ApoM-fc fusion proteins, complexes thereof with sphingosine 1-phosphate (s1p), and methods for treating vascular and non-vascular diseases

Abstract

The present disclosure relates to engineered phospholipid or lysophospholipid (e.g., sphingosine 1-phosphate (S1P)) chaperones derived from apolipoprotein M (ApoM)-Fc fusion proteins with extended half-life in vivo. The disclosed ApoM-Fc fusion proteins provide a safe, highly efficient and effective way of delivering S1P to endothelial cells and all tissues of the body.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 375,088, filed August 15, 2016, the entire contents of which are incorporated herein by reference.

[0003] Statement regarding federally funded research or development

[0004] This invention was made with government support from the National Institutes of Health (Grant No. HL-89934). The government holds certain rights to this invention.

[0005] By referencing and incorporating into the sequence list

[0006] The sequence list is incorporated herein by reference as a 6KB ASCII text file named 33974_Seq_ST25.txt, created on August 9, 2017, and filed with the U.S. Patent and Trademark Office via EFS-Web. Background Technology

[0007] Endothelial cell function is essential for normal cardiovascular homeostasis (Harrison, DG, Basic research in Cardiology, 89 Suppl 1, 87-102 (1994); Pober, JS & Sessa, WC, Nature Reviews. Immunology, 7, 803-815 (2007)). Many environmental and intrinsic risk factors for cardiovascular and cerebrovascular diseases lead to endothelial dysfunction. In fact, it is believed that dysfunctional endothelium triggers the development of vascular diseases (Girouard, H. & Iadecola, C, J. of App. Physiology, 100, 328-335 (2006)). On the other hand, multiple endogenous factors promote endothelial health and counteract risk factors (Libby, P. et al., J. of the Am. Coll. of Cardio. 54, 2129-2138 (2009)). One such factor is high-density lipoprotein (HDL), a type of multifunctional cyclic nanoparticle (Rosenson, RS et al., Nature Rev. Cardiology, 13, 48-60 (2016)).

[0008] Many epidemiological studies have shown that plasma HDL levels are associated with reduced risk of cardiovascular and cerebrovascular diseases (Hovingh, G.K. et al., Curr. Op. in Lipidology, 26, 127-132 (2015); Rader, D.J., Nature Med., 18, 1344-1346 (2012)) and improved outcomes after ischemic events (Makihara, N. et al., Cerebrovascular Diseases, 33, 240-247 (2012); Olsson, A.G. et al., European Heart Journal, 26, 890-896 (2005)). However, pharmacological increases in total HDL-cholesterol by cholesteryl ester transfer protein inhibitors or supplemental niacin did not reduce cardiovascular outcomes (Keene, D. et al., BMJ, 349, g4379 (2014)). Furthermore, HDL particles are heterogeneous, contain a large number of bioactive factors and modulate vascular, metabolic and immune functions (Rye, K.A., Journal of Lipid Research, 50 Suppl, S195-200 (2009)), suggesting that specific HDL particle subtypes modulate unique functions in the cardiovascular system. For example, HDL containing plasma apolipoprotein M (ApoM + HDL) was recently shown to be the physiological carrier of the bioactive lipid sphingosine 1-phosphate (S1P), which acts on G protein-coupled S1P receptors to inhibit inflammatory responses and maintain vascular barrier function (Christensen, P.M. et al., FASEB J., 30.6 (2016): 2351-2359 (2016); Christoffersen, C. et al., PNAS, 108, 9613-9618 (2011); Galvani, S. et al., Science Signaling, 8, ra79 (2015)). Regarding S1P-dependent immune effects, ApoM +HDL is not required for lymphocytes to exit secondary lymphoid organs, but rather suppresses lymphopoiesis in the bone marrow (Blaho, V.A. et al., Nature, 523, 342-346 (2015)). Mice lacking ApoM have altered lipoprotein metabolism in an LDL receptor null background and exhibit enhanced atherosclerosis. Furthermore, adenoviral expression of ApoM suppresses atherosclerosis in LDL receptor null mice (Wolffum, C. et al., Nature Med., 11, 418-422 (2005); Christoffersen, C. et al., J. of Biol. Chem., 283, 1839-1847 (2008)). Plasma ApoM is positively correlated with HDL, LDL and cholesterol, and negatively correlated with acute myocardial infarction, endotoxemia, diabetes, metabolic syndrome and BMI (Frej, C. et al., JCMM, 20.6 (2016): 1170-1181 (2016); Borup, A. et al., Current Opinion in Lipidology, 26, 48-55 (2015); Nielsen, L.B. et al., Trends in Endocrinology and Metabolism, 20, 66-71 (2009), Plomgaard, P. et al., Journal of Internal Medicine, 266, 258-267 (2009)). Taken together, these observations suggest that ApoM + HDL promotes endothelial function and this signaling pathway is impaired in cardiovascular, inflammatory and metabolic diseases.

[0009] Sphingosine 1 -phosphate (S1P), a phosphorylated metabolite of D-sphingosine, binds to five G protein-coupled receptors (S1P1 through S1P5) and modulates a variety of biological actions (Garcia et al., J. Clin. Invest, 108:689-701 (2001); Ishii et al., Annu. Rev. Biochem., 73:321-354 (2004)). In particular, the prototypic S1P1 receptor is required for vascular maturation during development and promotes endothelial cell migration, angiogenesis, and barrier function (Liu et al., J. Clin. Invest, 106:951-961 (2000); Paik et al., J. Biol Chem., 276:11830-11837 (2001); Lee et al., Cell, 99:301-312 (1999)). Thus, S1P is required to maintain the barrier properties of lung endothelium (Camerer et al., J. Clin. Invest, 119:1871-1879 (2009)). Plasma S1P, which originates from several cellular sources (Pappu et al., Science, 316:295-298 (2007); Venkataraman et al., Circ. Res., 102:669-676 (2008)), is associated with high-density lipoprotein (HDL) (about 65%) and albumin (about 35%) (Aoki et al., J. Biochem., 138:47-55 (2005); Argraves et al., J. Lipid Res., 48:2325-2333 (2007)). HDL-induced vasodilation and barrier-promoting and pro-survival effects on endothelium have been attributed to S1P signaling (Kimura et al., J. Biol Chem., 281:37457-37467 (2006); Nofer et al., J. Clin. Invest, 113:569-581 (2004); Argraves et al., J. Biol Chem., 283:25074-25081 (2008)). Thus, the endothelium-protective effects of HDL are largely due to the effects of S1P on endothelial S1P receptors.

[0010] S1 P chaperone apolipoprotein M (ApoM) is an approximately 22 kDa HDL-associated apolipoprotein and a member of the lipocalin protein family that is found primarily in the plasma HDL fraction (XU et al., J. Biol Chem., 274:31286-31290 (1999)). Mature ApoM (human apoM (SEQ ID NO: 9 (GenBank Accession No: NP_061974.2)) and murine ApoM (SEQ ID NO: 10 (GenBank Accession No: NP_061286.1))) retains a signal peptide (amino acids 1-21 of SEQ ID NO: 9 and SEQ ID NO: 10) that serves as a lipid anchor, linking ApoM to the phospholipid layer of the lipoprotein, thereby keeping it in circulation and preventing filtration of ApoM in the kidney (Christoffersen et al., J. Biol Chem., 283: 18765-18772 (2008)).

[0011] ApoM contains a lipid binding pocket associated with S1P and a tethered signal peptide that allows it to anchor to HDL particles (Axler, O. et al., FEBS Letters 582, 826-828 (2008)). The binding affinity of S1P to its receptors is higher than to ApoM, which can allow S1P to be released from the chaperone, followed by association with and activation of the receptor (Christoffersen, C. et al., PNAS, 108, 9613-9618 (2011); Sevvana, M. et al., Journal of Molecular Biology, 404, 363-371 (2010); Lee, M.J. et al., Science, 279, 1552-1555 (1998)). Recent studies have shown that HDL-bound S1P acts as a "biased agonist" to the endothelial S1P1 receptor, meaning that only a subset of downstream responses are activated (Galvani, S. et al., Science Signaling, 8, ra79 (2015)). HDL-bound S1P is important for endothelial survival, migration, angiogenesis, NO production, and suppression of inflammatory responses (Galvani, S. et al., Science Signaling, 8, ra79 (2015); Nofer, J.R. et al., JCI, 113, 569-581 (2004); Nofer, J.R. et al., JBC, 276, 34480-34485 (2001); Kimura, T. et al., JBC, 281, 37457-37467 (2006)). In addition, HDL-bound S1P can engage both HDL receptors (SR-B1, etc.) and S1P receptors to elicit specific biological responses, such as stimulation of NO synthesis, suppression of endothelial damage and inflammation (Sato, K., World Journal of Biological Chemistry, 1, 327-337 (2010)).

[0012] SUMMARY

[0013] In one aspect, the present disclosure provides a fusion protein comprising an apolipoprotein M (ApoM) polypeptide fused to a fragment crystallizable (Fc) region of an antibody.

[0014] In some embodiments, the ApoM polypeptide comprises amino acids 21-188 of SEQ ID NO: 9. In some embodiments, the ApoM polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0015] In some embodiments, the Fc region is fused to the amino terminus of the ApoM polypeptide. In some embodiments, the Fc region is fused to the carboxy terminus of the ApoM polypeptide.

[0016] In some embodiments, the Fc region is an Fc region selected from the group consisting of an IgG antibody, an IgM antibody, an IgA antibody, an IgE antibody, and an IgD antibody. In a particular embodiment, the Fc region is an IgGl-Fc.

[0017] In another aspect, the disclosure provides a composition comprising an ApoM fusion protein complexed with a phospholipid or a lysophospholipid.

[0018] In some embodiments, the phospholipid comprises phosphocholine. In some embodiments, the phospholipid comprises sphingosine 1 -phosphate (S1P).

[0019] In some embodiments, the composition is formed by mixing the fusion protein with the phospholipid or the lysophospholipid, incubating the mixture to allow complex formation, and purifying the complex.

[0020] In another aspect, the disclosure provides a method of treating a disorder in a subject, comprising administering to the subject a composition comprising an ApoM-Fc fusion protein complexed with a phospholipid or a lysophospholipid, wherein the disorder is selected from the group consisting of: hypertension, cardiac ischemia, cerebral ischemia, accelerated atherosclerosis, non-cardiac reperfusion injury, and peripheral vascular disease.

[0021] In some embodiments, the hypertension comprises a disorder selected from the group consisting of: primary resistant hypertension, secondary resistant hypertension, neurogenic hypertension, pregnancy hypertension (pre-eclampsia), diabetic pre-eclampsia, and hypertension of chronic kidney disease.

[0022] In some embodiments, the cardiac ischemia comprises a disease selected from the group consisting of: cardiac reperfusion injury, myocardial infarction, acute coronary syndrome, and angina pectoris.

[0023] In some embodiments, the non-cardiac reperfusion injury comprises an injury caused by ischemia selected from the group consisting of: liver ischemia, kidney ischemia, intestinal ischemia, and muscle ischemia.

[0024] In another aspect, the present disclosure provides a method of reducing side effects of fingolimod in a patient being treated with fingolimod, comprising administering to the patient an ApoM-Fc fusion protein. SUMMARY

[0026] FIGS. 1A-1E. Production, purification and characterization of S1P binding by ApoM-F c and ApoM-F c -TM fusion proteins. (A) (Left panel) Co-crystal structure of S1P bound to ApoM. The three residues R98, W100 and R116 that contact the headgroup region of S1P are labeled. (Right panel) Space-filling model of the headgroup region of S1P in the ApoM molecule. (B) ApoM-F c and ApoM-F c -TM fusion proteins were expressed and purified from conditioned medium of HEK293 or Sf9 cells as described. Purified material was separated by non-reducing or reducing 10% SDS-PAGE and detected by anti-ApoM immunoblotting. (C) Sf9-derived purified proteins (5 μg) were analyzed by reducing 10% SDS-PAGE and stained with Coomassie blue. (D) Binding of purified IgG1-Fc (Fc), ApoM-Fc or ApoM-Fc-TM to S1P was analyzed by fluorescence spectrofluorimetry (N=4, expressed as mean (+ S.D.)) as described. Data were analyzed by Student's t test and two-way ANOVA followed by Bonferroni post-hoc test, comparing ApoM-F c or ApoM-F c -TM to Fc alone (ApoM-F c (****, P<0.001) ApoM-F c -TM (n.s.; not significant)). (E) Purified ApoM-F c and ApoM-F c -TM (5 μM) were incubated with SIP for 24 to 48 hours or not, purified by gel filtration chromatography and analyzed for sphingolipids by electrospray ionization-tandem mass spectrometry (ESI-MS / MS) as described. The resulting data are expressed as mean (+ S.D.); N=4.

[0027] FIGS. 2A-2D. ApoM-F c activates S1P receptors. (A) Increasing doses of albumin (Alb)-S1P, ApoM-F c or ApoM-F c- TM were incubated for 24 hours with MEF cells isolated from S1P1-GFP signaling mice and analyzed by flow cytometry. (B) Quantitative analysis of the results from (A). N=3, expressed as mean (+ S.D.). Data were analyzed by two-way ANOVA followed by Bonferroni post-test. ApoM-F c or BSA-S1P were compared to ApoM-F c -TM. (****, P<0.001; ***, P<0.01) (C) ApoM-F c -S1P (6 to 12 μg / ml; 60 to 120 nM S1P) or ApoM-F c -TM (12 μg / ml) were used to treat CHO cells stably transduced with S1P1 or S1P2 or not for 5 min and analyzed for p44 / 42 ERK and Akt by immunoblotting. N=3; representative blots are shown. (D) ApoM-F c -S1P (20 μg / ml; 240 nM) or ApoM-F c -TM (20 μg / ml) were used to treat HUVECs (left panels) or CRISPR / Cas9-derived S1P1 KO HUVECs (middle and right panels) for the indicated times and analyzed for p44 / 42 ERK, Akt and eNOS activation by immunoblotting. ApoM-F c -S1P (20 μg / ml; 240 nM S1P) or S1P2 inhibitor JTE-013 (10 μM) or S1P3 inhibitor TY52156 (10 μM) or both were used to treat CRISPR / Cas9-derived S1P1 KO HUVECs and analyzed for p44 / 42 ERK and Akt activation by immunoblotting. N=2 to 3; representative blots are shown.

[0028] Figures 3A to 3C. ApoM-F c effects on S1P receptor endocytosis and endothelial cell barrier function. (A) Barrier function of HUVECs was analyzed by real-time measurement of TEER as described. At time 0, Alb-S1P (200 nM) or ApoM-Fc (20 μg / ml; 200 nM) or ApoM-F c -TM (20 μg / ml) were added. All data were compared to baseline ApoM-F c- TM (N=3; expressed as mean (+ s.e.m) t-test, ****, P<0.0001; two-way ANOVA, P<0.0001) (B) HUVECs or S1P1 KO HUVECs (S1P1-CRISPR) were treated with ApoM-F c (10 pg / ml; 100 nM) and barrier function was analysed by real-time measurement of TEER as described. (t-test, ****, P<0.0001; one-way ANOVA, P<0.0001) (C) U2OS cells expressing S1P1-GFP were treated with the indicated concentrations of FTY720-P, BSA-S1P, ApoM-F c or ApoM-F c - TM for 30 min at 37°C, fixed and receptor internalization was quantified as described. All data were compared to baseline ApoM-F c - TM (N=2, n=8; expressed as mean (+ s.e.m) t-test **, P<0.01; *, P<0.05, one-way ANOVA, P<0.01).

[0029] Figure 4A-4G. Effect of ApoM-Fc administration on plasma S1P levels and circulating hematopoietic cells. (A) WT mice (N=4) were treated with 4 mg / kg purified ApoM-F c or ApoM-F c - TM (N=4) and plasma ApoM levels were determined by immunoblot analysis as described. (B) Apom - / - mice (N=4, expressed as mean (+ S.D.)) were administered ApoM-F c S1P (4 mg / kg) and plasma sphingolipids 24 h after administration were quantified as described. (C) WT mice (N=4, expressed as mean (+ S.D.)) were administered 4 mg / kg purified ApoM-Fc-S1P or ApoM-Fc-TM by i.p. injection and plasma sphingolipids 24 h after administration were quantified as described. For B and C, data were analysed by two-tailed t-test (* p=0.05; ** p<0.01 **** p<0.005). (D) to (G) WT mice were administered PBS (N=5), 4 mg / kg purified ApoM-Fc-S1P (N=5) or ApoM-F c- TM (N=5) and blood was collected at 6 and 24 hours after injection. Blood cells were separated by centrifugation and lymphocytes (D), white blood cells (WBC) (E), red blood cells (RBC) (F) and platelets (G) were quantified by clinical grade cytometry. Observed changes in relative blood cell counts were not statistically significant as judged by two-way ANOVA.

[0030] Figures 5A to 5E. ApoM-F c Administration in mice elicited a sustained anti-hypertensive effect. (A) Vehicle (PBS) (N=2) or ApoM-F c (N=6) or ApoM-F c - SBP was measured in AngII-treated mice (N=4) over time. (B) AngII-treated mice were injected with S1P1 antagonist W146 (10 mg / kg) every 24 hours (arrow) and SBP was subsequently measured. (N=5, 5) (C) ApoM-F c or ApoM-F c - SBP was measured in AngII-treated mice (N=4) over time. (B) AngII-treated mice were injected with S1P1 antagonist W146 (10 mg / kg) every 24 hours (arrow) and SBP was subsequently measured. (N=5, 5) (C) ApoM-F c or ApoM-F c - SBP was measured in AngII-treated mice (N=4) over time. (B) AngII-treated mice were injected with S1P1 antagonist W146 (10 mg / kg) every 24 hours (arrow) and SBP was subsequently measured. (N=5, 5) (C) ApoM-F - / - - SBP was measured in AngII-treated mice (N=4) over time. (B) AngII-treated mice were injected with S1P1 antagonist W146 (10 mg / kg) every 24 hours (arrow) and SBP was subsequently measured. (N=5, 5) (C) ApoM-F All data are expressed as mean ± Standard Error of Mean (S.EM.); *P<0.05; **P<0.01; ***P<0.001 compared to wild type (WT) (A to D); statistical significance was determined by two-way ANOVA followed by Bonferroni post-hoc test or one-way ANOVA.

[0031] Figures 6A to 6H. ApoM-F cAdministration attenuates ischemia / reperfusion injury in heart and brain. WT mice were administered PBS or 4 mg / kg of ApoM-Fc (N=9) or ApoM-Fc-TM (N=9) by intravenous injection 30 minutes prior to myocardial ischemia / reperfusion surgery. Animals were subjected to 30 minutes of ischemia followed by 24 hours of reperfusion. (A) Quantitative measurement of area at risk (AAR) / left ventricle (LV) area and infarct / AAR area was performed in a blinded manner for the following cohorts: PBS (N=7), ApoM-F c -TM (N=9) and ApoM-F c (N=9). (B) Heart sections were stained with Ly6G and IB4 antibodies and neutrophil and capillary density quantified. N=9. (C) LV end-diastolic diameter (LVDd), LV end systolic (LVDs) diameter and fractional shortening (FS) were measured at the indicated time points after myocardial I / R injury (n=6). Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 compared to ApoM-F c group. (D) Mice were subjected to 60 minutes of focal cerebral ischemia by middle cerebral artery occlusion (MCAO). Immediately after reperfusion, mice received 4 mg / kg of ApoM-Fc (N=10), ApoM-F c -TM (N=10) or PBS (N=10) by intraperitoneal injection. (E) Infarct and edema proportions were calculated by image analysis and reported as a proportion of the non-ischemic hemisphere. Infarct proportion was corrected for edema. (F) Total infarct volume (in mm 3(G) Neurological deficit scores were assessed 23 hours after reperfusion. (H) Relative cerebral blood flow (rCBF) in the middle cerebral artery (MCA) region was measured by laser speckle during the MCAO procedure. Relative CBF (% of contralateral (CL)) is shown during occlusion (designated "I") and after reperfusion (designated "R"). Individual values and mean ± S.EM. are shown. *P < 0.05 (one-way non-parametric ANOVA followed by Dunn's test). DETAILED DESCRIPTION

[0033] DEFINITIONS

[0034] The term "about" as used herein means a variation of about ±10% relative to a given value.

[0035] The term "acute coronary syndrome" (ACS) refers to a syndrome in which part of the heart muscle does not function properly or dies due to a decrease in blood flow in the coronary arteries. The most common symptom of ACS is chest pain, often radiating to the left shoulder or angle of jaw, accompanied by nausea and sweating. Acute coronary syndrome is usually caused by one of three problems: ST elevation myocardial infarction (STEMI, 30% of cases), non ST elevation myocardial infarction (NSTEMI, 25% of cases) or unstable angina (38% of cases).

[0036] The term "angina pectoris" refers to chest pain or discomfort that occurs when a region of the heart muscle does not get enough oxygen.

[0037] The term "atherosclerosis" refers to a pathological process that leads to an abnormal accumulation of cholesterol and cholesteryl esters and related lipids in macrophages, smooth muscle cells and other types of cells, which causes one or several arteries and arterioles of the body and of the body organs to narrow and / or occlude, including but not limited to the coronary arteries, the aorta, the renal arteries, the carotid arteries and the arteries supplying blood to the limbs and to the central nervous system. The associated inflammatory response and mediators of this pathological process are also included in this definition.

[0038] The term "chronic kidney disease" (CKD) as used herein refers to a gradual loss of kidney function over a period of months or years. CKD has its general meaning in the art and is used to classify a number of disorders that affect the kidney, destruction of the renal parenchyma and loss of functional nephrons or glomeruli. It is further noted that CKD can be caused by different reasons, but the final pathway is still kidney fibrosis. Examples of CKD etiology include, but are not limited to, cardiovascular disease, hypertension, diabetes, glomerulonephritis, polycystic kidney disease, and kidney transplant rejection.

[0039] The term "fragment crystallizable region" (Fc region) refers to the carboxy-terminal region of an antibody heavy chain that can interact with cell surface receptors known as Fc receptors and some proteins of the complement system.

[0040] The term "fusion protein" or "fusion polypeptide" refers to a protein having at least two heterologous polypeptides covalently linked, either directly or through an amino acid linker. The polypeptides forming the fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of a fusion protein can be in any order, and can include more than one of either or both component polypeptides. The term "host cell" as used herein refers to a cell or cell line into which a recombinant expression vector for producing a polypeptide can be transfected for expression of the polypeptide.

[0041] The term "hypertension" refers to a condition in which the blood pressure within the blood vessels is higher than normal as blood circulates through the body. Normal blood pressure at rest is in the range of 100 to 140 millimeters of mercury (mm Hg) systolic and 60 to 90 mm Hg diastolic. For most adults, hypertension is present if the resting blood pressure is consistently at or above 140 / 90 mm Hg.

[0042] Hypertension is classified as "essential" (primary) or "secondary". Essential (primary) hypertension has no obvious cause. This can be due to, for example, family history or lifestyle. The majority of people with hypertension have essential hypertension. Secondary hypertension, on the other hand, is less common and is a result of another condition, for example: adrenal diseases, including Cushing's syndrome, hyperaldosteronism and pheochromocytoma; kidney disease, which can include polycystic kidney disease, kidney tumours, kidney failure, or narrowing or blockage of the main artery supplying blood to the kidney; drugs, such as corticosteroids (anti-inflammatory drugs, such as prednisone), non-steroidal anti-inflammatory drugs, weight loss drugs (such as phentermine), cold medicines, including decongestants, such as pseudoephedrine, birth control pills (oestrogen component) and migraine drugs; sleep apnoea; coarctation of the aorta, which is a birth defect of the narrowing of the aorta; pre-eclampsia, a condition associated with pregnancy; and thyroid and parathyroid problems.

[0043] The term "ischemia" as used herein refers to an insufficient or stoppage of blood flow to a part of the body caused by constriction or blockage of the blood supplying vessels.

[0044] The term "cerebral ischemia" refers to an absolute or relative shortage of blood supply to the brain, resulting in damage or dysfunction of brain tissue, especially central nervous cells.

[0045] The term "cardiac ischemia" refers to a reduction in blood flow to heart tissue, which can lead to arrhythmic conditions (such as ventricular arrhythmias and ventricular fibrillation) as well as cell death. Such arrhythmic conditions are the result of a desynchronized state of excitability between normal and ischemically damaged heart cells, which in turn causes disruption of normal ion transport channels within the cardiac tissue.

[0046] The term "myocardial infarction" (also known as "heart attack") as used herein refers to an acute cardiovascular event that occurs suddenly when a part of the heart is deprived of blood supply, and is defined by evidence of myocardial cell necrosis due to significant and sustained ischemia.

[0047] The term "peripheral vascular disease" or "PVD" refers to peripheral atherosclerotic disease or occlusive arteriosclerosis, which involves the blockage of blood supply to the extremities by atherosclerotic plaques and encompasses intermittent claudication (pain caused by too little blood flow).

[0048] The term "phospholipid" refers to compounds derived from fatty acids and phosphorus-containing compounds linked to glycerol or aminoalcohol sphingosine, which yield compounds with both lipid-soluble and water-soluble regions. The term "lysophospholipid" refers to a phospholipid derivative in which one or both acyl groups have been removed by hydrolysis.

[0049] The term "pre-eclampsia" as used herein refers to a condition that occurs during pregnancy whose primary symptom is high blood pressure in various forms, often accompanied by the presence of protein in the urine and edema (swelling). Pre-eclampsia (sometimes also referred to as toxemia of pregnancy) is associated with a more severe disease known as "eclampsia", which is pre-eclampsia accompanied by seizures. These conditions usually occur during the second half of pregnancy (after 20 weeks), although they can occur shortly after birth or before 20 weeks of gestation.

[0050] The term "resistant hypertension" refers to the maintenance of blood pressure above normal despite the concurrent use of three different classes of antihypertensive agents, one of which is a diuretic.

[0051] The term "recombinant protein" as used herein refers to a protein produced from the transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a host cell.

[0052] The term "reperfusion injury" refers to tissue damage that occurs when blood flow returns to a tissue after a period of ischemia.

[0053] The term "subject" or "patient" refers to an animal, including a human, that has been diagnosed with a disease and treated with the methods of the present application. The term "subject" or "patient" is intended to refer to both males and females, unless explicitly indicated otherwise.

[0054] The term "sphingosine 1 -phosphate" (SlP) refers to a signaling sphingolipid, also known as a lysosphingolipid. SlP is also known as a bioactive lipid mediator. Sphingolipids are a class of lipids characterized primarily by a specific aliphatic amino alcohol, i.e., sphingosine. SlP has the following chemical structure:

[0055]

[0056] and the chemical name of "(2S, 3R, 4E)-2-amino-4-octadecen-1,3-diol 1 -phosphate, D- erythro-sphingosine 1 -phosphate". SlP can be prepared according to protocols known in the art, for example, in Reimann, C. and S1P is extracted and purified from tissue samples, cells from heparinized blood, or plasma following the protocol described in M. H. (Bio-protocol, (2016), 6(10): el817. DOI: 10.21769 / BioProtoc.1817). Purified S1P is also available from various commercial sources, such as from Sigma-Aldrich (Cat#: S9666), from VWR (Cat#: AAJ66459-LB0), or from Tocris Bioscience (Cat#: 1370).

[0057] General Description

[0058] ApoM-Fc fusion proteins

[0059] Free ApoM, which is not associated with HDL, has a very short half-life (e.g., less than 15 minutes in vivo) (Faber, K. et al., Molecular Endocrinology, 20, 212-218, (2006)). In accordance with the present disclosure, the present inventors have discovered that when the apolipoprotein M (ApoM) protein is fused to a fusion partner, such as the fragment crystallizable (Fc) region of an antibody, its half-life is significantly improved (e.g., greater than 96 hours in vivo). Without being bound by any particular theory, the Fc domain improves the stability of ApoM by preventing its degradation by proteasomes. The ApoM-Fc fusion proteins disclosed herein have been shown to be functional and effective in raising plasma levels of bioactive phospholipids in vivo. The disclosed ApoM-Fc fusion proteins can be produced recombinantly in highly purified form, and the purified ApoM-Fc fusion proteins can be stored in buffered saline solutions for longer periods of time than purified native ApoM protein. The ApoM fusion proteins are also useful for delivering phospholipids to organs and tissues in need thereof.

[0060] Thus, in one aspect, the present disclosure provides ApoM-Fc fusion proteins. In some embodiments, the ApoM-Fc fusion proteins are provided as dimers. In some embodiments, the ApoM-Fc dimers are formed by disulfide bonds between the Fc regions.

[0061] According to the present disclosure, "ApoM" in the ApoM-Fc fusion protein refers to any ApoM polypeptide that includes a portion of the native mature ApoM protein that retains S1P binding ability. In one embodiment, the ApoM polypeptide in the ApoM-Fc fusion protein is a human ApoM polypeptide. In a specific embodiment, the ApoM polypeptide in the ApoM-Fc fusion protein comprises amino acids 21 to 188 of SEQ ID NO: 9. In some embodiments, the ApoM polypeptide in the ApoM-Fc fusion protein comprises a full-length, mature ApoM protein as in SEQ ID NO: 9. In some embodiments, the ApoM polypeptide in the ApoM-Fc fusion protein contains one or more deletions, additions, modifications, or substitutions of amino acid residues compared to the native ApoM protein or a fragment of the native ApoM protein, provided that the ApoM polypeptide retains S1P binding ability. For example, the ApoM polypeptide in the ApoM-Fc fusion protein has an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 9 or to amino acids 21 to 188 of SEQ ID NO: 9.

[0062] In some embodiments, the Fc region used herein includes at least CH2 and CH3 of an immunoglobulin. In some embodiments, the Fc region includes the hinge, CH2 domain, and CH3 domain of an immunoglobulin. The hinge can serve as a flexible spacer between the two portions of the Fc fusion protein, allowing each portion of the molecule to function independently. In some embodiments, the Fc region in the ApoM-Fc fusion protein is an Fc region of an immunoglobulin selected from the group consisting of IgG, IgM, IgA, IgE, and IgD. In a specific embodiment, the Fc region is the Fc region of IgG1 (IgG1-Fc). In some embodiments, the Fc region is fused to the amino terminus of ApoM; while in other embodiments, the Fc region is fused to the carboxy terminus of ApoM. In some embodiments, the Fc region is fused directly to the ApoM protein, i.e., without a linker. In other embodiments, the Fc region is fused to the ApoM protein via a linker. One skilled in the art can use any type of linker, provided that the linker allows for chemical linkage of the ApoM peptide to the Fc region. A suitable linker for use in the present disclosure can be a short peptide sequence that exists between protein domains. In some embodiments, the linker is composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other.

[0063] In some embodiments, ApoM-Fc fusion proteins can be produced recombinantly in a suitable host cell. Suitable host cells include bacteria, archaea, fungi (especially yeast), as well as plant, insect, and animal cells (especially mammalian cells). Specific examples of host cells include E. coli, B. subtilis, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Schizosaccharomyces pombe, Pichia pastoris, SF9 cells, C129 cells, 293 cells, Neurospora, as well as CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines. In one specific embodiment, ApoM-Fc fusion proteins can be expressed in E. coli, recovered from inclusion bodies, purified (e.g., by chromatography), and refolded as described by Sevvana et al. (J. Mol. Biol. 393:920-936 (2009)). In another specific embodiment, ApoM-Fc fusion proteins can be expressed in sf9 insect cells infected with a baculovirus strain containing a nucleic acid construct encoding the ApoM-Fc fusion protein.

[0064] In another embodiment, ApoM-Fc fusion proteins are produced using in vitro cell-free translation systems, which are commercially available from, e.g., Life Technology or any other suitable source. Both prokaryotic and eukaryotic cell-free translation systems can be used. Typically, cell-free translation systems utilize extracts prepared from cells that participate in high rates of protein synthesis, such as rabbit reticulocytes, wheat germ, and E. coli, which contain the macromolecular components required for translation (e.g., 70S or 80S ribosomes, tRNA, aminoacyl-tRNA synthetases, initiation, elongation, and termination factors). The extracts are typically supplemented with amino acids, energy sources (ATP, GTP), energy regeneration systems (creatine phosphate and creatine phosphate kinase for eukaryotic systems, and phosphoenolpyruvate and pyruvate kinase for E. coli lysates), and other cofactors (Mg 2+ , K + Some translation systems (e.g., reticulocyte lysates and wheat germ extracts) use RNA as a template, while others start with a DNA template, which is transcribed into RNA, which is then translated. All of these systems are suitable for in vitro synthesis of ApoM-Fc fusion proteins.

[0065] In some embodiments, the ApoM-Fc fusion protein is purified using suitable protein purification techniques. In a specific embodiment, the ApoM-Fc fusion protein is purified using chromatography. In some embodiments, the ApoM-Fc fusion protein purification comprises at least one of ion exchange chromatography, size exclusion chromatography, or expanded bed adsorption chromatography separation methods. In a specific embodiment, the chromatography method is lectin affinity chromatography, protein A affinity chromatography, gel filtration chromatography, or a combination thereof. In some embodiments, the ApoM-Fc fusion protein can be purified by affinity tag purification. In a specific embodiment, the ApoM-Fc fusion protein can be tagged with a poly-His tag and purified by nickel affinity purification. In another embodiment, the ApoM-Fc fusion protein can be tagged with a tag selected from a myc-tag, a FLAG tag, a HA tag, a GST tag, a NE tag, a V5 tag, or a VSV tag, or any other tag known in the art, and subsequently purified using an antibody against the tag.

[0066] The purity of the ApoM-Fc fusion protein can be assessed by a variety of methods, including but not limited to SDS-PAGE followed by silver staining and chromatography with multi-wavelength detection, as described in Frank J. et al., Anal Biochem. Apr; 162(l):65-73 (1987).

[0067] The purified ApoM-Fc fusion protein can be maintained in buffered saline for in vitro or in vivo use.

[0068] In some embodiments, the ApoM-Fc fusion protein can be covalently linked or bound to a carrier or a vehicle to further improve the desired biological properties of the protein. In a specific embodiment, the desired biological properties of the ApoM-Fc fusion protein include an improvement in bioavailability, or an improvement or prolongation of the plasma half-life of the protein in vivo. In a specific embodiment, the carrier comprises polyethylene glycol (PEG) or a nanoparticle.

[0069] Compositions comprising complexes of ApoM-Fc fusion proteins and phospholipids or lysophospholipids

[0070] The present disclosure also provides compositions comprising ApoM-Fc fusion proteins complexed with phospholipids or lysophospholipids.

[0071] As used herein, a complex is formed between the ApoM fusion protein and the phospholipid, wherein the fusion protein binds (i.e., is bound by) the phospholipid through non-covalent interactions. Phospholipids are naturally insoluble in water and form membranes or micelles in solution. Without being bound by any particular theory, the ApoM-Fc fusion protein increases the solubility of the phospholipid in solution by binding to and masking the hydrophobic portion of the phospholipid. Thus, the ApoM-Fc fusion protein allows the phospholipid to be transported in an aqueous medium (e.g., blood), thereby acting as a carrier for the phospholipid.

[0072] In some embodiments, the phospholipid can be selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, or a combination thereof.

[0073] In some embodiments, the lysophospholipid of the composition comprises a naturally occurring lysophospholipid, a synthetic lysophospholipid, or a combination thereof. In some embodiments, the lysophospholipid comprises a glycerol backbone bound to a polar group (e.g., a phosphocholine group), a free hydroxyl group at the 2 position of the glycerol backbone, and a saturated or unsaturated fatty acid residue bound to the glycerol backbone. In some embodiments, the fatty acid residue of the lysophospholipid has a C n - an alkyl chain or a C n - an alkenyl chain, wherein n > 4. In a particular embodiment, the lysophospholipid comprises an oxidized fatty acid residue.

[0074] In some embodiments, the complex between the ApoM-Fc fusion protein and the phospholipid or lysophospholipid can be formed by mixing the fusion protein with the phospholipid or lysophospholipid suspended in methanol, beta-cyclodextrin, or DMSO. In some embodiments, the fusion protein is mixed with the lipid at a ratio of about 1 :2 to 1 :50 (pmol fusion protein / pmol phospholipid or lysophospholipid). In some embodiments, the ratio (pmol fusion protein / pmol phospholipid or lysophospholipid) in methanol beta-cyclodextrin, or DMSO is about 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :20, 1 :30, 1 :40, or 1 :50. In a particular embodiment, the ratio is 1 :8. The fusion protein-lipid mixture can be incubated at about 4 to 37 °C for 24 to 48 hours. In some embodiments, the incubation temperature is about 2 °C, 4 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, or 40 °C. In some embodiments, the incubation time is about 24 hours, 30 hours, 36 hours, 42 hours, or 48 hours.

[0075] The ApoM-Fc fusion protein / phospholipid or lysophospholipid complex can be purified using known techniques, e.g., chromatography, to remove free / unbound lipid. In some particular embodiments, purification of the ApoM-Fc fusion protein-phospholipid complex includes at least one of ion exchange chromatography, size exclusion chromatography, or expanded bed adsorption chromatography. In some embodiments, the complex can be purified using a FPLC (fast protein liquid chromatography) machine. In one particular embodiment, the chromatography method is lectin affinity chromatography, protein A affinity chromatography, protein G affinity chromatography, or gel filtration chromatography.

[0076] The lipid content of the purified ApoM-Fc fusion protein-phospholipid or lysophospholipid complex can be analyzed by liquid chromatography / mass spectrometry (LC / MS / MS) as described by Sommer, U. et al. (Journal of Lipid Research, 47(4), 804-814 (2006)) and Christoffersen, C. et al. (PNAS, 108, 9613-9618, (2011)).

[0077] In some embodiments, binding of S1P to the ApoM-Fc fusion protein can be determined by intrinsic tryptophan fluorescence quenching in a fluorospectrophotometer as previously described (Sevvana, M. et al., Journal of Molecular Biology, 404, 363-371 (2010)).

[0078] In some embodiments, after purification to remove unbound lipid, the resulting composition includes ApoM-Fc fusion protein bound to lipid (i.e., complexed or "loaded") as well as ApoM-Fc fusion protein not bound to lipid ("empty"). In some embodiments, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more of the ApoM-Fc fusion protein in the composition is bound by phospholipid or lysophospholipid. The percentage of ApoM-Fc fusion protein in the composition that is bound by phospholipid or lysophospholipid can be determined by mass spectrometry, e.g., using electrospray ionization-tandem mass spectrometry (ESI-MS / MS).

[0079] In some embodiments, in the ApoM-Fc fusion protein-phospholipid or lysophospholipid complex, the ratio of ApoM-Fc fusion protein to phospholipid or lysophospholipid is about 1 :0.3, 1 :0.4, 1 :0.5, 1 :0.6, 1 :0.7, 1 :0.8, 1 :0.9, or 1 :1. The ratio of ApoM-Fc fusion protein:phospholipid or lysophospholipid in the fusion protein-lipid complex can be determined by mass spectrometry, for example, using electrospray ionization-tandem mass spectrometry (ESI-MS / MS).

[0080] Pharmaceutical compositions

[0081] A pharmaceutical composition comprising an ApoM-Fc fusion protein with or without a phospholipid bound thereto can be prepared using one or more physiologically acceptable carriers or excipients. As used herein, a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic agents, and the like. Except insofar as any conventional media, agent, diluent or carrier is incompatible with the active ingredient, its use in the practice of the application is suitable. The carrier can be a liquid, semi-solid (e.g., paste), or solid carrier. Examples of carriers include oils, water, saline solutions, alcohols, sugars, gels, lipids, liposomes, resins, porous matrices, adhesives, fillers, coatings, preservatives, and the like, or combinations thereof. In some embodiments, the carrier is a controlled release matrix, which is a material that allows the slow release of the active ingredient in a composition comprising an ApoM-Fc fusion.

[0082] According to the present application, the active ingredient of the pharmaceutical composition of the present application can be combined with the carrier in any convenient and useful way (e.g., by mixing, dissolving, suspending, emulsifying, encapsulating, adsorbing, etc.) and can be formulated into preparations suitable for injection, implantation, inhalation, ingestion, etc., such as tablets, capsules, powders, syrups, suspensions. Where appropriate, the pharmaceutical composition of the present application should be sterilized, for example, by filtration or by heat sterilization. To obtain a sterile powder, the sterilized solution can be vacuum-dried or freeze-dried as necessary.

[0083] Methods of treatment

[0084] In one aspect, the present disclosure provides a method of treating a disorder in a subject, wherein the disorder would benefit from an increase in plasma phospholipid levels, the method comprising administering to the subject a pharmaceutical composition comprising an Fc fusion protein complexed (or bound) to a phospholipid. Administration of the pharmaceutical composition results in the delivery of the bioactive phospholipid in the complex to the endothelial cells and all tissues of the body. The delivered bioactive phospholipid in turn promotes the impaired endothelial function in diseases including, but not limited to, cardiovascular, inflammatory, and metabolic diseases.

[0085] In some embodiments, the phospholipid is a sphingolipid. In a particular embodiment, the sphingolipid is sphingosine 1 -phosphate (S1P).

[0086] In some embodiments, the subject has a condition selected from the group consisting of hypertension, cardiac ischemia, cerebral ischemia, accelerated atherosclerosis, non-cardiac reperfusion injury, and peripheral vascular disease, which is a disease that would benefit from an increase in plasma levels of phospholipids. In some particular embodiments, the hypertension includes a condition selected from the group consisting of primary resistant hypertension, secondary resistant hypertension, neurogenic hypertension, gestational hypertension (preeclampsia), diabetic preeclampsia, and chronic kidney disease hypertension. In some embodiments, the cardiac ischemia includes a disease selected from the group consisting of cardiac reperfusion injury, myocardial infarction, acute coronary syndrome, and angina. In some embodiments, the non-cardiac reperfusion injury includes an injury due to ischemia of an organ or tissue other than the heart (cardiac) tissue. Examples of non-cardiac ischemia include hepatic ischemia, renal ischemia, intestinal ischemia, and muscle ischemia.

[0087] In other embodiments, the ApoM-Fc fusion protein in the pharmaceutical composition for administration is "empty", i.e., not bound by a phospholipid, and after administration, recruits and forms a complex with circulating S1P in the recipient. Plasma S1P is derived from several cellular sources (Pappu et al., Science 316:295-298 2007). For example, S1P is stored in human platelets at relatively high concentrations, which lack the enzymes responsible for its catabolism, and is released into the bloodstream upon activation by physiological stimuli (e.g., growth factors, cytokines, and receptor agonists and antigens).

[0088] In a particular embodiment, the ApoM-Fc fusion protein is administered to a patient being treated with FTY720 / Fingolimod / Gilenya TM (IUPAC name: 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol) or an analog thereof. FTY720 / Fingolimod / Gilenya TM is an approved therapeutic for multiple sclerosis, and can be used to treat other autoimmune indications, such as psoriasis, rheumatoid arthritis, uveitis, and type I diabetes. The most common side effects of Fingolimod are head colds, headache, and fatigue. However, Fingolimod has been associated with cases of potentially fatal infections, bradycardia, skin cancer, and hemorrhagic encephalitis (inflammation of the brain with hemorrhage). According to the present application, it is believed that the side effects of Fingolimod and analogs thereof can be reduced by the administered ApoM-Fc fusion protein, which will sequester excess circulating S1P molecules.

[0089] The pharmaceutical compositions of the present application, both the composition comprising empty ApoM-Fc fusion protein and the composition comprising ApoM-Fc fusion protein / phospholipid complex, can be administered to a subject by standard routes, including the oral, nasal, intratracheal, transdermal, parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous, or intramuscular) or rectal route. In addition, the composition comprising ApoM-Fc fusion can be introduced into the body by injection or by means of a surgically implanted or connected reservoir, in proximity to a preselected tissue or organ site, so that a significant amount of the active ingredient is able to diffuse through direct diffusion into that site, e.g., in a controlled release manner.

[0090] The dosage depends on the disease state or condition being treated and other clinical factors, such as the subject's body weight and condition, the subject's response to the treatment, the type of formulation, and the route of administration. The skilled artisan can determine the precise dosage which is therapeutically effective and non-harmful. Generally, the pharmaceutical composition can be administered in a unit dosage of about 0.5 μg to about 2 g per unit dosage form. Unit dosage form refers to physically discrete units suitable for use in a unit dosage to mammals: each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with any needed pharmaceutical carrier. The methods of the present application contemplate single as well as multiple administrations (given simultaneously or over an extended period of time).

[0091] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0092] The specific examples listed below are merely illustrative and are in no way limiting. Examples

[0093] Example 1: Materials and Methods

[0094] Generation of ApoM-Fc and Tri-mutant ApoM-Fc (ApoM-Fc-TM)

[0095] Generation of ApoM-Fc and Tri-mutant ApoM-Fc (ApoM-Fc-TM) 1-20PCR-derived cDNA of the ApoM open reading frame (ORF) (Xu, N., JBC, 274, 31286-31290 (1999)) was cloned into the pFUSE-mIgGl-Fc2 vector between the IL-2 signal peptide and the IgGl-Fc framework region (InVivogen; Cat. # pfuse-mglfc2) to generate the ApoM-IgGl-Fc fusion protein. Thus, a 507 bp open reading frame of human ApoM, lacking the signal peptide and having a stop codon replaced, was generated by PCR using the following primers:

[0096] Fwd: 5'-TATCCATGGGGATCTACCAGTGCCCTGAGCACAGT-3' (SEQ ID NO: 1)

[0097] Rev: 5'-TATGGATCCTCCGTTATTGGACAGCTCACAGGCCT-3' (SEQ ID NO: 2)

[0098] The forward primer inserted a new Ncol restriction site (bold and italic) starting at ApoM codon 21 and eliminated the non-cleavable ApoM signal peptide 23. The reverse primer inserted a new BamHI restriction site (bold and italic) and replaced the stop codon (TGA, codon 189) with a glycine codon by A>G substitution of TGA>GGA. The resulting PCR-derived DNA was purified and cut with Ncol-BamHI (New England Biolabs; Cat# R0193S, R3136S) double digestion and ligated (Quick Ligation Kit, New England Biolabs, Cat# M2200) into the open reading frame of pFUSE-mIgGl-Fc2 vector (InVivogen; Cat. # pfuse-mglfc2) digested with Ncol-BglII (New England Biolabs; Cat# R0193S, R0144S). BglII is a compatible cohesive end to BamHI and both sites are eliminated after ligation. The ligation was transformed into high efficiency competent DH5a (ThermoFisher Scientific; Cat# 18263012) and selected on Zeocin (25 pg / ml; ThermoFisher Scientific; Cat# R25005) Miller broth agar petri plates. Single colonies were picked and grown in 2 ml of Zeocin (25 pg / ml) Miller broth and DNA was isolated using GeneJET Plasmid Miniprep Kit (Thermo Scientific, Cat# K0503). Recombinant vectors were identified using diagnostic EcoRI (New England Biolabs, R3101S) DNA digest, which released a 430 bp DNA fragment, and positive clones were sequenced using the Cornell DNA Sequencing Core Facility. This clone produced the fusion gene ApoM-F c (pApoM-F c ). To produce a non-S1P binding negative control for these studies, the ApoM-F c triple mutant (pApoM-Fc-TM) was produced. Mutations were produced by site-specific mutagenesis at codons R98A, W100A and R116A using the QuikChange II XL Site-Directed Mutagenesis Kit according to the manufacturer's protocol and using the following primers:

[0099] For R98A and W100A: 5'-CGCCCTGCCATGGCGACTGAGCTC-3' (SEQ ID NO: 3)

[0100] For R116A: 5'-AATCATGCTGAATGCGACAGGCC-3' (SEQ ID NO: 4)

[0101] The mutated plasmids were transformed into bacteria as described above and selected on Zeocin agar plates. Clones were isolated and miniprepped as described above and sequenced as above. This resulted in the mutant fusion genes ApoM-F c -TM (pApoM-F c -TM).

[0102] ApoM-F c , ApoM-F c TM and IgG-F c 1 protein expression

[0103] To produce milligram quantities of properly folded glycosylated proteins for in vivo studies, the Bac-to-Bac baculovirus expression system (ThermoFisher / Invitrogen) was employed, which uses recombinant baculovirus to express soluble proteins in insect cell supernatant. The pApoM-Fc, pApoM-Fc triple mutant or original pFUSE-IgGl F c 2 (IgGl-F c ) plasmid was used as template for another round of PCR using primers reactive to the pFUSE-IgGl F c 2 vector:

[0104] Fwd: 5'-TATGGATCCATGTACAGGATGCAACTCCTGTCTT-3' (SEQ ID NO: 5)

[0105] Rev: 5'-TATTTATCATGTCTGGCCAGCTAGCGACACTGGG-3' (SEQ ID NO; 6)

[0106] The forward primer created a restriction site for BamHI (italicized) and the reverse primer created a restriction site for NheI (italicized). The resulting PCR-derived DNA cassette was purified and cleaved by double digestion with BamHI-NheI (New England Biolabs, Cat# R3136S, R5131S) and ligated into the baculovirus expression vector pFASTBAC1 (Invitrogen) which was restriction digested with BamHI-XbaI (New England Biolabs, R3136S, R5145S). Recombinant viral DNA was generated using the manufacturer’s protocol (Invitrogen; Bac-to-Bac Baculovirus Expression System) using DH10BAC E. coli bacteria and plated on Luria Broth (LB) agar plates using triple antibiotic selection with tetracycline (10 pg / ml), gentamycin (7 pg / ml), and kanamycin (50 pg / ml). Single colonies were selected and grown in LB broth supplemented with the same triple antibiotic combination. Recombinant plasmids were isolated by miniprep as described above and positive clones were sequenced at the Cornell University Sequencing Core Facility. The resulting baculovirus plasmids were designated pApoM-F c (Bac), pApoM-F c TM (Bac), or pIgG1-F c (Bac).

[0107] ApoM-F c , ApoM-F c TM, and IgG1-F c Production

[0108] pApoM-F c , pApoM-Fc-TM, or control pFUSE-mIgG1 Fc2 vector were assayed for protein expression by transfecting HEK 293T cells (AATC Cat.#: CRL-1573) using Lipofectamine 2000 reagent (ThermoFisher Scientific, Cat# 11668019). Normal cultures were maintained at 37°C in DMEM supplemented with 10% Fetal Bovine Serum (FBS). Recombinant pApoM-F cPlasmids were transfected into 3 separate cultures of 293T cells for 72 hours. For the last 18 hours, the media was replaced with serum-free OptiMem media (ThermoFisher Scientific Cat#31985-070). Supernatants were collected, clarified by centrifugation and stored at -80°C until use.

[0109] Recombinant ApoM-F c , ApoM-F c -TM or IgG1-F c Generation of baculovirus

[0110] Recombinant baculovirus DNA (1 to 3 pg) pApoM-F c (Bac), pApoM-F c -TM (Bac) or pi gG1-F c (Bac) was transfected into a single culture of insect cell line Sf9. After 5 days, the resulting culture supernatant containing viral particles was passaged at a 1 :50 dilution onto naive Sf9 cells and incubated for 5 days. This was repeated 3 times to generate a high titer stock of virus (>10 9 PFU / ml). To further amplify / maintain the virus stock, 150 mm tissue culture plates seeded with 3 x 105sf9 cells in complete media (Sf900-III; ThermoFisher Scientific, Cat. #12658027) were inoculated with 500 mI of virus supernatant from the previous virus amplification step (passage 5) and incubated at 27°C for 5 days. For protein production, 3 x 105High-Five 3 (ThermoFisher Scientific, Cat#PHG0143) cells were seeded into 150 mm tissue culture plates in complete media (Sf900-III; ThermoFisher Scientific, Cat#12658027) and subsequently infected with 1 ml of virus stock and incubated at 27°C for 4 to 5 days in a humidified incubator. Supernatants were collected and clarified by centrifugation at 3,000 x g for 10 min and stored at 4°C. 7 7 TM 2

[0111] Western blot analysis of recombinant ApoM-Fc

[0112] ​​​​Characterization of fusion proteins was confirmed using anti-ApoM specific immunoblot analysis. For most experiments, 10 to 20 μΐ of recombinant cell culture supernatant was heated to 95°C for 10 minutes in 5X Laemmli sample buffer. Separate samples with or without 100 mM dithiothreitol (Sigma-Aldrich) were prepared. Samples were separated on 12% SDS-PAGE gels (BioRad, Acrylamide, Cat# 1610156) and electroblotted to nitrocellulose membranes (BioRad, Cat# 1620115). Blots were blocked with 5% milk (Carnation) suspended in TBS-T (50 mM Tris base pH 8.0, 150 mM NaCl, 0.05% Tween-20) at RT for 1 hour and then incubated with rabbit anti-ApoM monoclonal antibody (Genetex GTX62234; Clone EPR2904) overnight (>12 hours) and washed with 5 changes of TBS-T over a 30 minute period. Blots were incubated in 1% milk-TBS-T supplemented with goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (1 :5000 (v / v); Jackson Labs) for 60 minutes and then washed 5 times in TBS-T over a 30 minute period at RT with gentle shaking. Blots were incubated with Immobilon Western Chemiluminescent HRP Substrate Millipore, Cat# WBKLS0500) and chemiluminescence was visualized using X-ray film (Denvillie Scientific, HyBlotCL E3018).

[0113] Purification of ApoM-Fc, ApoM-Fc-S1P, ApoM-Fc-TM and IgG1-Fc

[0114] Large scale purification of fusion proteins was performed on a BioRad NGC FPLC chromatography system using the following protocol:

[0115] Step 1) Clarification of 100 to 200 ml of culture supernatant containing fusion protein by ultracentrifugation at 42,000 RPM (>100,000 g; Sorvall Discovery 90, T-1250 rotor) in sterile polystyrene screw cap tubes.

[0116] Step 2) Collection of supernatant and concentration to 1 / 10 volume using Amicon Ultra-15 centrifugal filters (Ultracel-50K).

[0117] Step 3) The concentrated culture supernatant was replaced with 10 volumes of Concanavalin A lectin binding buffer (LBB) (Tris-HCl 50 mM pH 7.5, NaCl 300 mM, MnCl2 1.5 mM, CaCl2 1 mM, MgCl2 1 mM) in an Amicon Ultra-15 centrifugal filter (Ultracel-50K).

[0118] Step 4) The protein sample was applied to a 5 ml Bioscale MT-5 column pre-packed with Concanavalin A-Sepharose beads, which was previously washed with 20 bed volumes of LBB. The application rate was 0.2 ml / min and the average pressure was 30 psi.

[0119] Step 5) The column was washed with LBB at a flow rate of 0.4 ml / min until the column flow-through reached the buffer baseline, OD 280 - Typically 55 to 57 mAU.

[0120] Step 6) The protein was eluted from the column using LBB supplemented with 200 mM a-methyl-mannoside (elution buffer), which was filter-sterilized before use. Typically, 2 ml of elution buffer was applied to the column (0.4 ml / min) followed by a 15 to 30 min incubation to allow efficient displacement of the bound protein, after subtracting the system volume.

[0121] Step 7) The protein was eluted from the column in elution buffer at a rate of 0.4 ml / min. Fractions of 0.8 ml were collected on a BioFrac collector until the OD 280 Back to the elution buffer baseline (about 125 mAU).

[0122] Step 8) The positive fractions were pooled and concentrated 10-fold on an Arnicon Ultra-4 centrifugal filter (Ultracel-10K) and the elution buffer was replaced with 10 volumes of PBS-1 mM EDTA to remove the mannose.

[0123] Step 9) The approximate concentration of the fusion protein was determined by BCA protein assay (Thermo Fisher Scientific) of the sample combined with SDS-PAGE of 5 μg of the preparation. For some experiments, the fusion protein was mixed with S1 P resuspended in methanol at 1 :8 (μmoles fusion protein / μmoles S1 P) and incubated at 4°C under gentle shaking for 24 to 48 hours. The final concentration of methanol in the sample did not exceed 3% (vol / vol). The sample was concentrated on an Amicon Ultra-4 centrifugal filter (Ultracel-10K).

[0124] Step 10) 1 ml of the protein concentrate was injected on a Superose 6 Increase 10 / 300 GL column pre-equilibrated with PBS-1 mM EDTA and separated at a rate of 0.4 ml / min until peak fractions were collected.

[0125] Step 11) The protein positive fractions were pooled and concentrated in an Amicon Ultra-4 centrifugal filter (Ultracel-10K). The buffer was replaced with 10 volumes of sterile PBS and maintained a final concentration of 1 to 3 mg / ml.

[0126] Analysis of FPLC protein fractions

[0127] All fractions were analyzed by SDS-PAGE. 10 μl of each fraction was boiled in 5X Laemmli sample buffer supplemented with 100 mM DTT at 95°C and separated on a 12% SDS-PAGE gel. The gel was fixed in methanol:acetic acid:water (50%:10%:40%) and stained in the fixing solution containing 0.3% Coomassie blue (BioRad) and destained in the fixing solution.

[0128] ApoM-F based fluorescence quenching analysis to measure S1 P c Binding

[0129] Previous studies have shown that recombinant ApoM, expressed in bacteria, binds S1 P with a relative affinity of about 1 μM based on fluorescence quenching of the predicted tryptophan at position 47 (W47) of the human ApoM polypeptide (Sevvana, M. et al., Journal of Molecular Biology, 404, 363-371 (2010)). Therefore, ApoM-Fc, ApoM-F TM (Horiba) in the range of 250 to 400 nm of the emission spectrum were analyzed at 0.125, 0.25 and 0.5 μM. c -TM or IgG1-F cLipid-dependent fluorescence quenching was performed. Baseline emission was established and the emission maxima for each protein sample were determined. For quenching studies, each protein was stabilized for 5 minutes, and then S1P dissolved in methanol was added over a 60-minute period to a final concentration of 0.25 to 3.0 μM. Since the fusion protein was being evaluated and quenching in ApoM was of interest only, IgG1-F was used. c Used as a control for nonspecific quenching of the C-terminus of the fusion protein. From all suitable ApoM-F c and ApoM-F c -The emission fluorescence of IgG1-Fc was subtracted from the TM data. Data were collected and analyzed using FelixGX software.

[0130] Measurement of purified ApoM-F after injection of ApoM fusion protein c ApoM-F c -TM or S1P in plasma

[0131] 50 μg of purified ApoM-F was analyzed by liquid chromatography / mass spectrometry (LC / MS) using the Stony Brook University Lipidomics Core Facility. c ApoM-F c -S1P, ApoM-F c -TM or ApoM-F c -TM sphingolipid content. For plasma studies, Apomom was measured via buccal puncture. - / - Alternatively, C57BL / 6 mice were pre-bleeded and allowed to rest for 24 to 48 hours. Mice were then injected intraperitoneally with 100 μg (4 mg / kg) of Apom-F. c -S1P or ApoM-F c -TM, and after 24 hours, blood was collected in EDTA and centrifuged at 2000×g for 10 minutes to collect plasma. The sphingolipid content and types of 25 μl of plasma were analyzed by LC / MS.

[0132] S1P1 reports in vitro analysis of S1P-dependent signal transduction in cells.

[0133] Proia and colleagues established a mouse strain based on β-arrestin (β- arrestin) signaling to record S1P1 signaling (referred to as S1P1 GFP signaling mouse) (Kono, M. et al., JCI, 124, 2076-2086 (2014)). Basically, activation of S1P1 receptor by S1P leads to accumulation of histone-GFP fusion protein in the nucleus of the activated cells. A mouse embryonic fibroblast (MEF) cell line was established from a 10.5 day embryo. Using standard protocols, the embryo was isolated, MEF cells were isolated and transformed with SV40 large T antigen. The resulting transformed cells were selected for low endogenous GFP expression and maintained in DMEM supplemented with 10% charcoal-stripped fetal bovine serum, which contains very low levels of S1P. For functional assays, it was determined that addition of Fatty Acid Free (FAF) albumin-S1P (Alb-S1P) leads to nuclear GFP accumulation, with the earliest appearance at 6 hours after stimulation, with maximal signal at 24 hours. Treated cells were harvested by trypsinization and analyzed directly by FACS analysis, gating on GFP expression. Using this assay, S1P1 activation and GFP expression by ApoM-Fc-S1P or ApoM-Fc-TM was determined by FACS analysis from 0.12 to 1 μΜ. Data are expressed as percentage of GFP positive / total live cells analyzed.

[0134] Analysis of ApoM-F c or ApoM-F c Effect of ApoM-F

[0135] Stable transfected Chinese Hamster Ovary (CHO) cells were previously reported for analysis of S1P receptor-1 signaling using S1P1 cDNA cloned into a lentiviral vector, p(CHO-S1P1) (Christoffersen, C. et al., PNAS, 108, 9613-9618 (2011)). A separate CHO cell clone was established for S1P receptor-2 using the Tet-On vector system (CHO S1P2). Cells were maintained in Ham's F12 medium (Invitrogen) supplemented with 10% FBS. For analysis of signaling, seeded cultures were left to adhere overnight, washed twice in serum-free medium and subsequently incubated overnight in medium supplemented with 0.1% FAF-albumin. Medium was replaced and cells were incubated with either OptiMem medium alone or albumin-S1P (100 nM S1P), ApoM-Fc - S1P or ApoM-F c - TM together for 5 minutes. Cells were briefly washed with PBS and then lysed in PBS-NP-40 (PBS, 1% NP40, protease inhibitors (Sigma) and 1 mM NaVO3, 10 mM NaF, 10 mM β-glycerophosphate). Lysates were clarified by centrifugation at 13,000 g for 10 minutes at 4°C and the supernatant was mixed with 5X Laemmli buffer containing 100 mM DTT. Samples were separated on 12% SDS-PAGE gels (BioRad, Acrylamide, Cat. #1610156) and electrophoretically transferred to nitrocellulose membranes (BioRad, Cat. #1620115). Blots were blocked with 5% milk (Carnation) suspended in TBS-T (50 mM Tris base pH 8.0, 150 mM NaCl, 0.05% Tween-20) for 1 hour at RT and then incubated overnight (>12 hours) with 1 : 1000 mouse monoclonal antibody for p-p44 / 42 MAPK (T202 / Y204) (E10) 9106S, Cell Signaling) and washed with 5 changes of TBS-T over a 30 minute period. Blots were incubated in 1% milk-TBS-T supplemented with goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) (1 :5000; Jackson Labs) for 60 minutes and then washed 5 times in TBS-T over a 30 minute period with gentle shaking at RT. Blots were incubated with Immobilon Western Chemiluminescent HRP Substrate (Millipore, Cat #WBKLS0500) and chemiluminescence was visualized using X-ray film (Denvillie Scientific, HyBlotCL E3018). As a loading control, blots were stripped for 10 minutes with glycine pH 2.5 and reprobed with rabbit anti-ERK1 / 2 (Santa Cruz Biotechnology, Cat #sc-292838). Blots were stripped and reprobed for expression of pAkt (S473) (#9271L Cell Signaling) and total Akt (#9272S, Cell Signaling). Blots were incubated, washed and developed as described above.

[0136] HUVECs (ATCC Cat. #100-010) were maintained in supplemented EGM buffer and isolated prior to assay. Cells were starved for 4 hours in 0.1% FAF-albumin media and then assayed. For signaling experiments, HUVECs were incubated with Alb-S1P (100 to 400 nM S1P), ApoM-Fc - S1P (5 to 20 pg / ml) or ApoM-F c - TM (5 to 20 pg / ml) at 5, 15 or 30 min incubation of starved cells. Cells were lysed, the lysate was isolated and transferred for Western blotting as described above. In addition to MapK and Akt, blots were analyzed for activation of p-eNOS (sl 177) (#9571S Cell Signaling) and total eNOS expression (cat# 610296, BD Biosciences).

[0137] Generation of S1P1 knock-out HUVECs by CRISPR / Cas9

[0138] A guide RNA (gRNA) targeting the start codon of S1PR1 was designed and cloned into the lentiCRISPRv2 vector (kindly provided by Dr. Feng Zhang, Addgene plasmid #529619) using the following oligonucleotides:

[0139] 5'-CACCGCGGGACGCTGGTGGGCCCCA-3' (SEQ ID NO: 7) and

[0140] 5'-AAACTGGGGCCCACCAGCGTCCCGC-3' (SEQ ID NO: 8).

[0141] Lentiviral particles were produced using HEK 293T cells and infected into HUVECs. 48 hours after infection, 2 pg / ml puromycin was added for selection, HUVECs were analyzed for S1PR1 locus mutation by DNA sequencing and S1P1 protein expression was determined by immunoblotting analysis.

[0142] Measurement of S1P1 internalization

[0143] Human osteosarcoma cell line U2OS was generated to stably express S1P1 receptor fused to GFP and selected for high level expression. Cells were plated in 384-well plates and allowed to confluence. Cells were then starved for 2 hours in serum-free medium and treated with several concentrations of FTY720-P, albumin-S1P (10 to 100 nM), ApoM-F c - S1P (1 to 20 pg / ml) or ApoM-F cCells were stimulated with 1 to 20 μg / ml of 3-TM for 30 min. Cells were then fixed with 4% PFA for 15 min and permeabilized in PBS-0.1% Triton for 10 min. Nuclei were stained with DAPI for 5 min. Cells were then maintained in PBS, and the 384-well plate was imaged at 10X using ArrayScanVTI with spot detector software.

[0144] In vitro measurement of endothelial cell barrier function

[0145] Human umbilical vein endothelial cells (HUVECs) were maintained under standard conditions and analyzed between passages 4 and 8. Endothelial barrier function was assessed by measuring the resistance of the cell-covered electrodes using an endothelial cell impedance system (ECIS) instrument (Applied BioPhysics, Troy, NY, USA). HUVECs were measured at 1 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well on 0.1% fibronectin-coated electrodes (8W10E plates) (Kono, M. et al., JCI, 124, 2076-2086 (2014)). The confluent cells were starved in endothelial cell basal medium (EBM-2; Lonza, Basel, Switzerland) for 2 to 6 hours and treated with albumin-S1P (50 to 200 nM; S1P dissolved in 2% fatty acid-free albumin; Sigma-Aldrich) and ApoM-F... c -S1P or ApoM-F c -TM (all 0.2 to 0.4 μM) processing. The resistance was monitored and expressed as fractional resistance, normalized relative to the baseline at the start of the measurement.

[0146] mice and cell lines

[0147] Male C57Bl / 6 mice (6 to 8 weeks old) were acquired from Jackson Labs. ApoM knockout mice were maintained in a C57Bl / 6 background as previously reported (Christoffersen, C. et al., PNAS, 108, 9613-9618 (2011)). All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Weill-Cornell Medicine. Mycoplasma contamination was tested in all cell lines.

[0148] ApoM-F c Measurement of plasma half-life of ApoM-F

[0149] C57B1 / 6 mice (n=4) were administered (i.p.) 100 μg (4 mg / kg) of purified ApoM-F c or ApoM-F c -TM and analyzed at 2, 4, 6, 8, 24, 48, 72, 96, 120, 168 and 216 hours after injection. One μl of plasma was analyzed by SDS-PAGE and anti-ApoM immunoblotting as described above. Ponceau S staining was performed to ensure that loading of each lane was equal. Western blots for protein expression were quantified by scanning and analysis with ImageJ using un-injected plasma as a control. The maximum signal was observed between 4 and 6 hours and was used as a reference point to evaluate subsequent expression at different time points.

[0150] Effect of ApoM-Fc administration on blood cell counts in WT mice

[0151] C57B1 / 6 WT mice were injected with 100 μg (4 mg / kg) of ApoM-F c -S1P or ApoM-F c -TM or PBS (N=5 each). After 6 or 24 hours, blood was collected into 2 mM EDTA and the cellular fraction was isolated by centrifugation. Whole blood counts were determined by clinical cytometry (Cytometry Core, Memorial Sloan-Kettering Cancer Center).

[0152] ApoM-F c and ApoM-F c Analysis of the effect of triple mutants on systolic blood pressure (SBP) in normotensive mice

[0153] Systolic, diastolic and mean blood pressure were measured in conscious 12-week old male mice using the pneumatic tail-cuff method (MRBP System, Life Science, Woodland Hills, California). Briefly, animals were placed in a plastic chamber maintained at 34°C and a cuff with a pneumatic pulse sensor was attached to the tail. After 1 week of training, multiple measurements were taken for each mouse and averaged. Mice were given PBS (vehicle control), ApoM-F c -S1P or ApoM-F c- TM (4 mg / kg), and blood pressure was monitored at 1, 2, 4, 8, and 24 hours, and then every 24 hours until day 7.

[0154] Long-term infusion of AngII and ApoM-F c and ApoM-F c Analysis of the effect of triple mutants on hypertension

[0155] As previously described, AngII (500 ng / kg / min) was infused using osmotic minipumps (model ALZET 2004) (Cantalupo, A. et al., Nature Medicine, 21, 1028-1037, (2015)). Briefly, 10-week-old male C57B1 / 6 (WT) mice were subcutaneously implanted with minipumps. Blood pressure was monitored twice a week from day 0 to day 14 of AngII infusion. Systolic blood pressure was evaluated as described above. In another set of experiments, C57B1 / 6 mice were treated with ApoM-F c or ApoM-F c -TM and blood pressure was measured at different time points (1 hour to 216 hours) after injection. To determine the dose-response relationship, C57B1 / 6 mice were treated with PBS (vehicle control) or 30 μg (1.3 mg / kg) of ApoM-F c or ApoM-F c -TM. In addition, the S1P receptor-1 antagonist W146 (10 mg / kg) (8) was used in similar experiments. W146 was administered by intraperitoneal injection at time 0, and then at 24-hour intervals (24 to 96 hours, and then again at 168 hours).

[0156] After ApoM-F c -S1P or ApoM-F c Measurement of plasma nitrite in AngII-treated mice after ApoM-F

[0157] The improved Griess reaction as previously described was used to measure plasma nitrite in AngII-treated mice after ApoM-F c -S1P or ApoM-F c- 24 hours after TM infusion, NO levels were measured as nitrite in plasma from Angll-treated WT mice (Cantalupo, A. et al., Nature Medicine, 21, 1028-1037 (2015)). Briefly, after plasma proteins were precipitated with ZnS04(30% w / v), the supernatant was chemically reduced with acid-washed (0.24 M HC1) cadmium powder (Sigma-Aldrich). After centrifugation, the nitrite content of the samples was measured with Griess reagent (0.1% naphthyl ethylenediamine dihydrochloride in H20 and 1% sulfaniamide in 5% concentrated H3P04) and read at a wavelength of 550 nm. All samples were determined in duplicate and NO concentration was calculated according to the NaN02calibration curve.

[0158] In vivo myocardial ischemia / reperfusion (Ml / R)

[0159] Myocardial ischemia-reperfusion (Ml / R) injury model was employed as previously reported (Xu, Z., JoVE, doi: 10.3791 / 51329 (2014)). Briefly, mice were anesthetized, the chest cavity was opened through a small incision between the ribs and the heart was exposed. The left anterior descending artery (LAD) of the heart was identified and ligated by a slipknot suture for compression. After 45 minutes, the slipknot was removed and the incision was closed. After 24 hours, mice were sacrificed. To visualize the area at risk (AAR), the heart was perfused with Alcian blue dye through the aorta and coronary arteries and infarct size was evaluated by microscopic analysis of 1 mm cross sections of the heart. The heart was counterstained with 1% triphenyltetrazolium chloride (TTC) solution for 15 minutes. Images were observed by light microscopy and photographed. Infarct size and AAR (non-blue) and total left ventricle (LV) were evaluated after ImageJ analysis and expressed as percentage of infarct size under the suture (no Alcian blue perfusion) / total heart area (Shao, D. et al., Nature Communications, 5, 3315 (2014)). For all experiments, 4 mg / kg of ApoM-Fc was injected retro-orbitally 30 minutes before surgery. c - S1P or ApoM-F c - TM was administered at 100 μl IV to WT C57B1 / 6 mice.

[0160] Immunofluorescence staining and histological analysis of M(I / R)

[0161] At 24 hours after ischemia / reperfusion, mice were sacrificed and hearts were perfused with cold PBS, fixed in 4% PFA for 24 hours, and then embedded in OCT compound (Sakura Finetek, Torrence CA). 10 pm-thick frozen sections were cut and stained with primary antibody against Ly6G (Cat# 108401, BioLegend, San Diego, CA) and biotin-conjugated isolectin GS-IB4 (Cat# 121414, Invitrogen). Cy3-conjugated streptavidin (Invitrogen) and fluorescein isothiocyanate (FITC)-conjugated anti-rat antibody were used as secondary reagents. Images were visualized by confocal microscopy using an Olympus Fluoview FV10i.

[0162] Echocardiographic imaging studies

[0163] Cardiac size and function were analyzed by transthoracic echocardiography using a Vevo 770 imaging system (VisualSonics). Mice were lightly anesthetized with inhaled isoflurane (0.2% in O2). The left ventricular M-mode was used and all measurements were obtained over 3 to 6 consecutive heart cycles and analyzed using the mean values. Left ventricular end-diastolic (LVDd) and end-systolic (LVDs) dimensions were measured from the M-mode trace and fractional shortening (FS) was calculated as follows: [(LVDd - LVDs) / LVDd]. Diastolic measurements were made at the point of maximal cavity size and systolic measurements at the point of minimal cavity size using the leading-edge method of the American Society of Echocardiography (Zhang, Y. et al. JCI Insight, 1, doi:10.1172 / jci.insight.85484 (2016)).

[0164] Transient middle cerebral artery occlusion (tMCAO) and treatment

[0165] Transient focal cerebral ischemia was induced in mice by middle cerebral artery occlusion (tMCAO) as previously described (Kim, G. S. et al. Nature Communications, 6, 7893 (2015)). Thirty-three mice (male, 24 to 28 g, C57BL6) were used in this study. The exclusion criterion was the occurrence of subarachnoid hemorrhage. No animals were excluded from the study. Surgery and all behavioral and histological assessments were performed by a researcher blinded to the drug treatment. Mice were induced with 3% isoflurane vaporized in O2 and anesthesia was maintained with 1.5% isoflurane. Temperature was maintained at 36.5 ± 0.5 °C throughout the procedure, controlled by a homeothermic blanket (CMA 450 Temp Controller for mice, Harvard Apparatus, Holliston, MA). The left common carotid artery was exposed, and the occipital branch of the external carotid artery (ECA) was isolated and coagulated. The ECA was dissected distally and coagulated with the terminal lingual and maxillary artery branches. The internal carotid artery (ICA) was isolated and then the extracranial branches of the ICA were dissected. A silicone rubber-coated monofilament suture (monofilament size 6-0, diameter 0.09 to 0.11 mm, length 20 mm; coated diameter 0.23 ± 0.02 mm; coated length 5 mm, Doccol Corp., Sharon, MA) was introduced into the ECA lumen through the incision and subsequently gently advanced in the ICA lumen for approximately 9 to 9.5 mm to block MCA blood flow. For reperfusion, the suture was removed 60 minutes after MCAO. A 2-D laser speckle flowmeter (PeriCam PSI HR, Perimed, Jarfalla, Sweden) was used to confirm MCA occlusion and reperfusion. Immediately after removal of the suture, animals were randomized to receive intraperitoneal injections of PBS, apoM-F c or ApoM-TM-F c .

[0166] Physiological parameters (arterial 02saturation, heart rate, pulse expansion, and respiratory rate) were recorded before, during, and after tMCAO using Mouse Ox Plus (Starr Life Sciences Corp., Oakmon, PA). After surgery, all animals were kept in a small animal heating recovery chamber (IMS Vetcare Chamber Recovery Unit, Harvard Apparatus, Holliston, MA). After recovery, animals were returned to their cages with free access to food and water. Mortality was 1 / 11 in PBS-treated mice, 0 / 11 in apoM WT, and 1 / 11 in apoM-TM-treated mice.

[0167] Neurobehavioral testing

[0168] Neurological function was assessed 23 hours after reperfusion. Neurological deficits were graded from 0 to 4 as previously described (Menzies, S. A. et al., Neurosurgery, 31, 100-106; discussion 106-107 (1992); Belayev, L. et al., Stroke, 27, 1616-1622; discussion 1623 (1996); Mokudai, T. et al., Stroke, 31, 1679-1685 (2000)): 0, no observable deficit; 1, forelimb flexion; 2, forelimb flexion and decreased resistance to lateral push; 3, forelimb flexion, decreased resistance to lateral push, and unilateral circling; and 4, forelimb flexion, inability or difficulty to walk.

[0169] TTC staining and determination of infarct and edema ratios and infarct volume

[0170] Twenty-three hours after reperfusion, mice were anesthetized and decapitated. The brain was quickly removed from the skull, placed in a -20 °C freezer for 20 minutes, and then cut into 1.5 mm coronal sections using a rodent brain matrix. Sections were stained with 2% 2,3,5-chlorinated triphenyl tetrazolium chloride (TTC) (Sigma Co., St. Louis, MO) at 37 °C for 10 minutes and scanned. The infarct area on each section was determined by using image analysis software (Image J, the National Institutes of Health, Bethesda, MD) to obtain the infarct ratio, edema ratio, and infarct volume (in mm3) for each brain. 3Infarct area was calculated to correct for edema formation in the ischemic hemisphere by using the following equation (Swanson, R. A. et al., J. of Cerebral Blood Flow and Metabolism, 10, 290-293 (1990)): I = X - Y, where X is the area of the contralateral (non-ischemic) hemisphere and Y is the area of the intact region of the ipsilateral (ischemic) hemisphere. The infarct proportion was obtained after normalization by the contralateral hemisphere. The edema proportion was calculated with the following equation: E = (Z - X) / X, where Z is the area of the ipsilateral hemisphere.

[0171] Statistical analysis, randomization, and blinding for brain studies

[0172] All values reported are mean ± S.E.M. P values were calculated with GraphPad Prism software using one-way non-parametric ANOVA (Kruskal Wallis) followed by Dunn's test. The criterion for statistical significance was set at P < 0.05. All animal experiments used randomized treatment groups and were assessed in a blinded fashion (Lapchak, P. A. et al., J. of Neurology & Neurophysiology, 4 (2013)).

[0173] Other statistical analyses

[0174] All statistical analyses were performed with Prism, version 4.03 (GraphPad Software, Inc., La Jolla, CA, USA). Two groups were compared by using a two-tailed Student's t-test. Welch's correction for unequal variances was made where appropriate. Analysis of variance (ANOVA; one-way or two-way) was performed as indicated for Bonferroni's test or Tukey's post-test for multiple comparisons. Statistical significance was indicated at P < 0.05.

[0175] Example 2: Development of recombinant soluble ApoM to activate S1P receptors

[0176] ApoM has a very short half-life in the absence of HDL (Faber, K. et al., Molecular Endocrinology, 20, 212-218 (2006)). Therefore, the present inventors developed a strategy to stabilize ApoM in plasma by fusing it to the constant domain of an immunoglobulin (Fc). ApoM-Fc fusion proteins were expressed in both HEK293 and insect Sf9 cells. Robust expression and efficient secretion of ApoM-Fc into the conditioned medium was observed. Also, S1P binding mutants (R98A, W100A and R116A), hereinafter referred to as ApoM-Fc-TM, were prepared, which comprise mutations of three amino acid residues that contact the head region of the S1P molecule (Figure 1A). The purified proteins migrated as oligomers in non-reducing gels, but quantitatively reduced to monomers of 50 to 55 kD (Figure IB). A two-step purification procedure consisting of concanavalin A affinity chromatography followed by gel filtration chromatography yielded highly purified ApoM-F c fusion proteins. ApoM-F c -TM mutants were expressed and purified in a similar manner as ApoM-F c fusion proteins, with a yield of 6.4 + 1.4 μg / ml. Both ApoM-F c , ApoM-F c -TM proteins as well as IgG1-F c domains were purified to homogeneity (Figure 1C).

[0177] ApoM-F c bound to S1P with an EC 50 of 0.22 μM (95% confidence interval: 0.168 to 0.336), whereas ApoM-F c -TM and F c did not show significant binding activity (Figure ID). Furthermore, Sf9 isolated ApoM-F c contained 1.94 + 0.31 mol% S1P, presumably extracted from the cells and / or cell culture medium. ApoM-F c was incubated with S1P (1 :8 mol / mol) for 24 to 48 hours at 4°C and then purified by gel filtration chromatography, resulting in ApoM-F c containing 51.3 + 8.1 mol% S1P (Figure IE). Purified ApoM-F c -TM contained only 0.12 + 0.01 mol% S1P; furthermore, the S1P content did not increase stoichiometrically upon incubation with exogenous S1P in vitro, consistent with the fact that the mutant does not bind to lysophospholipids. ApoM-F cFor further signal transduction and biological experiments, as shown below.

[0178] Example 3: ApoM-F via S1P binding c Continuous activation of endothelial cell S1P receptors:

[0179] Given ApoM-F c Combined with S1P, the next step is to determine ApoM-F. c Does it activate the S1P receptor? ApoM-F c Activation of the β-custodial-based S1P1 reporter in a dose-dependent manner (Kono, M. et al., JCI, 124, 2076-2086 (2014)). However, ApoM-F c -TM was inactive, while albumin-S1P activated reporter activity with a similar dose-response relationship (Fig. 2A, Fig. 2B). In CHO cells expressing S1P receptor subtype 1 or 2, ApoM-F c Instead of ApoM-F c -TM activates extracellular receptor-activated kinase phosphorylation (ppERK) and pAKT phosphorylation, which are known to be mediated by G... i The pathway is activated by the S1P receptor ( Figure 2C In contrast, ApoM-F c The effect of -TM is negligible. In human endothelial cells, ApoM-F c Activation of ppERK, pAKT, and p-endothelial nitric oxide synthase (eNOS) via S1P1 and S1P3 receptors Figure 2D These data indicate that ApoM-F c Able to activate S1P 1-3 Receptors.

[0180] Activation of endothelial cell S1P1 and S1P3 receptors leads to the assembly of adhesive junctions and enhanced barrier function (McVerry, BJ et al., J. of Cellular Biochem., 92, 1075-1085 (2004)), which can be measured by increased trans-endothelial electrical resistance (TEER). HDL-bound S1P is more potent in promoting the vascular barrier in vitro and in vivo than albumin-S1P (Christensen, PM et al., FASEBJ. (2016); Christoffersen, C. et al., PNAS, 108, 9613-9618 (2011)). When ApoM-F c A sustained increase in TEER was observed when processing monolayer HUVECs. In contrast, ApoM-F c- TM did not increase TEER. While albumin-bound S1P also increased TEER, the increase was transient. ApoM-F c The ability to increase TEER was dependent on S1P1 signaling, as it was greatly attenuated in HUVECs designed to lack S1P1 by CRISPR / Cas9-mediated gene disruption (Fig. 3A, 3B). Moreover, even ApoM-F c induced internalization of S1P1 receptors, but to a lesser extent than FTY720-P or albumin-bound S1P (Fig. 3C). These data suggest that ApoM-F c induces sustained enhancement of endothelial cell barrier function by activating S1P receptors.

[0181] Example 4: ApoM-F c In vivo stability of bound S1P

[0182] ApoM-F c and ApoM-F c In vivo stability of -TM. ApoM-F c and ApoM-F c -TM showed plasma half-lives of 93.5 hours and 86.5 hours, respectively, indicating that they are highly stable in vivo (Fig. 4A). 24 hours after injection of 100 μg of ApoM-F c in Apom - / - and / or WT mice, plasma S1P and dihydro-S1P levels were increased by 76.3+13.7%; 52.9+12.9% and 29.9+10.1%; 38.9+4.1%, respectively. Plasma levels of sphingosine, dihydrosphingosine and ceramide, as well as cholesterol, were not affected (Fig. 4B, 4C). Injected ApoM-Fc did not bind to the HDL fraction and was present in the fraction of non-lipid proteins. These data suggest that ApoM-Fc stably binds S1P in vivo as a soluble protein, likely due to protection from phosphatase-mediated degradation. Albumin-bound S1P is rapidly degraded in vivo with an estimated half-life of 15 minutes (Venkataraman, K. et al., Circulation Research, 102, 669-676 (2008)). This property at least partially explains the sustained biological effects of ApoM-F c .

[0183] Example 5: ApoM-F c In vivo effects of bound S1P on hematopoietic cell trafficking

[0184] ApoM-F cApplication and the resulting elevation of plasma S1P can potentially activate lymphocyte S1P1 receptors to regulate lymphocyte egress and platelet formation (Cyster, J. G. & Schwab, S. R., Annual Review of immunology, 30, 69-94 (2012); Zhang, L. et al., The Journal of Experimental Medicine, 209, 2165-2181 (2012)). Therefore, circulating blood cells were quantified following ApoM-Fc application. As shown in Figure 4D, ApoM-F c or ApoM-F c -TM application did not change circulating levels of leukocytes, lymphocytes, platelets, and red blood cells, indicating that immune and hematopoietic S1P receptors were not activated by ApoM-Fc application. This is in sharp contrast to small molecule S1P1 modulators, which induce lymphopenia due to their functional antagonism in secondary lymphoid organs, thymus, and spleen (Cyster, J. G. & Schwab, S. R., Annual Review of immunology, 30, 69-94 (2012)). ApoM-Fc can not have access to hematopoietic S1P1 receptors in lymphoid and / or hematopoietic tissues.

[0185] Example 6: ApoM-F c Sustained blood pressure reduction in hypertensive mice following application

[0186] Endothelial dysfunction contributes to the pathophysiology of hypertension. Indeed, plasma S1P levels modulate vascular tone by stimulating eNOS activity (Nofer, J. R. et al., JCI, 113, 569-581 (2004); Cantalupo, A. et al., Nature Medicine, 21, 1028-1037 (2015)), while endothelial cell S1P1 stimulates eNOS activity through the protein kinase Akt (Christoffersen, C. et al., PNAS, 108, 9613-9618 (2011); Igarashi, J. et al., Biochimica et Biophysica Acta, 1781, 489-495 (2008)). Therefore, it was investigated whether ApoM-F c application modulated blood pressure in hypertensive mice engrafted with angiotensin-II permeable mini-pumps. In C56 / Bl6 mice, ApoM-F c but not ApoM-F c -TM application potently reduced blood pressure by about 40 mmHg 2 hours after treatment (Figure 5A). ApoM-F cThe effect persisted and the therapeutic efficacy was maintained for 192 hours. This significant and sustained reduction in blood pressure was completely abolished by co-administration with W146 (10 mg / kg), a competitive antagonist of S1P1 (Figure 5B). In hypertensive mice, plasma nitrite levels were reduced by ApoM-F c but not by ApoM-F c -TM (Figure 5C). Resting blood pressure in normal mice was transiently reduced by ApoM-F c but the magnitude and duration of the effect were not as high and short-lived (Figure 5D). Indeed, Apom - / - mice showed a significantly elevated resting blood pressure (Figure 5E). These data suggest that ApoM-F c administration activates the endothelial S1P1 / eNOS / NO axis to achieve a sustained antihypertensive effect. In contrast to ApoM-F c administration of small molecules targeting S1P1 receptors induce a mild increase in blood pressure in rodents and humans, in part because of their functional antagonism of endothelial S1P1 (Cantalupo, A. et al., Nature Medicine, 21, 1028-1037 (2015); Camm, J. et al., American Heart Journal, 168, 632-644 (2014)).

[0187] Example 7: Effect of S1P binding by ApoM-F c on cardiac function after myocardial infarction

[0188] HDL and S1P are known to inhibit ischemia / reperfusion (I / R) injury in rodent and pig models of myocardial infarction (Morel, S. et al., Cardiovascular Research, 109, 385-396 (2016); Sattler, K. et al., Journal of the American College of Cardiology, 66, 1470-1485 (2015); Theilmeier, G. et al., Circulation, 114, 1403-1409 (2006); Santos-Gallego, C.G. et al., Circulation, 133, 954-966 (2016)) and in rodent models of stroke (Lapergue, B. et al., Stroke, 44, 699-707 (2013); Wei, Y. et al., Annals of Neurology, 69, 119-129 (2011)). In stroke patients treated with reperfusion therapy, HDL cholesterol levels were associated with favorable outcome at 3 months (Makihara, N. et al., Cerebrovascular Diseases, 33, 240-247 (2012)). In the heart, therapeutic administration of S1P1 agonists also inhibited I / R injury (Levkau, B., Frontiers in Pharmacology, 6, 243 (2015)), even though a small molecule agonist (SEW2871) induced abnormal heart rhythms (Hofmann, U. et al., Cardiovascular Res., 83, 285-293 (2009); Tsukada, Y.T. et al., Journal of Cardiovascular Pharmacology, 50, 660-669 (2007)). Therefore, the inventors hypothesized that therapeutic administration of ApoM-F c exerts a protective effect on endothelial cells that would attenuate myocardial I / R injury. At 24 hours after reperfusion, ApoM-F c but not ApoM-F c administration reduced I / R injury (Figure 6A). Moreover, ApoM-F c attenuated significantly neutrophil accumulation into the infarct site, but did not change the vascular density in the infarct site, suggesting that ApoM-F c maintained endothelial homeostasis after myocardial I / R injury (Figure 6B). Echocardiography analysis 1 to 2 weeks after I / R injury showed that ApoM-Fc administration preserved significantly myocardial function (Figure 6C). These data indicate that therapeutic administration of ApoM-F c activates vascular S1P receptors to inhibit myocardial I / R injury.

[0189] To investigate the therapeutic potential of ApoM-F c The therapeutic potential of treatment in cerebral ischemia was investigated using a mouse model of transient focal cerebral ischemia, the middle cerebral artery occlusion (MCAO) model. After 60 minutes of ischemia, mice were treated with PBS, ApoM-F c or ApoM-TM-F c (I.P. injection, 4 mg / kg) at the time of reperfusion. Twenty-three hours after reperfusion, the edema and infarct ratio and infarct volume were calculated as previously described (Kim, G.S. et al., Nature Communications, 6, 7893 (2015)). As shown in Figures 6D-F, administration of ApoM-F c led to a significant reduction in both the infarct ratio (0.393 ± 0.026 for PBS-treated mice versus 0.238 ± 0.019 for ApoM-F c -treated mice; 39% reduction) and the total edema ratio (0.146 ± 0.013 for PBS-treated mice versus 0.052 ± 0.009 for ApoM-F c -treated mice, 64% reduction), which is the sum of cytotoxic and vasogenic edema. The infarct volume (52.4 ± 4.2 mm 3 ) was significantly lower in ApoM-F c -treated mice (corrected for edema) compared to PBS-treated mice (86.8 ± 5.8 mm 3 ). In contrast, treatment with ApoM-TM-F c tended to have a protective effect, but did not reach statistical significance. It was also found that neurological scores were significantly improved in ApoM-F c -treated mice compared to PBS-treated mice, but not in ApoM-TM-F c -treated mice (Figure 6G). Cerebral blood flow in the MCA region, monitored by laser speckle flowmetry during surgery, was similarly reduced during occlusion and similarly recovered after reperfusion in all three groups of mice (Figure 6H). These data indicate that, in experimental stroke, ApoM-F c treatment after reperfusion potently reduces total brain edema and infarct area, thereby improving stroke outcome. Physiological parameters (arterial oxygen saturation, heart rate, pulse expansion, and respiratory rate) measured before, during, and after reperfusion in mice treated with PBS, ApoM-F c or ApoM-TM-F c were not significantly changed.

[0190] The results disclosed herein show that ApoM-F c binds to S1P and activates S1P receptors of the endothelium. ApoM-F c is stable in vivo, thus allowing the continuous activation of S1P receptors in the vasculature. Interestingly, ApoM-F c treatment does not induce lymphopenia, suggesting that it does not access lymphocyte S1P receptors in secondary lymphoid organs. This indicates that ApoM-F c administration selectively targets endothelial S1P receptors and provides a novel strategy for the therapeutic modulation of S1P-dependent vascular pathologies in vivo. Indeed, ApoM-F c administration of the recombinant protein achieves a sustained reduction in blood pressure in hypertensive mice. This effect is dependent on S1P1 activation and involves NO synthesis. Therapeutic targeting of this pathway can be useful for the treatment of refractory hypertension syndromes. Moreover, the evidence provided herein indicates that ApoM-F c recombinant protein inhibits myocardial I / R injury in mouse models. Previous work has shown that HDL infusion or S1P1 agonists protect the myocardium from MI / R injury (Morel, S. et al., Cardiovascular Research, 109, 385-396 (2016); Sattler, K. et al., Journal of the American College of Cardiology, 66, 1470-1485, (2015); Theilmeier, G. et al., Circulation, 114, 1403-1409 (2006); Santos-Gallego, C.G. et al., Circulation, 133, 954-966 (2016)). Furthermore, in mouse models of stroke, HDL infusion and S1P1 receptor activators have shown to reduce neuronal I / R injury in animal models and in human clinical trials (Makihara, N. et al., Cerebrovascular Diseases, 33, 240-247 (2012); Keene, D. et al., BMJ, 349, g4379 (2014); Lapergue, B. et al., Stroke, 44, 699-707 (2013); Wei, Y. et al., Annals of Neurology, 69, 119-129 (2011); Kim, G.S. et al., Nature Communications, 6, 7893 (2015); Fu, Y. et al., PNAS, 111, 18315-18320 (2014); Zhu, Z. et al., Circulation, 132, 1104-1112 (2015)). With ApoM-Fc The ability of recombinant proteins to selectively activate vascular S1P receptors provides a significant advantage for small molecules targeting this pathway. Indeed, small molecule S1P1 inhibitors are not selective for the vasculature and short-term agonism evolves into chronic functional antagonism affecting many organ systems (Hla, T., Neurology, 76, S3-8 (2011)). Thus, it is proposed herein that ApoM-F c administered as a new treatment for diseases in which endothelial function is impaired. SEQUENCE LISTING <110> Children's Medical Center Corporation <120> ApoM-Fc fusion proteins, complexes thereof with sphingosine 1 -phosphate (S1P), and methods for treating vascular and non-vascular diseases <130> 33974 <150> 62 / 375,088 <151> 2016-08-15 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 1 tatccatggg gatctaccag tgccctgagc acagt 35 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 2 tatggatcct ccgttattgg acagctcaca ggcct 35 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 3 cgccctgcca tggcgactga gctc 24 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 4 aatcatgctg aatgcgacag gcc 23 <210> 5 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 5 tatggatcca tgtacaggat gcaactcctg tctt 34 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 6 tatttatcat gtctggccag ctagcgacac tggg 34 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 7 caccgcggga cgctggtggg cccca 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 8 aaactggggc ccaccagcgt cccgc 25 <210> 9 <211> 188 <212> PRT <213> Homo sapiens <400> 9 Met Phe His Gin lie Trp Ala Ala Leu Leu Tyr Phe Tyr Gly lie lie 1 5 10 15 Leu Asn Ser lie Tyr Gin Cys Pro Glu His Ser Gin Leu Thr Thr Leu 20 25 30 Gly Val Asp Gly Lys Glu Phe Pro Glu Val His Leu Gly Gin Trp Tyr 35 40 45 Phe lie Ala Gly Ala Ala Pro Thr Lys Glu Glu Leu Ala Thr Phe Asp 50 55 60 Pro Val Asp Asn lie Val Phe Asn Met Ala Ala Gly Ser Ala Pro Met 65 70 75 80 Gln Leu His Leu Arg Ala Thr lie Arg Met Lys Asp Gly Leu Cys Val 85 90 95 Pro Arg Lys Trp lie Tyr His Leu Thr Glu Gly Ser Thr Asp Leu Arg 100 105 110 Thr Glu Gly Arg Pro Asp Met Lys Thr Glu Leu Phe Ser Ser Ser Cys 115 120 125 Pro Gly Gly lie Met Leu Asn Glu Thr Gly Gin Gly Tyr Gin Arg Phe 130 135 140 Leu Leu Tyr Asn Arg Ser Pro His Pro Pro Glu Lys Cys Val Glu Glu 145 150 155 160 Phe Lys Ser Leu Thr Ser Cys Leu Asp Ser Lys Ala Phe Leu Leu Thr 165 170 175 Pro Arg Asn Gin Glu Ala Cys Glu Leu Ser Asn Asn 180 185 <210> 10 <211> 190 <212> PRT <213> Mus musculus <400> 10 Met Phe His Gin Val Trp Ala Ala Leu Leu Ser Leu Tyr Gly Leu Leu 1 5 10 15 Phe Asn Ser Met Asn Gin Cys Pro Glu His Ser Gin Leu Thr Ala Leu 20 25 30 Gly Met Asp Asp Thr Glu Thr Pro Glu Pro His Leu Gly Leu Trp Tyr 35 40 45 Phe lie Ala Gly Ala Ala Ser Thr Thr Glu Glu Leu Ala Thr Phe Asp 50 55 60 Pro Val Asp Asn lie Val Phe Asn Met Ala Ala Gly Ser Ala Pro Arg 65 70 75 80 Gln Leu Gln Leu Arg Ala Thr lie Arg Thr Lys Ser Gly Val Cys Val 85 90 95 Pro Arg Lys Trp Thr Tyr Arg Leu Thr Glu Gly Lys Gly Asn Met Glu 100 105 110 Leu Arg Thr Glu Gly Arg Pro Asp Met Lys Thr Asp Leu Phe Ser Ser 115 120 125 Ser Cys Pro Gly Gly lie Met Leu Lys Glu Thr Gly Gin Gly Tyr Gin 130 135 140 Arg Phe Leu Leu Tyr Asn Arg Ser Pro His Pro Pro Glu Lys Cys Val 145 150 155 160 Glu Glu Phe Gin Ser Leu Thr Ser Cys Leu Asp Phe Lys Ala Phe Leu 165 170 175 Val Thr Pro Arg Asn Gin Glu Ala Cys Pro Leu Ser Ser Lys 180 185 190

Claims

1. A fusion protein comprising an apolipoprotein M (ApoM) polypeptide fused to a fragment crystallizable (Fc) region of an antibody, wherein the Fc region is fused to the carboxy terminus of the ApoM polypeptide, and wherein the ApoM polypeptide comprises amino acids 21 to 188 of SEQ ID NO: 9, and wherein the ApoM polypeptide does not comprise the ApoM signal peptide of the native ApoM protein.

2. A fusion protein comprising an apolipoprotein M (ApoM) polypeptide fused to a fragment crystallizable (Fc) region of an antibody, wherein the Fc region is fused to the carboxy terminus of the ApoM polypeptide, and wherein the ApoM polypeptide comprises amino acids 21 to 188 of SEQ ID NO: 9, and wherein the ApoM polypeptide does not comprise amino acids 1 to 20 of SEQ ID NO:

9.

3. The fusion protein of claim 1 or claim 2, wherein the Fc region is an Fc region selected from the group consisting of an IgG antibody, an IgM antibody, an IgA antibody, an IgE antibody, and an IgD antibody.

4. The fusion protein of claim 3, wherein the Fc region is an IgGl-Fc.

5. A composition comprising the fusion protein of any one of claims 1 to 4 complexed with a phospholipid or a lysophospholipid.

6. The composition of claim 5, wherein the phospholipid comprises phosphocholine.

7. The composition of claim 5, wherein the phospholipid comprises sphingosine 1 -phosphate (S1P).

8. The composition of claim 5, wherein the composition is formed by mixing the fusion protein with the phospholipid or the lysophospholipid, incubating the mixture to allow complex formation, and purifying the complex.

9. The composition of claim 8, wherein the phospholipid comprises phosphocholine.

10. The composition of claim 8, wherein the phospholipid comprises sphingosine 1 -phosphate (S1P).

11. Use of the composition of any one of claims 5 to 10 in the manufacture of a medicament for treating a disorder in a subject, wherein the disorder is selected from the group consisting of hypertension, cardiac ischemia, cerebral ischemia, accelerated atherosclerosis, non-cardiac reperfusion injury, and peripheral vascular disease.

12. The use of claim 11, wherein the hypertension comprises a disorder selected from the group consisting of primary resistant hypertension, secondary resistant hypertension, neurogenic hypertension, and gestational hypertension.

13. The use of claim 11, wherein the hypertension comprises a disorder selected from the group consisting of diabetic pre-eclampsia and chronic kidney disease hypertension.

14. The use of claim 12, wherein the gestational hypertension is pre-eclampsia.

15. The use of claim 11, wherein the cardiac ischemia comprises a disease selected from the group consisting of cardiac reperfusion injury, myocardial infarction, acute coronary syndrome, and angina pectoris.

16. The use of claim 11, wherein the non-cardiac reperfusion injury comprises an injury caused by ischemia selected from the group consisting of hepatic ischemia, renal ischemia, intestinal ischemia, and muscle ischemia.

17. Use of an ApoM-Fc fusion protein according to any one of claims 1 to 4 for the manufacture of a medicament for reducing side effects of fingolimod in a patient being treated with fingolimod.

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