TGFβ inhibitors and uses thereof

Abstract

Novel inhibitors of TGFβ1 activation that have durable inhibitory effects are disclosed herein. Methods directed to the selection of appropriate TGFβ inhibitors for various disease states and patient populations are also provided.

Description

[Technical Field]

[0001] Related Applications This international application claims priority under 35 U.S.C. §119(e) to and the benefit of the following applications: U.S. Provisional Application No. 62 / 959,925, filed January 11, 2020; U.S. Provisional Patent Application No. 63 / 033,904, filed June 3, 2020; and U.S. Provisional Application No. 63 / 038,413, filed June 12, 2020, the contents of each of which are expressly incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on January 6, 2021, has the filename 127036-03920_SL.txt and is 209,944 bytes in size. [Background technology]

[0003] Transforming growth factor beta 1 (TGFβ1) is a member of the TGFβ superfamily of growth factors, along with two other structurally related isoforms, TGFβ2 and TGFβ3, each of which is encoded by a separate gene. TGFβs function as pleiotropic cytokines that regulate cell proliferation, differentiation, immunoregulation (e.g., adaptive immune response), and a variety of other biological processes in both homeostasis and disease settings. The three TGFβ isoforms signal through the same cell surface receptor and initiate similar canonical downstream signaling events, including the SMAD2 / 3 pathway.

[0004] TGFβ has been implicated in the pathogenesis and progression of several pathologies, including fibrosis, cancer, and immune disorders. Often, such conditions are associated with dysregulation of the extracellular matrix (ECM). For these and other reasons, TGFβ has become an attractive therapeutic target for the treatment of various proliferative disorders, including fibrotic conditions and cancer. However, observations from preclinical studies, including in rats and dogs, have revealed severe toxicity associated with antagonizing TGFβ signaling in vivo, and to date, no TGFβ therapeutics are commercially available that are considered both safe and effective.

[0005] Previously, applicants have described a class of monoclonal antibodies that function via a novel mechanism of action to modulate growth factor signaling (see, e.g., WO 2014 / 182676 ). These antibodies were designed to take advantage of the fact that TGFβ1 is expressed as a latent pro-protein complex consisting of a prodomain and growth factor and requires an activation step to release the growth factor from the latent complex. Rather than taking the traditional approach of directly targeting the soluble growth factor itself after activation (e.g., neutralizing antibodies), this novel class of inhibitory antibodies specifically targets the inactive pro-proprotein complex itself to preemptively block the activation step upstream of the ligand-receptor interaction. Without being bound by theory, this unique mechanism of action may offer the advantage of acting at its source, i.e., by targeting latent pro-TGFβ1 complexes within the disease microenvironment before activation occurs, thereby achieving both spatial and temporal benefits. Indeed, the advantage of locally targeting tissue / cell-tethered complexes at the source, as opposed to soluble active species (i.e., mature growth factors after release from the source), is further supported by recent studies. Ishihara et al. (Sci. Transl. Med. 11, eaau3259 (2019) "Targeted antibody and cytokine cancer immunotherapies through collagen affinity") reported that when systemically administered drugs were targeted to diseased tissues by conjugating them with collagen-binding moieties, they were able to enhance antitumor immunity and reduce treatment-related toxicity compared to their non-targeted counterparts.

[0006] Thus, monoclonal antibodies (so-called "activation inhibitors") that specifically bind to TGFβ1 in an isoform-selective manner and inhibit the activation step (i.e., release of mature growth factor from the latent complex) have been generated and are disclosed, for example, in WO2017 / 156500, WO2018 / 129329, PCT / US2019 / 041373, and PCT / US2019 / 041390, the contents of each of which are incorporated herein by reference in their entirety. These isoform-specific inhibitors have demonstrated both efficacy and safety in vivo. Summary of the Invention [Problem to be solved by the invention]

[0007] While the earlier studies described above have demonstrated the utility of antibodies capable of binding to each of the known pro-TGFβ1-presenting molecule complexes and capable of inhibitory activity both in vitro and in vivo, the inventors of the present application sought to generate improved inhibitors of TGFβ1 activation with increased affinity, potency, durability, and therapeutic efficacy. [Means for solving the problem]

[0008] Disclosed herein are isoform-selective inhibitors of TGFβ1 activation that have advantageous features. More particularly, TGFβ1 inhibitors include those with slow dissociation rates (i.e., off-rates, k オフ The present invention thus is based at least on the recognition that treatment of chronic and progressive diseases such as fibrosis may require inhibitors with good durability, which may be reflected in the dissociation rate of such antibodies.

[0009] The affinity of an antibody for its antigen is typically measured by the equilibrium dissociation constant, K D The experimentally measured ratio of off and on speeds (k オフ / k オン) to K D The value of k can be calculated. オフ The value represents the antibody dissociation rate, which indicates how quickly it dissociates from its bound antigen, while k オン The K value represents the antibody association rate, which gives an indication of how quickly the antibody binds to its antigen. The latter is typically concentration-dependent, while the former is concentration-independent. D The K value is related to the antibody concentration (the amount of antibody needed for a particular experiment). D The lower the value (lower concentration), the higher the affinity of the antibody. Higher affinity antibodies have lower k relative to the reference antibody. オフ speed, higher k オン The speed may be either constant or constant.

[0010] k オフ and k オン Both rates contribute to the overall affinity of a particular antibody for its antigen, and the relative importance or impact of each component may depend on the antibody's mechanism of action. For example, a neutralizing antibody that binds to a mature growth factor (e.g., a soluble, transient TGFβ1 ligand released from a latent complex) must compete with endogenous high-affinity receptors for ligand binding in vivo. Because the ligand-receptor interaction is a local event and the ligand is short-lived, such an antibody must be able to rapidly target and sequester the soluble growth factor before the ligand can find its cellular receptor in the tissue and thus activate the TGFβ1 signaling pathway. Therefore, for a ligand-targeted neutralizing antibody to be potent, it must have a fast, i.e., high, association rate (k オン ) may be particularly important.

[0011] In contrast, applicants have argued that antibodies that inhibit TGFβ1 signaling by preventing the release of mature growth factors from latent complexes ("activation inhibitors") may preferentially benefit from a slow dissociation rate once the antibody engages its target antigen (e.g., the pro-TGFβ1 complex). Unlike neutralizing antibodies, such antibodies do not directly compete with cellular receptors; rather, they act upstream of signal transduction by targeting inactive precursor forms (e.g., the latent pro-TGFβ1 complex) that are kept dormant in the tissue environment, thus preemptively preventing TGFβ1 activation. Such antibodies may exert their inhibitory activity by preventing the release of mature growth factors from latent complexes. For example, such antibodies may function like a "clamp" to anchor active growth factors within the prodomain cage structure, keeping them in an inactive (e.g., "latent") state. Indeed, structural analysis, including epitope mapping, has provided insights into the molecular mechanisms underlying the ability of these antibodies to block TGFβ1 activation. In this regard, the Latency Lasso region of the prodomain may be a particularly useful target.

[0012] Antibodies that can remain bound to the target upon target engagement (e.g., dissociate very slowly from the latent complex) are expected to be advantageous in achieving superior in vivo efficacy due to enhanced durability of potency and / or avidity. Based on this recognition, applicants of the present disclosure have developed antibodies that have particularly low k オフ Therefore, in accordance with the present invention, preferred antibodies have fast association rates (k オン ) in contrast to the slow dissociation rate (k オフ ) can be primarily attributed to high affinity (e.g., K in the subnanomolar to picomolar range). D In some embodiments, such antibodies bind to an epitope comprising at least a portion of latent Lasso.

[0013] Accordingly, the present disclosure provides an isoform-selective inhibitor of TGFβ1 activation, wherein the inhibitor is a monoclonal antibody or antigen-binding fragment thereof that selectively inhibits TGFβ1 activation, and wherein the monoclonal antibody has a monovalent dissociation rate of 10.0e-04 or less, and optionally a K of <1.0 nM, as measured by surface plasmon resonance (SPR)-based techniques. D and wherein the antibody or antigen-binding fragment binds to human LTBP1-proTGFβ1 and / or human LTBP3-proTGFβ1 with a CDR1 of 0.05 or less, and the antibody or antigen-binding fragment comprises the following six CDRs: H-CDR1 comprising GFTFADYA (SEQ ID NO: 276), H-CDR2 comprising ISGSG(X1)AT, where optionally, X1 is A or K (SEQ ID NO: 277), H-CDR3 comprising VSSG(X1)WD(X2)D, where optionally, X1 is H, D, or Q, and further optionally, X2 is F or Y (SEQ ID NO: 278). and X4 is S or T] (SEQ ID NO: 280), and an L-CDR3 comprising QQTY(X1)VPLT [wherein, optionally, X1 is T or G] (SEQ ID NO: 281).

[0014] In a preferred embodiment, the antibody comprises an H-CDR1 comprising GFTFADYA (SEQ ID NO: 276), an H-CDR2 comprising ISGSGAAT (SEQ ID NO: 282), an H-CDR3 comprising VSSG(X1)WD(X2)D (wherein, optionally, X1 = H or Q, and further optionally, X2 = Y or F) (SEQ ID NO: 283), an L-CDR1 comprising QSISSY (SEQ ID NO: 279), an L-CDR2 comprising AASGLES (SEQ ID NO: 284), and an L-CDR3 comprising QQTYGVPLT (SEQ ID NO: 285).

[0015] In particularly preferred embodiments, the antibody comprises the six CDR sequences of Ab42, Ab46, or Ab50.

[0016] The present invention includes compositions, such as pharmaceutical compositions (e.g., formulations, medicaments) suitable for administration to a human patient, comprising at least one antibody or fragment thereof according to the present disclosure and an excipient. Accordingly, an antibody or fragment thereof according to the present disclosure can be used in the manufacture of such a medicament.

[0017] The present invention further provides therapeutic uses of such antibodies. Accordingly, the TGFβ1-selective inhibitors (e.g., monoclonal antibodies or antigen-binding fragments thereof) of the present disclosure can be used in TGFβ1-related indications in subjects. TGFβ1-selective inhibitors can be particularly advantageous for treating diseases or disorders involving dysregulation of the extracellular matrix (ECM), including, for example, fibrotic disorders (such as organ fibrosis and fibrosis associated with chronic inflammation), proliferative disorders (cancer, such as solid tumors and myelofibrosis), diseases associated with endothelial-mesenchymal transition (EndMT), diseases associated with epithelial-mesenchymal transition (EMT), diseases associated with proteases, and diseases with abnormal gene expression of specific markers described herein. TGFβ1-selective inhibitors can be used in conjunction with another treatment as a combination therapy (e.g., add-on therapy). Methods for treating such diseases or disorders, comprising administering a TGFβ1-selective inhibitor to a subject as either a monotherapy or combination therapy, are encompassed by the present invention.

[0018] The present invention involves the selection of subjects or patients likely to respond to or benefit from TGFβ1 inhibitory therapy. Related diagnostic methods, as well as methods for monitoring or determining therapeutic response to TGFβ1 inhibitory therapy, are encompassed herein.

[0019] Processes and methods for identifying or selecting TGFβ1 selective inhibitors suitable for therapeutic use are encompassed by the present invention. In preferred embodiments, the selection involves selecting inhibitors that: i) have sub-nanomolar affinity (e.g., K) for each of the human LTBP1 / 3-proTGFβ1 complexes; D<1 nM), and ii) low dissociation rates (k ) measured by a suitable in vitro binding / kinetic assay, such as surface plasmon resonance (SPR), e.g., a BIACORE®-based system. オフ The present disclosure also includes one or more antibodies or antigen-binding fragments having particularly favorable kinetic criteria, characterized by a kinetic coefficient of reactivity (KR) of 1.00E-4, for example, ≤5.00E-4. The selected antibody or antibodies are evaluated in preclinical studies, including efficacy studies and toxicity / safety studies, using appropriate preclinical models. The effective dose of the antibody or antibodies, as determined in the efficacy studies, is below a level that results in undesirable toxicity, as determined in the toxicity / safety studies. Preferably, an antibody or antibodies having a therapeutic window of at least 3-fold, 6-fold, or more preferably 10-fold is selected. An effective amount of an antibody according to the present disclosure can be from about 0.1 mg / kg to about 30 mg / kg when administered once weekly. In a preferred embodiment, the maximum tolerated dose (MTD) of an antibody according to the present disclosure is >100 mg / kg when administered once weekly for at least four weeks.

[0020] The present disclosure includes the surprising discovery that simultaneous inhibition of TGFβ3 produced a pro-fibrotic effect in mice, contrary to the conventional wisdom that inhibition of multiple isoforms is required for an anti-fibrotic effect. This observation raises the possibility that non-selective TGFβ inhibitors (such as pan-inhibitors and TGFβ1 / 3 inhibitors) may actually exacerbate fibrosis. Advantageously, the antibodies disclosed herein are isoform-selective in that they specifically target the latent TGFβ1 complex and do so with a low dissociation rate. Thus, the present disclosure recognizes that when selecting a particular TGFβ inhibitor for a patient with a fibrotic condition (e.g., a disease involving ECM dysregulation, such as cardiovascular disease), careful consideration should be given to isoform selectivity to avoid the risk of exacerbating ECM dysregulation. Accordingly, the present disclosure includes a therapeutic method for treating a subject with a fibrotic condition, comprising selecting a TGFβ inhibitor that does not inhibit TGFβ3, optionally wherein the subject has organ fibrosis or cancer, and further optionally wherein the cancer is myelofibrosis. In some embodiments, the subject has or is at risk of developing cardiovascular disease. In some embodiments, the TGFβ inhibitor is TGFβ1 selective in that it does not inhibit TGFβ2 and TGFβ3. In some embodiments, the organ fibrosis is liver fibrosis, kidney fibrosis, or pulmonary fibrosis (e.g., IPF). In some embodiments, the liver fibrosis is associated with NASH. Patients at risk of developing conditions involving fibrosis or ECM dysregulation may include those suffering from metabolic conditions such as diabetes, obesity, and NASH. [Brief explanation of the drawings]

[0021] [Figure 1] Representative OCTET® binding curves showing the association and dissociation of Ab2 with six different antigen complexes. Ab2 specifically binds to the TGFβ1 small latent complex (SLC) in an isoform-selective manner, but does not specifically bind to the mature growth factor. [Figure 2]

[0023] Figure 1 shows representative Biacore binding curves showing the association and dissociation of Ab46 with a reference antibody. A summary of the binding kinetics is provided. [Figure 3] FIG. 1 provides four graphs showing dose-dependent binding by ELISA of five antibodies (hIgG4) to the indicated LLC. [Figure 4] FIG. 1 provides four graphs showing dose-dependent binding of five antibodies (hIgG4) to LLC by ELISA. [Figure 5A] Graph showing the effect of Ab2 or Ab3 on collagen gene (Col1a1 and Col3a1) expression in UUO mice. Mice were treated with Ab3 at 3, 10, or 30 mg / kg / week or Ab2 at 3 or 10 mg / kg / week. IgG alone was used as a control. [Figure 5B] Graph showing the effect of Ab3 or Ab2 on the expression of Fn1 and Loxl2 genes in UUO mice. Mice were treated with Ab3 at 3, 10, or 30 mg / kg / week or Ab2 at 3 or 10 mg / kg / week. IgG alone was used as a control. [Figure 6] FIG. 11 summarizes the statistical significance of changes in gene expression following treatment in the UUO model (vs. UUO+IgG). [Figure 7] Figures 7A and 7B are graphs showing the relative ratios of phosphorylated to total (phosphorylated and non-phosphorylated) SMAD2 / 3 in kidneys from a genetic model of Alport syndrome treated with and without antibodies Ab2 and Ab3. Figure 7C is a graph showing the effect of Ab3 and Ab2 on gene expression in kidneys from a genetic model of Alport syndrome. [Figure 8A] 1 is a graph showing serum exposure of Ab2 in a CDHFD mouse model at 6, 8, 10, and 12 weeks. [Figure 8B] 1 is a graph showing the effect of Ab2 on SMAD2 / 3 phosphorylation in liver tissue from mice treated with CDHFD. [Figure 8C] 1 is a graph showing the correlation between the decrease in phosphorylated SMAD2 / 3 and Ab2 exposure. [Figure 8D]1 is a graph showing a comparison of the effects of Ab3 and Ab2 on SMAD2 / 3 phosphorylation in liver tissue from mice treated with CDHFD. [Figure 8E] 1 is a graph showing the effect of Ab3 and Ab2 on liver fibrosis as measured by hydroxyproline levels. [Figure 8F] 1 is a graph showing the effect of Ab2 on α-Col1 by IHC in mice treated with CDHFD. [Figure 8G] 1 is a graph showing the correlation between Ab2 exposure levels and reduction in α-Col1 levels. [Figure 9] 1 is a graph showing the effect of Ab3 and Ab2 on picrosirius red staining (PRS) in a CCL4 mouse model of liver fibrosis. [Figure 10A] Figure 10A provides an HDX-MS heat map of the Ab3 Fab-LTBP3:proTGFβ1 complex. [Figure 10B] 10B shows the regions protected by Ab3 on the surface and ribbon structure of pro-TGF-β1, and discloses SEQ ID NOs: 258-260 and 304-309, respectively, in order of appearance. [Figure 10C] Figure 10C provides an HDX-MS heat map of the Ab2 Fab-proTGFβ1 C4S complex. [Figure 10D] Figure 10D shows Ab2-protected regions on the surface and ribbon structure of pro-TGFβ1. Region 1 overlaps with the so-called "latent lasso" within the prodomain of pro-TGFβ1, while region 3 is within the growth factor domain. Sequence alignment between the three isoforms is also provided. Figure 10D discloses SEQ ID NOs: 261, 259, 262, 310, 305, 311-312, 308, and 313, respectively, in order of appearance. [Figure 11] 11 provides the crystal structure of Ab2 Fab bound to proTGFβ1, showing contact residues on proTGFβ1 and Ab2. FIG. 11 discloses SEQ ID NOs: 314-316, respectively, in order of appearance. [Figure 12A] FIG. 1 shows microscopic cardiac findings from a pan-TGFβ antibody from a 1-week rat toxicity study. [Figure 12B] FIG. 1 shows microscopic cardiac findings from Ab3 compared to an ALK5 inhibitor or a pan-TGFβ antibody from a 4-week rat toxicity study. [Figure 12] 12C and 12D show microscopic heart, bone, and lung findings from Ab3 and Ab2 compared to an ALK5 inhibitor or a pan-TGFβ antibody from a 4-week rat toxicity study. [Figure 13] 13A-13D provide the relative expression of TGFβ isoforms. [Figure 13A] FIG. 1 shows TGFβ isoform expression versus normal control (by cancer type). [Figure 13B] FIG. 1 shows the frequency of TGFβ isoform expression for each type of human cancer. [Figure 13C] FIG. 1 shows the expression of TGFβ isoforms in individual tumor samples by cancer type. [Figure 13D] FIG. 1 shows TGFβ isoform expression in a mouse syngeneic cancer cell model system. [Figure 14] Figure 1 provides a set of graphs showing the change in tumor growth (tumor volume in mm3) measured over time (days) after administration of Ab3 at 30 mg / kg or 10 mg / kg or Ab2 at 3 mg / kg or 10 mg / kg in combination with anti-PD-1 in the MBT-2 tumor model (P<0.05, Mann-Whitney U test). Anti-PD-1 alone was used as a control. [Figure 15] Graph showing median tumor volume (mm3) at day 15 in mice treated with 30 mg / kg or 10 mg / kg Ab3 or 3 mg / kg or 10 mg / kg Ab2 in combination with anti-PD-1 in the MBT-2 tumor model (P<0.05, Mann-Whitney U test). [Figure 16]Figure 1 provides a graph showing median S91 tumor volume as a function of time. The combination arms represent treatment with two different isoform-selective TGFβ1 inhibitors (Ab3 and Ab2) at two dose levels, each in combination with anti-PD-1. [Figure 17] Figures 17A and 17B provide representative immunohistochemical sections of an S91 tumor model stained for CD8+ cell markers. Figure 17A shows a tumor section from an animal treated with anti-PD-1 alone. Figure 17B shows a tumor section from an animal treated with both anti-PD-1 and a representative context-independent TGFβ1 inhibitor. [Figure 18] Figures 18A and 18B provide representative immunohistochemical sections of S91 tumors stained with macrophage markers. Figure 18A shows a tumor section from an animal treated with anti-PD-1 alone. Figure 18B shows a tumor section from an animal treated with both anti-PD-1 and a representative context-independent TGFβ1 inhibitor. [Figure 19] FIG. 1 provides a graph showing the association and dissociation of Ab2 with TGFβ1 C4S at various pHs. [Figure 20] FIG. 1 provides two graphs showing picosirius red area (%) in liver sections of CDHFD mice treated with Ab2 or control. [Figure 21] FIG. 1 provides three representative PSR staining images from CDHFD mice treated with Ab2 or control. [Figure 22] FIG. 1 provides two graphs showing hydroxyproline content (μg / mg tissue) in the liver of CDHFD mice treated with Ab2 or control. [Figure 23] FIG. 1 provides two graphs showing type 1 collagen positive area in liver sections of CDHFD mice treated with Ab2 or control. [Figure 24] FIG. 1 provides a graph showing picosirius red area (%) in kidney sections of an adenine-induced rat renal fibrosis model. [Figure 25]Figure 1 provides five representative PSR staining images from CDHFD mice treated with control, Ab2, TGFβ3 inhibitor, or both (left). Also provided is a graph showing the picosirius red area (%) in liver sections from CDHFD mice treated with Ab2, TGFβ3 inhibitor, or both compared to control (right). [Figure 26] FIG. 1 provides immunocytochemical images of mouse hepatocytes visualized with two fluorescent labels: green indicates TGFβ1 and red indicates TGFβ3. [Figure 27]

[0023] Figure 1 provides graphs showing data from a cell-based potency assay with reporter cells. TGFβ activity is inhibited by increasing concentrations of Ab2 or Ab46. [Figure 28] Figure 1 provides a graph showing the ratio of phosphorylated to total SMAD2 / 3 in the median lobe of the liver of CDHFD mice. A significant decrease in pSMAD2 / 3 was seen in the median lobe at all tested doses of Ab46 (p<0.05 (t-test)). [Figure 29]

[0023] Figure 1 provides a graph showing the percent (%) positive phospho-SMAD2 (pSMAD2) nuclei in the liver of mice after 10 weeks of a choline-deficient high-fat diet (CDHFD). A significant decrease in % positive pSMAD2 nuclei was seen in mice treated with Ab46 at a dose of 30 mg / kg. [Figure 30] Figure 1 provides a graph showing the percent (%) positive phospho-SMAD2 (pSMAD2) nuclei in the liver (left (eft) lateral lobe) of rats after 12 weeks of a CDHF diet. Human IgG (HuNeg, negative control) or Ab46 was administered on days 1 and 3 after 12 weeks of CDHFD. An ALK5 inhibitor (ALK5i, positive control) was given to a control group of rats 2 hours before harvesting. Treatment with all doses of Ab46 (3 mg / kg, 10 mg / kg, and 30 mg / kg) resulted in suppression of SMAD2 phosphorylation, as did treatment with the positive control (ALK5i). [Figure 31]Figure 1 provides a graph showing the percent (%) positive phospho-SMAD2 (pSMAD2) nuclei in rat livers after 12 weeks of a CDHF diet. Human IgG (HuNeg, negative control) or Ab46 at doses of 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg were administered on day 1 after 12 weeks of CDHFD. An ALK5 inhibitor (ALK5i, positive control) was given to a control group of rats 2 hours before harvesting. Treatment with Ab46 at doses of 10 mg / kg and 30 mg / kg appeared to completely suppress SMAD2 phosphorylation, as did treatment with the positive control (ALK5i). DETAILED DESCRIPTION OF THE INVENTION

[0022] definition In order that this disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

[0023] Advanced cancer, advanced malignancy: As used herein, the term "advanced cancer" or "advanced malignancy" has the meaning understood in the relevant art, e.g., by an oncologist, in the context of diagnosing or treating a subject / patient with cancer. Advanced malignancies involving solid tumors can be locally advanced or metastatic. The term "locally advanced cancer" is used to describe a cancer (e.g., a tumor) that has grown outside the organ where it began but has not yet spread to distant parts of the body. Thus, this term includes cancer that has spread from where it began to nearby tissues or lymph nodes. In contrast, a "metastatic cancer" is a cancer that has spread from the part of the body where it began (the primary site) to other parts of the body (e.g., distant sites).

[0024] Affinity: Affinity is the strength of binding of a molecule (such as an antibody) to its ligand (such as an antigen). It is typically measured and reported by the equilibrium dissociation constant (KD). In the context of antibody-antigen interactions, KD is the ratio of the antibody's dissociation rate ("off rate" or Koff or Kdissoci), i.e., how quickly it dissociates from its antigen, to the antibody association rate ("on rate" or Kon), i.e., how quickly it binds to its antigen. For example, an antibody with an affinity of ≦5 nM has a KD value of 5 nM or less (i.e., an affinity of 5 nM or greater), as determined by a suitable in vitro binding assay. Suitable in vitro assays, such as biolayer interferometry (BLI) and solubility equilibrium titration (e.g., MSD-SET), can be used to measure the KD value of an antibody for its antigen.

[0025] Antibody: The term "antibody" includes any naturally occurring, recombinant, modified, or engineered immunoglobulin or immunoglobulin-like structure, or antigen-binding fragment or portion thereof, or derivatives thereof, as further described elsewhere herein. Unless expressly stated to the contrary, the term "antigen," as used herein, will encompass antigen-binding fragments and functional variants thereof. Thus, the term refers to an immunoglobulin molecule that specifically binds to a target antigen, including, for example, chimeric, humanized, fully human, and bispecific antibodies. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, although in some instances, they may contain fewer chains, such as antibodies naturally occurring in camels that contain only heavy chains. Antibodies can be derived from only a single source or can be "chimeric," i.e., different portions of the antibody can be derived from two different antibodies. Antibodies, or antigen-binding portions thereof, can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. As used herein, the term antibody includes monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, and antibody fusions (sometimes referred to herein as "antibody conjugates"). In some embodiments, the term also encompasses peptibodies.

[0026] Antigen: The term "antigen" broadly includes any molecule containing an antigenic determinant within the binding region to which an antibody or binding fragment specifically binds. An antigen can be a single unit molecule (such as a protein monomer or fragment) or a complex consisting of multiple components. An antigen provides an epitope, e.g., a molecule or portion of a molecule, or a complex of multiple molecules or portions of multiple molecules, that can be bound by a selective binding agent, such as an antigen-binding protein (including, e.g., an antibody). Thus, a selective binding agent can specifically bind to an antigen formed by two or more components in a complex. In some embodiments, an antigen can be used in an animal to generate antibodies capable of binding to the antigen. An antigen can possess one or more epitopes that can interact with various antigen-binding proteins, e.g., antibodies. In the context of the present disclosure, a suitable antigen is a complex (e.g., a multimeric complex consisting of associated components) containing a pro-TGF dimer associated with a presentation molecule. Each monomer of the pro-TGF dimer contains a prodomain and a growth factor domain separated by a furin cleavage sequence. Two such monomers form a pro-TGF dimer complex. This, in turn, is covalently bound to the presentation molecule via a disulfide bond involving a cysteine residue present near the N-terminus of each pro-TGF monomer. This multimeric complex formed by the pro-TGF dimer bound to the presentation molecule is generally referred to as the large latent complex. Antigen complexes suitable for screening antibodies or antigen-binding fragments include, for example, the presentation molecule component of the large latent complex. Such presentation molecule components can be full-length presentation molecules or fragments thereof. The minimum required portion of the presentation molecule typically contains at least 50 amino acids, more preferably at least 100 amino acids, of the presentation molecule polypeptide, including two cysteine residues capable of forming covalent bonds with the pro-TGFβ1 dimer.

[0027] Antigen-binding portion / fragment: As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TGFβ1). Antigen-binding portions include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. In some embodiments, an antigen-binding portion of an antibody may be derived from an intact antibody molecule using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Non-limiting examples of antigen-binding portions include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a single-chain Fv (scFv) molecule (see, e.g., Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Nat'l. Acad. Sci. USA 85:5879-5883); and (vi) a dAb fragment (see, e.g., Ward et al. (1989) Nature 341: (See, e.g., 544-546), and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs)). Other forms of single-chain antibodies, such as diabodies, are also encompassed.The term antigen-binding portion of an antibody includes a "single-chain Fab fragment," also known as "scFab," comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following orders from N-terminal to C-terminal: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL, and the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids.

[0028] Biased: In the context of the present disclosure, the term "bias" refers to skewed or heterogeneous affinities toward or for a subset of antigens to which an antibody can specifically bind. For example, an antibody is said to be biased if its affinity for one antigen complex and its affinity for another antigen complex are unequal (e.g., greater than a five-fold affinity difference). Preferred antibodies of the present disclosure include "matrix-biased" (or "LTBP-biased") antibodies that preferentially bind to EMC-associated complexes (LTBP1-proTGFβ1 and LTBP3-proTGFβ) such that the relative affinity between at least one of the matrix-associated complexes and at least one of the cell-associated complexes (GARP-proTGFβ1 and / or LRRC33-proTGFβ1 complexes) is greater than five-fold. In comparison, antibodies characterized as "unbiased" have approximately equal affinities for such antigen complexes (e.g., less than a five-fold affinity difference).

[0029] Binding region: As used herein, a "binding region" is a portion of an antigen (e.g., an antigen complex) that can form the interface of antibody-antigen interaction when bound to an antibody or fragment thereof. Upon antibody binding, the binding region becomes "protected" from surface exposure, which can be detected by an appropriate technique, such as HDX-MS. An antibody-antigen interaction can be mediated through multiple (e.g., two or more) binding regions. A binding region can comprise an antigenic determinant, or epitope.

[0030] Cancer: As used herein, the term "cancer" refers to a physiological condition in multicellular eukaryotic organisms that is typically characterized by uncontrolled cell growth and malignant tumors. The term broadly encompasses solid and liquid malignancies, including tumors, hematological cancers (e.g., leukemia, lymphoma, and myeloma), and myelofibrosis.

[0031] Cell-associated TGFβ1 / proTGFβ1: This term refers to membrane-bound (e.g., cell surface-tethered) TGFβ1 or its signaling complexes (e.g., pro / latent TGFβ1). Typically, such cells are immune cells. TGFβ1 presented by GARP or LRRC33 is cell-associated TGFβ1. GARP and LRRC33 are transmembrane presentation molecules expressed on the cell surface of certain cells. GARP-proTGFβ1 and LRRC33-proTGFβ1 can be collectively referred to as "cell-associated" (or "cell surface") proTGFβ1 complexes that mediate cell-associated (e.g., immune cell-associated) TGFβ1 activation / signaling. This term also includes recombinant, purified GARP-proTGFβ1 and LRRC33-proTGFβ1 complexes in solution that are not physically attached to a cell membrane (e.g., in vitro assays). The average KD values of antibodies (or fragments thereof) for the GARP-proTGFβ1 complex and the LRRC33-proTGFβ1 complex can be calculated to collectively represent the affinity for the cell-associated (e.g., immune cell-associated) proTGFβ1 complex. See, e.g., column (G) of the table. Human counterparts of the presenting molecules or presenting molecule complexes may be indicated by an "h" preceding the protein or protein complex, e.g., "hGARP," "hGARP-proTGFβ1," "hLRRC33," and "hLRRC33-proTGFβ1."

[0032] Checkpoint inhibitors: In the context of this disclosure, checkpoint inhibitors refer to immune checkpoint inhibitors and have the meaning understood in the art. Typically, the target is a receptor molecule on T cells or NK cells, or the corresponding cell surface ligand on antigen-presenting cells (APCs) or tumor cells. Immune checkpoints are activated in immune cells to prevent inflammatory immunity generated against "self." Therefore, shifting the balance of the immune system through checkpoint inhibition should enable it to become fully activated and detect and eliminate cancer. The best-known inhibitory receptors implicated in regulating the immune response are cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1), PD-L1, T-cell immunoglobulin domain and mucin domain-3 (TIM3), lymphocyte-activation gene 3 (LAG3), killer cell immunoglobulin-like receptors (KIR), glucocorticoid-inducible tumor necrosis factor receptor (GITR), and V-domain immunoglobulin (Ig)-containing 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. Keytruda® is an example of a PD-1 inhibitor. A treatment or treatment regimen using one or more immune checkpoint inhibitors may be referred to as checkpoint blockade therapy (CBT).

[0033] Clinical benefit: As used herein, the term "clinical benefit" is intended to include both the effectiveness and safety of a treatment. Thus, a therapeutic treatment that achieves a desired clinical benefit is both effective and safe (e.g., has tolerable or acceptable toxicity or adverse events).

[0034] Combination therapy: "Combination therapy" refers to a therapeutic regimen for a clinical indication that includes two or more therapeutic agents. Thus, this term refers to a therapeutic regimen in which a first treatment containing a first composition (e.g., active ingredient) is administered to a patient in conjunction with a second treatment containing a second composition (active ingredient) intended to treat the same or overlapping disease or clinical condition. The first and second compositions may act on the same cellular target or on distinct cellular targets. In the context of combination therapy, the phrase "in conjunction with" means that the therapeutic effect of the first treatment overlaps in time and / or space with the therapeutic effect of the second treatment 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. When a second treatment for treating the same disease is administered to a subject who has been treated with a first treatment for the treatment of the same disease, the second treatment may be referred to as "add-on therapy" or "adjunctive therapy."

[0035] Combinatorial or combinatorial epitope: A combinatorial epitope is an epitope recognized and bound by a combinatorial antibody at a site (i.e., antigenic determinant) formed by non-adjacent portions of one or more components of an antigen that are in close proximity in three-dimensional structure to form the epitope. Thus, the antibodies of the present invention can bind to an epitope formed by two or more components (e.g., portions or segments) of the pro / latent TGFβ1 complex. A combinatorial epitope can include amino acid residues from a first component of the complex, amino acid residues from a second component of the complex, etc. Each component can be from a single protein or from two or more proteins of the antigen complex. A combinatorial epitope is formed by structural contributions from two or more components (e.g., portions or segments, such as amino acid residues) of an antigen or antigen complex.

[0036] Compete or cross-compete: When used in the context of antigen-binding proteins (e.g., antibodies or antigen-binding portions thereof) that compete for the same epitope, the term "compete" refers to competition between the antigen-binding proteins, as determined in an assay in which the antigen-binding protein being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen-binding protein to a common antigen (e.g., TGFβ1 or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, for example, solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays, solid-phase direct biotin-avidin EIAs, solid-phase direct label assays, and solid-phase direct label sandwich assays. Typically, when a competing antigen-binding protein is present in excess, it inhibits (e.g., reduces) specific binding between the reference antigen-binding protein and 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 instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more. In some instances, binding is inhibited by at least 80-90%, at least 85%-95%, or at least 95-99%. In some instances, binding is inhibited by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, a first antibody or antigen-binding portion thereof and a second antibody or antigen-binding portion thereof are assayed, for example, by BLI (e.g., BIACORE® or OCTET®), to cross-block each other for the same antigen using standard test conditions, e.g., according to the manufacturer's instructions (e.g., performing the binding assay at room temperature, about 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 overlapping, but non-identical, epitopes.In further embodiments, a first antibody or fragment thereof and a second antibody or fragment thereof may have separate (different) epitopes that are in close proximity in three-dimensional space such that antibody binding is cross-blocked through steric hindrance. "Cross-blocking" means that binding of a first antibody to an antigen prevents binding of a second antibody to the same antigen, and similarly, binding of a second antibody to an antigen prevents binding of the first antibody to the same antigen.

[0037] Complementarity-determining region (CDR): As used herein, the term "CDR" refers to a complementarity-determining region in an antibody variable sequence. Three CDRs exist in each of the heavy and light chain variable regions, and are designated CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs present in a single variable region that can bind to an antigen. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., (1987; 1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 and Chothia et al., (1989) Nature 342:877-883) have described the Kabat CDRs in detail. It has been found that certain portions within CDRs adopt nearly identical peptide backbone conformations despite the high degree of diversity at the amino acid sequence level. These portions are designated L-CDR1, L-CDR2, and L-CDR3 or H-CDR1, H-CDR2, and H-CDR3, with "L" and "H" designating the light chain and heavy chain regions, respectively. These regions have boundaries that overlap with Kabat CDRs and can be called Chothia CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs are described by Padlan (1995) FASEB J. 9: 133-139 and MacCallum (1996) J. Mol. Biol. 262(5): 732-45. Still other CDR boundary definitions may not strictly follow one of the systems herein, but still overlap with Kabat CDRs.However, they may be shortened or lengthened in light of predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding (see, e.g., Lu X et al., MAbs. 2019 Jan; 11(1):45-57). The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use CDRs defined by Kabat or Chothia.

[0038] Conformational epitope: A conformational epitope is an epitope that is recognized and bound by a conformational antibody in a three-dimensional conformation, but not by an unfolded peptide of the same amino acid sequence. A conformational epitope may be called a conformation-specific epitope, a conformation-dependent epitope, or a conformation-sensitive epitope. The corresponding antibody or fragment thereof that specifically binds to such an epitope may be called 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 antigen complex.

[0039] Constant region / domain: An immunoglobulin constant domain refers to a heavy or light chain constant domain. The amino acid sequences of human IgG heavy and light chain constant domains are known in the art.

[0040] Context-biased: As used herein, a "context-biased antibody" refers to a type of conformational antibody that binds to an antigen with differential affinity when the antigen is associated with (i.e., bound or attached to) an interacting protein or fragment thereof. Thus, a context-biased antibody that specifically binds to an epitope within proTGFβ1 may bind to LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1 with different affinities. For example, an antibody is said to be "matrix-biased" if it has higher affinity for matrix-associated proTGFβ1 complexes (e.g., LTBP1-proTGFβ1 and LTBP3-proTGFβ1) than for cell-associated proTGFβ1 complexes (e.g., GARP-proTGFβ1 and LRRC33-proTGFβ1). The relative affinity of a [matrix-associated complex]:[cell-associated complex] can be obtained by taking the average KD value of the former and the average KD value of the latter and calculating the ratio of the two, as exemplified herein. Context-biased antibodies may be biased for or against one presenting molecule-proTGFβ1 complex compared to the other presenting molecule-proTGFβ1 complex, such that the affinity (measured by KD) for the former is more than 10-fold weaker or stronger, respectively, than the average for the latter.

[0041] Context-independent: According to the present disclosure, a "context-independent antibody" that binds to proTGFβ1 has equal affinity across the four known presentation molecule-proTGFβ1 complexes, i.e., LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. Context-independent antibodies may also be characterized as "unbiased" or "balanced." Typically, context-independent antibodies exhibit an affinity bias of no more than 5-fold, such that the relative ratio of measured KD values between matrix-associated and cell-associated complexes does not exceed 5, as measured by a suitable in vitro binding assay, such as surface plasmon resonance, biolayer interferometry (BLI), and / or lysis equilibrium titration (e.g., MSD-SET).

[0042] Dissociation rate: As used herein, the term dissociation rate has the meaning understood by those skilled in the relevant art (e.g., antibody technology) to refer to a kinetic parameter measured by how fast / slow a ligand (e.g., antibody or fragment) dissociates from its binding target (e.g., antigen). Dissociation rate also refers to the "off" rate ("k オフ The relative on / off rates (i.e., k オン and k オフ ) determines the overall strength, or affinity, of the interaction, typically expressed as the dissociation constant, or K D Therefore, equal affinity (e.g., K D value) indicates fast association (high k オン ), slow dissociation (low k オフ Monovalent interactions can be measured by using monovalent antigen-binding molecules / fragments such as fAbs (Fabs), while bivalent interactions can be measured by using bivalent antigen-binding molecules such as whole immunoglobulins (e.g., IgGs).

[0043] ECM-associated TGFβ1 / pro-TGFβ1: This term refers to TGFβ1 or its signaling complexes (e.g., pro / latent TGFβ1) that are components of (e.g., deposited within) the extracellular matrix. TGFβ1 presented by LTBP1 or LTBP3 is ECM-associated TGFβ1. LTBPs are critical for the correct deposition and subsequent bioavailability of TGFβ in the ECM, and fibrillin (Fbn) and fibronectin (FN) are thought to be the main matrix proteins mediating the association of LTBPs with the ECM. The average KD values of antibodies (or fragments thereof) against the LTBP1-pro-TGFβ1 complex and the LTBP3-pro-TGFβ1 complex can be calculated to collectively represent their affinity for the ECM-associated (or matrix-associated) pro-TGFβ1 complex. See, for example, column (D) in the table. Human counterparts of presentation molecules or presentation molecule complexes may be indicated by an "h" preceding the protein or protein complex, for example, "hLTBP1," "hLTBP1-proTGFβ1," "hLTBP3," and "hLTBP3-proTGFβ1."

[0044] Effective amount: The terms "effective" and "therapeutically effective" refer to a capacity or amount sufficient to produce a detectable change in a disease parameter, e.g., a slowing, cessation, reversal, reduction, or amelioration of a symptom or downstream effect of the disease. The term encompasses, but does not require, the use of an amount that completely cures the disease. According to some embodiments, an "effective amount" (or therapeutically effective amount, or therapeutic dose) is a dosage, concentration, or dosing regimen that achieves a statistically significant clinical benefit (e.g., efficacy) in a patient population. For example, for an antibody shown to be effective in preclinical models at a dose of 3 mg / kg to 30 mg / kg, the effective amount can be said to be about 3 to 30 mg / kg.

[0045] Effective tumor control: The term "effective tumor control" can be used to refer to the degree of tumor regression achieved in response to treatment, e.g., tumors regressing by a defined percentage (e.g., <25%) of the endpoint tumor volume. For example, in a particular model, an endpoint tumor volume of 2,000 mm3 If set to , assuming a threshold of <25%, the tumor is 500 mm 3 Effective tumor control is achieved when the tumor size is reduced to less than 100%. Thus, effective tumor control encompasses complete regression. Clinically, effective tumor control includes partial response (PR) and complete response (CR) based on art-recognized criteria such as RECIST 1.1 and the corresponding iRECIST. In some embodiments, effective tumor control in a clinical setting also includes stable disease, where a tumor that is typically expected to grow at a certain rate has been prevented from growing by treatment, even if shrinkage has not been achieved.

[0046] Effector T cells: As used herein, effector T cells are T lymphocytes that respond immediately and actively to stimuli, such as costimulation, and include, but are not limited to, CD4+ T cells (also called T helper or Th cells) and CD8+ T cells (also called cytotoxic T cells). Th cells assist other white blood cells in immune processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist in active immune responses. These cells can differentiate into one of several subtypes, including Th1, Th2, Th3, Th17, Th9, or Tfh, which secrete different cytokines to promote different types of immune responses. Signaling from APCs directs T cells toward specific subtypes. On the other hand, cytotoxic (killer) T cells (TC cells, CTL, T-killer cells, killer T cells) destroy virus-infected and cancer cells and have also been implicated in graft rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules, which are present on the surface of all nucleated cells. Cytotoxic effector cells (e.g., CD8+ cells) include, for example, perforin and granzyme B.

[0047] Epitope: The term "epitope," which may also be referred to as an antigenic determinant, is a molecular determinant (e.g., a polypeptide determinant) that can be specifically bound by a binding agent, immunoglobulin, or T-cell receptor. Epitope determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural and / or charge characteristics. The epitope recognized by an antibody or antigen-binding fragment of an antibody is the component of the antigen that interacts with the CDR (e.g., complementary site) of the antibody or fragment. An epitope can be formed by contributions from several amino acid residues that interact with the CDR of the antibody to generate specificity. An antigenic fragment can contain multiple epitopes. In certain embodiments, an antibody specifically binds to an antigen when it recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0048] 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 collagen, within a tissue or organ.

[0049] Fibrotic microenvironment: The term "fibrotic microenvironment" refers to the localized disease niche within tissues where fibrosis occurs in vivo. The fibrotic microenvironment may contain disease-related molecular signatures (such as a set of chemokines and cytokines), disease-related cell populations (such as activated macrophages and MDSCs), and disease-related ECM environments (altered ECM components and / or structures). The fibrotic microenvironment is believed to support the transition of fibroblasts to α-smooth muscle actin-positive myofibroblasts in a TGFβ-dependent manner. The fibrotic microenvironment may be further characterized by the infiltration of specific immune cells (such as macrophages and MDSCs).

[0050] GARP-TGFβ1 complex: As used herein, the term "GARP-TGFβ1 complex" (or "GARP-proTGFβ1 complex") refers to a protein complex comprising a precursor or latent form of transforming growth factor-β1 (TGFβ1) protein and glycoprotein-A repeat dominant protein (GARP), or a fragment or variant thereof. In some embodiments, the precursor or latent form of the TGFβ1 protein may be referred to as a "pro / latent TGFβ1 protein." In some embodiments, a GARP-TGFβ1 complex comprises GARP covalently linked to pro / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed by the presence of a cysteine residue near the N-terminus (e.g., amino acid position 4) of the proTGFβ1 dimeric complex. In other embodiments, a GARP-TGFβ1 complex comprises GARP non-covalently linked to pro / latent TGFβ1. In some embodiments, the GARP-TGFβ1 complex is a naturally occurring complex, for example, a GARP-TGFβ1 complex in a cell. The term "hGARP" refers to human GARP.

[0051] Human antibody: As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues (e.g., mutations introduced in vitro by random or site-specific mutagenesis or in vivo by somatic mutation), e.g., in the CDRs and particularly CDR3, not encoded by human germline immunoglobulin sequences. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0052] Humanized antibody: The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences have been altered to be more "human-like," i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Additionally, a "humanized antibody" refers to an antibody, or a variant, derivative, analog, or fragment thereof, that immunospecifically binds to an antigen of interest and comprises FR regions having substantially the amino acid sequence of a human antibody and CDR regions having substantially the amino acid sequence of a non-human antibody. As used herein in the context of CDRs, the term "substantially" refers to a CDR having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence CDR of a non-human antibody. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the FR regions being of a human immunoglobulin consensus sequence. In one embodiment, the humanized antibody also comprises at least a portion of an immunoglobulin Fc region, typically that of a human immunoglobulin. In some embodiments, the humanized antibody contains a light chain and at least the variable domains of a heavy chain. The antibody may also contain the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only a humanized heavy chain. In a specific embodiment, the humanized antibody contains only a humanized variable domain of the light chain and / or a humanized heavy chain.

[0053] Hydrogen / Deuterium Exchange Mass Spectrometry (HDX-MS): HDX-MS is a well-known technique used to confirm protein structure and protein-protein interactions in solution by measuring solvent accessibility. See, e.g., Wei et al. (2014) Drug Discovery Today 19(1): 95-102. "Hydrogen / Deuterium Exchange Mass Spectrometry for Probing Higher Order Structure of Protein Therapeutics: Methodology and Applications." HDX-MS techniques can be used to determine the region or regions of an antigen bound by an antibody (i.e., "binding region"). Such binding regions may therefore contain or form epitopes.

[0054] Immunosuppression, immunosuppressive: This term refers to the ability to suppress immune cells such as T cells, NK cells, and B cells. The gold standard for assessing immunosuppressive function is inhibition of T cell activity, which can include antigen-specific and non-specific suppression. Regulatory T cells (Tregs) and MDSCs can be considered immunosuppressive cells. M2-polarized macrophages (e.g., TAMs) can also be characterized as immunosuppressive.

[0055] Isoform-specific / selective: The terms "isoform specificity" or "isoform selectivity" refer to the ability of an agent to distinguish one isoform from other structurally related isoforms (i.e., selectivity). An isoform-specific TGFβ inhibitor exerts its inhibitory activity against one isoform of TGFβ at a given concentration, but not against other TGFβ isoforms. For example, an isoform-specific TGFβ1 antibody selectively binds to TGFβ1. A TGFβ1-specific inhibitor (antibody) preferentially targets (binds to and thereby inhibits) the TGFβ1 isoform with substantially higher affinity than TGFβ2 or TGFβ3. For example, selectivity in this context can refer to at least a 500- to 1000-fold difference in the respective affinities, as measured by in vitro binding assays such as OCTET® and BIACORE®. In some embodiments, selectivity is such that the inhibitor does not inhibit TGFβ2 and TGFβ3 when used at a dose effective to inhibit TGFβ1 in vivo. For example, an antibody may preferentially bind to TGFβ1 with an affinity of about 1 pM, while the same antibody may bind to TGFβ2 and / or TGFβ3 with an affinity of about 0.5-50 nM. For such inhibitors to be useful as therapeutic agents, the dosage to achieve the desired effect (e.g., a therapeutically effective amount) must fall within a window in which the inhibitor can effectively inhibit the TGFβ1 isoform without inhibiting TGFβ2 or TGFβ3. The terms "isoform-specific" and "isoform-selective" are used interchangeably herein.

[0056] Isolated: As used herein, an "isolated" antibody refers to an antibody that is substantially free of other antibodies having different antigen specificities. In some embodiments, an isolated antibody is substantially free of other unintended cellular material and / or chemicals.

[0057] Large latent complex: In the context of the present disclosure, the term "large latent complex" ("LLC") refers to a complex consisting of a pro-TGFβ1 dimer bound to a so-called presentation molecule. Thus, large latent complexes are presentation molecule-pro-TGFβ1 complexes such as LTBP1-pro-TGFβ1, LTBP3-pro-TGFβ1, GARP-pro-TGFβ1, and LRRC33-pro-TGFβ1. Such complexes can be formed in vitro using recombinant purified components capable of complex formation. For screening purposes, the presentation molecule used to form such an LLC does not need to be a full-length polypeptide. However, a portion of the protein capable of forming a disulfide bond with the pro-TGFβ1 dimer complex via a cysteine residue near its N-terminal region is typically required.

[0058] Latency-associated peptide (LAP): LAP is the so-called "prodomain" of pro-TGFβ1. As described in more detail herein, LAP consists of a "Straight Jacket" domain and an "Arm" domain. The Straight Jacket itself is further divided into an alpha-1 helix and a latency lasso domain.

[0059] Latent Lasso: As used herein, the "latent lasso," also known as the latency loop, is a domain adjacent to the alpha-1 helix and arm in the prodomain of pro-TGFb1. In its unmutated form, the latent lasso of human pro-TGFb1 comprises the amino acid sequence: LASPPSQGEVPPGPL (SEQ ID NO: 270). As used herein, the term "extended latent lasso region" refers to the latent lasso together with the motif immediately C-terminal to it, called the alpha-2 helix (α2-helix) of the prodomain. A proline residue at the C-terminus of the latent lasso provides an "elbow"-like vertical "turn" that connects the lasso loop with the α2-helix. Certain high-affinity TGFβ1 activation inhibitors confer inhibitory potency (e.g., the ability to block activation) by at least partially binding to the latent lasso or a portion thereof, and optionally, the portion of the latent lasso is ASPPSQGEVPPGPL (SEQ ID NO: 286). In some embodiments, antibodies of the present disclosure bind to the pro-TGFβ1 complex at ASPPSQGEVPPGPL (SEQ ID NO: 286), or a portion thereof. Certain high-affinity TGFβ1 activation inhibitors confer inhibitory efficacy (e.g., the ability to block activation) by at least partially binding to an extended latent Lasso, or a portion thereof, where optionally the extended latent Lasso portion is LASPPSQGEVPPGPLPEAVLALYNSTR (SEQ ID NO: 271).

[0060] Localized: In the context of this disclosure, the term "localized" (e.g., "localized tumor") refers to an anatomically isolated or isolatable abnormality, such as a solid malignant tumor, as opposed to a systemic disease. For example, certain leukemias may have both a localized (e.g., bone marrow) and a systemic (e.g., circulating blood cells) component to the disease.

[0061] LRRC33-TGFβ1 complex: As used herein, the term "LRRC33-TGFβ1 complex" (or "LRRC33-proTGFβ1 complex") refers to a complex between the proprotein or latent form of transforming growth factor-β1 (TGFβ1) protein and leucine-rich repeat-containing protein 33 (LRRC33, also known as Negative Regulator of Reactive Oxygen Species, or NRROS), or a fragment or variant thereof. In some embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 covalently linked to pro / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed by the presence of a cysteine residue near the N-terminus (e.g., amino acid position 4) of the proTGFβ1 dimeric complex. In other embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 non-covalently linked to pro / latent TGFβ1. In some embodiments, the LRRC33-TGFβ1 complex is a naturally occurring complex, for example, a LRRC33-TGFβ1 complex in a cell. The term "hLRRC33" refers to human LRRC33.

[0062] LTBP1-TGFβ1 complex: As used herein, the term "LTBP1-TGFβ1 complex" (or "LTBP1-proTGFβ1 complex") refers to a protein complex comprising the proprotein or latent form of transforming growth factor-β1 (TGFβ1) protein and latent TGF-beta binding protein 1 (LTBP1), or a fragment or variant thereof. In some embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 covalently linked to pro / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed by the presence of a cysteine residue near the N-terminus (e.g., amino acid position 4) of the proTGFβ1 dimeric complex. In other embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 non-covalently linked to pro / latent TGFβ1. In some embodiments, the LTBP1-TGFβ1 complex is a naturally occurring complex, e.g., a LTBP1-TGFβ1 complex in a cell. The term "hLTBP1" refers to human LTBP1.

[0063] LTBP3-TGFβ1 complex: As used herein, the term "LTBP3-TGFβ1 complex" (or "LTBP3-proTGFβ1 complex") refers to a protein complex comprising transforming growth factor-β1 (TGFβ1) protein and latent TGF-beta binding protein 3 (LTBP3) precursor or latent form, or a fragment or variant thereof. In some embodiments, the LTBP3-TGFβ1 complex comprises LTBP3 covalently bound to pro / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed by the presence of a cysteine residue near the N-terminus (e.g., amino acid position 4) of the proTGFβ1 dimeric complex. In other embodiments, the LTBP3-TGFβ1 complex comprises LTBP1 non-covalently bound to pro / latent TGFβ1. In some embodiments, the LTBP3-TGFβ1 complex is a naturally occurring complex, e.g., a LTBP3-TGFβ1 complex in a cell. The term "hLTBP3" refers to human LTBP3.

[0064] M2 or M2-like macrophages: M2 macrophages represent a subset of activated or polarized macrophages and include disease-associated macrophages in both fibrotic and tumor microenvironments. Cell surface markers of M2-polarized macrophages typically include CD206 and CD163 (i.e., CD206+ / CD163+). Applicant recently discovered that M2-polarized macrophages can also express cell surface LRRC33. M2 macrophage activation is primarily driven by IL-4, IL-13, IL-10, and TGFβ. They secrete the same cytokines that activate them (IL-4, IL-13, IL-10, and TGFβ). These cells have high phagocytic capacity and produce ECM components, angiogenic, and chemotactic factors. The release of TGFβ by macrophages can perpetuate myofibroblast activation, EMT, and EndMT induction in fibrotic tissues. For example, M2 macrophages are essential for TGFβ-driven pulmonary fibrosis and are also enriched in some tumors.

[0065] Matrix-associated pro-TGFβ1: LTBP1 and LTBP3 are presentation molecules that are components of the extracellular matrix (ECM). LTBP1-pro-TGFβ1 and LTBP3-pro-TGFβ1 can be collectively referred to as "ECM-associated" (or "matrix-associated") pro-TGFβ1 complexes that mediate ECM-associated TGFβ1 activation / signaling. This term also includes recombinant, purified LTBP1-pro-TGFβ1 and LTBP3-pro-TGFβ1 complexes in solution (e.g., in vitro assays) that are not physically attached to a matrix or substrate.

[0066] Maximum tolerated dose (MTD): The term MTD generally refers, in the context of safety / toxicity considerations, to the highest dose of a test substance (such as a TGFβ1 inhibitor) evaluated at the no observed adverse effect level (NOAEL). For example, based on a 4-week toxicity study, the NOAEL for Ab2 in rats was the highest dose evaluated (100 mg / kg), suggesting that the MTD for Ab2 is >100 mg / kg.

[0067] Mesoscale Discovery: "Mesoscale discovery" or "MSD" is a type of immunoassay that uses high-binding carbon electrodes to capture proteins (e.g., antibodies). The antibody can be incubated with a specific antigen, and the binding can be detected using a secondary antibody conjugated to an electrochemiluminescent label. Upon electrical signal, light intensity can be measured to quantify the analyte in the sample.

[0068] Myelofibrosis: "Myelofibrosis," also known as bone myelofibrosis, is a relatively rare myeloproliferative disorder (e.g., cancer) that belongs to a group of diseases called myeloproliferative disorders, including primary myelofibrosis and secondary myelofibrosis. Characterized by the proliferation of abnormal clones of hematopoietic stem cells in the bone marrow and other sites, myelofibrosis leads to fibrosis, or replacement of marrow by scar tissue. Myelofibrosis is characterized by mutations that cause upregulation or hyperactivation of the downstream JAK pathway.

[0069] Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells generated during various pathological conditions and are thought to represent the pathological state of activation of monocytes and relatively immature neutrophils. MDSCs include at least two classes of cells: i) "granulocytic" (G-MDSCs) or polymorphonuclear (PMN-MDSCs), which are phenotypically and morphologically similar to neutrophils, and ii) monocytic (M-MDSCs), which are phenotypically and morphologically similar to monocytes. MDSCs are characterized by a distinct set of genomic and biochemical features and can be identified by specific surface molecules. For example, human G-MDSCs / PMN-MDSCs typically express the cell surface markers CD11b, CD33, CD15, and CD66. Furthermore, human G-MDSCs / PMN-MDSCs may also express HLA-DR and / or arginase. In comparison, human M-MDSCs typically express the cell surface markers CD11b, CD33, and CD14. MDSCs also express CD39 and CD73 to mediate adenosine signaling, which is involved in organ fibrosis (liver fibrosis, pulmonary fibrosis), cancer, and bone marrow fibrosis. Furthermore, human M-MDSCs may also express HLA-DR. In addition to these cell surface markers, MDSCT is characterized by its ability to suppress immune cells, such as NK cells, and B cells. The immunosuppressive function of MDSCs can include the inhibition of antigen-nonspecific and antigen-specific functions. MDSCs can express cell surface LRRC33 and / or LRRC33-proTGFβ1.

[0070] Myofibroblasts: Myofibroblasts are cells with a specific phenotype of fibroblasts and smooth muscle cells and generally express vimentin, alpha-smooth muscle actin (α-SMA, human gene ACTA2), and palladin. In many pathologies involving extracellular matrix dysregulation (e.g., increased matrix stiffness), normal fibroblasts dedifferentiate into myofibroblasts in a TGFβ-dependent manner. Abnormal overexpression of TGFβ is common among myofibroblast-driven pathologies. TGFβ is known to promote myofibroblast differentiation, cell proliferation, and matrix production. Myofibroblasts or myofibroblast-like cells within a fibrotic microenvironment may be referred to as fibrosis-associated fibroblasts (or "FAFs"), while myofibroblasts or myofibroblast-like cells within a tumor microenvironment may be referred to as cancer-associated fibroblasts (or "CAFs").

[0071] Off speed (k オフ ): The off-rate is a kinetic parameter of how quickly or slowly an antibody (such as a mAb) or antigen-binding fragment (such as a fAb) dissociates from its antigen, and can also be referred to as the dissociation rate. The off-rate can be measured experimentally in suitable in vitro binding assays such as OCTET® and BIACORE®-based systems.

[0072] Pan-TGFβ inhibitor / pan-TGFβ inhibitor: The term "pan-TGFβ inhibitor" or "pan-TGFβ inhibitor" refers to any agent capable of inhibiting or antagonizing all three isoforms of TGFβ. Such inhibitors may be small molecule inhibitors of TGFβ isoforms. This term includes pan-TGFβ antibodies, which refer to any antibody capable of binding to each of the TGFβ isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3. In some embodiments, the pan-TGFβ antibody binds and neutralizes the activity of all three isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3. Antibody 1D11 (or the human analog fresolimumab (GC1008)) is a well-known example of a pan-TGFβ antibody that neutralizes all three isoforms of TGFβ. An example of a small molecule pan-TGFβ inhibitor is galunisertib (LY2157299 monohydrate, CAS number 700874-72-2), an antagonist of TGFβ receptor I kinase / ALK5, which mediates signaling of all three TGFβ isoforms.

[0073] Potency: As used herein, the term "potency" refers to the activity of a drug, such as an inhibitory antibody (or fragment) having inhibitory activity, in terms of the concentration or amount of drug to produce a defined effect. For example, an antibody that can produce a specific effect at a given dosage is more potent than another antibody that requires twice the amount (dosage) to produce the same effect. Potency may be measured in a cell-based assay, such as a TGFβ activation / inhibition assay, in which the degree of TGFβ activation, such as activation triggered by integrin binding, can be measured in a cell-based system in the presence or absence of a test substance (e.g., an inhibitory antibody). Typically, among those that can bind to the same or overlapping binding region of an antigen (e.g., cross-blocking antibodies), those with higher affinity (lower K D An antibody with a lower affinity (higher K D These antibodies tend to be more potent than antibodies with higher IgG values.

[0074] Predictive biomarkers: Predictive biomarkers provide information about the probability or likelihood of response to a particular treatment. Typically, predictive biomarkers are measured before and after treatment, and changes or relative levels of the marker in samples taken from a subject indicate or predict therapeutic benefit.

[0075] Presentation molecule: In the context of the present disclosure, a presentation molecule refers to an anchor protein that can form a covalent bond with a latent precursor protein (e.g., pro-TGFβ1) and "present" the inactive complex in an extracellular niche (such as the ECM or immune cell surface), thereby maintaining its latent form until an activation event occurs. Known presentation molecules for pro-TGFβ1 include LTBP1, LTBP3, GARP (also known as LRRC32), and LRRC33, which can form presentation molecule-pro-TGFβ1 complexes (LLCs), i.e., LTBP1-pro-TGFβ1, LTBP3-pro-TGFβ1, GARP-pro-TGFβ1, and LRRC33-pro-TGFβ1, respectively. In nature, LTBP1 and LTBP3 are components of the extracellular matrix (ECM), and thus LTBP1-proTGFβ1 and LTBP3-proTGFβ1 may collectively be referred to as "ECM-associated" (or "matrix-associated") proTGFβ1 complexes that mediate ECM-associated TGFβ1 signaling / activity. On the other hand, GARP and LRRC33 are transmembrane proteins expressed on the cell surface of certain cells, and thus GARP-proTGFβ1 and LRRC33-proTGFβ1 may collectively be referred to as "cell-associated" (or "cell surface") proTGFβ1 complexes that mediate cell-associated (e.g., immune cell-associated) TGFβ1 signaling / activity.

[0076] Protection (from solvent exposure): In the context of HDX-MS-based assessment of protein-protein interactions, such as antibody-antigen binding, the degree to which a protein (e.g., a region of a protein containing an epitope) is exposed to solvent, thereby allowing proton exchange to occur, is inversely correlated with the degree of binding / interaction. Thus, when an antibody described herein binds to a region of an antigen, the binding region is "protected" from exposure to solvent because the protein-protein interaction prevents the binding region from being accessible by the surrounding solvent. Thus, the protected region is indicative of the interaction site. Typically, the appropriate solvent is a physiological buffer.

[0077] Pro-TGFβ1: As used herein, the term "pro-TGFβ1" is intended to encompass the precursor form of an inactive TGFβ1 dimeric complex containing the prodomain sequence of TGFβ1 within the complex. Therefore, this term can include the pro- and latent forms of TGFβ1. The expression "pro / latent TGFβ1" can be used interchangeably. The "pro" form of TGFβ1 resides at the furin site prior to proteolytic cleavage. After cleavage, the resulting form is said to be the "latent" form of TGFβ1. The "latent" complex remains associated until further activation, such as an integrin-driven activation event. The pro-TGFβ1 complex consists of dimeric TGFβ1 precursor protein polypeptides linked by disulfide bonds. The latent dimeric complex is covalently bound to a single presentation molecule via the cysteine residue at position 4 (Cys4) of each of the pro-TGFβ1 polypeptides. The adjective "latent" can be used generally to describe the "inactive" state of TGFβ1 prior to integrin-mediated or other activation events. The pro-TGFβ1 polypeptide contains a prodomain (LAP) and a growth factor domain (SEQ ID NO: 24).

[0078] Regression: Regression of tumors or tumor growth can be used as an in vivo efficacy measure. In preclinical settings, median tumor volume (MTV) and regression response treatment efficacy measures can be determined from the tumor volume of animals remaining in the study on the final day. Treatment efficacy can also be determined from the incidence and magnitude of regression responses observed during the study. Treatment can cause partial regression (PR) or complete regression (CR) of tumors in animals. Complete regression achieved in response to treatment (e.g., administration of a drug) is referred to as a "complete response," and subjects achieving a complete response can be referred to as "complete responders." In some embodiments of preclinical tumor models, a PR response is defined as a tumor volume that is 50% or less of its day 1 volume at three consecutive measurements during the course of the study and is 13.5 mm or less at one or more of those three measurements. 3 In some embodiments, a CR response is defined as a tumor volume greater than or equal to 13.5 mm on three consecutive measurements during the course of the study. 3 A CR response is defined as a tumor volume that is less than 2,000 mm. In preclinical models, animals with a CR response at the end of the study may be further classified as tumor-free survivors (TFS). The term "effective tumor control" may be used to refer to the degree of tumor regression achieved in response to treatment, e.g., tumor volume is reduced to <25% of the endpoint tumor volume. For example, in certain models, an endpoint tumor volume of 2,000 mm 3 If the tumor is 500 mm 3 Effective tumor control is achieved when the tumor size is reduced to less than 100%. Therefore, effective tumor control encompasses complete regression and partial regression that reaches a threshold reduction. Similarly, regression of fibrosis can be used as an in vivo efficacy measure of treatment such as TGFβ1 inhibitors. Regression of fibrotic status can be determined based on standard criteria for assessing the severity of fibrotic symptoms according to disease stage.

[0079] Regulatory T cells: "Regulatory T cells" or Tregs are a type of immune cell characterized by expression of the biomarkers CD4, FOXP3, and CD25. Tregs, sometimes called suppressor T cells, represent a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T (Teff) cells. Tregs can arise in the thymus (so-called CD4+Foxp3+ "natural" Tregs), for example, following exposure to TGFβ or retinoic acid, or can differentiate from naive CD4+ T cells in the periphery. Tregs can express cell surface GARP-proTGFβ1.

[0080] Resistance (vs. treatment): Resistance to a particular treatment (e.g., CBT) can be due to an innate feature of a disease, such as cancer ("primary resistance"), or it can be due to an acquired phenotype that develops over time after treatment ("acquired resistance"). Patients who do not demonstrate a therapeutic response to treatment (e.g., non-responders or poor responders to treatment) are said to have primary resistance to treatment and may be characterized as primary non-responders. Patients who initially demonstrate a therapeutic response to treatment but later lose benefit (e.g., progress or relapse despite continued treatment) are said to have acquired resistance to treatment.

[0081] Response Evaluation Criteria in Solid Tumors (RECIST) and iRECIST: RECIST is a set of published rules that define when tumors in cancer patients improve ("respond"), stay the same ("stabilize"), or get worse ("progress") during treatment. The criteria were published in February 2000 by an international collaboration including the European Organization for Research and Treatment of Cancer (EORTC), the National Cancer Institute, and the National Cancer Institute of Canada Clinical Trials Group. A revised version of the RECIST guidelines (RECIST v1.1) has since been widely adopted (see Eisenhauera et al. (2009), "New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1)," Eur J Cancer 45: 228-247, incorporated herein). Response criteria are as follows: complete response (CR): disappearance of all target lesions; partial response (PR): at least a 30% reduction in the sum of the LD of target lesions, taking the baseline total LD as reference; stable disease (SD): no sufficient shrinkage to qualify as PR or sufficient increase to qualify as PD, taking the smallest total LD since treatment began; progressive disease (PD): at least a 20% increase in the sum of the LD of target lesions, taking the smallest total LD recorded since treatment began or the appearance of one or more new lesions as reference. On the other hand, iRECIST provides an improved set of criteria that takes immune-related responses into account. See www.ncbi.nlm.nih.gov / pmc / articles / PMC5648544 / . RECIST and iRECIST criteria are standardized and may be revised from time to time as more data becomes available, and are well understood in the art.

[0082] Solid tumor: The term "solid tumor" refers to a proliferative disorder that results in an abnormal growth or tissue mass that usually does not contain cysts or liquid areas. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Solid tumors typically consist of multiple cell types, including, but not limited to, cancerous (malignant) cells, stromal cells such as CAFs, and infiltrating leukocytes such as macrophages and lymphocytes. Solid tumors treated with TGFβ1 isoform-selective inhibitors such as those described herein are typically TGFβ1-positive (TGFβ1+) tumors.

[0083] Solubility equilibrium titration (SET): SET is an assay that can measure the binding between two molecules (such as an antigen and an antibody that binds to the antigen) at equilibrium in solution. For example, mesoscale discovery ("MSD")-based SET, or MSD-SET, is a useful method for determining the dissociation constant at equilibrium, particularly for high-affinity protein-protein interactions, such as the binding of a picomolar affinity antibody to its antigen (see, e.g., Ducata et al., (2015) J Biomolecular Screening 20(10): 1256-1267). SET-based assays are particularly useful for determining K values for antibodies with subnanomolar (e.g., picomolar) affinities.

[0084] Specific binding: As used herein, the terms "specific binding" or "specifically binds" mean that the interaction of an antibody, or antigen-binding portion thereof, with an antigen or amino acid residue is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope). For example, an antibody, or antigen-binding portion thereof, binds to a specific protein rather than to proteins in general. In some embodiments, an antibody, or antigen-binding portion thereof, binds to a target with at least about 10 -9 M, 10 -10 M, 10 -11 M, 10 -12In some embodiments, the terms "specific binding to an epitope of pro-TGFβ1," "specifically binds to an epitope of pro-TGFβ1," "specific binding to pro-TGFβ1," or "specifically binds to pro-TGFβ1" refer to an antibody that binds to pro-TGFβ1 and has a KD of 1.0×10 or less as determined by a suitable in vitro binding assay. -8 The term "antibody" refers to an antibody or antigen-binding portion thereof that has a dissociation constant (KD) of less than or equal to M. In one embodiment, the antibody or antigen-binding portion thereof is capable of specifically binding to both human and non-human (e.g., murine) orthologs of pro-TGFβ1.

[0085] Subject: In the context of therapeutic applications, the term "subject" refers to an individual receiving clinical care or intervention, such as treatment, diagnosis, etc. Suitable subjects include vertebrates, including, but not limited to, mammals (e.g., humans and non-human mammals). When the subject is a human subject, the term "patient" may be used interchangeably. In a clinical context, the term "patient population" or "patient subpopulation" is used to refer to a group of individuals within a set of criteria, such as clinical criteria (e.g., disease presentation, disease stage, susceptibility to a particular condition, responsiveness to treatment, etc.), medical history, health status, sex, age group, genetic criteria (e.g., carriers of particular mutations, polymorphisms, gene duplications, DNA sequence repeats, etc.), and lifestyle factors (e.g., diet, smoking, alcohol consumption, exercise, etc.).

[0086] Target Engagement: As used herein, the term target engagement refers to the ability of a molecule (e.g., a TGFβ inhibitor) to bind to its intended target (e.g., endogenous TGFβ) in vivo. In the case of an activated inhibitor, the intended target can be the large latent complex.

[0087] TGFβ1-related indications: "TGFβ1-related indications" refers to any disease, disorder, and / or condition associated with TGFβ1 expression, activity, and / or metabolism, or any disease, disorder, and / or condition that may benefit from inhibiting TGFβ1 activity and / or levels. Certain TGFβ1-related indications are primarily driven by TGFβ1 isoforms. TGFβ1-related indications include, but are not limited to, fibrotic conditions (such as organ fibrosis and tissue fibrosis associated with chronic inflammation), proliferative disorders (such as cancer, e.g., solid tumors and myelofibrosis), diseases associated with ECM dysregulation (such as conditions associated with matrix stiffening and remodeling), diseases associated with endothelial-mesenchymal transition (EndMT), diseases associated with epithelial-mesenchymal transition (EMT), diseases associated with proteases, and diseases with aberrant gene expression of certain markers described herein. These disease categories are not intended to be mutually exclusive.

[0088] TGFβ inhibitors: The term "TGFβ inhibitor" broadly refers to any agent capable of inhibiting the biological activity, signaling, or function of TGFβ growth factors (e.g., TGFβ1, TGFβ2, and / or TGFβ3). This term is not intended to limit the mechanism of action and includes, for example, neutralizing antibodies against TGFβ, receptor antagonists (e.g., kinase inhibitors), soluble ligand traps, and activation inhibitors. Non-selective TGFβ inhibitors are commonly referred to as "pan-inhibitors" of TGFβ. TGFβ inhibitors also include antibodies that can reduce the availability of latent pro-TGFβ, which can be activated in the niche, for example, by inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADPC), as well as antibodies that cause internalization of cell surface complexes containing latent pro-TGFβ, thereby removing the precursor from the plasma membrane without depleting the cell itself. Internalization may be a suitable mechanism of action of LRRC33-containing protein complexes (such as human LRRC33-proTGFβ1), resulting in reduced levels of cells expressing LRRC33-containing protein complexes on the cell surface.

[0089] The "TGF-β family" is a class within the TGF-β superfamily that, in humans, contains three structurally similar members encoded by separate genes: TGF-β1, TGF-β2, and TGF-β3. The three growth factors are known to signal through the same receptor.

[0090] Therapeutic window: The term "therapeutic window" refers to the range of doses / concentrations that produce a therapeutic response in a subject without causing significant / observable / unacceptable adverse effects (e.g., within the range of acceptable or tolerable adverse effects). The therapeutic window may be calculated as the ratio between the minimum effective concentration (MEC) and the minimum toxic concentration (MTC). By way of example, a TGFβ1 inhibitor that achieves in vivo efficacy at 10 mg / kg and exhibits tolerable or acceptable toxicity at 100 mg / kg provides a therapeutic window of at least 10-fold (e.g., 10×). In contrast, a pan-TGFβ inhibitor that is effective at 10 mg / kg but causes adverse effects at 5 mg / kg is said to have "dose-limiting toxicity." For example, Ab2 has been shown to be effective at doses ranging from approximately <3 to 30 mg / kg / week, and preclinical models, such as rats, have also shown no observable toxicity associated with pan-TGFβ inhibition at at least 100 mg / kg / week for 4 weeks. On this basis, Ab2 exhibits a minimum 3.3-fold and maximum 33-fold therapeutic window.

[0091] Toxicity: As used herein, the term "toxicity" or "toxicities" refers to unwanted in vivo effects in a subject (e.g., a patient) associated with a treatment administered to the subject (e.g., a patient), such as undesirable side effects and adverse events. "Tolerability" refers to the level of toxicity associated with a treatment or treatment regimen that a patient can reasonably tolerate without discontinuing treatment due to toxicity. Typically, toxicity studies are conducted prior to clinical development to evaluate the safety profile of a drug candidate (e.g., a monoclonal antibody treatment) in one or more preclinical models. Toxicity studies may help determine the "no observed adverse effect level (NOAEL)" and "maximum tolerated dose (MTD)" of a test substance, based on which a therapeutic window can be estimated. Preferably, a species shown to be sensitive to a particular intervention should be selected as the preclinical animal model in which to conduct safety / toxicity studies. In the case of TGFβ inhibition, suitable species include rats, dogs, and cynomolgus monkeys. Mice have been reported to be less sensitive to pharmacological inhibition of TGFβ, and certain studies have reported toxicity observed with pan-inhibition of TGFβ in mice, although potentially dangerous toxicities may not be evident in other species, including humans. Illustratively, in the context of the present disclosure, based on a 4-week toxicity study, the NOAEL for Ab2 in rats was the highest dose evaluated (100 mg / kg), suggesting an MTD of >100 mg / kg.

[0092] Treat / Treatment: The term "treat" or "treatment" includes therapeutic treatments, prophylactic treatments, and applications that reduce a subject's risk of developing a disorder or other risk factor. Thus, the term is intended broadly to mean causing a therapeutic benefit in a patient by, for example, slowing disease progression, reversing certain disease features, normalizing gene expression, enhancing or boosting the body's immunity, reducing or reversing immunosuppression, reducing, removing, or eradicating harmful cells or substances from the body, reducing disease burden (e.g., fibrosis and tumor burden), preventing recurrence or recurrence, extending refractory periods, and / or otherwise improving survival. The term includes therapeutic treatments, prophylactic treatments, and applications that reduce a subject's risk of developing a disorder or other risk factor. Treatment does not require a complete cure of the disorder, and encompasses embodiments that reduce symptoms or underlying risk factors. In the context of combination therapy, the term can also refer to: i) the ability of the second therapeutic agent to lower the effective dose of the first therapeutic agent to reduce side effects and increase tolerability; ii) the ability of the second treatment to make the patient more responsive to the first treatment; and / or iii) the ability to provide an additive or synergistic clinical benefit.

[0093] Tumor-associated macrophages (TAMs): TAMs are polarized / activated macrophages (M2-like macrophages) with a tumor-promoting phenotype. TAMs can be either monocytes / macrophages of marrow origin recruited to the tumor site or tissue-resident macrophages derived from erythro-myeloid precursors. The differentiation of monocytes / macrophages into TAMs is influenced by several 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). Generally, monocytes / macrophages can polarize into so-called "M1" or "M2" subtypes, the latter associated with a more tumor-promoting phenotype. In solid tumors, up to 50% of the tumor mass may correspond to macrophages, which are preferentially M2-polarized. Among tumor-associated monocyte and myeloid cell populations, M1 macrophages typically express cell surface HLA-DR, CD68, and CD86, while M2 macrophages typically express cell surface HLA-DR, CD68, CD163, and CD206. Tumor-associated M2-like macrophages (such as M2c and M2d subtypes) can express cell surface LRRC33 and / or LRRC33-proTGFβ1. M2-like macrophages may also be enriched in fibrotic microenvironments.

[0094] Tumor microenvironment: The term "tumor microenvironment (TME)" refers to the local disease niche in which a tumor (e.g., a solid tumor) resides in vivo. The TME can include disease-associated molecular signatures (a set of chemokines, cytokines, etc.), disease-associated cell populations (TAMs, CAFs, MDSCs, etc.), and disease-associated ECM milieu (altered ECM components and / or structure).

[0095] Variable region: The term "variable region" or "variable domain" refers to a portion of an antibody's light and / or heavy chain, typically comprising approximately the amino-terminal 120-130 amino acids in heavy chains and approximately the amino-terminal 100-110 amino acids in light chains. In certain embodiments, the variable regions of different antibodies vary significantly in amino acid sequence, even among antibodies of the same species. The variable regions of an antibody typically determine the specificity of a particular antibody for its target.

[0096] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as being modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±1%.

[0097] The indefinite articles "a" and "an," as used herein and in the claims, unless clearly indicated otherwise, should be understood to mean "at least one."

[0098] As used herein and in the claims, the term "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and non-conjunctively present in other cases. Unless clearly indicated otherwise, other elements, whether related or unrelated to the elements specifically identified by the "and / or" clause, may optionally be present. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements), etc.

[0099] The phrase "at least one," as used herein and in the claims, in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than the specifically identified elements in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer to, in one embodiment, at least one, optionally more than one, A, and no B (and optionally including elements other than B); in another embodiment, at least one, optionally more than one, B, and no A (and optionally including elements other than A); in yet another embodiment, at least one, optionally more than one, A, and at least one, optionally more than one, B (and optionally including other elements); etc.

[0100] The use of ordinal terms such as "first," "second," and "third" in the claims to modify claim elements does not, by itself, imply any priority, precedence, or order of one claim element over another, or any chronological order of performing acts of a method, but is merely used as a label to distinguish one claim element having a particular name from another element having the same name (other than the use of ordinal terms) to identify the claim element.

[0101] It is to be understood that the ranges provided herein are shorthand for all values within the range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, such as 10-20, 1-10, 30-40, etc.

[0102] Transforming growth factor-beta (TGFβ) The activity of transforming growth factor-beta (TGFβ) and subsequent partial purification of the soluble growth factor were first described in the late 1970s and early 1980s, thus initiating the TGFβ field approximately 40 years ago. To date, 33 gene products have been identified, constituting the large TGFβ superfamily. The TGFβ superfamily can be divided into at least three subclasses based on structural similarity: TGFβ, growth differentiation factors (GDFs), and bone morphogenetic proteins (BMPs). TGFβ subclass I consists of three highly conserved isoforms, TGFβ1, TGFβ2, and TGFβ3, which are encoded by three separate genes in humans.

[0103] TGF-β is thought to play a key role in diverse processes, including cell growth inhibition, extracellular matrix (ECM) remodeling, and immune homeostasis. The importance of TGF-β1 in T cell homeostasis is demonstrated by the observation that TGF-β1- / - mice succumb to multiple organ failure due to extensive immune activation and survive only 3-4 weeks (Kulkarni, AB et al., Proc Natl Acad Sci USA, 1993. 90(2): pp. 770-4; Shull, MM et al., Nature, 1992. 359(6397): pp. 693-9). The roles of TGF-β2 and TGF-β3 are less clear. While the three TGFβ isoforms have distinct temporal and spatial expression patterns, they signal through the same receptors, TGFβRI and TGFβRII, although in some cases, such as TGFβ2 signaling, type III receptors such as betaglycan are also required (Feng, XH, and R. Derynck, Annu Rev Cell Dev Biol, 2005. 21: pp. 659-93; Massague, J., Annu Rev Biochem, 1998. 67: pp. 753-91). Ligand-induced oligomerization of TGFβRI / II triggers phosphorylation of SMAD transcription factors, leading to the transcription of target genes such as Col1a1, Col3a1, ACTA2, and serpin1 (Massague, J., J. Seoane, and D. Wotton, Genes Dev, 2005. 19(23): pp. 2783-810). SMAD-independent TGFβ signaling pathways have also been described, for example, in cancer and in aortic lesions in Marfan mice (Derynck, R. and Y.E. Zhang, Nature, 2003. 425(6958): pp. 577-84; Holm, T.M. et al., Science, 2011. 332(6027): pp. 358-61).

[0104] The biological importance of the TGF-β pathway in humans is validated by genetic diseases. Kamurachi-Engelman disease results from an autosomal dominant mutation in the TGFB1 gene, resulting in bone dysplasia and constitutive activation of TGF-β1 signaling (Janssens, K. et al., J Med Genet, 2006. 43(1): pp. 1-11). Patients with Loeys / Dietz syndrome carry autosomal dominant mutations in components of the TGF-β signaling pathway, which cause aortic aneurysms, hypertelorism, and a bifid uvula (Van Laer, L., H. Dietz, and B. Loeys, Adv Exp Med Biol, 2014. 802: pp. 95-105). Because dysregulation of the TGF-β pathway has been implicated in multiple diseases, several drugs targeting the TGF-β pathway have been developed and tested in patients, but with limited success.

[0105] Dysregulation of TGFβ signaling has been implicated in a wide range of human diseases. Indeed, in some pathologies, such dysregulation may involve multiple aspects of TGFβ function. Affected tissues, such as fibrotic and / or inflammatory tissues and tumors, may create a local environment in which TGFβ activation can lead to disease exacerbation or progression, which may be mediated, at least in part, by interactions between multiple TGFβ-responsive cells that are activated in an autocrine and / or paracrine manner along with several other cytokines, chemokines, and growth factors that play a role in specific disease settings.

[0106] The present invention is further illustrated by the following examples, which should not be construed as limiting, and which refer to the following figures: Figures 1-31.

[0107] A novel, highly potent, TGFβ1-selective inhibitor The present disclosure provides antibodies that bind to each of the four known human LLCs (hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1) with high affinity (e.g., a K of less than 1 nM), as measured, for example, by surface plasmon resonance (SPR). D ) and slow dissociation rates (i.e., low k オフNovel monoclonal antibodies and antigen-binding fragments thereof are provided that can bind to TGFβ1 at the isoform (value). The novel antibodies and fragments are isoform-selective inhibitors of TGFβ1. Such antibodies or antigen-binding fragments thereof comprise H-CDR1, H-CDR2, and H-CDR3, L-CDR1, L-CDR2, and L-CFR3, wherein H-CDR1 comprises GFTFADYA (SEQ ID NO: 276), H-CDR2 comprises a sequence represented by the formula ISGSGX1AT, where optionally, X1 is A or K (SEQ ID NO: 277), and H-CDR3 comprises a sequence represented by the formula VSSGX1WDX2D, where optionally, X1 is H, D, or Q, and further optionally, X2 is F or Y (SEQ ID NO: 278). wherein L-CDR1 comprises a sequence represented by the formula QSISSY (SEQ ID NO: 279), L-CDR2 comprises a sequence represented by the formula AASXIX2X3X4, where optionally X1 is N, G, or V, further optionally X2 is L, N, or E, further optionally X3 is Q or E, and further optionally X4 is S or T (SEQ ID NO: 280), and L-CDR3 comprises a sequence represented by the formula QQTYX1VPLT, where optionally X1 is T or G (SEQ ID NO: 281). In a preferred embodiment, H-CDR2 comprises ISGSGAAT (SEQ ID NO: 282), H-CDR3 comprises VSSGHWDYD (SEQ ID NO: 287), L-CDR2 comprises AASGLES (SEQ ID NO: 284), and L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285). In some embodiments, the antibody or fragment binds to an epitope that includes one or more of the following amino acid residues of the pro-TGFβ1 polypeptide sequence: S35, G37, E38, V39, P40, P41, G42, P43, R274, K280, H283, and K309.In some embodiments, H-CDR1 may comprise the sequence GFTFADYA (SEQ ID NO: 276), H-CDR2 may comprise the sequence ISGSGAAT (SEQ ID NO: 282), and H-CDR3 comprises a sequence represented by the formula VSSGX1WDX2D (wherein, optionally, X1 is H or Q, and further optionally, X2 is Y or F) (SEQ ID NO: 283). L-CDR1 may comprise the sequence QSISSY (SEQ ID NO: 279), L-CDR2 may comprise the sequence AASGLES (SEQ ID NO: 284), and L-CDR3 may comprise the sequence QQTYGVPLT (SEQ ID NO: 285). In a preferred embodiment, H-CDR3 is VSSGHWDYD (SEQ ID NO: 287). In some embodiments, the antibody or fragment binds to an epitope that includes one or more of the following amino acid residues of the pro-TGFβ1 polypeptide sequence: S35, G37, E38, V39, P40, P41, G42, P43, R274, K280, H283, and K309.

[0108] The table below provides CDR sequences of useful variants.

[0109] [Table 1]

[0110] Optionally, one or more of the six CDRs may contain one or more (eg, one or two) amino acid changes.

[0111] In some embodiments, an antibody or antigen-binding fragment thereof selected for use or production according to the present disclosure comprises H-CDR1, H-CDR2, and H-CDR3, L-CDR1, L-CDR2, and L-CFR3, wherein H-CDR1 comprises GFTFADYA (SEQ ID NO: 276), H-CDR2 comprises a sequence represented by the formula ISGSGX1AT, where optionally, X1 is A or K (SEQ ID NO: 277), and H-CDR3 comprises a sequence represented by the formula VSSGX1WDX2D, where optionally, X1 is H, D, or Q, and further optionally, X2 is F or Y. and X4 is S or T] (SEQ ID NO: 280), and L-CDR3 comprises a sequence represented by the formula QQTYX1VPLT, where optionally, X1 is T or G] (SEQ ID NO: 281). In a preferred embodiment, H-CDR2 comprises ISGSGAAT (SEQ ID NO: 282), H-CDR3 comprises VSSGHWDYD (SEQ ID NO: 287), L-CDR2 comprises AASGLES (SEQ ID NO: 284), and L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285). In some embodiments, the antibody or fragment binds to an epitope comprising one or more of the following amino acid residues of the pro-TGFβ1 polypeptide sequence: S35, G37, E38, V39, P40, P41, G42, P43, R274, K280, H283, and K309.

[0112] The table below provides further CDR sequences of useful variants.

[0113] [Table 2]

[0114] In some embodiments, one or more of the six CDRs may contain one or more (eg, one or two) amino acid changes.

[0115] Non-limiting examples of preferred TGFβ1 activation inhibitors are provided in the table below and are referred to herein as Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, and Ab52. Each of these antibodies can be in the form of a whole immunoglobulin (such as IgG) or its antigen-binding fragment, such as a Fab fragment. The antigen-binding fragment can be used to create engineered constructs containing fragments or their derivatives, such as bispecific antibodies and other fusion proteins that function as TGFβ1 inhibitors. The six CDRs of each of the exemplary antibodies are listed in the table below.

[0116] [Table 3]

[0117] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), including V H comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) sequence identity to EVQLLESGGGLVQPGGSLRLSCAASGFTFADYAMTWVRQAPGKGLEWVSAISGSGAATYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSSGHWDYDYWGQGTLVTVSS (SEQ ID NO: 297), Lcomprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) sequence identity to DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASGLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGVPLTFGGGTKVEIK (SEQ ID NO: 298). In some embodiments, the antibody or fragment binds to an epitope comprising one or more of the following amino acid residues of the pro-TGFβ1 polypeptide sequence: S35, G37, E38, V39, P40, P41, G42, P43, R274, K280, H283, and K309.

[0118] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), including V H comprises EVQLLESGGGLVQPGGSLRLSCAASGFTFADYAMTWVRQAPGKGLEWVSAISGSGAATYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSSGHWDYDYWGQGTLVTVSS (SEQ ID NO: 297), L contains DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASGLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGVPLTFGGGTKVEIK (SEQ ID NO: 298).

[0119] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable domain (V H ) and the light chain variable domain (V L ), including V H comprises EVQLLESGGGLVQPGGSLRLSCAASGFTFADYAMTWVRQAPGKGLEWVSAISGSGAATYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSSGHWDFDYWGQGTLVTVSS (SEQ ID NO: 299), Lcontains DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYTVPLTFGGGTKVEIK (SEQ ID NO: 300).

[0120] The present invention includes nucleic acid sequences encoding any one of the amino acid sequences provided above. Encompassed herein are vectors (e.g., DNA plasmids, such as mammalian expression vectors and related nucleic acid preparations) comprising the nucleic acid sequences, cells transfected with the vectors, cell lines with stable expression of the nucleic acid, cell cultures comprising the cells, and optionally, the cell cultures comprise mammalian cells capable of large-scale production of protein constructs comprising antibodies or antigen-binding fragments of antibodies.

[0121] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof that selectively inhibits TGFβ1 activation comprises a heavy chain complementarity determining region 1 (CDRH1) having an amino acid sequence at least 95% identical to the sequence set forth in GFTFADYA (SEQ ID NO: 276), a heavy chain complementarity determining region 2 (CDRH2) having an amino acid sequence at least 95% identical to the sequence set forth in ISGSGAAT (SEQ ID NO: 282), a heavy chain complementarity determining region 3 (CDRH3) having an amino acid sequence at least 95% identical to the sequence set forth in VSSGHWDYD (SEQ ID NO: 287), a light chain complementarity determining region 1 (CDRL1) having an amino acid sequence at least 95% identical to the sequence set forth in QSISSY (SEQ ID NO: 279), a light chain complementarity determining region 2 (CDRL2) having an amino acid sequence at least 95% identical to the sequence set forth in AASGLES (SEQ ID NO: 284), and a light chain complementarity determining region 3 (CDRL3) having an amino acid sequence at least 95% identical to the sequence set forth in QQTYGVPLT (SEQ ID NO: 285).

[0122] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof that selectively inhibits TGFβ1 activation comprises a heavy chain complementarity determining region 1 (CDRH1) having an amino acid sequence at least 96% identical to the sequence set forth in GFTFADYA (SEQ ID NO: 276), a heavy chain complementarity determining region 2 (CDRH2) having an amino acid sequence at least 96% identical to the sequence set forth in ISGSGAAT (SEQ ID NO: 282), a heavy chain complementarity determining region 3 (CDRH3) having an amino acid sequence at least 96% identical to the sequence set forth in VSSGHWDYD (SEQ ID NO: 287), a light chain complementarity determining region 1 (CDRL1) having an amino acid sequence at least 96% identical to the sequence set forth in QSISSY (SEQ ID NO: 279), a light chain complementarity determining region 2 (CDRL2) having an amino acid sequence at least 96% identical to the sequence set forth in AASGLES (SEQ ID NO: 284), and a light chain complementarity determining region 3 (CDRL3) having an amino acid sequence at least 96% identical to the sequence set forth in QQTYGVPLT (SEQ ID NO: 285).

[0123] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof that selectively inhibits TGFβ1 activation comprises a heavy chain complementarity determining region 1 (CDRH1) having an amino acid sequence at least 98% identical to the sequence set forth in GFTFADYA (SEQ ID NO: 276), a heavy chain complementarity determining region 2 (CDRH2) having an amino acid sequence at least 98% identical to the sequence set forth in ISGSGAAT (SEQ ID NO: 282), a heavy chain complementarity determining region 3 (CDRH3) having an amino acid sequence at least 98% identical to the sequence set forth in VSSGHWDYD (SEQ ID NO: 287), a light chain complementarity determining region 1 (CDRL1) having an amino acid sequence at least 98% identical to the sequence set forth in QSISSY (SEQ ID NO: 279), a light chain complementarity determining region 2 (CDRL2) having an amino acid sequence at least 98% identical to the sequence set forth in AASGLES (SEQ ID NO: 284), and a light chain complementarity determining region 3 (CDRL3) having an amino acid sequence at least 98% identical to the sequence set forth in QQTYGVPLT (SEQ ID NO: 285).

[0124] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof that selectively inhibits TGFβ1 activation comprises a heavy chain complementarity determining region 1 (CDRH1) having an amino acid sequence at least 99% identical to the sequence set forth in GFTFADYA (SEQ ID NO: 276), a heavy chain complementarity determining region 2 (CDRH2) having an amino acid sequence at least 99% identical to the sequence set forth in ISGSGAAT (SEQ ID NO: 282), a heavy chain complementarity determining region 3 (CDRH3) having an amino acid sequence at least 99% identical to the sequence set forth in VSSGHWDYD (SEQ ID NO: 287), a light chain complementarity determining region 1 (CDRL1) having an amino acid sequence at least 99% identical to the sequence set forth in QSISSY (SEQ ID NO: 279), a light chain complementarity determining region 2 (CDRL2) having an amino acid sequence at least 99% identical to the sequence set forth in AASGLES (SEQ ID NO: 284), and a light chain complementarity determining region 3 (CDRL3) having an amino acid sequence at least 99% identical to the sequence set forth in QQTYGVPLT (SEQ ID NO: 285).

[0125] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof that selectively inhibits TGFβ1 activation comprises a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in GFTFADYA (SEQ ID NO: 276), a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in ISGSGAAT (SEQ ID NO: 282), a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in VSSGHWDYD (SEQ ID NO: 287), a light chain complementarity determining region 1 (CDRL1) having the amino acid sequence set forth in QSISSY (SEQ ID NO: 279), a light chain complementarity determining region 2 (CDRL2) having the amino acid sequence set forth in AASGLES (SEQ ID NO: 284), and a light chain complementarity determining region 3 (CDRL3) having the amino acid sequence set forth in QQTYGVPLT (SEQ ID NO: 285). In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V) comprising a sequence having, comprising, or consisting of at least 95% identity, 96% identity, 97% identity, 98% identity, 99% identity to SEQ ID NO: 297. H) and a light chain variable domain (V) comprising a sequence having, comprising, or consisting of at least 95% identity, 96% identity, 97% identity, 98% identity, 99% identity to SEQ ID NO: 298. L ) and

[0126] Binding kinetics of novel antibodies The novel antibodies and antigen-binding fragments thereof (e.g., Fabs) disclosed herein are characterized by enhanced binding properties. The antibodies and fragments can specifically bind to each of the presentation molecule-proTGFβ1 complexes (sometimes referred to as "large latent complexes" or LLCs, which are ternary complexes consisting of a proTGFβ1 dimer coupled to a single presentation molecule), i.e., LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. Recombinantly produced purified protein complexes can be used as antigens (e.g., antigen complexes) to screen, evaluate, or confirm the ability of antibodies to bind to the antigen complex in appropriate in vitro binding assays. Such assays are well known in the art and include, but are not limited to, biolayer interferometry (BLI)-based assays (e.g., OCTET®) and surface plasmon resonance (SPR)-based assays (e.g., BIACORE®).

[0127] Previously, we have reported that ATP has high affinity for LLC (e.g., subnanomolar K D ) were identified. Here, the aim was to specifically select antibodies and fragments that advantageously have a particularly slow dissociation rate, in order to achieve a particularly durable inhibitory effect.

[0128] Thus, selection of an appropriate TGFβ inhibitor for carrying out the methods and therapeutic uses according to the present disclosure may include performing in vitro binding assays to measure binding kinetics. In preferred embodiments, the antibody or antigen-binding fragment binds to each of the following large latent complexes with sub-nanomolar affinity, e.g., a K of 1.0 nM or less: Dand k less than or equal to 10E-4 (1 / sec) オフ The antibodies or fragments bind to the following murine LLC counterparts: hLTBP1-proTc, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. Preferably, the antibodies or fragments further bind to each of the murine LLC counterparts, i.e., mLTBP1-proTGFβ1, mLTBP3-proTGFβ1, mGARP-proTGFβ1, and mLRC33-proTGFβ1, with equal affinity to human LLC. In vitro binding kinetics can be readily determined by measuring the interaction of a test antibody (such as an antigen-binding fragment) with an appropriate antigen, such as large latent complex (LLC) or small latent complex (SLC). Suitable in vitro binding assays for determining binding kinetic parameters include BLI-based assays, such as OCTET®, and surface plasmon resonance-based assays, such as the BIACORE® system. An example of an Octet-based in vitro binding assay is provided in Figure 1. An example of an SPR-based in vitro binding assay is provided in Figure 2. Fab fragments of Ab46 and a reference antibody, both of which are inhibitors of TGFβ1 activation, were used in this experiment. As illustrated in Figure 2, the two Fabs have similar "on" rates (k オン ), indicating that they engage (i.e., associate) with antigen at similar rates. However, their "off" rates (k オフ ) is Ab46 t 1 / 2 over 130 minutes, while the reference antibody had a t of 2.4 minutes. 1 / 2 The difference is striking in that Ab46 only has a t value, because although the binding / association phase of the interaction occurs with similar kinetics, Ab46 "falls off" (e.g., dissociates) from the antigen relatively quickly for a much longer time (e.g., longer t 1 / 2 ), indicating that the antibody can remain bound to the antigen. Therefore, the difference in dissociation kinetics is related to its overall affinity (K D), which may result in enhanced potency (see below). Characterization of binding kinetics therefore provides useful information regarding the potential durability of the effect and resulting in vivo efficacy.

[0129] Therefore, the present invention provides a method for determining the kinetic energy of a 10.0e-4 (sec) -1 (i.e., subnanomolar), e.g., less than 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, or 50 pM.

[0130] The following table illustrates the binding kinetics of the listed antibodies (e.g., Fabs) obtained by an OCTET®-based binding assay. Experiments were performed with immobilized biotin-labeled antigen and Fab fragments (e.g., test antibodies) in solution.

[0131] [Table 4]

[0132] Of these antibodies, Ab42, Ab46, and Ab50 were selected for further evaluation. Surface plasmon resonance (SPR) was used to measure binding kinetics using a BIACORE® system. The association and dissociation kinetics of Ab42, Ab46, Ab50, and the Fab fragments of a reference antibody (denoted "Reference") with and without the antigen complex were measured, and the resulting equilibrium dissociation constants (KD) are provided below. Experiments were performed using eight LLCs: hLTBP1-proTGFβ1, mLTBP1-proTGFβ1, hLTBP3-proTGFβ1, mLTBP3-proTGFβ1, hGARP-proTGFβ1, mGARP-proTGFβ1, hLRRC33-proTGFβ1, and mLRRC33-proTGFβ1.

[0133] [Table 5]

[0134] [Table 6]

[0135] [Table 7]

[0136] [Table 8]

[0137] [Table 9]

[0138] [Table 10]

[0139] [Table 11]

[0140] [Table 12]

[0141] Potency, inhibitory activity The antibodies disclosed herein can be broadly characterized as "functional antibodies" for their ability to inhibit TGFβ1 signaling. As used herein, a "functional antibody" confers one or more biological activities by virtue of its ability to bind to an antigen (e.g., an antigen complex). Thus, functional antibodies broadly include those that can modulate the activity / function of a target molecule (i.e., an antigen). Such modulating antibodies include inhibitory antibodies (or inhibitory antibodies) and activating antibodies. The present disclosure is drawn to antibodies that can inhibit biological processes mediated by TGFβ1 signaling associated with multiple contexts of TGFβ1. Inhibitors used to practice the present invention, such as the antibodies described herein, are intended to be selective for TGFβ1 and not target or interfere with TGFβ2 and TGFβ3 when administered at therapeutically effective doses (doses that achieve sufficient efficacy within acceptable toxicity levels). The novel antibodies of the present disclosure have enhanced inhibitory activity (potency) compared to previously identified activation inhibitors of TGFβ1.

[0142] Pharmacodynamic (PD) effects can be measured to determine the relative potency of inhibitory antibodies. Commonly used PD measures of the TGFβ signaling pathway include, but are not limited to, SMAD2 / 3 phosphorylation and expression of downstream effector genes whose transcription is sensitive to TGFβ activation, such as those with TGFβ-responsive promoter elements (e.g., SMAD-binding elements). In some embodiments, antibodies of the present disclosure can completely block disease-induced SMAD2 / 3 phosphorylation in preclinical fibrosis models when administered to animals at a dose of 3 mg / kg or less. In some embodiments, antibodies of the present disclosure can reduce and / or completely block disease-induced SMAD2 / 3 phosphorylation. In some embodiments, antibodies of the present disclosure can reduce and / or completely block disease-induced SMAD2 phosphorylation (e.g., regardless of any changes in SMAD3). In some embodiments, reduction is measured as the ratio of phosphorylated SMAD2 / 3 to total SMAD2 / 3. In some embodiments, reduction is measured as the ratio of phosphorylated SMAD2 to total SMAD2. In some embodiments, antibodies of the present disclosure can reduce the nuclear translocation of phosphorylated SMAD2, as measured, for example, by IHC. Without being bound by theory, in some embodiments, measuring SMAD2 phosphorylation (but not SMAD3) can improve accurate detection of treatment-related effects. Denis et al., Development 143: 3481-90 (2016); Liu et al., J. Biol. Chem. 278: 11721-8 (2003); David et al., Oncoimmunology 6: e1349589 (2017). In some embodiments, antibodies of the present disclosure can significantly suppress fibrosis-induced expression of a panel of marker genes, including Acta2, Col1a1, Col3a1, Fn1, Itga11, Lox, and Loxl2, when administered to animals at a dose of 10 mg / kg or less in a UUO model of renal fibrosis.

[0143] In some embodiments, the potency of an inhibitory antibody can be measured in a suitable cell-based assay, such as the CAGA reporter assay described herein. Generally, cell-based potency assays can be performed using cultured cells, such as heterologous and primary cells. Cells expressing endogenous TGFβ1 and / or LTBP1, LTBP3, GARP, and LRRC33, and other target presentation molecules, can be used. Alternatively, exogenous nucleic acids encoding target proteins, such as TGFβ1 and / or LTBP1, LTBP3, GARP, and LRRC33, can be introduced into such cells, for example, by transfection (e.g., stable or transient transfection) or infection using a viral vector. In some embodiments, LN229 cells are used in such assays. Cells expressing TGFβ1 and a presentation molecule of interest (e.g., LTBP1, LTBP3, GARP, or LRRC33) are grown in culture and "present" the large latent complex either on the cell surface (if associated with GARP or LRRC33) or deposited within the ECM (if associated with LTBP). Activation of TGFβ1 can be initiated by an integrin expressed on the surface of another cell. The integrin-expressing cell can be the same cell co-expressing the large latent complex or a different cell type. Reporter cells incorporating a TGFβ-responsive element are added to the assay system. In this way, the degree of TGFβ activation can be measured by detecting a signal from the reporter cell (e.g., a TGFβ-responsive reporter gene, such as luciferase, coupled to a TGFβ-responsive promoter element) upon TGFβ activation. Using such cell-based assay systems, the inhibitory activity of an antibody can be determined by measuring a change (decrease) or difference in reporter signal (e.g., luciferase activity measured by fluorescence readout) in either the presence or absence of the test antibody.

[0144] Results from cell-based efficacy studies are illustrated below. In these studies, human LM229 cells were used to measure the potency of test antibodies in their ability to inhibit TGFβ signaling. These cells express endogenous LTBP-proTGFβ1. Assays were performed using i) monoclonal antibodies (hIgG4 immunoglobulin) and ii) Fab fragments of a panel of the same test antibodies, as indicated.

[0145] [Table 13]

[0146] The reference antibody used as a benchmark ("Reference Ab") was shown to have similar association kinetics to many of the novel antibodies tested, but the overall potency was significantly improved for the novel antibodies, likely due to them having much slower dissociation rates than the reference antibody.

[0147] In some embodiments, the inhibitory potency (e.g., IC ) of the novel antibodies of the present disclosure calculated based on a cell-based assay (such as the LN229 cell assay described elsewhere herein) is 50) may be less than 10 nM as measured against each of the hLTBP1-proTGFβ1 and hLTBP3-proTGFβ1 complexes. In some embodiments, the antibody has an IC50 of 5 nM or less (i.e., ≦5 nM) as measured against each of the hLTBP1-proTGFβ1 and hLTBP3-proTGFβ1 complexes. In preferred embodiments, the antibody has an IC50 of less than 1 nM as measured against at least one of the hLTBP1-proTGFβ1 and hLTBP3-proTGFβ1 complexes. In some embodiments, the antibody has an IC50 of less than 1 nM as measured against at least one of the hLTBP1-proTGFβ1 and hLTBP3-proTGFβ1 complexes and further against at least one of the mLTBP1-proTGFβ1 and mLTBP3-proTGFβ1 complexes. In some embodiments, antibodies of the disclosure have an IC50 of 10 nM or less (i.e., ≦10 nM) for each of hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes. In some embodiments, antibodies of the disclosure have an IC50 of 5 nM or less (i.e., ≦5 nM) (e.g., 1 nM or less) for each of hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes.

[0148] In some embodiments, efficacy can be evaluated as a measure of efficacy and / or pharmacodynamic effect in a suitable in vivo model.For example, if a first antibody is effective at a certain concentration in an in vivo model, and a second antibody is equally effective at a lower concentration than the first antibody in the same in vivo model, then the second antibody can be said to be more potent than the first antibody.Depending on the specific target indication, any suitable disease model known in the art, such as cancer model and fibrosis model, can be used to evaluate the relative efficacy of TGFβ1 inhibitors.Preferably, multiple doses or concentrations of each test antibody are included in such studies.

[0149] Similarly, pharmacodynamic (PD) effects can be measured to determine the relative potency of inhibitory antibodies. Commonly used PD measures of the TGFβ signaling pathway include, but are not limited to, SMAD2 / 3 phosphorylation and expression of downstream effector genes whose transcription is sensitive to TGFβ activation, such as those with TGFβ-responsive promoter elements (e.g., SMAD-binding elements). In some embodiments, antibodies of the present disclosure can completely block disease-induced SMAD2 / 3 phosphorylation in preclinical fibrosis models when administered to animals at a dose of 3 mg / kg or less. In some embodiments, antibodies of the present disclosure can significantly suppress fibrosis-induced expression of a panel of marker genes, including Acta2, Col1a1, Col3a1, Fn1, Itga11, Lox, and Lox12, in a UUO model of renal fibrosis when administered to animals at a dose of 10 mg / kg or less.

[0150] bond area In the context of the present disclosure, the "binding region" of an antigen provides the structural basis for antibody-antigen interaction. As used herein, "binding region" refers to the region of the interface between an antibody and an antigen where the antibody or fragment protects the binding region from solvent exposure when bound to the pro-TGFβ1 complex ("antigen") in physiological solution, as determined by a suitable technique, such as hydrogen-deuterium exchange mass spectrometry (HDX-MS).

[0151] The art is familiar with HDX-MS, a widely used technique for investigating protein conformation or protein-protein interactions in solution. This method relies on the exchange of hydrogen in protein backbone amides with deuterium present in solution. Measuring the rate of hydrogen-deuterium exchange can provide information about protein dynamics and conformation (reviewed in Wei et al. (2014) "Hydrogen / deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications." Drug Disc Today. 19(1): 95-102, incorporated by reference). The application of this technique is based on the premise that when an antibody-antigen complex is formed, the interface between the binding partners occludes solvent, thereby slowing or preventing the exchange rate due to steric exclusion of the solvent.

[0152] This technique can be used to determine the binding (and therefore protecting) regions of pro-TGFβ1. In some embodiments, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, and variants thereof (e.g., VH sequences having at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) and variants thereof (e.g., VH sequences having at least 90% sequence identity, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). The portion of pro-TGFβ1 identified as important for binding to any one of the following antibodies (those with VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) comprises at least a portion of the amino acid stretch SPPSQGEVPPGPLPEAVLALYNST (SEQ ID NO: 261) ("first binding region"), which largely overlaps with a protein domain within the latent lasso, commonly referred to as LAP. In some embodiments, the antibody binds to (and thus protects from) at least a portion of the amino acid sequence LREAVPE (SEQ ID NO: 259) ("second binding region") within the arm domain of LAP. In some embodiments, the antibody binds to (and thus protects from) at least a portion of the amino acid sequence WKWIHEPKGYHANFCLG (SEQ ID NO: 262) ("third binding region"), which largely overlaps with the so-called finger-1 within the growth factor domain. In some embodiments, the antibody binds to an epitope of the pro-TGFβ1 complex that includes one or more amino acid residues of SPPSQGEVPPGPLPEAVLALYNST (SEQ ID NO: 261) (the "first binding region"), LREAVPE (SEQ ID NO: 259) (the "second binding region"), and / or one or more amino acid residues of WKWIHEPKGYHANFCLG (SEQ ID NO: 262) (the "third binding region").

[0153] In some embodiments, additional residues, e.g., residues outside of the three identified binding regions protected by HD-X, may further contribute to achieving enhanced antibody-antigen binding of the novel antibodies disclosed herein. In some embodiments, the lysine (Lys) residue at position 309 (i.e., K309) within the growth factor domain of the pro-TGFβ1 polypeptide sequence is a lysine (Lys) residue of Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, and variants thereof (e.g., V having at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). H V having a sequence and at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). L In some embodiments, the proline (Pro) residue at position 43 (i.e., P43) in the prodomain of the pro-TGFβ1 polypeptide sequence is a V-like peptide having at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) with one or more of Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, and variants thereof (e.g., V-like peptides having at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). H V having a sequence and at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). LIn some embodiments, the antibody or fragment binds to an epitope comprising one or more of the following amino acid residues of the pro-TGFβ1 polypeptide sequence: S35, G37, E38, V39, P40, P41, G42, P43, R274, K280, H283, and K309. In some embodiments, the epitope is a combinatorial epitope (see below) comprising P43 and K309.

[0154] In some embodiments, the first binding region and / or the second binding region confer isoform selectivity on the antibody or fragment.

[0155] Advantageously, preferred inhibitory antibodies of the present disclosure can inhibit the release of mature growth factors from latent complexes, thereby reducing growth factor signaling. Such antibodies can target any epitope that, upon association with such an antibody, results in reduced growth factor release or activity. In some embodiments, antibodies of the present disclosure specifically bind to combinatorial epitopes, i.e., epitopes formed by two or more components / portions of an antigen or antigen complex. For example, combinatorial epitopes can be formed by contributions from amino acid residues from multiple portions of a single protein, i.e., multiple non-adjacent segments of the same protein. Alternatively, combinatorial epitopes can be formed from contributions from multiple protein components of an antigen complex. In some embodiments, antibodies of the present disclosure specifically bind to conformational epitopes (or conformation-specific epitopes), e.g., epitopes that are sensitive to the three-dimensional structure (i.e., conformation) of an antigen or antigen complex. In a preferred embodiment, the combinatorial epitope comprises amino acid residues within a cryptic lasso and amino acid residues within a growth factor domain.

[0156] [Table 14]

[0157] Antigen complexes and components The novel antibodies of the present disclosure specifically bind to each of the four known human large latent complexes (e.g., hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1) with a low dissociation rate, selectively inhibit TGFβ1 activation, and meet the safety criteria described herein. Screening for such antibodies (e.g., identification and selection) typically involves the use of appropriate recombinantly produced antigen complexes. Useful protein components that may comprise such antigen complexes are provided, including TGFβ isoforms and related polypeptides, fragments, and variants, presentation molecules (e.g., LTBP, GARP, LRRC33) and related polypeptides, fragments, and variants. These components may be expressed and purified to form protein complexes (e.g., large latent complexes) that can be used in antibody screening processes. Screening may include positive selection, in which desirable binders are selected from a pool or library of binders and non-binders, and negative selection, in which undesirable binders are removed from the pool.

[0158] In some embodiments, TGFβ1 comprises a naturally occurring mammalian amino acid sequence. In some embodiments, TGFβ1 comprises a naturally occurring human amino acid sequence. In some embodiments, TGFβ1 comprises a human, monkey, rat, or mouse amino acid sequence. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein do not specifically bind to TGFβ2. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein do not specifically bind to TGFβ3. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein do not specifically bind to TGFβ2 or TGFβ3. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein do not specifically bind to TGFβ2 and TGFβ3. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein specifically bind to TGFβ1 comprising the amino acid sequence set forth in SEQ ID NO: 24. The amino acid sequence of TGFβ2 and the amino acid sequence of TGFβ3 are set forth in SEQ ID NOs: 28 and 32, respectively. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein specifically bind to TGFβ1 comprising a non-naturally occurring amino acid sequence (also referred to herein as non-naturally occurring TGFβ1). For example, a non-naturally occurring TGFβ1 can include one or more recombinantly induced mutations compared to the amino acid sequence of a naturally occurring TGFβ1. In some embodiments, the amino acid sequence of TGFβ1, TGFβ2, or TGFβ3 comprises the amino acid sequence set forth in SEQ ID NOs:24-35, as shown in Table 10. In some embodiments, the amino acid sequence of TGFβ1, TGFβ2, or TGFβ3 comprises the amino acid sequence set forth in SEQ ID NOs:36-43, as shown in Table 11.

[0159] TGFβ1 (pro domain + growth factor domain) LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 24)

[0160] TGFβ2 (pro domain + growth factor domain) SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS (SEQ ID NO: 28)

[0161] TGFβ3 (pro domain + growth factor domain) SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS (SEQ ID NO: 32)

[0162] [Table 15] JPEG0007719779000016.jpg198166

[0163] [Table 16] JPEG0007719779000018.jpg231166JPEG0007719779000019.jpg236166JPEG0007719779000020.jpg139166

[0164] In some embodiments, the antigen-protein complex may be a so-called small latent complex (or SLC), which is composed of a dimeric complex of LAP (or prodomain) and growth factor domain. In other embodiments, the antigen-protein complex may be a so-called large latent complex (or LLC), which further comprises a presentation molecule bound to the SLC. Presentation molecules include LTBP proteins (e.g., LTBP1, LTBP2, LTBP3, and LTBP4), GARP proteins, LRRC33 proteins, or fragments thereof. When LLC is used as the antigen-protein complex, the minimal fragment required for practicing the embodiments disclosed herein typically contains at least 50 amino acids, preferably at least 100 amino acids, of the presentation molecule protein, including at least two cysteine residues capable of forming disulfide bonds with the pro-TGFβ1 complex. Specifically, these Cys residues form covalent bonds with cysteine residues present near the N-terminus of each monomer of the pro-TGFβ1 complex.

[0165] The antibodies or antigen-binding portions thereof described herein can bind to the latent LTBP1-TGFβ1 complex. In some embodiments, the LTBP1 protein is a naturally occurring protein or a fragment thereof. In some embodiments, the LTBP1 protein is a non-naturally occurring protein or a fragment thereof. In some embodiments, the LTBP1 protein is a recombinant protein. Such recombinant LTBP1 proteins may include LTBP1, alternatively spliced variants thereof, and / or fragments thereof. The recombinant LTBP1 protein may also be modified to include one or more detectable labels. In some embodiments, the LTBP1 protein includes a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP1 protein does not include a leader sequence (i.e., the leader sequence has been processed or cleaved). Such detectable labels may include, but are not limited to, a biotin label, a polyhistidine tag, a myc tag, an HA tag, and / or a fluorescent tag. In some embodiments, the LTBP1 protein is a mammalian LTBP1 protein. In some embodiments, the LTBP1 protein is a human, monkey, mouse, or rat LTBP1 protein. In some embodiments, the LTBP1 protein comprises the amino acid sequence set forth in SEQ ID NOs: 46 and 47 of Table 11. In some embodiments, the LTBP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 50 of Table 12.

[0166] The antibodies or antigen-binding portions thereof described herein can bind to the latent LTBP3-TGFβ1 complex. In some embodiments, the LTBP3 protein is a naturally occurring protein or a fragment thereof. In some embodiments, the LTBP3 protein is a non-naturally occurring protein or a fragment thereof. In some embodiments, the LTBP3 protein is a recombinant protein. Such recombinant LTBP3 proteins may include LTBP3, alternatively spliced variants thereof, and / or fragments thereof. In some embodiments, the LTBP3 protein includes a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP3 protein does not include a leader sequence (i.e., the leader sequence has been processed or cleaved). The recombinant LTBP3 protein may also be modified to include one or more detectable labels. Such detectable labels may include, but are not limited to, a biotin label, a polyhistidine tag, a myc tag, an HA tag, and / or a fluorescent tag. In some embodiments, the LTBP3 protein is a mammalian LTBP3 protein. In some embodiments, the LTBP3 protein is a human, monkey, mouse, or rat LTBP3 protein. In some embodiments, the LTBP3 protein comprises the amino acid sequence set forth in SEQ ID NOs: 44 and 45 of Table 11. In some embodiments, the LTBP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 51 of Table 12.

[0167] The antibodies or antigen-binding portions thereof described herein can bind to the latent GARP-TGFβ1 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 GARPs may be recombinant and are referred to herein as recombinant GARPs. Some recombinant GARPs may contain one or more modifications, truncations, and / or mutations compared to wild-type GARP. Recombinant GARPs may be modified to be soluble. In some embodiments, the GARP protein contains a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the GARP protein does not contain a leader sequence (i.e., the leader sequence has been processed or cleaved). In other embodiments, the recombinant GARP is modified to contain one or more detectable labels. In further embodiments, such detectable labels may include, but are not limited to, a biotin label, a polyhistidine tag, a flag tag, a myc tag, an HA tag, and / or a fluorescent tag. In some embodiments, the GARP protein is a mammalian GARP protein. In some embodiments, the GARP protein is a human, monkey, mouse, or rat GARP protein. In some embodiments, the GARP protein comprises the amino acid sequence set forth in SEQ ID NOs: 48-49 in Table 11. In some embodiments, the GARP protein comprises the amino acid sequence set forth in SEQ ID NOs: 52 and 53 in Table 13. In some embodiments, the antibodies or antigen-binding portions thereof described herein do not bind to TGFβ1 in a context-dependent manner; for example, binding to TGFβ1 occurs only when the TGFβ1 molecule is complexed with a specific presentation molecule, such as GARP. Instead, the antibodies and antigen-binding portions thereof bind to TGFβ1 in a context-independent manner. In other words, the antibodies or antigen-binding portions thereof bind to TGFβ1 when bound to any presentation molecule, i.e., GARP, LTBP1, LTBP3, and / or LRCC33.

[0168] The antibodies or antigen-binding portions thereof described herein can bind to the latent LRRC33-TGFβ1 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 and is referred to herein as recombinant LRRC33. Some recombinant LRRC33 proteins may contain one or more modifications, truncations, and / or mutations compared to wild-type LRRC33. Recombinant LRRC33 proteins may be modified to be soluble. For example, in some embodiments, the ectodomain of LRRC33 may be expressed with a C-terminal His tag to express a soluble LRRC33 protein (sLRRC33, e.g., see SEQ ID NO: 84). In some embodiments, the LRRC33 protein comprises a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LRRC33 protein does not comprise a leader sequence (i.e., the leader sequence is processed or cleaved). In other embodiments, the recombinant LRRC33 protein is modified to include one or more detectable labels. In further embodiments, such detectable labels may include, but are not limited to, a biotin label, a polyhistidine tag, a flag tag, a myc tag, an HA tag, and / or a fluorescent tag. In some embodiments, the LRRC33 protein is a mammalian LRRC33 protein. In some embodiments, the LRRC33 protein is a human, monkey, mouse, or rat LRRC33 protein. In some embodiments, the LRRC33 protein comprises the amino acid sequence set forth in SEQ ID NOs: 83, 84, and 101 in Table 13.

[0169] [Table 17] JPEG0007719779000022.jpg14166

[0170] [Table 18] JPEG0007719779000024.jpg120166

[0171] Safety / toxicity considerations histopathology, toxicology As mentioned above, known pan-inhibitors that antagonize all TGFβ isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3, have been documented to cause a variety of toxicities across multiple mammalian species. The most notable known toxicities include cardiovascular toxicity (such as valvular heart disease), epithelial hyperplasia, skin lesions, inflammation, and bleeding. More specifically, some of the observed toxicities associated with pan-TGFβ inhibitors (e.g., small molecule antagonists of TGFβR and non-selective neutralizing antibodies) reported in the literature include the following:

[0172] Cardiovascular toxicities associated with TGFβ inhibition include hyperplasia in the aortic, right AV, and left AV valves, inflammation in the aortic, left AV, and ascending aorta, bleeding in the ascending aorta, aortic, and left AV valves, and connective tissue degeneration in the ascending aorta (see, e.g., Strauber et al. (2014) "Nonclinical safety evaluation of a Transforming Growth Factor β receptor I kinase inhibitor in Fischer 344 rats and beagle dogs" J. Clin. Pract 4(3): 1000196).

[0173] Furthermore, neutralizing antibodies that bind all three TGFβ isoforms have been associated with specific epithelial toxicities, summarized in the table below.

[0174] [Table 19]

[0175] Applicants of the present disclosure have previously demonstrated an improved safety profile of monoclonal antibodies that selectively block the activation step of TGFβ1 by targeting the latent pro-TGFβ1 complex (see, e.g., WO2017 / 156500 and WO2018 / 129329), in which rat toxicity studies were described where no observable test article-related toxicity was observed when animals were dosed with up to 100 mg / kg / week of the inhibitor for 4 weeks.

[0176] Given the earlier recognition by the applicants of the present disclosure that the lack of isoform specificity of conventional TGFβ antagonists may underlie the toxicity associated with TGFβ inhibition (see, e.g., WO2017 / 156500), the inventors sought to further achieve broad-spectrum TGFβ1 inhibition for treating a variety of diseases that exhibit multifaceted TGFβ1 dysregulation, while maintaining the safety / tolerability profile of isoform-selective inhibitors. Thus, one of the goals of the work presented herein was to identify high-affinity inhibitors, particularly those with low dissociation rates (k オフ The goal was to increase the efficacy of such inhibitors while maintaining at least the same or an equivalent level of safety profile by specifically selecting a subset of antibodies having the following structure: (1) a nucleotide sequence similar to that of the nucleotide sequence ...

[0177] Thus, in some embodiments, novel antibodies according to the present disclosure have a maximum tolerated dose (MTD) of >100 mg / kg when dosed once weekly for at least 4 weeks (e.g., 4, 6, 8, 10, 12 weeks). In some embodiments, novel antibodies according to the present disclosure have a no observed adverse effect level (NOAEL) of up to 100 mg / kg when dosed once weekly for at least 4 weeks in rats. In some embodiments, antibodies have a NOAEL of at least 100 mg / kg / week when dosed for 4 or 12 weeks in mice. Suitable animal models for use in conducting safety / toxicity studies of TGFβ inhibitors and TGFβ1 inhibitors include, but are not limited to, rats, dogs, cynomolgus monkeys, and mice. In preferred embodiments, the minimum effective dose of the antibody based on appropriate preclinical efficacy studies is less than the NOAEL. More preferably, the minimum effective dose of the antibody is no more than about one-third the NOAEL. In particularly preferred embodiments, the minimum effective dose of the antibody is no more than about one-sixth the NOAEL. In some embodiments, the minimum effective amount of the antibody is about one-tenth of the NOAEL or less.

[0178] In some embodiments, the present invention encompasses isoform-selective antibodies capable of inhibiting TGFβ1 signaling that, when administered to a subject, do not cause cardiovascular or known epithelial toxicity at doses effective for treating TGFβ1-related indications. In some embodiments, the TGFβ inhibitor is TGFβ1 isoform-selective in that it does not inhibit TGFβ2. In some embodiments, the TGFβ inhibitor is TGFβ1 isoform-selective in that it does not inhibit TGFβ3. In some embodiments, the TGFβ inhibitor is TGFβ1 isoform-selective in that it does not inhibit TGFβ2 and TGFβ3. In some embodiments, the antibody has a minimum effective dose of about 3-10 mg / kg administered weekly, biweekly, or monthly. Preferably, the antibody causes no or minimal toxicity at doses that are at least 6 times the minimum effective dose (e.g., a 6-fold therapeutic window). More preferably, the antibody causes no or minimal toxicity at doses that are at least 10 times the minimum effective dose (e.g., a 10-fold therapeutic window). Even more preferably, the antibody causes no or minimal toxicity at doses that are at least 15 times the minimum effective dose (eg, a 15-fold therapeutic window).

[0179] Thus, selection of an antibody or antigen-binding fragment thereof for therapeutic use will depend on whether the antibody or antigen-binding fragment meets one or more criteria of a TGFβ inhibitor (e.g., a slow dissociation rate, e.g., a k of 10.0E-4 or less). オフconducting in vivo safety / toxicity studies in a suitable model to determine an amount of the antibody that is safe or toxic (e.g., an MTD, a NOAEL, or any art-recognized parameter for assessing safety / toxicity); and selecting an antibody or fragment that provides at least a 3-fold therapeutic window (preferably a 6-fold, more preferably a 10-fold, and even more preferably a 15-fold therapeutic window). The selected antibody or fragment may be used in the manufacture of a pharmaceutical composition comprising the antibody or fragment. Such a pharmaceutical composition may be used to treat a TGFβ1 indication in a subject as described herein. For example, the TGFβ1 indication may be a fibrotic and / or proliferative disorder. Preferably, the TGFβ inhibitor selected for therapeutic use or large-scale manufacture produces no observable adverse effects in treated animals after at least 4 weeks, e.g., 8 and 12 weeks of sustained exposure. In some embodiments, certain toxicities observed in histopathological analysis are considered non-adverse.

[0180] Immunosafety evaluation Cytokines play an important role in normal immune responses. However, when the immune system becomes overactivated, the positive feedback loop of cytokine production can lead to a "cytokine storm" or hypercytokinemia, a condition in which excessive cytokine production triggers an immune response that can damage organs, particularly the lungs and kidneys, and even result in death. Such a condition is characterized by significantly elevated levels of proinflammatory cytokines in serum. Historically, a phase I clinical trial of the anti-CD28 monoclonal antibody TGN1412 in healthy volunteers resulted in a life-threatening "cytokine storm" response resulting from the unexpected systemic and rapid induction of proinflammatory cytokines (Suntharalingam G et al., N Engl J Med. 2006 Sept. 7, 355(10):1018-28). This incident prompted increased awareness of the potential dangers associated with pharmacological stimulation of T cells.

[0181] Although TGFβ-directed therapies do not target specific T cell receptors or their ligands, it is contemplated that it would be prudent to conduct immune safety assessments including, for example, in vitro cytokine release assays, in vivo cytokine measurements from plasma samples of non-human primates treated with TGFβ inhibitors, and platelet assays using human platelets.

[0182] In some embodiments, selection of a TGFβ inhibitor for its therapeutic use and / or large-scale production includes evaluation of the ability of the TGFβ inhibitor to trigger cytokine release from cytokine-producing cells. Such evaluation may assay for one or more of the cytokines (e.g., pro-inflammatory cytokines), namely, IL-2, TNFα, IFNγ, IL-1β, CCL2 (MCP-1), and IL-6. In some embodiments, cytokine-producing cells may include peripheral blood mononuclear cell (PBMC) components from healthy donors. Cytokine responses following exposure to a TGFβ inhibitor (e.g., a disclosed antibody) herein may be compared to release following exposure to a control, e.g., an IgG isotype negative control, or any other appropriate control depending on the TGFβ inhibitor being tested. Cytokine activation may be assessed in plate-bound (e.g., immobilized) and / or soluble assay formats. Levels of IFNγ, IL-2, IL-1β, TNFα, IL-6, and CCL2 (MCP-1) should not exceed 10-fold, e.g., 8, 6, 4, or 2-fold activation in the negative control. In some embodiments, a positive control may also be used to confirm cytokine activation in the sample, e.g., in PBMCs. In some embodiments, these in vitro cytokine release results may be further confirmed in vivo in an animal model, e.g., a monkey toxicity study, e.g., a 4-week GLP or non-GLP repeated-dose monkey study.

[0183] Human platelets have been reported to express GARP, which can form TGFβ1 LLC (Tran et al., 2009. Proc. Nat'l. Acad. Sci. USA. 106(32): 13445-13450). In some embodiments, the antibodies disclosed herein do not significantly bind to GARP expressed on platelets. In some embodiments, platelet activation is assessed in vitro. In some embodiments, platelet aggregation, binding, and activation can be assessed in human whole blood or platelet-rich plasma from healthy donors. Platelet aggregation and binding after exposure to an antibody disclosed herein can be compared to exposure to a negative control, e.g., saline, or a reference article in a vehicle, e.g., a buffer solution. In selecting an appropriate TGFβ inhibitor for therapeutic use, a candidate drug should be evaluated to ensure that it does not trigger spontaneous or agent-induced activation. Furthermore, the drug should not interfere with the normal function of platelets (e.g., aggregation or clotting).

[0184] In certain embodiments, platelet aggregation and binding do not exceed the aggregation in a negative control by more than 10%. In some embodiments, platelet activation after exposure to an antibody disclosed herein can be compared to exposure to a positive control, such as adenosine diphosphate (ADP). Platelet activation status can be determined by surface expression of activation markers, such as CD62P (P-selectin) and GARP detectable by flow cytometry. Platelet activation should not exceed the activation in a negative control by more than 10%. In some embodiments, the results of in vitro platelet responses can be further confirmed in vivo in animal models, such as, for example, immunization-directed safety studies in non-human primates.

[0185] In some embodiments, selecting an antibody or antigen-binding fragment thereof for therapeutic use may include identifying an antibody or antigen-binding fragment that meets one or more criteria described herein, conducting in vivo efficacy studies in an appropriate preclinical model to determine an effective amount of the antibody or fragment, conducting in vivo safety / toxicity studies in an appropriate model to determine an amount of the antibody that is safe or toxic (e.g., an MTD, a NOAEL, or any art-recognized parameter for assessing safety / toxicity), and selecting an antibody or fragment that provides at least a 3-fold therapeutic window (preferably a 6-fold, more preferably a 10-fold, and even more preferably a 15-fold therapeutic window). In certain embodiments, the in vivo efficacy studies are conducted in two or more appropriate preclinical models that replicate the human condition. In some embodiments, such preclinical models include TGFβ1-positive fibrosis. In some embodiments, the preclinical model is selected from a liver fibrosis model, a kidney fibrosis model, a lung fibrosis model, a cardiac (cardiac) fibrosis model, and a skin fibrosis model.

[0186] Identifying an antibody or antigen-binding fragment thereof for therapeutic use may further include conducting an immune safety assay, which may include, but is not limited to, measuring cytokine release and / or determining the effect of the antibody or antigen-binding fragment on platelet binding, activation, and / or aggregation. In certain embodiments, cytokine release may be measured in vitro using PBMCs or in vivo using a preclinical model such as a non-human primate. In certain embodiments, the antibody or antigen-binding fragment thereof does not induce levels of IL-6, IFNγ, and / or TNFα greater than 10-fold compared to levels in an IgG control sample in an immune safety assessment. In certain embodiments, assessment of platelet binding, activation, and aggregation may be performed in vitro using PBMCs. In some embodiments, the antibody or antigen-binding fragment thereof does not induce a greater than 10% increase in platelet binding, activation, and / or aggregation compared to a buffer or isotype control in an immune safety assessment.

[0187] The selected antibody or fragment may be used in the manufacture of a pharmaceutical composition comprising the antibody or fragment. Such a pharmaceutical composition may be used to treat a TGFβ indication in a subject as described herein. For example, the TGFβ indication may be a fibrotic disorder such as organ fibrosis, e.g., liver fibrosis. Accordingly, the present invention includes a method for producing a pharmaceutical composition comprising a TGFβ inhibitor, comprising selecting a TGFβ inhibitor, which is tested for immune safety by an immune safety assessment including a cytokine release assay and optionally further including a platelet assay. The TGFβ inhibitor selected by this method does not trigger unacceptable levels of cytokine release compared to a control (e.g., an IgG control). Similarly, the TGFβ inhibitor selected by this method does not cause unacceptable levels of platelet aggregation, platelet activation, and / or platelet binding. Such a TGFβ inhibitor is then manufactured on a large scale, e.g., 250 L or more, e.g., 1000 L, 2000 L, 3000 L, 4000 L, or more, for commercial production of pharmaceutical compositions comprising the TGFβ inhibitor.

[0188] Isoform selectivity and mechanism of action of TGFβ inhibitors TGFβ inhibitors useful in carrying out various embodiments of the present invention are intended to pharmacologically interfere with one or more aspects of TGFβ1 function in vivo. The TGFβ inhibitor can be a TGFβ1 inhibitor, such as a TGFβ1 isoform-selective inhibitor, or a non-isoform-selective inhibitor. Non-isoform-selective inhibitors include, but are not limited to, low-molecular-weight ALK5 antagonists, neutralizing antibodies that bind to two or more of TGFβ1 / 2 / 3, such as GC1008 and variants, antibodies that bind to TGFβ1 / 3, and ligand traps, such as TGFβ1 / 3 inhibitors.

[0189] From a safety perspective, there is growing recognition that broad inhibition of TGFβ across isoforms may be responsible for observed toxicities, highlighting the fact that no successful TGFβ inhibitors have been developed to date. To avoid potentially dangerous adverse effects, several groups have recently begun work to identify inhibitors that target a subset, but not all, of the isoforms and still retain efficacy. However, from an efficacy perspective, the prevailing view in the field remains that it is advantageous to achieve therapeutic efficacy by inhibiting multiple isoforms of TGFβ. To accommodate this, toxicity management through "careful dosing regimens" has been proposed as a solution (Brennan et al., (2018) mAbs, 10:1, 1-17). Consistent with this premise, several groups are developing TGFβ inhibitors that target multiple isoforms. These include small molecular weight antagonists of the TGFβ receptor, e.g., ALK5 antagonists such as galunisertib (LY2157299 monohydrate, Eli Lilly), monoclonal antibodies (e.g., neutralizing antibodies) that inhibit all three isoforms ("pan-blocking" antibodies) (see, e.g., WO2018 / 134681), monoclonal antibodies that preferentially inhibit two of the three isoforms (e.g., antibodies against TGFβ1 / 2 (e.g., WO2016 / 161410) and TGFβ1 / 3 (e.g., WO2006 / 116002 and WO2020 / 051333), α V Integrin inhibitors, such as antibodies that bind to β3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrins and inhibit downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3 (e.g., PLN-74809, α V β1 / α VThese include small molecule, dual-selective inhibitors of β6 (Pliant Therapeutics, San Francisco, CA), and engineered molecules (e.g., fusion proteins) such as ligand traps (see, e.g., International Publication Nos. WO2018 / 029367, WO2018 / 129331, and WO2018 / 158727). Similarly, inhibitors of integrins such as αVβ6 also block integrin-dependent activation of both TGFβ1 and TGFβ3 and thus can be considered isoform-nonselective inhibitors of TGFβ signaling.

[0190] Previously, applicants have demonstrated that inhibition of TGFβ1 alone was sufficient to sensitize immunosuppressive tumors to checkpoint inhibitor treatment, even in tumors in which both TGFβ1 and TGFβ3 are co-expressed (see International Publication No. WO / 2020 / 014460). Similarly, TGFβ1-selective inhibitors have been shown to reduce fibrosis in preclinical models, including a mouse liver fibrosis model in which both TGFβ1 and TGFβ3 isoforms are co-expressed in fibrotic tissue, albeit in distinct cell types (herein). Surprisingly, inhibition of TGFβ3 promoted a pro-fibrotic phenotype. When TGFβ3 inhibitors were used alone, exacerbation of fibrosis was observed. Furthermore, when used in combination with a TGFβ1-selective inhibitor, the TGFβ3 inhibitor attenuated the anti-fibrotic effect of the TGFβ1-selective inhibitor, as evidenced by increased collagen accumulation in fibrotic liver. These results raise the possibility that in situations where fibrosis is a concern, inhibitory potency against TGFβ3 may be an undesirable feature of using TGFβ inhibitors as a treatment.

[0191] This unexpected finding has broader implications beyond the context of fibrosis, as profibrotic phenotypes (e.g., increased collagen deposition within the ECM) are associated not only with fibrosis but also with aspects of cancer progression, such as tumor invasion and metastasis. See, for example, Chakravarthy et al. (Nature Communications, (2018) 9:4692. "TGF-β-associated extracellular matrix genes link cancer-associated fibroblasts to immune evasion and immunotherapy failure"). Diseased tissues with dysregulated ECM, including fibrous tissue and stroma in various tumor types, can express both TGFβ1 and TGFβ3. To date, multiple groups have been working to develop TGFβ inhibitors that target both of these isoforms, including ligand traps, neutralizing antibodies, and integrin inhibitors. However, the findings presented herein suggest that such approaches may actually exacerbate disease.

[0192] Therefore, the present disclosure provides the teaching that a TGFβ inhibitor that does not specifically target TGFβ3 should be selected for the treatment of disorders involving ECM dysregulation, such as fibrosis and cancer. Preferably, such an inhibitor is a TGFβ1 isoform-selective inhibitor. A related method includes a method for selecting a TGFβ inhibitor for use in treating a fibrotic disorder in a subject, comprising testing the efficacy of one or more candidate inhibitors for their ability to inhibit TGFβ1, TGFβ2, and TGFβ3, and selecting an inhibitor that inhibits TGFβ1 but does not inhibit TGFβ3 for therapeutic use. A related treatment method can further include administering to the subject an inhibitor that inhibits TGFβ1 but does not inhibit TGFβ3 in an amount sufficient to treat a fibrotic disorder or to treat a subject who has or is at risk of developing a fibrotic disorder and / or cardiovascular disease. In some embodiments, the subject at risk of developing a fibrotic disorder may suffer from a metabolic disorder such as diabetes, obesity, and NASH. The proposed exclusion of patient subpopulations is intended to reduce the risk of initiating, promoting, or exacerbating pro-fibrotic effects.

[0193] In a preferred embodiment, the TGFβ inhibitor for use in treating fibrotic disorders is an isoform-selective activation inhibitor of TGFβ1 (a low kTGFβ1 inhibitor as disclosed herein) that is capable of targeting TGFβ1-containing latent complexes in vivo. オフ In some embodiments, the inhibitor is selected from the group consisting of Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, and Ab52. In preferred embodiments, the isoform-selective activation inhibitor of TGFβ1 is Ab2, Ab42, Ab46, Ab50, or a derivative thereof. Preferably, the isoform-selective activation inhibitor of TGFβ1 is Ab46, or an engineered molecule comprising an antigen-binding fragment thereof.

[0194] Thus, the antibodies of the present invention are intended to target the following complexes in disease sites (e.g., TME or fibrotic tissue), where the antibodies preemptively bind to the latent complexes, thereby preventing growth factor release: i) pro-TGFβ1 presented by GARP, ii) pro-TGFβ1 presented by LRRC33, iii) pro-TGFβ1 presented by LTBP1, and iv) pro-TGFβ1 presented by LTBP3. Typically, complexes (i) and (ii) are present on the cell surface because both GARP and LRRC33 are transmembrane proteins that can present or tether latent pro-TGFβ1 on the extracellular surface of cells expressing GARP or LRRC33, while complexes (iii) and (iv) are components of the extracellular matrix. In this way, the inhibitors embodied herein eliminate the need for full binding to endogenous high-affinity receptors to exert their inhibitory effects. Furthermore, targeting upstream of the ligand / receptor interaction may allow for a more durable effect because the target accessibility window is longer and more localized to the relevant tissue than conventional inhibitors, which target the active soluble growth factor only after it has been released from the latent complex.

[0195] Several studies have elucidated the mechanism of TGFβ1 activation. Three integrins, namely, αVβ6, α V β8, and α VIt has been demonstrated that β1 is the primary activator of latent TGFβ1 (Reed, NI et al., Sci Transl Med, 2015. 7(288): pp. 288ra79; Travis, MA and D. Sheppard, Annu Rev Immunol, 2014. 32: pp. 51-82; Munger, JS et al., Cell, 1999. 96(3): pp. 319-28). αV integrin binds with high affinity to the RGD sequence present in TGFβ1 and TGFβ1 LAP (Dong, X. et al., Nat Struct Mol Biol, 2014. 21(12): pp. 1091-6). Transgenic mice with a mutation in the TGFβ1 RGD site that prevents integrin binding but not secretion phenocopy TGFβ1- / - mice (Yang, Z. et al., J Cell Biol, 2007. 176(6): pp. 787-93). Mice lacking both β6 and β8 integrins recapitulate all essential phenotypes of TGFβ1 and TGFβ3 knockout mice, including multiorgan inflammation and cleft palate, confirming the essential role of these two integrins for TGFβ1 / 3 activation in development and homeostasis (Aluwihare, P. et al., J Cell Sci, 2009. 122(Pt 2): pp. 227-32). Key to integrin-dependent activation of latent TGFβ1 is covalent tethering with the presentation molecule. Disruption of the disulfide bond between GARP and TGFβ1 LAP by mutagenesis does not impair complex formation, whereas disruption of α VIt completely abolishes TGFβ1 activation by β6 (Wang, R. et al., Mol Biol Cell, 2012. 23(6): pp. 1129-39). Recent structures of latent TGFβ1 have revealed how integrins enable the release of active TGFβ1 from the latent complex; i.e., covalent binding of latent TGFβ1 to its presentation molecule anchors it to the ECM via LTBP or to the cytoskeleton via GARP or LRRC33. Integrin binding to the RGD sequence leads to a force-dependent change in the structure of LAP, allowing TGFβ1 to be released and bind to nearby receptors (Shi, M. et al., Nature, 2011. 474(7351): pp. 343-9). The importance of integrin-dependent TGFβ1 activation in disease has also been well validated. Small molecule inhibitors of αVβ1 protect against bleomycin-induced pulmonary fibrosis and carbon tetrachloride-induced liver fibrosis (Reed, NI et al., Sci Transl Med, 2015. 7(288): pp. 288ra79), and antibody-based α Vβ6 blockade or loss of integrin β6 expression suppresses bleomycin-induced pulmonary fibrosis and radiation-induced fibrosis (Munger, JS et al., Cell, 1999. 96(3): pp. 319-28; Horan, GS et al., Am J Respir Crit Care Med, 2008. 177(1): pp. 56-65). In addition to integrins, other mechanisms of TGFβ1 activation have been implicated, including activation by thrombospondin-1 and proteases such as thrombin, plasmin, matrix metalloproteinases (MMPs, e.g., MMP2, MMP9, and MMP12), cathepsin D, and kallikrein. Knockout of thrombospondin-1 recapitulates some aspects of the TGFβ1- / - phenotype in some tissues but is not protective in bleomycin-induced pulmonary fibrosis, which is known to be TGFβ-dependent (Ezzie, ME et al., Am J Respir Cell Mol Biol, 2011. 44(4): pp. 556-61). Furthermore, knockout of candidate proteases did not result in a TGFβ1 phenotype (Worthington, JJ, JE Klementowicz, and MA Travis, Trends Biochem Sci, 2011. 36(1): pp. 47-54). This could be explained by redundancy or by these mechanisms being critical in specific diseases rather than development and homeostasis.

[0196] The antibodies of the present disclosure work by interfering with the activation step of TGFβ1. In some embodiments, such inhibitors can inhibit integrin-dependent (e.g., mechanical or force-driven) activation of TGFβ1. In some embodiments, such inhibitors can inhibit protease-dependent or protease-induced activation of TGFβ1. The latter includes inhibitors that can inhibit the activation step of TGFβ1 in an integrin-independent manner. In some embodiments, such inhibitors can inhibit TGFβ1 activation regardless of the mode of activation, e.g., inhibit both integrin-dependent and protease-dependent activation of TGFβ1. Non-limiting examples of proteases that can activate TGFβ1 include serine proteases such as kallikrein, chymotrypsin, trypsin, elastase, plasmin, thrombin, and zinc metalloproteases (MMP family) such as MMP-2, MMP-9, MMP-12, MMP-13, and ADAM proteases (e.g., ADAM10 and ADAM17). Kallikreins include plasma kallikrein and tissue kallikrein, e.g., KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14, and KLK15. The data presented herein demonstrate examples of isoform-specific TGFβ1 inhibitors that can inhibit kallikrein-dependent activation of TGFβ1 in vitro. In some embodiments, the inhibitors of the invention prevent the release or dissociation of the latent complex active (mature) TGFβ1 growth factor.

[0197] In some embodiments, antibodies according to the present disclosure can induce internalization of complexes containing pro-TGFβ1 bound to LRRC33 or GARP on the cell surface. In some embodiments, the antibodies are inhibitors of cell-associated TGFβ1 (e.g., GARP-presented pro-TGFβ1 and LRRC33-presented pro-TGFβ1). The present invention includes antibodies or fragments thereof that specifically bind to such complexes (e.g., GARP-pro / latent TGFβ1 and LRRC33-pro / latent TGFβ1), thereby triggering internalization (e.g., endocytosis) of the complexes. This mechanism of action results in the removal or depletion of inactive TGFβ1 complexes from the cell surface (e.g., Tregs, macrophages, MDSCs, etc.), thus reducing the latent TGFβ1 available for activation.

[0198] TGFβ inhibitors suitable for therapeutic use A number of lines of evidence support the idea that many diseases manifest complex perturbations in TGFβ signaling, likely involving the participation of heterogeneous cell types that confer various effects of TGFβ function mediated by its interaction with so-called presentation molecules. At least four such presentation molecules have been identified, which can "present" TGFβ in various extracellular niches, enabling its activation in response to local stimuli. In one category, TGFβ is deposited within the ECM in association with ECM-associated presentation molecules such as LTBP1 and LTBP3, which mediate ECM-associated TGFβ activity. In another category, TGFβ is tethered to the surface of cells (e.g., immune cells) via presentation molecules such as GARP and LRRC33, which mediate specific immune functions. These presentation molecules show differential expression, localization, and / or function in various tissues and cell types, indicating that the initiating events and outcomes of TGFβ activation vary depending on the biological or pathological microenvironment. Based on the idea that many TGFβ effects may interact to contribute to disease progression, therapeutic agents that can antagonize multiple aspects of TGFβ function may offer greater efficacy.

[0199] In a preferred embodiment, the TGFβ inhibitor for use in treating fibrotic disorders is an isoform-selective activation inhibitor of TGFβ1 that can target TGFβ1-containing latent complexes in vivo (such as the low k オフ In a most preferred embodiment, the isoform-selective inhibitor of TGFβ1 activation is Ab2, Ab42, Ab46, Ab50, or a derivative thereof. Preferably, the isoform-selective inhibitor of TGFβ1 activation is Ab46, or an engineered molecule comprising an antigen-binding fragment thereof.

[0200] It is recognized that various diseases involve heterogeneous populations of cells as multiple sources of TGFβ1 that collectively contribute to disease pathogenesis and / or progression. Multiple TGFβ1-containing complexes ("contexts") likely coexist within the same disease microenvironment. Therefore, the ability to inhibit TGFβ1 in various biological contexts may be important.

[0201] However, under certain circumstances, so-called context-biased antibodies, which still specifically bind to all four antigen complexes but bind with stronger affinity to matrix-associated complexes than to cell-associated complexes, may be advantageous. This feature, i.e., the differential binding affinity of these antibodies to ECM complexes compared to immune cell complexes, may raise the possibility that such inhibitors may be particularly suitable as therapeutic agents for treating fibrotic conditions, such as organ fibrosis, in which affected patients undergo long-term treatment regimens to treat chronic symptoms. Under these circumstances, it is desirable to minimize unwanted inflammation triggered by immune stimulation.

[0202] In some embodiments, antibodies of the present disclosure have higher affinity for EMC complexes, such as hLTBP1-proTGFβ1 and hLTBP3-proTGFβ1, than for cell-associated complexes (KD of <1 nM), as determined, for example, by solubility equilibrium titration. It is contemplated that EMC-biased antibodies can preferentially target and inhibit EMC-associated TGFβ1 in vivo. Such antibodies may be advantageous for use in treating conditions with ECM dysregulation, such as abnormal ECM remodeling and / or stiffness. Typically, ECM dysregulation can be accompanied by an increase in the number of myofibroblasts or myofibroblast-like cells in disease environments, such as tumor and fibrotic microenvironments. Many of the abnormal features of the ECM are often manifested in a wide range of pathological conditions, including fibrosis, a proliferative disorder driven, at least in part, by the TGFβ1 pathway.

[0203] Context-biased antibodies with weaker binding to GARP-associated TGFβ complexes (e.g., human GARP-proTGFβ1) may be used in the treatment of conditions in which stimulating an immune response in a subject is undesirable and / or in situations in which the subject is expected to benefit from long-term TGFβ inhibitory therapy for managing chronic conditions such as many types of fibrosis. The rationale for the therapeutic use of TGFβ1 inhibitors with weaker binding affinity to GARP-proTGFβ1 is at least threefold.

[0204] First, GARP is primarily expressed on regulatory T cells, which play an important role in maintaining immune tolerance to self-antigens and preventing autoimmune diseases. Because Tregs generally suppress, slow, or downregulate the induction and proliferation of effector T cells, systemic inhibition of this function can lead to an overactive or exaggerated immune response in the host by neutralizing the "brake" normally provided by Treg cells. Therefore, the approach taken here (e.g., TGFβ1 inhibition without completely abolishing Treg function) aims to avoid the risk of inducing autoimmunity. Furthermore, patients who already have a propensity to develop an overactive immune response or autoimmunity may be particularly at risk of initiating or exacerbating such conditions if functional Tregs are not available. Therefore, inhibitors that at least partially preserve GARP-mediated TGFβ1 function may advantageously minimize such risks.

[0205] Second, evidence suggests that alterations in the Th17 / Treg ratio result in an imbalance of pro-fibrotic Th17 cytokines, which correlates with the severity of fibrosis, such as liver fibrosis (see, e.g., Shoukry et al. (2017) J Immunol 198 (Suppl 1): 197.12). We argue that abrogating perturbations of the GARP arm of TGFβ1 function may directly or indirectly exacerbate fibrotic conditions.

[0206] Third, regulatory T cells are essential for immune homeostasis and the prevention of autoimmunity. It has been argued that, particularly for TGFβ1 inhibitory therapies intended for long-term or chronic administration, it would be desirable to preserve at least some of the GARP-mediated TGFβ1 to avoid potential side effects resulting from complete disruption of normal Treg function in maintaining immune homeostasis (e.g., reviewed in Richert-Spuhler and Lund (2015) Prog Mol Biol Transl Sci. 136: 217–243). This strategy aims, at least in part, to preserve normal immune function, which is necessary, inter alia, for fighting infections.

[0207] TGFβ1-related indications The isoform-specific TGFβ1 inhibitors described herein can be used to treat TGFβ1-related indications in subjects. Various disease states have been suggested to involve dysregulation of TGFβ signaling as a contributing factor. Indeed, the pathogenesis and / or progression of certain human conditions are believed to be primarily driven or dependent on TGFβ1 activity. Furthermore, it is contemplated that cross-talk exists between TGFβ1-responsive cells. In some cases, the interplay between the pleiotropic activities of the TGFβ1 axis can trigger a cascade of events that leads to disease progression, exacerbation, and / or suppression of the host's ability to combat disease. For example, a particular disease microenvironment, such as the tumor microenvironment (TME), may be associated with TGFβ1 presented by multiple different presentation molecules, such as LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, LRRC33-proTGFβ1, and any combination thereof. It is possible that TGFβ1 activity in one context may in turn regulate or influence TGFβ1 activity in another context, and if dysregulated, this could lead to exacerbation of the disease state. Therefore, it is desirable to broadly inhibit multiple modes of TGFβ1 function (i.e., multiple contexts), while selectively limiting such inhibitory effects to TGFβ1 isoforms. The goal is not to disrupt homeostatic TGFβ signaling mediated by other isoforms, including TGFβ3, which plays an important role in what would be a cure.

[0208] Furthermore, recent observations in murine fibrosis models suggest deleterious effects of TGFβ3 inhibition in tissues with dysregulated ECM (see Example 17), raising the possibility that the role of TGFβ3 extends beyond homeostasis. Ample evidence suggests that ECM dysregulation is found in several pathologies, including fibrosis and cancer. Indeed, many of the key profibrotic genes are recognized as markers for various cancers. These markers include, for example, col1a1, col3A1, PAI-1, CCL2, ACTA2, FN-1, CTGF, and TGFB1. Therefore, the finding that simultaneous blockade of TGFβ3 appears to be detrimental in fibrosis may be applicable to a broader range of conditions associated with ECM dysregulation.

[0209] In addition to the potential concerns about inhibiting TGFβ3 discussed above, Takahashi et al. (Nat Metab. 2019, 1(2): 291-303) recently reported a beneficial role for TGFβ2 in metabolic regulation. The authors identified TGFβ2 as an exercise-induced adipokine that stimulated glucose and fatty acid uptake in vitro and tissue glucose uptake in vivo, which improved metabolism and reduced high-fat diet-induced inflammation in obese mice. Furthermore, the authors observed that lactate, a metabolite released from muscle during exercise, stimulated TGFβ2 expression in human adipocytes and that lactate-lowering agents reduced circulating TGFβ2 levels and reduced the exercise-stimulated improvement in glucose tolerance. These observations suggest that the therapeutic use of TGFβ inhibitors with inhibitory activity against the TGFβ2 isoform may be harmful, at least in metabolic terms.

[0210] Thus, the present disclosure provides TGFβ inhibitors that inhibit TGFβ1 but not TGFβ2 for use in treating a TGFβ-related indication (e.g., fibrosis) in a subject. In some embodiments, the subject benefits from improved metabolism, and optionally the subject has or is at risk of developing a metabolic disease, such as obesity, high-fat diet-induced inflammation, or glucose dysregulation (e.g., diabetes). In some embodiments, the TGFβ-related indication is cancer, and optionally the cancer has a solid tumor, such as locally advanced cancer and metastatic cancer. In some embodiments, the TGFβ-related indication is myelofibrosis. In some embodiments, the TGFβ-related indication is an immune disorder. In some embodiments, the TGFβ-related indication is fibrosis.

[0211] In some embodiments, the TGFβ inhibitor is selective for TGFβ1 in that it does not inhibit TGFβ2. In some embodiments, the TGFβ inhibitor is selective for TGFβ1 in that it does not inhibit TGFβ3. In preferred embodiments, the TGFβ inhibitor is selective for TGFβ1 in that it does not inhibit TGFβ2 and TGFβ3.

[0212] Related methods for selecting TGFβ inhibitors for therapeutic use are also encompassed herein. According to some embodiments, the TGFβ inhibitor is TGFβ-1 selective.

[0213] According to a preferred embodiment, a TGFβ1-selective inhibitor is selected for use in treating patients with fibrosis associated with fatty liver (e.g., nonalcoholic fatty liver disease (NAFLD)) or nonalcoholic steatohepatitis (NASH). This is based at least on the rationale that i) avoiding TGFβ3 inhibition may reduce the risk of worsening ECM dysregulation (which may increase fibrosis), and ii) avoiding TGFβ2 inhibition may reduce the risk of increasing metabolic burden in patients. The present invention expands on the idea of selecting the "right TGFβ1 inhibitor" for the "right patient population" to treat conditions with specific criteria and / or clinical features. With regard to identifying / selecting appropriate indications and / or patient populations in which inhibitors of TGFβ1 described herein are likely to have beneficial effects (e.g., clinical benefit), at least two questions can be asked: i) whether the disease is primarily driven by or dependent on (or at least co-dominant with) the TGFβ1 isoform over other isoforms in humans, and ii) whether the disease involves both matrix-associated and / or immune cell-associated TGFβ1 functions.

[0214] Differential expression of the three known TGFβ isoforms, namely, TGFβ1, TGFβ2, and TGFβ3, has been observed in various tissues under normal (healthy, homeostatic) and diseased conditions (note that "TGFB" is sometimes used to refer to the gene as opposed to the protein). Nevertheless, the concept of isoform selectivity has never been fully exploited, nor has it been achieved in a conventional manner to support pan-inhibition of TGFβ across multiple isoforms. Furthermore, isoform expression patterns may be differentially regulated not only in normal (homeostatic) versus abnormal (pathological) conditions, but also in various patient subpopulations. Because most preclinical studies are performed in a limited number of animal models, data obtained using such models may be biased, leading to misinterpretation of the data or erroneous conclusions regarding their applicability to the human condition (i.e., translatability).

[0215] Thus, the present invention recognizes that differential expression of TGFβ isoforms in preclinical animal models should be considered in predicting the efficacy of specific inhibitors and meaningfully interpreting preclinical data regarding translatability to human clinical conditions. As exemplified herein, TGFβ1 and TGFβ3 are codominant in certain murine syngeneic cancer models (e.g., EMT-6 and 4T1) widely used in preclinical studies (see Figure 13D). In contrast, numerous other cancer models (e.g., S91, B16, and MBT-2) express TGFβ1 almost exclusively, similar to what is observed in many human tumors, where TGFβ1 frequently appears to be the dominant isoform over TGFβ2 / 3 (see Figures 13B and 13C). Furthermore, the TGFβ isoform predominantly expressed under homeostatic conditions may not be the disease-associated isoform. For example, in normal lung tissue in healthy rats, tonic TGFβ signaling appears to be primarily mediated by TGFβ3. However, TGFβ1 appears to be significantly upregulated in pathologies such as pulmonary fibrosis. Taken together, it is beneficial to test or confirm the relative expression of TGFβ isoforms in clinical samples in order to select appropriate therapeutic agents to which patients are likely to respond.

[0216] As described herein, isoform-selective TGFβ1 inhibitors are particularly advantageous for treating diseases in which the TGFβ1 isoform is predominantly expressed compared to other isoforms (e.g., referred to as TGFβ1-dominant). As an example, a non-limiting list of human cancer clinical samples containing the relative expression levels of TGFB1 (left), TGFB2 (center), and TGFB3 (right) is provided in Figure 13C. Each horizontal lime across the three isoforms represents a single patient. As can be seen, overall TGFβ1 expression (TGFB1) is significantly higher than the other two isoforms across many tumor / cancer types in most of these human tumors / cancers, suggesting that TGFβ1-selective inhibition would be beneficial in these disease types. More recent bioinformatic analyses also support the idea that TGFβ1 is a key driver in several disease states.

[0217] In some embodiments, the TGFβ1-selective inhibitors disclosed herein are sufficient to treat diseases (e.g., fibrosis, solid tumors, etc.) despite co-expression of TGFβ1 and TGFβ3. In some embodiments, the antibody is selected from the group consisting of Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, and Ab52. In preferred embodiments, the isoform-selective inhibitor of TGFβ1 is Ab2, Ab42, Ab46, Ab50, or a derivative thereof. Preferably, the isoform-selective inhibitor of TGFβ1 activation is Ab46, or an engineered molecule comprising an antigen-binding fragment thereof.

[0218] In certain cases, it is beneficial to test or confirm the relative expression levels of the three TGFβ isoforms (i.e., TGFβ1 / TGFB1, TGFβ2 / TGFB2, and TGFβ3 / TGFB3) in clinical samples taken from individual patients. Such information may provide better predictions regarding the effectiveness of particular treatments in individual patients or patient populations, which can help ensure the selection of appropriate treatment regimens (e.g., personalized / customized treatments) to increase the likelihood of clinical response.

[0219] Thus, the present invention includes methods for selecting a patient population or subject likely to respond to a treatment comprising an isoform-specific TGFβ1 inhibitor according to the present disclosure. Such methods include providing a biological sample (e.g., a clinical sample) from the subject, determining (e.g., measuring or assaying) the relative levels of TGFβ1, TGFβ2, and TGFβ3 in the sample, and administering a composition comprising a TGFβ1 inhibitor to the subject if TGFβ1 is the predominant isoform compared to TGFβ2 and TGFβ3 and / or if TGFβ1 is significantly overexpressed or upregulated compared to a control. In some embodiments, such methods include obtaining information regarding the predetermined relative expression levels of TGFβ1, TGFβ2, and TGFβ3, identifying the subject as having a TGFβ1-positive, preferably TGFβ1-dominant, disease, and administering a TGFβ1 inhibitor to the subject. In some embodiments, such a subject has a disease (e.g., cancer) that is resistant to a treatment (e.g., cancer treatment). In some embodiments, such subjects are intolerant to treatment and therefore have discontinued or are likely to discontinue treatment.Adding a TGFβ1 inhibitor to a treatment regimen can reduce the dosage of the first treatment and still achieve clinical benefits in combination.Preferably, the TGFβ1 isoform-selective activation inhibitor is Ab46, or an engineered molecule comprising its antigen-binding fragment.

[0220] The relative levels of isoforms may be determined by RNA-based assays and / or protein-based assays that are well known in the art. In some embodiments, the administering step may also include another treatment, such as an immune checkpoint inhibitor, or other agents provided elsewhere herein. Such methods may optionally include a step of evaluating the therapeutic response by monitoring changes in the relative levels of TGFβ1 / TGFB1, TGFβ2 / TGFB2, and TGFβ3 / TGFB3 at two or more time points. In some embodiments, clinical samples (such as biopsies) are taken both before and after administration. In some embodiments, clinical samples (such as biopsies) are taken multiple times after treatment to evaluate in vivo effects over time.

[0221] In addition to the first question, which is driven by the aspect of isoform specificity, the second question examines the breadth of TGFβ1 function involved in a particular disease. This can be expressed by the number of TGFβ1 contexts, i.e., which presentation molecules mediate disease-related TGFβ1 function. Broad-context inhibitors specific for TGFβ1, such as context-independent inhibitors, are useful for treating diseases involving both the ECM and immune components of TGFβ1 function. Such diseases may be associated with dysregulation of the ECM and disruption of immune cell function or immune responses.

[0222] Whether a patient's specific condition is related to or driven by multiple aspects of TGFβ1 function can be assessed in clinical samples collected from patients by evaluating the expression profile of presentation molecules. Various assays, including RNA-based and protein-based assays, are known in the art and can be performed to obtain an expression profile. The relative expression levels (and / or changes / alterations) of LTBP1, LTBP3, GARP, and LRRC33 in a sample can indicate the source and / or context of TGFβ1 activity associated with the condition. For example, a biopsy sample collected from a solid tumor can show high expression of all four presentation molecules. For example, LTBP1 and LTBP3 may be highly expressed in CAFs within the tumor stroma, while GARP and LRRC33 may be highly expressed by disease-related immune cells such as Tregs, MDSCs, and infiltrating leukocytes, respectively. Similarly, LTBP1 and LTBP3 may be highly expressed in FAFs (e.g., myofibroblasts) within the fibrotic microenvironment, while LRRC33 may be highly expressed by fibrosis-associated immune cells such as M2 macrophages and MDSCs.

[0223] Thus, the present invention includes methods for determining (e.g., testing or confirming) the involvement of TGFβ1 in disease relative to TGFβ2 and TGFβ3. In some embodiments, the method further includes identifying the source (or context) of disease-associated TGFβ1. In some embodiments, the source / context is assessed by determining the expression of TGFβ-presenting molecules, e.g., LTBP1, LTBP3, GARP, and LRRC33, in a clinical sample taken from the patient. In some embodiments, a tissue biopsy is used. In some embodiments, the histopathological analysis may include digital pathology.

[0224] Isoform-selective TGFβ1 inhibitors such as those described herein can be used to treat a wide variety of diseases, disorders, and / or conditions associated with TGFβ1 dysregulation in human subjects (i.e., TGFβ1-related indications). As used herein, "disease (disorder or condition) associated with TGFβ1 dysregulation" or "TGFβ1-related indication" refers to any disease, disorder, and / or condition associated with the expression, activity, and / or metabolism of TGFβ1, or any disease, disorder, and / or condition that may benefit from inhibiting the activity and / or levels of TGFβ1. Preferably, the isoform-selective activation inhibitor of TGFβ1 is Ab46, or an engineered molecule comprising an antigen-binding fragment thereof.

[0225] The present invention includes the use of such isoform-specific TGFβ1 inhibitors in methods for treating diseases associated with TGFβ1 dysregulation in human subjects. Such inhibitors are typically formulated into pharmaceutical compositions further comprising pharmaceutically acceptable excipients. TGFβ is a key regulator of ECM composition, structure, and function. Advantageously, the inhibitor targets both ECM-associated TGFβ1 signaling and immune cell-associated TGFβ1 signaling, but does not target TGFβ2 and / or TGFβ3 signaling in vivo. In some embodiments, the inhibitor preferentially binds to ECM-associated pro-TGFβ1 complexes, thereby blocking TGFβ1 signaling in matrix niches. The disease may involve dysregulation or dysfunction of ECM components or functions, including increased collagen deposition. In some embodiments, dysregulation or dysfunction of ECM components or functions may further include increased stiffness and / or ECM reorganization. In some embodiments, dysregulation or dysfunction of ECM components or functions includes an increase in myofibroblasts within the disease site. In some embodiments, the dysregulation of the ECM comprises increased matrix stiffness. In some embodiments, the dysregulation of the ECM involves fibronectin and / or fibrillin. Preferably, the TGFβ1 isoform-selective activation inhibitor is Ab46, or an engineered molecule comprising an antigen-binding fragment thereof.

[0226] In some embodiments, the disease is characterized by dysregulation or dysfunction of myeloid cell proliferation or differentiation, and optionally, the dysregulation or dysfunction of myeloid cells includes monocyte recruitment to the disease site or differentiation into polarized M2 cells, and / or abnormal macrophage function. In some embodiments, the dysregulation of myeloid cells includes increased levels of MDSCs. Elevated MDSCs may include an increase in the number / frequency of circulating MDSCs, for example, in peripheral blood. Elevated MDSCs may be observed at disease sites such as fibrous tissues and solid tumors. The terms circulating and circulating (such as "circulating MDSCs" and "circulating MDSCs") may be used interchangeably.

[0227] In some embodiments, the disease is a fibrotic disorder or disease (such as organ fibrosis). In some embodiments, the present disclosure provides a method of using measurements of circulating MDSCs in treating fibrosis in a subject administered a TGFβ inhibitor. In some embodiments, the TGFβ inhibitor is TGFβ1 isoform selective in that it does not inhibit TGFβ2. In some embodiments, the TGFβ inhibitor is TGFβ1 isoform selective in that it does not inhibit TGFβ3. Preferably, the TGFβ inhibitor is TGFβ1 isoform selective in that it does not inhibit TGFβ2 and TGFβ3. Furthermore, in some embodiments, the circulating MDSC population can be used as an early predictive biomarker of the effectiveness of a treatment for a fibrotic disorder, for example, at a time point before other markers of treatment effectiveness can be detected.

[0228] In some embodiments, the disease is characterized by abnormal cellular differentiation involving epithelial-mesenchymal transition (EMT) and / or endothelial-mesenchymal transition (EndMT). In some embodiments, these processes occurring at the disease site (e.g., the TME and fibrotic microenvironment) result in an increase in myofibroblasts or myofibroblast-like cells at the site, including, for example, CAFs and FAFs.

[0229] In some embodiments, the disease is characterized by aberrant gene expression of one or more of the marker genes selected from the group consisting of PAI-1, ACTA2, CCL2, Col1a1, Col3a1, FN-1, CTGF, and TGFB1.

[0230] A therapeutically effective amount of such an inhibitor is administered to a subject suffering from or diagnosed with a disease.

[0231] In some embodiments, the dysregulation or dysfunction of fibroblast differentiation comprises an increase in 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 may produce CCL2 / MCP-1 and / or CXCL12 / SDF-1. In some embodiments, the myofibroblasts or myofibroblast-like cells are localized in fibrotic tissue.

[0232] In some embodiments, the dysregulation or dysfunction of regulatory T cells comprises increased Treg activity.

[0233] In some embodiments, the dysregulated or impaired effector T cell (Teff) proliferation or function comprises suppression of CD4+ / CD8+ cell proliferation.

[0234] In some embodiments, the dysregulated or impaired proliferation or differentiation of myeloid cells comprises increased proliferation of myeloid progenitor cells. Increased proliferation of myeloid cells can occur in the bone marrow.

[0235] In some embodiments, dysregulated or impaired monocyte differentiation comprises increased differentiation of bone marrow-derived and / or tissue-resident monocytes into macrophages at disease sites such as fibrous tissue and / or solid tumors.

[0236] In some embodiments, dysregulation or dysfunction of monocyte recruitment comprises increased recruitment of bone marrow-derived monocytes to disease sites, such as the TME, which leads to increased macrophage differentiation and M2 polarization, and subsequently an increase in TAMs.

[0237] In some embodiments, the dysregulation or impairment of macrophage function comprises increased polarization of macrophages towards the M2 phenotype.

[0238] In some embodiments, the dysregulated or impaired proliferation or differentiation of myeloid cells comprises an increase in the number of Tregs, MDSCs, and / or TANs.

[0239] TGFβ-related indications may include conditions involving an immune-negative disease microenvironment, such as tumors or cancerous tissues that suppress the body's normal defense mechanisms / immunity, in part by eliminating effector immune cells (e.g., CD4+ and / or CD8+ T cells). In some embodiments, such immune-negative conditions are associated with a poor response to treatment (e.g., cancer therapy). Non-limiting examples of cancer therapies to which patients respond poorly include, but are not limited to, checkpoint inhibitor therapy, cancer vaccines, chemotherapy, and radiation therapy. Without intending to be bound by a particular theory, it is contemplated that TGFβ inhibitors, such as those described herein, may help counter the ability of tumors to evade or eliminate anti-cancer immunity by restoring T cell (e.g., CD8+ cell) access by promoting T cell expansion and / or infiltration into tumors.

[0240] Thus, TGFβ inhibition may overcome treatment resistance (e.g., immune checkpoint resistance, cancer vaccine resistance, CAR-T resistance, chemotherapy resistance, radiotherapy resistance, etc.) in immune-eliminating disease environments (such as the TME) by removing and restoring blockade of effector T cell access and cytotoxic effector function. Such effects of TGFβ inhibition may further provide durable immune memory mediated, for example, by CD8+ T cells.

[0241] Non-limiting examples of TGFβ-related indications include fibrosis, including organ fibrosis (e.g., renal fibrosis, liver fibrosis, cardiac / cardiovascular fibrosis, muscle fibrosis, skin fibrosis, uterine fibrosis / endometriosis, and pulmonary fibrosis), scleroderma, Alport syndrome, cancer (including, but not limited to, hematological cancers such as leukemia, myelofibrosis, multiple myeloma, colon cancer, kidney cancer, breast cancer, malignant melanoma, glioblastoma), fibrosis associated with solid tumors (e.g., cancer desmoplasia such as desmoplastic melanoma, pancreatic cancer-associated desmoplasia, and breast cancer desmoplasia), stromal fibrosis (e.g., breast stromal fibrosis), radiation-induced fibrosis (e.g., radiation fibrosis syndrome), promoting rapid hematopoiesis after chemotherapy, bone healing, wound healing, dementia, myelofibrosis, myelodysplasia (e.g., myelodysplasic syndrome or MDS), renal disease (e.g., end-stage renal disease, kidney disease or ESRD), unilateral ureteral obstruction (UUO), tooth loss and / or degeneration, endothelial proliferative syndrome, asthma and allergies, gastrointestinal disorders, anemia of aging, aortic aneurysm, orphan indications (such as Marfan syndrome and Kamurati-Engelman disease), obesity, diabetes, arthritis, multiple sclerosis, muscular dystrophies (e.g., myotonic muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, and Emery-Dreifuss muscular dystrophy), amyotrophic lateral sclerosis (ALS), Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, metabolic syndrome, nutritional disorders, organ atrophy, chronic obstructive pulmonary disease (COPD), and anorexia.

[0242] TGFβ-related indications can also include conditions in which major histocompatibility complex (MHC) class I is deleted or deficient (e.g., downregulated), including genetic disorders in which one or more components of MHC-mediated signaling are impaired, as well as conditions in which MHC expression is altered by other factors such as cancer, infection, fibrosis, and drugs.

[0243] For example, downregulation of MHC class I in tumors is associated with tumor escape from immune surveillance. Indeed, immune evasion strategies aimed at avoiding T cell recognition, including loss of tumor MHC class I expression, are commonly found in malignant cells. Tumor immune evasion has been observed to have a negative effect on the clinical outcome of cancer immunotherapy, including treatment with antibodies that block immune checkpoint molecules (e.g., reviewed in Garrido et al., (2017) Curr Opin Immunol 39: 44-51. "The urgent need to recover MHC class I in cancers for effective immunotherapy," incorporated herein by reference). Therefore, isoform-selective TGFβ1 inhibitors encompassed by the present disclosure can be administered either as monotherapy or in conjunction with another treatment (e.g., checkpoint inhibitors, chemotherapy, radiation therapy, etc.) to release or boost anti-cancer immunity and / or enhance the responsiveness or efficacy of another treatment.

[0244] Downregulation of MHC class I proteins has also been associated with certain infectious diseases, including viral infections such as HIV. See, e.g., Cohen et al. (1999) Immunity 10(6): 661-671. "The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells," incorporated herein by reference. Thus, isoform-selective TGFβ1 inhibitors encompassed by the present disclosure can be administered either as monotherapy or in conjunction with another therapy (such as an antiviral therapy, a protease inhibitor therapy, etc.) to relieve or boost host immunity and / or enhance the responsiveness or effectiveness of another therapy.

[0245] fibrotic conditions In response to tissue injury caused by physical damage / trauma, toxic substances, and / or infection, a natural repair process is initiated that involves several cell types, including fibroblasts, several different types of immune cells, and resident epithelial and endothelial cells. However, if left unchecked, this process can lead to excessive accumulation of extracellular matrix (ECM) and fibrosis, which in turn can result in progressive loss of tissue function and organ failure (Caja et al., Int. J. Mol. Sci. 2018, 19, 1294).

[0246] Fibrosis can occur in several different organs, including the lungs, kidneys, liver, heart, and skin. Regardless of the organ, the fibrotic response is characterized by inflammation, altered epithelial-mesenchymal interactions, and fibroblast proliferation. One of the hallmarks of fibrosis is the differentiation of fibroblasts into myofibroblasts, which contributes significantly to the dysregulation of the ECM. However, it has been proposed that myofibroblasts can also be derived from other cell sources (e.g., endothelial cells, epithelial cells, and mesenchymal stem cells) (Kim, K.K. et al., Cold Spring Harb. Perspect. Biol., 2017; Okabe, H. Histol. Histophathol., 2016, 31, 141-148; and Li, C. et al., Nat Commun., 2016, 7, 11455). Furthermore, immune cells play an important role in the process by secreting cytokines and chemokines that promote myofibroblast differentiation, stimulate ECM deposition, and recruit additional immune cells to the injured tissue (Caja et al., Int. J. Mol. Sci. 2018, 19, 1294).

[0247] Similar to the activation of fibroblasts in fibrous tissue, activation of cancer-associated fibroblasts (CAFs) can occur in the tumor environment, resulting in excessive amounts of ECM. The ECM provides a scaffold for the infiltration of other cells (e.g., tumorigenic-promoting immune cells) and a substrate for cell migration. In other cases, excess ECM can act as a barrier to anti-tumorigenic immune cells.

[0248] TGFβ is recognized as a central orchestrator of the fibrotic response. TGFβ can promote myofibroblast differentiation, recruit immune cells, and influence epithelial and endothelial cell differentiation. In particular, TGFβ upregulates the production of ECM and basement membrane proteins, such as fibronectin, collagen, laminin, osteopontin, tenascin, elastin, and decorin. TGFβ-induced myofibroblast differentiation can result in further deposition of ECM proteins, secretion of matrix metalloproteinases (MMPs), and proliferation of myofibroblasts (Fabregat et al., FEBS J. 2016, 283, 2219-2232; Meng et al., Nat. Rev. Nephrol. 2016, 12, 325-338; and Kulkarni et al., Am. J. Respir. Cell Mol. Biol. 2016, 54, 751-760). Furthermore, TGFβ mediates phenotypic changes affecting contractile proteins and type I collagen in vascular smooth muscle cells (VSCM) and can activate myofibroblasts and other stromal cells to enhance the synthesis of collagen cross-linking proteins, such as the lysyl oxidase (LOX) family of matrix remodeling enzymes (Busnadiego et al., Mol. Cell. Biol. 2013, 33, 2388-2401). Furthermore, TGFβ has been shown to regulate both EMT and EndMT, which contributes to the differentiation of pro-fibrotic cell types such as myofibroblasts and CAFs. Furthermore, TGFβ has been shown to induce epithelial apoptosis, which may promote lung and liver fibrosis, among other tissues (Barbas-Filho et al., J. Clin. Pathol. 2001, 54, 132-138 and Wang et al., Dev. Dyn. 2017, 247, 492-508).

[0249] Whether innate or recruited, macrophages play an important role in the response to tissue injury and repair. However, upon receiving certain signals, they can become profibrotic. TGFβ has also been shown to activate M2 macrophages, which are proinflammatory, among other cytokines. Upon activation, these macrophages secrete their own cytokines, including TGFβ, ECM components, angiogenic factors, and chemotactic factors. M2 macrophages have been shown to be essential for TGFβ-driven pulmonary fibrosis (Murray et al., Int. J. Biochem. Cell Biol. 2011, 43, 154-162).

[0250] Therefore, according to the present invention, isoform-specific inhibitors of TGFβ1, such as those described herein, are used to treat fibrosis (e.g., 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 ameliorating fibrosis. More specifically, such antibodies and / or compositions are selective antagonists of TGFβ1 that can target TGFβ1 presented by various types of presentation molecules.

[0251] Antibodies targeting TGFβ reduce fibrosis in a number of preclinical models. Such antibodies and / or antibody-based compounds include LY2382770 (available from Eli Lilly, Indianapolis, IN, e.g., Creative Biolabs, CAT#TAB-605CL). Also included are those described in U.S. Patents Nos. 6,492,497, 7,151,169, 7,723,486, and 8,383,780, the contents of each of which are incorporated herein by reference in their entirety. Prior art TGFβ antagonists include, for example, agents that target and block integrin-dependent activation of TGFβ.

[0252] However, evidence suggests that such prior art agents may not mediate isoform-specific inhibition and may cause undesired effects by inadvertently blocking the normal function of TGFβ2 and / or TGFβ3. Indeed, the data presented herein support this idea. Normal (non-diseased) lung tissue contains relatively low but measurable levels of TGFβ2 and TGFβ3, but significantly less TGFβ1. In comparison, in certain pathologies, such as fibrosis, TGFβ1 is preferentially upregulated compared to other isoforms. Preferably, TGFβ antagonists for use in treating such conditions exert their inhibitory activity only against disease-induced or disease-related isoforms, while preserving the function of other isoforms normally expressed in tissues to mediate tonic signaling. Prior art inhibitors (LY2109761 (CAS No. 700874-71-1, Eli Lilly)), small molecule TGFβ receptor antagonists, and α V Both the β6 integrin-targeting monoclonal antibody and the β6 integrin-targeting monoclonal antibody have been shown to inhibit tonic signaling downstream of TGFβ in non-diseased rat BAL, raising the possibility that these inhibitors may cause unwanted side effects. Alternatively or additionally, agents that target and block integrin-dependent activation of TGFβ may block only a subset of integrins responsible for disease-associated TGFβ1 activation among the numerous integrins expressed by various cell types and playing a role in pathogenesis. Furthermore, even if such antagonists can selectively block integrin-mediated activation of TGFβ1 isoforms, they may be ineffective in blocking TGFβ1 activation initiated by other modes, such as protease-dependent activation. In contrast, isoform-specific inhibitors of TGFβ1, such as those described herein, aim to prevent the activation step of TGFβ1 regardless of the specific mode of activation while maintaining isoform selectivity. Preferably, the isoform-selective activation inhibitor of TGFβ1 is Ab46 or an engineered molecule comprising an antigen-binding fragment thereof.

[0253] It is further contemplated that isoform-specific TGFβ1 inhibitors that preferentially inhibit matrix-associated over cell-associated antigen complexes (i.e., exhibit context bias) may provide therapeutic benefits in certain clinical situations. For example, TGFβ1 inhibitors (those that target all four antigen complexes) may increase immune activation through targeting cell-associated TGFβ1 (e.g., GARP-TGFβ1 expressed on regulatory T cells). Immune activation may be disadvantageous for certain patients, such as those with autoimmune disease or at risk of sepsis. Thus, context-biased antibodies may be useful for treating diseases associated with matrix-associated TGFβ1 complexes (e.g., fibrosis) while minimizing immune activation.

[0254] As mentioned above, the inhibitory effect on TGFβ3 may be an undesirable feature when using TGFβ inhibitors as a treatment in situations where fibrosis is a concern.Therefore, in some embodiments, the TGFβ inhibitor is TGFβ1 isoform selective in that it does not inhibit TGFβ3.In some embodiments, the TGFβ inhibitor is TGFβ1 isoform selective in that it does not inhibit TGFβ2.In some embodiments, the TGFβ inhibitor is TGFβ1 isoform selective in that it does not inhibit TGFβ2 and TGFβ3.

[0255] It is further contemplated that isoform-specific TGFβ3 inhibitors may provide therapeutic benefits in certain disease states. For example, certain fibrotic diseases treated with TGFβ1 inhibitors may also be TGFβ3-positive (i.e., TGFβ1+ / TGFβ3+ fibrotic tissue), characterized in that diseased tissue (e.g., fibrotic tissue) expresses both isoforms. Thus, the present invention includes the use of isoform-selective TGFβ1 inhibitors in combination with isoform-selective TGFβ3 inhibitors in the treatment of such conditions (i.e., TGFβ1+ / TGFβ3+ fibrotic tissue). Such TGFβ3 inhibitors may be context-independent or context-biased.

[0256] Fibrotic indications for which the antibodies and / or compositions of the disclosure may be used therapeutically include, but are not limited to, pulmonary indications (e.g., idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disorder (COPD), allergic asthma, acute lung injury, eosinophilic esophagitis, pulmonary arterial hypertension, and chemical gas injury), renal indications (e.g., diabetic glomerulosclerosis, focal segmental glomeruloclerosis (FSGS), chronic kidney disease (CKD), fibrosis associated with kidney transplantation and chronic rejection, IgA nephropathy, diabetic kidney disease (DKD), hemolytic uremic syndrome), hepatic fibrosis (e.g., non-alcoholic steatohepatitis (NASH), such as alcoholic fibrosis, non-alcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC), primary biliary cirrhosis, and sclerosing cholangitis), chronic viral hepatitis, parasitemia, pulmonary fibrosis, and pulmonary fibrosis. and cancer or secondary fibrosis (e.g., myelofibrosis, head and neck cancer, M7 acute myocardial infarction, ulcerative colitis ... Other diseases, disorders, or conditions associated with fibrosis (including degenerative diseases) that may be treated using the compounds and / or compositions of the present disclosure include, but are not limited to, adenomyosis, endometriosis, Marfan syndrome, scleroderma syndrome, scleroderma, rheumatoid arthritis, myelofibrosis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, muscular dystrophies (such as DMD), Parkinson's disease, ALS, Dupuytren's contracture, Kamraci-Engelmann disease, nerve scarring, dementia, proliferative vitreoretinopathy, corneal injury, complications following glaucoma drainage surgery, and multiple sclerosis (MS).Many such fibrotic conditions are also associated with inflammation in the affected tissue, indicating the involvement of immune components. Such inflammation may be accompanied by abnormal immune cell populations, such as an increased number of Th17 cells, a decreased number of Treg cells, and / or both. In each case, affected patients may show an increased Th17 / Treg cell ratio.

[0257] In some embodiments, fibrotic indications that may be treated with the compositions and / or methods described herein include organ fibrosis, such as pulmonary fibrosis (e.g., IPF), renal fibrosis (e.g., fibrosis associated with CKD), liver fibrosis (e.g., associated with or caused by NASH), cardiac or cardiac tissue fibrosis, skin fibrosis (e.g., scleroderma), uterine (e.g., endometrium, myometrium) fibrosis, muscle (e.g., skeletal muscle) fibrosis, and bone marrow fibrosis. In some embodiments, such treatment may reduce or delay the need for an organ transplant in a patient. In some embodiments, such treatment may prolong patient survival.

[0258] In some embodiments, TGFβ1 inhibitors such as those disclosed herein may delay or reduce the need for organ transplants, hi some embodiments, the organ transplant is a lung transplant, a liver transplant, or a kidney transplant.

[0259] For the treatment of IPF, patients who may benefit from treatment include those with familial IPF and those with sporadic IPF. Administration of a therapeutically effective amount of a TGFβ1 isoform-specific inhibitor can reduce myofibroblast accumulation in lung tissue, reduce collagen deposition, reduce IPF symptoms, improve or maintain lung function, and prolong survival. In some embodiments, the inhibitor blocks the activation of ECM-associated TGFβ1 (e.g., pro / latent TGFβ1 presented by LTBP1 / 3) in the fibrotic environment within IPF. The inhibitor can optionally further block macrophage-associated TGFβ1 (e.g., pro / latent TGFβ1 presented by LRRC33), for example, the activation of alveolar macrophages. As a result, the inhibitor can suppress fibronectin release and other fibrosis-related factors. In some embodiments, the inhibitor blocks the activation of hepatic stellate cells.

[0260] According to some embodiments, patients with IPF who may benefit from treatment with a composition described herein (e.g., a therapeutically effective amount of an isoform-specific inhibitor of TGFβ1) are subjects who are candidates for lung transplantation. According to some embodiments, subjects with IPF who may benefit from treatment with a composition described herein (e.g., a therapeutically effective amount of an isoform-specific inhibitor of TGFβ1) are subjects who are not candidates for lung transplantation.

[0261] According to some embodiments, the composition described herein is administered to patients with IPF in combination with a second drug. According to some embodiments, the second drug is one or more of pirfenidone, nintedanib, and / or N-acetylcysteine. According to some embodiments, the therapeutically effective amount of TGFβ1 isoform-specific inhibitor is administered to patients with IPF in combination with one or more of pirfenidone, nintedanib, and / or N-acetylcysteine.

[0262] It is well established that hepatic stellate cell (HSC) activation is a central driver of fibrosis in liver injury. This process involves the transdifferentiation of quiescent vitamin A-storing cells into proliferative, fibrogenic myofibroblasts, the primary source of extracellular matrix (ECM) protein accumulation. However, this process has been shown to be mediated by many different pathways, including autophagy, endoplasmic reticulum stress, oxidative stress, retinol and cholesterol metabolism, epigenetics, and receptor-mediated signaling. Furthermore, inflammatory cells, including macrophages, hepatocytes, liver sinusoidal endothelial cells, natural killer cells, natural killer T cells, platelets, and B cells, have also been shown to modulate HSC activation (Tsuchida and Friedman, Nature Reviews Gastroenterology & Hepatology, volume 14, pages 397–411 (2017)). In just one specific example, Seki et al. demonstrated that activation of TLR4 (which recognizes LPS presented by bacteria) leads to upregulation of chemokine secretion, inducing Kupffer cell chemotaxis, and sensitizing HSCs to TGFβ-induced signals, allowing unrestrained activation of Kupffer cells (Seki et al., Nature Medicine volume 13, pages 1324-1332 (2007)).

[0263] It is well known that inflammation plays a major role in the development and progression of liver fibrosis. Specifically, liver injury leads to inflammation and recruitment of monocytes / macrophages (as well as lymphocytes, eosinophils, and plasma cells), which produce profibrotic factors, including TGFβ. Furthermore, studies have shown that both liver tissue-resident macrophages (Kupffer cells) and recruited macrophages derived from bone marrow play important roles in the progression of liver fibrosis, and that the TGFβ pathway can promote macrophage polarization and profibrotic function during liver fibrosis. Indeed, both Kupffer cells and recruited macrophages have been shown to activate HSCs and induce their transdifferentiation into myofibroblasts through a paracrine mechanism involving TGFβ. In turn, myofibroblasts produce and deposit ECM components, leading to fibrosis (Fabregat and Caballero-Diaz, Front Oncol. 2018;8:357).

[0264] However, myofibroblasts can also be derived from other sources, including portal vein and resident fibroblasts, bone marrow-derived fibrocytes, liver epithelial cells undergoing EMT, endothelial cells undergoing Endothelial Modulation (EMT), and vascular smooth muscle cells and pericytes. Indeed, TGFβ has been shown to regulate both Endothelial Modulation (EMT) and EMT, leading to an increase in myofibroblasts that drive liver fibrosis. (Pardali et al., Int J Mol Sci. 2017 Oct;18(10):2157). Therefore, targeting TGFβ has become an attractive therapeutic target for the treatment of fibrotic conditions.

[0265] TGFβ has been shown to play multiple roles in liver fibrosis and disease progression. For example, TGFβ has been shown to mediate the activation of HSCs into myofibroblasts. TGFβ has also been shown to mediate epithelial-mesenchymal transition (EMT) in hepatocytes, which may contribute to an increase in the myofibroblast population. Furthermore, TGFβ has been shown to induce changes in tumor cell plasticity (Fabregat and Caballero-Diaz, Front Oncol. 2018;8:357).

[0266] TGFβ can be found on many different cellular sources in the fibrotic and / or tumor microenvironment, thus suggesting TGFβ presentation by multiple different presentation molecules (e.g., LTBP1, LTBP3, GARP, and / or LRRC33). However, under certain circumstances, targeting specific sources of TGFβ over others may be beneficial. For example, Henderson et al. showed that deleting αv integrin in HSCs protected mice from CCL4-induced liver fibrosis (Henderson et al., Nat. Med. 2013, 19, 1617-16-24). Because integrins are the primary activators of LTBP-presented TGFβ, this result suggests that targeting LTBP-presented TGFβ may be sufficient to treat fibrosis under certain circumstances. However, because immune cells play an important role in the fibrotic response, TGFβ inhibitors that target TGFβ presented by most or all of the presentation molecule TGFβ complex may be beneficial.

[0267] In recent years, the treatment of liver fibrosis has become an area of interest due to its increasing prevalence worldwide. For example, nonalcoholic fatty liver disease (NAFLD) is associated with metabolic abnormalities such as obesity, insulin resistance, fasting hyperglycemia, dyslipidemia, and altered adipokine profiles. NAFLD is characterized by excessive lipid accumulation in hepatocytes and is a spectrum of diseases that progress from fatty liver (accumulation of lipid / fat droplets in hepatocytes) to nonalcoholic steatohepatitis (NASH), liver fibrosis, and ultimately cirrhosis in the most severe cases. NASH with fibrosis or cirrhosis increases the risk of developing hepatocellular carcinoma (HCC) (Starley BQ et al., Hepatology 2010; 51: 1820-1832). The progression of steatosis to NASH has been proposed to be controlled by a "multiple hit" model, where the first hit is insulin resistance and metabolic disturbances, which lead to hepatic steatosis, followed by oxidative stress, proinflammatory cytokine-mediated hepatocellular injury, altered lipid partitioning and hepatotoxicity mediated by free fatty acids, abnormal hepatic cholesterol loading, hyperinsulinemia, hyperleptinemia, and hypoadiponectinemia (Tilg H, Moschen AR, Hepatology 2010; 52: 1836-1846 and Yilmaz Y., Aliment Pharmacol Ther 2012; 36: 815-823).

[0268] Many animal models have been developed to study liver fibrosis. For example, a high-fat diet in mice has been shown to mimic both the histopathology and pathogenesis of human NAFLD. Furthermore, some genetic models, such as the db / db and ob / ob mouse models, also exhibit characteristics of human metabolic syndrome and NAFLD. Animal models for the study of NASH also exist, primarily consisting of various diet-induced models, including, but not limited to, a methionine- and choline-deficient diet (MCD), a high-cholesterol diet (HCD), a choline-deficient high-fat diet (CDHFD), a choline-deficient L-amino acid-deficient diet, a choline-deficient L-amino acid-deficient diet plus carbon tetrachloride, a high-fat diet plus streptozotocin, a high-fat plus high-cholesterol diet (HFHC), a high-fructose diet (HFD), and a high-fructose high-fat diet (HFHF). Genetic mouse models for the study of NASH include but are not limited to foz / foz mice, hepatocyte-specific PTEN-deficient mice, Db / db mice + diethylnitrosamine (DEN), and db / db mice + MCD.All the details of these models, including their respective advantages and disadvantages, are outlined in Jennie Ka Ching Lau et al., J Pathol 2017;241:36-44, the contents of which are incorporated herein by reference.

[0269] Other models useful for testing the effectiveness of isoform-specific TGFβ inhibitors in liver fibrosis include carbon tetrachloride (CCL4) model and choline-deficient high-fat diet (CDHFD) liver fibrosis model.Other models useful for testing the effectiveness of isoform-specific TGFβ inhibitors in liver fibrosis include bile duct ligation (BDL) model (see, for example, Tag et al., J Vis Exp. 2015; (96): 52438).

[0270] As described herein, based on a body of evidence suggesting the possible adverse effects of TGFβ2 / 3 inhibition, in situations where fibrosis is a concern, the inhibitory effect on TGFβ2 and / or TGFβ3 may be an undesirable feature of using TGFβ inhibitors as a treatment.According to some embodiments, the preferred inhibitors for use in the treatment of liver conditions such as NAFLD and NASH are TGFβ1 isoform selective.In some embodiments, TGFβ inhibitors are TGFβ1 isoform selective in that they do not inhibit TGFβ3.In some embodiments, TGFβ inhibitors are TGFβ1 isoform selective in that they do not inhibit TGFβ2.In some embodiments, TGFβ inhibitors are TGFβ1 isoform selective in that they do not inhibit TGFβ2 and TGFβ3.

[0271] Isoform-specific TGFβ1 inhibitors, such as those provided herein, may be used to treat fibrotic conditions of the liver, such as fatty liver (e.g., non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Fatty liver may or may not be inflammatory. Inflammation of the liver due to fatty liver (i.e., steatohepatitis) can develop into scarring (fibrosis), which then often progresses to cirrhosis (scarring that distorts the liver's structure and impairs its function). Thus, inhibitors may be used to treat such conditions. In some embodiments, the inhibitors block activation of ECM-associated TGFβ1 (e.g., pro / latent TGFβ1 presented by LTBP1 / 3) within the fibrotic environment of the liver. The inhibitors optionally also block activation of macrophage-associated TGFβ1 (e.g., pro / latent TGFβ1 presented by LRRC33), such as Kupffer cells (also known as stellate macrophages), and infiltrating monocyte-derived macrophages and MDSCs. As a result, the inhibitor may suppress fibrosis-related factors (e.g., fibrotic markers described herein). Administration of an inhibitor to subjects with such a condition 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 produce clinically meaningful results in a patient population treated with the inhibitor compared to a control population not treated with the inhibitor. In some embodiments, an effective amount of an inhibitor may achieve both a reduction in liver fat and a reduction in fibrosis (e.g., scarring) in NASH patients. In some embodiments, an effective amount of an inhibitor may achieve an improvement in at least one stage of fibrosis in NASH patients without worsening steatohepatitis. In some embodiments, an effective amount of an inhibitor may reduce the incidence of liver failure and / or liver cancer in NASH patients.

[0272] In some embodiments, an effective amount of an inhibitor may normalize levels of multiple inflammatory or fibrotic serum biomarkers compared to controls, assessed, for example, 12 to 36 weeks after initiation of treatment. In some embodiments, inflammatory or fibrotic biomarkers may be used to assess the severity of NAFLD (by measuring the level of hepatic steatosis), to select patients for treatment, and / or to monitor disease progression or treatment response. For example, blood biomarkers and panels may include, but are not limited to: i) fatty liver indices (BMI, waist circumference, serum triglycerides, and gamma-glutamyltransferase (GGT); ii) hepatic steatosis index (serum aspartate aminotransferase (AST): alanine aminotransferase (ALT) ratio, BMI, sex, and presence of diabetes mellitus); i) NAFLD liver fat score (serum ALT, HDL cholesterol, triglycerides, hemoglobin A) 1c , and white blood cell count), ii) SteatoTest (BioPredictive) (serum levels of total bilirubin, GGT, α2-macroglobin, haptoglobin, ALT, apolipoprotein AI, total cholesterol, triglycerides, glucose (adjusted for age and sex), and BMI), and iii) NAFLD Ridge score (ALT, HDL cholesterol, triglycerides, hemoglobin A 1c , serum levels of white blood cell count, and comorbidity data (and presence of hypertension).

[0273] In some embodiments, imaging biomarkers can be used to assess the level of hepatic steatosis.For example, imaging biomarkers can include but are not limited to ultrasound, control attenuation parameter (CAP), MRI estimated proton density fat fraction (MRI-PDFF) and magnetic resonance spectroscopy (MRS).

[0274] Although liver biopsy is the current standard for diagnosing NASH, the variability between pathologists limits the effectiveness of such diagnostic methods.Therefore, the use of the Fatty Liver Progression Prevention (FLIP) algorithm (including histological steatosis, activity and fibrosis score) can be used to improve the consistency of biopsy-based NASH diagnosis.In addition, many non-invasive biomarkers can also be useful for diagnosing and monitoring disease.Therefore, in some embodiments, inflammatory or fibrotic biomarkers can be used to evaluate the severity of NASH, select patients for treatment, and / or monitor disease progression or treatment response.Blood biomarkers can include: i) apoptosis markers such as CK18 fragments, pan-cytokeratins, and sFAS; ii) inflammatory markers such as CRP, TNF, IL-8, and CXCL10; iii) lipid oxidation products such as 11-HETE, 9-HODE, 13-HODE, 12-oxo-ODE, LA-13-HODE (oxNASH score), and 11,12-diHETrE; iv) lysosomal enzymes such as cathepsin D, and v) Combination panels such as the NASHTest (BioPredictive) and NASH diagnostic panels (including the presence of diabetes mellitus, gender, BMI, and serum levels of triglycerides, CK18 fragments, and total CK18).

[0275] In some embodiments, biomarkers and related panels can be useful in diagnosing fibrosis and / or cirrhosis, selecting patients for treatment, and / or monitoring disease progression or treatment response.For example, non-invasive tests for liver fibrosis and cirrhosis include but are not limited to AST:ALT ratio, AST:platelet ratio index, fibrosis-4 index (age, AST, ALT and platelet count), NAFLD fibrosis score (age, BMI, impaired fasting glucose and / or diabetes, AST ALT, platelet count, albumin), BARD score (AST, ALT, BMI and diabetes).

[0276] Specific fibrosis markers and panels may also be useful, including but not limited to hyaluronic acid, PIIPNP, Pro-C3, TIMP1, laminin, Enhanced Liver Fibrosis (ELF) panel (PIINP, hyaluronic acid, TIMP1), FibroTest (GGT, total bilirubin, α2m, apolipoprotein AI, and haptoglobin), and FibroMeter NAFLD (body weight, prothrombin index, ALT, AST, ferritin, and fasting blood glucose). Imaging biomarkers of liver fibrosis may include, but are not limited to, FibroScan (TE), point shear wave elastography (pSWE) (also known as acoustic radiation force impulse (ARFI)), 2D-3D SWE, magnetic resonance elastography (MRE), and multiparameter MRI.

[0277] In some embodiments, serum levels of liver enzymes such as alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate tramsaminase (AST), or G-glutamyltransferase (GGT) may be measured as indicators of fibrosis in the liver.

[0278] In some embodiments, gene and genome biomarkers may be useful for assessing the risk and severity of NAFLD, including the evaluation of various SNPs, cell-free ncRNA and miRNA.A comprehensive review of known gene and genome biomarkers, and the above-mentioned blood biomarkers, panels, imaging biomarkers and tests are summarized in VWS Wong et al., Nat Rev Gastroenterol Hepatol.2018 August,15(8):461-478, the contents of which are incorporated herein by reference.

[0279] In some embodiments, in patients with NASH, isoform-specific TGFβ1 inhibitors may be administered to patients receiving one or more additional therapies, including, but not limited to, myostatin inhibitors, which may generally enhance metabolic regulation in patients with clinical signs of metabolic syndrome, including NASH and NAFLD. In a preferred embodiment, the isoform-selective activation inhibitor of TGFβ1 is an engineered molecule, including Ab42, Ab46, Ab50, a derivative thereof, or an antigen-binding fragment thereof.

[0280] In some embodiments, in NASH patients, an isoform-specific TGFβ1 inhibitor may be administered to patients receiving an acetyl-CoA carboxylase inhibitor (ACCi) (e.g., Filsocostat (also known as GS-0976) or PF-05221304). Other therapeutic agents that may be useful in combination with the improved isoform-specific TGFβ1 inhibitors described herein include, but are not limited to, GLP-1 receptor agonists or analogs (e.g., semaglutide), farnesoid X receptor (FXR) agonists (e.g., GS-9674, also known as Cilofexor), ASK1 inhibitors (e.g., selonsertib), obeticholic acid, PPAR agonists (e.g., GFT505, also known as Elafibranor), nitazoxanide, ketohexokinase (KHK) inhibitors (e.g., PF-06835919), and / or diacylglycerol O-acyltransferase 2 (DGAT2) inhibitors (e.g., PF-06865571). In some embodiments, any one or more of the above-mentioned therapeutic agents can be combined with an isoform-specific TGFβ1 inhibitor of the present disclosure, for example, an isoform-specific TGFβ1 inhibitor in combination with an FXR agonist, an ACC inhibitor, and / or a GLP-1 analog.

[0281] In some embodiments, treatment with an isoform-specific TGFβ1 inhibitor, alone or in combination with one or more additional therapeutic agents, reduces liver fat F measured by MRI-PDF. In some embodiments, the reduction in liver fat is at least 20%, for example, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, or ≧50%. In some embodiments, treatment with an isoform-specific TGFβ1 inhibitor, alone or in combination with one or more additional therapeutic agents, reduces serum ALT and / or GGT by at least 20%, for example, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, or ≧50%. In some embodiments, treatment with an isoform-specific TGFβ1 inhibitor, alone or in combination with one or more additional therapeutic agents, reduces bile acid synthesis.

[0282] In some embodiments, NASH patients may have advanced liver fibrosis (stage F3 / F4). In some embodiments, such patients may have stage F3 advanced liver fibrosis. In some embodiments, such patients have stage F4 liver fibrosis characterized by cirrhosis. In some embodiments, NASH patients have or are at risk of developing hepatocellular carcinoma and / or esophageal varices.

[0283] Provided below is the fibrosis staging in nonalcoholic fatty liver disease according to the classification derived by the Nonalcoholic Steatohepatitis Clinical Research Network Pathology Committee.

[0284] [Table 20]

[0285] To enable assessment of various histologic features during treatment and encompass the full spectrum of NAFLD, the NASH Clinical Research Network (CRN) Pathology Committee conducted extensive univariate and multivariate analyses of the association between various histologic features observed in NASH and the diagnosis of NASH by the Pathology Committee. The results were a scoring system for both NASH activity (grade) and collagen deposition plus structural remodeling (stage). The grading system, the NASH Activity Score (NAS), was the unweighted sum of three histologic components: steatosis (0–3), lobular inflammation (0–3), and swelling and degeneration (0–2). It ranged from 0 to 8. The NAS includes features of active injury that are potentially reversible. Furthermore, the fibrosis staging system of Brunt et al. was further developed. In the NASH CRN system, stage 1 fibrosis scores were subdivided into delicate (1A) and dense (1B) perisinusoidal fibrosis, while stage 1C was defined as portal fibrosis without concurrent perisinusoidal fibrosis (reviewed by Staal, World J. Gastroenterol. 2015 Oct. 21;21(39):11077-11087, which is incorporated herein by reference).

[0286] Isoform-specific TGFβ1 inhibitors, such as those provided herein, can be used to treat renal fibrotic conditions, such as diseases characterized by extracellular matrix accumulation, in which significant increases in TGFβ expression in glomeruli and tubulointerstitium have been observed (IgA nephropathy, focal and segmental glomerulosclerosis, crescentic glomerulonephritis, lupus nephritis, and diabetic nephropathy). While glomerular and tubulointerstitial deposition of two matrix components induced by TGFβ, fibronectin (EDA+), and PAI-1 were significantly elevated in all diseases with matrix accumulation, correlation analysis revealed a close relationship primarily with the TGFβ1 isoform. Therefore, isoform-specific TGFβ1 inhibitors are useful as therapeutic agents for a range of human glomerular disorders in which TGFβ is associated with the pathological accumulation of extracellular matrix. In a preferred embodiment, the isoform-selective activation inhibitor of TGFβ1 is Ab42, Ab46, Ab50, a derivative thereof, or an engineered molecule comprising an antigen-binding fragment thereof.

[0287] In some embodiments, the fibrotic condition of the kidney is associated with chronic kidney disease (CKD). CKD is primarily caused by hypertension or diabetes and claims over a million lives. CKD patients require lifelong medical care, ranging from strict dietary habits and medications to dialysis and transplantation. In some embodiments, TGFβ1 inhibitor treatments described herein may reduce or delay the need for dialysis and / or transplantation. In some embodiments, such treatments may reduce the need (e.g., dosage, frequency) for other treatments. In some embodiments, isoform-specific TGFβ1 inhibitors may be administered to patients receiving one or more additional therapies, including, but not limited to, myostatin inhibitors, which may generally enhance metabolic regulation in patients with CKD.

[0288] Fibrotic conditions that can be treated with the TGFβ1 inhibitors of the present disclosure include conditions involving fibrosis and / or chronic inflammation. Such conditions may be neuromuscular disorders, including, but not limited to, Duchenne muscular dystrophy (DMD), as well as other genetic disorders such as multiple sclerosis (MS) and cystic fibrosis (CF). Through the inhibition of both ECM and immune cell-associated TGFβ1 arms, TGFβ1 inhibitors such as those described herein are believed to suppress fibrotic progression and restore M1 / M2 macrophage polarization. In a preferred embodiment, the isoform-selective activation inhibitor of TGFβ1 is an engineered molecule, including Ab42, Ab46, Ab50, derivatives thereof, or antigen-binding fragments thereof.

[0289] Useful models for studying CKD and renal fibrosis include, but are not limited to, NZB / W, MRL / lpr, and BXSB mouse strains, anti-GBM models, anti-Thy1 models, 5 / 6 nephrectomy, radiation nephropathy, puromycin aminonucleoside nephrosis (PAN) and adriamycin nephropathy, folate nephropathy, CyA nephropathy, DOCA salt nephropathy, HIV-associated nephropathy (HIVAN) transgenic mouse model, spontaneously hypertensive rats (SHR), buffalo / mna rats, Munich Wistar Fromter (MWF) rats, unilateral ureteral obstruction (UUO), Col4A knockout mice (Alport syndrome) (see Yang et al., Drug Discov Today Dis Models. 2010;7(1-2):13-19, the contents of which are incorporated herein by reference).

[0290] Organ fibrosis that can be treated using the methods provided herein includes cardiac (e.g., cardiovascular) fibrosis. In some embodiments, the cardiac fibrosis is associated with heart failure, e.g., chronic heart failure (CHF). In some embodiments, the heart failure can be associated with myocardial disease and / or metabolic disease. In some embodiments, an isoform-specific TGFβ1 inhibitor can be administered to a patient receiving one or more additional therapies, including, but not limited to, a myostatin inhibitor in patients with cardiac dysfunction associated with cardiac fibrosis and metabolic disorders. In a preferred embodiment, the isoform-selective activation inhibitor of TGFβ1 is an engineered molecule, including Ab42, Ab46, Ab50, derivatives thereof, or antigen-binding fragments thereof.

[0291] Useful genetic models for studying cardiac fibrosis include, but are not limited to, cardiomyocyte-specific FAK-KO mice, genetically modified SR-BI / apoE double KO (dKO) mice, syndecan-1 null mice, EC-SOD overexpressing mice, and PKC-δ knockout mice. Useful surgical mouse models for studying cardiac fibrosis include, but are not limited to, coronary artery ligation, ischemia-reperfusion models (open-chest and closed-chest), chronic ischemia models, ischemia-reperfusion models with ischemic preconditioning, Langendorff models, transverse aortic constriction (TAC), ascending aortic constriction, abdominal aortic constriction, pulmonary artery banding, TAC with distal left anterior coronary artery ligation, aortocaval fistula (ACF) models, and aortic regurgitation models (see Rai et al., Mol Cell Biochem. 2017 January; 424(1-2): 123-145, the contents of which are incorporated herein by reference).

[0292] In some embodiments, fibrotic conditions that may be treated with the compositions and / or methods described herein include desmoplasia. Desmoplasia occurs around neoplasms and can cause dense fibrosis around tumors (e.g., desmoplastic stroma) or scar tissue within the abdomen after abdominal surgery. In some embodiments, desmoplasia is associated with malignant tumors. Due to the dense formation around malignant tumors, conventional anti-cancer therapeutic agents (e.g., chemotherapy) may not effectively penetrate and reach cancer cells for clinical efficacy. Isoform-specific inhibitors of TGFβ1, such as those described herein, can be used to disrupt desmoplasia, which can slow fibrosis and aid in the effectiveness of anti-cancer treatments. In some embodiments, isoform-specific inhibitors of TGFβ1 can be used as monotherapy (described further below).

[0293] In some embodiments, the patient has a fibrous solid tumor (e.g., desmoplastic) and has been excluded or removed from the surgical candidate pool such that the fibrous solid tumor is considered unresectable or inoperable. Such patients may be candidates for receiving the TGFβ1 inhibitor therapy of the present disclosure. The TGFβ1 inhibitors of the present invention may render the tumor resectable or operable after administration, such that the patient may be a candidate for surgical resection.

[0294] To treat a patient with a fibrotic condition, a TGFβ1 isoform-specific inhibitor is administered to the subject in an amount effective to treat fibrosis. In a preferred embodiment, the isoform-selective activation inhibitor of TGFβ1 is an engineered molecule including Ab42, Ab46, Ab50, a derivative thereof, or an antigen-binding fragment thereof. 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 an antibody capable of blocking the activation of LTBP-mediated TGFβ1 localized (e.g., tethered) in the ECM and GARP-mediated TGFβ1 localized (e.g., tethered on) in immune cells. In some embodiments, the antibody is an antibody capable of blocking the activation of LTBP-mediated TGFβ1 localized in the ECM, monocytes / macrophages localized (e.g., tethered on) and LRRC33-mediated TGFβ1. In some embodiments, the LTBP is LTBP1 and / or LTBP3. In some embodiments, it may be beneficial to target and inhibit TGFβ1 presented by LRRC33 on pro-fibrotic M2-like macrophages in the fibrotic microenvironment.

[0295] Assays useful in determining the effectiveness of the antibodies and / or compositions of the present disclosure in altering fibrosis include, but are not limited to, histological assays for enumerating fibroblasts and basic immunohistochemical analyses known in the art.

[0296] In some embodiments, circulating LAP fragments may be used as serum markers of fibrogenesis. See, e.g., U.S. Patent No. 8,198,412, the contents of which are incorporated herein by reference.

[0297] Diseases involving ECM dysregulation The extracellular matrix is a cell-secreted network that surrounds cells and is primarily composed of proteoglycans and fibrous proteins, the most abundant of which is collagen. The novel antibodies disclosed herein can be used in the treatment of diseases associated with extracellular matrix dysregulation. Diseases associated with extracellular matrix dysregulation are typically myofibroblast-driven pathologies, including cancer, fibrosis, and cardiovascular disease (e.g., reviewed in Lampi and Reinhart-King (2018) "Targeting extracellular matrix stiffness to attenuate disease: From molecular mechanisms to clinical trials," Sci Transl Med 10(422): eaao0475). The progression of fibrotic conditions involves increased levels of matrix components deposited within the ECM and / or ECM maintenance / remodeling. TGFβ1 contributes, at least in part, to this process. This is supported by the observation that increased deposition of ECM components such as collagen can alter the mechanophysical properties of the ECM (e.g., matrix / substrate stiffness), and that this phenomenon is associated with TGFβ1 signaling. TGFβ1 inhibitors, such as those described herein, can block this process and be used to counteract disease progression involving ECM alterations, such as fibrosis, tumor growth, invasion, metastasis, and desmoplasia. The LTBP arm of such inhibitors can directly block ECM-associated pro / latent TGFβ complexes presented by LTBP1 and / or LTBP3, thereby preventing growth factor activation / release from the complex in the disease niche. In some embodiments, isoform-specific TGFβ1 inhibitors, such as those described herein, can normalize ECM stiffness to treat diseases involving integrin-dependent signaling. In some embodiments, the integrin includes an α11 chain, a β1 chain, or both.

[0298] Thus, the antibody can be administered to a subject diagnosed with a disease involving extracellular matrix dysregulation in an amount effective to treat the disease. A therapeutically effective amount of the antibody can be an amount sufficient to reduce the expression of one or more markers of myofibroblasts, such as α-SMA. The amount can be an amount sufficient to reduce the stiffness of the extracellular matrix of the affected tissue (e.g., fibrotic tissue). The amount can be an amount sufficient to reduce downstream effectors of TGFβ1, such as phosphorylation of SMAD2 and / or SMAD3. In a preferred embodiment, the isoform-selective activation inhibitor of TGFβ1 is an engineered molecule comprising Ab42, Ab46, Ab50, derivatives thereof, or antigen-binding fragments thereof.

[0299] Diseases involving endothelial-mesenchymal transition (EndMT) Similarly, TGFβ is also a key regulator of endothelial-mesenchymal transition (EndMT), which is observed in normal development, such as cardiogenesis. However, the same or similar phenomenon is also observed in many diseases, such as cancer stroma. In some disease processes, endothelial markers such as CD31 are downregulated upon TGFβ1 exposure, and α-SMA and fibronectin are induced instead of the expression of mesenchymal markers such as FSP-1. In fact, interstitial CAFs can be derived from vascular endothelial cells. Therefore, TGFβ1 isoform-specific inhibitors, such as those described herein, can be used to treat diseases initiated or driven by EndMT. In a preferred embodiment, the TGFβ1 isoform-selective activation inhibitor is an engineered molecule, including Ab42, Ab46, Ab50, derivatives thereof, or antigen-binding fragments thereof.

[0300] Diseases involving epithelial-mesenchymal transition (EMT) Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells with tight junctions switch to mesenchymal characteristics (phenotypes), such as loose cell-cell contacts. This process is observed in several normal biological processes and 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). It is generally believed that EMT signals are primarily induced by TGFβ. In a preferred embodiment, the TGFβ1 isoform-selective activation inhibitor is an engineered molecule comprising Ab42, Ab46, Ab50, a derivative thereof, or an antigen-binding fragment thereof.

[0301] Epithelial cells have also been proposed to give rise to myofibroblasts by undergoing the process of EMT in some fibrous tissues, such as the kidney, lung, and liver. EMT occurs when epithelial cells lose their cuboidal shape and expression of adhesion and tight junction proteins, resulting in weak cell-cell contacts and reorganization of their actin cytoskeleton. When cells acquire expression of mesenchymal proteins (fibronectin, vimentin, N-cadherin), they adopt a fibroblast-like structure that favors cell migration and invasion. EMT is induced by many growth factors, among which TGFβ is a very potent inducer, which regulates the expression and activity of several transcription factors known as EMT-TFs (e.g., Snail1 / Snail, Snail2 / Slug, ZEB1, ZEB2, Twist1 / Twist, etc.), which are responsible for executing the cell differentiation changes that constitute EMT. Gene and protein markers used to identify post-EMT mesenchymal cell generation in the context of fibrosis include FSP1 (fibroblast-specific protein 1), α-SMA, and type I collagen, as well as vimentin and desmin, whose expression increases concomitantly with decreased levels of epithelial markers (E-cadherin and certain cytokeratins). Cells co-expressing epithelial and mesenchymal markers represent intermediate stages of EMT (e.g., reviewed by Caja et al., Int. J. Mol. Sci. 2018, 19(5), 1294).

[0302] For example, many types of cancer appear to involve cellular transdifferentiation toward mesenchymal phenotypes (such as CAFs), which correlates with a worse prognosis. Therefore, isoform-specific inhibitors of TGFβ1, such as those described herein, may be used to treat diseases initiated or driven by EMT. Indeed, data exemplified herein (e.g., Figures 12 and 13) demonstrate that such inhibitors have the ability to suppress the expression of CAF markers, such as α-SMA, Col1 (type I collagen), and FN (fibronectin), in vivo. In a preferred embodiment, the isoform-selective inhibitor of TGFβ1 activation is an engineered molecule comprising Ab42, Ab46, Ab50, derivatives thereof, or antigen-binding fragments thereof.

[0303] Protease-related diseases Activation of TGF-β from its latent complex can be initiated by integrins in a force-dependent manner and / or by proteases. Evidence suggests that specific classes of proteases may be involved in the process, including, but not limited to, Ser / Thr proteases such as kallikrein, chymotrypsin, elastase, plasmin, and thrombin, as well as zinc metalloproteases from the ADAM family (e.g., ADAM10 and ADAM17) and the MMP family (e.g., MMP-2, MMP-9, and MMP-13). MMP-2 degrades type IV collagen, the most abundant component of basement membranes, raising the possibility that it may play a role in regulating ECM-associated TGF-β1. MMP-9 has been implicated in playing a central role in tumor progression, angiogenesis, stromal remodeling, and metastasis. Therefore, protease-dependent activation of TGF-β1 in the ECM may be important for cancer treatment.

[0304] Kallikreins (KLKs) are trypsin- or chymotrypsin-like serine proteases, including plasma kallikrein and tissue kallikrein. The ECM plays a role in tissue homeostasis, acting as a structural and signaling scaffold and a barrier that inhibits malignant growth. KLKs may play a role in the degradation of ECM proteins and other components that can promote tumor expansion and invasiveness. For example, KLK1 is highly upregulated in certain breast cancers and can activate pro-MMP-2 and pro-MMP-9. KLK2 activates latent TGF-β1, making prostate cancer cells adjacent to fibroblasts permissive for cancer growth. KLK3 has been widely studied as a diagnostic marker for prostate cancer (PSA). KLK3 can directly activate TGF-β1 by processing plasminogen to plasmin, which proteolytically cleaves LAP. KLK6 may be a potential marker for Alzheimer's disease.

[0305] All known activators of TGFβ1, such as plasmin, TSP-1, and αVβ6 integrin, directly interact with LAP. It is hypothesized that proteolytic cleavage of LAP destabilizes the LAP-TGFβ interaction, thereby releasing active TGFβ1. It has been suggested that the region containing 54-LSKLRL-59 (SEQ ID NO: 301) is important for maintaining the latent form of TGFβ1. Therefore, agents (e.g., antibodies) that stabilize the interaction or block the proteolytic cleavage of LAP may prevent TGFβ activation.

[0306] Many of these proteases associated with pathological conditions (e.g., cancer) function through distinct mechanisms of action. Thus, targeted inhibition of specific proteases or combinations of proteases may provide therapeutic benefits in treating conditions involving the protease-TGFβ axis. It is therefore contemplated that inhibitors (e.g., TGFβ1 antibodies) that selectively inhibit protease-induced activation of TGFβ1 may be advantageous in treating such diseases (e.g., fibrosis and cancer). Similarly, selective inhibition of TGFβ1 activation by one protease over another may also be preferred, depending on the condition being treated. In a preferred embodiment, the isoform-selective activation inhibitor of TGFβ1 is an engineered molecule comprising Ab42, Ab46, Ab50, a derivative thereof, or an antigen-binding fragment thereof.

[0307] Plasmin is a serine protease produced as a precursor form called plasminogen. Upon release, plasmin enters the circulation and is therefore detectable in serum. Elevated plasmin levels appear to correlate with cancer progression, likely through mechanisms involving the breakdown of extracellular matrices (e.g., basement membranes and interstitial barriers), which promotes tumor cell motility, invasion, and metastasis. Plasmin also influences adhesion, proliferation, apoptosis, tumor nutrition, oxygenation, angiogenesis, and VEGF activation (Didiasova et al., Int. J. Mol. Sci. 2014, 15, 21229-21252). Furthermore, plasmin can promote macrophage migration into the tumor microenvironment (Philips et al., Cancer Res. 2011, Nov. 1, 71(21):6676-83 and Choong et al., Clin. Orthop. Relat. Res. 2003, 415S, S46-S58). Indeed, tumor-associated macrophages (TAMs) are well-characterized drivers of tumorigenesis through their ability to promote tumor growth, invasion, metastasis, and angiogenesis.

[0308] Plasmin activity is primarily associated with ECM destruction. However, there is growing evidence that plasmin also regulates downstream MMP and TGF-beta activation. Specifically, it has been suggested that plasmin activates TGF-beta through proteolytic cleavage of the latent-associated peptide (LAP) derived from the N-terminal region of the TGF-beta gene product (Horiguchi et al., J. Biochem. 2012 Oct. 152(4):321-9), resulting in the release of active growth factors. Because TGF-beta 1 can promote cancer progression, it raises the possibility that plasmin-induced activation of TGF-beta may at least partially mediate this process. In a preferred embodiment, the TGF-beta 1 isoform-selective activation inhibitor is an engineered molecule comprising Ab42, Ab46, Ab50, a derivative thereof, or an antigen-binding fragment thereof.

[0309] TGFβ1 has also been shown to regulate the expression of uPA, a critical player in the conversion of plasminogen to plasmin (Santibanez, Juan F., ISRN Dermatology, 2013: 597927). uPA has independently been shown to promote cancer progression (e.g., adhesion, proliferation, and migration) by binding to its cell surface receptor (uPAR) and promoting the conversion of plasminogen to plasmin. Furthermore, studies have shown that the expression of uPA and / or plasminogen activator inhibitor-1 (PAI-1) is a predictor of poor prognosis in colorectal cancer (DQ Seetoo et al., Journal of Surgical Oncology, Vol. 82, No. 3, pp. 184-193, 2003), breast cancer (N. Harbeck et al., Clinical Breast Cancer, Vol. 5, No. 5, pp. 348-352, 2004), and skin cancer (Santibanez, Juan F., ISRN Dermatology, 2013: 597927). Therefore, without wishing to be bound by any particular theory, the interaction between plasmin, TGFβ1, and uPA may create a positive feedback loop that promotes cancer progression. Therefore, inhibitors that selectively inhibit plasmin-dependent TGFβ1 activation may be particularly suitable for the treatment of cancers dependent on the plasmin / TGFβ1 signaling axis.

[0310] Thrombin may be involved in the activation of GARP-associated TGFβ1. Platelets have been reported to express GARP-proTGFβ1. Thus, thrombin may mediate TGFβ1 activation by targeting this axis in an integrin-independent manner.

[0311] In one aspect of the present invention, the isoform-specific inhibitors of TGFβ1 described herein include inhibitors capable of inhibiting protease-dependent activation of TGFβ1. In some embodiments, the inhibitors can inhibit protease-dependent TGFβ1 activation in an integrin-independent manner. In some embodiments, such inhibitors can inhibit TGFβ1 activation regardless of the mode of activation, for example, inhibiting both integrin-dependent and protease-dependent activation of TGFβ1. In some embodiments, the protease is selected from the group consisting of serine proteases such as kallikrein, chymotrypsin, trypsin, elastase, plasmin, and thrombin, and zinc metalloproteases (MMP family) such as MMP-2, MMP-9, and MMP-13.

[0312] In some embodiments, the inhibitor can inhibit plasmin-induced activation of TGFβ1. In some embodiments, the inhibitor can inhibit plasmin- and integrin-induced TGFβ1 activation. In some embodiments, the inhibitor is a monoclonal antibody that specifically binds to TGFβ1. In some embodiments, the antibody is a monoclonal antibody that specifically binds to pro-TGFβ1. In some embodiments, the antibody binds to latent pro-TGFβ1, thereby inhibiting the release of mature growth factors from latent complexes. In some embodiments, the inhibitor of TGFβ1 activation suitable for use in the method of inhibiting plasmin-dependent activation of TGFβ1 is any one of the isoform-specific inhibitors disclosed herein. In a preferred embodiment, the isoform-selective activation inhibitor of TGFβ1 is Ab42, Ab46, Ab50, a derivative thereof, or an engineered molecule comprising an antigen-binding fragment thereof.

[0313] In some embodiments, the inhibitor (e.g., a TGFβ1 antibody) inhibits cancer cell migration. In some embodiments, the inhibitor inhibits monocyte / macrophage migration. In some embodiments, the inhibitor inhibits TAM accumulation.

[0314] In another aspect, provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a TGFβ1 inhibitor (e.g., a TGFβ1 antibody), wherein the inhibitor inhibits protease-induced activation of TGFβ1 (e.g., plasmin), thereby treating cancer in the subject.

[0315] In another aspect, provided herein is a method for reducing tumor growth in a subject in need thereof, comprising administering to the subject an effective amount of a TGFβ1 inhibitor (e.g., a TGFβ1 antibody), wherein the inhibitor inhibits protease-induced activation of TGFβ1 (e.g., plasmin), thereby reducing tumor growth in the subject.

[0316] Diseases involving abnormal gene expression Aberrant activation of the TGFβ1 signaling pathway in various disease states has been observed to be associated with altered gene expression of several markers. These gene expression markers (e.g., as measured by mRNA) include, but are not limited to, serpin1 (encoding PAI-1), MCP-1 (also known as CCL2), Col1a1, Col3a1, FN1, TGFβ1, CTGF, ACTA2 (encoding α-SMA), SNAI1 (encoding E-cadherin (Cdh1), MMP2 (a matrix metalloproteinase associated with EMT), MMP9 (a matrix metalloproteinase associated with EMT), TIMP1 (a matrix metalloproteinase associated with EMT), FOXP3 (a marker of Treg induction), CDH1 (a TGFβ1 marker), and SNAI1 (a marker of E-cadherin (Cdh1)). These include CDH2 (which drives EMT in fibrosis and metastasis by downregulating E-cadherin (a marker of epithelial cells) downregulated by TGFβ), and CDH2 (which drives EMT in fibrosis and metastasis by downregulating E-cadherin (a marker of epithelial cells) upregulated by TGFβ). Interestingly, many of these genes have been implicated as playing a role in a diverse set of pathologies, including various types of organ fibrosis, including myelofibrosis, and in many cancers. Indeed, a pathophysiological link between fibrotic states and abnormal cell proliferation, tumorigenesis, and metastasis has been suggested. See, for example, Cox and Erler, the contents of which are incorporated herein by reference. (2014) Clinical Cancer Research 20(14): 3637-43 "Molecular pathways: connecting fibrosis and solid tumor metastasis"; Shiga et al. (2015) Cancers 7:2443-2458 "Cancer-associated fibroblasts: their characteristics and their roles in tumor growth"; Wynn and Barron (2010) Semin. Liver Dis. 30(3): 245-257 "Macrophages: master regulators of inflammation and fibrosis".Without wishing to be bound by any particular theory, the inventors of the present disclosure contemplate that the TGFβ1 signaling pathway may indeed be a key link between these wide-ranging pathologies.

[0317] The ability of chemotactic cytokines (or chemokines) to mediate leukocyte recruitment (e.g., monocytes / macrophages) to damaged or diseased tissue has significant consequences in disease progression. Members of the CC chemokine family, such as monocyte chemoattractant protein 1 (MCP-1), also known as CCL2, macrophage inflammatory protein 1-alpha (MIP-1α), also known as CCL3, and MIP-1β, also known as CCL4, have been implicated in this process.

[0318] For example, MCP-1 / CCL2 is thought to play a role in both fibrosis and cancer. MCP-1 / CCL2 has been characterized as a profibrotic chemokine and a monocyte chemoattractant, and evidence suggests that it may be involved in both the initiation and progression of cancer. In fibrosis, MCP-1 / CCL2 has been shown to play an important role in the inflammatory phase of fibrosis. For example, neutralization of MCP-1 resulted in a dramatic reduction in glomerular crescent formation and type I collagen deposition. Similarly, passive immunotherapy with either anti-MCP-1 or anti-MIP-1 alpha antibodies has been shown to significantly reduce mononuclear phagocyte accumulation in bleomycin-challenged mice, suggesting that MIP-1 alpha and MCP-1 contribute to leukocyte recruitment during pulmonary inflammatory responses (Smith, Biol Signals. 1996 July-August; 5(4):223-31, "Chemotactic cytokines mediate leukocyte recruitment in fibrotic lung disease"). Elevated levels of MIP-1 alpha have been reported in patients with cystic fibrosis and multiple myeloma (see, for example, Mrugacz et al., J Interferon Cytokine Res. 2007 June; 27(6):491-5), supporting the idea that MIP-1 alpha is involved in local or systemic inflammatory responses.

[0319] Several lines of evidence point to the involvement of CC chemokines in tumor progression. For example, tumor-derived MCP-1 / CCL2 can promote a "pro-cancer" phenotype in macrophages. For example, in lung cancer, MCP-1 / CCL2 is produced by stromal cells and has been shown to promote metastasis. In human pancreatic cancer, tumors secrete CCL2, and immunosuppressive CCR2-positive macrophages infiltrate these tumors. Patients with tumors exhibiting high CCL2 expression and low CD8+ T cell infiltration have significantly reduced survival. It is contemplated that monocytes recruited to the injured or diseased tissue environment may subsequently be polarized in response to local cues (e.g., in response to tumor-derived cytokines), thereby further contributing to disease progression. These M2-like macrophages likely contribute to immune evasion by suppressing effector cells such as CD4+ and CD8+ T cells. In some embodiments, this process is mediated in part by LRRC33-TGFβ1 expressed by activated macrophages. In some embodiments, this process is mediated in part by GARP-TGFβ1 expressed by Tregs.

[0320] Similarly, the involvement of PAI-1 / serpin1 has been implicated in various cancers, angiogenesis, inflammation, and neurodegenerative diseases (e.g., Alzheimer's disease). Elevated expression of PAI-1 in tumors and / or serum correlates with poor prognosis (e.g., shorter survival, increased metastasis) in various cancers, such as breast cancer and bladder cancer (e.g., transitional cell carcinoma), as well as myelofibrosis. In the context of fibrotic conditions, PAI-1 has been recognized as a key downstream effector of TGFβ1-induced fibrosis, and increased PAI-1 expression has been observed in various forms of tissue fibrosis, including pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis (IPF)), renal fibrosis, liver fibrosis, and scleroderma. In some embodiments, this process is mediated in part by ECM-associated TGFβ1, e.g., via LTBP1 and / or LTBP3.

[0321] In some embodiments, the in vivo effects of TGFβ1 inhibitor treatment may be assessed by measuring changes in genetic markers. Suitable markers include TGFβ (e.g., TGFβ1, TGFβ2, and TGFβ3). Suitable markers may also include one or more presenting molecules for TGFβ (e.g., TGFβ1, TGFβ2, and TGFβ3), such as LTBP1, LTBP3, GARP (or LRRC32), and LRRC33. In some embodiments, suitable markers include mesenchymal transition genes (e.g., fibronectin, vimentin, N-cadherin, AXL, ROR2, WNT5A, LOXL2, TWIST2, TAGLN, and / or FAP), immunosuppressive genes (e.g., IL10, VEGFA, VEGFC), monocyte and macrophage chemotaxis genes (e.g., CCL2, CCL3, CCL4, CCL7, CCL8, and CCL13), and / or various fibrotic markers described herein. Preferred markers are plasma markers.

[0322] As shown in the Examples herein, isoform-specific inhibitors of TGFβ1 described herein can reduce the expression levels of many of these markers in mechanistic animal models such as UUO, which have been shown to be TGFβ1-dependent. Thus, such inhibitors may be used to treat diseases or disorders characterized by aberrant expression (e.g., overexpression / upregulation or underexpression / downregulation) of one or more gene expression markers.

[0323] Thus, in some embodiments, an isoform-specific inhibitor of TGFβ1 is used to treat a disease associated with overexpression of one or more of PAI-1 (encoded by serpin 1), MMP2, MMP9, MCP-1 (also known as CCL2), Col1a1, Col3a1, FN1, TGFβ1, CTGF, α-SMA, ITGA11, and ACTA2, wherein the treatment comprises 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 cancer, e.g., cancer involving solid tumors. In some embodiments, the disease is organ fibrosis, e.g., fibrosis of the liver, kidney, lung, muscle, skin, and / or heart or cardiovascular tissue. In some embodiments, the disease is Alport syndrome. In some embodiments, the inhibitor reduces expression of one or more of PAI-1 (encoded by serpin 1), MMP2, MMP9, MCP-1 (also known as CCL2), Col1a1, Col3a1, FN1, TGFβ1, CTGF, α-SMA, ITGA11, and ACTA2. In preferred embodiments, the TGFβ1-selective inhibitor is an engineered molecule comprising Ab42, Ab46, Ab50, derivatives thereof, or antigen-binding fragments thereof.

[0324] Another biomarker that can be used to evaluate the in vivo effect of TGFβ1 inhibitor treatment is blood urea nitrogen (BUN). Urea is naturally formed in the body as a by-product of protein breakdown. Urea travels from the liver to the kidneys, where it is filtered / removed from the blood. Therefore, BUN levels can increase in situations where a patient's kidneys are not functioning properly. For example, patients with renal fibrosis may exhibit increased BUN. Therefore, in some embodiments, BUN is measured to evaluate the in vivo effect of the TGFβ1 isoform-specific inhibitors described herein. In other embodiments, the TGFβ1 isoform-specific inhibitors are used in the treatment of diseases associated with increased BUN (e.g., renal fibrosis and / or acute or chronic kidney disease, injury, or failure). In certain embodiments, the disease associated with increased BUN is Alport syndrome.

[0325] Thus, the present disclosure includes methods for selecting candidate patients or patient populations that are likely to respond to TGFβ1 inhibitor therapy. Such methods may include testing a biological sample, such as a biopsy sample, taken from a patient (or patient population) for the expression of one or more of the markers described herein. Similarly, such genetic markers may be used to monitor a patient's responsiveness to treatment. Monitoring may include testing two or more biological samples taken from the patient, for example, before and after administration of treatment, and over time during the course of a treatment regimen, to evaluate changes in the gene expression levels of one or more of the markers that are indicative of treatment response or effectiveness.

[0326] In some embodiments, methods for selecting candidate patients or patient populations likely to respond to TGFβ1 inhibitor therapy may include identifying patients or patient populations previously tested for and exhibiting aberrant expression of genetic markers, such as those described herein. In some embodiments, the aberrant marker expression includes elevated levels of at least one of TGFβ1, LRRC33, GARP, LTBP1, LTBP3, CCL2, CCL3, PAI-1 / serpin1, MMP2, MMP9, Col1a1, Col3a1, FN1, CTGF, α-SMA, ITGA11, and ACTA2. In some embodiments, the patient or patient population (e.g., a biological sample obtained therefrom) exhibits elevated TGFβ1 activation, phospho-SMAD2 / 3, or a combination thereof. In some embodiments, the patient or patient population exhibits elevated BUN.

[0327] Circulating / circulating MDSCs as biomarkers MDSCs are a heterogeneous population of cells named for their myeloid origin and their primary immunosuppressive function (Gabrilovich. Cancer Immunol Res. 2017 January; 5(1): 3-8). MDSCs generally exhibit high plasticity and a strong ability to reduce the cytotoxic function of T cells and natural killer (NK) cells, including their ability to promote the expansion of T regulatory cells (Tregs) and, in turn, suppress T effector cell function (Gabrilovich et al., Nat Rev Immunol. (2012) 12:253-68). MDSCs are typically classified into two subsets based on the expression of their surface markers: monocytic (m-MDSCs) and granulocytic (G-MDSCs or PMN-MDSCs) (Consonni et al., Front Immunol. 2019 May 5; 10:949). Suppressive G-MDSCs can be characterized by the generation of reactive oxygen species (ROS) as their primary mechanism of immunosuppression. In contrast, M-MDSCs mediate immunosuppression primarily by upregulating the inducible nitric oxide synthase gene (iNOS), producing nitric oxide (NO) and a series of immunosuppressive cytokines (Youn and Garilovich, Eur J Immunol. 2010 Nov;40(11):2969-2975).

[0328] In some embodiments, the patient or patient population (e.g., a biological sample taken therefrom) exhibits elevated MDSCs. In some embodiments, the sample is a whole blood sample or a blood component (e.g., plasma or serum). In some embodiments, the sample is fresh whole blood or a blood component (e.g., from a sample that has not been previously frozen).

[0329] In some embodiments, the patient or patient population has a fibrotic disorder or disease (such as organ fibrosis). In some embodiments, the patient has NASH.

[0330] In certain embodiments, the TGFβ inhibitors described herein, e.g., isoform-selective inhibitors of activation of TGFβ1 such as Ab2, Ab42, Ab46, Ab50, or derivatives thereof, isoform-nonselective inhibitors, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants, antibodies that bind to TGFβ1 / 3, ligand traps, e.g., TGFβ1 / 3 inhibitors, and / or integrin inhibitors (e.g., For example, an antibody that binds to αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selectively inhibits TGFβ1 and / or TGFβ3, is administered such that the amount (e.g., dose) of TGFβ1 inhibitor administered is sufficient to reduce circulating MDSC levels by at least 10%, at least 15%, at least 20%, at least 25%, or more compared to baseline MDSC levels. Circulating MDSC levels can be measured before or after each treatment or each dose of TGFβ inhibitor, such that a decrease of at least 10%, at least 15%, at least 20%, at least 25%, or more can be an indicator or predictor of treatment efficacy. In some embodiments, circulating MDSC levels can be used to determine disease burden (e.g., as measured by changes in fibrosis before and after a treatment regimen). In certain embodiments, a decrease in circulating MDSC levels may be indicative of a decrease in disease burden (eg, a decrease in fibrosis).For example, circulating MDSC levels may be measured before and after administration of a dose of a TGFβ inhibitor described herein, e.g., an isoform-selective inhibitor of TGFβ1 activation such as Ab2, Ab42, Ab46, Ab50, or derivatives thereof, a non-isoform-selective TGFβ inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants, an antibody that binds to TGFβ1 / 3, a ligand trap, e.g., a TGFβ1 / 3 inhibitor, and / or an integrin inhibitor (e.g., an antibody that binds to αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selectively inhibiting TGFβ1 and / or TGFβ3), and a reduction in circulating MDSC levels may be indicative of or predictive of a pharmacological effect, e.g., a reduction in disease burden (e.g., reduced fibrosis). In certain embodiments, circulating MDSC levels may be measured before and after administration of a first dose of a TGFβ inhibitor described herein, such as an isoform-selective inhibitor of TGFβ1 activation such as Ab2, Ab42, Ab46, Ab50, or derivatives thereof, a non-isoform-selective inhibitor, such as a low molecular weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, such as GC1008 and variants, an antibody that binds to TGFβ1 / 3, a ligand trap, such as a TGFβ1 / 3 inhibitor, and / or an integrin inhibitor (e.g., an antibody that binds to αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selectively inhibiting TGFβ1 and / or TGFβ3). In some embodiments, a decrease in circulating MDSC levels is an indicator or predictor of pharmacological effect, further justifying the administration of a second or more doses of TGFβ inhibitor.In some embodiments, the first dose of TGFβ inhibitor is the first dose that the patient receives of the TGFβ inhibitor.In some embodiments, the first dose of TGFβ inhibitor is the first dose of a predetermined treatment regimen that includes multiple doses of TGFβ inhibitor.In another embodiment, circulating MDSC levels may be measured before and after combination treatment including a TGFβ inhibitor described herein, e.g., an isoform-selective activation inhibitor of TGFβ1, such as Ab2, Ab42, Ab46, Ab50, or a derivative thereof. In some embodiments, a decrease in circulating MDSC levels following treatment with a TGFβ inhibitor described herein, e.g., an isoform-selective inhibitor of TGFβ1 activation such as Ab2, Ab42, Ab46, Ab50, or derivatives thereof, a non-isoform-selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants, an antibody that binds to TGFβ1 / 3, a ligand trap, e.g., a TGFβ1 / 3 inhibitor, and / or an integrin inhibitor (e.g., an antibody that binds to αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selectively inhibiting TGFβ1 and / or TGFβ3), may justify continued treatment.

[0331] In some aspects, the present disclosure provides a method of treating fibrosis in a subject, comprising: selecting a TGFβ inhibitor that inhibits TGFβ1 but not one or both of TGFβ2 and TGFβ3; and administering the TGFβ inhibitor to a subject with a fibrotic condition in an amount sufficient to reduce circulating MDSC levels. In some embodiments, the circulating MDSC levels are determined from whole blood or blood components collected from the subject. In some embodiments, the circulating MDSC levels are reduced by at least 10%, optionally by at least 15%, 20%, 25%, or more.

[0332] In some embodiments, the TGFβ1 isoform-selective inhibitor described herein is used in a method for reducing circulating MDSC levels in a subject. In some embodiments, the circulating MDSC levels are determined from whole blood or blood components collected from the subject. In some embodiments, the circulating MDSC levels are reduced by at least 10%, optionally at least 15%, 20%, 25% or more. In some embodiments, the TGFβ inhibitor is administered in an amount sufficient to reduce circulating MDSC levels.

[0333] In certain embodiments of the present disclosure, levels of circulating MDSCs may be used to predict, determine, and monitor the pharmacological effect of a treatment comprising a dose of a TGFβ inhibitor described herein, e.g., an isoform-selective inhibitor of TGFβ1 activation such as Ab2, Ab42, Ab46, Ab50, or derivatives thereof, a non-isoform-selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants, an antibody that binds to TGFβ1 / 3, a ligand trap, e.g., a TGFβ1 / 3 inhibitor, and / or an integrin inhibitor (e.g., an antibody that binds to αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3), administered alone or in combination with another therapy. In certain embodiments, circulating MDSCs may be measured within 6 weeks after administration of the initial treatment (e.g., a (first) dose of a TGFβ inhibitor). In certain embodiments, circulating MDSC levels may be measured within 30 days after administration of the first dose of a TGFβ inhibitor. In some embodiments, MDSC levels may be measured within or about 3 weeks after administration of the first dose of a TGFβ inhibitor. In some embodiments, MDSC levels may be measured within or about 2 weeks after administration of the first dose of a TGFβ inhibitor. In some embodiments, MDSC levels may be measured within or about 10 days after administration of the first dose of a TGFβ inhibitor.

[0334] In some embodiments, such patients or patient populations have cancer, which may include solid tumors. The solid tumors may be TGFβ1-dominant tumors, in which TGFβ1 is the predominant isoform expressed in the tumor compared to other isoforms. In some embodiments, such patients or patient populations exhibit resistance to cancer therapies, such as chemotherapy, radiation therapy, and / or immune checkpoint therapies, such as anti-PD-1 (e.g., pembrolizumab and nivolumab), anti-PD-L1 (e.g., atezolizumab), anti-CTLA4 (e.g., ipilimumab), engineered immune cell therapy (e.g., CAR-T), and cancer vaccines. According to the present invention, isoform-specific TGFβ1 inhibitors, such as those disclosed herein, overcome resistance by removing immune suppression, allowing effector cells to access cancer cells, thereby achieving an anti-tumor effect.

[0335] Proliferative disorders (e.g., myeloproliferative disorders) In some embodiments, the antibodies described herein can be used to treat proliferative disorders. In some embodiments, the TGFβ1 isoform-selective inhibitors described herein are used to treat myeloproliferative disorders in subjects. In some embodiments, the proliferative disorder is cancer or myeloproliferative disorder. In some embodiments, the myeloproliferative disorder is myelofibrosis.

[0336] Myelofibrosis, also known as bone marrow fibrosis, is a relatively rare myeloproliferative disorder (cancer) that belongs to a group of diseases called myeloproliferative disorders. Myelofibrosis is classified as a Philadelphia chromosome-negative (-) branch of myeloproliferative neoplasms. Myelofibrosis is characterized by clonal myeloproliferation, abnormal cytokine production, extramedullary hematopoiesis, and myelofibrosis. The proliferation of abnormal clones of hematopoietic stem cells in the bone marrow and other sites leads to fibrosis, or replacement of the marrow by scar tissue. Unless otherwise specified, the term myelofibrosis refers to primary myelofibrosis (PMF), which can also be called chronic idiopathic myelofibrosis (cIMF) (the terms idiopathic and primary refer to the unknown or spontaneous origin of the disease in these cases). This contrasts with myelofibrosis occurring secondary to polycythemia vera or essential thrombocythemia. Myelofibrosis is a form of myeloid metaplasia, which refers to the change in cell types in the blood-forming tissues of bone marrow, and the two terms are often used interchangeably. The terms idiopathic myeloid metaplasia and myelofibrosis with myeloid metaplasia (MMM) are also used to refer to primary myelofibrosis. In some embodiments, hematological proliferative disorders that can be treated according to the present invention include myeloproliferative diseases such as myelofibrosis. The so-called "classic" group of BCR-ABL (Ph)-negative chronic myeloproliferative disorders includes essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF).

[0337] Myelofibrosis disrupts the body's normal blood cell production. The result is extensive scarring in the bone marrow, leading to severe anemia, weakness, fatigue, and often splenomegaly. Production of cytokines, such as fibroblast growth factor, by abnormal hematopoietic cell clones (especially megakaryocytes) leads to replacement of hematopoietic tissue in the bone marrow with connective tissue via collagen fibrosis. The reduction in hematopoietic tissue impairs the patient's ability to generate new blood cells, resulting in progressive pancytopenia, a deficiency of all blood cell types. However, fibroblast proliferation and collagen deposition are thought to be secondary phenomena, and fibroblasts themselves may not be part of the abnormal cell clone.

[0338] Myelofibrosis can be caused by abnormal blood stem cells in the bone marrow that give rise to mature and less differentiated cells that grow rapidly and take over the bone marrow, causing both fibrosis (scar tissue formation) and chronic inflammation.

[0339] 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, progressive scarring, or bone marrow fibrosis. Because hemopoietic cells are forced to migrate to other areas, particularly the liver and spleen, patients can develop extramedullary hematopoiesis, or blood cell formation occurring outside the bone marrow. This causes enlargement of these organs. In the liver, abnormal size is called hepatomegaly. Enlargement of the spleen is called splenomegaly, which can also contribute to pancytopenia, particularly thrombocytopenia, and anemia. Another complication of extramedullary hematopoiesis is poikilocytosis, or the presence of abnormally shaped red blood cells.

[0340] The main site of extramedullary hematopoiesis in myelofibrosis is the spleen, which is usually significantly enlarged in patients with myelofibrosis. As a result of the massive enlargement of the spleen, multiple subcapsular infarctions often occur in the spleen, which means that partial or complete tissue death occurs due to the interruption of oxygen supply to the spleen. At the cellular level, the spleen contains erythroid precursors, granulocyte precursors, and megakaryocytes, and megakaryocytes are remarkable for their number and abnormal shape. Megakaryocytes may be involved in causing the secondary fibrosis seen in this condition.

[0341] It has been suggested that TGFβ may be involved in the fibrotic aspects of the pathogenesis of myelofibrosis (see, for example, Agarwal et al., "Bone marrow fibrosis in primary myelofibrosis: pathogenic mechanisms and the role of TGFβ" (2016) Stem Cell Investig 3:5). Bone marrow pathology in primary myelofibrosis is characterized by fibrosis, neoangiogenesis, and osteosclerosis, and fibrosis is associated with increased production of collagen, which is deposited in the ECM.

[0342] Several biomarkers have been described, the alterations of which are indicative of or correlated with disease. In some embodiments, the biomarker is a cellular marker. Such disease-related biomarkers are useful for diagnosing and / or monitoring disease progression and treatment effectiveness (e.g., patient responsiveness to treatment). These biomarkers include several fibrotic and cellular markers. For example, in lung cancer, TGFβ1 concentrations in bronchoalveolar lavage (BAL) fluid have been reported to be significantly higher (approximately 2+-fold increase) in patients with lung cancer compared to patients with benign disease, which may also serve as a biomarker for diagnosing and / or monitoring the progression or treatment effect of lung cancer.

[0343] Because primary myelofibrosis is associated with abnormal megakaryocytic development, specific cell markers of megakaryocytes and their precursors in the stem cell lineage may serve as markers for diagnosing and / or monitoring disease progression and the effectiveness of treatment. In some embodiments, useful markers include, but are not limited to, cell markers of differentiated megakaryocytes (e.g., CD41, CD42, and Tpo R), cell markers of megakaryocyte-erythroid progenitors (e.g., CD34, CD38, and CD45RA-), cell markers of common myeloid progenitors (e.g., IL-3α / CD127, CD34, SCF R / c-kit, and Flt-3 / Flk-2), and cell markers of hematopoietic stem cells (e.g., CD34, CD38-, Flt-3 / Flk-2). In some embodiments, useful biomarkers include fibrotic markers. These include, but are not limited to, TGFβ1, PAI-1 (also known as serpin 1), MCP-1 (also known as CCL2), Col1a1, Col3a1, FN1, CTGF, α-SMA, ACTA2, Timp1, Mmp8, and Mmp9. In some embodiments, useful biomarkers are serum markers (e.g., proteins or fragments found and detected in serum samples).

[0344] Based on the finding that TGFβ is a component of the leukemic bone marrow niche, it is contemplated that targeting the bone marrow microenvironment with TGFβ inhibitors may be a promising approach to reduce leukemic cells expressing presentation molecules that regulate local TGFβ availability in affected tissues.

[0345] Indeed, due to the multifaceted nature of the pathology, which manifests TGFβ-dependent dysregulation in both myeloproliferative and fibrotic aspects (as the term "myofibrosis" itself suggests), TGFβ1 isoform-specific inhibitors that target matrix- and cell-associated TGFβ1 complexes, such as those described herein, may provide particularly advantageous therapeutic effects for patients suffering from myelofibrosis. It is contemplated that the LTBP arm of such inhibitors can target ECM-associated TGFβ1 complexes in the bone marrow, while the LRRC33 arm of the inhibitors can block myeloid cell-associated TGFβ1. Furthermore, the abnormal megakaryocyte biology associated with myelofibrosis may involve both GARP- and LTBP-mediated TGFβ1 activity. TGFβ1 isoform-specific inhibitors can target such complexes, thereby inhibiting the release of active TGFβ1 into the niche.

[0346] Therefore, such TGFβ1 inhibitors are useful for treating patients with polycythemia vera who have had an inadequate response or intolerance to other (or standard of care) treatments, such as hydroxyurea and JAK inhibitors. Such inhibitors include primary MF, post-polycythemia vera MF, and post-essential thrombocythemia MF. They are also useful for treating patients with intermediate or high-risk myelofibrosis (MF). In a preferred embodiment, the isoform-selective inhibitor of TGFβ1 is an engineered molecule, including Ab42, Ab46, Ab50, derivatives thereof, or antigen-binding fragments thereof.

[0347] Accordingly, one aspect of the present invention relates to a method for treating primary myelofibrosis. The method comprises administering to a patient suffering from primary myelofibrosis a therapeutically effective amount of a composition comprising a TGFβ inhibitor that causes a decrease in TGFβ availability. In some embodiments, an inhibitor of TGFβ1 activation is administered to a patient with myelofibrosis. Such antibodies may be administered at a dosage ranging from 0.1 to 100 mg / kg, e.g., 1 to 30 mg, e.g., 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, etc. For example, a suitable dosing regimen includes 1 to 20 mg / kg administered once weekly. Preferred routes of administration of pharmaceutical compositions comprising antibodies are intravenous or subcutaneous. If the composition is administered intravenously, the patient may receive the therapeutic agent for an appropriate duration per treatment, e.g., approximately 30-120 minutes (e.g., 30, 60, 75, 90, and 120 minutes), and then repeat every few weeks, e.g., every 3, 4, 6 weeks, etc., for a total of several cycles, e.g., 4, 6, 8, 10, 12, etc. In some embodiments, a patient may be treated with a composition comprising an inhibitory antibody via intravenous administration at a dose level of 1-10 mg / kg (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg / dose) every 28 days (4 weeks) for 6 or 12 cycles. In some embodiments, such treatment is administered as a chronic (long-term) therapy (e.g., continued indefinitely as long as deemed beneficial), instead of being discontinued after a set number of administration cycles.

[0348] Myelofibrosis is considered a type of leukemia, but is also characterized by signs of fibrosis. Because TGFβ regulates aspects of ECM homeostasis, and its dysregulation can lead to tissue fibrosis, it is desirable to inhibit ECM-associated TGFβ activity. Thus, the antibodies or fragments described herein inhibit pro-TGFβ presented by LTBPs (such as LTBP1 and LTBP3), and also inhibit pro-TGFβ presented by GARP and LRRC33.

[0349] Initial in vivo data indicate that TGFβ1 isoform-selective inhibitors, such as those described herein, can be used to treat myelofibrosis in a translatable murine model of primary myelofibrosis.Unlike the current standard of care JAK2 inhibitors, which only provide symptomatic relief but do not provide clinical or life-prolonging benefits, TGFβ1 isoform-selective inhibitors can achieve significant anti-fibrotic effects in the bone marrow of diseased mice and may also prolong survival, supporting the idea that TGFβ1 inhibitors may be effective in treating myeloproliferative disorders in human patients.

[0350] Suitable patient populations with myeloproliferative neoplasms that may be treated with the compositions and methods described herein include, but are not limited to, a) patient populations that are Philadelphia(+), b) patient populations that are Philadelphia(-), c) patient populations classified as "classical" (PV, ET, and PMF), d) patient populations that carry the mutation JAK2V617F(+), e) patient populations that carry JAK2V617F(-), f) patient populations that have JAK2 exon 12(+), g) patient populations that have MPL(+), and h) patient populations that have CALR(+).

[0351] In some embodiments, the patient population includes patients with intermediate-2 or high-risk myelofibrosis. In some embodiments, the patient population includes subjects with myelofibrosis who are refractory to or are n...

Claims

1. 1. An isoform-selective inhibitor of TGFβ1 activation, comprising: the inhibitor is a monoclonal antibody or antigen-binding fragment thereof that selectively inhibits TGFβ1 activation; The monoclonal antibody or antigen-binding fragment thereof exhibits a monovalent dissociation rate of 10.0e-4 (1 / sec) or less and a K of <1.0 nM with each of human LTBP1-proTGFβ1 and / or human LTBP3-proTGFβ1, as measured by a surface plasmon resonance (SPR)-based assay. Ｄ and combine them with The monoclonal antibody or antigen-binding fragment thereof has the following six CDRs: i) H-CDR1 comprising GFTFADYA (SEQ ID NO: 276); ii) ISGSG(X 1 )AT [where, X １ may be A or K] (SEQ ID NO: 277); iii) VSSG(X 1 )WD(X 2 )D [where, X １ can be H, D, or Q, and X ２ may be F or Y] (SEQ ID NO: 278); iv) L-CDR1 comprising QSISSY (SEQ ID NO: 279); v) AAS(X 1 )(X 2 )(X 3 )(X 4 ) [where, X １ can be N, G, or V, and X ２ can be L, N, or E, and X ３ may be Q or E, and X ４ may be S or T] (SEQ ID NO:280), and vi) QQTY(X 1 )VPLT [where, X １ may be T or G] (SEQ ID NO: 281) an isoform-selective inhibitor comprising:

2. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 is VSSG(X 1 )WD(X 2 )D [where, X １ may be H or Q, and X ２ may be Y or F] (SEQ ID NO: 283); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASGLES (SEQ ID NO:284); vi) L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285); The isoform-selective inhibitor of claim 1.

3. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGHWDFD (SEQ ID NO:289); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASNLES (SEQ ID NO: 292); vi) L-CDR3 comprises QQTYTVPLT (SEQ ID NO: 296); The isoform-selective inhibitor of claim 1.

4. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGHWDFD (SEQ ID NO:289); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASGLES (SEQ ID NO:284); vi) L-CDR3 comprises QQTYTVPLT (SEQ ID NO: 296); The isoform-selective inhibitor of claim 1.

5. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGHWDFD (SEQ ID NO:289); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASVNES (SEQ ID NO:293); vi) L-CDR3 comprises QQTYTVPLT (SEQ ID NO: 296); The isoform-selective inhibitor of claim 1.

6. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGHWDFD (SEQ ID NO:289); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASNEET (SEQ ID NO: 294); vi) L-CDR3 comprises QQTYTVPLT (SEQ ID NO: 296); The isoform-selective inhibitor of claim 1.

7. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGHWDFD (SEQ ID NO:289); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASNLES (SEQ ID NO: 292); vi) L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285); The isoform-selective inhibitor of claim 1.

8. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGHWDFD (SEQ ID NO:289); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASGLES (SEQ ID NO:284); vi) L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285); The isoform-selective inhibitor of claim 1.

9. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGHWDYD (SEQ ID NO:287); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASNLES (SEQ ID NO: 292); vi) L-CDR3 comprises QQTYTVPLT (SEQ ID NO: 296); The isoform-selective inhibitor of claim 1.

10. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGHWDYD (SEQ ID NO:287); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASGLES (SEQ ID NO:284); vi) L-CDR3 comprises QQTYTVPLT (SEQ ID NO: 296); The isoform-selective inhibitor of claim 1.

11. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGHWDYD (SEQ ID NO:287); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASNLES (SEQ ID NO: 292); vi) L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285); The isoform-selective inhibitor of claim 1.

12. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGHWDYD (SEQ ID NO:287); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASGLES (SEQ ID NO:284); vi) L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285); The isoform-selective inhibitor of claim 1.

13. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGDWDYD (SEQ ID NO:290); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASNLES (SEQ ID NO: 292); vi) L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285); The isoform-selective inhibitor of claim 1.

14. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGDWDYD (SEQ ID NO:290); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASGLES (SEQ ID NO:284); vi) L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285); The isoform-selective inhibitor of claim 1.

15. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGQWDYD (SEQ ID NO:291); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASNLES (SEQ ID NO: 292); vi) L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285); The isoform-selective inhibitor of claim 1.

16. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSGQWDYD (SEQ ID NO:291); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASGLES (SEQ ID NO:284); vi) L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285); The isoform-selective inhibitor of claim 1.

17. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGKAT (SEQ ID NO:288); iii) H-CDR3 comprises VSSGDWDYD (SEQ ID NO:290); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASNLQS (SEQ ID NO:295); vi) L-CDR3 comprises QQTYTVPLT (SEQ ID NO: 296); The isoform-selective inhibitor of claim 1.

18. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGKAT (SEQ ID NO:288); iii) H-CDR3 comprises VSSGDWDYD (SEQ ID NO:290); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AASGLES (SEQ ID NO:284); vi) L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285); The isoform-selective inhibitor of claim 1.

19. i) H-CDR1 comprises GFTFADYA (SEQ ID NO:276); ii) H-CDR2 comprises ISGSGAAT (SEQ ID NO:282); iii) H-CDR3 comprises VSSG(H / D / Q)WD(F / Y)D (SEQ ID NO:278); iv) L-CDR1 comprises QSISSY (SEQ ID NO:279); v) L-CDR2 comprises AAS(G / N)LES (SEQ ID NO: 303); vi) L-CDR3 comprises QQTY(G / T)VPLT (SEQ ID NO: 281); The isoform-selective inhibitor of claim 1.

20. A pharmaceutical composition comprising an isoform-selective inhibitor of TGFβ1 activation described in any one of claims 1 to 19, and a pharmaceutically acceptable excipient.

21. 21. An isoform-selective inhibitor of TGFβ1 according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 20 for use in the treatment of a fibrotic disorder in a subject.

22. 21. An isoform-selective inhibitor of TGFβ1 according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 20 for use in a method for reducing the level of circulating latent TGFβ in a subject.

23. 23. The isoform-selective inhibitor of TGFβ1 according to claim 22, wherein the level of circulating latent TGFβ is determined in a sample obtained from the subject.

24. The isoform-selective inhibitor of TGFβ1 according to claim 23, wherein the sample is a whole blood sample or a blood component.

25. The isoform-selective inhibitor of TGFβ1 according to claim 22, wherein the circulating latent TGFβ is circulating latent TGFβ1.

26. Use of an isoform-selective inhibitor of TGFβ1 described in any one of claims 1 to 19 or a pharmaceutical composition described in claim 20 in the manufacture of a pharmaceutical product comprising an antibody or its antigen-binding fragment and a pharmaceutically acceptable excipient.

27. 21. An isoform-selective inhibitor according to any one of claims 1 to 19, or a pharmaceutical composition according to claim 20, for use in treating a disease involving extracellular matrix (ECM) dysregulation in a subject, wherein the disease may be fibrosis.

28. 21. An isoform-selective inhibitor according to any one of claims 1 to 19, or a pharmaceutical composition according to claim 20, for use in treating a fibrotic condition in a subject.

29. 29. The isoform-selective inhibitor for use according to claim 28, wherein the fibrotic condition is liver fibrosis, kidney fibrosis, cardiac fibrosis, skin fibrosis, uterine fibrosis, muscle fibrosis, bone marrow fibrosis or lung fibrosis.

30. 30. The isoform-selective inhibitor for use according to claim 29, wherein the fibrotic condition is liver fibrosis associated with NASH.

31. 31. The isoform-selective inhibitor for use according to claim 30, wherein the liver fibrosis is stage 3 or stage 4 fibrosis.

32. An isoform-selective inhibitor for use as described in claim 30, wherein the skin fibrosis is scleroderma.

33. The treatment (i) determining the level of circulating latent TGFβ in the subject prior to administering a TGFβ inhibitor; (ii) determining the level of circulating latent TGFβ in the subject after administering a TGFβ inhibitor.

29. The isoform-selective inhibitor for use according to claim 28, further comprising:

34. An isolated monoclonal antibody or antigen-binding fragment thereof that selectively inhibits TGFβ1 activation, comprising: heavy chain complementarity determining region 1 (CDRH1) comprising GFTFADYA (SEQ ID NO: 276); heavy chain complementarity-determining region 2 (CDRH2) comprising ISGSGAAT (SEQ ID NO: 282); heavy chain complementarity-determining region 3 (CDRH3) comprising VSSGHWDYD (SEQ ID NO: 287); light chain complementarity determining region 1 (CDRL1) comprising QSISSY (SEQ ID NO: 279); a light chain complementarity-determining region 2 (CDRL2) comprising AASGLES (SEQ ID NO: 284); and Light chain complementarity-determining region 3 (CDRL3) comprising QQTYGVPLT (SEQ ID NO: 285) and an isolated monoclonal antibody or an antigen-binding fragment thereof, which comprises:

35. 35. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 34, wherein the VH comprises EVQLLESGGGLVQPGGSLRLSCAASGFTFADYAMTWVRQAPGKGLEWVSAISGSGAATYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSSGHWDYDYWGQGTLVTVSS (sequence number 297) and the VL comprises DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASGLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGVPLTFGGGTKVEIK (sequence number 298).

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