, and muscles collected 3, 7, and 14 days postinjury. Tissue from at least 4 mice could be used for each condition/time point. Co-staining experiments can also be performed to identify cell populations expressing different molecules (eg: CD11b for macrophages, FoxP3 for Tregs, MyoD for myogenic cells).

Example 19 Ab2 and Ab3 exhibit reduced toxicity compared to the ALK5 kinase inhibitor LY2109761 and pan-TGFe antibody.

To evaluate the toxicity of Ab2 and Ab3 compared to the small molecule TGF-β type I receptor kinase inhibitor LY2109761 (ALK5) and the anti-TGFe antibody (hIgG4), toxicity studies were performed in rats. The rat was chosen as the species for this safety study based on previous reports that rats are more sensitive to TGFe inhibition compared to mice. Similar toxicity observed in rats is also observed in other mammalian species such as dogs, non-human primates, and humans.

A. Phase I study.

Briefly, female F344/NHsd rats were treated with either 3 mg/kg Ab2 (group 1, n=5), 30 mg/kg (group 1, n=5), or 100 mg/kg (group 1, n=5). n=5); pan-TGFe antibody at a dose of 3 mg/kg (group 1, n=5), at a dose of 30 mg/kg (group 1, n=5) or at a dose of 100 mg/kg (group 1, n=5); LY2109761 at a dose of 200 mg/kg (group 1, n=5) or 300 mg/kg (group 1, n=5); or PBS (pH 7.4), vehicle control (group 1, n=5). Animals receiving either Ab2, pan-TGFe antibody, or vehicle control were administered once intravenously (on day 1), and rats receiving LY2109761 were gavaged once daily for 7 days (7 doses). The body weight of the animal was determined on days 1, 3 and 7 of the dosing phase. Animals were sacrificed on day 8 and necropsies were performed.

As shown in the survival data shown in FIG. 17A, Ab2 showed reduced toxicity compared to other treatment groups. All animals administered 300 mg/kg of the ALK5 kinase inhibitor LY2109761 were sacrificed in a moribund state or found dead on days 3, 6, or 7 of the study. Two animals administered 200 mg/kg LY2109761 were found dead on day 7 of the study. One animal treated with 100 mg/kg pan-TGFe antibody was found dead on day 6 of the study. All animals treated with up to 100 mg/kg Ab2 survived to final sacrifice.

Likewise, as shown in the survival data shown in FIG. 19A, rats treated with Ab3 exhibited reduced toxicity compared to other treatment groups. The animal injected with 100 mg/kg pan-TGFe antibody was found dead on day 6 of the study. All animals treated with up to 100 mg/kg Ab3 survived to final sacrifice.

In addition, the toxicity of the exposures was assessed by monitoring the body weight of the animals during the dosing phase. As shown in FIG. 18B-18E, animals treated with LY2109761 at a dose of 200 mg/kg or 300 mg/kg showed a decrease in body weight over the course of the study.

The organ weight of the animal was also assessed after killing. As shown in table. 11, an increase in heart weight was observed in animals administered >200 mg/kg LY2109761. An increase in heart weight was also observed in animals treated with >30 mg/kg pan-TGFe antibody. In animals injected with up to 100 mg/kg Ab2 or Ab3, no effect on organ weight was observed.

Table 11

Changes in organ weight in treatment groups

Treatment group Control fill L ^a LY2109761 Pan-TGFp antibody Dose size 0 200 300 3 thirty 100 (mg/mg/day) Heart Absolute mass (g) Body mass ratio 0.4084 112 NE 99 123 119 (%) Mass coefficient 0.3952 132 NE 96 122 122 brain (%) 26.3420 FROM NE 98 123 116

NE = not assessed due to early mortality.

^a Vehicle control = phosphate buffered saline (PBS), pH 7.4.

Note. Absolute mass and organ mass ratio (relative to body or brain) values for each treatment group are expressed as a percentage of the control mean.

While no macroscopic findings were observed in animals treated with up to 100 mg/kg Ab2 or pan-TGFe antibodies, abnormal sternum shape was observed in four animals of each treatment group receiving either 200 mg/kg or 300 mg/kg LY2109761 . 2.5 mL of clear fluid in the chest cavity and enlargement of the thymus due to excess fluid (ie, edema) were observed in one animal administered 300 mg/kg LY2109761, which was found dead on day 3 of the study.

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As shown in table. 12, at the microscopic level, animals treated with 200 mg/kg LY2109761 exhibited heart valve abnormalities (i.e., valvulopathy). Valvulopathy was characterized by cardiac valve thickening due to hemorrhage, endothelial hyperplasia, mixed inflammatory cell infiltrates, and/or stromal hyperplasia (see FIG. 18F, top right panel). Most animals had multiple affected valves. In addition, atrial abnormalities were found, including minimal or subtle mixed infiltrates of inflammatory cells, minimal hemorrhage, and/or minimal endothelial (endocardial) hyperplasia resulting in increased basophilic staining of the atrium in hematoxylin and eosin-stained sections. Myocardial abnormalities were also observed primarily at the base of the heart and consisted of minimal to mild degeneration/necrosis, minor hemorrhage, and/or minor mixed infiltrates of inflammatory cells. One animal treated with 300 mg/kg LY2109761 had mild necrosis with inflammation of the coronary artery. Additionally, two animals treated with 200 mg/kg LY2109761 had minimal mixed inflammatory cell infiltrates or aortic root hemorrhage.

Table 12 Microscopic cardiac abnormalities in animals receiving LY2109761

LY2109761

Dose size (mg/kg/day) 0 200 300 Heart Heart valves Valvulopathy Minimum 0 1 2 Weak 0 3 3 Moderate 0 1 0 Atrium Infiltration, mixed cellular Minimum 0 2 3 Weak 0 0 1 Hyperplasia, endothelium Minimum 0 1 3 Hemorrhage Minimum 0 1 2 Myocardium Degeneration/necroasis Minimum 0 0 1 Weak 0 1 1 Hemorrhage Weak 0 1 0 Infiltration, mixed cellular Weak 0 0 1 Coronary artery Necrosis with inflammation Weak 0 0 1 Aortic root Hemorrhage Minimum 0 1 0 Infiltration, mixed cellular Minimum 0 1 0

As shown in table. 13 and fig. 22, animals administered approximately 3 mg/kg panTGF antibody exhibited heart valve abnormalities (i.e., valvulopathy) similar to those described in animals administered LY2109761 as described above (see also FIG. 17F, lower left panel). Animals treated with 30 mg/kg pan-TGFe antibody exhibited atrial abnormalities similar to those described in animals treated with LY2109761. Animals treated with 100 mg/kg pan-TGFe antibody exhibited myocardial abnormalities similar to those described in animals treated with LY2109761, and animals treated with 30 mg/kg pan-TGFe antibody exhibited myocardial hemorrhage. One animal treated with 100 mg/kg pan-TGFe antibody had mild focal necrosis with coronary artery hemorrhage associated with mild perivascular infiltrates with mixed inflammatory cells. Bone abnormalities in animals treated with pan-TGFe antibody and LY2109761 consisted of macroscopic abnormal shape of the sternum and microscopic increased thickness of the hypertrophic zone in the sternal endplate and epiphysis of the femur and tibia; these disorders had a higher frequency and/or severity in animals treated with LY2109761 compared to the pan-TGFe antibody.

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Table 13

Microscopic cardiac abnormalities in animals receiving pan-TGFe antibody

HaH-TGFP antibody Dose size (mg/kg/day) 0 3 thirty 100 Heart Heart valves Valvulopathy Minimum 0 2 0 0 Weak 0 2 4 5 Moderate 0 0 1 0 Atrium Infiltrate, mixed cellular Minimal 0 0 1 2 Weak 0 0 1 1 Hyperplasia, endothelium Minimum 0 0 3 1 Hemorrhage Minimum 0 0 1 0 Myocardium Degeneration/necroasis Weak 0 0 0 2 Hemorrhage Minimum 0 0 2 1 Weak 0 0 1 1 Infiltrate, mixed cell, base Weak 0 0 0 1 Coronary artery Necrosis with hemorrhage Moderate 0 0 0 1 Infiltrate, mixed cell, perivascular Weak 0 0 0 1

Although minimal or minor heart valve abnormalities occurred in a small number of Ab2-treated animals, these abnormalities were considered unlikely to be related to the study device, with low frequency (per animal and number of heart valves per animal), lack of dose response, and/or or absence of concurrent bone disorders.

B. Phase II study.

In the second phase of the study, female rats were divided into groups and administered either Ab2 at a dose of 3 mg/kg (group 1, n=5), or at a dose of 30 mg/kg (group 1, n=5), or at a dose of 100 mg/kg. kg (group 1, n=5); Ab3 at a dose of 3 mg/kg (group 1, n=5), 30 mg/kg (group 1, n=5), 100 mg/kg (group 1, n=5) or 60 mg/kg (group 1, n=5) n=5); LY2109761 at a dose of 200 mg/kg (group 1, n=5); or PBS (pH 7.4) (group 1, n=5) as discussed above. Animals receiving Ab2, Ab3, or vehicle control were administered intravenously once a week for 4 weeks at a volume of 10 ml/kg, and rats receiving LY2109761 were administered an oral gavage once daily for five days. Animals were sacrificed and necropsies were performed.

Similar to observations in the phase 1 study, study device-related cardiac events were observed over a shorter period of time (ie, 5 days instead of 7 days) in animals administered 200 mg/kg LY2109761. Microscopic cardiac abnormalities were associated with increased cardiac mass in animals treated with 200 mg/kg LY2109761 or >>3 mg/kg pan-TGFe antibody.

Although minimal or minor heart valve abnormalities were observed in a small number of phase II animals treated with Ab2 or Ab3, these abnormalities were considered unlikely to be related to the study device, due to the low incidence (per animal and number of heart valves per animal), lack of dosing, response and/or lack of parallel dice results.

Additional tissues were evaluated in phase II; microscopic abnormalities were not attributed to Ab2 or Ab3. However, microscopic abnormalities have been found in the bones (sternum, femur, and tibia), liver, pancreas (arteries), thymus, thyroid, female reproductive tissues (ovary, uterus, cervix, and vagina), and mammary gland of phase II animals. which were administered 200 mg/kg LY2109761. Thymic abnormalities consisted of minimally or slightly decreased lymphocytes in the cortex, which correlated with a macroscopically small thymus and decreased thymic mass. Decreased thymic lymphocytes were consistent with a primary effect of the test device or represented a secondary stress effect (ie, increased levels of endogenous glucocorticoids). The minimal hypertrophy of thyroid follicular cells, which correlated with an increase in thyroid mass, corresponded to the induction of liver enzymes, which led to an increase in thyroxine metabolism. Increased liver mass in LY2109761-treated animals is suggestive of liver enzyme induction, but there was no microscopic correlate. Microscopic abnormalities in female reproductive tissues and mammary glands were consistent with decreased estrus cycles and correlated with decreased uterine weight. Some animals also exhibited mammary abnormalities characterized by lobular hyperplasia/hypertrophy of alveolar and/or ductal epithelial cells (i.e., masculinization), consistent with decreased estrogen levels.

C. Conclusion of the study.

In conclusion, animals treated with Ab2 and Ab3 at all study doses (3 mg/kg, 30 mg/kg, or 100 mg/kg) for 4 weeks did not exhibit toxic effects compared to background in any of the following parameters: degeneration or myocardial necrosis, atrial hemorrhage, myocardial hemorrhage, valve hemorrhage, valve endothelial hyperplasia, valve stromal hyperplasia, mixed infiltrate of inflammatory cells in cardiac valves, mineralization, necrosis with coronary artery hemorrhage, necrosis with inflammation in the aortic horse, necrosis or inflammatory cell infiltrate in cardiomyocytes and valvulopathy. Thus, exposure to isoform-specific inhibitors of TGFe1 activation unexpectedly resulted in significantly improved safety profiles, such as reduced mortality and reduced cardiotoxicity, compared with exposure to a pan-TGFe inhibitor (eg, ALK5 kinase inhibitor LY2109761 or pan-TGFe antibody).

Example 20 Selectivity for Ab3 isoforms in vivo.

To confirm isoform-selective inhibition of TGFe1 in vivo, a pharmacodynamic study was performed that assessed the effect of Ab3 on tonic phosphoSMAD2/3 levels in bronchoalveolar lavage (BAL) cells collected from healthy rats. The literature reports that under homeostatic conditions, BAL cells predominantly express TGFe2/3 but little TGFe1, whereas the latter becomes predominantly elevated under pathological conditions.

Healthy Sprague Dawley rats (approximately 6-8 weeks old, weighing 200-250 g at baseline; Charles River) were randomized by body weight to study groups and treated with the doses described below.

Animals received test antibodies (huNEG-mIgG1, anti-P6 integrin antibody, or Ab3) on days 1, 8, and 15 by intraperitoneal injection. Animals are sacrificed on day 16 to collect BAL and serum. One group of control animals was dosed with a single oral gavage (PO) of LY2109761 (a small molecule inhibitor of ALK5) at 100 mg/kg and euthanized 2 h (+/20 min) after dosing to collect BAL.

To collect BAL samples, the whole lung was washed three times with 5.0 ml of ice-cold Dulbecco's phosphate-buffered saline. The lavages were combined and immediately placed on wet ice until the next treatment. A small portion (100-150 μl) from each sample was set aside on ice for cell counting. The remaining samples were centrifuged at 1300 g (2-8°C) for >10 min. Cell pellets were immediately placed on ice. 250 μl of freshly prepared ice-cold pSMAD lysis buffer was used to lyse the pellets. Lysed samples were centrifuged at 14,000 g for 10 min (2-8°C). The resulting supernatant was aliquoted and immediately frozen in liquid nitrogen or dry ice.

Serum samples were processed by centrifugation at 2500 g, temperature 2-8°C, for 10 minutes. Serum samples were frozen at -70 to -90°C.

Phospho-SMAD2/3 assays were performed by ELISA (Cell Signaling Technologies) according to the manufacturer's instructions. The results were assessed by the ratio of phosphorylated SMAD2/3 to the total amount. As shown in FIG. 20, tonic SMAD2/3 signaling was significantly suppressed in animals treated with either the small molecule pan-TGFe inhibitor, LY2109761, or a monoclonal antibody to the β6 integrin chain, which blocks integrin-mediated activation of TGFe1/3. In comparison, animals treated with an antibody specific for the TGFe1 isoform, Ab3, maintained tonic phosphorylation levels in BAL cells, supporting the notion that Ab3 is able to selectively inhibit TGFe1 activation without impairing the homeostatic function of TGFe2 or TGFe3 in vivo.

Example 21 Ab3: a novel and highly specific TGFe1 inhibitory antibody with antifibrotic activity.

Transforming growth factor-β 1 (TGFe 1) has a variety of biological functions, including the regulation of immune responses and tissue homeostasis. Dysregulation of TGFe1 has been associated with a number of diseases, including renal fibrosis, where chronic activation is a major cause of the disease. However, due to the high homology between the growth factor TGFe1 and its relatives

- 110 045239 relatives of TGFe2 and TGFe3, true TGFei-specific inhibitors remain unclear. On the other hand, pan-TGFe inhibition may cause dose-limiting cardiac valvulopathy, leading to toxicity issues with long-term administration. TGFe are expressed as proproteins that are proteolytically cleaved into an N-terminal prodomain and a C-terminal growth factor. The prodomain remains non-covalently associated with the growth factor, preventing receptor binding. This latent TGFe complex resides on cells or in the extracellular matrix until the complex is activated by integrins, releasing the growth factor and allowing receptor binding. To identify TGFe1-specific antibodies, we targeted the prodomain, which has much lower homology to TGFe2 and TGFe3 than the growth factor. A monoclonal antibody Ab3 was identified that specifically binds to latent TGFe1, without detectable binding to latent TGFe2 or TGFe3. Ab3 has been shown to block activation of latent TGFei by integrins aVe6 or aVe8, providing a specificity not achieved by biological factors that target TGFe1 growth factor/receptor interactions. Ab3 binds and inhibits latent TGFe1 in complex with all four known TGFe presenting molecules, allowing latent TGFe1 to be targeted to multiple tissues. Ab3 blocks the activation of endogenous TGFe1 in a number of primary cells, including dermal myofibroblasts and hepatic stellate cells. Finally, the in vivo efficacy of TGFe1 inhibition by this novel mechanism was examined in the UUO model of renal fibrosis, showing that Ab3 suppresses fibrosis markers to levels similar to those achieved in pan-TGFe antibody-treated animals. Taken together, these data demonstrate that inhibition of latent TGFe1 activation is effective in a model of preclinical fibrosis and has a superior safety profile compared with pan-TGFe inhibition.

Example 22. Highly specific inhibition of TGFe1 activation by Ab1 antibody, which has antifibrotic activity.

Transforming growth factor-e 1 (TGFe 1) is a cytokine with important and diverse biological functions, including the regulation of immune responses and tissue homeostasis. TGFe are expressed as proproteins that are proteolytically cleaved into an N-terminal prodomain and a C-terminal growth factor. The secreted growth factor remains non-covalently bound to the prodomain, preventing receptor binding and signaling. Latent TGFe1 is covalently associated with presentation molecules through disulfide bonds, which link latent TGFe1 to the extracellular matrix or to the cell surface. To date, four TGFe-presenting molecules have been identified (LTBP1, LTBP3, GARP, and LRRC33). These presentation molecules play a critical role in the activation of the latent complex as they provide an anchor for integrins to exert a pulling force on latent TGFe1, thereby releasing active growth factor. Dysregulated TGFe1 activation has been associated with a number of pathologies, including fibrotic diseases, where chronic activation of TGFe1 drives myofibroblast transdifferentiation and overexpression of extracellular matrix proteins. The role of TGFe1 in controlling fibrosis has led to the development of a variety of therapeutic agents to inhibit its activity. However, inhibition by potent pan-TGFe antibodies has been found to cause dose-limiting cardiac valvulopathies, leading to concerns regarding the toxicity of this therapeutic approach. The alternative strategy of specifically targeting TGFe1 is complicated by the high homology between the growth factor TGFei and its close relatives TGFe2 and TGFe3. We targeted the prodomain of TGFe1, which has much lower homology to the prodomains of TGFe2 and TGFe3, and identified Ab3, a fully human monoclonal antibody that specifically binds to and inhibits activation of latent TGFe1 without apparent binding to latent TGFe2 or TGFe3. This novel mechanism allows for isoform specificity not achieved by biological agents that bind and block the growth factor/TGFe1 receptor interaction and prevent latent activation of TGFe1 by both aVe6 and aVe8 integrins. Ab3 binds and inhibits latent TGFe1 in a complex with all four known TGFe-presenting molecules, allowing latent TGFe1 to be targeted to many tissues. Ab3 inhibits endogenous TGFe1 in a number of primary cells in vitro, including dermal myofibroblasts and hepatic stellate cells. Additionally, the in vivo efficacy of TGFei inhibition through this novel mechanism was examined in a model of unilateral ureteral obstruction in renal fibrosis. Ab3 was found to suppress the induction of profibrotic genes to levels similar to those achieved in animals treated with the pan-TGFe antibody. Taken together, these data demonstrate that inhibition of latent TGFei activation is effective in a model of preclinical fibrosis and has a potentially superior safety profile compared with pan-TGFe inhibition.

Example 23. Bioinformatic analysis of the relative expressions of TGFei, TGFe2 and TGFe3.

To evaluate the expression of TGFe isoforms in cancer tumors, gene expression (RNAseq) data from publicly available datasets were examined. Using a publicly available online interface tool (Firebrowse) to study TGFe isoform expression in The Cancer Genome Atlas (TCGA), we first examined the differential expression of RNA encoding TGFe isoforms in both normal and cancer tissue. All tumor RNAseq datasets in the TCGA database for which normal tissue comparators were available were selected and gene expression of TGFB1, TGFB2 and TGFB3 was examined (Figure 21A). Data from the Firebrowse interface are reported as log2 reads per million kb.

(RPKM).

These data suggest that in most tumor types (gray), TGFB1 is the most widely expressed transcript of TGFe isoforms, with log2 (RPKM) values typically in the range of 4–6, versus 0–2 for TGFB2 and 2–4 for TGFB3. We also note that in some tumor types, the mean expression levels of both TGFB1 and TGFB3 are increased compared to normal reference samples (black), suggesting that increased expression of these TGFe isoforms may be associated with cancer cells. Due to the potential role of TGFe signaling in suppressing the host immune system in the cancer microenvironment, we were interested to note that TGFB1 transcripts were elevated in cancer types for which anti-PD1 or anti-PDL1 therapy is approved - these indications are marked in gray in fig. 21A.

Note that although RPKM > 1 is generally considered to be the minimum value associated with biologically relevant gene expression (Hebenstreit et al., 2011; Wagner et al., 2013), more stringent RPKM (or associated FPKM measure (see Conesa et al, 2016)) > 10 or > 30 to avoid false positives. For comparison, all three of these thresholds are shown in FIG. 21A.

The large interquartile ranges in Figs. 21A indicate significant variability in TGFe isoform expression among individual patients. To identify malignancies where at least a subset of the patient population has tumors that differentially express the TGFB1 isoform, RNAseq data from individual tumor samples in the TCGA dataset was analyzed, calculating the number of fragments per million kb. (FPKM). RPKM and FPKM are approximately equivalent, although FPKM is adjusted for double-count reads at opposite ends of the same transcript (Conesa et al., 2016). Tumor samples were scored as positive for TGFB1, TGFB2, or TGFB3 expression if the FPKM value of the transcript was >30, and the fraction of patients (expressed as %) of each cancer type that expressed each TGFe isoform was calculated (Figure 21B).

As shown in FIG. 21B, most tumor types in the TGCA dataset show a significant percentage of individual samples that are TGFB1-positive, with some cancer types, including acute myeloid leukemia, diffuse large B-cell lymphoma, and head and neck squamous cell carcinoma, expressing TGFB1 in more than 80% of all tumor samples. In accordance with the data in Fig. 21A, fewer cancer types are positive for TGFB2 or TGFB3, although some cancers show an equal or greater percentage of tumor samples that are positive for TGFB3, including invasive breast carcinoma, mesothelioma, and sarcoma. These data suggest that cancer types can be stratified by TGFe isoform expression and that such stratification may be useful in identifying patients who are candidates for treatment with TGFe isoform-specific inhibitors.

To further investigate this hypothesis, RNAseq log2 (FPKM) data from a subset of individual tumor samples were heat mapped (Figure 21C), setting a color threshold to reflect FPKM >30 as the minimum transcription level to score positive for the TGFB isoform.

Each sample is represented as a single row on a heat map, and samples are ordered by TGFB1 expression level (highest expression levels on top). According to the analysis in FIG. 21B, a significant number of samples from each cancer type are positive for TGFB1 expression. However, this notion also highlights the fact that many tumors exclusively express TGFB1 transcripts, particularly in esophageal cancer, bladder urothelium, lung adenocarcinoma, and cutaneous melanoma. Interestingly, this distortion of TGFB1 is not a feature of all cancers, as samples from invasive breast cancer show many more samples that are TGFB3 positive rather than TGFB1 positive. However, this analysis shows that the pi isoform is the predominant, and in most cases the only, member of the TGFe family present in tumors of a large number of cancer patients. Taken together with data indicating that TGFe signaling plays an essential role in immunosuppression in the cancer microenvironment, these data also indicate the utility of TGFe1-specific inhibition in the treatment of these tumors.

To identify mouse models in which the efficacy of TGFe1-specific inhibition as an antitumor agent could be tested, TGFe isoform expression was analyzed in RNAseq data from various cell lines used in mouse syngeneic tumor models. For this analysis, two views of the data were produced. First, similar to the data in FIG. 3, the present inventors generated a heat map of log2 values (FPKM) for tumors derived from each cell line (Fig. 21D, left). Since this assay was used to identify syngeneic models expressing high TGFB1 that are negative for TGFB2 and TGFB3, the present inventors were primarily interested in avoiding false negative values, and they set a positive threshold of FPKM > 1, significantly lower than in the representations in Fig. 21B and 21C.

As the data presentation in FIG. 1 clearly shows. 21D (left), a number of syngeneic tumors, including MC-38, 4T-1 and EMT6, express significant levels of both TGFe1 and TGFe3. In contrast, the A20 and EL4 models express TGFe1 almost exclusively, and the S91 and P815 tumors show a strong bias for TGFB1 expression.

To further evaluate the differential expression of TGFB1 compared to TGFB2 and/or TGFB3, minATGFb1 was calculated, defined as the lesser of log2(FPKM _TGFB1 ) log2(FPKM _TGFB2 ) or log2(FPKM _TGFB1 ) - log2(FPKM _TGFB3 ). The minATGFb1 value for each model is shown as a heat map in Fig. 21D (right) and highlights the conclusion from FIG. 21D (left) that syngeneic tumors from the A20, EL4, S91 and/or P815 cell lines may represent excellent models for studying the effectiveness of TGFe1-specific inhibitors.

Various features and embodiments of the present invention indicated in individual sections above are applicable, if necessary, to other sections accordingly. Therefore, functions specified in one section can be appropriately combined with functions specified in other sections.

Those skilled in the art will recognize or be able to determine, using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be covered by the following claims.

Claims (23)

1. The use of an antibody or an antigen-binding fragment thereof in the treatment of cancer, including solid tumors, where the antibody binds the following pro/latent TGFe 1 complexes: i) GARP pro-TGFe 1; ii) LTBP1 npo-TGFβ1; iii) LTBP3 pro-TGFe 1 and iv) LRRC33 pro-TGFe 1; wherein the antibody or antigen-binding fragment thereof does not bind TGFe2 or TGFe3 and the antibody or antigen-binding fragment thereof inhibits the release of mature TGFe1 from the pro/latent complex; a solid tumor contains infiltrated tumor-associated macrophages, tumor-associated neutrophils, or myeloid-derived suppressor cells expressing the LRRC33-pro-TGFe1 complex on the cell surface. 2. Use according to claim 1, wherein said antibody or antigen-binding fragment does not bind free mature TGFe1, which is not associated with the pro/latent complex. 3. Use according to any of the preceding claims, wherein the solid tumor is a desmoplastic tumor. 4. Use as claimed in any one of the preceding claims, wherein the antibody or antigen binding fragment thereof is a monoclonal antibody or antigen binding fragment thereof. 5. Use as claimed in any one of the preceding claims, wherein said antibody or antigen binding fragment thereof binds to the GARP-pro-TGFe1 complex, the LTBP1-pro-TGFe1 complex and the LTBP3-pro-TGFe1 complex with a dissociation constant (KD) between 50 pM and 100 nM . 6. Use as claimed in any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof binds to the GARP-pro-TGFe1 complex, the LTBP1-pro-TGFe1 complex, the LTBP3-pro-TGFe1 complex, and the LRRC33-pro-TGFe1 complex, each of which has a constant dissociation (KD) from 50 pM to 100 nM. 7. Use as claimed in any one of the preceding claims, wherein said antibody or antigen-binding fragment reduces the suppressor activity of regulatory T cells. 8. Use as claimed in any one of the preceding claims, wherein the antibody or antigen binding fragment thereof binds to the GARP-pro/latent TGFe1 complex and the LRRC33pro/latent TGFe 1 complex and triggers the internalization of said complex. 9. Use as claimed in any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof binds in a pH-dependent manner such that binding occurs at neutral or physiological pH, but said antibody or antigen-binding fragment dissociates at acidic pH. - 113 045239 10. Application according to any of the pi. 1-9, and a solid tumor includes expressing LRRC33 M2 macrophages and/or myeloid suppressor cells in the tumor microenvironment. I. Use according to claim 10, wherein the tumor microenvironment is an immunosuppressive niche from which host antitumor immune cells are excluded. 12. Application according to any of the pi. 1-11, wherein the solid tumor includes tumor-associated macrophages expressing the LRRC33-npo-TGFpl complex and/or regulatory T cells expressing the GARP-npo-TGFpl complex. 13. Application according to any of the pi. 1-12, wherein the solid tumor responds poorly or is resistant to cancer therapy selected from the group consisting of radiation therapy, chemotherapy and checkpoint inhibitor therapy. 14. Application according to any of the pi. 1-13, and a solid tumor is characterized by increased rigidity of the extracellular matrix. 15. Application according to any of the pi. 1-14, wherein cancer is associated with overexpression of one or more of the following: ΡΑΙ-l (also known as Serpine 1), MCP-1 (also known as CCL2), Coll al, Col3al, FN1, TGFpl, CTGF, α-SMA , ITGA11 and ACTA2. 16. Application according to π. 15, where cancer is associated with overexpression of ACTA2, CTGF and TGFpl. 17. Application according to any of the pi. 1-16, wherein the antibody or antigen-binding fragment thereof is used in combination therapy with a checkpoint inhibitor. 18. Use according to claim 17, wherein the checkpoint inhibitor comprises a PD-1 or PD(L)-1 antagonist. 19. Use according to any of the preceding claims, wherein the antibody or antigen-binding portion thereof is used in combination with chemotherapy. 20. Use according to any of the preceding claims, wherein the antibody or antigen-binding portion thereof is used in combination with radiation therapy. 21. A method for producing an antibody or antigen-binding fragment according to claim 1, including the steps of: screening potential antibodies or antigen-binding fragments thereof for their ability to inhibit TGFpi, which is associated with GARP, LRRC33, LTBP1 and LTBP3, and selecting antibodies or antigen-binding fragments thereof capable of inhibiting TGFpi, which is associated with GARP, LRRC33, LTBP1 and LTBP3. 22. The method of claim 21, further comprising screening the antibody or antigen binding fragment for TGFpi isoform specificity using TGF32 and/or TGFP3, wherein optionally the antibody or antigen binding fragment specifically binds and blocks activation of TGF31, but not TGF32 and/or TGF33. 23. The method of claim 21 or 22, wherein the antibody or antigen binding fragment binds to the GARP-προ TGFpi complex and the LRRC33-npo TGFpi complex and triggers internalization of said complex. 24. The method of claim 21, wherein the selection step comprises screening the antibody or antigen binding fragment for the ability to inhibit TGFpi activation on high stringency silica-based substrates.