WO2018136163A2 - Polypeptides de fusion apoa-1 en tandem - Google Patents

Polypeptides de fusion apoa-1 en tandem Download PDF

Info

Publication number
WO2018136163A2
WO2018136163A2 PCT/US2017/065078 US2017065078W WO2018136163A2 WO 2018136163 A2 WO2018136163 A2 WO 2018136163A2 US 2017065078 W US2017065078 W US 2017065078W WO 2018136163 A2 WO2018136163 A2 WO 2018136163A2
Authority
WO
WIPO (PCT)
Prior art keywords
fusion polypeptide
residues
seq
amino acid
polypeptide
Prior art date
Application number
PCT/US2017/065078
Other languages
English (en)
Other versions
WO2018136163A3 (fr
Inventor
Martha S. Hayden-Ledbetter
Jeffrey A. Ledbetter
Vince Montes
Original Assignee
Theripion, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Theripion, Inc. filed Critical Theripion, Inc.
Publication of WO2018136163A2 publication Critical patent/WO2018136163A2/fr
Publication of WO2018136163A3 publication Critical patent/WO2018136163A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • Apolipoprotein A-I ⁇ - ⁇ and High Density Lipoprotein (HDL)
  • Cardiovascular disease is the leading cause of mortality in many countries, accounting for approximately 16.7 million deaths each year world-wide. The most common consequences of cardiovascular disease are myocardial infarction and stroke, which have a common underlying etiology of atherosclerosis.
  • HDL high density lipoprotein
  • Apolipoprotein A-l (ApoA- 1) is the principal protein component of HDL. Phillips, Journal of Lipid Research 54:2034-2048, 2013. Human ApoA- 1 is a 243 amino acid protein, with a series of eight 22-mer and two 11-mer amphipathic a-helices spanning residues 44-243. Lund-Katz and Phillips, Subcell Biochem. 51 : 183-227, 2010.
  • the helices in the amino-terminal two-thirds of the molecule form a helix bundle structure, whereas the carboxyl-terminal region forms a separate, relatively disorganized domain important for lipid binding.
  • the interaction of the C-terminal segment with lipids induces conformational changes in the ApoA- 1 structure, increasing the a-helix content of the molecule and allowing subsequent opening of the N -terminal helix bundle. See id.
  • the lipid affinity of ApoA- 1 confers detergent-like properties, and it can solubilize phospholipids to form discoidal HDL particles containing a segment of phospholipid bilayer and two ApoA-1 molecules arranged in an anti-parallel, double-belt conformation around the edge of the disc.
  • the conformational adaptability of ApoA- 1 also confers stability to HDL particles, including discoidal particles of different sizes as well as spherical HDL particles. See id. These characteristics allow ApoA-1 to partner with ABCA1 in mediating efflux of cellular phospholipid and cholesterol and in producing stable HDL particles. See Phillips, supra; Lund-Katz and Phillips, supra.
  • CER-001 in development by Cerenis Therapeutics, is a recombinant ApoA-1 produced by mammalian cells and formulated with specific lipids to form pre- -like HDL particles. According to Cerenis, CER-001 met its primary end point of a reduction in carotid plaque volume measured by MR! in patients with familial hypercholesterolaemia in the MODE trial (NCTO 1412034). In the CHI-SQUARE trial (NCT01201837), Cerenis announced that CER-001 reduced plaque volume versus baseline in patients with acute coronary syndrome, but the reduction was not significant versus placebo.
  • ApoA- 1 has also been fused with an Fc domain (ApoA-l-Ig) and is available commercially from Creative Biomart (cat. No. APOA-I-33H) and from Life Technologies (Cat # 10686-HO2H-5). However, this ApoA-l -Ig molecule has very low functional activity ⁇ see Example 1).
  • Additional recombinant ApoA-1 fusion proteins include anti-CD20 scFv-ApoA-1 (Crosby et al , Biochem. Cell Biol. 10: 1139 bcb, 2015), IL-15-ApoA-l (Ochoa et al. Cancer Res. 73: 139- 149, 2013), and a trimeric ApoA- 1 fusion protein made by the addition of the trimerization domain of human tetranectin (Graversen et al, J. Cardiovascular Pharmacol. 51: 170-77, 2008). In these examples, the fusion was at the N -terminus of ApoA- 1.
  • TN-ApoA-1 The trimeric tetranectin- ApoA- 1 (TN-ApoA-1) was effective in reverse cholesterol efflux and its half-life in mice was increased to 12 hours versus three hours for monomeric ApoA-1. See Gaversen et al, supra. In an aggressive model of atherosclerosis (LDLR -/- mice fed a high-fat diet), trimeric TN-ApoA-1 slowed progression of lesions in the aortic roots. See id. Recent studies in nonhuman primates, however, showed that multiple infusions of lipidated TN-ApoA-1 were not well tolerated and resulted in high immunogemeity and lipid accumulation.
  • trimer fusion protein was complexed with phospholipids and injected at concentrations of 100 mg/kg and 400 mg/kg every four days for three weeks, followed by a six week recovery period.
  • lipidated TN-ApoA-1 After multiple infusions of lipidated TN-ApoA-1, clinical condition deteriorated and was accompanied by changes indicative of a progressive inflammatory response, increased levels of cytokines, C-reactive protein and vascular/perivascular infiltrates in multiple tissues. Rapid formation of antidrug antibodies occurred in ail animals receiving lipidated TN-ApoA- l. See id.
  • ApoA-1 therapy has also shown significant benefit in improving insulin sensitivity and glucose uptake (see Drew et al . Nature Reviews Endocrinology 8:237, 2012), and may be useful in patients with diabetes and with NASH (non-alcoholic steatohepatitis).
  • ApoA- 1 binds amyloid-beta and prevents neurotoxicity in cultured hippocampal neuronal cells. See Koldamova et al. Biochemistry 40:3553, 2001; Paula-Lima et al, Int. J. Biochem. Cell Biol. 41 : 1361, 2009.
  • ApoA- 1 polymorphisms are linked to risk for Alzheimer's disease and ApoA- 1 is found at decreased levels in patients with neurodegenerative disorders. See Keeney et al, Proteomics Clin. Appl. 7: 109- 122, 2013).
  • Efficacy of ApoA- 1 therapy has also been demonstrated in animal models of cancer.
  • MDSC myeloid-dcrivcd suppressor cells
  • ApoA-1 mimetic peptides have shown efficacy in a number of animal models of disease and have properties that make them attractive as potential therapeutic agents. See, e.g. , Reddy et al , Curt: Opin. Lipidol. 25:304-308, 2014 and White et al , J. Lipid. Res. 55:2007-2021, 2014. Peptide 4F has been tested in high risk patients with coronary artery disease.
  • ApoA-1 mimetic peptides that are resistant to oxidation have been described in the past several years. While these a-helical peptides show activity in animal models, they require daily dosing because of their short half-life.
  • RNase has been studied as a therapy for cancer and autoimmune disease.
  • natural onconase, frog RNase
  • recombinant human RNase 1 resistant to inhibition by cytoplasmic inhibitor see US Patent No. 8,569,457
  • targeting of RNase to tumor cells by conjugation of cytotoxic RNase (onconase) to anti-tumor antibodies has been reported. See Lui et ⁇ ., ⁇ . Cancer 13: 1186, 2014: Newton et al. Blood 97:528-535, 2001.
  • RNase therapy has also been studied in a mouse model of cardiovascular disease. See Simsekyilmaz et al , Circulation 129:598-606, 2014. They and others show that extracellular RNA accumulates at sites of vascular injury and that extracellular RNA causes production of inflammatory cytokines. See Fischer et al , Thromb. Haemost. 108:730-741, 2012. RNase therapy reduced neointima formation in a mouse model of accelerated cardiovascular disease, reduced plaque macrophage content, and inhibited leukocyte recruitment to injured carotid arteries in vivo. See Simsekyilmaz et al , supra.
  • RNase therapy has also been studied in models of acute stroke, where it was found to reduce infarction size. See Walberer et al , Curr. Neurovasc. Res. 6: 12-19, 2009. Thus systemic treatment with RNase 1 rescued mice from arterial thrombotic occlusion to limit cerebral edema and to serve as a potent anti -inflammatory regimen in vivo. In these RNase therapy studies, the RNase was given by continuous infusion using osmotic minipumps implanted subcutaneously because the half- life of RNase 1 is very short.
  • RNase therapy has also been studied in a mouse model of systemic lupus erythematosus (SLE). See Sun et al, J. Immunol 190:2536-2543, 2013. Overexpression of TLR7, an RNA sensor, causes a lupus-like disease with autoantibodies, kidney disease, and early mortality. Crossing these mice with mice that overexpress RNase A as a transgene resulted in progeny with increased survival, reduced lymphocyte activation, reduced kidney deposits of IgG and C3, and reduced hepatic inflammation and necrosis.
  • PON 1 Human Paraoxonase 1
  • PON 1 is a lipolactonase with efficient esterase activity and capable of hydroiyzing organophosphates. PON 1 prevents LDL and cell membrane oxidation and is considered to be atheroprotective. PON 1 is exclusively associated with HDL and contributes to the antioxidative function of HDL. See, e.g. , Mackness et al , Gene 567: 12-21, 2015. Reductions in HDL-PON 1 activit are present in a wide variety of inflammatory diseases where loss of PON I activity leads to dysfunctional HDL which can promote inflammation and atheroscierosis.
  • PO i has shown protective activity in multiple animal models. Overexpression of human PON I inhibited the development of atherosclerosis in mice with combined leptin and LDL receptor deficiency, a model of metabolic syndrome. See Mackness et al , Arterioscler. Thromb. Vase. Biol 26: 1545-50, 2006.
  • PON I transduction protected microglial cells in vitro from oxidative stress-induced inflammatory responses and protected against dopaminergic neuronal cell death in a Parkinson's disease model. See Kim et al, Biomaterials 64:45- 56, 2015,
  • mice were given recombinant adenovirus PON I or PONS and either was shown to protect against CCl(4)-induced liver injury. Overexpression of either human PON I or human PON3 reduced hepatic oxidative stress and strengthened the antioxidant capabilities in the liver. See Peng et al, Toxicol. Lett. 193: 159-166, 2010.
  • PON 1 was fused to the C-terminus of an Fc domain, and expressed as a bispecific molecule using an antibody to human insulin receptor (HIR).
  • HIR human insulin receptor
  • HIRMAb-PON l This molecule, termed HIRMAb-PON l, was stable after expression in CHO cells, and was shown in Rhesus monkeys to have a high blood brain barrier permeation but was rapidly cleared by the liver. See Boado et al., Biotechnol Bioeng. 108: 186-196, 2011.
  • Platelet-activating factor aeetyihydroiase is an LDL and HDL-associated enzyme that hydroiyzes short chain acyl groups of phospholipids such as platelet-activating factor and oxidized phospholipids to reduce their inflammatory properties.
  • PAF-AH Platelet-activating factor aeetyihydroiase
  • PAF-AH also enhanced liver recovery after paracetamol intoxication in the rat, and PAF is associated with liver toxicity from high doses of acetaminophen. See Grypioti et al., Dig. Dis. Sci. 52:2580-2590, 2007; Grypioti et al . Dig. Dis. Sci. 53: 1054- 1062, 2008.
  • a mutation in PAF-AH that causes a loss of function is present in 4% of Japanese, and PAF-AH was found to be an independent risk factor for cardiovascular disease and stroke in these individuals. See Blankenberg et al., J. Lipid Res. 44: 1381- 1386, 2003.
  • Cholesteryl ester transfer protein transports cholesteryl ester from high- density lipoproteins (HDL) to low density and very low density lipoproteins (LDL and VLDL).
  • HDL high- density lipoproteins
  • LDL and VLDL very low density lipoproteins
  • Torecetrapib was the first CETP inhibitor to advance to late stage clinical trials, and showed a significant effect on plasma lipoprotein levels, raising antiatherogenic HDL cholesterol levels while lowering proatherogenic LDL cholesterol levels. Torecetrapib binds deeply within CETP and shifts the bound cholesteryl ester in the N- terminal pocket of the hydrophobic tunnel and displaces phospholipid from the pocket. See Liu et al., J. Biol Chem.
  • the present invention provides a fusion polypeptide comprising, from an ammo-terminal position to a carboxyl-terminal position, ApoA l a -Ll -ApoA l b -L2-D, where ApoA 1 a and ApoA b are, respectively, first and second polypeptide segments, each of said segments having cholesterol efflux activity and which is independently selected from (i) a polypeptide comprising an amino acid sequence having at least 90% or at least 95% identity with amino acid residues 25-267 of SEQ ID NO:36 and (ii) an ApoA-1 mimetic; LI is a first polypeptide linker; L2 is a second polypeptide linker; and D is selected from a dimerizing domain and a domain that specifically binds to the neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • ApoAl a is a polypeptide comprising an amino acid sequence having at least 90% or at least 95% identity with amino acid residues 19-267 or 1-267 of SEQ ID NO:36.
  • both ApoA l a and ApoA b are polypeptides comprising an amino acid sequence having at least 90% or at least 95% identity with amino acid residues 25-267 of SEQ ID NO:36; in other variations, both ApoAl a and ApoA b are an ApoA -I mimetic.
  • L2 comprises at least two amino acid residues, at least three amino acid residues, or at least 16 amino acid residues.
  • L2 consists of from two to 60 amino acid residues, from three to 60 amino acid residues, from five to 40 amino acid residues, from 15 to 40 amino acid residues, or from 16 to 36 amino acid residues.
  • L2 linkers include linkers comprising a plurality of glycine residues and optionally comprising at least one serine residue; in some such embodiments, L2 includes the sequence [Gly-Gly-Gly-Gly-Ser] n ([SEQ ID NO: 15] n ), where n is a positive integer from I to 7.
  • L2 consists of 16 amino acid residues, 21 amino acid residues, 26 amino acid residues, 31 amino acid residues, or 36 amino acid residues; in some such embodiments, L2 has the amino acid sequence shown in residues 268-283 of SEQ ID NO:22, residues 268-288 of SEQ ID NO:26, residues 268-293 of SEQ ID NO:2, SEQ ID NO:54, or residues 268-303 of SEQ ID NO:24.
  • the first polypeptide segment comprises the amino acid sequence shown in residues 19-267 or 25-267 of SEQ ID NO:36
  • the second polypeptide comprises the amino acid sequence shown in residues 25-267 of SEQ ID M O:36.
  • L 1 comprises at least five amino acid residues.
  • L I consists of from five to 20 amino acid residues or from five to 15 amino acid residues (e.g. , 10 amino acid residues or 12 amino acid residues).
  • Particularly suitable L I linkers include linkers comprising a plurality of glycine residues and optionally comprising at least one serine residue.
  • L I has the amino acid sequence shown in SEQ ID O:66 (also shown as residues 268-279 of SEQ ID NO:56 or 268-279 of SEQ ID NO:96) or SEQ ID NO:68 (also shown as residues 268-279 of SEQ ID NO:58 or 268-279 of SEQ ID NO:98).
  • D is a dimerizing domain.
  • D is an immunoglobulin heavy chain constant region such as, for example, an immunoglobulin Fc region.
  • the Fc region is a human Fc region such as, e.g. , a human Fc variant comprising one or more amino acid substitutions relative to the wild-type human sequence.
  • Particularly suitable Fc regions include human l and ⁇ 3 Fc regions.
  • the Fc region is a human l Fc variant in which Eu residue C220 is replaced by serine; in some such embodiments Eu residues C226 and C229 are each replaced by serine, and/or Eu residue P238 is replaced by serine.
  • the Fc region is a human ⁇ ⁇ Fc variant in which Eu residue P331 is replaced by serine.
  • Fc variants may include an amino acid substitution that reduces glycosylation relative to the wild-type human sequence; in some such embodiments, Eu residue N297 is replaced with another amino acid.
  • the Fc region is an Fc variant comprising an amino acid substitution that increases or reduces binding affinity for an Fc receptor (e.g. , an amino acid substitution that increases or reduces binding affinity for at least one of FcyRI, FcyRII, and FcyRIII).
  • an Fc variant includes an amino acid substitution that increases or reduces binding affinity for the neonatal Fc receptor (FcRn).
  • Suitable Fc regions include (i) an Fc region comprising the amino acid sequence shown in residues 294-525 or 294-524 of SEQ ID NO:2 and (ii) an Fc region comprising the amino acid sequence shown in residues 294-525 or 294-524 of SEQ ID NO: 13.
  • D is a domain that specifically binds to the neonatal Fc receptor (FcRn).
  • D is an albumin (e.g. , human albumin).
  • the albumin comprises an amino acid sequence having at least 90% or at least 95% identity with amino acid residues 550- 1 134 of SEQ ID NO:62 (also shown as, e.g., residues 25-609 of SEQ ID NO:60); in some such embodiments, the albumin comprises the amino acid sequence shown in residues 550- 1134 of SEQ ID NO:62.
  • D is an Fc region such as, e.g., an Fc region as disclosed above.
  • the fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identity with (i) residues 19- 780, 19-779, 25-780, or 25-779 of SEQ ID NO: 6. (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19- 790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19-1134 or 25-1134 of SEQ ID NO:62.
  • the fusion polypeptide comprises the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID O:56, (ii) residues 19-780, 19-779, 25- 780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19-1134 or 25- 1134 of SEQ ID O:62.
  • the fusion polypeptide further includes a third polypeptide segment located carboxyl-terminal to D.
  • the third polypeptide segment is an RNase, a paraoxonase, a platelet-activating factor acetylhydrolase (PAF- AH), a cholesterol ester transfer protein (CETP), a lecithin-cholesterol acyltransferase (LCAT), or a polypeptide that specifically binds to amyloid beta ( ⁇ ) such as, e.g. , an ⁇ -specific scFv.
  • a fusion polypeptide comprising a third polypeptide segment as above may be represented by the formula ApoAl a -L l-ApoAl b -L2-D-L3-P (from an ammo-terminal position to a carboxyl-terminal position), where ApoAl a , LI, ApoAl b , L2, and D are each defined as above, where L,3 is a third polypeptide linker and is optionally present, and where P is the third polypeptide segment.
  • the fusion polypeptide comprises (as the portion corresponding to ApoAl a -L l-ApoAl b -L2-D) an amino acid sequence having at least 90% or at least 95% identity with (i) residues 19-780, 19-779, 25- 780, or 25-779 of SEQ ID NO:56, (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25- 790, or 25-789 of SEQ ID NO:98, or (v) residues 19- 1134 or 25- 1134 of SEQ ID NO:62.
  • the fusion polypeptide comprises (as the portion corresponding to ApoAl a -Ll- ApoAl b -L2-D) the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID O:56, (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19- 790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19- 1134 or 25-1134 of SEQ ID NO:62.
  • L3 has the amino acid sequence shown in residues 781 -798 of SEQ ID NO:75.
  • the present invention provides a fusion polypeptide comprising, from an amino-terminai position to a earboxyl-terminal position, ApoAl a -Ll-ApoAli,, where each of ApoA L, LI, and ApoAl b is defined as above.
  • the first polypeptide segment has the amino acid sequence shown in residues 19-267 or 25-267 of SEQ ID NO:36
  • the second polypeptide has the amino acid sequence shown in residues 25-267 of SEQ ID O:36.
  • the ApoAI a -Ll-ApoAl b fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identity with amino acid residues 19-522 or 25-522 of SEQ ID NO:64; in some such embodiments, the ApoA l a -L l-ApoAl b fusion polypeptide comprises the amino acid sequence shown in residues 19-522 or 25-522 of SEQ ID NO:64.
  • the ApoAI a -Ll-ApoAl b fusion further includes a third polypeptide segment located earboxyl-terminal to the second polypeptide segment, where the third polypeptide segment is selected from an RNase, a paraoxonase, a platelet-activating factor aeetyihydroiase (PAF-AH), a cholesterol ester transfer protein (CETP), a lecithin-cholesterol acyltransferase (LCAT), and a polypeptide that specifically binds to amyloid beta such as, e.g., an ⁇ -specific scFv.
  • the third polypeptide segment is selected from an RNase, a paraoxonase, a platelet-activating factor aeetyihydroiase (PAF-AH), a cholesterol ester transfer protein (CETP), a lecithin-cholesterol acyltransferase (LCAT), and a
  • the fusion polypeptide further includes a linker polypeptide located earboxyl-terminal to the second polypeptide segment and amino-terminai to the third polypeptide segment. In some embodiments, the fusion polypeptide further includes a dimerizing domain or a domain that specifically binds to the neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • the RNase is human RNAse 1 or a functional variant or fragment thereof.
  • the RNase has at least 90% or at least 95% identity with amino acid residues 544-675 or 548-675 of SEQ ID NO:4.
  • the RNase has the amino acid sequence shown in residues 544-675 or 548-675 of SEQ ID O:4.
  • the fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identify with (i) residues 19-930 or 25-930 of SEQ ID NO:75, (ii) residues 19-926 or 25-926 of SEQ ID NO:94, or (iii) residues 19-1280 or 25-1280 of SEQ ID NO:87; in some such embodiments, the fusion polypeptide comprises the amino acid sequence shown in (i) residues 19-930 or 25-930 of SEQ ID NO- 75. (ii) residues 19-926 or 25-926 of SEQ ID NO:94, or (iii) residues 19-1280 or 25-1280 of SEQ ID NO:87.
  • the paraoxonase is human paraoxonase 1 (PON 1) or a functional variant thereof.
  • the paraoxonase has at least 90% or at least 95% identity with amino acid residues 16-355 of SEQ ID NO: 12, amino acid residues 16-355 of SEQ ID NO:42, or amino acid residues 16-355 of SEQ ID NO:44.
  • the paraoxonase comprises the amino acid sequence shown in residues 16-355 of SEQ ID NO: 12, residues 16-355 of SEQ ID NO:42, or residues 16-355 of SEQ ID NO:44.
  • the fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identify with (i) residues 19-1138 or 25- 1138 of SEQ ID NO:77, (li) residues 19-1138 or 25- 1138 of SEQ ID NO.79.
  • the fusion polypeptide comprises the amino acid sequence shown in (i) residues 19-1138 or 25-1138 of SEQ ID NO:77, (ii) residues 19-1 138 or 25- 1 138 of SEQ ID NO:79, (iii) residues 19-1138 or 25-1138 of SEQ ID NO:81, or (iv) residues 19-1492 or 25-1492 of SEQ ID NO: 89.
  • the platelet-activating factor acetylhydrolase is a human PAF-AH or a functional variant thereof.
  • the platelet-activating factor acetylhydrolase has at least 90% or at least 95% identity with amino acid residues 22-441 of SEQ ID NO:32.
  • the platelet-activating factor acetylhydrolase comprises the amino acid sequence shown in residues 22-441 of SEQ ID NO: 32.
  • the fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identify with residues 19- 1218 or 25-1218 of SEQ ID NO:83; in some such embodiments, the fusion polypeptide comprises the amino acid sequence shown in residues 19- 1218 or 25-1218 of SEQ ID NO:83.
  • the cholesterol ester transfer protein is human CETP or a functional variant thereof.
  • the cholesterol ester transfer protein has at least 90% or at least 95% identity with amino acid residues 18-493 of SEQ ID NO:30.
  • the cholesterol ester transfer protein comprises the amino acid sequence shown in residues 1 -493 of SEQ ID NO:30.
  • the fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identify with residues 19-1274 or 25-1274 of SEQ ID NO:85; in some such embodiments, the fusion polypeptide comprises the amino acid sequence shown in residues 19-1274 or 25- 1274 of SEQ ID NO:85.
  • the fusion polypeptide is linked to a myeloperoxidase (MPO) inhibitor.
  • MPO myeloperoxidase
  • the present invention provides a dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, where each of said first and second fusion polypeptides is a fusion polypeptide comprising a dimerizing domain, as described above.
  • the present invention provides a polynucleotide encoding a fusion polypeptide as described above.
  • the present invention provides an expression vector comprising the following operabiy linked elements: a transcription promoter, a DNA segment encoding a fusion polypeptide as described above, and a transcription terminator. Also provided is a cultured cell into which has been introduced an expression vector as above, wherein the cell expresses the DNA segment.
  • the present invention provides a method of making a fusion polypeptide.
  • the method generally includes (a) cuiiuring a ceil into which has been introduced an expression vector as described above, where the cell expresses the DN A segment and the encoded fusion polypeptide is produced, and (b) recovering the fusion polypeptide.
  • the present invention provides a method of making a dimeric protein.
  • the method generally includes (a) culturing a cell into which has been introduced an expression vector as described above, where the encoded fusion polypeptide comprises a dimerizing domain and where the cell expresses the DMA segment and the encoded fusion polypeptide is produced as a dimeric protein, and (b) recovering the dimeric protein.
  • the present invention provides a composition comprising a fusion polypeptide as described above and a pharmaceutically acceptable carrier.
  • the present invention provides a composition comprising a dimeric protein as described above and a pharmaceutically acceptable carrier.
  • the present invention provides a method for treating a cardiovascular disease characterized by atherosclerosis.
  • the method generally includes administering to a subject having the cardiovascular disease an effective amount of a fusion polypeptide or dimeric fusion protein as described above.
  • the cardiovascular disease is selected from the group consisting of coronary heart disease and stroke.
  • the coronary heart disease is characterized by acute coronary syndrome.
  • the present invention provides a method for treating a neurodegenerative disease.
  • the method generally includes administering to a subject having the neurodegenerative disease an effective amount of a fusion polypeptide or dimeric fusion protein as described above.
  • the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, and multiple sclerosis.
  • the neurodegenerative disease is characterized by dementia; in some such variations, the neurodegenerative disease is Alzheimer's disease.
  • the present invention provides a method for treating a disease characterized by amyloid deposit.
  • the method generally includes administering to a subject having the disease characterized by amyloid deposit an effective amount of a fusion polypeptide or dimeric fusion protein as described above.
  • the disease is Alzheimer's disease.
  • the present invention provides a method for treating an autoimmune disease.
  • the method generally includes administering to a subject having the autoimmune disease an effective amount of a fusion polypeptide or dimeric fusion protein as described above.
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type 1 diabetes.
  • the present invention provides a method for treating an inflammatory disease.
  • the method generally includes administering to a subject having the inflammatory disease an effective amount of a fusion polypeptide or dimeric fusion protein as described above.
  • the inflammatory disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes, type 2 diabetes, obesity, non-alcoholic steatohepatitis, coronary heart disease, and stroke.
  • the inflammatory disease is an inflammatory lung disease such as, for example, asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, idiopathic pulmonary fibrosis, hyperoxia, hypoxia, or acute respiratory distress syndrome.
  • COPD chronic obstructive pulmonary disease
  • the present invention provides a method for treating an infectious disease.
  • the method generally includes administering to a subject having the infectious disease an effective amount of a fusion polypeptide or dimeric fusion protein as described above.
  • the infectious disease is characterized by a bacterial infection; in some such embodiments, the bacterial infection is a Pseudomonas aeruginosa infection.
  • the present invention provides a method for treating nephrotic syndrome (NS).
  • the method generally includes administering to a subject having nephrotic syndrome an effective amount of a fusion polypeptide or dimeric fusion protein as described above.
  • the subject's nephrotic syndrome is associated with a disease selected from the group consisting of a primary kidney disease (e.g., minimal-change nephropathy, focal glomerulosclerosis, membranous nephropathy, or IgA nephropathy), amyloidosis, systemic lupus erythematosus, type 1 diabetes, and type 2 diabetes.
  • a primary kidney disease e.g., minimal-change nephropathy, focal glomerulosclerosis, membranous nephropathy, or IgA nephropathy
  • amyloidosis e.g., systemic lupus erythematosus, type 1 diabetes, and type 2 diabetes.
  • the present invention provides a method for treating exposure to sulfur mustard gas or to an organophosphate.
  • the method generally includes administering to a subject exposed to the sulfur mustard gas or to the organophosphate an effective amount of a fusion polypeptide or dimeric fusion protein as described above.
  • the present invention provides a method for treating cancer.
  • the method generally includes administering to a subject having cancer an effective amount of a fusion polypeptide or dimeric fusion protein as described above.
  • the cancer is selected from the group consisting of malignant melanoma, renal cell carcinoma, non-small cell lung cancer, bladder cancer, and head and neck cancer.
  • the cancer treatment is a combination therapy.
  • the combination therapy includes a non-ApoA l -mediated immunomodulatory therapy such as, e.g. , an immunomodulatory therapy comprising an anti-PD- l/PD-L l therapy, an anti-CTLA-4 therapy , or both.
  • the combination therapy includes radiation therapy or chemotherapy.
  • the combination therapy includes a targeted therapy; in some such embodiments, the targeted therapy includes (i) a therapeutic monoclonal antibody targeting a specific cell-surface or extracellular antigen (e.g.
  • VEGF vascular endothelial growth factor
  • EGFR vascular endothelial growth factor
  • CTLA-4 PD- 1
  • PD-L 1 vascular endothelial growth factor 1
  • a small molecule targeting an intracellular protein such as, for example, an intracellular enzyme (e.g., a proteasome, a tyrosine kinase, a cyciin-dependent kinase, serine/threonine-protein kinase B-Raf (BRAF), or a MEK kinase).
  • an intracellular enzyme e.g., a proteasome, a tyrosine kinase, a cyciin-dependent kinase, serine/threonine-protein kinase B-Raf (BRAF), or a MEK kinase
  • the treatment is a combination therapy.
  • the combination therapy includes administering a cyclodextrin (e.g., 2- hydroxypropyl-P-cyciodextrin) to the subject.
  • the combination therapy comprises administering to the subject an agent selected from (i) a liver X receptor (LXR) agonist (e.g.
  • statin e.g., atorvastatin, simvastatin, rosuvastatin, or pravastatin
  • PCSK9 inhibitor e.g., evolocumab or alirocumab
  • a "polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides.”
  • a "protein” is a macromolecule comprising one or more polypeptide chains.
  • a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures: substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • amino-terminal (or “N -terminal”) and “carboxyi-terminai” (or “C- terminal”) are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyi-terminai to a reference sequence within a polypeptide is located proximal to the carboxyi terminus of the reference sequence, but is not necessarily at the carboxyi terminus of the complete polypeptide.
  • polynucleotide and “nucleic acid” are used synonymously herein and refer to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kiiobases ("kb").
  • the latter two terms may describe polynucleotides that are single-stranded or double-stranded. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may ⁇ be staggered as a result of enzymatic cleavage; thus ail nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
  • a “segment” is a portion of a larger molecule (e.g. , polynucleotide or polypeptide) having specified attributes.
  • a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment that, when read from the 5' to the 3' direction, encodes the sequence of amino acids of the specified polypeptide.
  • a polypeptide segment "having cholesterol efflux activity” and “comprising an amino acid sequence having at least 90% or at least 95% identity with amino acid residue 19-267 or 25-267 of SEQ ID NO:36” is a portion of the longer polypeptide fusion molecule that, in addition to the specified polypeptide segment having cholesterol efflux activity, includes other polypeptide segments (e.g. , linker(s), dimerizing domain) as described herein.
  • expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
  • additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a “secretory signal sequence” is a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • operably linked means that two or more entities are joined together such that they function in concert for their intended purposes.
  • DNA segments the phrase indicates, for example, that coding sequences are joined in the correct reading frame, and transcription initiates in the promoter and proceeds through the coding segment(s) to the terminator.
  • "operably linked” includes both covarrire (e.g. , by disulfide bonding) and non-covalently (e.g., by hydrogen bonding, hydrophobic interactions, or salt-bridge interactions) l inked sequences, wherein the desired function(s) of the sequences are retained.
  • recombinant when used with reference, e.g. , to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a ceil so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.
  • nucleic acid By the term “recombinant nucleic acid” herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid using, e.g. , polymerases and endonucleases, in a form not normally found in nature. In this manner, operable linkage of different sequences is achieved.
  • an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined are both considered recombinant for the purposes disclosed herein. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, ' . e.
  • a "recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.
  • heterologous when used with reference to portions of a nucleic acid, indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences, e.g., from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • heterologous when used in reference to portions of a protein, indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature ⁇ e.g. , two or segments of a fusion polypeptide).
  • immunoglobulin is a serum protein which functions as an antibody in a vertebrate organism.
  • Five classes of “immunoglobulin,” or antibody, protein (IgG, IgA, IgM, IgD, and IgE) have been identified in higher vertebrates.
  • IgG comprises the major class; it normally exists as the second most abundant protein found in plasma.
  • IgG consists of four subclasses, designated IgGl, IgG2, IgG3, and IgG4.
  • the heavy chain constant regions of the IgG class are identified with the Greek symbol ⁇ .
  • immunoglobulins of the IgGl subclass contain a ⁇ heavy chain constant region.
  • Each immunoglobulin heavy chain possesses a constant region that consists of constant region protein domains (CHI, hinge, CH2, and CH3) that are essentially invariant for a given subclass in a species.
  • DNA sequences encoding human and non-human immunoglobulin chains are known in the art. See, e.g., Ellison et al, DNA 1: 11-18, 1981; Ellison et al , Nuc. Acids Res. 10:4071-4079, 1982; Kenten et al , Proc. Natl Acad. Sc.: USA 79:6661-6665, 1982; Seno et al, Nuc. Acids Res. 11:719-726, 1983; Riechmann et al .
  • an "immunoglobulin hinge” is that portion of an immunoglobulin heavy chain connecting the CHI and CH2 domains.
  • the hinge region of human ⁇ corresponds approximately to Eu residues 216-230.
  • Fc fragment Fc region
  • Fc domain Fc domain
  • Fc fragment crystalline
  • Distinct protein fragments which were originally described by proteolytic digestion, can define the overall general structure of an immunoglobulin protein. As originally defined in the literature, the Fc fragment consists of the disulfide-linked heavy chain hinge regions, CH2, and CH3 domains.
  • the term also refers to a single chain consisting of CH3, CH2, and at least a portion of the hinge sufficient to form a disulfide-linked dimer with a second such chain.
  • the term Fc region further includes variants of naturally occurring sequences, where the variants are capable of forming dimers and including such variants that have increased or decreased Fc receptor-binding or complement-binding activi ty .
  • dimerizing domain refers to a polypeptide having affinity for a second polypeptide, such that the two polypeptides associate under physiological conditions to form a dimer.
  • the second polypeptide is the same polypeptide, although in some variations the second polypeptide is different.
  • the polypeptides may interact with each other through covalent and/or non-covalent association(s).
  • dimerizing domains include an Fc region; a hinge region; a CH3 domain; a CH4 domain; a CHI or CL domain; a leucine zipper domain ⁇ e.g., a jun/fos leucine zipper domain, see, e.g., Kostelney et al. , J.
  • a yeast GCN4 leucine zipper domain an isoleucine zipper domain; a dimerizing region of a dimerizing cell-surface receptor (e.g., interleukin-8 receptor (IL-8R); or an integrin heterodimer such as LFA- 1 or GPIllb/IIIa); a dimerizing region of a secreted, dimerizing ligand (e.g., nerve growth factor (NGF), neurotrophin-3 (NT-3), interleukin-8 (1L-8), vascular endothelial growth factor (VEGF), or brain- derived neurotrophic factor (BDNF); see, e.g. , Arakawa et al. , J.
  • NGF nerve growth factor
  • NT-3 neurotrophin-3
  • VEGF vascular endothelial growth factor
  • BDNF brain- derived neurotrophic factor
  • a preferred dimerizing domain in accordance with the present invention is an Fc region.
  • dimerized first and second fusion polypeptides refers to a multimer of two ("first" and “second") fusion polypeptides as disclosed herein linked together via a dimerizing domain.
  • a “dimer” or “dimeric protein” includes reference to such dimerized first and second fusion polypeptides in the context of higher order multimers that may form in spherical HDL particles (e.g., (rimers), such as through an interaction of dimerized first and second fusion polypeptides with another ApoA- 1 polypeptide that may be present (e.g. , through interaction with a naturally occurring, endogenous ApoA- 1 protein).
  • the term also includes reference to dimerized first and second fusion polypeptides in the context of higher order multimers that may be created by inclusion of an additional dimerizing domain in a first or second fusion polypeptide (e.g., a first fusion polypeptide comprising an immunoglobulin light chain and a second fusion polypeptide comprising an immunoglobulin heavy chain can heterodimerize via the interaction between the CHI and CL domains, and two such heterodimers may furtiier dimerize via the Fc region of the immunoglobulin heavy chain, thereby forming a tetramer).
  • a first fusion polypeptide comprising an immunoglobulin light chain and a second fusion polypeptide comprising an immunoglobulin heavy chain can heterodimerize via the interaction between the CHI and CL domains, and two such heterodimers may furtiier dimerize via the Fc region of the immunoglobulin heavy chain, thereby forming a tetramer.
  • domain that specifically binds to the neonatal Fc receptor (FcRn) or "FcRn-binding domain,” as used herein, means a polypeptide that (i) binds to FcRn with a high affinity at pH 5.8, typically with a binding affinity (K a ) of !0 6 M “1 or greater (e.g. , 10 7 M “! or greater, 10 8 M “ 1 or greater, or 10 9 M “1 or greater), and (ii) does not have affinity for FcRn at physiological pH (e.g. , pH 7.4).
  • K a binding affinity
  • the binding affinity of a polypeptide for FcRn can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51 :660, 1949). Typically, a FcRn-binding domain does not significantly cross-react with polypeptides related to FcRn.
  • a polypeptide does not significantly cross-react with a polypeptide related to FcRn if, for example, it detects FcRn, but not presently known FcRn-related polypeptides, using a molecular binding assay such as, e.g., a multiweil plate assay (e.g., ELISA), a filter assay, or a surface plasmon resonance assay.
  • a molecular binding assay such as, e.g., a multiweil plate assay (e.g., ELISA), a filter assay, or a surface plasmon resonance assay.
  • known related polypeptides include known members of the major histocompatibility complex (MHC) class 1 protein family.
  • An FcRn-binding domain is not mutually exclusive of a dimerizing domain, i.e. , a dimerizing domain can also be an FcRn-binding domain. Examples of FcRn-binding domains that are also dimerizing
  • linker or "polypeptide linker” is used herein to indicate two or more amino acids joined by peptide bond(s) and linking two discrete, separate polypeptide regions.
  • the linker is typically designed to allow the separate polypeptide regions to perform their separate functions (such as, e.g., where a dimerizing domain, linked to other polypeptide regions, associates with another, corresponding dimerization domain to form a dimer).
  • the linker can be a portion of a native sequence, a variant thereof, or a synthetic sequence.
  • Linkers are also referred to herein using the abbreviation "L.”
  • the use of a numerical identifier (e.g. , " 1" or "2”) with "L” is used herein to differentiate among multiple linkers within a polypeptide chain, which linkers may be the same or different with respect to amino acid sequence.
  • ApoA- 1 is understood to include naturally occurring ApoA- 1 polypeptides as well as functional variants, functional fragments, and mimetics thereof.
  • ApoA l Apo A- l
  • Apo A- 1 Apo A- 1
  • apo A- l are used herein synonymously with “ApoA- 1.”
  • the use of a subscript identifier (e.g. , "a” or "b") with "ApoA I " is used herein to differentiate among multiple ApoA- 1 polypeptide segments within a polypeptide chain, which ApoA- 1 polypeptide segments may be the same or different with respect to amino acid sequence.
  • first polypeptide segment and “second polypeptide segment” refer to two ApoA- 1 polypeptide segments that are joined in tandem.
  • first polypeptide segment is used interchangeably herein with “first ApoA- 1 polypeptide segment” or “first ApoA- 1 polypeptide”; and the term “second polypeptide segment” is used interchangeably herein with “second ApoA- i polypeptide segment” or “second ApoA- 1 polypeptide.”
  • the first ApoA- 1 polypeptide segment is located N-terminal to the second ApoA- 1 polypeptide segment.
  • the second ApoA- 1 polypeptide segment is also referred to herein as the "carboxyl-terminal ApoA- 1 polypeptide segment" of a tandem ApoA- 1 region.
  • tandem ApoA- 1 region as used herein in the context of a tandem ApoA- 1 fusion polypeptide, means the region of the fusion polypeptide corresponding to first and second ApoA- 1 polypeptides joined by a flexible linker.
  • C-terminal to the tandem ApoA- 1 region in reference to another portion of the fusion polypeptide, means that the referenced portion is located C-terminal to the second ApoA- polypeptide segment of the tandem ApoA- 1 region.
  • third polypeptide segment refers to a non-ApoA- 1 polypeptide segment located C-terminal to a tandem ApoA- 1 region and which is not a linker, dimerizing domain, or FcRn-binding domain.
  • bispecific tandem ApoA- 1 fusion polypeptides comprising a third polypeptide segment selected from an RNase, a paraoxonase, a platelet-activating factor acetylhydrolase (PAF- AH), a cholesterol ester transfer protein (CETP), a lecithin-cholesterol acyl transferase (LCAT), and a polypeptide that specifically binds to amyloid beta ( ⁇ )
  • the third polypeptide segment is located C- terminal to the tandem ApoA- 1 region, and such bispecific fusion polypeptide may include other polypeptide segments (e.g. , a dimerizing or FcRn-binding domain, and/or polypeptide linker(s)) between the tandem ApoA- 1 region and the third polypeptide segment.
  • RNase e.g., "RNase 1 "
  • paraoxonase e.g., "PONT”
  • PAF-AH platelet-activating factor acetylhydrolase
  • CETP cholesterol ester transfer protein
  • LCAT lecithin-cholesterol acyltransferase
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenolypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • ApoA- 1 fusion polypeptides of the present disclosure may be referred to herein by formulae such as, for example, "ApoA l a -L l-ApoA l b ,” “ApoA l a -L l-ApoA l b -L2-D,” “ApoA l a -L l- ApoA l b -L2-D-L3-P,” “ApoA l a -L l-ApoA l b -L2-[Fc region]," “ApoA l a -L l-ApoA l b -L2-albumin,” “ ApoA l a -L I -ApoA l b -L2-D-L3-RNase,” “ ApoA 1 a -L 1 -ApoA l b -L2-[Fc region]-L3-RNase 1
  • a term referring to a particular segment of a fusion polypeptide e.g. , "ApoA l ,” “D” (for dimerizing domain), “L I “ or “L2” (for first or second polypeptide linkers, respectively), “Fc region,” “RNase,” “paraoxonase,” etc. is understood to have the meaning ascribed to such term herein and is inclusive of the various embodiments as described herein.
  • the term “effective amount” refers to an amount of such molecule that is sufficient to modulate an autoimmune response in the subject so as to inhibit the occurrence or ameliorate one or more symptoms of the autoimmune disease.
  • An effective amount of an agent is administered according to the methods of the present invention in an "effective regime.”
  • the term “effective regime” refers to a combination of amount of the agent being administered and dosage frequency adequate to accomplish treatment or prevention of the disease.
  • the term "patient” or "subject,” in the context of treating a disease or disorder as described herein, includes mammals such as, for example, humans and other primates. The term also includes domesticated animals such as, e.g. , cows, hogs, sheep, horses, dogs, and cats.
  • the term "combination therapy” refers to a therapeutic regimen that involves the provision of at least two distinct therapies to achieve an indicated therapeutic effect.
  • a combination therapy may involve the administration of two or more chemically distinct active ingredients, or agents, for example, a soluble ApoA 1 fusion polypeptide or dimeric protein according to the present invention and another agent such as, e.g., another anti-inflammatory or immunomodulatory agent.
  • a combination therapy may involve the administration of a soluble ApoA 1 fusion polypeptide or dimeric protein according to the present invention, alone or in conjunction with another agent, as well as the delivery of another therapy (e.g., radiation therapy).
  • the distinct therapies constituting a combination therapy may be delivered, e.g., as simultaneous, overlapping, or sequential dosing regimens.
  • the active ingredients may be administered as part of the same composition or as different compositions.
  • the compositions comprising the different active ingredients may be administered at the same or different times, by the same or different routes, using the same or different dosing regimens, all as the particular context requires and as determined by the attending physician.
  • non-ApoAl-mediated immunomodulatory therapy in the context of treating cancer, means an immunomodulatory therapy that does not specifically target ApoA- 1 or ApoA- 1 -mediated signaling pathways.
  • targeted therapy in the context of treating cancer, refers to a type of treatment that uses a therapeutic agent to identify and attack a specific type of cancer cell, typically with less harm to normal cells.
  • a targeted therapy blocks the action of an enzyme or other molecule involved in the growtii and spread of cancer cells.
  • a targeted therapy either helps the immune system to attack cancer cells or delivers a toxic substance directly to cancer cells.
  • a targeted therapy uses a small molecule drag or a monoclonal antibody therapeutic agent.
  • Two amino acid sequences have " 100% amino acid sequence identity" if the amino acid residues of the two amino acid sequences are the same when aligned for maximal correspondence. Sequence comparisons can be performed using standard software programs such as those included in the LASERGENE bioinformatics computing suite, which is produced by DNASTAR (Madison, Wisconsin). Other methods for comparing amino acid sequences by determining optimal alignment are well-known to those of skill in the art. (See, e.g. , Peruski and Peruski, The Internet and the New Biology: Tools for Genomic and Molecular Research (ASM Press, Inc. 1997); Wu et al.
  • Two amino acid sequences are considered to have "substantial sequence identity” if the two sequences have at least 80%, at least 90%, or at least 95% sequence identity relative to each other.
  • Percent sequence identity is determined by conventional methods. See, e.g., Altschui et al, Bull. Math. Bio. 48:603, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1992. For example, two amino acid sequences can be aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff, supra, as shown in Table 1 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as: ([Total number of identical matches]/ [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100).
  • the trimmed initial regions are examined to determine whetiier the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch- Sellers algorithm. (Needleman and Wunsch, J. Mol. Biol. 48:444, 1970; Sellers, SI AM J. Appl. Math.
  • corresponding to when applied to positions of amino acid residues in a reference sequence to describe positions within a subject sequence, means corresponding positions in the subject sequence when the reference and subject sequences are optimally aligned.
  • FIG. 1 illustrates cholesterol efflux in BHK cell cultures expressing ApoA-1 molecules and recombinant fusions thereof.
  • ApoA-l -Fc fusion protein containing a 26 amino acid linker between ApoA- 1 and the Fc region demonstrated increased cholesterol efflux as compared to either an ApoA-l-Fc fusion protein with a two amino acid linker (ApoA- l(2)Fc (Theripion)) or an ApoA- l -Fc fusion protein without a linker (ApoA-l(O)Fc (Sino Biol)) and had activity similar to wild-type human ApoA-1 (Control ApoA-1).
  • ApoA-1 molecules were incubated for 2 hours with H3-choiesterol labeled BHK ceils induced for ABCA1 expression.
  • the Fc proteins were predicted to be dirners; however, the concentrations shown were calculated and normalized based on the mass of ApoA- 1 per molecule.
  • FIGS. 2A and 2B show schematic diagrams of certain embodiments of fusion proteins in accordance with the present disclosure, including component functional domains.
  • FIG. 2A depicts a schematic representation of a human ApoA- 1 joined at the carboxyl terminus, via a linker, to a human IgG Fc region (also referred to herein as a "THER fusion protein” or "THER molecule”).
  • FIG. 2A depicts a schematic representation of a human ApoA- 1 joined at the carboxyl terminus, via a linker, to a human IgG Fc region (also referred to herein as a "THER fusion protein" or "THER molecule”).
  • FIG. 2B depicts a schematic representation of a THER fusion protein further joined at the carboxyl terminus, via a linker, to an enzyme region (these fusions are also referred to herein as "Bifunctional Enzyme Lipid Transport” or "BELT” molecule; a BELT molecule may also be generally referred to herein as a THER fusion protein or molecule).
  • BELT Bifunctional Enzyme Lipid Transport
  • the linker sequence and the domain present at the carboxyl terminus of the fusion protein varies depending on the construct.
  • FIG. 3 shows a Western blot of culture supernatants (serum free) from transiently transfected 293T cells expressing five different THER molecules. Transfections and Western blot analysis were performed as described in Example 3, infra.
  • FIGS. 4A-4E show columnar graphs summarizing the initial screening of stable CHO clones expressing THERO (FIG. 4A), THER2 (FIG. 4B), THER4 (FIG. 4C), THER6 (FIG. 4D), and THER4RNA2 (FIG. 4E) ApoA-1 fusion protems and relative expression levels of the fusion proteins from 96 well culture supernatants ⁇ see Example 4, infra).
  • FIGS. 5A-5C show results from analysis of a subset of THER clones that expressed higher levels of fusion protein, assessing their cell growth pattern (FIG. 5A), relative cell viability (FIG. 5B), and expression of fusion protein (FIG. 5C) after six and ten days of culture (see Example 4, infra).
  • FIGS. 6A and 6B show nonreducing (FIG. 6A) and reducing (FIG. 6B) SDS-PAGE analysis of THER fusion proteins purified from CHO clone spent culture supernatants (see Example 4, infra).
  • FIG. 7 shows Native PAGE gel analysis of the purified THER fusion proteins. Samples were prepared and BLUE Native PAGE gels were run and stained as described in Example
  • FIG. 8 shows a graph summarizing the relative binding of the different fusion proteins in a sandwich ELISA, using an anti-IgG capture of fusion proteins and detection step with an HRP-conjugated anti-ApoA-1 antibody (see Example 5, infra),
  • FIG. 9 shows results from a kinetic enzyme assay measuring the RNase activity present in samples of serial dilutions of purified ApoA l-IgG-RNase bispecific fusion protein (THER4RNA2).
  • An RNASEALERTTM assay (IDT, Coralvilie, IA) was performed as described in Example 6, infra, using RNase A (“RNase”) as a positive control and ApoA-l-lnk26-hIgG ("THER4") as a negative control.
  • RNase RNase
  • THER4 ApoA-l-lnk26-hIgG
  • Each box displays the relative fluorescence units observed as a function of time during the course of a 45 minute assay, with a fixed concentration of a non- fluorescent RNA substrate that generates a fluorescent signal upon digestion of the RNA.
  • FIG. 10 shows a subset of the data shown in FIG. 9, comparing the RNase enzyme activity at the 4 pmol/ ⁇ protein dilution.
  • FIG. 11 shows the results of a BODIPY-cholesterol efflux assay using purified fusion proteins and differentiated human monocytic cell line, THP-1. Assays were performed in a 96 well plate format as described in Example 7, infra, and data are displayed as the mean efflux observed from 5 replicates, with baseline efflux (media alone) subtracted from ail samples.
  • FIG. 12 shows the results of a cholesterol efflux assay using the mouse monocyte- macrophage cell line J774 A.1 (ATCC, Manassas, VA). Both baseline and cAMP-stimulated efflux were assessed as described in Example 7, infra,
  • FIG. 13 shows a schematic diagram of a tandem ApoA- 1 dimeric fusion protein in accordance with an embodiment of the present disclosure. Specifically, FIG. 13 depicts a dimer of two polypeptide chains, each chain consisting of two ApoA-1 polypeptides joined in tandem via a linker and an Ig Fc region (Hinge-CH2-CH3) joined to the carboxvl terminus of the second (carboxyl terminal) ApoA-1 polypeptide via a second linker.
  • the tandem ApoA-1 polypeptides within each polypeptide chain are shown as a head-to-toe dimer.
  • FIG. 14 shows a schematic diagram of a tandem ApoA-1 fusion polypeptide in accordance with an embodiment of the present disclosure. Specifically, FIG. 14 depicts two ApoA- 1 polypeptides joined in tandem via a linker and an albumin polypeptide joined to the carboxvl terminus of the second (carboxyl terminal) ApoA-1 polypeptide via a second linker. The tandem ApoA-1 polypeptides are shown as a head-to-toe dimer.
  • FIG. 15 shows the results of a Western blot of tandem ApoA- 1 fusion proteins expressed from HEK 293T cell transient transfections. Tandem ApoA-1 fusion constructs were generated and transiently expressed in HEK 293 T cells as described in Example 11, infra. Supernatant samples were analyzed by SDS-PAGE electrophoresis. Serum free, phenol red free culture supernatants (10 ⁇ ) were mixed with 4 ⁇ 3-4 ⁇ LDS sample buffer and 1 ⁇ 3-10 ⁇ reducing agent, heated at 72 ° C for 10 minutes and loaded directly onto NuPAGE SDS-PAGE gels.
  • Electrophoresis was performed at 185 volts for approximately 75 minutes, blotted to nitrocellulose, and blots probed with horseradish -peroxidase conjugated anti-human TgG (HRP-anti-human IgG; Jackson Immunoresearch, West Grove, PA). Bands were visualized using ECL substrate (Pierce/ThermoFisher, Rockford, IL), and a ProteinSimple FluorChemE fluorescence imager (San Jose,CA).
  • FIG. 16 shows purified tandem ApoA-1 fusion proteins after reducing NuPAGE Bis- Tris SDS-PAGE electrophoresis and visualization with IMPERIALTM protein stain (Pierce, ThermoFisher, Dallas TX).
  • CHO DG44 stable clones expressing tandem ApoA- 1 fusion constructs were generated and the expressed fusion proteins were purified and analyzed by NuPAGE SDS- PAGE as described in Example 12, infra.
  • the migration positions of Precision Plus protein molecular weight markers BioRAD, Hercules, CA
  • the tandem apoA-l -Fc derivatives migrate at a molecular weight of approximately 85 kDa.
  • [1191 F G. 17 shows the results of a BODIPY-cholesteroi efflux assay using TOPFLUOR fluorescent cholesterol derivative, purified tandem ApoA- 1 fusion proteins, and differentiated human monocytic cell Sine, THP-1. Assays were performed in a 96-weil plate format as described in Example 13, infra, and data are displayed as the mean percent efflux observed from six replicates, with baseline efflux (media alone) shown. Statistical analysis of the efflux levels was performed using GraphPad Prism and two-tailed, unpaired t test P values are indicated on the graph.
  • the present invention provides compositions and methods relating to tandem ApoA-1 fusion polypeptides.
  • the fusion polypeptides generally comprise first and second polypeptide segments joined in tandem via a peptide linker, each of the first and second segments having cholesterol efflux activity and which is either an ApoA l polypeptide or functional variant or fragment thereof or, alternatively, an ApoA-1 mimetic.
  • the fusion polypeptide further includes a dimerizing domain with a second peptide linker between the amino-terminal end of the dimerizing domain and the carboxyl-terminal end of the second ApoA-1 polypeptide, variant, fragment, or mimetic, thereby allowing the fusion polypeptide to form stable dirners.
  • the fusion polypeptide further includes a domain that specifically binds to the neonatal Fc receptor (FcRn), with a second peptide linker between the amino-terminal end of the FcRn-binding domain and the carboxyl-terminal end of the second ApoA- 1 polypeptide, variant, fragment, or mimetic.
  • the fusion polypeptides are bispecific constructs further comprising a tliird polypeptide segment carboxyl-terminal to the second ApoA-1 polypeptide, variant, fragment, or mimetic and which confers a second biological activity.
  • Exemplary second polypeptides include RNases, paraoxonases, platelet-activating factor acetylhydrolases (PAF-AHs), cholesterol ester transfer proteins (CETPs), lecithin-cholesterol acyltransferases (LCATs), and polypeptides that specifically bind to amyloid beta, any of which may be a naturally occurring protein or a functional variant or fragment thereof.
  • PAF-AHs platelet-activating factor acetylhydrolases
  • CETPs cholesterol ester transfer proteins
  • LCATs lecithin-cholesterol acyltransferases
  • polypeptides that specifically bind to amyloid beta any of which may be a naturally occurring protein or a functional variant or fragment thereof.
  • the tandem ApoA- 1 fusion molecules of the present invention can be used, for example, to increase reverse cholesterol transport in a subject and provide therapeutic benefit in the treatment of various diseases.
  • ApoA- 1 the major protein of HDL, has already shown beneficial activity in clinical trials in patients with acute coronary syndrome.
  • the ApoA-1 fusion molecules of the present invention can be used to treat coronary heart disease, acute coronary syndrome, and other cardiovascular diseases characterized by atherosclerosis such as, e.g. , stroke. Fusion molecules of the present invention are also useful, for example, for the treatment of autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus), inflammatory diseases, type 2 diabetes, obesity, and neurodegenerative diseases (e.g.
  • autoimmune diseases e.g., rheumatoid arthritis, systemic lupus erythematosus
  • inflammatory diseases e.g., type 2 diabetes, obesity, and neurodegenerative diseases (e.g.
  • fusion proteins of the present invention are used to replace defective ApoA- 1 such as, for example, in the treatment of type 1 diabetes and dementia.
  • fusion proteins as disclosed herein are used to treat multiple sclerosis (MS).
  • MS multiple sclerosis
  • ApoA-1 levels have been shown to be low in patients with MS, and ApoA-1 deficient mice have been shown to exhibit more neurodegeneration and worse disease in experimental allergic encephalomyelitis (EAE), a model for MS, than wild-type animals.
  • EAE allergic encephalomyelitis
  • Tandem ApoA-l fusion molecules of the present invention may also be used in the treatment of infectious disease.
  • significant alterations in lipid metabolism and lipoprotein composition occur, including a reduction in ApoA-l and changes in HDL composition and size.
  • HDL can bind and neutralize Gram-negative LPS and Gram-positive lipoteichoic acid, promoting clearance of these inflammatory products.
  • Pharmacological studies support the benefit of recombinant ApoA-l during bacterial infection. See, e.g., Pirillo et al , Hanclh Exp Pharmacol 224:483-508, 2015.
  • Bifunctional ApoA-l fusion molecules of the present invention that contain a paraoxonase ⁇ e.g., PO 1) are particularly useful for therapy of patients infected with Pseudomonas aeruginosa, a gram negative bacterium. This is particularly important for immunocompromised patients, where infections with P. aeruginosa are common, P. aeruginosa secrete virulence factors and form biofiim in response to small signaling molecules called acyl-homoserine lactones in a concentration-dependent process called quorum sensing (QS). Paraoxonase 1 degrades acyl- homoserine lactones and was shown to protect from lethality from P.
  • a paraoxonase ⁇ e.g., PO 1
  • Fusion molecules of the present invention may also be used in the treatment of inflammatory disease.
  • ApoA-l fusion polypeptides and dimeric proteins as described herein may alter the phenotype of neutrophils, macrophages, and/or antigen-presenting cells to reduce proinflammatory responses.
  • Molecules of the present disclosure cause efflux of cholesterol from cell membranes, mediated by transporter molecules such as, e.g. , ABCA1.
  • Efflux of cholesterol from antigen-presenting ceils, including macrophages and dendritic cells can inhibit proinflammatory responses mediated by these cells, resulting in reduced production of inflammatory cytokines.
  • treatment with ApoA-1 was shown to inhibit the proinflammatory signaling in macrophages after stimulation of CD40 by altering the composition of lipid rafts. See Yin et al. , J. A therosclerosis and Thrombosis 19:923-36, 2012. ApoA- 1 was also shown to cause a decrease in TRAF-6 recruitment to lipid rafts, and a decrease in activation of NF-kB. See id.
  • Inflammatory lung diseases are among inflammatory diseases that may be treated with tandem ApoA-1 fusion molecules as described herein. Serum ApoA-1 was found to be positively correlated with FEV1 in patients with combined atopy and asthma, but not in atopic and nonatopic subjects without asthma. See Barochia et ⁇ ., ⁇ . J. Respir. Crit. Care Med. 191 :990- 1000, 2015. In another study, patients with idiopathic pulmonary fibrosis had low levels of ApoA-1 in bronchiolar lavage fluid compared to controls (P ⁇ 0.01). See Kim et al., Am. J. Respir. Crit. Care Med. 182:633-642, 2010. Further, intranasal treatment with ApoA-1 in mice treated with bleomycin was very effective in reducing the number of inflammatory cells and collagen deposition in the lungs. See id.
  • Obesity is another inflammatory disease amenable to treatment with tandem ApoA-1 fusion molecules in accordance with the present invention.
  • Evidence supports the use of ApoA- 1 and HDL to combat obesity. See. e.g., Mineo et al., Circ. Res. 111 : 1079-1090, 2012,
  • overexpression of ApoA- 1 or administration of the ApoA-1 mimetic peptide D-4F has been shown to decrease white adipose mass and insulin resistance and increase energy expenditure in mice fed a high-fat diet.
  • the ApoA-1 mimetic L-4F was shown to lower adiposity and inflammation and improve glucose tolerance. Id.
  • NS nephrotic syndrome
  • HDL filterable HDL
  • lipid- poor apo Al lipid- poor apo Al
  • ApoA-1 fusion molecules comprising an Fc region as described herein would bypass the need for reuptake in this usual manner, since the molecules are being recycled via FcRn due to the presence of the Fc domain.
  • Fusion molecules as described herein may also be used for therapy of patients with cancer. It is expected that ApoA-1 fusion polypeptides and dimeric proteins of the present invention, while reducing proinflammatory responses, enhance activation and tumor infiltration of CD8 + T-cells. Studies support the efficacy of ApoA-1 therapy in animal models of cancer and have shown that ApoA-1 therapy can cause a specific increase in CD8 + T cells in tumors. See, e.g. , Zamanian- Daryoush et al , J. Biol. Chem. 288:21237-21252, 2013. In some aspects, ApoA-1 fusion molecules of the present invention are useful in combination with one or more other anti-cancer therapies such as, for example, an anti-cancer immunotherapy.
  • a tandem ApoA-1 region is joined to a domain that specifically binds to the neonatal Fc receptor (FcRn) by providing a flexible linker between the C -terminus of the carboxyl -terminal ApoA-1 polypeptide, or variant, fragment, or mimetic thereof, and the N -terminus of the FcRn-binding domain.
  • the FcRn-binding domain is an immunoglobulin Fc region or a serum albumin (also referred to herein as "albumin"). The presence of an FcRn- binding domain extends ApoA- 1 half-life while retaining ApoA-1 reverse cholesterol efflux and eliminating the requirement for extensive lipid formulation.
  • an IgG Fc region to bind FcRn at low pH, but not at physiological pH, allows IgG Fc-containing fusions to bypass a lysosomal degradative fate following pinocytosis by cells, and to be instead transported with FcRn back to the cell surface where, under the influence of neutral pH, it dissociates from FcRn and is free to recycle.
  • Fc regions other FcRn-binding domains ⁇ e.g. , albumin or FcRn- specific single chain antibodies may also be used in accordance with the present disclosure to provide for extended half-life via a similar mechanism.
  • the present invention provides a way to stabilize an active ApoA-1 dimer while also controlling the maturation from pre-beta particles to discoid particles and spherical particles by providing a flexible linker between a dimerizing domain (e.g., an Fc domain) and the C- terminus of an ApoA-1 polypeptide, or functional variant, fragment, or mimetic thereof.
  • a dimerizing domain e.g., an Fc domain
  • a previous ApoA-l-Ig molecule not containing a linker exhibits low activity in cholesterol efflux assays compared to wild-type ApoAl.
  • dimerizing fusion polypeptides of the present disclosure retain ApoA-1 activity in cholesterol efflux assays and also allow for further improvements such as, e.g.
  • fusion of an RNase e.g., RNase 1
  • other polypeptide segments C -terminal to the dimerizing domain.
  • RNase e.g., RNase 1
  • Fc region as the dimerizing domain also allows for increased half-life of the dimer.
  • the invention provides tandem ApoA- 1 fusion molecules containing flexible linkers between the C-terminus of the carboxyi-terminai ApoA-1 polypeptide, or variant, fragment, or mimetic thereof, and the N -terminus of a dimerizmg domain such as, e.g. , an Fc domain.
  • Linkers are of sufficient length to allow ApoA-1, or the functional variant, fragment, or mimetic thereof, to mediate cholesterol efflux from cells, an initial and critical step in Reverse Cholesterol Transport (RCT).
  • RCT Reverse Cholesterol Transport
  • Linkers are typically between 2 and 60 amino acids in length. It is believed that ApoA- 1 fusion molecules with alternative linker lengths joining an ApoA-1 polypeptide and a dimerizmg domain have distinct functional properties by controlling the maturation of the HDL particle by constraining the C-terminus of ApoA-1. HDL discoid particles of intermediate size may have improved atheroprotective properties, and may have improved CNS transport properties. The molecules of this invention may change the progress of HDL maturation at these intermediate discoid stages, thereby improving efficacy of the fusion proteins of the invention relative to wild type ApoA-1 proteins.
  • the molecules of this invention are likely to affect the structure and composition of spherical HDL particles which are composed of trimeric ApoA-1 particles (see Silva et al , Natl. Acad. Sci. USA 105: 12176-12181, 2008). It is likely that molecules of this invention will interact with natural ApoA-1 in the formation of larger spherical HDL particles.
  • the dimerizmg domain is a immunoglobulin Fc region.
  • tandem ApoA- l-Fc fusion molecules of the present invention extend ApoA-1 half- life while retaining ApoA-I reverse cholesterol efflux and eliminating the requirement for extensive lipid formulation.
  • the presence of the Fc region allows purification using immobilized Protein A according to standard practices in and antibody and Fc fusion protein manufacturing.
  • spherical ApoA-1 particles that incorporate a third ApoA-1 monomer show the C-terminus of each monomer in a different position compared to the positions in dimeric discoid ApoA-1. See, e.g., Gogonea, supra.
  • the flexible linkers of the present disclosure are of sufficient length to allow ApoA- 1 to assume these positions without conformational constraint.
  • [tandem ApoA-l]-[linker]-[dimerizing domain] molecules of the present invention include an additional polypeptide segment fused carboxyi-terminai to the dimerizing domain. Such variations allow for the creation of bispecific molecules with ApoA-1 functional activity and a second biological activity.
  • bispecific fusion molecules comprising (i) first and second polypeptide segments joined in tandem via a linker, each segment having cholesterol efflux activity and which is either an ApoA l polypeptide or functional variant or fragment thereof or, alternatively, an ApoAl mimetic and (ii) a third polypeptide segment carboxyl- terminal to the second polypeptide segment, wherein the third polypeptide segment is selected from an RNase, a paraoxonase, a platelet-activating factor acetyihydrolase (PAF-AH), a cholesterol ester transfer protein (CETP), a lecithin-cholesterol acyltransferase (LCAT), and a polypeptide that specifically binds to amyloid beta.
  • PAF-AH platelet-activating factor acetyihydrolase
  • CETP cholesterol ester transfer protein
  • LCAT lecithin-cholesterol acyltransferase
  • Such third polypeptides may be a naturally occurring protein or a functional variant or fragment thereof.
  • a linker and dimerizing domain, or a linker and an FcRn-binding domain is included between the second and third polypeptides as summarized above.
  • the fusion polypeptide lacks a dimerizing or FcRn- binding domain.
  • the fusion molecule may be conjugated to PEG to provide extended half-life.
  • PEG may include bispecific molecules as described herein, such as, e.g., fusion molecules comprising an RNase, a paraoxonase, a platelet-activating factor acetyihydrolase (PAF- AH), a cholesterol ester transfer protein (CETP), a lecithin-cholesterol acyltransferase (LCAT), or a polypeptide that specifically binds to amyloid beta.
  • fusion molecules comprising an RNase, a paraoxonase, a platelet-activating factor acetyihydrolase (PAF- AH), a cholesterol ester transfer protein (CETP), a lecithin-cholesterol acyltransferase (LCAT), or a polypeptide that specifically binds to amyloid beta.
  • the third polypeptide segment is an RNase.
  • a preferred RNase is human RNase 1 or a functional variant or fragment thereof.
  • the RNase retains its sensitivity to inhibition by cytoplasmic inhibitor and has very low toxicity to cells, but is highly active extracellularly.
  • RNase has anti-inflammatory properties by digestion of inflammatory extracellular RNA and provides additional therapeutic benefit for treatment of various diseases, including cardiovascular diseases (e.g. , coronary artery disease, stroke), autoimmune diseases, inflammatory diseases, type 2 diabetes, infectious disease, and neurodegenerative diseases (e.g., Alzheimer' disease).
  • a bispecific tandem ApoA- 1 fusion molecule comprising an RNase segment as described herein may be used, e.g. , for treatment of an inflammatory disease such as, for example, an inflammatory lung disease.
  • an inflammatory disease such as, for example, an inflammatory lung disease.
  • TLR3, an RNA sensor has a major role in the development of ARDS-like pathology in the absence of a viral pathogen. See Murray et al . Am. J. Respir. Crit. Care Med. 178: 1227-1237, 2008. Oxygen therapy is a major therapeutic intervention in ARDS, but contributes to further lung damage and susceptibility to viral infection.
  • Oxygen therapy was a major stimulus for increased TLR3 expression and activation in cultured human epithelial ceils, and absence or blockade of TLR3 protected mice from lung injur ⁇ ' and inflammation after exposure to hyperoxic conditions. See Murray et al, supra. Another study has shown that TLR3 activation by extracellular RNA occurs in response to acute hypoxia, and that therapy in mice with RNaseA diminished lung inflammation after acute hypoxia. See Biswas et al, Eur. J. Immunol. 45: 3158-3173, 2015. A bispecific ApoA- l fusion molecule comprising an RNase segment as described herein may also be used, e.g.
  • RNA immune complexes and RNA receptors including TLR7
  • TLR7 RNA receptors
  • RNase overexpression in mouse models of SLE. See, e.g. , Sun et al , J. Immunol. 190:2536- 2543, 2013.
  • the third polypeptide segment is a paraoxonase.
  • a preferred paraoxonase is human paraoxonase 1 (PON 1) or a functional variant or fragment thereof.
  • Paraoxonase bispecific fusion molecules provide additional therapeutic benefit for the treatment of diseases amenable to ApoA-l -mediated therapy, including, for example, through its atheroprotective, antioxidant, anti-inflammatory, and/or neuroprotective properties.
  • PON1 may attached to a tandem ApoA-l fusion molecule of the present invention through its natural, high affinity binding to ApoA- l , which binding is mediated by Tyr71 of PON 1 (see Huang et al , J. Clin. Invest. 123:3815-3828, 2013). Incubating an ApoA-l fusion molecule with recombinant or natural PON 1 prior to administration will be sufficient to "load" PON 1 onto the ApoA-l fusion molecule.
  • a bispecific tandem ApoA-l fusion molecule comprising a paraoxonase segment as described herein may be used, e.g., for treatment of an autoimmune disease or an inflammatory disease.
  • a paraoxonase for treatment of autoimmune disease such as systemic lupus erythematosus (SLE).
  • SLE systemic lupus erythematosus
  • the autoantibody titer in many patients with systemic lupus erythematosus (SLE) is correlated with loss of activity of PON 1 (see Batukla et al , Ann. NY Acad. Sri.
  • SM sulfur mustard gas
  • COPD chronic obstructive pulmonary disease
  • bronchiectasis bronchiolar Savage fluid
  • Bispecific tandem ApoA- 1 fusion molecules comprising either an RNase segment or a paraoxonase segment as described herein may also be used, e.g. , for treatment of a neurological disease.
  • Such bispecific molecules are transported to the brain where they deliver a protective paraoxonase or RNase enzyme.
  • PON 1 is protective in the brain because of its antioxidant properties
  • RNase is protective by digesting extracellular RNA that promotes inflammation via stimulation of TLR7 and other RNA receptors.
  • Exemplary neurological diseases amenable to treatment using an ApoA- 1 /paraoxonase or ApoA l/RNase bispecific molecule of the present invention include multiple sclerosis, Parkinson's disease, and Alzheimer's disease.
  • Attachment of myeloperoxidase (MPO) inhibitors to tandem ApoA-1 fusion molecules of the present invention may be particularly desirable as a way to protect ApoA-1 from inactivation due to oxidation mediated by MPO, and can also similarly protect paraoxonase from MPO-mediated oxidation and inactivation in the context of a bispecific fusion polypeptide comprising a paraoxonase such as PON 1.
  • Myeioperoxidase-mediated oxidation of ApoA-1 promotes crossiinking of ApoA-1, and may be implicated in the mechanism that leads to amy loid deposition in atherosclerotic plaques in vivo. See Chan et al, J. Biol. Chem.
  • MPO inhibitors see Malle et al , Br J Pharmacol. 152: 838-854, 2007.
  • the attachment of a MPO inhibitor to a molecule of the present invention can also localize the MPO inhibition to selectively protect ApoA-1 from oxidation while preserving MPO activity important in anti -microbial activity.
  • the third polypeptide segment is selected from a cholesterol ester transfer protein (CETP), and a lecithin- cholesterol acyltransferase (LCAT).
  • CETP is involved in one of the major mechanisms by which HDL particles can deliver cholesterol to the liver during the process of reverse cholesterol transport (RCT), specifically, through unloading and transferring of cholesterol to LDL, which then transports cholesterol back to the liver via LDL receptors. This process of unloading requires CETP.
  • RCT reverse cholesterol transport
  • ApoA-1 molecules such as provided herein
  • adding other RCT components can provide an attractive and potentially synergistic therapeutic approach.
  • Providing more exogenous CETP in the form of a bispecific fusion molecule containing ApoA- 1 can enhance CETP activity and overall reverse cholesterol transport.
  • LCAT Lecithin-cholesterol acyltransferase
  • the third polypeptide segment is a polypeptide that specifically binds to amyloid beta ( ⁇ ).
  • the third polypeptide is a ⁇ -speeific single chain antibody such as, for example, an ⁇ - specific scFv.
  • a scFv specific for amyloid beta peptide is described, for example, by Cattepoel et al, PLoS One 6:e 18296, 2011.
  • the ⁇ -binding polypeptide is typically fused C- terminal to the tandem ApoA- 1 region, or C -terminal to the dimerizing domain or FcRn-binding domain, if present.
  • This bispecific fusion molecule has improved properties for therapy of patients with Alzheimer's disease.
  • the present invention provides a fusion polypeptide comprising, from an amino-terminai position to a carboxyl-terminal position, ApoAl a -Ll-ApoAl b - L2-D, where ApoAl 3 and ApoAl b are, respectively, first and second polypeptide segments, each of the first and second polypeptide segments having cholesterol efflux activity and which is independently selected from (i) a naturally occurring ApoA-1 polypeptide or a functional variant or fragment thereof and (ii) an ApoA-1 mimetic; LI is a first polypeptide linker; L2 is a second polypeptide linker; and D is selected from a dimerizing domain and a domain that specifically binds to the neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • ApoAl a and ApoAl b comprise or consist of the same amino acid sequence; in other embodiments, ⁇ I , and ApoA I b comprise or consist of different amino acid sequences.
  • the fusion polypeptide further includes an additional polypeptide segment located carboxyl-terminal to the dimerizing or FcRn-binding domain.
  • the additional (“third") polypeptide segment is (a) a naturally occurring RNase, paraoxonase, platelet-activating factor acetylhydrolase (PAF-AH), cholesterol ester transfer protein (CETP), or lecithin-cholesterol acyltransterase (LCAT); (b) a functional variant or fragment of any of the naturally occurring proteins specified in (a); or (c) a polypeptide that specifically binds to amyloid beta ( ⁇ ) such as, e.g. , an ⁇ -specific scFv.
  • PAF-AH platelet-activating factor acetylhydrolase
  • CETP cholesterol ester transfer protein
  • LCAT lecithin-cholesterol acyltransterase
  • Such a fusion polypeptide comprising a third polypeptide segment may be represented by the formula ApoA l a -L l-ApoA l b -L2-D-L2-P (from an ammo-terminal position to a carboxyl-terminal position), where ApoA l a , L I, ApoA l b , L2, and D are each as previously defined, where L3 is a third polypeptide linker and is optionally present, and where P is the third polypeptide segment.
  • the present invention provides a fusion polypeptide comprising, from an amino-terminal position to a carboxyl-terminal position, ApoA l a -Ll -ApoA 13 ⁇ 4, where ApoAl a and ApoAl b are, respectively, first and second polypeptide segments, each of the first and second polypeptide segments having cholesterol efflux activity and which is independently selected from (i) a naturally occurring ApoA-1 polypeptide or a functional variant or fragment thereof and (ii) an ApoA- 1 mimetic; and LI is a first polypeptide linker.
  • ApoAl a and ApoAl 3 ⁇ 4 comprise or consist of the same amino acid sequence; in other embodiments, ApoA l a and ApoA l b comprise or consist of different amino acid sequences.
  • the fusion polypeptide is a bispecific molecule comprising a third polypeptide segment located carboxyl-terminal to the second polypeptide segment, where the third polypeptide segment is (a) a naturally occurring RNase, paraoxonase, platelet-activating factor acetylhydrolase (PAF-AH), cholesterol ester transfer protein (CETP), or lecithin-cholesterol acyltransferase (LCAT); (b) a functional variant or fragment of any of the naturally occurring proteins specified in (a); or (c) a polypeptide that specifically binds to amyloid beta ( ⁇ )
  • the fusion polypeptide further includes a linker polypeptide located carboxyl-terminal to the second polypeptide segment and amino-terminal to the third polypeptide segment.
  • the fusion polypeptide further includes a dimerizing domain or a domain that specifically binds to the neonatal Fc receptor (FcRn), which can be located, for example, carboxyl-terminal to the second polypeptide segment and amino-terminal to the third polypeptide segment.
  • FcRn neonatal Fc receptor
  • variants of a particular naturally occurring protein specified above can be readily identified using routine assays for assessing the variant for a relevant biological or biochemical activity corresponding to the natural protein.
  • ApoA- 1 variants may be assayed for their ability to induce cholesterol efflux using known cholesterol efflux assays such as described herein. See, e.g., Examples 1 and 7, infra; see also Tang et at, J Lipid Res. 47: 107-14, 2006.
  • variants may be assayed for their ability to digest single or double-stranded RNA is known assays to assess ribonuclease activity. See, e.g.
  • Paraoxonase 1 (PON 1) variants may be assayed for phosphotriesterase activity using diethyl p-nitrophenol phosphate (paraoxon) as a substrate, or for arylesterase activity using phenyl acetate as a substrate. See, e.g., Graves and Scott, Curr Chem Genomics 2:51-61, 2008. Assays to assess relevant CETP and LCAT activities are also known.
  • assays for measuring LCAT and CETP enzyme activity are commercially available and include, e.g.. Cell Biolabs Cat. No. STA-615, Sigma-Aldrich Cat. No. MAK107, and Roar Biomedical Cat. No. RB-LCAT for LCAT, and Abeam Cat. No. ab65383 and Sigma- Aldrich Cat. No. MAK 106 for CETP, [151]
  • polypeptides such as, e.g., single chain antibodies may be assessed for binding activity using any of various known assays.
  • one assay system employs a commercially available biosensor instrument (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ), wherein a binding protein ⁇ e.g., ⁇ -binding candidate, such as an antibody) is immobilized onto the surface of a sensor chip, and a test sample containing a soluble antigen ⁇ e.g., ⁇ peptide) is passed through the cell. If the immobilized protein has affinity for the antigen, it will bind to the antigen, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • a binding protein ⁇ e.g., ⁇ -binding candidate, such as an antibody
  • a test sample containing a soluble antigen ⁇ e.g., ⁇ peptide
  • This system allows the determination of on- and off- rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
  • Use of this instrument is disclosed, e.g., by Karlsson ⁇ J. Immunol. Methods 145:229-240, 1991) and Cunningham and Wells (J. Mot Biol. 234:554-563, 1993).
  • ⁇ -binding polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity ⁇ see Scatchard, Ann. NY Acad. Sci. 51 : 660-672, 1949) and calorimetric assays ⁇ see Cunningham et at, Science 253:545-548, 1991 ; Cunningham et at, Science 254:821-825, 1991).
  • Naturally occurring polypeptide segments for use in accordance with the present invention ⁇ e.g., a naturally occurring ApoA- 1 polypeptide, RNase, paraoxonase, or platelet-activating factor acetylhydrolase
  • Naturally occurring variants such as, for example, allelic variants and interspecies homologs consistent with the disclosure.
  • Functional variants of a particular reference polypeptide are generally characterized as having one or more amino acid substitutions, deletions or additions relative to the reference polypeptide. These changes are preferably of a minor nature, that is conservative amino acid substitutions ⁇ see, e.g..
  • DNAs encoding affinity tags are available from commercial suppliers ⁇ e.g., Pharmacia Biotech, Piscataway, NJ).
  • Conservative substitutions may also be selected from, the following: 1 ) Alanine, Glycine; 2) Aspartate, Glutamate; 3) Asparagine, Glutamine; 4) Arginine, Lysine; 5) Isoleucine, Leucine, Methionine, Valine; 6) Phenylalanine, Tyrosine, Tryptophan; 7) Serine, Threonine; and 8) Cysteine, Methionine ⁇ see, e.g. , Creighton, Proteins (1984)).
  • Table 2 Co servative amino acid substitutions
  • Essential amino acids in a naturally occurring polypeptide can be identified according to procedures known in the ait, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081 -1085, 1989; Bass et al, Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991).
  • site-directed mutagenesis or alanine-scanning mutagenesis
  • Single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g.
  • cholesterol efflux for ApoA-1 variants to identify amino acid residues that are critical to the activity of the molecule, in addition, sites of relevant protein interactions can be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. The identities of essential amino acids can also be inferred from analysis of homologies with related proteins (e.g., species orthologs retaining the same protein function).
  • variant nucleotide and polypeptide sequences can also be generated through DNA shuffling.
  • DNA shuffling See, e.g., Stemmer, Nature 370:389, 1994; Stemmer, Proc. Nat 'l Acad. Sci. USA 91 : 10747, 1994; International Publication No. WO 97/20078.
  • variant DNA molecules are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PGR, resulting in randomly introduced point mutations.
  • This technique can be modified by using a family of parent DNA molecules, such as allelic variants or DNA molecules from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • a polypeptide fusion in accordance with the present invention can include a polypeptide segment corresponding to a "functional fragment" of a particular poiypeptide.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule encoding a given poiypeptide.
  • ApoA-1 -encoding DNA molecules having the nucleotide sequence of residues 70-816 of SEQ ID NO: l can be digested with ⁇ /31 nuclease to obtain a series of nested deletions. The fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for the ability to induce cholesterol efflux.
  • exonuciease digestion is to use oligonucieotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment.
  • particular fragments of a gene encoding a poiypeptide can be synthesized using the polymerase chain reaction.
  • polypeptides that (i) are substantially identical to a reference poiypeptide (e.g. , residues 19-267 or 25-267 of SEQ ID NO:36 for a human wild-type ApoA- 1 poiypeptide) and (ii) retain s the desired functional properties of the reference polypeptide.
  • a reference poiypeptide e.g. , residues 19-267 or 25-267 of SEQ ID NO:36 for a human wild-type ApoA- 1 poiypeptide
  • Polypeptide segments used within the present invention may be obtained from a variety of species. If the protein is to be used therapeutically in humans, it is preferred that human polypeptide sequences be employed. However, non-human sequences can be used, as can variant sequences.
  • polypeptide sequences from humans or non-human animals can be employed, although sequences from the same species as the patient may be preferred for in vivo veterinary use or for in vitro uses where species specificity of intermolecular reactions is present.
  • polypeptide segments for use within the present invention can be, without limitation, human, non-human primate, rodent, canine, feline, equine, bovine, ovine, porcine, lagomorph, and avian polypeptides, as well as variants thereof.
  • the first and/or second polypeptide segment is a human wild-type ApoA-1 poiypeptide or a functional variant or fragment thereof.
  • the first and/or second poiypeptide segment comprises an amino acid sequence having at least 80% identity with amino acid residues 19-267 or 25-267 of SEQ ID NO:36.
  • the first and/or second poiypeptide segment comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with amino acid residues 19-267 or 25-267 of SEQ ID NO:36.
  • the first and/or second polypeptide segment comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with amino acid residues 19-267 or 25- 267 of SEQ ID NO:36.
  • valine at the amino acid position corresponding to position 156 of mature human wild-type ApoA- 1 is replaced by lysine
  • arginine at the amino acid position corresponding to position 173 of mature human wild-type ApoA-1 is replaced by cysteine (also referred to herein, respectively, as V156K and R173C variants or mutations).
  • Position 156 of the mature human wild-type ApoA-1 corresponds to amino acid position 180 of SEQ ID NO:36
  • position 173 of mature human wild-type ApoA- 1 corresponds to amino acid position 197 of SEQ ID NO:36.
  • V156K and R173C mutations have improved activity and half-life in atherosclerotic mice compared to wild-type ApoA- 1. See Cho et al , Exp Mol Med 41 :417, 2009.
  • the first and/or second polypeptide segment is an ApoA-1 mimetic such as, for example, the 4F peptide (see Song et al, Int. J. Biol. Sci. 5:637-646, 2009).
  • ApoA- 1 mimetics are generally known in the art and are reviewed in Reddy et al, Ciirr. Opin. Lipidol. 25: 304-308, 2014.
  • the third polypeptide segment is an RNase.
  • the RNase is a human RNAse 1 or a functional variant or fragment thereof.
  • the third polypeptide segment comprises an amino acid sequence having at least 80% identity with amino acid residues 544-675 or 548-675 of SEQ ID NQ:4 (residues 548-675 of SEQ ID O:4 are also shown, e.g.
  • the third polypeptide segment comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with amino acid residues 544-675 or 548-675 of SEQ ID NO:4. In yet other embodiments, the third polypeptide segment comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with amino acid residues 544-675 or 548-675 of SEQ ID NO:4.
  • the third polypeptide segment is a paraoxonase.
  • the paraoxonase is a human paraoxonase 1 (PON 1) or a functional variant or fragment thereof.
  • the third polypeptide segment comprises an amino acid sequence having at least 80% identity with amino acid residues 16-355 of SEQ ID NO: 12, amino acid residues 16-355 of SEQ ID NQ:42, or amino acid residues 16-355 of SEQ ID NO:44.
  • the third polypeptide segment comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with amino acid residues 16-355 of SEQ ID NO: 12, amino acid residues 16-355 of SEQ ID NQ:42, or amino acid residues 16-355 of SEQ ID NO:44.
  • the third polypeptide segment comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with amino acid residues 16-355 of SEQ ID NO: 12, amino acid residues 16-355 of SEQ ID NO:42, or ammo acid residues 16-355 of SEQ ID NO:44.
  • the third polypeptide segment is a platelet-activating factor aeetylhydrolase (PAF-AH).
  • PAF-AH platelet-activating factor aeetylhydrolase
  • the platelet-activating factor aeetylhydrolase is a human PAF-AH or a functional variant or fragment thereof.
  • the third polypeptide segment comprises an amino acid sequence having at least 80% identity with amino acid residues 22-441 of SEQ ID NO: 32.
  • the third polypeptide segment comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with amino acid residues 22- 441 of SEQ ID NO:32, In yet other embodiments, the third polypeptide segment comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with ammo acid residues 22-441 of SEQ ID NO:32.
  • the third polypeptide segment is a cholesterol ester transfer protein (CETP),
  • the cholesterol ester transfer protein is a human CETP or a functional variant or fragment thereof.
  • the third polypeptide segment comprises an amino acid sequence having at least 80% identity with amino acid residues 18-493 of SEQ ID NO:30.
  • the third polypeptide segment comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with amino acid residues 18-493 of SEQ ID NO:30. In yet other embodiments, the third polypeptide segment comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with amino acid residues 18-493 of SEQ ID NO:30.
  • Polypeptide linkers for use in accordance with the present invention can be naturally- occurring, synthetic, or a combination of both.
  • the linker joins two separate polypeptide regions (e.g. , a dimerizing domain and an ApoA-1 polypeptide, or first and second ApoA-1 polypeptides) and maintains the linked polypeptide regions as separate and discrete domains of a longer polypeptide.
  • the linker can allow the separate, discrete domains to cooperate yet maintain separate properties (e.g., in the case of an Fc region dimerizing domain linked to an ApoA-1 polypeptide, Fc receptor (e.g., FcRn) binding may be maintained for the Fc region, while functional properties of the ApoA-1 polypeptide (e.g.
  • residues within the linker polypeptide are selected to provide an overall hydrophilic character and to be non-immunogenic and flexible.
  • a "flexible" linker is one that lacks a substantially stable higher-order conformation in solution, although regions of local stability are permissible. In general, small, polar, and hydrophilic residues are preferred, and bulky and hydrophobic residues are undesirable. Areas of local charge are to be avoided; if the linker polypeptide includes charged residues, they will ordinarily be positioned so as to provide a net neutral charge within a small region of the polypeptide. It is therefore preferred to place a charged residue adjacent to a residue of opposite charge.
  • preferred residues for inclusion within the linker polypeptide include Gly, Ser, Ala, Thr, Asn, and Gin; more preferred residues include Gly, Ser, Ala, and Thr; and the most preferred residues are Gly and Ser.
  • Phe, Tyr, Trp, Pro, Leu, He, Lys, and Arg residues will be avoided (unless present within an immunoglobulin hinge region of the linker), Pro residues due to their hydrophobicity and lack of flexibility, and Lys and Arg residues due to potential immunogenicity.
  • the sequence of the linker will also be designed to avoid unwanted proteolysis.
  • Linker LI typically comprises at least five amino acid residues ⁇ e.g., at least six, at least seven, at least eight, at least nine, at least 10, or at least 12 amino acid residues).
  • LI consists of from five to 20 amino acid residues, from five to 15 amino acid residues, from five to 12 amino acid residues, from 10 to 20 amino acid residues, from 10 to 15 amino acid residues, from 12 to 20 amino acid residues, or from 12 to 15 amino acid residues.
  • LI consists of from six to 20, from six to 15, from six to 12, from seven to 20, from seven to 15, from seven to 12, from eight to 20, from eight to 15, from eight to 12, from nine to 20, from nine to 15, from 9 to 12, from 1 1 to 20, or from 1 1 to 15 amino acid residues.
  • L I consists of 12 amino acid residues.
  • LI has the amino acid sequence shown in SEQ ID NO:66 (also shown as residues 268-279 of SEQ ID NO:56 or 268-279 of SEQ ID NO:96) or SEQ ID NO:68 (also shown as residues 268-279 of SEQ ID NO:58 or 268-279 of SEQ ID NO:98).
  • linker L2 comprises at least two or at least three amino acid residues ⁇ e.g. , at least five, at least 10, at least 16, at least 26, or at least 36 amino acid residues).
  • L2 consists of from two to 60 amino acid residues, from three to 60 amino acid residues, from five to 40 amino acid residues, or from 15 to 40 amino acid residues.
  • L2 consists of from two to 50, from, two to 40, from two to 36, from two to 35, from two to 30, from two to 26, from three to 50, from three to 40, from three to 36, from three to 35, from three to 30, from three to 26, from five to 60, from five to 50, from five to 40, from five to 36, from five to 35, from five to 30, from five to 26, from 10 to 60, from 10 to 50, from 10 to 40, from 10 to 36, from 10 to 35, from 10 to 30, from 10 to 26, from 15 to 60, from 15 to 50, from 15 to 36, from 15 to 35, from 15 to 30, or from 15 to 26 amino acid residues.
  • L2 consists of from 16 to 60, from 16 to 50, from 16 to 40, or from 16 to 36 amino acid residues. In yet other variations, L2 consists of from 20 to 60, from 20 to 50, from 20 to 40, from 20 to 36, from 25 to 60, from 25 to 50, from 25 to 40, or from 25 to 36 amino acid residues. In still other variations, L2 consists of from 26 to 60, from 26 to 50, from 26 to 40, or from 26 to 36 amino acid residues. In more specific variations, L2 consists of 16 amino acid residues, 21 amino acid residues, 26 amino acid residues, 31 amino acid residues, or 36 amino acid residues.
  • L2 comprises or consists of the amino acid sequence shown in residues 268-293 of SEQ ID NO:2, residues 268-288 of SEQ ID NO:26, residues 268-283 of SEQ ID NO:22, SEQ ID NO:54, or residues 268-303 of SEQ ID NO:24.
  • Exemplary L3 linkers comprise at least three amino acid residues and are typically up to 60 amino acid residues.
  • L3 linkers have a range of sequence lengths as described above for L2.
  • L3 comprises or consists of the amino acid sequence shown in residues 781 -798 of SEQ ID NO: 75 (also shown, e.g., as residues 781 -798 of SEQ ID NO:77).
  • L3 has the amino acid sequence shown in residues 781-798 of SEQ ID NO: 75.
  • polypeptide linkers comprise a plurality of glycine residues.
  • a polypeptide linker e.g., L I or L2 comprises a plurality of glycine residues and optionally at least one serine residue.
  • a polypeptide linker [e.g., L2) comprises the sequence Giy-Giy-Giy-Gly-Ser (SEQ ID M O: 15), such as, e.g. , two or more tandem repeats of the amino acid sequence of SEQ ID NO: 15.
  • a linker comprises the sequence [Gly-Gly-Gly-Gly-Ser] n ([SEQ ID NO: 15] n ), where n is a positive integer such as, for example, an integer from 1 to 5, from 2 to 5, from 3 to 5, from 1 to 6, from 2 to 6, from 3 to 6, from 4 to 6, from 1 to 7, from 2 to 7, from 3 to 7, or from 4 to 7.
  • n is 4.
  • n is 3.
  • n is 5.
  • n is 6.
  • a polypeptide linker comprises a series of glycine and serine residues (e.g. , [Gly-Gly-Gly-Gly-Ser] n , where n is defined as above) inserted between two other sequences of the polypeptide linker (e.g., any of the polypeptide linker sequences described herein).
  • a polypeptide linker includes glycine and serine residues (e.g. , [Giy- Gly-Gly-Gly-Ser], where n is defined as above) attached at one or both ends of anotlier sequence of the polypeptide linker (e.g. , any of the polypeptide linker sequences described herein).
  • a polypeptide linker e.g. , L2 comprises at least a portion of an upper hinge region (e.g. , derived from an IgGl, IgG2, IgG3, or IgG4 molecule), at least a portion of a middle hinge region (e.g.
  • a polypeptide linker comprises a non-naturally occurring immunoglobulin hinge region, e.g. , a hinge region that is not naturally found in an immunoglobulin and/or a hinge region that has been altered so that it differs in amino acid sequence from a naturally occurring immunoglobulin hinge region.
  • mutations can be made to a hinge region to make a polypeptide linker.
  • a polypeptide linker comprises a hinge domain that does not comprise a naturally occurring number of cysteines, i.e. , the polypeptide linker comprises either fewer cysteines or a greater number of cysteines than a naturally occurring hinge molecule.
  • dimerization domains are suitable for use in accordance with certain fusion polypeptide embodiments and dimeric fusion proteins as described herein.
  • the dimerizing domain is an immunoglobulin heavy chain constant region, such as an Fc region.
  • the Fc region may be a native sequence Fc region or a variant Fc region.
  • the Fc region lacks one or more effector functions (e.g. , one or both of ADCC and CDC effector functions).
  • the dimerizing domain is an Fc region of a human antibody with a mutation in the CH2 region so that the molecule is not glycosylated, including but not limited to N297 (EU numbering for human IgG heavy chain constant region) (corresponding to amino acid position 375 of SEQ ID M O:2).
  • the Fc region is human IgGl ( ⁇ ) with the three cysteines of the hinge region (C220, C226, C229) each changed to serine, and the proline at Eu position 238 of the CH2 domain changed to serine.
  • the Fc region is human ⁇ ⁇ with 297 changed to any other amino acid.
  • the Fc region is human l with one or more amino acid substitutions between Eu positions 292 and 300. In another embodiment, the Fc region is human ⁇ with one or more amino acid additions or deletions at any position between Eu residues 292 and 300. In another embodiment, the Fc region is human v l with an SCC hinge (i. e. , with cysteine C220 changed to serine and with a cysteine at each of Eu positions 226 and 229) or an SSS hinge (i. e. , each of the three cysteines at Eu positions 220, 226, and 229 changed to serine). In further embodiments, the Fc region is human ⁇ with an SCC hinge and a P238 mutation.
  • the Fc domain is human l with mutations that alter binding by Fc gamma receptors (I, II, III) without affecting FcRn binding important for half-life.
  • an Fc region is as disclosed in Ehrhardt and Cooper, Curr. Top. Microbiol. Immunol. 2010 Aug. 3 (Immunoregulatory Roles for Fc Receptor-Like Molecules); Davis et al, Ann. Rev. Immunol. 25:525-60, 2007 (Fc receptor-like molecules); or Swainson et al, J. Immunol. 184:3639-47, 2010.
  • the Fc region comprises an amino acid substitution that alters the antigen-independent effector functions of the fusion protein.
  • the Fc region includes an amino acid substitution that alters the circulating half-life of the resulting molecule.
  • Such antibody derivatives exhibit either increased or decreased binding to FcRn when compared to antibodies lacking these substitutions and, therefore, have an increased or decreased half-life in serum, respectively.
  • Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such antibodies have useful applications in methods of treating mammals where long half-life of the administered antibody is desired.
  • Fc variants with decreased FcRn binding affinity are expected to have shorter half-lives, and such antibodies are also useful, for example, for administration to a mammal where a shortened circulation time may be advantageous, e.g., where the starting antibody has toxic side effects when present in the circulation for prolonged periods.
  • Fc variants with decreased FcRn binding affinity are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in pregnant women.
  • other applications in which reduced FcRn binding affinity may be desired include those applications in which localization the brain, kidney, and/or liver is desired.
  • the antibodies of the invention exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature.
  • fusion proteins of the invention exhibit reduced transport across the blood brain barrier (BBB) from the brain, into the vascular space.
  • BBB blood brain barrier
  • a fusion protein with altered FcRn binding comprises an Fc region having one or more amino acid substitutions within the "FcRn binding loop" of the Fc domain. Exemplary amino acid substitutions which altered FcRn binding activity are disclosed in International PCT Publication No. WO 05/047327, which is incorporated by reference herein.
  • a fusion polypeptide of the present invention comprises an Fc variant comprising an amino acid substitution which alters the antigen-dependent effector functions of the polypeptide, in particular ADCC or complement activation, e.g. , as compared to a wild-type Fc region.
  • such fusion polypeptides exhibit altered binding to an Fc gamma receptor (FcyR, e.g. , CD 16).
  • FcyR Fc gamma receptor
  • Such fusion polypeptides exhibit either increased or decreased binding to FcvR when compared to wild-type polypeptides and, therefore, mediate enhanced or reduced effector function, respectively.
  • Fc variants with improved affinity for FcvRs are anticipated to enhance effector function, and such fusion proteins have useful applications in methods of treating mammals where target molecule destruction is desired.
  • Fc variants with decreased FcvR binding affinity are expected to reduce effector function, and such fusion proteins are also useful, for example, for treatment of conditions in which target cell destruction is undesirable, e.g., where normal cells may express target molecules, or where chronic administration of the antibody might result in unwanted immune system activation.
  • the fusion polypeptide comprising an Fc region exhibits at least one altered antigen-dependent effector function selected from the group consisting of opsonization, phagocytosis, complement dependent cytotoxicity, antigen-dependent cellular cytotoxicity (ADCC), or effector cell modulation as compared to a polypeptide comprising a wild-type Fc region.
  • antigen-dependent effector function selected from the group consisting of opsonization, phagocytosis, complement dependent cytotoxicity, antigen-dependent cellular cytotoxicity (ADCC), or effector cell modulation as compared to a polypeptide comprising a wild-type Fc region.
  • a fusion polypeptide comprising an Fc region exhibits altered binding to an activating FcyR (e.g. , Fcyl, FevIIa, or FcvRIIIa).
  • the fusion protein exhibits altered binding affinity to an inhibitor ⁇ ' FcyR (e.g. , FcyRIIb).
  • an inhibitor ⁇ ' FcyR e.g. , FcyRIIb.
  • a fusion polypeptide comprising an Fc region may also comprise an amino acid substitution that alters the glycosylation of the Fc region.
  • the Fc domain of the fusion protein may have a mutation leading to reduced glycosylation (e.g., N- or O-iinked glycosylation) or may comprise an altered glycoform of the wild-type Fc domain (e.g., a low fucose or fucose-free glycan).
  • the molecule has an amino acid substitution near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS. Exemplary' amino acid substitutions which reduce or alter glycosylation are disclosed in International PCT Publication No. WO 05/018572 and US Patent Application Publication No. 2007/01 1 1281, which are incorporated by reference herein.
  • a dimerizing domain is an Fc region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity with an amino acid sequence selected from sequence shown in (i) residues 294-525 or 294-524 of SEQ ID NO:2, or (ii) residues 294-525 or 294-524 of SEQ ID NO: 13.
  • the Fc region comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in (i) residues 294-525 or 294-524 of SEQ ID NO:2, or (ii) residues 294-525 or 294-524 of SEQ ID N O: 13.
  • a tandem ApoA-1 fusion polypeptide of the present disclosure includes an FcRn-binding domain.
  • FcRn-binding domains include Fc regions that retain FcRn-binding activity; in some such variations, the Fc region is an Fc region as described herein (e.g., as described above in the context of a dimerizing domain).
  • an FcRn-binding domain is an albumin (e.g., human albumin).
  • the albumin comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acid residues 550-1134 of SEQ ID NO:62; in other embodiments, the albumin comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with residues 550-1134 of SEQ ID NO:62.
  • suitable FcRn-binding domains include single-chain antibodies (e.g.
  • scFvs peptide aptamers
  • certain non-antibody protein structures having binding affinity for FcRn are readily created using, for example, display technologies that allow for selection of binding agents through screening of large expression libraries (e.g. , libraries of immunoglobulin domains or randomized peptides).
  • display technologies are generally well- known in the art and include, for example, phage display. See, e.g. , Antibody Engineering: A Practical Approach, McCafferty, Hoogenboom, and Chiswell Eds., IRL Press 1996.
  • non- antibody protein scaffold structures for generating FcRn-binding domains include, e.g., avimers, ankyrin repeats, and adnectins, as well as other proteins with domains that can be evolved to generate specific affinity for a desired molecular target. See, e.g. , Silverman et al, Nature Biotechnology 23: 1556- 1561, 2005; Zahnd et al , J. Mol. Biol. 369: 1015-1028, 2007; US Patent No, 7,115,396 to Lipovsek et al.
  • the fusion polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity with an amino acid sequence selected from sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (li) residues 19-780, 19- 779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv ) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19- 1134 or 25-1134 of SEQ ID NO:62.
  • the fusion polypeptide comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the ammo acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO 56. (li) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19- 789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19-1134 or 25-1134 of SEQ ID NO:62.
  • the portion of the fusion polypeptide corresponding to ApoAl a -Ll-ApoAl b -L2-D comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19-780, 19-779, 25- 780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v)
  • the portion of the fusion polypeptide corresponding to ApoAl 3 -Ll-ApoAl b -L2-D comprises or consists of an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25- 780, or 25-779 of SEQ ID NO:56, (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58,
  • the portion of the polypeptide corresponding to P comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the ammo acid sequence shown in residues 544-675 or 548-675 of SEQ ID NO:4.
  • the fusion polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in (i) residues 19-930 or 25-930 of SEQ ID NO:75, (ii) residues 19- 926 or 25-926 of SEQ ID NO:94, or (iii) residues 19-1280 or 25- 1280 of SEQ ID NO:87.
  • the portion of the fusion polypeptide corresponding to ApoAl a -Ll-ApoAl b -L2-D comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19-780, 19-779, 25- 780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96,
  • the portion of the polypeptide corresponding to P comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in (a) residues 16-355 of SEQ ID NO: 12, (b) residues 16-355 of SEQ ID NO:42, or (c) residues 16-355 of SEQ ID NO:44.
  • the portion of the fusion polypeptide corresponding to ApoAl a -Ll-ApoAl b -L2-D comprises or consists of an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID O:56, (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID O:58, (iii) residues 19- 790, 19-789, 25-790, or 25-789 of SEQ ID NG:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19- 1134 or 25-1134 of SEQ ID NO:62; in some such embodiments, the portion of the polypeptide corresponding to P (the paraoxonase) comprises or consists of an amino acid
  • the fusion polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in (i) residues 19-1138 or 25- 1138 of SEQ ID O:77, (ii) residues 19-1138 or 25- 1138 of SEQ ID NO: 79. (iii) residues 19- 1 138 or 25-1 138 of SEQ ID NO:81, or (iv) residues 19-1492 or 25- 1492 of SEQ ID NO:89.
  • the portion of the fusion polypeptide corresponding to ApoAl a -Ll-ApoAl b -L2-D comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity with the ammo acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID O:56, (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-
  • the portion of the polypeptide corresponding to P comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in residues 22-441 of SEQ ID NO:32.
  • the portion of the fusion polypeptide corresponding to ApoAl a - L l -Apo A l b -L2-D comprises or consists of an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in (i) residues 19- 780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID O:96, (iv) residues 19-
  • the portion of the polypeptide corresponding to P comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in residues 22-441 of SEQ ID NO:32.
  • the fusion polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in residues 19- 1218 or 25- 1218 of SEQ ID NO 83.
  • the portion of the fusion polypeptide corresponding to ApoA l a -Ll -Apo.Al -L2-D comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO: 6.
  • the portion of the polypeptide corresponding to P comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in residues 18-493 of SEQ ID NO:30.
  • the portion of the fusion polypeptide corresponding to ApoAl a -Ll-ApoAl b - L2-D comprises or consists of an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID O:56, (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID O:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO: 6.
  • the portion of the polypeptide corresponding to P comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in residues 18-493 of SEQ ID NO:30.
  • the fusion polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in residues 19-1274 or 25-1274 of SEQ ID NO:85.
  • the present invention also provides dimeric proteins comprising first and second polypeptide fusions, each of the polypeptide fusions comprising a dimerizing domain, as described above. Accordingly, in another aspect, the present invention provides a dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, where each of the first and second polypeptide fusions comprises, from an amino-terminal position to a carboxyi-terminai position, ApoAl a -Ll-ApoAl b -L2-D, where ApoAl a and ApoAl b are, respectively, first and second polypeptide segments, each of the first and second polypeptide segments having cholesterol efflux activity and which is independently selected from (i) a naturally occurring ApoA-1 polypeptide or a functional variant or fragment thereof and (ii) an ApoA-1 mimetic; LI is a first polypeptide linker: L2 is a second polypeptide linker; and D is a dimerizing domain.
  • each of the first and second fusion polypeptides further includes an additional polypeptide segment located carboxyi-terminai to the dimerizing domain.
  • the additional (“third") polypeptide segment is (a) a naturally occurring RNase, paraoxonase, platelet-activating factor acetylhydrolase (PAF-AH), cholesterol ester transfer protein (CETP), or lecithin-cholesterol acyltransferase (LCAT); (b) a functional variant or fragment of any of the naturally occurring proteins specified in (a); or (c) a polypeptide that specifically binds to amyloid beta ( ⁇ ) such as, e.g. , an ⁇ - specific scFv.
  • PAF-AH platelet-activating factor acetylhydrolase
  • CETP cholesterol ester transfer protein
  • LCAT lecithin-cholesterol acyltransferase
  • Such a fusion polypeptide comprising a third polypeptide segment may be represented by the formula ApoAl a -Ll-ApoA i b -L2-D-L3-P (from an amino-terminal position to a carboxyi- terminai position), where ApoAl a , LI, ApoAl b , L2, and D are each as previously defined, where L3 is a third polypeptide linker and is optionally present, and where P is the third polypeptide segment.
  • the present invention provides a dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, where each of the first and second fusion polypeptides comprises
  • A a tandem ApoA-1 polypeptide segment comprising, from an amino-terminal position to a carboxyi-terminai position, ApoAl a -Ll-ApoAl b , where ApoAl a and ApoA l b are, respectively, first and second polypeptide segments, each of the first and second polypeptide segments having cholesterol efflux activity and which is independently selected from (i) a naturally occurring ApoA-i polypeptide or a functional variant or fragment thereof and (ii) an ApoA-1 mimetic, and where LI is a first polypeptide linker;
  • PAF-AH platelet- activating factor acetylhydrolase
  • CETP cholesterol ester transfer protein
  • LCAT lecithin-cholesterol acyltransferase
  • ApoA l a and ApoAl b comprise or consist of the same amino acid sequence; in other embodiments, ApoAl a and ApoAl b comprise or consist of different amino acid sequences.
  • the dimerizing domain is located carboxyl-terminal to the second polypeptide segment and amino-terminal to the third polypeptide segment.
  • the fusion polypeptides of the present invention can further be conjugated to an effector moiety.
  • the effector moiety can be any number of molecules, including, e.g. , a labeling moiety such as a radioactive label or fluorescent label, a TLR ligand or binding domain, an enzyme, or a therapeutic moiety .
  • the effector moiety is a myeloperoxidase (MPO) inhibitor.
  • MPO inhibitors are generally known (see, e.g., Malle et a!., Br J Pharmacol. 152: 838-854, 2007) and may be readily conjugated to fusion polypeptides as described herein.
  • fusion polypeptides of the present invention are modified to extend half-life, such as, for example, by attaching at least one molecule to the fusion protein for extending serum half-life.
  • molecules for attachment may include, e.g., a polyethlyene glycol (PEG) group, serum albumin, transferrin, transferrin receptor or the transferrin-binding portion thereof, or a combination thereof.
  • PEG polyethlyene glycol
  • Methods for such modification are generally well-known in the art.
  • the word "attached” refers to a covalently or noncovalently conjugated substance. The conjugation may be by genetic engineering or by chemical means.
  • the present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the fusion polypeptides disclosed above.
  • the polynucleotides of the present invention include both single-stranded and double-stranded molecules.
  • Polynucleotides encoding various segments of a fusion polypeptide e.g., a dimerizing domain such as an Fc fragment; ApoAl and P polypeptide segments
  • DNA sequences encoding ApoA-1, RNases (e.g., RNase 1), paraoxonases (e.g., PON 1), platelet-activating factor acetylhydrolase (PAF-AH), cholesterol ester transfer protein (CETP), and lecithin-cholesterol acyl transferase (LCAT) are known in the art.
  • DNA sequences encoding various dimerizing or FcRn-binding domains e.g. , immunoglobulin heavy chain constant regions such as Fc fragments; albumin
  • variable regions of ⁇ -binding antibodies are also readily identifiable using techniques well- known in the art such as screening of recombinant antibody expression libraries (e.g. , phage display expression libraries). Additional DNA sequences encoding any of these polypeptides can be readily generated by those of ordinary skill in the art based on the genetic code. Counterpart RNA sequences can be generated by substitution of U for T. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among polynucleotide molecules encoding a given polypeptide.
  • DNA and RNA encoding functional variants and fragments of such polypeptides can also be obtained using known recombinant methods to introduce variation into a polynucleotide sequence, followed by expression of the encoded polypeptide and determination of functional activity (e.g. , cholesterol efflux) using an appropriate screening assay.
  • functional activity e.g. , cholesterol efflux
  • RNA and RNA are well-known in the art.
  • complementary DNA (cDNA) clones can be prepared from RNA that is isolated from a tissue or cell that produces large amounts of RNA encoding a polypeptide of interest.
  • Total RNA can be prepared using guanidine HC1 extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al. , Biochemistry 18:52-94, 1979).
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69: 1408- 1412, 1972).
  • Complementary DNA is prepared from poiy(A) + RNA using known methods.
  • genomic DNA can be isolated.
  • Methods for identifying and isolating cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequences disclosed herein, or parts thereof, for probing or priming a library .
  • Polynucleotides encoding polypeptides of interest are identified and isolated by, for example, hybridization or polymerase chain reaction ("PCR," Muliis, U.S. Patent 4,683,202).
  • PCR polymerase chain reaction
  • Expression libraries can be probed with antibodies to the polypeptide of interest, receptor fragments, or other specific binding partners.
  • the polynucleotides of the present invention can also be prepared by automated synthesis.
  • the production of short, double-stranded segments (60 to 80 bp) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. Longer segments (typically >300 bp) are assembled in modular form from single- stranded fragments that are from 20 to 100 nucleotides in length. Automated synthesis of polynucleotides is within the level of ordinary skill in the art, and suitable equipment and reagents are available from commercial suppliers.
  • materials and methods are provided for producing the polypeptide fusions of the present invention, including dimeric proteins comprising the fusion polypeptides.
  • the fusion polypeptides can be produced in genetically engineered host ceils according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic ceils (including cultured cells of multicellular organisms), particularly cultured mammalian cells.
  • a DNA sequence encoding a fusion polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • a secretory signal sequence is provided in the expression vector.
  • the secretory signal sequence may be that of the native ApoA- 1 polypeptide, or may be derived from another secreted protein (e.g., t-PA; see U.S. Patent No. 5,641,655) or synthesized de novo.
  • An engineered cleavage site may be included at the junction between the secretory peptide and the remainder of the polypeptide fusion to optimize proteolytic processing in the host cell.
  • the secretory signal sequence is operably linked to the DNA sequence encoding the polypeptide fusion, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide fusion into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g. , Welch et al , U.S. Patent No. 5,037,743; Holland et al, U.S. Patent No. 5, 143,830).
  • Secretory signal sequences suitable for use in accordance with the present invention include, for example, polynucleotides encoding amino acid residues 1- 18 of SEQ ID NO:36,
  • the present invention provides dimeric proteins comprising first and second fusion polypeptides as described above ⁇ e.g.
  • a dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, where each of the first and second fusion poly peptides comprises, from an amino-terminai position to a carboxyl-terminal position, ApoAl a -Ll-ApoA l b -L2-D or ApoAl a -Ll- ApoA l b -L2-D-L3-P as described herein).
  • Dimers may also be assembled in vitro upon incubation of component polypeptides under suitable conditions, in general, in vitro assembly will include incubating the protein mixture under denaturing and reducing conditions followed by refolding and reoxidation of the polypeptides to form dimers. Recovery and assembly of proteins expressed in bacterial cells is disclosed below.
  • Cultured mammalian cells are suitable hosts for use within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate- mediated transfection (Wigler et al, Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981 : Graham, and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al, EMBO J.
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al,, J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. , CHO-K1 , ATCC No. CCL 61; CHO-DG44, Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980) cell lines.
  • COS-1 ATCC No. CRL 1650
  • COS-7 ATCC No. CRL 1651
  • BHK ATCC No. CRL 1632
  • BHK 570 ATCC No. CRL 10314
  • 293 ATCC No. CRL 1573
  • Graham et al ATCC No. CCL 61
  • Suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, Virginia. Strong transcription promoters can be used, such as promoters from SV-40, cytomegalovirus, or myeloproliferative sarcoma virus. See, e.g., U.S. Patent No. 4,956,288 and U.S. Patent Application Publication No. 20030103986.
  • Other suitable promoters include those from metallothionein genes (U.S. Patents Nos. 4,579,821 and 4,601 ,978) and the adenovirus major late promoter.
  • Expression vectors for use in mammalian cells include pZP- 1, pZP- 9, and pZMP21, which have been deposited with the American Type Culture Collection, 10801 University Boulevard., Manassas, VA USA under accession numbers 98669, 98668, and PTA-5266, respectively, and derivatives of these vectors.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants.” Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
  • An exemplary selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
  • Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by cuSturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • An exemplary amplifiabie selectable marker is dihydrofoiate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g., hygromycin resistance, multidrug resistance, puromycin acetyl transferase
  • drug resistance genes e.g., hygromycin resistance, multidrug resistance, puromycin acetyl transferase
  • Cell-surface markers and other phenotypic selection markers can be used to facilitate identification of transfected cells (e.g., by fluorescence-activated cell sorting), and include, for example, CDS, CD4, nerve growth factor receptor, green fluorescent protein, and the like.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis vims (AcNPV). See King and Possee, The Baculovirus Expression System: A. Laboratory Guide, Chapman & Hall, London; O'Reilly et al , Baculovirus Expression Vectors: A Laboratory Manual, Oxford University Press., New York, 1994; and Richardson, Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Humana Press, Totowa, NJ, 1995. Recombinant baculovirus can also be produced through the use of a transposon- based system described by Luckow et al. (J. Virol.
  • This system which utilizes transfer vectors, is commercially available in kit form (BAC-TO-BAC kit; Life Technologies, Gaithersburg, MD).
  • the transfer vector e.g. , PFASTBAC i; Life Technologies
  • the transfer vector contains a Tn7 transposon to move the DNA encoding the protein of interest into a baculovirus genome maintained in E. coli as a large plasmid called a "hacmid.” See Hill-Perkins and Possee, J. Gen. Virol. 71 :971-976, 1990; Bonning et al, J. Gen. Virol. 75: 1551-1556, 1994; and Chazenbalk and Rapoport, J. Biol. Chew,.
  • a transfer vector encoding a polypeptide fusion is transformed into E. coli host cells, and the cells are screened for baemids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculoviais genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda ceils, such as Sf9 ceils.
  • Recombinant virus that expresses the polypeptide fusion is subsequently produced.
  • Recombinant viral stocks are made by methods commonly used the art.
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall army worm, Spodoptera frugiperda (e.g. , Sf or Sf21 ceils) or Trichoplusia ni (e.g. , HIGH FIVE cells; Invitrogen, Carlsbad, CA). See generally Glick and Pastemak, supra. See also U.S. Patent No. 5,300,435. Serum-free media are used to grow and maintain the cells. Suitable media formulations are known in the art and can be obtained from commercial suppliers.
  • the ceils are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 b ceils, at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
  • Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al. , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et ai, U.S. Patent No. 5,037,743; and Murray et ai, U.S. Patent No.
  • Transformed ceils are selected by phenotype determined by the selectable marker, commonly drag resistance or the ability to grow in the absence of a particular nutrient (e.g. , leucine).
  • An exemplary vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g. , Kawasaki, U.S. Patent No. 4,599,311; Kmgsman et al, U. S. Patent No.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well-known in the art (see, e.g., Sambrook et al, supra).
  • the polypeptide When expressing a fusion polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine HQ or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by diaiysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the protein may be recovered from the cytoplasm in soluble form and isolated without the use of denaturants.
  • the protein is recovered from the cell as an aqueous extract in, for example, phosphate buffered saline.
  • the extract is applied directly to a chromatographic medium, such as an immobilized antibody or heparin- Sepharose column.
  • Secreted polypeptides can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) and recovering the protein, thereby obviating the need for denaturation and refolding. See, e.g., Lu et al, J. Immunol Meth. 267:213-226, 2002.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
  • Proteins of the present invention are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See generally Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York, 1994. Proteins comprising an immunoglobulin heavy chain polypeptide can be purified by affinity chromatography on immobilized protein A. Additional purification steps, such as gel filtration, can be used to obtain the desired level of purit or to provide for desalting, buffer exchange, and the like.
  • fractionation and/or conventional purification methods can be used to obtain fusion polypeptides and dimeric proteins of the present invention purified from recombinant host cells, in general, ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
  • Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PE1, DEAE, QAE and Q derivatives are suitable.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl- Sepharose FF (Pharmacia), Toyopearl but ⁇ '! 650 (Toso Haas, Montgomery ville, PA), Octyl- Sepharose (Pharmacia) and the like; or poiyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Phenyl- Sepharose FF Pharmacia
  • Toyopearl but ⁇ '! 650 Toso Haas, Montgomery ville, PA
  • Octyl- Sepharose Pharmacia
  • poiyacrylic resins such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross- linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxy! groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well-known and widely used in the art, and are available from commercial suppliers. Selection of a particular method for polypeptide isolation and purification is a matter of routine design and is determined in part by the properties of the chosen support. See, e.g. , Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988); and Doonan, Protein Purification Protocols (The Humana Press 1996).
  • antibodies that specifically bind a fusion polypeptide or dimeric protein as described herein ⁇ e.g. , an antibody that specifically binds a polypeptide segment corresponding to ApoA-1) can be used to isolate large quantities of protein by immunoaffinity purification.
  • the proteins of the present invention can also be isolated by exploitation of particular properties.
  • immobilized metal ion adsorption (!MAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3: 1, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the rnetal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • !MAC immobilized metal ion adsorption
  • fusion of the polypeptide of interest and an affinity tag may be constructed to facilitate purification.
  • an affinity tag e.g., maltose -binding protein, an immunoglobulin domain
  • receptor- or ligand-binding properties of a fusion polypeptide or dimer thereof can be exploited for purification.
  • a fusion polypeptide comprising an ⁇ -binding polypeptide segment may be isolated by using affinity chromatography wherein amyloid beta ( ⁇ ) peptide is bound to a column and the fusion polypeptide is bound and subsequently eluted using standard chromatography methods.
  • the polypeptides of the present invention are typically purified to at least about 80% purity, more typically to at least about 90% purity and preferably to at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% purity with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • the polypeptides of the present invention may also be purified to a pharmaceutically pure state, which is greater than 99.9% pure. In certain preparations, purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • the tandem ApoA-1 fusion polypeptides and dimeric proteins of the present invention can be used to provide ApoA-1 -mediated therapy for the treatment of various diseases or disorders.
  • an additional polypeptide segment as described herein e.g., an RNase, paraoxonase, platelet-activating factor acetylhydrolase (PAF-AH), cholesterol ester transfer protein (CETP), or lecithin-cholesterol acyltransferase (LCAT)
  • the fusion polypeptides and dimeric proteins may further provide one or more additional biological activities for such treatment.
  • the present invention provides methods for treating a disease or disorder selected from a cardiovascular disease characterized by atherosclerosis, a neurodegenerative disease, a disease characterized by amyloid deposit, an autoimmune disease, an inflammatory disease, an infectious disease, obesity, metabolic syndrome, nephrotic syndrome, burns, exposure to sulfur mustard gas, exposure to an organophosphate, and cancer.
  • the methods generally include administering to a subject having the disease or disorder an effective amount of a fusion polypeptide or dimeric protein as described herein.
  • Atherosclerotic cardiovascular diseases amenable to treatment in accordance with the present invention include, for example, coronary heart disease and stroke.
  • the coronary heart disease is characterized by acute coronary syndrome.
  • the atherosclerotic cardiovascular disease is selected from cerebral artery disease (e.g. , extracranial cerebral artery disease, intracranial cerebral artery disease), arteriosclerotic aortic disease, renal artery disease, mesenteric artery disease, and peripheral artery disease (e.g., aortoiliac occlusive disease).
  • Neurodegenerative diseases amenable to treatment in accordance with the present invention include, for example, neurodegenerative diseases characterized by amyloid deposit and/or dementia.
  • An exemplary neurodegenerative disease characterized by amyloid deposit is Alzheimer's disease.
  • Exemplary neurodegenerative diseases characterized by dementia include Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS).
  • the neurodegenerative disease is an inflammatory disease such as, for example, a demyeiinating inflammatory disease of the CNS (e.g. , multiple sclerosis (MS), including, for example, spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS)).
  • MS multiple sclerosis
  • PPMS primary progressive MS
  • RRMS relapsing remitting MS
  • a fusion molecule for the neurodegenerative disease treatment is a polypeptide having the structure ApoA l a -L l -ApoA l b -L2-D-L3-RNase (e.g., ApoA l a - L l-ApoA l b -L2-[Fc region] -L3 -R ase 1 , ApoA l a -L l-ApoA l b -L2-albumin-L3-R ase l) or ApoA l a - L l-ApoA l b -L2-D-L3-paraoxonase (e.g., ApoA l a -L l-ApoA l -L2-[Fc region]-L3-PON
  • (A) the portion of the fusion polypeptide corresponding to ApoA l a -L l-ApoA l b -L2-D comprises or consists of an amino acid sequence having at least at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19- 780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19- 1 134 or 25- 1 1434 of SEQ ID NO:62: and (B) the portion of the polypeptide corresponding to P (the RNase or paraoxonase) comprises or consists of an amino acid
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-930 or 25-930 of SEQ ID NO:75, (ii) residues 19-926 or 25-926 of SEQ ID O:94, (in) residues 19-1280 or 25-1280 of SEQ ID NO:87, (iv) residues 19-1138 or 25-1138 of SEQ ID NO:77, (v) residues 19-1138 or 25- 1138 of SEQ ID NO:79, (vi) residues 19-1138 or 25-1138 of SEQ ID NO:81, or (vii) residues 19
  • Autoimmune diseases amenable to treatment in accordance with the present invention include, for example, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type 1 diabetes.
  • the autoimmune disease is selected from coeliac disease, neuritis, polymyositis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, vitiligo, Sjogren's syndrome, autoimmune pancreatitis, an inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), active chronic hepatitis, glomerulonephritis, lupus nephritis, scleroderma, antiphospholipid syndrome, autoimmune vasculitis, sarcoidosis, autoimmune thyroid diseases, Hashimoto's thyroiditis, Graves disease, Wegener's granulomatosis, myasthenia gravis, Addison'
  • the autoimmime disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, lupus nephritis, scleroderma, psoriasis, Sjogren's syndrome, type 1 diabetes, antiphospholipid syndrome, and autoimmune vasculitis.
  • a fusion molecule for treatment of an autoimmune disease is a polypeptide having the structure ApoAl a -Ll-ApoAl b -L2-D (e.g., ApoAl a -Ll-ApoAl b -L2-[Fc region], ApoAl a -Ll-ApoAl b -L2-albumin), ApoAl a -Ll-ApoAl b -L2-D-L3- Nase (e.g., ApoAl a -Ll- ApoAl b -L2-[Fc region]-L3-RNasel, ApoAl a -Ll-ApoAl b -L2-albumin-L3-RNasel), or ApoAl a -Ll- ApoAl b -L2-D-L3-paraoxonase (e.g., ApoAl a -Ll
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25- 790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19-1134 or 25-1134 of SEQ ID NO:62.
  • a fusion polypeptide having the structure ApoAl a -Ll-ApoAl -L2-D-L3-RNase or ApoAl a -Ll-ApoAl -L2-D- L3-paraoxonase for the treatment of an autoimmune disease (A) the portion of the fusion polypeptide corresponding to ApoAl a -Ll-ApoAl b -L2-D comprises or consists of an amino acid sequence having at least at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO: 6.
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-930 or 25-930 of SEQ ID NO:75, (ii) residues 19-926 or 25-926 of SEQ ID NO:94, (iii) residues 19-1280 or 25-1280 of SEQ ID NO:87, (iv) residues 19-1138 or 25-1138 of SEQ ID NO:77, (v) residues 19-1138 or 25-1138 of SEQ ID NO: 79. (vi) residues 19-1138 or 25-1138 of SEQ ID NO:81, or (vii) residues 19-1492 or 25-1492
  • a fusion molecule for the RA treatment is a polypeptide having the structure ApoAl a -Ll-ApoAl b -L2-D (e.g., ApoAl a -Ll-ApoAl b -L2-[Fc region], ApoAl a -Ll-ApoAl b -L2-albumin), or a dimeric protein formed by dimerization of the foregoing fusion polypeptide where D is a dimerizing domain.
  • ApoAl a -Ll-ApoAl b -L2-D e.g., ApoAl a -Ll-ApoAl b -L2-[Fc region], ApoAl a -Ll-ApoAl b -L2-albumin
  • D is a dimerizing domain.
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19-1134 or 25-1134 of SEQ ID NO:62.
  • a fusion molecule for the SLE treatment is a polypeptide having the structure ApoAl a -LI- ApoAl b -L2-D-L3-RNase (e.g., ApoAl a -Ll-ApoAl b -L2-[Fc region]-L3-RNasel, ApoAl a -Ll- ApoAl b -L2-albumin-L3-RNasel) or ApoAl a -Ll-ApoAl b -L2-D-L3-paraoxonase (e.g., ApoAl a -Ll- ApoAl b -L2-[Fc region]-L3-PON 1, ApoAl a -Ll-ApoAl b -L2-albumin-L3-PON 1)
  • ApoAl a -LI- ApoAl b -L2-D-L3-RNase
  • the portion of the fusion polypeptide corresponding to ApoAl a -Ll- ApoAl b -L2-D comprises or consists of an amino acid sequence having at least at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19-780, 19-779, 25-780, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or 25-779 of SEQ ID NO:58, or (v) residues 19- 1 134 or 25- 1 134 of SEQ ID NO:62; and (B) the portion of the polypeptide corresponding to P (the RNase or paraoxonase) comprises or consists of an amino acid
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-930 or 25-930 of SEQ ID NO:75, (ii) residues 19-926 or 25-926 of SEQ ID NO:94, (lii) residues 19- 1280 or 25- 1280 of SEQ ID NO: 87, (iv) residues 19- 1 138 or 25- 1 138 of SEQ ID NO:77, (v) residues 19- 1 138 or 25- 1 138 of SEQ ID NO:79, (vi) residues 19- 1 138 or 25- 1 138 of SEQ ID NO:79, (vi) residues 19- 1 138 or 25- 1 138 of SEQ ID NO:79, (vi) residues 19- 1 138 or 25- 1 138 of SEQ ID NO:79, (vi) residues 19- 1 138 or 25- 1 138 of SEQ ID NO:79, (vi) residues 19- 1 138 or 25- 1
  • a fusion molecule for the MS treatment is a polypeptide having the structure ApoA l a -L l-ApoA l b -L2- D-L3 -paraoxonase (e.g., ApoA l a -L l-ApoA lt,-L2-[Fc region] -L3 -PON 1, ApoA I a -L I-ApoA l ! ,-L2- albumin-L3-PON I), or a dimeric protein formed by dimerization of the foregoing fusion polypeptide where D is a dimerizing domain.
  • ApoA l a -L l-ApoA l b -L2- D-L3 -paraoxonase e.g., ApoA l a -L l-ApoA lt,-L2-[Fc region] -L3 -PON 1, ApoA I
  • the portion of the fusion polypeptide corresponding to ApoA l a -L l-ApoA l b -L2-D comprises or consists of an amino acid sequence having at least at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19-780, 19- 779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19- 1 134 or 25- 1 134 of SEQ ID N Q:62: and (B) the portion of the polypeptide corresponding to P (the paraoxonase) comprises or consists of
  • Inflammatory diseases amenable to treatment in accordance with the present invention include, for example, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes, type 2 diabetes, and obesity.
  • the inflammatory disease is an neurodegenerative inflammatory disease such as, for example, multiple sclerosis, Alzheimer's disease. or Parkinson's disease.
  • the inflammatory disease is an atherosclerotic disease (e.g. , coronary heart disease or stroke).
  • the inflammatory disease is selected from, hepatitis (e.g., non-alcoholic steatohepatitis), ankylosing spondylitis, arthritis (e.g., osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis), Crohn's disease, ulcerative colitis, dermatitis, diverticulitis, fibromyalgia, irritable bowel syndrome (IBS), and nephritis.
  • hepatitis e.g., non-alcoholic steatohepatitis
  • arthritis e.g., osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis
  • Crohn's disease e.g., ulcerative colitis, dermatitis, diverticulitis, fibromyalgia, irritable bowel syndrome (IBS), and nephritis.
  • the inflammatory disease is an inflammatory lung disease; in some such embodiments, the inflammatory lung disease is selected from asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, idiopathic pulmonary fibrosis, hyperoxia, hypoxia, and acute respiratory distress syndrome (ARDS).
  • COPD chronic obstructive pulmonary disease
  • ARDS acute respiratory distress syndrome
  • a patient having the inflammatory lung disease is a patient that has been exposed to sulfur mustard gas (SM).
  • SM sulfur mustard gas
  • a patient having the inflammatory lung disease is a patient that has been exposed to an organophosphate, such as an insecticide or other neurotoxin.
  • a fusion molecule for treatment of an inflammatory disease is a polypeptide having the structure ApoA l a -L l-ApoA l b -L2-D (e.g. , ApoA I a -L l-ApoA l b -L2-[Fe region], ApoA l a -L l-ApoA l -L2-albumin), ApoA l a -L l-ApoA I b - L2-D-L3-RNase (e.g.
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID N O:56,(ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID O:98, or (v) residues 19- 1 134 or 25- 1 134 of SEQ ID NO:62.
  • the portion of the fusion polypeptide corresponding to ApoA l a -L l-ApoA l b -L2-D comprises or consists of an amino acid sequence having at least at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID M O:56, (ii) residues 19-780, 19- 779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-7
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-930 or 25-930 of SEQ ID N O:75, (ii) residues 19-926 or 25-926 of SEQ ID NO:94, (in) residues 19- 1280 or 25- 1280 of SEQ ID NO: 87, (iv) residues 19- 1 138 or 25- 1 138 of SEQ ID NO:77, (v) residues 19- 1 138 or 25- 1 138 of SEQ ID NO:79, (vi) residues 19
  • a fusion molecule for the idiopathic pulmonary fibrosis treatment is a polypeptide having the structure ApoA l a -L I-ApoA l b -L2-D (e.g., ApoA I a -L l-ApoA l b -L2-[Fc region], ApoAl a -L l -ApoA l b -L2- albumin), or a dimeric protein formed by dimerization of the foregoing fusion polypeptide where D is a dimerizing domain.
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-780, 19-
  • a fusion molecule for the treatment is a polypeptide having the structure ApoA l a -L l -ApoA l b -L2-D-L3-paraoxonase (e.g.
  • (A) the portion of the fusion polypeptide corresponding to ApoA l a -L l -ApoA l b -L2-D comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-
  • the portion of the polypeptide corresponding to P comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in (i) residues 16-355 of SEQ ID NO: 12, (ii) residues 16-355 of SEQ ID NO:42, or (in) residues 16-355 of SEQ ID NO:44.
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19- 1 138 or 25- 1138 of SEQ ID NO:77, (ii) residues 19- 1 138 or 25- 1 138 of SEQ ID NO:79, (iii) residues 19- 1 138 or 25- 1 138 of SEQ ID NO: 81, or (iv) residues 19- 1492 or 25- 1492 of SEQ ID NO: 89.
  • a fusion molecule for the treatment is a polypeptide having the structure ApoA l a -L l -ApoA l b -L2-D-L3-RNase (e.g. , ApoA l a -L l-ApoA I b -L2-[Fc region]-L3- RNase l, ApoA l a -L I-ApoA l b -L2-albumin-L3-RNase l), or a dimeric protein formed by dimerization of the foregoing fusion polypeptide where D is a dimerizing domain.
  • ApoA l a -L l -ApoA l b -L2-D-L3-RNase e.g. , ApoA l a -L l-ApoA I b -L2-[Fc region]-L3- RNase l, ApoA l a -L I-
  • the portion of the fusion polypeptide corresponding to ApoA l a -L l-ApoA l b -L2-D comprises or consists of an amino acid sequence having at least at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25- 790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19- 1 134 or 25- 1 134 of SEQ ID N Q:62; and (B) the portion of the polypeptide corresponding to P (the RNase) comprises or consists of an amino acid sequence having
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-930 or 25-930 of SEQ ID NO: 75, (ii) residues 19-926 or 25-926 of SEQ ID NO:94, or (hi) residues 19- 1280 or 25- 1280 of SEQ ID NO: 87.
  • a fusion molecule for treatment of exposure to sulfur mustard gas (SM) or exposure to an organophosphate is a polypeptide having the structure ApoA l a -L l- ApoA l -L2-D-L3 -paraoxonase (e.g. , ApoA l a -L l -ApoA l b -L2-[Fc region] -L3 -PON 1, ⁇ ⁇ ⁇ .- ! . !
  • the portion of the fusion polypeptide corresponding to ApoA l a -Ll-ApoA l b -L2-D comprises or consists of an amino acid sequence having at least at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19- 780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (in) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID N O.
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-1138 or 25- 1138 of SEQ ID NG:77, (ii) residues 19-1138 or 25- 1 138 of SEQ ID NO 79. (in) residues 19- 1138 or 25-1138 of SEQ ID NO:81 , or (iv) residues 19-1492 or 25-1492 of SEQ ID NO: 89.
  • Infectious diseases amenable to treatment in accordance with the present invention include, for example, bacterial infections and parasitic infections.
  • the parasitic infection is a Trypanosoma briicei or Leishmania infection.
  • the bacterial infection is a Pseudomonas aeruginosa infection.
  • a fusion molecule for the Pseudomonas aeruginosa infection treatment is a polypeptide having the structure ApoA l a -Ll -ApoAl -L2-D-L3-paraoxonase (e.g., ApoAl a -Ll -ApoA l b -L2-[Fc region]-L3- PON 1, ApoAl a -Ll-ApoAl b -L2-aibumin-L3-PON l), or a dimeric protein formed by dimerization of the foregoing fusion polypeptide where D is a dimerizing domain.
  • ApoA l a -Ll -ApoAl -L2-D-L3-paraoxonase e.g., ApoAl a -Ll -ApoA l b -L2-[Fc region]-L3- PON 1, Ap
  • the portion of the fusion polypeptide corresponding to ApoAl a -L l -ApoAl -L2-D comprises or consists of an amino acid sequence having at least at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25- 790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19-1 134 or 25-1134 of SEQ ID NO:62; and (B) the portion of the polypeptide corresponding to P (the paraoxonase ) comprises or consists of an amino acid sequence having at
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-1138 or 25-1 138 of SEQ ID NO:77, (ii) residues 19-1138 or 25- 1138 of SEQ ID ; 79. (iii) residues 19-1 138 or 25-1138 of SEQ ID NO:81, or (iv) residues 19-1492 or 25-1492 of SEQ ID NO:89.
  • a fusion molecule for treatment of an infectious disease is a polypeptide having the structure ApoAl a -Ll-ApoAl b -L2-D (e.g., ApoAl a -L l-ApoAl -L2-[Fc region], ApoA l a -Ll-ApoAl b -L2-albumin), or a dimeric protein formed by dimerization of the foregoing fusion polypeptide where D is a dimerizing domain.
  • ApoAl a -Ll-ApoAl b -L2-D e.g., ApoAl a -L l-ApoAl -L2-[Fc region], ApoA l a -Ll-ApoAl b -L2-albumin
  • D is a dimerizing domain.
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19- 780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (lii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:98, or (v) residues 19- 1 134 or 25- 1 134 of SEQ ID NO: 62.
  • Cancers that may be treated in accordance with the present invention include, for example, the following: a cancer of the head and neck (e.g., a cancer of the oral cavity, orophyarynx, nasopharynx, hypopharynx, nasal cavity or paranasal sinuses, larynx, lip, or salivary gland); a lung cancer (e.g., non-small cell lung cancer, small cell carcinoma, or mesothelimia); a gastrointestinal tract cancer (e.g.
  • GIST gastrointestinal stromal tumor
  • pancreatic adenocarcinoma pancreatic acinar cell carcinoma
  • a cancer of the small intestine a cancer of the liver or biliary tree (e.g. , liver cell adenoma, hepatocellular carcinoma, hem angiosarcoma, extrahepatic or intrahepatic cholangiosarcoma, cancer of the ampulla of vater, or gallbladder cancer)
  • a breast cancer e.g.
  • a gynecologic cancer e.g., cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal carcinoma, vaginal cancer, vulvar cancer, gestational trophoblastic neoplasia, or uterine cancer, including endometrial cancer or uterine sarcoma
  • a cancer of the urinary tract e.g., prostate cancer; bladder cancer; penile cancer; urethral cancer, or kidney cancer such as, for example, renal cell carcinoma or transitional cell carcinoma, including renal pelvis and ureter
  • testicular cancer a cancer of the central nervous system (CNS) such as an intracranial tumor (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, primary CNS lymphoma, medulloblasto
  • CNS central nervous system
  • schwannoma schwannoma
  • chordoma craniopharyngioma
  • craniopharyngioma a chloroid plexus tumor (e.g. , chloroid plexus carcinoma); or other intracranial tumor of neuronal or glial origin) or a tumor of the spinal cord (e.g.
  • an endocrine neoplasm e.g., thyroid cancer such as, for example, thyroid carcinoma, medullary cancer, or thyroid lymphoma; a pancreatic endocrine tumor such as, for example, an insulinoma or glucagonoma; an adrenal carcinoma such as, for example, pheochromocytoma; a carcinoid tumor; or a parathyroid carcinoma); a skin cancer (e.g., squamous cell carcinoma; basal cell carcinoma; Kaposi's sarcoma; or a malignant melanoma such as, for example, an intraocular melanoma); a bone cancer (e.g., a bone sarcoma such as, for example, osteosarcoma, osteochondroma, or Ewing's sarcoma); multiple myeloma; a chloroma; a soft tissue sarcoma (e.g.,
  • an ApoA- 1 fusion molecule as described herein is administered to a cancer patient as one of the distinct therapies of a combination therapy such as, for example, a combination therapy comprising a non-ApoAl -mediated immunomodulatory therapy (e.g., a therapy comprising an immune checkpoint inhibitor), a radiation therapy, or a chemotherapy.
  • a combination therapy comprising a non-ApoAl -mediated immunomodulatory therapy (e.g., a therapy comprising an immune checkpoint inhibitor), a radiation therapy, or a chemotherapy.
  • a combination cancer therapy comprises a tandem ApoA- 1 fusion molecule as described herein and a targeted therapy such as, e.g., a therapeutic monoclonal antibody targeting a specific cell-surface or extracellular antigen, or a small molecule targeting an intracellular protein (e.g., an intracellular enzyme).
  • a targeted therapy such as, e.g., a therapeutic monoclonal antibody targeting a specific cell-surface or extracellular antigen, or a small molecule targeting an intracellular protein (e.g., an intracellular enzyme).
  • exemplary antibody targeted therapies include anti-VEGF (e.g. , hevacizumab), anti-EGFR (e.g., cetuximab), anti-CTLA-4 (e.g. , ipilimumab), anti- PD- 1 (e.g., nivolumab), and anti-PD-Ll (e.g., pembrolizumab).
  • Exemplary small molecule targeted therapies include proteasome inhibitors (e.g. , bortezomib), tyrosine kinase inhibitors (e.g. , imatinib), cyclin-dependent kinase inhibitors (e.g., seliciclib); BRAF inhibitors (e.g. , vemurafenib or dabrafenib); and MEK kinase inhibitors (e.g. , trametnib).
  • proteasome inhibitors e.g. , bortezomib
  • tyrosine kinase inhibitors e.g. , imatinib
  • cyclin-dependent kinase inhibitors e.g., seliciclib
  • BRAF inhibitors e.g. , vemurafenib or dabrafenib
  • MEK kinase inhibitors e.g. , trametnib
  • the combination therapy includes an anti-PD- 1 /PD-L 1 therapy, an anti-CTLA-4 therapy, or both.
  • tandem ApoA- 1 fusion molecules as described herein can increase the response rate to either anti-CTLA-4 or anti-PD- I/PD-L1 therapy, as well as the response rate to the combination of anti-CTLA-4 plus anti-PD- 1 PD-L 1 therapy. Fusion molecules of the invention may also be useful for reducing the toxicity associated with anti-CTLA-4, anti-PD- 1/PD-L 1 , or the combination thereof.
  • a cancer treated in accordance with the present invention is selected from malignant melanoma, renal cell carcinoma, non-small ceil lung cancer, bladder cancer, and head and neck cancer. These cancers have shown responses to immune checkpoint inhibitors anti-PD- 1 PD-L 1 and anti-CTLA-4. See Grimaldi et ciL , Expert Opin. Biol. Ther. 16:433-41, 2016; Gunturi et aL ' , Curr. Treat. Options Oncol. 15: 137-46, 2014; Topalian et ⁇ ., ⁇ . Rev. Cancer 16:275- 87, 2016. Thus, in some more specific variations, any of these cancers is treated with a tandem ApoA-1 fusion molecule as described herein in combination with an anti-PD- 1 PD-L 1 therapy, an anti-CTLA-4 therapy, or both.
  • a fusion molecule for treatment of a cancer is a polypeptide having the structure ApoA l a -Ll-ApoAl b -L2-D (e.g. , ApoAl a -Ll -ApoAl b -L2-[Fc region], ApoAl a - L 1 -ApoA l b -L2-aibumin), ApoA l a -L 1 -ApoA l b -L2-D-L3-RNase (e.g.
  • the fusion polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:56, (ii) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID NO:58, (iii) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID NO:96, (iv) residues 19-790, 19-789, 25-790, or 25-789 of SEQ ID O:98, or (v) residues 19- 1 134 or 25- 1 134 of SEQ ID NO:62.
  • a fusion polypeptide having the structure ApoA l a -L l-ApoA l b -L2-D-L3-RNase or ApoA l a -L l-ApoA I b -L2-D-L3 -paraoxonase for the treatment of a cancer (A) the portion of the fusion polypeptide corresponding to ApoA l a -L l-ApoA l b -L2-D comprises or consists of an amino acid sequence having at least at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in (i) residues 19-780, 19-779, 25-780, or 25-779 of SEQ ID N O: 6.
  • the portion of the polypeptide corresponding to P comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in (i) residues 544-675 or 548-675 of SEQ ID NO:4, (ii) residues 16-355 of SEQ ID NO: 12, (iii) residues 16-355 of SEQ ID NO:42, or (iv) residues 16-355 of SEQ ID NO:44, In some embodiments of a fusion
  • an ApoA- 1 fusion molecule of the present disclosure is administered in combination with a cyclodextrin.
  • a particularly suitable cyclodextrin for use in such a combination therapy is 2-hydroxypropyl-p- cyclodextrin.
  • 2-Hydroxypropyl-P-cyclodextrin (CD) is approved by the US FDA and is used to solubilize numerous lipophilic drugs for use in humans. See Gould et al , Food Chem Toxicol 43 : 1451, 2005; Loftsson et al , Expert Opin. Drug Deliv. 2:335, 2005. CD may be used to help solubilize molecules of the present invention.
  • CD is also known to increase cholesterol solubility, remove cholesterol from foam cells in vitro, and initiate anti-inflammatory mechanisms. rithnridcs ⁇ . ⁇ / ⁇ //.. J. Biol. Chem.. 271 :27450, 1996; ⁇ . . et al. , J. Lipid Res. 44: 1 156, 2003 ; Atger et al , J. Clin. Invest. 99:773, 1997.
  • CD has been shown to induce plaque regression in vivo in a mouse model of atherosclerosis (see Zimmer et al , Science Translational Medicine 8:333 333ra50, 2016), and was active through liver X receptor (LXR) activation and upreguiation of ABCA 1 and ABCG1 transporters.
  • Cyciodextrins such as 2-hydroxypropyl-p-cyelodextrin may, for example, show enhanced activity in reverse cholesterol efflux and inhibition of inflammation when used in combination with a tandem ApoA- 1 fusion molecule of the present invention and could increase activity of an ApoA- 1 fusion molecule at lower doses.
  • An ApoA-1 fusion molecule of the present disclosure may also be used in combination with an LXR agonist other than CD.
  • LXR agonists have shown reduction in cholesterol, but side effects include an increase in triglycerides and fatty liver. See Huang, J. Integr. Med. 12:76, 2014. Other studies have found neurotoxicity from in vivo use of LXR agonists. See Steffensen et al, Expert Opin. Ther. Targets 17:977, 2013. The combination of an LXR agonist with a fusion molecule of the present invention could reduce LXR side effects by increasing effectiveness at lower doses.
  • an ApoA- 1 fusion molecule of the present disclosure is administered in combination with a statin or with an inhibitor of proprotein convertase subtilisin/kexin type 9 (PCSK9).
  • exemplary statins for use in combination with a tandem ApoA-1 fusion molecule include atorvastatin, simvastatin, rosuvastatin, and pravastatin.
  • Exemplar ⁇ ' PCSK9 inhibitors for use in combination with a tandem ApoA-1 fusion molecule include evolocumab and aiirocumab.
  • a fusion polypeptide or dimeric protein as described herein is delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought.
  • an effective amount of the fusion polypeptide or dimeric protein is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.
  • Subjects for administration of fusion polypeptides or dimeric proteins as described herein include patients at high risk for developing a particular disease or disorder as well as patients presenting with an existing disease or disorder.
  • the subject has been diagnosed as having the disease or disorder for which treatment is sought. Further, subjects can be monitored during the course of treatment for any change in the disease or disorder (e.g. , for an increase or decrease in clinical symptoms of the disease or disorder). Also, in some variations, the subject does not suffer from another disease or disorder requiring treatment that involves administration of an ApoA- 1 protein.
  • compositions or medicants are administered to a patient susceptible to, or otherwise at risk of, a particular disease in an amount sufficient to eliminate or reduce the risk or delay the outset of the disease.
  • compositions or medicants are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease and its complications. An amount adequate to accomplish this is referred to as a therapeutically or pharmaceutically effective dose or amount.
  • agents are usually administered in several dosages until a sufficient response (e.g. , atherosclerosis regression or stabilization of existing plaques in coronary heart disease) has been achieved. Typically, the response is monitored and repeated dosages are given if the desired response starts to fade.
  • accepted screening methods may be employed to determine risk factors associated with a specific disease or to determine the status of an existing disease identified in a subject. Such methods can include, for example, determining whether an individual has relatives who have been diagnosed with a particular disease. Screening methods can also include, for example, conventional work-ups to determine familial status for a particular disease known to have a heritable component. Toward this end, nucleotide probes can be routinely employed to identify individuals carrying genetic markers associated with a particular disease of interest. In addition, a wide variety of immunological methods are known in the art that are useful to identify markers for specific diseases. Screening may be implemented as indicated by known patient symptomology, age factors, related risk factors, etc.
  • treatment using a fusion polypeptide or dimeric protein of the present invention may be implemented as an independent treatment program or as a follow-up, adjunct, or coordinate treatment regimen to other treatments.
  • a fusion polypeptide or dimeric protein in accordance with the present invention is formulated as a pharmaceutical composition.
  • a pharmaceutical composition comprising a fusion polypeptide or dimeric protein as described herein can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic molecule is combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition is said to be a "pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • Other suitable carriers are well-known to those in the art. See.
  • Formulations may further include one or more excipients, preservatives, soiubiiizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
  • a pharmaceutical composition comprising a fusion polypeptide or dimeric protein of the present invention is administered to a subject in an effective amount.
  • the fusion polypeptide or dimeric protein may be administered to subjects by a variety of administration modes, including, for example, by intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, and oral routes of administration.
  • the fusion polypeptide or dimeric protein may be administered to a subject in a single bolus delivery, via continuous delivery ⁇ e.g. , continuous transdermal deliver ⁇ ') over an extended time period, or in a repeated administration protocol (e.g. , on an hourly, daily, or weekly basis).
  • Effective dosages of the compositions of the present invention vary depending upon many different factors, including means of administration, target site, phy siological state of the patient, whether the patient is human or an animal, other medications administered, whether treatment is prophylactic or therapeutic, as well as the specific activity of the composition itself and its ability to elicit the desired response in the individual.
  • the patient is a human, but in some diseases, the patient can be a nonhuman mammal.
  • dosage regimens are adjusted to provide an optimum therapeutic response, i.e.
  • a therapeutically or prophylactically effective amount is also one in which any undesired collateral effects are outweighed by beneficial effects (e.g., in the case of treatment of atherosclerotic cardiovascular disease, where any undesired collateral effects are outweighed by any beneficial effects such as increase in HDL, atherosclerosis regression, and/or plaque stabilization).
  • a dosage typically ranges from about 0.1 ⁇ g to 100 mg/kg or 1 g/kg to about 50 mg/kg, and more usually 10 ⁇ ig to 5 mg/kg of the subject's body weight.
  • an effective amount of the agent is between about 1 g/kg and about 20 mg/kg, between about 10 g kg and about 10 mg/kg, or between about 0.1 mg/kg and about 5 mg/kg. Dosages within this range can be achieved by single or multiple administrations, including, e.g. , multiple administrations per day or daily, weekly, bi-weekly, or monthly administrations.
  • a regimen consists of an initial administration followed by multiple, subsequent administrations at weekly or bi-weekly intervals.
  • Another regimen consists of an initial administration followed by multiple, subsequent administrations at monthly or bi-monthly intervals.
  • administrations can be on an irregular basis as indicated by monitoring of clinical symptoms of the disease or disorder and/or monitoring of disease biomarkers or other disease correlates (e.g., HDL levels in the case of atherosclerotic cardiovascular disease).
  • Particularly suitable animal models for evaluating efficacy of a tandem ApoA-1 composition of the present invention for treatment of atherosclerosis include, for example, known mouse models that are deficient in the low density lipoprotein receptor (LDLR) or ApoE.
  • LDLR deficient mice develop atherosclerotic plaques after eating a high fat diet for 12 weeks, and human ApoA-1 (reconstituted with lipids) is effective in reducing plaques in this model.
  • ApoE deficient mice are also commonly used to study atherosclerosis, and human ApoA- 1 (reconstituted with lipids) works rapidly in this model.
  • Rabbits that are transgenic for hepatic lipase are another known atherosclerosis model for testing ApoA- 1 compositions.
  • CIA collagen-induced arthritis
  • RA rheumatoid arthritis
  • CIA shares similar immunological and pathological features with RA, making it an ideal model for evaluating efficacy of ApoA- 1 compositions. See, e.g., Charles-Schoeman et al, Clin Immunol. 127:234-44, 2008 (describing studies showing efficacy of the ApoA- 1 mimetic peptide, D-4F, in the CIA model).
  • Another known model for RA is PG-polysaccharide (PG-PS)-induced arthritis in female Lewis rats. In these mice, administration of ApoA- 1 protein or reconstituted HDLs reduced acute and chronic joint inflammation. Wu et al. , Arterioscler Thromb Vase Biol 34:543-551 , 2014.
  • Animal models for multiple sclerosis include, for example, experimental allergic encephalomyelitis (EAE) models that rely on the induction of an autoimmune response in the CNS by immunization with a CNS antigen (also referred to as an "encephalitogen" in the context of EAE), which leads to inflammation, demyelination, and weakness.
  • EAE experimental allergic encephalomyelitis
  • ApoA-1 deficient mice have been shown to exhibit more neurodegeneration and worse disease than wild-type animals in this model. See Meyers et al. , J. Neuroimmunol. 277: 176- 185, 2014.
  • Fusion molecules of the present invention can be evaluated for anti-tumor activity in animal tumor models such as, e.g. , B 16 melanoma, a poorly immunogenic tumor.
  • Multiple models of tumor immunotherapy have been studied. See Mgiow et al., Adv. Immunol. 130: 1-24, 2016.
  • the B 16 melanoma model has been studied extensively with checkpoint inhibitors anti-CTLA-4, anti-PD- I, and the combination thereof.
  • Anti-CTLA-4 alone has a potent therapeutic effect in this model only when combined with GM-CSF transduced tumor vaccine, or combined with anti-PD-1. See Weber, Semin. Oncol. 37:430-439, 2010; Ai et al., Cancer Immunol. Immunoiher.
  • Efficacy of a tandem ApoA- 1 fusion molecule for treatment of malignant melanoma is shown, for example, by slowed tumor growth following administration to B 16 melanoma mice that have formed palpable subcutaneous tumor nodules.
  • Efficacy of an ApoA-1 fusion molecule can be evaluated in B 16 melanoma mice either alone or, alternatively, in combination with another anti-cancer therapy (e.g., anti-CTLA-4, with or without tumor vaccine or with or without anti-PD-l/PD-Ll).
  • mice may be administered a fusion molecule of the present invention for a short period (for example, one week, administered in, e.g., two doses of about 40mg/kg three days apart), and tumor growth then monitored, typically for two to three weeks after injection with the fusion molecule.
  • Dosage of the pharmaceutical composition may be varied by the attending clinician to maintain a desired concentration at a target site.
  • local concentration of the agent in the bloodstream at the target tissue may be between about 1 -50 nanomoles of the composition per liter, sometimes between about 1.0 nanomole per liter and 10, 15, or 25 nanomoles per liter depending on the subject's status and projected measured response.
  • Higher or lower concentrations may be selected based on the mode of deliver ⁇ ', e.g., trans-epidermal delivery versus delivery to a mucosal surface.
  • Dosage should also be adjusted based on the release rate of the administered formulation, e.g., nasal spray versus powder, sustained release oral or injected particles, transdermal formulations, etc.
  • the release rate of the administered formulation e.g., nasal spray versus powder, sustained release oral or injected particles, transdermal formulations, etc.
  • siow-reiease particles with a release rate of 5 nanomolar would be administered at about twice the dosage of particles with a release rate of 10 nanomolar.
  • a pharmaceutical composition comprising a fusion polypeptide or dimeric protein as described herein can be furnished in liquid form, in an aerosol, or in solid form.
  • Liquid forms are illustrated by injectable solutions, aerosols, droplets, topological solutions and oral suspensions.
  • Exemplary solid forms include capsules, tablets, and controiied-release forms. The latter form is illustrated by miniosmotic pumps and implants. See, e.g. , Bremer et al. Pharm. Biotechnol.
  • Degradable polymer microspheres have been designed to maintain high systemic levels of therapeutic proteins.
  • Microspheres are prepared from degradable polymers such as poiy(lactide-co-glycoiide) (PLG), polyanhydrides, poly (ortho esters), nonbiodegradable ethyivinyl acetate polymers, in which proteins are entrapped in the polymer.
  • PLG poiy(lactide-co-glycoiide)
  • PEG poiy(lactide-co-glycoiide)
  • polyanhydrides polyanhydrides
  • poly (ortho esters) poly (ortho esters)
  • nonbiodegradable ethyivinyl acetate polymers in which proteins are entrapped in the polymer. See, e.g., Gombotz and Pettit, Bioconjugate Chem.
  • compositions as described herein may also be used in the context of combination therapy.
  • combination therapy is used herein to denote that a subject is administered at least one therapeutically effective dose of a fusion polypeptide or dimeric protein as described herein and another therapeutic agent.
  • compositions may be supplied as a kit comprising a container that comprises a fusion polypeptide or dimeric protein as described herein.
  • a therapeutic molecule can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection.
  • a kit can include a dry -powder disperse!-, liquid aerosol generator, or nebulizer for administration of a therapeutic protein.
  • Such a kit may further comprise written information on indications and usage of the pharmaceutical composition.
  • the present invention provides an ApoA-1 fusion polypeptide comprising or consisting of an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with (i) residues 19-671 or 25-671 of SEQ ID NO:91, or (ii) residues 19-671 or 25-671 of SEQ ID NO:92. Also provided is a dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, where each of the first and second fusion polypeptides is a fusion polypeptide as above.
  • the present invention provides (a) a polynucleotide encoding a fusion polypeptide as above; (b) an expression vector comprising a transcription promoter, a DMA segment encoding a fusion polypeptide as above, and a transcription terminator linked in operable combination; (c) a cultured cell into which has been introduced an expression vector as above, where the cell expresses the DNA segment; (d) a method of making a fusion polypeptide comprising cuituring a ceil into which has been introduced an expression vector as above, where the cell expresses the DMA segment and the encoded fusion polypeptide is produced, and recovering the fusion polypeptide; (e) a method of making a dimeric protein comprising cuituring a cell into which has been introduced an expression vector as above, where the cell expresses the DNA segment and the encoded fusion polypeptide is produced as a dimeric protein, and recovering the dimeric protein; and (f) a composition comprising a fusion polypeptide
  • the present invention provides a method of treating a disease, the method comprising administering to a subject having the disease an effective amount of a fusion polypeptide or dimeric fusion protein as above, where the disease is selected from (a) a cardiovascular disease characterized by atherosclerosis (e.g. , stroke or coronary heart disease such as, for example, coronary heart disease characterized by acute coronary syndrome); (b) a neurodegenerative disease (e.g. , Alzheimer's disease, Parkinson's disease, or multiple sclerosis); (c) a disease characterized by amyloid deposit (e.g. , Alzheimer's disease); (d) an autoimmune disease (e.g.
  • rheumatoid arthritis systemic lupus erythematosus, multiple sclerosis, or type 1 diabetes
  • an inflammatory disease e.g. , rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type I diabetes, type 2 diabetes, obesity, non-alcoholic steatohepatitis, coronary heart disease, stroke, or an inflammatory lung disease such as, for example, asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, idiopathic pulmonary fibrosis, hyperoxia, hypoxia, or acute respiratory distress syndrome);
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • bronchiectasis idiopathic pulmonary fibrosis
  • hyperoxia oxia
  • hypoxia hypoxia
  • acute respiratory distress syndrome e.g.
  • nephrotic syndrome e.g. , nephrotic syndrome associated with a disease selected from amyloidosis, systemic lupus erythematosus, type 1 diabetes, type 2 diabetes, or primary kidney disease such as, for example, minimal-change nephropathy, focal glomerulosclerosis, membranous nephropathy, or IgA nephropathy
  • NS nephrotic syndrome
  • exposure to sulfur mustard gas or to an organophosphate e.g. , malignant melanoma, renal ceil carcinoma, non-small cell lung cancer, bladder cancer, or head and neck cancer.
  • This construct contained a DMA segment encoding a 26 amino acid linker (residues 268-293 of SEQ ID NO:2) between the C -terminal end of human ApoA -i (residues 1 -267 of SEQ ID NO:2) and a human vl Fc variant (residues 294-525 of SEQ ID NO:2).
  • this fusion polypeptide Upon expression in mammalian ceils and cleavage of the secretory signal peptide (residues 1-18), and any potential cleavage of the propeptide (residues 19-24), this fusion polypeptide had a predicted amino acid sequence corresponding to residues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:2 (the C- terminal lysine of the Fc region is known to be frequently cleaved in the production of Fc-containing proteins).
  • the other construct contained ApoA-1 and Fc regions identical to those of the ApoA- l(26)Fc construct, but lacked a (gly4ser) linker between human ApoA-1 and the Fc regions; this construct is also referred to herein as Apo A- 1 (2)Fc (Theripion) or as THERO (for no (gly4ser) repeat units).
  • This construct does contain a two amino acid linker due to insertion of overlapping restriction sites between the ApoA-1 region and the hinge region of human IgGI.
  • Cholesterol efflux The cholesterol efflux activity of the ApoA-I fusion proteins were measured using an in vitro assay. See Tang et a!., J Lipid Res. 47: 107-14, 2006. In vitro cholesterol efflux assays were performed using radio-labelled cholesterol and BHK cells expressing a mifespristone-inducible human ABCA1. H3-cholesteroi was added to growth media in order to label cellular cholesterol 24 hours prior to treatments, and ABCA1 is induced using ⁇ mifepristone for 6-20 hours. Cholesterol efflux was measured by incubating cells with or without the fusion proteins for 2 hours at 37°C, chilled on ice, and medium and cells separated to measure radiolabeled cholesterol.
  • Wild-type human ApoA-1 protein was used as a positive control.
  • a commercially available ApoA-l-Fc protein, linked directly to Fc without any linker between the ApoA-1 and Fc regions (APOA1 Recombinant Human Protein, hlgGl-Fc tag; Sino Biological, Inc.), was also tested and is referred to herein as ApoA-l(0)Fc (Sino Biol). The results of this assay are shown in FIG. 1.
  • Cholesterol efflux was increased in cultures containing ApoA-l-Fc with a 26 amino acid linker (ApoA- l(26)Fc), compared to either ApoA-I-Fc with a two amino acid linker (ApoA- l(2)Fc (Theripion)) or ApoA-l-Fc without a linker (ApoA-l(O)Fc (Sino Biol)).
  • ApoA- l (26)Fc also had activity similar to wild-type human ApoA-1 (Control ApoA-1).
  • FIGS. 2A and 2B A basic schematic diagramming the position of functional domains is shown in FIGS. 2A and 2B for the design of the ApoAl fusion proteins.
  • Fusion gene constructs inserted into pUC -based vectors isolated by restriction enzyme digestion, and fragments encoding the fusion genes were subcloned into the mammalian expression vector pDG. Briefly, HindlU+Xbal flanking restriction sites were used for removal of each expression gene from the vector, subfragments isolated by gel electrophoresis, DNA extracted using QIAquick purification columns, and eluted in 30 microliters EB buffer.
  • Fragments were ligated into HindlU+Xbal digested pDG vector, and ligation reactions transformed into NEB 5-alpha, chemically competent bacteria. Clones were inoculated into 3 ml LB broth with 100 ⁇ g/ml ampicillin, grown at 37°C overnight with shaking at 200 rpm, and plasmid DNA prepared using the QIAGEN spin plasmid miniprep kits according to manufacturer's instructions.
  • Sequencing primers were obtained from IDT Integrated DNA Technologies (Coralville, IA) and included the following: pdgF-2: 5'-ggttttggcagtacatcaatgg-3' (SEQ ID NO: 16); pdgR-2: 5'-ctattgtcttcccaatcctccc-3' (SEQ ID NO: 17); higgras: 5'-accttgcacttgtactcctt-3' (SEQ ID NO: 18).
  • Plasmid DNA 800 ng
  • sequencing primers 25 pmol, or 5 ⁇ of a 5 pmol/ ⁇ stock
  • GENEWIZ South Plamfield, NJ
  • Chromatograms were then analyzed, sequences assembled into contigs, and sequence verified using Vector Nti Advance 11.5 software (Life Technologies, Grand Island, NY).
  • This example illustrates transfection of plasmid constructs and expression of ApoA-1 fusion proteins in a mammalian transient transfection system.
  • the Ig fusion gene fragments with correct sequence were inserted into the mammalian expression vector pDG, and DNA from positive clones was amplified using QIAGEN plasmid preparation kits (QIAGEN, Valencia, CA). Five different constructs were generated. These each included the native coding sequence of the human ApoA-1 gene (nucleotide sequence shown in SEQ ID NO:35, encoding the amino acid sequence shown in SEQ ID NO:36).
  • Each sequence included the wild-type signal peptide (nucleotides 1-54 of SEQ ID NO:35, encoding amino acids 1-18 of SEQ ID NO:36) and propeptide sequences (nucleotides 55-72 of SEQ ID NO:35, encoding amino acids 19-24 of SEQ ID NO:36) for apolipoprotein A-l .
  • the C-terminal Q (Gin) residues of the ApoA-1 sequence was linked via a variable length linker segment to the human IgGl hinge, CH2, and CH3 domains to create a single chain (ApoA- ⁇ -Ink- human IgGl Fc fusion gene/protein.
  • the hinge sequence of the human IgGl is mutated so that the three cysteines are substituted with serine residues, eliminating disulfide bond formation in this region or unpaired cysteines that might compromise proper folding of the fusion protein.
  • the P238 and P331 residues of CH2 are also mutated to serines to eliminate effector functions such as ADCC and complement fixation.
  • Each construct also included a linker sequence inserted between the carboxyl terminus of apolipoprotein A- l (ending with the sequence ... TKKLNTQ (SEQ ID NO:35 residues 261-267) ) and the beginning of the hinge sequence of the human Fc (starting with the motif ... EPKSSDKT... (SEQ ID NO:2 residues 294-301).
  • This linker sequence ranged from two amino acids (or four if the overlap with the flanking domains is included) to 36 amino acids in length, depending on the construct.
  • the shortest linker included only two overlapping restriction sites (Bglll and Xhol) with a linker length of six additional nucleotides or two additional non-native amino acids.
  • the restriction sites were incorporated into the coding sequence of the molecule so that only two additional amino acids needed to be added to the amino acid sequence.
  • the Bglll site of the linker overlaps with the codon for the C-terminal glutamine of ApoA-1, and three of the nucleotides encoding the Xhol site form the codon for the first amino acid of the hinge (E-giutamic acid).
  • the linker amino acid sequence (including the two overlapping amino acids) is shown in residues 267-270 of SEQ ID NO:20, which is encoded by nucleotides 816-825 of SEQ ID NO: 19.
  • the fusion gene and protein for this construct are identified as THERO (since there are no (giy4ser) repeat units present) or apoA- l-lnk(2)higG.
  • the nucleotide and amino acid sequences for THERO are listed as SEQ ID NO: 19 and SEQ ID NO:20. The figures use the THERO nomenclature to specify this construct.
  • the second construct included a linker that encodes two (gly4ser) sequences flanked by restriction sites (16 amino acid linker), and the fusion gene and protein for this construct are identified as THER2 (or apoA-l-lnk(16)-hIgGl or apoA- l-(g4s)2-hIgGl).
  • THER2 or apoA-l-lnk(16)-hIgGl or apoA- l-(g4s)2-hIgGl.
  • the nucleotide and amino acid sequences for THER2 are listed as SEQ ID NO:21 and SEQ ID NO:22, respectively.
  • the (gly4ser)2 linker sequence is shown in residues 268-283 of SEQ ID NO:22, and the encoding nucleotide sequence for the (glv4ser)2 linker is shown in residues 817-864 of SEQ ID NO:21.
  • the third construct included a linker that encodes four (gly4ser) sequences flanked by restriction sites (26 amino acid linker), and the fusion gene and protein for this construct is identified as THER4 (or apoA-l-(g4s)4-mthIgG or apoA- l-lnk(26)-mthIgG), where "4" in "THER4" refers to the number of (gly4ser) repeat units, and the number 26 refers to the total number of amino acids encoded in the non-native, introduced linker sequence.
  • the nucleotide and amino acid sequences for THER4 are listed as SEQ ID NO: 1 and SEQ ID NQ:2, respectively.
  • the (giy4ser)4 linker sequence is shown in SEQ ID NO:50 (residues 268-293 of SEQ ID NO:2), and the encoding nucleotide sequence for the (gly4ser)4 linker is shown in SEQ ID NO:49 (residues 817-894 of SEQ ID NO: 1 ).
  • the fourth construct included a linker that encodes six (giy4ser) sequences flanked by restriction sites (36 amino acid linker), and the fusion gene and protein for this construct is identified as THER6 (or apoA-l -(g4s)6-mthIgG or apoA- l-lnk(36)-mthIgG).
  • THER6 or apoA-l -(g4s)6-mthIgG or apoA- l-lnk(36)-mthIgG.
  • the nucleotide and amino acid sequences for THER6 are listed as SEQ ID NQ:23 and SEQ ID NO:24, respectively.
  • the (giv4ser)6 linker sequence is shown in SEQ ID NO:52 (residues 268-303 of SEQ ID NO:24), and the encoding nucleotide sequence for the (gly4ser)6 linker is shown in SEQ ID NO:51 (residues 817-924 of SEQ ID NO:23).
  • the fifth construct included a linker that encodes four (gly4ser) sequences flanked by restriction sites (26 amino acid linker), but in addition, the construct included a second linker and an enzyme sequence at the carboxyi terminus of the IgGI domain.
  • the (gly4ser)4 linker sequence is as described above for THER4 (nucleotide and amino acid sequences shown in SEQ ID NO:49 and SEQ ID NG:50, respectively).
  • the second linker is an 18 amino acid long sequence (VDGASSPVNVSSPSVQDI; amino acid residues 1- 18 of SEQ ID NO:8, encoded by nucleotides 1- 54 of SEQ ID NO:7) that includes an N-linked glycosylation site, followed by a sequence that encodes human RNase l enzyme activity.
  • the linker sequence is listed as the first 54 nucleotides of SEQ ID NO 7, or the first 18 amino acids of SEQ ID NO 8, followed by the RNase sequence.
  • the ApoA-1 -lnk-hIgGl segment is fused to the NLG-RNase, and this construct is identified as THER4RNA2.
  • the nucleotide and amino acid sequences are identified as SEQ ID NO:3 and SEQ ID NO:4, respectively.
  • FIG. 3 shows Western Blot analysis of culture supernatants from representative 293 T transient transfections. Positive and negative controls (CD40IgG and mock transfecti on/no DNA, respectively) were included in each transfection series.
  • Transfected samples are as indicated in FIG. 3; lanes from left to right are as follows: Lane #1 - rnock transfection; Lane #2 - CD401gG; Lane #3 - MW markers; Lane #4 - THERO; Lane #5 - THER2: Lane #6 - THER4; Lane #7 - THER6; Lane #8 - MW marker; Lane #9 - THER4RNA2.
  • the THERO, THER2, THER4, and THER6 fusion proteins run at a position above the 50 kDa molecular weight marker.
  • the predicted molecular weight for these fusion proteins should be approximately 55, 56, 56.6, and 57 kDa, respectively.
  • the increasing linker length is evident by altered mobility for each fusion protein.
  • the THER4RNA2 molecule is predicted to be approximately 73.2 kDa, while ApoA- 1 is predicted to ran at 28.6 kDa.
  • the CD40IgG control is expected to ran at approximately 55 kDa.
  • This example illustrates expression of different ApoA-1 Ig fusion genes in eukaryotic cell lines and characterization of the expressed fusion proteins by SDS-PAGE and by IgG sandwich ELISA.
  • the pDG vector is a modified version of pcDNA3 encoding the DHFR selectable marker with an attenuated promoter to increase selection pressure for the plasmid.
  • Plasmid D A 200 , ug
  • purified plasmid was linearized at a unique Ascl site (New England Biolabs, Ipswich, MA Catalog # R.0558), purified by phenol extraction (Sigma-Aldrich, St. Louis, MO), ethanol precipitated, washed, and resuspended in EX-CELL® 302 tissue culture media, (Catalog #14324, SAFC/Sigma Aldnch, St. Louis, MO).
  • Salmon sperm DNA (Sigma-Aldrich, St. Louis, MO) was added as carrier DNA just prior to phenol extraction and ethanol precipitation, Plasmid and carrier DNA were coprecipitated, and the 400 ,ug was used to transfect 2x10 ' CHO DG44 cells by electroporation.
  • EX-CELL 302 media (Catalog # 13424C, SAFC Biosciences, St. Louis, MO) containing glutamine (4 mM), pyruvate, recombinant insulin (1 ⁇ ig/ml), penicillin-streptomycin, and 2xDMEM nonessential amino acids (all from Life Technologies, Grand Island, NY), hereafter referred to as "EX-CELL 302 complete” media.
  • Media for uniransiected cells and cells to be transfected also contained HT (diluted from a lOOx solution of hypoxanthine and thymidine) (Invitrogen/Life Technologies, Grand Island, NY).
  • Electroporations were performed at 280 volts, 950 microFarads, using a BioRad (Hercules, CA) GENEPULSER® el ectr operation unit with capacitance extender, Electroporation was performed in 0.4 cm gap sterile, disposable cuvettes. Eiectroporated ceils were incubated for 5 minutes after electroporation prior to transfer of culture to non-selective EX-CELL 302 complete media in T75 flasks.
  • Transfected cells were allowed to recover overnight in non-selective media prior to selective plating in 96 well fiat bottom plates (Costar) at varying serial dilutions ranging from 250 cells/well (2500 cells/ml) to 2000 cells/well (20,000 ceils/mi).
  • Culture media for cell cloning was EX-CELL 302 complete media containing 50 nM methotrexate.
  • Transfection plates were fed at five day intervals with 80 ⁇ fresh media. After the first couple of feedings, media was removed and replaced with fresh media. Plates were monitored and individual wells with clones were fed until clonal outgrowth became close to confluent, after which clones were expanded into 24 well dishes containing i ml media. Aliquots of the culture supernatants from the original 96 well plate were harvested to a second 96 well plate prior to transfer and expansion of the cells in 24 well plates. This second plate was frozen until EL1SA analysis to estimate IgG concentrations.
  • Plates were washed three times in PBS/0.05 % Tween 20, and incubated with horseradish peroxidase conjugated F(ab'2)goat anti-human IgG (Jackson Immunoreseareh, West Grove, PA, Catalog # 109-036-098) at 1 :7500-1 : 10,000 in PBS/0.5% BSA, for 1-2 hours at room temperature. Plates were washed five times in PBS/0.05% Tween 20, and binding detected with SUREBLUE RESERVETM TMB substrate (KPL Labs, Gaithersburg, MD: catalog #53-00-02).
  • FIGS. 4A-4E show a series of columnar graphs representing the production levels obtained from each CHO clone of a transfection series.
  • the clones from the THERO, THER2, THER4, THER6, and THER4R A2 transfections are displayed as a group in each of the five panels shown.
  • Each clone was screened at least once by IgG sandwich ELISA to assess expression level of the fusion protein.
  • 5A-5C show three panels showing the results of 6 and 10 day assays of fusion protein expression from the CHO transfectants with the highest expression after initial screening.
  • Six and ten day assays were performed by setting up 5 ml cultures at 1x10 s viable cells/ml (5xi0 5 initial inoculum.) in T25 flasks. The cultures were grown for six days after which a 1 ml aliquot was removed and live and dead cells counted. Cells were then centrifuged and the culture supernatants saved for further analysis by IgG sandwich ELISA and other analyses.
  • Protein concentration of aliquots (2 ⁇ ) from each fraction were determined at 280nM using a Nanodrop (Wilmington DE) microsample spectrophotometer, with blank determination using 0.1 M citrate buffer, pH 3.2, 0.5M NaC0 3 at a 10: 1 v:v ratio.
  • Fractions containing fusion protein were pooled, and buffer exchange performed by dialysis using Spectrum Laboratories G2 (Ranch Dominguez, CA, Catalog #G235057, Fisher Scientific catalog # 08-607-007) FLOAT-A-LYZER® units (MWCO 20kDa) against D-PBS (Hyclone, ThermoFisher Scientific, Dallas, TX), pH 7.4. Dialysis was performed in sterile, 2.2 liter Coming roller bottles at 4°C overnight.
  • fusion proteins were analyzed by electrophoresis on SDS-Polyacrylamide 4- 12% Bis-Tris NuPAGE® gels (Life Technologies, Grand Island, NY). Fusion protein samples were heated at 72°C for 10 minutes in LDS sample buffer with and without reduction of disulfide bonds and applied to 4-12% BIS-Tris gels (Catalog #NP0301, LIFE Technologies, Grand Island, NY). Five micrograms of each purified protein was loaded on the gels. The proteins were visualized after electrophoresis by IMPERIALTM protein staining (Pierce Imperial Protein Stain Reagent, Catalog #24615, ThermoFisher Scientific/Pierce, Rockford, IL), and destaining in distilled water.
  • IMPERIALTM protein staining Pieris Imperial Protein Stain Reagent, Catalog #24615, ThermoFisher Scientific/Pierce, Rockford, IL
  • Lanes are as follows from left to right: Lane #1 - KALEIDOSCOPE prestained MW markers; Lane #2 - THERO; Lane #3 - TFIER2; Lane #4 - THER4; Lane #5 - THER6; Lane #6 - THER4R A2; Lane #7 - KALEIDOSCOPE Prestained MW markers. Approximate molecular weights are indicated on the figures.
  • fusion proteins were subjected to native PAGE analysis.
  • BLUE Native PAGE gels were run using 4-16% Bis-Tris NativePAGETM gels (Life Technologies/ThermoFisher) with cathode and anode buffers prepared according to manufacturer's instructions. Samples (4.5 ⁇ g each fusion protein) were prepared without heating, using 4X sample buffer, without detergents. Gels were run for 30 minutes at 150 volts, followed by 1 hour at 180 volts, and the final hour at 220 volts. Gels were washed in distilled water and incubated for two hours in IMPERIALTM Protein stain.
  • FIG. 7 shows a representative native gel using these conditions.
  • Molecular weight markers were GE Healthcare high molecular weight calibration markers, a mixture of six large, multicomponent proteins, resuspended in the loading buffer used for samples, again without added detergents.
  • Lane #1 - ORENCIA® (abatacept; CTLA4hIgG); Lane #2 - anti- mouse CD40 monoclonal antibody ICI O; Lane #3 - THER4RNA2; Lane #4 - GE Healthcare High MW calibration markers; Lane #5 - THER6; Lane #6 - THER4; Lane #7 - THER2; Lane #8 - THERO; Lane #9 - GE Healthcare high MW markers; Lane #10 - Athens Research Apo A-L
  • the native ApoAl-IgG fusion proteins run at an approximate molecular weight somewhere between the 140 and 233 kDa markers and with a similar mobility as ORENCIA' ⁇ (abatacept), a CTLA4Ig fusion protein with the same human IgGl Fc domain.
  • the THER4RNase bispecific fusion protein did not stain well with the IMPERIAL stain possibly due to the highly basic composition of the RNase domain, but appears to migrate in a more diffuse pattern with predominate visible band migrating between the 233 and 440 kDa standards.
  • An antigen binding EL1SA was performed to assess the ability of Ig fusion proteins, captured by immobilized anti-human IgG (Fc-specific) to bind to and be detected by a horseradish peroxidase conjugated antibody specific for human apolipoprotein A-l.
  • High protein-binding, 96- weii ELISA plates (NUNC MAXISORP® plates, ThermoFisher Scientific) were coated with 1.5 .tig/ml goat anti-human IgG (Jackson Immunoresearch). Plates were blocked overnight at 4°C with PBS/3% BSA. Serial dilutions of each THER fusion protein, starting at 5 , iig/ml, were incubated overnight at 4°C.
  • the plate was washed three times and then incubated with horseradish peroxidase conjugated anti -human apolipoprotein A-l (ThermoFisher Scientific, catalog # PAI-28965) diluted 1 : 1500. Plates were incubated at room temperature for 2 hours. Plates were washed four times, then SUREBLUE RESERVETM TMB substrate (Catalog #: 53-00-02, KPL, Gaithersburg, MD) was added to the plate at 80ul/well. Development was stopped by addition of 80 ⁇ /weil IN HQ. Samples were read at 450 nm using a SYNERGYTM HT Biotek plate reader (Biotek Instruments, Winooski, VT) and data analyzed using GEN 5TM 2.0 software.
  • FIG. 8 shows the results from a representative Apo A-l binding ELISA.
  • OD450 is plotted versus concentration of fusion protein.
  • THER 0, 2, 4, 6, and THER4RNA2 fusion proteins all exhibited similar dose-response curves, indicating that the molecules can each be captured by binding to the Ig tail and detected by binding of the Apo A- 1 domain to the antibody targeted to human Apo A- l.
  • Human apolipoprotein A-l (Athens Research & Technology, catalog # 16- 16-120101) was included as a control and was not captured by the anti-human Fc specific antibody. At higher concentrations, the molecule showed weak binding by the antibody targeted to Apo A-l, indicating that the Apo A-l may have bound weakly to the plastic without capture by the anti-Fc antibody.
  • RNASEALERTTM assay IDT, Coralville, IA
  • RNASEALERTTM Substrate is a synthetic RNA oligonucleotide that has a fluorescein (R) on one end and a dark quencher (Q) on the other end.
  • the substrate When intact, the substrate has little or no fluorescence, but when cleaved by an RNase, the substrate fluoresces green (490 nni excitation, 520 nm emission) and can be detected with the appropriately equipped fluorescence plate reader.
  • a positive signal in this assay shows increasing fluorescence signal over time due to cleavage of the substrate by RN ase present in the sample(s).
  • Microplates were incubated with RNASEALERT substrate (a fixed concentration of 20 pmol/ ⁇ ), IX RNASEALERT buffer, and fusion protein or enzyme controls dilutions added to each well of a 96 well plate.
  • Enzyme activity assays were performed in triplicate for each sample, and the kinetic assay allowed to proceed for 45 minutes, with successive readings every 60 seconds. The increasing fluorescence at each time point is displayed for each well as a trace of RFU/weil as a function of time in FIG. 9. Serial dilutions of enzyme/fusion protein included 20 pmol/ ⁇ , 13.4 pmol/ ⁇ , 8.9 pmol/ ⁇ , 6 pmol/ ⁇ , 4 pmol/ ⁇ , 2.7 pmol/ ⁇ , 1.8 pmol/ ⁇ , and no enzyme.
  • RNase A (Ambion/ThermoFisher, catalog #AM2270) was included as a positive control, and THER4 (apo A-l-ink26-higG) was included as a negative control for comparing to the THER4RN A2 fusion protein.
  • THER4 apo A-l-ink26-higG
  • FIG. 10 Two replicates of the RNaseA, THER4RNA2, and THER4, are shown. All enzymes are at 4 pmol/ ⁇ and the substrate is present at 20 pmol/ ⁇ .
  • THER0, TITER2, THER4, THER6, and THER4RNA2 fusion proteins were assessed for their ability to act as acceptor molecules for reverse cholesterol transport from pre-loaded monocyte/macrophage mammalian cell lines.
  • the first assay used the human monocytic/macrophage cell Sine THP-1 and a fluorescently labeled derivative of cholesterol, BODTPY-cholesterol or TOPFLUOR-cholesterol (cholesterol compound with fluorescent boron dipyrromethene difluoride linked to sterol carbon-24) (Avanti Polar Lipids, Alabaster, AL).
  • the ⁇ -1 ceils were grown in RPM1 with 4mM glutamine, 10% FBS and maintained in mid-logarithmic growth prior to plating.
  • the protocol was adapted from the procedures outlined in Sankaranararyanan et al. (J. Lipid Res. 52:2332-2340, 201 1) and Zhang et al. (ASSA Y and Drug Development Technologies: 136-146, 2011).
  • Cells were harvested and plated to 96-weil flat bottom tissue culture plates at 2xl0 6 cells/ml or 2xl0 5 cells/well in 100 ⁇ RPMI media containing 33 ng/ml PMA. Cells were maintained in culture for 36-48 hours to allow for differentiation to occur prior to the assay.
  • Culture media was aspirated and plates were washed in IxPBS. Labeling media consisting of the following components (Phenol Red free RPMI with media supplements, 0.2% FBS, with ACAT inhibitor at 2 ⁇ / ⁇ 1, Sandoz 58-035 (Sigma-Aidrich, St. Louis MO), LXR agonist TO-901317 at 2.5 ⁇ (Sigma-Aldrich, St. Louis, MO), 35 ng/ml PMA (Sigma- Aldrich, St. Louis, MO), and 1.25 mM methyl beta-cyelodextrin (Sigma-Aldrich, St. Louis, MO), 50 ⁇ cholesterol (Sigma-Aldrich, St.
  • TOPFLUOR cholesterol (Avanti Polar Lipids, Alabaster, AL) was added at a volume of 100 ⁇ /weli and incubated at 37°C, 5% C0 2 for 10-12 hours.
  • Equilibration media RPMI complete with 10% FBS, 33 ng/ml PMA (100 ⁇ /well), was added to each well and incubated for 8 hours prior to incubation with acceptors. Labeling/equilibration media was aspirated from plates, and plates were washed twice with 200 ⁇ /weii PBS + 0.15% BSA. Efflux acceptor reagents were added to individual wells in efflux buffer and incubated for two hours prior to assay.
  • Efflux buffer was phenol red-free RPMI with growth supplements and 0.15% BSA. Samples were run in sets of 6-12 per condition/acceptor, and a minimum of five replicates used for statistical analysis. APO A- l was run as a positive control, and efflux media alone was used as the background negative control (baseline efflux). The efflux reaction was allowed to proceed for two hours, after which culture media was harvested to black, flat bottom 96-well plates (media reading). Cell lysates were prepared by addition of 100 ⁇ 0.1 N NaOH to each well of the efflux plate, and incubation for 15 minutes on a plate shaker at 4°C.
  • Ceil lysates were transferred to black, 96-well plates (iysate reading), and fluorescence for media and iysate samples measured using a SYNERGYTM HT plate reader with excitation at 485 nm and emission at 528 nm. Efflux was calculated as the ratio of the fluorescence measurements: (media/(media+lysate) x 100). The specific efflux was calculated by subtracting the baseline readings of the samples with no acceptor present from the total efflux/sample for each tested acceptor. Data analysis was performed using GraphPad Prism v 4.0 Software (San Diego, CA). The assay results are shown in FIG. 11.
  • the second assay used the mouse macrophage cell line J774A.1 (ATCC, Manassas, VA) to assess reverse cholesterol transport (RCT) using a radioactive derivative of cholesterol, [ ⁇ ]- choiesterol as described by Sankaranararyanan et a I. (J. Lipid Res. 52:2332-2340, 2011) and Yancey et al. (J. Lipid Res. 45:337-346, 2004).
  • J774 cells (3.5x10 s per well in 24 well plates) were incubated for 24 hours in 0.25 ml RPMI media supplemented with 5% FBS, ACAT inhibitor Sandoz 58-035 (2 ⁇ g/mi), and 4 ⁇ / ⁇ of [ J H] -cholesterol.
  • ACAT inhibitor was present at all times during the assay.
  • Cells were equilibrated 16-24 hours in media with or without cAMP (0.3mM) prior to incubation with acceptors. The presence of c AMP upregulates the ABCA I molecule.
  • apoA- 1-IgG-R ase expression constructs described above additional molecules which physically link the ApoA- 1 phospholipid transport function to the active sites of other enzyme domains are constructed.
  • One such molecule contains a segment corresponding to human paraoxanase 1 (PON 1), with nucleotide and encoded amino acid sequences as shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
  • PON 1 human paraoxanase 1
  • This arylesterase enzyme is present in human serum exclusively associated with high density lipoprotein (HDL), and inhibits oxidation of low density lipoprotein molecules. This protection from oxidation also inhibits development of vascular and coronary diseases.
  • the mature protein form of PON 1 is unique in that it retains its amino terminal signal peptide after secretion (amino acid residues I to 15 of SEQ ID NO: 12, encoded by nucleotide residues 1 to 45 of SEQ ID NO: 11).
  • Expression of a mutant form of PON 1 with a cleavable amino terminus demonstrated that PON 1 associates with lipoproteins through its amino terminus by binding to phospholipids directly rather than first binding to ApoA-1. See Sorenson et al. , Arteriosclerosis, Thrombosis, and Vascular Biology 19:2214-2225, 1999. Removal of the signal sequence was found to eliminate binding of the PON 1 moiety to phospholipids, proteoliposomes, and serum lipoproteins.
  • wild-type PON 1 does not bind directly to ApoA-1. See Sorenson et al, supra. These PON 1 signal sequence mutants showed reduced enzyme activity, possibly due to inability to bind the optimal phospholipid substrates. Nevertheless, a recombinant, active form of human PON 1 has been expressed in bacteria that is missing this signal sequence. See Stevens et al, Proc. Natl. Acad. Sci. USA 105: 12780- 12784, 2008. The presence of ApoA-1 does appear to stabilize arylesterase activity of the enzyme.
  • PONT has been expressed at the carboxyl terminus of an insulin receptor targeted antibody (see Boado et al., Mol. Pharm. 5: 1037- 1043, 2008; Boado et al , Biotechnology and Bioengineering 108: 186-196, 2011); however, the amino terminal signal peptide was included in this fusion protein.
  • the fusion gene and protein described here provides a novel method of PON l fusion protein expression, eliminating the requirement for the signal peptide by a direct physical coupling of the truncated enzyme to the apo A-l domain, thereby preserving and stabilizing both the binding function and arylesterase activity of PO l.
  • PON l Alternative forms of PON l are also used to construct bifunctional fusion molecules linking PON l to Apo A- l.
  • a sequence polymorphism that affects enzyme activity for different substrates is present at position 192 of the PON l sequence. See Steven et al, supra.
  • the amino acid at this position may be glutamine (Q) or arginine (R) in humans, or a lysine (K) in rabbits.
  • the arginine allele at position 192 has been reported to have a higher catalytic activity in vitro and in vivo.
  • THER4PON 1 Q192K nucleotide and amino acid sequences shown in SEQ ID NO:45 and SEQ ID NO:46, respectively
  • THER4PO 1 Q192R nucleotide and amino acid sequences shown in SEQ ID O:47 and SEQ ID NO:48, respectively
  • THER2PON 1 Q192K or THER2PON 1 Q192R a THER2 form of the fusion gene/protein is indicated as THER2PON 1 Q192K or THER2PON 1 Q192R.
  • the PON l amino terminal signal sequence amino acids 1- 15 of SEQ ID NO: 12 is removed.
  • Bispecific Enzyme Lipoprotein Transfer Proteins comprising PO l are screened for arylesterase/PON l activity using the nontoxic substrates 4-(chloromethyl)phenyl acetate (CM A) and phenyl acetate (see Richter et al, supra). These substrates are preferable for screening activity since the substrate and reaction product are relatively nontoxic compared to organophosphate pesticides.
  • the CMP A substrate Sigma-Aldrich, Inc. St Louis, MO
  • serial dilutions ef fusion protein is incubated with serial dilutions ef fusion protein and rates of CMP A hydrolysis assayed at 280 nm for 4 minutes at 25 C using ultraviolet transparent 96-well plates (Costar, Cambridge, Mass).
  • Dilutions are run in triplicate or quadruplicate and substrate concentration fixed at 3mmo3/L in 20mM Tris-HCl (pH 8.0), 1.0 mM CaC3 2 .
  • arylesterase assays are performed on phenyl acetate as substrate. The rates of PA hydrolysis are measured at 270nm, for 4 minutes under both high and low salt conditions.
  • One such molecule contains a segment corresponding to human PAFAH (lipoprotein- associated phospholipase A2, human phospholipase A2 group VII, platelet activating factor acetyl hydrolase), with nucleotide and encoded amino acid sequences as shown in SEQ ID NO:31 and SEQ ID NQ:32, respectively (see also GenBank accession number NM_005084 (transcript variant 1)).
  • PAFAH lipoprotein- associated phospholipase A2
  • human phospholipase A2 group VII platelet activating factor acetyl hydrolase
  • the PAFAH amino acid sequence is encoded by nucleotides 270 to 1592 of SEQ ID NO:31, with the STOP codon at nucleotides 1593 to 1595,
  • the fusion gene and protein are designed fusing the PAFAH coding sequence at the carboxyl end of the human IgG with the N-linked glycosvlation linker inserted between the two molecules.
  • the THER4PAFAH nucleotide and encoded amino acid sequences are shown as SEQ ID NO:33 and SEQ ID NO:34, respectively.
  • PAFAH sequence without the 21 amino acid signal peptide (MVPPKLHVLFCLCGCLAVVYP; residues 1 -21 of SEQ ID NO:32) is fused to the NLG linker at amino acid position 544 in SEQ ID NO 34.
  • Another such molecule contains a segment corresponding to human CETP or cholesteryl ester transfer protein (CETP), transcript variant 1, with nucleotide and encoded amino acid sequences as shown in SEQ ID NO:29 and SEQ ID NO:30, respectively (see also GenBank accession number NM_000078).
  • the CETP protein is encoded by nucleotides 58 to 1537 of SEQ ID NO:29.
  • the fusion gene and protein are designed fusing the CETP coding sequence at the carboxyl end of the human IgG with the N-linked glycosvlation linker inserted between the two molecules.
  • the THER4CETP (or human apo A-I-(g4s)4-hIgG-NLG-CETP) nucleotide and encoded amino acid sequences are shown as SEQ ID NO:39 and SEQ ID NO:40, respectively.
  • the nucleotides (57-107 of SEQ ID NO 29) encoding the signal peptide (amino acids 1-17 of SEQ ID NO 30) are removed in order to create the fusion gene between the NLG linker sequence and the CETP mature peptide.
  • the fusion junction between these two protein domains is located at amino acid 544 of SEQ ID NO 40.
  • Two ApoAl a -L l-ApoAl b -L2-Fc constructs are generated: one construct has the nucleotide sequence shown in SEQ ID NO:55 and encodes the fusion polypeptide of SEQ ID NO:56; the second construct has the nucleotide sequence shown in SEQ ID NO:57 and encodes the fusion polypeptide of SEQ ID NO:58.
  • Each construct includes native coding sequence of the human ApoA- 1 gene (nucleotide sequence shown in SEQ ID NO:35, encoding the amino acid sequence shown in SEQ ID NO:36).
  • the ApoAl a segment of each construct includes the wild-type signal peptide sequence (nucleotides 1-54 of SEQ ID NO:35, encoding amino acids 1-18 of SEQ ID NO:36) and wild-type propeptide sequence (nucleotides 55-72 of SEQ ID NO:35, encoding amino acids 19-24 of SEQ ID NO:36) for apolipoprotein A-l, while the ApoAl b segment does not include the signal peptide and propeptide sequences.
  • cDNA constructs are designed in cassette form, synthesized by PCR amplification, and the sequences assembled by subcloning using engineered restriction sites incorporated into the molecule design.
  • cassettes are assembled in the desired configuration, fusion gene constructs are assembled in pUC-based vectors and final restriction fragments encoding the fusion genes are subcloned into the mammalian expression vector pDG. Briefly, Hindlll+Xbal or EcoRV flanking restriction sites are used for removal of each expression gene from the vector, subfragments isolated by gel electrophoresis, DNA extracted using QI A quick purification columns, and eluted in 30 microliters EB buffer.
  • Fragments are ligated into Hindlll+Xbal or EcoKV digested pDG vector, and ligation reactions transformed into NEB 5 -alpha, chemically competent bacteria. Clones are inoculated into 3 ml LB broth with 100 ⁇ g/ml ampicillin, grown at 37°C overnight with shaking at 200 rpm, and plasmid DNA is prepared using the QIAGEN spin plasmid miniprep kits according to manufacturer's instructions.
  • Sequencing primers are obtained from IDT Integrated DNA Technologies (Coralville, IA) and include the following: pdgF-2: 5 '-ggttttggcagtacatc aatgg-3 ' (SEQ ID NO: 16); pdgR-2: 5'-ctattgtcttcccaatcctccc-3' (SEQ ID NO: 17); higgras: 5'-accttgcacttgtactcctt-3' (SEQ ID NO: 18).
  • Plasmid DNA (800 ng) and sequencing primers (25 pmol, or 5 ⁇ of a 5 pmol/ ⁇ stock) are mixed and submitted for DNA sequencing by GENEWIZ (South Plainfield, NJ). Chromatograms are then analyzed, sequences assembled into contigs, and sequence verified using Vector Nti Advance 11.5 software (Life Technologies, Grand Island, N Y). Clones with the correct sequence are then amplified and plasmid DNA is used for transient transfection of HEK293T cells using Polyfect reagent (QIAGEN, Valencia, CA), according to manufacturer's instructions.
  • Transfection media is changed after 24 hours to a low fluorescence, phenol red free media, Fiuorobrite DMEM (Life Technologies, Grand Island, NY), containing growth supplements (glutamine, pyruvate, non-essential amino acids, and pen/strep), but no serum. Serum-free transfection supernatants are harvested after 48-72 hours. Proteins are analyzed by SDS PAGE, Western blotting, and ELISA analysis. In addition, for proteins expressed at sufficient levels as determined by ELISA, undiluted and serial dilutions of culture supernatants are filter-sterilized and used in BODIPY cholesterol efflux assays to assess the relative functional activity of the molecules produced.
  • transient transfections are scaled up in order to harvest enough culture supernatant for affinity chromatography prior to functional studies.
  • Culture supernatants are also analy zed for the presence of the apo A- l domain using monoclonal antibodies against human apo A- l
  • Additional tandem apo A-l constructs are designed and generated in a similar manner.
  • One construct is an ApoA l a -Ll-ApoA l b -L2-albumin fusion gene having the nucleotide sequence shown in SEQ ID NO:61 and encoding the fusion polypeptide of SEQ ID NO:62.
  • An ApoAl a -L l-ApoAl b -L2 -albumin cDNA is designed with appropriate restriction sites in modular cassette form to facilitate assembly of the final fusion gene.
  • Human albumin is amplified by PGR amplification of cDNA derived from human PBMC RNA using a 5' and 3' primer set having the nucleotide sequences shown in SEQ ID NO 69 and SEQ ID NO 70, respectively.
  • the amplified cDNA is then digested with the appropriate restriction enzymes and inserted in place of the human Fc domain present in the ApoA l a -Ll-ApoAl b -L2-Fc construct described above.
  • tandem ApoA- 1 bispecific fusion constructs are also designed and generated: (a) an ApoA l a -Ll-ApoAl b -L2-Fc-L3-RNase construct having the nucleotide sequence shown in SEQ ID NO:74 and encoding the fusion polypeptide of SEQ ID NO:75; (b) an ApoAl a -Ll- ApoAl b -L2-Fc-L3-RNase construct having the nucleotide sequence shown in SEQ ID NO:93 and encoding the fusion polypeptide of SEQ ID NO:94; (c) an ApoA I a -Ll-ApoAl -L2-Fc-L3-PON l constmct having the nucleotide sequence shown in SEQ ID NO:76 and encoding the fusion polypeptide of SEQ ID NO: 77: (d) an ApoAl a -L l -Apo
  • oligonucleotides are designed for amplification of appropriate segments which read through into the next segment by removal of STOP codons, cDNA encoding the RNase, PON 1, CETP, and PAF-AH domains are amplified by PCR and then subcloned into the fusion gene using restriction sites incorporated into the sequences.
  • Each construct includes native coding sequence of the human ApoA-1 gene (nucleotide sequence shown in SEQ ID NO:35, encoding the amino acid sequence shown in SEQ ID NO:36).
  • the ApoAl a segment of each construct includes the wild-type signal peptide sequence (nucleotides 1 -54 of SEQ ID NO:35, encoding amino acids 1-18 of SEQ ID NO:36) and wild-type propeptide sequence (nucleotides 55-72 of SEQ ID NO:35, encoding amino acids 19-24 of SEQ ID NO:36) for apolipoprotein A- l, while the ApoAl b segment does not include the signal peptide and propeptide sequences.
  • Different combinations of Li and L2 linkers were used for each fusion construct as shown in Table 4 below.
  • Plasmid DNA was used for transient transfection of HEK293T cells using Polyfect reagent (QIAGEN, Valencia, CA), according to manufacturer's instructions. Briefly, HEK293T cells were trypsinized and cells counted prior to plating at a cell density of lxlO 6 cells/plate in 60mm tissue culture dishes. Cells were grown overnight and treated with polyfec plasmid DNA (4 ⁇ tg plasmid and 40 ⁇ poiyfect per transfection) complexes according to manufacturer's instructions, in DMEM with high glucose and containing 10% FBS.
  • Transfection media was changed after 24 hours to a low fluorescence, phenol red free media, Fluorobrite DMEM (Life Technologies, Grand Island, NY), containing growth supplements (glutamine, pyruvate, non-essential amino acids, and pen/strep), but no seram. Serum-free transfection supematants were harvested after 48 hours.
  • Electrophoresis was conducted at 185 volts for 80 minutes, followed by Western blotting to nitrocellulose using an XCeli electroblot module and MOPS-NuPAGE transfer buffer (Invitrogen/Life Technologies, Grand Island, NY) according to manufacturer's instructions.
  • Western blots were blocked at 4°C overnight with agitation, in D-PBS/5% nonfat milk, followed by incubation with HRP-goat anti-human TgG (Jackson Tmmunoresearch, West Grove, PA). Blots were washed five times in BLOTTO (D-PBS/5% nonfat milk), followed by treatment for 60 seconds with ECL substrate reagent (Pierce/ThermoFisher, Rockviiie, IL).
  • Plasmid DN A (200 ⁇ g) was prepared using Q1AGEN HISPEED® maxiprep kits, and purified plasmid was linearized at a unique Ascl site (New England Bioiabs, Ipswich, MA Catalog # R0558), purified by phenol extraction (Sigma-Aldrich, St. Louis, MO), ethanoi precipitated, washed, and resuspended in EX-CELL® 302 tissue culture media, (Catalog #14324, SAFC/Sigma Aldrich, St. Louis, MO). Salmon sperm DNA (Sigma-Aldrich, St. Louis, MO) was added as carrier DNA just prior to phenol extraction and ethanoi precipitation. Plasmid and carrier DNA were coprecipitated, and the 400 ,ug was used to transfect 2xl0 7 CHO DG44 ceils by eiectroporation.
  • EX-CELL 302 media (Catalog # 13424C, SAFC Biosciences, St. Louis, MO) containing giutamine (4 mM), pyruvate, recombinant insulin (1 ⁇ g/ml), penicillin-streptomycin, and 2xDMEM nonessential amino acids (ail from Life Technologies, Grand island, NY), hereafter referred to as "EX-CELL 302 complete” media.
  • Media for untransiected ceils and cells to be transfected also contained HT (diluted from a lOOx solution of hypoxanthine and thymidine) (Invitrogen/Life Technologies, Grand Island, NY).
  • Electroporations were performed at 280 volts, 950 microFarads, using a BioRad (Hercules, CA) GENEPULSER® eiectroporation unit with capacitance extender. Eiectroporation was performed in 0.4 cm gap sterile, disposable cuvettes. Eiectroporated ceils were incubated for 5 minutes after eiectroporation prior to transfer of culture to non-selective EX-CELL 302 complete media in T75 flasks.
  • Transfected cells were allowed to recover overnight in non-selective media prior to selective plating in 96 well flat bottom plates (Costar) at varying serial dilutions ranging from 250 cells/well (2500 ceils/mi) to 2000 cells/well (20,000 ceils/mi).
  • Culture media for cell cloning was EX-CELL 302 complete media containing 50 nM methotrexate.
  • Transfection plates were fed at five day intervals with 80 ⁇ fresh media. After the first couple of feedings, media was removed and replaced with fresh media. Plates were monitored and individual wells with clones were fed until clonal outgrowth became close to confluent, after which clones were expanded into 24 well dishes containing 1 ml media. Aliquots of the culture supernatants from the original 96 well plate were also harvested and frozen for later ELISA analysis to estimate IgG concentrations.
  • Plates were washed three times in PBS/0.05 % Tween 20, and incubated with horseradish peroxidase conjugated F(ab'2)goat anti-human IgG (Jackson Immunoresearch, West Grove, PA, Catalog # 109-036-098) at 1 :7500-1 : 10,000 in PBS/0.5% BSA, for 1-2 hours at room temperature. Plates were washed five times in PBS/0.05% Tween 20, and binding detected with SUREBLUE RESERVETM TMB substrate (SeraCare, Gaithersburg, MD).
  • cultures were grown for ten days and culture supernatants from spent cultures were harvested by centrifugation, culture media filtered, and pH adjusted with sodium carbonate/bicarbonate solution to pTI 8.0.
  • Proteins from transfected CHO cells were purified by protein A affinity chromatography, using a modified purification strategy to preserve protein function.
  • Culture supernatants were incubated in sterile 50 ml conical tubes with 0.5-1.0 ml protein A slurry (Repligen, Waltham, MA), per tube, with gentle rotation at 4°C, for 48-72 hours. Protein A slurry was harvested from each tube by centrifugation at 2500 rpm for 10 minutes, and culture media removed to a separate bottle.
  • Eluted fractions (1.0 ml/tube) were collected into microfuge tubes and aliquots from each fraction assessed for protein level using a nanodrop (ThermoFisher, Waltham, MA) spectrophotometer. Positive fractions were pooled and dialyzed in fioat-a-lyzer dialysis units (Spectrum Labs/Repligen, Collinso Dominguez, CA). Dialysis buffer contained 0.9% NaCl, 2.5 mM HEPES buffer, ImM CaCL, and 5 niM sodium bicarbonate, pH 7.5. Proteins were harvested after dialysis, concentrated using Millipore Sigma (Burlington, MA) centrifugal concentrators, and sterile filtered through 0.2 ⁇ PES syringe filter units. Protein concentrations were then determined from the OD280 assayed using a Nanodrop spectrophotometer with dialysis buffer as a blank. Once tandem molecules were purified, they were assessed by SDS-PAGE analysis and in functional assays.
  • FIG. 16 shows fusion proteins purified from stable CHO DG44 clones, visualized after electrophoresis by IMPERIALTM protein staining (Pierce Imperial Protein Stain Reagent, Catalog #24615, ThermoFisher Scientific/Pierce, Rockford, IL), and destaining in distilled water. Molecular weight markers were included on the same gel (KALEIDOSCOPETM Precision Plus Prestained Standards, Catalog #161-0324, Bio-Rad, Hercules, CA).
  • the L2 linker (located between the second apoAl cassette and the human Fc domain) has the amino acid sequence shown in SEQ ID NO 50.
  • the LI linker designated as "L la” is the linker sequence shown in SEQ ID NO 66
  • the LI linker designated as "Lib” is the linker sequence shown in SEQ ID NO 68.
  • a single purified protein from the Lla fusion protein and two different purifications from separate clones of the Lib form were analyzed on the gel shown. Each of these tandem apoA-1 constructs migrated on reducing NuPAGE gels at approximately 85 kDa as expected.
  • tandem apoA- 1 constructs were assessed using the human monocytic/macrophage cell line THP-1 and a fluoreseently labeled derivative of cholesterol, BODIPY-cholesterol or TOPFLUOR-choiesterol (cholesterol compound with fluorescent boron dipyrromethene difluoride linked to sterol carbon-24) (Avanti Polar Lipids, Alabaster, AL).
  • THP-1 cells were grown in RPMI with 4mM glutamine, 10% FBS and maintained in mid-logarithmic growth prior to plating. The protocol was adapted from the procedures outlined in Sankaranararyanan et al. (J. Lipid Res.
  • Labeling media consisted of the following components; phenol red free RPMI with media supplements, 0.2% FBS, with ACAT inhibitor at 2 ⁇ , Sandoz 58-035 (Sigma-Aldrieh, St. Louis MO), LXR agonist TO-901317 at 2.5 ⁇ (Sigma- Aldrich, St. Louis, MO), 50 ng/ml PMA (Sigma-Aidrich, St. Louis, MO), and 1.25 mM methyl beta- cyclodextrin (Sigma-Aidrich, St. Louis, MO), 50 ⁇ cholesterol (Sigma-Aidrich, St. Louis, MO), and 25 ⁇ TOPFLUOR cholesterol (Avanti Polar Lipids, Alabaster, AL).
  • Labeling media was added at a volume of 100 ⁇ /well and incubated at 37°C, 5% C0 2 for 2 hours.
  • Equilibration media phenol red free RPM1 complete media with 10% FBS, 50 ng/mi PMA (100 ⁇ /weil), was added to each well and incubated for approximately 8 hours (overnight) at 37°C, 5% C0 2 , After overnight incubation, equilibration media containing a nonbinding fusion protein with an identical Fc domain was added to each well of the assay (at a final concentration of 50 ⁇ g/ml) in order to block FcR binding sites present on the surface of the THP-1 cells.
  • This control Fc molecule includes an LDL receptor (LDL ) EGF A-B domain fused to the human IgG J Fc, and is described in US Patent Application Publication No. US 2014/0120091 A l, which is incorporated by reference herein. Plates were incubated for 1 hour at 37°C, 5% C0 2 to permit binding, then the labeling/equilibration media was aspirated from plates, and plates were washed twice with 200 ⁇ /weii PBS + 0.15% BSA. Efflux acceptor reagents were added to individual wells in efflux buffer and incubated for two hours prior to efflux measurements. Acceptors were added to efflux buffer at concentrations ranging from 100 nM to l ⁇ , depending on the assay.
  • LDL LDL receptor
  • Efflux buffer was phenol red-free Fluorobrite lM DMEM (Life Technologies, Grand Island, NY), containing growth supplements and 0.15% BSA. Samples were run in sets of six replicates per condition/acceptor, and the average of the six replicates was used to estimate the degree of cholesterol efflux to each acceptor. THER4 was run as a positive control, and efflux media alone was used as the background negative control (baseline efflux-identified as media alone on figure). The efflux reaction was allowed to proceed for two hours, after which culture media was harvested to black, flat bottom 96-well plates (media reading).
  • Ceil lysates were prepared by addition of 100 ⁇ 0.1 N NaOH to each well of the efflux pl ate, and incubation for 15-30 minutes on a plate shaker at 4°C. Cell lysates were transferred to black, 96-well plates (lysate reading), and fluorescence for media and lysate samples measured using a SYNERGYTM HT plate reader with excitation at 485 nm and emission at 528 nm. Percent efflux was calculated as the ratio of the fluorescence measurements: (media/(media+lysate) x 100). Data analysis was performed using GraphPad Prism v 4.0 Software (San Diego, CA).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des compositions et des procédés relatifs aux polypeptides de fusion ApoA-1. Dans des modes de réalisation particuliers, les polypeptides de fusion comprennent des premier et second segments polypeptidiques reliés en tandem par l'intermédiaire d'un lieur, chaque segment correspondant à un polypeptide ApoA-1 ou un mimétique d'ApoA-1. Les polypeptides de fusion peuvent également comprendre un domaine de dimérisation ou un domaine qui se lie spécifiquement au récepteur Fc néonatal (par exemple, une région Fc ou une albumine), qui est typiquement lié côté carboxyle-terminal au second segment polypeptidique par l'intermédiaire d'un lieur flexible. Dans certains modes de réalisation, le polypeptide de fusion comprend en outre un segment polypeptidique supplémentaire qui confère une seconde activité biologique (par exemple, RNase, paraoxonase, acétylhydrolase du facteur d'activation plaquettaire, protéine de transfert d'ester de cholestérol, lécithine-cholestérol acyltransférase ou polypeptide qui se lie de manière spécifique à l'amyloïde bêta). L'invention concerne également des protéines dimériques comprenant des premier et second polypeptides de fusion ApoA-1 comprenant un domaine de dimérisation tel que décrit dans la présente invention. Les polypeptides de fusion et les protéines dimériques sont utiles dans des méthodes thérapeutiques.
PCT/US2017/065078 2016-12-09 2017-12-07 Polypeptides de fusion apoa-1 en tandem WO2018136163A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201662432128P 2016-12-09 2016-12-09
US62/432,128 2016-12-09
US201762467288P 2017-03-06 2017-03-06
US62/467,288 2017-03-06

Publications (2)

Publication Number Publication Date
WO2018136163A2 true WO2018136163A2 (fr) 2018-07-26
WO2018136163A3 WO2018136163A3 (fr) 2018-09-27

Family

ID=62152610

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/065078 WO2018136163A2 (fr) 2016-12-09 2017-12-07 Polypeptides de fusion apoa-1 en tandem

Country Status (1)

Country Link
WO (1) WO2018136163A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022178078A1 (fr) * 2021-02-19 2022-08-25 Theripion, Inc. Polypeptides de fusion paraoxonase et compositions et méthodes associées

Citations (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4486533A (en) 1982-09-02 1984-12-04 St. Louis University Filamentous fungi functional replicating extrachromosomal element
US4579821A (en) 1981-11-23 1986-04-01 University Patents, Inc. Control of DNA sequence transcription
US4599311A (en) 1982-08-13 1986-07-08 Kawasaki Glenn H Glycolytic promotersfor regulated protein expression: protease inhibitor
US4601978A (en) 1982-11-24 1986-07-22 The Regents Of The University Of California Mammalian metallothionein promoter system
US4615974A (en) 1981-08-25 1986-10-07 Celltech Limited Yeast expression vectors
US4656134A (en) 1982-01-11 1987-04-07 Board Of Trustees Of Leland Stanford Jr. University Gene amplification in eukaryotic cells
US4661454A (en) 1983-02-28 1987-04-28 Collaborative Research, Inc. GAL1 yeast promoter linked to non galactokinase gene
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4713339A (en) 1983-01-19 1987-12-15 Genentech, Inc. Polycistronic expression vector construction
US4784950A (en) 1985-04-17 1988-11-15 Zymogenetics, Inc. Expression of factor VII activity in mammalian cells
US4845075A (en) 1985-02-25 1989-07-04 Zymogenetics, Inc. Biologically active B-chain homodimers
US4870008A (en) 1983-08-12 1989-09-26 Chiron Corporation Secretory expression in eukaryotes
US4882279A (en) 1985-10-25 1989-11-21 Phillips Petroleum Company Site selective genomic modification of yeast of the genus pichia
US4931373A (en) 1984-05-25 1990-06-05 Zymogenetics, Inc. Stable DNA constructs for expression of α-1 antitrypsin
US4935349A (en) 1986-01-17 1990-06-19 Zymogenetics, Inc. Expression of higher eucaryotic genes in aspergillus
US4956288A (en) 1988-04-22 1990-09-11 Biogen, Inc. Method for producing cells containing stably integrated foreign DNA at a high copy number, the cells produced by this method, and the use of these cells to produce the polypeptides coded for by the foreign DNA
US4977092A (en) 1985-06-26 1990-12-11 Amgen Expression of exogenous polypeptides and polypeptide products including hepatitis B surface antigen in yeast cells
US4990446A (en) 1984-12-06 1991-02-05 Labofina, S.A. Promoters for the expression of foreign genes in yeast, plasmids comprising them, and use thereof for the production of polypeptides
US5037743A (en) 1988-08-05 1991-08-06 Zymogenetics, Inc. BAR1 secretion signal
US5063154A (en) 1987-06-24 1991-11-05 Whitehead Institute For Biomedical Research Pheromone - inducible yeast promoter
US5139936A (en) 1983-02-28 1992-08-18 Collaborative Research, Inc. Use of the GAL1 yeast promoter
US5143830A (en) 1986-05-15 1992-09-01 Holland Ian B Process for the production of a polypeptide
US5162228A (en) 1988-12-28 1992-11-10 Takeda Chemical Industries, Ltd. Gylceraldehyde-3-phosphate dehydrogenase gene and promoter
US5162222A (en) 1989-07-07 1992-11-10 Guarino Linda A Use of baculovirus early promoters for expression of foreign genes in stably transformed insect cells or recombinant baculoviruses
WO1994006463A1 (fr) 1992-09-14 1994-03-31 Pfizer Inc Cellules immortalisees et leurs utilisations
US5300435A (en) 1991-09-16 1994-04-05 Boyce Thompson Institute For Plant Research, Inc. Trichoplusia ni cell line which supports replication of baculoviruses
WO1997020078A1 (fr) 1995-11-30 1997-06-05 Maxygen, Inc. Procede d'elaboration de polynucleotides presentant des caracteristiques desirees par selection iterative et recombinaison
US5641655A (en) 1994-11-30 1997-06-24 Zymogenetics, Inc. Methods for producing thrombopoietin polypeptides using a mammalian tissue plasminogen activator secretory peptide
US5716808A (en) 1995-11-09 1998-02-10 Zymogenetics, Inc. Genetic engineering of pichia methanolica
US5736383A (en) 1996-08-26 1998-04-07 Zymogenetics, Inc. Preparation of Pichia methanolica auxotrophic mutants
US5854039A (en) 1996-07-17 1998-12-29 Zymogenetics, Inc. Transformation of pichia methanolica
US5856456A (en) 1992-11-20 1999-01-05 Enzon, Inc. Linker for linked fusion polypeptides
US5888768A (en) 1996-08-26 1999-03-30 Zymogenetics, Inc. Compositions and methods for producing heterologous polypeptides in Pichia methanolica
US20030103986A1 (en) 2001-05-24 2003-06-05 Rixon Mark W. TACI-immunoglobulin fusion proteins
WO2005018572A2 (fr) 2003-08-22 2005-03-03 Biogen Idec Ma Inc. Anticorps ameliores possedant une fonction d'effecteur modifiee et procedes de fabrication associes
WO2005047327A2 (fr) 2003-11-12 2005-05-26 Biogen Idec Ma Inc. Variants de polypeptide se liant au recepteur fc neonatal (fcrn), proteines de liaison fc dimeres et techniques associees
WO2005063815A2 (fr) 2003-11-12 2005-07-14 Biogen Idec Ma Inc. Variants de polypeptides de liaison au recepteur fc$g(g) et procede apparentes
US7115396B2 (en) 1998-12-10 2006-10-03 Compound Therapeutics, Inc. Protein scaffolds for antibody mimics and other binding proteins
US20070111281A1 (en) 2005-05-09 2007-05-17 Glycart Biotechnology Ag Antigen binding molecules having modified Fc regions and altered binding to Fc receptors
US8937157B2 (en) 2011-04-29 2015-01-20 University Of Washington Therapeutic nuclease compositions and methods

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002213843B2 (en) * 2000-11-10 2008-02-07 F. Hoffmann-La Roche Ltd. Apolipoprotein analogues

Patent Citations (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4615974A (en) 1981-08-25 1986-10-07 Celltech Limited Yeast expression vectors
US4579821A (en) 1981-11-23 1986-04-01 University Patents, Inc. Control of DNA sequence transcription
US4656134A (en) 1982-01-11 1987-04-07 Board Of Trustees Of Leland Stanford Jr. University Gene amplification in eukaryotic cells
US4599311A (en) 1982-08-13 1986-07-08 Kawasaki Glenn H Glycolytic promotersfor regulated protein expression: protease inhibitor
US4486533A (en) 1982-09-02 1984-12-04 St. Louis University Filamentous fungi functional replicating extrachromosomal element
US4601978A (en) 1982-11-24 1986-07-22 The Regents Of The University Of California Mammalian metallothionein promoter system
US4713339A (en) 1983-01-19 1987-12-15 Genentech, Inc. Polycistronic expression vector construction
US4661454A (en) 1983-02-28 1987-04-28 Collaborative Research, Inc. GAL1 yeast promoter linked to non galactokinase gene
US5139936A (en) 1983-02-28 1992-08-18 Collaborative Research, Inc. Use of the GAL1 yeast promoter
US4870008A (en) 1983-08-12 1989-09-26 Chiron Corporation Secretory expression in eukaryotes
US4931373A (en) 1984-05-25 1990-06-05 Zymogenetics, Inc. Stable DNA constructs for expression of α-1 antitrypsin
US4990446A (en) 1984-12-06 1991-02-05 Labofina, S.A. Promoters for the expression of foreign genes in yeast, plasmids comprising them, and use thereof for the production of polypeptides
US4845075A (en) 1985-02-25 1989-07-04 Zymogenetics, Inc. Biologically active B-chain homodimers
US4683202B1 (fr) 1985-03-28 1990-11-27 Cetus Corp
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4784950A (en) 1985-04-17 1988-11-15 Zymogenetics, Inc. Expression of factor VII activity in mammalian cells
US4977092A (en) 1985-06-26 1990-12-11 Amgen Expression of exogenous polypeptides and polypeptide products including hepatitis B surface antigen in yeast cells
US4882279A (en) 1985-10-25 1989-11-21 Phillips Petroleum Company Site selective genomic modification of yeast of the genus pichia
US4935349A (en) 1986-01-17 1990-06-19 Zymogenetics, Inc. Expression of higher eucaryotic genes in aspergillus
US5143830A (en) 1986-05-15 1992-09-01 Holland Ian B Process for the production of a polypeptide
US5063154A (en) 1987-06-24 1991-11-05 Whitehead Institute For Biomedical Research Pheromone - inducible yeast promoter
US4956288A (en) 1988-04-22 1990-09-11 Biogen, Inc. Method for producing cells containing stably integrated foreign DNA at a high copy number, the cells produced by this method, and the use of these cells to produce the polypeptides coded for by the foreign DNA
US5037743A (en) 1988-08-05 1991-08-06 Zymogenetics, Inc. BAR1 secretion signal
US5162228A (en) 1988-12-28 1992-11-10 Takeda Chemical Industries, Ltd. Gylceraldehyde-3-phosphate dehydrogenase gene and promoter
US5162222A (en) 1989-07-07 1992-11-10 Guarino Linda A Use of baculovirus early promoters for expression of foreign genes in stably transformed insect cells or recombinant baculoviruses
US5300435A (en) 1991-09-16 1994-04-05 Boyce Thompson Institute For Plant Research, Inc. Trichoplusia ni cell line which supports replication of baculoviruses
WO1994006463A1 (fr) 1992-09-14 1994-03-31 Pfizer Inc Cellules immortalisees et leurs utilisations
US5856456A (en) 1992-11-20 1999-01-05 Enzon, Inc. Linker for linked fusion polypeptides
US5641655A (en) 1994-11-30 1997-06-24 Zymogenetics, Inc. Methods for producing thrombopoietin polypeptides using a mammalian tissue plasminogen activator secretory peptide
US5716808A (en) 1995-11-09 1998-02-10 Zymogenetics, Inc. Genetic engineering of pichia methanolica
WO1997020078A1 (fr) 1995-11-30 1997-06-05 Maxygen, Inc. Procede d'elaboration de polynucleotides presentant des caracteristiques desirees par selection iterative et recombinaison
US5854039A (en) 1996-07-17 1998-12-29 Zymogenetics, Inc. Transformation of pichia methanolica
US5736383A (en) 1996-08-26 1998-04-07 Zymogenetics, Inc. Preparation of Pichia methanolica auxotrophic mutants
US5888768A (en) 1996-08-26 1999-03-30 Zymogenetics, Inc. Compositions and methods for producing heterologous polypeptides in Pichia methanolica
US7115396B2 (en) 1998-12-10 2006-10-03 Compound Therapeutics, Inc. Protein scaffolds for antibody mimics and other binding proteins
US20030103986A1 (en) 2001-05-24 2003-06-05 Rixon Mark W. TACI-immunoglobulin fusion proteins
WO2005018572A2 (fr) 2003-08-22 2005-03-03 Biogen Idec Ma Inc. Anticorps ameliores possedant une fonction d'effecteur modifiee et procedes de fabrication associes
WO2005047327A2 (fr) 2003-11-12 2005-05-26 Biogen Idec Ma Inc. Variants de polypeptide se liant au recepteur fc neonatal (fcrn), proteines de liaison fc dimeres et techniques associees
WO2005063815A2 (fr) 2003-11-12 2005-07-14 Biogen Idec Ma Inc. Variants de polypeptides de liaison au recepteur fc$g(g) et procede apparentes
US20070111281A1 (en) 2005-05-09 2007-05-17 Glycart Biotechnology Ag Antigen binding molecules having modified Fc regions and altered binding to Fc receptors
US8937157B2 (en) 2011-04-29 2015-01-20 University Of Washington Therapeutic nuclease compositions and methods

Non-Patent Citations (232)

* Cited by examiner, † Cited by third party
Title
"Antibody Engineering: A Practical Approach", 1996, IRL PRESS
"Baculovirus Expression Protocols. Methods in Molecular Biology", 1995, HUMANA PRESS
"Chromatography: Principles & Methods", PHARMACIA LKB BIOTECHNOLOGY, 1988
"Current Protocols in Molecular Biology", 1993, GREEN AND WILEY AND SONS
"GenBank", Database accession no. J00228
"GenBank", Database accession no. NM_005084
"Guide to Human Genome Computing", 1998, ACADEMIC PRESS, INC.
"Meth. Enzymol.", vol. 182, 1990, pages: 529
"Methods in Gene Biotechnology", 1997, CRC PRESS, INC., article "Information Superhighway and Computer Databases of Nucleic Acids and Proteins", pages: 123 - 151
"Pharmacia LKB Biotechnology", 1988, article "Affinity Chromatography: Principles & Methods"
"Remington's Pharmaceutical Sciences", 1995, MACK PUBLISHING COMPANY
AHME ET AL., EXCLI JOURNAL, vol. 12, 2013, pages 719 - 732
AHMED ET AL., EXCLI JOURNAL, vol. 12, 2013, pages 719 - 732
AI ET AL., CANCER IMMUNOL. IMMUNOTHER., vol. 64, 2015, pages 885 - 92
ALFTHAN ET AL., PROTEIN ENG., vol. 8, 1995, pages 725 - 731
ALTSCHUL ET AL., BULL. MATH. BIO., vol. 48, 1986, pages 603
AMSTER ET AL., NUC. ACIDS RES., vol. 8, 1980, pages 2055 - 2065
ANSEL; POPOVICH: "Pharmaceutical Dosage Forms and Drug Delivery Systems", 1990, LEA & FEBIGER
ARAKAWA, J. BIOL. CHEM., vol. 269, 1994, pages 27833 - 27839
ATGER ET AL., J. CLIN. INVEST., vol. 99, 1997, pages 773
BAROCHIA ET AL., AM. J. RESPIR. CRIT. CARE MED., vol. 191, 2015, pages 990 - 1000
BARTH ET AL., TRENDS CARDIOVASC. MED., vol. 11, 2001, pages 26 - 31
BARTUS ET AL., SCIENCE, vol. 281, 1998, pages 1161
BASS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 4498 - 4502
BATUKLA ET AL., ANN. NY ACAD. SCI., vol. 1108, 2007, pages 137 - 146
BEAMING ET AL., J. GEN. VIROL., vol. 75, 1994, pages 1551 - 1556
BIELICKI, CURR. OPIN. LIPIDOL., vol. 27, 2016, pages 40 - 46
BISWAS ET AL., EUR. J. IMMUNOL., vol. 45, 2015, pages 3158 - 3173
BLACK ET AL., J. IMMUNOL., vol. 195, 2015, pages 4685 - 4698
BLANKENBERG ET AL., J. LIPID RES., vol. 44, 2003, pages 1381 - 1386
BOADO ET AL., BIOTECHNOL. BIOENG., vol. 108, 2011, pages 186 - 196
BOADO ET AL., OL. PHARM., vol. 5, 2008, pages 1037 - 1043
BOADO, BIOTECHNOLOGY AND BIOENGINEERING, vol. 108, 2011, pages 186 - 196
BOSS ET AL., NUC. ACIDS RES., vol. 12, 1984, pages 3791 - 3806
BOTHWELL ET AL., NATURE, vol. 298, 1982, pages 380 - 382
BOWIE; SAUER, PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 2152 - 2156
BREINER ET AL., GENE, vol. 18, 1982, pages 165 - 174
BREMER ET AL., PHARM. BIOTECHNOL., vol. 10, 1997, pages 239
BREMER ET AL.: "Protein Delivery: Physical Systems", 1997, PLENUM PRESS, article "Protein Delivery with Infusion Pumps", pages: 239 - 254
CATTEPOEL ET AL., PLOS ONE, vol. 6, 2011, pages e 18296
CHAN, J. BIOL. CHEM., vol. 290, 2015, pages 10958 - 71
CHARLES-SCHOEMAN ET AL., ANN. RHEUM. DIS., vol. 71, 2012, pages 1157 - 1162
CHARLES-SCHOEMAN ET AL., CLIN. IMMUNOL., vol. 127, 2008, pages 234 - 244
CHARLES-SCHOEMAN, ARTHRITIS RHEUM., vol. 60, 2009, pages 2870 - 2879
CHARLES-SCHOEMAN, CLIN IMMUNOL., vol. 127, 2008, pages 234 - 44
CHAZENBALK; RAPOPORT, J. BIOL. CHEM., vol. 270, 1995, pages 1543 - 1549
CHIRGWIN ET AL., BIOCHEMISTRY, vol. 18, 1979, pages 52 - 94
CHO ET AL., EXP MOL MED, vol. 41, 2009, pages 417
CICCARONE ET AL., FOCUS, vol. 15, 1993, pages 80
CLIMIE ET AL., PROC. NATL. ACAD. SCI. US4, vol. 87, 1990, pages 633 - 637
CORSARO; PEARSON, SOMATIC CELL GENETICS, vol. 7, 1981, pages 603
CREIGHTON, PROTEINS, 1984
CROSBY ET AL., BIOCHEM. CELL BIOL., vol. 10, 2015
CUNNINGHAM ET AL., SCIENCE, vol. 253, 1991, pages 545 - 548
CUNNINGHAM ET AL., SCIENCE, vol. 254, 1991, pages 821 - 825
CUNNINGHAM; WELLS, J. MO/. BIOL., vol. 234, 1993, pages 554 - 563
CUNNINGHAM; WELLS, SCIENCE, vol. 244, 1989, pages 1081 - 1085
DAVIS ET AL., ANN. REV. IMMUNOL., vol. 25, 2007, pages 525 - 60
DERBYSHIRE ET AL., GENE, vol. 46, 1986, pages 145
DOONAN: "Protein Purification Protocols", 1996, THE HUMANA PRESS
DREW ET AL., NATURE REVIEWS ENDOCRINOLOGY, vol. 8, 2012, pages 237
EHRHARDT; COOPER, CURR. TOP. ALICROBIOL. IMMUNOL., 3 August 2010 (2010-08-03)
ELLISON ET AL., DNA, vol. 1, 1981, pages 11 - 18
ELLISON ET AL., NUC. ACIDS RES., vol. 10, 1982, pages 4071 - 4079
EREN, CHOLESTEROL, 2013, pages 792090
ESTIN ET AL., ADV. EXP. MED. BIOL., vol. 660, 2010, pages 183 - 193
FARES ET AL., ENDOCRINOLOGY, vol. 139, 1998, pages 2459 - 2464
FIORAVANTI ET AL., J. IMMUNOL., vol. 188, 2012, pages 3988 - 3992
FISCHER ET AL., THROMB. HAEMOST., vol. 108, 2012, pages 730 - 741
FORD ET AL., PROTEIN EXPRESSION AND PURIFICATION, vol. 2, 1991, pages 95 - 107
GARDNER ET AL., FRONTIERS IN PHARMACOLOGY, 20 November 2015 (2015-11-20)
GLEESON ET AL., J. GEN. MICROBIOL., vol. 132, 1986, pages 3459 - 3465
GLICK; PASTERNAK: "Molecular Biotechnology", 1994, ASM PRESS, article "Principles & Applications of Recombinant DNA"
GOGONEA, FRONTIERS PHARMACOL., vol. 6, 2016, pages 318
GOLMANESH ET AL., IMMUNOPHARMACOL. IMMUNOTOXICAL., vol. 35, 2013, pages 419 - 425
GOMBOTZ; PETTIT, BIOCONJUGATE CHEM., vol. 6, 1995, pages 332
GOULD ET AL., FOOD CHEM TOXICOL, vol. 43, 2005, pages 1451
GRAHAM ET AL., J. GEN. VIROL., vol. 36, 1977, pages 59 - 72
GRAHAM; VAN DER EB, VIROLOGY, vol. 52, 1973, pages 456
GRAVERSEN ET AL., J. CARDIOVASCULAR PHARMACOL., vol. 51, 2008, pages 170 - 77
GRAVES; SCOTT, CURR CHEM GENOMICS, vol. 2, 2008, pages 51 - 61
GREF ET AL., PHARM. BIOTECHNOL., vol. 10, 1997, pages 167
GRIMALDI ET AL., EXPERT OPIN. BIOL. THER., vol. 16, 2016, pages 433 - 41
GRYPIOTI ET AL., DIG. DIS. SCI., vol. 52, 2007, pages 2580 - 2590
GRYPIOTI ET AL., DIG. DIS. SCI., vol. 53, 2008, pages 1054 - 1062
GUNTURI ET AL., CURR. TREAT. OPTIONS ONCOL., vol. 15, 2014, pages 137 - 46
HAANEN ET AL., PROG. TUMOR RES., vol. 42, 2015, pages 55 - 66
HALLEWELL ET AL., J. BIOL. CHEM., vol. 264, 1989, pages 5260 - 5268
HAWLEY-NELSON ET AL., FOCUS, vol. 15, 1993, pages 73
HENIKOFF; HENIKOFF, PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 10915
HEWING ET AL., ARTERIOSCLER. THROMB. VASE. BIOL., vol. 34, 2014, pages 779 - 89
HILL-PERKINS; POSSEE, J. GEN. VIROL., vol. 71, 1990, pages 971 - 976
HUANG ET AL., J. CLIN. INVEST., vol. 123, 2013, pages 3815 - 3828
HUANG, ARCH BIOCHERN BIOPHYS, vol. 570, 2015, pages 14 - 22
HUANG, J. INTEGR. A1ED., vol. 12, 2014, pages 76
ISO-O ET AL., MOLECULAR THERAPY, vol. 13, 2006, pages 118 - 126
ITAKURA ET AL., ANN. REV. BIOCHEM., vol. 53, 1984, pages 323 - 356
JUN ET AL., LANCET, vol. 375, 2010, pages 1875
KARABINA ET AL., BIOCHIM. BIOPHYS. ACTA, vol. 1761, 2006, pages 1351 - 1358
KARLIN ET AL., J. MOL. EVOL., vol. 22, 1985, pages 195 - 208
KARLSSON, J. IMMUNOL. METHODS, vol. 145, 1991, pages 229 - 240
KEENEY ET AL., PROTEOMICS CLIN APPL., vol. 7, 2013, pages 109 - 122
KEENEY ET AL., PROTEOMICS CLIN. APPL., vol. 7, 2013, pages 109 - 122
KEMPEN ET AL., J. LIPID. RES., vol. 54, 2013, pages 2341 - 2353
KENTEN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 79, 1982, pages 6661 - 6665
KHANDEKAR ET AL., J. BIOL. CHEM., vol. 272, 1997, pages 32190 - 32197
KIM ET AL., AM. J. RESPIR. CRIT. CARE MED., vol. 182, 2010, pages 633 - 642
KIM ET AL., BIOMATERIALS, vol. 64, 2015, pages 45 - 56
KINDSVOGEL ET AL., DNA, vol. 1, 1982, pages 335 - 343
KING; POSSEE: "The Baculovirus Expression System: A Laboratory Guide", CHAPMAN & HALL
KINGWELL ET AL., CIRCULATION, vol. 128, 2013, pages 1112
KISS ET AL., ANN. NY ACAD. SCI., vol. 108, 2007, pages 83 - 91
KOLDAMOVA ET AL., BIOCHEMISTRY, vol. 40, 2001, pages 3553
KONDO ET AL., EUR. J. IMMUNOL., vol. 23, 1993, pages 245 - 249
KOREN-GLUSER ET AL., ATHEROSCLEROSIS, vol. 219, 2011, pages 510 - 518
KOSMAS ET AL., CLINICAL MEDICAL INSIGHTS: CARDIOLOGY, vol. 10, 2016, pages 37 - 42
KOSTELNEY ET AL., J. IMMUNOL., vol. 148, 1992, pages 1547 - 1553
KOUTSIS ET AL., MULT. SCLER., vol. 15, 2009, pages 174 - 179
KRITHARIDES ET AL., J. BIOL. CHEM., vol. 271, 1996, pages 27450
LEHMANN ET AL., J. IMMUNOL, vol. 189, 2012, pages 1448 - 58
LEWIS ET AL., JBIOL. CHEM., vol. 285, 2010, pages 36958 - 36968
LIBONATI; SORRENTINO, METHODS ENZYMOL., 2001, pages 341234 - 248
LIU ET AL., J. BIOL. CHEM., vol. 287, 2012, pages 37321 - 37329
LIU ET AL., J. LIPID RES., vol. 44, 2003, pages 1156
LOFTSSON ET AL., EXPERT OPIN. DRUG DELIV., vol. 2, 2005, pages 335
LU ET AL., J. IMMUNOL. ALETH., vol. 267, 2002, pages 213 - 226
LUCKOW ET AL., J. VIROL., vol. 67, 1993, pages 4566 - 4579
LUI, MOL. CANCER, vol. 13, 2014, pages 1186
LUND-KATZ; PHILLIPS, SUBCELL BIOCHEM., vol. 51, 2010, pages 183 - 227
MACKNESS ET AL., ARTERIOSCLER. THROMB. VASE. BIOL., vol. 26, 2006, pages 1545 - 50
MACKNESS ET AL., GENE, vol. 567, 2015, pages 12 - 21
MALLE ET AL., BR J PHARMACOL., vol. 152, 2007, pages 838 - 854
MCMAHON ET AL., ATHRITIS RHEUM., vol. 60, 2009, pages 2428 - 2437
MENINI ET AL., REDOX REP., vol. 19, 2014, pages 49 - 58
MEYERS ET AL., J. NEUROIMMUNOL., vol. 277, 2014, pages 176 - 185
MILLER, F100RESEARCH, vol. 3, 2014, pages 124
MINEO ET AL., CIRC. RES., vol. 111, 2012, pages 1079 - 1090
MIZUGUCHI ET AL., J. BIOL. CHEM., vol. 290, 2015, pages 20947 - 20959
MURRAY, AM. J. RESPIR. CRIT. CARE MED., vol. 178, 2008, pages 1227 - 1237
NANJEE ET AL., ARTERIOSCLER THROMB VASE BIOL, vol. 16, 1996, pages 1203 - 1214
NEEDLEMAN; WUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 444
NER ET AL., DNA, vol. 7, 1988, pages 127
NEUMANN, EMBO J., vol. 1, 1982, pages 841 - 845
NEWTON ET AL., BLOOD, vol. 97, 2001, pages 528 - 535
NGIOW ET AL., ADV. IMMUNOL., vol. 130, 2016, pages 1 - 24
NILSSON ET AL., EMBO J., vol. 4, 1985, pages 1075
NILSSON ET AL., METHODS ENZYMOL., vol. 198, 1991, pages 3
NISSEN ET AL., JAMA, vol. 290, 2003, pages 2292
OBICI ET AL., AMYLOID, vol. 13, 2006, pages 191 - 205
OCHOA ET AL., CANCER RES., vol. 73, 2013, pages 139 - 149
O'REILLY ET AL.: "Baculovirus Expression Vectors: A Laboratory Manual", 1994, OXFORD UNIVERSITY PRESS
PADLAN, MOL. IMMUNOL., vol. 31, 1994, pages 169 - 217
PAULA-LIMA ET AL., INT. J. BIOCHEM. CELL BIOL., vol. 41, 2009, pages 1361
PEARSON, METH. ENZYMOL., vol. 183, 1990, pages 63
PEARSON; LIPMAN, PROC. NAT'L ACAD SCI. USA, vol. 85, 1988, pages 2444
PENG ET AL., TOXICOL. LETT., vol. 193, 2010, pages 159 - 166
PERUSKI; PERUSKI: "The internet and the New Biology: Tools for Genomic and Molecular Research", 1997, ASM PRESS, INC.
PHILLIPS, JOURNAL OF LIPID RESEARCH, vol. 54, 2013, pages 2034 - 2048
PIRILLO ET AL., HANDB EXP PHARMACOL., vol. 224, 2015, pages 483 - 508
PRIETO ET AL., PROTEIN J., vol. 31, 2012, pages 681 - 688
PROC. NATL. ACAD. SCI. US, vol. 4, no. 69, 1972, pages 1408 - 1412
PUTNAM: "The Plasma Proteins", vol. V, 1987, ACADEMIC PRESS, INC., pages: 49 - 140
PUTNEY, CURR. OPIN. CHEM. BIOL., vol. 2, 1998, pages 548
PUTNEY; BURKE, NATURE BIOTECHNOLOGY, vol. 16, 1998, pages 153
RADZIEJEWSKI ET AL., BIOCHEM., vol. 32, 1993, pages 1350
RANADE: "Drug Delivery Systems", 1995, CRC PRESS, article "Implants in Drug Delivery", pages: 95 - 123
RANADE: "Drug Delivery Systems", 1995, CRC PRESS, article "Role of Polymers in Drug Delivery", pages: 51 - 93
RANADE; HOLLINGER: "Drug Delivery Systems", 1996, CRC PRESS
RAYMOND ET AL., YEAST, vol. 14, 1998, pages 11 - 23
REDDY ET AL., CURR. OPIN. LIPIDOL., vol. 25, 2014, pages 304 - 308
REGENESS-LECHNER ET AL., TOXILOGICAL SCIENCES, vol. 150, 2016, pages 378 - 89
REIDHAAR-OLSON; SAUER, SCIENCE, vol. 241, 1988, pages 53 - 57
RICHTER ET AL., CIRCULATION CARDIOVASCULAR GENETICS, vol. 1, 2008, pages 147 - 152
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 327
ROBINSON; SAUER, BIOCHEMISTRY, vol. 35, 1996, pages 109 - 116
ROSENBLAT ET AL., BIOFACTORS, vol. 37, 2011, pages 462 - 467
ROSKOS; MASKIEWICZ: "Protein Delivery: Physical Systems", 1997, PLENUM PRESS, article "Degradable Controlled Release Systems Useful for Protein Delivery", pages: 45 - 92
ROTH ET AL., BIOORG MED CHEM, vol. 22, 2014, pages 6422 - 6429
ROUSSET ET AL., J PHARMACOL EXP THER., vol. 335, 2010, pages 140 - 8
RUSCONI; KOHLER, NATURE, vol. 314, 1985, pages 330 - 334
RYAN ET AL., PROTEIN EXPR. PURIF, vol. 27, 2003, pages 98 - 103
SAM BROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SANKARANARARYANAN ET AL., J. LIPID RES., vol. 52, 2011, pages 2332 - 2340
SCATCHARD, ANN. NY ACAD. SCI., vol. 51, 1949, pages 660
SCATCHARD, ANN. NY ACAD. SCI., vol. 51, 1949, pages 660 - 672
SCOPES: "Protein Purification: Principles and Practice", 1994, SPRINGER-VERLAG
SELLERS, SIAM J. APPL. MATH., vol. 26, 1974, pages 787
SENO ET AL., NUC. ACIDS RES., vol. 11, 1983, pages 719 - 726
SHAO ET AL., J. BIOL. CHEM., vol. 281, 2006, pages 9001 - 4
SILVA ET AL., NATL. ACAD. SCI. USA, vol. 105, 2008, pages 12176 - 12181
SILVEMAN ET AL., NATURE BIOTECHNOLOGY, vol. 23, 2005, pages 1556 - 1561
SIMSEKYILMAZ ET AL., CIRCULATION, vol. 129, 2014, pages 598 - 606
SINKAR ET AL., J. BIOSCI. (BANGALORE, vol. 11, 1987, pages 47 - 58
SKAGGS ET AL., CLIN. IMMUNOL., vol. 137, 2010, pages 147 - 156
SMALLSHAW ET AL., PROTEIN ENG., vol. 12, 1999, pages 623 - 630
SMITH; JOHNSON, GENE, vol. 67, 1988, pages 31
SMYTHIES ET AL., AM. J. PHYSIOL. CELL PHYSIOL., vol. 298, 2010, pages 1538 - 48
SONG ET AL., INT. J. BIOL. SCI., vol. 5, 2009, pages 637 - 646
SORENSON, ARTERIOSCLEROSIS, THROMBOSIS, AND VASCULAR BIOLOGY, vol. 19, 1999, pages 2214 - 2225
SOUBHYE ET AL., J MED CHEM, vol. 53, 2010, pages 8747 - 8759
SOUBHYE ET AL., J MED CHEM, vol. 56, 2013, pages 3943 - 58
SPAHR ET AL., PROTEIN SCI., vol. 22, 2013, pages 1739 - 53
STAFFORINI, CARDIOVASC. DRUGS THER., vol. 23, 2009, pages 73 - 83
STEFFENSEN, EXPERT OPIN. THER. TARGETS, vol. 17, 2013, pages 977
STEMMER, NATURE, vol. 370, 1994, pages 389
STEMMER, PROC. NAT'L ACAD SCI. USA, vol. 91, 1994, pages 10747
STEVENS ET AL., PROC. NATL. ACAD. SEI. USA, vol. 105, 2008, pages 12780 - 12784
SU ET AL., PROC. NATL. ACAD. SCI. USA, vol. 107, 2010, pages 19997 - 20002
SULKOWSKI, TRENDS IN BIOCHEM., vol. 3, 1985, pages 1
SUN ET AL., J. IMMUNOL., vol. 190, 2013, pages 2536 - 2543
SWAINSON ET AL., J. IMMUNOL., vol. 184, 2010, pages 3639 - 47
TANG ET AL., J LIPID RES., vol. 47, 2006, pages 107 - 14
TARAVATI ET AL., IMMUNOPHARMACOL. IMMUNOTOXICOL., vol. 34, 2012, pages 706 - 713
TOPALIAN, NAT. REV. CANCER, vol. 16, 2016, pages 275 - 87
URLAUB ET AL., PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216 - 4220
VAN DER LOO ET AL., IMMUNOGENETICS, vol. 42, 1995, pages 333 - 341
WALBERER ET AL., CURR. NEUROVASC. RES., vol. 6, 2009, pages 12 - 19
WANG ET AL., CYTOKINE, vol. 49, 2010, pages 194 - 2000
WATSON ET AL., J. CLIN. INVEST., vol. 95, 1995, pages 774 - 782
WEBER, SEMIN. ONCOL., vol. 37, 2010, pages 430 - 439
WHITE ET AL., J. LIPID. RES., vol. 55, 2014, pages 2007 - 2021
WIGLER ET AL., CELL, vol. 14, 1978, pages 725
WU ET AL., ARTERIOSCLER. THROMB. BASE. BIOL., vol. 34, 2014, pages 543 - 551
WU, ARTERIOSCLER THROMB VASE BIOL, vol. 34, 2014, pages 543 - 551
YANCEY ET AL., J. LIPID RES., vol. 45, 2004, pages 337 - 346
YEWEY ET AL.: "Protein Delivery: Physical Systems", 1997, PLENUM PRESS, article "Delivery of Proteins from a Controlled Release Injectable Implant", pages: 93 - 117
YIN ET AL., J. ATHEROSCLEROSIS AND THROMBOSIS, vol. 19, 2012, pages 923 - 36
ZAHND ET AL., J. MOL. BIOL., vol. 369, 2007, pages 1015 - 1028
ZAMANIAN-DARYOUSH ET AL., J. BIOI. CHEM., vol. 288, 2013, pages 21237 - 21252
ZAMANIAN-DARYOUSH ET AL., J. BIOL. CHEM., vol. 288, 2013, pages 21237 - 21252
ZHANG ET AL., ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES, 2011, pages 136 - 146
ZIMMER ET AL., SCIENCE TRANSLALIONAL MEDICINE, vol. 8, 2016, pages 333

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022178078A1 (fr) * 2021-02-19 2022-08-25 Theripion, Inc. Polypeptides de fusion paraoxonase et compositions et méthodes associées
US20240101982A1 (en) * 2021-02-19 2024-03-28 Theripion, Inc. Paraoxonase fusion polypeptides and related compositions and methods

Also Published As

Publication number Publication date
WO2018136163A3 (fr) 2018-09-27

Similar Documents

Publication Publication Date Title
US11472864B2 (en) Polynucleotides encoding APOA1-PON1 fusion polypeptides
JP7191076B2 (ja) Ｆｇｆ２１突然変異体及びその使用
CN105612175B (zh) 基于IL-15和IL-15Rαsushi结构域的调节因子
JP2022095925A (ja) ヒトヒアルロニダーゼｐｈ２０の変異体及び薬物を含む皮下投与用医薬組成物
EP3896083B1 (fr) Protéines de fusion fc comprenant de nouveaux lieurs et arrangements
CN107964539B (zh) 治疗性核酸酶组合物和方法
KR20180120245A (ko) 유도성 결합 단백질 및 사용 방법
JP7102670B2 (ja) 抗ｐｄ‐ｌ１抗体とｉｌ‐７との融合
EP3355908A1 (fr) Traitement de troubles liés à l'acide biliaire
WO2018136163A2 (fr) Polypeptides de fusion apoa-1 en tandem
NZ738963B2 (en) Apoa-1 fusion polypeptides and related compositions and methods
US20230015291A1 (en) Dnase fusion polypeptides and related compositions and methods
US20220112258A1 (en) Bifunctional molecules with il-7 activity
WO2024050058A2 (fr) Variants ul141, leurs compositions et leurs procédés d'utilisation pour l'immunothérapie anticancéreuse

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17870650

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17870650

Country of ref document: EP

Kind code of ref document: A2