Treatment of cancer-promoted atherosclerosis

Abstract

Cancer can increase risk of atherosclerosis in an individual. Specific genes are upregulated in endothelial cells when tumors are present. Methods and agents that target these upregulated genes are useful in the prevention and treatment of cancer-promoted atherosclerosis.

Description

TREATMENT OF CANCER-PROMOTED ATHEROSCLEROSISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 1 19 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 569,592, filed March 25, 2024 thedisclosure of which application is herein incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOP ENT

[0002] This invention was made with Government support under contracts HL141851 , HL144475 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] Atherosclerosis and cancer are common human diseases that result from a combination of genetic predisposition and exposure to risk factors. Atherosclerotic cardiovascular disease (ASCVD) remains the primary cause of morbidity and mortality worldwide. In general, atherosclerosis is believed to be a complex disease involving multiple biological pathways. Variations in the natural history of the atherosclerotic disease process, as well as differential response to risk factors and variations in the individual response to therapy, reflect in part differences in genetic background and their intricate interactions with the environmental factors that are responsible for the initiation and modification of the disease. Atherosclerotic disease is also influenced by the complex nature of the cardiovascular system itself where anatomy, function and biology all play important roles in health as well as disease.

[0004] The interaction between ASCVD, and cancer is highly challenging to study, because ASCVD and cancer share multiple risk factors. For example risk factors like smoking, a diet high in saturated fat, a higher intake of sugar and high-glycemic-index foods, a sedentary lifestyle, and a lack of exercise may contribute both to cancer and the ASCVD. Further, genetic predisposition, under the influence of environmental and atherogenic factors, may be implicated in both cardiovascular diseases and cancer.

[0005] New studies suggest the presence of cancer or atherosclerosis may impact each other. Epidemiological studies show that cancer patients have a markedly higher risk of developing atherosclerosis than those without malignancy. However, a direct role of cancer on atherosclerosis has not been established, and it is unclear if therapies can be developed that might lessen the burden of CVD amongst cancer patients and survivors. Identification and targeting of cancer processes that affect atherosclerosis are of great interest and are addressed herein.SUMMARY OF THE INVENTION

[0006] Methods and compositions are provided for the prevention and treatment of cancer- promoted ASCVD in a subject. The effect of cancer on atherogenesis is shown, where specific genes are upregulated in aortic cells, e.g. endothelial cells, when tumors are present. In particular, a panel of genes is shown to be upregulated in aortic endothelial cells when tumors are present, across multiple cancer types. The upregulated genes include Wnt9b, Gm39822, Nppc, Hba-a1 , Ret, Pthlh, Sema3g, and Lrg1 , which may be referred to as cancer-promoted atherosclerosis (CPA) genes. Methods and agents that target one or more of these upregulated genes are useful in the prevention and treatment of cancer-promoted atherosclerosis.

[0007] In an embodiment, an effective dose of an agent that modulates activity of a CPA gene or gene product is administered to an individual with cancer in a dose and for a period of time effective to stabilize, prevent or reduce atherosclerotic plaque in the individual. In some embodiments the agent inhibits activity of a CPA gene or gene product. In some embodiments, multiple CPA genes are targeted. In some embodiments, LRG1 is targeted in combination with, for example and without limitation, NPPC and / or Sema3G.

[0008] LRG1 is an angiogenic factor associated with tumor metastasis and cancer mortality. In addition to upregulation of LRG1 , it was found that tumor-bearing animals had increased serum levels of TNFa and IL-6. Both TNFoc and IL-6 are shown to stimulate LRG1 gene expression in human aortic endothelial cells (ECs), and to increase angiogenesis. Tumors increased atherogenesis and plaque vulnerability by regulating LRG1 expression in diseased vessels in animals. LRG1 mediates tumor-promoted atherosclerosis by stimulating angiogenesis and intraplaque neovessel formation, increasing plaque vulnerability. Blocking the interaction between TNFoc and LRG1 can reduce the risk of ASCVD in cancer patients.

[0009] In some embodiments the individual has been diagnosed with cancer prior to treatment. In some embodiments the cancer is a solid tumor, e.g. carcinoma, melanoma, glioma, sarcoma, lymphoma, etc. In some embodiments the cancer is a carcinoma, e.g. lung carcinoma, breast carcinoma, colon carcinoma, etc. In some embodiments the individual is undergoing treatment for the cancer. In some embodiments the individual has completed treatment for the cancer. The individual may have one or more risk factors for the development of atherosclerosis, in addition to cancer. In some embodiments an individual selected for treatment has cancer and has 1 , 2, 3, 4, or more risk factors for atherosclerosis, including elevated levels of LDL cholesterol, low levels of HDL cholesterol, high blood pressure, type 2 diabetes, smoking, and obesity.

[0010] In some embodiments, methods and compositions are provided for the prevention and / or treatment of cancer-promoted ASCVD in a subject by inhibition of LRG1 activity. Insuch methods, an effective dose of an agent that inhibits LRG1 activity is administered to an individual with cancer, where the dose is effective to reduce LRG1 activity relative to an untreated control. The agent may be an agent that binds to and inhibits LRG1 . The agent may be an agent that binds to and inhibits an activator of LRG1 , including TNFoc and IL-6, or that binds to and inhibits a receptor of TNFa or IL-6. In some embodiments the inhibitor is an antibody. In some embodiments the inhibitor is a soluble receptor, e.g. TNFR1 , etc. In some embodiments the inhibitor acts at the genetic level, e.g. an RNAi, an anti-sense oligonucleotide, a genome editing agent, etc. In some embodiments the inhibitor is a small molecule drug. The agent that inhibits LRG1 activity may be administered systemically, e.g. by parenteral administration, oral administration, etc.

[0011] In some embodiments the methods of treatment further comprise monitoring the level of atherosclerosis, e.g. plaque formation, vascular inflammation, LRG1 activity, etc. in a subject following treatment. In some embodiments an individual is treated according to the results of such an assessment, e.g. therapy is continued or discontinued based on the results, or dosages are altered according to the results. In some embodiments, following treatment the indicia of atherosclerosis, e.g. plaque formation, vascular inflammation, etc. is reduced by at least 10% relative to an untreated control. In some embodiments, the indicia is reduced by at least 20%, at least 30%, at least 50% or more.

[0012] Another aspect of the present invention relates to the use of an agent that inhibits cancer-promoted atherosclerosis in the manufacture of a medicament to stabilize, prevent or reduce atherosclerotic plaque, wherein the medicament is administered to an individual having, or at risk of having, cancer-promoted atherosclerosis. Still another aspect of the present invention provides a kit to stabilize, prevent or reduce atherosclerotic plaque in an individual with cancer. The kit includes an effective dose of an agent that modulates activity of a CPA gene or gene product, e.g. LRG1 , in an amount sufficient to stabilize, prevent or reduce atherosclerotic plaque. The kit may also instructions for use, reagents for monitoring atherosclerotic disease, and the like.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGS. 1 A-1 B. a) The timeline for tumor inoculation in an atherosclerotic mouse model. Apoe C57BL / 6 mice were fed a high-fat diet for 12 weeks, followed by inoculation of MC38 colorectal tumor cells or buffer subcutaneously at the beginning of the last 4 weeks away from the harvesting time point, b) The relative plaque size of the aortic sinus from Apoe1mice bearing tumors versus tumor-free control mice, n = 19 for the control mice, n = 21 for the tumor-bearing mice. Student t-test was performed, p-value = 0.13.

[0014] FIGS. 2A-2B. a) The relative area of the intima of the tandem stenosis (TS) segment of the carotid artery between Apoe1mice bearing tumors and tumor-free control mice, n = 11 for the control mice and n = 13 for the tumor-bearing mice. Student t-test was performed, p- value = 0.1500. b) The relative area of the media of the tandem stenosis (TS) segment of the carotid artery, n = 11 for the control mice and n = 13 for the tumor-bearing mice. Mann-Whitney test was performed, p-value = 0.1500. c) The ratio of intima versus media of the tandem stenosis (TS) segment of the carotid artery, n = 1 1 for the control mice and n = 13 for the tumor-bearing mice. Welch’s t-test was performed, p-value = 0.0964.

[0015] FIGS. 3A-3D. Representative immunofluorescence images of the cross-sections of the carotid arteries after tandem stenosis of control mice (a) and tumor mice (b). The sections were stained for CD31 (an endothelial cell marker), TER-119 (a red blood cell marker), and DARI (a DNA marker). The average signal area of CD31 (c) and TER-1 19 (d) was quantified across the tandem stenosis samples of the batch of mice from Figure 2. The Mann-Whitney test was performed, p-value = 0.0129 for the CD31 signal, p-value = 0.0156 for the TER-1 19 signal.

[0016] Fig. 4. Venn Diagram of overlapped genes among various datasets that were upregulated in the aortic arches of mice with MC38 colon cancer than control mice without tumors. Each dataset (A, B, C, D, and E) represents a unique combination of genetic background, diet, and age. A: 18-week-old, wildtype (WT) mice fed with a chow diet (CD); B: 18-week-old, Apoe1mice fed with a high-fat diet (HFD); C: 21 -29 weeks old, Apoe1mice fed with HFD; D: 18-week-old, WT mice fed with HFD; E: 18-week-old, Apoe7mice fed with CD.

[0017] FIG. 5. Venn Diagram of overlapped genes among various datasets that were upregulated in the aortic arches of mice with different types of cancer than control mice without tumors.

[0018] FIGS. 6A-6C. Olink analysis was performed on the serum samples collected from mice bearing tumors (MC38 colon, B16F10 melanoma cells, Lewis lung carcinoma, or E0771 breast) or tumor-free mice, a) The enrichment analysis of upregulated proteins detected by Olink. b) and c) Relative protein levels (TNF or IL-6) detected by Olink. “ p-value < 0.01 ; “* p-value < 0.001 .

[0019] FIGS. 7A-7B. a) The relative mRNA level of Lrg1 in cultured human aortic endothelial cells being treated with various doses of TNF. b) The relative mRNA level of Lrg1 in cultured human aortic endothelial cells being treated with TNF and an anti-TNF antibody.

[0020] FIGS. 8A-8B. a) The relative mRNA level of Lrg1 in cultured human aortic endothelial cells being treated with conditional media from primary colon epithelial cells or colon cell line, b) The parameters of tube formation assays for endothelial cells treated with conditional media from primary colon cells or the cell line. One-way ANOVA was performed. The parameters oftube formation assays for endothelial cells being treated with conditional media from colon cell line plus IgG or anti-TNF antibody. * p-value < 0.05, “ p-value < 0.01 , “* p-value < 0.001 , **** p-value < 0.0001.

[0021] FIG. 9. The relative intima area, relative media area, and the intima versus media ratio of the tandem stenosis segment of mice treated with anti-TNF antibody (Etanercept) or IgG, and inoculated with tumors or buffer.

[0022] FIGS. 10A-10C. LRG1 upregulation in human carotid plaques of cancer patients, (a) Representative immunohistochemistry images showing plaque hemorrhage (Elastica van Gieson and Glycophorin A staining) and LRG1 expression in carotid plaques from patients, (b) Quantification of LRG1 mRNA expression in carotid plaques from cancer patients compared to non-cancer matched controls, (c) LRG1 immunostaining intensity in carotid plaques from cancer patients versus non-cancer matched controls, (n = 15 per group).

[0023] FIGS. 1 1A-11 C. Cancer patients have higher circulating TNF levels in blood, (a) Circulating TNF levels in patients with any type of cancer compared to cancer-free individuals.(b) Circulating TNF levels in patients with colorectal cancer compared to cancer-free individuals, (c) Association between serum TNF levels and major adverse cardiovascular events (MACE), showing that patients with higher TNF levels have an increased risk of MACE.

[0024] FIGS. 12A-12C. TNF inhibition or LRG1 knockdown block tumor-induced intraplaque neovascularization. Targeting TNF and LRG1 reduces tumor-induced intraplaque neovascularization in the tandem stenosis model. Neovessel formation was assessed using three endothelial and angiogenesis markers: (a) CD31 , (b) von Willebrand factor (VWF), and(c) isolectin B4 (IB4).DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to methods of treating a subject for cancer-promoted atherosclerosis.

[0026] Coronary artery disease (CAD) is a narrowing or blockage of the arteries and vessels that provide oxygen and nutrients to the heart. It is caused by atherosclerosis, an accumulation of fatty materials on the inner linings of arteries. The resulting blockage restricts blood flow to the heart. When the blood flow is completely cut off, the result is a heart attack. CAD is the leading cause of death for both men and women in the United States.

[0027] Atherosclerosis (also referred to as arteriosclerosis, atheromatous vascular disease, arterial occlusive disease) as used herein, refers to a cardiovascular disease characterized by plaque accumulation on vessel walls and vascular inflammation. The plaque consists of accumulated intracellular and extracellular lipids, smooth muscle cells, connective tissue,inflammatory cells, and glycosaminoglycans. Inflammation occurs in combination with lipid accumulation in the vessel wall, and vascular inflammation is with the hallmark of atherosclerosis disease process.

[0028] Myocardial infarction is an ischemic myocardial necrosis usually resulting from abrupt reduction in coronary blood flow to a segment of myocardium. In the great majority of patients with acute Ml, an acute thrombus, often associated with plaque rupture, occludes the artery that supplies the damaged area. Plaque rupture occurs generally in vessels previously partially obstructed by an atherosclerotic plaque enriched in inflammatory cells. Altered platelet function induced by endothelial dysfunction and vascular inflammation in the atherosclerotic plaque presumably contributes to thrombogenesis. Myocardial infarction can be classified into ST-elevation and non-ST elevation Ml (also referred to as unstable angina). In both forms of myocardial infarction, there is myocardial necrosis. In ST-elevation myocardial infraction there is transmural myocardial injury which leads to ST-elevations on electrocardiogram. In non-ST elevation myocardial infarction, the injury is sub-endocardial and is not associated with ST segment elevation on electrocardiogram. Myocardial infarction (both ST and non-ST elevation) represents an unstable form of atherosclerotic cardiovascular disease. Acute coronary syndrome encompasses all forms of unstable coronary artery disease. Heart failure can occur as a result of myocardial dysfunction caused by myocardial infraction.

[0029] Angina refers to chest pain or discomfort resulting from inadequate blood flow to the heart. Angina can be a symptom of atherosclerotic cardiovascular disease. Angina may be classified as stable, which follows a regular chronic pattern of symptoms, unlike the unstable forms of atherosclerotic vascular disease. The pathophysiological basis of stable atherosclerotic cardiovascular disease is also complicated but is biologically distinct from the unstable form. Generally stable angina is not myocardial necrosis.

[0030] Atherosclerosis is influenced by various risk factors. Perhaps the most prominent risk factor is high levels of low-density lipoprotein (LDL) cholesterol in the blood. When LDL cholesterol levels are elevated, it can lead to the deposition of cholesterol in the arterial walls, initiating the formation of plaque. Additionally, low levels of high-density lipoprotein (HDL) cholesterol, often termed "good" cholesterol, can contribute to atherosclerosis by reducing the efficiency of cholesterol clearance from the arteries, allowing for its accumulation.

[0031] Another significant risk factor for atherosclerosis is hypertension, or high blood pressure. Elevated blood pressure puts added stress on the arterial walls, causing them to become stiff and damaged over time. This damage creates an environment conducive to the development of atherosclerotic plaques. Furthermore, hypertension can lead to complications such as aneurysms and ruptures in weakened arterial walls, exacerbating the progression ofatherosclerosis. Controlling blood pressure through lifestyle modifications and medication can help mitigate this risk.

[0032] Further, lifestyle factors such as smoking, poor diet, physical inactivity, and obesity play critical roles in the development and progression of atherosclerosis. Smoking, for instance, not only damages the lining of blood vessels but also promotes the formation of plaque and increases the risk of blood clots. A diet high in saturated and trans fats, along with excessive consumption of sugars and refined carbohydrates, can contribute to elevated cholesterol levels and obesity, both of which are associated with atherosclerosis. Sedentary behavior and obesity further compound the risk by promoting conditions such as insulin resistance and inflammation, which can accelerate plaque formation and arterial damage.

[0033] An individual suitable for treatment to reduce development of cancer-promoted atherosclerosis typically has, or had, cancer, which cancers include, without limitation, adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, cervical cancer, childhood Non-Hodgkin's lymphoma, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g. Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, children's leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, melanoma skin cancer, non-melanoma skin cancers, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer (e.g. uterine sarcoma), transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid carcinoma, bronchial adenoma, choriocarinoma, head and neck cancers, teratocarcinoma, or Waldenstrom's macroglobulinemia. Solid cancers may be a carcinoma, glioblastoma, lymphoma, sarcoma, melanoma, etc.

[0034] Chronic inflammation increases the risk of many cancers. For example, COPD (chronic obstructive pulmonary disease), alpha-1 antitrypsin deficiency, and pulmonary fibrosis increase susceptibility to lung cancer. People whose lungs are scarred by other lung diseases(eg, tuberculosis) are at increased risk of lung cancer. Also, active smokers who take betacarotene supplements may have an increased risk of developing lung cancer.

[0035] Individuals may be treated for cancer, e.g. chemotherapy may include Abitrexate (Methotrexate Injection), Abraxane (Paclitaxel Injection), Adcetris (Brentuximab Vedotin Injection), Adriamycin (Doxorubicin), Adrucil Injection (5-FU (fluorouracil)), Afinitor (Everolimus) , Afinitor Disperz (Everolimus) , Alimta (PEMET EXED), Alkeran Injection (Melphalan Injection), Alkeran Tablets (Melphalan), Aredia (Pamidronate), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arzerra (Ofatumumab Injection), Avastin (Bevacizumab), Bexxar (Tositumomab), BiCNU (Carmustine), Blenoxane (Bleomycin), Bosulif (Bosutinib), Busulfex Injection (Busulfan Injection), Campath (Alemtuzumab), Camptosar (Irinotecan), Caprelsa (Vandetanib), Casodex (Bicalutamide), CeeNU (Lomustine), CeeNU Dose Pack (Lomustine), Cerubidine (Daunorubicin), Clolar (Clofarabine Injection), Cometriq (Cabozantinib), Cosmegen (Dactinomycin), CytosarU (Cytarabine), Cytoxan (Cytoxan), Cytoxan Injection (Cyclophosphamide Injection), Dacogen (Decitabine), DaunoXome (Daunorubicin Lipid Complex Injection), Decadron (Dexamethasone), DepoCyt (Cytarabine Lipid Complex Injection), Dexamethasone Intensol (Dexamethasone), Dexpak Taperpak (Dexamethasone), Docefrez (Docetaxel), Doxil (Doxorubicin Lipid Complex Injection), Droxia (Hydroxyurea), DTIC (Decarbazine), Eligard (Leuprolide), Ellence (Ellence (epirubicin)), Eloxatin (Eloxatin (oxaliplatin)), Elspar (Asparaginase), Emcyt (Estramustine), Erbitux (Cetuximab), Erivedge (Vismodegib), Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Injection), Eulexin (Flutamide), Fareston (Toremifene), Faslodex (Fulvestrant), Femara (Letrozole), Firmagon (Degarelix Injection), Fludara (Fludarabine), Folex (Methotrexate Injection), Folotyn (Pralatrexate Injection), FUDR (FUDR (floxuridine)), Gemzar (Gemcitabine), Gilotrif (Afatinib), Gleevec (Imatinib Mesylate), Gliadel Wafer (Carmustine wafer), Halaven (Eribulin Injection), Herceptin (Trastuzumab), Hexalen (Altretamine), Hycamtin (Topotecan), Hycamtin (Topotecan), Hydrea (Hydroxyurea), Iclusig (Ponatinib), Idamycin PFS (Idarubicin), Ifex (Ifosfamide), Inlyta (Axitinib), Intron A alfab (Interferon alfa- 2a), Iressa (Gefitinib), Istodax (Romidepsin Injection), Ixempra (Ixabepilone Injection), Jakafi (Ruxolitinib), Jevtana (Cabazitaxel Injection), Kadcyla (Ado-trastuzumab Emtansine), Kyprolis (Carfilzomib), Leukeran (Chlorambucil), Leukine (Sargramostim), Leustatin (Cladribine), Lupron (Leuprolide), Lupron Depot (Leuprolide), Lupron DepotPED (Leuprolide), Lysodren (Mitotane), Marqibo Kit (Vincristine Lipid Complex Injection), Matulane (Procarbazine), Megace (Megestrol), Mekinist (Trametinib), Mesnex (Mesna), Mesnex (Mesna Injection), Metastron (Strontium-89 Chloride), Mexate (Methotrexate Injection), Mustargen (Mechlorethamine), Mutamycin (Mitomycin), Myleran (Busulfan), Mylotarg (GemtuzumabOzogamicin), Navelbine (Vinorelbine), Neosar Injection (Cyclophosphamide Injection), Neulasta (filgrastim), Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (Sorafenib), Nilandron (Nilandron (nilutamide)), Nipent (Pentostatin), Nolvadex (Tamoxifen), Novantrone (Mitoxantrone), Oncaspar (Pegaspargase), Oncovin (Vincristine), Ontak (Denileukin Diftitox), Onxol (Paclitaxel Injection), Panretin (Alitretinoin), Paraplatin (Carboplatin), Perjeta (Pertuzumab Injection), Platinol (Cisplatin), Platinol (Cisplatin Injection), PlatinolAQ (Cisplatin), PlatinolAQ (Cisplatin Injection), Pomalyst (Pomalidomide), Prednisone Intensol (Prednisone), Proleukin (Aldesleukin), Purinethol (Mercaptopurine), Reclast (Zoledronic acid), Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab), RoferonA alfaa (Interferon alfa-2a), Rubex (Doxorubicin), Sandostatin (Octreotide), Sandostatin LAR Depot (Octreotide), Soltamox (Tamoxifen), Sprycel (Dasatinib), Sterapred (Prednisone), Sterapred DS (Prednisone), Stivarga (Regorafenib), Supprelin LA (Histrelin Implant), Sutent (Sunitinib), Sylatron (Peginterferon Alfa-2b Injection (Sylatron)), Synribo (Omacetaxine Injection), Tabloid (Thioguanine), Taflinar (Dabrafenib), Tarceva (Erlotinib), Targretin Capsules (Bexarotene), Tasigna (Decarbazine), Taxol (Paclitaxel Injection), Taxotere (Docetaxel), Temodar (Temozolomide), Temodar (Temozolomide Injection), Tepadina (Thiotepa), Thalomid (Thalidomide), TheraCys BCG (BCG), Thioplex (Thiotepa), TICE BCG (BCG), Toposar (Etoposide Injection), Torisel (Temsirolimus), Treanda (Bendamustine hydrochloride), Trelstar (Triptorelin Injection), Trexall (Methotrexate), Trisenox (Arsenic trioxide), Tykerb (lapatinib), Valstar (Valrubicin Intravesical), Vantas (Histrelin Implant), Vectibix (Panitumumab), Velban (Vinblastine), Velcade (Bortezomib), Vepesid (Etoposide), Vepesid (Etoposide Injection), Vesanoid (Tretinoin), Vidaza (Azacitidine), Vincasar PFS (Vincristine), Vincrex (Vincristine), Votrient (Pazopanib), Vumon (Teniposide), Wellcovorin IV (Leucovorin Injection), Xalkori (Crizotinib), Xeloda (Capecitabine), Xtandi (Enzalutamide), Yervoy (Ipilimumab Injection), Zaltrap (Ziv-aflibercept Injection), Zanosar (Streptozocin), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zoladex (Goserelin), Zolinza (Vorinostat), Zometa (Zoledronic acid), Zortress (Everolimus), Zytiga (Abiraterone), Nimotuzumab and immune checkpoint inhibitors such as nivolumab, pembrolizumab / MK-3475, pidilizumab and AMP-224 targeting PD-1 ; and BMS-935559, MEDI4736, MPDL3280A and MSB0010718C targeting PD-L1 and those targeting CTLA-4 such as ipilimumab.

[0036] Antibiotics, e.g. antibiotics with the classes of aminoglycosides; carbapenems; and the like; penicillins, e.g. penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc. penicillins in combination with p-lactamase inhibitors, cephalosporins, e.g. cefaclor, cefazolin, cefuroxime, moxalactam, etc:; tetracyclines; cephalosporins; quinolones; lincomycins; macrolides; sulfonamides; glycopeptides including the anti-infective antibiotics vancomycin, teicoplanin, telavancin, ramoplanin and decaplanin. Derivatives of vancomycininclude, for example, oritavancin and dalbavancin (both lipoglycopeptides). Telavancin is a semi-synthetic lipoglycopeptide derivative of vancomycin. Other vancomycin analogs are disclosed, for example, in WO 2015022335 A1 and Chen et al. (2003) PNAS 100(10): 5658- 5663, each herein specifically incorporated by reference. Non-limiting examples of antibiotics include vancomycin, linezolid, azithromycin, daptomycin, colistin, eperezolid, fusidic acid, rifampicin, tetracyclin, fidaxomicin, clindamycin, lincomycin, rifalazil, and clarithromycin.

[0037] Radiotherapy means the use of radiation, usually X-rays, to treat illness. X-rays were discovered in 1895 and since then radiation has been used in medicine for diagnosis and investigation (X-rays) and treatment (radiotherapy). Radiotherapy may be from outside the body as external radiotherapy, using X-rays, cobalt irradiation, electrons, and more rarely other particles such as protons. It may also be from within the body as internal radiotherapy, which uses radioactive metals or liquids (isotopes) to treat cancer.

[0038] Tumor necrosis factor alpha (TNF-a) is a pro-inflammatory cytokine produced by macrophages and lymphocytes that mediates inflammation in a number of conditions. The strategies for inhibiting TNF that have been most extensively studied to date consist of monoclonal anti-TNFa antibodies, anti-TNF receptor antibodies, and soluble TNF receptors (sTNF-R). Some specific examples of anti-TNF agents that are in current clinical use are listed below.

[0039] Infliximab (IFX) is a recombinant lgG1 monoclonal antibody specific for TNF-a that hinders the cytokine from triggering the cellular TNF receptor complex. IFX needs to be administered by intravenous infusion and has a terminal half-life of 8-10 days. Conventionally it is administered every 4-8 weeks and the dosage varies from 3 to 6 (to 10) mg / kg. Combination therapies of the present invention may use a dose or dosing schedule that provides for a lower dose or reduced dosing schedule relative to conventional schedules.

[0040] Adalimumab is a monoclonal antibody of recombinant immunoglobulin (lgG1 ) containing only human sequences of peptides. It is an antagonist of TNF-a, which is able to prevent the binding of TNF-a to its receptors. It has a half-life of 10-20 days. The conventional dose of ADA is 25 mg s.c. twice a week. Combination therapies of the present invention may use a dose or dosing schedule that provides for a lower dose or reduced dosing schedule relative to conventional schedules.

[0041] Golimumab is a human anti-TNF-a monoclonal antibody that is generated and matured in an in vivo system. GOLI has a high affinity and specificity for human TNF-a and effectively neutralizes TNF-a bioactivity in vitro. Conventional dosing is from 50 to 100 mg. Combination therapies of the present invention may use a dose or dosing schedule that provides for a lower dose or reduced dosing schedule relative to conventional schedules.

[0042] Etanercept is a genetically engineered protein comprising two molecules of the extracellular domain of TNF receptor II (p75) and the Fc portion of lgG1 . Due to its half-life of 3-5.5 days, ETN is conventionally administered subcutaneously (s.c), either on a weekly basis (50 mg) or twice a week (25 mg). Combination therapies of the present invention may use a dose or dosing schedule that provides for a lower dose or reduced dosing schedule relative to conventional schedules.

[0043] Certolizumab is a pegylated anti-TNF-alpha monoclonal antibody. Certolizumab is a humanized antibody fragment (Fab') that is attached to polyethylene glycol to allow for less frequent administration. Certolizumab has a high affinity for human TNF-alpha, selectively targeting TNF-alpha in inflamed tissue. Although the presence of a Fab' portion allows certolizumab to retain the potency of the entire antibody, certolizumab is unable to bind phagocytic cells or to lyse cells because of a lack of an Fc portion. The elimination half-life of certolizumab was demonstrated to be 31 1 hours.

[0044] lnterleukin-6 (IL-6) is a cytokine involved in various inflammatory and immune responses. Dysregulation of IL-6 signaling has been implicated in several diseases, including rheumatoid arthritis, inflammatory bowel disease, and certain types of cancer. Inhibiting IL-6 signaling can be an effective therapeutic strategy in managing these conditions. Inhibitors of IL-6 that have been used in clinical practice include, for example, Tocilizumab (Actemra), which is a humanized monoclonal antibody that binds to both soluble and membrane-bound IL-6 receptors, thereby inhibiting IL-6 signaling. It is approved for the treatment of rheumatoid arthritis, juvenile idiopathic arthritis, giant cell arteritis, systemic juvenile idiopathic arthritis, and cytokine release syndrome induced by CAR T-cell therapy. Tocilizumab has shown efficacy in reducing disease activity and improving symptoms in these conditions.

[0045] Sarilumab (Kevzara): Sarilumab is another humanized monoclonal antibody that specifically targets the IL-6 receptor. It competitively inhibits IL-6 signaling by blocking the binding of IL-6 to its receptor. Sarilumab is approved for the treatment of rheumatoid arthritis in adults who have not responded adequately to conventional therapies. Like tocilizumab, sarilumab has demonstrated effectiveness in reducing inflammation and improving clinical outcomes in rheumatoid arthritis.

[0046] Siltuximab (Sylvant): Siltuximab is a chimeric monoclonal antibody that specifically binds to IL-6, preventing it from binding to its receptor. It is approved for the treatment of multicentric Castleman disease, a rare lymphoproliferative disorder characterized by excessive production of IL-6. Siltuximab has been shown to reduce disease symptoms and improve quality of life in patients with multicentric Castleman disease.

[0047] Leucine-rich a-2 glycoprotein 1 (LRG1 ) is a secreted member of the family of leucine- rich repeat (LRR) proteins. LRG1 is a multifunctional pathogenic signalling molecule which, amongst other activities, modulates the TGFp pathway in a highly context-dependent manner. LRG1 was first described as an important player in pathological angiogenesis but, since then, evidence for a much wider range of biological functions has accumulated.

[0048] LRG1 consists of a single polypeptide chain of 312 amino acid residues and contains 8 LRRs. LRRs are protein-ligand interaction motifs, typically arranged in repetitive stretches of variable length. Each LRR consists of 19-29 amino acids, comprising a well-conserved N- terminal stretch of 9-12 amino acids, which is rich in the hydrophobic amino acid leucine, and a C-terminal domain that varies in length, sequence, and structure. Multiple repeats are typically arranged together to form a horseshoe shaped solenoid protein domain with a concave surface providing a platform for protein-protein interactions. The negatively charged leucine-rich N-terminal stretches of the repeats form p-strands located towards the inside of the horseshoe shaped domain and represent ideal binding sites for cationic proteins such as TGFp. The refseq for human LRG1 may be accessed at Genbank, NM_052972 (RNA) and NP_443204 (protein).

[0049] Inhibitors of LRG1 include, for example, antibodies and antibody drug conjugates. See, for example, Wang et al. (2013) Nature 499(7458):306-1 1 , herein specifically incorporated by reference. For example the anti-LRG1 hinge-stabilised lgG4 monoclonal antibody Magacizumab has been used both as the free antibody, and as a conjugate with monomethyl auristatin E (MMAE). Anti-human polyclonal antibody (Proteintech) is also known in the art.

[0050] Additional CPA genes include Wnt9b, Gm39822, Nppc, Hba-a1 (hemoglobin subunit alpha 1 ), Ret, Pthlh, and Sema3g. Wnt-9b is a member of the Wnt family, which are glycolipoproteins that are involved with signaling and developmental processes. Like other Wnt genes, Wnt-9b codes for the Wnt-9b protein which participates in the canonical Wnt / p- catenin signaling pathway. The refseq for human Wnt9b may be accessed at Genbank at NM_003396 or NM_001320458 (RNA) and NP_001307387 or NP_003387 (protein).

[0051] Natriuretic peptide precursor C, also known as NPPC, is cleaved to the 22 amino acid peptide C-type natriuretic peptide (CNP). Natriuretic peptides comprise a family of 3 structurally related molecules: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), encoded by a gene symbolized NPPC. These peptides possess potent natriuretic, diuretic, and vasodilating activities and are implicated in body fluid homeostasis and blood pressure control. CNP is a selective agonist for the B-type natriuretic receptor (NPRB) whereas ANP and BNP are selective for the A-type natriuretic receptor(NPRA). The refseq for human NPPC may be accessed at Genbank at NM_024409 (RNA) and NP_077720 (protein).

[0052] CNP is mainly synthesized and released by vascular endothelial cells in response to TNF-a, TGF-p, or shear forces. NPRC, a member of the natriuretic peptide system, is recognized as a clearance receptor of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and c-type natriuretic peptide (CNP), which acts through endocytosis and lysosomal degradation to modulate their physiological function and maintain the balance of the natriuretic peptide system.

[0053] RET is a proto-oncogene encoding a receptor tyrosine kinase for members of the glial cell line-derived neurotrophic factor (GDNF) family of extracellular signalling molecules. The natural alternative splicing of the RET gene results in the production of 3 different isoforms of the protein RET. RET51 , RET43 and RET9 contain 51 , 43 and 9 amino acids in their C- terminal tail respectively. Common to each isoform is a domain structure. Each protein is divided into three domains: an N-terminal extracellular domain with four cadherin-like repeats and a cysteine-rich region, a hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase domain, which is split by an insertion of 27 amino acids. Within the cytoplasmic tyrosine kinase domain, there are 16 tyrosines (Tyrs) in RET9 and 18 in RET51 . Tyr1090 and Tyr1096 are present only in the RET51 isoform. Inhibitors of RET include multikinase inhibitors such as cabozantinib or vandetanib, selective inhibitors such as selpercatinib and pralsetinib; and TPX-0046, a macrocyclic inhibitor of RET and Src intended to inhibit mutations providing resistance to current inhibitors. The refseq for human RET may be accessed at Genbank at NM_000323, NM_020629, NM_020630, NM_020975 and NM_001355216 (RNA); NP_065681 , NP_066124, NP_001342145, and NP_066124.1 (protein).

[0054] Parathyroid hormone-related protein (PTHrP) is a proteinaceous hormone and a member of the parathyroid hormone family secreted by mesenchymal stem cells. It can be secreted by cancer cells. PTHrP is related in function to parathyroid hormone (PTH). When a tumor secretes PTHrP, this can lead to hypercalcemia. PTHrP shares the same N-terminal end as parathyroid hormone and therefore it can bind to the same receptor, the Type I PTH receptor (PTHR1 ). PTHrP can simulate most of the actions of PTH including increases in bone resorption and distal tubular calcium reabsorption, and inhibition of proximal tubular phosphate transport. PTHrP lacks the normal feedback inhibition as PTH. The refseq for human PTHrP may be accessed at Genbank at NM_002820, NM_198964, NM_198965, and NM_198966 (RNA); and NP_002811 , NP_945315, NP_945316, and NP_945317 (protein).

[0055] Semaphorin 3G is activated by PPAR-gamma, and the resulting protein product plays a role in endothelial cell migration. Expression of this gene also inhibits tumor cell migration and invasion. Class 3 semaphorins (Sema3s), including 7 family members (Sema3A-3G), are secreted proteins able to regulate intercellular crosstalk and control cell morphological changes, migration and directional growth via autocrine, paracrine and endocrine mechanisms. Sema3s were initially characterized for their roles in controlling axon guidance in developing nervous system but are gradually found to function in regulating cardiovascular development and pathology such as aortic malformation, angiogenesis and myocardial infarction. Sema3G is expressed mainly in endothelial cells (ECs) and acts in a paracrine or autocrine manner. Intercellular crosstalk between EC and VSMC is also crucial for the homeostasis of vasculature. ECs respond to adverse environmental stimuli and release various vasoactive molecules that influence VSMCs phenotypic change, thus triggering the progression of cardiovascular diseases such as atherosclerosis and fibrosis.

[0056] Certain agents that act on CPA genes and genes products are discussed above. Such agents may include, for example, antibodies. Antibodies, also referred to as immunoglobulins, conventionally comprise at least one heavy chain and one light, where the amino terminal domain of the heavy and light chains is variable in sequence, hence is commonly referred to as a variable region domain, or a variable heavy (VH) or variable light (VH) domain. The two domains conventionally associate to form a specific binding region, although as well be discussed here, a variety of non-natural configurations of antibodies are known and used in the art.

[0057] A “functional” or “biologically active” antibody or antigen-binding molecule is one capable of exerting one or more of its natural activities in structural, regulatory, biochemical or biophysical events. For example, a functional antibody or other binding molecule may have the ability to specifically bind an antigen and the binding may in turn elicit or alter a cellular or molecular event such as signaling transduction or enzymatic activity. A functional antibody or other binding molecule may also block ligand activation of a receptor or act as an agonist or antagonist. The capability of an antibody or other binding molecule to exert one or more of its natural activities depends on several factors, including proper folding and assembly of the polypeptide chains.

[0058] The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain only antibodies, three chain antibodies, single chain Fv, nanobodies, etc., and also include antibody fragments, so long as they exhibitthe desired biological activity (Miller et al (2003) Jour, of Immunology 170:4854-4861 ). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0059] The term antibody may reference a full-length heavy chain, a full length light chain, an intact immunoglobulin molecule; or an immunologically active portion of any of these polypeptides, i.e., a polypeptide that comprises an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass of immunoglobulin molecule, including engineered subclasses with altered Fc portions that provide for reduced or enhanced effector cell activity. The immunoglobulins can be derived from any species. In one aspect, the immunoglobulin is of largely human origin.

[0060] The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al (1991 ) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

[0061] The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region may comprise amino acid residues from a “complementarity determining region” or “CDR”, and / or those residues from a “hypervariable loop”. “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0062] Variable regions of interest include 3 CDR sequences, which may be obtained from available antibodies with the desired specificity, or may be obtained from antibodies developed for this purpose. One of skill in the art will understand that a number of definitions of the CDRsare commonly in use, including the Kabat definition (see “Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions.” Mol Immunol. 2010;47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of the structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature. 1989;342:877-883). Alternative CDR definitions of interest include, without limitation, those disclosed by Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001 ;309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol. 2008;181 :6230-6235; Almagro “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132-143; and Padlanet al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139., each of which is herein specifically incorporated by reference.

[0063] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0064] Antibodies specifically include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81 :6851 - 6855). Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc) and human constant region sequences.

[0065] An “intact antibody chain” as used herein is one comprising a full length variable region and a full length constant region. An intact “conventional” antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1 , hinge, CH2 and CH3 for secreted IgG. Other isotypes, such as IgM or IgA may have different CH domains. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (ADCP); and down regulation of cell surface receptors. Constant region variants include those that alter the effector profile, binding to Fc receptors, and the like.

[0066] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different “classes.” There are five major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, IgA, and lgA2. The heavychain constant domains that correspond to the different classes of antibodies are called a, 5, s, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modifications or hingeless forms (Roux et al (1998) J. Immunol. 161 :4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US 2005 / 0048572; US 2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called K and A, based on the amino acid sequences of their constant domains.

[0067] “Treatment”, “treating”, “treat” and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom. Those in need of treatment include individuals already diagnosed with cancer, who may have additional risk factors for development of atherosclerosis.

[0068] The terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. Preferably, the mammal is human.

[0069] An "effective amount" is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of an agent is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease state, e.g. atherosclerosis or atherosclerotic plaque. For example, in an animal model the percent of aortic surface area with atherosclerotic plaque may be reduced 10%, 20%, 25%, 50%, 75% or more relative to a control treated animal. Similar effects may be obtained with indicia appropriate for human patients, including without limitation C-reactive protein [CRP] and fibrinogen; lipoprotein-associated phospholipase A2 [Lp-PLA2] and myeloperoxidase [MPO]; growth differentiation factor-15 [GDF-15]) inflammatory markers; ambulatory arterial stiffness, IVUS imaging, and the like. See, for example Krintus et al. (2013) Crit Rev Clin Lab Sci. 11 :1 -17; Kollias et al. (2012) Atherosclerosis 224(2):291 -301 ; and Kollias et al. (201 1 ) Int. J. Cardiovasc. Imaging 27(2):225-37, each herein specifically incorporated by reference.

[0070] Other measurements of vascular disease include, for example, echocardiography and angiography, which have traditionally been the primary imaging modalities for diagnosing cardiac disease. Computed tomography (CT) and magnetic resonance (MR) imaging are used with increasing frequency because they improve tissue characterization. Intravascular ultrasonography, CT, and MR imaging are frequently used to detect atherosclerotic plaque, wall thickening, and luminal stenosis or enlargement; quantify the extent of the disease; and identify complications such as aneurysm, dissection, and thrombus.

[0071] Diagnostic tools that are more directly reflective of vascular inflammation have been sought. Positron emission tomography (PET) performed with fluorine 18 fluorodeoxyglucose (FDG) has the unique ability to depict metabolically active disease, and in this respect, it complements other cross-sectional imaging modalities, which provide predominantly anatomic information. Because whole-body imaging with combined Positron Emission Tomography (18F-FDG PET) combined with computed tomography (CT) (hereafter, PET / CT) is used with increasing frequency to evaluate noncardiovascular disease processes, it may be the first imaging study in which cardiovascular disease is identified. FDG PET / CT has become a valuable imaging modality for diagnosing various conditions in patients who present with systemic symptoms that are difficult to localize and diagnose with a clinical examination androutine imaging procedures. These methods can be used in both preclinical and clinical studies for the evaluation of inflammation in the arterial wall. Technical progress to extend the CV applications of18F-FDG PET / CT include improved image acquisition, measurements, and reconstruction protocols. This has allowed a number of clinical trials to provide results of18F- FDG PET / CT in detecting atherosclerotic plaque inflammation, discriminating stable from unstable plaques, predicting CV prognosis, and monitoring response to CV-related therapies.

[0072] 18F-FDG PET has been used to assess the impact of statin treatment on arterial wall inflammation in interventional studies. For this purpose, arterial18F-FDG uptake is expressed as the Target-to-Background Ratio (TBR), that is a measure of the blood-normalized standardized uptake value (SUV).18F-FDG is taken up mostly by macrophages within the atherosclerotic plaques, although other cells (i.e., endothelial cells, vascular smooth muscle cells, neutrophils, lymphocytes) may participate in tracer uptake. TBR, as a measure of SUV, has been demonstrated to be a reproducible index for quantification of18F-FDG uptake in the inflamed arterial wall. While many atherosclerotic plaques are not metabolically active at FDG PET, focal intense activity within atherosclerotic plaques may be a marker of lesions that are vulnerable to disruption and have more inflammatory cellular components.

[0073] In some embodiments, efficacy of a therapy disclosed herein is monitored by18F-FDG PET / CT, including specifically the determination of TBR as a function of SUV, where decreased uptake, e g. up to about 5% decrease, up to about 10% decrease, up to about 25% decrease, up to about 50% decrease, or more, is indicative of therapeutic efficacy.

[0074] As used herein, endpoints for treatment will be given a meaning as known in the art and as used by the Food and Drug Administration.

[0075] Overall survival is defined as the time from randomization until death from any cause, and is measured in the intent-to-treat population. Survival is considered the most reliable cancer endpoint, and when studies can be conducted to adequately assess survival, it is usually the preferred endpoint. This endpoint is precise and easy to measure, documented by the date of death. Bias is not a factor in endpoint measurement. Survival improvement should be analyzed as a risk-benefit analysis to assess clinical benefit. Overall survival can be evaluated in randomized controlled studies. Demonstration of a statistically significant improvement in overall survival can be considered to be clinically significant if the toxicity profile is acceptable, and has often supported new drug approval. A benefit of the methods of the invention can include increased overall survival of patients.

[0076] Endpoints that are based on tumor assessments include DFS, ORR, TTP, PFS, and time-to-treatment failure (TTF). The collection and analysis of data on these time-dependent endpoints are based on indirect assessments, calculations, and estimates (e.g., tumormeasurements). Disease-Free Survival (DFS) is defined as the time from randomization until recurrence of tumor or death from any cause. The most frequent use of this endpoint is in the adjuvant setting after definitive surgery or radiotherapy. DFS also can be an important endpoint when a large percentage of patients achieve complete responses with chemotherapy.

[0077] Objective Response Rate . ORR is defined as the proportion of patients with tumor size reduction of a predefined amount and for a minimum time period. Response duration usually is measured from the time of initial response until documented tumor progression. Generally, the FDA has defined ORR as the sum of partial responses plus complete responses. When defined in this manner, ORR is a direct measure of drug antitumor activity, which can be evaluated in a single-arm study.

[0078] Time to Progression and Progression-Free Survival. TTP and PFS have served as primary endpoints for drug approval. TTP is defined as the time from randomization until objective tumor progression; TTP does not include deaths. PFS is defined as the time from randomization until objective tumor progression or death. The precise definition of tumor progression is important and should be carefully detailed in the protocol.

[0079] Combination Therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, two or more agents may be administered simultaneously; in some embodiments, such agents may be administered sequentially; in some embodiments, such agents are administered in overlapping dosing regimens.

[0080] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under orwith different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0081] Composition: A "composition" or a "pharmaceutical composition" according to this invention refers to the combination of two or more agents as described herein for coadministration or administration as part of the same regimen. It is not required in all embodiments that the combination of agents result in physical admixture, that is, administration as separate co-agents each of the components of the composition is possible; however many patients or practitioners in the field may find it advantageous to prepare a composition that is an admixture of two or more of the ingredients in a pharmaceutically acceptable carrier, diluent, or excipient, making it possible to administer the component ingredients of the combination at the same time.

[0082] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of" (or which "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of" (or "consists of") the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.

[0083] Determine: Many methodologies described herein include a step of “determining”. Those of ordinary skill in the art, reading the present specification, will appreciate that such “determining” can utilize or be accomplished through use of any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves consideration and / or manipulation of data orinformation, for example utilizing a computer or other processing unit adapted to perform a relevant analysis. In some embodiments, determining involves receiving relevant information and / or materials from a source. In some embodiments, determining involves comparing one or more features of a sample or entity to a comparable reference.

[0084] Dosage Form: As used herein, the term “dosage form” refers to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Each unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

[0085] Dosing Regimen: As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0086] The term “sample” with respect to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations. The definition also includessample that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc.

[0087] The terms “specific binding,” “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides; high affinity binding of a ***a polypeptide to ***; etc.) In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD (dissociation constant) of 105M or less (e.g., 106M or less, 107M or less, 108M or less, 109M or less, 1010M or less, 1011M or less, 1012M or less, 1013M or less, 1014M or less, 1015M or less, or 1016M or less). "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD.

[0088] The term “specific binding member” as used herein refers to a member of a specific binding pair (i.e., two molecules, usually two different molecules, where one of the molecules, e.g, a first specific binding member, through non-covalent means specifically binds to the other molecule, e.g., a second specific binding member).

[0089] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.

[0090] A "variant" polypeptide means a biologically active polypeptide as defined below having less than 100% sequence identity with a native sequence polypeptide. Such variants include polypeptides wherein one or more amino acid residues are added at the N- or C- terminus of, or within, the native sequence; from about one to forty amino acid residues are deleted, and optionally substituted by one or more amino acid residues; and derivatives of the above polypeptides, wherein an amino acid residue has been covalently modified so that the resulting product has a non-naturally occurring amino acid. Ordinarily, a biologically active variant will have an amino acid sequence having at least about 90% amino acid sequence identity with a native sequence polypeptide, preferably at least about 95%, more preferably at least about 99%. The variant polypeptides can be naturally or non-naturally glycosylated, i.e., the polypeptide has a glycosylation pattern that differs from the glycosylation pattern found in the corresponding naturally occurring protein. The variant polypeptides can have post- translational modifications not found on the natural protein.

[0091] A "fusion" polypeptide is a polypeptide comprising a polypeptide or portion (e.g., one or more domains) thereof fused or bonded to heterologous polypeptide. A fusion soluble CRT protein, for example, will share at least one biological property in common with a nativesequence soluble CRT polypeptide. Examples of fusion polypeptides include immunoadhesins, as described above, which combine a portion of the polypeptide of interest with an immunoglobulin sequence, and epitope tagged polypeptides, which comprise a soluble polypeptide of interest or portion thereof fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with biological activity of the polypeptide of interest. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 6-60 amino acid residues.

[0092] A "functional derivative" of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence polypeptide. The term "derivative" encompasses both amino acid sequence variants of polypeptide and covalent modifications thereof. For example, derivatives and fusion of soluble CRT find use as CRT mimetic molecules.

[0093] Small molecule: As used herein, the term "small molecule" refers to organic compounds, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Typically, small molecules have a molecular weight of less than about 1500 g / mol. Also, small molecules typically have multiple carbon-carbon bonds.Methods

[0094] Methods are provided for treating or reducing the promotion of atherosclerosis by cancer, by administering an effective combined dose of an agent that modulates activity of a CPA gene or gene product as disclosed here. In some embodiments the agent inhibits activity of LRG1 .

[0095] In some embodiments the individual has been diagnosed with cancer prior to treatment. In some embodiments the cancer is a solid tumor, e.g. carcinoma, melanoma, glioma, sarcoma, lymphoma, etc. In some embodiments the cancer is a carcinoma, e.g. lung carcinoma, breast carcinoma, colon carcinoma, etc. In some embodiments the individual is undergoing treatment for the cancer. In some embodiments the individual has completed treatment for the cancer.

[0096] The individual may have one or more risk factors for the development of atherosclerosis, in addition to cancer. In some embodiments an individual selected for treatment has cancer and has 1 , 2, 3, 4, or more risk factors for atherosclerosis, includingelevated levels of LDL cholesterol, low levels of HDL cholesterol, high blood pressure, type 2 diabetes, smoking, and obesity.

[0097] In some embodiments, methods and compositions are provided for the prevention and treatment cancer-promoted ASCVD in a subject by inhibition of LRG1 activity. An effective dose of an agent that inhibits LRG1 activity is administered to an individual with cancer, in a dose effective to reduce LRG1 activity, relative to an untreated control. The agent may be an agent that binds to and inhibits LRG1. The agent may be an agent that binds to and inhibits an activator of LRG1 , including TNFa, or IL-6, or that binds to an inhibits a receptor of TNFa or IL-6. In some embodiments the inhibitor is an antibody. In some embodiments the inhibitor is a soluble receptor, e.g. TNFR1 , etc. In some embodiments inhibitor acts at the genetic level, e.g. an RNAi, an anti-sense oligonucleotide, a genome editing agent, etc. In some embodiments the inhibitor is a small molecule drug. An agent that inhibits LRG1 activity may be administered systemically, e.g. parenteral administration, oral administration, etc.

[0098] In some embodiments the agent inhibits TNFa signaling. In such embodiments the agent binds to an inhibits TNFa. In some embodiments the agent is selected from Infliximab, Adalimumab; Golimumab; Etanercept; and Certolizumab. The methods may be performed in the absence of administering an agent that interferes with CD47 signaling, e.g. see US 1 1 ,253,588, herein specifically incorporated by reference.

[0099] In some embodiments the agent binds to and inhibits IL-6 or IL-6 receptor. In some embodiments the agent is selected from Tocilizumab; Sarilumab; and Siltuximab.

[0100] In some embodiments the agent binds to and inhibits LRG1. In some embodiments the agent is an antibody. In some embodiments the antibody is Magacizumab.

[0101] Effective doses of the therapeutic entity of the present invention vary depending upon many different factors, including the nature of the agent, means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but nonhuman mammals may also be treated, e.g. companion animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc., and the like. Treatment dosages can be titrated to optimize safety and efficacy.

[0102] In certain embodiments, multiple therapeutically effective doses are administered according to a daily dosing regimen, or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By "intermittent" administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, once a week, once every two weeks, once every three weeks, once a month, and so forth. For example, in some embodiments, an antibody is administered onceevery two to four weeks for an extended period of time, such as for 1 , 2, 3, 4, 5, 6, 7, 8, 10, 15, 24 months, and so forth. By "twice-weekly" or "two times per week" is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By "thrice weekly" or "three times per week" is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as "intermittent" therapy. In accordance with the methods of the present invention, a subject can receive intermittent therapy for one or more weekly or monthly cycles until the desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below.

[0103] In certain embodiments, multiple therapeutically effective doses are administered according to a daily dosing regimen, or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By "intermittent" administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, once a week, once every two weeks, once every three weeks, once a month, and so forth. For example, in some embodiments, an antibody is administered once every two to four weeks for an extended period of time, such as for 1 , 2, 3, 4, 5, 6, 7, 8, 10, 15, 24 months, and so forth. By "twice-weekly" or "two times per week" is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By "thrice weekly" or "three times per week" is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as "intermittent" therapy. In accordance with the methods of the present invention, a subject can receive intermittent therapy for one or more weekly or monthly cycles until the desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below.

[0104] The therapeutic dose may be at least about 0.01 pg / kg body weight, at least about 0.05 pg / kg body weight; at least about 0.1 pg / kg body weight, at least about 0.5 pg / kg body weight, at least about 1 pg / kg body weight, at least about 2.5 pg / kg body weight, at least about 5 pg / kg body weight, at least about 10 pg / kg body weight, at least about 25 pg / kg body weight; at least about 50 pg / kg body weight, at least about 100 pg / kg body weight, at least about 500j g / kg body weight; and not more than about 100 mg / kg body weight, not more than about 50 mg / kg, not more than about 25 mg / kg, not more than about 10 mg / kg, not more than about 5 mg / kg, not more than about 1 mg / kg. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent. The dosage may also be varied for localized administration, e.g. intranasal, inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v., and the like.

[0105] For the treatment of disease, the appropriate dosage of the agent will depend on the severity and course of the disease, whether the agent is administered for preventive purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The agent is suitably administered to the patient at one time or over a series of treatments.

[0106] Suitable agents can be provided in pharmaceutical compositions suitable for therapeutic use, e.g. for human treatment. In some embodiments, pharmaceutical compositions of the present invention include one or more therapeutic entities of the present invention or pharmaceutically acceptable salts, esters or solvates thereof. In some other embodiments, the agent is combined with a second therapeutic agent, e.g., drugs useful in the treatment of atherosclerosis. Such combinations may include, without limitation, statins. Statins are inhibitors of HMG-CoA reductase enzyme. These agents are described in detail; for example, mevastatin and related compounds as disclosed in U.S. Pat. No. 3,983,140; lovastatin (mevinolin) and related compounds as disclosed in U.S. Pat. No. 4,231 ,938; pravastatin and related compounds as disclosed in U.S. Pat. No. 4,346,227; simvastatin and related compounds as disclosed in U.S. Pat. Nos. 4,448,784 and 4,450,171 ; fluvastatin and related compounds as disclosed in U.S. Pat. No. 5,354,772; atorvastatin and related compounds as disclosed in U.S. Pat Nos. 4,681 ,893, 5,273,995 and 5,969,156; and cerivastatin and related compounds as disclosed in U.S. Pat. Nos. 5,006,530 and 5,177,080. Additional agents and compounds are disclosed in U.S. Pat. Nos. 5,208,258, 5,130,306, 5,1 16,870, 5,049,696, RE 36,481 , and RE 36,520. Statins include the salts and / or ester thereof.

[0107] Therapeutic formulations comprising one or more agents of the invention are prepared for storage by mixing the agent having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. The agent composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factors known to medical practitioners. The "therapeutically effective amount" of the agent to be administered will be governed by such considerations, and is the minimum amount necessary to treat or prevent atherosclerosis.

[0108] The agent can be administered by any suitable means, including topical, oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intrathecal or subcutaneous administration. In addition, the agent can be suitably administered by pulse infusion, particularly with declining doses of the agent.

[0109] The combination of agents may be used in the same dosages and with administration routes as used hereinbefore as individual agents, or about from 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 90, 95, to 99% of the heretofore employed dosages.

[0110] An agent is often administered as a pharmaceutical composition comprising an active therapeutic agent and another pharmaceutically acceptable excipient. The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically- acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0111] In still some other embodiments, pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized Sepharose™, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0112] A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations. Suitable covalent-bond carriers include proteins such as albumins, peptides, and polysaccharides such as aminodextran, each of which have multiple sites for the attachment of moieties. A carrier may also bear an anti-*** agent by non-covalent associations, such as non-covalent bonding or by encapsulation. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding agents, or will be able to ascertain such, using routine experimentation.

[0113] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0114] The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0115] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-1 19, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0116] Toxicity of the agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD5o (the dose lethal to 50% of the population) or the LD o (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0117] Cell-based systems may be used to identify compounds and combinations of compounds that act to reduce cancer-promoted atherosclerosis. For example, such cell systems may be exposed to a compound at a sufficient concentration and for a time sufficient to elicit such an amelioration of cardiovascular disease symptoms in the exposed cells. After exposure, the cells are examined to determine whether one or more of the cardiovascular disease cellular phenotypes has been altered to resemble a more normal or more wild type, non-cardiovascular disease phenotype.

[0118] In addition, animal-based disease systems, such as those described, above may be used to identify compounds capable of ameliorating disease symptoms. Such animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies, and interventions, which may be effective in treating disease. For example, animal models may be exposed to a compound, suspected of exhibiting an ability to ameliorate cardiovascular disease symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of disease symptoms in the exposed animals. The response of the animals to the exposure may be monitored by assessing the reversal of disorders associated with disease, for example, by counting the number of atherosclerotic plaques and / or measuring their size before and after treatment.

[0119] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50(the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0120] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds liespreferably within a range of circulating concentrations that include the ED5o with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0121] The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.EXPERIMENTAL

[0122] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0123] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0124] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.Example 1 Cancer-promoted Atherosclerosis

[0125] Tumors were inoculated into animals that provide a model for atherosclerosis. The relative plaque size of the of the aortic sinus was measured with development of the tumor. A 13% increase in the plaque size of the aortic sinus in tumor-bearing mice was found compared to tumor-free mice. This data showed the atheroprone mice implanted with tumors mildly tended to have bigger atherosclerotic plaques than tumor-free mice.

[0126] Shown in FIG. 1 a is the timeline for tumor inoculation in an atherosclerotic mouse model. Apoe1C57BL / 6 mice were fed a high-fat diet for 12 weeks, followed by inoculation of MC38 colorectal tumor cells or buffer subcutaneously at the beginning of the last 4 weeks away from the harvesting time point. FIG. 1 B shows the relative plaque size of the aortic sinus from Apoe1mice bearing tumors versus tumor-free control mice, n = 19 for the control mice, n = 21 for the tumor-bearing mice. Student t-test was performed, p-value = 0.13.

[0127] Mice were anesthetized and perfused with phosphate buffer saline (PBS). Organs including the aortic sinus were harvested and fixed with 4% paraformaldehyde (PFA) overnight. Tissue samples were incubated with 30% sucrose overnight before being embedded in optimal cutting temperature (OCT). Embedded samples were sectioned at 7um thick and mounted on slides.

[0128] Shown in FIGS. 2A-2C are quantification by Imaged software of images of aortic sinuses captured using a microscope. Shown in FIG. 2A is the relative area of the intima of the tandem stenosis (TS) segment of the carotid artery between Apoe' mice bearing tumors and tumor-free control mice, n = 1 1 for the control mice and n = 13 for the tumor-bearing mice. Student t-test was performed, p-value = 0.1500. FIG. 2B, the relative area of the media of the tandem stenosis (TS) segment of the carotid artery, n = 11 for the control mice and n = 13 for the tumor-bearing mice. Mann-Whitney test was performed, p-value = 0.1500. FIG. 2C, the ratio of intima versus media of the tandem stenosis (TS) segment of the carotid artery, n = 11 for the control mice and n = 13 for the tumor-bearing mice. Welch’s t-test was performed, p- value = 0.0964.

[0129] A 45.3% increase in the plaque size of the tandem stenosis segment and 33.3% increase in the intima / media ratio was observed in tumor-bearing mice compared to tumor- free mice. We observed a strong tendency of tumor-bearing mice to have larger plaques in the carotid artery after the tandem stenosis surgery.

[0130] For an in vivo model, Apoe1C57BL / 6 mice were fed a high-fat diet and then subjected to tandem stenosis surgery on the right carotid artery. 3 weeks post-surgery, the mice were injected with MC38 tumor cells before being harvested 4 weeks later.

[0131] In a tandem stenosis model, mice were anesthetized and the right carotid artery was dissected after introducing an incision on the neck. The tandem stenosis was conducted with the distal end 1 mm away from the carotid bifurcation and the proximal end 3 mm away from the distal end. The stenosis was introduced using a 6-0 blue braided polyester fiber suture and a 150-um needle around the carotid artery. The needle was later removed.

[0132] For tissue processing, mice were anesthetized and perfused with phosphate buffer saline (PBS). Organs including the aortic sinus were harvested and fixed with 4% paraformaldehyde (PFA) overnight. Tissue samples were incubated with 30% sucrose overnight before being embedded in optimal cutting temperature (OCT). Embedded samples were sectioned at 7um thick and mounted on slides.

[0133] Oil Red O staining was performed. The aortic sinus sample slides were incubated with absolute propylene glycol for 5 minutes and then stained in Oil Red O solution for 15 minutes. Then the slides were rinsed in 2 changes of distilled water and mounted with an aqueous mounting medium. Images of aortic sinuses were captured using a Leica Thunder microscope. The areas of the plaques were quantified using the Imaged software.

[0134] FIGS 3A-3b provide representative immunofluorescence images of the cross-sections of the carotid arteries after tandem stenosis of control mice (a) and tumor mice (b). The sections were stained for CD31 (an endothelial cell marker), TER-1 19 (a red blood cell marker), and DAPI (a DNA marker). The average signal area of CD31 (c) and TER-1 19 (d) was quantified across the tandem stenosis samples of the batch of mice from FIG. 2. The Mann-Whitney test was performed, p-value = 0.0129 for the CD31 signal, p-value = 0.0156 for the TER-1 19 signal.

[0135] The tumor-bearing mice had significantly more intraplaque signals of CD31 and TER- 1 19 than tumor-free control mice, indicating that there were more intraplaque neovessels and hemorrhage in mice implanted with tumors. Since more intraplaque neovessels and hemorrhage indicate more plaque raptures, it infers tumors may promote plaque instability and atherogenesis.

[0136] Immunofluorescence. The cross-sections of the tandem stenosis segments were permeated with 0.1 % Triton X-100 and blocked by Rodent Block M. Sections were incubated with primary anti-CD31 antibody or anti-TER-1 19 antibody overnight. After being washed with PBS 3 times, the section samples were stained with secondary antibodies for 1 hour. After washing with PBS 3 times, Fluoroshield with DAPI was used to mount slides.

[0137] FIG. 4 provides a Venn Diagram of overlapped genes among various datasets that were upregulated in the aortic arches of mice with MC38 colon cancer than control mice without tumors. Each dataset (A, B, C, D, and E) represents a unique combination of genetic background, diet, and age. The datasets are: A: 18-week-old, wildtype (WT) mice fed with achow diet (CD); B: 18-week-old, Apoe1mice fed with a high-fat diet (HFD); C: 21 -29 weeks old, Apoe1mice fed with HFD; D: 18-week-old, WT mice fed with HFD; E: 18-week-old, Apoe 7 mice fed with CD.

[0138] MC38 colon cancer cell implantation consistently induces Lrg1 expression in the aorta of mice regardless of the genetic background (Apoe1or WT), diet (HFD or CD), and age.

[0139] Timeline for the in vivo model. Apoe1or wildtype C57BL / 6 mice were fed a high-fat diet or a chow diet starting at 6 weeks old and then the mice were implanted with MC38 colon tumors before being harvested 4 weeks from the 18-week-old harvesting endpoint (for A, B, D, E). In dataset C, the HFD started when the mice were 9-17 weeks old. The length of HFD was 12 weeks for all the datasets.

[0140] Bulk RNA sequencing. The aortic arch samples were subjected to RNA extraction using Trizol. Then the total RNAs were sent to Novogene Co. (Sacramento, CA, USA) for sample quality control, library preparation, and sequencing. FastQC was used to perform quality control of the sequencing data. STAR was used to align reads to the mouse reference genome. After counting the number of reads, DESeq2 was used to generate lists of differentially regulated genes among different datasets.

[0141] FIG. 5 provides a Venn diagram of overlapped genes among various datasets that were upregulated in the aortic arches of mice with different types of cancer than control mice without tumors. Each dataset contains mice with a certain type of tumor versus control littermates without tumors. B and C: MC38 colon cells; G: B16F10 melanoma cells; H: LLC Lewis lung carcinoma cells; I: E0771 breast cells.

[0142] Tumors consistently upregulated a set of genes in the aortic arch of Apoe^ C57BL / 6 mice fed a 12-week high-fat diet. Lrg1 is among the upregulated gene list across all the datasets, which indicates that Lrg1 upregulation in the aorta is a general effect of tumor implantation.

[0143] Timeline for the in vivo model. Apoe1or wildtype C57BL / 6 mice were fed a 12-week high-fat diet or a chow diet starting at 6 weeks old and then the mice were implanted with MC38 colon cells, B16F10 melanoma cells, Lewis lung carcinoma cells, or E0771 breast cells, before being harvested 4 weeks from the 18-week-old harvesting endpoint (for B, G, H, and I). In dataset C, the HFD started when the mice were 9-17 weeks old.

[0144] Bulk RNA sequencing. The aortic arch samples were subjected to RNA extraction using Trizol. Then the total RNAs were sent to Novogene Co. (Sacramento, CA, USA) for sample quality control, library preparation, and sequencing. FastQC was used to perform quality control of the sequencing data. STAR was used to align reads to the mouse reference genome. After counting the number of reads, DESeq2 was used to generate lists of differentially regulated genes among different datasets.

[0145] In FIG. 6, Olink analysis was performed on the serum samples collected from mice bearing tumors (MC38 colon, B16F10 melanoma cells, Lewis lung carcinoma, or E0771 breast) or tumor-free mice, a) The enrichment analysis of upregulated proteins detected by Olink. b) and c) Relative protein levels (TNF or IL-6) detected by Olink. “ p-value < 0.01 ; “* p-value < 0.001 .

[0146] Olink reveals differentially expressed circulating proteins in blood. After performing functional enrichment of those upregulated proteins in mice implanted with tumors than control mice, the TNF receptor activity-related pathway was one of the top hits among the gene ontology for molecular function. After examining the Olink data for individual circulating proteins, we found TNF and IL-6 those two cytokines that were reported to upregulate Lrg1 were upregulated in the tumor-bearing mice, indicating TNF and IL-6 might driven the gene expression changes of the aorta after tumor implantation.

[0147] Olink analysis. The serum samples were stored at -80 Celsius degrees before being sent to the Stanford Human Immune Monitoring Center for Olink Target 96 Mouse Exploratory analysis. Shown in FIG. 6A is the relative mRNA level of Lrg1 in cultured human aortic endothelial cells being treated with various doses of TNF. In FIG. 6B is the relative mRNA level of Lrg1 in cultured human aortic endothelial cells being treated with TNF and an anti- TNF antibody.

[0148] We found TNF was able to induce Lrg1 expression in human aortic endothelial cells, and the TNF-induced upregulation of Lrg1 can be blocked by treating the cells with an anti- TNF antibody.

[0149] Cell culture. Human aortic endothelial cells (P2-P5) were used. In the dose-response experiment, 0.0625 - 1 ng / ml of TNF was used to treat cells for 4 hours before harvesting the cells. In the in vitro antibody neutralizing experiment, 0.25ng / ml TNF or vehicle, and 1 .g / ml anti-TNF antibody or IgG was used to treat cells for 4 hours before cell harvesting.

[0150] RT-qPCR. Cells were harvested by extracting RNAs using the RNA Extraction Kit from Zymo Research. Then the RNA samples were reverse transcribed with a High-Capacity cDNA Reverse Transcription Kit. Then the qPCR was performed using TaqMan Universal PCR Master Mix with TaqMan probes for LRG1 and GAPDH (internal control).

[0151] FIG. 7A shows the relative mRNA level of Lrg1 in cultured human aortic endothelial cells being treated with conditional media from primary colon epithelial cells or colon cell line. In FIG. 7B are parameters of tube formation assays for endothelial cells treated with conditional media from primary colon cells or the cell line. One-way ANOVA was performed. In FIG. 7C are parameters of tube formation assays for endothelial cells being treated with conditional media from colon cell line plus IgG or anti-TNF antibody. * p-value < 0.05, ” p- value < 0.01 , *** p-value < 0.001 , *“* p-value < 0.0001 .

[0152] The conditional media collected from the HCT1 16 colon cell line was able to stimulate both the expression of Lrg1 and the tube formation more than the conditional media from primary colon epithelial cells or the basal media. Treating the HCT1 16 conditional media with an anti-TNF antibody decreased the tube formation.

[0153] Tube formation assay. Conditional media was prepared by incubating primary colon epithelial cells or HCT116 colon cell line with basal media for 12 hours before collecting. Human aortic endothelial cells were seeded on growth factor reduced Matrigel before being treated with conditional media for 9 hours.

[0154] Shown in FIG. 8 are the relative intima area, relative media area, and the intima versus media ratio of the tandem stenosis segment of mice treated with anti-TNF antibody (Etanercept) or IgG, and inoculated with tumors or buffer.

[0155] Consistent with the tandem stenosis model in Figure 2, we found there was a strong tendency for a bigger plaque size in the mice implanted with tumors compared to tumor-free mice between the IgG-treated groups. However, treating the mice with the anti-TNF antibody blocked the trend of difference between mice with or without tumor implantation.

[0156] Timeline for the in vivo model. Apoe1C57BL / 6 mice were fed a high-fat diet and then subjected to tandem stenosis surgery on the right carotid artery. 3 weeks post-surgery, the mice were injected with MC38 tumor cells before the endpoint which was 4 weeks later. Starting from 2 days before the tumor implantation, 2 mg / kg of Etanercept or IgG control was injected into mice every other day until the endpoint.Example 2LRG Expression in Cancer Patients

[0157] Carotid plaque samples were obtained from cancer and cancer-free patients who underwent carotid endarterectomy, sourced from the Munich Vascular Biobank. Samples were matched for age, sex, symptomatic status, cardiovascular risk factors, and medication use. Total RNA was extracted from a subset of plaques, followed by RT-qPCR to quantify LRG1 mRNA levels. Another subset of plaques was processed for staining of plaque stability markers and LRG1 expression.

[0158] As shown in FIGS. 10A-10C, LRG1 is upregulated in human carotid plaques of cancer patients. The increased expression of LRG1 in carotid plaques of cancer patients, compared to cancer-free individuals, aligns with findings from animal models, supporting its role in plaque pathology and cancer-associated vascular alterations.

[0159] Olink proteomics data from the UK Biobank (n = 52,704) were analyzed for circulating TNF levels. Among these, 7,968 individuals had a cancer diagnosis, including 774 patients with colorectal cancer. The cohort was stratified into four quartiles based on TNF levels. Linearregression was conducted to assess the association between TNF levels and MACE across quartiles. Definition of MACE: Major adverse cardiovascular events (MACE) were defined as acute myocardial infarction (AMI), acute coronary syndrome (ACS), ischemic heart disease (IHD), and stroke.

[0160] As shown in FIG. 1 1 , cancer patients have higher circulating TNF levels in blood. Elevated circulating TNF levels in cancer patients is consistent with findings from mouse tumor models, demonstrating human relevance. The observed association between TNF and MACE supports the causal role of TNF in tumor-induced atherosclerosis, reinforcing its potential human pathological relevance.Example 3Inhibition of TNF / LRG in an Animal Model

[0161] The tandem stenosis and anti-TNF antibody-treated mouse models are as described in Example 1 .

[0162] For the LRG1 knockdown model, wild-type C57BL / 6 mice were injected with AAV- PCSK9 and placed on a high-fat diet for two weeks. AAV-mediated LRG1 shRNA (1.9 x 1012GC / ml) was then administered via intravenous injection. Three weeks post-LRG1 shRNA injection, tandem stenosis surgery was performed, followed by tumor implantation three weeks later.

[0163] As shown in FIG. 12, TNF inhibition or LRG1 knockdown blocks tumor-induced intraplaque neovascularization. Neovessel formation was assessed using three endothelial and angiogenesis markers: CD31 , von Willebrand factor (VWF), and isolectin B4 (IB4).

[0164] Significance:

[0165] These findings demonstrate that targeting TNF and LRG1 -mediated neovascularization can effectively mitigate tumor-accelerated atherosclerosis in vivo.

Claims

What is claimed is:1 . A method of treating a subject with cancer to reduce cancer-promoted atherosclerosis, the method comprising: administering to the subject an effective dose of an agent that modulates activity of one or more cancer promoted atherosclerosis (CPA) genes.

2. The method of claim 1 , wherein the CPA gene comprises Lrg 1 , Wnt9b, Gm39822, Nppc, Hba-a1 , Ret, Pthlh, or Sema3g.

3. The method of claim 1 or claim 2, wherein the agent inhibits the activity of LRG1 .

4. The method of claim 3, wherein the agent binds to and inhibits LRG1 .

5. The method of claim 4, wherein the agent is an antibody.

6. The method of claim 5, wherein the antibody is Magacizumab.

7. The method of claim 3, wherein the agent binds to an inhibits TNFoc.

8. The method of claim 7, wherein the agent is selected from Infliximab, Adalimumab;Golimumab; Etanercept; and Certolizumab.

9. The method of claim 3, wherein the agent binds to and inhibits IL-6 or IL-6 receptor.

10. The method of claim 9, wherein the agent is selected from Tocilizumab; Sarilumab; and Siltuximab.1 1 . The method of any of claims 1 -10, wherein the subject is a human.

12. The method of any of claims 1 -1 1 , wherein the individual has been diagnosed with cancer prior to treatment.

13. The method of any of claims 1 -12, wherein the cancer is a solid tumor.

14. The method of any of claims 1 -13, wherein the individual is undergoing treatment for the cancer.

15. The method of any of claims 1 -13, wherein the individual has completed treatment for the cancer.

16. The method of any of claims 1 -15, wherein the individual has one or more risk factors for the development of atherosclerosis, in addition to cancer.

17. The method of claim 16, wherein the individual has 2 or more risk factors for atherosclerosis.

18. The method of claim 17, wherein the risk factors include elevated levels of LDL cholesterol, low levels of HDL cholesterol, high blood pressure, type 2 diabetes, smoking, and obesity.

19. The method of any of claims 1 -18, further comprising monitoring the level of atherosclerosis in the individual following treatment.

20. The method of claim 19, wherein the level of atherosclerosis is monitored by plaque formation, vascular inflammation, or LRG1 .

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