Paraoxonase fusion polypeptides and related compositions and methods

Abstract

Compositions and methods related to paraoxonase fusion polypeptides are disclosed. In some embodiments, the fusion is a bispecific molecule comprising a first biologically active polypeptide linked amino-terminally to biologically active paraoxonase (e.g., human PON1 or a variant thereof), and the first biologically active polypeptide is DNase, RNase, SOD1, CTLA-4 extracellular domain, CD40 extracellular domain, or a polypeptide that specifically binds and neutralizes inflammatory cytokines. The bispecific fusion may further comprise a second biologically active polypeptide (e.g., a dimerization domain or a domain that specifically binds to neonatal Fc receptor (FcRn)) linked carboxyl-terminally to the first biologically active polypeptide and amino-terminally to paraoxonase.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 151,236 and 63 / 151,272, filed February 19, 2021. Each of the aforementioned applications is incorporated herein by reference in its entirety. Reference to sequence listing

[0002] This application contains a Sequence Listing, which has been provided via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on February 16, 2022, is titled "TRP_0410PC_20220216_Seq_Listing_ST25" and is 552,734 bytes in size. [Background technology]

[0003] Background of the Invention Pulmonary Disease and Fibrosis Air pollution, primarily caused by atmospheric ozone levels, is a global health problem that is predicted to increase in the future (Zhang et al., Front. Immunol. 10:2518, 2019). Ozone is a strong oxidant, and tropospheric (ground-level) ozone causes lung inflammation and damage. Ozone pollution has also been linked to vascular and neurological diseases (Zhang et al., supra).

[0004] Pulmonary disease is already a significant health and economic burden worldwide. It is the third leading cause of death in the United States. Including chronic obstructive pulmonary disease (COPD) and asthma, costs are projected to exceed $80 billion annually by 2020 (Foster et al., J. COPD 3:2011-2018, 2006; Nurmagambetov et al., Annals of Am. Thoracic Soc., Center for Disease Control and Prevention, 2017). Current treatments include nebulized steroids, anticholinergics, and bronchodilators for COPD, and leukotriene modulators and long-acting beta-agonists for asthma. Humanized antibodies given by subcutaneous or intravenous injection have been approved as add-on treatments for patients with difficult-to-control asthma; these antibodies deplete IgE (omalizumab), inhibit IL-5 (mepolizumab and reslizumab), or deplete eosinophils (benralizumab). New targets and approaches for improved treatment of lung diseases are needed.

[0005] Pulmonary inflammation is associated with the infiltration of neutrophils, eosinophils, macrophages, and lymphocytes into the lung lumen and tissues. Extracellular DNA derived from neutrophil extracellular traps (NETS) is present in the lungs of patients with asthma, cystic fibrosis, and COPD (Liu et al., Chinese Med. Journal 130:730-736, 2017; Twaddell et al., CHEST 156:774-782, 2019; Uddin et al., Front. Immunol. 10:47, 2019; Yamashita et al., J. Immunol. 191:949-960, 2013). Furthermore, eosinophils contribute to extracellular traps in patients with asthma (Dworski et al., J. Allergy Clinical Immunol. 127:1260-1266, 2011). TRAPs contain inflammatory mediators, including myeloperoxidase and neutrophil elastase, which thicken lung mucus and promote tissue damage (Uddin et al., supra). NET-associated myeloperoxidase and elastase are elevated in sputum and bronchoalveolar lavage fluids from cystic fibrosis patients and correlate with the severity of lung disease (see generally Kahn et al., Genes 10:183, 2019). DNase 1 can digest NETS and reduce mucus viscosity, allowing for improved lung clearance (ibid.).

[0006] Extracellular DNA, which can be digested by DNase 1, has been found to be important for silica-induced pulmonary fibrosis (see Benmerzoug et al., Nature Comm. 9:5226, 2018). Silica induces cell death and the release of self-DNA in the alveolar space, inducing the stimulator of interferon genes (STING) inflammatory pathway, which leads to fibrosis (ibid.). P. aeruginosa infection

[0007] P. aeruginosa is a ubiquitous Gram-negative bacillus that causes severe opportunistic infections in immunocompromised individuals, burn patients, and patients with lung diseases such as COPD and cystic fibrosis (CF) (see Mulcahy et al., Microb. Ecol. 68:1-12, 2014). It is particularly troublesome on medical surfaces such as ventilators and is among the most common nosocomial pathogens and frequently isolated from patients with ventilator-associated pneumonia. Because it is a major pathogen in adults with cystic fibrosis (CF) and is associated with increased clinical exacerbations, eradication of P. aeruginosa has important implications for CF treatment and other P. aeruginosa infections (see, e.g., Martinez-Solano et al., Chronic Infect. Dis. 47:1526-1533, 2008; Meyer-Hamblett et al., Clin Inf. Dis. 59:624-631, 2014; Gallego et al., BMC Pulmonary Med. 14:103, 2014).

[0008] Pulmonary failure, a result of persistent bacterial lung infection, particularly with P. aeruginosa, accounts for 80% of deaths in CF patients. Infection with P. aeruginosa in CF increases the risk of premature death by nearly threefold (see McColley et al., Pediatric Pulmonology 52:909-915, 2017). Despite intensive research focused on mitigating bacterial lung infections, additional approaches for effective treatment in CF lungs are still needed.

[0009] Quorum sensing (QS) is a key mechanism that aids P. aeruginosa persistence in CF lungs and significantly contributes to its pathogenesis. QS is mediated by small molecules produced by bacteria, specifically acylhomoserine lactones (or AHLs), which enable coordination of activity across the community. Acylhomoserine lactones share a basic structure consisting of a homoserine lactone ring (HSL) and an acyl chain that can vary in length and degree of saturation. Variant side chains confer specificity to the QS mechanism, enabling bacteria to recognize and respond to their specific AHLs. Nearly one-quarter of the expressed P. aeruginosa proteome is regulated by QS. In P. aeruginosa, QS plays a key role in the expression and regulation of numerous virulence factors. QS also plays an important role in biofilm formation. The biofilm essentially encases the bacterial community in a matrix of polysaccharides, extracellular DNA, and proteins, protecting it from the host immune response and external antimicrobial agents such as antibiotics, facilitating the survival of P. aeruginosa (Mulcahy et al., supra). Paraoxonase 1 (PON1)

[0010] Paraoxonase 1 (PON1) is an antioxidant enzyme that inhibits the oxidation of low-density lipoprotein (LDL) and protects its function (Aviram et al., J. Clin. Invest. 101:1581-1590, 1998). PON1 has multiple substrates, including organophosphates and acylhomoserine lactones, quorum-sensing molecules produced by Pseudomonas aeruginosa. PON1 activity is important in protecting against atherosclerosis and ischemic stroke (Litvinov et al., N. Am. J. Med. Sci. 4:523-532, 2012). Furthermore, PON1 activity is reduced in patients with intestinal inflammation, including those with irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD), such as Crohn's disease (Oran et al., J. Pak. Med. Assoc. 64:820-822, 2014; Szczelkik et al., Molecules 23:2603, 2018; Rothem et al., Free Rad. Biol. Med. 43:730-739, 2007). Studies in mouse models of colitis have demonstrated that treatment with N-acetylcysteine ​​stimulates the upregulation of PON1, and increased PON1 activity is associated with the amelioration of colonic damage (You et al., Dig. Dis. Sci. 54:1643-1650, 2009).

[0011] PON1 is also defective in several lung diseases. There is strong evidence that PON1 is important in protecting against lung inflammation and that low PON1 activity is associated with lung disease. Oxidative stress is a key factor in the pathogenesis of asthma (see Sahiner et al., WAO Journal 4:151-158, 2011), and the antioxidant activity of PON1 is impaired in asthmatic patients. In children with asthma, PON1 levels were significantly lower than in controls (p<0.001) (Emin et al., Allergol. Immunopathol. (Madr) 43:346-352, 2014). Additional studies have reported that PON1 activity is significantly lower in asthmatic patients (p<0.024) (Sarioglu et al., Iran J. Asthma Immunol. 14:60-66, 2015). Another study showed that PON1 activity is lower in asthma patients during active disease, but increases during disease remission (Tolgyesi et al., Internat. Immunol. 21:967-975, 2009). This study also used gene profiling in an experimental mouse asthma model to identify downregulation of PON1 as a potential target for monitoring disease activity (see ibid.). Furthermore, a mouse asthma model showed that PON1 overexpression reduced airway inflammation and remodeling and reduced inflammatory cytokines (Chen et al., J. Cell Biochem. 119:793-805, 2018).

[0012] Compared with controls, patients with COPD were found to have significantly lower PON1 activity and increased oxidative stress, as evidenced by lower levels of reduced thiol groups (Rumora et al., J. COPD 11:539-545, 2014). Another study found that the polymorphism C>T at -108 in the PON1 promoter was strongly associated with COPD (Rajkovic et al., J. Clin. Path. 71:963-970, 2018). Both the TT genotype and the T allele at -108 were strongly associated (p<0.001), suggesting that the -108T allele may be a factor in the reduced expression of PON1 in COPD patients (ibid.).

[0013] PON1 activity has been reported to be reduced in patients with sarcoidosis and interstitial lung disease, especially during active disease (Ivanisevic et al., Eur. J. Clin. Invest. 46:418-424, 2016; Uzun et al., Curr. Med. Res. Opin. 24:1651-1657, 2008). Another study found lower PON1 activity during hypoxia (Okur et al., Sleep Breath. 17:365-371, 2013). PON1 activity was absent in patients with lung disease exposed to sulfur mustard gas, even if the exposure occurred decades earlier (Golmanesh et al., Immunopharmacol. and Immunotoxicol. 35:419-425, 2013).

[0014] It has been reported that PON1 and myeloperoxidase exert a reciprocal regulation, in that excessive amounts of PON1 inhibit myeloperoxidase, whereas excessive amounts of myeloperoxidase inhibit PON1 (Huang et al., J. Clin. Invest. 123:3815-3828, 2013). Huang et al. found that both PON1 and myeloperoxidase tightly bound to apoA-I. This provides one mechanism for decreased PON1 activity under inflammatory conditions in which neutrophils and eosinophils produce excessive amounts of myeloperoxidase. However, under normal conditions, PON1 is in excess and suppresses the activity of myeloperoxidase (see ibid.).

[0015] Recombinant PON1 (rPON1) has been reported to inhibit PMA-induced differentiation of the monocytic cell line THP-1 (Rosenblat et al., Atherosclerosis 219:49-56, 2011). Mice IP-injected with rPON1 had reduced monocyte differentiation into macrophages and reduced expression of CD11b and CD36. Total cellular peroxidation in peritoneal macrophages was reduced by 18% (ibid.).

[0016] PON1 therapy has been studied in experimental models. One study showed that recombinant human PON1 injection protected guinea pigs from sarin and soman inhalation toxicity (Valiyaveettil et al., Biochem. Pharmacol. 81:800-809, 2011; Valiyaveettil et al., Toxicol. Letters 202:203-208, 2011). Another study showed that treatment with PON1 protected mice from organophosphate poisoning (Bajaj et al., Appl. Biochem. Biotechnol. 180:165, 2016; Stevens et al., Proc. Natl. Acad. Sci. USA 105:12780-12784, 2008). These studies used recombinant PON1 purified from bacterial inclusion bodies or produced in T. ni larvae using a baculovirus vector. Both groups also expressed PON1 192K, a variant derived from the rabbit sequence that helps stabilize and increase PON1 enzymatic activity (see Bajaj et al., supra; Stevens et al., supra).

[0017] Expression of PON1 in bacteria has also been used to select mutants with enhanced enzymatic activity against organophosphates and nerve agents (see Aharoni et al., Proc. Natl. Acad. Sci. USA 101:482-487, 2004; Goldsmith et al., Chemistry and Biology 19:456, 2012). Engineering of PON1 was performed by directed evolution to find variants with improved soluble expression in bacteria. One evolved PON1 has been crystallized, and the PON1 structure has been described (see Harel et al., Nature Struct. Mol. Biol. 11:412-419, 2004). Another widely used PON1 variant is G3C9, a hybrid PON1 formed by shuffling mouse, rat, rabbit, and human PON1 sequences (see Aharoni et al., Proc. Natl. Acad. Sci. USA 101:482-487, 2004). Other PON1 variants with increased organophosphatase activity but reduced esterase activity have been described (see Aharoni et al., supra). Further evolution of G3C9 has also been reported to increase activity against the G-type nerve agents tabun, sarin, soman, and cyclosarin (Gupta et al., Nat. Chem. Biol. 7:120-125, 2011; Goldsmith et al., Chemistry and Biology 19:456-466, 2012). Compared to human PON1, G3C9 has amino acid differences at 55 positions, and more evolved PON1 variants have even more amino acid differences (Goldsmith et al., supra).

[0018] PON1 and the PON1 variant G3C9 have been tested in a mouse model of experimental colitis (Yamashita et al., J. Immunol. 191:949-960, 2013). PON1 treatment was effective in TNBS-induced colitis and was equivalent to anti-TNFα treatment, which is currently the most effective clinically used treatment in humans. However, CD4 + CD45RB high In a chronic colitis model using cell transfection, PON1 was ineffective, whereas G3C9 showed strong efficacy. The efficacy of G3C9 was equivalent to that of anti-TNFα in the chronic colitis model (ibid.). Another study found that PON1 activity correlated with the severity of Crohn's disease (Sczceklik et al., Molecules 23:2603, 2018).

[0019] Another approach to PON1 therapy has been to inject HDL-like particles formed from reconstituted apoA-I plus PON1, a therapy called BL-3050 (Gaidukov et al., BMC Clinical Pharmacol. 9:18, 2009). In these studies, both bacterially expressed G3C9 and the BL-3050 complex were protective in a mouse model of organophosphate poisoning (ibid.).

[0020] PON1 has been expressed as a fusion protein linked to the C-terminus of an insulin receptor-specific mAb heavy chain (see, e.g., Boado et al., Mol. Pharm. 5:1037, 2008). Expression of this hybrid heavy chain with an insulin receptor-specific light chain resulted in the secretion of a bifunctional antibody that binds to the insulin receptor and has PON1 enzymatic activity (ibid.). This hybrid molecule was expressed in mammalian cells (COS and CHO) (ibid.; see Boado et al., Biotechnol. and Bioengineering, 108:186, 2011). This hybrid molecule was also tested in non-human primates and found to cross the blood-brain barrier; however, the molecule was rapidly cleared from peripheral blood into peripheral tissues, primarily the liver (Boado et al. (2011), supra). There have been no reports of clinical trials of this molecule. Expression levels of this fusion protein in CHO cells were low (5-10 mg / liter) even after multiple transfections and subcloning (see id.).

[0021] PON1 has also been fused with a membrane transduction domain, termed PEP-1-PON1 (Kim et al., Biomaterials 64:45-56, 2015). This molecule inhibited the inflammatory response of microglial cell lines and protected against dopaminergic cell death in vivo in a mouse model of Parkinson's disease (ibid.).

[0022] The paraoxonase enzyme is capable of hydrolyzing QS molecules that are acyl homoserine lactones (AHLs), including C12HSL (N-3-oxo-dodecanoyl-L-homoserine lactone), the major QS molecule from P. aeruginosa. Studies of human PON1 have shown that it has pleiotropic enzymatic activity, hydrolyzing a variety of substrates, including lactones (Billecke et al., Drug Metabolism and Disposition 28:1335-1342, 2000; Khersonsky and Tawfik, Biochemistry 44:6371-6382, 2005; Gaidukov and Tawfik, J. Lipid Res. 48:1637-1646, 2007; Bajaj et al., Protein Science 22:1799-1807, 2013). One study in Drosophila melanogaster demonstrated protection from Pseudomonas aeruginosa lethality by expression of a human PON1 transgene capable of hydrolyzing the key quorum-sensing molecule N-3-oxodecanoylhomoserine lactone (3OC12-HSL) (Stoltz et al., J. Clin. Invest. 118:3123-3131, 2008). DNase1

[0023] DNase1 is a calcium and magnesium-dependent endonuclease encoded by the human DNase1 gene.DNase1 cleaves single-stranded and double-stranded DNA and chromatin.Chromatin cleavage occurs at highly sensitive sites where chromatin is open and accessible, and is used in genomics to identify regions that are likely to contain active genes (Boyle et al., Cell 132:311-322, 2008).DNase1 helps degrade DNA during apoptosis (Samejima et al., Nat. Rev. Mol. Cell Biol. 6:677-688, 2008), and also degrades neutrophil extracellular traps (NETS) (Hakkim et al., Proc. Natl. Acad. Sci. USA 107:9813, 2010). DNase 1 can digest biofilm DNA, resulting in a reduction in biofilm biomass and increasing antibiotic-mediated biofilm killing (Tetz et al., Antimicrob. Agents Chemother. 53:1204-1209, 2009).

[0024] The accumulation of extracellular DNA from dead and dying cells was long ago identified as a driver of disease in patients with systemic lupus erythematosus (SLE). DNase1 was administered to SLE patients at Rockefeller beginning in 1959. However, bovine DNase1 was used, and treatment was limited by its immunogenicity (see Valle et al., Autoimmunity Rev. 7:359, 2008). Reduced levels of DNase1 and DNase1 mutations with reduced activity have been reported in patients with SLE (see Valle et al., supra). Reduced DNase1 activity in urine was found in patients with lupus nephritis, and reduced DNase1 activity was a biomarker of disease progression (Pedersen et al., J. Pathol. Clin. Res. 4:193, 2018). In SLE patients, defective clearance of apoptotic debris and accumulation of chromatin contribute to the breakdown of tolerance and the development of autoimmune disease.

[0025] To combat infectious agents, activated neutrophils extrude DNA bound to cytoplasmic and granule proteins, called neutrophil extracellular traps (NETs), a process also known as "NETosis" (see Brinkmann, J. Innate Immun. 10:422-431, 2018). However, if NETs are not targeted and effectively removed, they often cause concomitant tissue damage and inflammation. NETs have been implicated in driving disease progression in several incurable and serious diseases. Evidence for the importance of NETs in psoriasis, cystic fibrosis, SLE, RA, type 1 diabetes, sepsis, small-vessel vasculitis, IBD, type 2 diabetes, and obesity has recently been reviewed (Mutua and Gershwin, Clin. Rev. Allergy Immunol., 2020, DOI 10.1007 / s12016-020-08804-7). See also Michailidou, Front. Immunol. 11:619705, 2020 (reviewing the involvement of neutrophils and NETs in vasculitis). NETs also play an important role in cardiovascular disease and thrombosis, as well as in cancer, where they promote tumor progression and metastasis (Brinkmann, supra). In addition to their importance in cystic fibrosis, NETs also play a role in the clinical pathology of other respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD) (see Liu et al., Chinese Med. Journal 130:730-736, 2017; Twaddell et al., CHEST 156:774-782, 2019; Uddin et al., Front. Immunol. 10:47, 2019). Recently, NETs have been found to contribute to immune thrombosis in COVID-19 acute respiratory distress syndrome (Middleton et al., Blood 136:1169, 2020; Zuo et al., JCI Insight 5:e138999, 2020; Barnes et al., J. Exp. Med. 217:e20200652, 2020).

[0026] DNase1 is capable of digesting NETs and works with another extracellular DNase called DNase1L3 to remove them. Mice deficient in both DNase1 and DNase1L3 were unable to remove NETs and died rapidly due to massive intravascular clot formation and organ damage after neutrophil activation (Brinkmann, supra). The contribution of NETs to thrombosis has already led to clinical trials of wild-type DNase1 (Pulmozyme®) taken by inhalation therapy in COVID-19 patients (NCT04359654).

[0027] Recombinant wild-type human DNase 1 (Pulmozyme®) is produced by Genentech and was first approved for cystic fibrosis (CF) to digest DNA and reduce congestion in the lungs. High levels of DNA, primarily derived from NETs, ​​are pathogenic in CF lungs (Law et al., J. Inflammation, 14:1-8, 2017). Bacterial DNA from Pseudomonas aeruginosa biofilm formation is also present in many CF patients and can also be digested by DNase 1 (Whitchurch et al., Science 295:1487, 2002).

[0028] Pulmozyme is delivered directly to the lungs by nebulization and taken daily. Pulmozyme has been studied in patients with lung diseases other than CF, but is not approved for alternative use (Torbic et al., J. Pharmacy Practice 29:480, 2016). Pulmozyme was tested in a mouse lupus model (Verthelyi et al., Lupus 7:223, 1998) and in a Phase I clinical trial in SLE patients (Davis et al., Lupus 8:68-76, 1999) without any reduction in disease measurements. Systemic administration of pulmozyme therapy may be limited due to the enzyme's inhibition by globular actin, which is abundant in the blood and lungs (Ulmer et al., Proc. Natl. Acad. Sci. USA 93: 8225, 1996), as well as its short half-life.

[0029] Attempts have been made to enhance DNase 1 for CF and SLE patients. Site-directed mutagenesis studies of the DNA-binding interface identified 27 amino acid positions critical for enzyme activity and another 13 positions peripherally involved in DNA interactions with minimal effect on enzyme activity (see Pan et al., Prot. Sci. 7:628-636, 1998). This group showed that increasing the local electrostatic attraction to DNA by adding specific additional positively charged residues not only improved binding affinity to DNA, but also improved functional activity and eliminated salt inhibition (Pan et al., Biochemistry 36:6624-6632, 1997; Pan and Lazarus, J. Biol. Chem. 273:11701-11708, 1998). Consistent with this observation, alanine substitution of two arginines (Arg-41 and Arg-111) that make critical contacts with DNA significantly reduced the DNA cleavage activity of human DNase 1 (Pan et al., 1998, supra). Furthermore, the introduction of positively or negatively charged residues at sites distal to the DNA interface did not alter the specific activity (Ulmer et al., Proc. Natl. Acad. Sci. USA 93:8225-8229, 1996).

[0030] The hyperactive DNase 1 variants showed significant enhancement (up to 10,000-fold) under conditions of low DNA concentration and short DNA length (Pan and Lazarus, supra). However, under conditions optimal for wild-type DNase 1, the best variant showed only 7-fold higher activity and contained a single amino acid change (N74K). The N74K hyperactive variant showed reduced dependence on calcium (Pan and Lazarus, Prot. Sci. 8:1780-1788, 1999).

[0031] Ulmer et al. (supra) and Pan et al. (1998, supra) showed that substitution of Ala-114 with phenylalanine (A114F) eliminated the inhibition of DNase 1 by G-actin.

[0032] Actin-resistant and hyperactive DNase 1 mutant enzymes are further described in US Pat. Nos. 6,348,343 and 6,391,607.

[0033] DNASE1 is polymorphic, and alleles encoding enzymes with reduced activity are associated with an increased risk of autoimmune disease. However, the naturally occurring allele G105R was found to encode a DNase1 variant with a three-fold increase in activity (Yasuda et al., Int. J. Biochem. Cell. Biol. 42:1216-1225, 2010). This is a minority allele found in African populations and can confer resistance to autoimmune disease (ibid.).

[0034] DNase 1 therapy using actin / salt-resistant and hyperactive DNase 1 has great potential. However, despite screening thousands of clones, it was not possible to obtain stable CHO cells that constitutively express enhanced DNase 1 (see Lam et al., Biotech. Progress 32: 523-533, 2017). This was thought to be due to the toxicity of the enzyme during high expression in CHO cells. Lam et al. (supra) described an inducible expression system in CHO cells as one potential solution to the problem of producing enhanced DNase 1 enzyme.

[0035] Dwyer et al. (J. Biol. Chem. 274:9738-43, 1999) described a DNase I-Fc fusion protein with impaired enzymatic activity due to dimerization with the Fc domain. Dwyer et al. added a short linker (up to 7 aa) between the DNase I and Fc domain without improving activity (see ibid.). U.S. Patent No. 8,841,416 to Ledbetter et al. describes a DNase I-Fc fusion with a 21 amino acid linker and at least equivalent activity to a recombinant DNase I control.

[0036] Wild-type DNase1 (Pulmozyme) treatment is FDA-approved for CF, but there are no CF mouse models in which DNase1 is active. Pulmozyme was approved for CF treatment without testing for activity in appropriate in vivo disease models (Greene, Hum. Exp. Toxicol., May:13 Suppl 1:S1-42, 1994). More recently, wild-type DNase1 treatment has been studied in several in vivo models with promising results. DNase1 was found to significantly reduce airway resistance but not inflammation in an acute asthma model (da Cunha et al., Exp. Lung Res. 42:66, 2016). In a silica-induced lung inflammation model, DNase1 treatment prevented DNA-mediated STING activation and blocked the downstream type I IFN response (Benmerzoug et al., Nat. Comm. 9:5226, 2018). In a model of acute lung injury, DNase 1 treatment protected mice from pulmonary edema and pulmonary vascular permeability. DNase 1 treatment also reduced NET formation and platelet sequestration in the lung (Caudrillier et al., J. Clin. Invest. 122:2661, 2012).

[0037] Studies in mouse tumor models have shown that NETs promote metastasis and that injection of DNase 1 or DNase 1-coated nanoparticles reduced tumor metastasis (Cools-Lartigue et al., J. Clin. Invest. 123:3446, 2013; Park et al., Sci. Translational Med. 8:361ra138, 2016). Another study showed that a combination of DNase 1 and proteases significantly inhibited the growth of human colon cancer cells in a nude mouse model (Trejo-Becirril et al., Integrative Cancer Therapies 15:NP35-NP43, 2016). In cancer patients, increased levels of citrullinated histone H3, a marker of neutrophil NET formation, strongly predicted poor clinical outcomes (Thalin et al., PLoS ONE 13:e0191231, 2018).

[0038] In a mouse DSS colitis model (Babicova et al., Folia Biolica (Praha) 64:10, 2018), DNase1 treatment caused a reduction in TNFα and myeloperoxidase in the colon. Another study (Li et al., J. Crohns Colitis 2020, 14:240-253, 2020) found that DNase1 treatment reduced cytokine production and attenuated accelerated thrombus formation and platelet activation after DSS-induced colitis.

[0039] DNase1 treatment in a hindlimb ischemia-reperfusion model resulted in increased perfusion, a decrease in infiltrating inflammatory cells, and a reduction in local markers of thrombosis (Albadawi et al., J. Vasc. Surg. 64:484, 2016). In a rat model of ischemia-reperfusion-induced acute kidney injury, DNase1 treatment demonstrated significant renal protective effects. Exogenous administration of DNase1 ameliorated both functional and histological features of acute injury in the kidneys of ischemic rats (Peer et al., Am. J. Nephrol. 43:195, 2016).

[0040] DNase 1 treatment prevented organ damage and protected against mortality when given 4 or 6 hours after injury in a cecal ligation and puncture sepsis model in mice (Mai et al., Shock 44:166, 2015). Extracellular DNA derived from NETs has been recognized as a scaffold for thrombus formation, and infusion of DNase 1 prevented thrombosis in a model of inferior vena cava stenosis (Brill et al., J. Thrombosis Haemostasis 10:136, 2012). RNase

[0041] RNase 1 is a member of the RNase A superfamily, which in humans consists of RNases 1-8. RNase A was first isolated from bovine pancreas, where it primarily functions as a digestive enzyme. In humans, RNase 1 digests single- and double-stranded RNA and RNA / DNA hybrids. RNase 1 is produced by multiple cell types in humans and is thought to be primarily involved in vascular homeostasis. RNase 1 plays a major role in polynucleotide digestion, removing inflammatory extracellular RNA. Furthermore, RNase 1 possesses antiviral activity.

[0042] RNase therapy has been studied in multiple disease models. For example, the growth of the human lung tumor line A549 in athymic mice was inhibited by PEG-RNase1 (Rutkoski et al., Translational Oncology 6:392-397, 2013). RNase treatment also demonstrated antitumor activity and altered microRNA profiles in a syngeneic mouse tumor model bearing Lewis lung carcinoma (Mironova et al., Oncotarget. 8:78796-78810, 2017). RNase1 has been reported to bind to the tumor-associated antigen Globo H, which is expressed on the surface of breast adenocarcinoma cells (Eller et al., ACS Central Sci. 1:181-190, 2015).

[0043] Furthermore, RNase treatment has been found to be effective in animal models of heart transplantation (Kleinert et al., J. Am. Heart Assn. doi: 10.1021, 2016), atherogenesis (Simsekyilmaz et al., Circulation 129: 598-606, 2014), and myocardial infarction (Steiger et al., JAMA 6:e004541, 2017). RNase1 treatment also reduced cerebral edema and infarct size in an acute stroke model (Walberer et al., Curr. Neovas. Res. 6:12-19, 2009).

[0044] RNase treatment was found to prevent postoperative cognitive decline in aging mice (Chen et al., PLoS One 10:e0134307, 2015).

[0045] RNase therapy has also been studied in a mouse model of systemic lupus erythematosus (SLE) (Sun et al., J. Immunol. 190:2536-2543, 2013). Overexpression of the RNA sensor TLR7 causes a lupus-like disease accompanied by autoantibodies, kidney disease, and early death. Crossing these mice with mice transgeneically overexpressing RNase A resulted in offspring with increased survival, reduced lymphocyte activation, reduced renal deposition of IgG and C3, and reduced liver inflammation and necrosis (ibid.).

[0046] RNase-Ig (see U.S. Patent No. 8,937,157), in which human RNase 1 is fused to a mutated human IgG1 Fc domain containing P238S and P331S mutations, is being tested in phase I and phase II clinical trials in patients with systemic lupus erythematosus (SLE) and Sjögren's syndrome. Superoxide dismutase (SOD)

[0047] Superoxide dismutase (SOD) is an antioxidant enzyme that converts oxygen free radicals into H2O2 and O2. There are three human SOD enzymes. SOD1, also known as Cu / Zn SOD, is a cytosolic enzyme that is a homodimer stabilized by intrachain disulfide bonds and metal binding (Doucette et al., J. Biol. Chem. 279:54558-54566, 2004). SOD2 is a tetramer that localizes in mitochondria and uses manganese in its active site. SOD3 is a secreted tetramer that contains copper and zinc. For a review of the roles of SOD, glutathione peroxidase, and catalase in overall protection from oxidative damage, see Ighodaro and Akinolye, Alexandria J. Med. 54:287-293, 2018.

[0048] SOD3 is the major extracellular antioxidant enzyme in the lung, where it protects the extracellular matrix during lung injury. Genetic variants in SOD3 are associated with reduced lung function and rapid progression of COPD in adults (for a review, see Ganguly et al., Physiol. Genomics 37:260-267, 2009).

[0049] SOD1 has been fused with a cell membrane transduction domain to deliver SOD1 to the cytoplasm. For example, Eum et al. (Free Rad. Biol. Med. 37:1656-1669, 2004) created a fusion of SOD with a 21-amino acid peptide called PEP-1 and expressed this fusion protein in bacteria. They demonstrated that the PEP-1-SOD molecule delivered SOD across the cell membrane and was effective in preventing neuronal death after ischemic stroke in vivo after IP injection. SOD1 and variant forms of SOD1 have also been fused with enhanced green fluorescent protein (EGFP) tags to create fusion proteins that can be detected in fluorescence-based binding and chaperone assays (Ganesan et al., Cell Death and Differentiation 15:312-321, 2008). Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4)

[0050] Cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) is an immunoregulatory membrane receptor that downregulates T cell responses by inhibiting the costimulatory interaction of CD28-B7 (Carreno et al., J. Immunol. 165:1352-1356, 2000). The extracellular domain of CTLA-4 fused to a human IgG1 mutant Fc domain (abatacept, Orencia™) has been approved by the FDA for the treatment of RA due to its ability to suppress the activation of CD28 receptors on T cells (Linsley et al., J. Exp. Med. 174:561-569, 1991; see Emma Hitt, FDA Approves Subcutaneous Abatacept for RA, Medscape Medical News, Aug. 2, 2011). Abatacept inhibits T cell production of inflammatory cytokines and inhibits B cell class switching and IgG antibody responses.

[0051] Abatacept treatment has been shown to prevent skin fibrosis and induce regression of established inflammation-driven fibrosis (Ponsoye et al., Ann. Rheum. Dis. 75:2142-2149, 2016). Furthermore, abatacept significantly reduced fibrogenic marker levels, T cell proliferation, and M1 / M2 macrophage infiltration in the lesioned lungs of Fra-2 mice, indicating that abatacept may be useful in treating pulmonary fibrosis in humans (Boleto et al., Arthritis Research and Therapy 20:197, 2018). According to the Pulmonary Fibrosis Foundation, abatacept is currently being tested in a phase 2 clinical trial in patients with interstitial fibrosis (trial number NCT03215927) and is also being tested in a phase 3 clinical trial for the treatment of relapsing non-severe granulomatosis with polyangiitis (Wegener's granulomatosis) (trial number NCT02108860).Co-stimulation inhibitors are being evaluated as potential candidates for suppressing the damage and repair processes associated with pulmonary inflammation. CD40-CD154

[0052] CD40 is a member of the TNFα receptor superfamily and is expressed on lymphocytes, hematopoietic cells, and structural cells, including fibroblasts, epithelial cells, and endothelial cells (Sime and O'Reilly, Clin. Immunol. 99:308-319, 2001). The CD40 ligand (CD40L or CD154) is expressed on activated T lymphocytes, activated platelets, mast cells, and eosinophils. CD154 expressed by activated T cells provides CD40 on B cells with an important signal required for class switching and IgG production. However, CD40 expressed on the surface of structural cells, such as endothelial cells, can be stimulated by binding to CD154, resulting in the production of inflammatory cytokines (ibid.). Activated platelets express CD154 on their surface and are also the major source of soluble CD154 in the blood.

[0053] The CD40-CD154 signaling pathway is involved in lung injury and disease processes, including fibrosis (Kaufman et al., J. Immunol. 172:1862-1871, 2004). Interaction between the CD40 receptor and CD154 can induce cellular activation, including the production of proinflammatory cytokines, the expression of cell adhesion molecules, and the induction of cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2) (Zhang, J. Immunol. 160:1053-1057, 1998). These events can then shift the balance between type 1 and type 2 cytokine responses in favor of type 2 fibrogenic responses. Inhibition of the CD40-CD154 pathway has been shown to have therapeutic benefit in animal lung disease models. For example, the monoclonal anti-mouse CD40L antibody MR1 was effective in protecting against hyperoxic lung injury and radiation-induced lung injury and fibrosis in mouse models (Adawi et al., Clin. Immunol. Immunopathol. 89:222-230, 1998; Adawi et al., Am. J. Pathol. 152:651-657, 1998). Furthermore, molecules that suppress the CD40-CD40L pathway, such as the CXXC5 zinc finger protein, which is involved in epigenetic and transcriptional regulation, have been shown to ameliorate bleomycin-induced pulmonary fibrosis in mice (Cheng et al., BioMed. Research Intl. Volume 2020, Article ID 7840652, 2020; Xiong et al., J. Cell. Mol. Med. 23:740-749, 2018), indicating the important role of the CD40-CD40L pathway in disease progression. Tumor necrosis factor alpha (TNFα)

[0054] Tumor necrosis factor alpha (TNFα) cytokine is a pleiotropic pro-inflammatory mediator produced by various cell types, including macrophages and monocytes. Macrophages are the primary source of TNFα in the lung; however, epithelial cells, eosinophils, and mast cells can also release TNFα upon activation (Malaviya et al., Pharmacol. Ther. 180:90-98, 2017; Aggarwal et al., Blood 119:651-665, 2012). Increased levels of TNFα have been associated with several pulmonary inflammatory diseases, including asthma, chronic obstructive pulmonary disease (COPD), acute lung injury (ALI) / acute respiratory distress syndrome (ARDS), sarcoidosis, and interstitial pulmonary fibrosis (IPF) (Malaviya et al., supra).

[0055] In addition to lung pathology, TNFα is elevated in and involved in inflammatory bowel diseases, such as Crohn's disease (CD) and ulcerative colitis (UC). Monoclonal antibody therapy against tumor necrosis factor-alpha (anti-TNF) has revolutionized the care of patients with inflammatory bowel disease (IBD) (Pouillon et al., Expert Opinion on Biological Therapy 16:1277-1290, 2016; Peyrin-Biroulet et al., The Lancet 372:67-81, 2008). Transforming growth factor-β (TGF-β)

[0056] TGF-β cytokines play an important role in immune response and regulation (Travis and Sheppard, Annu. Rev. Immunol. 32:51-82, 2014). Three major isoforms of TGF-β have been identified in mammals: TGF-β1, TGF-β2, and TGF-β3 (Fernandez and Eickelberg, Proc. Amer. Thorac. Soc. 9:111-116, 2012). Although many cell types express TGF-β, it is expressed in an inactive form complexed with other molecules and requires activation to exert its functional effects. Latent TGF-β is activated upon cleavage from its preprotein form into cytokines with pleiotropic activities, including stimulation of cytokines associated with chemotaxis, proliferation, and inflammation. TGF-β regulates cell proliferation, differentiation, migration, adhesion, survival, epithelial-mesenchymal transition (EMT), and collagen and extracellular matrix synthesis. It is important for angiogenesis, wound healing, and immune regulation, but can also drive cancer, metastasis, diabetes, and fibrotic disease progression (Varga and Pasche, Curr. Opin. Rheum. 20:720-728, 2008). TGF-β has also been shown to be important in regulating adaptive T cell responses, and recent studies suggest that TGF-β is a key mediator of fibrosis in different organs, including the lung. Clinical trials using several antibodies targeting TGF-β isoforms, such as fresolimumab (GC-1008), have been completed or are underway for cancer treatment (melanoma, glioma, breast cancer, and non-small lung cancer) and fibrosis. Additionally, antibodies targeting integrins such as αβ, which are involved in or important in TGF-β activation, are also being used in phase 2 clinical trials to determine their effectiveness against idiopathic pulmonary fibrosis (IPF) (Fernandez and Eickelberg, supra). Enhanced DNase1 expression in mammalian cells Wild-type DNase 1, such as the FDA-approved drug Pulmozyme®, is inhibited by G-actin in lungs and plasma. Enhanced DNase 1 has been engineered by Genentech to create DNase 1 that is resistant to actin inhibition and has increased enzymatic activity (Pan and Lazarus, Prot. Sci. 8:1780-8, 1999). However, this enhanced DNase 1 is toxic in mammalian cells, and stable CHO cells could not be established despite screening thousands of clones. Inducible expression in CHO cells was one potential solution reported (Lam et al., Biotech. Progress 32: 523-33, 2017). [Prior art documents] [Non-patent literature]

[0057] [Non-Patent Document 1] Zhang et al., Front. Immunol. 10:2518, 2019 [Non-patent document 2] Foster et al., J. COPD 3:2011-2018, 2006 [Non-patent document 3] Nurmagambetov et al., Annals of Am. Thoracic Soc., Center for Disease Control and Prevention, 2017 [Non-patent document 4] Liu et al., Chinese Med. Journal 130:730-736, 2017 [Non-patent document 5] Twaddell et al., CHEST 156:774-782, 2019 [Non-patent document 6] Uddin et al., Front. Immunol. 10:47, 2019 [Non-Patent Document 7] Yamashita et al., J. Immunol. 191:949-960, 2013 [Non-licensed document 8] Dworski et al., J. Allergy Clinical Immunol. 127:1260-1266, 2011

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[0058] Summary of the Invention In one aspect, the invention provides a fusion polypeptide comprising, from amino-terminal to carboxyl-terminal positions, T-L1-X-L2-P, where T is a first biologically active polypeptide selected from DNase, RNase, superoxide dismutase 1 (SOD1), cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) extracellular domain, CD40 extracellular domain, and a polypeptide that specifically binds to and neutralizes tumor necrosis factor alpha (TNFα) or transforming growth factor beta (TGF-β); L1 is a first polypeptide linker, and optionally L2 is a polypeptide linker. X is an immunoglobulin heavy chain constant region, the immunoglobulin heavy chain constant region capable of dimerizing and specifically binding to the neonatal Fc receptor (FcRn); L2 is a second polypeptide linker, L2 is optionally present; and P is a biologically active paraoxonase, wherein the paraoxonase has at least 80% or at least 90% identity to the amino acid sequence set forth in residues 16-355 or 26-355 of SEQ ID NO:6 and does not contain an amino-terminal leader sequence corresponding to residues 1-15 of SEQ ID NO:6. In some embodiments, the biologically active paraoxonase has at least 95% identity to the amino acid sequence set forth in (i) residues 16-355 or 26-355 of SEQ ID NO:6, (ii) residues 16-355 or 26-355 of SEQ ID NO:124, or (iii) residues 16-355 or 26-355 of SEQ ID NO:126.

[0059] In another aspect, the invention provides a fusion polypeptide comprising, from amino-terminal to carboxyl-terminal positions, T-L1-X-L2-P, wherein T is a first biologically active polypeptide selected from DNase, RNase, superoxide dismutase 1 (SOD1), cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) extracellular domain, CD40 extracellular domain, and a polypeptide that specifically binds to and neutralizes tumor necrosis factor alpha (TNFα) or transforming growth factor-beta (TGF-β); and L1 is a first biologically active polypeptide selected from the group consisting of DNase, RNase, superoxide dismutase 1 (SOD1), cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) extracellular domain, CD40 extracellular domain, and a polypeptide that specifically binds to and neutralizes tumor necrosis factor alpha (TNFα) or transforming growth factor-beta (TGF-β); and P is a biologically active paraoxonase, wherein the paraoxonase has at least 80% or at least 90% identity to the amino acid sequence set forth in residues 16-355 or 26-355 of SEQ ID NO: 6 and does not contain the amino-terminal leader sequence corresponding to residues 1-15 of SEQ ID NO: 6. In some variations, the second biologically active polypeptide is present and is selected from a dimerization domain and a domain that specifically binds to neonatal Fc receptor (FcRn). In some embodiments, the biologically active paraoxonase has at least 95% identity to the amino acid sequence set forth in (i) residues 16-355 or 26-355 of SEQ ID NO:6, (ii) residues 16-355 or 26-355 of SEQ ID NO:124, or (iii) residues 16-355 or 26-355 of SEQ ID NO:126.

[0060] In certain embodiments of the above fusion polypeptides comprising DNase as the first biologically active polypeptide and in which L1 is present, the DNase has at least 90% or at least 95% identity to the amino acid sequence set forth in (i) residues 21-280 of SEQ ID NO: 18, (ii) residues 21-280 of SEQ ID NO: 152, or (iii) residues 21-290 of SEQ ID NO: 136. In some embodiments, wherein the DNase has at least 90% or at least 95% identity to the amino acid sequence set forth in residues 21-280 of SEQ ID NO: 18 or residues 21-280 of SEQ ID NO: 152, the DNase contains at least one amino acid substitution at a position corresponding to an amino acid in human wild-type DNase 1 (SEQ ID NO: 120) selected from N74, G105, and A114, wherein (1) the amino acid substitution at a position corresponding to N74 in human DNase 1 (the N74 substitution), when present, increases DNA binding compared to human DNase 1; (2) the amino acid substitution at a position corresponding to G105 in human DNase 1 (the G105 substitution), when present, increases DNA binding compared to human DNase 1; and (3) the amino acid substitution at a position corresponding to A114 in human DNase 1 (the A114 substitution), when present, decreases G-actin-induced inhibition of endonuclease activity compared to human DNase 1. In some variations, the fusion polypeptide contains both an N74 substitution and a G105 substitution, both a G105 substitution and an A114 substitution, or each of an N74 substitution, a G105 substitution and an A114 substitution. Particularly suitable amino acid substitutions at these positions are a lysine at the position corresponding to N74 in human DNase 1, an arginine at the position corresponding to G105 in human DNase 1, and / or a phenylalanine at the position corresponding to A114 in human DNase 1. In some embodiments, where the fusion polypeptide contains both an N74 substitution and a G105 substitution, the fusion polypeptide does not contain an A114 substitution. In some embodiments, where the DNase has at least 90% or at least 95% identity to amino acid residues 21-290 of SEQ ID NO: 136, each of the amino acids at positions corresponding to R80, R95, and N96 of SEQ ID NO: 136 is an alanine or a serine.In more specific variations, the DNase has the amino acid sequence set forth in (i) residues 21-280 of SEQ ID NO:18, (ii) residues 21-280 of SEQ ID NO:20, (iii) residues 21-280 of SEQ ID NO:152; (iv) residues 21-290 of SEQ ID NO:138, or (v) residues 21-290 of SEQ ID NO:140. In some embodiments of the fusion polypeptides comprising the above-described DNase, L1 comprises at least 15 amino acid residues or at least 26 amino acid residues (e.g., an L1 linker of 26-60 amino acid residues or 26-36 amino acid residues). In particular variations, L1 comprises three or more (e.g., four or more) tandem repeats of the amino acid sequence of SEQ ID NO:119; in some such embodiments, L1 has the amino acid sequence set forth in SEQ ID NO:12 or SEQ ID NO:14.

[0061] In some embodiments of the above-described fusion polypeptides comprising an RNase as the first biologically active polypeptide, the RNase has at least 90% or at least 95% identity to the amino acid sequence set forth in residues 29-156 of SEQ ID NO:22. In more particular variations, the RNase has the amino acid sequence set forth in residues 29-156 of SEQ ID NO:22. In certain embodiments, L1 is present and comprises at least two amino acid residues (e.g., an L1 linker of 10-36 amino acid residues). In particular variations, L1 comprises two or more tandem repeats of the amino acid sequence of SEQ ID NO:119; in some such embodiments, L1 has the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:12.

[0062] In some embodiments of the above-described fusion polypeptides comprising SOD1 as the first biologically active polypeptide, the SOD1 has at least 90% or at least 95% identity to the amino acid sequence set forth in residues 2-154 of SEQ ID NO: 32. In some such embodiments, the SOD1 contains at least one of the following amino acid substitutions compared to human SOD1 (SEQ ID NO: 32): an alanine at the position corresponding to C7 of human SOD1 (a C7 substitution); and a serine at the position corresponding to C112 of human SOD1 (a C112 substitution). In certain variations, the fusion polypeptide contains both a C7 substitution and a C112 substitution. In more specific variations, the SOD1 has the amino acid sequence set forth in residues 23-175 of SEQ ID NO: 54. In certain embodiments, L1 is present and comprises at least two amino acid residues (e.g., an L1 linker of 10-36 amino acid residues). In particular variations, L1 comprises two or more tandem repeats of the amino acid sequence of SEQ ID NO: 119; in some such embodiments, L1 has the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12.

[0063] In some embodiments of the above fusion polypeptides comprising a CTLA-4 extracellular domain as the first biologically active polypeptide, the CTLA-4 extracellular domain has at least 90% or at least 95% identity to the amino acid sequence set forth in residues 21-144 of SEQ ID NO: 66. In more particular variations, the CTLA-4 extracellular domain has the amino acid sequence set forth in residues 21-144 of SEQ ID NO: 66.

[0064] In some embodiments of the above-described fusion polypeptides comprising a CD40 extracellular domain as the first biologically active polypeptide, the CD40 extracellular domain has at least 90% or at least 95% identity to the amino acid sequence set forth in (i) residues 21-188 of SEQ ID NO: 74, (ii) residues 21-188 of SEQ ID NO: 78, (iii) residues 21-188 of SEQ ID NO: 82, (iv) residues 21-188 of SEQ ID NO: 86, or (v) residues 21-188 of SEQ ID NO: 90. In some such embodiments, 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: 68) selected from E64, K81, P85, and L121, wherein the at least one amino acid substitution increases CD40 ligand binding compared to human CD40. Particularly suitable amino acid substitutions 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 L121 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 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. In more specific variations, the CD40 extracellular domain has the amino acid sequence set forth in (i) residues 21-188 of SEQ ID NO: 74, (ii) residues 21-188 of SEQ ID NO: 78, (iii) residues 21-188 of SEQ ID NO: 82, (iv) residues 21-188 of SEQ ID NO: 86, or (v) residues 21-188 of SEQ ID NO: 90.

[0065] In certain embodiments of the fusion polypeptide, the first biologically active polypeptide is a polypeptide that specifically binds to and neutralizes TNFα or TGF-β, hi some such embodiments, the first biologically active polypeptide is a single-chain antibody, such as a single-chain Fv (scFv) or a single-domain antibody (sdAb).

[0066] In some embodiments of the above-described fusion polypeptides comprising a single chain antibody that specifically binds and neutralizes TNFα, the single chain antibody comprises a complementarity determining region (CDR) CDR-H1 TNFα , CDR-H2 TNFα and CDR-H3 TNFα and the set of VH CDRs have three or fewer amino acid substitutions compared to the set of reference CDRs CDR-H1, CDR-H2 and CDR-H3 of the VH domain having the amino acid sequence set forth in SEQ ID NO: 108 (e.g., a set of VH CDRs having zero amino acid substitutions compared to CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO: 108, thereby TNFα , CDR-H2 TNFα and CDR-H3 TNFα are CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 108, respectively. Each VH CDR can be defined, for example, according to the Chothia definition, the Kabat definition, the AbM definition, the IMGT database definition, or the contact definition of a CDR. In some variations, each VH CDR is defined according to the IMGT database definition of a CDR, whereby the reference CDRs CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 108 correspond to residues 26-33, 50-59, and 97-110 of SEQ ID NO: 108, respectively; in some such embodiments, CDR-H1 TNFα , CDR-H2 TNFα and CDR-H3 TNFα is the VH CDR of SEQ ID NO: 108 according to the IMGT database definition, thereby CDR-H1 TNFα has the amino acid sequence shown in residues 26 to 33 of SEQ ID NO: 108, and CDR-H2 TNFαhas the amino acid sequence shown in residues 50 to 59 of SEQ ID NO: 108, and CDR-H3 TNFα has the amino acid sequence shown in residues 97 to 110 of SEQ ID NO:108.

[0067] In other, non-mutually exclusive embodiments of the above fusion polypeptides comprising a single chain antibody that specifically binds and neutralizes TNFα, the single chain antibody comprises a complementarity determining region (CDR) CDR-L1 TNFα , CDR-L2 TNFα and CDR-L3 TNFα and a VL domain comprising the set of VL CDRs having three or fewer amino acid substitutions compared to a set of reference CDRs CDR-L1, CDR-L2 and CDR-L3 of a VL domain having the amino acid sequence set forth in SEQ ID NO: 110 (e.g., a set of VL CDRs having zero amino acid substitutions compared to CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 110, thereby TNFα , CDR-L2 TNFα and CDR-L3 TNFα are CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 110, respectively. Each VL CDR can be defined, for example, according to the Chothia definition, the Kabat definition, the AbM definition, the IMGT database definition, or the contact definition of a CDR. In some variations, each VL CDR is defined according to the IMGT database definition of a CDR, with reference CDRs CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 110 corresponding to residues 27-32, 50-52, and 89-97, respectively, of SEQ ID NO: 110; in some such embodiments, CDR-L1 TNFα , CDR-L2 TNFα and CDR-L3 TNFα is the VL CDR of SEQ ID NO: 110 according to the IMGT database definition, thereby CDR-L1 TNFα has the amino acid sequence set forth in residues 27 to 32 of SEQ ID NO: 110; CDR-L2 TNFα has the amino acid sequence set forth in residues 50 to 52 of SEQ ID NO: 110; and CDR-L3 TNFαhas the amino acid sequence shown in residues 89 to 97 of SEQ ID NO:110.

[0068] In some embodiments of the above-described fusion polypeptides comprising a single chain antibody that specifically binds and neutralizes TNFα, the single chain antibody comprises a VH domain having at least 90% or at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 108 and / or the single chain antibody comprises a VL domain having at least 90% or at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 110. In more particular variations, the single chain antibody comprises a VH domain having the amino acid sequence set forth in SEQ ID NO: 108 and / or the single chain antibody comprises a VL domain having the amino acid sequence set forth in SEQ ID NO: 110.

[0069] In some embodiments of the above-described fusion polypeptides comprising a single chain antibody that specifically binds and neutralizes TGF-β, the single chain antibody comprises a complementarity determining region (CDR) CDR-H1 TGF-β , CDR-H2 TGF-β and CDR-H3 TGF-β and the set of VH CDRs have three or fewer amino acid substitutions compared to the set of reference CDRs CDR-H1, CDR-H2 and CDR-H3 of the VH domain having the amino acid sequence set forth in SEQ ID NO: 112 (e.g., a set of VH CDRs having zero amino acid substitutions compared to CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO: 112, thereby TGF-β , CDR-H2 TGF-β and CDR-H3 TGF-βare CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 112, respectively). Each VH CDR can be defined, for example, according to the Chothia definition, the Kabat definition, the AbM definition, the IMGT database definition, or the contact definition of a CDR. In some variations, each VH CDR is defined according to the Kabat definition of a CDR, whereby the reference CDRs CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 112 correspond to residues 31-35, 50-66, and 99-106 of SEQ ID NO: 112, respectively; in some such embodiments, CDR-H1 TGF-β , CDR-H2 TGF-β and CDR-H3 TGF-β is the VH CDR of SEQ ID NO: 112 according to the Kabat definition, thereby CDR-H1 TGF-β has the amino acid sequence set forth in residues 31 to 35 of SEQ ID NO: 112, and CDR-H2 TGF-β has the amino acid sequence shown in residues 50 to 66 of SEQ ID NO: 112, and CDR-H3 TGF-β has the amino acid sequence shown in residues 99-106 of SEQ ID NO:112.

[0070] In other, non-mutually exclusive embodiments of the above fusion polypeptides comprising a single chain antibody that specifically binds and neutralizes TGF-β, the single chain antibody comprises a complementarity determining region (CDR) CDR-L1 TGF-β , CDR-L2 TGF-β and CDR-L3 TGF-β and a VL domain comprising the set of VL CDRs having three or fewer amino acid substitutions compared to a set of reference CDRs CDR-L1, CDR-L2 and CDR-L3 of a VL domain having the amino acid sequence set forth in SEQ ID NO: 114 (e.g., a set of VL CDRs having zero amino acid substitutions compared to CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 114, thereby TGF-β , CDR-L2 TGF-β and CDR-L3 TGF-βare CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 114, respectively). Each VL CDR can be defined, for example, according to the Chothia definition, the Kabat definition, the AbM definition, the IMGT database definition, or the contact definition of a CDR. In some variations, each VL CDR is defined according to the Kabat definition of a CDR, and the reference CDRs CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 114 correspond to residues 24-40, 56-62, and 95-102, respectively, of SEQ ID NO: 114; in some such embodiments, CDR-L1 TGF-β , CDR-L2 TGF-β and CDR-L3 TGF-β is the VL CDR of SEQ ID NO: 114 according to the Kabat definition, whereby CDR-L1 TGF-β has the amino acid sequence set forth in residues 24 to 40 of SEQ ID NO: 114; CDR-L2 TGF-β has the amino acid sequence set forth in residues 56 to 62 of SEQ ID NO: 114; and CDR-L3 TGF-β has the amino acid sequence shown in residues 95-102 of SEQ ID NO:114.

[0071] In some embodiments of the above-described fusion polypeptides comprising a single chain antibody that specifically binds and neutralizes TGF-β, the single chain antibody comprises a VH domain having at least 90% or at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 112 and / or the single chain antibody comprises a VL domain having at least 90% or at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 114. In more specific variations, the single chain antibody comprises a VH domain having the amino acid sequence set forth in SEQ ID NO: 112 and / or the single chain antibody comprises a VL domain having the amino acid sequence set forth in SEQ ID NO: 114.

[0072] In some embodiments of the above-described fusion polypeptides comprising a single chain antibody that specifically binds and neutralizes TNFα or TGF-β, the single chain antibody has at least 90% or at least 95% identity to the amino acid sequence set forth in (i) residues 21-268 of SEQ ID NO:92, (ii) residues 21-268 of SEQ ID NO:96, (iii) residues 21-269 of SEQ ID NO:100, or (iv) residues 21-269 of SEQ ID NO:104. In more particular variations, the single chain antibody has the amino acid sequence set forth in (i) residues 21-268 of SEQ ID NO:92, (ii) residues 21-268 of SEQ ID NO:96, (iii) residues 21-269 of SEQ ID NO:100, or (iv) residues 21-269 of SEQ ID NO:104.

[0073] In another aspect, the invention provides a fusion polypeptide comprising, from amino-terminal to carboxyl-terminal position, X-L2-P, where X is an immunoglobulin heavy chain constant region, which is capable of dimerizing and specifically binding to the neonatal Fc receptor (FcRn); L2 is a polypeptide linker, L2 is optionally present; and P is biologically active paraoxonase, wherein the paraoxonase has at least 80% or at least 90% identity to the amino acid sequence set forth in residues 16-355 or 26-355 of SEQ ID NO:6, and does not contain an amino-terminal leader sequence corresponding to residues 1-15 of SEQ ID NO:6, and the fusion polypeptide does not contain a biologically active polypeptide N-terminal to the immunoglobulin heavy chain constant region. In some embodiments, the biologically active paraoxonase has at least 95% identity to the amino acid sequence set forth in (i) residues 16-355 or 26-355 of SEQ ID NO:6, (ii) residues 16-355 or 26-355 of SEQ ID NO:124, or (iii) residues 16-355 or 26-355 of SEQ ID NO:126.

[0074] In some embodiments of the above-described fusion polypeptides comprising the formula T-L1-X-L2-P or X-L2-P, the amino acid at the position corresponding to Q192 of the human paraoxonase 1 Q192 isoform (hPON1-Q192; SEQ ID NO: 4) is lysine or arginine. In other, non-mutually exclusive variations, the amino acid at the position corresponding to H115 of hPON1-Q192 is tryptophan. In particular variations, the paraoxonase has an amino acid sequence selected from (i) residues n through 355 of SEQ ID NO: 6, (ii) residues n through 355 of SEQ ID NO: 124, and (iii) residues n through 355 of SEQ ID NO: 126, where n is an integer between 16 and 26, inclusive.

[0075] In certain embodiments of the above-described fusion polypeptides comprising the formula T-L1-X-L2-P or X-L2-P, L2 is present and comprises at least 8 amino acid residues. In some such embodiments, L2 consists of 12 to 25 amino acid residues. A particularly suitable L2 linker has the amino acid sequence set forth in SEQ ID NO:56.

[0076] In some embodiments of the above-described fusion polypeptides having the formula T-L1-X-L2-P or X-L2-P, where X is an immunoglobulin heavy chain constant region, the immunoglobulin heavy chain constant region is an immunoglobulin Fc region. In some such embodiments, the Fc region is a human Fc region, such as a human Fc variant containing one or more (e.g., 1 to 10) amino acid substitutions compared to the wild-type human sequence. Particularly suitable Fc regions include human γ1 and γ4 Fc regions. In some variations, the Fc region is a human γ1 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 containing the above Fc regions, the Fc region is a human γ1 Fc variant in which Eu residue P331 is replaced by serine. In some embodiments, the Fc region has at least 90% or at least 95% identity to the amino acid sequence set forth in (i) residues 1-232 or 1-231 of SEQ ID NO:26, (ii) residues 1-232 or 1-231 of SEQ ID NO:28, (iii) residues 159-390 or 159-389 of SEQ ID NO:42, (iv) residues 1-232 or 1-231 of SEQ ID NO:116, or (v) residues 1-232 or 1-231 of SEQ ID NO:118. In more specific variations, the Fc region has the amino acid sequence set forth in (i) residues 1-232 or 1-231 of SEQ ID NO:26, (ii) residues 1-232 or 1-231 of SEQ ID NO:28, (iii) residues 159-390 or 159-389 of SEQ ID NO:42, (iv) residues 1-232 or 1-231 of SEQ ID NO:116, or (v) residues 1-232 or 1-231 of SEQ ID NO:118.

[0077] In some embodiments of the above-described fusion polypeptides having the formula T-L1-X-L2-P or X-L2-P, where X is 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 to the amino acid sequence set forth in (i) residues 16-232 or 16-231 of SEQ ID NO:26, (ii) residues 16-232 or 16-231 of SEQ ID NO:116, or (iii) residues 16-232 or 16-231 of SEQ ID NO:118. In more particular variations, the immunoglobulin heavy chain constant region comprises the amino acid sequence set forth in (i) residues 16-232 or 16-231 of SEQ ID NO:26, (ii) residues 16-232 or 16-231 of SEQ ID NO:116, or (iii) residues 16-232 or 16-231 of SEQ ID NO:118.

[0078] In some embodiments of the above-described fusion polypeptides having the formula T-L1-X-L2-P, wherein the first biologically active polypeptide is a DNase, the fusion polypeptide comprises an amino acid sequence set forth in (i) residues 21-896 of SEQ ID NO:2, (ii) residues 21-896 of SEQ ID NO:128, (iii) residues 21-896 of SEQ ID NO:130, (iv) residues 21-906 of SEQ ID NO:148, (v) residues 21-896 of SEQ ID NO:158, (vi) residues 21-906 of SEQ ID NO:160, (vii) residues 21-906 of SEQ ID NO:162, or (viii) residues 21-906 of SEQ ID NO:164. In some such embodiments, the fusion polypeptide comprises an amino acid sequence set forth in (i) residues 21-896 of SEQ ID NO:2, (ii) residues 21-896 of SEQ ID NO:128, (iii) residues 21-896 of SEQ ID NO:130, (iv) residues 21-906 of SEQ ID NO:148, (v) residues 21-896 of SEQ ID NO:158, (vi) residues 21-906 of SEQ ID NO:160, (vii) residues 21-906 of SEQ ID NO:162, or (viii) residues 21-906 of SEQ ID NO:164.

[0079] In some embodiments of the above-described fusion polypeptides having the formula T-L1-X-L2-P, wherein the first biologically active polypeptide is a DNase, the polypeptide region corresponding to T-L1-X (the "T-L1-X region") comprises the amino acid sequence set forth in SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:153, or SEQ ID NO:154. In certain variations of a T-L1-X region comprising the amino acid sequence set forth in SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:153, or SEQ ID NO:154, (i) the amino acid at each of positions 315 and 316 is alanine; (ii) the amino acid at position 319 is serine; (iii) the amino acid at position 378 is alanine, glutamine, or glycine, and the amino acid at position 346 is optionally alanine; (iv) the amino acid at position 410 is alanine, glycine, or serine; (v) the amino acid at position 412 is serine or alanine; (vi) the amino acid at position 333 is tyrosine, the amino acid at position 335 is threonine, and the amino acid at position 337 is glutamic acid; and / or (vii) the amino acid at position 509 is leucine and the amino acid at position 515 is serine. In other, non-mutually exclusive variations of the T-L1-X region comprising the amino acid sequence set forth in SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 153, or SEQ ID NO: 154, the amino acid sequence corresponding to positions 261 to 296 is (i) [Gly-Gly-Gly-Gly-Ser] n wherein n is an integer from 4 to 7; (ii) [Gly-Gly-Gly-Ser] n wherein n is an integer from 5 to 9, and (iii) [Gly-Gly-Ser] n wherein n is an integer between 6 and 12; in some such embodiments, the amino acid sequence corresponding to positions 261 to 296 is selected from the group consisting of: (i) [Gly-Gly-Gly-Gly-Ser] n (ii) [Gly-Gly-Gly-Ser], wherein n is an integer of 4 to 6. nwherein n is an integer from 5 to 7, and (iii) [Gly-Gly-Ser] n wherein n is an integer between 6 and 10. In still other, non-mutually exclusive variations of a T-L1-X region comprising the amino acid sequence set forth in SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 153, or SEQ ID NO: 154, the amino acid at position 261 is aspartic acid, the amino acid at position 262 is leucine, the amino acid at position 263 is serine, the amino acid at position 294 is threonine, the amino acid at position 295 is glycine, and / or the amino acid at position 296 is leucine. In other, non-mutually exclusive variations of a T-L1-X region comprising the amino acid sequence set forth in SEQ ID NO: 149 or SEQ ID NO: 153, the amino acids at positions 74 and 105 are each independently lysine or arginine. In other non-mutually exclusive variations of the T-L1-X region comprising the amino acid sequence set forth in SEQ ID NO: 149, the amino acid at position 114 is phenylalanine.

[0080] In some embodiments of the above-described fusion polypeptide having the formula T-L1-X-L2-P, wherein the first biologically active polypeptide is an RNase, the fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identity to the amino acid sequence set forth in (i) residues 21-764 of SEQ ID NO:40, or (ii) residues 21-740 of SEQ ID NO:48; in some such embodiments, the fusion polypeptide comprises the amino acid sequence set forth in (i) residues 21-764 of SEQ ID NO:40, or (ii) residues 21-740 of SEQ ID NO:48.

[0081] In some embodiments of the above-described fusion polypeptide having the formula T-L1-X-L2-P, and wherein the first biologically active polypeptide is SOD1, the fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identity to the amino acid sequence set forth in (i) residues 23-781 of SEQ ID NO:50, (ii) residues 23-781 of SEQ ID NO:52, or (iii) residues 23-791 of SEQ ID NO:54; in some such embodiments, the fusion polypeptide comprises the amino acid sequence set forth in (i) residues 23-781 of SEQ ID NO:50, (ii) residues 23-781 of SEQ ID NO:52, or (iii) residues 23-791 of SEQ ID NO:54.

[0082] In some embodiments of the above fusion polypeptides having the formula T-L1-X-L2-P, and wherein the first biologically active polypeptide is a CTLA-4 extracellular domain, the fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identity to the amino acid sequence set forth in residues 21-736 of SEQ ID NO:66; in some such embodiments, the fusion polypeptide comprises the amino acid sequence set forth in residues 21-736 of SEQ ID NO:66.

[0083] In some embodiments of the above-described fusion polypeptide having the formula T-L1-X-L2-P, wherein the first biologically active polypeptide is a CD40 extracellular domain, the fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identity to the amino acid sequence set forth in (i) residues 21-804 of SEQ ID NO:74, (ii) residues 21-804 of SEQ ID NO:78, (iii) residues 21-804 of SEQ ID NO:82, (iv) residues 21-804 of SEQ ID NO:86, or (v) residues 21-804 of SEQ ID NO:90; in some such embodiments, the fusion polypeptide comprises the amino acid sequence set forth in (i) residues 21-804 of SEQ ID NO:74, (ii) residues 21-804 of SEQ ID NO:78, (iii) residues 21-804 of SEQ ID NO:82, (iv) residues 21-804 of SEQ ID NO:86, or (v) residues 21-804 of SEQ ID NO:90.

[0084] In some embodiments of the above-described fusion polypeptide having the formula T-L1-X-L2-P, wherein the first biologically active polypeptide is a single-chain antibody that specifically binds to and neutralizes TNFα, the fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identity to (i) residues 21-860 of SEQ ID NO:94, or (ii) residues 21-860 of SEQ ID NO:98; in some such embodiments, the fusion polypeptide comprises the amino acid sequence set forth in (i) residues 21-860 of SEQ ID NO:94, or (ii) residues 21-860 of SEQ ID NO:98.

[0085] In some embodiments of the above-described fusion polypeptide having the formula T-L1-X-L2-P, wherein the first biologically active polypeptide is a single-chain antibody that specifically binds to and neutralizes TGF-β, the fusion polypeptide comprises an amino acid sequence having at least 90% or at least 95% identity to the amino acid sequence set forth in (i) residues 21-861 of SEQ ID NO:102, or (ii) residues 21-861 of SEQ ID NO:106; in some such embodiments, the fusion polypeptide comprises the amino acid sequence set forth in (i) residues 21-861 of SEQ ID NO:102, or (ii) residues 21-861 of SEQ ID NO:106.

[0086] In some embodiments of the above-described fusion polypeptide having the formula X-L2-P, the fusion polypeptide has at least 90% or more identical amino acid sequence to that set forth in (i) residues 21-613 of SEQ ID NO: 122, (ii) residues 21-613 of SEQ ID NO: 132, (iii) residues 21-613 of SEQ ID NO: 134, (iv) residues 21-610 of SEQ ID NO: 142, (v) residues 21-610 of SEQ ID NO: 144, or (vi) residues 21-610 of SEQ ID NO: 146. comprises an amino acid sequence having at least 95% identity; in some such embodiments, the fusion polypeptide comprises the amino acid sequence set forth in (i) residues 21-613 of SEQ ID NO:122, (ii) residues 21-613 of SEQ ID NO:132, (iii) residues 21-613 of SEQ ID NO:134, (iv) residues 21-610 of SEQ ID NO:142, (v) residues 21-610 of SEQ ID NO:144, or (vi) residues 21-610 of SEQ ID NO:146.

[0087] In another aspect, the 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 either (i) a fusion polypeptide having the formula T-L1-X-L2-P, where X is a dimerization domain (e.g., an immunoglobulin heavy chain constant region, such as an immunoglobulin Fc region), or (ii) a fusion polypeptide having the formula X-L2-P.

[0088] In another aspect, the present invention provides a polynucleotide encoding the above-described fusion polypeptide.

[0089] In yet another aspect, the present invention provides an expression cassette comprising a DNA segment encoding the fusion polypeptide operably linked to a promoter. In certain variations, the encoded fusion polypeptide has the formula T-L1-X-L2-P, and the first biologically active polypeptide (i) has at least 90% or at least 95% identity to amino acid residues 21-280 of SEQ ID NO: 18 or residues 21-280 of SEQ ID NO: 152, and (ii) is a DNase containing at least one of the N74, G105, and A114 substitutions (e.g., a DNase containing two or all three substitutions). Also provided are cultured cells into which the expression cassette has been introduced, wherein the cultured cells express the DNA segment. In a related aspect, the present invention provides a stable cell line comprising the expression cassette within its genomic DNA, 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.

[0090] In another aspect, the present invention provides a vector comprising the above-described expression cassette.

[0091] In another aspect, the present invention provides a method for producing a fusion polypeptide. The method generally includes (i) culturing cells into which the expression cassette has been introduced, so that the cells express the DNA segment and produce the encoded fusion polypeptide, and (ii) recovering the fusion polypeptide. In some variations, the cultured cells are the stable cell line described above.

[0092] In yet another aspect, the present invention provides a method for producing a dimeric protein. The method generally includes (i) culturing cells into which the expression cassette has been introduced, such that the cells express 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 cells are the stable cell line described above.

[0093] In another aspect, the present invention provides a composition comprising the fusion polypeptide described above and a pharmaceutically acceptable carrier.

[0094] In another aspect, the present invention provides a composition comprising the dimeric protein and a pharmaceutically acceptable carrier.

[0095] In some embodiments of the above compositions, the composition is formulated for pulmonary delivery by nebulization.

[0096] In yet another aspect, the present invention provides a method for treating an inflammatory disease. The method generally comprises administering an effective amount of the fusion polypeptide or dimeric protein to a subject with the inflammatory disease. In some embodiments, the inflammatory disease is an inflammatory lung disease, such as 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.g., idiopathic pulmonary fibrosis (IPF) or sarcoidosis); in some variations, the inflammatory lung disease is characterized by Pseudomonas aeruginosa infection. In other embodiments, the inflammatory disease is selected from 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 inflammatory disease is an inflammatory skin disease, such as, for example, psoriasis or atopic dermatitis.

[0097] In another aspect, the present invention provides a method for treating an autoimmune disease. The method generally comprises administering an effective amount of the fusion polypeptide or dimeric protein to a subject with an autoimmune disease. 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, type 1 diabetes, vasculitis, and systemic sclerosis.

[0098] In another aspect, the present invention provides a method for treating biofilm formation by Gram-negative bacteria. The method generally comprises administering an effective amount of the fusion polypeptide or dimeric protein to a subject suffering from biofilm formation. In some embodiments, the Gram-negative bacteria is Pseudomonas aeruginosa.

[0099] In another aspect, the present invention provides methods for treating exposure to sulfur mustard gas or an organophosphate. The methods generally comprise administering an effective amount of the fusion polypeptide or dimeric protein described above to a subject exposed to sulfur mustard gas or an organophosphate. 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.

[0100] In yet another aspect, the present invention provides a method for treating a neurological disease. The method generally comprises administering an effective amount of the fusion polypeptide or dimeric protein to a subject with a neurological disease. 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 Alzheimer's disease.

[0101] In another aspect, the present invention provides a method for treating cardiovascular disease. This method generally comprises administering an effective amount of the fusion polypeptide or dimeric protein to a subject with cardiovascular disease. In some embodiments, the cardiovascular disease is a disease characterized by atherosclerosis, such as coronary heart disease or ischemic stroke. In some variations, the coronary heart disease is characterized by acute coronary syndrome.

[0102] In another aspect, the present invention provides a method for treating chronic liver disease.This method generally comprises administering an effective amount of the above-mentioned fusion polypeptide or dimeric protein to a subject with chronic liver disease.In some embodiments, chronic liver disease is selected from non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease (ALD), portal hypertension or complications after liver transplantation.In some variations, non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH).

[0103] In another aspect, the present invention provides a method for treating a fibrotic disease. The method generally comprises administering an effective amount of the fusion polypeptide or dimeric protein to a subject with a fibrotic disease. In some embodiments, the fibrotic disease is selected from the group consisting of systemic sclerosis, systemic lupus erythematosus (SLE), inflammatory lung disease, chronic liver disease, and chronic kidney disease (e.g., lupus nephritis, IgA nephropathy, or membranous glomerulonephritis).

[0104] In yet another aspect, the present invention provides a method for treating a disease or disorder characterized by NETosis. In some embodiments, the method generally comprises administering to a subject with a disease or disorder characterized by NETosis an effective amount of a fusion polypeptide having the above formula T-L1-X-L2-P, or a dimeric protein formed by dimerization of the fusion polypeptide, wherein the fusion polypeptide or dimeric protein comprises DNase as a first biologically active polypeptide.

[0105] In other embodiments of the methods for treating a disease or disorder characterized by NETosis, the methods are combination therapy methods generally comprising administering to a subject having a disease or disorder characterized by NETosis (a) an effective amount of a fusion polypeptide having the formula X-L2-P, or a dimeric protein formed by dimerization of the fusion polypeptide, and (b) an effective amount of a biologically active DNase. In certain embodiments of the combination therapy methods, the DNase has at least 90% or at least 95% identity to the amino acid sequence set forth in (i) residues 21-280 of SEQ ID NO:18, (ii) residues 21-280 of SEQ ID NO:152, or (iii) residues 21-290 of SEQ ID NO:136. In some embodiments, wherein the DNase has at least 90% or at least 95% identity to the amino acid sequence set forth in residues 21-280 of SEQ ID NO: 18 or residues 21-280 of SEQ ID NO: 152, the DNase contains at least one amino acid substitution at a position corresponding to an amino acid in human wild-type DNase 1 (SEQ ID NO: 120) selected from N74, G105, and A114, wherein (1) the amino acid substitution at a position corresponding to N74 in human DNase 1 (the N74 substitution), when present, increases DNA binding compared to human DNase 1; (2) the amino acid substitution at a position corresponding to G105 in human DNase 1 (the G105 substitution), when present, increases DNA binding compared to human DNase 1; and (3) the amino acid substitution at a position corresponding to A114 in human DNase 1 (the A114 substitution), when present, decreases G-actin-induced inhibition of endonuclease activity compared to human DNase 1. In some variations, the DNase contains both an N74 substitution and a G105 substitution, or each of an N74 substitution, a G105 substitution, and an A114 substitution. Particularly suitable amino acid substitutions at these positions are a lysine at the position corresponding to N74 in human DNase 1, an arginine at the position corresponding to G105 in human DNase 1, and / or a phenylalanine at the position corresponding to A114 in human DNase 1.In some embodiments in which the DNase has at least 90% or at least 95% identity to the amino acid sequence set forth in residues 21-290 of SEQ ID NO: 136, each of the amino acids at positions corresponding to R80, R95, and N96 of SEQ ID NO: 136 is alanine or serine. In more particular variations, the DNase has the amino acid sequence set forth in (i) residues 21-280 of SEQ ID NO: 18, (ii) residues 21-280 of SEQ ID NO: 20, (iii) residues 21-280 of SEQ ID NO: 152, (iv) residues 21-290 of SEQ ID NO: 138, or (v) residues 21-290 of SEQ ID NO: 140.

[0106] In certain embodiments of the methods of combination therapy for treating a disease or disorder characterized by NETosis, the DNase is D-L1-X from the amino-terminal position to the carboxyl-terminal position. d wherein D is a DNase, L1 is a polypeptide linker (e.g., a linker comprising at least 15 amino acid residues or at least 26 amino acid residues), and X dis an immunoglobulin Fc region. The immunoglobulin Fc region can be an Fc region as described above for a fusion polypeptide having the formula T-L1-X-L2-P or X-L2-P. In some variations, the DNase fusion polypeptide has at least 90% or at least 95% identity to the amino acid sequence set forth in (i) residues 21-538 or 21-537 of SEQ ID NO:60, (ii) residues 21-548 or 21-547 of SEQ ID NO:62, (iii) residues 21-538 or 21-537 of SEQ ID NO:155, (iv) residues 21-548 or 21-547 of SEQ ID NO:156, (v) residues 21-548 or 21-547 of SEQ ID NO:138, or (vi) residues 21-548 or 21-547 of SEQ ID NO:140. in some such embodiments, the DNase fusion polypeptide comprises the amino acid sequence set forth in (i) residues 21-538 or 21-537 of SEQ ID NO:60, (ii) residues 21-548 or 21-547 of SEQ ID NO:62, (iii) residues 21-538 or 21-537 of SEQ ID NO:155, (iv) residues 21-548 or 21-547 of SEQ ID NO:156, (v) residues 21-548 or 21-547 of SEQ ID NO:138, or (vi) residues 21-548 or 21-547 of SEQ ID NO:140. In certain variations, the DNase comprises the amino acid sequence set forth in SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:153, or SEQ ID NO:154.

[0107] In some embodiments of the methods for treating a disease or disorder characterized by NETosis, the disease or disorder is an inflammatory lung disease, such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis (CF), bronchiectasis, hypoxia, acute respiratory distress syndrome (ARDS) (e.g., COVID-19-associated ARDS), or interstitial lung disease (e.g., idiopathic pulmonary fibrosis (IPF) or sarcoidosis). In other embodiments, the disease or disorder is an inflammatory skin disease, such as psoriasis or atopic dermatitis. In other embodiments, the disease or disorder is an autoimmune disease, such as systemic lupus erythematosus (SLE) (e.g., SLE with lupus nephritis), rheumatoid arthritis (RA), psoriasis, antiphospholipid syndrome, type 1 diabetes mellitus, vasculitis, or systemic sclerosis. In other embodiments, the disease or disorder is an autoinflammatory disease, such as inflammatory bowel disease (IBD) (e.g., Crohn's disease or ulcerative colitis) or gout. In other embodiments, the disease or disorder is a neurological disease or disorder, such as a chronic neurodegenerative disease (e.g., Alzheimer's disease or multiple sclerosis), a central nervous system infection (e.g., meningitis, encephalitis, or cerebral malaria), or ischemic stroke. In other embodiments, the disease or disorder is a metabolic disease, such as type 2 diabetes or obesity. In other embodiments, the disease or disorder is a cardiovascular disease, such as a cardiovascular disease characterized by atherosclerosis (e.g., coronary heart disease or ischemic stroke). In yet other embodiments, the disease or disorder is selected from thrombosis, sepsis, and ischemia-reperfusion. In other embodiments, the disease or disorder is a chronic liver disease, such as nonalcoholic steatohepatitis (NASH). In still other embodiments, the disease or disorder is a fibrotic disease, such as systemic sclerosis, systemic lupus erythematosus (SLE), an inflammatory lung disease, a chronic liver disease, or a chronic kidney disease (e.g., lupus nephritis, IgA nephropathy, or membranous glomerulonephritis).

[0108] In yet other embodiments of the method for treating a disease or disorder characterized by NETosis, the disease or disorder is cancer. In certain variations, the cancer treatment is a combination therapy. In some combination therapy embodiments, the combination therapy includes immunomodulatory therapy, including anti-PD-1 / PD-L1 therapy, anti-CTLA-4 therapy, CAR T cell therapy, or a combination thereof (e.g., both anti-PD-1 / PD-L1 therapy and anti-CTLA-4 therapy). In other combination therapy embodiments, the combination therapy includes radiation therapy or chemotherapy. In some combination therapy embodiments, the combination therapy includes a targeted therapy; in some such embodiments, the targeted therapy includes (i) a therapeutic monoclonal antibody that targets a specific cell surface or extracellular antigen (e.g., VEGF, EGFR, CTLA-4, PD-1, or PD-L1), or (ii) a small molecule that targets an intracellular protein, such as an intracellular enzyme (e.g., a proteasome, a tyrosine kinase, a cyclin-dependent kinase, a serine / threonine-protein kinase B-Raf (BRAF), or a MEK kinase).

[0109] In another aspect, the present invention provides a method for reducing lipid oxidation in a subject. The method generally comprises administering to the subject an effective amount of the fusion polypeptide or dimeric protein described above. In some embodiments, the method further reduces NETosis in the subject by administering to the subject (a) an effective amount of a fusion polypeptide having the formula X-L2-P described above, or a dimeric protein formed by dimerization of the fusion polypeptide, and (b) a biologically active DNase (e.g., a fusion polypeptide having the formula D-L1-X described above). d The combination therapy generally involves administering an effective amount of a DNase fusion polypeptide having the formula:

[0110] In another aspect, the present invention provides methods for protecting a subject from aging. The methods generally comprise administering to the subject an effective amount of the fusion polypeptide or dimeric protein described above. In some embodiments, the methods comprise administering to the subject (a) an effective amount of a fusion polypeptide having the formula X-L2-P described above, or a dimeric protein formed by dimerization of the fusion polypeptide, and (b) a biologically active DNase (e.g., a fusion polypeptide having the formula D-L1-X described above). d The combination therapy generally involves administering an effective amount of a DNase fusion polypeptide having the formula:

[0111] These and other aspects of the present invention will become evident upon reference to the following detailed description of the invention. definition

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions are described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0113] The terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0114] A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of fewer than about 50 amino acid residues may also be referred to as "peptides."

[0115] A "protein" is a macromolecule containing one or more polypeptide chains. Proteins may also contain non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents may be added to proteins by the cells in which they are produced and vary depending on the type of cell. Proteins are defined herein in terms of their amino acid backbone structure; substituents, such as carbohydrate groups, are generally not specified, but may be present nonetheless.

[0116] The terms "amino-terminus" (or "N-terminus") and "carboxyl-terminus" (or "C-terminus") are used herein to indicate positions within a polypeptide. Where the context allows, these terms are used with respect to a particular sequence or portion of a polypeptide to indicate proximity or relative position. For example, a particular sequence located 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.

[0117] The terms "polynucleotide" and "nucleic acid" are used synonymously herein and refer to single- or double-stranded polymers of deoxyribonucleotide or ribonucleotide bases read from the 5' end to the 3' end. Polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The size of a polynucleotide is expressed in base pairs (abbreviated as "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context permits, the latter two terms can describe polynucleotides that are single- or double-stranded. Those skilled in the art will recognize that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends may be staggered as a result of enzymatic cleavage; therefore, not all nucleotides within a double-stranded polynucleotide molecule are paired. Such unpaired ends generally do not exceed 20 nt in length.

[0118] A "segment" is a portion of a larger molecule (e.g., a polynucleotide or polypeptide) having a specified property. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, e.g., a plasmid or plasmid fragment, that, when read in the 5' to 3' direction, encodes the amino acid sequence of the specified polypeptide. Also, for example, with respect to the fusion polypeptides described herein, different components of the fusion polypeptide (e.g., DNase, RNase, superoxide dismutase (SOD), linker (s), immunoglobulin Fc region, paraoxonase) can each be referred to as a polypeptide segment.

[0119] The term "biologically active," when used with reference to a polypeptide segment of a fusion molecule described herein, refers to 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 signal transduction pathway in a eukaryotic cell, induction of cell proliferation, induction of cell differentiation, and the like. When used specifically with reference to a polypeptide segment that is a DNase, RNase, paraoxonase (PON), or superoxide dismutase (SOD), "biologically active" means that the polypeptide exhibits the same type of enzymatic activity as the corresponding naturally occurring enzyme (e.g., the same type of enzymatic activity as full-length wild-type human DNase1 / DNase1L3, RNase1, PON1, or SOD1, respectively), allowing for differences in the degree of activity, enzyme kinetics, and the like. An immunoglobulin Fc region, as referred to herein, is understood to be "biologically active" at least due to its dimerization activity and FcRn-binding activity.

[0120] Unless the context clearly indicates otherwise, references herein to "paraoxonase" (e.g., "paraoxonase 1" or "PON1"), "DNase" (e.g., "DNase 1" or "DNase 1L3"), "RNase" (e.g., "RNase 1"), "superoxide dismutase" (e.g., "superoxide dismutase 1" or "SOD1"), "CTLA-4 extracellular domain" and "CD40 extracellular domain" are understood to include any of the above naturally occurring polypeptides, as well as functional variants and functional fragments thereof.

[0121] The term "allelic variant" is used herein to refer to any of two or more alternative forms of a gene that occupies the same chromosomal locus. Allelic variants arise naturally through mutation and can result in phenotypic polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. The term allelic variant is also used herein to refer to proteins encoded by allelic variants of a gene.

[0122] The term "enhanced DNase 1," as used herein, refers to a hyperactive and / or actin-resistant variant of naturally occurring DNase 1 (e.g., human DNase 1), which is a variant having (a) at least one amino acid substitution that reduces G-actin-induced inhibition of endonuclease activity compared to naturally occurring DNase 1, and / or (b) at least one amino acid substitution that increases DNA binding compared to naturally occurring DNase 1. In some embodiments, the enhanced DNase 1 contains at least one amino acid substitution that reduces G-actin-induced inhibition of endonuclease activity. In some embodiments, the enhanced DNase 1 contains both at least one amino acid substitution that reduces G-actin-induced inhibition of endonuclease activity and at least one amino acid substitution that increases DNA binding. In some preferred embodiments, the enhanced DNase 1 is a hyperactive variant of wild-type human DNase 1 (the mature amino acid sequence set forth in SEQ ID NO: 120).

[0123] The term "linker" or "polypeptide linker" is used herein to refer to two or more amino acids connected by a peptide bond(s) and linking two separate, distinct polypeptide regions. The linker is typically designed to allow the separate polypeptide regions (e.g., a DNase or paraoxonase 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") in conjunction with "L" is used herein to distinguish between linkers connecting different fusion components: "L1" refers to a linker connecting the C-terminus of a first biologically active polypeptide that is not paraoxonase (e.g., DNase, RNase, or SOD1) to the N-terminus of another polypeptide segment, such as an immunoglobulin Fc region, and "L2" refers to a linker connecting the N-terminus of a biologically active paraoxonase (e.g., PON1) to the C-terminus of another polypeptide segment, such as an immunoglobulin Fc region. For polypeptide chains containing both an L1 linker and an L2 linker, these linkers may be the same or different in terms of amino acid sequence. In some variations in which the carboxyl terminus of a first biologically active polypeptide is linked directly (i.e., without an intervening biologically active polypeptide) to the amino terminus of a biologically active paraoxonase via a single polypeptide linker, such a polypeptide linker may be referred to as either L1 or L2.

[0124] The term "expression cassette" is used to denote a DNA construct containing 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, enhancers, polyadenylation signals, etc.

[0125] The term "vector" is used to refer to the polynucleotide produced by recombinant DNA technology, which is used to deliver genetic material into cells, where the vector can replicate.As is well known in the art, vector can refer to, for example, plasmid, cosmid, virus vector, artificial chromosome, cloning vector or expression vector.The term "expression vector" is used to refer to the vector that contains an expression cassette.

[0126] The term "promoter" is used herein in its art-recognized sense to refer to 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.

[0127] 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 the secretory pathway of the cell in which it is synthesized. The larger polypeptide is typically cleaved to remove the secretory peptide during transit through the secretory pathway.

[0128] "Operably linked" means that two or more entities are joined together so that they function cooperatively for their intended purpose. When referring to DNA segments, the phrase indicates, for example, that coding sequences are joined in the correct reading frame, so that transcription initiates at 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 bonds) and non-covalently (e.g., by hydrogen bonds, hydrophobic interactions, or salt bridge interactions) linked sequences, provided that the desired function(s) of the sequences are maintained.

[0129] The term "recombinant," when used with reference to, for example, 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, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all. As used herein, the term "recombinant nucleic acid" refers to a nucleic acid in a form not normally found in nature, generally originally formed in vitro by the manipulation of nucleic acid using, for example, polymerases and endonucleases. In this manner, operative linkage of different sequences is achieved. Thus, both an isolated nucleic acid in linear form or an expression vector formed in vitro by ligating DNA molecules that are not normally connected are considered recombinant for purposes disclosed herein. It is understood that when a recombinant nucleic acid is made and reintroduced into a host cell or organism, the recombinant nucleic acid replicates non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than through in vitro manipulation; however, such a nucleic acid, once produced recombinantly, may subsequently be replicated non-recombinantly but is still considered recombinant for the purposes disclosed herein. Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e., through expression of a recombinant nucleic acid as indicated above.

[0130] The term "heterologous," when used in reference to a portion of a nucleic acid, indicates that the nucleic acid contains two or more subsequences that are not normally found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, e.g., having two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, when used in reference to a portion of a protein, the term "heterologous" indicates that the protein contains two or more subsequences that are not normally found in the same relationship to each other in nature (e.g., two or more segments of a fusion polypeptide).

[0131] 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, consisting of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions together are responsible for antigen binding, and the constant regions are responsible for antibody effector functions. Immunoglobulins typically function as antibodies in vertebrate organisms. Five classes of immunoglobulin proteins (IgG, IgA, IgM, IgD, and IgE) have been identified in higher vertebrates. IgG constitutes the major class; it is usually present as the second most abundant protein found in plasma. In humans, IgG consists of four subclasses, designated IgG1, IgG2, IgG3, and IgG4. The heavy chain constant region of the IgG class is identified by the Greek symbol γ. For example, immunoglobulins of the IgG1 subclass contain a γ1 heavy chain constant region. Each immunoglobulin heavy chain possesses a constant region consisting of constant region protein domains (CH1, hinge, CH2, and CH3; IgG3 also contains a CH4 domain) that are essentially invariant for a given subclass within a species.DNA sequences encoding human and non-human immunoglobulin chains are known in the art (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., Nature 314:330-334, 1985). 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 number J00228). For reviews 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 in its general sense to refer to an intact antibody, its component chains, or fragments of the chains, depending on the context.

[0132] The term "antibody," as used herein, refers to an immunoglobulin molecule, or a fragment and / or engineered variant thereof, capable of specifically binding to an antigen. The term "antibody" includes intact monoclonal antibodies and antigen-binding antibody fragments, such as F(ab')2 and Fab fragments. Also included are genetically engineered intact antibodies and fragments, such as chimeric antibodies, humanized antibodies, single-chain Fv fragments, single-chain antibodies, diabodies, minibodies, and the like. Thus, the term "antibody" is used expansively to include any protein that contains an antigen-binding site and is capable of binding to that antigen.

[0133] The term "genetically engineered antibody" refers to an antibody whose amino acid sequence has been altered from that of a native (i.e., naturally occurring) antibody. Due to the applicability of recombinant DNA technology in generating antibodies, there is no need to be limited to the amino acid sequence found in natural antibodies; antibodies can be redesigned to obtain desired characteristics. The possible variations are many, ranging from, for example, changing just one or a few amino acids in the variable or constant region to complete redesign. Generally, the constant region will be altered to improve or alter characteristics, such as complement fixation, interaction with cells, and other effector functions. Typically, the variable region will be altered to improve antigen binding characteristics, improve variable region stability, or reduce the risk of immunogenicity.

[0134] An "antigen-binding site" is a portion of an antibody sufficient to bind to its antigen. A minimal such region is typically an immunoglobulin variable domain or fragment thereof, or a genetically engineered variant of an immunoglobulin variable domain or fragment thereof. Single-domain binding sites can be generated from camelid antibodies (see Muyldermans and Lauwereys, J. Mol. Recog. 12:131-140, 1999; Nguyen et al., EMBO J. 19:921-930, 2000) or from VH domains of other species to produce single-domain antibodies ("dAbs"; Ward et al., Nature 341:544-546, 1989; U.S. Patent No. 6,248,516 to Winter et al.). In certain variations, the antigen-binding site is a polypeptide region having only two complementarity-determining regions (CDRs) of a naturally occurring or non-naturally occurring (e.g., mutagenized) heavy chain variable domain or light chain variable domain, or a combination thereof (see, e.g., Pessi et al., Nature 362:367-369, 1993; Qiu et al., Nature Biotechnol. 25:921-929, 2007).

[0135] Full-length immunoglobulin "light chains" (about 25 Kd or 214 amino acids) are encoded at the N-terminus by a variable region gene (encoding about 110 amino acids) and at the C-terminus by a kappa or lambda constant region gene. Full-length immunoglobulin "heavy chains" (about 50 Kd or 446 amino acids) are encoded by a variable region gene (encoding about 116 amino acids) and a gamma, mu, alpha, delta, or epsilon constant region gene (encoding about 330 amino acids), the latter defining the antibody's isotype as IgG, IgM, IgA, IgD, or IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids (see generally, Fundamental Immunology (Paul, ed., Raven Press, NY, 2nd ed. 1989), Ch. 7).

[0136] An immunoglobulin light or heavy chain variable region consists of a framework region interrupted by three hypervariable regions. The term "hypervariable region," also referred to herein as "complementarity-determining region" ("CDR"), refers to the amino acid residues of an antibody responsible for antigen binding. CDRs can be defined according to any of several known analytical methods. Examples of such methods include, for example, the Kabat definition, the Chothia definition, the AbM definition, the IMGT database definition, and the contact definition. The Kabat definition is a standard for numbering residues in an antibody and is typically used to identify CDR regions (see, e.g., Johnson & Wu, Nucleic Acids Res. 28:214-8, 2000). The Chothia definition is similar to the Kabat definition, but it takes into account the location of certain structural loop regions (see, e.g., Chothia et al., J. Mol. Biol. 196:901-17, 1986; Chothia et al., Nature 342:877-83, 1989). The AbM definition uses an integrated suite of computer programs written by the Oxford Molecular Group to model antibody structure (see, e.g., Martin et al., 1989, Proc. Natl. Acad. Sci. USA 86:9268-9272, 1989; "AbM TM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd.). The AbM definition uses a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl. 3:194-198, 1999, to model the tertiary structure of antibodies from primary sequence. The IMGT database definition takes into account and combines framework (FR) and complementarity determining region (CDR) definitions, structural data from X-ray diffraction studies, and characterization of hypervariable loops to number variable regions / structures (see LeFranc, Immunol. Today 18:509, 1997). The contact definition is based on analysis of available complex crystal structures (e.g., MacCallum et al., J. Mol. Biol. 5:732-45, 1996). CDRs L1, L2, and L3 of the VL domain are also referred to herein as CDR-L1, CDR-L2, and CDR-L3, respectively; CDRs H1, H2, and H3 of the VH domain are also referred to herein as CDR-H1, CDR-H2, and CDR-H3, respectively.

[0137] "Framework regions" are variable domain residues other than hypervariable region residues (e.g., other than residues of CDRs as defined by the Kabat, Chothia, AbM, IMGT database, or contact definitions of CDRs). The sequences of framework regions of different light or heavy chains are relatively conserved within a species. Thus, a "human framework region" is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework regions of naturally occurring human immunoglobulins. The framework regions of an antibody, which are the combined framework regions of the constituent light and heavy chains, function to position and align the CDRs.

[0138] An "immunoglobulin hinge" is the portion of an immunoglobulin heavy chain that connects the CH1 and CH2 domains. The human γ1 hinge region corresponds approximately to Eu residues 216-230.

[0139] The terms "Fc fragment," "Fc region," or "Fc domain," as used herein, are synonymous and refer to the portion of an immunoglobulin responsible for binding to antibody receptors on cells and the C1q component of complement (in the absence of any amino acid changes that eliminate such binding activity compared to the naturally occurring sequence). Fc stands for "fragment crystalline," a fragment of an antibody that readily forms protein crystals. Distinct protein fragments originally described by proteolytic digestion may define the overall general structure of immunoglobulin proteins. As originally defined in the literature, the Fc fragment consists of the disulfide-linked heavy chain hinge region, CH2 domain, and CH3 domain. 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 that are capable of forming dimers at least via dimerization of the CH3 domains and that have increased or decreased Fc receptor binding or complement fixation activity while retaining binding to the neonatal Fc receptor (FcRn) at least sufficient to confer improved half-life (compared to the fusion partner in the absence of the Fc region) to the fusion partner in vivo. The abbreviated term "Fc" may also be used herein to indicate "Fc region" when referring to a fusion polypeptide by its general amino- to carboxyl-terminal configuration (e.g., "DNase1-L1-Fc-L2-PON1").

[0140] As used herein, the term "single-chain antibody" refers to an antibody having an antigen-binding site contained within a single polypeptide chain (e.g., variable regions from both the heavy and light chains within a single polypeptide chain). The term "single-chain Fv" refers to a single-chain antibody that contains variable regions from both the heavy and light chains but lacks a constant region. Generally, a single-chain Fv further comprises a polypeptide linker between the VH and VL domains that enables it to form the desired structure that enables antigen binding. Single-chain antibodies are discussed in detail, for example, by Pluckthun in *The Pharmacology of Monoclonal Antibodies*, vol. 113 (Rosenburg and Moore eds., Springer-Verlag, New York, 1994), pp. 269-315 (see also International PCT Publication No. WO 88 / 01649; U.S. Patent Nos. 4,946,778 and 5,260,203; Bird et al., Science 242:423-426, 1988). Single-chain antibodies can also be bispecific and / or humanized.

[0141] The term "alternative scaffold protein" refers to a non-antibody protein in which one or more regions can be diversified to produce one or more binding domains that specifically bind to a target molecule (e.g., an inflammatory cytokine, e.g., TNFα or TGF-β). In some embodiments, the binding domains bind to the target molecule with specificity and affinity similar to that of an antibody. Exemplary alternative scaffolds include those derived from fibronectin (e.g., Adnectin™), β-sandwich (e.g., iMab), lipocalin (e.g., Anticalin®), EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2 (e.g., Kunitz domain), Protein A (e.g., Affibody®), ankyrin repeat (e.g., DARPin), gamma-B-crystallin / ubiquitin (e.g., Affilin), CTLD3 (e.g., Tetranectin), Fynomer, and Avimer. Additional information regarding alternative scaffolds is provided in Binz et al., Nat. Biotechnol. 23:1257-1268, 2005; Skerra, Current Opin. in Biotech. 18:295-304, 2007; and Silacci et al., J. Biol. Chem. 289:14392-14398, 2014.

[0142] The terms "anti-TNFα" or "anti-TGFβ," as used herein, when referring to a fusion polypeptide by its general amino- to carboxyl-terminal structure (e.g., "anti-TNFα-L1-Fc-L2-PON1" or "anti-TGFβ-L1-Fc-L2-PON1"), refer to a polypeptide (e.g., a single-chain antibody) that specifically binds to and neutralizes TNFα or TGF-β, respectively.

[0143] "Dimerization domain," as used herein, refers to a polypeptide that has affinity for a second polypeptide such that the two polypeptides associate to form a dimer under physiological conditions. Typically, the second polypeptide is the same polypeptide, but in some variations, the second polypeptide is different. These polypeptides may interact with each other through covalent and / or non-covalent association(s). Examples of dimerization domains include an Fc region; a hinge region; a CH3 domain; a CH4 domain; a CH1 or CL domain; a leucine zipper domain (e.g., the jun / fos leucine zipper domain, see, e.g., Kostelney et al., J. Immunol., 148:1547-1553, 1992; or the yeast GCN4 leucine zipper domain); an isoleucine zipper domain; the dimerization region of a dimerizing cell surface receptor (e.g., interleukin-8 receptor (IL-8R); or an integrin heterodimer, e.g., LFA-1 or GPIIIb / IIIa); a secreted, dimerizing ligand (e.g., nerve growth factor (NGF), neurotrophin-3 (NT-3), interleukin-8 (IL-8), vascular endothelial growth 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 a polypeptide containing at least one cysteine ​​residue (e.g., about 1, 2, or 3 to about 10 cysteine ​​residues) such that disulfide bond(s) can form between the polypeptide and a second polypeptide containing at least one cysteine ​​residue (hereinafter "synthetic hinge"). A preferred dimerization domain according to the present invention is an Fc region.

[0144] The term "dimer" or "dimeric protein," as used herein, refers to a multimer of two ("first" and "second") fusion polypeptides disclosed herein linked together via a dimerization domain. Unless the context clearly indicates otherwise, "dimer" or "dimeric protein" includes reference to a dimerized first and second fusion polypeptide in terms of a higher-order multimer that may be created by including additional dimerization domains in the 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 through interactions between the CH1 and CL domains, and two such heterodimers can further dimerize via the Fc regions of the immunoglobulin heavy chains, thereby forming a tetramer).

[0145] The term "domain that specifically binds to the neonatal Fc receptor (FcRn)" or "FcRn-binding domain," as used herein, refers to a domain that specifically binds to the neonatal Fc receptor (FcRn) with (i) high affinity at pH 5.8, typically at pH 10 6 M -1 or higher (e.g., 10 7 M -1 or higher, 10 8 M -1 or higher, or 10 9 M -1 or higher) binding affinity (K a) and (ii) has no affinity for FcRn at physiological pH (e.g., pH 7.4). The binding affinity of a polypeptide to FcRn can be easily determined by one skilled in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660, 1949). Typically, an FcRn-binding domain does not significantly cross-react with polypeptides related to FcRn. A polypeptide does not significantly cross-react with polypeptides related to FcRn, for example, when a molecular binding assay, such as a multiwell plate assay (e.g., ELISA), a filter assay, or a surface plasmon resonance assay, is used to detect FcRn but not currently known FcRn-related polypeptides. Examples of known related polypeptides include known members of the major histocompatibility complex (MHC) class I protein family. FcRn-binding domains do not mutually exclude dimerization domains, i.e., a dimerization domain can also be an FcRn-binding domain. Examples of FcRn-binding domains that are also dimerization domains include Fc regions that retain FcRn-binding activity.

[0146] Fusion polypeptides of the disclosure may be referred to herein by formulas such as, for example, "DNase1-L1-Fc," "DNase1L3-L1-Fc," "DNase1-L1-Fc-L2-PON1," "DNase1L3-L1-Fc-L2-PON1," "RNase1-L1-Fc-L2-PON1," "SOD1-L1-Fc-L2-PON1," "anti-TNFα-L1-Fc-L2-PON1," or "anti-TGFβ-L1-Fc-L2-PON1." In each such case, unless the context clearly dictates otherwise, terms referring to particular segments of the fusion polypeptide (e.g., "DNase 1," "DNase 1L3," "PON1," "RNase 1," "SOD1," "L1," or "L2" (in place of the first or second polypeptide linker, respectively), "Fc" (in place of "Fc region"), etc.) will be understood to have the meaning ascribed to such terms herein, including the various embodiments described herein.

[0147] The term "disease or disorder characterized by NETosis," as used herein, means a disease or disorder in which NETosis—a process by which activated neutrophils shed DNA associated with cytoplasmic and granule proteins, called neutrophil extracellular traps (NETs)—plays at least some role in the clinical pathology.

[0148] The term "effective amount" refers to the amount of such molecule that is sufficient to inhibit the occurrence of disease or alleviate one or more symptoms of disease when administering soluble fusion polypeptide or dimeric protein to a subject as described herein for the treatment of disease.For example, specifically for the treatment of inflammatory lung disease by administering fusion protein to a subject as described herein, the term "effective amount" refers to the amount of such molecule that is sufficient to modulate the inflammatory response in a subject, so as to inhibit the occurrence of inflammatory lung disease or alleviate one or more symptoms of inflammatory lung disease.An effective amount of drug is administered in an "effective regimen" according to the method of the present invention.The term "effective regimen" refers to the combination of the amount of drug administered and the dosage frequency that is appropriate to achieve the treatment or prevention of disease.

[0149] The term "patient" or "subject" includes mammals, such as humans and other primates, with respect to treating a disease or disorder as described herein. The term also includes domesticated animals, such as cattle, pigs, sheep, horses, dogs, and cats.

[0150] The term "combination therapy" refers to a treatment regimen that involves providing at least two distinct therapies to achieve the indicated therapeutic effect.For example, combination therapy can involve the administration of two or more chemically distinct active ingredients or drugs, such as the soluble PON1 fusion polypeptide or dimeric protein according to the present invention, and another drug, such as another anti-inflammatory agent or immunomodulator.Alternatively, combination therapy can involve the administration of the soluble PON1 fusion polypeptide or dimeric protein according to the present invention, alone or in combination with another drug, and the delivery of another therapy (for example, radiation therapy).The distinct therapies that constitute combination therapy can be delivered, for example, as simultaneous, overlapping, or sequential dosing regimens.With regard to the administration of two or more chemically distinct drugs, it is understood that these active ingredients can be administered as part of the same composition or as different compositions. When administered as separate compositions, the compositions containing different active ingredients may all be administered at the same or different times, by the same or different routes, using the same or different dosing regimens, as the particular context requires and as determined by the attending physician.

[0151] The term "targeted therapy," in reference to treating cancer, typically refers to a type of treatment that uses therapeutic agents to identify and attack specific types of cancer cells while causing less harm to normal cells. In some embodiments, targeted therapy blocks the action of enzymes or other molecules involved in the growth and spread of cancer cells. In other embodiments, targeted therapy helps the immune system attack cancer cells or delivers toxic substances directly to cancer cells. In certain variations, targeted therapy uses small molecule drugs or monoclonal antibodies as therapeutic agents.

[0152] The phrase "protecting against aging," as used herein, refers to the inhibition or reduction of any of the broad aspects of aging, including, for example, age-related changes in systemic inflammation or disease risk as indicated by accepted biomarkers. Protection from aging can also include the treatment of age-related diseases, such as chronic inflammatory, autoimmune, neurodegenerative, cardiovascular, or fibrotic diseases, when the disease is present in the subject.

[0153] 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 maximum correspondence. Sequence comparison can be performed using standard software programs, such as those included in the LASERGENE bioinformatics computing suite created by DNASTAR (Madison, Wisconsin). Other methods for comparing amino acid sequences by determining optimal alignment are well known to those skilled in the art (see, for example, 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 to each other.

[0154] 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 score using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff, supra. Percent identity is then calculated as follows: ([total number of identical matches] / [length of the longer sequence + number of gaps introduced in the longer sequence to align the two sequences])(100).

[0155] Those skilled in the art will appreciate that there are many established algorithms available for aligning two amino acid sequences. The Pearson and Lipman "FASTA" similarity search algorithm is a suitable protein alignment method for testing the level of identity shared by the amino acid sequence disclosed herein and a second amino acid sequence. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444, 1988 and Pearson, Meth. Enzymol. 183:63, 1990. Briefly, FastA first characterizes sequence similarity by identifying the region shared by a query sequence (e.g., residues 16-355 or 26-355 of SEQ ID NO: 6) and a test sequence with either the highest density of identity (when the ktup variable is 1) or identity pair (when ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. Then, the 10 regions with the highest density of identity are re-scored by comparing the similarity of all pairwise amino acids using an amino acid substitution matrix, and the ends of these regions are "trimmed" to include only the 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 on the length of the sequence and the ktup value), the trimmed initial regions are examined to determine whether these regions can be connected to form an approximate alignment with gaps.Finally, the highest-scoring regions of the two amino acid sequences are aligned using a modified version 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 insertion and deletion. Example parameters for FASTA analysis are: ktup=1, gap opening penalty=10, gap extension penalty=1 and substitution matrix=blosum62.These parameters can be introduced into the FASTA program by modifying the scoring matrix file ("SMATRIX") as described in Appendix 2 of Pearson, Meth. Enzymol. 183:63, 1990.

[0156] The term "corresponding to," when applied to an amino acid residue position in a reference sequence to describe a position in a subject sequence, means the corresponding position in the subject sequence when the reference and subject sequences are optimally aligned.

[0157] 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%.

[0158] When aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the invention encompasses not only the entire group recited as a whole, but also each member of the group individually and all possible subgroups of the main group. The invention also envisions the explicit exclusion of one or more of any of the group members as embodiments. [Brief explanation of the drawings]

[0159] [Figure 1]Figure 1 shows Western blots of culture supernatants (serum-free) from transiently transfected HEK293 cells expressing DNase1-Fc and DNase1-Fc-PON1 fusion proteins. Transfection and Western blot analysis were performed as described in Example 1, below. From left to right: Lane 1—LICOR Chameleon™ molecular weight marker; Lanes 2, 3, and 4—transfection supernatants from three different individual clones (#1, #2, and #4) of hDNase™-(g4s)4-Fc-PON1-Q192K; Lane 5—THER4-RNase (control); Lane 6—LICOR Chameleon MW marker; Lane 7—hDNase™-(g4s)4-Fc #4; Lane 8—hDNase™-(g4s)6-Fc #2; Lane 9—THER4 (control); Lane 10—mock transfection.

[0160] [Figure 2] Figure 2 shows reducing SDS-PAGE analysis of DNase 1-Fc and DNase 1-Fc-PON1 fusion proteins purified from CHO clone spent culture supernatant. Stable CHO cell generation, Protein A purification of the fusion proteins, and SDS-PAGE were performed as described in Example 1, below. From left to right: Lane 1 - THER4PON1-K (control); Lane 2 - hDNase™-(g4s)4-Fc-PON1-K; Lane 3 - hDNase™-(g4s)4-Fc; Lane 4 - hDNase™-(g4s)6-Fc; Lane 5 - LI-COR Chameleon® MW marker; Lane 6 - BioRad Precision Plus Kaleidoscope MW marker.

[0161] [Figure 3-1] Figures 3A and 3B show raw data from a modified SYTOX™ Green fluorescent assay assessing the nuclease activity of DNase 1 fusion proteins at two different substrate concentrations, 220 μM or 66 μM salmon sperm DNA (see Example 2 below). [Figure 3-2] Same as above.

[0162] [Figure 4-1] 4A and 4B show Michaelis-Menten plots of data from two different DNase activity experiments using a modified SYTOX™ Green fluorescent assay (see Example 2 below). [Figure 4-2] Same as above.

[0163] [Figure 5-1] 5A and 5B show the results from a SYTOX™ Green fluorescence assay comparing the relative actin-induced inhibition of DNase activity of recombinant human DNase 1 and DNase 1-Fc fusion protein (see Example 2 below). [Figure 5-2] Same as above.

[0164] [Figure 6-1] 6A and 6B show Michaelis-Menten and Lineweaver-Burk plots, respectively, for a representative experiment measuring the arylesterase activity of a DNase1-Fc-PON1 fusion protein (see Example 3 below). [Figure 6-2] Same as above.

[0165] [Figure 7] FIG. 7 shows the results of a fluorometric assay measuring the organic phosphatase activity of the DNase1-Fc-PON1 fusion protein and an organic phosphatase positive control enzyme (see Example 3 below).

[0166] [Figure 8] 8 and 9 show Western blot analysis of PON1 fusion proteins immunoprecipitated from HEK293 transfection supernatants. Transfection, protein A immunoprecipitation, and Western blot analysis were performed as described in Example 12 below. [Figure 9]8 and 9 show Western blot analysis of PON1 fusion proteins immunoprecipitated from HEK293 transfection supernatants. Transfection, protein A immunoprecipitation, and Western blot analysis were performed as described in Example 12 below.

[0167] [Figure 10] 10 shows reducing SDS-PAGE analysis of PON1 fusion proteins immunoprecipitated from HEK293 transfection supernatants. Transfection, protein A immunoprecipitation, and SDS-PAGE were performed as described in Example 12 below.

[0168] [Figure 11] Figures 11-13 show the arylesterase activity (Figure 11), organic phosphatase activity (Figure 12), and lactonase activity (Figure 13) of PON1 fusion proteins assayed directly from HEK293 culture supernatants (see Example 13 below). [Figure 12] Figures 11-13 show the arylesterase activity (Figure 11), organic phosphatase activity (Figure 12), and lactonase activity (Figure 13) of PON1 fusion proteins assayed directly from HEK293 culture supernatants (see Example 13 below).

[0169] [Figure 13] Figures 11-13 show the arylesterase activity (Figure 11), organic phosphatase activity (Figure 12), and lactonase activity (Figure 13) of PON1 fusion proteins assayed directly from HEK293 culture supernatants (see Example 13 below).

[0170] [Figure 14] FIG. 14 shows high-affinity binding of immobilized CD40 ligand to CD40-Fc-PON1 fusion protein present in HEK293 transfection supernatant (see Example 14 below).

[0171] [Figure 15] FIG. 15 shows high-affinity binding of immobilized TNFα to anti-TNFα scFv-Fc-PON1 fusion protein present in HEK293 transfection supernatant (see Example 14 below). DETAILED DESCRIPTION OF THE INVENTION

[0172] Description of the invention I. Overview The present invention provides compositions and methods relating to paraoxonase fusion polypeptides. In certain embodiments, the fusion polypeptide comprises a first biologically active polypeptide linked amino-terminally to a biologically active paraoxonase, the first biologically active polypeptide being selected from DNase, RNase, superoxide dismutase 1 (SOD1), the cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) extracellular domain, the CD40 extracellular domain, and a polypeptide that specifically binds to and neutralizes a proinflammatory cytokine (e.g., a polypeptide that specifically binds to and neutralizes tumor necrosis factor alpha (TNFα) or transforming growth factor-beta (TGF-β)). In some embodiments, the fusion polypeptide further comprises a second biologically active polypeptide linked carboxyl-terminally to the first biologically active polypeptide and amino-terminally to the paraoxonase. The second biologically active polypeptide can be, for example, a dimerization domain or a domain that specifically binds to neonatal Fc receptor (FcRn). A particularly suitable second biologically active polypeptide is a dimerizing and FcRn-binding immunoglobulin heavy chain constant region, such as an immunoglobulin Fc region.

[0173] In other embodiments, the fusion polypeptide comprises a biologically active paraoxonase linked carboxyl terminally to a dimerization domain or a domain that specifically binds to FcRn (e.g., a dimerization- and FcRn-binding immunoglobulin heavy chain constant region, such as an immunoglobulin Fc region), and the fusion polypeptide does not comprise a biologically active polypeptide amino terminally to the dimerization domain or the FcRn-binding domain.

[0174] In other embodiments, the fusion polypeptide comprises a biologically active paraoxonase linked amino-terminally to a dimerization domain or a domain that specifically binds to FcRn (e.g., a dimerization- and FcRn-binding immunoglobulin heavy chain constant region, such as an immunoglobulin Fc region).

[0175] A preferred paraoxonase in the fusion polypeptide of the present disclosure is a paraoxonase 1 (PON1) polypeptide. Particularly suitable PON1 polypeptides lack an amino-terminal leader sequence corresponding to the naturally occurring non-cleavable leader sequence of PON1 (generally corresponding to residues 1-15 of human PON1 (SEQ ID NO: 4)). Association of PON1 with high-density lipoprotein (HDL) in serum primarily relies on direct binding of the hydrophobic non-cleavable leader to phospholipids (see Sorenson et al., Arterioscler. Thromb. Vasc. Biol. 19:2214-2225, 1999). Removal of this leader sequence removes PON1 from HDL binding and facilitates retargeting of PON1 to other sites for therapeutic benefit in accordance with the present disclosure. For example, with respect to fusion polypeptides containing DNase or RNase described herein, retargeting PON1 to DNA or RNA directs the molecule to sites of inflammatory NETs and cell death. In addition to targeting DNA or RNA at these sites, the DNase-PON1 or RNase-PON1 fusion molecules described herein can help direct the PON1 enzyme to myeloperoxidase, another important inflammatory NET target. Also, the fusion polypeptides described herein containing superoxide dismutase 1 (SOD1) provide molecules through which PON1 can digest hydrogen peroxide (HO) produced by SOD1's action on superoxide, thereby providing an improved antioxidant for, for example, lung therapy. Furthermore, fusion molecules containing other biologically active polypeptides according to the present invention (e.g., the CTLA-4 extracellular domain, the CD40 extracellular domain, or polypeptides that specifically bind and neutralize inflammatory cytokines) can target PON1 to other sites of pro-inflammatory and / or immune cell activation for therapeutic benefit, as described herein.

[0176] The paraoxonase fusion molecule of the present invention can be used to treat various diseases or disorders through its antioxidant, anti-inflammatory, atheroprotective and / or neuroprotective properties, including, for example, the treatment of autoimmune diseases or inflammatory diseases.For example, research supports the use of paraoxonase for the treatment of autoimmune diseases, such as systemic lupus erythematosus (SLE).The autoantibody titer in many patients with systemic lupus erythematosus (SLE) correlates with the loss of PON1 activity (see Batukla et al., Ann. NY Acad. Sci. 1108:137-146, 2007), and the SLE disease activity assessed by SLEDAI and the SLICC / ACR injury index assessed by SLE disease-related organ damage are negatively correlated with PON1 activity (see Ahmed et al., EXCLI Journal 12:719-732, 2013). Other studies support the use of paraoxonase for the treatment of inflammatory diseases, such as inflammatory lung diseases.For example, studies strongly suggest that PON1 is important in protecting against lung inflammation, and further indicate that low PON1 activity is associated with lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), and interstitial lung diseases (e.g., idiopathic pulmonary fibrosis (IPF) or sarcoidosis) (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., Internat. Immunol. 21:967-975, 2009; Chen et al., J. Cell Biochem. 119:793-805, 2013). 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 low PON1 activity correlates with the severity of Crohn's disease (Sczceklik et al., Molecules 23:2603, 2018).Furthermore, recombinant PON1 treatment 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 et al., J. Immunol. 191:949-960, 2013).

[0177] The paraoxonase fusion molecules described herein can also be used, for example, for the treatment of neurological diseases.For example, PON1 is protective in the brain due to its antioxidant properties.The neuroprotective role of PON1 is supported by research showing that PON1 activity is reduced in patients with Alzheimer's disease and other dementias (see, for example, Menini et al., Redox Rep. 19:49-58, 2014).In addition, research using PON1 fusions containing protein transduction domains to transduce PON1 into cells and tissues has shown that PON1 transduction protects microglial cells from oxidative stress-induced inflammatory responses in vitro and protects against dopaminergic neuronal cell death in Parkinson's disease models (see, for example, Kim et al., Biomaterials 64:45-56, 2015).

[0178] Bispecific fusions further comprising DNase provide additional therapeutic benefits for the treatment of diseases for which PON-mediated therapy is applicable, including, for example, diseases or disorders characterized by NETosis. NETs, ​​which cause tissue damage by themselves or by increasing pro-inflammatory responses (see Mutua and Gershwin, Clin. Rev. Allergy Immunol., 2020), are involved in various serious diseases and conditions. For example, NETs may play a role in enhancing inflammation seen in autoimmune diseases, including systemic lupus erythematosus (SLE) (e.g., SLE with lupus nephritis), rheumatoid arthritis, and psoriasis, and are also associated with autoinflammatory diseases, such as inflammatory bowel disease (IBD) (see Ibid.).NETs also play a role in sepsis, metabolic diseases (e.g., type 2 diabetes and obesity), neurological diseases (e.g., Alzheimer's disease, multiple sclerosis, meningitis, cerebral malaria, and ischemic stroke), infectious diseases, cardiovascular disease and thrombosis, tumor progression and metastasis, liver diseases (e.g., alcohol-related liver disease (ALD), portal hypertension, non-alcoholic fatty liver disease (NAFLD), e.g., non-alcoholic steatohepatitis (NASH), and complications after liver transplantation), and various inflammatory lung diseases, including, for example, cystic fibrosis, asthma, chronic obstructive pulmonary disease (COPD), and COVID-19-associated acute respiratory distress syndrome (ARDS) (e.g., ibid.; Brinkmann, J. Innate Immun. 10:422-431, 2018; Liu et al., Chinese Med. Journal 130:730-736, 2019). 2017;Twaddell et al., CHEST 156:774-782, 2019;Uddin et al., Front. Immunol. 10:47, 2019;Middleton et al., Blood 136:1169, 2020;Zuo et al., JCI Insight 5:e138999, 2020;Barnes et al. al., J. Exp. Med. 217:e20200652, 2020; Hilscher and Shah, Semin. Liver Dis. 40:171-179, 2020).The use of DNase for the treatment of diseases or disorders characterized by NETosis is further supported by, for example, the use of recombinant wild-type DNase (Pulmozyme®) for the treatment of cystic fibrosis, the ongoing clinical trial of Pulmozyme in COVID-19 patients (NCT04359654), and several animal model studies, including, for example, models of acute asthma, silica-induced pulmonary inflammation, acute lung injury, cancer, colitis, ischemia-reperfusion, sepsis, and thrombosis (e.g., da Cunha et al., Exp. Lung Res. 42:66, 2016; Benmerzoug et al., Nat. Comm. 9:5226, 2018; Caudrillier et al., J. Clin. Invest. 122:2661, 2012; Cools-Lartigue et al., J. Clin. Invest. 123:3446, 2013). 2013;Park et al., Sci. Translational Med. 8:361ra138, 2016;Trejo-Becirril et al., Integrative Cancer Therapies 15:NP35-NP43, 2016;Thalin et al., PloS ONE 13:e0191231, 2018;Babicova et al., Folia Biolica (Praha) 64:10, 2018;Li et al., J. Crohns Colitis 2020, 14:240-253, 2020;Albadawi et al., J. Vasc. Surg. 64:484, 2016;Peer et al., Am. J. Nephrol. 43:195, 2016;Mai et al. al., Shock 44:166, 2015; Brill et al., J. Thrombosis Haemostasis 10:136, 2012; Manda-Handzlik and Demkow, Cells 8:1477, 2019; Delgado-Rizo et al., Front. Immunol. 8:81, 2017).

[0179] The bispecific fusions described herein, further comprising DNase, are also useful for treating antiphospholipid syndrome (APLS). Antiphospholipid syndrome is an autoimmune disease characterized by antibodies against phospholipids, which causes a hypercoagulable state. Thrombosis occurs in both arteries and veins, leading to pregnancy complications, including miscarriage and stillbirth. Antiphospholipid syndrome can be primary (occurring in the absence of any other associated disease) or secondary (occurring together with another autoimmune disease, such as SLE). In rare cases, APLS leads to rapid organ failure due to systemic thrombosis; this is called fulminant antiphospholipid syndrome (CAPS or Asherson syndrome) and is associated with a high risk of death. Patients with acute respiratory distress associated with COVID-19 have antiphospholipid antibodies that promote thrombosis in vitro and in mouse thrombosis models (Zuo et al., JCI Insight 5:e138999, 2020; Zuo et al., Sci. Trans. Med. 12:eabd3876, 2020). Antiphospholipid antibodies stimulate neutrophil NET formation, providing a scaffold for thrombus formation. Digestion of NETs with the DNase fusions described herein can prevent thrombus formation and dissolve existing clots, helping to restore blood flow. The DNase fusion molecules of the present invention can also be used in conjunction with thrombolytic drugs, including, for example, tPA, urokinase, and streptokinase.

[0180] For example, the paraoxonase fusion molecules described herein, including those further comprising DNase, can also be used to treat inflammatory skin diseases. Many studies have reported the involvement of reactive oxygen species and lipid peroxides in the pathogenesis of inflammatory skin diseases (see, for example, Simonetti et al., Antioxidants 10:697, 2021). For example, significantly lower levels of paraoxonase 1 (PON1) and significantly higher levels of myeloperoxidase (MPO) and lipid peroxides have been found in atopic dermatitis (see, supra), and increased MPO and decreased PON1 activity have also been reported in children with psoriasis (see, Bacchetti et al., Archives of Dermatological Research 312:33-39). In psoriasis and atopic dermatitis, neutrophil activation during inflammation leads to the eruption of MPO-bound NETs, ​​causing lipid oxidation and impairing the PON1 enzyme, which normally protects lipids from oxidation by digesting lipid peroxides. Digestion of NETs with DNase1 releases the bound MPO, allowing its clearance and removing a key driver of oxidation. Treatment with PON1 restores protection from oxidative damage and rebalances the redox potential in patients. Therefore, PON1 and DNase treatment are particularly effective as a combination.

[0181] The paraoxonase fusion molecules described herein, including those further comprising DNase, can also be used to protect against aging. Oxidative stress is known to be a major cause of aging. In particular, oxidized lipids are important targets for aging due to their promotion of inflammation through uptake by macrophage scavenger receptors and damage to vascular endothelial cells. For a discussion of the relationship between lipid oxidation and aging, see, for example, Moldogazieva et al. (Oxidative Medicine and Cellular Longevity, Volume 2019, Article ID 3085756) and Barrera et al. (Antioxidants 7:102, 2018). See also Hajri (Frontiers In Bioscience, Landmark 23:1822-1847, 2018), which describes the toxic effects of oxidized lipids on inflammation and cardiac function. PON1 is known to protect lipids from oxidation by digesting lipid peroxides. Thus, there is a direct correlation between increased PON1 enzyme levels and slowed aging. Furthermore, PON1 enzyme activity is known to be deficient in multiple age-related diseases, including cardiovascular disease, autoimmune disease, and pulmonary disease, indicating a clinical need for increased PON1. Furthermore, embodiments further comprising DNase are particularly useful for slowing aging due to DNase's ability to digest NETs. NETs are a major driver of inflammation and oxidative stress because they provide a scaffold for myeloperoxidase (MPO) and elastase produced by neutrophils and other cells. Digestion of NETs by DNase allows the release and clearance of inflammatory enzymes. DNase also eliminates inflammatory activation through other DNA-sensing pathways (e.g., STING, TLR9).

[0182] Bispecific fusions further comprising an RNase as described herein have anti-inflammatory properties by digesting extracellular RNA from dead and dying cells, including, for example, extracellular RNA associated with NETs. Extracellular RNA synergizes with, for example, TLR2 agonists to enhance macrophage inflammatory responses, and digestion of this RNA prevents TLR2-mediated inflammation (see Noll et al., PloS ONE 12:e0190002, 2017).

[0183] RNase-containing fusions provide additional therapeutic benefits for the treatment of various diseases, including, for example, autoimmune diseases, inflammatory diseases (e.g., inflammatory lung diseases), type 2 diabetes, infectious diseases, cardiovascular diseases (e.g., coronary artery disease, stroke), neurodegenerative diseases (e.g., Alzheimer's disease) and cancer. For example, some studies support the use of RNase for the treatment of inflammatory lung diseases. One study has shown that the RNA sensor TLR3 plays a major role in the development of ARDS-like pathology in the absence of viral pathogens (see Murray et al., Am. J. Respir. Crit. Care Med. 178:1227-1237, 2008). Oxygen therapy is the main therapeutic intervention for ARDS, but it contributes to further lung damage and susceptibility to viral infection. Oxygen treatment is a major stimulus for increased TLR3 expression and activation in cultured human epithelial cells, and the absence or blockade of TLR3 protected mice from lung injury and inflammation after exposure to hyperoxic conditions (Murray et al., supra). Another study showed that TLR3 activation by extracellular RNA occurs in response to acute hypoxia, and that treatment with Rnase A in mice attenuated lung inflammation after acute hypoxia (Biswas et al., Eur. J. Immunol. 45: 3158-3173, 2015).

[0184] Other studies support the use of RNase for the treatment of autoimmune diseases such as systemic lupus erythematosus (SLE). Studies have shown, for example, the role of RNA immune complexes and RNA receptors, including TLR7, in SLE disease pathogenesis, as well as the protective effect of RNase overexpression in a mouse model of SLE (see, for example, Sun et al., J. Immunol. 190:2536-2543, 2013).

[0185] The RNase-containing fusion molecule described herein can digest the RNA contained in exosomes.For a review of exosomes in lung diseases, see Hough et al., Allergy 72:534-544, 2017.RNase1 can translocate through the cell membrane into the cytosol due to its basic charge (see Chao et al., Biochem. 50:8374-8382, 2011; Haigas et al., J. Cell Sci. 116:313-324, 2003; Johnson et al., Biochem. 46:10308-10316, 2007).Cytoplasmic inhibitors bind to RNase1 with high affinity and protect intact cells from RNase toxicity. However, extracellular RNA, including RNA in extracellular vesicles, is not protected by cytoplasmic inhibitors (see Mironova et al., Oncotarget 8: 78796-78810, 2017).

[0186] The RNase-containing fusion molecules described herein can further target microRNAs (miRNAs), which are small extracellular non-coding RNAs involved in post-transcriptional gene regulation. MiRNA dysregulation has been associated with a wide range of diseases, including lung disease, proliferative vascular disease, cardiac disorders, kidney disease, diabetes mellitus, fibrosis, and cancer (Rajasekaran et al., Front. Pharmacol. 6:254, 2015; Thum et al., Nature 456:980-984, 2008; Kato et al., Clin. J. Am. Soc. Nephrol. 4:1255-1266, 2009; Lee and Dutta, Annu. Rev. Pathol. 4:199-227, 2009; Kumar et al., Protein Cell. 3:726-738, 2012; Noetel et al., Front. Physiol. 3:49, 2012; Zampetaki and Mayr, Circ. Res. 110:508-522, 2012). (See, e.g., 2012). For example, microRNA-33 is overexpressed in granulomatous lung tissue in mouse models and in BAL fluid from patients with sarcoidosis (Barna et al., Am. J. Respir. Cell Mol. Biol. 54:865-871, 2016). MicroRNA-33 downregulates the cholesterol transporters ABCA1 and ABCG1, promoting cholesterol accumulation in alveolar macrophages and foam cell formation. Another target is microRNA-466, which was shown to be secreted into exosomes in a mouse model of acute respiratory distress syndrome (ARDS), where it activated the NLRP3 inflammasome (Shikano et al., BMC Pulmonary Med. 19:110, 2019).

[0187] Other targets of the RNase-containing fusions described herein include viral RNA, such as proviral microRNAs released into the lungs during influenza virus infection (see Scheller et al., J. Inf. Dis. 219:540-543, 2019), or HIV single-stranded RNA, which has been reported to stimulate macrophage foam cell formation via TLR8 binding (see Bernard et al., PloS ONE 9:e104039, 2014). Other viral RNA targets include coronavirus RNA (e.g., SARS-CoV-2 RNA).

[0188] Research also supports the use of RNases for the treatment of, for example, cancer, ischemia / reperfusion injury, graft rejection, atherosclerotic plaque formation, myocardial infarction, acute stroke, and postoperative cognitive decline (e.g., Rutkoski et al., Translational Oncology 6:392-397, 2013; Mironova et al., Oncotarget. 8:78796-78810, 2017; Eller et al., ACS Central Sci. 1:181-190, 2015; Kleinert et al., J. Am. Heart Assn. doi: 10.1021, 2016; Simsekyilmaz et al., Circulation 129: 598-606, 2014; Steiger et al., JAMA 6:e004541, 2017; Walberer et al., Curr. Neovas. Res. 6:12-19, 2009; Chen et al., PloS One 10:e0134307, 2015).

[0189] The bispecific paraoxonase fusion molecules described herein, including superoxide dismutase 1 (SOD1), provide additional therapeutic benefit in protecting against oxidative stress. The role of SOD1 in protecting against oxidative damage has been reviewed, for example, by Ighodaro and Akinolye, Alexandria J. Med. 54:287-293, 2018. In its native form, SOD1 is an intracellular enzyme (see, ibid.). According to the present disclosure, an SOD1 polypeptide segment is used as an extracellular enzyme that functions in conjunction with the paraoxonase segment of the fusion molecule to provide improved antioxidants for therapy. For example, paraoxonase can digest hydrogen peroxide (HO), a product of the SOD1 enzymatic reaction. Furthermore, the digestion of HO suppresses the activity of myeloperoxidase, which is associated with NETs. The coordinated activity of SOD1 and paraoxonase improves protection from tissue damage and inflammation in patients with diseases associated with NET production. SOD1-PON molecules according to the present invention are particularly useful, for example, in the treatment of inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), sarcoidosis, and interstitial lung disease. In some preferred embodiments, the SOD1-PON fusion further comprises a dimerization domain, such as an immunoglobulin Fc region; such variants are particularly compatible with SOD1, which in its native form is a dimer stabilized by disulfide bonds.

[0190] The bispecific paraoxonase fusion molecules containing the CTLA-4 extracellular domain described herein are particularly useful in the treatment of inflammatory and autoimmune diseases, including, for example, rheumatoid arthritis (RA) and inflammatory lung diseases. Oxidized cholesterol activates inflammatory responses in macrophages and endothelial cells (see Miller and Shyy, Trends Endocrinol. Metabol. 28:143-152, 2017), and PON1 can suppress this inflammation by protecting lipoproteins from oxidation. The immunosuppressive properties of soluble CTLA-4 (e.g., abatacept or other CTLA4-Fc) are compatible with paraoxonase enzyme activity, resulting in CTLA4-PON molecules that retain the activity of both components and have improved activity in fibrotic lung diseases and autoimmune / inflammatory diseases. CTLA4-PON1 molecule binds to CD80 and CD86, which are expressed on activated antigen-presenting cells, including monocytes and dendritic cells, where PON1 enzyme remains active.This molecule is effective in inhibiting the activation of T cells, macrophages and dendritic cells, and also reduces oxidative stress in tissues, such as inflamed lungs.

[0191] The bispecific paraoxonase fusion molecules comprising the CD40 extracellular domain described herein provide additional therapeutic benefit by suppressing the pro-inflammatory activation events associated with the CD40-CD154 signaling pathway. The CD40-CD154 pathway is involved in fibrotic diseases, including, for example, fibrosis in inflammatory lung diseases and injury (see, e.g., Kaufman et al., J. Immunol. 172:1862-1871, 2004), and research supports the use of molecules that inhibit this pathway in the treatment of fibrotic and inflammatory lung diseases (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. Research Intl. Volume 2020, Article ID 7840652, 2020; Xiong et al., J. Cell. Mol. Med. 23:740-749, 2018). The CD40-PON1 fusions of the present invention provide molecules that inhibit both inflammation and adaptive immunity, including molecules with improved activity in, for example, lung diseases and autoimmune / inflammatory diseases.For example, CD40-PON1 fusions can provide improved activity in lung diseases where CD40 activation exacerbates the inflammatory process and paraoxonase levels are low or absent, including asthma, COPD, hypoxia, and interstitial lung diseases (e.g., idiopathic pulmonary fibrosis (IPF) or sarcoidosis).CDO40-PON1 fusions are also useful for treating, for example, liver diseases (e.g., chronic liver diseases, such as alcohol-related liver disease (ALD), portal hypertension, non-alcoholic steatohepatitis (NASH), or complications after liver transplantation) and kidney diseases (e.g., chronic kidney diseases, such as lupus nephritis, IgA nephropathy, or membranous glomerulonephritis).

[0192] In some embodiments, the paraoxonase fusion molecules of the invention comprise a variant CD40 extracellular domain with increased binding to CD154. For example, one or more single amino acid substitutions can be made at specific residues to improve binding to CD40 ligand (see, e.g., U.S. Patent Application Publication No. 2014 / 0120091, incorporated herein by reference). Such CD40 extracellular domain variants can be fused to a human IgG1(γ1) Fc variant with impaired FcγR binding to provide a CD40-Fc variant that neither induces platelet activation nor aggregation in vitro, thereby avoiding toxicity from simultaneous binding to CD40 and FcγRIIa on human platelets. Platelet activation and thrombosis have been shown to occur due to cross-linking of FcRs on platelets with antigens on the platelet surface, such as VEGF, CD40, and CD40L (see Taylor et al., Blood 96:4254, 2020; Meyer et al., J. Thromb. Haemost. 7:151, 2009).

[0193] Bispecific fusion molecules containing polypeptides that specifically bind and neutralize TNFα or TGF-β described herein provide additional therapeutic benefits through inhibition of inflammatory responses, immune responses, and / or other cellular responses mediated by these cytokines. Paraoxonase fusions containing anti-TNFα components can be used to treat, for example, inflammatory or autoimmune diseases, such as rheumatoid arthritis (RA), inflammatory lung diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), or interstitial lung diseases (e.g., idiopathic pulmonary fibrosis or sarcoidosis), inflammatory bowel disease (IBD) (e.g., Crohn's disease or ulcerative colitis), or inflammatory skin diseases (e.g., psoriasis or atopic dermatitis). Paraoxonase fusions containing anti-TGF-β components can be used to treat, for example, inflammatory diseases, fibrotic diseases, or type 1 diabetes. In some embodiments, the inflammatory or fibrotic disease for treatment using the anti-TGFβ-PON fusions of the present invention is an inflammatory lung disease, such as asthma, chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), or interstitial lung disease (e.g., idiopathic pulmonary fibrosis (IPF) or sarcoidosis). In some variations for the treatment of inflammatory lung diseases, fusion molecules comprising the anti-TNFα or anti-TGF-β polypeptides described herein are delivered directly to the lungs by nebulization rather than systemically, which can achieve efficient therapeutic activity with fewer unwanted side effects that may result from the pleiotropic activities of these cytokines.

[0194] In some aspects, the present invention provides compositions and methods for producing the fusion polypeptides and dimeric proteins described herein, including compositions and methods for constitutive expression of DNase-PON fusions in stable cell lines, such as CHO. Previous attempts to obtain stable CHO cells constitutively expressing actin / salt-resistant, hyperactive DNase1 were unsuccessful despite screening thousands of clones (see Lam et al., Biotech. Progress 32: 523-533, 2017). In certain embodiments, the DNase-PON fusions containing the enhanced DNase1 described herein are easily expressed in stable systems with minimal screening, thereby overcoming this problem in the art. The ability to easily generate stable cell lines expressing certain enhanced DNase1-PON molecules of the present disclosure indicates that these molecules offer advantages in the feasibility and cost of industrial-scale biopharmaceutical manufacturing. II. Fusion Polypeptides and Dimeric Proteins

[0195] In one aspect, the invention provides a fusion polypeptide comprising, from amino-terminal to carboxyl-terminal, T-L1-X-L2-P, where T, L1, X, L2, and P are defined as follows: T is a first biologically active polypeptide selected from DNase, RNase, superoxide dismutase 1 (SOD1), cytotoxic T-lymphocyte-associated molecule-4 (CLTA-4) extracellular domain, CD40 extracellular domain, and a polypeptide that specifically binds to and neutralizes a proinflammatory cytokine; L1 is an optional first polypeptide linker; X is an optional second biologically active polypeptide; L2 is an optional second polypeptide linker; and P is biologically active paraoxonase. In some embodiments of the fusion polypeptide in which X is present, X is selected from a dimerization domain and a domain that specifically binds to neonatal Fc receptor (FcRn). Typically, the biologically active paraoxonase is a naturally occurring paraoxonase 1 (PON1) polypeptide or a functional variant or fragment thereof. In variations including DNase, RNase, SOD1, the CLTA-4 extracellular domain, or the CD40 extracellular domain, any of the above biologically active polypeptides can be selected from the identified naturally occurring polypeptides or functional variants or fragments thereof. In certain variations including polypeptides that specifically bind and neutralize inflammatory cytokines (e.g., TNFα or TGF-β), the anti-cytokine polypeptide is a single-chain antibody or alternative scaffold-binding protein.

[0196] In another aspect, the invention provides a fusion polypeptide comprising, from amino-terminal to carboxyl-terminal position, X-L2-P, where X is a biologically active polypeptide selected from a dimerization domain and a domain that specifically binds to the neonatal Fc receptor (FcRn), L2 is an optional polypeptide linker, and P is a biologically active paraoxonase, wherein the fusion polypeptide does not contain a biologically active polypeptide amino-terminal to X. Typically, the biologically active paraoxonase is a naturally occurring paraoxonase 1 (PON1) polypeptide or a functional variant or fragment thereof.

[0197] In yet another aspect, the present invention provides a fusion polypeptide comprising, from amino-terminal to carboxyl-terminal, P-L1-X, where P is a biologically active paraoxonase, L1 is a polypeptide linker, and X is a biologically active polypeptide selected from a dimerization domain and a domain that specifically binds to the neonatal Fc receptor (FcRn). Typically, the biologically active paraoxonase is a naturally occurring paraoxonase 1 (PON1) polypeptide or a functional variant or fragment thereof.

[0198] Functional variants of the above-identified naturally occurring paraoxonase or other biologically active polypeptides can be readily identified using conventional assays to evaluate the variants for their corresponding biological activity. For example, paraoxonase 1 (PON1) variants can be assayed for phosphotriesterase activity using diethyl p-nitrophenol phosphate (paraoxon) as a substrate or for arylesterase activity using phenyl acetate as a substrate (see, e.g., Graves and Scott, Curr Chem Genomics 2:51-61, 2008; see also Example 3 below). Furthermore, DNase 1 and DNase 1L3 variants, including hyperactive DNase 1 variants, exhibit (i) a reduced A methyl green dye release from hydrolyzed DNA. 260 (ii) a DNA-methyl green assay that measures the reduction of A due to DNA absorption (see, e.g., Sinicropi et al., Anal. Biochem. 222:351-358; Pan and Lazarus, J. Biol. Chem. 273:11701-11708, 1998); 260Nuclease activity can be assayed for using the Kunitz hyperchromicity assay (see, e.g., Kunitz, J. Gen. Physiol. 33:349-362, 1950; Pan and Lazarus, supra), in which fluorescence increases as a function of degradation; (iii) a plasmid digestion assay (see, e.g., Pan and Lazarus, supra), in which either supercoiled or linear plasmid DNA is used as a substrate and the disappearance of the substrate and / or the appearance of digestion products (e.g., the appearance of linear products, or relaxed products from supercoiled DNA) are measured; or (iv) the SYTOX™ Green fluorescence assay (see Example 2 below), in which the decrease in fluorescence is measured as DNA labeled with SYTOX Green dye is hydrolyzed. DNase 1 variants can also be assayed for G-actin-induced inhibition of nuclease activity using DNase pre-incubated with G-actin before the addition of substrate DNA (see, e.g., Pan et al., J. Biol. Chem. 273:18374-18381, 1998). In the case of RNases such as human RNase 1, variants can be assayed for their ability to digest single- or double-stranded RNA in known assays for assessing ribonuclease activity (see, e.g., Libonati and Sorrentino, Methods Enzymol. 341:234-248, 2001).

[0199] SOD1 variants can be assayed using a commercially available colorimetric superoxide dismutase activity kit (ThermoFisher™ catalog number: EIASODC). This kit is designed to measure all forms of superoxide dismutase activity, including Cu / Zn, Mn, and Fe superoxide dismutase activity, in a variety of sample types. Reactive oxygen species, including superoxide, hydroxyl radicals, and hydrogen peroxide, are produced during normal cellular aerobic metabolism, and the free radicals are eliminated or converted to other products by several pathways involving SOD enzymes. This assay quantitatively measures SOD activity and uses bovine erythrocyte SOD standards to generate a standard curve. A similar spectrophotometric kit is also available from Cayman Chemicals (product number 706002), and an SOD activity assay using this kit is described in Mason et al., Free Radic. Biol. Med. 77:130-138, 2014.

[0200] Soluble CTLA-4 and CD40 variants, as well as anti-inflammatory cytokine polypeptides (e.g., single-chain antibodies), can be evaluated for the desired binding activity to their respective targets (e.g., to CD80 / CD86 in the case of CTLA-4, or to gp139 in the case of CD40) using any of a variety of known assays. For example, one assay system uses a commercially available biosensor instrument (BIAcore™, Pharmacia Biosensor, Piscataway, NJ), in which candidate binding polypeptides (e.g., candidate CTLA-4-Fc, CD40-Fc, or anti-cytokine antibodies) are covalently coupled to dextran fibers attached to a gold film within a flow cell using amine or sulfhydryl chemistry. A test sample containing a soluble target molecule (e.g., CD80-Fc, CD86-Fc, CD40L-Fc (gp139-Fc), or a soluble cytokine) is passed through the cell. If the immobilized protein has affinity for the target molecule, the protein will bind to the target and cause a change in the refractive index of the medium, which is detected as a change in the surface plasmon resonance of the gold film.This system allows the determination of the on-rate and off-rate from which the binding affinity can be calculated, and the evaluation of the stoichiometry of binding.The use of this instrument is disclosed, for example, by Karlsson (J. Immunol. Methods 145:229-240, 1991) and Cunningham and Wells (J. Mol. Biol. 234:554-563, 1993).The binding activity of candidate polypeptides can also be evaluated using other assay systems known in the art. Such systems include Scatchard analysis (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) for determining binding affinity.

[0201] The activity of soluble CTLA-4 and CD40 variants can also be assessed for function in suitable in vitro cellular assays. For example, soluble CTLA-4 can be expressed on CD80- or CD86-expressing cells, such as B cells, dendritic cells, or CD80-expressing cells. + or CD86 + It inhibits T cell responses to stimulation by CHO cells (see, e.g., U.S. Patent No. 5,434,131; Linsley et al., J. Exp. Med. 174:561-569, 1991). Soluble CD40 blocks the 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 can 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 the NF-kB response element fused to secreted embryonic alkaline phosphatase (SEAP). Binding of the CD40 receptor by its ligand or antibody results in NF-kB activation and inducible expression of SEAP (see Jerome et al., Anal. Biochem. 585:113402, 2019).

[0202] The neutralization of the activity of inflammatory cytokine binding polypeptides can also be evaluated using cellular assays known in the art.For example, anti-TNFα binding proteins can be assayed for neutralization of activity using commercially available TNFα reporter cell systems available from Invivogen (San Diego, CA).These cell systems are HEK-293 cells transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene linked to a TNFα-responsive promoter (Gregory et al., Nature 488(7412):508-511, 2012; Idress et al., Molecules 25:922, 2020).In addition, anti-TGF-β binding proteins can be assayed for neutralization of activity using a similar reporter cell system available from Invivogen (San Diego, CA), which contains a TGF-β-responsive element to drive the expression of SEAP. The use of this reporter system has been described by van Noort et al. (Cancer Res. 74:5690-5699, 2014). A similar reporter gene assay was reported for TNFα antagonists by Lallemand et al. (J. Immunol. Methods 373:229-239, 2011). This study utilized human erythroleukemia K562 cells transfected with a reporter construct incorporating a TNFα response element linked to the firefly luciferase gene. TNFα was premixed with potential antagonists for 30 minutes prior to incubation with the reporter cell line. Residual TNFα activity was quantified by comparison to a standard curve of TNFα alone.

[0203] Naturally occurring polypeptide segments (e.g., naturally occurring paraoxonase, DNase, RNase, or SOD1) for use in accordance with the present disclosure include naturally occurring variants consistent with the present disclosure, such as allelic variants and interspecies homologs.

[0204] Functional variants of a particular reference polypeptide (e.g., wild-type human paraoxonase 1 (PON1) set forth at residues 16-355 or 26-355 of SEQ ID NO:4, wild-type DNase 1 set forth at residues 23-282 of SEQ ID NO:16, or enhanced DNase 1 set forth at residues 21-280 of SEQ ID NO:18) are generally characterized as having one or more amino acid substitutions, deletions, or additions compared to the reference polypeptide. These changes are preferably minor, i.e., conservative amino acid substitutions (see, e.g., Table 1 below, which lists several exemplary conservative amino acid substitutions) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically from 1 to about 30 amino acids; and small amino- or carboxyl-terminal extensions. Conservative substitutions may also be selected from: 1) alanine, glycine; 2) aspartic acid, glutamic acid; 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 [Table 1]

[0205] Important amino acids in naturally occurring polypeptides can be identified by 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 resulting mutant molecules are tested for biological activity (e.g., phosphotriesterase or arylesterase activity for PON1 variants, or nuclease activity for DNase1 variants) to identify amino acid residues important for the activity of the molecule. Furthermore, relevant protein interaction sites can be determined by analyzing crystal structures determined by techniques such as nuclear magnetic resonance, crystallography, or photoaffinity labeling. The identity of important amino acids can also be inferred from the analysis of homology with related proteins (e.g., species orthologs that retain the same protein function).

[0206] 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 a method for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptides, and then sequencing the mutagenized polypeptides to determine the range of substitutions that can be tolerated at each position. Another method that can be used is region-specific mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0207] Variant nucleotide and polypeptide sequences can also be generated through DNA shuffling (see, e.g., Stemmer, Nature 370:389, 1994; Stemmer, Proc. Nat'l Acad. Sci. USA 91:10747, 1994; International PCT Publication No. WO97 / 20078). Briefly, variant DNA molecules are generated by in vitro homologous recombination, which involves random fragmentation of parental DNA, followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parental 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 deleterious changes.

[0208] As previously discussed, polypeptide fusions according to the present invention may contain polypeptide segments corresponding to "functional fragments" of a particular polypeptide. Conventional deletion analysis of nucleic acid molecules can be performed to obtain functional fragments of nucleic acid molecules encoding a given polypeptide. As an illustration, a PON1-encoding DNA molecule having the nucleotide sequence from residues 46 to 1099 can be digested with Bal31 nuclease to obtain a series of nested deletions. The fragments are then inserted into an expression vector in the appropriate reading frame, and the expressed polypeptide is 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 the production of the desired fragment. Alternatively, specific fragments of a gene encoding a polypeptide can be synthesized using the polymerase chain reaction.

[0209] Thus, using methods such as those discussed above, one skilled in the art can prepare a variety of polypeptides that (i) are substantially identical to a reference polypeptide (e.g., PON1 set forth at residues 16-355 or 26-355 of SEQ ID NO: 6, human wild-type DNase 1 set forth at residues 23-282 of SEQ ID NO: 16, or enhanced DNase 1 set forth at residues 21-280 of SEQ ID NO: 18), and (ii) retain the desired functional properties of the reference polypeptide.

[0210] Polypeptide segments used within the present invention (e.g., polypeptide segments corresponding to paraoxonase, DNase, RNase, or dimerization or FcRn-binding domains, such as Fc fragments) can be obtained from various species. When proteins are used therapeutically in humans, human polypeptide sequences are preferably used. However, non-human sequences, as well as variant sequences, can be used. For other uses, including in vitro diagnostic and veterinary uses, polypeptide sequences from humans or non-human animals can be used, although sequences from the same species as the patient may be preferred for in vivo veterinary use or for in vitro use if species specificity of intermolecular reactions exists. 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, and variants thereof.

[0211] In some embodiments, the paraoxonase segment is human paraoxonase 1 (PON1) 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 to amino acid residues 16-355 or 26-355 of SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:4, and (b) does not contain the amino-terminal leader sequence corresponding to residues 1-15 of SEQ ID NO:6 (also designated as residues 1-15 of SEQ ID NO:8 or SEQ ID NO:4). In some such embodiments, the biologically active paraoxonase has at least 96%, at least 97%, at least 98%, or at least 99% identity to 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:6, SEQ ID NO:8, or SEQ ID NO:4.

[0212] In some variations of the above PON1 or functional variant or fragment thereof, the amino acid at the position corresponding to Q192 of human paraoxonase 1 Q192 isoform (PON1-Q192; SEQ ID NO: 4) is lysine or arginine. In other, non-mutually exclusive variations, the paraoxonase contains at least one of the following amino acid substitutions compared to hPON1-Q192: (i) aspartic acid at the position corresponding to 18R; (ii) arginine or glycine at the position corresponding to N19; (iii) glutamine at the position corresponding to H20; (iv) lysine at the position corresponding to Q21; (v) glutamic acid or phenylalanine at the position corresponding to Y24; (vi) phenylalanine at the amino acid corresponding to L28; (vii) valine at the position corresponding to A30; (viii) histidine at the position corresponding to L31; (ix) threonine at the position corresponding to Q35; (x) valine at the position corresponding to I48; (xi) aspartic acid at the position corresponding to E49; (xii) asparagine at the position corresponding to T50; (xiii) leucine or isoleucine at the position corresponding to M55; (xiv) at the position corresponding to L69 (xv) a methionine at a position corresponding to K75; (xvi) an aspartic acid at a position corresponding to N78; (xvii) an aspartic acid at a position corresponding to N80; (xviii) a lysine at a position corresponding to S81; (xix) a serine at a position corresponding to P82; (xx) a valine at a position corresponding to T96; (xxi) a serine at a position corresponding to L98; (xxii) a glutamic acid at a position corresponding to G101; (xxiii) an asparagine at a position corresponding to S105; (xxiv) a threonine at a position corresponding to K106; (xxv) a leucine at a position corresponding to F107; (xxvi) an isoleucine at a position corresponding to V109; (xxvii) a threonine at a position corresponding to S111; (xxviii) a tryptophan or alanine at a position corresponding to H115; (xxix) a threonine at a position corresponding to A126; (xxx) a valine at a position corresponding to M127;(xxxi) arginine at the position corresponding to H134; (xxxii) glutamine at the position corresponding to D136; (xxxiii) serine at the position corresponding to A137; (xxxiv) serine at the position corresponding to K138; (xxxv) valine at the position corresponding to L143; (xxxvi) serine at the position corresponding to N166; (xxxvii) valine at the position corresponding to L167; (xxxviii) alanine at the position corresponding to G180; (xxxix) glutamic acid at the position corresponding to Y185; (xl) (xli) a glutamine at the position corresponding to F186; (xli) a lysine or alanine at the position corresponding to L187; (xlii) a lysine at the position corresponding to Y190; (xxliii) a glutamine at the position corresponding to L191; (xliv) a lysine at the position corresponding to Q192; (xlv) a lysine at the position corresponding to W194; (xlvi) a histidine at the position corresponding to Y197; (xlvii) a glutamic acid at the position corresponding to L198; (xlviii) a glutamine at the position corresponding to L200; (xlix) W2 (l) a lysine at the position corresponding to Y204; (li) a phenylalanine at the position corresponding to V206; (lii) an asparagine at the position corresponding to S211; (liii) an aspartic acid at the position corresponding to E212; (liv) a serine or methionine at the position corresponding to F222; (lv) an aspartic acid at the position corresponding to N265; (lvi) a valine at the position corresponding to E276; (lvii) a glutamine at the position corresponding to M289; (lviii) a serine or methionine at the position corresponding to I291 (lix) glutamic acid or tyrosine at the position corresponding to F293; (lx) proline at the position corresponding to S296; (lxi) lysine at the position corresponding to E297; (lxii) glycine at the position corresponding to A301; (lxiii) aspartic acid at the position corresponding to N309; (lxiv) serine at the position corresponding to T312; (lxv) valine at the position corresponding to Q319; (lxvi) serine at the position corresponding to T332; and alanine at the position corresponding to S335.

[0213] In some variations, the paraoxonase is a variant of human PON1 identified as G3C9 (Aharoni et al., Proc. Natl. Acad. Sci. USA 101:482-487, 2004; Harel et al., Nat. Struct. Mol. Biol. 11:412-419, 2004; Mukherjee and Gupta, J. Toxicol. 2020:1-16, 2020; Goldsmit et al., Chemistry and Biology 19:456-466, 2012), or a functional variant or fragment thereof. GC39 contains 55 amino acid substitutions from the human isoform, improving catalytic activity against nerve agents and improving soluble expression in bacteria. The full-length form of this paraoxonase sequence variant is listed in SEQ ID NO: 123 (nucleotide) and SEQ ID NO: 124 (amino acid). In some embodiments, GC39 paraoxonase or functional variants thereof for use in accordance with the present invention have at least 90% or at least 95% identity to amino acid residues 16-355 or 26-355 of SEQ ID NO: 124; in some such embodiments, GC39 paraoxonase or functional variants thereof have at least 96%, at least 97%, at least 98%, or at least 99% identity to 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: 124. PON1 fusions comprising G3C9 or an active variant thereof (e.g., M-IIG1, discussed below) may be particularly useful in some short-term therapeutic applications, such as treating exposure to sulfur mustard gas or organophosphates.

[0214] In some embodiments of the fusion polypeptide, the paraoxonase is a modified or further variant form of GC39 identified as IIG1 (also referred to herein as M-IIG1; see Goldsmith et al., Chemistry & Biology 19, 456-466, 2012), or a functional variant or fragment thereof. The full-length sequence of M-IIG1 is set forth in SEQ ID NO: 125 (nucleotide) and SEQ ID NO: 126 (amino acid). In some embodiments, the M-IIG1 paraoxonase or functional variant thereof for use in accordance with the invention has at least 90% or at least 95% identity to amino acid residues 16-355 or 26-355 of SEQ ID NO:126; in some such embodiments, the M-IIG1 paraoxonase or functional variant thereof has at least 96%, at least 97%, at least 98%, or at least 99% identity to 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:126.

[0215] In more specific variations, the paraoxonase has an amino acid sequence selected from (i) residues 16-355 of SEQ ID NO:6, (ii) residues 17-355 of SEQ ID NO:6, (iii) residues 18-355 of SEQ ID NO:6, (iv) residues 19-355 of SEQ ID NO:6, (v) residues 20-355 of SEQ ID NO:6, (vi) residues 21-355 of SEQ ID NO:6, (vii) residues 22-355 of SEQ ID NO:6, (viii) residues 23-355 of SEQ ID NO:6, (ix) residues 24-355 of SEQ ID NO:6, (x) residues 25-355 of SEQ ID NO:6, and (xi) residues 26-355 of SEQ ID NO:6 (i.e., an amino acid sequence selected from residues n-355 of SEQ ID NO:6, where n is an integer between 16 and 26, inclusive, inclusive). In other variations, the paraoxonase has an amino acid sequence selected from (i) residues 16-355 of SEQ ID NO:8, (ii) residues 17-355 of SEQ ID NO:8, (iii) residues 18-355 of SEQ ID NO:8, (iv) residues 19-355 of SEQ ID NO:8, (v) residues 20-355 of SEQ ID NO:8, (vi) residues 21-355 of SEQ ID NO:8, (vii) residues 22-355 of SEQ ID NO:8, (viii) residues 23-355 of SEQ ID NO:8, (ix) residues 24-355 of SEQ ID NO:8, (x) residues 25-355 of SEQ ID NO:8, and (xi) residues 26-355 of SEQ ID NO:8 (i.e., an amino acid sequence selected from residues n-355 of SEQ ID NO:8, where n is an integer between 16 and 26, inclusive, inclusive). In still other variations, the paraoxonase has an amino acid sequence selected from (i) residues 16-355 of SEQ ID NO:4, (ii) residues 17-355 of SEQ ID NO:4, (iii) residues 18-355 of SEQ ID NO:4, (iv) residues 19-355 of SEQ ID NO:4, (v) residues 20-355 of SEQ ID NO:4, (vi) residues 21-355 of SEQ ID NO:4, (vii) residues 22-355 of SEQ ID NO:4, (viii) residues 23-355 of SEQ ID NO:4, (ix) residues 24-355 of SEQ ID NO:4, (x) residues 25-355 of SEQ ID NO:4, and (xi) residues 26-355 of SEQ ID NO:4 (i.e., an amino acid sequence selected from residues n-355 of SEQ ID NO:4, where n is an integer between 16 and 26, inclusive, inclusive).In still other variations, the paraoxonase has an amino acid sequence selected from (i) residues 16-355 of SEQ ID NO:124, (ii) residues 17-355 of SEQ ID NO:124, (iii) residues 18-355 of SEQ ID NO:124, (iv) residues 19-355 of SEQ ID NO:124, (v) residues 20-355 of SEQ ID NO:124, (vi) residues 21-355 of SEQ ID NO:124, (vii) residues 22-355 of SEQ ID NO:124, (viii) residues 23-355 of SEQ ID NO:124, (ix) residues 24-355 of SEQ ID NO:124, (x) residues 25-355 of SEQ ID NO:124, and (xi) residues 26-355 of SEQ ID NO:124 (i.e., an amino acid sequence selected from residues n-355 of SEQ ID NO:124, where n is an integer between 16 and 26, inclusive, inclusive). In yet another variation, the paraoxonase has an amino acid sequence selected from (i) residues 16-355 of SEQ ID NO:126, (ii) residues 17-355 of SEQ ID NO:126, (iii) residues 18-355 of SEQ ID NO:126, (iv) residues 19-355 of SEQ ID NO:126, (v) residues 20-355 of SEQ ID NO:126, (vi) residues 21-355 of SEQ ID NO:126, (vii) residues 22-355 of SEQ ID NO:126, (viii) residues 23-355 of SEQ ID NO:126, (ix) residues 24-355 of SEQ ID NO:126, (x) residues 25-355 of SEQ ID NO:126, and (xi) residues 26-355 of SEQ ID NO:126 (i.e., an amino acid sequence selected from residues n-355 of SEQ ID NO:126, where n is an integer between 16 and 26, inclusive, inclusive).

[0216] In some embodiments of a fusion polypeptide comprising DNase as the first biologically active polypeptide, the DNase is human wild-type DNase 1 or a functional variant or fragment thereof. For example, in some embodiments, the DNase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to (i) amino acid residues 23-282 of SEQ ID NO: 16, (ii) amino acid residues 21-280 of SEQ ID NO: 18, (iii) amino acid residues 21-280 of SEQ ID NO: 20, or (iv) amino acid residues 21-280 of SEQ ID NO: 152. In more specific embodiments, the DNase comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to (i) amino acid residues 23-282 of SEQ ID NO: 16, (ii) amino acid residues 21-280 of SEQ ID NO: 18, (iii) amino acid residues 21-280 of SEQ ID NO: 20, or (iv) amino acid residues 21-280 of SEQ ID NO: 152.

[0217] In certain preferred embodiments, the DNase segment is a hyperactive and / or actin-resistant variant of naturally occurring DNase 1 ("enhanced DNase 1"), such as an enhanced human DNase 1 variant. For example, in some variations, the DNase contains at least one amino acid substitution at a position corresponding to an amino acid in mature wild-type human DNase 1 (SEQ ID NO: 120) selected from N74, G105, and A114, such that (1) the amino acid substitution at a position corresponding to N74 of human DNase 1 (the N74 substitution), when present, increases DNA binding of the DNase compared to human DNase 1, (2) the amino acid substitution at a position corresponding to G105 of human DNase 1 (the G105 substitution), when present, increases DNA binding of the DNase compared to human DNase 1, and (3) the amino acid substitution at a position corresponding to A114 of human DNase 1 (the A114 substitution), when present, reduces G-actin-induced inhibition of endonuclease activity of the DNase compared to human DNase 1. Suitable amino acids for substitution at these positions include lysine at the position corresponding to N74 of human DNase 1, arginine at the position corresponding to G105 of human DNase 1, and / or phenylalanine at the position corresponding to A114 of human DNase 1. In some variations, the enhanced human DNase 1 variant contains at least two of the N74, G105, and A114 substitutions (e.g., at least substitutions at positions corresponding to both N74 and G105 or both G105 and A114 of human DNase 1). In some embodiments, the enhanced human DNase 1 contains each of the N74, G105, and A114 substitutions. In other variations, the enhanced human DNase 1 contains the N74 and G105 substitutions, but not the A114 substitution.In certain embodiments, the enhanced DNase 1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to (i) amino acid residues 21-280 of SEQ ID NO: 18, (ii) amino acid residues 21-280 of SEQ ID NO: 20, or (iii) residues 21-280 of SEQ ID NO: 152. In some variations, the enhanced DNase 1 comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid residues 21-280 of SEQ ID NO: 18, wherein the enhanced DNase 1 contains each of the N74, G105, and A114 substitutions, but does not contain any other amino acid substitutions that increase DNA binding of DNase compared to human DNase 1 and / or that decrease G-actin-induced inhibition of endonuclease activity of DNase compared to human DNase 1. In other variations, the enhanced DNase 1 comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid residues 21-280 of SEQ ID NO: 152, wherein the enhanced DNase 1 contains each of the N74 and G105 substitutions, but does not contain any other amino acid substitutions that increase DNA binding of DNase compared to human DNase 1, and / or does not contain the A114 substitution or any other amino acid substitutions that decrease G-actin-induced inhibition of DNase endonuclease activity compared to human DNase 1. Fusions comprising enhanced DNase that lack actin resistance are particularly beneficial for allowing higher expression of the fusion molecule and for reduced toxicity to host cells (e.g., CHO).

[0218] In other embodiments of fusion polypeptides comprising DNase as the first biologically active polypeptide, the DNase is a truncated form of human wild-type DNase 1L3 lacking the carboxyl-terminal nuclear localization signal (referred to herein as NLS2, corresponding to amino acid residues 291-305 of SEQ ID NO: 136), or a functional variant or fragment of such a truncated form of human DNase 1L3. For example, in some embodiments, the DNase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to (i) amino acid residues 21-290 of SEQ ID NO: 136, (ii) amino acid residues 21-290 of SEQ ID NO: 138, or (iii) amino acid residues 21-290 of SEQ ID NO: 140. In more specific embodiments, the DNase comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to (i) amino acid residues 21-290 of SEQ ID NO: 136, (ii) amino acid residues 21-290 of SEQ ID NO: 138, or (iii) amino acid residues 21-290 of SEQ ID NO: 140. In certain preferred embodiments, including truncated forms of human DNase 1L3 or functional variants thereof, the DNase is a variant DNase 1L3 containing one or more amino acid substitutions that inactivate a nuclear localization signal in the N-terminal half of the molecule (referred to herein as NLS1, corresponding to amino acid residues 80-96 of SEQ ID NO: 136) compared to wild-type human DNase 1L3 (SEQ ID NO: 136). In some such embodiments, each of the amino acids at positions corresponding to R80, R95, and N96 of SEQ ID NO: 136 is alanine or serine (e.g., each of R80, R95, and N96 is alanine, or each of R80, R95, and N96 is serine). In more specific embodiments of the above truncated forms of DNase 1L3 variants in which the wild-type NLS1 consensus sequence has been removed, the DNase has the amino acid sequence set forth in (i) amino acid residues 21-290 of SEQ ID NO: 138 or (ii) amino acid residues 21-290 of SEQ ID NO: 140.

[0219] In some embodiments of fusion polypeptides comprising an RNase as the first biologically active polypeptide, the RNase is human wild-type RNase 1 or a functional variant or fragment thereof. For example, in some embodiments, the RNase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to amino acid residues 29-156 of SEQ ID NO: 22. In more particular variations, the RNase comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acid residues 29-156 of SEQ ID NO: 22.

[0220] In some embodiments of fusion polypeptides comprising superoxide dismutase 1 (SOD1) as the first biologically active polypeptide, SOD1 is human wild-type SOD1 or a functional variant or fragment thereof. For example, in some embodiments, SOD1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to amino acid residues 2-154 of SEQ ID NO: 32. In more particular variations, SOD1 comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acid residues 2-154 of SEQ ID NO: 32. In certain variations, the amino acid at the position corresponding to C7 of human SOD1 (SEQ ID NO:32) is not a cysteine ​​and / or the amino acid at the position corresponding to C112 of human SOD1 is not a cysteine, and in some such embodiments, the amino acid at the position corresponding to C7 of human SOD1 is alanine and / or the amino acid at the position corresponding to C112 of human SOD1 is serine. In more specific variations, SOD1 comprises the amino acid sequence set forth in residues 2-154 of SEQ ID NO:32 (also set forth as residues 23-175 of SEQ ID NO:52 or residues 23-175 of SEQ ID NO:54), in which the amino acid at position C7 is changed to alanine and the amino acid at position C112 is serine.

[0221] In some embodiments of fusion polypeptides comprising a CTLA-4 extracellular domain as the first biologically active polypeptide, 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 to amino acid residues 21-144 of SEQ ID NO: 66. 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 to amino acid residues 21-144 of SEQ ID NO: 66.

[0222] In some embodiments of fusion polypeptides comprising a CD40 extracellular domain as the first biologically active polypeptide, 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 to (i) residues 21-188 of SEQ ID NO:74, (ii) residues 21-188 of SEQ ID NO:78, (iii) residues 21-188 of SEQ ID NO:82, (iv) residues 21-188 of SEQ ID NO:86, or (v) residues 21-188 of SEQ ID NO:90. In more specific embodiments, the CD40 extracellular domain comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity to (i) residues 21-188 of SEQ ID NO: 74, (ii) residues 21-188 of SEQ ID NO: 78, (iii) residues 21-188 of SEQ ID NO: 82, (iv) residues 21-188 of SEQ ID NO: 86, or (v) residues 21-188 of SEQ ID NO: 90.

[0223] 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: 68) selected from E64, K81, P85, and L121, which at least one amino acid substitution increases CD40 ligand binding compared to human CD40. Particularly suitable amino acid substitutions 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 L121 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 least at positions corresponding to K81 and L121 of human CD40, or substitutions at least at positions corresponding to E64, K81, and P85 of human CD40). In some embodiments, the amino acid at the position corresponding to K81 in human CD40 is selected from threonine, histidine, and serine. In other embodiments, the amino acid at the position corresponding to K81 in human CD40 is histidine, and the amino acid at the position corresponding to L121 in human CD40 is proline. In still other embodiments, the amino acid at the position corresponding to E64 in human CD40 is tyrosine, the amino acid at the position corresponding to K81 in human CD40 is threonine, and the amino acid at the position corresponding to P85 in human CD40 is tyrosine. In specific variations, the CD40 extracellular domain comprises the amino acid sequence set forth in (i) residues 21-188 of SEQ ID NO:74, (ii) residues 21-188 of SEQ ID NO:78, (iii) residues 21-188 of SEQ ID NO:82, (iv) residues 21-188 of SEQ ID NO:86, or (v) residues 21-188 of SEQ ID NO:90.

[0224] In some embodiments of fusion polypeptides comprising as a first biologically active polypeptide a polypeptide that specifically binds to and neutralizes an inflammatory cytokine, the inflammatory cytokine is a member of the tumor necrosis factor (TNF) superfamily, a member of the transforming growth factor (TGF) superfamily, an interleukin, a chemokine, a colony-stimulating factor (CSF), or an interferon. In more specific embodiments, the inflammatory cytokine is tumor necrosis factor alpha (TNFα), transforming growth factor-beta (TGF-β), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-17A (IL-17A), interleukin-17F (IL-17F), or a combination thereof. 17F), interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-31 (IL-31), interleukin-33 (IL-33), interleukin-36 alpha (IL-36α), interleukin-36 beta (IL-36β), interleukin IL-36 gamma (IL-36γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon gamma (IFNγ). In some variations, the polypeptide that specifically binds and neutralizes the inflammatory cytokine is an antibody. In alternative variations, the polypeptide that specifically binds and neutralizes the inflammatory cytokine is an alternative scaffold protein.

[0225] In certain embodiments of a fusion polypeptide comprising a polypeptide that specifically binds and neutralizes TNFα (an "anti-TNFα polypeptide"), the anti-TNFα polypeptide competes for binding to TNFα with an antibody having the same heavy and light chain variable domains (VH and VL) as the anti-TNFα monoclonal antibody adalimumab. The amino acid sequences of adalimumab VH and VL are set forth herein in SEQ ID NO: 108 and SEQ ID NO: 110, respectively (GenBank Accession Nos. LQ961187 and LQ961186 for the heavy and light chain variable regions, respectively; see also International PCT Publication No. WO2014 / 159579).

[0226] In certain embodiments of a fusion polypeptide comprising a polypeptide that specifically binds to and neutralizes TGF-β (an "anti-TGF-β polypeptide"), the anti-TGF-β polypeptide competes for binding to TGF-β with an antibody having the same heavy and light chain variable domains (VH and VL) as the anti-TGF-β monoclonal antibody fresolimumab. The amino acid sequences of fresolimumab VH and VL are set forth herein in SEQ ID NO: 112 and SEQ ID NO: 114, respectively (GenBank Accession Nos. JC232803.1 and JC232805 for the heavy and light chain variable regions, respectively; see also International PCT Publication No. WO2013 / 065869).

[0227] The ability of a binding protein to compete with an antibody having the VH and VL domains of adalimumab or fresolimumab for binding to TNFα or TGF-β, respectively, can be determined by an assay in which a test TNFα binding protein (e.g., an antibody) or functional binding fragment thereof prevents or inhibits the specific binding to TNFα of a reference antibody having the VH and VL domains of adalimumab (i.e., having the VH and VL domains of SEQ ID NO: 108 and SEQ ID NO: 110, respectively), or an assay in which a test TNF-β binding protein (e.g., an antibody) or functional binding fragment thereof prevents or inhibits the specific binding to TNF-β of a reference antibody having the VH and VL domains of fresolimumab (i.e., having the VH and VL domains of SEQ ID NO: 112 and SEQ ID NO: 114, respectively). Typically, such assays involve the use of purified target protein or cells bearing the target protein bound to a solid surface, an unlabeled test protein (i.e., a TNFα- or TGF-β-binding protein, or a candidate TNFα- or TGF-β-binding protein), and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test protein. Typically, the test protein is present in excess and / or allowed to bind first. Soluble TNFα- or TGF-β-binding proteins identified by competitive assays ("competing TNFα- or TGF-β-binding proteins") include proteins that bind to the same epitope bound by the reference antibody, proteins that bind to epitopes that overlap with the epitope bound by the reference antibody, and proteins that bind to epitopes that are sufficiently close to the epitope bound by the reference antibody to create steric hindrance.Typically, when a competing TNFα-binding or TGF-β-binding protein (e.g., a competing anti-TNFα or anti-TGF-β antibody) is present in excess, it will inhibit specific binding of a reference antibody to a TNFα or TGF-β target protein by at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, and in some cases, binding is inhibited by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or more. Conversely, if the reference antibody is bound, it will preferably inhibit binding of a subsequently added competing TNFα-binding or TGF-β-binding protein (e.g., a competing anti-TNFα or anti-TGF-β antibody) by at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, and in some cases binding is inhibited by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or more.

[0228] In certain preferred embodiments, the polypeptide specifically binding to and neutralizing inflammatory cytokines for use in the present invention is a single-chain antibody. Particularly suitable single-chain antibodies include single-chain Fvs (scFvs) and single-domain antibodies (sdAbs). Single-chain antibodies can be derived, for example, from an identified intact native monoclonal antibody or antibody fragment with the desired specificity. Methods for preparing and isolating monoclonal antibodies and their antigen-binding antibody fragments are well known in the art. See, for example, Current Protocols in Immunology (Cooligan et al. eds., John Wiley and Sons, Inc. 2006); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, NY, 2nd ed. 1989); and Monoclonal Hybridoma Antibodies: Techniques and Applications (Hurrell ed., CRC Press, Inc., Boca Raton, FL, 1982). Antigen-binding fragments, including scFvs, can be prepared, for example, using phage display libraries according to methods known in the art. As will be apparent to those skilled in the art, these methods are equally applicable to the production of antibodies against inflammatory cytokines for use according to the present invention.

[0229] The amino acid sequence of native antibody variable regions can be altered by applying recombinant DNA technology. Thus, antibodies can be redesigned to obtain desired characteristics. Typically, the variable region will be altered to improve antigen binding characteristics, improve the stability of the variable region, or reduce the risk of immunogenicity. Phage display technology can also be used. See, for example, Huse et al., Science 246:1275-1281, 1989; Ladner et al., U.S. Patent No. 5,571,698.

[0230] For therapeutic antibodies intended for human use, it is usually desirable to humanize the non-human regions of the antibody according to known procedures. Methods for producing humanized antibodies are disclosed, for example, in U.S. Patent Nos. 5,530,101, 5,821,337, 5,585,089, 5,693,762, and 6,180,370. Methods for producing humanized antibodies are also disclosed, for example, in U.S. Patent No. 7,732,578. Typically, a humanized anti-inflammatory cytokine antibody comprises the complementarity-determining regions (CDRs) of a mouse donor immunoglobulin and the heavy and light chain frameworks of a human acceptor immunoglobulin. In many cases, framework residues in the human framework regions will be replaced with corresponding residues from the donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions (see Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323, 1988).

[0231] The present invention also encompasses the use of fully human antibodies, such as those selected from human antibody expression libraries (e.g., phage display libraries); those made in human heavy chain loci and human light chain loci transgenic non-human animals (e.g., mice) in which the corresponding endogenous immunoglobulin loci have been inactivated; or those prepared by immortalizing human B lymphocytes that produce antibodies against inflammatory cytokine target antigens.

[0232] Antibodies for use according to the invention may be, for example, 5 x 10 -4 Under M, 10 -4 Less than M, 5 x 10 -5 Under M, 10 -5 Less than M, 5 x 10 -6 Under M, 10-6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Under M, 10 -9 Less than M, 5 x 10 -10 Under M, 10 -10 Less than M, 5 x 10 -11 Under M, 10 -11 Less than M, 5 x 10 -12 Under M, 10 -12 Less than M, 5 x 10 -13 Under M, 10 -13 Less than M, 5 x 10 -14 Under M, 10 -14 Less than M, 5 x 10 -15 Less than M or 10 -15 The dissociation constant (K d ) have binding affinity.

[0233] Antibodies for use according to the present invention include variants with one or more amino acid substitutions, deletions, or additions compared to a reference antibody, such that the variants retain one or more biological properties of the reference antibody. In certain embodiments, the antibody is a variant with one or more amino acid substitutions, deletions, or additions compared to a reference anti-TNFα neutralizing antibody having the VH and / or VL sequences as set forth in SEQ ID NO: 108 and SEQ ID NO: 110, respectively, such that the antibody retains the reference antibody's ability to specifically bind to TNFα and neutralize TNFα activity. In other embodiments, the antibody is a variant with one or more amino acid substitutions, deletions, or additions compared to a reference anti-TNF-β neutralizing antibody having the VH and / or VL sequences as set forth in SEQ ID NO: 112 and SEQ ID NO: 114, respectively, such that the antibody retains the reference antibody's ability to specifically bind to TNF-β and neutralize TNF-β activity. Those skilled in the art can easily generate variants with one or more amino acid substitutions, deletions, or additions compared to a reference antibody. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical and enzymatic techniques, are known to those skilled in the art.

[0234] In some embodiments of the above-described fusion polypeptides comprising a single-chain antibody that specifically binds and neutralizes TNFα ("anti-TNFα single-chain antibody"), the anti-TNFα single-chain antibody comprises one or more CDRs of the anti-TNFα monoclonal antibody adalimumab. Thus, in certain variations, the anti-TNFα single-chain antibody comprises the heavy chain CDRs (at least one of the CDR-H1, CDR-H2, and CDR-H3 regions) of the adalimumab VH domain (SEQ ID NO: 108) and / or the light chain CDRs (at least one of the CDR-L1, CDR-L2, and CDR-L3 regions) of the adalimumab VL domain (SEQ ID NO: 110). In typical embodiments, the anti-TNFα single-chain antibody has two or three CDRs of the adalimumab VH domain (SEQ ID NO: 108) and / or two or three CDRs of the adalimumab VL domain (SEQ ID NO: 110). In some variations in which the anti-TNFα single chain antibody has at least one CDR of an adalimumab VH domain, the antibody further comprises at least one CDR of an adalimumab VL domain, and in some such embodiments, the antibody has all three heavy chain CDRs and all three light chain CDRs of adalimumab (i.e., CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 108, and CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 110). One or more CDRs may be defined, for example, according to the Chothia definition, the Kabat definition, the AbM definition, the IMGT database definition, or the contact definition of a CDR. According to the IMGT database definition of CDRs, CDR-H1, CDR-H2, and CDR-H3 of adalimumab correspond to residues 26-33, 50-59, and 97-110, respectively, of SEQ ID NO: 108, and CDR-L1, CDR-L2, and CDR-L3 of adalimumab correspond to residues 27-32, 50-52, and 89-97, respectively, of SEQ ID NO: 110. According to the Kabat definition of CDRs, CDR-H1, CDR-H2, and CDR-H3 of adalimumab correspond to residues 31-35, 50-66, and 99-110, respectively, of SEQ ID NO: 108, and CDR-L1, CDR-L2, and CDR-L3 of adalimumab correspond to residues 24-34, 50-56, and 89-97, respectively, of SEQ ID NO: 110.In particular variations of the above anti-TNFα single chain antibodies, the single chain antibodies comprise a VH domain and / or a VL domain with a human immunoglobulin framework region, or a variant thereof having at least 85%, at least 90%, or at least 95% amino acid sequence identity to a human immunoglobulin framework region.

[0235] In some embodiments, the anti-TNFα single chain antibody comprises (a) a heavy chain variable domain that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 108, and / or (b) a light chain variable domain that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 110.

[0236] In some embodiments, the anti-TNFα single chain antibody for use according to the invention comprises the heavy chain CDR CDR-H1 TNFα , CDR-H2 TNFα , and CDR-H3 TNFα and this set of VH CDRs has 10 or fewer amino acid substitutions compared to the set of reference CDRs CDR-H1, CDR-H2 and CDR-H3 of the adalimumab VH domain (SEQ ID NO: 108). In another non-mutually exclusive embodiment, the anti-TNFα single chain comprises the light chain CDRs CDR-L1 TNFα , CDR-L2 TNFα , and CDR-L3 TNFαand this set of VL CDRs has 10 or fewer amino acid substitutions compared to the reference set of CDRs CDR-L1, CDR-L2, and CDR-L3 of the adalimumab VL domain (SEQ ID NO: 110). In certain embodiments, the anti-TNFα single chain antibody comprises both the above sets of heavy and light chain CDRs. Particularly preferred anti-TNFα single chain antibodies comprise CDRs CDR-H1 TNFα , CDR-H2 TNFα , and CDR-H3 TNFα a heavy chain variable domain comprising CDR CDR-L1 TNFα , CDR-L2 TNFα , and CDR-L3 TNFα and a light chain variable domain comprising: In particular variations of the above anti-TNFα antibodies, each of the VH and VL domains comprises a human immunoglobulin framework region or a variant thereof having at least 85%, at least 90%, or at least 95% amino acid sequence identity to a human immunoglobulin framework region.

[0237] In some embodiments of the above-described fusion polypeptides comprising a single-chain antibody that specifically binds and neutralizes TGF-β ("anti-TGF-β single-chain antibody"), the anti-TGF-β single-chain antibody comprises one or more CDRs of the anti-TGF-β monoclonal antibody fresolimumab. Thus, in certain variations, the anti-TGF-β single-chain antibody comprises the heavy chain CDRs (at least one of the CDR-H1, CDR-H2, and CDR-H3 regions) of the fresolimumab VH domain (SEQ ID NO: 112) and / or the light chain CDRs (at least one of the CDR-L1, CDR-L2, and CDR-L3 regions) of the fresolimumab VL domain (SEQ ID NO: 114). In typical embodiments, the anti-TGF-β single-chain antibody has two or three CDRs of the fresolimumab VH domain (SEQ ID NO: 112) and / or two or three CDRs of the fresolimumab VL domain (SEQ ID NO: 114). In some variations in which the anti-TGF-β single chain antibody has at least one CDR of the fresolimumab VH domain, the antibody further comprises at least one CDR of the fresolimumab VL domain, and in some such embodiments, the antibody has all three heavy chain CDRs and all three light chain CDRs of fresolimumab (i.e., CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 112, and CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 114). One or more CDRs may be defined, for example, according to the Chothia definition, the Kabat definition, the AbM definition, the IMGT database definition, or the contact definition of a CDR. According to the Kabat definition of CDRs, CDR-H1, CDR-H2, and CDR-H3 of fresolimumab correspond to residues 31-35, 50-66, and 99-106, respectively, of SEQ ID NO: 112, and CDR-L1, CDR-L2, and CDR-L3 of fresolimumab correspond to residues 24-40, 56-62, and 95-102, respectively, of SEQ ID NO: 114.In particular variations of the above anti-TGF-β single chain antibodies, the single chain antibodies comprise a VH domain and / or a VL domain having a human immunoglobulin framework region, or a variant thereof having at least 85%, at least 90%, or at least 95% amino acid sequence identity to a human immunoglobulin framework region.

[0238] In some embodiments, the anti-TGF-β single chain antibody comprises (a) a heavy chain variable domain that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 112, and / or (b) a light chain variable domain that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 114.

[0239] In some embodiments, the anti-TGF-β single chain antibody for use according to the invention comprises the heavy chain CDR CDR-H1 TGF-β , CDR-H2 TGF-β , and CDR-H3 TGF-β and this set of VH CDRs has 10 or fewer amino acid substitutions compared to the set of reference CDRs CDR-H1, CDR-H2 and CDR-H3 of the fresolimumab VH domain (SEQ ID NO: 112). In other non-mutually exclusive embodiments, the anti-TGF-β single chain comprises the light chain CDRs CDR-L1 TGF-β , CDR-L2 TGF-β , and CDR-L3 TGF-βand this set of VL CDRs has 10 or fewer amino acid substitutions compared to the reference set of CDRs CDR-L1, CDR-L2, and CDR-L3 of the fresolimumab VL domain (SEQ ID NO: 114). In certain embodiments, the anti-TGF-β single chain antibody comprises both the above sets of heavy and light chain CDRs. Particularly suitable anti-TGF-β single chain antibodies comprise CDRs CDR-H1 TGF-β , CDR-H2 TGF-β , and CDR-H3 TGF-β a heavy chain variable domain comprising CDR CDR-L1 TGF-β , CDR-L2 TGF-β , and CDR-L3 TGF-β and a light chain variable domain comprising: In particular variations of the above anti-TGF-β antibodies, each of the VH and VL domains comprises a human immunoglobulin framework region or a variant thereof having at least 85%, at least 90%, or at least 95% amino acid sequence identity to a human immunoglobulin framework region.

[0240] Anti-inflammatory cytokine antibodies for use according to the present invention include affinity matured embodiments. Affinity matured antibodies can be produced by procedures known in the art (see, e.g., Marks et al., Bio / Technology 10:779-783, 1992; Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813, 1994; Schier et al., Gene 169:147-155, 1995; Yelton et al., J. Immunol. 155:1994-2004, 1995; Jackson et al., J. Immunol. 154:3310-9, 1995; Hawkins et al., J. Mol. Biol. 226:889-896, 1992; and PCT Publication No. WO2004 / 058184). One method for adjusting antibody affinity is called "library scanning mutagenesis." Library scanning mutagenesis is generally performed as follows: One or more amino acid positions within at least one CDR (e.g., within two, three, four, five, or six CDRs) are replaced with two or more (e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty) amino acids using art-recognized methods. This generates a small library of clones (in some embodiments, one for each amino acid position analyzed), each with a complexity of two or more members (when two or more amino acids are substituted per position). Generally, the library also includes clones containing the native (unsubstituted) amino acid. A small number of clones from each library, e.g., about 20-80 clones (depending on the complexity of the library), are screened for binding affinity to the target polypeptide (or other binding target), and candidates with increased, identical, decreased, or no binding are identified.Binding affinity can be determined, for example, using Biacore™ surface plasmon resonance analysis, which detects differences in binding affinity of about 2-fold or greater. Biacore™ detects binding affinities where the starting antibody has a relatively high affinity, for example, a K of about 10 nM or less. D This is especially useful if you already have a binding in

[0241] In some embodiments of fusion polypeptides comprising an anti-TNFα single chain antibody as the first biologically active polypeptide, the anti-TNFα single chain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to (i) amino acid residues 21-268 of SEQ ID NO: 92 or (ii) amino acid residues 21-268 of SEQ ID NO: 96. In more particular variations, the anti-TNFα single chain antibody comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to (i) amino acid residues 21-268 of SEQ ID NO: 92 or (ii) amino acid residues 21-268 of SEQ ID NO: 96.

[0242] In some embodiments of fusion polypeptides comprising an anti-TGF-β single chain antibody as the first biologically active polypeptide, the anti-TGF-β single chain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to (i) amino acid residues 21-269 of SEQ ID NO: 100 or (ii) amino acid residues 21-269 of SEQ ID NO: 104. In more particular variations, the anti-TGF-β single chain antibody comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to (i) amino acid residues 21-269 of SEQ ID NO: 100 or (ii) amino acid residues 21-269 of SEQ ID NO: 104.

[0243] Polypeptide linkers for use according to the present invention can be naturally occurring, synthetic, or a combination of both. The linker connects two separate polypeptide regions (e.g., an Fc region and paraoxonase, or an Fc region and DNase) while maintaining the linked polypeptide regions as separate, distinct domains of a longer polypeptide. The linker can allow the separate, distinct domains to function together but maintain their separate properties (e.g., in the case of an Fc region linked to paraoxonase or DNase, Fc receptor (e.g., FcRn) binding to the Fc region can be maintained, while the functional properties of paraoxonase (e.g., organic phosphatase or arylesterase activity) or DNase (e.g., DNA binding and nuclease activity) will be maintained). For examples of the use of naturally occurring and 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.

[0244] Typically, the residues in 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 conformation in solution, although local stable regions are tolerated. Generally, small, polar, and hydrophilic residues are preferred, while bulky, hydrophobic residues are undesirable. Areas of local charge should be avoided. If the linker polypeptide contains charged residues, these residues will usually be positioned to impart a net neutral charge within a small region of the polypeptide. Therefore, it is preferable to place a charged residue adjacent to a residue of the opposite charge. Generally, preferred residues for inclusion in a linker polypeptide include Gly, Ser, Ala, Thr, Asn, and Gln, more preferred residues include Gly, Ser, Ala, and Thr, and most preferred residues are Gly and Ser. Generally, Phe, Tyr, Trp, Pro, Leu, Ile, Lys, and Arg residues will be avoided (except when present within the immunoglobulin hinge region of the linker), Pro residues will be avoided due to their hydrophobicity and lack of flexibility, and Lys and Arg residues will be avoided due to potential immunogenicity. The sequence of the linker will also be designed to avoid undesired proteolysis.

[0245] In certain embodiments, linker L1 comprises at least two or at least three amino acid residues (e.g., at least 5, at least 10, at least 16, at least 26, or at least 36 amino acid residues). In particular variations, L1 consists of 2 to 60 amino acid residues, 3 to 60 amino acid residues, 5 to 40 amino acid residues, or 15 to 40 amino acid residues. In other variations, L1 consists of 2 to 50, 2 to 40, 2 to 36, 2 to 35, 2 to 30, 2 to 26, 3 to 50, 3 to 40, 3 to 36, 3 to 35, 3 to 30, 3 to 26, 5 to 60, 5 to 50, 5 to 40, 5 to 36, 5 to 35, 5 to 30, 5 to 26, 10 to 60, 10 to 50, 10 to 40, 10 to 36, 10 to 35, 10 to 30, 10 to 26, 15 to 60, 15 to 50, 15 to 36, 15 to 35, 15 to 30, or 15 to 26 amino acid residues. In other variations, L1 consists of 16-60, 16-50, 16-40, or 16-36 amino acid residues. In still other variations, L1 consists of 20-60, 20-50, 20-40, 20-36, 25-60, 25-50, 25-40, or 25-36 amino acid residues. In still other variations, L1 consists of 26-60, 26-50, 26-40, or 26-36 amino acid residues. In more specific variations, L1 consists of 16, 21, 26, 31, or 36 amino acid residues. In some embodiments, L1 comprises or consists of the amino acid sequence set forth in residues SEQ ID NO:10, residues 268-288 of SEQ ID NO:12, or SEQ ID NO:14.

[0246] In typical variations of fusion polypeptides comprising DNase as the first biologically active polypeptide, L1 is present and comprises at least 15 amino acid residues. For example, the L1 linker joining the carboxyl terminus of DNase to the amino terminus of another fusion component (e.g., an immunoglobulin Fc region) can comprise at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 amino acid residues. In more specific variations, L1 consists of 15 to 60 amino acid residues, 15 to 51 amino acid residues, 15 to 50 amino acid residues, 15 to 46 amino acid residues, 15 to 45 amino acid residues, 15 to 41 amino acid residues, 15 to 40 amino acid residues, 15 to 36 amino acid residues, 26 to 60 amino acid residues, 26 to 51 amino acid residues, 26 to 50 amino acid residues, 26 to 46 amino acid residues, 26 to 45 amino acid residues, 26 to 41 amino acid residues, 26 to 40 amino acid residues, or 26 to 36 amino acid residues. In even more specific variations, L1 that links DNase consists of 26 amino acid residues, 27 amino acid residues, 28 amino acid residues, 29 amino acid residues, 30 amino acid residues, 31 amino acid residues, 32 amino acid residues, 33 amino acid residues, 34 amino acid residues, 35 amino acid residues, or 36 amino acid residues. In some embodiments, the L1 that links the DNase comprises or consists of the amino acid sequence set forth in SEQ ID NO:12 or SEQ ID NO:14.

[0247] In some variations of fusion polypeptides comprising RNase or SOD1 as the first biologically active polypeptide, L1 is present and comprises at least two amino acid residues. For example, the L1 linker joining the carboxyl terminus of RNase or SOD1 to the amino terminus of another fusion component (e.g., an immunoglobulin Fc region) can comprise at least three, at least five, or at least 10 amino acid residues. In more specific variations, L1 that links RNase or SOD1 consists of 2 to 60 amino acid residues, 2 to 51 amino acid residues, 2 to 50 amino acid residues, 2 to 46 amino acid residues, 2 to 45 amino acid residues, 2 to 41 amino acid residues, 2 to 40 amino acid residues, 2 to 36 amino acid residues, 10 to 60 amino acid residues, 10 to 51 amino acid residues, 10 to 50 amino acid residues, 10 to 46 amino acid residues, 10 to 45 amino acid residues, 10 to 41 amino acid residues, 10 to 40 amino acid residues, or 10 to 36 amino acid residues. In more specific variations, L1 that links RNase or SOD1 consists of 16 or 26 amino acid residues. In some embodiments, L1 that links RNase or SOD1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12.

[0248] In some variations of the fusion polypeptides comprising the formula P-L1-X described herein, L1 comprises at least 20 amino acid residues. For example, the L1 linker joining the carboxyl terminus of paraoxonase to the amino terminus of another fusion component (e.g., an immunoglobulin Fc region) can 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, L1 consists of 20 to 60 amino acid residues, 20 to 50 amino acid residues, 20 to 45 amino acid residues, 20 to 40 amino acid residues, 20 to 36 amino acid residues, 26 to 60 amino acid residues, 26 to 50 amino acid residues, 26 to 45 amino acid residues, 26 to 40 amino acid residues, 26 to 36 amino acid residues, 36 to 60 amino acid residues, 36 to 50 amino acid residues, 36 to 45 amino acid residues, or 36 to 40 amino acid residues. In some embodiments, L1 linking the carboxyl terminus of paraoxonase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 14.

[0249] Exemplary L2 linkers contain at least three amino acid residues and typically up to 60 amino acid residues. In certain variations, the L2 linker contains at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid residues. In more specific variations, L2 consists of 6-30, 6-25, 6-20, 7-30, 7-25, 7-20, 8-30, 8-25, 8-20, 9-30, 9-25, 9-20, 10-30, 10-25, 10-20, 11-30, 11-25, 11-20, 12-30, 12-25, or 12-20 amino acid residues. In some embodiments, L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:56.

[0250] In certain embodiments, the polypeptide linker comprises multiple glycine residues. For example, in some embodiments, the polypeptide linker (e.g., L1) comprises multiple glycine residues and optionally at least one serine residue. In certain variations, the polypeptide linker (e.g., L1) comprises the sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 29), such as two or more tandem repeats of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the linker comprises the sequence [Gly-Gly-Gly-Gly-Ser] n ([SEQ ID NO: 29] n ), wherein n is a positive integer, such as 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. n In a specific variation of a polypeptide linker comprising the formula [Gly-Gly-Gly-Gly-Ser], n is 4. n In another specific variation of a polypeptide linker comprising the formula [Gly-Gly-Gly-Gly-Ser], n is 6. n In yet another specific variation of a polypeptide linker comprising: n is 5. In certain embodiments, the polypeptide linker comprises a series of glycine and serine residues (e.g., [Gly-Gly-Gly-Gly-Ser]) inserted between two other sequences of the polypeptide linker (e.g., inserted between Asp-Leu-Ser at the N-terminus of the linker and Thr-Gly-Leu at the C-terminus of the linker). n , where n is defined above. In other embodiments, the polypeptide linker comprises glycine and serine residues (e.g., [Gly-Gly-Gly-Gly-Ser]) attached to one or both ends of another sequence of the polypeptide linker. n , where n is defined as above.

[0251] In some embodiments of the above fusion polypeptides in which X is present, X is a dimerization domain. Various dimerization domains are suitable for use with certain fusion polypeptide embodiments and dimeric fusion proteins as described herein. In certain embodiments of fusion polypeptides comprising a dimerization domain, the dimerization domain is an immunoglobulin heavy chain constant region. The immunoglobulin heavy chain constant region can be a native sequence constant region or a variant constant region. In a typical variant, the immunoglobulin heavy chain constant region can bind to the neonatal Fc receptor (FcRn) with sufficient affinity to provide the fusion polypeptide with improved half-life in vivo. A particularly suitable immunoglobulin heavy chain constant region for use according to 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).

[0252] In some embodiments of the fusion polypeptide comprising an immunoglobulin Fc region, the immunoglobulin Fc region is a human IgG Fc region with amino acid substitutions in the CH2 region relative to the wild-type human IgG sequence that prevent the molecule from being glycosylated, including, but not limited to, an amino acid substitution at N297 (Eu numbering of the human IgG heavy chain constant region) (corresponding to amino acid position 82 of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 116, or SEQ ID NO: 118). In another embodiment, the Fc region is human IgG1(γ1) with each of the three cysteines in the hinge region (C220, C226, C229) changed to a non-cysteine ​​residue (e.g., serine) and, optionally, with the proline at position 238 in the CH2 domain changed to a non-proline residue (e.g., serine or aspartic acid). In another embodiment, the Fc region is human γ1 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 a non-proline residue (e.g., serine or aspartic acid). In another embodiment, the Fc region is human γ1 with N297 changed to a non-asparagine residue (e.g., alanine, glutamine, or glycine). In another embodiment, the Fc region is human γ1 with one or more amino acid substitutions between EU positions 292-300. In another embodiment, the Fc region is human γ1 with one or more amino acid additions or deletions anywhere between residues 292-300. In another embodiment, the Fc region is human γ1 with an SCC hinge (i.e., cysteine ​​C220 is changed to serine and there is a cysteine ​​at each of EU positions 226 and 229) or with an SSS hinge (i.e., each of the three cysteines at EU positions 220, 226, and 229 are changed to serine). In a further embodiment, the Fc region is human γ1 with an SCC hinge and an amino acid substitution at P238. In another embodiment, the Fc domain is human γ1 with an amino acid substitution that alters binding by Fc gamma receptors (I, II, III) without affecting FcRn binding, which is important for half-life.In further embodiments, the Fc region is an Fc region 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.

[0253] In some embodiments, the Fc region contains amino acid substitutions that alter the antigen-independent effector function of the fusion protein. In some such embodiments, the Fc region contains amino acid substitutions that alter the circulating half-life of the resulting molecule. Such Fc variants exhibit either increased or decreased binding to FcRn compared to Fc regions lacking these substitutions, thereby resulting in an increased or decreased serum half-life, respectively, of the resulting molecule. Fc variants with improved affinity for FcRn are predicted to have longer serum half-lives, and such Fc variants are useful in methods of treating mammals where a longer half-life of the administered Fc fusion is desired and where increased transport into the circulation via the lungs is desired. On the other hand, Fc variants with reduced FcRn binding affinity are predicted to have shorter half-lives, and such variants are also useful, for example, when a shortened circulation time would be advantageous for administration to a mammal (e.g., when the fusion protein causes toxic side effects if present in the circulation for an extended period of time). For the treatment of conditions characterized by NETosis, fine-tuning the half-life of DNase-Fc fusion proteins by altering FcRn binding may be useful because NETs, ​​while contributing to pathogenesis, can also be beneficial in certain circumstances. Digestion of pathogenic NETs may not require a long half-life, thereby allowing more rapid clearance of DNase, potentially allowing neutrophils to form NETs for their protective antibacterial function. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta and therefore may be useful for treating diseases or disorders in pregnant women. Additionally, other applications in which reduced FcRn binding affinity may be desirable include those in which localization to the brain, kidney, and / or liver is desired. In one exemplary embodiment, the fusion molecules of the present invention exhibit reduced transport from the vasculature across the epithelium of kidney glomeruli. In another embodiment, the fusion molecules of the present invention exhibit reduced transport across the blood-brain barrier (BBB) ​​from the brain to the vascular space. In one embodiment, a fusion molecule with altered FcRn binding comprises an Fc region with one or more amino acid substitutions in the "FcRn binding loop" of the Fc domain.Exemplary amino acid substitutions that alter FcRn binding activity are disclosed in International PCT Publication No. WO05 / 047327, which is incorporated herein by reference. Exemplary amino acid substitutions that increase FcRn binding activity are also described, for example, by Wang et al., Protein Cell 9:63-73, 2018 (see, e.g., Table 1). In some embodiments, Fc variants with increased FcRn binding activity have amino acid substitutions at EU positions 252, 254, and 256 (e.g., M252Y, S254T, and T256E). In other variations, Fc variants with increased FcRn binding activity have amino acid substitutions at EU positions 428 and 434 (e.g., M428L and N434S).

[0254] In other embodiments, fusion polypeptides of the invention comprise Fc variants containing amino acid substitutions that alter one or more antigen-dependent effector functions of the polypeptide, particularly antibody-dependent cellular cytotoxicity (ADCC) or complement activation, e.g., compared to a wild-type Fc region. In exemplary embodiments, such fusion polypeptides exhibit altered binding to Fc gamma receptors (FcγRs, e.g., CD16). Such fusion polypeptides exhibit either increased or decreased binding to FcγRs compared to the wild-type polypeptide, and thus mediate enhanced or decreased effector function, respectively. Fc variants with improved affinity for FcγRs are expected to enhance effector function, and such variants are useful in methods of treating mammals where target molecule destruction is desired. On the other hand, Fc variants with reduced FcγR binding affinity are expected to reduce effector function, and such fusion proteins are also useful, for example, in the treatment of conditions where target cell destruction is undesirable, e.g., when normal cells may express the target molecule or when chronic administration of the fusion molecule may result in undesired immune system activation. In one embodiment, a fusion polypeptide comprising an Fc region exhibits at least one altered antigen-dependent effector function selected from the group consisting of opsonization, phagocytosis, complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or effector cell modulation, compared to a polypeptide comprising a wild-type Fc region. In typical embodiments of DNase fusion molecules comprising Fc variants with altered antigen-dependent effector functions, the Fc variants have one or more reduced effector functions compared to the corresponding wild-type Fc region.

[0255] In one embodiment, a fusion polypeptide comprising an Fc region exhibits altered binding to an activating FcγR (e.g., FcγI, FcγIIa, or FcγRIIIa). In another embodiment, the fusion protein exhibits altered binding affinity to an inhibitory FcγR (e.g., FcγRIIb). Exemplary amino acid substitutions that alter FcR or complement binding activity are disclosed in International PCT Publication No. WO 05 / 063815, which is incorporated herein by reference. Exemplary Fc variants with reduced effector function are described, for example, by Tam et al., Antibodies 6:12, 2017 (describing variants of human IgG1 (γ1) and IgG4 (γ4)); Wang et al., Protein Cell 9:63-73, 2018 (see, e.g., Table 1); Lo et al., J. Biol. Chem. 292:3900-3908, 2017; Idusogie et al., J. Immunol. 164:4178-4184, 2000 (each of which is incorporated herein by reference). Suitable Fc variants that reduce antigen-dependent effector function include, for example, variants with amino acid substitutions at EU positions 238 and / or 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 IgG1, F234A / L235A in IgG4) have been shown to reduce FcγR binding and reduce cytokine storm when introduced into anti-CD3 mAbs (see, e.g., Wang et al., supra), and amino acid substitutions at EU position 329 (e.g., P329A) are highly effective at reducing C1q binding (see, e.g., Lo et al., supra). Other exemplary approaches to removing ADCC and CDC effector functions are to create hybrid Fc domains derived from human IgG2 (Eu positions 118-260) and IgG4 (Eu positions 261-447), or to engineer human IgG2 to contain amino acid substitutions selected from IgG4.See Wang et al., supra.

[0256] Fusion polypeptides comprising an Fc region may also contain amino acid substitutions that alter the glycosylation of the Fc region. For example, the Fc domain of the fusion protein may have a mutation that leads to reduced glycosylation (e.g., N-linked or O-linked glycosylation) or may contain an altered glycoform of the wild-type Fc domain (e.g., a low-fucose glycan or a fucose-free glycan). In another embodiment, the molecule has an amino acid substitution near or within a glycosylation motif, e.g., an N-linked glycosylation motif containing the amino acid sequence NXT or NXS. Exemplary amino acid substitutions that reduce or alter glycosylation are disclosed in International PCT Publication No. WO 05 / 018572 and U.S. Patent Application Publication No. 2007 / 0111281, which are incorporated herein by reference.

[0257] Particularly suitable amino acid substitutions that reduce glycosylation and also reduce Fc ADCC and CDC effector functions include amino acid substitutions at EU position 297 (e.g., N297A, N297Q, or N297G). See, e.g., Wang et al., supra. The N297 substitution can also be paired with a substitution at position 265 (e.g., D265A) to further reduce CDC. See, e.g., Lo et al., supra.

[0258] It will be understood by those skilled in the art that, unless the context clearly dictates otherwise, the various embodiments of the Fc variants described herein can be combined in the fusion polypeptides of the invention.

[0259] In some embodiments, the immunoglobulin Fc region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to an amino acid sequence selected from the sequence set forth in (i) residues 1-232 or 1-231 of SEQ ID NO:26, (ii) residues 1-232 or 1-231 of SEQ ID NO:28, (iii) residues 159-390 or 159-389 of SEQ ID NO:42, (iv) residues 1-232 or 1-231 of SEQ ID NO:116, or (v) residues 1-232 or 1-231 of SEQ ID NO:118. In still 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 to the amino acid sequence set forth in (i) residues 1-232 or 1-231 of SEQ ID NO:26, (ii) residues 1-232 or 1-231 of SEQ ID NO:28, (iii) residues 159-390 or 159-389 of SEQ ID NO:42, (iv) residues 1-232 or 1-231 of SEQ ID NO:116, or (v) residues 1-232 or 1-231 of SEQ ID NO:118.

[0260] In some embodiments, the 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 to an amino acid sequence selected from the sequence set forth in (i) residues 16-232 or 16-231 of SEQ ID NO: 26, (ii) residues 16-232 or 16-231 of SEQ ID NO: 116, or (iii) residues 16-232 or 16-231 of SEQ ID NO: 118. In still 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 to the amino acid sequence set forth in (i) residues 16-232 or 16-231 of SEQ ID NO: 26, (ii) residues 16-232 or 16-231 of SEQ ID NO: 116, or (iii) residues 16-232 or 16-231 of SEQ ID NO: 118.

[0261] In some variations of the above fusion polypeptides in which X is present, X is an FcRn-binding domain. Particularly suitable FcRn-binding domains include immunoglobulin heavy chain constant regions that retain FcRn-binding activity, such as immunoglobulin Fc regions; in some such variations, the Fc region is an Fc region described herein (e.g., above in the context of the dimerization domain). In other embodiments, the FcRn-binding domain is albumin (e.g., human albumin). Still other suitable FcRn-binding domains include single-chain antibodies (e.g., scFvs), peptide aptamers, or alternative scaffold proteins that have binding affinity for FcRn; such alternative FcRn-binding molecules are readily generated, for example, using display technologies that allow for the selection of binders by screening 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, for example, avimers, ankyrin repeats, and adnectins, as well as other proteins with domains that can be evolved to generate specific affinities for desired molecular targets (see, e.g., Silverman et al., Nature Biotechnology 23:1556-1561, 2005; Zahnd et al., J. Mol. Biol. 369:1015-1028, 2007; Lipovsek et al., U.S. Patent No. 7,115,396).

[0262] In some embodiments of the paraoxonase-containing DNase fusion polypeptide 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 to the amino acid sequence set forth in (i) residues 21-896 or 1-896 of SEQ ID NO:2, (ii) residues 21-896 or 1-896 of SEQ ID NO:128, (iii) residues 21-896 or 1-896 of SEQ ID NO:130, (iv) residues 21-906 or 1-906 of SEQ ID NO:148, residues 21-896 or 1-896 of SEQ ID NO:158, (vi) residues 21-906 or 1-906 of SEQ ID NO:160, (vii) residues 21-906 or 1-906 of SEQ ID NO:162, or (viii) residues 21-906 or 1-906 of SEQ ID NO:164. In still 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 to the amino acid sequence set forth in (i) residues 21-896 or 1-896 of SEQ ID NO:2, (ii) residues 21-896 or 1-896 of SEQ ID NO:128, (iii) residues 21-896 or 1-896 of SEQ ID NO:130, (iv) residues 21-906 or 1-906 of SEQ ID NO:148, (v) residues 21-896 or 1-896 of SEQ ID NO:158, (vi) residues 21-906 or 1-906 of SEQ ID NO:160, (vii) residues 21-906 or 1-906 of SEQ ID NO:162, or (viii) residues 21-906 or 1-906 of SEQ ID NO:164.

[0263] In some embodiments of the paraoxonase-containing RNase fusion polypeptide 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 to an amino acid sequence selected from (i) residues 21-764 or 1-764 of SEQ ID NO:40, or (ii) residues 21-740 or 1-740 of SEQ ID NO:48. In still 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 to (i) residues 21-764 or 1-764 of SEQ ID NO:40, or (ii) residues 21-740 or 1-740 of SEQ ID NO:48.

[0264] In some embodiments of the paraoxonase fusion polypeptide containing superoxide dismutase 1 (SOD1) 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 to an amino acid sequence selected from the sequence set forth in (i) residues 23-781 or 1-781 of SEQ ID NO:50, (ii) residues 23-781 or 1-781 of SEQ ID NO:52, or (iii) residues 23-791 or 1-791 of SEQ ID NO:54. In still 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 to the amino acid sequence set forth in (i) residues 23-781 or 1-781 of SEQ ID NO:50, (ii) residues 23-781 or 1-781 of SEQ ID NO:52, or (iii) residues 23-791 or 1-791 of SEQ ID NO:54.

[0265] In some embodiments of the paraoxonase fusion polypeptide comprising the CTLA-4 extracellular domain 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 to an amino acid sequence selected from the sequence set forth in residues 21-736 or 1-736 of SEQ ID NO: 66. In still 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 to the amino acid sequence set forth in residues 21-736 or 1-736 of SEQ ID NO: 66.

[0266] In some embodiments of the paraoxonase fusion polypeptide comprising the CD40 extracellular domain 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 to an amino acid sequence selected from the group consisting of: (i) residues 21-804 or 1-804 of SEQ ID NO:74; (ii) residues 21-804 or 1-804 of SEQ ID NO:78; (iii) residues 21-804 or 1-804 of SEQ ID NO:82; (iv) residues 21-804 or 1-804 of SEQ ID NO:86; or (v) residues 21-804 or 1-804 of SEQ ID NO:90. In still 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 to the amino acid sequence set forth in (i) residues 21-804 or 1-804 of SEQ ID NO:74, (ii) residues 21-804 or 1-804 of SEQ ID NO:78, (iii) residues 21-804 or 1-804 of SEQ ID NO:82, (iv) residues 21-804 or 1-804 of SEQ ID NO:86, or (v) residues 21-804 or 1-804 of SEQ ID NO:90.

[0267] In some embodiments of a paraoxonase fusion polypeptide comprising a polypeptide that specifically binds and neutralizes TNFα 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 to an amino acid sequence selected from (i) residues 21-860 or 1-860 of SEQ ID NO:94, or (ii) residues 21-860 or 1-860 of SEQ ID NO:98. In still 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 to (i) residues 21-860 or 1-860 of SEQ ID NO:94, or (ii) residues 21-860 or 1-860 of SEQ ID NO:98.

[0268] In some embodiments of a paraoxonase fusion polypeptide comprising a polypeptide that specifically binds and neutralizes TGF-β 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 to an amino acid sequence selected from (i) residues 21-861 or 1-861 of SEQ ID NO: 102, or (ii) residues 21-861 or 1-861 of SEQ ID NO: 106. In still 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 to (i) residues 21-861 or 1-861 of SEQ ID NO: 102, or (ii) residues 21-861 or 1-861 of SEQ ID NO: 106.

[0269] In some embodiments of a paraoxonase fusion polypeptide comprising the formula X-L2-P described herein, the fusion polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence set forth in (i) residues 21-613 or 1-613 of SEQ ID NO:122, (ii) residues 21-613 or 1-613 of SEQ ID NO:132, (iii) residues 21-613 or 1-613 of SEQ ID NO:134, (iv) residues 21-610 or 1-610 of SEQ ID NO:142, (v) residues 21-610 or 1-610 of SEQ ID NO:144, or (vi) residues 21-610 or 1-610 of SEQ ID NO:146. In still 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 to the amino acid sequence set forth in (i) residues 21-613 or 1-613 of SEQ ID NO:122, (ii) residues 21-613 or 1-613 of SEQ ID NO:132, (iii) residues 21-613 or 1-613 of SEQ ID NO:134, (iv) residues 21-610 or 1-610 of SEQ ID NO:142, (v) residues 21-610 or 1-610 of SEQ ID NO:144, or (vi) residues 21-610 or 1-610 of SEQ ID NO:146.

[0270] The present invention also provides a dimeric protein comprising a first and a second polypeptide fusion, each of the polypeptide fusions comprising a dimerization domain as described above. Thus, in another aspect, the present invention provides a dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, each of the first and second fusion polypeptides comprising, from amino-terminal to carboxyl-terminal, T-L1-X-L2-P, where T is a first biologically active polypeptide selected from DNase, RNase, superoxide dismutase 1 (SOD1), cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) extracellular domain, CD40 extracellular domain, and a polypeptide that specifically binds to and neutralizes a proinflammatory cytokine (e.g., a polypeptide that specifically binds to and neutralizes tumor necrosis factor alpha (TNFα) or transforming growth factor-beta (TGF-β)); L1 is an optional first polypeptide linker; X is a dimerization domain; L2 is an optional second polypeptide linker; and P is a biologically active paraoxonase. In another aspect, the invention provides a dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, each of which comprises, from amino- to carboxyl-terminal position, X-L2-P, where X is a dimerization domain, L2 is an optional polypeptide linker, and P is a biologically active paraoxonase. In another aspect, the invention provides a dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, each of which comprises, from amino- to carboxyl-terminal position, P-L1-X, where P is a biologically active paraoxonase, L1 is a polypeptide linker, and X is a dimerization domain. III. Materials and Methods for Making Polypeptide Fusions and Dimeric Proteins

[0271] 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 the fusion polypeptide (e.g., Fc fragment; DNase and P polypeptide segments) can be generated and linked together using known methods for recombinant manipulation of nucleic acids to form polynucleotides encoding the fusion polypeptides described herein.

[0272] DNA sequences encoding fusion components according to the present disclosure (e.g., DNase (e.g., DNase 1), paraoxonase (e.g., PON1), RNase (e.g., RNase 1), superoxide dismutase 1 (SOD1), CTLA-4 and CD40 extracellular domains, and immunoglobulin Fc regions) are generally known in the art. Exemplary DNA sequences are disclosed herein (see Sequence Listing). Additional DNA sequences encoding any of these polypeptides can be readily generated by one of skill in the art based on the genetic code. Corresponding RNA sequences can be generated by substituting T for U. Given the degeneracy of the genetic code, one of skill in the art will readily recognize that there can be considerable sequence variation between 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 for introducing mutations into polynucleotide sequences, followed by expression of the encoded polypeptide and determination of functional activity (e.g., nuclease activity) using an appropriate screening assay.

[0273] Methods for preparing DNA and RNA are well known in the art.For example, complementary DNA (cDNA) clones can be prepared from RNA isolated from tissues or cells that produce large amounts of RNA encoding the desired polypeptide.Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation on 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 by poly(A) digestion using known methods. + It is prepared from RNA. Alternatively, 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, including the use of the sequences disclosed herein or portions thereof to probe or prime libraries. Polynucleotides encoding the polypeptide of interest are identified and isolated, for example, by hybridization or polymerase chain reaction ("PCR," Mullis, U.S. Pat. No. 4,683,202). Expression libraries can be probed with antibodies, receptor fragments, or other specific binding partners against the polypeptide of interest.

[0274] Polynucleotides of the invention can also be prepared by automated synthesis. Production of short double-stranded segments (60-80 bp) is technically straightforward and can be accomplished by synthesizing complementary strands and then annealing them. Longer segments (typically >300 bp) are assembled modularly from single-stranded fragments of 20-100 nucleotides in length. Automated synthesis of polynucleotides is within the ordinary skill level of those 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, DC, 1994; Itakura et al., Ann. Rev. Biochem. 53:323-356, 1984; and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-637, 1990.

[0275] In another aspect, materials and methods are provided for producing the polypeptide fusions of the present invention, including dimeric proteins comprising fusion polypeptides. Fusion polypeptides can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are cell types that can be transformed or transfected with exogenous DNA and grown in culture, including 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 various 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.

[0276] Generally, for the production of fusion polypeptides in host cells, the DNA sequence encoding the fusion polypeptide is operably linked to other genetic elements required for its expression, typically including a transcription promoter and terminator, in an expression cassette. Typically, the expression cassette is contained in an expression vector for delivery to host cells. The vector will also generally contain one or more selectable markers and one or more origins of replication, although those skilled in the art will understand that in certain systems, selectable markers may be provided by another vector, and replication of foreign DNA may be achieved by integrating the expression cassette into the host cell genome, for example, during the generation of stable cell lines. The 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 have been described in the literature and are available through commercial suppliers.

[0277] A secretory signal sequence is provided in the expression cassette to direct the DNase fusion polypeptide into the secretory pathway of the host cell. The encoded secretory peptide can be that of the corresponding native protein (e.g., the native DNase secretory peptide as set forth in amino acid residues 1-22 of SEQ ID NO: 16, the native RNase secretory peptide as set forth in amino acid residues 1-28 of SEQ ID NO: 38, or the native CD40 secretory peptide as set forth in amino acid residues 1-20 of SEQ ID NO: 68), or can be derived from another secreted protein (e.g., t-PA; see U.S. Patent No. 5,641,655), or can be synthesized de novo. An engineered cleavage site can be included at the junction between the secretory peptide and the remainder of the polypeptide fusion to optimize proteolytic processing in the host cell. The secretory signal sequence is operably linked to the DNA sequence encoding the polypeptide fusion, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide fusion into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be located elsewhere within the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830). Secretory signal sequences suitable for use according to the present invention include, for example, polynucleotides encoding the human VK3 leader peptide (SEQ ID NO: 58).

[0278] Expression of a fusion polypeptide comprising a dimerization domain described herein through a host cell secretory pathway is expected to result in the production of a dimeric protein. Thus, in another aspect, the present invention provides a dimeric protein comprising a first and second fusion polypeptide described above (e.g., a dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, each of which comprises, from amino-terminal to carboxyl-terminal positions, T-L1-X-L2-P, X-L2-P, or P-L1-X as described herein). Dimers can also be assembled in vitro by incubation of the component polypeptides under suitable conditions. Generally, in vitro assembly will involve incubating a 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.

[0279] Cultured mammalian cells are suitable hosts for use in the present invention. Methods for introducing foreign 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). Production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, in Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells include 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, such as promoters from SV-40, cytomegalovirus, or myeloproliferative sarcoma virus, can be used. See, for example, U.S. Patent No. 4,956,288 and U.S. Patent Application Publication No. 20030103986. Other suitable promoters include those from metallothionein genes (U.S. Patent 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, deposited at 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.

[0280] Drug selection is commonly used to select cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants." Cells cultured in the presence of a selection agent that are capable of transmitting the gene of interest to their progeny—through integration of the expression cassette into their genomic DNA—are called "stable transfectants." An exemplary selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of neomycin-type drugs, such as G-418. Selection systems can also be used to increase the expression level of the gene of interest, a process called "amplification." Amplification is achieved by culturing transfectants in the presence of low levels of the selection agent, then increasing the amount of the selection agent to select for cells that produce high levels of the introduced gene product. An exemplary amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g., hygromycin resistance, multidrug 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); these markers include, for example, CD8, CD4, nerve growth factor receptor, green fluorescent protein, etc.

[0281] In some aspects, the present invention provides stable cell lines containing within their genomic DNA an expression cassette encoding a paraoxonase fusion polypeptide described herein, wherein the stable cell line constitutively expresses the encoded paraoxonase fusion. Stable cell lines can be generated by methods commonly known in the art, which generally involve identifying single stable cell clones from polyclonal colonies 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 a Chinese hamster ovary (CHO) cell line. In some preferred embodiments of stable cell lines constitutively expressing a DNase-PON fusion polypeptide according to the present disclosure, the DNase is an enhanced DNase 1 described herein, and in some such embodiments, the DNase-PON fusion polypeptide is an enhanced DNase 1-Fc-PON1 fusion polypeptide comprising 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 to the amino acid sequence set forth in (i) residues 21-896 or 1-896 of SEQ ID NO:2, (ii) residues 21-906 or 1-906 of SEQ ID NO:148, (iii) residues 21-896 or 1-896 of SEQ ID NO:158, or (iv) residues 21-906 or 1-906 of SEQ ID NO:160. In some variations, a stable cell line is a mammalian cell line (e.g., CHO) capable of producing a fusion polypeptide (e.g., a DNase1-Fc-PON1 fusion polypeptide) at a concentration of at least 25 mg / L of cell culture, at least 50 mg / L, at least 100 mg / L, at least 200 mg / L, or at least 500 mg / L. Typically, the DNase1-Fc fusion polypeptides described herein can be expressed at levels of approximately 25-100 mg / L from the initial isolated clone. Recloning of the initial culture can often stabilize and increase expression by 2-3 fold.Amplification of expression levels can also be achieved by further seeding the cells at low density while increasing the level of methotrexate from an initial concentration of 50 nM up to as high as 1 uM. Once the cells have adapted, further rounds of limiting dilution cloning are required to maintain high expression levels.

[0282] Other higher eukaryotic cells, including insect cells, plant cells, and avian cells, can also be used as hosts. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells is 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.

[0283] Insect cells can be infected with recombinant baculovirus, typically 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 Vectors: A Laboratory Manual, Oxford University Press, New York, 1994; and Richardson, Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Humana Press, Totowa, NJ, 1995). Recombinant baculovirus can also be produced using the 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 transfer DNA encoding a protein of interest into the baculovirus genome maintained in E. coli as a large plasmid called a "bacmid" (Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556, 1994; Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995). Using techniques known in the art, the transfer vector encoding the polypeptide fusion is introduced into E. coli host cells to cause transformation, and the cells are screened for bacmids containing an interrupted lacZ gene, which indicates a recombinant baculovirus.The bacmid DNA containing the recombinant baculovirus genome is isolated using common techniques and used to transfect Spodoptera frugiperda cells, such as Sf9 cells. Recombinant viruses expressing polypeptide fusions are then produced. Recombinant virus stocks are produced by methods commonly used in the art.

[0284] For protein production, recombinant viruses are used to infect host cells, typically cell lines derived from the fall armyworm, 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. Pat. No. 5,300,435). Serum-free medium is used to grow and maintain the cells. Suitable media formulations are known in the art and available from commercial suppliers. Cells are cultured at approximately 2-5×10 5 Cell seeding density ranges from 1 to 2 x 10 6 The cells are grown to a density at which point the recombinant virus stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically approximately 3. The procedures used are generally described in available laboratory manuals (e.g., King and Possee, supra; O'Reilly et al., supra; Richardson, supra).

[0285] Fungal cells, including yeast cells, can also be used in the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed, for example, by Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are selected by a phenotype determined by a selectable marker, typically 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 POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformants to be selected by growth on glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, for example, 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 those from alcohol dehydrogenase genes. See also U.S. Patent 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. Pat. No. 4,882,279; and Raymond et al., Yeast 14:11-23, 1998. Aspergillus cells can be used according to the method of McKnight et al., U.S. Pat. No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Pat. No. 4,486,533. Production of recombinant proteins in Pichia methanolica is disclosed in U.S. Pat. Nos. 5,716,808; 5,736,383; 5,854,039; and 5,888,768.

[0286] 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 fusion polypeptides in bacteria such as E. coli, the polypeptides 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, for example, using guanidine HCl or urea. The denatured polypeptides can then be refolded and dimerized by diluting the denaturant, for example, by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. Alternatively, the protein may be recovered from the cytoplasm in a soluble form and isolated without the use of a denaturant. Protein is recovered from cells as an aqueous extract, for example, in phosphate-buffered saline.To capture the target protein, the extract is directly applied to a chromatographic medium, for example, immobilized antibody or heparin-Sepharose column.Secreted polypeptides can be recovered from the periplasmic space in a soluble and functional form by disrupting cells (for example, by sonication or osmotic shock) and recovering the protein, thereby eliminating the need for denaturation and refolding.See, for example, Lu et al., J. Immunol. Meth. 267:213-226, 2002.

[0287] Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components necessary for the growth of the selected host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally contain a carbon source, a nitrogen source, essential amino acids, vitamins, and minerals. The medium may also contain components such as growth factors or serum as needed. The growth medium will generally select cells containing exogenously added DNA, for example, by drug selection or essential nutrient deprivation complemented by a selectable marker carried on the expression vector or co-transfected into the host cells.

[0288] In some variations, host cells for production of the actin-resistant DNase 1-PON fusion polypeptides described herein (e.g., actin-resistant DNase 1-L1-Fc-L2-PON1 fusion polypeptides comprising 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 to the amino acid sequence set forth in residues 21-896 or 1-896 of SEQ ID NO:2 or (ii) residues 21-906 or 1-906 of SEQ ID NO:148) are cultured in the presence of a DNase 1 inhibitor to reduce DNase toxicity that is enhanced during its expression. DNase 1 inhibitors are commonly known in the art and include, for example, the inhibitor rutin (see, e.g., Kolarevic et al., Chem. Biodiversity 16:e1900060, 2019). In certain embodiments, the host cells cultured in the presence of a DNase 1 inhibitor are mammalian cells, such as CHO.

[0289] The proteins of the present invention are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See generally, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York, 1994. Proteins containing immunoglobulin Fc regions can be purified by affinity chromatography using immobilized protein A. Additional purification steps, such as gel filtration, can be used to obtain the desired level of purity, or desalting, buffer exchange, etc.

[0290] For example, purified fusion polypeptides and dimeric proteins of the present invention can be obtained from recombinant host cells using fractionation and / or conventional purification methods. Generally, ammonium sulfate precipitation and acid or chaotrope extraction can be used to fractionate samples. Exemplary purification steps include hydroxyapatite, size exclusion, FPLC, and reverse-phase high-performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silica, and the like. PEI, DEAE, QAE, and Q derivatives are preferred. Exemplary chromatographic media include media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), and Octyl-Sepharose (Pharmacia); or polyacrylic acid resins, such as Amberchrom CG 71 (Toso Haas). Suitable solid supports include glass beads, silica-based resins, cellulose resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins, etc., which are insoluble under the conditions in which they will be used. These supports can be modified with reactive groups that allow for attachment of proteins through amino, carboxyl, sulfhydryl, hydroxyl, and / or carbohydrate moieties.

[0291] 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 chemistry. These and other solid media are well known and widely used in the art and are available from commercial suppliers. The 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 selected support. See, for example, Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988); Doonan, Protein Purification Protocols (The Humana Press 1996).

[0292] Additional variations in protein isolation and purification can be devised by those skilled in the art. For example, antibodies that specifically bind to the fusion polypeptides or dimeric proteins described herein (e.g., antibodies that specifically bind to a polypeptide segment corresponding to a DNase) can be used to isolate large amounts of protein by immunoaffinity purification.

[0293] Proteins of the present invention can also be isolated by exploiting specific properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those containing 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 are adsorbed to this matrix with different affinities depending on the metal ion used and are eluted by competitive elution, lowering the pH, or using strong chelating agents. Other purification methods include the purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (see, e.g., M. Deutscher, (ed.), Meth. Enzymol. 182:529, 1990). In additional embodiments of the present invention, fusions of the polypeptide of interest with affinity tags (e.g., maltose-binding protein) can be constructed to facilitate purification. Additionally, the receptor- or ligand-binding properties of the fusion polypeptide or its dimer can be exploited for purification.

[0294] In some variations, DNase 1-PON fusion molecules containing the enhanced but non-actin-resistant DNase 1 described herein (i.e., a DNase that does not contain any amino acid substitutions that, compared to human DNase 1, reduce G-actin-induced inhibition of DNase endonuclease activity) are treated with ethylenediamine (EDA) after purification to reduce actin binding of the molecule and thereby obtain a DNase-Fc fusion molecule that is actin-resistant. The EDA-treated DNase 1-PON fusion molecule is then further processed to remove EDA using methods commonly known in the art (see, e.g., U.S. Pat. No. 11,225,648). Such embodiments are advantageous for achieving higher expression of DNase 1-PON fusions for pharmaceutical use, since expression of the non-actin-resistant form of enhanced DNase 1 is less toxic to host cells (e.g., CHO). In certain embodiments, the purified DNase1-PON to be treated with EDA comprises 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 to the amino acid sequence set forth in (i) residues 21-896 of SEQ ID NO: 158 or (ii) residues 21-906 of SEQ ID NO: 160.

[0295] The polypeptides of the present invention are typically purified to at least about 80% purity with respect to contaminating macromolecules, particularly other proteins and nucleic acids, more typically at least about 90% purity, preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% purity, and are free of infectious and pyrogens. The polypeptides of the present invention can also be purified to a pharmaceutically pure state, that is, greater than 99.9% pure. In certain preparations, the purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. IV. Methods of Use and Pharmaceutical Compositions

[0296] The fusion polypeptides and dimeric proteins of the present invention can be used to administer therapies for the treatment of various diseases or disorders. Paraoxonase fusion polypeptides are particularly useful, for example, for the treatment of inflammatory, autoimmune, neurological, cardiovascular, and / or fibrotic diseases and disorders. In embodiments relating to bispecific fusions further comprising a first biologically active polypeptide (e.g., DNase, RNase, or SOD1) described herein, the fusion polypeptides and dimeric proteins may further provide one or more additional biological activities for treatment. For example, paraoxonase fusion polypeptides comprising DNase or RNase are particularly useful, for example, for the treatment of diseases and disorders characterized by NETosis. In addition, paraoxonase fusion polypeptides comprising the CTLA-4 or CD40 extracellular domains are particularly useful, for example, for the treatment of diseases or disorders characterized by an abnormal adaptive immune response. As yet another example, paraoxonase fusion polypeptides comprising the CD40 extracellular domain, or a polypeptide that specifically binds and neutralizes TGF-β, are particularly useful, for example, in treating diseases and disorders characterized by a fibrotic inflammatory response.

[0297] In some aspects, the present invention provides methods for treating a disease or disorder selected from inflammatory diseases, autoimmune diseases, neurological diseases, infectious diseases, metabolic diseases, cardiovascular diseases, liver diseases, fibrotic diseases, thrombosis, sepsis, ischemia-reperfusion, biofilm formation by Gram-negative bacteria, exposure to sulfur mustard gas, exposure to organophosphates, and cancer. The methods generally comprise administering to a subject having the disease or disorder an effective amount of a fusion polypeptide or dimeric protein described herein.

[0298] Inflammatory diseases suitable for treatment according to the present invention include, for example, inflammatory lung diseases such as asthma, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), bronchiectasis, hypoxia, interstitial lung disease (e.g., idiopathic pulmonary fibrosis (IPF) or sarcoidosis), and acute respiratory distress syndrome (ARDS). In certain embodiments involving 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, the patient with the inflammatory lung disease has been exposed to sulfur mustard gas (SM). In other variations, the patient with the inflammatory lung disease is a patient 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).

[0299] Other inflammatory diseases suitable for treatment by the present invention include autoinflammatory diseases (i.e., disorders of innate immune system activation characterized by seemingly non-inflammatory episodes and a relative lack of overt 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) disorders, hereditary angioedema (HAE), atypical hemolytic uremic syndrome, familial Mediterranean fever (FMF), TNF receptor-associated periodic fever syndrome (TRAPS), cryopyrin-associated periodic fever syndrome (CAPS), NOD2-associated autoinflammatory disease (NAID), and Blau syndrome.

[0300] In still other embodiments, the inflammatory disease or disorder for treatment according to the present invention is selected from rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes, type 2 diabetes, hepatitis (e.g., nonalcoholic steatohepatitis (NASH)), ankylosing spondylitis, psoriasis, psoriatic arthritis, dermatitis (e.g., atopic dermatitis), diverticulitis, irritable bowel syndrome, and nephritis. In yet other variations, the inflammatory disease or disorder is an inflammatory skin disease, e.g., psoriasis or atopic dermatitis.

[0301] In a more particular embodiment of the method for treating an inflammatory disease, the fusion molecule for treating an inflammatory disease has the structure DNase1-L1-Fc-L2-PON1, DNase1L3-L1-Fc-L2-PON1, RNase1-L1-Fc-L2-PON1, RNase1-Fc-L2-PON1, SOD1-L1-Fc-L2-PON1, SOD1-Fc-L2-PON1, CTLA4-L1-Fc-L2-PON1, CTLA4-Fc-L2-PON1, CD40-L1-Fc-L2-PON1, CD40-Fc-L2-PON1, anti-TNFα-L In some such embodiments, the fusion polypeptide is a polypeptide having the sequence of: (i) residues 21-896 of SEQ ID NO:2; (ii) residues 21-764 of SEQ ID NO:40; (iii) residues 21-740 of SEQ ID NO:48; (iv) residues 21-740 of SEQ ID NO:49; (v) residues 21-896 of SEQ ID NO:50; (vi) residues 21-740 of SEQ ID NO:51; (vii) residues 21-896 of SEQ ID NO:52; (viii) residues 21-740 of SEQ ID NO:53; (viii) residues 21-740 of SEQ ID NO:54; (viii) residues 21-740 of SEQ ID NO:55; (viii) residues 21-740 of SEQ ID NO:56; (viii) residues 21-740 of SEQ ID NO:57; (viii) residues 21-740 of SEQ ID NO:58; (viii) residues 21-740 of SEQ ID NO:59 ... (v) residues 23 to 781 of SEQ ID NO: 50, (v) residues 23 to 781 of SEQ ID NO: 52, (vi) residues 23 to 791 of SEQ ID NO: 54, (vii) residues 21 to 736 of SEQ ID NO: 66, (viii) residues 21 to 804 of SEQ ID NO: 74, (ix) residues 21 to 804 of SEQ ID NO: 78, (x) residues 21 to 804 of SEQ ID NO: 82, (xi) residues 21 to 804 of SEQ ID NO: 86, (xii) residues 21 to 804 of SEQ ID NO: 90, (xiii) residues 21 to 860 of SEQ ID NO: 94, (xiv) residues 21 to 860 of SEQ ID NO: 98, (xv) residues 21 to 861 of SEQ ID NO: 102, (xvi (xvii) residues 21-613 of SEQ ID NO: 122, (xviii) residues 21-610 of SEQ ID NO: 142, (xix) residues 21-906 of SEQ ID NO: 148, (xx) residues 21-896 of SEQ ID NO: 158, (xxi) residues 21-906 of SEQ ID NO: 160, (xxii) residues 21-906 of SEQ ID NO: 162, or (xxiii) residues 21-906 of SEQ ID NO: 164.In some embodiments of the methods for treating autoinflammatory diseases, the fusion molecule is a polypeptide having the structure DNase 1-L1-Fc-L2-PON1, DNase 1L3-L1-Fc-L2-PON1, RNase 1-L1-Fc-L2-PON1, or RNase 1-Fc-L2-PON1, or a dimeric protein formed by dimerization of any of the foregoing fusion polypeptides. In some embodiments of the methods for treating rheumatoid arthritis, psoriatic arthritis, or juvenile idiopathic arthritis, the fusion molecule is a polypeptide having the structure CTLA4-L1-Fc-L2-PON1 or CTLA4-Fc-L2-PON1, or a dimeric protein formed by dimerization of either of the foregoing fusion polypeptides. In some embodiments of the methods for treating inflammatory bowel disease (IBD) (e.g., Crohn's disease or ulcerative colitis), the fusion molecule is a polypeptide having the structure DNase1-L1-Fc-L2-PON1, DNase1L3-L1-Fc-L2-PON1, anti-TNFα-L1-Fc-L2-PON1, or anti-TNFα-Fc-L2-PON1, or a dimeric protein formed by dimerization of any of the foregoing fusion polypeptides. In some embodiments of the methods for treating inflammatory skin diseases (e.g., psoriasis or atopic dermatitis), the fusion molecule is a polypeptide having the structure DNase1-L1-Fc-L2-PON1, DNase1L3-L1-Fc-L2-PON1, anti-TNFα-L1-Fc-L2-PON1, anti-TNFα-Fc-L2-PON1, Fc-L2-PON1, or PON1-L1-Fc, or a dimeric protein formed by dimerization of any of the foregoing fusion polypeptides.

[0302] Autoimmune diseases suitable for treatment according to 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 celiac 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 disease, Hashimoto's thyroiditis, Graves' disease, Wegener's granulomatosis, myasthenia gravis, Addison's disease, autoimmune uveoretinitis, pemphigus vulgaris, primary biliary cirrhosis, pernicious anemia, sympathetic ophthalmia, uveitis, autoimmune hemolytic anemia, pulmonary fibrosis, chronic beryllium disease, and idiopathic pulmonary fibrosis. In some variations involving treatment of vasculitis, the vasculitis is selected from small- and medium-sized vessel vasculitis, while in other variations, the vasculitis is large-vessel vasculitis.

[0303] In a more particular embodiment of the method for treating an autoimmune disease, the fusion molecule for treating an autoimmune disease has the structure DNase1-L1-Fc-L2-PON1, DNase1L3-L1-Fc-L2-PON1, RNase1-L1-Fc-L2-PON1, RNase1-Fc-L2-PON1, SOD1-L1-Fc-L2-PON1, SOD1-Fc-L2-PON1, CTLA4-L1-Fc-L2-PON1, CTLA4-Fc-L2-PON1, CD40-L1-Fc-L2-PON1, CD40-Fc-L2-PON1, anti-TNFα In some such embodiments, the fusion polypeptide is a polypeptide having anti-TNFα-Fc-L2-PON1, anti-TGFβ-L1-Fc-L2-PON1, anti-TGFβ-L1-Fc-L2-PON1, anti-TGFβ-Fc-L2-PON1, Fc-L2-PON1, or PON1-L1-Fc, or a dimeric protein formed by dimerization of any of the foregoing fusion polypeptides, and in some such embodiments, the fusion polypeptide is a polypeptide having (i) residues 21-896 of SEQ ID NO:2, (ii) residues 21-764 of SEQ ID NO:40, (iii) residues 21-740 of SEQ ID NO:48, (iv) residues 21-740 of SEQ ID NO:49, (v) residues 21-896 of SEQ ID NO:50, (vi) residues 21-764 of SEQ ID NO:51, (vii) residues 21-740 of SEQ ID NO:52, (viii) residues 21-740 of SEQ ID NO:53, (viii) residues 21-740 of SEQ ID NO:54, (viii) residues 21-740 of SEQ ID NO:55, (viii) residues 21-740 of SEQ ID NO:56, (viii) residues 21-740 of SEQ ID NO:57, (viii) residues 21-740 of SEQ ID NO:58, (viii) residues 21-740 of SEQ ID NO:59, (viii) residues 21-740 of SEQ ID NO:60, (viii) residues 21-740 of SEQ ID NO:61, (viii) residues 21-740 of SEQ ID NO:62, (viii) residues (v) residues 23 to 781 of SEQ ID NO: 50, (v) residues 23 to 781 of SEQ ID NO: 52, (vi) residues 23 to 791 of SEQ ID NO: 54, (vii) residues 21 to 736 of SEQ ID NO: 66, (viii) residues 21 to 804 of SEQ ID NO: 74, (ix) residues 21 to 804 of SEQ ID NO: 78, (x) residues 21 to 804 of SEQ ID NO: 82, (xi) residues 21 to 804 of SEQ ID NO: 86, (xii) residues 21 to 804 of SEQ ID NO: 90, (xiii) residues 21 to 860 of SEQ ID NO: 94, (xiv) residues 21 to 860 of SEQ ID NO: 98, (xv) residues 21 to 861 of SEQ ID NO: 102, (xv (xvii) residues 21-613 of SEQ ID NO: 122, (xviii) residues 21-610 of SEQ ID NO: 142, (xix) residues 21-906 of SEQ ID NO: 148, (xx) residues 1-896 of SEQ ID NO: 158, (xxi) residues 21-906 of SEQ ID NO: 160, (xxii) residues 21-906 of SEQ ID NO: 162, or (xxiii) residues 21-906 of SEQ ID NO: 164, comprising, or consisting of, an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence set forth in SEQ ID NO: 106, (xvii) residues 21-613 of SEQ ID NO: 122, (xviii) residues 21-610 of SEQ ID NO: 142, (xix) residues 21-906 of SEQ ID NO: 148, (xx) residues 1-896 of SEQ ID NO: 158, (xxi) residues 21-906 of SEQ ID NO: 160, (xxii) residues 21-906 of SEQ ID NO: 162, or (xxiii) residues 21-906 of SEQ ID NO: 164.In some embodiments of the methods for treating systemic lupus erythematosus, rheumatoid arthritis, psoriasis, type 1 diabetes, or small vasculitis, the fusion molecule is a fusion polypeptide having the structure DNase 1-L1-Fc-L2-PON1 or DNase 1L3-L1-Fc-L2-PON1, or a dimeric protein formed by dimerization thereof. In some embodiments of the methods for treating systemic lupus erythematosus, Sjogren's syndrome, or type 1 diabetes, the fusion molecule is a polypeptide having the structure RNase 1-L1-Fc-L2-PON1 or RNase 1-Fc-L2-PON1, or a dimeric protein formed by dimerization of either of the foregoing fusion polypeptides. In some embodiments of the methods for treating rheumatoid arthritis or psoriatic arthritis, the fusion molecule is a polypeptide having the structure CTLA4-L1-Fc-L2-PON1 or CTLA4-Fc-L2-PON1, or a dimeric protein formed by dimerization of either of the foregoing fusion polypeptides.

[0304] Neurological diseases suitable for treatment according to the present invention include, for example, neurodegenerative diseases characterized by inflammation of the CNS, such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis (ALS). In some embodiments, the neurological disease is a neurological disease characterized by dementia, such as Alzheimer's disease. In a more particular variation of the method for treating multiple sclerosis (MS), the MS is spino-optic MS, primary progressive MS (PPMS), or relapsing-remitting MS (RRMS). In other embodiments, the neurological disease for treatment according to the present invention is a CNS infection, such as meningitis, encephalitis, or cerebral malaria. In more particular variations of the methods for treating meningitis, the meningitis is bacterial meningitis, and in some such embodiments, the CNS infection is a S. pneumoniae, N. meningitis, S. aureus, E. coli, A. baumannii, S. oralis, S. capitis, or S. epidermidis infection. Other neurological diseases or disorders suitable for treatment with the fusion molecules described herein include, for example, acute brain injury, such as ischemic stroke. In still other embodiments, the neurological disease is an intracranial tumor selected from brain cancer, e.g., astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, primary CNS lymphoma, medulloblastoma, germ cell tumor, pineal neoplasm, meningioma, pituitary tumor, nerve sheath tumor (e.g., schwannoma), chordoma, craniopharyngioma, and choroid plexus tumor (e.g., choroid plexu...

Claims

1. A fusion polypeptide comprising, from the amino terminal position to the carboxyl terminal position, T-L1-X-L2-P, wherein T is Cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) extracellular domain, the CD40 extracellular domain, and A single chain antibody that specifically binds to and neutralizes an inflammatory cytokine, the single chain antibody being a single chain Fv (scFv). a first biologically active polypeptide selected from the group consisting of: L1 is a first polypeptide linker, L1 is optionally present; X is an immunoglobulin heavy chain constant region, said immunoglobulin heavy chain constant region capable of dimerizing and specifically binding to the neonatal Fc receptor (FcRn); L2 is a second polypeptide linker comprising at least eight amino acid residues; and P is a biologically active paraoxonase, said paraoxonase having at least 95% identity to the amino acid sequence set forth in residues 16-355 or 26-355 of SEQ ID NO:6 and not containing the amino-terminal leader sequence corresponding to residues 1-15 of SEQ ID NO:

6. Fusion polypeptides.

2. 2. The fusion polypeptide of claim 1, wherein the paraoxonase has the amino acid sequence set forth in residues n to 355 of SEQ ID NO:6, where n is an integer between 16 and 26, inclusive.

3. The fusion polypeptide of claim 1, wherein L2 consists of 12 to 25 amino acid residues.

4. The fusion polypeptide of claim 1, wherein the first biologically active polypeptide is the CTLA-4 extracellular domain.

5. The fusion polypeptide of claim 4, wherein the CTLA-4 extracellular domain has at least 95% identity to the amino acid sequence set forth in residues 21-144 of SEQ ID NO:

66.

6. The fusion polypeptide of claim 5, wherein the CTLA-4 extracellular domain has the amino acid sequence set forth in residues 21-144 of SEQ ID NO:

66.

7. The fusion polypeptide of claim 1 , wherein the first biologically active polypeptide is the CD40 extracellular domain.

8. the CD40 extracellular domain is (i) residues 21-188 of SEQ ID NO:74; (ii) residues 21-188 of SEQ ID NO:78; (iii) residues 21-188 of SEQ ID NO: 82; (iv) residues 21-188 of SEQ ID NO: 86, or (v) residues 21-188 of SEQ ID NO:90 8. The fusion polypeptide of claim 7, which has at least 95% identity with the amino acid sequence shown in

9. The fusion polypeptide of claim 8, 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: 68) selected from the group consisting of E64, K81, P85 and L121, and the at least one amino acid substitution increases CD40 ligand binding compared to human CD40.

10. 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 a histidine and the amino acid at the position corresponding to L121 of human CD40 is a 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; The fusion polypeptide of claim 9.

11. the CD40 extracellular domain is (i) residues 21-188 of SEQ ID NO:74; (ii) residues 21-188 of SEQ ID NO:78; (iii) residues 21-188 of SEQ ID NO: 82; (iv) residues 21-188 of SEQ ID NO: 86, or (v) residues 21-188 of SEQ ID NO:90 9. The fusion polypeptide of claim 8 having the amino acid sequence shown in

12. The fusion polypeptide of claim 1 , wherein the first biologically active polypeptide is a single chain antibody that specifically binds to and neutralizes a proinflammatory cytokine.

13. The fusion polypeptide of claim 12, wherein the proinflammatory cytokine is TNFα.

14. The single chain antibody comprises: Complementarity determining region (CDR) CDR-H1 ＴＮＦα , CDR-H2 ＴＮＦα and CDR-H3 ＴＮＦα wherein CDR-H1 TNFα, CDR-H2 TNFα and CDR-H3 TNFα are the VH CDRs of SEQ ID NO: 108; and A VL domain comprising complementarity determining regions (CDRs) CDR-L1 TNFα, CDR-L2 TNFα and CDR-L3 TNFα, wherein CDR-L1 TNFα, CDR-L2 TNFα and CDR-L3 TNFα are the VL CDRs of SEQ ID NO:

110. The fusion polypeptide of claim 13, comprising:

15. The fusion polypeptide of claim 13, wherein the single chain antibody comprises a VH domain having at least 95% identity with the amino acid sequence set forth in SEQ ID NO: 108 and / or the single chain antibody comprises a VL domain having at least 95% identity with the amino acid sequence set forth in SEQ ID NO:

110.

16. The single chain antibody comprises: (i) residues 21 to 268 of SEQ ID NO:92; or (ii) residues 21-268 of SEQ ID NO:96 14. The fusion polypeptide of claim 13, having at least 95% identity with the amino acid sequence shown in

17. The single chain antibody comprises: (i) residues 21 to 268 of SEQ ID NO:92; or (ii) residues 21-268 of SEQ ID NO:96 17. The fusion polypeptide of claim 16 having the amino acid sequence shown in

18. The fusion polypeptide of claim 1 , 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 Fc region is a human gamma 1 Fc region or a human gamma 4 Fc region.

21. The fusion polypeptide of claim 4, comprising an amino acid sequence having at least 95% identity to the amino acid sequence set forth in residues 21 to 736 of SEQ ID NO:

66.

22. The fusion polypeptide of claim 4, comprising the amino acid sequence set forth in residues 21 to 736 of SEQ ID NO:

66.

23. (i) residues 21-804 of SEQ ID NO:74; (ii) residues 21-804 of SEQ ID NO:78; (iii) residues 21-804 of SEQ ID NO: 82; (iv) residues 21-804 of SEQ ID NO: 86, or (v) residues 21-804 of SEQ ID NO:90 8. The fusion polypeptide of claim 7, comprising an amino acid sequence having at least 95% identity to the amino acid sequence shown in

24. (i) residues 21-804 of SEQ ID NO:74; (ii) residues 21-804 of SEQ ID NO:78; (iii) residues 21-804 of SEQ ID NO: 82; (iv) residues 21-804 of SEQ ID NO: 86, or (v) residues 21-804 of SEQ ID NO:90 24. The fusion polypeptide of claim 23, comprising the amino acid sequence shown in

25. (i) residues 21-860 of SEQ ID NO:94, or (ii) residues 21-860 of SEQ ID NO:98 14. The fusion polypeptide of claim 13, comprising an amino acid sequence having at least 95% identity to the amino acid sequence shown in

26. (i) residues 21-860 of SEQ ID NO:94, or (ii) residues 21-860 of SEQ ID NO:98 26. The fusion polypeptide of claim 25, comprising the amino acid sequence shown in

27. 27. A dimeric protein comprising a first fusion polypeptide and a second fusion polypeptide, each of the first and second fusion polypeptides being a fusion polypeptide according to any one of claims 1 to 26.

28. 27. A polynucleotide encoding a fusion polypeptide according to any one of claims 1 to 26.

29. 27. An expression cassette comprising a DNA segment encoding a fusion polypeptide according to any one of claims 1 to 26, wherein the DNA segment is operably linked to a promoter.

30. 30. A cultured cell into which has been introduced the expression cassette of claim 29, said cultured cell expressing said DNA segment.

31. 1. A method for making a fusion polypeptide comprising the steps of: Culturing a cell into which the expression cassette of claim 29 has been introduced, wherein the cell expresses the DNA segment and the encoded fusion polypeptide is produced; and Recovering the fusion polypeptide. A method comprising:

32. 1. A method for producing a dimeric protein, comprising the steps of: Culturing a cell into which the expression cassette of claim 29 has been introduced, wherein the cell expresses the DNA segment and the encoded fusion polypeptide is produced as a dimeric protein; and Recovering the dimeric protein. A method comprising:

33. A vector comprising the expression cassette of claim 29.

34. A fusion polypeptide according to any one of claims 1 to 26; and Pharmaceutically acceptable carriers A composition comprising:

35. The dimeric protein of claim 27; and Pharmaceutically acceptable carriers A composition comprising:

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