WO2024259220A1

WO2024259220A1 - Pon3 and evolved pon1 fusion polypeptides

Info

Publication number: WO2024259220A1
Application number: PCT/US2024/033987
Authority: WO
Inventors: Jeffrey A. Ledbetter; Martha S. Hayden-Ledbetter
Original assignee: Theripion Inc
Current assignee: Theripion Inc
Priority date: 2023-06-15
Filing date: 2024-06-14
Publication date: 2024-12-19
Anticipated expiration: 2025-12-15

Abstract

Compositions and methods relating to paraoxonase 3 (PON3) and evolved paraoxonase 1 (PON1) fusion polypeptides are disclosed. The fusion polypeptides include a PON3 or evolved PON1 polypeptide linked carboxyl-terminal or amino-terminal to a dimerizing domain or a domain that specifically binds to the neonatal Fc receptor (FcRn). In certain variations, a dimerizing or FcRn-binding domain is an immunoglobulin Fc region. In some embodiments comprising the PON3 or evolved PON1 linked carboxyl-terminal to the dimerizing or FcRn-binding domain, the fusion polypeptide further includes a biologically active polypeptide (e.g., a CTLA-4 extracellular domain or a CD40 extracellular domain) linked amino-terminal to the dimerizing or FcRn-binding domain. Also disclosed are dimeric proteins comprising first and second PON3 or evolved PON1 fusion polypeptides as disclosed herein. The fusion polypeptides and dimeric proteins are useful in methods for therapy.

Description

PON3 AND EVOLVED PON1 FUSION POLYPEPTIDES

CROSS-REFERENCE TO RELATED APPLICATION

[1] This application claims the benefit of U.S. Provisional Application No. 63/508,318, filed June 1 , 2023, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

[2] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML Copy, created on June 13, 2024, is named "4346-P5WO_Seq_Listing_ST26.xml" and is 142,762 bytes in size.

BACKGROUND OF THE INVENTION

Paraxonase 3 (PON3)

[3] The paraoxonase gene family consists of three genes located on the long arm of chromosome seven. All three gene products are antioxidant enzymes that protect lipids from oxidation. For reviews, see Kowalska et al., Ann. Clin. Lab. Sci. 45:226-233, 2015; Litvinov et al., North Am. J. Med. Sci. 4:523-532, 2012. Paraoxonase 2 (PON2) is the ancestral gene with PON 1 and PON3 arising by gene duplication. The three paraoxonase enzy mes show about 70% amino acid sequence identity and each has unique functions and overlapping substrate specificities. PON1 and PON3 are bound and carried by HDL-cholesterol while PON2 is associated with the mitochondrial membrane. Importantly, the binding of PON 1 and PON3 to HDL-cholesterol enhances enzy me activity and is thought to be essential for enzyme function, acting as a transporter and stabilizer. Each PON shows a unique specificity towards substrates in vitro. PON3 is 100-fold more potent than PON1 in protecting LDL-cholesterol from oxidation, but is present in much lower (0.5%) amounts (Draganov et al., J. Biol. Chem. 275:33435, 2000). (For reviews, see Priyanka et al., Biomolecules 2019, 9, 817; doi: 10.3390/biom9120817. PMID 31816846; Vercic et al., Molecules 2020, 25, 5980; doi: 10.3390/molecules. PMID 33348669: Mohammed et al.. Antioxidants 2022, 11, 590. https://doi.org/10.3390/antioxl l030590, PMID 35326240.) [4] P0N3 has lactonase and arylesterase activity but does not also have orghanophosphatase activity like P0N1. P0N1 is more active than PON3 towards dihydrocoumarin, a small lactone, while PON3 is more active towards the larger, bulky- statin lactones such as lovastatin and simvastatin (Mohammed et al., supra). PON3 has a larger active site and can hydrolyze bioactive lactones formed during activation of the arachidonic acid pathway (Teiber et al., Biochemical and Biophysical Research Communications 505 (2018) 87e92). These lactones may represent natural substrates of PON3. Other natural substrates include oxidized HDL and LDL-cholesterol, homocysteine thiolactone, and N-acyl homoserine lactones that are bacterial quorum-sensing molecules.

[5] PON3 has been found to be active in regulating growth and differentiation of cancer cells. In multi-drug resistant esophageal tumor cells, the PON3 gene is down regulated by hypermethylation in the promoter region. Decreasing or increasing PON3 expression with siRNA or by transfection of GFP-PON3 in esophageal tumor cell lines provided evidence that PON3 levels regulate sensitivity to chemotherapeutic drugs (see Huang et al.. Cancer Cell Int. 18: 1-11, 2018). In prostate tumor cells, hypermethylation and downregulation of the PON3 gene were associated with smoking and poor prognosis (see Shui et al., Cancer 122:2168, 2016).

[6] Analysis of Oncomine database found that PON3 mRNA expression is decreased in hepatocellular carcinoma (see Jin etal., Tumor Biol. 37: 14193, 2016). Other liver datasets confirmed that PON3 mRNA is significantly decreased in hepatocellular carcinomas. PON3 expression levels strongly correlated with cumulative and overall survival rate. Dataset analysis also shows that PON3 mRNA is decreased in 13 of 20 cancer ty pes, and increased in one (sarcomas) (see id.).

[7] Another study found that PON3 mRNA and protein are decreased in hepatocellular carcinomas, and that decreased PON3 expression predicted shorter progression- free survival (see Cai et al., Oncotarget 7:70045, 2016). This study showed that PON3 regulates cell proliferation in vitro and in vivo, and PON3 expression increases cell cycle arrest.

[8] In oral squamous cell carcinoma, PON3 was found to promote proliferation and invasion (see Zhu et al., Biomedicine and Pharmacotherapy 85:712, 2017). The increased expression in some cancer cells has indicated that PON3 may contribute to tumor growth bydecreasing mitochondrial ROS-induced apoptosis (see Witte et al., J. Lipids Volume 2012, Article ID 342806). This means that PON3 may have suppressive or enhancing effects on tumor cells depending on the cell type.

[9] Other studies have shown that (a) potent PON3 antioxidant activity protects LDL-cholesterol from oxidation and prevents inflammatory' foam cell formation (see Draganov et al.. J. Biol. Chem. 275: 33435, 2000; Aviram et al., Free Rad. Biol. Med. 37: 1304, 2004); (b) PON3 is depleted from the high-density lipoproteins of autoimmune disease patients with subclinical atherosclerosis (Marsillach et al., J. Proteome Res. 14:2046, 2015); (c) human PON3 transgenic mice have decreased obesity and atherosclerosis (see Shih et al., Circ. Res. 100: 1200-1207, 2007); (d) adenovirus-mediated PON3 expression protects against the progression of atherosclerosis in Apo E knockout mice (see Ng et al., Arterioscler. Thromb. Vase. Biol. 27:1368-1374, 2007; Zhang et al.. Gene Ther. 17:626-633, 2010); (e) PON3 knockout mice are susceptible to atherosclerosis, obesity, and gallstones (see Shih et al., Arterioscler. Thromb. Vase. Biol. 27: 1368-1374, 2007); and (f) transgene-expressed human PON3 has protective effects against CC14-induced subacute liver injury in mice (see Peng et al., Biomed. Pharmacother. 63:592-598, 2009).

Evolved PON1

[10] Enzymes that hydrolyze nerve agents and organophosphates are desirable for prophylactic protection from toxicity. Human butyrylcholinesterase is promising since it is non-immunogenic and has an extended half-life (see Lockridge, Pharmacology and Therapeutics 148:34, 2015). Butyrylcholinesterase binds and inhibits nerve agents in a stochiometric rather than enzymatic manner (see Cerasoli et al. , Biochemical Pharmacology 171 : 113670, 2020). Human butyrylcholinesterase inhibits cocaine and has been studied in man as a therapy for cocaine addiction. It is currently purified from plasma where it is present at 3.5 to 9.3 mg/liter (see id.). While butyrylcholinesterase has many of the desirable properties for therapy including low immunogenicity and long half-life, it is limited by its stochiometric rather than enzymatic activity, and by its limitations in manufacturing. As the best current candidate, it is being lyophylized and stockpiled by the US military (see id.).

[11] Human PON1 purified from plasma or expressed as a recombinant protein can digest nerve agents and has been shown to protect guinea pigs from the toxic effects of nerve agent exposure (see Valiyaveettil et al., Biochemical Pharmacology 81:800, 2011; Valiyaveettil et al.. Toxicology Letters 202: 203, 2011). However, the short half-life and low activity of wild-type human P0N1 against nerve agents are limitations to this approach (see Valiyaveettil et al., Toxicology Letters 210:87, 2012; Hodgins et al., Chem. Biol. Interact. 203: 177, 2013).

[12] Recombinant human PON1, including the noncleaved leader sequence, has been fused with domain three of human albumin (see Dobariya et al. , Enzyme Microb. Technol. 165: 110209, 2023). This fusion protein was expressed in bacteria and refolded to generate active protein. Rats exposed to one LD50 OP poison (paraoxon) exhibited partial protection from organophosphate poisoning when this fusion protein was given two hours prior to exposure.

[13] The organophosphatase activity of PON1 has been optimized by mutagenesis, yielding evolved PON1 variants such as IF 11, with greatly enhanced activity towards organophosphates and G-type nerve agent but decreased arylesterase and lactonase activity (see Goldsmith et al., Chem. Biol. 19:456, 2012). The evolved PON1 enzy mes have up to 60 mutations versus human PON 1 and have improved ability to protect from nerve agent toxicityin animals when administered shortly (one hour) before exposure (see Warek et al., Arch. Toxicol. 88: 1257, 2014). Another study used adeno-associated virus gene therapy to express an evolved PON1 (1F11) in the liver (see Betapudi et al., Sci. Trans. Med. 12:0356, 2020). Betapudi et al. found high and persistent expression and durable protection from nerve agents for over four months.

SUMMARY OF THE INVENTION

[14] In one aspect, the present invention provides a fusion polypeptide comprising, from an amino terminal position to a carboxyl terminal position. X-Ll-P, wherein X is a dimerizing domain or a domain that specifically binds to the neonatal Fc receptor (FcRn); LI is a polypeptide linker, wherein LI is optionally present; and P is a biologically active paraoxonase, wherein the paraoxonase has at least 90% or at least 95% identity wi th the amino acid sequence shown in residues 21-354 or 31-354 of SEQ ID NO:30 and does not contain an amino terminal leader sequence corresponding to residues 1-20 of SEQ ID NO:30. In specific variations, the paraoxonase has an amino acid sequence selected from residues n-354 of SEQ ID NO:30, wherein n is an integer from 21 to 31, inclusive. [15] In another aspect, the present invention provides a fusion polypeptide comprising, from an amino terminal position to a carboxyl terminal position, X-Ll-P, wherein X is a dimerizing domain or a domain that specifically binds to the neonatal Fc receptor (FcRn); LI is a polypeptide linker, wherein LI is optionally present; and P is a biologically active paraoxonase, wherein the paraoxonase comprises an amino acid sequence as shown in (i) residues 16-355 or 26-355 of SEQ ID NO:55 or (ii) residues 16-355 or 26-355 of SEQ ID NO:56, and wherein the paraoxonase does not contain an amino terminal leader sequence corresponding to residues 1-15 of SEQ ID NO: 2. In some variations, the paraoxonase has an amino acid sequence selected from the group consisting of residues n-355 of SEQ ID NO:55, wherein n is an integer from 16 to 26, inclusive. In other embodiments, the paraoxonase has an amino acid sequence selected from the group consisting of residues n-355 of SEQ ID NO:56, wherein n is an integer from 16 to 26, inclusive.

[16] In certain embodiments of a fusion polypeptide as above, LI is present and comprises at least eight amino acid residues. In some such embodiments, LI consists of from 12 to 25 amino acid residues. A particularly suitable LI linker has the amino acid sequence shown in SEQ ID NO: 12.

[17] In some embodiments of a fusion polypeptide as above, the fusion polypeptide does not comprise a biologically active polypeptide N-terminal to the dimerizing domain or domain that specifically binds to FcRn. In other variations, the fusion polypeptide further comprises a biologically active polypeptide N-terminal to the dimerizing domain or domain that specifically binds FcRn, wherein the fusion polypeptide comprises, from an aminoterminal position to a carboxyl-terminal position, T-L2-X-L1-P, wherein X, LI, and P are as defined above, L2 is a second polypeptide linker, wherein L2 is optionally present, and T is the biologically active polypeptide. In some such embodiments, the biologically active polypeptide N-terminal to the dimerizing or FcRn-binding domain is selected from a cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) extracellular domain and a CD40 extracellular domain.

[18] In some embodiments of a fusion polypeptide as above comprising a CTLA-4 extracellular domain as the biologically active polypeptide N-terminal to the dimerizing or FcRn-binding domain, the CTLA-4 extracellular domain has at least 90% or at least 95% identity with the amino acid sequence shown in residues 21-144 of SEQ ID NO:26. In a specific variation, the CTLA-4 extracellular doman has the amino acid sequence shown in residues 21-144 of SEQ ID NO:26.

[19] In some embodiments of a fusion polypeptide as above comprising a CD40 extracellular domain as the biologically active polypeptide N-terminal to the dimerizing or FcRn-binding domain, the CD40 extracellular domain has at least 90% or at least 95% identity with the amino acid sequence shown in residues 21-188 of SEQ ID NO:42. In a specific variation, the CD40 extracellular doman has the amino acid sequence shown in residues 21- 188 of SEQ ID NO:42. In other variations, the CD40 extracellular domain contains at least one amino acid substitution at a position corresponding to an amino acid of human CD40 (SEQ ID NO:46) selected from the group consisting of E64, K81, P85, and L 121, wherein the at least one amino acid substitution increases CD40 ligand binding relative to human CD40. Particularly suitable amino acid subsitutions at these positions are tyrosine at the position corresponding to E64 of human CD40, threonine, histidine, or serine at the position corresponding to K81 of human CD40. tyrosine at the position corresponding to P85 of human CD40, and/or proline at the position corresponding to LI 21 of human CD40. In some variations, the amino acid at the position corresponding to K81 of human CD40 is selected from threonine, histidine, and serine; the amino acid at the position corresponding to K81 of human CD40 is histidine and the amino acid at the position corresponding to L 121 of human CD40 is proline; or the amino acid at the position corresponding to E64 of human CD40 is tyrosine, the amino acid at the position corresponding to K81 of human CD40 is threonine, and the amino acid at the position corresponding to P85 of human CD40 is tyrosine.

[20] In some embodiments of a fusion polypeptide, X is a dimerizing domain that specifically binds to FcRn. In some embodiments, X is an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region is capable of forming dimers and specifically binding FcRn. A particularly suitable immunoglobulin heavy chain constant region is an immunoglobulin Fc region. In some embodiments comprising an immunoglobulin Fc region, the Fc region is a human Fc region such as. e g., a human Fc variant comprising one or more (e.g. , from one to 10) amino acid substitutions relative to the wild-type human sequence. Particularly suitable Fc regions include human y 1 and y4 Fc regions. In some variations, the Fc region is a human yl 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. In further variations comprising an Fc region as above, the Fc region is a human yl Fc variant in which Eu residue P331 is replaced by serine. In still further variations comprising an Fc region as above, the Fc region is a human yl Fc variant in which Eu residue M252 is replaced by tyrosine, Eu residue S254 is replaced by threonine, and/or Eu residue T256 is replaced by glutamate. In some embodiments, the Fc region has at least 90% or at least 95% identity with the amino acid sequence shown in (i) residues 1-232 or 1-231 of SEQ ID NO:6, (ii) residues 1-232 or 1-231 of SEQ ID NO:8, (iii) residues 1-232 or 1-231 of SEQ ID NO:48, (iv) residues 1-232 or 1-231 of SEQ ID NO:50, (v) residues 1-232 or 1-231 of SEQ ID NO:52, or (vi) residues 1-232 or 1-231 of SEQ ID NO:54. In more specific variations, wherein the immunoglobulin heavy chain constant region comprises the amino acid sequence shown in (i) residues 1-232 or 1-231 of SEQ ID NO:6, (ii) residues 1-232 or 1-231 of SEQ ID NO:8, (iii) residues 1-232 or 1-231 of SEQ ID NO:48, (iv) residues 1-232 or 1-231 of SEQ ID NO:50, (v) residues 1-232 or 1-231 of SEQ ID NO:52, or (vi) residues 1-232 or 1-231 of SEQ ID NO:54.

[21] In some embodiments of a fusion polypeptide as above comprising an immunoglobulin heavy chain constant region, the immunoglobulin heavy chain constant region comprises an amino acid sequence having at least 90% or at least 95% identity with the amino acid sequence shown in (i) residues 16-232 or 16-231 of SEQ ID NO:6, (ii) residues 16-232 or 16-231 of SEQ ID NO:48, or (iii) residues 16-232 or 16-231 of SEQ ID NO:52. In more specific variations, the immunoglobulin heavy chain constant region comprises the amino acid sequence shown in (i) residues 16-232 or 16-231 of SEQ ID NO:6, (ii) residues 16-232 or 16- 231 of SEQ ID NO:48, or (iii) residues 16-232 or 16-231 of SEQ ID NO:52.

[22] In certain embodiments of a fusion polypeptide as above, the fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identity with (i) residues 21-607 or 24-607 of SEQ ID NO:32. (ii) residues 21-604 of SEQ ID NO:34, (iii) residues 21- 613 or 24-613 of SEQ ID NO: 18, (iv) residues 21-610 of SEQ ID NO:20, (v) residues 21-739 of SEQ ID NO:26, (vi) residues 21-736 of SEQ ID NO:28, (vii) residues 21-613 or 24-613 of SEQ ID NO:36, (viii) residues 21-610 of SEQ ID NO:38. (ix) residues 21-736 of SEQ ID NO:40, (x) residues 21-803 of SEQ ID NO:42. (xi) residues 21-803 of SEQ ID NO:44, or (xii) residues 21-739 of SEQ ID NO:58. In some such embodiments, the fusion polypeptide comprises an amino acid having at least 96%, at least 97%, at least 98%, or at least 99% identity with (i) residues 21-607 or 24-607 of SEQ ID NO:32, (ii) residues 21-604 of SEQ ID NO:34, (iii) residues 21-613 or 24-613 of SEQ ID NO: 18, (iv) residues 21-610 of SEQ ID NO:20, (v) residues 21-739 of SEQ ID NO:26, (vi) residues 21-736 of SEQ ID NO:28, (vii) residues 21- 613 or 24-613 of SEQ ID NO:36, (viii) residues 21-610 of SEQ ID NO:38, (ix) residues 21- 736 of SEQ ID NO:40, (x) residues 21-803 of SEQ ID NO:42. (xi) residues 21-803 of SEQ ID NO:44. or (xii) residues 21-739 of SEQ ID NO:58. In still more specific variations, the fusion polypeptide comprises the amino acid sequence shown in with (i) residues 21-607 or 24-607 of SEQ ID NO:32, (ii) residues 21-604 of SEQ ID NO:34, (iii) residues 21-613 or 24-613 of SEQ ID NO: 18, (iv) residues 21-610 of SEQ ID NO:20, (v) residues 21-739 of SEQ ID NO:26, (vi) residues 21-736 of SEQ ID NO:28. (vii) residues 21-613 or 24-613 of SEQ ID NO:36, (viii) residues 21 -610 of SEQ ID NO:38, (ix) residues 21 -736 of SEQ ID NO:40, (x) residues 21-803 of SEQ ID NO:42, (xi) residues 21-803 of SEQ ID NO:44, or (xii) residues 21-739 of SEQ ID NO:58.

[23] In another aspect, the present invention provides a dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, wherein each of the first and second fusion polypeptides is a fusion polypeptide as above wherein X is a dimerizing domain (e.g, an immunoglobulin heavy chain constant region such as, for example, an immunoglobulin Fc region).

[24] In another aspect, the present invention provides a polynucleotide encoding a fusion polypeptide as described above.

[25] In still another aspect, the present invention provides an expression cassette comprising a DNA segment encoding a fusion polypeptide as described above and which is operably linked to a promoter. Also provided is a cultured cell into which has been introduced an expression cassette as described above, wherein the cell expresses the DNA segment. In a related aspect, the present invention provides a stable cell line comprising, within its genomic DNA. an expression cassette as described above, wherein the stable cell line constitutively expresses the DNA segment. In some embodiments, the stable cell line is a Chinese hamster ovary (CHO) cell line.

[26] In another aspect, the present invention provides a vector comprising an expression cassette as described above.

[27] In another aspect, the present invention provides a method of making a fusion polypeptide. The method generally includes (i) culturing a cell into which has been introduced an expression cassette as described above, wherein the cell expresses the DNA segment and the encoded fusion polypeptide is produced, and (ii) recovering the fusion polypeptide. In some variations, the cultured cell is a stable cell line as described above.

[28] In yet another aspect, the present invention provides a method of making a dimeric protein. The method generally includes (i) culturing a cell into which has been introduced an expression cassette as described above, wherein the cell expresses the DNA segment and the encoded fusion polypeptide is produced as a dimeric protein, and (ii) recovering the dimeric protein. In some variations, the cultured cell is a stable cell line as described above.

[29] In another aspect, the present invention provides a composition comprising a fusion polypeptide as described above and a pharmaceutically acceptable carrier.

[30] In another aspect, the present invention provides a composition comprising a dimeric protein as described above and a pharmaceutically acceptable carrier.

[31] In some embodiments of a composition as described above, the composition is formulated for delivery to the lung by nebulization.

[32] In still another aspect, 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 protein as described above. In some embodiments, the inflammatory’ disease is an inflammatory⁷ lung disease such as, for example, chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis (CF), bronchiectasis, hypoxia, acute respiratory distress syndrome (ARDS) (e.g, COVID-19-associated ARDS), and interstitial lung disease (e , idiopathic pulmonary fibrosis (IPF) or sarcoidosis); in some variations, the inflammatory⁷ lung disease is characterized by Pseudomonas aeruginosa infection. In other embodiments, the inflammatory disease is selected from an inflammatory bowel disease (IBD) (e.g, Crohn’s disease or ulcerative colitis), systemic lupus erythematosus (SLE) (e.g, SLE with lupus nephritis), type 1 diabetes, and type 2 diabetes. In still other embodiments, the inflammatory⁷ disease is an inflammatory⁷ skin disease such as. for example, psoriasis or atopic dermatitis.

[33] In another aspect, 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 protein as described above. In some embodiments, the autoimmune disease is selected from systemic lupus erythematosus (SLE) (e.g., SLE with lupus nephritis), Sjogren’s syndrome, rheumatoid arthritis, psoriasis, psoriatic arthritis, antiphospholipid syndrome, ty pe 1 diabetes, vasculitis, and systemic sclerosis.

[34] In another aspect, the present invention provides a method for treating biofilm formation by a gram-negative bacteria. The method generally includes administering to a subject having the biofilm formation an effective amount of a fusion polypeptide or dimeric protein as described above. In some embodiments, the gram-negative bacteria is Pseudomonas aeruginosa.

[35] In yet another aspect, the present invention provides a method for treating a neurological disease. The method generally includes administering to a subject having the neurological disease an effective amount of a fusion polypeptide or dimeric protein as described above. In some embodiments, the neurological disease is selected from Parkinson’s disease and Alzheimer’s disease. In some embodiments, the neurological disease is a disease characterized by dementia such as, for example, Alzheimer’s disease.

[36] In another aspect, the present invention provides a method for treating a cardiovascular disease. The method generally includes administering to a subject having the cardiovascular disease an effective amount of a fusion polypeptide or dimeric protein as described above. In some embodiments, the cardiovascular disease is a disease characterized by atherosclerosis such as, e.g, coronary' heart disease or ischemic stroke. In some variations, the coronary' heart disease is characterized by acute coronary' syndrome.

[37] In another aspect, the present invention provides a method for treating a chronic liver disease. The method generally includes administering to a subject having the chronic liver disease an effective amount of a fusion polypeptide or dimeric protein as describe above. In some embodiments, the chronic liver disease is selected from nonalcoholic fatty' liver disease (NAFLD), alcohol-associated liver disease (ALD), portal hypertension, or a complication following liver transplantation. In some variations, the nonalcoholic fatty liver disease is nonalcoholic steatohepatitis (NASH).

[38] In another aspect, the present invention provides a method for treating a fibrotic disease. The method generally includes administering to a subject having the fibrotic disease an effective amount of a fusion polypeptide or dimeric protein as described above. In some embodiments, the fibrotic disease is selected from the group consisting of systemic sclerosis, systemic lupus erythematosus (SLE), an inflammatory lung disease, a chronic liver disease, and a chronic kidney disease (e.g, lupus nephritis, IgA nephropathy, or membranous glomerulonephritis).

[39] In another aspect, the present invention provides a method for treating exposure to sulfur mustard gas or 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 protein as described above, wherein the biologically active paraoxonase of said fusion polypeptide or dimeric protein is the paraoxonase comprising an amino acid sequence as shown in (i) residues 16-355 or 26-355 of SEQ ID NO:55 or (ii) residues 16-355 or 26-355 of SEQ ID NO:56. In some embodiments, the organophosphate is an insecticide selected from parathion, malathion, chlorpyrifos, diazinon, dichlorvos, phosmet, fenitrothion, terbufos, tetrachlorvinphos, azamethiphos, and azinphos-methyl. In some embodiments the organophosphate is a nerve agent selected from tabun, sarin, soman, and cyclosarin.

[40] These and other aspects of the invention will become evident upon reference to the following detailed description of the invention.

DEFINITIONS

[41] Unless defined otherw ise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art pertinent to the methods and compositions described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[42] The terms “a,” “an,” and “the” include plural referents, unless the context clearly indicates otherwise.

[43] A “polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 50 amino acid residues may also be referred to as “peptides.”

[44] 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.

[45] The terms “amino-terminal'’ (or “N-terminaF’) and “carboxyl-terminal” (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 carboxyl- terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.

[46] The terms “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 kilobases (“kb”). Where the context allows, 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 all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.

[47] A “segment” is a portion of a larger molecule (e.g., polynucleotide or polypeptide) having specified attributes. For example, 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. Also, for example, in the context of a fusion polypeptide as described herein, different components of the fusion polypeptide (e.g., a paraoxonase, linker(s), an immunoglobulin Fc region, a CTLA-4 extracellular domain) may each be referred to as a polypeptide segment.

[48] The term “biologically active,” when used in reference to a polypeptide segment of a fusion molecule as described herein, means a polypeptide that causes a measurable or detectable physiological, biochemical, or molecular effect in a biological system. Biological activities include, for example, enzymatic activity, antigen-binding, binding to a cell-surface receptor, dimerization, activation of a signaling pathway in a eukaryotic cell, induction of cell proliferation, induction of cell differentiation, and the like. When used in specific reference to a polypeptide segment that is an enzyme (e.g., a paraoxonase 1 (P0N1) or paraoxonase 3 (P0N3)), “biologically active” means that the polypeptide exhibits the same ty pe of enzymatic activity as a corresponding, naturally occurring enzy me (e.g.. the same type of enzymatic activity as a full-length, wild-type human PON1 or P0N3, respectively), allowing for differences in degree of activity', enzyme kinetics, and the like. An immunoglobulin Fc region, as referenced herein, is understood to be “biologically active” at least by virtue of its dimerizing and FcRn-binding activities.

[49] Unless the context clearly indicates otherwise, reference herein to “paraoxonase 1” (“P0N1”), “paraoxonase 3” (“P0N3”), “CTLA-4 extracellular domain.” and “CD40 extracellular domain” is understood to include naturally occurring polypeptides of any of the foregoing, as well as functional variants and functional fragments thereof.

[50] The term “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 phenoty pic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered ammo acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.

[51] The term “linker” or “polypeptide linker” is used herein to indicate a polypeptide segment of one or more amino acids (e.g., two or more amino acids) linking two discrete, separate polypeptide regions. The linker is typically designed to allow the separate polypeptide regions (such as. e.g., a paraoxonase or CTLA-4 extracellular domain polypeptide linked to an Fc region) to perform their separate functions. 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 linkers joining different fusion components: “LI” refers to a linker joining the N-terminus of a biologically active paraoxonase to the C-terminus of second biologically active polypeptide (for example, a dimerizing domain or domain that specifically binds to the neonatal Fc receptor (FcRn), such as, e.g., an immunoglobulin Fc region), and “L2” refers to a linker joining a biologically active polypeptide that is not a paraoxonase (e.g, a CTLA-4 or CD40 extracellular domain) to the N-terminus of another polypeptide segment such as, e.g, a dimerizing domain or domain that specifically binds to FcRn (e.g., an immunoglobulin Fc region). In the context of a polypeptide chain containing both LI and L2 linkers, the linkers may be the same or different with respect to amino acid sequence. In some variations in which the amino-terminus of a biologically active paraoxonase is linked directly to the carboxyl-terminus of a biologically active polypeptide that is not a paraoxonase or dimerizing/FcRn- binding domain via a single polypeptide linker (z.e. , with no intervening biologically active polypeptide), such polypeptide linker may be referred to as either LI or L2.

[52] The term “expression cassette” is used to denote a DNA construct that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription in an appropriate host cell. Such additional sequences include a promoter and, typically, a transcription terminator, and may also include one or more selectable markers, an enhancer, a polyadenylation signal, etc.

[53] The term “vector” is used to denote a polynucleotide produced by recombinant DNA techniques for delivering genetic material into a cell, where it can be replicated. As is well-known in the art, it may refer, e.g, to a plasmid, a cosmid, a viral vector, an artificial chromosome, a cloning vector, or an expression vector. The term “expression vector” is used to denote a vector comprising an expression cassette.

[54] The term “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.

[55] 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. A “secretory peptide” is also referred to as a “leader sequence” or “leader peptide.” In naturally occurring P0N1 and P0N3, the leader sequence is not cleaved and is also referred to herein as a “noncleaved leader sequence” or “noncleaved leader peptide.” [56] “Operably linked” means that two or more entities are joined together such that they function in concert for their intended purposes. When referring to 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. When referring to polypeptides, “operably linked” includes both covalently (e.g., by disulfide bonding) and non-covalently (e.g, by hydrogen bonding, hydrophobic interactions, or salt-bridge interactions) linked sequences, wherein the desired function(s) of the sequences are retained.

[57] The term “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 cell so modified. Thus, for example, 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. 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. Thus 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. i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes disclosed herein. Similarly, a “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.

[58] The term “heterologous,” when used with reference to portions of a nucleic acid, indicates that the nucleic acid comprises tw o or more subsequences that are not normally found in the same relationship to each other in nature. For instance, 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. Similarly, “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 more segments of a fusion polypeptide).

[59] As used herein, the term “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin gene(s). One form of immunoglobulin constitutes the basic structural unit of an intact, native antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions. Immunoglobulins typically function as antibodies in a vertebrate organism. Five classes of immunoglobulin 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. In humans, 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 y. For example, immunoglobulins of the IgGl subclass contain a yl heavy’ chain constant region. Each immunoglobulin heavy chain possesses a constant region that consists of constant region protein domains (CHI, hinge, CH2, and CH3; IgG3 also contains a CH4 domain) 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., Nucleic Acids Res. 10:4071-4079, 1982; Kenten et al., Proc. Natl. Acad. Sci. USA 79:6661-6665, 1982; Seno et al., Nuc. Acids Res. 11:719-726, 1983; Riechmann et al., Nature 332:323-327, 1988; Amster et al., Nuc. Acids Res. 8:2055-2065, 1980; Rusconi and Kohler, Nature 314:330-334, 1985; Boss et al., Nuc. Acids Res. 12:3791-3806, 1984; Bothwell et al., Nature 298:380-382, 1982; van der Loo et al., Immunogenetics 42:333-341, 1995; Karlin et al., J. Mol. Evol. 22: 195-208, 1985; Kindsvogel et al.. DNA 1:335-343, 1982; Breiner et al., Gene 18: 165-174, 1982; Kondo et al., Eur. J. Immunol. 23:245-249. 1993; and GenBank Accession No. J00228.) For a review of immunoglobulin structure and function see Putnam, The Plasma Proteins, Vol V, Academic Press, Inc., 49-140, 1987; and Padlan, Mol. Immunol. 31: 169-217, 1994. The term “immunoglobulin” is used herein for its common meaning, denoting an intact antibody, its component chains, or fragments of chains, depending on the context. [60] An ‘’immunoglobulin hinge” is that portion of an immunoglobulin heavy chain connecting the CHI and CH2 domains. The hinge region of human yl corresponds approximately to Eu residues 216-230.

[61] The terms “Fc fragment,” “Fc region.” or “Fc domain,” as used herein, are synonymous and refer to the portion of an immunoglobulin that is responsible for binding to antibody receptors on cells and the Clq component of complement (in the absence of any amino acid changes, relative to the naturally occurring sequence, to remove such binding activity). Fc stands for “fragment crystalline,” the fragment of an antibody that will readily form a protein crystal. 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. As used herein, 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. As used herein, the term Fc region further includes variants of naturally occurring hinge-CH2-CH3 sequences, wherein the variants are capable of forming dimers at least through dimerization of the CH3 domain and including such variants that have increased or decreased Fc receptor-binding or complement-binding activity while retaining at least sufficient binding to the neonatal Fc receptor (FcRn) to confer improved halflife to a fusion partner in vivo (relative to the fusion partner in the absence of the Fc region). The abbreviated term “Fc” may also be used herein to denote “Fc region” when referring to a fusion polypeptide by its general amino- to carboxyl-terminal structure (e.g., “CTLA4-L2-Fc- L1-PON1”).

[62] “Dimerizing domain,” as used herein, refers to a polypeptide having affinity for a second polypeptide, such that the two polypeptides associate under physiological conditions to form a dimer. Typically, 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). Examples of 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. Immunol., 148: 1547-1553, 1992; or 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 GPIIIb/IIIa); a dimerizing region of a secreted, dimerizing ligand (e.g., nerve growth factor (NGF), neurotrophin-3 (NT-3), interleukin-8 (IL- 8), vascular endothelial grow th factor (VEGF), or brain-derived neurotrophic factor (BDNF); see, e.g., Arakawa et al., J. Biol. Chem. 269:27833-27839, 1994. and Radziejewski et al., Biochem. 32: 1350, 1993); and apolypeptide comprising at least one cysteine residue (e.g.. from about one, two, or three to about ten cysteine residues) such that disulfide bond(s) can form between the polypeptide and a second polypeptide comprising at least one cysteine residue (hereinafter "a synthetic hinge’'). A preferred dimerizing domain in accordance with the present invention is an Fc region.

[63] The term “dimer” or “dimeric protein” as used herein, refers to a multimer of two (“first” and “second”) fusion polypeptides as disclosed herein linked together via a dimerizing domain. Unless the context clearly indicates otherwise, a “dimer” or “dimeric protein” 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 further dimerize via the Fc region of the immunoglobulin heavy chain, thereby forming a tetramer).

[64] The term “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 (Ka) of 10⁶ M'¹ or greater (e.g., 10⁷ M'¹ or greater, 10⁸ M'¹ or greater, or 10⁹ M'¹ or greater), and (ii) does not have affinity⁷ for FcRn at physiological pH (e.g. , pH 7.4). 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 multiwell plate assay (e.g., ELISA), a filter assay, or a surface plasmon resonance assay. Examples of known related polypeptides include known members of the major histocompatibility complex (MHC) class I protein family. Another example of an FcRn-binding domain is albumin or a fragment of albumin such as the third domain region (see, e.g., Zhao et al., Biomed Res. Int. 2013, Article ID 107238). 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 domains include Fc regions that retain FcRn-binding activity.

[65] Fusion polypeptides of the present disclosure may be referred to herein byformulae such as. for example, “Fc-Ll-PON3,” “Fc-Ll-[PON1 1F11],” “Fc-Ll-[PON1 2G1].” CTLA4-L2-Fc-Ll-PON3,” “CD40-L2-Fc-Ll-PON3,” “CTLA4-L2-Fc-Ll-[PON1 1F11],” “CD40-L2-Fc-Ll-[PON1 1F11],” “CTLA4-L2-Fc-Ll-PON1 2G1],” or “CD40-L2-Fc-Ll- PON1 2G1].” In each such case, unless the context clearly dictates otherwise, a term referring to a particular segment of a fusion polypeptide (e.g, “PON3.” “PON1 1F11,” “PON1 2G1.” “CTLA4” (for CTLA-4 extracellular domain), “CD40” (for CD40 extracellular domain), “LI” or “L2” (for first or second polypeptide linkers, respectively), “Fc” (for “Fc region”), etc.) is understood to have the meaning ascribed to such term herein and is inclusive of the various embodiments as described herein.

[66] The term “effective amount,” in the context of treatment of a disease byadministration of a soluble fusion polypeptide or dimeric protein to a subject as described herein, refers to an amount of such molecule that is sufficient to inhibit the occurrence or ameliorate one or more symptoms of the disease. For example, in the specific context of treatment of an inflammatory lung disease by administration of a fusion protein to a subject as described herein, the term “effective amount” refers to an amount of such molecule that is sufficient to modulate an inflammatory response in the subject so as to inhibit the occurrence or ameliorate one or more symptoms of the inflammatory lung 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.

[67] 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.

[68] 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. For example, a combination therapy may involve the administration of two or more chemically distinct active ingredients, or agents, for example, a soluble PON3 fusion polypeptide or dimeric protein according to the present invention and another agent such as, e.g. , another antiinflammatory or immunomodulatory agent. Alternatively, a combination therapy may involve the administration of, e.g., a soluble PON3 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. In the context of the administration of two or more chemically distinct agents, it is understood that the active ingredients may be administered as part of the same composition or as different compositions. When administered as separate 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.

[69] The term ’’targeted therapy,” in the context of treating cancer, refers to a type of treatment that uses a therapeutic agent to identity⁷ and attack a specific type of cancer cell, typically with less harm to normal cells. In some embodiments, a targeted therapy blocks the action of an enzyme or other molecule involved in the growth and spread of cancer cells. In other embodiments, a targeted therapy either helps the immune system to attack cancer cells or delivers a toxic substance directly to cancer cells. In certain variations, a targeted therapy uses a small molecule drug or a monoclonal antibody as a therapeutic agent.

[70] The phrase “protect from aging,” as used herein, refers to inhibition or mitigation of any of broad aspects of aging, including, for example, age-related changes in systemic inflammation or disease risk, as indicated by accepted biomarkers. Protection from aging may also include treatment of an age-related disease where the disease is present in a subject, such as, for example, a chronic inflammatory, autoimmune, neurodegenerative, cardiovascular, or fibrotic disease.

[71] 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 compansons 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 w ell-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. (eds.), “Information Superhighway and Computer Databases of Nucleic Acids and Proteins/’ in Methods in Gene Biotechnology 123-151 (CRC Press, Inc.

1997); Bishop (ed.), Guide to Human Genome Computing (2nd ed., Academic Press, Inc.

1998).) 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.

[72] Percent sequence identity is determined by conventional methods. (See, e.g, Altschul 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 penalty⁷ of 10, a gap extension penalty⁷ of 1, and the “BLOSUM62” scoring matrix of Henikoff and Henikoff, supra. 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).

[73] Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The “FASTA” similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and a second amino acid sequence. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat’lAcad. Sci. USA 85:2444, 1988, and by Pearson, Meth. Enzymol. 183:63, 1990. Briefly, FASTA first characterizes sequence similarity by identify ing regions shared by the query⁷ sequence (e.g., residues 21-354 or 31-354 of SEQ ID NO:30) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity⁷ of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are “trimmed” to include only those residues that contribute to the highest score. If there are several regions with scores greater than the “cutoff’ value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, 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, SIAM J. Appl. Math. 26:787 , 1974), which allows for amino acid insertions and deletions. Illustrative parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=blosum62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file (“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63, 1990.

[74] The term “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.

[75] When such a value is expressed as “about” X or “approximately” X, the stated value of X will be understood to be accurate to ±10%.

[76] Where aspects or embodiments of the present invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. The present invention also envisages the explicit exclusion of one or more of any of the group members as embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[77] FIGs. 1A and IB show schematic diagrams of certain embodiments of fusion proteins in accordance with the present disclosure, including component functional domains. FIG. 1 A depicts a schematic representation of a PON1 (human or evolved) or PON3 joined at the amino terminus, via a linker, to a human IgG Fc region (including, in certain embodiments, an IgGl Fc variant containing M252Y/S254T/T256E (“YTE”) substitutions relative to wildtype). FIG. IB depicts a schematic representation of a PON1 or PON3 fusion protein as shown in FIG. 1A further joined at the Fc amino terminus to a human CTLA-4 extracellular domain (“huCTLA4EC”). Exemplary PON1 polypeptides include human PON1 Q192K (“PON1-K”) and evolved PON1 variants 1F11 (“PON1-1F11”) and 2G1 (“PON1-2G1”). An exemplary linker joining the amino terminus of a PON1 or PON3 to an Fc is a peptide linker containing an N-linked glycosylation site (an “NGS” linker; see, e.g.. SEQ ID NO: 12). [78] FIG. 2 shows a Western blot of culture supernatants (serum free) from transiently transfected HEK293 cells expressing P0N1 and PON3 fusion proteins. Transfections and Western blot analysis were performed as described in Example 1, infra. From left to right: Lane 1 - Chameleon DUO molecular weight markers (LICOR, ), Lane 2 - Fc-YTE-PON3, Lane 3 - Fc-PON3, Lane 4 - Fc-YTE-Pl-lFl l, Lane 5 - Fc-YTE-Pl-M2G1, Lane 6 - Fc-YTE-Pl-2G1, Lane 7 - LICOR Chameleon DUO MW Markers, Lane 8 - Fc- M2G1, Lane 9 - Fc-G3C9, Lane 10 - Fc-2G1; Lane 11 - CTLA4-Fc-Pl-2G1; Lane 12 - MOCK. The approximate molecular weight (kDa) of each marker band is indicated on the right side of the Western blot.

[79] FIG. 3 shows the results of a fluorometric assay measuring the organophosphatase activity of monospecific and bispecific PON1 fusion proteins (CTLA4-Fc- P1-1F11, CTLA4-Fc-Pl-2G1, CTLA4-Fc-Pl-M2G1, Fc-Pl-K, Fc-Pl-lFll) and an organophosphatase positive control enzyme (see Example 2, infra).

[80] FIG. 4 shows the results of a fluorometric assay measuring the organophosphatase activity of PON1 and PON3 fusion proteins (Fc-YTE-Pl-2G1, Fc-YTE- Pl-IF 11, Fc-YTE-PON3) and an organophosphatase positive control enzyme (see Example 2, infra).

[81] FIG. 5 shows high affinity binding of immobilized CD80 to a CTLA4-Fc-PON 1 fusion protein (CTLA4-Fc-PON1-K) present in HEK293 transfection supernatant (see Example 2, infra). A human CTLA4Ig fusion protein (Orencia®) and an ApoAl-Fc-PONl fusion protein (APOA-l-(g4s)4-Fc-Pl-K) were included as positive and negative controls, respectively.

[82] FIG. 6 shows hydrolysis of dihydrocoumarin (DHC) by HEK 293T cell transient transfection supernatants of paraoxonase fusion proteins (Fc-PON3, Fc-YTE-PON3, TR-43) (see Example 5, infra). Samples containing mock transfected supernatant (MOCK) or DHC substrate alone (DHC) were used as negative controls. DESCRIPTION OF THE INVENTION

I Overview

[83] The present invention provides compositions and methods relating to fusion polypeptides comprising a biologically active paraoxonase that is either a paraoxonase 3 (PON3) polypeptide or functional variant or fragment thereof or, alternatively, an evolved paraoxonase 1 (PON1) polypeptide or functional variant or fragment thereof. The biologically active paraoxonase lacks an N-terminal hydrophobic sequence corresponding to the N-terminal hydrophobic sequence of wild-type PON3 or PON 1 and which is thought to be essential for the enzyme to be stable, active, and properly transported to cell membranes.

[84] In certain preferred embodiments, the P0N3 or evolved P0N1 polypeptide is joined at its N-terminus to a dimerizing domain or a domain that specifically binds to the neonatal Fc receptor (FcRn). which may be a dimerizing and FcRn-binding immunoglobulin heavy chain constant region such as, e.g, an immunoglobulin Fc region. In some embodiments, the fusion polypeptide does not include a biologically active polypeptide N- terminal to the dimerizing or FcRn-binding domain. In other embodiments, the fusion polypeptide is a multi-specific construct further comprising a biologically active polypeptide N-terminal to the dimerizing or FcRn-binding domain. Exemplary biologically active polypeptides include extracellular domains of cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) extracellular domain and CD40, either of which may be a naturally occurring protein or a functional variant or fragment thereof.

[85] In other embodiments, the PON3 or evolved P0N1 polypeptide is joined at its C-terminus to a dimerizing domain or domain that specifically binds to FcRn (for example, a dimerizing and FcRn-binding immunoglobulin heavy chain constant region such as, e.g.. an immunoglobulin Fc region).

[86] Paraoxonase fusion molecules of the present invention may be used for the treatment of various diseases or disorders through its antioxidant, anti-inflammatory, atheroprotective, and/or neuroprotective properties, including, e.g., treatment of an autoimmune or inflammatory disease. For example, there is evidence that PON3 therapy would be very beneficial in treatment of metabolic disease, including, e.g., obesity, kidney disease, liver disease, and heart disease. (See, e.g., Draganov et al., J. Biol. Chem. 275: 33435, 2000; Aviram et al. , Free Rad. Biol. Med. 37: 1304, 2004; Marsillach et al. , J. Proteome Res. 14:2046, 2015; Shih et al., Circ. Res. 100: 1200-1207, 2007); Ng et al., Arterioscler. Thromb. Vase. Biol. 27: 1368-1374, 2007; Zhang et al., Gene Ther. 17:626-633, 2010; Shih et al., Arterioscler. Thromb. Vase. Biol. 27: 1368-1374, 2007; Peng et al., Biomed. Pharmacother. 63:592-598, 2009.) Oxidized LDL-cholesterol promotes cardiovascular disease, metabolic disease, and oxidative stress (see Mahdi Garelnabi, Srikanth Kakumanu, and Dmitry Litvinov (2012), “Role of Oxidized Lipids in Atherosclerosis,” in Oxidative Stress and Diseases, Dr. Volodymyr Lushchak (Ed.), ISBN: 978-953-51-0552-7, DOI: 10.5772/32999, available from InTechOpen website). LDL-cholesterol is normally transported to the liver where it binds the LDLR receptor and is excreted. However, at sites of vascular injury or inflammation, LDL-cholesterol is oxidized by myeloperoxidase secreted from activated neutrophils and myeloid cells. Oxidized LDL-cholesterol enters the vascular intima where it binds and is taken up byscavenger receptors expressed by macrophages, leading to inflammatory foam cells. The protective activity of PON3 against metabolic and vascular disease may be primarily due to its ability to protect LDL-cholesterol from oxidation.

[87] PON3 fusion proteins of the invention will also be useful for the therapy of cancer. The divergent effects on different cancer cell types means that PON3 therapy may be most useful for treatment of some tumors such as. e.g., hepatocellular carcinoma, esophogeal tumors, and protate tumors. (See, e.g., Huang et al., Cancer Cell Int. 18: 1-11, 2018; Shui et al., Cancer 122:2168, 2016; Jin et al.. Tumor Biol. 37: 14193, 2016; Cai et al., Oncotarget 7:70045, 2016; Zhu et al., Biomedicine and Pharmacotherapy 85:712, 2017.) PON3 fusion proteins as disclosed herein may inhibit tumor growth directly or may sensitize tumor cells to chemotherapy or immunotherapy agents.

[88] In addition, evolved PON1 fusion molecules of the present invention will be particularly useful for treatment against exposure to sulfur mustard gas and organophosphates. The PON 1 1F11 and 2G1 fusion proteins of the invention have a long half-life in the blood and will provide protection from nerve agents for an extended period (e.g., several months) after a single injection. Another advantage of the present IF 11 and 2G1 fusion proteins is their ease of manufacturing and purification. High expression in mammalian cells is seen, and the proteins are readily purified using Protein A chromatography that is standard for recombinant antibodies.

[89] Studies also support use of a paraoxonase for treatment of autoimmune disease such as systemic lupus erythematosus (SLE). The autoantibody titer in many patients with systemic lupus erythematosus (SLE) is correlated with loss of activity of P0N1 (see Batukla etal.,Ann. NY Acad. Sei. 1108:137-146, 2007), and SLE-disease activity' assessed by SLED Al and SLE disease related organ damage assessed by SLICC/ACR damage index are negatively correlated with PON 1 activity (see Ahmed et al.. EXC LI Journal 12:719-732. 2013). Other studies support use of a paraoxonase for treatment of inflammatory disease such as inflammatory' lung diseases. Studies strongly suggest, for example, that PON1 is important in protection from lung inflammation, and further show that low PON1 activity' is associated with lung diseases such as. e.g.. asthma, chronic obstructive pulmonary disease (COPD). and interstitial lung disease (e.g., idiopathic pulmonary' fibrosis (1PF) or sarcoidosis) (see, e.g., Sahiner et al., WAO Journal 4: 151-158, 2011; Emin et al., Allergol. Immunopathol. (Madr) 43:346-352, 2014; Sarioglu et al., Iran J. Asthma Immunol. 14:60-66, 2015; Tolgyesi et al., Internal. Immunol. 21:967-975. 2009; Chen et al., J. Cell Biochem. 119:793-805. 2018; Rumora et al., J. COPD 11 :539-545, 2014; Rajkovic et al., J. Clin. Path. 71 :963-970, 2018; Ivanisevic et al., Eur. J. Clin. Invest. 46:418-424, 2016; Uzun et al., Curr. Med. Res. Opin. 24: 1651-1657, 2008; Okur et al., Sleep Breath. 17:365-371, 2013; Golmanesh et al., Immunopharmacol. And Immunotoxicol. 35:419-425, 2013). Another study showed that loyv PON1 activity is correlated with the severity of Crohn’s disease (Sczceklik et al.. Molecules 23:2603, 2018). In addition, recombinant PON1 therapy has shown efficacy in animal models of organophosphate poisoning and colitis (see, e.g, Valiyaveettil et al., Biochem. Pharmacol. 81:800-809, 2011; Valiyaveettil et al., Toxicol. Letters 202:203-208, 2011; Bajaj et al., Appl. Biochem. Biotechnol. 180: 165, 2016; Stevens et al., Proc. Natl. Acad. Sci. USA 105: 12780- 12784, 2008; Yamashita e/ o/.. J. Immunol. 191 :949-960, 2013).

[90] Paraoxonase fusion molecules as described herein may also be used, e.g., for treatment of a neurological disease. For example, PON1 is protective in the brain because of its antioxidant properties. A neuroprotective role of PON1 is supported by studies showing that PON1 activity is decreased in patients with Alzheimer’s disease and other dementias (see, e.g., Menini et al.. Redox Rep. 19:49-58. 2014). In addition, a study using a PON1 fusion containing a protein transduction domain to transduce PON1 into cells and tissues showed that PON1 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). [91] A bispecific paraoxonase fusion molecule comprising a CTLA-4 extracellular domain as described herein is particularly useful for treatment of inflammatory and autoimmune disease, including, e.g., rheumatoid arthritis (RA) and inflammatory lung disease. Oxidized cholesterol activates inflammatory responses in macrophages and endothelial cells (see Miller and Shyy, Trends Endocrinol. Metabol. 28: 143-152, 2017), and PON1 and PON3 can suppress this inflammation by protecting lipoproteins from oxidation. The immunosuppressive properties of a soluble CTLA-4 (e.g.. Abatacept or other CTLA4-Fc) are compatible with paraoxonase enzyme activity so that a CTLA4-PON molecule will retain the activity of both components and have improved activity in fibrotic lung disease and autoimmune/inflammatory disease. A CTLA4-PON3 molecule, for example, will bind to CD80 and CD86 that are expressed on activated antigen presenting cells, including monocytes and dendritic cells, where the PON3 enzyme will remain active. This molecule is effective in inhibiting activation of T cells, macrophages, and dendritic cells, and will also reduce oxidative stress in tissues such as, e.g., inflamed lungs.

[92] A bispecific paraoxonase fusion molecule comprising a CD40 extracellular domain as described herein provides additional therapeutic benefit by suppressing the proinflammatory activation events associated with the CD40-CD154 signaling pathway. The CD40-CD154 pathway has been implicated, for example, in fibrotic disease, including fibrosis in inflammatory lung disease and injury (see, e.g., Kaufman etal., J. Immunol. 172: 1862-1871, 2004), and studies support the use of molecules that suppress this pathway for treatment of fibrotic and inflammatory lung disease (see, e.g., Adawi et al., Clin. Immunol. Immunopathol. 89:222-230, 1998; Adawi et al., Am. J. Pathol. 152:651-657, 1998; Cheng et al., BioMed. Researching. Volume 2020, Article ID 7840652, 2020; Xiong Zo/., J. Cell. Mol. Med. 23:740- 749, 2018). CD40-PON fusions of the present invention provide molecules that inhibit both inflammation and adaptive immunity, including e.g., molecules with improved activity in lung disease and autoimmune/inflammatory disease. For example, a CD40-PON1 fusion may provide improved activity in lung diseases where CD40 activation exacerbates inflammatory processes, and where paraoxonase levels are low or absent, including asthma, COPD, hypoxia, and interstitial lung disease. CD40-PON1 fusions are also beneficial, for example, for treatment of liver disease and kidney disease.

[93] Bispecific paraoxonase molecules comprising an extracellular domain of CTLA-4 or CD40 are also beneficial in reducing potential immunogenicity of a paraoxonase polypeptide such as, e.g., an evolved P0N1. For example, immunogenicity of 1F11 and 2G1 fusion proteins could occur due to the number of mutations relative to the wild-type protein. 1F11, for example, has 60 mutations versus human PON1. To prevent immunogenicity, bifunctional CTLA4-PON or CD40-PON fusion molecules (e.g., CTLA4-Fc-[PON 1 1F11] or CD40-Fc-[PON1 1F11]) will block the CD28 or CD40 pathways that are essential for T celldependent antibody responses. The anti-inflammatory properties of evolved PON1 fusion molecules containing a CTLA-4 or CD40 extracellular domain, as described herein, will improve the outcome in individuals exposed to nerve agents.

[94] PON1 and PON3 have hydrophobic N-terminal sequences that bind phospholipids and HDL-cholesterol. This hybrophobic sequence is a noncleaved leader sequence (or “noncleaved leader peptide”). This interaction is thought to be needed to stabilize and transport the enzy me. Studies with PON1 have shown that the N-terminal hydrophobic sequence is required for association with phospholipids and ApoA-I (see Sorenson et al., Arterioscler. Thromb. Vase. Biol. 19:2214-2225, 1999). Deletion of the N-terminal hydrophobic sequence resulted in a significant (17-fold) reduction in enzyme activity. Sorenson et al. also found that ApoA-I binding to PON1 stabilized the enzy me to inactivation by heat treatment. (See id) PON1 is anchored on the cell surface by its N-terminus and is taken up by binding to phospholipids and ApoA-I. ApoA-I then transports PON1 and can deposit the enzyme on the surface of peripheral cells. (See Deakin et al., J. Biol. Chem. 277:4301-4308, 2002). Binding of PON1 to ApoA-I also increases its cholesterol efflux capacity (see White et al., Curr. Opin. Lipidol. 28:397-402, 2017). Oxidative stress and myeloperoxidase decrease the association of PON 1 with ApoA-I and cause dysfunctional HDL (see Bachetti et al., Curr. Med. Chem. 28:2842, 2021; Aggarwal et al., J. Biol. Chem. 297: 101019, 2021).

[95] Studies with PON3 show- that that PON3 possesses properties very similar to those of PON1 : the enzy me’s lactonase activity is selectively stimulated by binding to ApoA- I-HDL, with a concomitant increase in its stability (see Khersonsky et al.. Biochemistry 48:6644-6654, 2009). In view of these studies, and commonly in the paraoxonase literature, the N-terminal hydrophobic domain is thought to be essential for the enzyme to be stable, active, and properly transported to cell membranes.

[96] The PON3 and evolved PON1 fusion molecules of the present invention utilize PON polypeptides that lack an amino-terminal hydrophobic leader sequence corresponding to the naturally occurring, non-cleaved leader sequence of PON3 or P0N1 (generally corresponding to residues 1-20 of human PON3 (SEQ ID NO:30) or residues 1-15 of human PON1 (SEQ ID NO:2), respectively). Truncation of the PON3 or PON1 N-terminus in the fusion protein deletes the normal association with HDL-cholesterol. Further, in certain embodiments, attachment to an FcRn-binding domain (e.g., an immunoglobulin Fc region) stabilizes the enzyme and gives the molecule a long half-life in the blood. Fusion molecules of the present disclosure retain PON3 and PON1 activity while also — in the case of FcRn- binding embodiments — conferring stability in the absence of the hydrophobic N-terminus. In addition, PON3 and evolved PON1 fusion molecules comprising other biologically active polypeptides (e.g, a CTLA-4 extracellular domain or a CD40 extracellular domain) can target PON3 or evolved PON1 to other sites in vivo (e.g, sites of proinflammatory and/or immune cell activation) for therapeutic benefit such as described herein.

II. Fusion Polypeptides and Dimeric Proteins

[97] In one aspect, the present invention provides a fusion polypeptide comprising, from an amino-terminal position to a carboxyl-terminal position. X-Ll-P, wherein X is a dimerizing domain or a domain that specifically binds to the neonatal Fc receptor (FcRn); LI is a polypeptide linker, wherein LI is optionally present; and P is a biologically active paraoxonase. In certain embodiments, the biologically active paraoxonase is a naturally occurring paraoxonase 3 (PON3) polypeptide or a functional variant or fragment thereof. In other embodiments, the paraoxonase is an evolved PON 1 polypeptide corresponding a PON 1 as shown in SEQ ID NO:55 (also referred to herein as “PON1 1F11” or “1FH”) or a PONl as shown in SEQ ID NO:56 (also referred to herein as “PON1 2G1” or “2G1”). In some embodiments, the fusion polypeptides are bispecific constructs further comprising a biologically active polypeptide amino-terminal to the dimerizing or FcRn-binding domain, wherein the fusion polypeptide comprises, from an amino-terminal position to a carboxyl- terminal position, T-L2-X-L1-P, wherein X, LI, and P are defined as above, L2 is a second polypeptide linker, wherein L2 is optionally present, and T is the biologically active polypeptide. Exemplary biologically active polypeptides include the extracellular domains of CTLA-4 and CD40, which may be a naturally occurring protein or a functional variant or fragment thereof.

[98] In another aspect, the present invention provides a fusion polypeptide comprising, from an amino-terminal position to a carboxyl-terminal position, P-L2-X, wherein P is a biologically active paraoxonase, L2 is a polypeptide linker, and X is biologically active polypeptide selected from a dimerizing domain and a domain that specifically binds to the neonatal Fc receptor (FcRn). In certain embodiments, the biologically active paraoxonase is a naturally occurring paraoxonase 3 (PON 3) polypeptide or a functional variant or fragment thereof. In other embodiments, the paraoxonase is an evolved PON1 polypeptide corresponding PON1 1F11 or PONl 2G1.

[99] Functional variants of a naturally occurring PON3 or other biologically active polypeptides specified above can be readily identified using routine assays for assessing the variant for the corresponding biological activity. For example, P0N3 and PON1 variants may be assayed for phosphotri esterase 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). PON3 and PON1 variants may also be assayed for lactonase activity using dihydrocoumarin as a substrate (see, e.g., Example 3, infra). In addition, PON1 variants may be assayed for organophosphatase activity using, for example, a proprietary organophosphatase substrate contained in the EnzCHEK paraoxonase assay kit (Molecular Probes/ThermoFisher Scientific, Waltham, MA) (see, e.g., Example 2, infra).

[100] Soluble CLTA-4 and CD40 variants (as well as other polypeptides in which the desired biological activity⁷ includes its binding to a known target molecule, such as, e.g., an antibody with specific binding against a target antigen), may be assessed for desired binding activity against their respective targets (e.g., against CD80/CD86 in the case of CTLA-4, or against gp39 or CD154 in the case of CD40) using any of various known assays. For example, one assay system employs a commercially available biosensor instrument (BIAcore™, Pharmacia Biosensor, Piscataway, NJ), wherein a candidate binding polypeptide (e.g., a candidate CTLA-4-Fc, CD40-Fc. or antibody) is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample containing a soluble target molecule (e.g., CD80-Fc, CD86-Fc, CD40L-Fc (gpl39-Fc), or target antigen) is passed through the cell. If the immobilized protein has affinity for the target molecule, it will bind to the target causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry⁷ of binding. Use of this instrument is disclosed, e.g., by Karlsson (J. Immunol. Methods 145:229-240, 1991) and Cunningham and Wells (J. Mol. Biol. 234:554-563, 1993). Binding activity of candidate polypeptides can also be assessed with 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 al.. Science 253:545-548, 1991; Cunningham et al.. Science 254:821-825, 1991).

[101] Activity of soluble CTLA-4 and CD40 variants can also be assessed for function in appropriate in vitro cellular assays. For example, soluble CTLA-4 will inhibit T cell responses to stimulation by CD80- or CD86-expressing cells, such as B cells, dendritic cells, or CD80⁺ or CD86⁺ CHO cells (see, e.g, US Patent No. 5.434,131; Linsley etal.,J. Exp. Med. 174:561-569, 1991). Soluble CD40 will block stimulation of CD40 positive cells by CD154 (CD40L, gp39). CD40-responsive cells can be B cells, monocytes, or dendritic cells. The response can vary' depending on the cell type, and may include proliferation, suppression of antibody production, or inflammatory cytokine production (see, e.g., Noelle et al., Proc. Natl. Acad. Sci. USA 89:6550-6554. 1992; Grammer et al.. J. Immunol. 154:4996-5010. 1995). A CD40L reporter cell line is also available from Invivogen (San Diego, CA). This reporter system uses HEK293 cells transfected with human CD40 and an NF-kB response element fused to secreted embryonic alkaline phosphatase (SEAP). Binding of the CD40 receptor by its ligand or antibodies leads to NF-kB activation and inducible expression of the SEAP. (See Jerome et al., Anal. Biochem. 585: 113402, 2019.)

[102] Naturally occurring polypeptide segments for use in accordance with the present disclosure (e.g., a naturally occurring PON3, CTLA-4, or CD40) include naturally occurring variants such as, for example, allelic variants and interspecies homologs consistent with the disclosure.

[103] Functional variants of a particular reference polypeptide (e.g., a wild-type human paraoxonase 3 (PON3) as shown in residues 21-354 or 31-354 of SEQ ID NO:30, a CTLA-4 extracellular domain as shown in residues 21-144 of SEQ ID NO:26, or a CD40 extracellular domain as shown in residues 21-188 of SEQ ID NO: 42) 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., Table 1. infra, which lists some exemplary conservative amino acid substitutions) and other substitutions that do not significantly affect the folding or activity' of the protein or polypeptide; small deletions, ty pically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions. Conserv ative 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 1: Conservative amino acid substitutions

Basic Acidic Polar Hydrophobic Aromatic Small

Arginine Glutamate Glutamine Leucine Phenylalanine Glycine

Lysine Aspartate Asparagine Isoleucine Tryptophan Alanine

Histidine Valine Tyrosine Serine

Methionine Threonine

Methionine

[104] Essential amino acids in a naturally occurring polypeptide can be identified according to procedures known in the art, 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). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (<?.g., phosphotriesterase or arylesterase activity for PON1 variants, or nuclease activity for DNasel 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 analy sis of homologies with related proteins (e.g., species orthologs retaining the same protein function).

[105] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer Science 241 :53-57, 1988 or Bowie and Sauer Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989. Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Another method that can be used is region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7: 127, 1988).

[106] Variant nucleotide and polypeptide sequences can also be generated through DNA shuffling. (See, e.g.. Stemmer, Nature 370:389, 1994; Stemmer, Proc. Natl. Acad. Sci. USA 91: 10747, 1994; International PCT Publication No. WO 97/20078.) Briefly, variant DNA molecules are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, 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.

[107] As previously discussed, a polypeptide fusion in accordance with the present invention can include a polypeptide segment corresponding to a “functional fragment” of a particular polypeptide. Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule encoding a given polypeptide. As an illustration, PON 1 -encoding DNA molecules having the nucleotide sequence of residues 46- 1099 can be digested with ZA//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 nuclease activity. One alternative to exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment. Alternatively, particular fragments of a gene encoding a polypeptide can be synthesized using the polymerase chain reaction.

[108] Accordingly , using methods such as discussed above, one of ordinary' skill in the art can prepare a variety of polypeptides that (i) are substantially identical to a reference polypeptide e.g., a PON3 as shown in residues 21-354 or 31-354 of SEQ ID NO:30, a CTLA- 4 extracellular domain as shown in residues 21-144 of SEQ ID NO:26, or a CD40 extracellular domain as shown in residues 21-188 of SEQ ID NO:42) and (ii) retains the desired functional properties of the reference polypeptide.

[109] Polypeptide segments used within the present invention (e.g., polypeptide segments corresponding to a paraoxonase. CTLA-4 extracellular domain, CD40 extracellular domain, or dimerizing or FcRn-binding domain such as, e.g., an Fc fragment) 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. For other uses, including in vitro diagnostic uses and veterinary uses, 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. Thus, 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.

[HO] In some embodiments, the paraoxonase segment is a human paraoxonase 3 (PON3) or a functional variant or fragment thereof. For example, in some embodiments, the paraoxonase (a) has at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acid residues 21-354 or 31-354 of SEQ ID NO:30 and (b) does not contain an amino-terminal leader sequence corresponding to residues 1-20 of SEQ ID NO:30. In some such embodiments, the biologically active paraoxonase has at least 96%, at least 97%, at least 98%, or at least 99% identity with amino acid residues 21-354, 22-354, 23-354, 24-354, 25-354, 26-354, 27-354, 28-354. 29-354, 30-354, or 31-354 of SEQ ID NO:30. In more specific variations, the paraoxonase has an amino acid sequence selected from (i) residues 21-354 of SEQ ID NO:30, (ii) residues 17-354 of SEQ ID NO:30, (iii) residues 18-354 of SEQ ID NO:30, (iv) residues 19-354 of SEQ ID NO:30, (v) residues 20-354 of SEQ ID NO:30, (vi) residues 21-354 of SEQ ID NO:30, (vii) residues 22-354 of SEQ ID NO:30, (viii) residues 23-354 of SEQ ID NO:30, (ix) residues 24-354 of SEQ ID NO: 30, (x) residues 25-354 of SEQ ID NO: 30, and (xi) residues 26-354 of SEQ ID NO:30 (z.e., an amino acid sequence selected from residues n-354 of SEQ ID NO:30, wherein n is an integer from 21 to 31, inclusive).

[Hl] In some variations, the paraoxonase is an evolved human PON1 identified as 1F11 (see Goldsmith et al. , Chemistry & Biology 19, 456-466, 2012), or a functional variant or fragment thereof. The full length form of this paraoxonase sequence variant is shown in SEQ ID NO:55 (amino acid). In some embodiments, a 1F11 paraoxonase or functional variant thereof for use in accordance with the present invention has at least 90% or at least 95% identity with amino acid residues 16-355 or 26-355 of SEQ ID NO:55; in some such embodiments, the 2G1 paraoxonase or functional variant thereof has at least 96%. at least 97%. at least 98%, or at least 99% identity with amino acid residues 16-355, 17-355, 18-355, 19-355, 20-355, 21- 355, 22-355, 23-355, 24-355, 25-355, or 26-355 of SEQ ID NO:55. In more specific variations, the paraoxonase has an amino acid sequence selected from (i) residues 16-355 of SEQ ID NO:55. (ii) residues 17-355 of SEQ ID NO:55. (hi) residues 18-355 of SEQ ID NO:55, (iv) residues 19-355 of SEQ ID NO:55, (v) residues 20-355 of SEQ ID NO:55, (vi) residues 21- 355 of SEQ ID NO:55, (vii) residues 22-355 of SEQ ID NO:55, (viii) residues 23-355 of SEQ ID NO:55, (ix) residues 24-355 of SEQ ID NO:55, (x) residues 25-355 of SEQ ID NO:55, and (xi) residues 26-355 of SEQ ID NO:55 (/.e., an amino acid sequence selected from residues n- 355 of SEQ ID NO: 55, wherein n is an integer from 16 to 26, inclusive).

[112] In some variations, the paraoxonase is an evolved human PON1 identified as 2G1 (see Goldsmith el al., supra), or a functional variant or fragment thereof. The full length form of this paraoxonase sequence variant is shown in SEQ ID NO:56 (amino acid). In some embodiments, a 2G1 paraoxonase or functional variant thereof for use in accordance with the present invention has at least 90% or at least 95% identity with amino acid residues 16-355 or 26-355 of SEQ ID NO:56; in some such embodiments, the 2G1 paraoxonase or functional variant thereof has at least 96%, at least 97%, at least 98%, or at least 99% identity with amino acid residues 16-355, 17-355, 18-355, 19-355, 20-355, 21-355, 22-355, 23-355, 24-355, 25- 355, or 26-355 of SEQ ID NO:56. In more specific variations, the paraoxonase has an amino acid sequence selected from (i) residues 16-355 of SEQ ID NO:56, (ii) residues 17-355 of SEQ ID NO:56, (iii) residues 18-355 of SEQ ID NO:56, (iv) residues 19-355 of SEQ ID NO:56, (v) residues 20-355 of SEQ ID NO:56, (vi) residues 21-355 of SEQ ID NO:56, (vii) residues 22- 355 of SEQ ID NO:56. (viii) residues 23-355 of SEQ ID NO:56, (ix) residues 24-355 of SEQ ID NO:56, (x) residues 25-355 of SEQ ID NO:56, and (xi) residues 26-355 of SEQ ID NO:56 (i.e., an amino acid sequence selected from residues n-355 of SEQ ID NO:56, wherein n is an integer from 16 to 26, inclusive).

[113] PON1 fusions comprising 1F11 or 2G1, or an active variant thereof, may be particularly useful in some short term therapy applications such as, e.g., treatment of exposure to sulfur mustard gas or exposure to an organophosphate.

[114] In some embodiments of a fusion polypeptide comprising a CTLA-4 extracellular domain N-terminal to the dimerizing or FcRn-binding domain, the extracellular domain is a human wild-type CTLA-4 extracellular domain or a functional variant or fragment thereof. For example, in some embodiments, the CTLA-4 extracellular domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity with amino acid residues 21-144 of SEQ ID NO:26. In more particular variations, the CTLA- 4 extracellular domain comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with amino acid residues 21-144 of SEQ ID NO:26.

[115] In some embodiments of a fusion polypeptide comprising a CD40 extracellular domain N-terminal to the dimerizing or FcRn-binding domain, the extracellular domain is a human wild-type CD40 extracellular domain or a functional variant or fragment thereof. For example, in some embodiments, the CD40 extracellular domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity with residues 21-188 of SEQ ID NO:42. In more particular embodiments, the CD40 extracellular domain comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity⁷ with residues 21-188 of SEQ ID NO:42.

[116] In some embodiments, a paraoxonase fusion molecule of the present invention comprises a variant CD40 extracellular domain having increased binding to CD154 (CD40L, gp39). For example, one or more single amino acid substitutions can be made at specific residues to improve binding to CD40 ligand (see. e.g., US Patent Application Publication No. 2014/0120091, incorporated by reference herein). Such CD40 extracellular domain variants may be fused to a human IgGl (yl) Fc variant with impaired FcyR binding to provide CD40- Fc variants that do not induce platelet activation or aggregation in vitro, avoiding toxicity from simultaneous binding to CD40 and FcyRIIa on human platelets. Activation of platelets and thrombosis has been shown to occur due to crosslinking of FcR on platelets with antigens on the platelet surface such as VEGF, CD40, and CD154 (see Taylor et al. , Blood 96:4254, 2020; Meyer el al., J. Thromb. Haemost. 7:151, 2009).

[117] In certain preferred embodiments, the CD40 extracellular domain contains at least one amino acid substitution at a position corresponding to an amino acid of wild-type human CD40 (SEQ ID NO:46) selected from E64, K81, P85, and L121, wherein the at least one amino acid substitution increases CD40 ligand binding relative to human CD40. Particularly suitable amino acid substitutions at these positions are ty rosine at the position corresponding to E64 of human CD40, threonine, histidine, or serine at the position corresponding to K.81 of human CD40. tyrosine at the position corresponding to P85 of human CD40, and/or proline at the position corresponding to LI 21 of human CD40. In some variations, the CD40 extracellular domain contains at least two of the positions corresponding to E64, K81, P85, and L121 of human CD40 (e.g., substitutions at at least the positions corresponding to K81 and L121 of human CD40, or substitutions at at least the positions corresponding to E64, K81, and P85 of human CD40). In some embodiments, the amino acid at the position corresponding to K81 of human CD40 is selected from threonine, histidine, and serine. In other embodiments, the amino acid at the position corresponding to K81 of human CD40 is histidine and the amino acid the position corresponding to L121 of human CD40 is proline. In yet other embodiments, the amino acid at the position corresponding to E64 of human CD40 is tyrosine, the amino acid at the position corresponding to K81 of human CD40 is threonine, and the amino acid at the position corresponding to P85 of human CD40 is tyrosine.

[118] 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., an Fc region and a paraoxonase, or an Fc region and a CTLA-4 or CD40 extracellular domain) 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 linked to a paraoxonase or a CTLA-4 extracellular domain, Fc receptor (e.g., FcRn) binding may be maintained for the Fc region, while functional properties of the paraoxonase (e.g., organophosphatase or arylesterase activity) or CTLA-4 extracellular domain (e.g. , CD80/CD86 binding) will be maintained. For examples of the use of naturally occurring as well as artificial peptide linkers to connect heterologous polypeptides, see, e.g., Hallewell et al., J. Biol. Chem. 264, 5260-5268, 1989; Alfthan et al.. Protein Eng. 8, 725-731, 1995; Robinson and Sauer, Biochemistry 35, 109-116, 1996; Khandekar et al. , J. Biol. Chem. 272, 32190-32197, 1997; Fares et al. , Endocrinology 139, 2459-2464, 1998; Smallshaw et al. , Protein Eng. 12, 623-630, 1999; U.S. Patent No. 5,856,456.

[119] Typically, residues within the linker polypeptide are selected to provide an overall hydrophilic character and to be non-immunogenic and flexible. As used herein, 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. In general, 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. In general. 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 hydrophobicity⁷ and lack of flexibility⁷, and Lys and Arg residues due to potential immunogenicity. The sequence of the linker will also be designed to avoid unwanted proteolysis.

[120] Exemplary LI linkers comprise at least three amino acid residues and are typically up to 60 amino acid residues. In certain variations, LI linkers comprise at least four, at least five, at least six, at least seven, at least eight, at least 9, or at least 10 amino acid residues. In more specific variations, LI consists of from six to 30, from six to 25, from six to 20, from seven to 30, from seven to 25, from seven to 20, from eight to 30, from eight to 25, from eight to 20. from nine to 30, from nine to 25, from nine to 20, from 10 to 30, from 10 to 25. from 10 to 20, from 11 to 30, from 11 to 25, from 11 to 20, from 12 to 30, from 12 to 25, or from 12 to 20 amino acid residues. In some embodiments, LI comprises or consists of the amino acid sequence shown in SEQ ID NO: 12.

[121] In certain embodiments, an 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). In particular variations, 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. In other variations, 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. In other variations. 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. In some embodiments, L2 comprises or consists of the amino acid sequence shown in residues 1-16 of SEQ ID NO: 10, residues 145- 146 of SEQ ID NO:22, residues 145-146 of SEQ ID NO:24, residues 145-149 of SEQ ID NO:26, or residues 189-213 of SEQ ID NO:42.

[122] In some variations of a fusion polypeptide comprising the formula P-L2-X as described herein, L2 comprises at least 20 amino acid residues. For example, an L2 linker joining the carboxy 1-terminus of a paraoxonase to the amino-terminus of another fusion component (e.g., an immunoglobulin Fc region) may comprise at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, or at least 36 amino acid residues. In certain variations, L2 consists of from 20 to 60 amino acid residues, from 20 to 50 amino acid residues, from 20 to 45 amino acid residues, from 20 to 40 amino acid residues, from 20 to 36 amino acid residues, from 26 to 60 amino acid residues, from 26 to 50 amino acid residues, from 26 to 45 amino acid residues, from 26 to 40 amino acid residues, from 26 to 36 amino acid residues, from 36 to 60 amino acid residues, from 36 to 50 amino acid residues, from 36 to 45 amino acid residues, or from 36 to 40 amino acid residues. In some embodiments, L2 linking the carboxyl-terminus of a paraoxonase comprises or consists of the amino acid sequence shown in residues 1-26 of SEQ ID NO: 10.

[123] In certain embodiments, a polypeptide linker comprises a plurality of glycine residues. For example, in some embodiments, a polypeptide linker (e.g, L2) comprises a plurality of glycine residues and optionally at least one serine residue. In particular variations, a polypeptide linker (e.g., L2) comprises the sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:65), such as, e.g, two or more tandem repeats of the amino acid sequence of SEQ ID NO:65. In some embodiments, a linker comprises the sequence [Gly-Gly-Gly-Gly-Ser]_n ([SEQ ID NO:65]_n), where n is a positive integer such as, for example, an integer from 2 to 8, from 2 to 7, from 2 to 6, from 3 to 8, from 3 to 7, from 3 to 6, from 4 to 8, from 4 to 7, or from 4 to 6. In a specific variation of a polypeptide linker comprising the formula [Gly-Gly-Gly-Gly-Ser]_n, n is 4. In another specific variation of a polypeptide linker comprising the formula [Gly-Gly- Gly-Gly-Ser]n, n is 6. In yet another specific variation of a polypeptide linker comprising the formula [Gly-Gly-Gly-Gly-Ser]_n, n is 5. In certain embodiments, 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., inserted between Asp-Leu-Ser at the N-terminal end of the linker and Thr-Gly-Leu at the C-terminal end of the linker). In other embodiments, a polypeptide linker includes glycine and serine residues (e.g, [Gly-Gly-Gly-Gly-Ser]_n, where n is defined as above) attached at one or both ends of another sequence of the polypeptide linker.

[124] In some embodiments of a fusion polypeptide as above, X is a dimerizing domain. Various dimerization domains are suitable for use in accordance with certain fusion polypeptide embodiments and dimeric fusion proteins as described herein. In certain embodiments of a fusion polypeptide comprising a dimerizing domain, the dimerizing domain is an immunoglobulin heavy chain constant region. The immunoglobulin heavy chain constant region may be a native sequence constant region or a variant constant region. In typical variations, an immunoglobulin heavy chain constant region is capable of binding to the neonatal Fc receptor (FcRn) with sufficient affinity to confer improved half-life to the fusion polypeptide in vivo. A particularly suitable immunoglobulin heavy chain constant region for use in accordance with the present invention is an immunoglobulin Fc region. In some embodiments, the heavy chain constant region lacks one or more effector functions (e.g., one or both of ADCC and CDC effector functions).

[125] In some embodiments of a fusion polypeptide comprising an immunoglobulin Fc region, the immunoglobulin Fc region is a human IgG Fc region having, relative to the wildtype human IgG sequence, an amino acid substitution in the CH2 region so that the molecule is not glycosylated, including but not limited to an amino acid substitution at N297 (Eu numbering for human IgG heavy chain constant region) (corresponding to amino acid position 82 of SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:48, SEQ ID NO:52, or SEQ ID NO:54). In another embodiment, the Fc region is human IgGl (yl) with the three cysteines of the hinge region (C220, C226, C229) each changed to a non-cysteine residue (e.g., serine) and, optionally, the proline at position 238 of the CH2 domain changed to a non-proline residue (e.g, serine or aspartate). In another embodiment, the Fc region is human yl with cysteine C220 changed to a non-cysteine residue (e.g. serine) and, optionally, the proline at position 238 of the CH2 domain changed to anon-proline residue (e.g, serine or aspartate). In another embodiment, the Fc region is human yl with N297 changed to a non-asparagine residue (e.g, alanine, glutamine, or glycine). In another embodiment, the Fc region is human yl with one or more amino acid substitutions between Eu positions 292 and 300. In another embodiment, the Fc region is human yl with one or more amino acid additions or deletions at any position between residues 292 and 300. In another embodiment, the Fc region is human yl with an SCC hinge (z.e., with cysteine C220 changed to serine and with a cysteine at each of Eu positions 226 and 229) or an SSS hinge (z.e., each of the three cysteines at Eu positions 220, 226, and 229 changed to serine). In further embodiments, the Fc region is human yl with an SCC hinge and an amino acid substitution at P238. In another embodiment, the Fc domain is human yl with amino acid substitutions that alter binding by Fc gamma receptors (I, II, III) without affecting FcRn binding important for half-life. In further embodiments, 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.

[126] In some embodiments, the Fc region comprises an amino acid substitution that alters the antigen-independent effector functions of the fusion protein. In some such embodiments, the Fc region includes an ammo acid substitution that alters the circulating halflife of the resulting molecule. Such Fc variants exhibit either increased or decreased binding to FcRn when compared to an Fc region lacking these substitutions and, therefore, confer increased or decreased half-life, respectively, of the resulting molecule in serum. Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such Fc variants have useful applications in methods of treating mammals where long half-life of the administered Fc fusion is desired and where increased transport through the lungs to the circulation is desired. In contrast, Fc variants with decreased FcRn binding affinity are expected to have shorter half-lives, and such variants are also useful, for example, for administration to a mammal where a shortened circulation time may be advantageous (e.g, where the fusion protein has toxic side effects when present in the circulation for prolonged periods). Fc variants with decreased FcRn binding affinity⁷ are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in pregnant women. In addition, other applications in which reduced FcRn binding affinity⁷ may be desired include those applications in which localization to the brain, kidney, and/or liver is desired. In one exemplary⁷ embodiment, the fusion molecules of the invention exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature. In another embodiment, the fusion molecules of the invention exhibit reduced transport across the blood brain barrier (BBB) from the brain, into the vascular space. In one embodiment, a fusion molecule 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 that alter FcRn binding activity are disclosed in International PCT Publication No. WO 05/047327, which is incorporated by reference herein. Exemplary amino acid substitutions that increase FcRn binding activity are also described, e.g., by Wang et al., Protein Cell 9:63-73, 2018 (see, e.g., Table 1). In some embodiments, an Fc variant with increased FcRn binding activity has an amino acid substitution at each of Eu positions 252, 254, and 256 (e.g., M252Y, S254T, and T256E). In other variations, an Fc variant with increased FcRn binding activity has an amino acid substitution at each of Eu positions 428 and 434 (e.g., M428L and N434S).

[127] In other embodiments, a fusion polypeptide of the present invention comprises an Fc variant comprising an amino acid substitution that alters one or more antigen-dependent effector functions of the polypeptide, in particular antibody-dependent cellular cytotoxicity⁷ (ADCC) or complement activation, e.g. , as compared to a wild t pe Fc region. In an exemplary embodiment, such fusion polypeptides exhibit altered binding to an Fc gamma receptor (FcyR, e.g, CD16). Such fusion polypeptides exhibit either increased or decreased binding to FcyR when compared to wild-type polypeptides and, therefore, mediate enhanced or reduced effector function, respectively . Fc variants with improved affinity for FcyRs are anticipated to enhance effector function, and such variants have useful applications in methods of treating mammals where target molecule destruction is desired. In contrast, Fc variants with decreased FcyR binding affinity⁷ 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 fusion molecule might result in unwanted immune system activation. In one embodiment, 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 cytotoxicity⁷ (CDC), antibody-dependent cellular cytotoxicity (ADCC), or effector cell modulation as compared to a polypeptide comprising a wild-type Fc region. In typical embodiments of a DNase fusion molecule comprising an Fc variant with altered antigen-dependent effector function, the Fc variant has one or more reduced effector functions relative to the corresponding wild-type Fc region.

[128] In one embodiment, a fusion polypeptide comprising an Fc region exhibits altered binding to an activating FcyR (e.g, Fcyl, Fcylla, or FcyRIIIa). In another embodiment, the fusion protein exhibits altered binding affinity to an inhibitory FcyR (e.g., FcyRIIb). Exemplary⁷ amino acid substitutions which alter FcR or complement binding activity' are disclosed in International PCT Publication No. WO 05/063815, which is incorporated by reference herein. Exemplary Fc variants with reduced effector function are also described, e.g. , by Tam et al., Antibodies 6: 12, 2017 (describing variants of human IgGl (yl) and IgG4(y4)); Wang etal., Protein Cell 9:63-73, 2018 see, e.g., Table 1); Lo etal.,J. Biol. Chem. 292:3900- 3908, 2017; Idusogie et al., J. Immunol. 164:4178-4184, 2000 (each of which is incorporated by reference herein). Suitable Fc variants that reduce antigen-dependent effector function include, for example, variants having an amino acid substitution at Eu position 238 and/or position 331 e.g., P238S and/or P331S or P331A). In addition, amino acid substitutions at Eu positions 234 and 235 of human Fc (e.g., L234A/L235A in IgGl, or F234A/L235A in IgG4) reduce FcyR binding and have been shown to reduce cytokine storm when introduced into anti- CD3 mAb {see, e.g., Wang et al, supra), and an amino acid substitution at Eu position 329 (e.g, P329A) is highly effective at reducing Clq binding {see, e.g., Lo et al., supra). Other exemplary' approaches to removing ADCC and CDC effector functions is to make hybrid Fc domains derived from human IgG2 (Eu positions 118-260) and IgG4 (Eu positions 261-447), or to modify human IgG2 to contain selected amino acid substitutions from IgG4. See Wang et al. , supra.

[129] A fusion polypeptide comprising an Fc region may also comprise an amino acid substitution that alters the glycosylation of the Fc region. For example, the Fc domain of the fusion protein may have a mutation leading to reduced glycosylation {e.g., N- or O-linked glycosylation) or may comprise an altered glycoform of the wild-type Fc domain {e.g, a low fucose or fucose-free glycan). In another embodiment, 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. 2007/0111281, which are incorporated by reference herein.

[130] Particularly suitable amino acid substitutions to reduce glycosylation and which also reduce ADCC and CDC effector functions of Fc include amino acid substitutions at Eu position 297 {e.g., N297A, N297Q, or N297G). See, e.g., Wang et al., supra. N297 substitutions may also be paired with substitions at position 265 (e.g., D265A) to further reduce CDC. See, e.g., Lo et al., supra.

[131] It will be understood by those of skill in the art that various embodiments of Fc variants as described herein can be combined in the fusion polypeptides of the present invention, unless the context clearly indicates otherwise.

[132] In some embodiments, an immunoglobulin Fc region 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 1-232 or 1-231 of SEQ ID NO:6, (ii) residues 1-232 or 1-231 of SEQ ID NO:8, (iii) residues 1-232 or 1-231 of SEQ ID NO:48, (iv) residues 1-232 or 1-231 of SEQ ID NO:50, (v) residues 1-232 or 1-231 of SEQ ID NO:52, or (vi) residues 1-232 or 1-231 of SEQ ID NO:54. In yet other embodiments, the Fc region comprises 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 1-232 or 1-231 of SEQ ID NO:6, (ii) residues 1-232 or 1-231 of SEQ ID NO:8, (iii) residues 1-232 or 1-231 of SEQ ID NO:48, (iv) residues 1-232 or 1-231 of SEQ ID NO:50. (v) residues 1-232 or 1-231 of SEQ ID NO 52, or (vi) residues 1-232 or 1-231 of SEQ ID NO:54.

[133] In some embodiments, an immunoglobulin heavy chain constant region 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 16- 232 or 16-231 of SEQ ID NO: 6, (ii) residues 16-232 or 16-231 of SEQ ID NO: 8, or (iii) residues 16-232 or 16-231 of SEQ ID NO:48. In yet other embodiments, the immunoglobulin heavy chain constant region comprises 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 16-232 or 16-231 of SEQ ID NO:6, (ii) residues 16-232 or 16-231 of SEQ ID NO:8, or (iii) residues 16-232 or 16-231 of SEQ ID NO:48.

[134] In some variations of a fusion polypeptide as above, X is an FcRn-binding domain. Particularly suitable FcRn-binding domains include immunoglobulin heavy chain constant regions that retain FcRn-binding activity such as, e.g., an immunoglobulin Fc region; 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). In other embodiments, an FcRn-binding domain is an albumin (e.g., human albumin) or a fragment thereof (e.g, domain III of albumin; see, e.g., see, e.g., Zhao et al., supra). Yet other suitable FcRn-binding domains include singlechain antibodies (e.g., scFvs), peptide aptamers, or alternative scaffold proteins having binding affinity for FcRn; such alternative FcRn-binding molecules 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, randomized peptides, or other protein structures). Such 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. Alternative scaffold proteins 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.).

[135] In some embodiments of a paraoxonase 3 (PON3) fusion polypeptide as described above, the fusion polypeptide comprises 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 21-607, 24-607, or 1-607 of SEQ ID NO:32, or (li) residues 21-604 or 1-604 of SEQ ID NO:34. In other embodiments, 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 amino acid sequence shown in (i) residues 21-607, 24-607, or 1-607 of SEQ ID NO:32, or (ii) residues 21-604 or 1-604 of SEQ ID NO:34.

[136] In some embodiments of an evolved paraoxonase 1 (PON1) fusion polypeptide as described above, 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 the sequence shown in (i) residues 21-613, 24-613, or 1-613 of SEQ ID NO: 18, (ii) residues 21-610 or 1-610 of SEQ ID NO:20, (iii) residues 21-739 or 1-739 of SEQ ID NO:26, (iv) residues 21-736 or 1-736 of SEQ ID NO:28, (v) residues 21-613, 24-613, or 1-613 of SEQ ID NO:36, (vi) residues 21-610 or 1-610 of SEQ ID NO:38, (vii) residues 21-736 or 1-736 of SEQ ID NO:40, (viii) residues 21-803 of SEQ ID NO:42. (ix) residues 21-803 of SEQ ID NO:44, or (x) residues 21-739 of SEQ ID NO:58. In yet other embodiments, 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 amino acid sequence shown in (i) residues 21-613. 24-613, or 1-613 of SEQ ID NO: 18, (ii) residues 21-610 or 1-610 of SEQ ID NO:20, (iii) residues 21-739 or 1-739 of SEQ ID NO:26, (iv) residues 21-736 or 1-736 of SEQ ID NO:28, (v) residues 21-613, 24-613, or 1-613 of SEQ ID NO:36, (vi) residues 21-610 or 1-610 of SEQ ID NO:38, (vii) residues 21-736 or 1-736 of SEQ ID NO:40, (viii) residues 21-803 of SEQ ID NO:42, (ix) residues 21-803 of SEQ ID NO:44. or (x) residues 21-739 of SEQ ID NO:58.

[137] 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, wherein each of the first and second fusion polypeptides comprises, from an amino-terminal position to a carboxyl-terminal position, X-Ll-P, wherein X is a dimerizing domain; LI is a polypeptide linker that is optionally present; and P is a biologically active paraoxonase. In some embodiments, each of the first and second fusion polypeptides is a bispecific contruct further comprising a biologically active polypeptide amino-terminal to the dimerizing domain, wherein each of the first and second fusion polypeptides comprises, from an amino-terminal position to a carboxyl-terminal position, T-L2-X-L1-P, wherein X, LI, and P are defined as above, L2 is a second polyeptide linker, wherein L2 is optionally present, and T is the biologically active polypepetide. In another aspect, the present invention provides a dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, wherein each of the first and second fusion polypeptides comprises, from an amino-terminal position to a carboxyl-terminal position, P-L2-X, wherein P is a biologically active paraoxonase, L2 is a polypeptide linker, and X is a dimerizing domain.

III. Materials and Methods for Making Polypeptide Fusions and Dimeric Proteins

[138] 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., an Fc fragment; paraoxonase and CTLA-4 polypeptide segments) can be generated and linked together to form a polynucleotide encoding a fusion polypeptide as described herein using known methods for recombinant manipulation of nucleic acids. [139] DNA sequences encoding fusion components in accordance with the present disclosure (for example, paraoxonases (e.g., PON3, PON1 1F11), CTLA-4 and CD40 extracellular domains, and immunoglobulin Fc regions) are generally known in the art. Exemplary DNA sequenes are disclosed herein (see Sequence Listing). 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, paraoxonase enzyme activity or CD80/CD86 binding activity) using an appropriate screening assay.

[140] Methods for preparing DNA and RNA are well known in the art. For example, 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 poly(A)⁺ RNA using known methods. In the alternative, 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,” Mullis, U.S. Patent 4,683,202). Expression libraries can be probed with antibodies to the polypeptide of interest, receptor fragments, or other specific binding partners.

[141] 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. See generally Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA. ASM Press, Washington, D.C., 1994; Itakura et al., Ann. Rev. Biochem. 53:323-356. 1984; and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-637, 1990.

[142] In another aspect, 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 cells 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 cells (including cultured cells of multicellular organisms), particularly cultured mammalian cells. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al.. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., eds., Current Protocols in Molecular Biology, Green and Wiley and Sons, NY, 1993.

[143] In general, for production of a fusion polypeptide in a host cell, a DNA sequence encoding the fusion polypeptide is operably linked to other genetic elements required for its expression, typically including a transcription promoter and terminator, within an expression cassette. Typically, the expression cassette is contained within an expression vector for delivery into a host cell. 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 of an expression cassette into the host cell genome such as, e.g., in the generation of stable cell tines. 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.

[144] To direct a PON fusion polypeptide into the secretory pathway of a host cell, a secretory signal sequence is provided in the expression cassette. The encoded secretory peptide may be that of a corresponding native protein (e.g., a native CD40 secretory peptide as shown in amino acid residues 1-20 of SEQ ID NO:46), 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, z.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide fusion into the secretory⁷ pathway of the host cell. Secretory⁷ 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). Secretory⁷ signal sequences suitable for use in accordance with the present invention include, for example, polynucleotides encoding the human VK3 leader peptide (SEQ ID NO: 14).

[145] Expression of fusion polypeptides comprising a dimerizing domain as described herein, via a host cell secretory pathway, is expected to result in the production of dimeric proteins. Accordingly, in another aspect, 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, wherein each of the first and second fusion polypeptides comprises, from an amino-terminal position to a carboxyl- terminal position, X-Ll-P, P-L2-X, or T-L2-X-L1-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.

[146] Cultured mammalian cells are particularly suitable hosts for use within the present invention. Mammalian cells (e.g., CHO, COS, 293T) can express and secrete properly folded, active fusion proteins, whereas expression of fusion proteins in bacteria typically requires refolding of inactive protein. Fusion proteins expressed in mammalian cells also typically retain proper glycosylation, which can stabilize proteins and avoid potential immunogenicity. In addition, fusion proteins comprising an Fc region are particularly amenable to known antibody expression and purification technologies using mammalian systems such as, e.g., CHO, which is advantageous for increased manufacturability. [147] 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. 1 :841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., supra), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993). The production of recombinant polypeptides in cultured mammalian cells is disclosed by, for example, Levinson et al., U.S. Patent No. 4,713.339; Hagen et al., U.S. Patent No. 4,784.950; Palmiter et al., U.S. Patent No. 4,579,821 ; and Ringold, U.S. PatentNo. 4,656,134. 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. Additional 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. PatentNo. 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 Blvd., Manassas, VA USA under accession numbers 98669, 98668, and PTA-5266, respectively, and derivatives of these vectors.

[148] 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 — by virtue of integration of the expression cassette into its genomic DNA — 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 culturing 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 exemplary⁷ amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. 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, CD8, CD4, nen e growth factor receptor, green fluorescent protein, and the like.

[149] In some aspects, the present invention provides a stable cell line containing, within its genomic DNA, an expression cassette encoding a paraoxonase fusion polypeptide as described herein, wherein the stable cell line constitutively expresses the encoded paraoxonase fusion. Stable cell lines can be generated by methods generally known in the art, which generally include the identification of single stable cell clones from a polyclonal colony of stable transfectants by limited dilution and expansion. Protein expression of selected clones can then be assessed to identify high-expressing clones for expansion. In some embodiments, the stable cell line is a mammalian cell line such as, e.g., a Chinese hamster ovary (CHO) cell line. Recloning of the initial cultures can often stabilize and increase expression by 2-3 fold. Amplification of the expression level can also be achieved, e.g., by further plating of cells at low density in increasing levels of an appropriate selection agent e.g., methotrexate from an initial concentration of 50 nM up to as much as 1 pM). Once cells have adapted, further rounds of limiting dilution cloning are required to maintain high expression levels.

[150] Other higher eukary otic cells can also be used as hosts, including insect cells, plant cells and avian cells. The use of Agrobaclerium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11 :47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al. , U. S. Patent No. 5, 162,222 and International PCT Publication No. WO 94/06463.

[151] Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) (see King and Possee, The Baculovirus Expression System: A Laboratory Guide, Chapman & Hall, London; O'Reilly et al., Baculovirus Expression Lectors: A Laboratory Manual. Oxford University Press., New York, 1994; and Richardson, Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Humana Press, Totow a, NJ, 1995). Recombinant baculovirus can also be produced through the use of a transposon-based system described by Luckow et al. (J. Virol. 67:4566- 4579, 1993). 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, PFASTBAC1; Life Technologies) contains a Tn7 transposon to move the DNA encoding the protein of interest into a baculovirus genome maintained in E. coll as a large plasmid called a “bacmid” (see Hill-Perkins and Possee, J. Gen. Virol. 71 :971-976, 1990; Bonning eta/., J. Gen. Virol. 75: 1551-1556, 1994; Chazenbalk and Rapoport, J. Biol. Chem. 270: 1543-1549, 1995). Using techniques known in the art. a transfer vector encoding a polypeptide fusion is transformed into E. coli host cells, and the cells are screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, such as S19 cells. Recombinant virus that expresses the polypeptide fusion is subsequently produced. Recombinant viral stocks are made by methods commonly used in the art.

[152] For protein production, the recombinant virus is used to infect host cells, typically a cell line derived from the fall army worm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g.. HIGH FIVE cells; Invitrogen, Carlsbad, CA) (see generally Glick and Pasternak, 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 cells are grown up from an inoculation density' of approximately 2-5 x 10⁵ cells to a density' of 1-2 x 10⁶ cells, at which time a recombinant viral stock is added at a multiplicity’ of infection (MOI) of 0.1 to 10, more typically near 3. Procedures used are generally described in available laboratory manuals (e.g. , King and Possee, supra,' O’Reilly et al., supra:. Richardson, supra).

[153] 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 pastor is, 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. PatentNo. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug 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 POTI 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; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936; and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii , and Candida maltosa are known in the art. See, e.g., Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986; Cregg, U.S. Patent No. 4,882,279; and Raymond et al., Yeast 14: 11-23. 1998. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486.533. Production of recombinant proteins in Pichia methanolica is disclosed in U.S. Patents Nos. 5.716,808; 5,736,383; 5,854.039; and 5,888,768.

[154] 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). 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 HC1 or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the alternative, 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. To capture the protein of interest, 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.

[155] 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. A variety of 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.

[156] 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 Y ork, 1994. Proteins comprising an immunoglobulin Fc region 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 purity or to provide for desalting, buffer exchange, and the like.

[157] For example, 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. PEI, 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 butyl 650 (Toso Haas. Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic 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, hydroxyl groups and/or carbohydrate moi eties.

[158] 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); Doonan, Protein Purification Protocols (The Humana Press 1996).

[159] Additional variations in protein isolation and purification can be devised by those of skill in the art. For example, 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 a paraoxonase or CLTA-4 extracellular domain) can be used to isolate large quantities of protein by immunoaffinity purification.

[160] The proteins of the present invention can also be isolated by exploitation of particular properties. For example, immobilized metal ion adsorption (IMAC) 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 metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography {see. e.g., M. Deutscher, (ed.), Meth. Enzymol. 182:529, 1990). Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag {e.g., maltose-binding protein) may be constructed to facilitate purification. Moreover, receptor- or ligand-binding properties of a fusion polypeptide or dimer thereof can be exploited for purification. [161] 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.

IV. Methods of Use and Pharmaceutical Compositions

[162] The fusion polypeptides and dimeric proteins of the present invention can be used to provide therapy for the treatment of various diseases or disorders. The paraoxonase fusion polypeptides are particularly useful, e.g., for treatment of inflammatory, autoimmune, neurological, cardiovascular, and/or fibrotic diseases and disorders. In aspects relating to bispecific fusions further comprising a first biologically active polypeptide as described herein (e.g, a CTLA-4 or CD40 extracellular domain), the fusion polypeptides and dimeric proteins may further provide one or more additional biological activities for treatment. For example, paraoxonase fusion molecules comprising a CTLA-4 or CD40 extracellular domain are particularly useful, e.g., for treatment of diseases or disorders characterized by an aberrant adaptive immune response. In addition, paraoxonase fusion polypeptides comprising a CD40 extracellular domain are particularly useful, e.g, for treatment of diseases and disorders characterized by a fibrotic inflammatory response.

[163] In some aspects, the present invention provides methods for treating a disease or disorder selected from an inflammatory disease, an autoimmune disease, a neurological disease, an infectious disease, a metabolic disease, a cardiovascular disease, a liver disease, a fibrotic disease, biofilm formation by a gram-negative bacteria, 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.

[164] Inflammatory diseases amenable to treatment in accordance with the present invention include, for example, inflammatory lung diseases such as, for example, asthma, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), bronchiectasis, hypoxia, interstitial lung disease (e.g., idiopathic pulmonary fibrosis (IPF) or sarcoidosis), and acute respiratory⁷ distress syndrome (ARDS). In particular embodiments comprising treatment of ARDS, the ARDS is associated with COVID- 19. In some embodiments, the inflammatory lung disease is characterized by Pseudomonas aeruginosa infection. In some variations, a patient having the inflammatory lung disease is a patient that has been exposed to sulfur mustard gas (SM). In other variations, a patient having the inflammatory lung disease is a patient that has been exposed to an organophosphate, such as an insecticide (e.g, parathion, malathion, chlorpyrifos, diazinon, dichlorvos, phosmet, fenitrothion, terbufos, tetrachlorvinphos, azamethiphos, or azinphos-methyl) or other neurotoxin (e.g., tabun, sarin, soman, or cyclosarin).

[165] Other inflammatory' diseases amenable to treatment in accordance with the present invention include autoinflammatory diseases (z.e., innate immune system activation disorders characterized by seemingly unprovoked episodes of inflammation and a relative lack of obvious autoimmune pathology). Exemplary' autoinflammatory diseases include inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis), Behcet’s disease, systemic onset juvenile idiopathic arthritis (JIA), gout, pseudogout, storage (Gaucher’s) disorders, hereditary angioedema (HAE), atypical hemolytic uremic syndrome, familial Mediterranean fever (FMF), TNF -receptor associated periodic fever syndrome (TRAPS), cryopyrin-associated periodic syndromes (CAPS)), NOD2-associated autoinflammatory disease (NAID), and Blau syndrome.

[166] In yet other embodiments, an inflammatory disease or disorder for treatment in accordance with the present invention is selected from rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes, type 2 diabetes, hepatitis (e.g. , non-alcoholic steatohepatitis (NASH)), ankylosing spondylitis, psoriasis, psoriatic arthritis, dermatitis (e.g., atopic dermatitis), diverticulitis, irritable bowel syndrome, and nephritis. In still other variations, the inflammatory disease or disorder is an inflammatory' skin disease such as, e.g, psoriasis or atopic dermatitis.

[167] Autoimmune diseases amenable to treatment in accordance with the present invention include, for example, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, multiple sclerosis, type 1 diabetes, vasculitis, and systemic sclerosis (also known as scleroderma). In other embodiments, the autoimmune disease is selected from coeliac disease, neuritis, polymyositis, juvenile rheumatoid arthritis, psoriatic arthritis, vitiligo, Sjogren’s syndrome, autoimmune pancreatitis, autoimmune hepatitis, glomerulonephritis, lupus nephritis, scleroderma, antiphospholipid syndrome, autoimmune vasculitis, sarcoidosis, autoimmune thyroid diseases. Hashimoto’s thyroiditis, Graves disease, Wegener's granulomatosis, myasthenia gravis, Addison’s disease, autoimmune uveoretinitis, pemphigus vulgaris, primary biliary cirrhosis, pernicious anemia, sympathetic opthalmia, uveitis, autoimmune hemolytic anemia, pulmonary fibrosis, chronic beryllium disease, and idiopathic pulmonary fibrosis. In some variations comprising treatment of vasculitis, the vasculitis is selected from small vessel vasculitis and medium vessel vasculitis; in other variations, the vasculitis is large vessel vasculitis.

[168] Neurological diseases amenable to treatment in accordance with the present invention include, for example, neurodegenerative diseases characterized by inflammation in the CNS such as, e.g, multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, or amylotrophic lateral sclerosis (ALS). In some embodiments, the neurological disease is a neurodegenerative disease characterized by dementia such as, e.g, Alzheimer’s disease. In more specific variations of a method for treating multiple sclerosis (MS), the MS is spino- optical MS, primary progressive MS (PPMS), or relapsing remitting MS (RRMS). In other emodiments, a neurological disease for treatment in accordance with the present invention is a CNS infection such as. e.g, meningitis, encephalitis, or cerebral malaria. In more particular variations of a method for treating meningitis, the meningitis is a bacterial meningitis; in some such embodiments, the CNS infection is an S. pneumoniae, N. meningitis, S. aureus, E. coli, A. baumanii, S. oralis, S. capitis, or S. epidermidis infection. Other neurological diseases or disorders amenable to treatment with fusion molecules as described herein include, for example, acute brain injury such as, e.g., ischemic stroke. In still other embodiments, the neurological disease is a brain cancer such as, e.g., an intracranial tumor selected from astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, primary CNS lymphoma, medulloblastoma, germ cell tumor, pineal gland neoplasm, meningioma, pituitary tumor, tumor of the nerve sheath (e.g, schwannoma), chordoma, craniopharyngioma, and a choroid plexus tumor (e.g. , choroid plexus carcinoma).

[169] In more particular embodiments of a method for treating an inflammatory, autoimmune, or neurological disease as above, a fusion molecule for the inflammatory, autoimmune, or neurological disease treatment is a polypeptide having the structure Fc-Ll- P0N3, CTLA4-L2-Fc-Ll-PON3, CTLA4-Fc-Ll-PON3, CD40-L2-Fc-Ll-PON3, or CD40- Fc-Ll-PON3. or a dimeric protein formed by dimerization of any of the foregoing fusion polypeptides. In some such embodiments comprising a fusion polypeptide having the structure Fc-Ll-PON3. the fusion polypeptide comprises or consists 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% identity⁷ with the amino acid sequence shown in (i) residues 21-607 or 24-607 of SEQ ID NO:32 or (ii) residues 21-604 of SEQ ID NO:34. In some embodiments of a method for treating rheumatoid arthritis, psoriatic arthritis, or juvenile idiopathic arthritis, the fusion molecule is a polypeptide having the structure CTLA4-L2-Fc-Ll -PON3 or CTLA4-Fc-Ll-PON3, or a dimeric protein formed by dimerization of either of the foregoing fusion polypeptides.

[170] Infectious diseases amenable to treatment in accordance with the present invention include, for example, bacterial infections, viral infections, fungal infections, and parasitic infections. In some embodiments comprising treatment of a parasitic infection, the infection is a Trypanosoma brucei. Leishmania. Plasmodium falciparum, or Toxoplasma gondii infection. In some embodiments comprising treatment of a bacterial infection, the infection is a Staphylococcus aureus, Streptococcus pneumoniae, or Mycobacterium tuberculosis infection. In other embodiments, the bacterial infection is a Pseudomonas aeruginosa infection. In yet other embodiments, the bacterial infection is a Borrelia burgdorferi infection (Lyme disease). In some embodiments comprising treatment of a viral infection, the infection is an influenza virus (e.g., influenza A virus) or respiratory syncytial virus (RSV) infection. In still other embodiments, the infection is a CNS infection such as, for example, meningitis (e.g., a bacterial meningitis), encephalitis, or cerebral malaria.

[171] In some embodiments of a method for treating biofilm formation by a gram negative bacteria, the gram negative bacteria is Pseudomonas aeruginosa.

[172] Metabolic diseases that may be treated in accordance with the present invention include, for example, type 2 diabetes and obesity'.

[173] Cardiovascular diseases that may be treated in accordance with the present invention include, for example, cardiovascular diseases characterized by atherosclerosis. In some embodiments, the cardiovascular disease characterized by atherosclerosis is coronary' heart disease or ischemic stroke. In more particular variations comprising treatment of coronary heart disease, the coronary heart disease is characterized by acute coronary syndrome. [174] In some embodiments of a method for treating an infectious, metabolic, or cardiovascular disease as above, or for treating biofilm formation by a gram negative bacteria as above, a fusion molecule for the treatment is a polypeptide having the structure Fc-Ll- PON3, or a dimeric protein formed by dimerization of the foregoing fusion polypeptide. In some such embodiments, the fusion polypeptide comprises or consists 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 the amino acid sequence shown in (i) residues 21-607 or 24-607 of SEQ ID NO 32 or (ii) residues 21-604 of SEQ ID NO:34.

[175] In some embodiments of a method for treating exposure to sulfur mustard gas (SM) or exposure to an organophosphate (e.g., tabun, sarin, soman, or cyclosarin), a fusion molecule for such exposure treatment is a polypeptide having the structure Fc-Ll-[PON1 1F11], FC-L1-[PON1 2G1], CTLA4-L2-FC-L1-[PON1 1F11], CTLA4-L2-Fc-Ll-[PON1 2G1], CTLA4-Fc-Ll-[PON1 1F11], CTLA4-Fc-Ll-[PON1 2G1], CD40-L2-Fc-Ll-[PON1 1F11], CD40-L2-Fc-Ll-[PON1 2G1], CD40-L2-Fc-Ll-[PON1 2G1], CD40-Fc-Ll-[PONI 1F11], or CD40-Fc-Ll-[PON1 2G1], or a dimeric protein formed by dimerization of any of the foregoing fusion polypeptides. In some such embodiments, the fusion polypeptide comprises or consists 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 the amino acid sequence shown in (i) residues 21 -613 or 24-613 of SEQ ID NO : 18, (ii) residues 21 -610 of SEQ ID NO : 20, (iii) residues 21 - 739 of SEQ ID NO:26, (iv) residues 21-736 of SEQ ID NO:28, (v) residues 21-613 or 24-613 of SEQ ID NO:36, (vi) residues 21-610 of SEQ ID NO:38, (vii) residues 21-736 of SEQ ID NO:40, (viii) residues 21-803 of SEQ ID NO:42, (ix) residues 21-803 of SEQ ID NO:44. or (x) residues 21-739 of SEQ ID NO:58.

[176] Liver diseases or disorders that may be treated in accordance with the present invention include chronic liver diseases such as, e.g., nonalcoholic fatty liver disease (NAFLD), alcohol-associated liver disease (ALD), portal hypertension, and complications following liver transplantation. In some variations comprising treatment of nonalcoholic fattyliver disease (NAFLD), the NAFLD is nonalcoholic steatohepatitis (NASH).

[177] Fibrotic diseases or disorders amenable to treatment in accordance with the present invention include systemic sclerosis, systemic lupus erythematosus, inflammatory lung diseases, chronic liver diseases, and chronic kidney diseases. In some variations comprising treatment of an inflammatory- lung disease, the fibrotic disease is cystic fibrosis, chronic obstructive pulmonary disease, interstitial lung disease (e.g., idiopathic pulmonary fibrosis or sarcoidosis), acute respiratory⁷ distress syndrome, or asthma. In some variations comprising treatment of a chronic liver disease, the fibrotic disease is nonalcoholic steatohepatitis. alcohol- associated liver disease, portal hypertension, or a complication following liver transplantation. In some variations comprising treatment of a chronic kidney disease, the fibrotic disease is lupus nephritis, IgA nephropathy, or membranous glomerulonephritis.

[178] In particular variations of a method for treating a liver or fibrotic disease or disorder as above, a fusion molecule for the liver or fibrotic disease or disorder treatment is a polypeptide having the structure Fc-Ll-PON3, CD40-L2-Fc-Ll-PON3. or CD40-Fc-Ll- PON3, or a dimeric protein formed by dimerization of any of the foregoing fusion polypeptides. In some such embodiments comprising a fusion polypeptide having the structure Fc-Ll-PON3, the fusion polypeptide comprises or consists 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 the amino acid sequence shown in (i) residues 21-607 or 24-607 of SEQ ID NO:32 or (ii) residues 21-604 of SEQ ID NO:34.

[179] 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 cavity⁷, orophyarynx, nasopharynx, hypophary nx, nasal cavity⁷ or paranasal sinuses, larynx, lip, or salivary gland); a lung cancer (e.g., non-small cell lung cancer, small cell carcinoma, or mesothelioma); a gastrointestinal tract cancer (e.g, colorectal cancer, gastric cancer, esophageal cancer, or anal cancer); gastrointestinal stromal tumor (GIST); 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, hemangiosarcoma, extrahepatic or intrahepatic cholangiocarcinoma, cancer of the ampulla of vater. or gallbladder cancer); a breast cancer (e.g., metastatic breast cancer or inflammatory breast cancer); 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 nerv ous system (CNS) such as an intracranial tumor (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, primary CNS lymphoma, medulloblastoma, germ cell tumor, pineal gland neoplasm, meningioma, pituitary tumor, tumor of the nen e sheath (e.g., schwannoma), chordoma, craniopharyngioma, a choroid plexus tumor (e.g., choroid plexus carcinoma), or other intracranial tumor of neuronal or glial origin) or a tumor of the spinal cord (e.g., schwannoma, meningioma); 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., a fibrous tumor or fibrohistiocytic tumor); a tumor of the smooth muscle or skeletal muscle; a blood or lymph vessel perivascular tumor (e.g., Kaposi’s sarcoma); a synovial tumor; a mesothelial tumor; a neural tumor; a paraganglionic tumor; an extraskeletal cartilaginous or osseous tumor; and a pluripotential mesenchymal tumor.

[180] In some embodiments of a method for treating a cancer as above, a fusion molecule for the treatment is a polypeptide having the structure Fc-Ll-PON3, or a dimeric protein formed by dimerization of the foregoing fusion polypeptide. In some such embodiments, the fusion polypeptide comprises or consists 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 the amino acid sequence shown in (i) residues 21-607 or 24-607 of SEQ ID NO: 32 or (ii) residues 21-604 of SEQ ID NO:34.

[181] In some embodiments, a paraoxonase 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 an immunomodulatory therapy (e.g. , a CAR T cell therapy (see, e.g., June et al., Science 359: 1361-1365. 2018) or a therapy comprising an immune checkpoint inhibitor), a radiation therapy, or a chemotherapy.

[182] In certain embodiments, a combination cancer therapy comprises a PON3 (e.g, Fc-Ll-PON3) 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). Exemplary antibody targeted therapies include anti-VEGF (e.g., bevacizumab), 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).

[183] In some cancer combination therapy variations comprising an immune checkpoint inhibitor, the combination therapy includes an anti-PD-1 /PD-L1 therapy, an anti- CTLA-4 therapy, or both. In certain aspects, a PON3 (e.g, Fc-Ll-PON3) fusion molecule as described herein can increase the response rate to either anti-CTLA-4 or anti-PD-1 /PD-L1 therapy, as well as the response rate to the combination of anti-CTLA-4 plus anti-PD-l/PD-Ll therapy. Fusion molecules of the invention may also be useful for reducing the toxicity⁷ associated with anti-CTLA-4, anti-PD-1 /PD-L1, or the combination thereof.

[184] In certain variations, a cancer treated in accordance with the present invention is selected from malignant melanoma, renal cell carcinoma, non-small cell lung cancer, bladder cancer, and head and neck cancer. These cancers have shown responses to immune checkpoint inhibitors anti-PD-l/PD-Ll and anti-CTLA-4. See Grimaldi et al., Expert Opin. Biol. Ther. 16:433-41, 2016; Gunturi et al., Curr. Treat. Options Oncol. 15: 137-46, 2014; Topalian et al., Nat. Rev. Cancer 16:275-87, 2016. Thus, in some more specific variations, any of these cancers is treated with a PON3 (e.g, Fc-Ll-PON3) fusion molecule as described herein in combination with an anti-PD-l/PD-Ll therapy, an anti-CTLA-4 therapy, or both.

[185] In other aspects, the present invention provides methods for reducing lipid oxidation in a subject. The method generally includes administering to the subject an effective amount of a fusion polypeptide or dimeric protein as described herein, wherein one or more oxidized lipids in the subject are reduced. In some embodiments, the method reduces one or more oxidized lipids associated with the presence of a disease or disorder in the subject (e.g, a disease or disorder discussed above). In other embodiments, the one or more oxidized lipids are associated with a risk of developing such a disease or disorder; in particular variations, treatment with the fusion molecule reduces the risk of developing the disease or disorder in the subject. [186] In another aspect, the present invention provides a method for protecting a subject from aging. The method generally includes administering to the subject an effective amount of a fusion polypeptide of a fusion polypeptide or dimeric protein as described herein. In some embodiments, the subject has an age-related disease or disorder (e.g.. an inflammatory disease, an autoimmune disease, a neurodegenerative disease, a cardiovascular disease, or a fibrotic disease). In other embodiments, the subject is at risk of developing such an age-related disease or disorder, and treatment with the fusion molecule reduces the risk of the disease or disorder in the subject.

[187] In some embodiments of a method for treating a disease or disorder, reducing lipid oxidation, or protecting from aging as above, the method is a combination therapy comprising administering to the patient (a) an effective amount of a paraoxonase fusion polypeptide having the formula X-Ll-P or T-L2-X-L1-P as described above, or a dimeric protein formed by dimerization of the fusion polypeptide, and (b) an effective amount of a biologically active DNase. Such embodiments are particularly useful, e.g.. for treatment of diseases and disorder characterized by NETosis (z.e., the process wherein activated neutrophils extrude DNA bound with cytoplasmic and granule proteins, called neutrophil extracellular traps (NETs)). In some variations, the method comprises administration of a paraoxonase fusion polypeptide having the structure Fc-Ll-PON3, or a dimeric protein formed by dimerization thereof; in some such embodiments, the paraoxonase fusion polypeptide comprises or consists 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 the amino acid sequence shown in (i) residues 21-607 or 24-607 of SEQ ID NO:32 or (ii) residues 21-604 of SEQ ID NO:34. The biologically active DNase may be, for example, a DNasel or DNaselL3 polypeptide as described in International PCT Publication No. WO 2022/178078, incorporated by reference herein (e.g., a polypeptide corresponding to the DNase component of a DNase- containing paraoxonase fusion as described in WO 2022/178078). In some embodiments, the DNase is contained within a fusion polypeptide (for example, a fusion polypeptide comprising, from an amino terminal position to a carboxyl terminal position, D-L2-Xd, wherein D is the DNase, L2 is a polypeptide linker (e.g., an L2 linker as described herein), and Xd is an immunoglobulin Fc region as described herein). DNase fusion molecules suitable for use in combination with a paraoxonase fusion molecule as described herein are also described in International PCT Publication No. WO 2022/178090, incorporated by reference herein. [188] In some embodiments of a method for treating a disease or disorder, reducing lipid oxidation, or protecting from aging as above, the method is a combination therapy comprising administering to the patient (a) an effective amount of a paraoxonase fusion polypeptide having the formula X-Ll-P or T-L2-X-L1-P as described above, or a dimeric protein formed by dimerization of the fusion polypeptide, and (b) an effective amount of a biologically active apolipoprotein A-l (ApoAl) (e.g, an ApoAl-Fc fusion polypeptide as described in International PCT Publication No. WO 2017/044424 (incorporated by reference herein), or a dimeric protein formed by dimerization of the fusion polypeptide).

[189] In some embodiments of a method for treating a disease or disorder, reducing lipid oxidation, or protecting from aging as above, the method is a combination therapy comprising administering to the patient (a) an effective amount of a paraoxonase 3 (PON3) fusion polypeptide having the formula X-Ll-P or T-L2-X-L1-P as described above, or a dimeric protein formed by dimerization of the fusion polypeptide, and (b) an effective amount of a biologically active paraoxonase I (PON1). In some variations, the method comprises administration of a PON3 fusion polypeptide having the structure Fc-Ll-PON3, or a dimeric protein formed by dimerization thereof; in some such embodiments, the PON3 fusion polypeptide comprises or consists 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 the amino acid sequence shown in (i) residues 21-607 or 24-607 of SEQ ID NO:32 or (ii) residues 21-604 of SEQ ID NO: 34. The biologically active PON1 may be, for example, a PON1 polypeptide as described in International PCT Publication No. WO 2022/178078, incorporated by reference herein (e.g., a polypeptide corresponding to the PON1 component of a paraoxonase fusion as described in WO 2022/178078). In some embodiments, the PON1 is contained within a fusion polypeptide (for example, a fusion polypeptide comprising, from an amino terminal position to a carboxyl terminal position, Xd-Ll-Pi, wherein Xd is an immunoglobulin Fc region as described herein, LI is a polypeptide linker (e.g., an LI linker as described herein), and Pi is the PON1 polypeptide). PON1 fusion molecules suitable for use in combination with a PON3 fusion molecule as described herein are described in WO 2022/178090, supra. PON3/PON1 combination therapy may be particularly advantageous since the two enzymes have overlapping but distinct substrate specificities. In certain variations, a PON3/PON1 combination therapy comprises administration of PON3 and PON1 at a PONLPON3 ratio of from about 5:l to about 200: 1, from about 10: 1 to about 200: 1, from about 5: l to about 100: 1, from about 10: 1 to about 100: 1, from about 5: 1 to about 50: 1, or from about 10: 1 to about 50: 1.

[190] For therapeutic use, 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. In accordance with the disclosure herein, 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.

[191] 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. In certain embodiments, 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 a protein selected from a paraoxonase, a CTLA-4 extracellular domain, or a CD40 extracellular domain.

[192] In prophylactic applications, pharmaceutical 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 onset of the disease. In therapeutic applications, 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. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient response (e.g., enhanced alveolar fluid clearance in inflammatory lung disease) has been achieved. Typically, the response is monitored and repeated dosages are given if the desired response starts to fade.

[193] To identify subject patients for treatment according to the methods of the invention, 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 variety⁷ of immunological methods are know n 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. These methods allow the clinician to routinely select patients in need of the methods described herein for treatment. In accordance with these methods, 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.

[194] For administration, 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, e.g, Gennaro (ed.), Remington 's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995). Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.

[195] 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, intraarticular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, and oral routes of administration. For prevention and treatment purposes, the fusion polypeptide or dimeric protein may be administered to a subject in a single bolus delivery⁷, via continuous delivery⁷ (e.g., continuous transdermal delivery) over an extended time period, or in a repeated administration protocol (e.g.. on an hourly, daily, or weekly basis). [196] In some embodiments, a pharmaceutical composition comprising a fusion molecule or dimeric protein of the present invention is formulated for delivery to the lung by nebulization. Previous studies of pulmonary delivery of Fc fusion proteins, including erythropoietin-Fc, interferon Betala-Fc. and FSH-Fc, have shown that the immunoglobulin transport FcRn pathway is active in the lungs and provides 20% to 50% bi oavai I ability (see Bitonti et al., Proc. Natl. Acad. Sci. USA 101 :9763-9768, 2004; Bitonti and Dumont, Adv. Drug Deliv. Rev. 58: 1106, 2006; Valle et al., J. Interferon Cytokine Res. 32: 178-184, 2012). Pulmonary delivery of FcRn-binding paraoxonase fusion molecules as described herein (e.g., paraoxonase fusion molecules containing an Fc region) could act both locally and in circulation and peripheral organs. In certain embodiments wherein the treatment is a combination therapy with a paraoxonase fusion molecule and a DNase, both the paraoxonase fusion molecule (e.g., an Fc-Ll-PON3 fusion as described herein) and the DNase (e.g.. a DNase-Fc fusion such as described, e.g., in International PCT Publication No. WO 2022/178090, incorporated by reference herein) are delivered by a nebulizer. In some variations comprising delivery to the lung by a nebulizer, the disease or disorder to be treated is selected from an inflammatory lung disease (e.g, cystis fibrosis, interstitial lung disease (e.g, idiopathic pulmonary fibrosis or sarcoidosis), acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), asthma, exposure to sulfur mustard gas, or exposure to an organophosphate), biofilm formation by a gram-negative bacteria (e.g. , Pseudomonas aeruginosa), and sepsis. In other, non-mutually exclusive variations, an FcRn-binding fusion molecule for delivery’ to the lung by nebulization (e.g., an Fc-Ll-PON3, Fc-Ll-[PON 1F11], or CTLA4-L2-Fc-Ll-[PON1 1 Fl 1 ] fusion as described herein) comprises an Fc variant with increased FcRn-binding affinity relative to the corresponding wild-type Fc, thereby increasing fusion molecule half-life and increasing concentration in the blood after inhalation with a nebulizer.

[197] Multiple biologies have been successfully formulated to retain biologic activity⁷ after nebulization (see, e.g., Hertel etal.,Adv. DrugDeliv. Rev. 93:79-94, 2015). Formulations for delivery of a paraoxonase fusion molecule of the present invention may include an excipient suitable for pulmonary delivery⁷ such as, e.g. , Polysorbate 80 (PS80) or Polysorbate 20 (PS20), surfactants that are included in many biopharmaceutical formulations. Such stabilizing excipients protect proteins from degradation at the air-liquid interface when applied above their critical micelle concentration (for example, PS80 above 0.01% was effective in stabilizing G- CSF, LDH, rhConIFN, and t-Pa; and PS20 applied at 0.04% was effective for protection of Fc- gamma Rllb). Another suitable stabilizing excipient is HP-beta-cyclodextrin (HP-beta-CD) applied at, e.g., 0.35% or above (see Hertel et al., supra). In some variations, a stabilizing excipient is not required for nebulized delivery of a paraoxonase fusion molecule (e.g., Fc-Ll- PON3, Fc-Ll-[PON 1F11]. or CTLA4-L2-Fc-Ll-[PON 1 1F11]) as descnbed herein (for example, wild-type human DNase 1 (Pulmozyme®) has been shown to not aggregate and remain stable after nebulization with either jet or vibrating mesh (VM) nebulizers without requiring excipients, see Cipo\\a el al.. Pharm. Res. 11:491-498, 1994; Scherer etal., J. Pharm. Set. 100:98-109, 2011).

[198] Determination of effective dosages is typically based on animal model studies followed up by human clinical trials and is guided by determining effective dosages and administration protocols that significantly reduce the occurrence or severity of the subject disease or disorder in model subjects. Effective doses of the compositions of the present invention vary depending upon many different factors, including means of administration, target site, physiological 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. Usually, the patient is a human, but in some diseases, the patient can be a nonhuman mammal. Typically, dosage regimens are adjusted to provide an optimum therapeutic response, i.e., to optimize safety and efficacy. Accordingly, 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 inflammatory lung disease, where any undesired collateral effects are outweighed by any beneficial effects such as, for example, improved alveolar fluid clearance, improved lung physiology and function, etc ). For administration of a fusion polypeptide or dimeric protein of the present invention, a dosage typically ranges from about 0.1 pg to 100 mg/kg or 1 pg/kg to about 50 mg/kg, and more usually 10 pg to 5 mg/kg of the subject’s body weight. In more specific embodiments, an effective amount of the agent is between about 1 pg/kg and about 20 mg/kg, between about 10 pg/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. For example, in certain variations, 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 bimonthly intervals. Alternatively, 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.

[199] In some specific embodiments comprising delivery by inhalation with a nebulizer (for example, for treatment of an inflammatory lung disease such as, e.g, cystic fibrosis or insterstitial lung disease), a fusion polypeptide or dimeric protein of the present invention is administered to the lung by a nebulizer at a dose of from about 1 mg to about 5 mg or from about 2 mg to about 4 mg per day. In other specific embodiments comprising systemic administration (for example, for treatment of systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, or inflammatory bowel disease), a fusion polypeptide or dimeric protein of the present invention is administered (e.g, by intravenous or subcutaneous injection) at a dose of from about 50 mg to about 1,000 mg, from about 50 mg to about 800 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 100 mg to about 400 mg, from about 100 mg to about 300 mg, from about 150 mg to about 250 mg, or about 200 mg every⁷ month to six weeks. For combination therapy with a paraoxonase fusion molecule and a DNase as described herein, each of the paraoxonase fusion molecule (e.g, an Fc-Ll- PON3 fusion as described herein) and the DNase (e.g., a DNase-Fc fusion as described in WO 2022/178090, supra) may be administered at these doses. For combination therapy with a PON3 fusion molecule and a PON1 as described herein, each of the PON3 fusion molecule (e.g, an Fc-Ll-PON3 fusion as described herein) and the PON1 (e.g., a PON1 fusion as described in WO 2022/178078, supra) may be administered at these doses. In some embodiments comprising a PON3/PON1 combination therapy, the ratio of the PON1 dose to the PON3 dose is from about 5: 1 to about 200: 1, from about 10: 1 to about 200: 1, from about 5:1 to about 100: 1, from about 10: 1 to about 100:1, from about 5: l to about 50: 1, or from about 10: 1 to about 50: 1.

[200] Particularly suitable animal models for evaluating efficacy of a paraoxonase fusion composition of the present invention for treatment of inflammatory lung disease include, for example, a murine ovalbumin-induced acute asthma model as described by da Cunha et al. (Exp. Lung Res. 42:66, 2016) (showing significantly reduced airway resistance with wild-type (wt) DNasel treatment), a murine silica-induced lung inflammation model as described by Benmerzoug et al. (Nat. Comm. 9:5226, 2018) (showing prevention of DNA-mediated STING activation and blockade of the downstream type I IFN response with wt DNase 1 treatment), and a murine model of transfusion-related acute lung injury as described by Caudrillier et al. (J. Clin. Invest. 122:2661. 2012) (showing protection from lung edema and lung vascular permeability as well as reduced NET formation and platelet sequestration in the lung with wl DNasel treatment).

[201] Suitable animal models for evaluating efficacy of a paraoxonase fusion composition as described herein for treatment of exposure to sulfur mustard gas or an organophosphate include, for example, a guinea pig model as describe by Valiyaveettil et al. Biochem. Pharmacol. 81:800-809. 2011; Toxicol. Letters 202:203-208, 2011) (showing protection from sarin and soman inhalation toxicity with recombinant human PON1 injection) and mouse models as described by Bajaj etal. (Appl. Biochem. Biotechnol. 180: 165-176, 2016) and Stevens et al. (Proc. Natl. Acad. Sci. USA 105:12780-12784, 2008) (showing protection from organophosphate poisoning using a recombinant Q192K variant of PON1).

[202] Particularly suitable animal models for evaluating efficacy of a paraoxonase composition as described herein for treatment of an inflammatory’ bowel disease include a TNBS-induced colitis model and a chronic colitis model with CD4⁺CD45RB^hlgh cell transfer in mice. See, e.g., Yamashitaef al., J. Immunol. 191 :949-960, 2013 (showing efficacy of PON1 therapy in the TNBS-induced colitis model and a PON1 variant (G3C9) in the chronic colitis model). Another suitable model is a dextran sulfate sodium (DSS)-induced colitis model in mice. See, e.g., Babicovaet al.. Folia Biolica (Praha) 64: 10, 2018 (show ing reduction in TNF a and myeloperoxidase in the colon with wt DNasel treatment of DSS-induced colitis); Li et al., J. Crohns Colitis 2020, 14:240-253, 2020 (showing decreased cytokine production and attenuated accelerated thrombus formation and platelet activation with DNasel treatment of DSS-induced colitis). In the DSS-induced colitis model, a PON1 or PON3 fusion composition may be evaluated, e.g., by dosing with the PON fusion molecule IV, 100 pg/animal, 2-3 times per week for two weeks prior to analysis.

[203] Also known is the collagen-induced arthritis (CIA) model for rheumatoid arthritis (RA) (see, e.g., Brand et al., Nat. Protoc. 2:1269-1275, 2007). CIA shares similar immunological and pathological features with RA, making it an ideal model for evaluating efficacy of paraoxonase compositions. Another suitable model for RA is PG-PS (proteoglycan- polysaccharidej-induced arthritis in Lewis rats (see, e.g. , Esser et cd. , Arthritis and Rheumatism 28: 1402-1411, 1985; Brooks et al.. Proc. Inti. Soc. Mag. Reason. Med. 11: 1526, 2003). [204] Suitable animal models for multiple sclerosis (MS) 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 "encephahtogen” in the context of EAE). which leads to inflammation, demyelination, and weakness (see, e.g., Constantinescu et al., British Journal of Pharmacology 164: 1079-1106, 2011).

[205] Fusion molecules of the present invention can also be evaluated for anti-tumor activity in animal tumor models. For example, efficacy of a PON3 fusion treatment in reducing tumor metastasis can be evaluated in mouse models as described by. e.g., Cools-Lartigue et al. (J. Clin. Invest. 123:3446, 2013) (showing reduction in metastasis of injected tumor (lung carcinoma) cells with systemic administration of wt DNasel in a model of severe postoperative infection) and Park et al. (Sei. Translational Med. 8:361ral38, 2016) (showing reduction in metastasis of breast cancer cells to the lung with systemic administration of wt DNasel -coated nanoparticles). Also known is a model utilizing nude mice subcutaneously grafted with the human colon cancer cell line SW480 as described, e.g., by Trejo-Becirril et al. (Integrative Cancer Therapies 15:NP35-NP43, 2016) (showing inhibition of tumor growth with a combination of DNasel and proteases). Other suitable models include a human lung tumor xenograft model described by Rutkoski et al. (Translational Oncology 6:392-397. 2013) (showing inhibition of tumor growth in mice using PEG-RNasel ) and a syngeneic mouse tumor model with Lewis lung carcinoma described by Mironova et al., Oncotarget 8:78796-78810, 2017 (showing anti-tumor activity with RNase therapy). These models are readily amenable to use in evaluating anti-tumor therapy with administration of PON3 fusion molecules disclosed herein.

[206] Another known animal tumor model is B16 melanoma, a poorly immunogenic tumor. Multiple models of tumor immunotherapy have been studied. See Ngiow et al., Adv. Immunol. 130: 1-24, 2016. The B16 melanoma model has been studied extensively with checkpoint inhibitors anti-CTLA-4, anti-PD-1, 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. Immunother. 64:885-92, 2015; Haanen et al.. Prog. Tumor Res. 42:55-66, 2015. Efficacy of a PON3 fusion molecule for treatment of malignant melanoma is shown, for example, by slowed tumor growth following administration to Bl 6 melanoma mice that have formed palpable subcutaneous tumor nodules. Efficacy of a paraoxonase fusion molecule can be evaluated in Bl 6 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). For example, tumor rejection in B16 melanoma mice using a combination of a PON3 fusion molecule as described herein and anti-CTLA-4, in the absence of tumor vaccine, demonstrates an enhanced response to anti- CTLA-4 using the paraoxonase fusion therapy. In exemplary studies to evaluate paraoxonase fusion molecules comprising human protein sequences, which are functionally active in mice but are expected to be immunogenic in these models (and thereby likely to result in formation of neutralizing antibodies after 7-10 days), 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.

[207] Dosage of the pharmaceutical composition may be varied by the attending clinician to maintain a desired concentration at a target site. For example, if an intravenous mode of delivery⁷ is selected, 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 delivery, e.g, trans-epi dermal delivery versus delivery⁷ 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. To achieve the same serum concentration level, for example, slow-release particles with a release rate of 5 nanomolar (under standard conditions) would be administered at about twice the dosage of particles with a release rate of 10 nanomolar. Dosing may also vary, e.g, depending on the activity of the fusion molecule being administered.

[208] 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 controlled-release forms. The latter form is illustrated by miniosmotic pumps and implants. See, e.g.. Bremer et al., Pharm. Biotechnol. 10:239, 1997; Ranade. ‘Implants in Drug Delivery,” in Drug Delivery Systems 95-123 (Ranade and Hollinger, eds., CRC Press 1995); Bremer et al., “Protein Deliver}⁷ with Infusion Pumps,” in Protein Delivery: Physical Systems 239-254 (Sanders and Hendren, eds., Plenum Press 1997); Yewey et al., “Deliver}⁷ of Proteins from a Controlled Release Injectable Implant,” in Protein Delivery: Physical Systems 93-117 (Sanders and Hendren, eds.. Plenum Press 1997). Other solid forms include creams, pastes, other topological applications, and the like.

[209] Degradable polymer microspheres have been designed to maintain high systemic levels of therapeutic proteins. Microspheres are prepared from degradable polymers such as poly(lactide-co-glycolide) (PLG). polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetate polymers, in which proteins are entrapped in the polymer. See, e.g., Gombotz and Pettit, Bioconjugate Chem. 6:332, 1995; Ranade, “Role of Polymers in Drug Deliver}⁷,” in Drug Delivery Systems 51-93 (Ranade and Hollinger, eds., CRC Press

1995); Roskos and Maskiewicz, “Degradable Controlled Release Systems Useful for Protein Delivery.” in Protein Delivery: Physical Systems 45-92 (Sanders and Hendren, eds., Plenum Press 1997); Bartus et al.. Science 281: 1161, 1998; Putney and Burke, Nature Biotechnology 16: 153, 1998; Putney, Curr. Opin. Chem. Biol. 2:548, 1998. Polyethylene glycol (PEG)-coated nanospheres can also provide carriers for intravenous administration of therapeutic proteins. See. e.g., Gref et al., Pharm. Biotechnol. 10: 167, 1997.

[210] Other dosage forms can be devised by those skilled in the art, as shown by. e.g. , Ansel and Popovich, Pharmaceutical Dosage Forms and Drug Delivery Systems (Lea & Febiger, 5th ed. 1990); Gennaro (ed.), Remington ’s Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995), and Ranade and Hollinger, Drug Delivery Systems (CRC Press

1996).

[211] In some embodiments comprising systemic administration of a fusion polypeptide or dimeric protein as described herein, the fusion molecule is formulated in a buffered saline (for example, saline buffered with 2mM carbonate, pH 7.5, plus 1 mM calcium chloride).

[212] Pharmaceutical compositions as described herein may also be used in the context of combination therapy. For example, for a combination therapy comprising administration of a paraoxonase fusion molecule and a DNase as described herein, each of the paraoxonase fusion molecule and the DNase (e.g.. a DNase-Fc fusion as described in WO 2022/178090, supra) may be formulated in a buffered saline (for example, saline buffered with 2mM carbonate, pH 7.5, plus 1 mM calcium chloride), either separately or as a mixture.

[213] Pharmaceutical 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. Alternatively, such a kit can include a dry-powder disperser, 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.

[214] The invention is further illustrated by the following non-limiting examples.

Example 1: Construction and Expression of Fc-PON3, Fc-PONl and CTLA4-Fc-PON1 Fusion Proteins

[215] Various Fc fusion genes were assembled in a pUC based vector, and assembled genes screened by DNA sequencing prior to further manipulations. Assembled genes included both monospecific PON3 and PON1 fusions (Fc-Ll-PON3 and Fc-Ll-PONl) and bispecific PON1 fusions comprising a CTLA-4 extracellular domain (CTLA4-L2-Fc-Ll-PON1), constructed to reduce immunogenicity of the evolved PON1 molecules. The PON1 component sequences included human PON1 Q192K and evolved PON1 variants 1F11, 2G1, M2G1, and G3C9. The assembled PON1 fusion constructs generated are as follows: hVK3LP-[SCChinge-P238S-P331S Fc]-NGS-PON3 (nucleotide and amino acid sequences as shown in SEQ ID NO:31 and SEQ ID NO:32) (also referred to hereinbelow as Fc-PON3); hVK3LP-[SCChinge-P238S-M252Y-S254T-T256E-P331S Fc]-NGS-PON3 (nucleotide and amino acid sequences as shown in SEQ ID NO: 33 and SEQ ID NO:34) (also referred to hereinbelow as Fc-YTE-PON3; hVK3LP-[SCChinge-P238S-P331S Fc]-NGS-[PON1 Q192K] (nucleotide and amino acid sequences as shown in SEQ ID NO: 141 and SEQ ID NO: 142 of International PCT Publication No. WO 2022/178078, incorporated by reference herein) (also referred to hereinbelow as Fc-Pl-K); hVK3LP-[SCChinge-P238S-P331S Fc]-NGS-[PON1 1F11] (nucleotide and amino acid sequences as shown in SEQ ID NO: 17 and SEQ ID NO: 18) (also referred to hereinbelow as Fc-Pl-lFl 1); hVK3LP-[SCChinge-P238S-M252Y-S254T-T256E-P331S Fc]-NGS-[PON1 1F11] (nucleotide and amino acid sequences as shown in SEQ ID NO: 19 and SEQ ID NO:20) (also referred to hereinbelow as Fc-YTE-Pl-lFl 1; hVK3LP-[SCChinge-P238S-P331S Fc]-NGS-[PON1 2G1] (nucleotide and amino acid sequences as shown in SEQ ID NO:35 and SEQ ID NO:36) (also referred to hereinbelow as Fc-2G1); hVK3LP-[SCChinge-P238S-M252Y-S254T-T256E-P331S Fc]-NGS-[PON1 2G1] (nucleotide and amino acid sequences as shown in SEQ ID NO:37 and SEQ ID NO:38) (also referred to hereinbelow as Fc-YTE-Pl-2G1); hVK3LP-[CTLA-4 EC]-[SCChinge-P238S-P331S Fc]-NGS-[PON1 Q192K] (nucleotide and amino acid sequences as shown in SEQ ID NO:21 and SEQ ID NO:22) (also referred to hereinbelow as CTLA4-Fc-Pl-K); hVK3LP-[CTLA-4 ECJ-lSCChinge-P238S-M252Y-S254T-T256E-P331S FcJ- NGS-[PON1 Q192K] (nucleotide and amino acid sequences as shown in SEQ ID NO:23 and SEQ ID NO:24) (also referred to hereinbelow as CTLA4-FcYTE-Pl-K); hVK3LP-[CTLA-4 EC]-[SCChinge-P238S-P331S Fc]-NGS-[PON1 1F11] (nucleotide and amino acid sequences as shown in SEQ ID NO:25 and SEQ ID NO:26) (also referred to hereinbelow as CTLA4-Fc-Pl-1F11); hVK3LP-[CTLA-4 EC]-[SCChinge-P238S-M252Y-S254T-T256E-P331S Fc]- NGS-[PON1 IF 11] (nucleotide and amino acid sequences as shown in SEQ ID NO:27 and SEQ ID NO:28); hVK3LP-[CTLA-4 EC]-[SCChmge-P238S-P331S Fc]-NGS-[PON1 2G1] (nucleotide and amino acid sequences as shown in SEQ ID NO: 57 and SEQ ID NO:58) (also referred to hereinbelow as CTLA4-Fc-Pl-2G1); hVK3LP-[CTLA-4 EC]-[SCChinge-P238S-M252Y-S254T-T256E-P331S Fc]- NGS-[PON1 2G1] (nucleotide and amino acid sequences as shown in SEQ ID NO: 39 and SEQ ID NO:40); hVK3LP-[SCChinge-P238S-P331S Fc]-NGS-[PON1 M2G1] (nucleotide and amino acid sequences as shown in SEQ ID NO:61 and SEQ ID NO:62) (also referred to hereinbelow as Fc-M2G1); hVK3LP-[SCChinge-P238S-M252Y-S254T-T256E-P331S Fc]-NGS-[PON 1 M2G1] (nucleotide and amino acid sequences as shown in SEQ ID NO: 59 and SEQ ID NO:60) (also referred to hereinbelow as Fc-YTE-Pl-M2G1); hVK3LP-[SCChinge-P238S-P331S Fc]-NGS-[PON1 G3C9] (nucleotide and amino acid sequences as shown in SEQ ID NO: 143 and SEQ ID NO: 144 of International PCT Publication No. WO 2022/178078) (also referred to hereinbelow as Fc-G3C9); hVK3LP-[CTLA-4 EC]-[SCChinge-P238S-P331S Fc]-NGS-[PON1 M2G1] (nucleotide and amino acid sequences as shown in SEQ ID NO: 63 and SEQ ID NO:64) (also referred to hereinbelow^ as CTLA4-Fc-Pl-M2G1).

[216] Fusion gene cassettes containing the desired sequences were then inserted into a multiple cloning site of the mammalian expression vector pDG, a pcDNA3 plasmid derivative containing a CMV promoter to drive expression of the fusion gene. Plasmid DNA was prepared using QIAGEN (Germantown. MD) mini or maxiprep plasmid DNA kits. Purified plasmid DNA was transfected into HEK293 cells plated at approximately 50-75% confluence, using Polyfect (QIAGEN, Germantown. MD) transfection reagent according to the manufacturer’s instructions. Culture media was changed to DMEM Fluorobrite™ (Life Technologies, Carlsbad, CA) serum free media on the day after transfections, and transfected cells incubated for an additional 48 hours prior to harvest of culture supernatants. Culture supernatants were filtered through 0.2 pm PES syringe filters prior to analysis. FIG. 1 shows a schematic diagram of the structure of the Fc-PONl (human or evolved) and Fc-PON3 sequence variants, as well as the CTLA4-Fc-PON1 constructs.

[217] FIG. 2 show s the results of Western blot analysis of fusion protein expression from a representative set of HEK293 transient transfections. Culture supernatants were immunoprecipitated with 60 pl protein A agarose (Repligen, Waltham MA) washed in gentle antigen-antibody binding buffer pH 8.0, (Pierce/Life Technologies, Carlsbad, CA) and 0.5 ml 293 transfection supernatants in Fluorobrite DMEM added to each microfuge tube. Immunoprecipitates were rotated overnight at 4 °C, centrifuged at 3000 rpm, and washed in Binding Buffer pH 8.0. prior to addition of 60 pl 2X LDS sample buffer (Life Technologies, Carlsbad, CA), samples heated for 10 minutes at 72 °C, the samples centrifuged for 1 minute at 3000 rpm in a microfuge, and 15 pl sample in loading buffer (one fourth of each sample) from each sample was loaded onto 4-12% Bis-Tris NuPAGE gels (Life Technologies, Carlsbad. CA). Protein molecular weight markers were included on each gel (Chameleon® DUO molecular weight markers; LI-COR Biosciences, Lincoln, NE and/or Precision Plus Kaleidoscope MW markers; BIORAD, Hercules, CA). SDS-PAGE gels were run in NuPAGE MOPS running buffer (Life Technologies, Carlsbad, CA) under reducing conditions at 185 volts for approximately 1-1.5 hours. Gels were blotted to nitrocellulose membranes using the XCell blot module (Life Technologies, Carlsbad, CA) and NUPAGE-MOPS (Life Technologies, Carlsbad, CA) transfer buffer containing 10% methanol. Blots were blocked in LI-COR Odyssey Intercept™ blocking buffer, followed by incubation with diluted goat antihuman IgG (Fc specific) secondary antibodies (Jackson Immunoresearch. WestGrove, PA). Secondary antibody was conjugated with Alexafluor 790 near IR dye (LI-COR Odyssey detection) and diluted in blocking buffer 1:45,000 at 4 °C and rocked overnight. Blots were washed four times with Tris buffered saline (TBS) containing 0.1% Tween 20. Blots were then rinsed in TBS buffer and scanned with a LICOR Odyssey™ scanner. For the Western Blot shown in FIG. 2. transfection samples were loaded as follows: Lane I - Chameleon DUO molecular weight markers (LICOR, ), Lane 2 - Fc-YTE-PON3, Lane 3 - Fc-PON3, Lane 4 - Fc-YTE-Pl-lFl l, Lane 5 Fc-YTE-Pl-M2G1, Lane 6 Fc-YTE-Pl-2G1, Lane 7 LICOR Chameleon DUO MW Markers, Lane 8 - Fc-M2G1, Lane 9 - Fc-G3C9, Lane 10 - Fc-2G1; Lane 11 - CTLA4-Fc-Pl-2G1: Lane 12 - MOCK. The approximate molecular weight (kDa) of each marker band is indicated on the right side of the Western blot.

[218] As shown in FIG. 2. all of the PON3 and PON1 fusion proteins were well- expressed in mammalian cells. Expression levels of the evolved PON1 fusion molecules, including the bispecific fusion further containing CTLA-4, were particularly and unexpectedly robust. Example 2; In Vitro Assessment of PON 1 and PON3 Fusion Proteins for Functional Activity

[219] Functional activity⁷ of the PON1, PON1 evolved, and PON3 fusion proteins was assessed with a series of in vitro assays. For several of the assays, protein activity was often analyzed directly from culture supernatants of transiently transfected HEK 293 cells. For other in vitro assays, fusion proteins were first purified from culture supernatants (either HEK 293 or CHO DG44 transfected cells) prior to assessment of functional activity. Fusion proteins were purified from culture supernatants by protein A affinity chromatography. Culture supernatants were filtered through 0.2 pm PES express filters (Nalgene, Rochester, NY) and subjected to affinity chromatography using slow rotation of culture supernatants with Protein A-agarose (IPA 300 crosslinked agarose) slurry in 50 ml sterile, conical centrifuge tubes at 4 °C (Repligen, Waltham, MA). Fusion protein bound to protein A agarose was recovered by centrifugation, and culture supernatants removed, replaced, and the incubation process repeated until the desired volume of supernatant was processed. The final protein A agarose slurry⁷ was then loaded into sterile, acid-washed econocolumns (BioRad, Hercules, CA) to wash the resin. Columns were then washed with several column volumes of column wash buffer (Gentle Ag- Ab binding buffer, Pierce/ThermoFisher, Waltham, MA) to remove any residual culture supernatant, prior to elution. Bound protein was then eluted from the resin using gentle Ag/Ab elution buffer (Pierce/ThermoFisher, Waltham, MA). Fractions (0.8-1.0 ml) were collected and protein concentration of aliquots (2 pl) from each fraction were determined at 280nM using a Nanodrop (Wilmington DE) microsample spectrophotometer, with blank determination using elution buffer alone. Fractions containing fusion protein were pooled, and buffer exchange was 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 [0.9% sodium chloride, 5mM sodium bicarbonate, ImM HEPES buffer, ImM calcium chloride, pH 7.5], Dialysis was performed in sterile. 2.2-liter Coming roller bottles at 4 °C overnight. After dialysis, protein was filtered using 0.2 pM filter units, and aliquots tested for endotoxin contamination using Pyrotell LAL gel clot system single test vials (STV) (Catalog # G2006, Associates of Cape Cod, East Falmouth, MA).

[220] FIGs. 3 and 4 show similar assessments measuring organophosphatase activity of the PON1 or PON3 moiety in bispecific and monospecific paraoxonase fusion proteins. In these assays, the substrate used was a proprietary organophosphatase substrate contained in the EnzCHEK paraoxonase assay kit (Molecular Probes/ThermoFisher Scientific, Waltham, MA). This assay is a very' sensitive fluorometric assay for the organophosphatase activity of paraoxonase that uses excitation/emission maxima of 360/450nm to measure the conversion of a fluorogenic organophosphate analog provided with the kit. The assay can either be set up as a kinetic assay or terminated after a particular period of time for an endpoint assay. The change in relative fluorescence units (RFU) per unit time is converted to the units of paraoxonase in the sample using the standard curve generated from the fluorescent standard and the conversion factor that 1 U unit of paraoxonase generates 1 nmol of fluorescent product per minute at 37 °C. The amount of paraoxonase present in the fusion protein samples can be compared to the paraoxonase positive control provided with the kit.

[221] FIGs. 3 and 4 show graphical representations of the organophosphatase enzyme kinetics present in serial dilutions of culture supernatant from several of the evolved Fc-PONl and FcYTE- PON1 and PON3 fusion proteins expressed in HEK 293 transient transfections and from a purified batch of the Fc-PON 1 -K (Fc-P 1 -K) fusion protein. The organophosphatase activity for fusion protein samples was compared to the RFU curves generated by organophosphatase positive controls in the average to above average activity' range provided by the manufacturer. Culture supernatants from mock HEK293 transfections and from CTLA4-Fc-PON1 evolved fusion proteins were also included in the assays. FIG. 3 shows the organophosphatase kinetics for the Fc-PONl evolved variants and the CTLA4-Fc-PON1 evolved variants. The organophosphate specific enzyme activity' in the multispecific fusion proteins was not affected by the presence of the CTLA-4 extracellular domain, indicating that these molecules have utility for in vivo use to suppress potential immunogenicity of the sequence-modified evolved PON1 domains.

[222] FIG. 4 shows the organophosphate specific enzyme activity of the evolved PON1 variants and the PON3 variant fused to the Fc domain containing the YTE sequence modification. The Fc-PON3 and Fc-YTE-PON3 fusion proteins do not possess detectable organophosphate specific enzyme activity in this assay. However, the evolved PON1 sequence variants 2G1 and 1F11 both exhibited high levels of organophosphate specific enzyme activity’, significantly higher than the positive control “high” enzyme provided by the kit manufacturer.

[223] In addition to the paraoxonase enzyme activity, the functional properties of a CTLA-4 extracellular domain positioned at the amino terminus of the CTLA4-Fc-PON1 fusion proteins was also assessed. These molecules are functional for the P0N1 enzy me activity'. They are also capable of binding to the target ligand. FIG. 5 shows the results of an antigen binding assay to CD80, a ligand for the human CTLA-4 extracellular domain present in the fusion proteins. The antigen binding ELISA assay was performed as follows: Human CD80 protein was obtained from BioLegend (San Diego, CA). The human CD80 was diluted in 0. 1 M carbonate buffer (pH 9.0) to a concentration of 2 pg/ml and 100 pl/well aliquoted to each well to be used in the assay in a 96 well NUNC immulon II MaxiSorp plate (ThermoFisher Scientific, Waltham, MA). Plates were incubated overnight at 4 °C, prior to blocking in 200 pl/well PBS/2.0% BSA. overnight at 4 °C. Plates were washed in PBS, 0.5% Tween-20, 0.005% Kathon (wash buffer), and serial dilutions of the HEK 293 transfection supernatants added to wells of the plate. As a positive control for antigen binding, a human CTLA4Ig fusion protein (Orencia®) was also serially diluted on the plate. An ApoAl-Fc-PONl fusion protein (APOA-l-(g4s)4-Fc-Pl-K nucleotide and amino acid sequences shown in SEQ ID NO:45 and SEQ ID NO:46 of PCT Publication No. WO 2017/044424) was also included as a negative control. Plates were incubated with supernatant or antibody dilutions at 4 °C, overnight. Plates were washed four times in wash buffer, then incubated with horseradish peroxidase conjugated goat anti-human IgG Fc specific, at a dilution of 1: 10.000 (Jackson Immunoresearch, West Grove, PA). FIG. 5 shows the CD80 binding curves for the CTLA4Ig and the transfection supernatants, by plotting the absorbance at 450 nm as a function of dilution.

Exampe 3; In Vitro Assessment of PON1 and PON3 Fusion Proteins for Lactonase Activity

[224] Lactonase activity of Fc-PON3 and Fc-PONl evolved variant fusion proteins is assessed using an assay measuring enzyme against dihydrocoumarin. In these assays, the substrate used is dihydrocoumarin (DHC) (Millipore-Sigma, St. Louis, MO) at a final concentration of 1 mM in IX reaction buffer. Reaction buffer for the DHC assays is 50mM Tris HC1 (pH 7.4), 1 mM CaCb. HEK293 culture supernatants are diluted 1 :4 in reaction buffer and 25 pl added to individual wells of 96 well UV STAR (Greiner BioOne, Thomas Scientific) microtiter plates. Substrate dihydrocoumarin (DHC) solution in reaction buffer is added to each well (75 pl) to generate a final volume of 100 pl. Hydrolysis of the substrate is monitored at OD 270 for 15 minutes. Exampe 4; Dihydrocoumarin Hydrolysis by Paraoxonase Fusion Proteins

[225] Lactonase activity (LACase) of paraoxonase fusion proteins is measured using dihydrocoumarin (DHC) as the substrate, according the modified method described by Billecke et al. (Drug Metab. Dispos. 24: 1335-1342, 2000) and Aviram and Rosenblat (Methods Mol. Biol. 477:259-276, 2013). A stock solution of 100 mM DHC is prepared in DMSO for the assay and diluted in reaction buffer (RB) to the appropriate final concentration of substrate. Lactonase (LACase) activity is measured in a 96-well UV transparent microtiter plate containing 1.0 mM DHC, 50 mM Tris-HCl buffer pH 7.4, 100 mM NaCl, 2 mM CaCh in a total volume of 150 ml. The reaction is initiated by the addition of 75 pL of serial dilutions of purified PON1 or PON3 fusion protein in reaction buffer to 75 ml DHC (2mM in reaction buffer, for a final concentration of 1 mM), and the increase of absorbance at 270nm is monitored every 30 seconds for 15 minutes at 37 °C. A molar extinction coefficient of 1295 M 'em ¹ is utilized to calculate the rate of hydrolysis. One unit of LACase activity is equivalent to 1 pmol of DHC hydrolyzed/min/mg of fusion protein. To determine enzyme kinetics, the concentration of enzyme is fixed at 100 nM, and the final concentration of DHC substrate in the reaction is titrated as follows: 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM. Reactions are performed in duplicate or triplicate, depending on the assay. For assays using culture supernatants, a DHC concentration of 1 mM is used with serial dilutions of the culture supernatant in Fluorobrite DMEM media (ThermoFisher, Dallas, TX) in RB.

Exampe 5: Dihydrocoumarin Hydrolysis by Fc-PON3 and Fc-YTE-PON3

[226] PON3 fusion proteins were assessed for lactonase activity using a dihydrocoumarin hydrolysis assay as described in Example 4. The PON3 fusion proteins tested in this study were Fc-PON3 and Fc-YTE-PON3 as described in Example 1 (having the amino acid sequences of residues 21-607 of SEQ ID NO:32 (Fc-PON3) and residues 21-604 of SEQ ID NO:34 (Fc-YTE-PON3)). For this assay, an Fc-PONl fusion protein as described in International Patent Publication WO 2022/178078 was also used (“TR-43”; having the amino acid sequence of residues 21-613 of WO 2022/178078 SEQ ID NO:122).

[227] HEK 293T cells were plated in 60 mm tissue culture dishes at a concentration of 1.2 x 10⁶ cells/ml, and grown overnight at 37 °C, 5% CO2. Plasmid DNA (4 mg) from different PON fusion protein constructs was added to DMEM growth media without supplements or FBS (to a total volume of 150 ml), and 40 ml polyfect transfection reagent added to each tube, then vortexed for 10 seconds to mix. Tubes were incubated at room temperature without disturbance for 10 minutes to allow complex formation. Plasmid complexes were diluted with 1.0 ml growth medium: DMEM with supplements and FBS (10%), prior to addition to the HEK 293T cells. HEK 293T transfections were allowed to proceed for 24 hours at 37 °C, 5% CO2. After 24 hours, transfection plates were washed and growth media replaced with Fluorobrite DMEM containing growth supplements (glutamine, sodium pyruvate, penicillin/streptomycin, and non-essential amino acids) but without FBS and left at 37 °C, 5% CO2 for 48 hours prior to harvest of culture supernatants and subsequent analysis. Transfected culture supernatants were harvested and filtered through 0.2 mm PES syringe filter units to remove debris prior to use in assays. Culture supernatants were diluted in enzyme reaction buffer (RB), containing 50 mM Tris-HCl (pH 7.4). 100 mM NaCl, and 2 mM CaCh. Based on prior transfection results, different expression levels in the transfected plates had been observed for the PON1 and PON3 plasmids, so different dilutions were tested in initial experiments. For PON1 fusion proteins, supernatants were diluted either 2.5X or 5X in RB prior to addition to equal volume of 1.0 mM dihydrocoumarin (DHC) in reaction buffer (RB), to give a final dilution of 5X and 10X. For the PON3 fusion proteins, supernatants were diluted either 2X or 4X in RB prior to addition of an equal volume of 1.0 mM DHC in RB, to give a final dilution of 4X and 8X. All final reaction volumes were 150 ml. Immediately after addition of substrate to enzyme dilutions in wells of UV transparent plates, DHC hydrolysis was monitored by the change in absorbance at 270 nm in a VarioSkan LUX plate reader at 37 °C for 15 minutes. The hydrolysis reactions were usually complete within 5 minutes of assay initiation. Starting absorbance levels varied due to the rapidity of hydrolysis and the time lag between initiation of the assay and the beginning of reads on the instrument. FIG. 6 shows a subset of the raw data of elapsed time (in seconds) versus the change in absorbance at 270 nm. Both the PON1 and PON3 fusion proteins show significant DHC hydrolysis activity, while the mock transfected supernatant and DHC (substrate alone) samples showed only background hydrolysis. Example 6; Lactonase Activity of Paraoxonase Fusion Proteins Against TBBL and Homocysteine Thiolactone

[228] Paraoxonase fusion proteins are assessed for the ability to hydrolyze homocysteine thiolactone to homocysteine (HTase activity) or 5-thiobutyl butyrolactone (TBBL) to mercaptobutryric acid, as a measure of lactonase activity. Two different methods are used to assess lactonase activity. The first method uses a colorimetric reagent called CU(NC)2⁺, which absorbs light at 450 nm. The CUPRAC or (Cu(Nc)2²⁺) reagent is reduced to a highly colored Cu(I)-neocuproine complex (Cu(Nc)2⁺) by the homocysteine produced in the enzyme reaction, and the result is quantified by absorbance at 450 nm. This method is similar to that used by Obeid and Hadwan (Anal. Biochem. 631: 114365, 2021). The second method uses a fluorescent molecule N-(9-acridinyl)maleimide (NAM) which binds to free thiol groups to produce a highly fluorescent product. This method is described be Al Talebi et al. (Microchem. J. 195: 109431, 2023). The NAM method requires a shorter incubation time of 5 minutes, while the CUPRAC method requires 10 minutes to complete.

[229] The substrate used in both cases is either homocysteine thiolactone or TBBL. Enzyme reactions are set up in volumes appropriate for microplate assays, usually a final reaction volume of 150 ml. All reactions are performed in triplicate. Substrate solutions are prepared and aliquoted in a volume of 75 pl to each well, to which 75 pl/well enzyme solution is added for a final reaction volume of 150 pl. Substrate solution is prepared by dissolving 7.68 mg homocysteine thiolactone (5 mM) in 10 ml HEPES buffer (25 mM, pH 7.2). Homocysteine standard is prepared by dissolving 5.4 mg homocysteine in 10 ml HEPES buffer (25 mM, pH 7.2), to give a final concentration of 4 mM. Alternatively, L-homocysteine solution at 10 mM is obtained from MedChem Express LLC, catalog # HY-W010347) (Monmouth Junction, NJ). L-homocysteine thiolactone (hydrochloride) at 10 mM, catalog # HY-101404A, is also purchased from MedChem Express LLC (Monmouth Junction, NJ). TBBL is purchased from Enamine (Kyiv, Ukraine, Catalog number EN300-7462676; 5- (butylsulfanyl)oxolan-2-one). TBBL is resuspended in DMSO or acetonitrile at a concentration of 240 mM. Stock solutions are diluted to 1 mM with reaction buffer just prior to use.

[230] For detection of activity using colorimetric assays, copper (11) chloride solution is prepared and diluted to a concentration of 10 rnM ( 1 O'² M) from CuCh 2H2O and water by dissolving 0.4262 g CuCU2H2O in 250 ml H2O. Ammonium acetate (NH4AC) buffer is prepared to a final concentration of 1.816 M (pH 7.0), by dissolving 35 g NH4AC in water, and adjusting the volume to 250 ml. A neocuproine or Nc (2,9-dimethyl-l,10-phenanthroline) solution is prepared at a final concentration of 7.5 mM by dissolving 0.039 g Nc in 25 ml ethanol. Working CUPRAC indicator reagent is freshly prepared from these solutions of Cu(II):Nc:NH4Ac at a ratio of 1 : 1 : 1 (v/v/v).

[231] For more sensitive fluorometric assays of lactonase activity, the fluorescent reagent N-(9-acridinyl)maleimide or NAM, catalog # HY-W011618, is obtained from MedChem Express (Monmouth Junction, NJ), and is resuspended in DMSO at a concentration of 5 mg/ml. The lactonase activity is measured by incubating the enzyme containing samples in a reaction buffer containing 50 mM Tris-HCl pH 8.0, 1 mM CaCh, containing appropriate dilutions of substrate at 37 °C, for 5-15 minutes. The NAM reagent is then used to develop the fluorescent end product. The protocol uses thiol fluorometry to assess lactonase activity, measuring the fluorometric intensity at excitation/emission wavelengths of 360 and 432 nm. Once the enzyme reaction is complete, the concentration of the produced thiol groups should be directly correlated with the fluorescence intensity⁷ observed.

Example 7: Statin Hydrolysis by Paraoxonase Fusion Proteins

[232] For hydrolysis of statins, a class of larger lactones used for cardiovascular disease therapy, a different measurement strategy is used based on release of H⁺ ions, so that detection of enzyme activity relies on changes in pH of the solution detected with the cresol- purple indicator dye in Bicine buffer. The hydrolysis of lactones, via the opening of the lactone ring, increases the acidity of the solution by generating a proton. A pH indicator, cresol purple (pKa 8.3 at 25 °C) is used to monitor the acidification of the medium induced by the hydrolysis of the lactone ring as previously described for hydrolysis reactions of aliphatic lactones. The time course of the hydrolysis of the lactones is recorded at 577 nm in the activity buffer (50 mM Bicine pH 8.3, 150 mM NaCl, 0.2 mM cresol purple, 0.5% DMSO, and 1 mM CaCh). Kinetic parameters can be estimated by comparison with a standard calibration curve obtained using acetic acid.

[233] Alternatively, statin hydrolysis can be determined by HPLC analysis, monitoring the changes in column retention time for the different species. Statin substrates are prepared at 10-20 mM in reaction buffer containing 2% acetonitrile and incubated with PON1 fusion protein samples at room temperature for 1 minute. Reactions are stopped by addition of an equal volume of acetonitrile containing 0.2% acetic acid, and analyzed by reverse-phase HPLC on an analytical C18 column (250 mm x 4.6 mm, 5 urn particles; Vydac) using a 65- 80% linear gradient of acetonitrile in water (1% per min), with both solvents containing 0.2% acetic acid. Quantification of the extent of hydrolysis is based on the ratio of peak areas of hydroxy acid (elution at ~7.3 min) with lactone (elution at ~ 15.2 min) detected at 236 nm. This procedure is similar to that described by Gaidukov and Tawfik (Biochemistry 44: 11843- 11854, 2005).

Example 8; Assessment of HDL Oxidation, LDL Oxidation, and PON1 or PON3 Inhibition

[234] ('it- -Induced LDL Oxidation'. Oxidation of LDL is carried out in a 96 well UV ELISA plate at 37 °C and measured using a VarioSkan LUX plate reader. To each well, 15 pL of PON1 or PON3 fusion protein is added so that the final concentration of fusion protein is 5 mg/ml, 10 pg/ml, or 20 pg/ml, in reaction buffer or RB (0.9% NaCl, 1.5 mM sodium bicarbonate, 2 mM CaCh, pH 7.4). Then, stock solutions of LDL (final concentration of 100 mg/ml) or HDL (final concentration of 100 mg/ml) are diluted in Tris-HCl pH 7.4, ImM CaCh) and added to the reactions at a final concentration of LDL or HDL of 100 mg/ml. Samples are incubated together at 37°C, for 2 hours prior to treatment with oxidizing agent(s). Any additional test compounds such as antioxidant chemicals are also added at this time (final concentration of 20 pg/mL in RB pH 7.4 with 0.1% v/v DMSO) and incubated for 2 hours at 37 °C . After the two hour preincubation, CuSCL (5 pM final concentration) is added to test wells and RB was added to a final volume of 150 pL. LDL oxidation is determined by direct spectrophotometry, via continuous absorbance monitoring of conjugated diene formation at 234 nm (every 5 min for 3 h). Lag time and rate constant (K) of the propagation phase are calculated via nonlinear regression using the Gompertz growth equation. Percent inhibition of LDL or HDL oxidation is calculated as: 100 - (K(LDL or HDL + sample)/K(LDL or HDL) * 100).

[235] In addition to direct monitoring of conjugated diene formation, a TBARS assay is used on treated samples to determine the ability of PON 1 fusion proteins to inhibit production of malonaldehyde in the lipid oxidation reactions. The Lipid Peroxidation (MDA) Assay Kit (Colorimetric/Fluorometric) (Abeam, Eugene OR;Waltham MA; Catalog # abl 18970) provides a convenient tool for sensitive detection of malondialdehyde (MDA). In the lipid peroxidation assay protocol, the MDA in the sample reacts with thiobarbituric acid (TBA) to generate a MDA-TBA adduct. The MDA-TBA adduct can be easily quantified colorimetrically (OD = 532 nm) or fluorometrically (Ex/Em = 532/553 nm). This assay detects MDA levels as low as 1 nmol/well colorimetrically and 0.1 nmol/well fluorometrically. Results are compared to a standard curve generated from the MDA standard provided with the kit, according to the kit directions. Treated LDL or HDL samples are mixed with 500 ml of 42 mM H2SO4 in a microcentrifuge tube. Phosphotungstic acid solution (12 ml) is added to the solution and mixed by vortexing. Samples are incubated at room temperature, 5 minutes, then centrifuged at 13,000 x g for 3 minutes. Pellets are collected and resuspended on ice with 100 ml ddEhO containing 2 ml BHT stock/BHT (lOOx). The final volume is adjusted to 200 ml with ddEBO. To these samples, 600 ml developer VII/TBA reagent is added. Samples are incubated at 95 °C, 60 minutes , then cooled to room temperature in an ice bath for 10 minutes. For analysis, 200 ml is aliquoted from the reaction mix containing the MDA-TBA adduct and added to a 96-well microplate. Absorbance is immediately read at OD 532 nm for colorimetric analysis, or RFU at Ex/Em=532/553 nm for fluorometric assay. MDA concentration in the test samples is then calculated using the OD readings from the standard curve to estimate MDA amounts and correcting for the dilution.

Example 9; Effect of Fc-PON3 and Fc-PONl (TR-43) on Differentiation and Activation of the Monocytic Cell Line THP-1

[236] The human monocytic leukemia cell line, THP-1 (ATCC TIB-202, Manassas, VA) is cultured in RPMI 1640 supplemented with 10% FBS, glutamine, non-essential amino acids, and Pen/Strep (Gibco/BRL; ThermoFisher). To measure the effects of TR-43 and Fc- PON3 on differentiation, THP-1 cells are plated at 5 X 10⁵ cells/ml into 12 or 24 well tissue culture plates. PMA is added at a final concentration of 0 or 100 ng/ml, TR-43 or Fc-PON3 are added at final concentrations of 0, 5, 10, or 20 mg/ml, and cells are cultured for 24 hours. Differentiation is measured by cell adherence and by measurement of cell surface expression of CD14. CD1 lb, CD80. and CD86 using flow cytometry. Flow cytometry is performed on a Cytoskan flow cytometer and data analyzed using FlowJo (TreeStar, Ashland OR) software. Fluorescent antibodies against CD14, CDl lb, CD80 and CD86 are obtained from Biolegend (San Diego, CA).

[237] To measure the effects of TR-43 and Fc-PON3 on THP-1 activation, cells are first differentiated by PMA addition for 24 hours as above without addition of TR-43 (Fc- P0N1-K) or Fc-PON3. Then, LPS is added at 0 or 100 ng/ml final concentration, and Fc- PON1-K (TR-43) or Fc-PON3 is added at concentrations as above, and cells are cultured for another 24 hours. Cells and culture supernatants are then analyzed further by similar procedures as described in Wu et al. (Food Funct. 15:4207-4222, 2024). Culture supernatants are harvested for further analysis using cytokine ELISA kits obtained from Biolegend (San Diego, CA) or Cytokine Bead Array analyzer kits from Becton Dickinson (Franklin Lakes, NJ) BD CB A Human Inflammatory Cytokines Kit (Catalog # 551811), or Biolegend LEGENDplex pre-defined human inflammation Panel 1 (Catalog # 740809). In addition, THP-1 cells from the different treatment groups are harvested for further analysis by flow cytometry, cell lysis, and protein and mRNA isolation. Cytokines including IL-6. IL-8. TNF-a, IL-ip. and IL- 10 are measured by cytokine ELISAs/cytometric bead arrays from culture supernatants and/or by qPCR on THP-1 mRNA isolated from cell lysates. Cell lysis, RNA extraction, reverse transcription and qPCR are performed using QIAGEN kits and according to manufacturer's instructions. TaqMAN premix (ThermoFisher Scientific, Waltham MA) and cytokine specific appropriately labeled primer sets are used to perform qPCR according to suggested protocols from ThermoFisher.

[238] Oxidative stress is detected by measuring the lipid oxidation product MDA (ABC AM, Eugene OR;Waltham MA) in cell lysates in RIPA buffer. Superoxide anion, O2‘, is measured by staining the treated THP-1 cells with the fluorescent probe dihydroethidium (DHE). Nitric oxide, (NO) is measured by staining the cells with the fluorescent probe 3-amino 4-aminomethyl 2,7 fluorescein diacetate (DAF-FM DA). Staining reagents are obtained from Molecular Probes/Invitrogen (ThermoFisher Scientific, Waltham MA).

[239] The present inventors expect that in THP-1 activation assays, Fc-PON 3 will be more potent than Fc-PONl (e.g., TR-43) in protection from oxidation and in inhibition of inflammatory cytokine production. Oxidative stress is very high in rheumatoid arthritis and SLE patients. Therefore, the present inventors believe that Fc-PON3 may be superior to TR- 43 in these diseases and other diseases exhibiting high oxidative stress. [240] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes.

Claims

CLAIMS What is claimed is:

1. A fusion polypeptide comprising, from an amino terminal position to a carboxyl terminal position, X-Ll-P, wherein:

X is a dimerizing domain or a domain that specifically binds to the neonatal Fc receptor (FcRn);

LI is a polypeptide linker, wherein LI is optionally present; and

P is a biologically active paraoxonase, wherein the paraoxonase has at least 95% identity with the amino acid sequence shown in residues 21-354 or 31-354 of SEQ ID NO:30 and does not contain an amino terminal leader sequence corresponding to residues 1-20 of SEQ ID NO:30.

2. The fusion polypeptide of claim 1, wherein the paraoxonase has an amino acid sequence selected from the group consisting of residues n-354 of SEQ ID NO:30, wherein n is an integer from 21 to 31, inclusive.

3. The fusion polypeptide of claim 1 or 2, wherein LI is present and comprises at least eight amino acid residues.

4. The fusion polypeptide of claim 3, wherein LI consists of from 12 to 25 amino acid residues.

5. The fusion polypeptide of claim 3, wherein LI has the amino acid sequence shown in SEQ ID NO: 12.

6. The fusion polypeptide of any one of claims 1 to 5, wherein the fusion polypeptide does not comprise a biologically active polypeptide N-terminal to the dimerizing domain or domain that specifically binds to FcRn.

7. The fusion polypeptide of any one of claims 1 to 5, wherein the fusion polypeptide further comprises a biologically active polypeptide N-terminal to the dimerizing domain or domain that specifically binds FcRn, wherein said biologically active polypeptide is selected from the group consisting of a cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) extracellular domain, and a CD40 extracellular domain, and wherein the fusion polypeptide comprises, from an amino-terminal position to a carboxyl-terminal position. T-L2-X-L1-P, wherein X, LI, and P are as defined in claim 1, L2 is a second polypeptide linker, wherein L2 is optionally present, and T is the biologically active polypeptide.

8. The fusion polypeptide of claim 7, wherein the biologically active polypeptide is the CTLA-4 extracellular domain.

9. The fusion polypeptide of claim 8, wherein the CTLA-4 extracellular domain has at least 95% identity⁷ with the amino acid sequence shown in residues 21-144 of SEQ ID NO:26.

10. The fusion polypeptide of claim 9, wherein the CTLA-4 extracellular domain has the amino acid sequence shown in residues 21-144 of SEQ ID NO:26.

11. The fusion polypeptide of claim 7, wherein the biologically active polypeptide is the CD40 extracellular domain.

12. The fusion polypeptide of claim 11, wherein the CD40 extracellular domain has at least 95% identity with the amino acid sequence shown in residues 21-188 of SEQ ID NO:42.

13. The fusion polypeptide of claim 12, wherein the CD40 extracellular domain has the amino acid sequence shown in residues 21-188 of SEQ ID NO:42.

14. The fusion polypeptide of claim 12, wherein the CD40 extracellular domain contains at least one amino acid substitution at a position corresponding to an amino acid of human CD40 (SEQ ID NO:46) selected from the group consisting of E64, K81, P85, and L121, wherein the at least one amino acid substitution increases CD40 ligand binding relative to human CD40.

15. The fusion polypeptide of claim 14, wherein the amino acid at the position corresponding to K81 of human CD40 is selected from the group consisting of threonine, histidine, and serine; the amino acid at the position corresponding to K81 of human CD40 is histidine and the amino acid the position corresponding to LI 21 of human CD40 is proline; or the amino acid at the position corresponding to E64 of human CD40 is tyrosine, the amino acid at the position corresponding to K81 of human CD40 is threonine, and the amino acid at the position corresponding to P85 of human CD40 is tyrosine.

16. The fusion polypeptide of any one of claims 1 to 15, wherein X is a dimerizing domain, optionally wherein said dimerizing domain specifically binds to FcRn.

17. The fusion polypeptide of claim 16, wherein X is an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region is capable of forming dimers and specifically binding FcRn.

18. The fusion polypeptide of claim 17, wherein the immunoglobulin heavy chain constant region is an immunoglobulin Fc region.

19. The fusion polypeptide of claim 18, wherein the Fc region is a human Fc region.

20. The fusion polypeptide of claim 19, wherein the human Fc region is an Fc variant comprising one or more amino acid substitutions relative to the wild-type human sequence.

21. The fusion polypeptide of claim 19 or 20, wherein the Fc region is a human yl Fc region or a human y4 Fc region.

22. The fusion polypeptide of claim 20, wherein the Fc region is a human y 1 Fc variant in which Eu residue C220 is replaced by serine.

23. The fusion polypeptide of claim 22, wherein Eu residues C226 and C229 are each replaced by serine.

24. The fusion polypeptide of claim 23, wherein Eu residue P238 is replaced by serine.

25. The fusion polypeptide of 20, wherein the Fc region is a human yl Fc variant in which Eu residue P331 is replaced by serine.

26. The fusion polypeptide of any one of claims 22 to 24, wherein Eu residue P331 is replaced by serine.

27. The fusion polypeptide of any one of claims 22 to 26, wherein Eu residue M252 is replaced by tyrosine, Eu residue S254 is replaced by threonine, and/or Eu residue T256 is replaced by glutamate.

28. The fusion polypeptide of claim 17, wherein the immunoglobulin heavy chain constant region comprises an amino acid sequence having at least 95% identity with the amino acid sequence shown in

(i) residues 16-232 or 16-231 of SEQ ID NO:6,

(ii) residues 16-232 or 16-231 of SEQ ID NO:48, or

(iii) residues 16-232 or 16-231 of SEQ ID NO:52.

29. The fusion polypeptide of claim 28, wherein the immunoglobulin heavy chain constant region comprises the amino acid sequence shown in

(i) residues 16-232 or 16-231 of SEQ ID N0:6,

(n) residues 16-232 or 16-231 of SEQ ID NO:48, or

(iii) residues 16-232 or 16-231 of SEQ ID NO:52.

30. The fusion polypeptide of claim 18, wherein the Fc region has the amino acid sequence shown in

(i) residues 1 -232 or 1 -231 of SEQ ID NO: 6,

(n) residues 1 -232 or 1 -231 of SEQ ID NO: 8,

(iii) residues 1-232 or 1-231 of SEQ ID NO:48,

(iv) residues 1-232 or 1-231 of SEQ ID NO: 50,

(v) residues 1-232 or 1-231 of SEQ ID NO: 52, or

(vi) residues 1-232 or 1-231 of SEQ ID NO: 54.

31. The fusion polypeptide of claim 17, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% identity with the amino acid sequence shown in

(i) residues 21-607 or 24-607 of SEQ ID NO:32, or

(ii) residues 21-604 of SEQ ID NO:34.

32. The fusion polypeptide of claim 31, wherein the fusion polypeptide comprises the amino acid sequence shown in

(i) residues 21 -607 or 24-607 of SEQ ID NO : 32, or

(ii) residues 21-604 of SEQ ID NO:34.

33. A dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, wherein each of the first and second fusion polypeptides is a fusion polypeptide as defined in any one of claims 1 to 32.

34. A fusion polypeptide comprising, from an amino terminal position to a carboxyl terminal position. X-Ll-P, wherein:

LI is a polypeptide linker, wherein LI is optionally present; and

P is a biologically active paraoxonase, wherein the paraoxonase comprises an amino acid sequence as shown in

(i) residues 16-355 or 26-355 of SEQ ID NO:55, or

(ii) residues 16-355 or 26-355 of SEQ ID NO:56, and wherein the paraoxonase does not contain an amino terminal leader sequence corresponding to residues 1-15 of SEQ ID NO:2.

35. The fusion polypeptide of claim 34, wherein the paraoxonase has an amino acid sequence selected from the group consisting of residues n-355 of SEQ ID NO:55 and residues n-355 of SEQ ID NO:56, wherein n is an integer from 16 to 26, inclusive.

36. The fusion polypeptide of claim 34 or 35, wherein LI is present and comprises at least eight amino acid residues.

37. The fusion polypeptide of claim 36, wherein LI consists of from 12 to 25 amino acid residues.

38. The fusion polypeptide of claim 36, wherein LI has the amino acid sequence shown in SEQ ID NO: 12.

39. The fusion polypeptide of any one of claims 34 to 38, wherein the fusion polypeptide does not comprise a biologically active polypeptide N-terminal to the dimerizing domain or domain that specifically binds to FcRn.

40. The fusion polypeptide of any one of claims 34 to 38, wherein the fusion polypeptide further comprises a biologically active polypeptide N-terminal to the dimerizing domain or domain that specifically binds FcRn, wherein said biologically active polypeptide is selected from the group consisting of a cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) extracellular domain, and a CD40 extracellular domain, and wherein the fusion polypeptide comprises, from an amino-terminal position to a carboxyl-terminal position. T-L2-X-L1-P, wherein X, LI, and P are as defined in claim 1, L2 is a second polypeptide linker, wherein L2 is optionally present, and T is the biologically active polypeptide.

41. The fusion polypeptide of claim 40, wherein the biologically active polypeptide is the CTLA-4 extracellular domain.

42. The fusion polypeptide of claim 41, wherein the CTLA-4 extracellular domain has at least 95% identity with the amino acid sequence shown in residues 21-144 of SEQ ID NO:26.

43. The fusion polypeptide of claim 42, wherein the CTLA-4 extracellular domain has the amino acid sequence shown in residues 21 -144 of SEQ ID NO:26.

44. The fusion polypeptide of claim 40, wherein the biologically active polypeptide is the CD40 extracellular domain.

45. The fusion polypeptide of claim 44, wherein the CD40 extracellular domain has at least 95% identity⁷ with the amino acid sequence shown in residues 21-188 of SEQ ID NO:42.

46. The fusion polypeptide of claim 45, wherein the CD40 extracellular domain has the amino acid sequence shown in residues 21-188 of SEQ ID NO:42.

47. The fusion polypeptide of claim 45, wherein the CD40 extracellular domain contains at least one amino acid substitution at a position corresponding to an amino acid of human CD40 (SEQ ID NO:46) selected from the group consisting of E64, K81, P85, and L121, wherein the at least one amino acid substitution increases CD40 ligand binding relative to human CD40.

48. The fusion polypeptide of claim 47, wherein the amino acid at the position corresponding to K81 of human CD40 is selected from the group consisting of threonine, histidine, and serine; the amino acid at the position corresponding to K81 of human CD40 is histidine and the amino acid the position corresponding to L121 of human CD40 is proline; or the amino acid at the position corresponding to E64 of human CD40 is tyrosine, the amino acid at the position corresponding to K81 of human CD40 is threonine, and the amino acid at the position corresponding to P85 of human CD40 is tyrosine.

49. The fusion polypeptide of any one of claims 34 to 48, wherein X is a dimerizing domain, optionally wherein said dimerizing domain specifically binds to FcRn.

50. The fusion polypeptide of claim 49, wherein X is an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region is capable of forming dimers and specifically binding FcRn.

51. The fusion polypeptide of claim 50, wherein the immunoglobulin heavy chain constant region is an immunoglobulin Fc region.

52. The fusion polypeptide of claim 51 , wherein the Fc region is a human Fc region.

53. The fusion polypeptide of claim 52, wherein the human Fc region is an Fc variant comprising one or more amino acid substitutions relative to the wild-type human sequence.

54. The fusion polypeptide of claim 52 or 53, wherein the Fc region is a human yl Fc region or a human y4 Fc region.

55. The fusion polypeptide of claim 53, wherein the Fc region is a human y 1 Fc variant in which Eu residue C220 is replaced by serine.

56. The fusion polypeptide of claim 55, wherein Eu residues C226 and C229 are each replaced by serine.

57. The fusion polypeptide of claim 56, wherein Eu residue P238 is replaced by serine.

58. The fusion polypeptide of 53, wherein the Fc region is a human yl Fc variant in which Eu residue P331 is replaced by serine.

59. The fusion polypeptide of any one of claims 55 to 57, wherein Eu residue P331 is replaced by serine.

60. The fusion polypeptide of any one of claims 55 to 59, wherein Eu residue M252 is replaced by tyrosine, Eu residue S254 is replaced by threonine, and/or Eu residue T256 is replaced by glutamate.

61. The fusion polypeptide of claim 50, wherein the immunoglobulin heavy chain constant region comprises an amino acid sequence having at least 95% identity with the amino acid sequence shown in

(i) residues 16-232 or 16-231 of SEQ ID NO : 6,

(ii) residues 16-232 or 16-231 of SEQ ID NO:48, or

(iii) residues 16-232 or 16-231 of SEQ ID NO:52.

62. The fusion polypeptide of claim 61, wherein the immunoglobulin heavy chain constant region comprises the amino acid sequence shown in

(i) residues 16-232 or 16-231 of SEQ ID NO:6,

(n) residues 16-232 or 16-231 of SEQ ID NO:48, or

(iii) residues 16-232 or 16-231 of SEQ ID NO:52.

63. The fusion polypeptide of claim 51, wherein the Fc region has the amino acid sequence shown in

(i) residues 1 -232 or 1 -231 of SEQ ID NO: 6,

(ii) residues 1 -232 or 1 -231 of SEQ ID NO: 8,

(iii) residues 1-232 or 1-231 of SEQ ID NO:48,

(iv) residues 1-232 or 1-231 of SEQ ID NO: 50.

(v) residues 1 -232 or 1 -231 of SEQ ID NO: 52, or

(vi) residues 1-232 or 1-231 of SEQ ID NO: 54.

64. The fusion polypeptide of claim 50, wherein the fusion polypeptide comprises an amino acid sequence having at least 97% identity with the amino acid sequence shown in

(i) residues 21-613 or 24-613 of SEQ ID NO: 18,

(n) residues 21 -610 of SEQ ID N 0 : 20.

(iii) residues 21-739 of SEQ ID NO:26,

(iv) residues 21-736 of SEQ ID NO:28,

(v) residues 21-613 or 24-613 of SEQ ID NO:36,

(vi) residues 21-610 of SEQ ID NO:38.

(vn) residues 21-736 of SEQ ID NQ:40,

(viii) residues 21-803 of SEQ ID NO:42,

(ix) residues 21-803 of SEQ ID NO:44, or

(x) residues 21-739 of SEQ ID NO:58.

65. The fusion polypeptide of claim 64, wherein the fusion polypeptide comprises the amino acid sequence shown in

(i) residues 21-613 or 24-613 of SEQ ID NO: 18,

(n) residues 21-610 of SEQ ID NO:20,

(iii) residues 21-739 of SEQ ID NO:26,

(iv) residues 21-736 of SEQ ID NO:28,

(v) residues 21-613 or 24-613 of SEQ ID NO:36,

(vi) residues 21-610 of SEQ ID NO:38.

(vii) residues 21-736 of SEQ ID NO:40,

(viii) residues 21-803 of SEQ ID NO:42,

(ix) residues 21-803 of SEQ ID NO:44, or

(x) residues 21-739 of SEQ ID NO:58.

66. A dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, wherein each of the first and second fusion polypeptides is a fusion polypeptide as defined in any one of claims 46 to 65.

67. A polynucleotide encoding the fusion polypeptide of any one of claims 1 to 31 and 34 to 65.

68. An expression cassette comprising a DNA segment encoding the fusion polypeptide of any one of claims 1 to 32 and 34 to 65, wherein the DNA segment is operably- linked to a promoter.

69. A cultured cell into which has been introduced the expression cassette of claim 68, wherein the cell expresses the DNA segment.

70. A method of making a fusion polypeptide, the method comprising: culturing a cell into which has been introduced the expression cassette of claim 68, wherein the cell expresses the DNA segment and the encoded fusion polypeptide is produced; and recovering the fusion polypeptide.

71. An expression cassette comprising a DNA segment encoding the fusion polypeptide of any one of claims 16 to 32 and 46 to 65, wherein the DNA segment is operably linked to a promoter.

72. A method of making a dimeric protein, the method comprising: culturing a cell into which has been introduced the expression cassette of claim 71, wherein the cell expresses the DNA segment and the encoded fusion polypeptide is produced as a dimeric protein; and recovering the dimeric protein.

73. A vector comprising the expression cassette of claim 68 or 71.

74. A composition comprising: a fusion polypeptide of any one of claims 1 to 32; and a pharmaceutically acceptable carrier.

75. A composition comprising: a dimeric protein of claim 33; and a pharmaceutically acceptable carrier.

76. A composition comprising: a fusion polypeptide of any one of claims 34 to 65; and a pharmaceutically acceptable carrier.

77. A composition comprising: a dimeric protein of claim 66; and a pharmaceutically acceptable carrier.

78. A method for treating an inflammatory disease, the method comprising: administering to a subj ect having the inflammatory disease an effective amount of a fusion polypeptide of any one of claims 1 to 32 or a dimeric protein of claim 33.

79. The method of claim 78, wherein the inflammatory disease is an inflammatory lung disease.

80. The method of claim 79, wherein the inflammatory lung disease is selected from the group consisting of chronic obstructive pulmonary disease (COPD), asthma, Cystic Fibrosis (CF), bronchiectasis, hypoxia, acute respiratory distress syndrome (ARDS), and interstitial lung disease.

81. The method of claim 80, wherein the interstitial lung disease is selected from the group consisting of idiopathic pulmonary fibrosis (IPF) and sarcoidosis.

82. The method of claim 79, wherein the inflammatory lung disease is characterized by Pseudomonas aeruginosa infection.

83. The method of claim 78, wherein the inflammatory disease is selected from the group consisting of an inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), type 1 diabetes, and type 2 diabetes.

84. The method of claim 78, wherein the inflammatory disease is an inflammatory skin disease.

85. The method of claim 84, wherein the inflammatory skin disease is selected from the group consisting of psoriasis and atopic dermatitis.

86. A method for treating an autoimmune disease, the method comprising: administering to a subject having the autoimmune disease an effective amount of a fusion polypeptide of any one of claims 1 to 32 or a dimeric protein of claim 33.

87. The method of claim 86, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), Sjogren’s syndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, antiphospholipid syndrome, type 1 diabetes, vasculitis, and systemic sclerosis.

88. A method for treating biofilm formation by a gram-negative bacteria, the method comprising: administering to a subj ect having the biofilm formation an effective amount of a fusion polypeptide of any one of claims 1 to 32 or a dimeric protein of claim 32 [33.

89. The method of claim 88, wherein the gram-negative bacteria is Pseudomonas aeruginosa.

90. A method for treating a neurological disease, the method comprising: administering to a subject having the neurological disease an effective amount of a fusion polypeptide of any one of claims 1 to 32 or a dimeric protein of claim 33.

91. The method of claim 90, wherein the neurological disease is characterized by dementia.

92. The method of claim 90, wherein the neurological disease is selected from the group consisting of Parkinson’s disease and Alzheimer’s disease.

93. A method for treating a cardiovascular disease, the method comprising: administering to a subj ect having the cardiovascular disease an effective amount of a fusion polypeptide of any one of claims 1 to 32 or a dimeric protein of claim 33.

94. The method of claim 93, wherein the cardiovascular disease is characterized by atherosclerosis.

95. The method of claim 94, wherein the cardiovascular disease is selected from the group consisting of coronary heart disease and ischemic stroke.

96. A method for treating a chronic liver disease, the method comprising: administering to a subject having the chronic liver disease an effective amount of a fusion polypeptide of any one of claims 1 to 32 or a dimeric protein of claim 33.

97. The method of claim 96, wherein the chronic liver disease is nonalcoholic steatohepatitis (NASH).

98. A method for treating a fibrotic disease, the method comprising; administering to a subject having the fibrotic disease an effective amount of a fusion polypeptide of any one of claims 1 to 32 or a dimeric protein of claim 33.

99. The method of claim 98, wherein the fibrotic disease is selected from the group consisting of systemic sclerosis, systemic lupus ery thematosus, an inflammatory⁷ lung disease, a chronic liver disease, and a chronic kidney disease.

100. A method for treating exposure to sulfur mustard gas or an organophosphate, the method comprising: administering to a subject exposed to the sulfur mustard gas or to the organophosphate an effective amount of a fusion polypeptide of any one of claims 34 to 65 or a dimeric protein of claim 66.

101. The method of claim 100. wherein the organophosphate is selected from the group consisting of tabun, sarin, soman, and cyclosarin.