Methods of treating hepatic fibrosis, inflammation, or related diseases using ANGPTL4 antagonists

Abstract

The present invention relates generally to methods for the treatment of liver fibrosis and / or inflammation or related diseases, such as non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), using Angiopoietin-like 4 protein (ANGPTL4) antagonists, more particularly ANGPTL4 antagonists directed against the C-terminal fibrinogen-like domain of ANGPTL4.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Singapore Patent Application No. 10202250125X filed on June 10, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present invention generally relates to methods for treating liver fibrosis and / or inflammation or related diseases such as non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), using angiopoietin-like 4 protein (ANGPTL4) antagonists, more specifically ANGPTL4 antagonists targeting the C-terminal fibrinogen-like domain of ANGPTL4 (cANGPTL4) to inhibit the activity or expression of cANGPTL4. Background Art

[0004] Nonalcoholic fatty liver disease (NAFLD) is an ongoing epidemic that affects nearly 30% of the world’s population (1). NAFLD encompasses all fatty liver disease states, spanning a spectrum of liver pathologies associated with metabolic dysregulation, beginning with hepatic steatosis and progressing through nonalcoholic steatohepatitis (NASH) to cirrhosis (2). Global disease prevalence is expected to rise in line with rising obesity and diabetes (3). Despite the enormous burden of NAFLD, there are still no Food and Drug Administration (FDA)-approved pharmacological treatments. The current standard of care is lifestyle modification to reduce weight, which is often difficult to achieve and rarely sustained (4). Once the disease progresses to cirrhosis, these measures become less effective (5). Despite decades of recognition of these challenges and clinical realities, our understanding of the causes of this multiorgan disease remains severely lacking, and our ability to treat it remains woefully inadequate.

[0005] Patients with NASH and fibrosis are at increased risk for liver-related mortality (6). As fibrosis is the strongest predictor of adverse clinical outcomes, NASH represents the optimal treatment state in which most clinical symptoms begin to manifest. Reversal of fibrosis has the greatest clinical impact on the disease and is a key marker for currently defined clinical trial endpoints. Chronic liver inflammation is a driving force in the progression of NASH to fibrosis / cirrhosis. The evolution of NAFLD in humans and mice is accompanied by an increase in activated cytotoxic CD8+ T cells and IFN-γ-producing CD4+ T cells in the liver. In addition to T cells, B cells can also be detected in the inflammatory infiltrate of liver biopsies from NASH patients. Hepatic infiltration of B cells and T cells occurs concurrently with worsening liver damage and lobular inflammation (7, 8). Not surprisingly, as fibrosis decreases, a decrease in the recruitment of activated CD4+ T cells and CD8+ T cells to the liver is observed (9). Given the emerging role of adaptive immunity in the progression of NASH, modulating the recruitment and activation of lymphocytes provides a novel approach to ameliorate NASH-associated fibrosis. To date, the development of NASH therapeutics has focused primarily on modulating metabolic dysfunction, oxidative stress, and innate immunity (10). Progress in targeting adaptive immunity in NASH will also rely on a better understanding of the role of B and T cells in the chronic inflammatory processes that drive the progression of NASH fibrosis.

[0006] Therefore, there is still a need in the art to identify new therapeutic targets for treating non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH), more specifically treating, preventing, delaying or improving liver fibrosis or inflammation, or diseases associated with liver fibrosis or inflammation. Summary of the invention

[0007] In a first aspect, a method for treating, improving, delaying or preventing liver fibrosis or a disease associated with liver fibrosis in a subject is provided, the method comprising administering an effective amount of angiopoietin-like 4 protein (ANGPTL4) antagonist to the subject.

[0008] In various embodiments, the disease associated with liver fibrosis is selected from non-alcoholic steatohepatitis (NASH), autoimmune hepatitis, idiopathic liver fibrosis, non-alcoholic fatty liver disease (NAFLD), cholestatic liver disease, alcoholic hepatitis, and viral hepatitis.

[0009] In various embodiments, the disease associated with liver fibrosis is non-alcoholic steatohepatitis (NASH), or wherein liver fibrosis is associated with non-alcoholic steatohepatitis (NASH).

[0010] In various embodiments, the disease associated with liver fibrosis is non-alcoholic fatty liver disease (NAFLD) or wherein liver fibrosis is associated with non-alcoholic fatty liver disease (NAFLD).

[0011] In various embodiments, the ANGPTL4 antagonist is an antisense molecule; or an aptamer; or an siRNA molecule; or an anti-ANGPTL4 antibody.

[0012] In various embodiments, the ANGPTL4 antagonist is directed against the C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4).

[0013] In various embodiments, the ANGPTL4 antagonist neutralizes, blocks, inhibits, reduces or interferes with the expression and / or activity of the C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4).

[0014] In various embodiments, the ANGPTL4 antagonist is an anti-cANGPTL4 antibody, preferably a monoclonal antibody, more preferably a neutralizing monoclonal antibody.

[0015] In various embodiments, the anti-cANGPTL4 antibody is a monoclonal antibody that binds to the same epitope as mAb 11F6C4.

[0016] In various embodiments, the anti-cANGPTL4 antibody is a humanized antibody, preferably humanized mAb 11F6C4.

[0017] In various embodiments, the anti-cANGPTL4 antibody specifically binds to the C-terminal fibrinogen-like domain of ANGPTL4.

[0018] In various embodiments, the ANGPTL4 antagonist is a siRNA molecule, preferably an ANGPTL4-siRNA molecule.

[0019] In various embodiments, the siRNA molecules target the mRNA sequence encoding ANGPTL4.

[0020] In various embodiments, the subject is a human.

[0021] In another aspect, provided is a method for treating, ameliorating or preventing liver inflammation in a subject having liver fibrosis or a disease associated with liver fibrosis, the method comprising administering an effective amount of an angiopoietin-like 4 protein (ANGPTL4) antagonist.

[0022] In another aspect, a method for regulating an immune response in a subject having liver fibrosis or a disease associated with liver fibrosis is provided, the method comprising administering an effective amount of an angiopoietin-like 4 protein (ANGPTL4) antagonist, wherein the administration induces a population of regulatory T cells (preferably CD8 + The number, activity and / or effector function of T cells and / or CD4+ T cells are reduced.

[0023] In another aspect, an in vitro method of reducing fibrosis in a liver sample is provided, comprising contacting liver cells or tissue with an angiopoietin-like 4 protein (ANGPTL4) antagonist.

[0024] In another aspect, an in vitro method of reducing hepatic inflammation in a liver sample is provided, comprising contacting the liver sample with an angiopoietin-like 4 protein (ANGPTL4) antagonist.

[0025] In another aspect, an in vitro method for regulating an immune response in a liver sample is provided, comprising: contacting the liver sample with an angiopoietin-like 4 protein (ANGPTL4) antagonist, wherein the contacting induces a regulatory T cell or activated T cell population (preferably CD8 + The number, activity and / or effector function of T cells and / or CD4+ T cells are reduced.

[0026] In various embodiments, the ANGPTL4 antagonist is directed against the C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Various embodiments may be better understood with reference to the following detailed description, taken in conjunction with the non-limiting examples and accompanying drawings.

[0028] Figure 1Physiological and metabolic parameters of control mice and LIDPAD mice are shown: A. Liver weight normalized to body weight after 1 to 48 weeks of feeding; B. Area under the curve (AUC) of intraperitoneal glucose tolerance test (IGTT) at designated time points; C. Proton density fat fraction (PDFF) images of livers from control mice and LIDPAD mice at 4, 8, and 16 weeks after dietary intervention. The PDFF metric represents increased liver fat accumulation. Liver PDFF of control mice and LIDPAD mice was quantified at all imaging time points (right). Excessive lipid deposition was observed during 8 to 12 weeks of LIDPAD intervention. Subcutaneous fat growth was determined between various time points; D. Representative macroscopic and microscopic images of livers obtained from LIDPAD mice. Tissue sections were stained with hematoxylin and eosin (H&E) to show general features of the liver, with Sirius red (PSR) and Masson's trichrome (MT) to highlight collagen deposition, and with Oil Red O (ORO) to detect the presence of lipids. Scale bar represents 100 μm. Graph shows cumulative histological SAF scores of livers of LIDPAD mice at the indicated weeks after feeding in the table (right). Each row corresponds to one liver analyzed. For 1A-B, n=7-10 per group. Data are presented as mean±SEM. **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05 (unpaired t-test, ANOVA, Welch's t-test or covariance test followed by post hoc comparisons, as appropriate). ns indicates not significant. For 1C, n=5 per group, * p<0.05 (Mann–Whitney test after pixel thresholding at 60%).

[0029] Figure 2 shows metabolic parameters of control mice and LIDPAD mice: A. Body weight changes of mice fed with control diet and LIDPAD (left) and the number of animals measured at each time point over 48 weeks (left); B. IGTT curves of mice fed with LIDPAD and control diet from 1 week to 48 weeks. n = 7-10 per group. Data are presented as mean ± SEM. **** p<0.0001, *** p<0.001, ** p<0.01, * p < 0.05 (unpaired t test, ANOVA, Welch's t test, or covariance test followed by post hoc comparisons, as appropriate).

[0030] Figure 3Physiological parameters of control mice and LIDPAD mice are shown. AB. Respiratory exchange ratio (RER) of mice fed with control diet and LIDPAD diet per hour (A). Gray and white areas represent alternating dark and light cycles from 7 am to 19:00 pm every day. The average RER (B) was calculated to determine any statistical differences between control mice and LIDPAD mice; CE. The physiological status of mice was recorded when the mice performed voluntary exercise. Metabolic rate (C) is shown with gray and white areas representing dark and light cycles. After the introduction of voluntary exercise, the respiratory exchange ratio (D) of LIDPAD mice remained below 0.8, and the daily energy expenditure (E) of LIDPAD mice remained relatively unchanged. Data are expressed as mean ± SEM. *** p<0.001 (unpaired t-test).

[0031] Figure 4 Histological images of islets and kidneys of control mice and LIDPAD mice are shown: A. Representative H&E images of islets; B. Quantification and analysis of islet area in control mice and LIDPAD mice from 1 week to 48 weeks using ImageJ. n = 12-28 regions of interest (≥ 5 islets / animal). Data are expressed as median ± interquartile range; C. Comparison of kidney weight (two kidneys) of control mice and LIDPAD mice at 48 weeks. Data information: n = 8 per group. Data are expressed as mean ± SEM, *** p<0.001, ** p<0.01, * p<0.05 (Welch's t test) ns indicates not significant; D. Representative images of kidneys stained with H&E to show general morphology. Periodic acid-Schiff (PAS) staining was used to highlight the basement membrane, and Masson's trichrome staining was used to identify collagen deposition.

[0032] Figure 5 shows representative macroscopic and microscopic images of livers obtained from control mice and LIDPAD mice: A. Tissue sections were stained with hematoxylin and eosin (H&E) to show general features of the liver, stained with Sirius red (PSR) and Masson's trichrome (MT) to highlight collagen deposition, and stained with Oil Red O (ORO) to detect the presence of lipids. The scale bar represents 100 μm. The graph shows the cumulative histological SAF scores of the livers of control mice within the specified weeks after feeding in the list (right). Each row corresponds to an analyzed liver; B. Representative histological images of the livers of mice fed with LIDPAD for 16 weeks, stained with H&E and PSR to indicate the pathological features of the liver. * Indicates macrovesicular fatty degeneration; Indicates microvesicular fatty degeneration; § indicates balloon-shaped hepatocytes; § indicates areas of lobular inflammation; Indicates pericellular fibrotic areas; # indicates periportal fibrosis. Scale bar represents 100 μm.

[0033] Figure 6 Transcriptomic and molecular analysis of livers from control and LIDPAD mice: A. Heatmap of differentially expressed genes (DEGs) from 1 to 48 weeks, and corresponding gene ontology (GO) biological process terms guided by hierarchical clustering; B. Gene ontology and pathway analysis across multiple time points was used to track the temporal changes of DEGs, and only recurring GO terms across at least two time points were characterized to increase the confidence of the data as true positives; C. Transcriptome profiles of LIDPAD mice plotted with homologous genes of human genes associated with NAFLD progression. The percentage of genes associated with each histological feature of NASH patients. The charts are arranged in order of increasing severity (from left to right). The transcriptomics-based staging of LIDPAD livers was included as a reference; D. GSEA was plotted using FDR and gene set size, showing terms from Reactome, WP, KEGG, Biocarta, and GO.

[0034] Figure 7 Liver ALT levels and serum cytokine levels are shown: A. Blood chemistry readings for control mice and LIDPAD mice based on liver function tests (LFTs) consisting of alanine aminotransferase (ALT). Data are median ± interquartile range; B. Lipid analysis highlights differences in cholesterol and squalene between control mice and LIDPAD mice; C. Heat map of serum cytokine arrays after 1 to 48 weeks of LIDPAD feeding, with colors based on row values. Data are expressed as log (fold difference in mean concentration) between LIDPAD mice and control mice; D. Representative images of liver biopsies from NASH patients. Scale bar: 200 μm. For AC: n = 3-7 per group. ** p<0.01, * p<0.05 (C) Two-way ANOVA with Sidak's multiple comparisons and D with Welch's t-test). ns indicates not significant.

[0035] Figure 8The metabolic effects of Angptl4 neutralizing antibodies on NASH mice are shown: AB. Body weight (A) and IGTT AUC (B) of mice in different treatment groups. AUC: area under the curve; IGTT, intraperitoneal glucose tolerance test; CD. Representative immunofluorescence images of the livers of the designated treatment groups after 8 and 12 weeks of feeding (C). CD45 indicates immune infiltration, while α-SMA indicates fibroblast activation. The scale bar represents 100 μm. The bar graph shows the quantification of CD45 and α-SMA fluorescence intensity in immunofluorescence sections (D); E. FACS analysis shows the proportion of CD45+ immune cells in the liver in the designated groups; F. Representative liver histology images of immunodeficient NSG mice fed with LIPDAD diet for 52 weeks. Data are expressed as mean ± SD. * p<0.05, *** p<0.001. n=6-9 mice per group.

[0036] Fig. 9 The effects of Angptl4 deficiency on liver pathology and transcriptome are shown: A. Representative images of microscopic images of livers obtained from control mice, LIDPAD-fed mice, LIDPAD-fed mice treated with neutralizing mAb against cAngptl4, and LIDPAD-fed Angptl4LysM- / - mice after 8 and 12 weeks of feeding. Tissue sections were stained with hematoxylin and eosin (H&E) to show general features of the liver and with Sirius red (PSR) to highlight collagen deposition. The scale bar represents 100 μm. The chart shows a list of cumulative histological SAF scores for livers of the specified groups at the specified weeks after feeding (right). Each row corresponds to one analyzed liver; B. The chart shows liver triglyceride concentrations normalized to protein levels. Data are expressed as mean ± SEM. *** p<0.001, * p<0.05 (unpaired t-test). n=5 per group; C. Principal component analysis (PCA) of liver transcriptomes of designated and treated groups at 8 and 12 weeks; D. Heatmap of differentially expressed genes (DEGs) at 8 and 12 weeks, and the corresponding gene ontology (GO) biological process terms guided by hierarchical clustering of liver transcriptomes of designated and treated groups.

[0037] Fig.10The intrahepatic immune status during LIDPAD-induced NAFLD is shown: A. Overall UMAP representation of total intrahepatic CD45+ immune population (left). Dot plots represent molecular markers used to identify major immune cell types (right); BC. Relative abundance of total CD45+ immune cell types (B) and T cell populations (C) in the liver of control mice, LIDPAD-fed mice, LIDPAD-fed mice treated with neutralizing monoclonal antibodies (mAbs) against cAngptl4, and LIDPAD-fed Angptl4LysM- / - mice after 8 and 12 weeks of feeding.

[0038] Fig.11 Figure 3 shows the distribution of intrahepatic B cells and T cells: UMAP of adaptive immune subsets of AB cells and T cells (left). Dot plots represent molecular markers used to identify specific subsets of B cells and T cells (right); B. Based on the differential transcriptomes of different treatment groups, GSEA showed highly enriched gene sets in CD8+ T cells during NASH progression from 8 to 12 weeks of feeding. Normalized enrichment scores (NES) are color-coded, where NES>0 (red) and NES<0 (blue) indicate gene functions enriched in upregulated genes and downregulated genes, respectively. Point size represents the -log of representative enrichment tests. 10 FDR.

[0039] Fig.12Figure 2 shows that cAngptl4 mAb inhibits intrahepatic CD4 T cell activation through eIF2α-mediated translation attenuation: A. The ratio of intrahepatic naive CD4 T cells and activated CD4 T cells in control mice, LIDPAD-fed mice, LIDPAD-fed mice treated with a neutralizing monoclonal antibody (mAb) against cAngptl4, and LIDPAD-fed Angptl4LysM- / - mice after 8 and 12 weeks of feeding; B. Pseudo-time analysis, sorting CD4 T cells according to activation status. The vertical line represents the median pseudo-time score of each group; C. The percentage of intrahepatic naive CD4 T cells (CD45+Lin-CD3+CD4+CD62L+CD69-) and activated CD4 T cells (CD45+Lin-CD3+CD4+CD69+) in the designated groups after 12 weeks of feeding based on FACS. Lineage markers (Lin) include CD11b, NK1.1, and Ly6G; D E. Based on the differential transcriptome of naive CD4 T cells in different treatment groups, GSEA showed highly enriched gene sets in CD4+ T cells during NASH progression from 8 to 12 weeks of feeding (D) and signature genes involved in regulating eIF2α phosphorylation (E). Normalized enrichment scores (NES) >0 and <0 indicate gene sets enriched by up-regulated genes and down-regulated genes, respectively. For 12D, NES are color-coded and the point size indicates the -log10FDR of the representative enrichment test.

[0040] Fig.13 The gating strategy for FACS analysis to identify intrahepatic naive and activated CD4+ T cells and CD8+ T cells is shown.

[0041] Fig.14Figure 2 shows that cAngptl4 mAb inhibits intrahepatic CD8+ T cell activation: A. The ratio of naive and activated CD8 T cells in the liver of control mice, LIDPAD-fed mice, and LIDPAD-fed mice treated with a neutralizing monoclonal antibody (mAb) against cAngptl4 after 8 and 12 weeks of feeding; B. Pseudo-time analysis, sorting CD8 T cells according to activation status. The vertical line represents the median pseudo-time score of each group; C. The percentage of naive CD8 T cells (CD45+Lin-CD3+CD8+CD62L+CD69-) and activated CD8 T cells (CD45+Lin-CD3+CD8+CD69+) in the liver of the designated group after 12 weeks of feeding based on FACS. Lineage markers (Lin) include CD11b, NK1.1, and Ly6G; D E . Based on the differential transcriptome of naive CD8 T cells in different treatment groups, GSEA showed highly enriched gene sets in CD8+ T cells during NASH progression from 8 to 12 weeks of feeding (D) and signature genes involved in regulating eIF2α phosphorylation (E). Normalized enrichment scores (NES) >0 and <0 indicate gene sets enriched by up-regulated genes and down-regulated genes, respectively. For 14D, NES are color-coded and point size indicates -log of representative enrichment test 10 FDR.

[0042] Fig.15 Different roles of CD4+T cells and CD8+T cells in NASH severity and liver fibrosis are shown. GSEA of CD4+T cells and CD8+T cells and gene sets in two major processes, namely eukaryotic translation and immune response. Normalized enrichment scores (NES) >0 and <0 indicate gene sets enriched by up-regulated genes and down-regulated genes, respectively. FDR is color-coded.

[0043] Figure 16 shows A. In naive CD4+ T cells, Eif2ak4 signaling was specifically activated by cANGPTL4 monoclonal antibody, while the gene characteristics of effector CD4+ (i.e., Th1 and Th17) and CD8+ T cells in activated T cells were significantly inhibited regardless of how the disease progressed; B. cANGPTL4 monoclonal antibody weakened interferon-γ signaling and antigen processing activity in activated CD4+ T cells, both of which are related to the immune regulatory ability of CD4+ T cells.

[0044] Figure 17 shows the kinome regulation pathway of the Angptl4-eIF2α phosphorylation pathway: AB. Using LPS as a stimulus, kinase inhibitors were screened in CD4+T cells from wild-type and Angptl4-KO mice. The heat map illustrates the expression levels of target genes of eIF2α and phosphorylated eIF2α (i.e., Ddit3, Atf4, and Hspa5) relative to DMSO-treated wild-type CD4+T cells (A). Using a hierarchical clustering approach, kinase inhibitors and their molecular targets (in brackets) were divided into three groups with different activities on phospho-eIF2α signaling and Angptl4 dependence based on expression profiles. The schematic (B) outlines six scenarios to demonstrate the role of different protein kinases in the Angptl4-eIF2α axis. The reduced and grayed terms and arrows indicate that Angptl4-eIF2α activity is inhibited, while the enlarged terms and arrows indicate overactivity. In short, Angptl4 deficiency in CD4+ T cells was associated with upregulation of eIF2α and phosphorylated eIF2α target genes, indicating an inhibitory effect of Angptl4 on eIF2α expression and its downstream signaling. For protein kinases that do not regulate the eIF2α pathway, their inhibitors did not alter the expression of eIF2α and target genes (e.g., CK2, ASK, AMPK, CaMK, and CDK4 / 6) in wild-type and Angptl4-knockout CD4+ T cells. When the inhibitors upregulated eIF2α and target genes in wild-type but not Angptl4-knockout CD4+ T cells (which reflects the de-repression of eIF2α in the absence of Angptl4), the regulatory activities of the corresponding molecular targets on the eIF2α pathway were Angptl4-dependent (e.g., MAPK, PI3K, Src, Syk-Btk, etc.). For kinase inhibitors that further upregulate eIF2α and target genes, the regulatory activity of molecular targets is Angptl4-independent regardless of Angptl4 status (e.g., PKD1 / 2 / 3, JAK1 / 2 / 3, Aurora A / B / C, etc.); CD. mRNA expression of eIF2α in wild-type and Angptl4 knockout CD4+ T cells stimulated with and without LPS (C) and immunoblotting of eIF2α and phosphorylated eIF2α (D). For (C), 18s was used as an endogenous reference gene, while for (D), β-tubulin used as a loading control was from the same sample; EF. Efficiency of knocking down eIF2α in splenic lymphocytes of Angptl4 knockout mice using SMARTpool siRNA (E). 18s was used as an endogenous reference gene. Activation status of cell-tracking labeled adoptively transferred CD4+ T cells (top) and host CD4+ T cells (bottom) in the liver of LIDPAD-fed mice after 12 weeks of feeding (F).Adoptively transferred cells were treated with 10 nM nonspecific small interfering RNA (si-scrambled) or si-eIF2α. FACS was used to determine naive CD4 T cells (CD45+Lin-CD3+CD4+CD62 L+CD69-) and activated CD4 T cells (CD45+Lin-CD3+CD4+CD69+); G. Schematic diagram summarizing the Angptl4-eIF2α pathway and its role in T lymphocyte activation during fibrosis in the LIDPAD-induced NAFLD mouse model. Angptl4 stimulates kinases that inhibit phosphorylated eIF2α signaling, eliminating its inhibitory effect on T cell activation, i.e., reduced phosphorylated eIF2α signaling is associated with more T cell activation. DETAILED DESCRIPTION

[0045] The following detailed description mentions the specific details and embodiments that can practice the present invention by way of example. These embodiments describe enough details to enable those skilled in the art to implement the present invention. Without departing from the scope of the present invention, other embodiments may be used and logical changes may be made. Various embodiments are not necessarily mutually exclusive, because some embodiments may be combined with one or more other embodiments to form new embodiments.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Unless the context clearly indicates otherwise, the singular terms "a", "an" and "the" include plural forms. Similarly, unless the context clearly indicates otherwise, the word "or" is intended to include "and". The term "comprising" means "including". In the event of a conflict, the present specification (including term explanations) shall prevail. As used herein, "about" in relation to a numerical value refers to ±10% or ±5% of the cited numerical value.

[0047] The inventors of the present application have discovered the key mediating role of angiopoietin-like 4 (ANGPTL4) in liver fibrosis and / or inflammation and related liver diseases such as non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). In particular, the C-terminal fibrinogen-like domain of ANGPTL4 (cANGPTL4) has been identified as playing a key role in the development and severity of liver inflammation and liver fibrosis associated with NAFLD or NASH.

[0048] In this regard, cANGPTL4 represents a new therapeutic inhibitory target in the treatment, prevention or improvement of liver fibrosis, liver inflammation or diseases associated with said liver fibrosis and / or inflammation, more specifically liver fibrosis and / or inflammation associated with NASH. As exemplified by the experimental data disclosed herein, systemic inhibition of cANGPTL4 activity using a neutralizing monoclonal antibody showed a potent inhibitory effect on T cell activation and functionality by impairing global protein translation, especially in CD4+T cells. In essence, inhibition of cANGPTL4 activity was shown to reshape immune cell subsets and attenuate the recruitment of adaptive immune cells in the intrahepatic NASH environment. cANGPTL4 inhibition regulates the translation initiation pathway in CD4+T cells, subsequently disrupting the maturation of CD4+T cells and CD8+T cells into their effector counterparts, changing the ratio of activated CD4+T cells and CD8+T cells in the liver, resulting in the loss of immune and inflammatory activity of these cell subsets.

[0049] Therefore, inhibition of cANGPTL4 could translate into reduced inflammation and remodeling of the immune landscape, thereby improving liver fibrosis and delaying the progression of liver diseases such as NASH.

[0050] In addition, cANGPTL4 was shown to shape the activation of the intrahepatic T cell landscape during fibrosis by regulating eIF2α signaling. Single immune cell analysis and liver transcriptomics during fibrosis and kinase inhibitor screening confirmed that ANGPTL4 orchestrates hyperactivation of intrahepatic adaptive immunity through eIF2α signaling. Consistently, inhibition of cANGPTL4 (e.g., immune blockade) reduced T cell hyperactivation and delayed disease progression. Therefore, cANGPTL4 is a key determinant in shaping intrahepatic adaptive immunity during liver fibrosis.

[0051] These findings support the potential clinical use of targeting cANGPTL4 by gene knockdown / knockout, immunoneutralization or other related methods to inhibit cANGPTL4 activity for the treatment, prevention, improvement or delay of liver fibrosis, liver inflammation or diseases related to liver fibrosis. In particular, the inhibition of cANGPTL4 activity represents a new direction for the treatment, prevention or improvement of liver fibrosis and / or liver inflammation associated with NASH, and therefore may be important for the treatment or prevention of NAFLD.

[0052] Therefore, provided is a method for treating, improving, delaying or preventing liver fibrosis or a disease associated with liver fibrosis in a subject, the method comprising administering an effective amount of angiopoietin-like 4 protein (ANGPTL4) antagonist to the subject.

[0053] As used herein, the term "liver fibrosis" refers to a disorder in which scar tissue (fibrous tissue) is excessively accumulated in the liver, usually caused by chronic liver damage or inflammation that interferes with liver function. Scar tissue blocks blood flow to the liver portal through the organ and interferes with normal function. Due to inflammation, damage to the liver parenchyma causes activation of stellate cells, which increases fibrosis and obstructs blood flow in the circulation by producing myofibroblasts. The production of myofibroblasts accelerates the loss of liver function and may lead to death. Liver fibrosis can be caused by various factors, including chronic viral hepatitis B or hepatitis C infection, excessive drinking, non-alcoholic fatty liver disease (NAFLD) or non-alcoholic fatty hepatitis (NASH), autoimmune hepatitis, certain drugs, hereditary diseases and other less common diseases. The severity of liver fibrosis can vary, from mild scarring that may not cause obvious symptoms to large-area scarring that may damage liver function and cause complications such as cirrhosis, liver failure and portal hypertension. In various embodiments, indicators of diseases or conditions associated with liver fibrosis include increased levels of one or more of the following molecules: alpha-smooth muscle actin (alpha-SMA), connective tissue growth factor (CTGF), vascular cell adhesion factor 1 (VCAM-1), collagen, Yes-associated protein (YAP), phosphorylated YAP, serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), and liver hydroxyproline. As used herein, "chronic liver injury" refers to liver injury caused by chronic injury due to infection, exposure to drugs or toxic compounds, alcohol, impurities in food, abnormal accumulation of normal substances in the blood, autoimmune processes, genetic defects, or other factors. Chronic liver injury can lead to liver fibrosis and cirrhosis.

[0054] As used herein, "disease associated with liver fibrosis" refers to a class of diseases characterized by chronic liver damage and fibrosis. Exemplary "diseases or conditions associated with liver fibrosis" include autoimmune hepatitis, congenital liver fibrosis, nonalcoholic steatohepatitis (NASH), metabolic-associated fatty liver disease (MAFLD), nonalcoholic fatty liver disease (NAFLD), cholestatic liver disease, alcoholic hepatitis, or viral hepatitis.

[0055] In various embodiments, the disease associated with liver fibrosis is selected from non-alcoholic steatohepatitis (NASH), autoimmune hepatitis, innate liver fibrosis, metabolic associated fatty liver disease (MAFLD), non-alcoholic fatty liver disease (NAFLD), cholestatic liver disease, alcoholic hepatitis and viral hepatitis.

[0056] In various embodiments, the disease associated with liver fibrosis is non-alcoholic fatty liver disease (NAFLD).

[0057] As used herein, the terms "non-alcoholic fatty liver disease" and "NAFLD" are used interchangeably and refer to a series of conditions that affect people who rarely drink or do not drink alcohol, characterized at least in part by being caused by excessive fat (e.g., steatosis) stored in liver cells, rather than by drinking alcohol. NAFLD is generally associated with risk factors such as obesity, insulin resistance, hyperglycemia, high cholesterol, and metabolic syndrome. The characteristics of NAFLD can be any combination of the characteristics of steatosis, fibrosis, hepatomegaly, fatigue, abdominal pain, abdominal swelling, enlarged blood vessels, enlarged breasts, enlarged spleen, red palms, and jaundice. NAFLD refers to a series of conditions that may vary in severity or degree, from simple steatosis (fat accumulation in the liver without inflammation or liver damage) to non-alcoholic steatohepatitis (NASH) with inflammation and hepatocellular damage, depending on the progression of the disease in a particular subject. NAFLD can be subdivided into non-alcoholic fatty liver disease (NAFL). Non-alcoholic fatty liver disease is a type of NAFLD, a condition in which fat accumulates in liver cells. The risk of NAFL progressing to cirrhosis is very small. Simple fatty liver does not usually damage the liver, but it is a condition that can be identified by liver biopsy. Simple fatty liver is not associated with any other liver abnormalities (such as scarring or inflammation). It is common in patients who are overweight or have diabetes. When fat occupies at least 5% of the liver, the patient has fatty liver. As used herein, the term "steatosis" refers to the accumulation of fat in liver cells.

[0058] In various embodiments, NAFLD is independent of obesity and diabetes.

[0059] Recently, the term NAFLD has been proposed to be replaced by metabolic-associated fatty liver disease (MAFLD) (11). MAFLD is a newer term that reflects the evolving understanding of the disease and emphasizes the close association between fatty liver disease and metabolic health. According to the proposed criteria, MAFLD is diagnosed when there is evidence of hepatic fat accumulation accompanied by any of the following three criteria: overweight / obesity, type 2 diabetes, or insulin resistance, and evidence of liver inflammation or liver damage. MAFLD encompasses a wider range of patients who may have fatty liver disease, including those who would not be traditionally diagnosed with NAFLD due to lack of alcohol consumption but who still have metabolic risk factors. In summary, NAFLD refers to a condition in which excess fat accumulates in the liver of individuals who drink little or no alcohol, while MAFLD is a newer term that reflects a broader understanding of the disease, emphasizes the close association between fatty liver disease and metabolic health, and includes a wider range of patients who may have fatty liver disease.

[0060] In various embodiments, the disease associated with liver fibrosis is metabolic associated fatty liver disease (MAFLD). In various embodiments, the disease associated with liver fibrosis is NAFLD and MAFLD.

[0061] In various embodiments, MALFD is lean MAFLD. As used herein, the term "lean MAFLD" refers to a subtype or variant of metabolic-associated fatty liver disease (MAFLD) characterized by the presence of hepatic fat accumulation and metabolic risk factors, but occurring in subjects with a normal body mass index (BMI) or lower body fat levels. Typically, less than 25 kg / m is considered to be within the normal or "lean" range. 2 Lean MAFLD was diagnosed in individuals with a BMI of

[0062] In various embodiments, the disease associated with liver fibrosis is non-alcoholic fatty liver disease (NASH). In various embodiments, liver fibrosis can be associated with non-alcoholic fatty liver disease (NASH). As used herein, the term "NASH" is a more severe form of NAFLD, characterized by lipid accumulation, inflammation, hepatocyte ballooning and varying degrees of fibrosis in the liver, and is considered to be the main cause of liver cirrhosis of unknown cause. When the results of liver function tests (such as alanine aminotransferase (ALT) or aspartate aminotransferase (AST)) are found to be elevated in the routine blood test items of the object, it is usually first suspected that it suffers from NASH. When further evaluation does not find obvious liver disease causes and when X-rays or imaging studies of the liver show fat, it is suspected to be NASH. The only way to provide a clear diagnosis of NASH and to separate it from simple fatty liver is liver biopsy. When fat, inflammation and hepatocyte damage are observed from biopsy, it is diagnosed as NASH. If the tissue shows fat without inflammation and damage, it is diagnosed as NAFL or NAFLD.

[0063] As highlighted above and shown in the working examples, cANGPTL4 inhibition was shown to modulate the translation initiation pathway in CD4+ T cells, subsequently impairing the maturation of CD4+ T cells and CD8+ T cells into their effector counterparts, altering the ratio of activated CD4+ T cells to CD8+ T cells in the liver, leading to loss of immune and inflammatory activity of these cell subsets.

[0064] Therefore, a method for treating, preventing, delaying or improving liver inflammation in a subject suffering from liver fibrosis or a disease associated with liver fibrosis is also provided, the method comprising administering an effective amount of angiopoietin-like 4 protein (ANGPTL4) antagonist.

[0065] As used herein, the term "hepatic inflammation" refers to inflammation of liver tissue. Chronic hepatic inflammation occurs when the liver continues to be inflamed for a period of time, usually more than six months. Chronic hepatitis can be caused by persistent viral infection (such as chronic hepatitis B or hepatitis C), autoimmune hepatitis, metabolic diseases (such as non-alcoholic fatty liver disease or NASH), or other causes. Chronic hepatic inflammation can lead to liver fibrosis.

[0066] In various embodiments, the liver inflammation is chronic liver inflammation. In various embodiments, the liver inflammation is associated with NAFLD or NASH, such that the subject suffers from said NAFLD or NASH.

[0067] In treating, ameliorating, preventing or delaying liver inflammation according to the methods disclosed herein, one effect that can be seen is a reduction in the number of regulatory T cells or activated T cell populations (e.g., CD4+ T cells or CD8+ T cells). Therefore, the methods disclosed herein can also be considered as methods of influencing or altering the immune response in a subject to which an ANGPTL4 antagonist is administered. The subject may have liver inflammation, where immune modulation of CD4+ T cells or CD8+ T cells is a desired outcome.

[0068] In various embodiments, methods and administration of an ANGPTL4 antagonist reduce CD8 + T cells and / or CD4+ T cells (more specifically activated CD8 + In various embodiments, the reduction occurs in the liver, i.e., in the liver cells or tissues of the subject.

[0069] Therefore, also provided is a method for regulating an immune response in a subject suffering from liver fibrosis or a disease associated with liver fibrosis, the method comprising administering an effective amount of angiopoietin-like 4 protein (ANGPTL4) antagonist, wherein administration induces a decrease or inhibition of the number, activity and / or effector function of a regulatory T cell population (e.g., CD8+ T cells and / or CD4+ T cells). In various embodiments, this decrease or inhibition occurs in the liver, i.e., in liver cells or tissues.

[0070] In addition, a method for reducing T cell activation (preferably CD8 + T cell and / or CD4+ T cell activation) method, the method comprising administering an effective amount of angiopoietin-like 4 protein (ANGPTL4) antagonist. In various embodiments, the reduction of T cell activation occurs in the liver, that is, in liver cells or tissues.

[0071] “ANGPTL4” or “Angptl4” refers to the angiopoietin-like 4 polypeptide or protein, as well as its naturally occurring alleles, secreted and processed forms. ANGPTL4 is a secreted glycoprotein that belongs to a family of nine structurally similar Angptl proteins (12). The action of Angptl4 depends on its proteolytic cleavage, which releases the N-terminal coiled-coil domain (nAngptl4) and the C-terminal fibrinogen-like domain (cAngptl4) (12). nAngptl4 present in plasma participates in lipid metabolism by inhibiting binding to lipoprotein lipase and inhibiting the release of peripheral triglycerides (13). cAngptl4 interacts with extracellular matrix proteins and is localized in different tissues (14). cAngptl4 is associated with a variety of inflammatory-related diseases, such as tissue damage and cancer (15-17). cANGPTL4 regulates inflammation by enhancing vascular permeability, cytokine secretion and monocyte differentiation (18-20).

[0072] The term "ANGPTL4" is also used to refer to fragments (e.g., subsequences, truncated forms, etc.) of polypeptides comprising, for example, an N-terminal fragment, a coiled-coil domain, a C-terminal fragment, a fibrinogen-like domain. Unless otherwise indicated, the term "ANGPTL4" refers to angiopoietin-like protein 4 from any vertebrate or mammalian source, including, but not limited to, human, cow, chicken, rodent, mouse, rat, pig, sheep, primate, monkey, and guinea pig. The term also refers to fragments and variants of native ANGPTL4 that maintain at least one in vivo or in vitro activity of native ANGPTL4. The term encompasses the full-length unprocessed precursor form of ANGPTL4.

[0073] ANGPTL4 is present in humans and mice. For example, human ANGPTL4 is a 406 amino acid protein, while mouse ANGPTL4 is a 410 amino acid protein. In various embodiments, ANGPTL4 is human ANGPTL4 or murine ANGPTL4.

[0074] ANGPTL4 polypeptides and nucleic acids encoding ANGPTL4 are readily known and available to those skilled in the art, and include, for example, human ANGPTL4, which has NCBI gene ID 51129, NCBI transcript reference sequence: NM_139314, Uniprot ID Q9BY76, and Ensembl gene ID ENSG00000167772; or murine ANGPTL4, which has NCBI gene ID 57875, NCBI transcript reference sequence: NM_020581, Uniprot ID Q9Z1P8, and Ensembl gene IDENSMUSG00000002289. For ease of reference, the sequences of human and murine ANGPTL4 are included in Table 1 below.

[0075] Table 1: Exemplary human and mouse sequences of ANGPTL4

[0076]

[0077]

[0078]

[0079] In this regard, the C-terminal fibrinogen-like domain of human ANGPTL4 (cANGPTL4) may comprise or consist of amino acids 186 to 406 (more preferably amino acids 179 to 401) of the amino acid sequence shown in SEQ ID NO: 1, wherein the position numbering is according to SEQ ID NO: 1. Specifically, the C-terminal fibrinogen-like domain of human ANGPTL4 (cANGPTL4) may comprise or consist of the amino acid sequence shown in SEQ ID NO: 2.

[0080] Furthermore, the C-terminal fibrinogen-like domain of mouse ANGPTL4 (cANGPTL4) may comprise or consist of amino acids 186 to 410 (more preferably amino acids 183 to 405) of the amino acid sequence shown in SEQ ID NO: 4, wherein the position numbering is according to SEQ ID NO: 4. Specifically, the C-terminal fibrinogen-like domain of murine ANGPTL4 (cANGPTL4) may comprise or consist of the amino acid sequence shown in SEQ ID NO: 5.

[0081] Therefore, the ANGPTL4 antagonists described herein are molecules that can neutralize, block, inhibit, eliminate, reduce or interfere with ANGPTL4 activity or its expression, including the binding of ANGPTL4 to ANGPTL4 receptors. For example, ANGPTL4 antagonists may include anti-ANGPTL4 antibodies and antigen-binding fragments thereof, oligonucleotide molecules, small molecules, receptor molecules and derivatives that specifically bind to ANGPTL4 and isolate ANGPTL4 from binding to one or more receptors, anti-ANGPTL4 receptor antibodies and ANGPTL4 receptor antagonists (e.g., small molecule inhibitors of receptors). Other ANGPTL4 antagonists also include ANGPTL4 antagonist variants, antisense molecules (e.g., ANGPTL4-SiRNA), RNA aptamers and ribozymes against ANGPTL4 or its receptors that directly or indirectly inhibit ANGPTL4 activity, or conjugates or fusion proteins thereof. In various embodiments, the antagonist ANGPTL4 antibody is an antibody that inhibits or reduces ANGPTL4 activity by binding to a specific subsequence or region of ANGPTL4.

[0082] In various embodiments, the ANGPTL4 antagonist is directed against the expression or activity of the C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4), which C-terminal fibrinogen-like domain of angiopoietin-like 4 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 5.

[0083] In various embodiments, the ANGPTL4 antagonist is a small molecule. Small molecule antagonists can be identified and chemically synthesized using known methods (see, e.g., PCT Publication Nos. WO / 2000 / 00823 and WO / 2000 / 39585). In general, small molecule antagonists are typically less than about 2000 Daltons, or less than about 1500 Daltons, 750 Daltons, 500 Daltons, 250 Daltons, or 200 Daltons in size, wherein such organic small molecule is capable of binding, preferably specifically binding, to a target polypeptide (i.e., in this case ANGPTL4, or more specifically cANGPTL4). Such small molecule antagonists can be identified without undue experimentation using well-known techniques. In this regard, it should be noted that techniques for screening organic small molecule libraries for molecules capable of binding to a polypeptide target are well known in the art (see, e.g., PCT Publication Nos. WO / 2000 / 00823 and WO / 2000 / 39585).

[0084] In various embodiments, the antagonist of ANGPTL4 is an antibody against the C-terminal region of the angiopoietin-like 4 protein (cANGPTL4), and can be referred to as an anti-cANGPTL4 antibody. The anti-cANGPTL4 antibody inhibits or reduces the activity of cANGPTL4 by binding to a specific target subsequence or region of the C-terminal fibrinogen-like domain of the ANGPTL4 protein.

[0085] As used herein, the term "antibody" refers to a protein consisting of one or more polypeptide chains substantially encoded by all or part of known immunoglobulin genes. Known immunoglobulin genes (e.g., in humans) include kappa (κ), lambda (λ), and heavy chain loci, which together constitute a large number of variable region genes; and constant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε), and alpha (α), which encode IgM, IgD, IgG (IgG1, IgG2, IgG3, and IgG4), IgE, and IgA (IgA1 and IgA2) isotypes, respectively. As used herein, the term "antibody" is meant to include full-length antibodies and antibody fragments, and may refer to natural antibodies from any organism, engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic, or other purposes. However, the antibody fragments or variants referred to herein always include the heavy chain variable region and the light chain variable region as disclosed herein. Therefore, such fragments and variants include known scFv fragments or scFv antibodies. The antibody can be formulated as a pharmaceutical composition or a drug. The terms "antibody" and "immunoglobulin" are used interchangeably herein and refer to polypeptides encoded by immunoglobulin genes. As used herein, "IgG" refers to polypeptides belonging to the antibody class substantially encoded by the recognized immunoglobulin gamma gene. In humans, such antibodies include IgG1, IgG2, IgG3, and IgG4. In mice, such antibodies include IgG1, IgG2a, IgG2b, IgG3.

[0086] An antibody directed against the C-terminal region of angiopoietin-like 4 protein (cANGPTL4) means that the antibody specifically recognizes and binds to cANGPTL4. As used herein, "specifically binds" and "specific binding" refer to the binding of an antibody to its target, i.e., cANGPTL4, based on the recognition of an epitope on the target molecule. An anti-cANGPTL4 antibody is an antibody that binds to cANGPTL4 with sufficient affinity and specificity, and generally does not bind to other homologs of ANGPTL4, e.g., ANGPTL3. The anti-cANGPTL4 antibody recognizes and binds to the target molecule cANGPTL4 with a higher binding affinity than other compounds that may be present. In various embodiments, "specific binding" may mean that the affinity with which the antibody binds to the target molecule cANGPTL4 is at least about 10 times greater than the affinity with which the antibody binds to a molecule unrelated to the target molecule (e.g., albumin). 6 times, preferably at least about 10 7 times, more preferably at least 10 8 times, most preferably at least about 10 9 Typically, specific binding refers to an affinity that is about 10 times that of nonspecific binding. 6 times to about 10 9 In some embodiments, specific binding may be characterized by an affinity that is 10 times that of nonspecific binding. 9 Binding affinity can be determined by any suitable method. Such methods are known in the art and include, but are not limited to, surface plasmon resonance and isothermal titration calorimetry. For example, binding affinity can be determined by competitive ELISA or by using BIACORE TM 、KINEXA TM or PROTEON TM The Kd is measured. In various embodiments, the antibody uniquely recognizes and binds to the target cANGPTL4.

[0087] Since the amino acid sequence of the C-terminal region of angiopoietin-like 4 protein (cANGPTL4) is known (e.g., SEQ ID NO: 2 or SEQ ID NO: 5), it will be easy for those skilled in the art to develop or identify antibodies that specifically bind to cANGPTL4 with sufficient affinity and specificity.

[0088] In various embodiments, the antibody is a monoclonal antibody, more preferably, the anti-cANGPTL4 antibody is an anti-cANGPTL4 monoclonal antibody that specifically binds to the C-terminal region of the Angiopoietin-like 4 protein, more preferably, the anti-cANGPTL4 antibody binds to a region or epitope within the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5.

[0089] In various embodiments, the antibody specifically binds to an epitope or region within the C-terminal region of the Angiopoietin-like 4 protein, wherein the C-terminal region of the Angiopoietin-like 4 protein comprises or consists of an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 5.

[0090] To determine the specific region or epitope on cANGPTL4 to which the antibody binds, epitope mapping may be performed. As used herein, the term "epitope" refers to the general biochemical meaning of a portion of a target protein / antigen that can be recognized and specifically bound by a particular antibody. In certain embodiments, an epitope includes a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments may have specific three-dimensional structural characteristics and / or specific charge characteristics. Antibody epitopes typically include at least 3, more typically at least 5, or 8 to 10 amino acids in a unique spatial configuration. Methods of epitope determination or "epitope mapping" are well known in the art and may be used in conjunction with the provisional disclosure to identify epitopes on cANGPTL4 to which an antibody can bind with sufficient affinity and specificity. In particular, as used herein, "epitope mapping" refers to the identification and definition of an epitope recognized by an antibody. Various methods can be used, including but not limited to synthetic peptides (where the sequence of the protein is known), phage display libraries (see epitope libraries), protein footprinting (using monoclonal antibodies to protect proteins from proteolytic degradation), isolation and characterization of peptides bound to MHC, or expression cloning. Once the epitope on the antigen is determined, antibodies that bind to the same epitope can be competitively screened. A method to achieve this goal is to perform competition studies to find antibodies that compete for binding to the antigen. A high-throughput method for screening antibodies based on antibody cross-competition is described in WO / 2003 / 48731A1.

[0091] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., each antibody contained in the population is identical except for possible naturally occurring mutations that may be present in a small amount. Monoclonal antibodies are highly specific for a single antigenic site. In addition, unlike conventional (polyclonal) antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins. The modifier "monoclonal" refers to that the antibody has the characteristics obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of antibodies by any ad hoc method. Monoclonal antibodies can include "chimeric" antibodies and humanized antibodies.

[0092] Monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuously cultured cell lines. These techniques include, but are not limited to, hybridoma technology, Koehler and Milstein (1975), Nature, 256: 495-7; and U.S. Patent No. 4,376,110, human B cell hybridoma technology (Kosbor et al., (1983), Immunology Today, 4: 72; Cote et al., (1983), Proc. Natl. Acad. Sci. USA, 80: 2026-30), and EBV-hybridoma technology (Cole et al. (1985), in Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., New York, pp. 77-96). The preparation of monoclonal antibodies specific for target compounds is also described in Harlow and Lane, edited (1988) Antibodies-A Laboratory Manual. Cold Spring Harbor Laboratory, Chapter VI. Such antibodies can be any class of immunoglobulins, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridomas producing mAbs can be cultured in vitro or in vivo. The production of high titers of mAbs in vivo makes it a very efficient production method.

[0093] In various embodiments, the anti-cANGPTL4 monoclonal antibody is a neutralizing anti-cANGPTL4 monoclonal antibody, such as mAb 3F4F5 (56, 57). As used herein, the term "neutralizing antibody" or "neutralizing antibody" refers to an antibody that reduces at least one activity of a polypeptide of cANGPTL4 comprising an epitope to which the antibody specifically binds. In various embodiments, the neutralizing antibody reduces the activity in vitro and / or in vivo.

[0094] In various embodiments, the neutralizing monoclonal antibody specifically binds to the same epitope in cANGPTL4 as mAb 3F4F5.

[0095] In various embodiments, the antibody is a humanized antibody, more preferably, the anti-cANGPTL4 antibody is a humanized anti-cANGPTL4 antibody. In various embodiments, the humanized anti-cANGPTL4 antibody is derived from a monoclonal anti-cANGPTL4 antibody.

[0096] For example, International Patent Publication No. WO 2014 / 027959A1 discloses a murine monoclonal antibody (referred to as mAb 11F6C4) against human cANGPTL4, the entire contents of which are incorporated herein. mAb 11F6C4 has been found to target an epitope located within the C-terminus of human ANGPTL4 (cANGPTL4) and does not affect the mitochondrial activity and glucose regulation of the protein. For ease of reference, the amino acid sequences associated with the VH and VL domains of mAb 11F6C4, as disclosed in WO 2014 / 027959A1, are listed in Table 2 below.

[0097] Table 2: VH and VL domains of murine mAb 11F6C4

[0098]

[0099] International patent publication WO2021 / 221565A1 discloses a humanized antibody against the C-terminal region (cANGPTL4) of angiopoietin-like 4 protein, the entire contents of which are incorporated herein. More specifically, it is a humanized form of mAb11F6C4 derived from the mouse monoclonal antibody mAb11 F6C4 described in WO2014 / 027959A1. In this regard, mouse mAb11 F6C4 is humanized because it contains a human framework region (FR) and all 6 CDRs of the mouse antibody mAb11F6C4 described in International Patent Publication WO2014 / 027959A1. For ease of reference, as disclosed in WO2021 / 221565A1, the amino acid sequences associated with the VH domain and VL domain of humanized mAb11F6C4 are listed in Table 3 below.

[0100] Table 3: VH and VL domains of humanized mAb 11F6C4

[0101]

[0102] In various embodiments, the monoclonal antibody is derived from the murine monoclonal antibody mAb11F6C4. In various embodiments, the monoclonal antibody specifically binds to the same epitope in cANGPTL4 as murine mAb11F6C4. In various embodiments, the monoclonal antibody is murine monoclonal antibody mAb11F6C4 or is derived from murine monoclonal antibody mAb11F6C4, comprising: a heavy chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof; and a light chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof.

[0103] The variant remains unchanged relative to the CDR region defined in WO2014 / 027959A1, i.e. any changes occur in the framework region. In various embodiments, the variant has at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.15 and SEQ ID NO.16. The variation is usually an amino acid substitution or deletion that occurs in the framework region rather than the CDR. It is possible, for example, to have a short truncation of the C-terminus or N-terminus of the variable domain, which is usually 1, 2, 3 or 4 amino acids in length, and / or a single amino acid substitution.

[0104] In various embodiments, the monoclonal antibody is or is derived from the humanized monoclonal antibody mAb11F6C4. In various embodiments, the monoclonal antibody specifically binds to the same epitope in cANGPTL4 as the humanized mAb11F6C4.

[0105] In various embodiments, the monoclonal antibody is a humanized antibody against the C-terminal region of angiopoietin-like 4 protein (cANGPTL4), the humanized antibody comprising a heavy chain variable domain and a light chain variable domain having a human framework region. In various embodiments, the humanized antibody comprises:

[0106] (i) a heavy chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, and a light chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof; or

[0107] (ii) a heavy chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof, and a light chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10 or a variant thereof; or

[0108] (iii) a heavy chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof, and a light chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof; or

[0109] (iv) a heavy chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 13 or a variant thereof, and a light chain variable domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof.

[0110] The variant remains unchanged relative to the CDR region defined in WO2021 / 221565A1, i.e. any changes occur in the framework region. In various embodiments, the variant has at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.7 to SEQ ID NO.14. The variation is usually an amino acid substitution or deletion that occurs in the framework region rather than the CDR. It is possible, for example, to have a short truncation of the C-terminus or N-terminus of the variable domain, which is usually 1, 2, 3 or 4 amino acids in length, and / or a single amino acid substitution. In various embodiments, in the variant of the heavy chain variable domain, the positions corresponding to positions 2, 10, 11, 12, 15, 16, 19, 23, 24, 43, 46, 68, 72, 77, 78, 83, 84, 86, 87, 89, 90, 99, 114 and 115 in SEQ ID NO:7 (i.e., using the position numbering of SEQ ID NO:7) are invariant (except for the CDR regions). In various embodiments, in the variant of the light chain variable domain, the positions corresponding to positions 1, 3, 4, 9, 10, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 40, 46, 47, 49, 62, 64, 78, 80, 81, 82, 83, 84, 87, 88, 89, 104, 107 and 108 in SEQ ID NO:8 (i.e., using the position numbering of SEQ ID NO:8) are unchanged (except for the CDR regions).

[0111] As used herein, the term "humanization" associated with antibodies generally refers to antibodies comprising a human framework region (FR) and one or more complementary determining regions (CDRs) from a non-human (usually mouse or rat) antibody. The non-human antibody providing CDR is called a "donor", and the human immunoglobulin providing the framework region is called an "acceptor". Humanization mainly relies on transplanting donor CDRs onto the acceptor (people) VL framework and VH framework (Winter US 5,225,539). This strategy is called "CDR transplantation". It is generally necessary to "reverse mutation" selected acceptor framework residues to the corresponding donor residues to regain the affinity lost in the initial transplant construct (US 5,693,762). Humanized antibodies are also preferably at least a portion of an immunoglobulin constant region (usually a constant region of a human immunoglobulin), and therefore generally will comprise a human Fc region. A variety of techniques and methods for humanizing and reshaping non-human antibodies are well known in the art (see Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited therein). Other methods for humanizing or reducing the immunogenicity of non-human antibody variable regions may include surface modification methods, such as described in Roguska et al. (1994, Proc Natl Acad Sci USA 91 969-973). In various embodiments, a selection-based method can be used to humanize and / or affinity mature the antibody variable region, that is, to increase the affinity of the variable region for its target antigen. Other humanization methods may involve transplanting only the CDR portion, including but not limited to the methods described in Tan et al., 2002, J Immunol 169 1119-1125, De Pascalis et al., 2002, J Immunol 169 3076-3084. Structure-based methods may be used for humanization and affinity maturation, such as described in U.S. Pat. No. 7,117,096 and related applications.

[0112] In various embodiments, the antibodies can have various structures, including but not limited to antibody fragments that recognize and specifically bind to cANGPTL4. Antibody fragments include but are not limited to bispecific antibodies, miniantibodies, domain antibodies, synthetic antibodies, antibody mimetics, chimeric antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each. Specific antibody fragments include, but are not limited to: (i) Fab fragments consisting of VL, VH, CL and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region; (v) isolated CDR regions; (vi) F(ab')2 fragments: bivalent fragments comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv), in which the VH domain and the VL domain are linked by a peptide linker that allows the two domains to associate to form an antigen binding site; (viii) bispecific single-chain Fv dimers; and (ix) "diabodies" or "tribodies", multivalent or multispecific fragments constructed by gene fusion. Antibody fragments can be modified. For example, the molecule can be stabilized by incorporating disulfide bonds linking the VH domain and the VL domain. Examples of antibody formats and architectures are described in Holliger & Hudson, 2006, Nature Biotechnology 23 (9): 1126-1136 and Carter 2006, Nature Reviews Immunology 6: 343-357 and references cited therein. Antibody fragments can be produced by known techniques. For example, such fragments include, but are not limited to, F(ab')2 fragments that can be produced by pepsin digestion of antibody molecules and Fab fragments that can be produced by reducing the disulfide bonds of F(ab')2 fragments. The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is typically truncated at any site in the Fc region to prevent cross-linking of the heavy chain. Optionally, the associated cysteine ​​residue is replaced or deleted by another amino acid residue to prevent cross-linking.

[0113] In various embodiments, the antagonist of ANGPTL4 is an aptamer to the C-terminal region of the angiopoietin-like 4 protein (cANGPTL4), and may be referred to as a cANGPTL4 aptamer.

[0114] As used herein, the term "aptamer" refers to a nucleic acid molecule characterized by the ability to bind to a target molecule (i.e., cANGPTL4) with high specificity and high affinity, and is a non-naturally occurring molecule. Aptamers for cANGPTL4 can be identified and / or generated by the systematic evolution of ligands by exponential enrichment (SELEX™) method. Aptamers targeting angiopoietin-like 4 (ANGPTL4) with high affinity and specificity obtained by SELEX technology have been previously disclosed (57).

[0115] In this regard, aptamers can be DNA or RNA molecules, and can be single-stranded or double-stranded. Aptamers can comprise chemically modified nucleic acids, for example, in which sugars and / or phosphates and / or bases are chemically modified. Such modifications can improve the stability of the aptamer or make the aptamer more resistant to degradation. Aptamers can be synthesized by methods well known to those skilled in the art. For example, aptamers can be synthesized chemically, for example, on a solid support. Solid phase synthesis can use phosphoramidite chemistry. Aptamers can be considered the nucleic acid equivalent of monoclonal antibodies, and typically have a K in the nM or pM range. d , for example, less than one of 500nM, 100nM, 50nM, 10nM, 1nM, 500pM, 100pM. As with monoclonal antibodies, aptamers can be used in almost any situation where binding to a target is desired, including for therapeutic and diagnostic applications in vitro or in vivo. Aptamers can be formulated as pharmaceutical compositions or drugs.

[0116] In various embodiments, the antagonist of ANGPTL4 is an oligonucleotide molecule directed against the C-terminal region of angiopoietin-like 4 protein (cANGPTL4). In particular, the oligonucleotide molecule inhibits or prevents the expression of ANGPTL4, and thus inhibits or prevents the expression of cANGPTL4. Therefore, in this article, the phrase "directed against cANGPTL4" refers to inhibiting or interfering with the expression and production of cANGPTL4 protein by inhibiting or interfering with the expression of nucleic acid encoding ANGPTL4.

[0117] As used herein, the term "oligonucleotide molecule" refers to a nucleic acid molecule, particularly RNA, used to regulate gene expression. These include antisense oligonucleotides, targeted degradation of mRNA by small interfering RNA (siRNA), post-transcriptional gene silencing (PTG), developmental regulatory sequence-specific translation inhibition of mRNA by microRNA (miRNA), and targeted transcriptional gene silencing. Antisense oligonucleotides are oligonucleotides, preferably single-stranded oligonucleotides, that target and bind to a target oligonucleotide (e.g., mRNA) by binding to a complementary sequence. When the target oligonucleotide is mRNA, the binding of the antisense oligonucleotide to the mRNA blocks the translation of the mRNA and the expression of the gene product. Antisense oligonucleotides can be designed to bind to positive genomic nucleic acids and inhibit the transcription of target nucleotide sequences. In view of the known nucleic acid sequences of ANGPTL4 and cANGPTL4 (e.g., known mRNA sequences that can be used for human and mouse ANGPTL4), oligonucleotides can be designed to inhibit or silence the expression of ANGPTL4 (more specifically, cANGPTL4). Such oligonucleotides may be of any length, but may preferably be shorter, e.g., less than 100 nucleotides, e.g., 10 to 40 nucleotides, or 20 to 50 nucleotides, and may comprise a nucleotide sequence that is fully complementary or nearly complementary (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary) to a nucleotide sequence of a corresponding length in a target oligonucleotide (e.g., ANGPTL4 mRNA). Inhibition of ANGPTL4 expression will preferably result in a decrease in the amount of ANGPTL4, thereby resulting in a decrease in the amount of cANGPTL4 expressed by the cell. For example, in a given cell, inhibition of ANGPTL4 by administration of a suitable oligonucleotide molecule will result in a decrease in the amount of ANGPTL4 expressed by the cell relative to untreated cells. Inhibition may be partial. Preferably, the degree of inhibition is at least 50%, more preferably at least one of 60%, 70%, 80%, 85% or 90%. Inhibition levels between 90% and 100% are considered expression "silencing" or functional "silencing". The role of RNAi machinery and small RNAs in epigenetic gene silencing targeting heterochromatin complexes and specific chromosomal loci has been demonstrated. Double-stranded RNA (dsRNA)-dependent post-transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific homologous genes to silence them in a short period of time. RNAi acts as a signal to promote the degradation of mRNAs with sequence identity. 20-nt siRNAs are generally long enough to induce gene-specific silencing, but short enough to escape host responses. The reduction in expression of the target gene product can be extensive, with a few siRNA molecules inducing 90% silencing.

[0118] In various embodiments, the oligonucleotide molecule is selected from RNA sequences of "short or small interfering RNA" (siRNA) or "micro RNA" (miRNA), depending on its source. Both types of sequences can be used to downregulate gene expression by binding to complementary RNA and triggering mRNA elimination (RNAi) or preventing the translation of mRNA into protein. siRNA is derived from the processing of long double-stranded RNA and is generally of exogenous origin when found in nature. Microinterfering RNA (miRNA) is an endogenously encoded small non-coding RNA obtained by processing short hairpins. Both siRNA and miRNA can inhibit the translation of mRNA carrying a partially complementary target sequence without cleaving the RNA, and degrade mRNA carrying a completely complementary sequence. The oligonucleotide molecules can be formulated as pharmaceutical compositions or drugs.

[0119] For example, International Patent Publication No. WO 2011046515A1 discloses siRNAs against ANGPTL4 protein capable of neutralizing or knocking down the protein to interrupt or reduce cell proliferation and methods for preparing siRNAs. International Patent Publication No. WO 2011046515A1 discloses four groups of siRNAs against human ANGPTL4, the entire contents of which are incorporated herein, as defined in Table 4 below, for ease of reference.

[0120] Table 4: Examples of siRNA targeting ANGPTL4

[0121]

[0122] * -The "u" in the above RNA sequence is replaced by "t" in the sequence listing, but both refer to uracil.

[0123] Thus, the oligonucleotide molecule downregulates the expression of ANGPTL4, and thus downregulates the expression of cANGPTL4. The siRNA ligand is typically double-stranded, and in order to optimize the effectiveness of RNA-mediated downregulation of target gene function, the length of the siRNA molecule is preferably selected to ensure that the RISC complex that mediates siRNA recognition of the mRNA target correctly recognizes the siRNA, and that the siRNA is short enough to reduce the host response. The miRNA ligand is typically single-stranded and has a partially complementary region that enables the ligand to form a hairpin structure. miRNA is an RNA gene that is transcribed from DNA but not translated into protein. The DNA sequence encoding the miRNA gene is longer than the miRNA. The DNA sequence includes the miRNA sequence and an approximate reverse complementary sequence. When the DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence forms a partially double-stranded RNA fragment with its reverse complementary base pair. Typically, the RNA ligand used to mimic the effect of siRNA or miRNA has 10 to 40 ribonucleotides (or synthetic analogs thereof), more preferably 17 to 30 ribonucleotides, more preferably 19 to 25 ribonucleotides, and most preferably 21 to 23 ribonucleotides. In some embodiments of the present invention using double-stranded siRNA, the molecule can have a symmetrical 3' overhang, such as one or two (ribo) nucleotides, typically UU with 3' overhang dTdT. Based on common knowledge in the art, those skilled in the art can easily design suitable siRNA and miRNA sequences, such as using resources such as Ambion siRNA finder. siRNA sequences and miRNA sequences can be synthetically produced and exogenously added to cause gene downregulation or produced using an expression system (e.g., vector). In various embodiments, siRNA is synthesized in an artificial synthetic manner.

[0124] It should be understood that ANGPTL4 antagonists are oligonucleotide molecules that can be introduced into a subject through gene therapy. Gene therapy refers to a therapy performed by administering an oligonucleotide molecule to a subject. In gene therapy applications, genes are introduced into cells to achieve in vivo synthesis of gene products with therapeutic effects, such as for replacing defective genes. "Gene therapy" includes traditional gene therapy that achieves lasting effects through a single treatment, as well as the administration of gene therapy agents involving one-time or repeated administration of therapeutically effective DNA or mRNA. Antisense RNA and DNA can be used as therapeutic agents that block the expression of certain genes in the body (e.g., ANGPTL4-siRNA). Specifically, short antisense oligonucleotides can be introduced into cells and act as inhibitors in cells. There are a variety of techniques that can be used to introduce nucleic acids into living cells. The techniques will vary depending on whether the nucleic acid is transfected into in vitro cultured cells or into in vivo cells of the target subject. Techniques suitable for transfecting nucleic acids into cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. Currently preferred in vivo gene transfection techniques include transfection with viral (usually retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210 (1993)). For example, in vivo nucleic acid transfection techniques include transfection with viral vectors (such as adenovirus, herpes simplex virus type I, lentivirus, retrovirus or adeno-associated virus) and lipid-based systems.

[0125] Thus, in various embodiments, the ANGPTL4 antagonist is directed against a nucleotide sequence encoding ANGPTL4, including the C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4).

[0126] In various embodiments, the ANGPTL4 antagonist is an siRNA molecule. In various embodiments, the siRNA molecule is an ANGPTL4-siRNA molecule, wherein the siRNA molecule targets an mRNA sequence encoding ANGPTL4. Since the mRNA sequence encoding ANGPTL4 is known, those skilled in the art can easily identify and screen for siRNA molecules that specifically target and interfere with ANGPTL4 expression with sufficient specificity.

[0127] In various embodiments, the ANGPTL4 antagonist is an ANGPTL4-siRNA molecule that targets the mRNA sequence encoding ANGPTL4 and its C-terminal fibrinogen-like domain.

[0128] In various embodiments, the ANGPTL4 antagonist is an ANGPTL4-siRNA molecule comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 17, or SEQ ID NO. 18, or SEQ ID NO. 19, or SEQ ID NO. 20.

[0129] As used herein, the term "treat, improve, delay or prevent" refers to achieving one or more of the following in a subject: (a) reducing the severity of a particular condition; (b) limiting or preventing the development of a condition; (c) eliminating a particular condition; (d) limiting or preventing the recurrence of a particular condition; (e) alleviating a condition and / or its symptoms; and (f) delaying the onset of a condition. Any one or more of these effects may be achieved in a subject who has previously suffered from, is currently suffering from, or is suspected of developing liver fibrosis or liver fibrosis-related liver disease. Specifically, the therapeutic term "treat, improve or prevent" may refer to reducing the likelihood of a particular condition or disease state (e.g., non-alcoholic fatty hepatitis) occurring in a subject who is not currently experiencing the condition or disease state or is not suffering from the condition or disease state. These terms do not necessarily mean complete or absolute prevention or treatment. For example, "preventing NASH" refers to reducing the likelihood of NASH occurring in a subject who is not currently experiencing or diagnosed with NASH. For example, preventing NASH may reduce the likelihood of NASH occurring in a subject who is currently diagnosed with mild NAFLD but is not currently diagnosed with NASH. These terms may also refer to delaying the onset of a particular condition or disease state (e.g., NASH) in a subject not currently experiencing or afflicted with that condition or disease. To "treat, ameliorate, or prevent" a disease or condition, a method need only reduce the likelihood of onset of the disease or condition and / or delay its onset, rather than completely blocking any likelihood of its onset.

[0130] Administration of the ANGPTL4 antagonist can be performed in a variety of ways, including but not limited to oral, subcutaneous, intravenous, intranasal, intraaural, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, parenteral, rectal or intraocular administration. The ANGPTL4 antagonist can be formulated accordingly depending on the mode of administration. In various embodiments, the ANGPTL4 antagonist is formulated as a composition or contained in a composition, more specifically a pharmaceutical composition or medicament for administration to a subject. It should be understood that the mode of administration will vary depending on the ANGPTL4 antagonist used and its dosage form as a medicament.

[0131] Protein therapeutics are typically delivered by intravenous infusion or bolus injection. In various embodiments, an ANGPTL4 antagonist or a composition comprising an ANGPTL4 antagonist may also be delivered using such methods. For example, administration may be by intravenous infusion using 0.9% sodium chloride as an infusion vehicle or carrier.

[0132] Medicaments or compositions comprising ANGPTL4 antagonists can be prepared for storage by mixing the ANGPTL4 antagonist with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed., 1980), for example, in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are pharmaceutically acceptable, i.e., non-toxic to the receptor, at the amounts and concentrations employed, and include: buffers, such as phosphates, citrates, acetates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives, such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin Protein, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; sweeteners and other flavoring agents; fillers such as microcrystalline cellulose, lactose, corn and other starches; binders; additives; colorants; salt-forming counterions such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TM , or polyethylene glycol (PEG).

[0133] In various embodiments, the ANGPTL4 antagonist or a composition / medicament comprising the same may be administered simultaneously with one or more other therapeutic regimens or agents. The additional therapeutic regimen or agent may be used to improve the efficacy or safety of the ANGPTL4 antagonist. In addition, the additional therapeutic regimen or agent may be used to treat the same disease or comorbidity, rather than to alter the effect of the ANGPTL4 antagonist. The ANGPTL4 antagonist of the present invention may be administered in combination with one or more other prophylactic or therapeutic agents, including but not limited to anti-obesity drugs and anti-diabetic drugs known in the art. The terms "in combination with..." and "co-administered" are not limited to administering the ANGPTL4 antagonist or therapeutic agent at exactly the same time. Rather, it means that the ANGPTL4 antagonist and the other one or more agents are administered sequentially within a time interval, so that they can act together to provide an increased benefit compared to treatment with only the ANGPTL4 antagonist or the other one or more agents. A skilled medical practitioner may determine one or more appropriate doses of each therapeutic agent, as well as the appropriate time of administration and method of administration, empirically or by considering the pharmacokinetics and mode of action of the agent.

[0134] In various embodiments, the pharmaceutical composition or medicament comprising the ANGPTL4 antagonist may be in a water-soluble form, for example comprising certain ingredients in the form of pharmaceutically acceptable salts (e.g., acid addition salts and / or base addition salts). Compositions or medicaments for in vivo administration are generally sterile. This is readily achieved by known methods.

[0135] In various embodiments, when the ANGPTL4 antagonist is an antibody, the antibody can be formulated as immunoliposomes or microcapsules. Techniques for preparing such formulations are disclosed in Remington's Pharmaceutical Sciences (16th edition, Osol, A. ed., 1980).

[0136] Therefore, the invention also provides use of angiopoietin-like 4 protein (ANGPTL4) antagonists in the manufacture of a medicament for treating, improving, delaying or preventing liver fibrosis or a disease associated with liver fibrosis in a subject.

[0137] In addition, provided is a use of angiopoietin-like 4 protein (ANGPTL4) antagonist in the manufacture of a medicament for treating, preventing, delaying or ameliorating liver inflammation in a subject suffering from liver fibrosis or a disease or condition associated with liver fibrosis.

[0138] As used herein, the term "effective amount" refers to an amount of an ANGPTL4 antagonist that, when administered to a subject to treat a condition or disease, will produce the desired therapeutic effect (e.g., "treat, ameliorate, or prevent"). It is understood that the "effective amount" will vary depending on the ANGPTL4 antagonist used, the severity of the condition, disease, and / or its symptoms, and the age, weight, etc. of the subject being treated. A sufficient therapeutic effect is not necessarily produced by administering one dose (or dosage), but may only be produced after a series of doses are administered. Therefore, an effective amount may be administered in one or more administrations. The dosage and administration of the ANGPTL4 antagonist can be determined by a person of ordinary skill in the art of clinical pharmacology or pharmacokinetics. The effective amount of an ANGPTL4 antagonist (e.g., an antibody described herein) for therapeutic use will depend, for example, on the therapeutic goal, the route of administration, and the condition of the subject. Therefore, it is necessary for the therapist to adjust the dosage and modify the route of administration as needed to obtain the best therapeutic effect. In one embodiment, the dosage and frequency of administration are selected to be effective in treatment or prevention. As is known in the art, adjustments may need to be made depending on protein degradation, systemic versus local delivery, and rate of synthesis of new proteases, as well as age, weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition, and will be determined by one skilled in the art through routine experimentation.

[0139] As used herein, the term "subject" in the context of therapeutic methods refers to warm-blooded animals, preferably mammals, more preferably humans. The terms "subject", "individual" and "patient" are used interchangeably herein and refer to humans or non-human animals. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. In various embodiments, the object is non-human. In various embodiments, the object is human. The object may be waiting for or receiving medical care, or will be the object of a medical procedure, or is in the process of monitoring the development of liver fibrosis or liver inflammation or liver fibrosis-related diseases. Objects include those who have suffered from or are suffering from liver fibrosis or liver inflammation or objects suffering from liver fibrosis and / or liver inflammation-related diseases, as well as objects susceptible to liver fibrosis or liver inflammation or objects related to liver fibrosis, or objects for whom liver fibrosis or liver inflammation or diseases related to liver fibrosis should be prevented or delayed. In various embodiments, the subject is a human having or at risk of having liver fibrosis or liver inflammation or a disease associated with liver fibrosis, more preferably NAFLD, NASH or a related disease phenotype.

[0140] However, in another embodiment, in addition to administering an ANGPTL4 antagonist in vivo to a subject (e.g., a patient or individual) in need of treatment, it is also contemplated that the ANGPTL4 antagonist can be used in an in vitro method of treating a liver sample having fibrosis and / or inflammation and / or a liver sample obtained from a subject having a disease associated with liver fibrosis.

[0141] All embodiments disclosed above in relation to methods of treatment and ANGPTL4 antagonists apply analogously to the in vitro methods described below.

[0142] Thus, there is provided an in vitro method of reducing fibrosis in a liver sample, the method comprising the step of contacting the liver sample with an angiopoietin-like 4 protein (ANGPTL4) antagonist.

[0143] In another embodiment, an in vitro method of reducing liver inflammation in a liver sample is provided, the method comprising the step of contacting the liver sample with an angiopoietin-like 4 protein (ANGPTL4) antagonist.

[0144] In another embodiment, an in vitro method for modulating an immune response in a liver sample is provided, the method comprising contacting the liver sample with an angiopoietin-like 4 protein (ANGPTL4) antagonist, wherein the contact induces a decrease in the number, activity and / or effector function of a regulatory T cell population (preferably CD8+ T cells and / or CD4+ T cells) in the liver sample.

[0145] In various embodiments of the in vitro methods, the ANGPTL4 antagonist is directed against the C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4).

[0146] The terms "sample" or "biological sample" as used interchangeably herein include any suitable sample separated from a subject. Suitable samples include, but are not limited to, samples containing tissues, cells, and / or biological fluids separated from a subject. Examples of samples include, but are not limited to, tissues, cells, biopsies, blood, lymph, serum, plasma, urine, saliva, mucus, and tears. In one embodiment, the sample includes a serum sample, a blood sample, or a plasma sample. The sample can be obtained directly from a subject or a control (e.g., by blood sampling or tissue sampling), or from a third party (e.g., from an intermediary such as a healthcare provider or a laboratory technician). The sample can be obtained from a subject suffering from liver fibrosis, liver inflammation, or a disease or condition associated with liver fibrosis.

[0147] The "contacting" step in the in vitro method generally refers to any suitable method of delivering, exposing or bringing together the sample and antagonist under appropriate conditions in a controlled experimental environment. It will be appreciated by those skilled in the art that the "contacting" step may vary depending on the nature of the sample and antagonist involved, wherein contacting may involve physically mixing the sample with the antagonist in a test tube, culture dish or other container, or contacting may include applying the antagonist to the sample by techniques such as pipetting, incubation or surface fixation, or contacting may include introducing the antagonist into the sample (i.e., cells) by, for example, transfection. The purpose of the "contacting" step is to allow the antagonist to interact with the sample and exert its inhibitory effect on the target molecule (ANGPTL4). In various embodiments, contacting occurs in solution, wherein the sample and ANGPTL4 antagonist are mixed in a common solution and allowed to freely bind, or contacting may occur intracellularly or in a cell-free environment.

[0148] In various embodiments, the liver sample comprises hepatocytes. In various embodiments, the cells are derived from a hepatocyte cell line or obtained from a subject suffering from liver fibrosis, liver inflammation, or a disease associated with liver fibrosis. In this regard, the cells or cell lines can also be maintained or grown in a cell culture dish under suitable conditions and can be contacted with the ANGPTL4 antagonist (i.e., antibody) in such an environment.

[0149] In various embodiments, the contacting step can include culturing the cell or cell line in cell culture in the presence of the ANGPTL4 antagonist, or transfecting the cell or cell line with the ANGPTL4 antagonist.

[0150] In various embodiments, the liver sample is liver tissue. In various embodiments, the liver tissue is obtained from a subject having liver fibrosis, liver inflammation, or a disease or condition associated with liver fibrosis.

[0151] The present invention is further illustrated by the following examples. However, it should be understood that the present invention is not limited to the illustrated embodiments.

[0152] Example

[0153] Materials and methods

[0154] Animals, feeding regimens and treatments. Male wild-type C57BL / 6J mice (Invivos, Singapore), LysMCre-Angptl4- / - mice and NOD scidγ mice (NSG, Jackson Laboratory) between 8 and 9 weeks of age were given LIDPAD (liver disease progression diet, modified by Teklad diet TD.88137, Envigo, USA) or control diet (Teklad custom diet, modified by AIN-93M, Envigo, USA) (Table 5, below) and water ad libitum. LysMCre-Angptl4- / - mice have site-specific knockout of Angptl4 expression, mainly in myeloid immune cells. After 4 weeks of induction of NAFLD with LIDPAD, a neutralizing monoclonal antibody (clone 3F4F5) against mouse cAngptl4 was administered intraperitoneally at 10 mg / kg twice a week. Animals were housed in standard cages for up to 48 weeks, with temperature and humidity controlled at 30°C (thermoneutral zone) and 49.9%, respectively, with a 12-h dark-light cycle. Body weight and food consumption were measured weekly. Animals were euthanized using CO2 before blood and tissue collection. Organs were fixed with 4% PFA or snap-frozen with liquid nitrogen. All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) (SingHealth IACUC: #2014 / SHS / 1008; NTU-IACUC: A18031, A10032, A18033, A18042, A20055).

[0155] Table 5: Dietary nutritional information

[0156]

[0157] Patient samples. Ten patients with NAFLD and five patients with HCC scheduled for liver surgery at the Fifth Affiliated Hospital of Sun Yat-sen University were recruited to provide residual liver tissue for surgical resection. Patients had no previous or current liver infection or other active infectious diseases. NAFLD liver tissue samples were obtained from the NAFLD area of ​​the liver of the 10 NAFLD patients. Normal liver tissue samples were obtained from normal tissue surrounding the liver tumor of the five HCC patients. The obtained samples were then cut into two halves and preserved in formaldehyde for histological analysis or stored in a −80°C freezer before RNA extraction. This study was approved by the Institutional Ethics Review Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (Zhuhai, China) (Approval No. L136-1), and written consent was obtained from all participants.

[0158] Liver histopathology. Liver samples were fixed with 4% paraformaldehyde and embedded in paraffin. Tissue sections were cut into 5 μm thick sections, dewaxed and rehydrated, and then stained with H&E, PSR and MT by Leica Autostainer XL (Leica, Germany). Frozen tissue samples were prepared for ORO staining and stained according to an isopropanol-based protocol. Images were captured using Axioscan Z1 (Zeiss, Germany) at a bright field setting of 20 times magnification. Blinded histological evaluation was performed using the fatty degeneration, activity and fibrosis (SAF) scoring system as described below.

[0159] RNA sequencing was performed using TRIzol reagent (Life Technologies, US) and Total RNA was extracted from frozen liver samples and patient samples obtained from all mouse groups using the Total RNA Kit (Omega Biotek, US). RNA samples were sequenced using the Illumina HiSeq platform to generate 50-bp paired-end reads. Raw reads were aligned to the Mus musculus genome assembly GRCm38 or human GRCh38 from Ensembl (21) using HISAT2 (22). Uniquely aligned reads were analyzed using FeatureCounts (23) to generate a gene count matrix, which was then subjected to differential expression analysis using DESeq2 (24). Genes that met the criteria of log2 fold difference > ±1 and adjusted p value < 0.05 were considered differentially expressed genes (DEGs). DEGs were stratified by expression and analyzed using Gene Ontology and Ingenuity Pathway Analysis (QIAGEN) to identify top enriched pathways. Raw sequences have been deposited in GEO with accession numbers GSE159911 and GSE214504.

[0160] Single-cell isolation of intrahepatic immune cells. The isolation of intrahepatic immune cells was performed as previously described, but with some modifications (25). Briefly, mice were anesthetized with 100 mg / kg ketamine and 10 mg / kg xylazine, injected with 3 μg of anti-mouse CD45-FITC antibody (Miltenyi Biotec, Germany) retrobulbarly and left for 3 minutes, and then euthanized by cardiac puncture. The liver was harvested and dissociated into a single cell suspension on a GentleMACS Octo dissociator (Miltenyi Biotec) using a liver dissociation kit. The cell suspension was filtered through a 70 μm filter. Lymphocytes in the supernatant were enriched by Percoll density gradient centrifugation (1.07 g / mL) and treated with RBC lysis buffer to deplete red blood cells. The lymphocyte fraction was stained with anti-CD45-APC (pan-hematopoietic cell marker) and propidium iodide (live / dead dye) before sorting on a FACSAria Fusion cell sorter (BD Biosciences, USA). Live intrahepatic immune cells (CD45-FITCCD 45-APC+PI-) were collected, washed twice, and counted before single-cell library preparation. Single-cell RNA sequencing and annotation of scRNAseq immune cell subsets were described below.

[0161] FACS. Intrahepatic immune cells were isolated as described above. Single cells were obtained by passing through a 70 μM filter, blocked with 3% BSA containing FcR blocker, and stained with antibodies (Table 6). Fig.13 The gating strategy was used to analyze the ratio of naive (CD45+Lin-CD3+CD4+CD8-CD62L+CD69-) CD4+ T cells and activated (CD45+Lin-CD3+CD4+CD8-CD69+) CD4+ T cells, and naive (CD45+Lin-CD3+CD4-CD8+CD62L+CD69-) CD8+ T cells and activated (CD45+Lin-CD3+CD4-CD8+CD69+) CD8+ T cells.

[0162] Table 6: Antibodies used for FACS

[0163]

[0164] Kinase inhibitor screening. Using CD4+T cell magnetic separation columns (Miltenyi Biotech), spleen CD4+T cells were isolated from lymphocytes of wild-type mice and Angptl4- / - mice (the same method as above). The separated CD4+T cells were cultured in AIM-V medium (Thermo Fisher Scientific, USA) supplemented with 5% wild-type mouse serum and Angplt4- / - mouse serum. CD4+T cells were treated overnight with 28 kinase inhibitors (SYN-2103; SYN kinase, Victoria, Australia & Target Mol, Massachusetts, USA) at specified concentrations (Table 7), and then treated with 1 μg / mL LPS for 2 hours. By qPCR analysis, the log2 (difference fold) relative expression of target genes of eIF2α and eIF2α-mediated downstream genes (i.e., DDIT3, HSPA5, and ATF4) under each kinase inhibitor treatment was compared with the wild-type case treated with LPS.

[0165] Table 7: List of kinase inhibitors used in drug screening, molecular targets and concentrations used.

[0166]

[0167]

[0168] Adoptive transfer. Splenic lymphocytes were harvested from Angptl4 knockout mice as described above. Knockdown of eIF2α (or Eif2s1) was performed using 10 nM Accell mouse Eif2s1 SMARTpool siRNA (Horizon Discovery, UK) based on published protocols (28). Control cells were transfected with nonspecific small interfering RNA. After knockdown, lymphocytes were labeled with 1 μM CellTrackerTMGreen CMFDA dye (Thermo Fisher Scientific, USA) for 45 minutes. A total of 1x10 7 The cells were injected intravenously into mice fed with LIDPAD diet for 12 weeks to generate NASH environment. Intrahepatic immune cells were isolated by FACS to examine donor CD4+ T cells (labeled with Cell Tracker Green; FITC 高 ) and recipient CD4+ T cells (unlabeled; FITC 低 ) is the ratio of immature cells to activated cells.

[0169] In vivo MRI. Prior to in vivo imaging, animals were first anesthetized using 2.5% to 3% isoflurane in combination with medical oxygen and medical air in a dedicated mouse chamber. During imaging, a reduction in isoflurane concentration to 1% to 2% was induced to maintain a respiratory rate of 80 cycles / min to 90 cycles / min. The respiratory rate and body temperature of the animals were monitored by a physiological monitoring system. Respiratory-linked gating with a 50 ms trigger delay was used during imaging. In vivo MRI was performed using a 40 mm transmit / receive body coil (Bruker 9.4T Biospec). High-resolution anatomical T1-weighted images were acquired by a fast low-angle acquisition (FLASH) sequence with a repetition time (TR) of 337 ms, an echo time (TE) of 2.5 ms, a flip angle (FA) of 30°, an acquisition repetition number (AV) of 3 times, an image matrix size of 256x256, and a field of view (FOV) of 40x40 mm. High-resolution anatomical T2-weighted images were acquired using the rapid imaging with refocused echo (RARE) sequence with a TR of 5175 ms, TE of 30 ms, FA of 90°, AV of 2, image matrix size of 256 × 256, and FOV of 40 × 40 mm. Proton density fat fraction imaging of the liver was performed in LIDPAD mice (L) and control mice (C) at weeks 4 (n: L = 3, C = 3), 8 (n: L = 5, C = 4), and 16 (n, L = 4, C = 6) using a multi-echo Dixon (m-Dixon) pulse sequence with the following parameters: TR of 12 ms, TE of 1.85 ms, 2.08 ms, 2.32 ms, 2.56 ms, 2.80 ms, 3.04 ms, 3.27 ms, 3.51 ms, FA of 5°, slices of 30, slice thickness of 1 mm, image matrix size of 256x256, and FOV of 40x40 mm. The m-Dixon imaging data were processed and liver PDFF images were generated using the Fat-Water Toolbox. The same region of interest (ROI) was drawn in the liver of all animals by matching the anatomical location. Liver PDFF values ​​were expressed as mean percentages and statistical analysis was performed using SPSS software V23 (Mann-Whitney test).

[0170] Histological scoring of liver sections. The fatty degeneration score (S) ranges from 0 to 3 (S0: <5%; S1: 5%-33%, mild; S2: 34%-66%, moderate; S3>67%, marked). The activity grade (A, 0 to 4) is a combination of hepatocyte ballooning (0 to 2) and lobular inflammation (0 to 2). Ballooning is scored as 0 (normal polygonal hepatocytes), 1 (round, non-swollen hepatocytes, reticular cytoplasm), and 2 (2-fold swollen hepatocytes with clear cytoplasm and aggregated intermediate fibers). Lobular inflammation is scored as 0 (none), 1 (≤2 lesions per 20x field of view), and 2 (>2 lesions per 20x field of view). Fibrosis was scored as follows: stage 0 (F0): absent; stage 1 (F1): 1a or 1b, respectively, fine or dense 3-zone perisinusoidal fibrosis, 1c only periportal fibrosis; stage 2 (F2): 3-zone perisinusoidal fibrosis and periportal fibrosis; stage 3 (F3): bridging fibrosis; and stage 4 (F4): cirrhosis. The presence or absence of portal inflammation was detected.

[0171] Histological preparation of kidney samples. Kidneys were fixed with buffered formaldehyde solution, processed and embedded in paraffin. Four micron thick tissue sections were stained with H&E, Periodic Acid-Schiff (PAS) staining kit (ab150680) and MT stain according to the manufacturer's protocol. All slide images were captured using Carl Zeiss Axio slide scanner Z1.

[0172] Pancreas harvest, H&E staining, and islet size determination. Harvested pancreatic specimens were fixed in 4% paraformaldehyde (PFA) for 16 h and then cryoprotected in 30% sucrose for an additional 24 h. The specimens were embedded in optimal cutting temperature compound (OCT, Sakura, Japan), sectioned and stained with H&E. The stained slides were imaged using a Carl Zeiss Axio slide scanner Z1 at 20x magnification. Magnified tif images of islets were recorded and annotated together with a digital ruler. Islets were marked and their size analyzed using ImageJ software. The scale of each image was adjusted, and the ROI of each islet was manually marked for area calculation. Islet area was then collated and evaluated on GraphPad Prism. Data are presented as mean ± SEM (≥5 islets / animal).

[0173] Insulin tolerance test. After fasting for 4 hours, mice were injected intraperitoneally (ip) with 1U / kg body weight insulin (Lilly, USA). Blood glucose concentration was measured 120min after injection from the tail vein using a blood glucose meter (Accucheck Performa, Roche, USA). All measurements were performed from 10 am to 12 noon the next day.

[0174] Intraperitoneal glucose tolerance test. After 16 hours of overnight fasting, mice were injected intraperitoneally with 2.0 g / kg body weight glucose. Blood glucose concentration was measured 120 min after injection from the tail vein using a glucometer (Accucheck Performa, Roche, USA). All measurements were performed from 10 am to 12 noon the next day.

[0175] Serum cytokine array. Blood samples collected from mice via the heart were placed at room temperature to coagulate for 30 minutes and then transferred to ice. The coagulated samples were spun in a centrifuge at 12,000×g for 1 min to separate the serum. The supernatant was carefully recovered and stored. Subsequently, the mouse cytokine 32 rediscovery assay was performed by Eve Technologies (Calgary, Canada).

[0176] Calorimetric chamber. Mice were pre-acclimated for 5 days in the test room at 30°C, including 3 days in the acclimation cage. The climate chamber was maintained at a constant temperature of 30°C and 50% humidity. Lights were turned on at 7 am, the illumination in the room was 80 lux, and lights were turned off at 7 pm. Animals had ad libitum access to food (corresponding LIDPAD or control) and filtered water. Oxygen and carbon dioxide sensors were calibrated with a calibration gas mixture (calibration report: good air equivalent calibration gas: CO2 in N2: 0.050 ± 0.0005%, O2: 20.900 ± 0.1%; CO2 span: CO2 in N2: 1.000 ± 0.001%, O2: 20.000 ± 0.1%). The sample air flow was adjusted to 0.37 L / min. A high-precision weighing station was combined with a leak-proof and spill-proof container to record body weight, food intake, and water intake. Spontaneous activity was recorded using a two-layer infrared beam frame around each cage. Recording began when the animals first entered the chamber, but measures were considered from the second day of entry. From the fifth day onwards, a running wheel was added to the cage. Adaptation to the running wheel was maintained for 2.5 days before data were considered. Measurements were taken every 15 minutes and are presented as hourly or hourly averages.

[0177] Lipid analysis was performed using liquid chromatography-mass spectrometry. Lipids were extracted from mouse liver tissue using liquid-liquid extraction as described with slight modifications (26). Briefly, 10 mg to 20 mg of tissue was mechanically homogenized in a mixture of methanol and MTBE using a bead mill (Precellys). The lysate was further sonicated in an ultrasonic bath with ice for 10 min. Phase separation was induced by the addition of water and then incubated at room temperature for 10 min. The samples were then centrifuged at 1,000 × g for 10 min. The upper organic phase was collected and the aqueous phase was re-extracted. The pooled organic phases were vacuum dried using a Speedvac and the lipid extracts were stored at −80°C before further analysis. An internal standard containing d7-cholesterol (Avanti Lipids) was spiked into the samples and sterols were analyzed using atmospheric pressure chemical ionization (APCI) triple quadrupole mass spectrometry (SCIEX Qtrap 6500+) with pre-liquid chromatography (Agilent Technologies). Separation of sterols was achieved using a Poroshell 120SB-C18 column (3.0 x 50 mm, 2.7 μM, Agilent Technologies), a modification of a previous method (27). For mass spectrometry analysis, the instrument was operated in multiple reaction mode (MRM). For quantification, the area under the curve was obtained for each sterol measured and normalized to the internal standard and tissue weight. External calibration was performed for cholesterol and squalene to adjust the response factor of squalene relative to cholesterol.

[0178] Immunofluorescence staining. Liver sections were dewaxed, rehydrated, and heat antigen retrieval was performed in sodium citrate buffer (10 mM, 0.05% Tween-20, pH 6) using Aptum Biologics 2100 Antigen Retriever (Aptum Biologics, UK). Sections were blocked with 5% (v / v) fetal bovine serum for 1 hour and labeled overnight at 4°C with anti-mouse CD45 or anti-mouse α-smooth muscle actin (α-SMA) antibodies (Cell Signaling Technology, USA). Sections were then treated with AlexaFluor 680-conjugated secondary antibodies and counterstained with Hoechst 33342. Microscopic images of sections were captured with Zeiss AxioScan Z1.

[0179] Single-cell RNA sequencing (scRNA-seq). Single-cell droplet libraries and cDNA libraries were prepared using the Chromium Single Cell 3'V3 kit (10XGenomics, USA) according to the manufacturer's instructions. The cDNA library was sequenced on NovaSeq6000 (Illumina, USA). The raw sequencing reads were aligned to the mm10 mouse reference genome using Cell Ranger (v3.1.0) to generate a single-cell count matrix, which was normalized, integrated and annotated using Seurat (v3.0) (28). The obtained count matrix was merged and normalized using SCTransform. Subsequently, cells with less than 250 genes, mitochondrial genes greater than 20%, and genes with less than 500 UMI counts were screened out. The mean housekeeping gene count was used as a quality control standard. The data set was not corrected for cell cycle differences because visual inspection of the first two principal components of cell cycle genes did not show cell cycle bias. Overall, a total of 32,551 cells were obtained under 8 conditions. Subsequently, the datasets were integrated using the Seurat v4 analysis pipeline. The kBET metric revealed that batch effects were effectively eliminated. Principal component analysis (PCA) was performed on highly variable genes, and the first 35 PCA components were used to construct UMAP. The Louvain algorithm was used for unsupervised cell clustering. Differentially expressed gene (DEG) analysis of each marker relative to the remaining markers was performed using FindAllMarkers to detect clustered markers. EnrichR (29) was used to functionally annotate the differentially expressed gene set. Trajectory analysis and pseudo-temporal DEG analysis were performed using Slingshot (30) and Trade-seq (31). Single-cell sequencing data were deposited in GEO (accession number: GSE214172).

[0180] Annotation of scRNAseq immune cell subsets. Cell type annotation was automated using the SingleR package (32) against the ImmGen (33) database due to its excellent performance in a recent benchmark evaluation (34). Subsequently, the differentially expressed genes at the top of the rows for each cluster were manually curated for cell type and compared to the automated annotation to ensure the fidelity of the annotation (35). T lymphocytes were identified using expression of the canonical Cd3 marker, where T lymphocytes were separated into α-β T cell receptor (TCR) T lymphocytes and gd-T cells (expressing γ-δ TCR) using the Trac marker or the Trdc marker, respectively. In addition, conventional CD4+ T cells and CD8+ T cells were identified using the Cd4, Cd8a, and Cd8b1 markers. Naive T cells within these populations were identified using L-selectin (Sell). Cells that did not clearly express CD4 and CD8 markers were identified as double-negative T cells (dnT). T-intrinsic-like cell types and NKT-cell types are enriched for innate-like markers, such as Nkg7 and the Klr gene family. Mouse hepatic NKT cells specifically express the Cxcr6 marker, indicating that they can recognize Cd1d+ antigen-presenting cells (36).

[0181] B lymphocytes were identified using enrichment for the canonical Ms4a1, Cd19, and Cd79a markers. B lymphocytes also express H2-Aa, a major histocompatibility class 2 (MHC-II) molecule (37) that is functionally involved in antigen presentation to CD4+ T cells during activation. Antibody-producing plasma cells were identified using the Igha marker. Intrinsic lymphoid cells (ILCs) are differentiated from NK cells that express high levels of Xcl1 and Cxcr6 and low levels of Nkg7 (38). Neutrophils were identified using the S100a8 and S100a9 markers (36). Others have reported technical information loss issues with the canonical Ly6g mRNA marker for identifying neutrophils in scRNA sequencing (38). Monocytes express Fcgr1, Fcgr3, and Mafb and lack the Itgax activation marker. Kupffer cells are differentiated from monocyte-derived macrophages (mo-macs) and express only Clec4f + and Csf1r + (39). Finally, Clec10a and H2-Aa were used to identify classical dendritic cells (cDC), while Tcf4 and Irf8 markers were used to identify plasmacytoid DC (pDC) (40,41).

[0182] result

[0183] Example 1: LIDPAD mice exhibit transitional stages that reflect human NAFLD and extrahepatic complications.

[0184] Wild-type C57BL / 6J male mice housed in the thermoneutral zone (30°C) were fed a modified high-fat cholesterol refined ingredient diet (herein referred to as LIDPAD mice, liver disease progression exacerbation diet), or were fed a matched refined ingredient control diet to ensure comparability (Table 5). LIDPAD mice showed an increase in body weight and liver weight as early as 1 week on the diet, and their body weight and liver weight gradually increased compared with the control group ( Figure 1 A. Figure 2A Whole-body caloric analysis revealed that LIDPAD mice displayed impaired metabolic flexibility, less distinct circadian oscillations between carbohydrate and lipid utilization, and a stable low respiratory exchange ratio (RER 0.70-0.75), indicating that LIDPAD mice preferred lipids as a fuel compared with control mice ( Figure 3 A to Figure 3 B) When the running wheel was introduced, the daily energy expenditure shift in LIDPAD mice was also lower, indicating that LIDPAD mice exercised less than control mice ( Figure 3 C to Figure 3 E).

[0185] LIDPAD mice developed glucose intolerance within 8 weeks, which corresponded to changes in islet size ( Figure 1 B. Figure 2B The islet area of ​​the control group did not change significantly from week 1 to week 48, with an average islet area of ​​0.5 μm 2 x10 4 ( Figure 4 A to Figure 4 B). In contrast, in LIDPAD mice, islet area increased significantly (1.5-fold) between weeks 4 and 16, corresponding to the time when glucose intolerance becomes evident, suggesting compensation by islet cell hyperplasia, consistent with islet compensation in prediabetes. Starting at week 40, smaller islets (≤0.5 μm) began to appear in LIDPAD mice compared with control mice. 2 x10 4 ), although islet area remained widely distributed, which may correspond to deterioration of islet mass due to long-term insulin resistance, reflecting the association between NAFLD and the development of type 2 diabetes. Renal hypertrophy characterized by enlarged interstitial spaces, increased immune cell infiltration, and mesangial expansion was also observed in LIDPAD mice at week 48 ( Figure 4 C to Figure 4 D). Kidney sections also show fibrosis, suggesting previous kidney damage. Given the impaired ability of these mice to metabolize glucose, the enlarged kidneys may be due to diabetic nephropathy.

[0186] LIDPAD mice and control mice underwent magnetic resonance imaging-based hepatic proton density fat fraction (PDFF) measurements at 4, 8, and 16 weeks after starting the diet. At all imaging time points, LIDPAD mice had significantly increased hepatic fat fraction ( Figure 1 C). LIDPAD-fed mice exhibited a change in hepatic PDFF from 12.9% at 4 weeks post-diet to 30.13% at 16 weeks post-diet. The hepatic fat fraction of LIDPAD mice increased 2.2-fold (12.9% to 28.9%) as measured by imaging time points from week 4 to week 8. Hepatic lipid accumulation slowed from week 8 to week 16, with PDFF increasing by only 1.3% ( Figure 1 C). The liver fat fraction of control mice increased slightly as expected at the imaging time points of weeks 4, 8, and 16, with PDFF changing from 7.4% to 12.5% ​​( Figure 1 C).

[0187] Liver histology remains the gold standard for diagnosing NASH. Liver sections from LIDPAD mice and control mice fed different weeks of diet were scored by a pathologist using the steatosis, activity, and fibrosis (SAF) system. Hepatic lipid accumulation was evident as early as 1 week of LIDPAD feeding but was absent in the livers of control mice ( Figure 1 D. Figure 5A ), consistent with the MRI imaging data. After 4 weeks, the livers of LIDPAD mice were pale and enlarged with intracellular lipid vacuoles, and all liver specimens had SAF scores of 2 or 3. By weeks 8 to 12, LIDPAD mice developed extensive steatosis, varying degrees of intralobular inflammation, and lymphocyte and macrophage infiltration, similar to what is observed in human NASH. Specifically, characteristic features of NASH (i.e., hepatocyte ballooning) were visible at week 8 ( Figure 1 D. Figure 5B Fibrosis was prevalent in 50% to 80% of mice ( Figure 1 D). With prolonged feeding of LIDPAD, increased severity of liver fibrosis and more extensive lipid accumulation were observed, except at weeks 32 and 48 ( Figure 1 D. Figure 5A These reductions in steatosis may be due to reduced lipid accumulation as fibrosis becomes more prevalent. Liver samples from weeks 16 to 40 demonstrated sustained and consistent increases in all aspects of steatosis, ballooning, inflammation, and fibrosis ( Figure 1 D. Figure 5A , Figure 5B Starting at week 32, hepatocellular nodules were observed in ∼13% of mice, which is comparable to the ∼2% to 12% rate of human hepatocellular carcinoma (42).

[0188] In summary, LIDPAD mice exhibited a wide range of physiological and physical activity impairments. The LIDPAD mouse model exhibited the entire process of NAFLD disease progression, with fatty degeneration starting at early time points (weeks 1 to 4) and NASH appearing after week 8. Starting from week 12, 80% of mice developed fibrosis. A portion of LIDPAD mice eventually developed end-stage sequelae such as cirrhosis and hepatocellular nodules, reflecting the disease progression and morbidity of NAFLD patients.

[0189] Example 2: Transcriptome analysis and cytokine profiling of the liver highlights hepatic inflammation and fibrosis.

[0190] Transcriptomic and biochemical analyses were performed to identify key cellular mechanisms supporting the histological changes observed in NAFLD. Hierarchical clustering of differentially expressed genes (DEGs) in all LIDPAD liver samples revealed three gene groups based on their expression trends, with distinct gene ontology profiles ( Figure 6 A and Figure 6 B). Among the early events highlighted were dysregulation of cholesterol and lipid homeostasis and an acute phase response, indicative of hepatic inflammation in response to lipotoxicity. At weeks 1 to 4, LIDPAD mice also had a hepatic transcriptomic profile with altered cellular responses to LPS, indicative of a compromised intestinal barrier. As NASH progressed, genes involved in collagen assembly and inflammatory response pathways emerged and were enhanced, consistent with the histological and pathological scores of NASH ( Figure 6 A and Figure 6 B).

[0191] These findings were confirmed by a series of biochemical measurements. Starting from the 8th week of the diet, a significant increase in the level of alanine aminotransferase (ALT), a biomarker of liver damage, was observed in LIDPAD mice ( Figure 7 A). Although there was a trend toward a slight increase in ALT in control mice with aging, this increase was not statistically significant. As determined by LC-MS, both free cholesterol and squalene levels were elevated in LIDPAD mice compared with control mice ( Figure 7 B), confirming transcriptomic analysis. Basal amounts of cholesterol were detected in the liver (site of cholesterol synthesis) of control mice, while squalene levels were close to absent ( Figure 7 B). The increase in free cholesterol can be attributed to the cholesterol in the LIDPAD diet. The increase in squalene (a precursor of cholesterol) concentrations suggests that the sterol biosynthesis pathway is affected by LIDPAD.

[0192] Various studies have identified serum cytokines in the evaluation of NAFLD patients (43). Multiplex immunoassay analysis of serum revealed that no single cytokine remained consistently elevated throughout the progression of steatosis to cirrhosis. We observed distinct but overlapping cytokine profiles in the early, mid, and late stages ( Figure 7 C). At weeks 1 to 4, we observed an increase in low-grade chronic inflammatory cytokines (IL-6 and IL-13) and inflammatory chemokines (MIP-1α and eotaxin), consistent with the onset of steatosis and liver damage. At weeks 8 to 12, the inflammatory response was sustained by a new set of cytokines (TNF-α and IL-1α) and chemokines (MCP-1 and MIG), further promoting the infiltration of immune cells. After 12 weeks, the early and intermediate cytokines decreased and were replaced by another set of cytokines important for the maturation of adaptive immune cells (IL-7, IL-9, IL-10, and IL-2) ( Figure 7 C) Although cohort studies have identified similar serum biomarkers, they have not addressed temporal changes with disease progression. Therefore, a different panel of cytokines / chemokines appears to be useful for monitoring NAFLD progression.

[0193] Next, the LIDPAD model results were compared with a human meta-analysis of a transcriptomic signature of NAFLD that identified 218 genes associated with histological progression (44). The expression of most genes detected in the LIDPAD model showed the same signature consistency compared with human gene expression ( Figure 6 C). Guided by histological examination and major temporal gene expression changes, LIDPAD feeding for 1, 8, 16, and 32 weeks revealed key transitions corresponding to the progression of human NAFLD ( Figure 6 C). Transcriptomics-guided staging of 10 NASH patients was performed to assess the robustness of the LIDPAD transcriptomic progression signature, which revealed that liver biopsies in most patients (6 of 10) corresponded to weeks 12 to 16 of the LIDPAD mouse model, i.e., NASH with fibrosis ( Figure 6 C). Histological evaluation of matched liver specimens showed some degree of concordance based on the ordered SAF staging ( Figure 7 D). Gene set enrichment analysis (GSEA) of the human NASH transcriptome revealed that biological processes such as liver inflammation, fibrosis, and vascular perturbations are dominant in human NASH livers ( Figure 6 D).

[0194] Example 3: Angptl4 deficiency alters the intrahepatic immune cell landscape to delay liver fibrosis.

[0195] To understand the role of Angptl4 in the immunopathogenesis of fibrosis, the effects of Angptl4 deficiency during LIDPAD-induced NASH were investigated in two models: myeloid cell-specific Angptl4 mutant mice (Angptl4LysM- / -) and mice treated with a neutralizing antibody (mAb) against cAngptl4 (28, 32). Mice fed LIDPAD for 4 weeks were treated with the mAb. These mice continued to gain weight, similar to control mice. The mAb did not alter the glucose intolerance induced by LIDPAD feeding ( Figure 8 A and Figure 8 B). Likewise, Angptl4LysM- / - mice gained weight but had significantly better glucose tolerance in all cases. These observations suggest that immunoneutralization of cAngptl4 after bolus administration does not affect glucose utilization, whereas genetic Angptl4 deletion in myeloid cells improves systemic glucose tolerance.

[0196] At 8 weeks of LIDPAD treatment, the livers of wild-type mice showed steatosis and inflammation, and the prevalence and severity of NASH and fibrosis increased by week 12. Notably, hepatic inflammation and fibrosis were reduced in mAb-treated mice and Angptl4LysM- / - mice ( Fig. 9 A and Fig. 9 B. Figure 8 C to E). Hepatic transcriptomic profiles confirmed the ameliorative effect of targeting Angptl4 to slow the progression of NASH. Principal component analysis (PCA) revealed that the hepatic transcriptome of mAb-treated LIDPAD mice occupied a space between control mice and LIDPAD mice, and the difference between mAb-treated LIDPAD mice and LIDPAD mice decreased with prolonged feeding time, indicating that the former had a slower onset ( Fig. 9 C). With prolonged feeding, the hepatic transcriptome of Angptl4LysM- / - mice formed distinct clusters from the other groups of mice switched to LIDPAD, indicating that the combined effects of gene ablation and high-calorie feeding affect the liver differently. DEG analysis identified five functional clusters ( Fig. 9 D). As expected, the LIDPAD diet significantly activated hepatic inflammatory responses and ECM remodeling activity, which became stronger with prolonged feeding time. cAngptl4 mAb effectively reduced these pro-inflammatory and pro-fibrotic events, leading to a slower progression. Although most NASH-related immune responses were suppressed in Angptl4LysM- / - mice, different sets of pro-inflammatory genes related to fibrinolysis, complement activation, and the adaptive immune system were triggered in these mice, highlighting the interrelationship between innate and adaptive immune responses.

[0197] This analysis revealed an immunomodulatory role for cAngptl4 and the benefit of targeting cAngptl4 to delay diet-induced NASH.

[0198] Example 4: Single-cell transcriptomics reveals adaptive immune cell remodeling during fibrosis.

[0199] After long-term (1 year) LIPDAD feeding, immunodeficient NSG mice developed steatosis with only mild fibrosis, confirming the essential and important role of immune cells in fibrosis ( Figure 8 F). To gain insight into the immune cell landscape as NASH livers develop fibrosis, scRNA-seq was performed on intrahepatic immune cells from LIDPAD and control livers at 8 and 12 weeks. 19 immune cell subsets were identified, of which B lymphocytes and T lymphocytes comprised the majority of the intrahepatic immune cell population. B lymphocytes (differentiated and mature plasma cells) and T lymphocytes (8 major cell subsets) accounted for ~43.2% and ~41.2% of the total intrahepatic immune cells, respectively. Six myeloid-derived immune cell subsets, namely neutrophils, monocytes, Kupffer cells, monocyte-derived macrophages (mo-macs), classical dendritic cells (cDCs), and plasmacytoid DCs (pDCs), accounted for nearly 10% of CD45+ cells in the liver ( Fig.10 A and Fig.10 B, Table 8).

[0200] Table 8: Relative abundance of intrahepatic immune cell subsets.

[0201]

[0202] Total T cells and total B cells include the cells in bold or Represents subgroup.

[0203] Compared with the control diet, most myeloid lineage cells were slightly increased in NASH livers at weeks 8 and 12 ( Fig.10 B, Table 8). On the other hand, B lymphocytes increased at week 8 and decreased at week 12. The proportions of CD8+ T cells, CD4+ T cells, dnT cells, and NK T cells showed an opposite trend to that of the control diet, i.e., decreased at week 8 and increased at week 12, suggesting that these cells are involved in fibrosis. High-resolution analysis of T cell subsets revealed four CD8+ T cell subclusters, five CD4+ T cell subclusters, three dnT cell subclusters, five subclusters of innate-like T cells and NK T cells, and two gdT cell clusters ( Fig.10 C. Fig.11 A, Table 9). CD4+T cells and CD8+T cells showed dynamic changes in their proportions under different conditions, accounting for approximately two-thirds of the T lymphocyte population ( Fig.10C). We explored the fate of T cells to further understand their role. As the disease progressed from week 8 to week 12, more naive CD4+ T cells were activated as demonstrated by scRNA sequencing and FACS analysis ( Fig.12 A to Fig.12 C. Fig.13 Pseudo-temporal analysis revealed two major peaks along the CD4+ T cell differentiation trajectory, corresponding to activation states ( Fig.12 B). Similar shifts in CD8+ T cells in the liver were also observed ( Fig.14 A to Fig.14 C) In conclusion, the hallmark of fibrosis progression is a remodeling of the lymphoid immune cell landscape, particularly an increase in activated CD4+ T cells and CD8+ T cells.

[0204] Table 9: Relative abundance of T cell subsets

[0205]

[0206] In LIDPAD-fed Angptl4 LysM- / - mice, there was a higher neutrophil persistence and macrophage: monocyte ratio than in LIDPAD-fed mice, indicating a prolonged innate inflammatory response. This also reveals the relationship between innate and adaptive immune responses during fibrosis. The direction of change in the relative abundance of T cells and B cells was opposite to that of LIDPAD mice, similar to that of control mice, i.e., total B cells increased and T cells decreased from week 8 to week 12 (Table 8). However, compared with LIDPAD-fed mice, no change in the activation state of CD4+T cells and CD8+T cells was observed. mAb treatment of LIDPAD-fed mice reduced the abundance of many immune cells (e.g., neutrophils, MO-MAC and monocytes) to levels similar to those of control mice. Compared with LIDPAD mice, mAb treatment reduced the relative abundance of T cells and B cells from week 8 to week 12 (Tables 8 and 9). Importantly, in contrast to Angptl4 LysM- / - mice and LIDPAD-fed mice, mAb treatment effectively blocked the increase in activated CD4+ T cells and CD8+ T cells from week 8 to week 12 ( Fig.12 A to Fig.12 C. Fig.14 A to Fig.14 C). Therefore, activation of CD4+ and CD8+ T cells is a key event in fibrosis and blocking cAngptl4 reduces fibrosis.

[0207] GSEA of CD4+ T cells and CD8+ T cells revealed gene sets involved in two major processes, namely eukaryotic translation and immune response ( Fig.12 D. Fig.14D). During fibrosis, downregulation of genes involved in eukaryotic translation was observed, accompanied by upregulation of genes involved in inflammatory responses in CD4+ T cells ( Fig.12 D). In CD8+ T cells, the eukaryotic translation gene set became non-significant at week 12, whereas genes involved in cell recognition and cytokine-mediated responses were upregulated ( Fig.14 D) These observations indicate that regulation of eukaryotic translation activity is critical for the proinflammatory phenotype of both T cell lineages.

[0208] mAb treatment inhibited the eukaryotic translation machinery in both CD4+ and CD8+ T cells, accompanied by a substantial suppression of inflammatory activity in both CD4+ and CD8+ T cells at week 12 ( Fig.12 D. Fig.14 D to Fig.14 E). The effect of mAb on eIF2α signaling was observed in CD8+ T cells at week 8 ( Fig.14 E), which may be due to the complex activation mechanism of CD8+ T cells that requires the input of CD4+ T helper cells. Using curated gene sets from StringDB and Reactome, we identified a stimulatory effect of cAngptl4 mAb on phosphorylated eukaryotic initiation factor-2α (phospho-eIF2α)-mediated signaling, which is often associated with global translational attenuation and may contribute to the inhibition of T cell activation in mAb-treated mice ( Fig.12 E).

[0209] Example 5: cANGPTL4 deficiency regulates T cell activation by inhibiting translation initiation in CD4+ T cells

[0210] To understand how cANGPTL4 mediates T cell activation, GSEA was performed on CD4+ T cells and CD8+ T cells, revealing gene sets that were primarily involved in two processes, eukaryotic translation and immune response. With increasing disease severity, downregulation of genes involved in eukaryotic translation was observed, accompanied by upregulation of genes involved in immune response in CD4+ T cells. Both gene sets became non-significant at week 12 and were replaced by genes involved in cell recognition and cytokine-mediated responses, which are typically observed in CD8+ T cells ( Fig.15 These observations suggest that CD4+ T cells and CD8+ T cells play different roles in NASH severity and liver fibrosis.

[0211] After treatment with cANGPTL4 monoclonal antibody, the EIF2AK4 signaling response, which inhibits global protein translation, was significantly activated in CD4+ T cells. In contrast, the pro-inflammatory signaling pathway showed an opposite trend.

[0212] Many immune-related gene sets and inflammation-related gene sets, including cytokine-cytokine receptor interactions and IL-4 signaling pathway, IL-1 signaling pathway, IL-13 signaling pathway, and IFNγ signaling pathway, were continuously upregulated in CD4+ T cells and CD8+ T cells during NASH progression from week 8 to week 12. Anti-cANGPTL4 monoclonal antibody effectively inhibited pro-inflammatory signaling activity and antigen processing activity in T cells.

[0213] When zoomed in on T cells in different activation states, we found that Eif2ak4 signaling was specifically activated by cANGPTL4 monoclonal antibody in naive CD4+ T cells, whereas the gene signatures of effector CD4+ T cells (i.e., Th1 and Th17) and CD8+ T cells were significantly suppressed in activated T cells, regardless of disease progression ( Fig.16A ). Activation of Eif2ak4 signaling in naive CD4+ T cells impedes global protein synthesis, preventing their subsequent maturation into more specialized lineages. Similarly, differentiation of effector CD8+ T cells is impeded, as CD4+ T cells are key mediators of CD8+ T cell activation. Functionally, cANGPTL4 monoclonal antibodies reduced interferon-γ signaling activity and antigen processing activity in activated CD4+ T cells, both of which are associated with the immunoregulatory capacity of CD4+ T cells ( Fig. 16B In activated CD8+ T cells, although interferon-γ signaling was not affected by cANGPTL4 deficiency, the defense response was suppressed.

[0214] Example 6: cAngptl4-mediated eIF2α signaling regulates CD4+ T cell activation

[0215] Phosphorylation of eIF2α reduces global translation, allowing cells to conserve resources and initiate reconfiguration of gene expression to effectively manage stress conditions. Attempts have been made to explain how cAngptl4 alters phosphorylated eIF2 signaling. Active phosphorylated eIF2α signaling stimulates the expression of three typical target genes, namely, activating transcription factor 4 (ATF4), heat shock protein family A member 5 (HSPA5), and DNA damage-induced transcript 3 (DDIT3). Since protein kinases are ubiquitously involved in cellular signaling activities, kinase inhibitor screening was performed using LPS-activated wild-type T cells and Angptl4- / - CD4+ T cells to map the regulatory pathway of eIF2α phosphorylation by Angptl4 (based on the readout of target gene expression). Unsupervised hierarchical clustering of the results revealed three kinase target clusters corresponding to (i) uninvolved kinases, (ii) Angptl4-independent kinases, and (iii) Angptl4-dependent kinases ( Fig.17A). In wild-type cells, one-fourth of the kinase inhibitors had minimal effects on the expression of these target genes compared to DMSO treatment, indicating that these kinases are not involved in phosphorylated eIF2α-mediated signaling. In DMSO-treated Angptl4- / - cells, target gene expression was already higher than in DMSO-treated wild-type cells, indicating that Angptl4 deficiency activates phosphorylated eIF2α-mediated signaling of these target genes. As expected, inhibition of these unrelated kinases did not alter target gene expression compared to DMSO-treated Angptl4- / - cells ( Fig.17A and Fig. 17B ).

[0216] Twenty-one kinase inhibitors activated target gene expression in wild-type cells, indicating that these kinases inhibit phosphorylated eIF2α-mediated signaling ( Fig.17A and Fig. 17B ). Among them, 9 kinase inhibitors targeting Angptl4-independent kinases derepressed phosphorylated eIF2α-mediated signaling and increased target gene expression. Compared with DMSO treatment, these inhibitors further increased the expression of target genes in Angptl4- / - cells. Compared with DMSO-treated Angptl4- / - cells, the remaining 12 kinase inhibitors did not change the expression of target genes in Angptl4- / - cells and were identified as Angptl4-dependent ( Fig.17A and Fig. 17B ). Angptl4 deficiency does not activate Angptl4-dependent kinases, while ineffective inhibition by corresponding kinase inhibitors produces minimal changes in target gene expression ( Fig.17A and Fig. 17B ). In general, the expression profile of eIF2α is consistent with that of the target gene, revealing a positive correlation between eIF2α and phosphorylated eIF2α signaling. α The expression of eIF2α was higher than that of wild-type cells, and LPS could further increase the expression of eIF2α ( Fig. 17C ). Immunoblotting analysis showed that phosphorylated eIF2α was higher in Angptl4- / - CD4 T cells than in wild-type cells, regardless of exposure to LPS ( Fig.17D ). The results confirmed that Angptl4 inhibited phosphorylated eIF2α by suppressing eIF2α expression, and kinase inhibitor screening revealed three major kinase pathways in the Angptl4-eIF2α axis, namely MAPK, PI3K and Src.

[0217] To confirm the in vivo role of phosphorylated eIF2α signaling on activated CD4+ T cells, the effect of eIF2α deficiency on the fate of ANGPTL4- / -CD4+ T cells with high endogenous eIF2α was monitored in LIDPAD mice. CellTracker-labeled donor Angptl4- / -CD4+ T cells treated with si-eIF2α were intravenously injected into LIDPAD recipient mice ( Fig.17E As expected, an increase in activated host CD4+ T cells (unlabeled) was detected in the livers of LIDPAD-fed mice. The absence of eIF2α in labeled donor CD4+ T cells significantly increased the percentage of activated CD4+ T cells compared to si-scrambled donor CD4+ T cells ( Fig.17F ). Apparently, reducing eIF2α expression inhibits phosphorylated eIF2α signaling, leading to more T cell activation. In summary, the key role of Angptl4-eIF2α signaling in T cell activation is systematically demonstrated, which contributes to increased inflammation and fibrosis ( Figure 17G ).

[0218] discuss

[0219] In summary, a human-relevant diet-induced NAFLD model has been conceived, providing the opportunity to reliably translate mouse findings into insights for human NAFLD drug development. Using this model, we have demonstrated domain-specific roles for cAngptl4 and phosphorylated eIF2α signaling in suppressing T lymphocyte (particularly CD4+ T cell) activation during fibrosis.

[0220] The pathogenesis of NAFLD is a multifactorial and multistep process involving poorly characterized gene-environment interactions and multi-organ involvement (45).

[0221] First, a physiologically relevant refined diet-induced NAFLD model termed LIDPAD was described that recapitulates the key transition phases of human NAFLD, including the full range of histological and transcriptomic changes. Understanding the network of multisystem interactions requires physiologically relevant animal models that reflect the characteristics and comorbidities of human NASH (46). A modified mouse model of diet-induced liver disease was established using a refined liver disease progression-aggravating diet (LIDPAD). Species differences between the LIDPAD model and humans were minimized by thermoneutral mouse housing conditions, while the refined diet mitigated batch-to-batch compositional differences (i.e., metabolic confounding factors). A key feature of the NAFLD model is its rapid disease progression, which allows the full range of anthropometric, physiological, histological, and transcriptomic abnormalities of human pathogenesis to be acquired in a relatively short time frame, i.e., simple steatosis from weeks 1 to 4, chronic inflammation from weeks 4 to 8, and fibrosis from weeks 12 to 16. LIDPAD mice rapidly gain weight and develop impaired glucose homeostasis. Noninvasive longitudinal imaging by magnetic resonance imaging and temporal transcriptome analysis of the liver demonstrated that steatosis transitioned to NASH at week 4 of the diet, with fibrosis developing between weeks 12 and 16. Transcriptomic-guided staging using biopsy-confirmed NASH patients highlights the relevance of the LIDPAD mouse model to human NAFLD. This model supports the idea that the liver-pancreas axis promotes compensatory islet cell proliferation, a phenomenon similar to that observed in animal models of insulin resistance (47) and in humans, with a clear correlation between BMI and β-cell mass (48), followed by steatosis and development of type 2 diabetes (49). Furthermore, LIDPAD mice exhibit NAFLD-associated vasculopathy, with enhanced expression of CXCL12 in endothelial cells in the aorta and hepatic vasculature (50). These various metabolic dysfunctions and extrahepatic comorbidities are absent in the current NAFLD model.

[0222] Secondly, using the LIDPAD model, we characterized the intrahepatic immune cell landscape during NASH at single-cell resolution, revealing the importance of adaptive immunity in disease progression.

[0223] Single-cell analysis of the intrahepatic immune landscape of a human-relevant diet-induced NAFLD mouse model revealed an integral role for adaptive immune cell remodeling in disease progression characterized by an increase in activated T lymphocytes. During fibrosis, CD4+ T cell activation was shown to be dependent on eIF2α-mediated eukaryotic translation initiation.

[0224] Understanding the time frame of the transition from fatty liver to NASH in the LIDPAD model allows for in-depth study of the role of immune cells in the progression of the disease to fibrosis. Histological examination revealed an increase in cellular infiltration of the adaptive immune system at the 8th week of LIDPAD feeding, a phenomenon that persisted at more advanced stages. Single-cell monitoring of intrahepatic immune cells in the livers of LIDPAD mice undergoing this transition showed an overexpression of adaptive immune cells, including B and T lymphocytes. These abnormal immune responses were primarily coordinated by CD4+ helper T cells, which were highly activated in the early and late NASH stages in the LIDPAD model. This confirms the finding that T lymphocytes are critically involved in the progression of NAFLD (50-52). Recent studies have also identified an indispensable role for activated CD4+ T cells in fibrotic sites in NASH-driven liver inflammation and fibrosis (53). Mechanistically, the results disclosed herein reveal that Angptl4-eIF2α signaling is critical for the activation of CD4+ T cells. CD4+ T cells also mediate NASH exacerbation by regulating the activation of CD8+ T cells, NKT cells, and fibrogenic HSCs. Mechanistically, CD8+ cytotoxic T cells drive the transformation of NAFL to NASH by increasing proinflammatory cytokine secretion and auto-aggressive killing of hepatocytes (54). Different CD4+ T cell subsets exhibit distinct roles in liver fibrosis. Th2- and Th17-derived cytokines stimulate collagen production and TGFβR-dependent liver fibrosis (55). Notably, NAFLD patients also exhibit increased serum proinflammatory and profibrotic cytokine signatures, along with an increase in activated CD4+ T cells (55). Overall, the multifaceted effects of CD4+ T cell responses remain an intrinsic driver of NASH exacerbation.

[0225] Finally, we demonstrate that cANGPTL4 orchestrates the remodeling of overactive adaptive immunity, which exacerbates liver inflammation and fibrosis, and suggest that inhibition of cANGPTL4 is a promising approach to ameliorate NASH.

[0226] The ANGPLT4-eIF2α signaling pathway was further deciphered and found to be essential for the activation of CD4+ T cells ( Figure 17G Immunoneutralization of cANGPTL4 effectively reduced the immune response that promotes NASH and delayed liver fibrosis.

[0227] These results specifically focus on the exacerbation of NASH with fibrosis and confirm the immunomodulatory role of cANGPTL4 in the adaptive immune response within the liver through the ANGPTL4-eIF2α axis. Little is known about the role of active phosphorylated eIF2α signaling in the immunopathology of NAFLD. Studies have shown that active eIF2α supports the maturation and clonal expansion of CD4+ T cells, which subsequently mediate the activation of other lymphocytes, thereby generating an inflammatory and fibrotic liver environment in NASH. In fact, inhibition of components in the translation initiation complex abolished the activation and differentiation of CD4+ T cells, which was observed in NAFLD mice carrying CD4+ T cells with eIF2α knockdown by adoptive transfer. These results also show that fibrosis is reduced in myeloid-specific Angptl4 knockout mice, probably due to reduced T cell infiltration rather than their activation state. Immunomodulation of T cells provides an exciting avenue to alleviate the chronic inflammation that leads to NASH. Studies have shown that immunotherapy with mAbs against cANGPTL4 effectively reduces T lymphocyte activation and delays liver fibrosis in NASH by modulating phosphorylated eIF2α signaling.

[0228] The invention is described broadly and generically herein. Each of the narrower species and subgeneric classifications falling within the generic disclosure also forms a part of the invention. This includes the generic description of the invention with a proviso or negative limitation excluding any subject matter from the generic class, whether or not the excluded material is specifically described in this application. Other embodiments are also within the scope of the following claims.

[0229] Those skilled in the art will readily recognize that the present invention is well suited to achieve the stated objects and obtain the results and advantages mentioned and the advantages inherent therein. In addition, it will be apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The methods and uses described herein currently represent preferred embodiments and are exemplary and are not intended to limit the scope of the invention. Those skilled in the art will recognize that changes and other uses therein will occur, which are all included in the spirit of the invention and are defined by the scope of the claims. The listing or discussion of previously published documents in this specification should not be considered as an admission that the document is part of the prior art or common general knowledge.

[0230] The invention described illustratively in this application can be appropriately implemented after removing any one or more elements or limitations not specifically disclosed in this application. Therefore, it should be understood that although the present invention has been specifically disclosed through preferred embodiments and optional features, those skilled in the art can still modify and change the present invention disclosed herein, and such modifications and changes are considered to be within the scope of the present invention.

[0231] The contents of all documents and patent documents cited herein are incorporated by reference in their entirety.

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Claims

1. A method for treating, improving, preventing or delaying liver fibrosis or a disease associated with liver fibrosis in a subject, the method comprising administering an effective amount of angiopoietin-like 4 protein (ANGPTL4) antagonist to the subject.

2. The method of claim 1, wherein the disease associated with liver fibrosis is selected from non-alcoholic steatohepatitis (NASH), autoimmune hepatitis, congenital liver fibrosis, non-alcoholic fatty liver disease (NAFLD), cholestatic liver disease, alcoholic hepatitis and viral hepatitis.

3. The method according to claim 1 or 2, wherein the disease associated with liver fibrosis is non-alcoholic steatohepatitis (NASH), or wherein the liver fibrosis is associated with non-alcoholic steatohepatitis (NASH).

4. The method according to any one of claims 1 to 3, wherein the disease associated with liver fibrosis is non-alcoholic fatty liver disease (NAFLD), or wherein the liver fibrosis is associated with non-alcoholic fatty liver disease (NAFLD).

5. The method according to any one of claims 1 to 4, wherein the ANGPTL4 antagonist is an antisense molecule; or an aptamer; or a siRNA molecule; or an anti-ANGPTL4 antibody.

6. The method of any one of claims 1 to 5, wherein the ANGPTL4 antagonist is directed against the C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4).

7. The method of any one of claims 1 to 6, wherein the ANGPTL4 antagonist neutralizes, blocks, inhibits, reduces or interferes with the expression and / or activity of the C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4).

8. The method according to any one of claims 1 to 7, wherein the ANGPTL4 antagonist is an anti-cANGPTL4 antibody, preferably a monoclonal antibody, more preferably a neutralizing monoclonal antibody.

9. The method of claim 8, wherein the anti-cANGPTL4 antibody is a monoclonal antibody that binds to the same epitope as mAb 11F6C4.

10. The method according to claim 8 or 9, wherein the anti-cANGPTL4 antibody is a humanized antibody, preferably humanized mAb 11F6C4.

11. The method of any one of claims 8 to 10, wherein the anti-cANGPTL4 antibody specifically binds to the C-terminal fibrinogen-like domain of ANGPTL4.

12. The method according to any one of claims 1 to 7, wherein the ANGPTL4 antagonist is a siRNA molecule, preferably an ANGPTL4-siRNA molecule.

13. The method of claim 12, wherein the siRNA molecule targets an mRNA sequence encoding ANGPTL4.

14. The method of any one of claims 1 to 13, wherein the subject is a human.

15. A method for treating, ameliorating, delaying or preventing liver inflammation in a subject suffering from liver fibrosis or a disease associated with liver fibrosis, the method comprising administering an effective amount of angiopoietin-like 4 protein (ANGPTL4) antagonist.

16. A method for regulating an immune response in a subject suffering from liver fibrosis or a disease associated with liver fibrosis, the method comprising administering an effective amount of angiopoietin-like 4 protein (ANGPTL4) antagonist, wherein the administration induces a population of regulatory T cells or activated T cells, preferably CD8 + The number, activity and / or effector function of T cells and / or CD4+ T cells is reduced.

17. An in vitro method for reducing fibrosis in a liver sample, comprising: The hepatic cells or tissue are contacted with an ANGPTL4 antagonist.

18. An in vitro method for reducing hepatic inflammation in a liver sample, comprising: The liver sample is contacted with an ANGPTL4 antagonist.

19. An in vitro method for modulating an immune response in a liver sample, comprising: The liver sample is contacted with an angiopoietin-like 4 protein (ANGPTL4) antagonist, wherein the contacting induces a regulatory T cell or activated T cell population, preferably CD8 + The number, activity and / or effector function of T cells and / or CD4+ T cells is reduced.

20. The method of any one of claims 15 to 19, wherein the ANGPTL4 antagonist is directed against the C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4).

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