Methods and materials for treating atherosclerosis

Abstract

Provided herein are methods and materials for treating atherosclerosis and / or atherosclerosis-related conditions (e.g., atherosclerotic cardiovascular disease) in a mammal. For example, provided herein are methods and materials for using one or more HB-EGF inhibitors (e.g., CRM197) to treat a mammal (e.g., a human) having atherosclerosis and / or atherosclerosis-related conditions (e.g., atherosclerotic cardiovascular disease).

Description

[0001] Atorney Docket No. 44807-0499WO1 / C18224

[0002] METHODS AND MATERIALS FOR TREATING ATHEROSCLEROSIS

[0003] CLAIM OF PRIORITY

[0004] This application claims the benefit of European Patent Application Serial No. EP24205717.2, filed on October 10, 2024, the entire contents of which are hereby incorporated by reference.

[0005] TECHNICAL FIELD

[0006] This document relates to methods and materials for treating atherosclerosis and / or atherosclerosis-related conditions (e.g., atherosclerotic cardiovascular disease) in a mammal (e.g., a human). For example, this document provides methods and materials for using one or more HB-EGF inhibitors (e.g., CRM197) to treat a mammal (e.g., a human) having atherosclerosis and / or atherosclerosis-related conditions (e.g., atherosclerotic cardiovascular disease).

[0007] BACKGROUND

[0008] Despite recent advances in lipid lowering therapies, atherosclerosis remains the leading cause of death worldwide. Atherosclerosis and / or atherosclerosis-related conditions such as atherosclerotic cardiovascular disease (ASCVD) pose a major public health problem. For example, coronary artery disease (CAD), a major type of ASCVD, accounts for 1 in every 4 deaths in the United States. Each year, approximately 659,041 people die from heart disease. Thus, there is an unmet clinical need for improved therapeutic approaches for the treatment of atherosclerosis and / or atherosclerosis-related conditions.

[0009] SUMMARY

[0010] This document provides methods and materials for treating atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). For example, this document provides methods and materials for administering one or more heparin binding EGF like growth factor (HB-EGF) inhibitors (e.g., CRM197) to a mammal (e.g., a human) having atherosclerosis and / or an Atorney Docket No. 44807-0499WO1 / C18224 atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) to treat (e.g., reduce the extent or severity of) the mammal.

[0011] As described herein, reducing the activity of epidermal growth factor receptor (EGFR) pathway by inhibiting HB-EGF can diminish atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). Having the ability to treat atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) as described herein (e.g., by administering one or more HB-EGF inhibitors) provides a unique and unrealized opportunity to treat diseases and disorders characterized by atherosclerosis.

[0012] Provided herein are methods for treating atherosclerosis in a subject in need thereof, the method including: (a) determining or having determined that the subject expresses binding epidermal growth factor (HB-EGF); and (b) administering to the subject a therapeutically effective amount of an HB-EGF inhibitor. Also provided herein are methods for treating atherosclerosis in a subject expressing HB-EGF, the method including administering to the subject a therapeutically effective amount of an HB-EGF inhibitor.

[0013] In some embodiments, the subject has atherosclerotic cardiovascular disease (ASCVD), aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis.

[0014] Also provided herein are methods for reducing the risk of developing atherosclerosis in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of an HB-EGF inhibitor. In some embodiments, the subject is at risk of developing atherosclerosis or an atherosclerotic condition. In some embodiments, the HB-EGF inhibitor includes an inhibitory antibody, an inhibitory nucleic acid molecule, a small molecule inhibitor, an inhibitor of HB-EGF cleavage, an inhibitor of HB-EGF activity in a cell nucleus. In some embodiments, the small molecule inhibitor includes CRM197. In Atorney Docket No. 44807-0499WO1 / C18224 some embodiments, the inhibitory antibody includes U3-1565. In some embodiments, the inhibitory nucleic acid molecule includes an RNAi, a siRNA, an antisense oligonucleotides (ASO), and / or a shRNA. In some embodiments, the inhibitor of HB-EGF cleavage includes an a disintegrin and metalloprotease (ADAM) inhibitor. In some embodiments, methods and materials described herein further include administering to the subject one or more additional anti-atherosclerotic agents. In some embodiments, the anti-atherosclerotic agent includes statins, proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, ezetimibe, bempedoic acid, niacin, cholesterol sequestrants, glucagon-like peptide-1 (GLP1) agonists, anti-platelet agents, anti -thrombotic agents, and / or any combinations thereof.

[0015] Also provided herein are methods for diagnosing atherosclerosis in a subject, where the method includes determining levels of HB-EGF in a sample from a subject, where elevated levels of HB-EGF in the sample compared to a control sample identifies that the subject has atherosclerosis. In some embodiments, the control sample is a biological sample isolated from a healthy subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample includes blood, plasma, urine, serum, or saliva. In some embodiments, administering the therapeutically effective amount of the HB- EGF inhibitor to the subject reduces the uptake of low-density lipoprotein (LDL), vascular inflammation, atherosclerotic plaque formation, smooth muscle cell proliferation and phenotype switching, and / or endothelial dysfunction.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Atorney Docket No. 44807-0499WO1 / C18224

[0017] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1. Individuals with a history of LDL-Cholesterol (LDL-C) levels greater than 175 mg / dL and / or ApoB levels greater than 129 mg / dL without other known atherosclerosis risk factors were recruited and grouped into those with ASCVD and those without ASCVD. Peripheral blood mononuclear cells (PBMCs) were collected for downstream analysis.

[0020] Figure 2. Whole genome sequencing quality control. Variant calling analysis showed that approximately 99% of the reads were aligned in all individuals. The total number of variants in all individuals was around 4 million. The total number of variants after merging all the values in individuals with and without ASCVD was close to 13 million.

[0021] Figures 3A-3B. Polygenic risk score (PRS) analysis did not show a difference in common variant risk score associated with coronary artery disease (CAD). Figure 3A) Principal component analysis of the individual genotypes clustered together with individuals from the 1000 Genomes reference panel is shown. The vast majority of individuals clustered were in the appropriate group according to their self-reported ancestry. The White Caucasian ancestry group had the highest number of individuals. Figure 3B) PRS analysis showed no relative change in the cumulative common variant genetic risk score of CAD in the atherosclerotic condition and the no atherosclerotic condition. The top panel shows PRS associated with CAD, adjusted for ancestry using the first two genotype principal components, the middle panel shows PRS associated with CAD in individuals with White Caucasian Ancestry defined by PCA, and the bottom panel shows PRS associated with CAD in all individuals.

[0022] Figures 4A-4C. UMAP clustering of Single cell RNAseq (ScRNAseq) analysis. Figure 4A) ScRNAseq analysis identified eleven cell type clusters. All the data were processed in 5 batches with 3 individuals per batch. The resulting data were subjected to deconvolution and followed by normalization and integration using SCT transform. The result is shown as a plot of all resulting cells in a Uniform Manifold Approximation and Atorney Docket No. 44807-0499WO1 / C18224

[0023] Projection (UMAP) by using SEURAT (tool). Figures 4B-4C) UMAP clustering showed the presence of cells in all clusters in equivalent proportions across all groups of race (Figure 4B), sex (Figure 4B), and age (Figure 4C).

[0024] Figure 5. Comparison of cell proportion between individuals with and without ASCVD. The analysis showed no significant difference between the two conditions in any of the cell type clusters involved. Among all the clusters, the cluster of natural killer cells exhibited the highest level of difference in cell proportion between the two conditions.

[0025] Figure 6. The CD14+monocyte cluster showed the highest number of significantly differentially expressed genes between the Non-ASCVD and ASCVD groups. Pseudobulk approach was used with a false discovery rate (FDR) of 0.05.

[0026] Figure 7. Epidermal growth factor receptor (EGFR) pathway showed the highest enrichment in monocytes of the ASCVD group compared to the non-ASCVD group. Enriched pathways in monocytes were analyzed using Fast Gene Set Enrichment Analysis (FGSEA) with Gene Ontology and Reactome.

[0027] Figure 8. Volcano plot shows upregulation of heparin binding EGF like growth factor (HB-EGF and amphiregulin AREG), both ligands of EGFR, in monocyte clusters in the ASCVD group.

[0028] Figure 9. Analysis of gene expression in monocytes of known EGFR ligands. EGFR ligands including AREG, EREG, HB-EGF, and EGF were upregulated in the ASCVD group.

[0029] Figure 10. Enzyme-linked immunosorbent assay (ELISA) was performed to assess the expression levels of HB-EGF and AREG in individuals with and without ASCVD. Individuals with ASCVD showed higher levels of circulating HB-EGF polypeptide compared to the non-ASCVD group (p=0.04, for the comparison of HB-EGF levels, after adjusting for age, sex, race, and eGFR). The same was not observed for AREG (p=0.49 for the comparison of AREG levels after adjusting for age, sex, race, and eGFR). The same individuals whose serum samples were used for scRNAseq analysis were also tested to measure the levels of HB-EGF and AREG in individuals with and without ASCVD.

[0030] Figure 11. The role of HB-EGF in ASCVD was tested by silencing the expression of HB-EGF in monocyte-derived macrophages in vitro. Knocking down HB-EGF reduced LDL uptake and disrupted the formation of foam cells in these macrophages. Atorney Docket No. 44807-0499WO1 / C18224

[0031] Figure 12. EGFR inhibition reduced arterial inflammation in an independent patient cohort. Arterial inflammation was measured by target-to-background ratio (TBR = aortic standardized uptake value [SUV] / vena cava SUV). Individuals were stratified into tertiles based on arterial inflammation at baseline. Despite the progression of malignancy, the results showed decreased arterial inflammation following the initiation of the EGFR inhibitor treatment in individuals within the second and third highest tertiles.

[0032] Figures 13A-13B. Sequence kernel association test (SKAT) analysis of rare variant analysis in the sample cohort showed that there was no significant genes associated with ASCVD. Figure 13A) Following the PRS analysis, the analysis of the rare variants and their association with ASCVD was tested. Shown are the top 15 genes identified in rare variant analyses with their nominal p-values. Variant effect predictor (VEP) was used to perform annotation of the genes and the variants. The right panel shows genes considered by VEP with high impact. The left panel shows genes considered by VEP with high and moderate impact. Figure 13B) Enriched pathways from the resulting SKAT analysis showed Reactome Signaling by EGFR as one of the top 20 enriched pathways.

[0033] Figures 14A-14E. Figure 14A) UMAP of single cell RNA-seq in PBMCs from 17 individuals. Figure 14B) Differential cell type proportions between participant groups using single cell RNA-seq of PBMCs. Figure 14C) Volcano plot for differential gene expression between the participant groups in single cell RNA-seq using a pseudobulk approach among all cell clusters. Figure 14D) Pathway enrichment analysis for the differentially expressed genes between participant groups among all PBMC cells. Datapoints above the black horizontal line indicate positive enrichment, while those below indicate negative enrichment. Figure 14E) Number of significant genes in differential expression between participant groups in single cell RNA seq of PBMCs

[0034] Figures 15A-15D. Figure 15A) Volcano plot of differential gene expression in the CD 14+ monocyte cluster between the participant groups in single cell RNA-seq using a pseudobulk approach. Figure 15B) Heatmap of the genes included in the EGFR pathway among the participant groups. Figure 15C) Pathway enrichment analysis for the differentially expressed genes in CD 14+ monocytes. Figure 15D) Serum ELISA results for HB-EGF and AREG between the participant groups. Atorney Docket No. 44807-0499WO1 / C18224

[0035] Figures 16A-16D. Figure 16A) Genotype principal components plot of the cohort of participants combined with the 1000 Genomes v3 cohort. Figure 16B) CAD Polygenic risk score comparison between the participant groups. Figure 16C) Colocalization plot between CAD genome-wide association studies (GWAS) and expression quantitative trait locus (eQTL) Gen data for HB-EGF eQTLs in whole blood. Figure 16D) Pathway enrichment analyses for the Sequence Kernel Association Test-optimal test (SKAT-O) variant analysis results.

[0036] Figures 17A-17E. Figure 17A) Single cell RNA-seq expression data for HB-EGF in human coronary arteries from Wirka et al., Nat Med, 25(8): 1280-1289 (2019) (data obtained from the PlaqView2 database with author provided cell type annotations). Figure 17B) Single cell RNA-seq expression data for HB-EGF in human peripheral blood mononuclear cells from the cohort. For Figures 17A and 17B, the colors of the data points indicate the strength of expression, and the size of each data point indicates the percentage of cells expressing the gene. Figures 17C-17D) Representative figures (Figure 17C) and fluorescence quantification of dil-ac-LDL uptake for THP-1 derived macrophages that were treated with HB-EGF siRNA or scramble control (Figure 17D). Figure 17E) Quantification data of foam cell formation assay for THP-1 derived macrophages that were treated with HB- EGF siRNA or scramble control.

[0037] Figure 18. PET-CT results of atherosclerotic lesions in aortas of participants with atherosclerosis that received EGFR inhibitor therapy pre- and post- treatment with the EGFR inhibitor. Inflammation was measured at the same location pre- and post- treatment based on the Target to background SUV ratio with the background set at the IVC.

[0038] Figures 19A-19B. Figure 19A) Oil Red O staining for cholesterol on whole aortas of mice with monocyte / macrophage HB-EGF overexpression (HB-EGF) or controls (CT) that were treated with a single AAN -Pcsk9 injection and 9 weeks of high-fat diet (HFD). Figure 19B) H & E stained cross sections of aortic roots from CT and HB-EGF mice treated as described in Figure 19 A.

[0039] Figure 20. To evaluate the effects of HB-EGF not only in macrophages, but also in other cell types involved in atherosclerosis and inflammation levels, such as vascular smooth muscle cells (VSMCs), macrophages were co-cultured with VSMCs using a transwell Atorney Docket No. 44807-0499WO1 / C18224 system. The results showed that THP-1 macrophages promoted VSMC proliferation, and this effect was reduced when HB-EGF was silenced, demonstrating the contributing role of HB- EGF in this process. MTS assay was used to measure the proliferation. ** p < 0.01; ns = not significant.

[0040] Figure 21. qPCR of human aortic vascular smooth muscle cells exposed to HB-EGF or vehicle control. The results showed decreased expression of contractile VSMC markers (CNN1), increased expression of synthetic markers (SOX9), and a trend towards increased proliferation (MKI67). ** p < 0.01 .

[0041] DETAILED DESCRIPTION

[0042] Lipids are known to play a major role in atherosclerotic plaque development. Besides lipids, several non-lipid mechanisms including pathways related to immune response can contribute to disease susceptibility and development. It has been shown that patients can develop atherosclerosis despite having LDL cholesterol levels within the guideline- recommended targets. Despite the link between atherogenic lipoproteins and atherosclerotic cardiovascular disease (ASCVD), there are people with significant hyperlipidemia who do not develop atherosclerosis. Inhibiting EGFR can decrease vascular inflammation; however, EGFR inhibition is associated with adverse effects of skin toxicity. On the contrary, inhibiting heparin binding EGF like growth factor (HB-EGF), an EGFR ligand can circumvent this problem while reducing vascular inflammation and atherosclerotic plaque formation.

[0043] Compared to patients with similar lipid levels who are susceptible to atherosclerosis, this document demonstrates that protected individuals exhibit reduced epidermal growth factor receptor (EGFR) pathway activity in circulating classical monocytes. Provided herein are methods and materials for treating atherosclerosis or atherosclerosis-related conditions in a subject (e.g., a mammal). In some embodiments, a mammal (e.g., a human) having an atherosclerosis-related condition can be a human having one or more diseases and / or disorders characterized by atherosclerosis). Non-limiting examples of atherosclerosis-related conditions include atherosclerotic cardiovascular disease (ASCVD), e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, Atorney Docket No. 44807-0499WO1 / C18224 abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis. In some embodiments, this document provides methods and materials for using one or more HB-EGF inhibitors to treat (e.g., reduce the extent or severity of) or reduce the risk of developing atherosclerosis or atherosclerosis-related conditions. For example, an HB-EGF inhibitor can be an inhibitory antibody, an inhibitory nucleic acid, and / or a small molecule inhibitor. In some embodiments, an HB-EGF inhibitor can be CRM197. In some embodiments, the HB- EGF inhibitor provided herein can be combined with other anti-atherosclerotic agents (e.g., such as statins, proprotein convertase subtil i si n / kexin type 9 (PCSK9) inhibitors, ezetimibe, bempedoic acid, niacin, cholesterol sequestrants, glucagon-like peptide- 1 (GLP1) agonists, anti-platelet agents, anti -thrombotic agents, and / or any combinations thereof). In some embodiments, one or more HB-EGF inhibitors can be administered to a mammal (e.g., a human) having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). Also provided herein are methods and materials for diagnosing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a mammal (e g., a human). Methods and materials provided herein can be used to treat (e.g., reduce the extent or severity of) or reduce the risk of developing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in any appropriate mammal including, without limitation, humans, monkeys, canines / dogs, cats / felines, pigs, horses, cows, sheep, goats, rabbits, mice, and rats. For example, a human having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can be treated with one or more HB-EGF inhibitors as described herein. Atorney Docket No. 44807-0499WO1 / C18224

[0044] A therosclerosis

[0045] Provided herein are methods and materials for treating (e.g., reducing the extent or severity of) atherosclerosis or atherosclerosis-related conditions and / or reducing the risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject. Also provided herein are methods and materials for diagnosing atherosclerosis or atherosclerosis-related conditions in a subject. Atherosclerosis is a condition caused by buildup of plaque in the inner lining of the artery that leads to thickening and hardening of the artery. In some embodiments, the plaque buildup comprises buildup of fats, cholesterol, cellular waste products, calcium, fibrin, or other substances in and on the artery walls. Risk factors for atherosclerosis are well known in the art and include, without limitation, high cholesterol and triglyceride levels, history of smoking, tobacco use, diabetes mellitus, obesity, high blood pressure, physical inactivity, high saturated fat diet, sleep apnea, high levels of C-reactive protein (CRP), high levels of inflammation marker, or family history. In some embodiments, a subject with atherosclerosis and / or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has vascular inflammation, atherosclerotic plaque formation, smooth muscle cell proliferation and phenotype switching, and / or endothelial dysfunction. Methods of assessing these risk factors for a given subject are also well known in the art.

[0046] In some embodiments, a subject (e.g., a mammal) that can be diagnosed and / or treated according to methods provided herein as having atherosclerosis or atherosclerosis- related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can have low-density lipoprotein (LDL) cholesterol levels within the guideline recommended range. A guideline recommended range of LDL can be the range or levels of LDL in a subject that is considered healthy or optimal according to medical guidelines. A guideline recommended range of LDL may differ from one individual to another depending on the individual’s age, sex, health history (e.g., diseases), family Atorney Docket No. 44807-0499WO1 / C18224 history, genetics, and / or lifestyle choices. For example, the recommended range of LDL levels in people 20 years of age and older can be about less than lOOmg / dL (e.g., about less than 50mg / dL, about less than 55mg / dL, about less than 60mg / dL, about less than 65mg / dL, about less than 70mg / dL, about less than 75mg / dL, about less than 80mg / dL, about less than 85mg / dL, or about less than 90mg / dL). The recommended range of LDL levels in people 19 years of age and younger can be about less than 1 lOmg / dL (e.g., about less than 50mg / dL, about less than 55mg / dL, about less than 60mg / dL, about less than 65mg / dL, about less than 70mg / dL, about less than 75mg / dL, about less than 80mg / dL, about less than 85mg / dL, about less than 90mg / dL, about less than 95mg / dL, about less than lOOmg / dL, or about less than 105mg / dL). In some embodiments, a subject (e.g., a mammal) that can be diagnosed and / or treated according to methods provided herein as having atherosclerosis or atherosclerosis- related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can have low-density lipoprotein (LDL) cholesterol levels higher than the guideline recommended range. For example, a higher than the guideline recommended range can be about higher than lOOmg / dL (e.g., from about lOOmg / dL to about 129mg / dL, from about 130mg / dL to about 159mg / dL, from about 160mg / dL to about 189mg / dL, or higher than about 190mg / dL).

[0047] In some embodiments, a subject (e.g., a mammal) that can be diagnosed and / or treated according to methods provided herein as having atherosclerosis or atherosclerosis- related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can have HB-EGF levels within a range that is comparable to a control sample (e.g., HB-EGF level in a sample from a healthy subject). In some embodiments, a subject (e.g., a mammal) that can be diagnosed and / or treated according to methods provided herein as having atherosclerosis or atherosclerosis-related conditions can have higher HB-EGF level compared to a control sample (e.g., HB-EGF level in a sample from a healthy subject).

[0048] In some embodiments, methods provided herein can include determining that a mammal (e.g., a human) has or is at risk of developing atherosclerosis or atherosclerosis- Atorney Docket No. 44807-0499WO1 / C18224 related conditions (e.g., the mammal can have one or more diseases and / or disorders characterized by atherosclerosis such as plaque buildup in the artery). Any appropriate method can be used to determine that a mammal has atherosclerosis or an atherosclerosis- related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). For example, imaging techniques (e.g., CT scans, positron emission tomography (PET) scans, ultrasound imaging, MRI scans, and / or X rays), symptoms examination, physical examination, neurological examination, laboratory tests (e.g., blood, urine, serum, and / or spinal fluid tests), electrocardiograms, exercise stress tests, and / or genetic tests (e.g., whole genome sequencing and / or whole exome sequencing), and / or coronary angiograms can be used to determine that a human or other mammal has atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis).

[0049] Heparin binding EGF like growth factor (HB-EGF)

[0050] In some embodiments, methods and materials provided herein can include inhibiting EGFR pathway activity in a subject (e.g., a mammal). For example, methods and materials provided herein can include an agent that can inhibit EGFR pathway activity (e.g., an inhibitor of an EGFR ligand and / or an inhibitor of an EGFR). EGFR is a Type I transmembrane glycoprotein of 170kDa molecular weight. EGFR signaling is initiated by binding of its ligand (e.g., HB-EGF) which triggers a downstream signal transduction cascade that contributes to a wide variety of cellular functions, including cell proliferation and survival. In some embodiments, methods and materials provided herein include an EGFR ligand inhibitor. For example, an EGFR ligand inhibitor can be a heparin binding EGF like growth factor (HB-EGF) inhibitor. HB-EGF is produced as a precursor transmembrane polypeptide that can be cleaved by a disintegrin and metalloprotease 17 (ADAM 17). The N-terminus of HB-EGF is then released and acts as a soluble EGFR ligand, while its C-terminus activates B-cell lymphoma 6 (BCL6) and cell cycle signaling in the nucleus. In some embodiments, methods and materials described herein Atorney Docket No. 44807-0499WO1 / C18224 can be used to treat (e.g., reduce the extent or severity of) or reduce the risk of developing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject (e.g., a mammal) expressing HB-EGF.

[0051] In some embodiments, methods and materials provided herein for treating or reducing the risk of developing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject include one or more HB-EGF inhibitors. In some embodiments, an HB-EGF inhibitor is an inhibitor of an HB-EGF polypeptide. An inhibitor of an HB-EGF polypeptide can be an inhibitor of HB-EGF polypeptide activity (e.g., anti-HB-EGF antibodies such as neutralizing anti-HB-EGF antibodies and small molecules that target an HB-EGF polypeptide) or an inhibitor of HB-EGF polypeptide expression (e.g., inhibitory nucleic acid molecule). For example, an inhibitor of an HB-EGF polypeptide can be U3-1565. In some embodiments, U3-1565 is used to treat (e.g., reduce the extent or severity of) or reduce the risk of developing atherosclerosis or atherosclerosis- related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject (e.g., a mammal). In some embodiments, the subject (e.g., a mammal) has atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject is at high risk of developing atherosclerosis or atherosclerosis- related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level higher than the guideline recommended Atorney Docket No. 44807-0499WO1 / C18224 range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level within the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has HB-EGF level within a range that is comparable to a control sample (e.g., HB-EGF level in a sample from a healthy subject). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has higher HB-EGF level compared to a control sample (e.g., HB-EGF level in a sample from a healthy subject).

[0052] In some embodiments, an inhibitory nucleic acid molecule is designed to induce RNA interference (RNAi) of HB-EGF polypeptide expression such as antisense oligonucleotides (ASOs), siRNA molecules, and shRNA molecules. In some embodiments, an inhibitory nucleic acid molecule can include a nucleic acid sequence that is from about 8 to about 80 nucleotides in length (e.g., from about 13 nucleotides to about 80 nucleotides, from about 12 nucleotides to about 50 nucleotides, from about 12 nucleotides to about 30 nucleotides, from about 15 nucleotides to about 30 nucleotides, from about 20 nucleotides to about 30 nucleotides, from about 20 nucleotides to about 24 nucleotides, or from about 16 nucleotides to about 20 nucleotides). In some cases, an inhibitory nucleic acid molecule can include a nucleic acid sequence that is about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length. Additional inhibitors of an HB-EGF polypeptide (e.g., nucleic acid molecules designed to induce RNAi against HB-EGF polypeptide expression) can be designed based on any appropriate nucleic acid (e.g., a mRNA) encoding an HB-EGF polypeptide sequence. Examples of nucleic acids encoding an Atorney Docket No. 44807-0499WO1 / C18224

[0053] HB-EGF polypeptide sequence include, without limitation, those set forth in National Center for Biotechnology Information (NCBI) accession nos. NM_001945, NC_000005.10, and NP 001936. In some embodiments, an inhibitory nucleic acid molecule described herein is used to treat (e.g., reduce the extent or severity of) or reduce the risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject (e.g., a mammal). In some embodiments, the subject (e.g., a mammal) has atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject is at high risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level higher than the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level within the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has HB-EGF level within a range that is comparable to a control sample (e.g., HB-EGF level in a sample from a healthy subject). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve Atorney Docket No. 44807-0499WO1 / C18224 stenosis) has higher HB-EGF level compared to a control sample (e.g., HB-EGF level in a sample from a healthy subject).

[0054] In some embodiments, an HB-EGF inhibitor to be used in accordance with materials and methods provided herein is a small molecule inhibitor (e.g., CRM197). In some embodiments, the small molecule inhibitor is CRM197. In some embodiments, CRM197 is used to treat (e.g., reduce the extent or severity of) or reduce the risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject (e.g., a mammal). In some embodiments, the subject (e.g., a mammal) has atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject is at high risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level higher than the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level within the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has HB-EGF level within a range that is comparable to a control sample (e.g., HB-EGF level in a sample from a healthy subject). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral Atorney Docket No. 44807-0499WO1 / C18224 artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has higher HB-EGF level compared to a control sample (e.g., HB-EGF level in a sample from a healthy subject).

[0055] In some embodiments, an HB-EGF inhibitor to be used in accordance with materials and methods provided herein is an inhibitor of HB-EGF cleavage (e.g., an ADAM inhibitor). In some embodiments, the inhibitor of HB-EGF cleavage is an ADAM inhibitor. In some embodiments, the inhibitor of HB-EGF cleavage prevents the release of N-terminus of HB- EGF as a soluble EGFR ligand. In some embodiments, the inhibitor of HB-EGF cleavage prevents the release of C-terminus of HB-EGF (e.g., translocation into the nucleus). Examples of ADAM inhibitors include, without limitation, ADAM10 inhibitors, ADAM12 inhibitors, and / or ADAM17 inhibitors. Such ADAM10 inhibitors, ADAM12 inhibitors, and / or ADAMI 7 inhibitors used in materials and methods provided herein can be generally known in the art. In some embodiments, an inhibitor of HB-EGF cleavage is used to treat (e.g., reduce the extent or severity of) or reduce the risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject (e.g., a mammal). In some embodiments, the subject (e.g., a mammal) has atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject is at high risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level higher than the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery Atorney Docket No. 44807-0499WO1 / C18224 disease, and / or aortic valve stenosis) has LDL level within the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has HB-EGF level within a range that is comparable to a control sample (e.g., HB-EGF level in a sample from a healthy subject). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has higher HB-EGF level compared to a control sample (e.g., HB-EGF level in a sample from a healthy subject).

[0056] In some embodiments, an HB-EGF inhibitor to be used in accordance with materials and methods provided herein is an inhibitor of HB-EGF activity in a cell nucleus. In some embodiments, the inhibitor of HB-EGF activity in a cell nucleus prevents the cleaved C-terminus of HB-EGF from interacting (e.g., activating) with BLC6 in the nucleus. In some embodiments, the inhibitor of HB-EGF activity in a cell nucleus prevents the cleaved C-terminus of HB-EGF from activating cell cycle signaling pathways in the nucleus. Such inhibitors of HB-EGF activity in a cell nucleus used in materials and methods provided herein can be generally known in the art. In some embodiments, an inhibitor of HB-EGF activity in a cell nucleus is used to treat (e.g., reduce the extent or severity of) or reduce the risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject (e.g., a mammal). In some embodiments, the subject (e.g., a mammal) has atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject is at high risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, Atorney Docket No. 44807-0499WO1 / C18224 abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level higher than the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level within the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has HB-EGF level within a range that is comparable to a control sample (e.g., HB-EGF level in a sample from a healthy subject). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has higher HB-EGF level compared to a control sample (e.g., HB-EGF level in a sample from a healthy subject).

[0057] In some embodiments, one or more HB-EGF inhibitors (e g., an HB-EGF inhibitory antibody or an HB-EGF inhibitory nucleic acid) described herein can be administered to a subject (e.g., a mammal) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with one or more (e.g., one, two, three, four, or five) additional EGFR pathway inhibitors (e.g., HB-EGF inhibitors, or EGFR / EGFR ligand inhibitors) described herein. In some embodiments, the additional EGFR pathway inhibitors include HB-EGF inhibitors and / or EGFR / EGFR ligand inhibitors. In some embodiments, the subject (e.g., a mammal) has atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic Atorney Docket No. 44807-0499WO1 / C18224 aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject is at high risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level higher than the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level within the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has HB-EGF level within a range that is comparable to a control sample (e.g., HB- EGF level in a sample from a healthy subject). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has higher HB-EGF level compared to a control sample (e.g., HB-EGF level in a sample from a healthy subject).

[0058] In some embodiments, one or more HB-EGF inhibitors (e.g., an HB-EGF inhibitory antibody or an HB-EGF inhibitory nucleic acid) described herein can be administered to a subject (e.g., a mammal) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with one or more (e.g., one, two, three, four, or five) additional HB-EGF inhibitors (e g., an HB-EGF inhibitory nucleic acid molecule or an HB-EGF inhibitory polypeptide) described herein. For example, methods and materials provided herein for treating or Atorney Docket No. 44807-0499WO1 / C18224 reducing the risk of developing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can include administering CRM197 to a mammal (e.g., a human) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with an HB-EGF inhibitory antibody (e.g., an inhibitor of HB-EGF polypeptide activity). In some cases, methods and materials provided herein for treating or reducing the risk of developing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can include administering CRM197 to a mammal (e.g., a human) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with an HB-EGF inhibitory nucleic acid molecule (e.g., an RNAi, siRNA, ASO, and / or shRNA). In some cases, methods and materials provided herein for treating or reducing the risk of developing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can include administering CRM197 to a mammal (e.g., a human) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with one or more additional HB-EGF small molecule inhibitors. In some cases, methods and materials provided herein for treating or reducing the risk of developing atherosclerosis or an atherosclerosis-related condition Atorney Docket No. 44807-0499WO1 / C18224

[0059] (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can include administering an HB-EGF inhibitory antibody (e.g., an inhibitor of HB-EGF polypeptide activity) to a mammal (e.g., a human) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with an HB-EGF inhibitory nucleic acid molecule (e.g., an RNAi, siRNA, ASO, and / or shRNA). In some cases, methods and materials provided herein for treating or reducing the risk of developing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can include administering an HB-EGF inhibitory antibody (e.g., an inhibitor of HB-EGF polypeptide expression or activity) to a mammal (e.g., a human) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with an HB-EGF small molecule inhibitor. In some cases, methods and materials provided herein for treating or reducing the risk of developing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can include administering an HB-EGF inhibitory nucleic acid molecule (e g., an RNAi, siRNA, ASO, and / or shRNA) to a mammal (e.g., a human) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with an HB-EGF small molecule inhibitor. In some embodiments, the subject (e.g., a mammal) has atherosclerosis or Atorney Docket No. 44807-0499WO1 / C18224 atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject is at high risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level higher than the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level within the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has HB-EGF level within a range that is comparable to a control sample (e.g., HB-EGF level in a sample from a healthy subject). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has higher HB-EGF level compared to a control sample (e g., HB-EGF level in a sample from a healthy subject).

[0060] In some embodiments, one or more HB-EGF inhibitors (e.g., an HB-EGF inhibitory antibody or an HB-EGF inhibitory nucleic acid) described herein can be administered to a subject (e.g., a mammal) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with one or more (e.g., one, two, three, four, or five) EGFR / EGFR ligand inhibitors Atorney Docket No. 44807-0499WO1 / C18224

[0061] (e.g., an EGFR inhibitory nucleic acid molecule or an EGFR inhibitory polypeptide) described herein. Examples of EGFR / EGFR ligand inhibitors include, without limitations, erlotinib, gefitinib, osimertinib, afatinib, dacomitinib, mobocertinib, vandetanib, cetuximab, panitumumab, and necitumumab. In some embodiments, the EGFR / EGFR ligand inhibitors are well known to people of ordinary skill in the art. In some cases, methods and materials provided herein for treating or reducing the risk of developing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject described herein can include administering CRM197 to a mammal (e.g., a human) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with one or more (e.g., one, two, three, four, or five) EGFR / EGFR ligand inhibitors provided herein. In some cases, methods and materials provided herein for treating or reducing the risk of developing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can include administering an HB-EGF inhibitory antibody (e.g., an inhibitor of HB-EGF polypeptide activity) to a mammal (e.g., a human) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with an EGFR / EGFR ligand inhibitor provided herein. In some cases, methods and materials provided herein for treating or reducing the risk of developing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can include administering an HB-EGF inhibitory nucleic acid (e.g., an RNAi, siRNA, ASO, and / or Atorney Docket No. 44807-0499WO1 / C18224 shRNA) to a mammal (e.g., a human) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with an EGFR / EGFR ligand inhibitor provided herein. In some cases, methods and materials provided herein for treating or reducing the risk of developing atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can include administering an HB-EGF small molecule inhibitor (e.g., an inhibitor of HB-EGF polypeptide expression or activity) to a mammal (e.g., a human) having atherosclerosis and / or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) or at high risk for atherosclerosis and / or an atherosclerosis-related condition, together with an EGFR / EGFR ligand inhibitor provided herein. In some embodiments, the subject (e.g., a mammal) has atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject is at high risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level higher than the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level within the guideline recommended range as described herein. In some Atorney Docket No. 44807-0499WO1 / C18224 embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has HB-EGF level within a range that is comparable to a control sample (e.g., HB-EGF level in a sample from a healthy subject). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has higher HB-EGF level compared to a control sample (e.g., HB-EGF level in a sample from a healthy subject).

[0062] In cases where one or more HB-EGF inhibitors (e.g., CRM197) described herein are used in combination with one or more additional EGFR pathway inhibitors (e.g., HB-EGF inhibitors, EGFR / EGFR ligand inhibitors) described herein, the additional HB-EGF inhibitor can be administered at the same time (e.g., in a single composition containing CRM197 and the HB-EGF inhibitory nucleic acid molecule) or independently to a subject described herein. For example, CRM197 described herein can be administered first, and the additional EGFR pathway inhibitors (e.g., HB-EGF inhibitors, EGFR / EGFR ligand inhibitors) described herein can be administered second, or vice versa. In cases where one or more HB-EGF inhibitors (e.g., CRM197) described herein are used in combination with one or more additional EGFR pathway inhibitors (e g., HB-EGF inhibitors, EGFR / EGFR ligand inhibitors) described herein, the additional EGFR pathway inhibitors (e.g., HB-EGF inhibitors, EGFR / EGFR ligand inhibitors) described herein can be administered at the same time or independently of the administration of one or more HB-EGF inhibitors (e.g., CRM197) described herein. For example, one or more additional EGFR pathway inhibitors (e g., HB-EGF inhibitors, EGFR / EGFR ligand inhibitors) described herein can be administered before, during, or after the one or more HB-EGF inhibitors (e.g., CRM197) are administered. In some embodiments, the subject (e.g., a mammal) has atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject is at high risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic Atorney Docket No. 44807-0499WO1 / C18224 aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level higher than the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level within the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has HB-EGF level within a range that is comparable to a control sample (e.g., HB-EGF level in a sample from a healthy subject). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has higher HB-EGF level compared to a control sample (e.g., HB-EGF level in a sample from a healthy subject).

[0063] Pharmaceutical composition

[0064] EGFR pathway inhibitors (e.g., an HB-EGF inhibitor) described herein can be formulated into a composition (e.g., a pharmaceutical acceptable composition) for administration to a subject (e.g., a mammal) having atherosclerosis or an atherosclerosis- related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). For example, a therapeutically effective amount of one or more HB-EGF inhibitors described herein can be formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. A pharmaceutical composition can be formulated for administration in solid or liquid form including, without Atorney Docket No. 44807-0499WO1 / C18224 limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.

[0065] As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, sterile aqueous or non-aqueous solutions, suspensions, emulsions, and the like, compatible with pharmaceutical administration. Examples of nonaqueous solvents include, without limitation, propylene glycol, polyethylene glycol, vegetable oils, and organic esters. Aqueous carriers include, without limitation, water, alcohol, saline, and buffered solutions. Acceptable carriers also can include physiologically acceptable aqueous vehicles (e.g., physiological saline) or other known carriers for oral administration. Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include oral, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), or inhaled administration. In some embodiments, administering comprises oral administration, injection, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, inhalation, or any combinations thereof.

[0066] In some embodiments, one or more HB-EGF inhibitors when formulated as a pharmaceutical composition can be formulated in an ingestible form or an injectable form or a topical form. For example, the pharmaceutical composition can be in the form of a liquid, solution, suspension, tablet, powder, granule, pill, capsule, gel, cream, mist, atomized vapor, aerosol, soft gelatin capsule, or hard gelatin capsule. For oral administration, tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents, fdlers, lubricants, disintegrants, or wetting agents. The tablets can be coated by methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspension, or they can be presented as a dry product for constitution with saline or other suitable liquid vehicle before use. Liquid preparations also can contain pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles, preservatives, buffer salts, flavoring agents, coloring agents, and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the compound. In Atorney Docket No. 44807-0499WO1 / C18224 some embodiments, the pharmaceutical composition (e.g., an inhibitory molecule) provided herein is formulated for oral ingestion, injection, subcutaneous injection, or tissue-specific targeting.

[0067] The pharmaceutical compositions (e g., one or more HB-EGF inhibitors) suitable for injectable use can include sterile aqueous solutions (e.g., water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The composition can be sterile and fluid to the extent that easy syringability exists. In some cases, the composition are stable under the conditions of manufacture and storage and are preserved against the contaminating action of microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). In some cases, the composition can include isotonic agents (e g., sugars, polyalcohols such as mannitol, sorbitol, sodium chloride). Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0068] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the active compound into a sterile vehicle, which can contain a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation can include vacuum drying and freeze-drying, which can yield a powder of the Atorney Docket No. 44807-0499WO1 / C18224 active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0069] The pharmaceutical composition (e.g., an HB-EGF inhibitory) provided herein can be administered by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.

[0070] Methods of use

[0071] Provided herein are methods for treating atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject (e.g., a mammal). In some embodiments, methods provided herein include determining or having determined that the subject (e.g., a mammal) expresses HB-EGF. In some embodiments, methods provided herein include administering to the subject a therapeutically effective amount of one or more HB-EGF inhibitors (e.g., CRM197). Also provided herein are methods for treating atherosclerosis or atherosclerosis- related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject (e.g., a mammal) expressing HB-EGF.

[0072] In some embodiments, methods provided herein include administering to the subject (e.g., a mammal) a therapeutically effective amount of one or more HB-EGF inhibitors (e.g., CRM197), alone or in combination. Methods and materials provided herein can be used to diagnose atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject (e.g., a mammal). In some embodiments, methods provided herein include determining a level of HB-EGF in a sample collected from the subject (e.g., a mammal). In Atorney Docket No. 44807-0499WO1 / C18224 some embodiments, the level of HB-EGF is elevated in the sample compared to a control sample (e.g., a sample obtained from a healthy human). In some embodiments, an elevated level of HB-EGF identifies that the subject (e.g., a mammal) has atherosclerosis. In some embodiments, the level of HB-EGF in a sample is comparable to the level of HB-EGF in a control sample (e.g., a sample from a healthy control mammal). In some embodiments, samples collected from the subject (e.g., a mammal) include biological sample (e.g., blood, serum, plasma, urine, serum, or saliva).

[0073] In some embodiments, the subject (e.g., a mammal) has atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject is at high risk of developing atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level higher than the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has LDL level within the guideline recommended range as described herein. In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) has HB-EGF level within a range that is comparable to a control sample (e.g., HB-EGF level in a sample from a healthy subject). In some embodiments, the subject with atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery Atorney Docket No. 44807-0499WO1 / C18224 disease, and / or aortic valve stenosis) has higher HB-EGF level compared to a control sample (e.g., HB-EGF level in a sample from a healthy subject).

[0074] A “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. For example, an effective amount is an amount that achieves a desired therapeutic effect, e.g., an amount sufficient to treat (e.g., reduce the extent or severity of) a disease and / or to reduce risk of development of disease or disease symptoms (also referred to as a prophylactically effective amount). A therapeutically effective amount can be administered in one or more administrations, applications, or dosages. A therapeutically effective amount of a therapeutic compound (e.g., an effective dosage) depends on the therapeutic compounds selected. For example, a therapeutically effective amount of a pharmaceutical composition (e.g., one or more HB-EGF inhibitors) as provided herein can be effective to improve at least one symptom of atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). The pharmaceutical composition (e.g., one or more HB-EGF inhibitors) provided herein can be administered one or more times per year (e.g., one time per year, two times per year, three times per year , four times per year , or five times per year) to one or more times per month (e.g., one time per month, two times per month, three times per month, four times per month, or five times per month), including once every other month, once every three months, or twice a month. In some embodiments, the pharmaceutical composition (e.g., one or more HB-EGF inhibitors) provided herein can be administered one or more times per week (e.g., seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once per day, twice per day, or thrice per day). The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat (e.g., reduce the extent or severity of) a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments. Various factors can influence the actual amount used for a particular application. For example, the frequency of administration, duration of Atorney Docket No. 44807-0499WO1 / C18224 treatment, combination of other agents, site of administration, stage of disease (if present), and the anatomical configuration of the treated area may require an increase or decrease in the actual amount administered.

[0075] An effective duration for administering a pharmaceutical composition (e.g., one or more HB-EGF inhibitors) provided herein can be any duration that reduces the symptoms related to atherosclerosis or atherosclerosis-related conditions (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) and / or inhibits the activity of EGFR pathway in a subject (e.g., a mammal) without producing significant toxicity to the subject (e.g., a mammal). In some embodiments, an effective duration for administering a pharmaceutical composition (e.g., one or more HB-EGF inhibitors) provided herein can be any duration that reduces the uptake of low-density lipoprotein (LDL), vascular inflammation, atherosclerotic plaque formation, smooth muscle cell proliferation and phenotype switching, and / or endothelial dysfunction in a subject (e.g., a mammal) without producing significant toxicity to the subject (e.g., a mammal). In some embodiments, an effective duration can vary from several days to several weeks, to several months, or longer. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the effective amount, frequency of administration, use of route of administration, and severity of the subject’s condition.

[0076] Dosage, toxicity and therapeutic efficacy of the pharmaceutical composition (e.g., one or more HB-EGF inhibitors) disclosed herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compositions that exhibit high therapeutic indices are advantageous. While compositions that exhibit toxic side effects may be used, care should be taken to minimize and reduce side effects. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations Atorney Docket No. 44807-0499WO1 / C18224 that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compositions used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models. Such information can be used to more accurately determine useful doses in humans.

[0077] In some embodiments, one or more (e.g., one, two, three, four, or more) HB-EGF inhibitors can be effective to reduce the uptake of low-density lipoprotein (LDL), vascular inflammation, atherosclerotic plaque formation, smooth muscle cell proliferation and phenotype switching, and / or endothelial dysfunction.

[0078] In some embodiments, one or more (e.g., one, two, three, four, or more) HB-EGF inhibitors can be effective to reduce expression of HB-EGF in a subject (e.g., a mammal) described herein having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) by, for example 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, methods and materials described herein can be effective to reduce the level of EGFR pathway activation in a subject (e.g., a mammal) having atherosclerosis or atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis).

[0079] In some embodiments, one or more (e.g., one, two, three, four, or more) HB-EGF inhibitors can be used to reduce arterial plaque formation in a subject (e.g., a mammal) described herein. For example, one or more HB-EGF inhibitors can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) to reduce arterial plaque formation within the mammal. In some cases, methods and materials described herein can be effective to reduce arterial plaque formation in a mammal having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve Atorney Docket No. 44807-0499WO1 / C18224 stenosis) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent . In some cases, methods and materials described herein can be effective to eliminate arterial plaque formation in a mammal having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis).

[0080] In some embodiments, one or more (e.g., one, two, three, four, or more) HB-EGF inhibitors can be used to reduce the LDL levels in a subject (e.g., a mammal) described herein. For example, one or more HB-EGF inhibitors can be administered to a mammal (e g., a human) in need thereof (e.g., a human having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) to reduce the LDL levels within the mammal. In some cases, methods and materials described herein can be effective to reduce the LDL levels by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent in a mammal having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis).

[0081] In some embodiments, one or more (e.g., one, two, three, four, or more) HB-EGF inhibitors can be used to reduce uptake of LDL in a subject (e.g., a mammal) described herein. For example, one or more HB-EGF inhibitors can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) to reduce the uptake of LDL levels within the mammal. In some cases, methods and materials described herein can be effective to reduce the uptake of LDL levels by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent in a mammal having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis). Atorney Docket No. 44807-0499WO1 / C18224

[0082] In some embodiments, one or more (e.g., one, two, three, four, or more) HB-EGF inhibitors can be used to reduce vascular inflammation in a subject (e.g., a mammal) described herein. For example, one or more HB-EGF inhibitors can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) to reduce vascular inflammation within the mammal. In some cases, methods and materials described herein can be effective to reduce vascular inflammation in a mammal having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the methods and materials described herein can be effective to eliminate vascular inflammation in a mammal having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis).

[0083] In some embodiments, one or more (e.g., one, two, three, four, or more) HB-EGF inhibitors can be used to reduce the severity of one or more symptoms of atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) in a subject (e.g., a mammal) described herein. For example, one or more HB-EGF inhibitors can be administered to a mammal (e g., a human) in need thereof (e.g., a human having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) to reduce the severity of one or more symptoms of atherosclerosis or atherosclerosis-related condition. Examples of symptoms of atherosclerosis include, without limitation, coronary heart disease (e.g., coronary arterial disease (CAD)), angina, heart palpitation, cold sweats, dizziness, extreme tiredness, shortness of breath, nausea, pain, Atorney Docket No. 44807-0499WO1 / C18224 heaviness, transient ischemic attack (TIA), weight loss, weakness, and / or numbness. In some cases, methods and materials described herein can be effective to reduce symptoms of atherosclerosis in a mammal having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent .

[0084] In some embodiments, one or more HB-EGF inhibitors described herein can be used as the sole active agent(s) to treat (e.g., reduce the extent or severity of) a subject (e.g., a mammal) having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) as described herein.

[0085] In some cases, one or more HB-EGF inhibitors described herein can be administered to a subject (e.g., a mammal) having atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) as described herein together with one or more (e.g., one, two, three, four, or more) additional agents / therapies (e.g., anti-atherosclerotic agents) used to treat (e.g., reduce the extent or severity of) atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) as described herein. Examples of additional agents / therapies that can be used to treat (e.g., reduce the extent or severity of) atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis) can include anti-atherosclerotic agents (e.g., such as statins, proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, ezetimibe, bempedoic acid, niacin, cholesterol sequestrants, glucagon-like peptide-1 (GLP1) agonists, anti-platelet agents, anti -thrombotic agents, and / or any combinations thereof). In cases where one or more HB-EGF inhibitors described herein are used in combination with one or more additional agents used to treat Atorney Docket No. 44807-0499WO1 / C18224

[0086] (e.g., reduce the extent or severity of) atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis), the one or more additional agents can be administered at the same time (e.g., in a single composition containing one or more HB-EGF inhibitors described herein and the one or more additional agents) or independently. For example, one or more HB-EGF inhibitors described herein can be administered first, and the one or more additional agents administered second, or vice versa. Tn cases where one or more HB-EGF inhibitors described herein are used in combination with one or more additional therapies used to treat (e.g., reduce the extent or severity of) atherosclerosis or an atherosclerosis-related condition (e.g., ASCVD, aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis), the one or more additional therapies can be performed at the same time or independently of the administration of HB-EGF inhibitors described herein. For example, one or more HB- EGF inhibitors described herein can be administered before, during, or after the one or more additional therapies are performed.

[0087] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0088] EXAMPLES

[0089] Example 1: Targeting HB-EGF to treat atherosclerosis

[0090] Patients may develop atherosclerosis despite having LDL cholesterol levels within the guideline-recommended targets. It had been shown that certain individuals appear to be protected against atherosclerosis and don’t develop clinical or subclinical disease despite a long-term exposure to extremely elevated LDL cholesterol. This study showed that compared to patients with similar lipid levels who were susceptible to atherosclerosis, the protected individuals had reduced EGFR pathway activity in circulating classical monocytes. This effect was shown to be driven by diminished levels of monocyte-derived production and subsequent release of the EGFR ligand HB-EGF. Furthermore, the results showed that HB- Atorney Docket No. 44807-0499WO1 / C18224

[0091] EGF silencing in monocyte-derived macrophages in vitro led to decreased macrophage LDL uptake, foam cell formation, and proliferation of human coronary artery smooth muscle cells in a transwell co-culture model. Patients who received a clinically indicated EGFR inhibitor for cancer therapy had significantly decreased vascular inflammation as measured by the FDG-PET post-treatment in atherosclerotic regions. The skin toxicity effects of EGFR inhibitors make the EGFR inhibition unappealing for treatment and prevention of atherosclerosis in the general population. On the other hand, HB-EGF inhibition can reduce vascular inflammation and atherosclerotic plaque formation without the adverse effects of EGFR inhibitors by leaving the primary skin EGFR ligands, EGF and TGF-a, intact.

[0092] Example 2: Targeting HB-EGF to treat atherosclerotic cardiovascular disease (ASCVD)

[0093] This Example demonstrates that EGFR pathway in CD14+monocytes was upregulated in the ASCVD group. For example, individuals with ASCVD showed higher serum HB-EGF levels compared to the individuals without ASCVD. Further, it was shown that EGFR inhibition was associated with decreased levels of arterial inflammation.

[0094] RESULTS

[0095] To identify the differences in the immune pathways of hyperlipidemic individual with and without ASCVD, the following tests were conducted. Individuals with a history of LDL- C and / or ApoB levels greater than 95thpercentile of the general population. These individuals can be categorized into two groups: 1) those with clinical ASCVD and 2) those without subclinical or clinical ASCVD (> 55 years old). Seven individuals with ASCVD and eight individuals without ASCVD were recruited for the study. Comparative analysis of the PBMC transcriptomes showed no major difference in cell composition between the groups. In cell-type stratified differential expression analyses, the most striking difference was observed in the transcriptome of CD14+monocytes, revealing upregulation of EGFR signaling pathway in individuals with premature ASCVD. Two EGFR ligands (HB-EGF and AREG) were among the top upregulated genes in monocytes of individuals with ASCVD Among those, HB-EGF polypeptide level was significantly elevated in sera of individuals with ASCVD (Mean±SD of 120.4±59.3 pg / mL for individuals with ASCVD and 91 ,3±40.5 Atorney Docket No. 44807-0499WO1 / C18224 pg / mL for individuals without ASCVD, p=0.04). Whole genome sequencing and rare variant analysis of these two groups confirmed genetic changes in EGFR signaling between individuals with and without ASCVD. In six individuals with malignancy and significant atherosclerosis, arterial inflammation assessed by FDG-PET was significantly decreased after treatment with an EGFR inhibitor (target-to-background (TBR) from l.OCHO. 17 before to 0.84±0.21 after treatment, p=0.01).

[0096] Whole Genome Sequencing Analysis

[0097] To analyze the whole genome sequencing results, variant calling analysis was performed. The results showed that approximately 99% of the reads were aligned in all individuals. The total number of variants in all individuals was found to be around 4 million. The total number of variants after merging all the values in individuals with and without ASCVD was close to 13 million.

[0098] Single Cell RNAseq Analysis

[0099] UMAP clustering was performed to analyze the ScRNAseq data. ScRNAseq analysis identified eleven cell type clusters (Figure 4A). All the data were processed in 5 batches with 3 individuals per batch. The resulting data were subjected to deconvolution, followed by normalization and integration using SCT transform. UMAP clustering showed all the clusters had equivalent proportions of cells across all groups of race, sex, and age (Figures 4B-4C).

[0100] The comparison of cell proportions between individuals with and without ASCVD showed no significant difference between the two conditions in any of the cell type clusters involved. The cluster of natural killer cells exhibited the highest level of difference in cell proportion between the two conditions (Figure 5). The CD14+monocyte cluster showed the highest number of significantly differentially expressed genes between the non-ASCVD group and the ASCVD group (Figure 6).

[0101] FGSEA with Gene Ontology and Reactome analysis showed EGFR pathway as the top enriched pathway in monocytes of the ASCVD group compared to the non-ASCVD group (Figure 7). The results further showed upregulation of HB-EGF a . AREG, both ligands of EGFR, in monocyte clusters of the ASCVD group (Figure 8). Gene expression Atorney Docket No. 44807-0499WO1 / C18224 analysis of known EGFR ligands in monocytes showed upregulation oi' AREG. EREG, HB- EGF, and EGF in the ASCVD group (Figure 9).

[0102] Knocking down HB-EGF reduced LDL uptake in vitro

[0103] ELISA was performed to assess the expression levels of HB-EGF and AREG in individuals with and without ASCVD. The same individuals whose serum samples were used for scRNAseq analysis were also tested to measure the levels of HB-EGF and AREG. The ASCVD group showed higher levels of circulating HB-EGF polypeptide compared to the non-ASCVD group (Figure 10). The same was not observed for AREG (Figure 10).

[0104] To test the role of HB-EGF in ASCVD, the expression of HB-EGF was silenced in monocyte-derived macrophages in vitro. Knocking down HB-EGF showed reduced LDL uptake and disrupted formation of foam cells in these macrophages (Figure 11).

[0105] EGFR inhibition reduced arterial inflammation

[0106] EGFR inhibition reduced arterial inflammation in an independent patient cohort (Figure 12). Arterial inflammation was measured by target-to-background ratio (TBR = aortic standardized uptake value [SUV] / vena cava SUV). Individuals were stratified into tertiles based on arterial inflammation at baseline. Despite the progression of malignancy, the results showed decreased arterial inflammation following the initiation of the EGFR inhibitor treatment in individuals within the second and third highest tertiles (Figure 12).

[0107] Rare variant analysis

[0108] The SKAT analysis of the rare variant analysis showed no significant genes associated with ASCVD. The top 15 genes identified in the rare variant analyses with their nominal p-values are shown in Figure 13A. VEP was used to perform annotation of the genes and the variants. Enriched pathways from the resulting SKAT analysis showed Reactome Signaling by EGFR as one of the top 20 enriched pathways (Figure 13B).

[0109] METHODS

[0110] Patients with LDL-cholesterol (LDL-C) and / or apolipoprotein B levels >95thpercentile of the U.S. adult population included in this study. Two groups of patients with Atorney Docket No. 44807-0499WO1 / C18224 hyperlipidemia were recruited: (a) those with premature ASCVD, and (b) those of age >55 years without clinical or subclinical ASCVD. Individuals with recent history of an ASCVD event, those with additional ASCVD risk factors, and patients with immune modulating conditions were excluded from the study. Exclusion criteria include ASCVD within 6 months of enrollment, history of smoking, Lp (a) levels > 75nmol / L, triglyceride levels > 500 mg / dL, presence of genetic variants associated with familial hypercholesterolemia (FH) (LDLR, APOB, PCSK9), uncontrolled hypertension (systolic blood pressure (SBP) > 140MM Hg or diastolic blood pressure (DBP) > 90mm Hg), uncontrolled diabetes mellitus with HbAl c levels >7%, BMI > 35 kg / m2, Hear Failure New York Heart Association (HF NYHA) class II-IV, creatinine clearance < 60 mL / min / 1.73 m2, connective tissue diseases, active malignancies, active infections, hematologic diseases, and / or substance abuse. PBMCs were isolated and single cell RNA-seq was performed. Differential expression analysis was performed on PBMC subsets between the groups, following a pseudobulk approach controlling for age, sex, race, and hidden confounders using surrogate variable analysis (SV A). Whole genome sequencing was performed in all individuals targeting a 30x coverage. Rare variant analysis was performed using SKAT-O. HB-EGF level was measured in serum samples from the same individuals.

[0111] 18F-FDG-PET imaging analysis

[0112] To assess the effects of EGFR inhibition on arterial inflammation, FDG-PET images were clinically obtained prior to and following the initiation of an EGFR inhibitor (EGFRi) treatment in an independent cohort of individuals with malignancy. Individuals included in this study had undergone EGFRi treatment initiation after baseline and before a follow-up PET scan, duration of the EGFRi treatment is more than 1 month, underwent EGFRi treatment within one month of follow-up PET scan, less than 2 years duration between the baseline PET scan and the EGFRi treatment initiation, the presence of atherosclerotic plaque at baseline scan, and / or the present of arterial inflammation at baseline scan (TBR>0.8). Atorney Docket No. 44807-0499WO1 / C18224

[0113] Example 3: Heparin-Binding EGF-Like Growth Factor (HB-EGF) Links Monocyte

[0114] Activation to Atherosclerotic Cardiovascular Disease (ASCVD) in Hypercholesterolemia

[0115] Monocytes and macrophages play central roles in atherogenesis, orchestrating inflammatory cascades, promoting foam cell formation, and mediating vascular injury. Activation of these cells in response to a chronic pro-inflammatory state may contribute to the residual ASCVD risk observed despite optimal lipid-lowering therapy. Yet, the molecular signals governing monocyte activation and atherogenic behavior in the context of isolated hypercholesterolemia remain poorly defined.

[0116] This Example examines molecular and cellular mechanisms of ASCVD in a cohort of individuals with severe hyperlipidemia and no other traditional cardiovascular risk factors, and demonstrates that HB-EGF-mediated EGFR signaling is a novel driver of residual inflammatory risk in ASCVD, and a potential therapeutic target in hyperlipidemic populations. For example, single-cell RNA sequencing, whole-genome sequencing, serum biomarker profiling, and PET / CT imaging were used to characterize immune cell phenotypes, genetic variation, and vascular inflammation. Further, it was shown that HB- EGF, a monocyte-derived EGFR ligand, was significantly upregulated in ASCVD and promoted key features of atherogenesis, including foam cell formation and vascular smooth muscle cell phenotypic switching.

[0117] RESULTS

[0118] Single cell RNA sequencing revealed a novel transcriptional landscape of inflammatory programs associated with ASCVD

[0119] Fifteen patients were enrolled in a cross-sectional discovery cohort study (8 with ASCVD and 7 without ASCVD). The mean ± SD age of the overall cohort was 60.9 ± 8.1 years; 13.3% were Asian, and 73.3% were women. Median (QI, Q3) historical LDL-C was 215.9 (201.0, 274.5) mg / dL, and median (QI, Q3) historical ApoB was 144.0 (136.5, 156.0). Compared to individuals without ASCVD, those with ASCVD were significantly younger (p = 0.022). There were no significant differences in sex, race, or lipid parameters between the groups. Use of lipid-lowering and cardiovascular medications was similar across the groups, (Table 1). Attorney Docket No. 44807-0499WO1 / C18224

[0120] The scRNA-seq analysis of peripheral blood mononuclear cells (PBMCs) demonstrated an expected distribution of all known major immune cell clusters (Figure 14A). Differential cell composition analysis between groups did not reveal any significant differences in the relative abundance of cell subsets, with the exception of a non- significant trend towards increased circulating NK cells in individuals with ASCVD (Figure 14B) (p = 0.1159 using Speckle). These results persisted even after adjusting for age, sex and genotype principal components. Pseudobulk differential expression analysis in individual cell subsets across all clusters identified 193 genes that were differentially regulated between the patient groups at an FDR < 0.05. Pathway analysis in different subsets identified common patterns of pathways across immune cell populations, including upregulation of translation initiation, elongation and non-sense mediated decay pathways in all lymphocyte subsets in individuals with ASCVD, and downregulation of interferon gamma pathways in most immune subpopulations in individuals with ASCVD (Figure 14C-D). Comparison between cell subsets identified CD14+ monocytes as the subset with the highest number of significant genes that were differentially expressed between the groups at FDR < 0.05 (Figure 14E). Given these findings and the well-established role of monocyte-derived macrophages in atherosclerosis pathogenesis, this cell population was examined for subsequent analysis.

[0121] Table 1. Baseline Characteristics Attorney Docket No. 44807-0499WO1 / C18224

[0122] EGFR signaling in CD 14+ monocytes was associated with ASCVD in patients with severe hypercholesterolemia

[0123] Gene set enrichment analysis of the differentially expressed genes in CD14+ monocytes between the groups revealed EGFR signaling as the most differentially regulated pathway (Normalized enrichment score equals to 2.15 (positive score denotes upregulation in ASCVD) (Figure 15C). Evaluation of genes belonging to the EGFR pathway identified AREG and HB-EGF as the most significantly upregulated genes in individuals with ASCVD (Figure 15B). The same genes were found to be among the most significantly upregulated genes across all differentially expressed genes in CD 14+ monocytes (Figure 15 A). Enzyme Atorney Docket No. 44807-0499WO1 / C18224 linked immunosorbent assay (ELISA) performed on serum from the same cohort participants confirmed upregulation of circulating HB-EGF but not AREG (Figure 15D) in individuals with ASCVD. Given the role of HB-EGF as a ligand that binds to EGFR to trigger the signaling cascade, and the findings of overall enrichment of genes downstream of EGFR in individuals with ASCVD, it suggests an underappreciated role of HB-EGF in modulating EGFR signaling, thereby contributing to increased ASCVD risk. The cross-sectional analyses of the transcriptome and secretome are inherently unable to distinguish cause from consequence; therefore, additional analyses were performed to establish a causal role of HB- EGF and EGFR signaling in atherosclerosis.

[0124] Genetic analyses highlighted a causal role of EGFR signaling and HB-EGF in ASCVD

[0125] To examine the causal role of EGFR signaling and HB-EGF, whole genome sequencing was performed on participants in the cohort. Since the recruitment and phenotyping process eliminated all known risk factors for ASCVD, residual risk might be, at least in part, driven by either common or rare genetic variation. Further, if EGFR signaling and / or HB-EGF itself are causally responsible for the different ASCVD phenotypes in the cohort, that would suggest existing genotype variants that regulate the expression or function of HB-EGF or other genes within the EGFR cascade. To test if EGFR signaling and HB-EGF have any causal role in ASCVD, it was first evaluated whether the phenotype is driven by polygenic risk that is not specifically associated with EGFR signaling. It was found that Polygenic Risk Scores (PRS) derived from common variants were not significantly different between individuals with and without ASCVD (Figure 16A). This result did not change even after adjusting for genotype principal components and in sensitivity analyses limited to European ancestry individuals, thus, arguing against a broad polygenic background driving the observed phenotype variability. This was followed by a rare variant analysis to estimate the effect of coding variants of individual genes on ASCVD status. Given the sample size, the study might be underpowered to detect individual genes at a genome-wide significance threshold. Therefore, the variants within EGFR pathway genes were examined. The analysis showed that EGFR pathway genes were enriched for rare variants in individuals with ASCVD in the cohort (Figure 16D). This finding demonstrated a putative causal role of the Atorney Docket No. 44807-0499WO1 / C18224

[0126] EGFR pathway in the ASCVD phenotype within the patient population. However, no rare coding variants were identified within HB-EGF itself, which suggests the possibility that either (a) HB-EGF is transcriptionally regulated by non-coding variants, which were unable to ascertain with the limited powered cohort, (b) HB-EGF expression is influenced by variants in genes upstream of HB-EGF, or (c) HB-EGF is not a part of the causal pathway.

[0127] To further evaluate whether HB-EGF is causally linked to ASCVD, it was tested whether common expression quantitative trait locus (eQTL) variants for HB-EGF in whole blood colocalize with GWAS loci for coronary artery disease using data from two large independent cohorts: eQTLGen and CARDIOGRAM. The results showed significant association between HB-EGF eQTLs and CAD; however, it did not meet the genome-wide significance threshold and has therefore not been previously detected in discovery GWAS. Further, this signal colocalized strongly with the HB-EGF eQTL signal suggesting a shared causal variant (Figure 16C). These findings identified the overall EGFR signaling and HB- EGF specifically as novel candidate targets for ASCVD pathogenesis.

[0128] HB-EGF regulates proatherogenic responses in monocytes and vascular smooth muscle cells in vitro

[0129] To evaluate the potential proatherogenic effects of HB-EGF in vitro, THP-1 cells were treated with PMA and the direct effects of HB-EGF in monocyte-derived macrophages were examined. These macrophages were exposed to siRNA against HB-EGF and nontargeting scramble siRNA for 48 hours. Subsequently, dil-ox-LDL uptake and foam cell formation were measured. The results showed that HB-EGF silencing decreased oxidized LDL uptake and lowered rate of foam cell formation (Figure 17D-E). The opposite effects were observed upon exposure of THP-1 derived macrophages to recombinant HB-EGF when compared to vehicle control. These results showed that HB-EGF directly modulates macrophage function to promote pro-atherogenic foam cell generation. Further, since HB- EGF is secreted by monocytes and macrophages, its effects need not be limited to macrophages themselves but could also affect other cells within the atherosclerotic milieu. The analysis of the published single cell RNA-seq data of human atherosclerotic arteries showed that the primary cell type that expressed EGFR, the presumed HB-EGF target, within Atorney Docket No. 44807-0499WO1 / C18224 the artery were VSMCs. Transition of VSMCs from their quiescent contractile state to a proliferative synthetic phenotype is considered a hallmark of atherosclerotic plaque progression. Therefore, human coronary artery VSMCs were co-cultured with THP-1 derived macrophages treated with siRNA against HB-EGF or siRNA scramble using a transwell coculture approach. It was found that co-culture with THP-1 macrophages increased VSMC proliferation, and the effect was almost entirely reversed upon silencing of HB-EGF in the macrophages (Figure 20). To evaluate whether HB-EGF directly regulates VSMC phenotype and proliferation, human coronary artery VSMCs were exposed to recombinant HB-EGF or the vehicle control. QPCR of those groups revealed a down-regulation of contractile VSMC markers (ACTA2, CNN1), upregulation of synthetic VSMC markers (KLF4, SOX9), and upregulation of proliferation markers (MKI67) upon HB-EGF exposure (Figures 21). These results demonstrate the multifaceted proatherogenic functional role of secreted HB-EGF in the microenvironment of atherosclerotic plaques.

[0130] EGFR inhibition attenuated arterial inflammation in patients with cancer receiving EGFR inhibitors

[0131] Since EGFR inhibitors are clinically used as cancer therapies, to investigate any potential unintended effects of these medications on arterial inflammation and atherosclerosis, sequential pre- and post-EGFR inhibitor treatment18F-FDG PET / CT data were retrospectively analyzed from an independent cohort of patients with malignancy who received EGFR inhibitors for their cancer and had evidence of atherosclerosis in their baseline scans. Intriguingly the results showed a significant reduction in arterial inflammation following therapy (target-to-background ratio (TBR) ± SD 1.08 ± 0.11 for the pre-EGFR inhibitor treatment scans and 0.96 ± 0.17 for the post-EGFR inhibitor scans, p = 0.007). These results demonstrate a causal role of EGFR on vascular inflammation in atherosclerotic plaques and suggest the EGFR cascade as a potential target for therapies against ASCVD. Atorney Docket No. 44807-0499WO1 / C18224

[0132] METHODS

[0133] Study Population

[0134] This was a single-center and a cross-sectional study. Adults with hypercholesterolemia were recruited and stratified based on the presence or absence of ASCVD. ASCVD was defined by a prior history of myocardial infarction, coronary revascularization, or a coronary artery calcium (CAC) score exceeding the 95th percentile for age and sex. The comparator group included individuals with no clinical history of ASCVD and a CAC score of 0 or no sonographic evidence of carotid atherosclerosis, defined by the absence of carotid intima-media thickness. To isolate hypercholesterolemia-associated disease, individuals with diabetes mellitus, hypertension, class II / III obesity (BMI >35 kg / m2), smoking history, chronic kidney disease, elevated lipoprotein(a) [Lp(a) >75 nmol / L], monogenic dyslipidemia (e.g., pathogenic variants in LDLR, APOB, or PCSK9), active infection, malignancy, or use of immunosuppressive medications were excluded. Participants who were within 1 year of a clinical ASCVD event were also excluded. All participants provided written informed consent.

[0135] PBMC Isolation and Processing

[0136] Peripheral blood was collected in sodium heparin tubes and diluted to 1 : 1.5 with phosphate-buffered saline (PBS, IX). Diluted blood was gently layered over Lymphoprep™ (StemCell Technologies) in 50 mL conical tubes at a 5:4 blood-to-reagent ratio. Samples were centrifuged at 400 / g for 40 minutes at room temperature with the brake off. The mononuclear cell layer was collected, washed with PBS, and centrifuged at 450*g for 10 minutes, followed by a second wash in PBS and centrifugation at 400xg for 5 minutes. The final pellet was resuspended in complete RPMI medium (RPMI 1640 supplemented with 10% fetal bovine serum and GlutaMAX™) and filtered through a 70 pm cell strainer. Cells were counted using a hemocytometer and trypan blue (1 : 1.25 dilution). PBMCs were cryopreserved in freezing medium (50% complete RPMI + 10% DMSO) at a final concentration of 2-4* 10® cells / mL. Atorney Docket No. 44807-0499WO1 / C18224

[0137] Single-Cell RNA Sequencing

[0138] PBMCs were isolated from fresh whole blood as described above and 3’ mRNA library preparation was performed according to the lOx Genomics protocol that targets 50,000 reads per cell and 10,000 cells per single cell capture. Samples were multiplexed using cholesterol-modified hashtag oligos (CMOs) with 3 samples loaded in each single cell capture. Sequencing was performed at the lOx Genomics Chromium platform. Sequencing data were aligned with Cell Ranger and demultiplexed via Souporcell with cluster assignment verified using CMOs. Downstream analysis was performed in Seurat (v4.0). Dimensionality reduction was performed using UMAP with an adapted resolution of 0.5. Differences in contributions to various clusters were analyzed based on sex, age, and ethnicity, as well as variations in cell proportions. Clusters were identified using broadly accepted canonical markers and validated by projection onto a previously defined reference dataset (OneKlK) as implemented in Seurat MapQuery. Differential gene expression was performed in individual cell clusters using pseudobulk analysis with edgeR v4.2.2. Speckle vl.4.0 was used to examine differences in cell proportions across various experimental groups. Differences were considered significant if their FDR-adjusted p-value was below 0.05. Pathway enrichment analysis was performed with FGSEA using Reactome and Gene Ontology pathways.

[0139] Genomic analysis with CAFEH (colocalization and fine-mapping in the presence of allelic heterogeneity)

[0140] CAFEH, a Bayesian multi-trait colocalization framework, was used to assess the association between the gene of interest and ASCVD. CAFEH was applied together with the GWAS (Van Der Harst et al., Cir Res 122(3): 433-443(2018)) for CAD and expression quantitative trait locus (eQTL) data from eQTLGen. CAFEH was used to calculate the top component colocalization probability, utilizing LD (linkage disequilibrium reference panel) from the 1000 Genomes Project. Atorney Docket No. 44807-0499WO1 / C18224

[0141] Genetic Analysis

[0142] WGS was performed using the NovaSeq Illumina platform targeting a 30x coverage genome-wide. Reads were aligned to the GRCh38 human reference genome using bwa-mem and variant calling was performed following the Genome Analysis Toolkit 4 (GATK4) pipeline. PRS for coronary artery disease were calculated using a previously validated 6- million variant PRS, harmonized to hg38, and computed with PLINK2 (Purcell et al., The Amer Jour of Hum Gen, 81(3): 559-575(2007)). Rare variant burden testing was performed using the SKAT-0 method after variant annotation with Variant Effect Predictor (VEP) and filtering for predicted high-impact variants.

[0143] Variant calling was performed following standard best practices

[0144] Raw FASTQ files underwent quality control using FastQC, followed by adapter trimming with Skewer when needed. Alignment to the human reference genome GRCh38 was performed with Burrrows Wheeler Aligner (BWA). After alignment, PCR duplicates were removed, and base quality scores were recalibrated using Genome Analysis Toolkit (GATK). Alignment and insert size metrics were collected, and variants were called using GATK’s HaplotypeCaller, resulting in approximately 4 million variants per individual and around 13 million after merging all samples across both conditions.

[0145] Polygenic risk scores (PRS) were computed to quantify individuals ’ inherited risk based on the cumulative effect of common genetic variants

[0146] Each variant was weighted by its reported effect size, and scores were calculated using PLINK2. Summary statistics from Khera et al., Nat Gen 50(9): 1219-1224 (2018) for coronary artery disease (CAD) were used, and harmonization with the genotype data was ensured. Genotype files were derived from the combined set of healthy and disease samples (n = 15). Principal component analysis (PCA) was performed on genotype data to adjust for population stratification. Atorney Docket No. 44807-0499WO1 / C18224

[0147] Rare variant analysis was performed using the SKA T

[0148] Variant files from healthy and disease samples were first merged using bcftools. Annotation of variants was conducted with the VEP for Homo sapiens (GRCh38), using rsID notation and the web-based interface. Functional consequences were classified according to VEP impact categories (e.g., HIGH, MODERATE). Analyses were performed using two filtering strategies: one restricted to variants with HIGH predicted impact, and another including both HIGH and MODERATE. After filtering, SNP Set Data (SSD) files were generated to run the SKAT association tests.

[0149] Functional Assays

[0150] THP-1 cells were differentiated into macrophages by treating them with PMA for 24 hours as per the manufacturer’s recommendations. Adherent THP-1 derived macrophages were transfected with siRNA targeting HB-EGF or a scrambled control. Functional endpoints included Dil-LDL uptake and Oil Red O staining for foam cell formation.

[0151] Dil-LDL Uptake.

[0152] Monocytes were seeded at 2-3 x 105cells per well in 24-well plates in RPMI 1640 medium supplemented with 10% fetal bovine serum and GlutaMAX™. Cells were incubated with 10 pg / mL Dil-labeled low-density lipoprotein (Dil-LDL; Thermo Fisher Scientific) at 37 °C for 4 hours. After washing with PBS, cells were fixed with 4% paraformaldehyde. Intracellular fluorescence was visualized using fluorescence microscopy, and mean fluorescence intensity (MFI) was quantified across multiple fields using ImageJ software (Kruth HS., Curr Opin Lipidol, 22:368-373(2011)).

[0153] Oil Red O Foam Cell Assay.

[0154] Monocytes were incubated with 50 pg / mL native LDL for 48 hours in the same medium conditions. Cells were then fixed with 4% paraformaldehyde, stained with filtered Oil Red O solution (0.3% in isopropanol, diluted 3:2 with distilled water), and counterstained with hematoxylin. Brightfield microscopy was used to visualize lipid-laden foam cells, and the percentage of Oil Red O-positive cells was quantified (Liang CP et al., J Biol Chem, 279:50092-50099(2004)). This protocol aligns with standard methods integrating Dil-LDL Atorney Docket No. 44807-0499WO1 / C18224 uptake and Oil Red O staining to assess macrophage lipid handling (de la Llera-Moya M et al., J Lipid Res 42: 1969-1978(2001)).

[0155] Serum HB-EGF and Amphiregulin Levels

[0156] Serum concentrations of HB-EGF and Amphiregulin were measured from aliquots collected prior to enrollment and stored at -80 °C. Quantification was performed using commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems), run on a fully automated ELISA platform (DS2, Dynex Technologies, Inc). Samples were assayed in duplicate according to the manufacturer’s protocol.

[0157] PET Imaging

[0158] A separate cohort of patients with malignancy undergoing treatment with EGFR inhibitors underwent serial18F-FDG PET / CT imaging. These individuals were not part of the primary lipid clinic study. Arterial inflammation was quantified as the target-to-background ratio (TBR), defined as mean arterial18F-FDG uptake divided by venous background activity, and was measured before and following the initiation of an EGFR inhibitor. Analyses were stratified by baseline TBR. The median time (QI, Q3) from EGFR inhibitor treatment initiation to the baseline scan was 1.0 (0.6, 3.5) month, and the median time (QI, Q3) between the baseline and follow-up scans was 18.0 (8.0, 35.0) months. Among the 21 patients with underlying malignancy who were included in this analysis, 76.2% had lung malignancy (n=16), 4.8% had colorectal carcinoma (n=l), 4.8% had breast carcinoma (n=l), and 14.3% had squamous cell carcinoma (n=3).

[0159] OTHER EMBODIMENTS

[0160] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 44807-0499WO1 / C18224WHAT IS CLAIMED IS:

1. A method for treating atherosclerosis in a subject in need thereof, the method comprising: a) determining or having determined that the subject expresses binding epidermal growth factor (HB-EGF); and b) administering to the subject a therapeutically effective amount of an HB-EGF inhibitor.

2. A method for treating atherosclerosis in a subject expressing HB-EGF, the method comprising administering to the subject a therapeutically effective amount of an HB- EGF inhibitor.

3. The method of any one of claims 1 or 2, wherein the subject has atherosclerotic cardiovascular disease (ASCVD), aortic aneurysm, cerebral atherosclerosis, carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, peripheral artery disease, and / or aortic valve stenosis.

4. A method for reducing the risk of developing atherosclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an HB-EGF inhibitor.

5. The method of claim 4, wherein the subject is at risk of developing atherosclerosis or an atherosclerotic condition.

6. The method of any one of claims 1-5, wherein the HB-EGF inhibitor comprises an inhibitory antibody, an inhibitory nucleic acid molecule, a small molecule inhibitor, an inhibitor of HB-EGF cleavage, an inhibitor of HB-EGF activity in a cell nucleus.

7. The method of claim 6, wherein the small molecule inhibitor comprises CRM197.Attorney Docket No. 44807-0499WO1 / C182248. The method of claim 6, wherein the inhibitory antibody comprises U3-1565.

9. The method of claim 6, wherein the inhibitory nucleic acid molecule comprises an RNAi, a siRNA, an antisense oligonucleotides (ASO), and / or a shRNA.

10. The method of claim 6, wherein the inhibitor of HB-EGF cleavage comprises an a disintegrin and metalloprotease (ADAM) inhibitor.

11. The method of any one of the above claims, further comprising administering to the subject one or more additional anti-atherosclerotic agents.

12. The method of claim 11, wherein the anti-atherosclerotic agent comprises statins, proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, ezetimibe, bempedoic acid, niacin, cholesterol sequestrants, glucagon-like peptide-1 (GLP1) agonists, anti-platelet agents, anti -thrombotic agents, and / or any combinations thereof.

13. A method for diagnosing atherosclerosis in a subject, wherein the method comprises determining levels of HB-EGF in a sample from a subject, wherein elevated levels of HB-EGF in the sample compared to a control sample identifies that the subject has atherosclerosis.

14. The method of claim 13, wherein the control sample is a biological sample isolated from a healthy subject.

15. The method any one of claims 1-14, wherein the subject is a mammal.

16. The method of claim 15, wherein the mammal is a human.Attorney Docket No. 44807-0499WO1 / C1822417. The method of claim 13, wherein the sample is a biological sample.

18. The method of any one of claims 14 or 17, wherein the biological sample comprises blood, plasma, urine, serum, or saliva.

19. The method any one of claims 1-18, wherein administering the therapeutically effective amount of the HB-EGF inhibitor to the subject reduces the uptake of low- density lipoprotein (LDL), vascular inflammation, atherosclerotic plaque formation, smooth muscle cell proliferation and phenotype switching, and / or endothelial dysfunction.

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