WO2023034803A1 - Methods for treatment of fibrosis - Google Patents

Abstract

Described herein are methods and compositions for use in treating fibrosis by inhibiting Leukemia Inhibitory Factor (LIF) and its receptor (LIFR).

Description

METHODS FOR TREATMENT OF FIBROSIS

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Application Serial No. 63/238,335, filed on August 30, 2021. The entire contents of the foregoing are incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. AR073833 awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD

Described herein are methods and composition for use in treating fibrosis by inhibiting Leukemia Inhibitory Factor (LIF) and its receptor (LIFR).

BACKGROUND

Fibrosis is a disease characterized by excessive deposition of collagen matrix that leads to tissue scaring with subsequent end organ functional failure. Diseases characterized by fibrosis include cirrhosis, renal fibrosis, idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases, systemic sclerosis (scleroderma), cardiomyopathies, keloids, Crohn’s Disease bowel structure, and post-surgical bowel obstruction. Fibrosis is a leading cause of morbidity and mortality.

SUMMARY

As shown herein, LIFR signaling is important for pro-fibrotic activation of fibroblasts derived from many tissues, thus the present methods can be used to treat fibrosis in a subject, including fibrosis in multiple tissues such as lung, liver, kidney, gut, heart, and skin.

Provided herein are methods for treating disorders associated with fibrosis in a subject, the method comprising administering a therapeutically effective amount of an inhibitor of LIF-LIFR signaling to a subject in need thereof. Also provided are inhibitors of LIF-LIFR signaling for use in treating disorders associated with fibrosis in a subject. In some embodiments, the fibrosis is in lung, liver, kidney, gut, heart, or skin of the subject.

In some embodiments, the fibrosis is in the liver and the disorder is chronic HCV (hepatitis C virus) infection, alcohol-induced, nonalcoholic steatohepatitis (NASH), autoimmune hepatitis, or primary biliary cirrhosis. As an example, systemic administration, e.g., intravenous administration, can be used.

In some embodiments, the fibrosis is in the kidney and the disorder is renal fibrosis, nephrogenic systemic fibrosis, chronic kidney disease, or renal anemia. As an example, systemic administration, e.g., intravenous administration, can be used.

In some embodiments, the fibrosis is in the lung and the disorder is idiopathic pulmonary fibrosis (IPF), cystic fibrosis, pulmonary hypertension, thromboembolic disease, emphysema, non-specific interstitial pneumonia (NSIP), chronic obstructive pulmonary disease (COPD), idiopathic pleuroparenchymal fibroelastosis, idiopathic lymphocytic interstitial pneumonia, or respirator bronchiolitis-ILD. As an example, administration by inhalation or insufflation can be used.

In some embodiments, the fibrosis is in the skin and the disorder is scleroderma, systemic scleroderma, hypertrophic scar, systemic sclerosis, or keloids. As an example, systemic or topical administration can be used.

In some embodiments, the fibrosis is in the heart and the disorder is cardiac fibrosis, hypertrophic cardiomyopathy, cardiac dysfunction, valvular disease, arrhythmia, myocardial infarction, sarcoidosis, myocarditis, toxic cardimyopathies, chronic renal insufficiency, hypertension, diabetes, non-ischemic dilated cardiomyopathy, hypertrophic cardiomyopathy, sarcoidosis, chronic renal insufficiency, amyloidosis, or Anderson-Fabry disease. As an example, systemic administration, e.g., intravenous administration, can be used.

In some embodiments, the fibrosis is in the gut and the disorder is intestinal fibrosis, enteropathies, inflammatory bowel disease, Crohn’s Disease bowel structure, or post-surgical bowel obstruction. As an example, systemic administration, e.g., intravenous or oral administration, can be used.

In some embodiments, is an antibody that binds to LIF, LIFR, or GP130 (the LIFR co-receptor) and inhibits LIF -LIFR signaling, or an antigen-binding fragment thereof that inhibits LIF -LIFR signaling. In some embodiments, the antibody is 1C7, 12D3, or MSC-1, or an antigen-binding fragment thereof. In some embodiments, the antibody is a monoclonal antibody, non-human animal antibody, humanized antibody, chimeric antibody, human antibody, minibody, bispecific antibody, amino acid sequence-modified antibodies, modified antibody conjugated to other molecules, or sugar chain-modified antibody that includes the variable heavy chain (HC) and light chain (LC) complementarity determining regions (CDRs) of mAb 1C7, 12D3, or MSC-1, or the entire variable HCs and LCs of mAb 1C7, 12D3, or MSC-1.

In some embodiments, the inhibitor is an inhibitory nucleic acid targeting LIF or LIFR mRNA. In some embodiments, the inhibitory nucleic acid is an antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a short, hairpin RNA (shRNA); or combinations thereof.

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 to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGs. 1A-B. TGFpi stimulation upregulates LIF and drives pro-fibrotic gene expression in fibroblasts. Four different human primary fibroblast cell lines were derived from diseased human lung tissues. Fibroblasts were incubated with TGFpi (TGFb, 5ng/ml) for 24 hours. COL1A1, COL1A2, POSTN, and ACTA2 expression (A) or LIF expression (B) was measured by qPCR and normalized with GAPDH. p value was calculated using the paired Student’s t test (*p < 0.05, **p < 0.01).

FIGs. 2A-D. Inhibition of LIFR signaling with a LIFR blocking antibody prevents TGFpi-induced fibrotic gene expression; blocking autocrine LIF does not affect IL11 expression. Human primary fibroblasts were derived from diseased tissues from human lung (A), heart (B), or skin (C). Fibroblasts were stimulated with TGFpi (5ng/ml) and an antibody against LIFR (LIFR mAb) or an isotype control (Ctrl IgG). (A) COL1A1, COL1A2, POSTN, and ACTA2, and (B) IL11 expression was measured by qPCR and normalized with GAPDH. p value was calculated using the paired Student’s t test (*p < 0.05, **p < 0.01, ns: not significant).

FIG. 3. Inhibition of LIFR signaling with LIFR and LIF silencing siRNA prevents TGFpi-induced fibrotic gene expression. Human primary fibroblasts were derived from diseased human lung tissues. Fibroblasts were transfected with siRNAs against LIFR and LIF (LIFR + LIF) or a control (Ctrl) siRNA. After 48 hours following siRNA transfection, fibroblasts were stimulated with TGFpi (5ng/ml) and RNA samples were collected 24 hours post TGFb stimulation. COL1 Al, COL1 A2, POSTN, and ACTA2 expression was measured by qPCR and normalized with GAPDH. p value was calculated using the paired Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001).

FIG. 4. Inhibition of LIFR signaling prevents major inducers of fibrotic gene expression following IL-4, IL-13 and TGFb stimulation. Human primary fibroblasts were derived from diseased human lung tissues. Fibroblasts were transfected with siRNAs against LIFR and LIF (LIFR + LIF) or a control (Ctrl) siRNA. After 48 hours following siRNA transfection, fibroblasts were stimulated with IL-4 (50ng/mL), IL-13 (50ng/mL), TGFpi (5ng/ml), or unstimulated (basal) and samples were collected 72 hours post stimulation. The protein levels of COL1A1, POSTN and a-SMA were measured by ELISA. Error bars represent SD of quadruplicate technical replicates, p value was calculated using the one-way ANOVA test (**p < 0.01, ***p < 0.001).

FIGs. 5A-C. JAK1 and JAK2 inhibitors prevent major inducers of fibrotic gene expression following TGFb, IL-4, and IL-13 stimulation. Human primary fibroblasts were derived from diseased human lung tissues. Fibroblasts were treated with various JAK inhibitors for 0.5 hour before being stimulated with (A) TGFpi (5ng/ml), (B) IL-4 (50ng/mL), or (C) IL- 13 (50ng/mL) and samples were collected 24 hours post stimulation. COL1 Al and POSTN expression was measured by qPCR and normalized with GAPDH. Error bars represent SD of quadruplicate technical replicates. 8uM and 24uM denote the concentrations of an inhibitor, uM = pM. FIGs. 6A-B. LIFR signaling activates JAK1 and JAK2 and JAK2 is responsible for fibrotic gene regulation. Human primary fibroblasts were derived from diseased human lung tissues. (A) Fibroblasts were transfected with siRNAs against LIFR and LIF (LIFR+LIF), IL11R and IL11 (IL11R+IL11) or a control (Ctrl) siRNA. After 72 hours following siRNA transfection, whole cell lysates were collected, and samples were run on an SDS-PAGE gel. Immunoblotting was done with antibodies against phosphorylated JAK1 (Tyrl034/1035) (p-JAKl), phosphorylated JAK2 (Tyrl 007/1008) (p-JAK2), and beta-Tubulin (used as loading controls). (B) Fibroblasts were transfected with siRNAs against JAK2 or a control (Ctrl) siRNA. After 48 hours following siRNA transfection, fibroblasts were stimulated with TGFpl (5ng/ml). COL1A1, COL1A2, POSTN, and ACTA2 expression was measured by qPCR and normalized with GAPDH.

FIG. 7. Targeting LIFR in human precision cut lung slices (PCLS) leads to reduction of key fibrotic gene expression. Human PCLSs were incubated with siRNAs against LIFR or a control (Ctrl) siRNA on day 0. On day 4, RNA samples were collected and LIFR, COL1A1, COL1A2, COL3A1, POSTN, and IL11R expression was measured by qPCR and normalized with HPRT. Data were representative of PCLSs from 2 different donors. Error bars were calculated based on data from 2 slices per condition.

FIGs. 8A-B. Inhibition of LIFR signaling with LIFR and LIF silencing siRNA prevents TGFpi-induced fibrotic gene expression. Human primary fibroblasts were derived from diseased human lung tissues from two different donors. Using a different siRNA against LIFR (LIFR, #2), fibroblasts were transfected with siRNAs against LIFR (LIFR, #1) and LIF (LIFR #1 + LIF), or LIFR (LIFR, #2) and LIF (LIFR #2 + LIF), or a control (Ctrl) siRNA. After 48 hours following siRNA transfection, fibroblasts were stimulated with TGFpi (5ng/ml) and RNA samples were collected 24 hours post TGFb treatment. COL1A1, COL1A2, POSTN, and ACTA2 expression was measured by qPCR and normalized with GAPDH. LIFR, #1 siRNA was used in FIG. 3.

FIG. 9. Inhibition of LIFR signaling with LIFR and LIF silencing siRNA prevents TGFpi-induced fibrotic gene expression. Human primary fibroblasts were derived from diseased human lung tissues. Fibroblasts were transfected with siRNAs against LIFR and LIF (LIFR + LIF) or a control (Ctrl) siRNA. After 48 hours following siRNA transfection, fibroblasts were stimulated with TGFpi (5ng/ml) and RNA samples were collected 24 hours post TGFb stimulation. COL3A1, COL5A2, COL6A2, COL6A3, MMP2, CTGF, and FN1 expression was measured by qPCR and normalized with GAPDH. p value was calculated using the paired Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001).

FIG. 10A-B. Inhibition of JAK2 prevents TGFpi-induced fibrotic gene expression. Human primary fibroblasts were derived from diseased human lung tissues from two different donors. Using a different siRNA against JAK2 (JAK2, #2), fibroblasts were transfected with siRNAs against JAK2 (JAK2, #1) or JAK2 (JAK2, #2) or a control (Ctrl) siRNA. After 48 hours following siRNA transfection, fibroblasts were stimulated with TGFpi (5ng/ml) and RNA samples were collected 24 hours post TGFb treatment. COL1 Al, COL1 A2, POSTN, and ACTA2 expression was measured by qPCR and normalized with GAPDH. JAK2, #1 siRNA was used in FIG.. 6.

FIG. 11. Blocking autocrine LIF signaling is more effective in diseased fibroblasts. Human lung fibroblasts were stimulated with TGFpi (5ng/ml) in the presence of an antibody against LIF-R or an isotype control IgG. In the presence of an antibody blocking LIF-R, TGFpi induced LIF expression was more suppressed in fibroblasts derived from diseased tissues compared to healthy ones. Percent suppression is calculated based on the fold change in LIF induction after TGFpi compared to the unstimulated conditions in the presence of an antibody against LIF-R or an isotype control IgG. This suggests that fibroblasts from fibrotic tissues are more susceptible to LIF-R blockage than those from healthy tissues.

FIG. 12. Inhibition of LIFR signaling prevents major inducers of fibrotic gene expression following IL-4, IL-13 and TGFb stimulation. Human primary fibroblasts were derived from lung cancer donors also known as cancer associated fibroblasts (CAF). Fibroblasts were transfected with siRNAs against LIFR and LIF (LIFR + LIF) or a control (Ctrl) siRNA. After 48 hours following siRNA transfection, fibroblasts were stimulated with IL-4 (50ng/mL), IL-13 (50ng/mL), TGFpi (5ng/ml), or unstimulated (basal) and samples were collected 72 hours post stimulation. The protein levels of COL1 Al, POSTN and a-SMA were measured by ELISA. Error bars represent SD of triplicate technical replicates. FIG. 13. JAK1 and JAK2 inhibitors prevent major inducers of fibrotic gene expression following TGFb, IL-4, and IL-13 stimulation. Human primary fibroblasts were derived from lung cancer donors also known as cancer associated fibroblasts (CAF). Fibroblasts were treated with various JAK inhibitors for 0.5 hour before being stimulated with (A) TGFpi (5ng/ml), (B) IL-4 (50ng/mL), or (C) IL- 13 (50 ng/mL) and samples were collected 24 hours post stimulation. COL1 Al and POSTN expression was measured by qPCR and normalized with GAPDH. Error bars represent SD of triplicate technical replicates. 8uM and 24uM denote the concentrations of an inhibitor, uM = pM.

FIG. 14. CXCL12 levels in fibroblasts. Primary fibroblasts were derived from human lung tissue of 7 individuals including healthy (2 donors) and early IPF (5 donors). Fibroblasts were cultured in 2% FBS media and supernatants were collected after 24 and 48 hours (H). Chart shows fibroblasts derived from early IPF donors have a more sustained production of CXCL12.

FIG. 15. Inhibition of LIFR signaling prevents CXCL12 production. (A) Human lung fibroblasts derived from fibrotic lung diseased donors (V6, V9, VI 1) were incubated with an antibody against LIFR (mAb) or an isotype control (IgG). In the presence of an antibody blocking LIF-R, fibroblasts have reduced levels of CXCL12. This shows that LIF-R is regulating CXCL12 production where its ligand LIF acts in an autocrine feed forward loop. This experiment was repeated with fibroblasts derived from different individuals with similar results, p value was calculated using the one-tailed and paired Student’s t test (*p < 0.05). (B) Human lung fibroblasts derived from fibrotic lung diseased donors (162, V6, VI 1) were transfected with an siRNA against LIF-R (LIFR) or a control siRNA (Ctrl). Fibroblasts expressing a LIF-R siRNA have reduced levels of CXCL12 compared to fibroblasts expressing a control siRNA. This further shows that LIF-R is involved in CXCL12 regulation, p value was calculated using the one-tailed and paired Student’s t test (**p < 0.025).

FIGs. 16A-B. Inhibiting JAK1/JAK2 suppresses CXCL12 gene expression. (A) Human lung fibroblasts derived from fibrotic lung diseased donors were treated with various JAK inhibitors for 48 hours. Fibroblasts treated with ruxolitinib or baricitinib had strongest reduction in CXCL12 levels. JAK1 and JAK2 are the primary targets of ruxolitinib and baricitinib. This suggests that JAK1 and JAK2 act downstream of LIFR to regulate CXCL12. (B) Human lung fibroblasts derived from fibrotic lung diseased donors were transfected with an siRNA against JAK1 or JAK2 or a control siRNA (Ctrl). Fibroblasts expressing a JAK1 or JAK2 siRNA have reduced levels of CXCL12 compared to fibroblasts expressing a control siRNA. This further confirms that JAK1 and JAK2 are involved in CXCL12 regulation, p value was calculated using the one-way ANOVA test (**p < 0.025).

FIGs. 17A-B. Blocking CXCL12 signaling suppresses T cell recruitment. (A) Higher levels of CXCL12 in the fibroblast culture supernatants resulted in more CD4 and CD8 T cells recruitment in a transwell assay. Mostly, only CD4 and CD8 T cells expressing the CXCR4 receptor which is the chemotactic receptor for CXCL12 were recruited. (B) Human peripheral blood mononuclear cells were incubated with an antibody against CXCR4 (mAb) or an isotype control (IgG). In the presence of an antibody neutralizing CXCR4, fibroblasts conditioned media resulted in less CXCR4 positive CD4 and CD8 T cells recruitment in a transwell assay, p value was calculated using the one-way ANOVA test (*p < 0.05, **p<0.025)

FIG. 18. CXCL12 expressing cells are closely localized with CXCR4 expressing cells in parenchyma region of the lung tissue from early ILD patients. In situ hybridization assay using RNAscope probes for CXCR4 and CXCL12 was performed on human lung tissue slides from both ILD patient and control subjects. All slides were scanned with 40x magnitude using slide scanner (VS 120, Olympus Life Science). Compared to control subject (Control), more cells in parenchyma region of human lung tissue from patient with early ILD (uILD) expressed CXCR4 and CXCL12. Also, CXCL12 and CXCR4 expression are in close proximity with each other. This provides in vivo evidence to support the finding that fibroblasts in the parenchyma region making CXCL12 in diseased lung to recruit CXCR4+ expressing cells including CD4 and CD8 T cells in vivo.

FIGs. 19A-B. Inhibition of LIFR signaling prevents CXCL12 production. (A) Human lung fibroblasts derived from lung cancer donors also known as cancer associated fibroblasts (CAF) were incubated with an antibody against LIFR (mAb) or an isotype control (IgG). In the presence of an antibody blocking LIF-R, fibroblasts have reduced levels of CXCL12. This shows that LIF-R is regulating CXCL12 production where its ligand LIF acts in an autocrine feed forward loop. This experiment was repeated with fibroblasts derived from different individuals with similar results, p value was calculated using the one-tailed and paired Student’s t test (*p < 0.05). (B) Human lung fibroblasts derived from lung cancer donors were transfected with an siRNA against LIF-R (LIFR) or a control siRNA (Ctrl).

Fibroblasts expressing a LIF-R siRNA have reduced levels of CXCL12 compared to fibroblasts expressing a control siRNA. This further shows that LIF-R is involved in CXCL12 regulation, p value was calculated using the one-tailed and paired Student’s t test (**p < 0.025).

FIGs. 20A-B. Inhibiting JAK1/JAK2 suppresses CXCL12 gene expression. (A) Human lung fibroblasts derived from lung cancer donors also known as cancer associated fibroblasts (CAF) were treated with various JAK inhibitors for 48 hours. Fibroblasts treated with ruxolitinib or baricitinib had strongest reduction in CXCL12 levels. JAK1 and JAK2 are the primary targets of ruxolitinib and baricitinib. This suggests that JAK1 and JAK2 act downstream of LIFR to regulate CXCL12. (B) Human lung fibroblasts derived lung cancer donors were transfected with an siRNA against JAK1 or JAK2 or a control siRNA (Ctrl). Fibroblasts expressing a JAK1 or JAK2 siRNA have reduced levels of CXCL12 compared to fibroblasts expressing a control siRNA. This further confirms that JAK1 and JAK2 are involved in CXCL12 regulation, p value was calculated using the one-way ANOVA test (**p < 0.025).

DETAILED DESCRIPTION

Fibroblasts have been implicated as the primary cells driving fibrosis in many organs including lung, heart, and skin (Schafer et al., 2017, Weiskirchen et al., 2019). We discovered an autocrine LIF-LIFR amplification loop in fibroblasts that amplifies pro-fibrotic pathways shared by major upstream pro-fibrotic stimuli including TGFb, IL-4 and IL-13, suggesting that shutting down this single autocrine loop can deactivate the pathological activation of fibroblasts in fibrosis regardless of the fibrogenic stimulus. In other words, LIFR is an attractive therapeutic target as it is a “common downstream pathway” of fibroblasts and myofibroblasts in fibrosis.

Further, we found that this mechanism regulates fibroblasts derived from multiple tissues including lung, heart, skin, and cancer. Thus, targeting LIFR will benefit fibrotic diseases in many organs such as the heart (i.e., maladaptive fibrosis and remodeling after myocardial infarction, or hypertrophic cardiomyopathy), kidney, skin (e.g., scleroderma and keloids), liver (cirrhosis), and lung (IPF, scleroderma and or other fibrotic interstitial lung diseases). Furthermore, fibrosis has been recognized as a major problem rendering immunotherapies, such as checkpoint inhibitors, ineffective in many solid tumors by blocking access of T cells to cancer cells as occurs in pancreatic, colon, lung and other cancers (Tauriello et al., 2018, Mariathasan et al., 2018, Holmgaard et al., 2018). Combining anti-fibrotic treatments with cancer immunotherapies are under active investigation. Thus, targeting LIFR for cancer fibrosis will also benefit cancer immunotherapies when used in combination with these drugs.

The present methods target a fibroblast intrinsic single LIFR-dependent autocrine amplification loop that is required for driving fibrosis downstream of a variety of upstream pro-fibrotic signals. In some embodiments, the methods use a monoclonal antibody that binds to LIF and blocks LIF-LIFR signaling on fibroblasts and myofibroblasts to treat fibrosis. The antibody can be used, e.g., in an out-patient setting as with many other biologies. Alternatively, inhibitory nucleic acids targeting LIF or LIFR can be used.

Although LIF-LIFR signaling has been implicated in the production of inflammatory cytokines in fibroblasts, a role for LIFR signaling in fibrosis per se has not previously been identified.

Since LIFR acts as a “common downstream pathway” of many major pro- fibrotic activators including TGFb, IL-4 and IL-13, blocking LIFR signaling alone will suppress pro-fibrotic programs induced by these activators. Further, blocking this pathway is effective in stromal cells derived from a range of relevant tissues. The present results help address why many attempts to create new anti-fibrotic drugs have failed as they only block a single upstream pro-fibrotic activator. LIFR has more selective expression on stromal cells including fibroblasts compared to immune cells, and further selective targeting can be achieved with a bivalent recombinant protein drug 1) targeting attachment to fibroblasts via specific markers (e.g FAPa, PDGFRa or podoplanin) and the second arm of the drug 2) blocking LIFR signaling.

In addition, blocking LIFR signaling is more effective in diseased fibroblasts compared to those derived from healthy individuals. This suggests that these methods target LIFR signaling specifically, and its major impact will be on activated fibroblasts reducing potential adverse side effects.

As shown herein, LIFR signaling is important for pro-fibrotic activation of fibroblasts derived from many tissues, thus the present methods can be used to treat fibrosis in a subject, including fibrosis in multiple tissues such as lung, liver, kidney, gut, heart, and skin.

Methods of Treatment

The methods described herein include methods for the treatment of disorders associated with fibrosis, e.g., in lung, liver, kidney, gut, heart, and skin. Exemplary conditions include lung fibrosis: e.g., idiopathic pulmonary fibrosis (IPF) , cystic fibrosis, pulmonary hypertension, thromboembolic disease, emphysema, non-specific interstitial pneumonia (NSIP), chronic obstructive pulmonary disease (COPD), idiopathic pleuroparenchymal fibroelastosis, idiopathic lymphocytic interstitial pneumonia, respirator bronchiolitis-ILD; cardiac fibrosis: e.g., hypertrophic cardiomyopathy, cardiac dysfunction, valvular disease, arrhythmia, myocardial infarction, sarcoidosis, myocarditis, toxic cardimyopathies, chronic renal insufficiency, hypertension, diabetes, non-ischemic dilated cardiomyopathy, hypertrophic cardiomyopathy, sarcoidosis, chronic renal insufficiency, amyloidosis, Anderson -Fabry disease; liver fibrosis: e.g., chronic HCV (hepatitis C virus) infection, alcohol-induced, nonalcoholic steatohepatitis (NASH), autoimmune hepatitis, primary biliary cirrhosis; renal fibrosis: e,g., nephrogenic systemic fibrosis, chronic kidney disease, renal anemia; skin fibrosis: e.g., scleroderma, systemic scleroderma, hypertrophic scar, systemic sclerosis; or intestinal fibrosis: e.g., enteropathies, inflammatory bowel disease.

Generally, the methods include administering a therapeutically effective amount of an inhibitor of LIF-LIFR signaling as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. Inhibitors of LIF-LIFR signaling include antibodies that bind to LIFR and inhibit LIF-LIFR signaling, as well as inhibitory nucleic acids targeting LIF or LIFR mRNA. As used in this context, to “treat” means to ameliorate at least one symptom of the disorders associated with fibrosis, e.g., to improve function of the affected organ, e.g., to improve lung function, liver function, heart function, kidney function, and/or skin function. The methods can reduce fibrosis in the subject.

In some embodiments, the subjects treated using a method described herein do not have an inflammatory or autoimmune disorder such as rheumatoid arthritis, Crohn’s disease, systemic lupus erythematosus, Castleman’s disease, Behcet’s disease, or systemic juvenile idiopathic arthritis, coronary artery or neurologic disease, or cancer.

Antibodies

The methods described herein can include the use of antibodies to the LIFR protein and blocks binding without activating LIF-LIFR signalling. The term “antibody” as used herein refers to an immunoglobulin molecule or an antigenbinding portion thereof. Examples of antigen-binding portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments, which retain the ability to bind antigen. The antibody can be polyclonal, monoclonal, recombinant, chimeric, deimmunized or humanized, fully human, non-human, (e.g., murine), or single chain antibody. In some embodiments the antibody has effector function and can fix complement. In some embodiments, the antibody has reduced or no ability to bind an Fc receptor. For example, the antibody can be an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region. Methods for making antibodies and fragments thereof are known in the art, see, e.g., Harlow et. al., editors, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice, (N.Y. Academic Press 1983); Howard and Kaser, Making and Using Antibodies: A Practical Handbook (CRC Press; 1st edition, Dec 13, 2006); Kontermann and Diibel, Antibody Engineering Volume 1 (Springer Protocols) (Springer; 2nd ed., May 21, 2010); Lo, Antibody Engineering: Methods and Protocols (Methods in Molecular Biology) (Humana Press; Nov 10, 2010); and Diibel, Handbook of Therapeutic Antibodies: Technologies, Emerging Developments and Approved Therapeutics, (Wiley-VCH; 1 edition September 7, 2010). Antibodies useful in the present methods include those that bind specifically to (i.e., do not bind to targets other than) LIF, LIFR, or gpl30, and inhibit LIFR signaling.

Exemplary inhibitory antibodies that bind to the LIFR protein, including those that block binding of LIF to LIFR without activating LIF-LIFR signaling, include 1C7 (directed against the Dllg region of LIFR, impairs the binding of LIF to the gpl90 subunit of LIFR) and 12D3 (directed against domain D2, interferes with the reconstitution of the high affinity LIF receptor complex), both of which are described in Taupin et al., J Biol Chem. 2001 Dec 21;276(51):47975-81, and are commercially available; and MSC-1, which is described in WO2021110873, US10206999, US11390670, US20220064279A1, and US20210130453. Exemplary inhibitory antibodies that bind to gpl30 include MAB628 (RnD Systems), B-R3 (Abeam), and BK5 (Cell Sciences).

In some embodiments, the antibodies are unmodified (native) monoclonal antibodies, non-human animal antibodies, humanized antibodies, chimeric antibodies, human antibodies, minibodies, bi-specific antibodies (e.g., that bind oAp and a receptor involved in RMT, such as the transferrin receptor (TfR), insulin receptor (IR), or the low-density lipoprotein receptor-related protein-1 (LRP-1)), amino acid sequence-modified antibodies, modified antibodies conjugated to other molecules (for example, polymers such as polyethylene glycol), and sugar chain-modified antibodies, as well as antigen-binding fragments thereof. The term “antigen-binding fragment” or “antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, 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 CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1969) Nature 341 :544-546), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. j., et al. (1994) Structure 2:1121- 1123). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540- 41354-5).

Still further, an antibody or antigen-binding fragment thereof can be part of a larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C- terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31 : 1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

The term “chimeric antibody” refers to antibodies that comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

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

The term “humanized antibody” refers to antibodies that comprise heavy and light chain variable region sequences from a nonhuman species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., 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 nonhuman VH and VL sequences to replace the corresponding nonhuman CDR sequences. In particular, the term “humanized antibody” is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a nonhuman antibody. As used herein, the term “substantially” in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Preferably, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In other embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.

The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgG 1, IgG2, IgG3 and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains can be selected to optimize desired effector functions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework can be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In a preferred embodiment, such mutations, however, will not be extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term “consensus framework” refers to the framework region in the consensus immunoglobulin sequence. Further, as used herein, the term “consensus immunoglobulin sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29: 128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L. L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21 :364-370) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. (See also Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991). The human antibodies of the present invention, however, may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). (See also Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990).

The term “chimeric antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.

Recombinant human antibodies of the present invention have variable regions, and may also include constant regions, derived from human germline immunoglobulin sequences. (See Kabat et al. (1991) supra.) In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. In certain embodiments, however, such recombinant antibodies are the result of selective mutagenesis or backmutation or both.

Inhibitory Nucleic Acids

Inhibitory nucleic acids useful in the present methods and compositions include antisense oligonucleotides, ribozymes, siRNA compounds, single- or doublestranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics that hybridize to at least a portion of the target LIF or LIFR nucleic acid and modulate its function. In some embodiments, the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a short, hairpin RNA (shRNA); or combinations thereof. See, e.g., WO 2010040112. Exemplary human LIF and LIFR sequence include the following:

LIF is also known as LIF interleukin 6 family cytokine.

In some embodiments, the inhibitory nucleic acids are 10 to 50, 10 to 20, 10 to 25, 13 to 50, or 13 to 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies inhibitory nucleic acids having complementary portions of 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 nucleotides in length, or any range therewithin. In some embodiments, the inhibitory nucleic acids are 15 nucleotides in length. In some embodiments, the inhibitory nucleic acids are 12 or 13 to 20, 25, or 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies inhibitory nucleic acids having complementary portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any range therewithin (complementary portions refers to those portions of the inhibitory nucleic acids that are complementary to the target sequence).

The inhibitory nucleic acids useful in the present methods are sufficiently complementary to the target RNA, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. "Complementary" refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a RNA, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.

Routine methods can be used to design an inhibitory nucleic acid that binds to the target sequence with sufficient specificity. In some embodiments, the methods include using bioinformatics methods known in the art to identify regions of secondary structure, e.g., one, two, or more stem-loop structures, or pseudoknots, and selecting those regions to target with an inhibitory nucleic acid. For example, “gene walk” methods can be used to optimize the inhibitory activity of the nucleic acid; for example, a series of oligonucleotides of 10-30 nucleotides spanning the length of a target RNA can be prepared, followed by testing for activity. Optionally, gaps, e.g., of 5-10 nucleotides or more, can be left between the target sequences to reduce the number of oligonucleotides synthesized and tested. GC content is preferably between about 30-60%. Contiguous runs of three or more Gs or Cs should be avoided where possible (for example, it may not be possible with very short (e.g., about 9-10 nt) oligonucleotides).

In some embodiments, the inhibitory nucleic acid molecules can be designed to target a specific region of the RNA sequence. For example, a specific functional region can be targeted, e.g., a region comprising a known RNA localization motif (i.e., a region complementary to the target nucleic acid on which the RNA acts). Alternatively or in addition, highly conserved regions can be targeted, e.g., regions identified by aligning sequences from disparate species such as primate (e.g., human) and rodent (e.g., mouse) and looking for regions with high degrees of identity. Percent identity can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), e.g., using the default parameters.

Once one or more target regions, segments or sites have been identified, e.g., within a target sequence known in the art or provided herein, inhibitory nucleic acid compounds are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity (i.e., do not substantially bind to other non-target RNAs), to give the desired effect. In the context of this disclosure, hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Complementary, as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a RNA molecule, then the inhibitory nucleic acid and the RNA are considered to be complementary to each other at that position. The inhibitory nucleic acids and the RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridisable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the inhibitory nucleic acid and the RNA target. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a RNA, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.

It is understood in the art that a complementary nucleic acid sequence need not be 100% complementary to that of its target nucleic acid to be specifically hybridisable. A complementary nucleic acid sequence for purposes of the present methods is specifically hybridisable when binding of the sequence to the target RNA molecule interferes with the normal function of the target RNA to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target RNA sequences under conditions in which specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

In general, the inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to a target region within the target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to the target region within an RNA. For example, an antisense compound in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity. Percent complementarity of an inhibitory nucleic acid with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Inhibitory nucleic acids that hybridize to an RNA can be identified through routine experimentation. In general, the inhibitory nucleic acids must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target.

In some embodiments, the inhibitory nucleic acids used in the methods described herein are modified, e.g., comprise one or more modified bonds or bases. A number of modified bases include phosphorothioate, methylphosphonate, peptide nucleic acids, or locked nucleic acid (LNA) molecules. Some inhibitory nucleic acids are fully modified, while others are chimeric and contain two or more chemically distinct regions, each made up of at least one nucleotide. These inhibitory nucleic acids typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. In some embodiments, the nucleic acid includes one or more of phosphorothioate linkages, base methylation, and numerous 2'-substitutions in the furanose ring, such as 2'-fluoro, O-methyl, or methoxy ethyl. Chimeric inhibitory nucleic acids of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. In some embodiments, the oligonucleotide is a gapmer (contain a central stretch (gap) of DNA monomers sufficiently long to induce RNase H cleavage, flanked by blocks of LNA modified nucleotides; see, e.g., Stanton et al., Nucleic Acid Then 2012. 22: 344-359; Nowotny et al., Cell, 121 : 1005-1016, 2005; Kurreck, European Journal of Biochemistry 270: 1628-1644, 2003; Fluiter et al., Mol Biosyst. 5(8):838-43, 2009). In some embodiments, the oligonucleotide is a mixmer (includes alternating short stretches of LNA and DNA; Naguibneva et al., Biomed Pharmacother. 2006 Nov; 60(9):633-8; 0rom et al., Gene. 2006 May 10; 3720: 137- 41). Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference.

For further disclosure regarding inhibitory nucleic acids, please see US2010/0317718 (antisense oligos); US2010/0249052 (double-stranded ribonucleic acid (dsRNA)); US2009/0181914 and US2010/0234451 (LNAs); US2007/0191294 (siRNA analogues); US2008/0249039 (modified siRNA); and WO2010/129746 and W02010/040112 (inhibitory nucleic acids). See also Kulkarni et al., Nat Nanotechnol. 2021 Jun; 16(6): 630-643; Duffy et al., BMC Biol. 2020 Sep 2; 18(1): 112; and Smith and Zain, Annu Rev Pharmacol Toxicol. 2019 Jan 6;59:605-630.

Compositions

Also provided herein are compositions comprising the inhibitors, e.g., inhibitory nucleic acids targeting LIF or LIFR, or anti-LIFR antibodies, for use in a method described herein, e.g., wherein the active agent in the composition comprises or consists of an antibody described herein, e.g., mAb 1C7, 12D3, or MSC-1, or antigen-binding fragments thereof, as an active ingredient. In some embodiments the antibody is a monoclonal antibody, non-human animal antibody, humanized antibody, chimeric antibody, human antibody, minibody, bispecific antibody, amino acid sequence-modified antibodies, modified antibody conjugated to other molecules (for example, polymers such as polyethylene glycol), or sugar chain-modified antibody that includes the HC and LC CDRs of mAb 1C7, 12D3, or MSC-1, or the entire HCs and LCs of mAb 1C7, 12D3, or MSC-1.

In some embodiments, the compositions are pharmaceutical compositions. Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous delivery; intracerebroventricular, intracerebral, or intrathecal injection; or injection into CSF.

Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems; the compounds can also be delivered, e.g., using a pump, e.g., a surgically implanted reservoir pump. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Nanoparticles (e.g., liposomes, polymeric nanoparticles, dendrimers, clathrin nanoparticles, or metallic nanoparticles), engineered bi-specific antibodies can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. Alternatively, viral vectors (such as AAV) encoding the antibodies can be delivered, e.g., comprising nucleic acids (preferably codon-optimized for use in humans) encoding a therapeutic antibody as described herein. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Dosage

An “effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments.

Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1.

Materials and Methods

The following materials and methods were used in the examples below.

Cell Lines and Reagents. Human lung fibroblasts were isolated from discarded tissues. Each line was derived from a unique donor and used experimentally between passages 5 and 8. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gemini), 2 mM L-glutamine, 50 pM 2-mercaptoethanol, antibiotics (penicillin and streptomycin), and essential and nonessential amino acids (Life Technologies). Human precision cut lung slices (PCLS) were purchased from AnaBios and were grown in DMEM/F-12 media (Gibco) supplemented with antibiotics and antimycotic reagents. Cardiac and skin fibroblasts were purchased from Lonza.

The following antibodies were used: anti-LIFR (clone 1C7, Sigma), control IgGl (MOPC21 clone), anti-a-tubulin (Sigma); anti-pJAKl, anti-pJAK2 (Cell Signaling Technologies). Other reagents were purchased from the following vendors: COL1A1 (Collagen 1A1), POSTN (Periostin) ELISA kits (R&D Systems); ACTA2 (aSMA) ELISA kit (Abeam); TGFb, IL-4, IL-13 (Peprotech). siRNAs were purchased from Life Technologies (for human cell lines) and from Horizon Discovery (for human PCLS). All JAK inhibitors were purchased from SelleckChem. qPCR primers were purchased from Integrated DNA Technologies. All human sample research was approved by the Brigham and Women’s Hospital Institutional Review Board. Cell Stimulation and Antibody Blocking Assays. Fibroblasts were plated on day 1 at 10,000 cells per well in 96-well plates in 10% FBS containing media. Cells were serum-starved on day 2 by changing to 1% FBS-containing media. Cells were stimulated as indicated on day 3 or blocking antibodies were added 1 hour prior to cytokine stimulation on day 3. siRNA Silencing. Fibroblasts were transfected with an siRNA by reverse transfection at 30 nM using the RNAi Max reagent (Life Technologies) in 10% FBS containing media. Cells were then switched to serum-starving media containing 2% FBS on day 2. Cells were stimulated as indicated on day 3. For human PCLS, siRNA was added to the media on day 0. On day 4, PCLS were snap-frozen in liquid nitrogen before RNA samples were extracted.

Quantitative Real-Time PCR. mRNA collection and cDNA synthesis from fibroblasts was carried out using the Power SYBR Green Cells-to-CT Kit (Life Technologies). mRNA samples were extracted from grounded PCLS using the RNeasy Fibrous Tissue Mini Kit (Qiagen) and cDNA synthesis was carried out using the QuantiTect Reverse Transcription Kit (Qiagen). qPCR reactions were performed using the Brilliant III Ultra-Fast SYBR reagent (Agilent). Relative transcription level was calculated by using the AACt method with GAPDH (for fibroblast cells) and HPRT (for PCLS) as the normalization control.

Western Blotting. Total cell lysates were collected by washing cells once with cold PBS followed by addition of lysate buffer (50mM HEPES pH 7.5, 5% glycerol, 100 mM NaCl, 0.1% SDS, 1% Triton X-100, supplemented with protease inhibitors and phosphatase inhibitors sodium orthovanadate, sodium fluoride, and beta-glycerol phosphate). Cells were lysed for 30 minutes on ice followed by centrifugation at 15000rpm for 15 minutes at 4°C. Protein concentration was measured by the microBCA kit (Pierce). Equal amounts of total protein (~20 pg per lane) were separated on an 8% SDS-PAGE gel. Proteins were transferred onto a PVDF membrane and blocked with 5% BSA in PBS and probed with primary antibodies overnight at 4°C, followed by secondary antibodies conjugated with HRP. Membranes were developed with the Clarity Western ECL Substrate (Bio-rad) and scanned with the ChemiDoc Imaging System (Bio-rad). Example 1.1. TGFb induces a robust expression of fibrotic genes as well as LIE in lung fibroblasts

Multiple human primary lung fibroblast samples derived from donors with chronic lung conditions were stimulated with TGFb for 24 hours. We observed that the expression of key fibrotic genes including COL1A1, COL1A2, POSTN and ACTA2 as measured by qPCR was significantly upregulated across all fibroblast lines (FIG. 1 A). Notably, we also observed that expression of LIF was strongly upregulated (FIG. IB). Previously, we showed than following stimulation of fibroblasts with inflammatory activators such as IL- lb, TNF and IL- 17, LIF expression was induced, and it acts as an autocrine ligand to sustain the pro-inflammatory gene expression in fibroblasts. Because of this, we asked whether LIF may play an autocrine role in regulating the fibrotic gene expression induced by TGFb.

Example 1.2. Blocking LIFR signaling prevents TGFb-induced upregulation of fibrotic genes

To interfere with LIFR signaling, we used a LIFR antibody that blocks the binding of LIF to LIFR. In the presence of the LIFR blocking antibody, expression of fibrotic genes including COL1A1, COL1A2, POSTN and ACTA2 following TGFb stimulation as measured by qPCR was significantly reduced compared to when fibroblasts were incubated with an isotype control antibody (FIG. 2A). Furthermore, we observed similar results that inhibition of LIFR signaling prevents TGFb-induced upregulation of fibrotic genes in human fibroblasts derived from heart and skin (FIGs. 2B-C). On the other hand, blocking LIFR did not affect IL11 expression (FIG. 2D). This suggests that LIFR is important for TGFb-induced fibrotic gene expression, and LIF plays an autocrine role which is independent of IL 11 autocrine.

Also, we observed that blocking autocrine LIF signaling is more effective in diseased fibroblasts. In the presence of an antibody blocking LIF-R, TGFpi induced LIF expression was more suppressed in fibroblasts derived from diseased tissues compared to healthy ones (FIG. 11). This suggests that fibroblasts from fibrotic tissues are more susceptible to LIF-R blockage than those from healthy tissues

To further confirm this observation, we used siRNA to silence the expression of both LIFR and LIF. We observed that fibroblasts transfected with LIFR and LIF siRNA also had significantly reduced expression of TGFb-induced fibrotic genes including COL1A1, COL1A2, POSTN and ACTA2 as measured by qPCR compared to cells expressing a control siRNA (FIG. 3). In addition, using a different siRNA against LIFR, we obtained similar observations (FIGs. 8A-B). Furthermore, other fibrotic genes including COL3A1, COL5A2, COL6A2, COL6A3, MMP2, CTGF and FN1 were also significantly suppressed by LIFR silencing (FIG. 9). Taken together, these results strongly indicate that autocrine LIF is a key driver of TGFb-induced fibrotic gene expression.

Example 1.3. LIFR signaling broadly regulates major pro-fibrotic activators including IL-4, IL-13 and TGFb

Besides TGFb, IL-4 and IL- 13 signaling have also been implicated in driving strong fibrotic gene expression in fibroblasts. Since the autocrine LIF/LIFR is important for TGFb signaling, we asked if it is also essential for the IL-4 and IL- 13 signaling. To examine the role of LIF/LIFR following IL-4 and IL- 13 stimulation of fibroblasts, we used siRNA to silence the expression of both LIFR and LIF. We observed that fibroblasts transfected with LIFR and LIF siRNA had significantly less collagen 1A1, periostin and alpha-smooth muscle actin protein levels as measured by ELISA after IL-4 and IL-13 stimulation compared to cells expressing a control siRNA (FIG. 4). Furthermore, we observed similar results that inhibition of LIFR signaling prevents IL-4, IL- 13 and TGFb-induced upregulation of fibrotic genes in human fibroblasts derived from lung cancer donors also known as cancer associated fibroblasts (CAF) (FIG. 12). These results imply that autocrine LIF is also involved in sustaining IL-4 and IL- 13 -induced fibrotic gene expression. In other words, LIFR signaling broadly regulates major pro-fibrotic activators including IL-4, IL- 13 and TGFb.

Example 1.4. Inhibiting JAK1/JAK2 suppresses IL-4, IL-13, and TGFb-induced fibrotic gene expression

LIFR belongs to the IL-6 receptor family. When activated by LIF, it binds to the co-receptor gpl30 and transduces the signal via activation of JAK kinases. To examine the role of JAK kinases in fibrotic gene regulation, we used several JAK inhibitors including baricitinib, ruxolitinib, and tofacitinib. Baricitinib and ruxolitinib inhibit JAK1 and JAK2 while tofacitinib inhibits JAK3. We observed that both baricitinib and ruxolitinib displayed a dose-dependent inhibition of fibrotic genes including COL1 Al and POSTN as measured by qPCR while tofacitinib did not (FIGs. 5A-C). Furthermore, we observed similar results that inhibition of JAK1/JAK2 prevents IL-4, IL- 13 and TGFb-induced upregulation of fibrotic genes in human fibroblasts derived from lung cancer donors also known as cancer associated fibroblasts (CAF) (FIG. 13). This suggests that JAK1 and/or JAK2 play an important role in fibrotic gene regulation following IL-4, IL- 13 and TGFb stimulation.

Example 1.5. LIFR signaling activates JAK1 and JAK2, and JAK2 regulates fibrotic gene expression

Next, we asked which JAK is regulated by LIFR signaling. We used siRNA to silence the expression of LIFR and LIF and examined JAK activation by Western blotting. We observed that silencing of LIFR and LIF resulted in a reduction in both phospho-STATl (p-STATl) and phospho-STAT2 (pSTAT2) compared to the control siRNA samples (FIG. 6A). This suggests that LIFR signaling activates JAK1 and JAK2. Since IL11 was previously implicated in fibrotic gene regulation, we also silenced the IL11/IL11R autocrine signaling. Interestingly, silencing of IL11R and IL11 only affected pJAK2 and not pJAKl. Taken together, this implicates JAK2 as a key JAK kinase in fibrotic signaling.

To confirm the role of JAK2, we silenced its expression by siRNA. We observed that fibroblasts transfected with a JAK2 siRNA had significantly reduced levels of fibrotic genes including COL1A1, COL1A2, POSTN, and ACTA2 after TGFb stimulation as measured by qPCR (FIG. 6B). Using another JAK2 siRNA, we obtained similar results (FIGs. 10A-B). This provides further evidence that JAK2 is involved in transducing LIFR and IL11R signaling.

Example 1.6. Blocking LIFR signaling reduces fibrosis in human lung tissue slices

To examine the role of LIFR signaling in regulating fibrosis in vivo, we used the human precision cut lung slices (PCLS). PCLSs were generated by slicing agarose-filled, intact human lung. These slices have the thickness of between 300 - 350 pm. They retain the native 3D cellular environment of the tissue and remain viable over a period of up to 10 days, thus providing an ideal model for translational research and drug screening. Using siRNA to silence LIFR expression in human PCLS, we were able to effectively knock down LIFR expression in the tissues (FIG. 7). More importantly, silencing LIFR on PCLS also resulted in reduction of major fibrotic genes including COL1 Al, COL1 A2, COL3A1, and POSTN, while it did not affect IL11R expression. This suggests that targeting LIFR in human diseased lung tissues will lead to a reduction in fibrosis.

Example 2. CXCL12 in Early Fibrosis

Materials and Methods

The following materials and methods were used in the examples below.

Cell Lines and Reagents. Human lung fibroblasts were isolated from discarded tissues. Each line was derived from a unique donor and used experimentally between passages 5 and 8. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gemini), 2 mM L-glutamine, 50 pM 2-mercaptoethanol, antibiotics (penicillin and streptomycin), and essential and nonessential amino acids (Life Technologies).

The following antibodies were used: anti -LIFR (clone 1C7) (Sigma), control IgGl (MOPC21 clone) (Sigma), anti-a-tubulin (Sigma); anti-pJAKl, anti-pJAK2 (Cell Signaling Technologies); flow cytometry antibodies for CD45, CD3, CD4, CD8 and CXCR4 (BioLegend or eBioscience). Other reagents were purchased from the following vendors: CXCL12 ELISA kits and CXCR4 antibody (R&D Systems). siRNAs were purchased from Life Technologies (for human cell lines) and from Horizon Discovery (for human PCLS). All JAK inhibitors were purchased from SelleckChem. qPCR primers were purchased from Integrated DNA Technologies. All human sample research was approved by the Brigham and Women’s Hospital Institutional Review Board.

Cell Stimulation and Antibody Blocking Assays. Fibroblasts were plated on day 1 at 10,000 cells per well in 96-well plates in 10% FBS containing media. Cells were serum-starved on day 2 by changing to 1% FBS-containing media. Cells were stimulated as indicated on day 3 or blocking antibodies were added 1 hour prior to cytokine stimulation on day 3. siRNA Silencing. Fibroblasts were transfected with an siRNA by reverse transfection at 30 nM using the RNAi Max reagent (Life Technologies) in 10% FBS containing media. Cells were then switched to serum-starving media containing 2% FBS on day 2. Quantitative Real-Time PCR. mRNA collection and cDNA synthesis from fibroblasts was carried out using the Power SYBR Green Cells-to-CT Kit (Life Technologies). qPCR reactions were performed using the Brilliant III Ultra-Fast SYBR reagent (Agilent). Relative transcription level was calculated by using the AACt method with GAPDH (for fibroblast cells) as the normalization control.

Example 2.1 CXCL12 level is upregulated in early IPF fibroblasts

Primary fibroblasts were derived from human lung tissue of 7 individuals including healthy (2 donors) and early IPF (5 donors). Fibroblasts were cultured in 2% FBS media and supernatants were collected after 24 and 48 hours. We observed that fibroblasts derived from early IPF donors had a more sustained production of CXCL12 (FIG. 14).

Example 2.2 Inhibition of LIFR signaling prevents CXCL12 production

Human lung fibroblasts derived from fibrotic lung diseased donors were incubated with an antibody against LIFR (mAb) or an isotype control (IgG). In the presence of an antibody blocking LIFR, fibroblasts had reduced levels of CXCL12. This shows that LIFR was regulating CXCL12 production where its ligand LIF acts in an autocrine feed forward loop (FIG. 15 A).

To further confirm this observation, we used siRNA to silence the expression of LIFR. Human lung fibroblasts derived from fibrotic lung diseased donors were transfected with an siRNA against LIFR (LIFR) or a control siRNA (Ctrl). Fibroblasts expressing a LIFR siRNA had reduced levels of CXCL12 compared to fibroblasts expressing a control siRNA. This further shows that LIFR is involved in CXCL12 regulation (FIG. 15B). Furthermore, we observed similar results that inhibition of LIFR signaling prevents CXCL12 production in human lung fibroblasts derived from lung cancer donors also known as cancer associated fibroblasts (CAF) (FIG. 19). Taken together, these results strongly indicate that autocrine LIF is a key driver of CXCL12 expression in early IPF fibroblasts.

Example 2.3 Inhibiting JAK1/JAK2 suppresses CXCL12 gene expression

LIFR belongs to the IL-6 receptor family. When activated by LIF, it binds to the co-receptor gpl30 and transduces the signal via activation of JAK kinases. To examine the role of JAK kinases in CXCL12 gene regulation, we used several JAK inhibitors. Human lung fibroblasts derived from fibrotic lung diseased donors were treated with various JAK inhibitors for 48 hours. Fibroblasts treated with ruxolitinib or baricitinib had the strongest reduction in CXCL12 levels (FIG. 16A). Baricitinib and ruxolitinib inhibit JAK1 and JAK2. This suggests that JAK1 and JAK2 act downstream of LIF-R to regulate CXCL12.

To confirm the role of JAK1 or JAK2, we silenced JAK1 or JAK2 expression by siRNA. We observed that fibroblasts transfected with either JAK1 or JAK2 siRNA had significantly reduced levels of CXCL12 (FIG. 16B). Furthermore, we observed similar results that inhibition of JAK1 or JAK2 with inhibitors or siRNA prevented CXCL12 production in human lung fibroblasts derived from lung cancer donors also known as cancer associated fibroblasts (CAF) (FIGs. 20A-B). This provides further evidence that JAK1 and JAK2 are involved in transducing LIFR signaling and CXCL12 regulation.

Example 2.4 Blocking CXCL12 signaling suppresses T cell recruitment

To see the effect of CXCL12 for recruiting T cells toward fibroblasts, we set up a trans-well assay with PBMCs on the upper chamber and supernatant from primary fibroblasts on the lower chamber. As a result, both CD4 and CD8 T cells could migrate toward the lower chambers and most of the migrated cells were expressing CXCR4 (FIG. 17A). To confirm the effect of CXCL12 - CXCR4 axis, we treated anti-CXCR4 neutralizing antibodies to PBMCs before migration. As a result, the migratory effect of both CD4 and CD8 T cells was inhibited (FIG. 17B).

Example 2.5 CXCL12 expressing cells are closely localized with CXCR4 expressing cells in parenchyma region of the lung tissue from early ILD patients

To examine the expression and localization pattern of CXCL12 and CXCR4 in human lung tissues, we performed an in situ hybridization assay using RNAscope probes for CXCR4 and CXCL12 on human lung tissue slides from both ILD patient and control subjects. All slides were scanned with 40x magnitude using slide scanner (VS 120, Olympus Life Science).

Compared to control subject (Control), more cells in the parenchyma region of human lung tissue from a patient with early ILD (uILD) expressed CXCR4 and CXCL12. Also, CXCL12 and CXCR4 expression were in close proximity with each other (FIG. 18). This provides in vivo evidence that fibroblasts in the parenchyma region making CXCL12 in diseased lung to recruit CXCR4+ expressing cells including CD4 and CD8 T cells in vivo.

References

Holmgaard et al., 2018. Targeting the TGFbeta pathway with galunisertib, a TGFbetaRI small molecule inhibitor, promotes anti-tumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint blockade. J Immunother Cancer, 6, 47.

Mariathasan et al., 2018. TGFbeta attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells. Nature, 554, 544-548.

Nguyen et al., 2017. Autocrine Loop Involving IL-6 Family Member LIF, LIF Receptor, and STAT4 Drives Sustained Fibroblast Production of Inflammatory Mediators. Immunity, 46, 220-232.

Schafer et al., 2017. IL-11 is a crucial determinant of cardiovascular fibrosis. Nature, 552, 110-115.

Tauriello et al., 2018. TGFbeta drives immune evasion in genetically reconstituted colon cancer metastasis. Nature, 554, 538-543.

Weiskirchen et al., 2019. Organ and tissue fibrosis: Molecular signals, cellular mechanisms and translational implications. Mol Aspects Med, 65, 2-15.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a disorder associated with fibrosis in a subject, the method comprising administering a therapeutically effective amount of an inhibitor of LIF-LIFR signaling to a subject in need thereof.

2. The method of claim 1, wherein the fibrosis is in lung, liver, kidney, gut, heart, or skin of the subject.

3. The method of claim 1, wherein the fibrosis is in the liver and the disorder is chronic HCV (hepatitis C virus) infection, chronic HBV (hepatitis B virus) infection, alcohol-induced (alcoholic liver disease), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver (NAFL), autoimmune hepatitis, iron overload, cirrhosis, or primary biliary cirrhosis.

4. The method of claim 1, wherein the fibrosis is in the kidney and the disorder is renal fibrosis, nephrogenic systemic fibrosis, chronic kidney disease, or renal anemia.

5. The method of claim 1, wherein the fibrosis is in the lung and the disorder is idiopathic pulmonary fibrosis (IPF) , cystic fibrosis, pulmonary hypertension, thromboembolic disease, emphysema, non-specific interstitial pneumonia (NSIP), chronic obstructive pulmonary disease (COPD), idiopathic pleuroparenchymal fibroelastosis, idiopathic lymphocytic interstitial pneumonia, or respirator bronchi oliti s-ILD .

6. The method of claim 1, wherein the fibrosis is in the skin and the disorder is scleroderma, systemic scleroderma, hypertrophic scar, systemic sclerosis, or keloids.

7. The method of claim 1, wherein the fibrosis is in the heart and the disorder is cardiac fibrosis, hypertrophic cardiomyopathy, cardiac dysfunction, valvular disease, arrhythmia, myocardial infarction, sarcoidosis, myocarditis, toxic cardimyopathies, chronic renal insufficiency, hypertension, diabetes, non-ischemic dilated cardiomyopathy, hypertrophic cardiomyopathy, sarcoidosis, chronic renal insufficiency, amyloidosis, or Anderson-Fabry disease.

35 The method of claim 1, wherein the fibrosis is in the gut and the disorder is intestinal fibrosis, enteropathies, inflammatory bowel disease, Crohn’s Disease bowel structure, or post-surgical bowel obstruction. The method of claims 1-8, wherein the inhibitor is an antibody that binds to LIF, LIFR, or GP130 and inhibits LIF-LIFR signaling, or an antigen -binding fragment thereof. The method of claim 9, wherein the antibody is 1C7, 12D3, or MSC-1, or an antigen-binding fragment thereof. The method of claim 10, wherein the antibody is a monoclonal antibody, nonhuman animal antibody, humanized antibody, chimeric antibody, human antibody, minibody, bispecific antibody, amino acid sequence-modified antibodies, modified antibody conjugated to other molecules, or sugar chain-modified antibody that includes the variable heavy chain (HC) and light chain (LC) complementarity determining regions (CDRs) of mAb 1C7, 12D3, or MSC-1, or the entire variable HCs and LCs of mAb 1C7, 12D3, or MSC-1. The method of claims 1-8, wherein the inhibitor is an inhibitory nucleic acid targeting LIF or LIFR mRNA. The method of claim 12, wherein the inhibitory nucleic acid is an antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a short, hairpin RNA (shRNA); or combinations thereof.

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