Compositions and methods for treating pulmonary inflammation

Abstract

Therapies, including combination therapies, for the treatment of pulmonary inflammation, including interstitial lung disease (ILD), are provided, comprising the use of at least one histidyl-tRNA synthetase (HRS) polypeptide, or an expressible polynucleotide encoding said HRS polypeptide, alone or in combination with at least one immunomodulatory agent.Embodiments of the present disclosure relate to therapies, including combination therapies, for the treatment of pulmonary inflammation, including interstitial lung disease (ILD), comprising the use of at least one histidyl-tRNA synthetase (HRS) polypeptide, or an expressible polynucleotide encoding said HRS polypeptide, alone or in combination with at least one immunomodulatory agent. [Selected figure] Figure 3

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority under 35 U.S.C.§119(e) to U.S. Patent Application No. 62 / 487,812, filed on April 20, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Statement Regarding the Sequence Listing The sequence listing associated with this application is provided in text format instead of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is ATYR_131_01WO_ST25.txt. The text file is approximately 276 KB, was created on April 18, 2018, and was electronically filed via EFS - Web. Background

[0003] Embodiments of the present disclosure relate to therapies, including combination therapies for the treatment of lung inflammation, including interstitial lung disease (ILD), comprising the use of at least one histidyl - tRNA synthetase (HRS) polypeptide, or an expressible polynucleotide encoding said HRS polypeptide, alone or in combination with at least one immunomodulatory agent.

Background Art

[0004] Interstitial lung disease (ILD) is a heterogeneous group of disorders that affect the lung interstitium where inflammation of heterogeneous disorders is the main pathogenesis. Among ILD designations, generally, there are many fibrotic lung conditions that are recognized as having measurable inflammatory elements, including both innate and adaptive immune mechanisms that contribute to the pathogenesis at some level.

[0005] Patients suffering from ILD often experience progressive and debilitating respiratory symptoms and have a significantly higher mortality and increased death rate compared to the general population. As a group, these conditions represent a high unmet medical need for which there are few effective treatments without significant undesirable side effects.

Summary of the Invention

Means for Solving the Problems

[0006] Embodiments of the present disclosure are, in suitable parts, therapeutic compositions comprising: (a) a histidyl-tRNA synthetase (HRS) polypeptide, or an expressible polynucleotide encoding an HRS polypeptide; and (b) an immunomodulatory agent.

[0007] In some embodiments, the HRS polypeptide comprises, consists of, or consists essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table H1, Table H2, and Table H4.

[0008] In some embodiments, the HRS polypeptide is 500 - 506 amino acids in length, is at least 90% identical to SEQ ID NO: 8 (HRS(1 - 506)) or SEQ ID NO: 9 (HRS(2 - 506)), and lacks residues 507 - 509 of SEQ ID NO: 1. In some embodiments, the HRS polypeptide comprises, consists of, or consists essentially of SEQ ID NO: 8 (HRS(1 - 506)). In some embodiments, the HRS polypeptide comprises, consists of, or consists essentially of SEQ ID NO: 9 (HRS(2 - 506)).

[0009] In some embodiments, the HRS polypeptide is conjugated to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises an Fc region, forming an HRS-Fc fusion polypeptide.

[0010] In some embodiments, the HRS-Fc fusion polypeptide comprises, consists of, or consists essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence selected from Table H8. In some embodiments, the HRS polypeptide is at least about 80%, 85%, 90%, or 95% protein-based pure and has less than about 5% aggregation.

[0011] In some embodiments, (a) is an expressible polynucleotide encoding an HRS polypeptide, optionally a modified mRNA polynucleotide, which optionally comprises one or more unnatural bases and / or unnatural internucleotide linkages.

[0012] In some embodiments, the HRS polypeptide has off-target activity, optionally anti-inflammatory activity.

[0013] In some embodiments, the immunomodulatory agent is selected from one or more of pirfenidone, nintedanib, sphingosine-1-phosphate (S1P) and / or S1P receptor (S1PR) modulators, steroids, optionally glucocorticoids, calcineurin inhibitors, mammalian target of rapamycin (mTOR) inhibitors, indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitors, inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors, cytokines and / or cytokine receptor inhibitors, B cell receptor inhibitors, kinase inhibitors, and cytostatic agents, optionally methotrexate.

[0014] In some embodiments, the S1P and / or S1PR modulator is amiselimod (S1PR antagonist), fingolimod (S1PR 1 functional antagonist), sonelixizumab (S1P-specific monoclonal antibody), KRP203 (S1PR 1 agonist), SEW2871 (S1PR 1 agonist), siponimod (S1PR 1and S1PR 5 modulator), RPC1063 (S1PR 1 modulator), ONO-4641 (S1PR 1 and S1PR 5 agonist), JTE-013 (S1PR 2 antagonist), GSK2018682 (S1PR 1 agonist), ponesimod (S1PR 1 agonist), suramin (selective S1PR 3 and S1PR 5 antagonist), VPC23019 (aryl-amide analog; competitive S1PR 1 and S1PR 3 antagonist), and W146 (selective S1PR 1 antagonist), an antisense or RNAi agent targeting S1PR, and an antibody or antigen-binding fragment or small molecule that specifically binds to S1P and / or S1PR, and optionally, amiselimod is in a dosage unit in the range of about 0.1 mg to about 10 mg, or about 0.1, 0.2, 0.3, 0.337, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg, about 0.1, 0.2, 0.3, 0.337, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg or less, or at least about 0.1, 0.2, 0.3, 0.337, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg of dosage unit, or optionally, amiselimod is in a dosage unit in the range of about 0.1 mg / kg to about 10 mg / kg, or about 0.1, 0.2, 0.3, 0.337, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg, about 0.1, 0.2, 0.3, 0.337, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg or less, or at least about 0.1, 0.2, 0.3, 0.337, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / kg.

[0015] In some embodiments, the steroid is selected from betamethasone, budesonide, cortisol (hydrocortisone), cortisone, deflazacort, deoxycorticosterone, dexamethasone, fludrocortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, and triamcinolone.

[0016] In some embodiments, the calcineurin inhibitor is selected from cyclosporine, pimecrolimus, tacrolimus, an antisense or RNAi agent targeting calcineurin or a subunit thereof, and an antibody or antigen-binding fragment or small molecule that specifically binds to calcineurin or a subunit thereof.

[0017] In some embodiments, the mTOR inhibitor is an ATP-competitive mTOR kinase inhibitor, an mTORC1 / mTORC2 dual inhibitor, and / or an mTOR / PI3K dual inhibitor, or the mTOR inhibitor is selected from everolimus, rapamycin, deforolimus, temsirolimus, dactolisib, BGT226, SF1126, PKI-587, NVPBE235, sapitinib, AZD8055, AZD2014, an antisense or RNAi agent targeting mTOR, and an antibody or antigen-binding fragment or small molecule that specifically binds to mTOR, one or more of which.

[0018] In some embodiments, the IDO inhibitor is selected from indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat, an antisense or RNAi agent targeting IDO, and an antibody or antigen-binding fragment or small molecule that specifically binds to IDO.

[0019] In some embodiments, the IMPDH inhibitor is selected from one or more of mycophenolic acid (mycophenolate mofetil), ribavirin, and 6TGMP (6-thioguanine monophosphate), an antisense or RNAi agent targeting IMPDH, and an antibody or antigen-binding fragment or small molecule that specifically binds to IMPDH.

[0020] In some embodiments, the cytokine inhibitor is an inhibitor of cytokines selected from interleukin-1 (IL-1) including IL-1α and IL-1β, interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-20 (IL-20), interleukin-33 (IL-33), tumor necrosis factor (TNF), interferon gamma (IFN-γ), transforming growth factor-β (TGF-β), and granulocyte-macrophage colony-stimulating factor (GM-CSF), and / or a cytokine receptor selected from one or more of IL-1R, IL-6R, IL-8R, IL-11R, IL-12R, IL-17R, IL-18R, IL-20R, ST2 (interleukin 1 receptor-like 1, IL1RL1), TNFR such as TNFR1, interferon-gamma receptor (IFNGR), and TGF-β receptor such as TGFβR1 (ALK5) or TGFβR2, and further, the cytokine and / or cytokine receptor inhibitor is selected from an antisense or RNAi agent targeting the cytokine and / or cytokine receptor, and an antibody or antigen-binding fragment or small molecule that specifically binds to the cytokine or cytokine receptor.

[0021] In some embodiments, the cytokine and / or cytokine receptor inhibitor is selected from one or more of adalimumab, anakinra, basiliximab, canakinumab, certolizumab, daclizumab, etanercept, golimumab, infliximab, ixekizumab, mepolizumab, reslizumab, rilonacept, secukinumab, serilumab, sirukumab, tocilizumab, and ustekinumab.

[0022] In some embodiments, the kinase inhibitor is an inhibitor of a kinase selected from one or more of Janus kinase (JAK including JAK1, JAK2, JAK3, TYK2), epidermal growth factor receptor (EGFR), receptor tyrosine-protein kinase erbB-2 (Her2 / neu or ERBB2), Bcr-Abl, c-SRC, mitogen-activated protein kinase (MAP) kinase, anaplastic lymphoma kinase (ALK), spleen tyrosine kinase (SYK), Bruton tyrosine kinase (BTK), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR including VEGFR1, VEGFR2, VEGFR3), fibroblast growth factor receptor (FGFR), B-Raf, RET proto-oncogene, platelet-derived growth factor receptor (PDGF-R), tropomyosin receptor kinase (Trk including TrkA, TrkB, TrkC), and c-Met. Further, the kinase inhibitor is selected from antisense or RNAi agents targeting the kinase, and antibodies or antigen-binding fragments or small molecules that specifically bind to the kinase.

[0023] In some embodiments, the kinase inhibitor is selected from one or more of nintedanib, baricitinib, fedratinib, filgotinib, ganitnib, lestaurtinib, momelotinib, pacritinib, peficitinib, ruxolitinib, tofacitinib, padacitinib, afatinib, axitinib, bosutinib, cetuximab, cobimetinib, crizotinib, cabozantinib, dasatinib, entrectinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, neratinib, nilotinib, pazopanib, pegaptanib, sorafenib, sunitinib, SU6656, toceranib, vandetanib, batatinib, and bemrafenib.

[0024] In some embodiments, the B cell receptor inhibitor is selected from an antisense or RNAi agent targeting CD20, and an antibody or antigen-binding fragment or small molecule that specifically binds to CD20, or the B cell receptor inhibitor is optionally selected from one or more of ibritumomab tiuxetan, obinutuzumab, ofatumumab, ocrelizumab, rituximab, tositumomab, and belzutifan.

[0025] In some embodiments, the antisense agent is about 10 to 40 bases in length and is optionally selected from morpholino oligonucleotide (PMO), peptide nucleic acid (PNA), 2’O-methyl phosphorothioate oligonucleotide, tricyclo-phosphorothioate oligonucleotide, and locked nucleic acid (LNA).

[0026] In some embodiments, the antisense agent specifically hybridizes to a target region within the pre-mRNA or mRNA target sequence encoding the target protein, and the target region is selected from one or more of the AUG start codon of the mRNA, the region upstream of the AUG start codon, the region downstream of the AUG codon, the 3’ or 5’ splice site of the pre-processed mRNA, the branch point, the 3’ untranslated region (UTR), and the polyadenylation signal sequence.

[0027] In some embodiments, the RNAi agent comprises a sense strand that is substantially identical to the mRNA target sequence encoding the target protein, and optionally, an antisense strand that is complementary or substantially complementary to the mRNA target sequence encoding the target protein, and optionally, the RNAi agent is a double-stranded short interfering RNA (siRNA) oligonucleotide, or optionally, the RNAi agent, optionally, the siRNA oligonucleotide is encoded by a viral vector.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, optionally, a humanized antibody, or optionally, an Fv fragment or single-chain Fv (sFv) polypeptide.

[0029] In some embodiments, the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or weight-by-weight basis and is substantially free of aggregates.

[0030] In some embodiments, the composition is substantially endotoxin-free.

[0031] Certain compositions contain lipid nanoparticles.

[0032] In some embodiments, the composition is in a syringe, optionally, an injectable syringe. In some embodiments, the composition is a capsule, e.g., an oral capsule.

[0033] A method of treating lung inflammation in a subject in need thereof, the method comprising administering to the subject (a) a histidyl-tRNA synthetase (HRS) polypeptide, or an expressible polynucleotide encoding the HRS polypeptide, and (b) an immunomodulatory agent.

[0034] In some embodiments, (a) and (b) are administered separately and optionally as defined herein. In some embodiments, (a) and (b) are administered together, optionally as a therapeutic composition described herein.

[0035] In some embodiments, the HRS polypeptide comprises an Fc region, forming an HRS-Fc fusion polypeptide. For example, this HRS-Fc fusion polypeptide comprises, consists of, or consists essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table H8. In some embodiments, the HRS polypeptide comprises, consists of, or consists essentially of SEQ ID NO: 157 (Fc-HRS(2-60) or HRS FC1 ).

[0036] In some embodiments, the immunomodulatory agent alters one or more pharmacokinetic characteristics of the HRS polypeptide as compared to the HRS polypeptide alone. In certain embodiments, one or more altered pharmacokinetic characteristics of the HRS polypeptide are an increase in serum concentration, an increase in serum half-life, an increase in bioavailability, an increase in exposure (AUC), an increase in serum concentration, and / or a decrease in clearance.

[0037] In some embodiments, the immunomodulatory agent is pirfenidone or nintedanib.

[0038] In some embodiments, the HRS polypeptide comprises, consists of, or consists essentially of SEQ ID NO: 157 (Fc-HRS(2-60) or HRSF C1 ), and the immunomodulatory agent is pirfenidone. In some embodiments, pirfenidone increases the serum concentration of the HRS polypeptide in a subject by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200% or more as compared to the HRS polypeptide alone.

[0039] In some embodiments, pirfenidone is in individual dosage units in the range of about 50 to about 1000 mg, or about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg, about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg or less, or at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,In individual dosage units of 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg, optionally, it is administered in one, two, or three capsules for oral administration.

[0040] In some embodiments, the dosage of pirfenidone is in a daily dosage unit in the range of about 100 to about 4000 mg / day, or about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day, about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500,2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day or less, or at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day in a single daily dosage unit, optionally, orally administered in about 1, 2, 3, 4, 5, 6, 7, 8, 9 capsules.

[0041] In some embodiments, pirfenidone is taken as 3 capsules per individual dosage, optionally, as 3 capsules of about 267 mg each for oral administration, in an individual dosage of about 800 mg (e.g., 801 mg). In some embodiments, pirfenidone is taken as 3 capsules per individual dosage, optionally, as 9 capsules of about 267 mg each for three times daily oral administration, in a single daily dosage unit of about 2400 mg / day (e.g., 2403 mg / day).

[0042] In some embodiments, nintedanib is administered in individual dosage units in the range of about 10 to about 500 mg, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg or less, or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg of individual dosage units, optionally, in about one, two, or three capsules.

[0043] In some embodiments, nintedanib is in a daily dosage unit in the range of about 20 to about 1000 mg / day, or about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg / day, about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg / day or less, or at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,In a daily dosage unit of 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg / day, it is optionally administered in about 1, 2, 3, 4, 5, or 6 capsules.

[0044] In some embodiments, nintedanib is administered in a daily dosage unit in the range of about 100 - 150 mg or in the range of about 200 - 300 mg / day, optionally in a dosage of once or twice a day. In some embodiments, nintedanib is administered in a daily dosage unit of about 100 - 150 mg or about 200 - 300 mg / day, optionally in a dosage of once or twice a day.

[0045] In some embodiments, the subject has or is at risk of having interstitial lung disease (ILD). In some embodiments, the ILD is idiopathic or associated with connective tissue disease, autoimmune disease, exposure to inhaled substances or drugs, infection, or malignancy.

[0046] In some embodiments, the ILD is selected from or associated with one or more of idiopathic interstitial pneumonia, idiopathic pulmonary fibrosis, sarcoidosis, Hamman-Rich syndrome, anti-synthetase syndrome, idiopathic eosinophilic pneumonia, alveolar hemorrhage syndrome, alveolar proteinosis, asbestosis, silicosis, berylliosis, rheumatoid arthritis, lupus erythematosus, chronic graft-versus-host disease with lung injury, scleroderma (systemic) or systemic sclerosis, polymyositis, dermatomyositis, chronic lung disease, asthma, bronchitis (respiratory bronchiolitis), pneumonia, hypersensitivity pneumonia, chronic hypersensitivity pneumonia, respiratory distress syndrome, Still's disease, acute lung injury, microscopic polyangiitis, pulmonary edema, pulmonary Langerhans cell histiocytosis, acute inhalation exposure, drug-induced lung disease, desquamative interstitial pneumonia, and / or cystic fibrosis.

[0047] In some embodiments, the ILD is associated with one or more of surfactant protein B deficiency (mutation in SFTPB), surfactant-protein C deficiency (mutation in SFTPC), ABCA3-deficiency (mutation in ABCA3), brain-lung-thyroid syndrome (mutation in TTF1), or congenital alveolar proteinosis (mutations in CSFR2A, CSFR2B), alveolar capillary dysplasia (mutation in FoxF1), mutations in telomerase reverse transcriptase (TERT), mutations in telomerase RNA component (TERC), mutations in regulators of telomere elongation helicase 1 (RTEL1), and / or mutations in poly(A)-specific ribonuclease (PARN).

[0048] In some embodiments, the drug is selected from one or more of antibiotics, chemotherapeutic agents, antiarrhythmic agents, and statins. In some embodiments, the infectious disease is selected from one or more of Pneumocystis pneumonia (PCP), tuberculosis, Chlamydia trachomatis, and respiratory syncytial virus (RSV), idiopathic organizing pneumonia. In some embodiments, the malignancy is angiosarcoma or lymphoma.

[0049] In some embodiments, the subject in need thereof has a condition selected from one or more of allergic asthma, non-allergic asthma, allergic bronchial IgE-mediated asthma, bronchial asthma, essential asthma, true asthma, intrinsic asthma caused by pathophysiological disturbances, extrinsic asthma caused by environmental factors, essential asthma of unknown or unclear cause, non-allergic asthma, bronchitis-like asthma, emphysematous asthma, exercise-induced asthma, allergen-induced asthma, cold-air-induced asthma, occupational asthma, infectious asthma caused by bacterial, fungal, protozoal, or viral infections, non-allergic asthma, initial asthma, wheezing infant syndrome and bronchiolitis, chronic or acute bronchoconstriction, chronic bronchitis, peripheral airway obstruction, and emphysema.

[0050] In some embodiments, the subject in need thereof has an obstructive or inflammatory airway disease. In some embodiments, the obstructive or inflammatory airway disease is selected from one or more of chronic eosinophilic pneumonia, chronic obstructive pulmonary disease (COPD), COPD including chronic bronchitis, COPD characterized by irreversible progressive airway obstruction, emphysema or dyspnea, and acute respiratory distress syndrome (ARDS).

[0051] In some embodiments, the subject in need of treatment of lung inflammation has a condition associated with exacerbation of airway hypersensitivity resulting from other drug therapies, airway diseases associated with pulmonary hypertension, bronchitis or acute bronchitis, acute laryngotracheobronchitis, arachidonic acid bronchitis, catarrhal bronchitis, croupous bronchitis, dry bronchitis, infectious asthmatic bronchitis, productive bronchitis, staphylococcal or streptococcal bronchitis, alveolar bronchitis, acute lung injury, bronchiectasis, or cylindrical bronchiectasis, cystic bronchiectasis, fusiform bronchiectasis, capillary bronchiectasis, cystoid bronchiectasis, dry bronchiectasis, or follicular bronchiectasis.

[0052] In some embodiments, the subject in need thereof has an Ashcroft score of 1, 2, 3, 4, 5, 6, 7, or 8.

[0053] Certain embodiments increase the average life expectancy of a subject in need thereof, optionally by about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 years, or more.

[0054] Certain embodiments improve one or more of the clinical symptoms or parameters of lung inflammation in a subject in need thereof.

[0055] In some embodiments, one or more clinical symptoms or parameters are selected from one or more of pulmonary fibrosis, inflammatory cell infiltration in the lung, respiratory function, and body weight.

[0056] Certain embodiments improve pulmonary fibrosis in a subject in need thereof, optionally by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000%, or more, as measured over a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months or more.

[0057] Certain embodiments improve pulmonary fibrosis in a subject in need thereof, as measured by a decrease in the Ashcroft score, optionally by a decrease of 1, 2, 3, 4, 5, 6, 7, or 8 grades compared to an initial score.

[0058] Certain embodiments optionally reduce inflammatory cell infiltration in the lungs by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000% or more when measured over a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months or more.

[0059] Certain embodiments optionally improve respiratory function by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000% or more when measured over a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months or more. In some embodiments, the improved respiratory function is selected from one or more of an increase in expiratory time, an increase in inspiratory time, a decrease in maximal expiratory flow, a decrease in maximal inspiratory flow, a decrease in respiratory minute volume (RMV), and a decrease in respiratory rate.

[0060] (a) a histidyl-tRNA synthetase (HRS) polypeptide, or an expressible polynucleotide encoding said HRS polypeptide, and (b) an immunomodulatory agent, is also included in a patient care kit.

[0061] In certain patient care kits, (a) and (b) are in separate compositions and are optionally defined as described herein. In some patient care kits, (a) and (b) are in the same composition and are defined as described herein.

[0062] In some embodiments, the immunomodulatory agent is pirfenidone or nintedanib.

[0063] In some embodiments, pirfenidone is in individual dosage units in the range of about 50 to about 1000 mg (optionally, in about one, two, or three capsules for oral administration), or (optionally, in one, two, or three capsules for oral administration) about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg, about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg or less, or at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or an individual dosage unit of 1000 mg.,

[0064] In some embodiments, pirfenidone is in a daily dosage unit in the range of about 100 to about 4000 mg / day (optionally, in about 3, 4, 5, 6, 7, 8, or 9 capsules for oral administration), or (optionally, in about 3, 4, 5, 6, 7, 8, or 9 capsules for oral administration) about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day, about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950,960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day or less, or at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day in individual dosage units.

[0065] In certain embodiments, pirfenidone is in individual dosage units of about 800 mg (e.g., 801 mg), for example, taken as three capsules per individual dosage, as three capsules of about 267 mg each for oral administration. In certain embodiments, pirfenidone is in a daily dosage unit of about 2400 mg / day (e.g., 2403 mg / day), for example, taken as three capsules per individual dosage, as nine capsules of about 267 mg each for three times a day oral administration.

[0066] In some embodiments, nintedanib is in individual dosage units in the range of about 10 to about 500 mg (optionally, in about 1, 2, or 3 capsules for oral administration), or (optionally, in about 1, 2, or 3 capsules for oral administration) about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg or less, or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg of individual dosage units.

[0067] In some embodiments, nintedanib is in a daily dosage unit in the range of about 20 to about 1000 mg / day (optionally, in about 1, 2, 3, 4, 5, or 6 capsules), or (optionally, in about 1, 2, 3, 4, 5, or 6 capsules) about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg / day, about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg / day or less, or at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,It is in a daily dosage unit of 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg / day.

[0068] In some embodiments, nintedanib is in a daily dosage unit in the range of about 100 - 150 mg, or in the range of about 200 - 300 mg / day, and optionally, is administered once or twice a day. In some embodiments, nintedanib is in a dosage unit in the range of about 100 - 150 mg, or in the range of about 200 - 300 mg / day, and optionally, is administered once or twice a day.

[0069] A method of altering one or more pharmacokinetic characteristics of an HRS-Fc fusion polypeptide in a subject, comprising administering to the subject an HRS-Fc fusion polypeptide, or an expressible polynucleotide encoding an HRS-Fc fusion polypeptide, optionally in combination with pirfenidone, is also included. In some embodiments, the HRS-Fc fusion polypeptide comprises, consists of, or consists essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table H8. In some embodiments, the HRS polypeptide comprises, consists of, or consists essentially of SEQ ID NO: 157 (Fc-HRS(2 - 60) or HRS FC1 )).

[0070] A method of performing it in a subject in need of treatment of lung inflammation described herein, the method also including administering to the subject an HRS-Fc fusion polypeptide, or an expressible polynucleotide encoding the HRS-Fc fusion polypeptide. In some embodiments, the HRS-Fc fusion polypeptide comprises, consists of, or consists essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from Table H8. In some embodiments, the HRS polypeptide comprises, consists of, or consists essentially of FC1 (SEQ ID NO: 157 (Fc-HRS(2-60) or HRS BRIEF DESCRIPTION OF THE DRAWINGS

[0071]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0072] In the practice of the present invention, unless otherwise indicated, conventional methods of molecular biology and recombinant DNA techniques within the scope of the art are used, many of which are described below for illustrative purposes. Such techniques are well explained in the references. For example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2000), DNA Cloning: A Practical Approach, vol. I&II (D. Glover, ed.), Oligonucleotide Synthesis (N. Gait, ed., 1984), Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed., 2004), Nucleic Acid Hybridization (B. Hames&S. Higgins, eds., 1985), Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009), Transcription and Translation (B. Hames&S. Higgins, eds., 1984), Animal Cell Culture (R. Freshney, ed., 1986), Freshney, R. I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th Ed. Hoboken NJ, John Wiley&Sons, B. Perbal, A Practical Guide to Molecular Cloning (3rd Edition 2010), Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd Edition 2005). Poly(ethylene glycol), Chemistry and Biological Applications, ACS, Washington, 1997, Veronese, F., and J. M. Harris, Eds.,Refer to Peptide and protein PEGylation, Advanced Drug Delivery Reviews, 54(4)453-609(2002), Zalipsky, S., et al., “Use of functionalized Poly(Ethylene Glycols) for modification of polypeptides” in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications.

[0073] Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods, materials, compositions, reagents, cells, or the like similar or equivalent to those described herein can be used in the practice or testing of the subject matter of this disclosure, but the preferred methods and materials are described herein. All publications and references, including but not limited to patent documents and patent application documents cited herein, are incorporated herein by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference as being fully described. Any patent applications for which this application claims priority are also incorporated herein by reference in their entirety for publications and references in the manner described above.

[0074] For the purposes of this disclosure, the following terms are defined as follows.

[0075] The articles “a” and “an” are used herein to refer to one or more than one (i.e., at least one) for the grammatical purpose of the article. By way of example, “a component” means one component or more than one component.

[0076] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length of reference.

[0077] "Antagonist" or "inhibitor" refers to a biological structure or chemical agent that prevents or otherwise reduces the physiological action of another agent or molecule. In some cases, an antagonist specifically binds to another agent or molecule. Complete and partial antagonists are included.

[0078] "Agonist" refers to a biological structure or chemical agent that increases or enhances the physiological action of another agent or molecule. In some cases, an agonist specifically binds to another agent or molecule. Complete and partial agonists are included.

[0079] The term "anergy" refers to the functional inactivation of the response of T cells or B cells to restimulation by an antigen.

[0080] As used herein, the term "amino acid" is intended to mean both naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics. Naturally occurring amino acids include, for example, the 20 (L)-amino acids utilized during protein biosynthesis, as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, etc., which are known to those skilled in the art. Amino acid analogs include modified forms of both naturally occurring and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid, or derivatization of the amino acid. Amino acid mimetics include organic structures that exhibit functionally similar properties such as, for example, the charge and charge spacing characteristic of a reference amino acid. For example, an organic structure that mimics arginine (Arg or R) has a positively charged moiety located within a similar molecular space and may have a mobility comparable to that of the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include structures that are constrained to maintain the optimal spacing and charge interactions of the amino acid or amino acid functional group. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0081] As used herein, a "risky" subject that gives rise to a disease or adverse reaction may or may not have a detectable disease or symptoms of a disease, and may or may not exhibit a detectable disease or symptoms of a disease prior to the treatment methods described herein. "Risky" indicates that the subject has one or more risk factors that are measurable parameters correlated with the subject developing a disease described herein and known in the art. A subject having one or more of these risk factors has a higher probability of developing a disease or adverse reaction than a subject not having one or more of these risk factors.

[0082] As used herein, the term "coding sequence" means any nucleic acid sequence that contributes to the coding of a polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to the coding of a polypeptide product of a gene.

[0083] As used herein, the term "binding" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges.

[0084] As used herein, the term "clonal deletion" refers to the removal (e.g., loss or death) of self-reactive T cells. Clonal deletion can occur primarily in the thymus, in the periphery, or in both.

[0085] Throughout this disclosure, unless the context requires otherwise, the words "comprise", "comprises", and "comprising" are to be construed as implying the inclusion of the stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0086] "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or essential and that no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase and being limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements may or may not be present, as necessary, depending on whether or not they substantially affect the activity or action of the listed elements.

[0087] The terms "endotoxin-free" or "substantially endotoxin-free" generally refer to a composition, solvent, and / or container that contains at most trace amounts (e.g., amounts that do not have a clinically harmful physiological effect on a subject), preferably undetectable amounts of endotoxin. Endotoxins, also known as endotoxins, can be found in gram-positive bacteria such as Listeria monocytogenes, but endotoxins are toxins associated with certain microorganisms, such as bacteria, typically gram-negative bacteria. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligo-saccharides (LOS) found in the outer membranes of various gram-negative bacteria, which exhibit central pathogenic characteristics in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans can cause, among other harmful physiological effects, fever, decreased blood pressure, and activation of inflammation and coagulation.

[0088] Therefore, in pharmaceutical products, even small amounts can cause harmful effects in humans, so it is often desirable to remove most or all traces of endotoxin from drug products and / or drug containers. Since temperatures above 300°C are typically required to degrade most endotoxins, a depyrogenation oven can be used for this purpose. For example, depending on the main packaging material such as a syringe or vial, a combination of a glass temperature of 250°C and a holding time of 30 minutes is often sufficient to achieve a 3 log reduction in endotoxin levels. Other methods of removing endotoxin, such as chromatography and filtration methods, as described herein and known in the art, are contemplated.

[0089] Endotoxins can be detected using conventional techniques known in the art. For example, the Limulus amebocyte lysate assay, which utilizes blood from horseshoe crabs, is a very sensitive assay for detecting the presence of endotoxins. In this test, very low levels of LPS can cause detectable coagulation of Limulus lysate due to a powerful enzyme cascade that amplifies this reaction. Endotoxins can also be quantified by an enzyme-linked immunosorbent assay (ELISA). The endotoxin level can be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU / mg of the active compound so as to be substantially endotoxin-free. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to about 1 - 10 EU.

[0090] As used herein, the terms "contacting a cell", "introducing", or "delivering" include delivery of the agents (e.g., polypeptide agents, polynucleotide agents) described herein to a cell by conventional methods in the art, such as transfection (e.g., liposomes, calcium phosphate, polyethyleneimine), electroporation (e.g., nucleofection), microinjection), or administration to a subject.

[0091] The terms "cell-penetrating peptide" (CPP) or "peptide moiety that enhances cellular uptake" are used interchangeably and refer to cationic cell-penetrating peptides, also sometimes called "transport peptides", "carrier peptides", or "peptide transduction domains". In some embodiments, the peptide, when administered systemically, induces penetration into about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells of a given cell culture population, or at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells (e.g., muscle cells) within the cells, or in vivo enables translocation of macromolecules within multiple tissues (e.g., muscle tissue) in other administration forms. In some embodiments, the CPP is a CPP of the formula -[(C(O)CHR’NH) m R’’ where R’ is the side chain of a naturally occurring amino acid or its 1- or 2-carbon analog, R’’ is selected from hydrogen or acyl, and m is an integer up to 50. Further CPPs are known in the art and are disclosed, for example, in U.S. Patent Application No. 2010 / 0016215, which is incorporated by reference in its entirety. In some embodiments, m is an integer selected from 1 to 50, and when m is 1, the moiety is a single amino acid or its derivative. Any of the polynucleotide agents (e.g., antisense, RNAi agents) described herein can be conjugated to a CPP to improve uptake, for example, into target cells, such as muscle cells.

[0092] The term "half maximal effective concentration" or "EC50" refers to the concentration of a drug (e.g., an HRS polypeptide or other drug) described herein that induces a response intermediate between baseline and maximum after some specified exposure time, and thus, the EC50 of a stepwise dose response curve represents the concentration of a compound at which 50% of its maximal effect is observed. EC50 also represents the plasma concentration required to obtain 50% of the maximal effect in vivo. Similarly, "EC90" refers to the concentration of a drug or composition at which 90% of its maximal effect is observed. "EC90" can be calculated from the "EC50" and the Hill slope, or can be determined directly from the data using conventional knowledge in the art. In some embodiments, the EC50 of a drug is less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 nM. In some embodiments, the biotherapeutic composition will have an EC50 value of about 1 nM or less.

[0093] "Homology" refers to the percentage of amino acids that are identical or that constitute conservative substitutions. Homology can be determined using a sequence comparison program such as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). In this way, sequences of similar or substantially different lengths to those sequences cited herein can be compared by inserting gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0094] The term "innate immune response" refers to the response of immune cells (including macrophages and natural killer cells (NK)) that defend the host from infection by pathogens, as well as the related mechanisms that regulate cytokine expression and release (e.g., interferon and interferon-signaling), induce cell death, and inhibit protein synthesis.

[0095] "Isolated" means a material that is substantially or essentially free of the components that are normally associated with it in its natural state. For example, as used herein, an "isolated polynucleotide", "isolated oligonucleotide", or "isolated oligonucleotide" can refer to a polynucleotide that has been purified or removed from the sequences that flank the polynucleotide in its naturally occurring state, e.g., a DNA fragment that has been removed from the sequences that flank the fragment in the genome. The term "isolating" when it relates to a cell refers to the purification of cells (e.g., fibroblasts, lymphocytes) from a source subject (e.g., a subject suffering from a polynucleotide repeat disorder). In the context of mRNA or protein, "isolating" refers to the recovery of mRNA or protein from a source, e.g., a cell.

[0096] The term "modulate" optionally includes "increase" or "decrease" of one or more quantifiable parameters by a defined and / or statistically significant amount. "Increase" or "increasing", "enhance" or "enhancing", or "stimulate" or "stimulating" generally refers to the ability of one or more agents or compositions to produce or cause a greater physiological response (i.e., downstream effect) in a cell or subject as compared to the response caused by no agent / compound or a control compound. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include an increase in skeletal muscle mass in a tissue or subject in need thereof. An "increased" or "enhanced" amount is typically a "statistically significant" amount and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 times, or more (e.g., 500, 1000 times), the amount produced by no agent / compound (absence of agent) or a control compound, including all integers and decimal points between and above 1 (e.g., 1.5, 1.6, 1.7, 1.8). The terms "decrease" or "inhibit" generally may relate to the ability of one or more agents or compositions to "reduce" a relevant physiological or cellular response such as the expression of a target gene or symptoms of a disease or condition described herein when measured by routine techniques of diagnostic art. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include a decrease or amelioration of lung inflammation or symptoms or pathology of ILD described herein. A "decrease" in the response may be "statistically significant" as compared to the response produced by no agent or composition or a control agent or composition and may include a decrease of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all intermediate integers.

[0097] In certain embodiments, the “purity” of any given agent in the composition can be specifically defined. For example, a particular composition, for example and without limitation, when measured by high performance liquid chromatography (HPLC), a well-known form of column chromatography frequently used in biochemistry and analytical chemistry to separate, identify, and quantify compounds, may contain an agent that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all minor species in between.

[0098] “Lipid nanoparticles” or “solid lipid nanoparticles” refer to one or more spherical nanoparticles having an average diameter of about 10 to about 1000 nanometers and containing a solid lipid core matrix capable of solubilizing lipophilic molecules. The lipid core is stabilized by surfactants (e.g., emulsifiers) and can include one or more of triglycerides (e.g., tristearin), diglycerides (e.g., glyceryl behenate), monoglycerides (e.g., glycerol monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate), including combinations thereof. Lipid nanoparticles are described, for example, in Petrilli et al., Curr Pharm Biotechnol. 15:847-55, 2014, as well as U.S. Patent Nos. 6,217,912; 6,881,421; 7,402,573; 7,404,969; 7,550,441; 7,727,969; 8,003,621; 8,691,750; 8,871,509; 9,017,726; 9,173,853; 9,220,779; 9,227,917; and 9,278,130, which are incorporated by reference in their entirety.

[0099] As used herein, "nucleobase" (Nu), "base pairing moiety" or "base" are used interchangeably and refer to purine or pyrimidine bases (uracil, thymine, adenine, cytosine, and guanine) found in natural DNA or RNA, as well as analogs of naturally occurring purines and pyrimidines that confer improved properties, such as binding affinity to oligonucleotides. Exemplary analogs include hypoxanthine (the base component of the nucleoside inosine), 2,6-diaminopurine, 5-methylcytosine, C5-propynyl-modified pyrimidines, 9-(aminoethoxy)phenoxazine (G-clamp), and the like.

[0100] Further examples of base pairing moieties include uracil, thymine, adenine, cytosine, guanine, and hypoxanthine, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pseudoisocytosine, and pseudouracil, etc., in which their respective amino groups are protected with an acyl protecting group, as well as other modified nucleobases such as 8-substituted purines, 8-substituted xanthines, or 8-substituted hypoxanthines (the latter two being natural degradation products), but are not limited thereto. Modified nucleobases disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196, and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313 are also contemplated.

[0101] Further examples of base pairing moieties include, but are not limited to, size-expanded nucleobases with one or more benzene rings added. Nucleobase substitutions described in the Glen Research catalog (www.glenresearch.com), Krueger AT et al, Acc.Chem.Res., 2007, 40, 141 - 150, Kool, ET, Acc.Chem.Res., 2002, 35, 936 - 943, Benner S.A., et al., Nat.Rev.Genet., 2005, 6, 553 - 543, Romesberg, F.E., et al., Curr.Opin.Chem.Biol., 2003, 7, 723 - 733, Hirao, I., Curr.Opin.Chem.Biol., 2006, 10, 622 - 627 are contemplated to be useful for the synthesis of the oligonucleotides described herein. Examples of size-expanded nucleobases are shown below. [Chemical formula]

[0102] Nucleobases covalently linked to ribose, sugar analogs, or morpholino include nucleosides. A "nucleotide" consists of a nucleoside together with one phosphate group. The phosphate groups are covalently linked to adjacent nucleotides to form an oligomer.

[0103] The terms "polypeptide" and "protein" are used interchangeably herein and refer to polymers of amino acid residues as well as variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non - naturally occurring amino acids such as chemical analogs of the corresponding naturally occurring amino acids, as well as to amino acid polymers that are naturally occurring amino acid polymers.

[0104] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA, and DNA. This term typically refers to nucleotides in polymeric form that are at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. This term includes single-stranded and double-stranded forms of DNA. The terms "isolated DNA" and "isolated polynucleotide", and "isolated nucleic acid" refer to isolated molecules that do not include genomic DNA of a particular species. Thus, an isolated DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences but is isolated substantially away from, or purified such that it is not present in, the genomic DNA of the species from which the DNA segment is obtained. Also included are non-coding polynucleotides (e.g., primers, probes, oligonucleotides) that do not encode polypeptides. Also included are recombinant vectors, such as expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, viruses, etc.

[0105] Additional coding or non-coding sequences may be present within the polynucleotides described herein, but need not be present, and the polynucleotides may be bound to other molecules and / or support materials, but need not be bound. Thus, a polynucleotide or expressible polynucleotide may be combined with other sequences, such as expression control sequences, regardless of the length of the coding sequence itself.

[0106] "Expression control sequence" includes nucleic acids or regulatory sequences of the corresponding amino acids, such as promoters, leaders, enhancers, introns, recognition motifs for RNA, or DNA-binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES), secretion signals, intracellular localization signals, etc., or the intracellular or cellular site of the coding sequence in the host cell. Exemplary expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0107] "Promoter" is a DNA regulatory region that can bind RNA polymerase in a cell and initiate transcription of a downstream (3' direction) coding sequence. As used herein, the promoter sequence is bound by the transcription start site at its 3' end, extends upstream (5' direction), and contains the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. Within the promoter sequence, a transcription start site (conventionally defined by mapping with nuclease S1) and a protein-binding domain (consensus sequence) responsible for binding of RNA polymerase can be found. Eukaryotic promoters do not necessarily contain a "TATA" box and a "CAT" box, but often can. Prokaryotic promoters include a Shine-Dalgarno sequence in addition to the -10 and -35 consensus sequences.

[0108] Numerous promoters, including structural, inducible, and repressive promoters from a variety of different sources, are well known in the art. Representative sources include, for example, viral, mammalian, insect, plant, yeast, and bacterial cell types), and suitable promoters from these sources are readily available or can be synthetically generated based on publicly available sequences, either online or from depositories such as, for example, the ATCC, as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3’ or 5’ direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter. Inducible promoters include the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci. (1996) 93(8):3346-3351), the T-RExTM system (Invitrogen Carlsbad, CA), LacSwitch® (Stratagene, (San Diego, CA), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al. Nuc. Acid Res. (1999) 27(22):4324-4327, Nuc. Acid Res. (2000) 28(23):e99, U.S. Pat. No. 7,112,715, as well as Kramer & Fussenegger, Methods Mol. Biol. (2005) 308:123-144) or any promoter known in the art suitable for expression in a desired cell.

[0109] An expressible polynucleotide includes cDNA, RNA, mRNA, or other polynucleotides that contain at least one coding sequence and optionally at least one expression control sequence, such as transcription and / or translation regulatory elements, and can express the encoded polypeptide (e.g., the HRS polypeptide) when introduced into a cell, such as a cell in a subject.

[0110] In some embodiments, the expressible polynucleotide is a modified RNA or a modified mRNA polynucleotide, such as a non-naturally occurring RNA analog. In certain embodiments, the modified RNA or mRNA polypeptide contains one or more modified or non-natural bases, such as nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T), and / or uracil (U). In some embodiments, the modified mRNA contains one or more modified or non-natural internucleotide linkages. Expressible RNA polynucleotides for delivering the encoded therapeutic polypeptide are described, for example, in Kormann et al., Nat Biotechnol. 29:154-7, 2011, and U.S. Patent Application Nos. 2015 / 0111248, 2014 / 0243399, 2014 / 0147454, and 2013 / 0245104, which are incorporated by reference in their entirety.

[0111] In some embodiments, the various viral vectors that can be used to deliver expressible polynucleotides include adenoviral vectors, herpes viral vectors, vaccinia viral vectors, adeno-associated virus (AAV) vectors, and retroviral vectors. In some cases, the retroviral vectors are derivatives of murine or avian retroviral vectors or are lentiviral vectors. Examples of retroviral vectors into which a single foreign gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous sarcoma virus (RSV). Many additional retroviral vectors can incorporate multiple genes. All of these vectors can move or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting the polypeptide sequence of interest into the viral vector along with another gene encoding a ligand for a receptor on a particular target cell, for example, the vector can achieve target specificity. A retroviral vector can achieve target specificity, for example, by inserting a polynucleotide encoding a protein. Exemplary targeting can be achieved by using an antibody that targets the retroviral vector. One of ordinary skill in the art knows or can readily identify the particular polynucleotide sequences that can be delivered to the retroviral genome that enable target-specific delivery of the retroviral vector without undue experimentation.

[0112] In certain cases, the expressible polynucleotides described herein are engineered to localize within the cell, potentially within a particular compartment such as the nucleus, or are engineered to be secreted from the cell or translocate to the plasma membrane of the cell. In an exemplary embodiment, the expressible polynucleotide is engineered for nuclear localization.

[0113] Also included are biologically active "variants" and "fragments" of the polypeptides described herein, and polynucleotides encoding the same. A "variant" contains one or more substitutions, additions, deletions, and / or insertions as compared to a reference polypeptide or polynucleotide (see, e.g., the tables and sequence listings). A variant polypeptide or polynucleotide contains an amino acid or polynucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity or homology to the reference sequence described herein, and substantially retains the activity of that reference sequence. Also included are sequences that consist of, or differ from, the reference sequence by the addition, deletion, insertion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acids or nucleotides, and that substantially retain the activity of that reference sequence. In certain embodiments, the addition or deletion includes C-terminal and / or N-terminal additions and / or deletions.

[0114] The term "sequence identity" or, for example, "50% sequence identity relative to" as used herein, refers to the degree to which two sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a window of comparison. Thus, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which the identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to obtain the number of matched positions, dividing this number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for aligning the comparison window can be performed by the computerized execution of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by the best alignment (i.e., the one that yields the highest percentage of homology over the comparison window) generated by any of a variety of methods inspected and selected. Reference may also be made to programs of the BLAST family, as disclosed by, for example, Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0115] "Statistically significant" means that the result is unlikely to occur by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability of the observed event occurring assuming the null hypothesis is true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In a simple example, this significance level is defined at a p-value of 0.05 or less.

[0116] The term "solubility" refers to the property of the agents provided herein to dissolve in a liquid solvent to form a homogeneous solution. Solubility is typically expressed as a concentration by any of the mass of solute per unit volume of solvent (g of solute per kg of solvent, g / dL (100 mL), mg / ml, etc.), molarity, molality, mole fraction, or other similar descriptions of concentration. The maximum equilibrium amount of solute that can dissolve per amount of solvent is the solubility of that solute in that solvent under specific conditions including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH or other pHs, such as pH 5.0, pH 6.0, pH 7.0, or pH 7.4. In certain embodiments, solubility is measured in water or a physiological buffer, such as PBS or NaCl (with or without NaP). In certain embodiments, solubility is measured at a relatively low pH (e.g., pH 6.0) and a relatively high salt (e.g., 500 mM NaCl and 10 mM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent), such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25 °C) or about body temperature (37 °C). In certain embodiments, the agent has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / ml at room temperature or 37 °C.

[0117] "Subject" or "subject in need thereof" includes mammalian subjects such as human subjects.

[0118] "Substantially" or "essentially" means almost entirely or completely, e.g., 95% or more of a given amount.

[0119] "Therapeutic response" refers to improvement of symptoms (whether persistent or not) based on administration of a therapeutic response.

[0120] As used herein, the term "target" refers to an RNA region, specifically, the RNA region of a target gene described herein. The target may include coding and non-coding sequences, 5' upstream sequences, 3' downstream sequences, and other RNA sequences described herein.

[0121] The term "target sequence" refers to a portion of the target RNA to which an antisense or RNAi agent is directed, e.g., the sequence to which an antisense oligonucleotide hybridizes by Watson-Crick base pairing of complementary sequences, or the sequence corresponding to the sense strand of an RNAi agent.

[0122] As used herein, "quantify", "quantification", or other related terms refer to determining the amount, mass, or concentration of a nucleic acid, polynucleotide, oligonucleotide, peptide, polypeptide, or protein in a unit volume.

[0123] As used herein, the terms "therapeutically effective amount", "therapeutic dose", "prophylactically effective amount", or "diagnostically effective amount" are the amount of a drug required to produce a desired biological response after administration. Similarly, the terms "antisense therapy" or "RNAi therapy" include therapies that maintain an average steady-state concentration of an antisense or RNAi agent in a patient's plasma or other tissue compartment (e.g., muscle tissue) above a minimally effective therapeutic level.

[0124] As used herein, "treatment" of a subject (e.g., a mammal, e.g., a human) or a cell is any type of intervention used in an attempt to alter the natural course of an individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, which may be performed either prophylactically or either after the onset of a pathological event or after contact with a pathogen. "Preventive" treatment is also included, which can be directed at reducing the rate of progression of a disease or condition being treated, delaying the onset of a disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily indicate complete eradication, cure, or prevention of a disease or condition or its associated symptoms.

[0125] The term "wild-type" refers to that which is most frequently observed in a population and thus refers to the "normal" or "wild-type" form of a gene or gene product (e.g., a polypeptide), as appropriately designed.

[0126] Histidyl-tRNA synthetase (HRS) polypeptides and polynucleotides Certain embodiments include histidyl-tRNA synthetase polypeptides ("HRS" or "HisRS" polypeptides) comprising conjugates (e.g., Fc conjugates), variants, and fragments thereof, as well as expressible polynucleotides encoding HRS polypeptides. Histidyl-tRNA synthetase belongs to the class II tRNA synthetase family and has three highly conserved sequence motifs. Class I and II tRNA synthetases are widely recognized to be responsible for the specific binding of an amino acid to its cognate tRNA in a two-step reaction: the amino acid (AA) is first activated by ATP to form AA-AMP, which is then transferred to the acceptor end of the tRNA. Full-length histidyl-tRNA synthetase typically exists either as a cytosolic homodimer or as an alternatively spliced mitochondrial form.

[0127] A specific biological fragment or alternatively spliced isoform of eukaryotic histidyl-tRNA synthetase, or in some contexts the intact full-length synthetase, modulates certain therapeutically relevant cellular signaling pathways and / or has anti-inflammatory properties. These activities, distinct from the classical role of tRNA synthetases in protein synthesis, are referred to herein as "non-canonical activities". For example, HRS polypeptides such as the N-terminal fragments of histidyl-tRNA synthetase provided herein (e.g., HRS1-48, HRS1-60) can exert anti-inflammatory signals, inter alia, by blocking in vivo the migration, activation, or differentiation of inflammatory cells (e.g., monocytes, macrophages, T cells, B cells, NK cells, dendritic cells) associated with sites of active inflammation. In addition, certain mutations or deletions (e.g., HRS1-506, HRS1-60), as compared to the full-length HRS polypeptide sequence, confer increased activity and / or improved pharmacological properties. The sequences of certain exemplary HRS polypeptides are provided in Table H1 below.

Table H1-1

Table H1-2

Table H1-3

Table H1-4

Table H1-5

Table H1-6

Table H1-7

Table H1-8

Table H1-9

Table H1-10

Table H1-11

Table H1-12

Table H1-13

Table H1-14

Table H1-15

Table H1-16

Table H1-17

Table H1-18

Table H1-19

Table H1-20

Table H1-21

Table H1-22

Table H1-23

Table H1-24

Table H1-25

Table H1-26

Table H1-27

Table H1-28

[0128] Thus, in certain embodiments, the HRS polypeptide comprises, consists of, or consists essentially of a mammalian HRS amino acid sequence (e.g., SEQ ID NOs: 1-117) in Table H1 or an active variant or fragment thereof. In some embodiments, the HRS polypeptide comprises, consists of, or consists essentially of a human HRS amino acid sequence (e.g., SEQ ID NOs: 1-109) in Table H1 or an active variant or fragment thereof. In some embodiments, an expressible polynucleotide encodes an HRS polypeptide that comprises, consists of, or consists essentially of an amino acid sequence (e.g., SEQ ID NOs: 1-117) in Table H1, e.g., a human HRS sequence (SEQ ID NOs: 1-109) in Table H1 or an active variant or fragment thereof.

[0129] As described above, the HRS polypeptide can be modified in various ways, including amino acid substitutions, deletions, truncations, additions, and insertions. Methods for such manipulations are generally well known in the art. For example, amino acid sequence variants of the HRS reference polypeptide can be prepared by mutations in DNA. Methods for mutagenesis and nucleotide sequence alteration are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al. (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, J.D. et al. (“Molecular Biology of the Gene”, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and the references cited therein. Guidance for effecting amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

[0130] Biologically active truncated and / or variant HRS polypeptides may contain conservative amino acid substitutions at various positions along their sequences, as compared to the reference HRS amino acid residues. “Conservative amino acid substitutions” are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains, which can generally be subclassified as follows, are defined in the art: Acidic: This residue has a negative charge due to the loss of an H ion at physiological pH, and this residue is attracted to an aqueous solution so as to seek a surface position in the conformation of the peptide in which the residue is included when the peptide is present in an aqueous medium at physiological pH. Amino acids having acidic side chains include glutamic acid and aspartic acid.

[0131] Basic: This residue has a positive charge due to association with H ions at physiological pH or within 1 or 2 pH units thereof (e.g., histidine), and this residue is attracted by an aqueous solution so as to seek a surface position in the conformation of the peptide containing this residue when the peptide is present in an aqueous medium at physiological pH. Amino acids with basic side chains include arginine, lysine, and histidine.

[0132] Charged: These residues are charged at physiological pH, and thus include amino acids with acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine, and histidine).

[0133] Hydrophobic: These residues are uncharged at physiological pH, and this residue is repelled by an aqueous solution so as to seek an internal position in the conformation of the peptide containing this residue when the peptide is present in an aqueous medium. Amino acids with hydrophobic side chains include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine, and tryptophan.

[0134] Neutral / Polar: These residues are uncharged at physiological pH, and this residue is not sufficiently repelled by an aqueous solution so as to seek an internal position in the conformation of the peptide containing this residue when the peptide is present in an aqueous medium. Amino acids with neutral / polar side chains include asparagine, glutamine, cysteine, histidine, serine, and threonine.

[0135] This description also characterizes certain amino acids as "small" because the side chain of a particular amino acid, even if lacking a polar group, is not large enough to confer hydrophobicity. Except for proline, "small" amino acids are those having four or fewer carbons if at least one polar group is present on the side chain and three or fewer carbons if none is present. Amino acids with small side chains include glycine, serine, alanine, and threonine. The genetically encoded secondary amino acid proline is a special case due to its known effect on the secondary conformation of the peptide chain. The structure of proline differs from that of all other naturally occurring amino acids in that its side chain is attached to both the nitrogen of the α-amino group and the α-carbon. Several amino acid similarity matrices are well known in the art (see, for example, the PAM120 matrix and the PAM250 matrix, as disclosed, for example, by Dayhoff et al., 1978, A model of evolutionary change in proteins). However, the matrices for determining distance relationships by M. O. Dayhoff, (ed.), Atlas of protein sequence and structure, Vol. 5, pp. 345-358, National Biomedical Research Foundation, Washington DC, and Gonnet et al. (Science, 256:14430-1445, 1992) include proline in the same group as glycine, serine, alanine, and threonine. Thus, proline is classified as a "small" amino acid.

[0136] The degree of attraction or repulsion required for classification as polar or non-polar is arbitrary, and thus the amino acids specifically contemplated by the present invention are classified as one or the other. Most amino acids not specifically named can be classified based on their known behavior.

[0137] Amino acid residues can be further subclassified as cyclic or acyclic, and aromatic or non-aromatic, with respect to the side-chain substituents of the residues, and as small or large. A residue is considered small if it contains a total of four or fewer carbon atoms, including the carboxyl carbon, provided that there are additional polar substituents, or if it contains three or fewer otherwise. Small residues are, of course, always non-aromatic. Depending on their structural properties, amino acid residues can fall into more than one class. For the naturally occurring protein amino acids, the subclassifications according to this scheme are presented in Table A.

Table A-1

Table A-2

[0138] The conserved amino acid substitutions also include a classification based on side chains. For example, the group of amino acids having aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic-hydroxyl side chains includes serine and threonine; the group of amino acids having amide-containing side chains includes asparagine and glutamine; the group of amino acids having aromatic side chains includes phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains includes lysine, arginine, and histidine; and the group of amino acids having sulfur-containing side chains includes cysteine and methionine. For example, substitutions such as leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or similar substitutions of an amino acid with an amino acid structurally related thereto would not have a significant impact on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional truncated and / or variant HRS polypeptide can be readily determined by assaying its non-canonical activity, as described herein. Conserved substitutions are shown in Table B under the heading of exemplary substitutions. Amino acid substitutions falling within the scope of the present invention are generally achieved by selecting substitutions that do not significantly differ in (a) the structure of the peptide backbone in the region of substitution, (b) the charge or hydrophobicity of the molecule at the target site, (c) the bulk of the side chain, or (d) its effect on maintaining the biological function. After the substitutions are introduced, these variants are screened for biological activity.

Table B-1

Table B-2

[0139] Alternatively, similar amino acids for performing conservative substitutions can be grouped into three categories based on the identity of their side chains. As described in Zubay, G., Biochemistry, third edition, Wm. C. Brown Publishers (1993), the first group includes glutamic acid, aspartic acid, arginine, lysine, and histidine, all of which have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, and asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, and methionine.

[0140] In some embodiments, the HRS polypeptide has one or more cysteine insertions or substitutions, e.g., one or more non-cysteine residues are substituted with cysteine residues (e.g., to promote thiol-based conjugation of the Fc fragment, to promote thiol-based binding of PEG or other molecules, for example, to alter stability). In some embodiments, one or more cysteine substitutions are near the N-terminus and / or C-terminus of the HRS polypeptide, or other surface-exposed regions of the HRS polypeptide. Particular embodiments include the case where one or more of the residues within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids of the N-terminus and / or C-terminus of the HRS polypeptide are substituted with cysteine residues. In some embodiments, cysteine residues can be added to the HRS polypeptide via creation of an N-terminal or C-terminal fusion protein. Such fusion proteins can be of any length, but are typically about 1-5, or about 5-10, about 10-20, or about 20-30 amino acids in length.

[0141] Specific examples of cysteine-modified proteins based on the HRS polypeptide HRS(1-60) are shown in Table H2. This approach can be applied to the HRS polypeptides of Table H1 and other HRS polypeptides described herein.

Table H2

[0142] Thus, in certain embodiments, the HRS polypeptide comprises, consists of, or consists essentially of the amino acid sequences (SEQ ID NOs: 118-120) in Table H2 or active variants or fragments thereof. In some embodiments, an expressible polynucleotide encodes an HRS polypeptide that comprises, consists of, or consists essentially of the amino acid sequences (e.g., SEQ ID NOs: 118-120) in Table H2 or active variants or fragments thereof.

[0143] In some embodiments, the HRS polypeptide may have variants in which endogenous or naturally occurring cysteine residues are mutated to alternative amino acids or deleted. In some embodiments, the insertion or substitution of cysteine residues into the HRS polypeptide is combined with the elimination of other surface-exposed reactive cysteine residues. Thus, in some embodiments, the HRS polypeptide comprises one or more substitutions and / or deletions, including combinations thereof, at any one or more of, for example, Cys83, Cys174, Cys191, Cys196, Cys224, Cys235, Cys379, Cys455, Cys507, and / or Cys509 (as defined by SEQ ID NO: 1) to remove naturally occurring cysteine residues.

[0144] Certain embodiments include an HRS polypeptide of Table H1 having one or more mutations or deletions of any of Cys83, Cys174, Cys191, Cys196, Cys224, Cys235, Cys379, Cys455, or deletions of Cys507 and Cys509 due to deletion of, for example, the 3 amino acids at the C-terminus (Δ507 - 509). Exemplary mutations at these positions include, for example, mutations from cysteine to serine, alanine, leucine, valine, or glycine. In certain embodiments, the amino acid residues for specific cysteine substitutions can be selected from naturally occurring substitutions found in HRS orthologs from other species and organisms. Exemplary substitutions of this type are shown in Table H3.

Table H3

[0145] In some embodiments, the naturally occurring cysteines selected for mutagenesis are selected based on their surface exposure. Thus, in one aspect, the cysteine residues selected for substitution are selected from Cys224, Cys235, Cys507, and Cys509. In some embodiments, the last 3 (C-terminal) residues of SEQ ID NO: 1 are deleted such that residues 507 - 509 are missing. In some embodiments, these cysteines are selected for mutation or deletion so as to eliminate intramolecular cysteine pairs, such as Cys174 and Cys191.

[0146] Specific examples of cysteine mutations / substitutions (shown in bold underlined) to reduce surface-exposed cysteine residues include those listed below in Table H4.

Table H4-1

Table H4-2

[0147] Thus, in certain embodiments, the HRS polypeptide comprises, consists of, or consists essentially of the amino acid sequences (SEQ ID NOs: 121-127) in Table H4 or active variants or fragments thereof. In some embodiments, the expressible polynucleotide encodes an HRS polypeptide that comprises, consists of, or consists essentially of the amino acid sequences (e.g., SEQ ID NOs: 121-127) in Table H4 or active variants or fragments thereof.

[0148] In some embodiments, such cysteine substitution variants are modified to engineer, insert, or otherwise introduce a new surface-exposed cysteine residue at a defined surface-exposed position, where the introduced residue does not substantially interfere with the non-canonical activity of the HRS polypeptide. Specific examples include, for example, the insertion (or re-insertion) of additional cysteine residues at either the N-terminus or C-terminus of any of the above-described reduced-cysteine HRS polypeptides. In some embodiments, such N-terminal or C-terminal surface-exposed cysteine insertion includes the re-insertion of the last one, last two, or last three naturally occurring C-terminal amino acids of full-length human HRS into the reduced-cysteine variant of the HRS polypeptide, e.g., the re-insertion of all or a portion of the sequence CIC (Cys Ile Cys). Exemplary reduced-cysteine variants include, for example, any combination of mutations (or deletions thereof) at residues Cys174, Cys191, Cys224, and Cys235 in any of the HRS polypeptides of Table H1, as well as or deletions or substitutions of Cys507 and Cys509 (based on the numbering of full-length human cytoplasmic HRS (SEQ ID NO: 1)).

[0149] For some types of site-specific conjugation or attachment to heterologous molecules such as the Fc region or PEG or other heterologous molecules, the HRS polypeptide can have one or more glutamine substitutions, and one or more naturally occurring (non-glutamine) residues are substituted with glutamine, for example, to facilitate the transglutaminase-catalyzed attachment of the molecule to the amide group of glutamine. In some embodiments, the glutamine substitutions are introduced in the vicinity of the N-terminus and / or C-terminus of the HRS polypeptide. Certain embodiments include cases where one or more of the residues within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids of the N-terminus and / or C-terminus of the HRS polypeptide are substituted with glutamine residues. These and related HRS polypeptides can also include the removal of any naturally occurring glutamine residues and, if desired, substitutions (e.g., conservative substitutions) to thereby adjust the degree of site-specific conjugation or attachment.

[0150] For some types of site-specific conjugation or attachment to heterologous molecules such as the Fc region or PEG or other heterologous molecules, the HRS polypeptide can have one or more lysine substitutions, and one or more naturally occurring (non-lysine) residues are substituted with lysine, for example, to facilitate a bond based on acylation or alkylation of the molecule to the amide group of lysine. These methods also typically result in the attachment of the molecule to the N-terminal residue. In some embodiments, the lysine substitutions are in the vicinity of the N-terminus and / or C-terminus of the HRS polypeptide. Certain embodiments include cases where one or more of the residues within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids of the N-terminus and / or C-terminus of the HRS polypeptide are substituted with lysine residues. These and related HRS polypeptides can also include the removal of any naturally occurring lysine residues and, if desired, substitutions (e.g., conservative substitutions) to thereby adjust the degree of site-specific conjugation or attachment.

[0151] Site-specific conjugation to an HRS polypeptide can also be carried out by substituting one or more solvent-accessible surface amino acids of the HRS polypeptide. For example, suitable solvent-accessible amino acids can be determined based on the predicted solvent accessibility using the published crystal structure of an exemplary HRS polypeptide and the SPIDDER server (http: / / sppider.cchmc.org / ) (see Xu et al., Structure. 20:1470-7, 2012 and U.S. Patent Application No. 61 / 674,639). Based on this analysis, some of the amino acids on the surface can potentially be used as mutation sites for introducing functional groups suitable for conjugation or binding. A surface accessibility score for amino acids based on the crystal structure can be calculated, where a higher score indicates better accessibility. In certain embodiments, a higher score (e.g., >40) is preferred. Thus, in some embodiments, amino acid positions having a surface accessibility score greater than 40 can be used to introduce cysteine, lysine, glutamine, or other non-naturally occurring amino acids.

[0152] In certain embodiments, the solvent-accessible surface amino acids can be selected from the group consisting of alanine, glycine, and serine and substituted with a naturally occurring amino acid, such as, but not limited to, cysteine, glutamine, or lysine, or a non-naturally occurring amino acid optimized for site-specific conjugation or binding.

[0153] Certain embodiments include site - specific conjugation or attachment to an HRS polypeptide at any amino acid position by substituting a non - naturally occurring amino acid that contains a functional group that forms a covalent bond with a functional group attached to a heterologous molecule such as an Fc region or PEG or other heterologous molecule. The non - naturally occurring amino acid can be inserted or substituted, for example, at the N - terminus and / or C - terminus of the HRS polypeptide, at the N - terminus and / or C - terminus, or at solvent - accessible surface amino acid residues, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids of one or more of the residues.

[0154] In certain embodiments, non - naturally occurring amino acids include any amino acid, modified amino acid, or amino acid analog other than selenocysteine, and the 20 alpha - amino acids encoded by the following 20 genes: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, but are not limited thereto. The general structure of an alpha - amino acid is exemplified by the following formula:

Chemical formula

[0155] A non-natural amino acid is typically any structure having the above formula, where the R group is any substituent other than the substituents used in the 20 natural amino acids. For the structures of the 20 natural amino acids, refer to biochemistry textbooks such as Biochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York. It should be noted that the non-natural amino acids disclosed herein can be naturally occurring compounds other than the above 20 alpha-amino acids. The non-natural amino acids disclosed herein typically differ from natural amino acids only in the side chain, so these non-natural amino acids form amide bonds with other amino acids, whether natural or non-natural, in the same manner as formed in naturally occurring proteins. However, these non-natural amino acids have side chain groups that distinguish them from natural amino acids. For example, R in the above formula can optionally be alkyl-, aryl-, aryl halide, vinyl halide, alkyl halide, acetyl, ketone, aziridine, nitrile, nitro, halide, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynyl, ether, thioether, epoxide, sulfone, boronic acid, boronic acid ester, borane, phenylboronic acid, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic-, pyridyl, naphthyl, benzophenone, a constrained ring such as cyclooctyne, thioester, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, carboxylic acid, alpha-ketocarboxylic acid, alpha or beta unsaturated acid and amide, glyoxylamide, or an organosilane group, or any combination thereof.

[0156] Specific examples of non-natural amino acids include p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, β-O-GlcNAc-L-serine, tri-O-acetyl-GalNAc-α-threonine, α-GalNAc-L-threonine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, those listed below, or those listed elsewhere in the present invention, etc., but are not limited thereto.

[0157] Therefore, a non-natural amino acid containing a functional group that forms a covalent bond with any preferred functional group of a desired molecule (e.g., Fc region, PEG) can be selected. Once selected, the non-natural amino acid can be either purchased from a supplier or chemically synthesized. Any number of non-natural amino acids can be incorporated into the target molecule, which can vary according to the number of desired molecules to be conjugated. These molecules can be conjugated to all or only some of the non-natural amino acids. Furthermore, the same or different non-natural amino acids can be incorporated into the HRS polypeptide depending on the desired outcome. In certain embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-natural amino acids are incorporated into the HRS polypeptide, and any or all of them can be conjugated to a molecule containing the desired functional group.

[0158] In certain embodiments, the use of non-natural amino acids can be utilized to modify (e.g., increase) selected non-canonical activities of the HRS polypeptide or to alter the in vivo or in vitro half-life of the protein. Non-natural amino acids can also be used to facilitate (selective) chemical modification (e.g., pegylation) of the HRS protein, as described herein. For example, certain non-natural amino acids enable the selective attachment of an Fc region or a polymer such as PEG to a given protein, thereby improving its pharmacokinetic properties.

[0159] Specific examples of amino acid analogs and mimetics can be found, for example, in Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341, Academic Press, Inc., New York, N.Y. (1983), the entire contents of which are incorporated herein by reference. Other examples include over-alkylated amino acids, particularly over-methylated amino acids. See, for example, Combinatorial Chemistry, Eds. Wilson and Czarnik, Ch. 11, p. 235, John Wiley & Sons Inc., New York, N.Y. (1997), the entire contents of which are incorporated herein by reference. Still other examples include amino acids in which the amide moiety (and thus the amide backbone of the resulting peptide) is replaced, for example, by a sugar ring, steroid, benzodiazepine, or carbo cycle. See, for example, Burger’s Medicinal Chemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620, John Wiley & Sons Inc., New York, N.Y. (1995), the entire contents of which are incorporated herein by reference. Methods for synthesizing peptides, polypeptides, peptidomimetics, and proteins are well known in the art (see, for example, U.S. Patent No. 5,420,109; M. Bodanzsky, Principles of Peptide Synthesis (1st ed. & 2d rev. ed.), Springer-Verlag, New York, N.Y. (1984 & 1993), see Chapter 7; Stewart and Young, Solid Phase Peptide Synthesis, (2d ed.), Pierce Chemical Co., Rockford, Ill. (1984), each of which is incorporated herein by reference).Accordingly, the HRS polypeptide can be composed of naturally occurring amino acids, non-naturally occurring amino acids, as well as amino acid analogs and mimetics.

[0160] In certain embodiments, the HRS polypeptide comprises, consists of, or consists essentially of a minimal active fragment of a full-length HRS polypeptide that can modulate anti-inflammatory activity in vivo or has the activity to block antibodies or autoreactive T cells. In some embodiments, such minimal active fragments comprise, consist of, or consist essentially of a WHEP domain (e.g., about amino acids 1-43 of SEQ ID NO: 1) or an active variant or fragment thereof. In some aspects, this minimal active fragment comprises, consists of, or consists essentially of an aminoacylation domain (e.g., about amino acids 54-398 of SEQ ID NO: 1) or an active variant or fragment thereof. In some aspects, this minimal active fragment comprises, consists of, or consists essentially of an anticodon-binding domain (i.e., about amino acids 406-501 of SEQ ID NO: 1) or an active variant or fragment thereof.

[0161] In certain embodiments, this HRS polypeptide is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 501, 502, 503, 504, 505, 506, 507, 508, or 509 amino acids in length, at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 501, 502, 503, 504, 505, 506, 507, 508, or 509 amino acids in length, and / or up to about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 501, 502, 503, 504, 505, 506, 507, 508, or 509 amino acids in length, including all integers in the range therebetween, and comprising, consisting of, or essentially consisting of the amino acid sequences in Table H1, Table H2, or Table H4.,

[0162] In certain embodiments, this HRS polypeptide has at least one non-canonical activity, such as anti-inflammatory activity, or cross-reactivity with autoantibodies or autoreactive T cells from a subject having a disease associated with autoantibodies to histidyl-tRNA synthetase (e.g., Jo-1 antibody). Assays for determining anti-inflammatory activity, including conventional measurement-based cytokine release from in vitro cells and animal studies, are well established in the art (see, e.g., Wittmann et al., J Vis Exp. (65):e4203.doi:10.3791 / 4203,2012; Feldman et al., Mol Cell. 47:585-95,2012; Clutterbuck et al., J Proteomics. 74:704-15,2011; Giddings and Maitra, J Biomol Screen. 15:1204-10,2010; Wijnhoven et al., Glycoconj J. 25:177-85,2008; and Frow et al., Med Res Rev. 24:276-98,2004) and can be readily used to profile and optimize anti-inflammatory activity. Exemplary in vivo experimental systems are also described in the accompanying examples.

[0163] In some embodiments, this HRS polypeptide does not significantly compete for the binding of disease-associated autoantibodies (e.g., Jo-1 antibody) to wild-type histidyl-tRNA synthetase in a competitive ELISA up to a concentration of up to about 1-5×10-7 M or more. Thus, in some embodiments, this HRS polypeptide has a lower affinity for disease-associated autoantibodies than wild-type histidyl-tRNA synthetase (SEQ ID NO: 1) as measured in a competitive ELISA. In some embodiments, this HRS polypeptide has an apparent affinity for disease-associated autoantibodies (e.g., Jo-1 antibody) that is at least about 10-fold less, or at least about 20-fold less, or at least about 50-fold less, or at least about 100-fold less than the affinity of disease-associated autoantibodies for wild-type human (SEQ ID NO: 1).

[0164] It will be appreciated that in any of the HRS polypeptides, the N-terminal acid of the HRS polypeptide (e.g., N-terminal Met) can be deleted.

[0165] In other embodiments, fusion proteins of the HRS polypeptide to other (non-HARS) proteins (e.g., heterologous proteins or polypeptides) are also included, and these fusion proteins can modulate the biological activity, secretion, antigenicity, targeting, biological lifespan, ability to cross the cell membrane or blood-brain barrier, or pharmacokinetic properties of the HRS polypeptide. Examples of fusion proteins that improve pharmacokinetic properties ( "PK modifiers") include, without limitation, human albumin (Osborn et al.: Eur. J. Pharmacol. 456(1-3):149-158, (2002)), antibody Fc domains, polyGlu or polyAsp sequences, and fusions to transferrin. Further, a conformationally disordered polypeptide sequence consisting of the amino acids Pro, Ala, and Ser ( "PASylation"), or a fusion with hydroxyethyl dextran (sold under the trademark HESYLATION®), provides a simple way to increase the hydrodynamic volume of the HRS polypeptide. This additional extension adopts a bulky random structure, which significantly increases the size of the resulting fusion protein. By this means, the generally rapid clearance of the smaller HRS polypeptide via renal filtration is retarded by several orders of magnitude. Further, it has also been shown that the use of IgG fusion proteins enables some fusion proteins to cross the blood-brain barrier (Fu et al., (2010) Brain Res. 1352:208-13).

[0166] Examples of fusion proteins that modulate the antigenicity or immunomodulatory properties of an HRS polypeptide include, for example, MHC class I and II proteins, b-2 microglobulin, portions of LFA-3, portions of the Fc region of the heavy chain, and fusions to T cell binding ligands including conjugates and derivatives thereof, examples of such fusion proteins are described, for example, in EP1964854, U.S. Patent Nos. 5,468,481, 5,130,297, 5,635,363, 6,451,314, and US2009 / 0280135.

[0167] Furthermore, in some embodiments, the HRS polypeptide may include a synthetic or naturally occurring secretory signal sequence derived from another well-characterized secreted protein. In some embodiments, such a protein can be processed by proteolytic cleavage to form the HRS polypeptide in situ. In some embodiments, the HRS polypeptide may include a heterologous proteolytic cleavage site to enable in situ expression and production of the HRS polypeptide at an intracellular or extracellular location. Other fusion proteins may include, for example, fusion of the HRS polypeptide to ubiquitin to provide a new N-terminal amino acid, or use of a secretory signal to mediate high-level secretion of the HRS polypeptide into the extracellular medium, or N-terminal or C-terminal epitope tags to improve purification or detection, and fusions to cell-penetrating peptides.

[0168] In certain embodiments, the use of non-natural amino acids can be utilized to modify (e.g., increase) selected non-canonical activities of the HRS polypeptide or to alter the in vivo or in vitro half-life of the protein. Non-natural amino acids can also be used to facilitate (selective) chemical modification (e.g., pegylation) of the HRS protein, as described elsewhere herein. For example, certain non-natural amino acids enable the selective attachment of polymers such as PEG to a given protein, thereby improving its pharmacokinetic properties.

[0169] Certain embodiments include an HRS-Fc conjugate comprising at least one Fc region covalently bound to one or more HRS polypeptides. Examples of HRS-Fc conjugates include fusion proteins and various forms of chemically cross-linked proteins. Wild-type sequences from any number of species, as well as variants, fragments, hybrids, and chemically modified forms thereof, can be used in the HRS-Fc conjugate. The HRS-Fc polypeptide may also optionally include one or more linkers that typically separate the Fc region from the HRS polypeptide, including peptide linkers and chemical linkers, as described herein and known in the art. In any of these HRS-Fc conjugates, the native N- or C-terminal amino acids of the HRS polypeptide, or the native N- or C-terminal amino acids in the Fc domain, may be deleted and / or replaced with non-native amino acids, for example, to facilitate expression and / or cloning, or to function as a linker sequence between the two proteins.

[0170] The HRS-Fc conjugate polypeptide can provide various advantages as compared to an unconjugated or unmodified HRS polypeptide, e.g., a corresponding HRS polypeptide of the same or similar sequence that does not have an Fc region attached thereto. By way of mere example, covalent attachment of one or more Fc regions can alter (e.g., increase, decrease) the solubility, half-life (e.g., in serum, in selected tissues, in vitro under storage conditions, e.g., at room temperature or under refrigeration), dimerization or multimerization properties, biological activity(ies) of the HRS polypeptide as compared to an unmodified HRS polypeptide of the same or similar sequence, e.g., by providing Fc region-related effector functions (e.g., activation of the classical complement cascade, interaction with immune effector cells via Fc receptors (FcRs), compartmentalization of immunoglobulins), cellular uptake, intracellular trafficking, tissue distribution, and / or bioavailability. In certain embodiments, the Fc region can confer effector functions related to complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or antibody-dependent cell-mediated phagocytosis (ADCP), which are thought to play a role in eliminating certain target cells such as tumor cells and infected cells.

[0171] Certain embodiments use an HRS-Fc fusion protein. A "fusion protein" is defined elsewhere in this specification and is well known in the art, similar to the methods for making fusion proteins (see, e.g., U.S. Patent Nos. 5,116,964, 5,428,130, 5,455,165, 5,514,582, 6,406,697, 6,291,212, and 6,300,099 for general disclosures and methods regarding Fc fusion proteins). In an HRS-Fc fusion protein, the Fc region can be fused to the N-terminus, C-terminus, or both of the HRS polypeptide. In some embodiments, one or more Fc regions can be fused internally to the HRS sequence, for example, by placing the Fc region between a first HRS sequence (e.g., domain) and a second HRS sequence (e.g., domain), where the first HRS sequence is fused to the N-terminus of the Fc region and the second HRS sequence is fused to the C-terminus of the Fc region. In certain embodiments, the first and second HRS sequences are identical. In other embodiments, the first and second HRS sequences are different (e.g., they include different functional domains of the HRS polypeptide). Certain HRS-Fc fusion proteins can also include additional heterologous protein sequences, i.e., non-Fc regions and non-HRS polypeptide sequences.

[0172] The term "HRS-Fc" can, but does not necessarily, denote an N-terminal or C-terminal attachment of the Fc region to the HRS polypeptide. For example, in certain cases, the term "Fc-HRS" denotes the fusion of the Fc region to the N-terminus of the HRS polypeptide, and the term "HRS-Fc" denotes the fusion of the Fc region to the C-terminus of the HRS polypeptide. However, either term can be used more generally to refer to any fusion protein or conjugate of the Fc region and the HRS polypeptide.

[0173] In some embodiments, the HRS-Fc fusion protein may optionally include tandem repeat copies of an HRS polypeptide coupled to a single Fc domain, separated by a linker peptide. Exemplary tandem repeat HRS-Fc fusion proteins are provided in Table H5. The preparation and sequences of specific tandem repeat HRS-Fc conjugates are illustrated in the Examples. [Table H5]

[0174] Certain embodiments relate to HRS-Fc conjugates, e.g., HRS-Fc conjugates in which one or more Fc regions are chemically conjugated or cross-linked to an HRS polypeptide. In these and related aspects, the Fc region can be conjugated to the HRS polypeptide at the N-terminal region (e.g., the first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc. amino acids), internal region (between the N-terminal and C-terminal regions), and / or C-terminal region (e.g., within the last 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc. amino acids). The polypeptide can be conjugated or cross-linked to other polypeptides according to various conventional techniques in the art. For example, certain techniques use the carboxyl-reactive carbodiimide cross-linking agent EDC (or EDAC) that covalently binds via D, E, and C-terminal carboxyl groups. Other techniques use activated EDC that covalently binds via K and N-terminal amino groups). Still other techniques use m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or sulfo-MBS that covalently binds via the thiol groups of cysteine residues (see also U.S. Patent Application No. 2007 / 0092940 for cysteine-engineered Ig regions that can be used for thiol conjugation). Such cross-linked proteins can also include linkers that are cleavable or otherwise releasable (e.g., enzymatically cleavable linkers, hydrolysable linkers), and non-cleavable linkers (i.e., physiologically stable linkers). Certain embodiments can use a non-peptide polymer (e.g., a PEG polymer; HRS-N-PEG-N-Fc conjugate) as a cross-linking agent between the Fc region and the HRS polypeptide, as described, for example, in U.S. Patent Application No. 2006 / 0269553. See also U.S. Patent Application No. 2007 / 0269369 for an exemplary description of Fc region conjugation sites.

[0175] In certain embodiments, a variant or otherwise modified Fc region can be used that has altered properties or biological activities compared to the wild-type Fc region, as discussed in more detail below. Examples of modified Fc regions include, for example, those having a sequence mutated by substitution, insertion, deletion, or truncation of one or more amino acids compared to the wild-type sequence, hybrid Fc polypeptides consisting of domains from different immunoglobulin classes / subclasses, Fc polypeptides having an altered glycosylation / sialylation pattern, and Fc polypeptides modified or derivatized, for example, by biotinylation (see, e.g., U.S. Patent Application No. 2010 / 0209424), phosphorylation, sulfation, etc., or any combination of the foregoing. Such modifications can be used to alter (e.g., increase, decrease) the binding properties of the Fc region to one or more specific FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcRn), its pharmacokinetic properties (e.g., stability or half-life, bioavailability, tissue distribution, volume of distribution, concentration, elimination rate constant, elimination rate, area under the curve (AUC), clearance, C max , t max , C min , variability), its immunogenicity, its complement fixation or activation, and / or its CDC / ADCC / ADCP-related activities of the Fc region compared to the corresponding wild-type Fc sequence among other properties described herein.

[0176] The "Fc region" of the HRS-Fc conjugate provided herein is typically derived from the heavy chain of an immunoglobulin (Ig) molecule. A typical Ig molecule consists of two heavy chains and two light chains. The heavy chain can be divided into at least three functional regions: the Fd region, the Fc region (fragment crystallizable region), and the hinge region, the latter being found only in IgG, IgA, and IgD immunoglobulins. The Fd region contains the variable (V H ) and constant (CH 1 ) domains of the heavy chain and the variable (V L ) and constant (CL )Together with the domain, it forms an antigen-binding fragment or Fab region.

[0177] The Fc regions of IgG, IgA, and IgD immunoglobulins contain the heavy-chain constant domains 2 and 3, designated as CH 2 and CH 3 regions, respectively. The Fc regions of IgE and IgM immunoglobulins contain the heavy-chain constant domains 2, 3, and 4, designated as CH 2 , CH 3 , and CH 4 regions, respectively. The Fc region is mainly responsible for immunoglobulin effector functions, including, for example, complement binding and binding to cognate Fc receptors on effector cells.

[0178] The hinge region (found in IgG, IgA, and IgD) acts as a flexible spacer so that the Fab portion can move spatially freely with respect to the Fc region. In contrast to the constant regions, the hinge region varies in both sequence and length among immunoglobulin classes and subclasses and is structurally diverse. The hinge region may also contain one or more glycosylation sites, including several structurally distinct types of sites for carbohydrate binding. For example, IgA1 contains five glycosylation sites within a 17-amino acid segment of the hinge region, conferring significant resistance of the hinge region polypeptide to intestinal proteases. Residues in the hinge-proximal region of the CH 2 domain can also affect the specificity of the interaction between the immunoglobulin and its respective Fc receptor (see, for example, Shin et al., Intern. Rev. Immunol. 10:177-186, 1993).

[0179] Thus, the term "Fc region" or "Fc fragment" or "Fc" as used herein refers to the CH 2 region, CH 3 region, and / or CH 4Refers to a protein that includes one or more of the regions. The "Fc region" may also include one or more hinge regions of the constant region of the heavy chain of an immunoglobulin. In certain embodiments, the Fc region does not include one or more of the CH 1 、C L 、V L 、and / or V H regions.

[0180] This Fc region can be derived from the CH 2 region, CH 3 region, CH 4 region, and / or hinge region of any one or more immunoglobulin classes, including but not limited to IgA, IgD, IgE, IgG, IgM, including their subclasses and combinations. In some embodiments, the Fc region is derived from an IgA immunoglobulin, including subclasses IgA1 and / or IgA2. In certain embodiments, the Fc region is derived from an IgD immunoglobulin. In specific embodiments, the Fc region is derived from an IgE immunoglobulin. In some embodiments, the Fc region is derived from an IgG immunoglobulin, including subclasses IgG1, IgG2, IgG2, IgG3, and / or IgG4. In certain embodiments, the Fc region is derived from an IgM immunoglobulin.

[0181] Certain Fc regions demonstrate specific binding to one or more Fc-receptors (FcRs). Examples of classes of Fc receptors include Fcγ receptors (FcγRs), Fcα receptors (FcαRs), Fcε receptors (FcεRs), and the neonatal Fc receptor (FcRn). For example, certain Fc regions increase binding (or affinity therefor) to one or more FcγRs as compared to FcαR, FcεR, and / or FcRn. In some embodiments, the Fc region increases binding to FcαR as compared to one or more FcγRs, FcεRs, and / or FcRn. In other embodiments, the Fc region increases binding to FcαR (e.g., FcαRI) as compared to one or more FcγRs, FcαRs, and / or FcRn. In certain embodiments, the Fc region increases binding to FcRn as compared to one or more FcγRs, FcαRs, and / or FcεRs. In certain embodiments, the binding (or affinity) of the Fc region to one or more selected FcRs is typically about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more (including all integers therebetween) increased as compared to its binding (or affinity therefor) to one or more different FcRs.

[0182] Examples of FcγRs include FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. FcγRI (CD64) is expressed on macrophages and dendritic cells and plays a role in phagocytosis, respiratory burst, cytokine stimulation, and dendritic cell endocytic transport. The expression of FcγRI is upregulated by both GM-CSF and gamma interferon (γ-IFN) and downregulated by interleukin-4 (IL-4). FcγRIIa is expressed on polymorphonuclear leukocytes (PMNs), macrophages, dendritic cells, and mast cells. FcγRIIa plays a role in phagocytosis, respiratory burst, and cytokine stimulation. The expression of FcγRIIa is upregulated by GM-CSF and γ-IFN and decreased by IL-4. FcγIIb is expressed on B cells, PMNs, macrophages, and mast cells. FcγIIb inhibits immunoreceptor tyrosine-based activation motif (ITAM)-mediated responses and is thus an inhibitory receptor. The expression of FcγRIIc is upregulated by intravenous immunoglobulin (IVIG) and IL-4 and decreased by γ-IFN. FcγRIIc is expressed on NK cells. FcγRIIIa is expressed on natural killer (NK) cells, macrophages, mast cells, and platelets. This receptor is involved in phagocytosis, respiratory burst, cytokine stimulation, platelet aggregation and degranulation, and NK-mediated ADCC. The expression of FcγRIII is upregulated by C5a, TGF-β, and γ-IFN and downregulated by IL-4. FcγRIIIb is a GPI-linked receptor expressed on PMNs.

[0183] Certain Fc regions increase binding to FcγRI as compared to FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb. Some embodiments increase binding to FcγRIIa as compared to FcγRI, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb. Certain Fc regions increase binding to FcγRIIb as compared to FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb. Certain Fc regions increase binding to FcγRIIc as compared to FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and / or FcγRIIIb. Some Fc regions increase binding to FcγRIIIa as compared to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, and / or FcγRIIIb. Certain Fc regions increase binding to FcγRIIIb as compared to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, and / or FcγRIIIa.

[0184] FcαR includes FcαRI (CD89). FcαRI is found on the surface of neutrophils, eosinophils, monocytes, certain macrophages (e.g., Kupffer cells), and certain dendritic cells. FcαRI consists of two extracellular Ig-like domains, is a member of both the immunoglobulin superfamily and the multichain immune recognition receptor (MIRR) family, and signals by associating with two FcRγ signaling chains.

[0185] FcεR includes FcεRI and FcεRII. The high-affinity receptor FcεRI is a member of the immunoglobulin superfamily, is expressed on epithelial Langerhans cells, eosinophils, mast cells, and basophils, and plays a major role in the control of allergic responses. FcεRI is also expressed on antigen-presenting cells and regulates the production of pro-inflammatory cytokines. The low-affinity receptor FcεRII (CD23) is a C-type lectin that can function as a membrane-bound or soluble receptor. FcεRII regulates the proliferation and differentiation of B cells and blocks IgE binding to eosinophils, monocytes, and basophils. Certain Fc regions increase binding to FcεRI compared to FcεRII. Other Fc regions increase binding to FcεRII compared to FcεRI. The following Table H6 summarizes the characterization of certain FcRs.

Table H6-1

Table H6-2

[0186] Fc regions can be derived from immunoglobulin molecules of any animal, including vertebrates such as mammals such as cows, goats, pigs, dogs, mice, rabbits, hamsters, rats, guinea pigs, non-human primates, and humans. The amino acid sequences of CH 2 , CH 3 , CH 4 , and the hinge region from exemplary wild-type human IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM immunoglobulins are shown in Table H7.

Table H7-1

Table H7-2

Table H7-3

Table H7-4

Table H7-5

[0187] Thus, the Fc region of the HRS-Fc conjugate can include, consist of, or consist essentially of one or more of the human Fc region amino acid sequences of Table H7, including their variants, fragments, homologs, orthologs, paralogs, and combinations. Certain exemplary embodiments include an Fc region in the range of about 20-50, 20-100, 20-150, 20-200, 20-250, 20-300, 20-400, 50-100, 50-150, 50-200, 50-250, 50-300, 50-400, 100-150, 100-200, 100-250, 100-300, 100-350, 100-400, 200-250, 200-300, 200-350, or 200-400 amino acids in length, optionally including, consisting of, or consisting essentially of any one or more of the sequences in Table H7. Certain embodiments include an Fc region of up to about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400 or more amino acids, optionally including, consisting of, or consisting essentially of any one or more of the sequences in Table H7.

[0188] Certain Fc regions include, consist of, or consist essentially of the human IgA1 sequences of Table H7, in any order reading from the N-terminus to the C-terminus, including their combinations, as well as their variants and fragments. Certain Fc regions include, consist of, or consist essentially of the human IgA1 sequences of Table H7. Certain Fc regions include, consist of, or consist essentially of the human IgA1 sequences of Table H7. Certain Fc regions include, consist of, or consist essentially of the human IgA1 sequences of Table H7.

[0189] Some Fc regions contain, consist of, or consist essentially of the human IgA2 sequences of Table H7, in any order read from the N-terminus to the C-terminus, including their combinations, as well as their variants and fragments. Certain Fc regions contain, consist of, or consist essentially of the human IgA2 sequences of Table H7. Certain Fc regions contain, consist of, or consist essentially of the human IgA2 sequences of Table H7. Certain Fc regions contain, consist of, or consist essentially of the human IgA2 sequences of Table H7.

[0190] Certain Fc regions include, consist of, or consist essentially of the human IgD sequences of Table H7, in any order reading from the N-terminus to the C-terminus, including their combinations, as well as variants and fragments of these sequences and combinations. Certain Fc regions include, consist of, or consist essentially of the human IgE sequences of Table H7, in any order reading from the N-terminus to the C-terminus, including their combinations, as well as variants and fragments of these sequences and combinations. Certain Fc regions include, consist of, or consist essentially of the human IgG1 sequences of Table H7, in any order reading from the N-terminus to the C-terminus, including their combinations, as well as variants and fragments of these sequences and combinations. Certain Fc regions include, consist of, or consist essentially of the human IgG2 sequences of Table H7, in any order reading from the N-terminus to the C-terminus, including their combinations. Certain Fc regions include, consist of, or consist essentially of the human IgG3 sequences of Table H7, in any order reading from the N-terminus to the C-terminus, including their combinations. Certain Fc regions include, consist of, or consist essentially of the human IgG4 sequences of Table H7, in any order reading from the N-terminus to the C-terminus, including their combinations. Certain Fc regions include, consist of, or consist essentially of the human IgM sequences of Table H7, in any order reading from the N-terminus to the C-terminus, including their combinations, as well as variants and fragments of these sequences and combinations.

[0191] Exemplary HRS-Fc fusion conjugates are provided in Table H8 below.

Table H8-1

Table H8-2

Table H8-3

Table H8-4

Table H8-5

Table H8-6

[0192] Thus, in certain embodiments, the HRS polypeptide is fused or otherwise conjugated to an Fc region, or comprises, consists of, or consists essentially of the amino acid sequences (SEQ ID NOs: 157-172) in Table H8 or active variants or fragments thereof. In some embodiments, the expressible polynucleotide encodes an HRS polypeptide that comprises, consists of, or consists essentially of the amino acid sequences (e.g., SEQ ID NOs: 157-172) in Table H8 or active variants or fragments thereof.

[0193] As described above, certain embodiments use variants, fragments, hybrids, and / or otherwise modified forms of Fc regions described herein and known in the art. Variants are included that have one or more amino acid substitutions, insertions, deletions, and / or truncations as compared to a reference sequence such as any one or more of the reference sequences in Table H7 or Table H8. Polypeptide and polynucleotide variants are described elsewhere herein.

[0194] Hybrid Fc regions, e.g., Fc domains from immunoglobulins of different species, different Ig classes, and / or different Ig subclasses (e.g., hinge, CH 2 , CH 3 , CH 4 ), are included. General examples include CH 2 / CH 3The following combinations of domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgE / IgA1, IgE / IgA2, IgE / IgD, IgE / IgE, IgE / IgG1, IgE / IgG2, IgE / IgG3, IgE / IgG4, IgE / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM, IgM / IgA1, IgM / IgA2, IgM / IgD, IgM / IgE, IgM / IgG1, IgM / IgG2, IgM / IgG3, IgM / IgG4, IgM / IgM (or fragments or variants thereof), or consisting of or essentially consisting of the same, optionally comprising a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, or IgG4, and / or a CH 4 domain-containing hybrid Fc region is included. In certain embodiments, this hinge, CH 2 , CH 3 , and CH 4The domain is from human Ig.

[0195] Further examples include the following combinations of CH 2 / CH 4 domains: IgA1 / IgE, IgA2 / IgE, IgD / IgE, IgE / IgE, IgG1 / IgE, IgG2 / IgE, IgG3 / IgE, IgG4 / IgE, IgM / IgE, IgA1 / IgM, IgA2 / IgM, IgD / IgM, IgE / IgM, IgG1 / IgM, IgG2 / IgM, IgG3 / IgM, IgG4 / IgM, IgM / IgM (or fragments or variants thereof), and optionally a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4, and / or a CH 3 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, wherein a hybrid Fc region is included. In certain embodiments, the hinge, CH 2 , CH 3 , and CH 4 domains are from human Ig.

[0196] In certain examples, the following combinations of CH 3 / CH 4 domains: IgA1 / IgE, IgA2 / IgE, IgD / IgE, IgE / IgE, IgG1 / IgE, IgG2 / IgE, IgG3 / IgE, IgG4 / IgE, IgM / IgE, IgA1 / IgM, IgA2 / IgM, IgD / IgM, IgE / IgM, IgG1 / IgM, IgG2 / IgM, IgG3 / IgM, IgG4 / IgM, IgM / IgM (or fragments or variants thereof), and optionally a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4, and / or a CH 2A hybrid Fc region is included, which includes a domain. In certain embodiments, the hinge, CH 2 and CH 3 and CH 4 domains are from human Ig.

[0197] Specific examples include the following combinations of hinge / CH 2 domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), and optionally, CH 3 domains from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or CH 4 domains from IgE and / or IgM, and a hybrid Fc region is included. In certain embodiments, the hinge, CH2 、 CH 3 、 and CH 4 domains are from human Ig.

[0198] In certain examples, the following combinations of hinge / CH 3 domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), include, consist of, or consist essentially of, optionally, CH 2 domains from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or CH 4 domains from IgE and / or IgM are included. In certain embodiments, the hinge, CH 2 、 CH 3 、 and CH 4The domain is from human Ig.

[0199] Some examples include the following combinations of hinge / CH 4 domains: IgA1 / IgE, IgA1 / IgM, IgA2 / IgE, IgA2 / IgM, IgD / IgE, IgD / IgM, IgG1 / IgE, IgG1 / IgM, IgG2 / IgE, IgG2 / IgM, IgG3 / IgE, IgG3 / IgM, IgG4 / IgE, IgG4 / IgM (or fragments or variants thereof), optionally including CH 2 domains from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or CH 3 domains from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, including a hybrid Fc region.

[0200] Specific examples of hybrid Fc regions derived from combinations of IgG subclasses or combinations of human IgD and IgG can be found, for example, in WO2008 / 147143.

[0201] Also included are derivatized or otherwise modified Fc regions. In certain embodiments, the Fc region can be modified, for example, by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc., as compared to the wild-type or naturally occurring Fc region. In certain embodiments, the Fc region can contain the wild-type or natural glycosylation pattern, or alternatively, can contain increased glycosylation as compared to the native form, decreased glycosylation as compared to the native form, or can be completely deglycosylated. As an example of a modified Fc glycoform, decreased glycosylation of the Fc region reduces binding to the C1q region of the first complement component C1, decreases in ADCC-related activity, and / or decreases in CDC-related activity. Thus, certain embodiments use a deglycosylated or non-glycosylated Fc region. For an exemplary generation of a non-glycosylated Fc region, see, for example, WO2005 / 047337. Another example of an Fc region glycoform can be generated by substituting the Q295 position with a cysteine residue (see, for example, US Patent Application No. 2010 / 0080794) according to the Kabat et al. numbering system. Certain embodiments can include an Fc region that contains a mature core carbohydrate structure in which about 80-100% of the glycoprotein in the Fc region lacks fructose (see, for example, US Patent Application No. 2010 / 0255013). Some embodiments can include an Fc region optimized by substitution or deletion to reduce the level of fucosylation, for example, to increase affinity for FcγRI, FcγRIa, or FcγRIIIa and / or to improve phagocytosis by FcγRIIa-expressing cells (see US Patent Application No. 2010 / 0249382 and US Patent Application No. 2007 / 0148170).

[0202] As another example of a modified Fc glycoform, the Fc region may contain oligomannose-type N-glycans and optionally have one or more of the following: increased ADCC activity, increased binding affinity for FcγRIIIA (and certain other FcRs), similar or increased binding specificity for the target of the HRS polypeptide, similar or higher binding affinity for the target of the HRS polypeptide, and / or similar or lower binding affinity for the mannose receptor (see, e.g., U.S. Patent Application No. 2007 / 0092521 and U.S. Patent No. 7,700,321). As another example, enhanced affinity of the Fc region for FcγR has been achieved using engineered glycoforms generated by expression of the antibody in engineered or variant cell lines (see, e.g., Umana et al., Nat Biotechnol. 17:176-180, 1999, Davies et al., Biotechnol Bioeng. 74:288-294, 2001, Shields et al., J Biol Chem. 277:26733-26740, 2002, Shinkawa et al., 2003, J Biol Chem. 278:3466-3473, 2003, and U.S. Patent Application No. 2007 / 0111281). Certain Fc region glycoforms contain an increased proportion of N-glycosidic linked complex glycans that do not have a fucose at position 1 of the fucose linked to the 6-position of N-acetylglucosamine at the reducing end of the sugar chain (see, e.g., U.S. Patent Application No. 2010 / 0092997). Certain embodiments may include an Fc region of IgG glycosylated by at least one galactose moiety connected to each terminal sialic acid moiety by an α-2,6 linkage, and optionally, this Fc region has higher anti-inflammatory activity compared to the corresponding wild-type Fc region (see U.S. Patent Application No. 2008 / 0206246).Certain ones of these and related modified glycosylation approaches result in a substantial enhancement of the ability of the Fc region to selectively bind to FcRs such as FcγRIII, the ability to mediate ADCC, and the ability to modify other properties of the Fc region, as described herein.

[0203] Certain variants, fragments, hybrids, or otherwise modified Fc regions can have altered binding to one or more FcRs compared to the corresponding wild-type Fc sequence (e.g., same species, same Ig class, same Ig subclass). For example, such Fc regions can have increased binding to one or more of the Fcγ receptor, Fcα receptor, Fcε receptor, and / or neonatal Fc receptor compared to the corresponding wild-type Fc sequence. In other embodiments, the variant, fragment, hybrid, or modified Fc region can have decreased binding to one or more of the Fcγ receptor, Fcα receptor, Fcε receptor, and / or neonatal Fc receptor compared to the corresponding wild-type Fc sequence. Specific FcRs are described elsewhere herein.

[0204] Specific examples of Fc variants having altered (e.g., increased, decreased) FcR binding can be found, for example, in U.S. Pat. Nos. 5,624,821 and 7,425,619, U.S. Patent Application Nos. 2009 / 0017023, 2009 / 0010921, and 2010 / 0203046, and WO2000 / 42072 and WO2004 / 016750. Certain examples include one or more substitutions at positions 298, 333, and / or 334, such as S298A, E333A, and / or K334A (based on the EU index numbering of Kabat et al.), which have been shown to increase binding to the activating receptor FcγRIIIa and reduce binding to the inhibitory receptor FcγRIIb. These mutations can be combined to obtain double and triple mutant variants having further improved binding to FcR. Certain embodiments include the S298A / E333A / K334A triple mutant having increased binding to FcγRIIIa, decreased binding to FcγRIIb, and increased ADCC (see, e.g., Shields et al., J Biol Chem. 276:6591-6604, 2001 and Presta et al., Biochem Soc Trans. 30:487-490, 2002). See also engineered Fc glycoforms having increased binding to FcR as disclosed by Umana et al., supra; and U.S. Pat. No. 7,662,925. Some embodiments include an Fc region comprising one or more substitutions selected from 434S, 252Y / 428L, 252Y / 434S, and 428L / 434S (see U.S. Patent Application Nos. 2009 / 0163699 and 2006 / 0173170) based on the EU index numbering of Kabat et al.

[0205] A particular variant, fragment, hybrid, or modified Fc region may have altered effector function as compared to the corresponding wild-type Fc sequence. For example, such Fc regions may have increased complement binding or activation, increased Clq binding affinity, increased CDC-related activity, increased ADCC-related activity and / or increased ADCP-related activity. In other embodiments, such Fc regions may have decreased complement binding or activation, decreased Clq binding affinity, decreased CDC-related activity, decreased ADCC-related activity and / or decreased ADCP-related activity as compared to the corresponding wild-type Fc sequence. By way of merely illustrative example, Fc regions may include deletions or substitutions at complement binding sites such as the C1q binding site and / or deletions or substitutions at the ADCC site. Examples of such deletions / substitutions are described, for example, in U.S. Patent No. 7,030,226. Many Fc effector functions such as ADCC can be assayed according to conventional techniques in the art (see, e.g., Zuckerman et al., CRC Crit Rev Microbiol. 7:1-26, 1978). Effector cells useful for such assays include, but are not limited to, natural killer (NK) cells, macrophages, and other peripheral blood mononuclear cells (PBMC). Alternatively, or additionally, certain Fc effector functions can be evaluated in vivo, for example, by using the animal models described in Clynes et al. PNAS. 95:652-656, 1998.

[0206] Certain variant hybrids or modified Fc regions may have altered stability or half-life compared to the corresponding wild-type Fc sequence. In certain embodiments, such Fc regions may have an increased half-life compared to the corresponding wild-type Fc sequence. In other embodiments, the variant hybrid or modified Fc region may have a decreased half-life compared to the corresponding wild-type Fc sequence. The half-life can be measured in vitro (e.g., under physiological conditions) or in vivo according to conventional techniques in the art, such as radiolabeling, ELISA, or other methods. In vivo measurements of stability or half-life can be made in one or more body fluids, including blood, serum, plasma, urine, or cerebrospinal fluid, or in a given tissue, such as liver, kidney, muscle, central nervous system tissue, bone, etc. As an example, modifications to the Fc region that alter its ability to bind to FcRn can alter its half-life in vivo. Assays for measuring in vivo pharmacokinetic properties (e.g., in vivo mean elimination half-life) and non-limiting examples of Fc modifications that alter its binding to FcRn are described, for example, in U.S. Patent Nos. 7,217,797 and 7,732,570; and U.S. Patent Application Nos. 2010 / 0143254 and 2010 / 0143254.

[0207] Further non-limiting examples of modifications for altering stability or half-life include, according to the Kabat et al. numbering system, CH 2 domains 251-256, 285-290, and 308-314, and CH 3One or more of the amino acid residues selected from 385-389 and 428-436 in the domain contain substitutions / deletions. See U.S. Patent Application No. 2003 / 0190311. Specific examples include, including any combinations thereof, substitution of leucine at position 251, substitution of tyrosine, tryptophan, or phenylalanine at position 252, substitution of threonine or serine at position 254, substitution of arginine at position 255, substitution of glutamine, arginine, serine, threonine, or glutamate at position 256, substitution of threonine at position 308, substitution of proline at position 309, substitution of serine at position 311, substitution of aspartate at position 312, substitution of leucine at position 314, substitution of arginine, aspartate, or serine at position 385, substitution of threonine or proline at position 386, substitution of arginine or proline at position 387, substitution of proline, asparagine, or serine at position 389, substitution of methionine or threonine at position 428, substitution of tyrosine or phenylalanine at position 434, substitution of histidine, arginine, lysine, or serine at position 433, and / or substitution of histidine, tyrosine, arginine, or threonine at position 436. Such modifications optionally increase the affinity of the Fc region for FcRn compared to the corresponding wild-type Fc region, thereby increasing the half-life.

[0208] Certain variant hybrids or modified Fc regions may have altered solubility compared to the corresponding wild-type Fc sequence. In certain embodiments, such Fc regions may have increased solubility compared to the corresponding wild-type Fc sequence. In other embodiments, the variant hybrid or modified Fc region may have decreased solubility compared to the corresponding wild-type Fc sequence. Solubility can be measured according to conventional techniques in the art, for example, in vitro (e.g., under physiological conditions). Exemplary solubility measurements are described elsewhere in this specification.

[0209] Further examples of variants include one or more of positions 250, 314, or 428 of the heavy chain or any combination thereof, e.g., positions 250 and 428, or positions 250 and 314, or positions 314 and 428, or positions 250, 314, and 428, having conservative or non-conservative substitutions (as described elsewhere herein) in the Fc region of IgG (see, e.g., U.S. Patent Application No. 2011 / 0183412). In certain embodiments, the residue at position 250 is substituted with glutamic acid or glutamine and / or the residue at position 428 is substituted with leucine or phenylalanine. As another exemplary example of an IgG Fc variant, any one or more of the amino acid residues at positions 214-238, 297-299, 318-322, and / or 327-331 can be used as suitable targets for modification (e.g., conservative or non-conservative substitution, deletion). In certain embodiments, the CH 2 domains of the IgG Fc variant include amino acid substitutions at positions 228, 234, 235, and / or 331 to attenuate the effector functions of the Fc region (see, e.g., human IgG4 having Ser228Pro and Leu235Ala mutations) (see U.S. Patent No. 7,030,226). Here, the numbering of residues in the heavy chain is the EU index numbering (see Kabat et al., “Sequences of Proteins of Immunological Interest,” 5 th th Ed., National Institutes of Health, Bethesda, Md. (1991)). Certain ones of these and related embodiments optionally alter (e.g., increase, decrease) FcRn binding and / or serum half-life without accompanying reduction of effector functions such as ADCC-related activity or CDC-related activity.

[0210] Additional examples include variant Fc regions that contain one or more amino acid substitutions at positions 279, 341, 343, or 373 of the wild-type Fc region or any combination thereof (see, e.g., U.S. Patent Application 2007 / 0224188). The wild-type amino acid residues at these positions for human IgG are valine (279), glycine (341), proline (343), and tyrosine (373). The substitutions can be conservative or non-conservative, or can include non-naturally occurring amino acids or mimetics as described herein. Alone or in combination with these substitutions, certain embodiments can also use variant Fc regions that contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions selected from: 235G, 235R, 236F, 236R, 236Y, 237K, 237N, 237R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 244L, 245R, 247A, 247D, 247E, 247F, 247M, 247N, 247Q, 247R, 247S, 247T, 247W, 247Y, 248F, 248P, 248Q, 248W, 249L, 249M, 249N, 249P, 249Y, 251H, 251I, 251W, 254D, 254E, 254F, 254G, 254H, 254I, 254K, 254L, 254M, 254N, 254P, 254Q, 254R, 254V, 254W, 254Y, 255K, 255N, 256H, 256I, 256K, 256L, 256V, 256W, 256Y, 257A, 257I, 257M, 257N, 257S, 258D, 260S, 262L, 264S, 265K, 265S, 267H, 267I, 267K, 268K, 269N, 269Q, 271T, 272H, 272K, 272L, 272R, 279A, 279D, 279F, 279G, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R, 279S, 279T, 279W, 279Y, 280T, 283F, 283G, 283H, 283I, 283K, 283L, 283M, 283P, 283R, 283T, 283W, 283Y, 285N, 286F, 288N, 288P, 292E, 292F, 292G, 292I, 292L, 293S, 293V,301W, 304E, 307E, 307M, 312P, 315F, 315K, 315L, 315P, 315R, 316F, 316K, 317P, 317T, 318N, 318P, 318T, 332F, 332G, 332L, 332M, 332S, 332V, 332W, 339D, 339E, 339F, 339G, 339H, 339I, 339K, 339L, 339M, 339N, 339Q, 339R, 339S, 339W, 339Y, 341D, 341E, 341F, 341H, 341I, 341K, 341L, 341M, 341N, 341P, 341Q, 341R, 341S, 341T, 341V, 341W, 341Y, 343A, 343D, 343E, 343F, 343G, 343H, 343I, 343K, 343L, 343M, 343N, 343Q, 343R, 343S, 343T, 343V, 343W, 343Y, 373D, 373E, 373F, 373G, 373H, 373I, 373K, 373L, 373M, 373N, 373Q, 373R, 373S, 373T, 373V, 373W, 375R, 376E, 376F, 376G, 376H, 376I, 376L, 376M, 376N, 376P, 376Q, 376R, 376S, 376T, 376V, 376W, 376Y, 377G, 377K, 377P, 378N, 379N, 379Q, 379S, 379T, 380D, 380N, 380S, 380T, 382D, 382F, 382H, 382I, 382K, 382L, 382M, 382N, 382P, 382Q, 382R, 382S, 382T, 382V, 382W, 382Y, 385E, 385P, 386K, 423N, 424H, 424M, 424V, 426D, 426L, 427N, 429A, 429F, 429M, 430A, 430D, 430F, 430G, 430H, 430I, 430K, 430L, 430M, 430N, 430P, 430Q, 430R, 430S, 430T, 430V, 430W, 430Y, 431H, 431K, 431P, 432R, 432S, 438G, 438K, 438L, 438T, 438W, 439E, 439H, 439Q, 440D, 440E, 440F, 440G, 440H, 440I, 440K, 440L, 440M, 440Q, 440T, 440V, or 442K. As described above, the numbering of residues in the heavy chain isThe EU indexing is as described by Kabat et al. (see above). Such variant Fc regions typically confer altered effector function or altered serum half-life to the HRS polypeptide to which the variant Fc region is operably linked. Preferably, the altered effector function is an increase in ADCC, a decrease in ADCC, an increase in CDC, a decrease in CDC, an increase in Clq binding affinity, a decrease in Clq binding affinity, an increase in FcR (preferably FcRn) binding affinity or a decrease in FcR (preferably FcRn) binding affinity as compared to the corresponding Fc region lacking such amino acid substitutions.,

[0211] Further examples include variants of the Fc region that contain amino acid substitutions at one or more of positions 221, 222, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 283, 285, 286, 288, 290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 302, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and / or 428 (see, e.g., U.S. Patent No. 7,662,925). In certain embodiments, the variant Fc region comprises at least one amino acid substitution selected from the group consisting of P230A, E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V, L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R, and T335Y. In other certain embodiments, the variant Fc region comprises V264I, F243L / V264I, L328M, I332E, L328M / I332E, V264I / I332E, S298A / I332E, S239E / I332E, S239Q / I332E, S239E, A330Y, I332D, L328I / I332E, L328Q / I332E,V264T, V240I, V266I, S239D, S239D / I332D, S239D / I332E, S239D / I332N, S239D / I332Q, S239E / I332D, S239E / I332N, S239E / I332Q, S239N / I332D, S239N / I332E, S239Q / I332D, A330Y / I332E, V264I / A330Y / I332E, A330L / I332E, V264I / A330L / I332E, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T, L328D / I332E, L328V / I332E, L328T / I332E, L328I / I332E, S239E / V264I / I332E, S239Q / V264I / I332E, S239E / V264I / A330Y / I332E, S239D / A330Y / I332E, S239N / A330Y / I332E, S239D / A330L / I332E, S239N / A330L / I332E, V264I / S298A / I332E, S239D / S298A / I332E, S239N / S298A / I332E, S239D / V264I / I332E, S239D / V264I / S298A / I332E, S239D / V264I / A330L / I332E, S239D / I332E / A330I, P230A, P230A / E233D / I332E, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, K326I, K326T, T335D, T335R, T335Y, V240I / V266I, S239D / A330Y / I332E / L234I, S239D / A330Y / I332E / L235D, S239D / A330Y / I332E / V240I, S239D / A330Y / I332E / V264T, S239D / A330Y / I332E / K326E, and S239D / A330Y / I332E / K326T, and comprises at least one amino acid substitution selected from the group consisting of. Further, in certain embodiments, this variant Fc region is N297D / I332E, F241Y / F243Y / V262T / V264T / N297D / I332E,It comprises a series of substitutions selected from the group consisting of S239D / N297D / I332E, S239E / N297D / I332E, S239D / D265Y / N297D / I332E, S239D / D265H / N297D / I332E, V264E / N297D / I332E, Y296N / N297D / I332E, N297D / A330Y / I332E, S239D / D265V / N297D / I332E, S239D / D265I / N297D / I332E, and N297D / S298A / A330Y / I332E. In certain embodiments, this variant Fc region comprises an amino acid substitution at position 332 (EU index, Kabat et al., using the numbering above). Examples of substitutions include 332A, 332D, 332E, 332F, 332G, 332H, 332K, 332L, 332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, and 332Y. The numbering of residues in the Fc region is according to the EU index of Kabat et al. Among other properties described herein, such variant Fc regions may have increased affinity for FcγR, increased stability, and / or increased solubility compared to the corresponding wild-type Fc region.,

[0212] Further examples include the following amino acid substitutions: 224N / Y, 225A, 228L, 230S, 239P, 240A, 241L, 243S / L / G / H / I, 244L, 246E, 247L / A, 252T, 254T / P, 258K, 261Y, 265V, 266A, 267G / N, 268N, 269K / G, 273A, 276D, 278H, 279M, 280N, 283G, 285R, 288R, 289A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 305A, 306I / F, 311R, 312N, 315D / K / S, 320R, 322E, 323A, 324T, 325S, 326E / R, 332T, 333D / G, 335I, 338R, 339T, 340Q, 341E, 342R, 344Q, 347R, 351S, 352A, 354A, 355W, 356G, 358T, 361D / Y, 362L, 364C, 365Q / P, 370R, 372L, 377V, 378T, 383N, 389S, 390D, 391C, 393A, 394A, 399G, 404S, 408G, 409R, 411I, 412A, 414M, 421S, 422I, 426F / P, 428T, 430K, 431S, 432P, 433P, 438L, 439E / R, 440G, 441F, 442T, 445R, 446A, 447E, wherein optionally, the variant has an altered recognition of Fc ligand and / or an altered effector function compared to the parental Fc polypeptide, and the residue numbering is according to the EU index of Kabat et al. Specific examples of these and related embodiments include the following sets of substitutions: (1) N276D, R292Q, V305A, I377V, T394A, V412A, and K439E, (2) P244L, K246E, D399G, and K409R, (3) S304G, K320R, S324T, K326E, and M358T, (4) F243S, P247L, D265V, V266A, S383N, and T411I, (5) H224N, F243L, T393A, and H433P, (6) V240A, S267G, G341E, and E356G, (7) M252T, P291L, P352A, R355W, N390D, S408G, S426F, and A431S, (8) P228L, T289A,Variants Fc regions comprising L365Q, N389S, and 5440G, (9) F241L, V273A, K340Q, and L441F, (10) F241L, T299A, I332T, and M428T, (11) E269K, Y300H, Q342R, V422I, and G446A, (12) T225A, R301c, S304G, D312N, N315D, L351S, and N421S, (13) S254T, L306I, K326R, and Q362L, (14) H224Y, P230S, V323A, E333D, K338R, and S364C, (15) T335I, K414M, and P445R, (16) T335I and K414M, (17) P247A, E258K, D280N, K288R, N297D, T299A, K322E, Q342R, S354A, and L365P, (18) H268N, V279M, A339T, N361D, and S426P, (19) C261Y, K290E, L306F, Q311R, E333G, and Q438L, (20) E283G, N315K, E333G, R344Q, L365P, and S442T, (21) Q347R, N361Y, and K439R, (22) S239P, S254P, S267N, H285R, N315S, F372L, A378T, N390D, Y391C, F404S, E430K, L432P, and K447E, and (23) E269G, Y278H, N325S, and K370R, or consisting of the foregoing, wherein residue numbering is according to the Kabat et al. EU index (see, e.g., U.S. Patent Application No. 2010 / 0184959).

[0213] Another specific example of an Fc variant includes the Fc sequence of Table H7, wherein Xaa at position 1 is Ala or absent, Xaa at position 16 is Pro or Glu, Xaa at position 17 is Phe, Val, or Ala, Xaa at position 18 is Leu, Glu, or Ala, Xaa at position 80 is Asn or Ala, and / or Xaa at position 230 is Lys or absent (see, for example, U.S. Patent Application No. 2007 / 0253966). Certain of these Fc regions and related HRS-Fc conjugates have an increased half-life, reduced effector activity, and / or are significantly less immunogenic than the wild-type Fc sequence.

[0214] The variant Fc region can also have one or more mutated hinge regions, as described, for example, in U.S. Patent Application No. 2003 / 0118592. For example, one or more cysteines in the hinge region can be deleted or substituted with a different amino acid. The mutated hinge region can contain no cysteine residues or can contain one, two, or three fewer cysteine residues than the corresponding wild-type hinge region. In some embodiments, Fc regions having this type of mutated hinge region exhibit a reduced ability to dimerize compared to the wild-type Ig hinge region.

[0215] As described above, HRS-Fc conjugates, such as HRS-Fc fusion proteins, typically have altered (e.g., improved, increased, decreased) pharmacokinetic properties compared to the corresponding HRS polypeptide. Examples of pharmacokinetic properties include stability or half-life, bioavailability (the fraction of drug absorbed), tissue distribution, volume of distribution (the apparent volume into which a drug distributes immediately after intravenous injection and reaches equilibrium between plasma and peripheral tissues), concentration (the initial or steady-state concentration of drug in plasma), elimination rate constant (the rate at which a drug is removed from the body), elimination rate (the rate of infusion required to balance elimination), area under the curve (AUC or exposure; the integral of the concentration-time curve after a single dose or at steady state), clearance (the volume of plasma cleared of drug per unit time), C max (peak plasma concentration of drug after oral administration), t max (C max (time to reach C min (lowest concentration reached by the drug before the next dose is administered), and variability (peak-trough variability within a single dosing interval at steady state). In some embodiments, these improved properties are achieved without significantly altering the secondary structure and / or reducing the off-target biological activity of the HRS polypeptide. In fact, some HRS-Fc conjugates have increased off-target biological activity.

[0216] Thus, in some embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has a plasma or serum pharmacokinetic AUC profile that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 200, 300, 400, or 500 times greater than the corresponding unmodified or differently modified HRS polypeptide when administered to a mammal under the same or comparable conditions. In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has a stability (e.g., as measured by half-life) that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% greater than the corresponding unmodified or differently modified HRS polypeptide when compared in PBS at pH 7.4 under similar conditions at room temperature, e.g., over about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or 1, 2, 3, 4 weeks, etc.

[0217] In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has a biological half-life at pH 7.4, 25° C., e.g., physiological pH, human body temperature (e.g., in vivo, in serum, in a given tissue, in a given species such as rat, mouse, monkey, or human), or any intervening half-life of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours, about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90 hours, about 96 hours, about 120 hours, or about 144 hours or more, or at least about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours, about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90 hours, about 96 hours, about 120 hours or about 144 hours or more.

[0218] In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has greater bioavailability after subcutaneous (SC) administration compared to the corresponding unmodified HRS polypeptide. In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more bioavailability compared to the corresponding unmodified HRS polypeptide.

[0219] In certain embodiments, the HRS-Fc fusion polypeptide has substantially the same secondary structure as the corresponding unmodified or differently modified HRS polypeptide, as determined via UV circular dichroism analysis. In certain embodiments, the HRS-Fc fusion polypeptide has substantially the same activity as the corresponding unmodified or differently modified HRS polypeptide in an assay of anti-inflammatory activity. In other embodiments, the HRS-Fc fusion polypeptide has an activity that is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more than 20-fold greater than the activity of the corresponding unmodified or differently modified HRS polypeptide in an assay of anti-inflammatory activity.

[0220] In certain embodiments, a peptide linker sequence can be used to separate the HRS polypeptide from the Fc region or PEG by a distance sufficient to ensure that each polypeptide folds into its desired secondary and tertiary structures. Such peptide linker sequences can be incorporated into the conjugate or fusion protein using standard techniques well known in the art.

[0221] A particular peptide linker sequence can be selected based on the following exemplary factors: (1) the ability to adopt a flexible extended conformation, (2) the inability to adopt a secondary structure that can interact with functional epitopes on the first polypeptide and the second polypeptide, (3) physiological stability, and (4) the absence of hydrophobic or charged residues that can react with the functional epitopes of the polypeptide, or other characteristics. See, for example, George and Heringa, J Protein Eng. 15:871-879, 2002.

[0222] The linker sequence can generally be from 1 to about 200 amino acids in length. Particular linkers can have an overall amino acid length of about 1 to 200 amino acids, 1 to 150 amino acids, 1 to 100 amino acids, 1 to 90 amino acids, 1 to 80 amino acids, 1 to 70 amino acids, 1 to 60 amino acids, 1 to 50 amino acids, 1 to 40 amino acids, 1 to 30 amino acids, 1 to 20 amino acids, 1 to 10 amino acids, 1 to 5 amino acids, 1 to 4 amino acids, 1 to 3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more amino acids.

[0223] The peptide linker can use any one or more naturally occurring amino acids, non-naturally occurring amino acids, amino acid analogs, and / or amino acid mimetics described elsewhere in this specification and known in the art. Certain amino acid sequences that can be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985, Murphy et al., PNAS USA. 83:8258-8262, 1986, U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. Specific peptide linker sequences contain Gly residues, Ser residues, and / or Asn residues. Other near-neutral amino acids such as Thr and Ala can also be used in the peptide linker sequence if desired.

[0224] Certain exemplary linkers include Gly, Ser, and / or Asn-containing linkers such as the following: [G] x , [S] x , [N] x , [GS] x , [GGS] x , [GSS] x , [GSGS] x (SEQ ID NO: 173), [GGSG] x (SEQ ID NO: 174), [GGGS] x (SEQ ID NO: 175), [GGGGS] x (SEQ ID NO: 176), [GN] x , [GGN] x , [GNN] x , [GNGN] x (SEQ ID NO: 177), [GGNG] x (SEQ ID NO: 178), [GGGN] x (SEQ ID NO: 179), [GGGGN] x (SEQ ID NO: 180) linkers are included, wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. Other combinations of these and related amino acids will be apparent to those skilled in the art.

[0225] Further examples of linker peptides include, but are not limited to, the following amino acid sequences: Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 181), Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 182), Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 183), Asp-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Lys-Asp-Ala-Ala-Ala-Arg-Glu-Ala-Ala-Ala-Arg-Asp-Ala-Ala-Ala-Lys-(SEQ ID NO: 184), and Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Arg-(SEQ ID NO: 185).

[0226] Further non-limiting examples of linker peptides include DGGGS (SEQ ID NO: 186), TGEKP (SEQ ID NO: 187) (see, e.g., Liu et al., PNAS. 94:5525-5530, 1997), GGRR (SEQ ID NO: 188) (Pomerantz et al. 1995), (GGGGS) n (SEQ ID NO: 176) (Kim et al., PNAS. 93:1156-1160, 1996), EGKSSGSGSESKVD (SEQ ID NO: 189) (Chaudhary et al., PNAS. 87:1066-1070, 1990), KESGSVSSEQLAQFRSLD (SEQ ID NO: 190) (Bird et al., Science. 242:423-426, 1988), GGRRGGGS (SEQ ID NO: 191), LRQRDGERP (SEQ ID NO: 192), LRQKDGGGSERP (SEQ ID NO: 193), LRQKd(GGGS) 2It includes ERP (SEQ ID NO: 194). In certain embodiments, this linker array includes a Gly3 linker array containing three glycine residues. In certain embodiments, the flexible linker can be rationally designed using a computer program capable of modeling both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS. 90:2256-2260, 1993 and PNAS. 91:11099-11103, 1994), or by phage display methods.

[0227] This peptide linker can be physiologically stable or can include a releasable linker, such as a physiologically degradable linker or an enzymatically cleavable linker (e.g., a proteolytically cleavable linker). In certain embodiments, one or more releasable linkers can result in a shorter half-life and more rapid clearance of the conjugate. These and related embodiments can be used, for example, to enhance the solubility and blood circulation lifetime of the HRS polypeptide in the bloodstream, while also delivering an HRS polypeptide substantially free of the Fc region into the bloodstream following linker degradation. These aspects are particularly useful when the HRS polypeptide demonstrates reduced activity when permanently conjugated to the Fc region. By using the linkers provided herein, such HRS polypeptides can maintain their therapeutic activity when in the conjugated form. As another example, a large, relatively inactive HRS-Fc conjugate polypeptide can be administered, which is then degraded in vivo (via a cleavable linker) to produce a bioactive HRS polypeptide that possesses a portion of the Fc region or is entirely lacking in the Fc region. In these and other ways, the properties of the HRS-Fc conjugate polypeptide can be more efficiently individualized to balance the bioactivity and circulatory half-life of the HRS polypeptide over time.

[0228] In certain embodiments, this linker peptide comprises an autocatalytic or self-cleaving peptide cleavage site. In one particular embodiment, the self-cleaving peptide comprises polypeptide sequences obtained from Potyviruses and Cardioviruses 2A peptides, FMDV (Foot-and-Mouth Disease Virus), Equine rhinitis A virus, Thosea asigna virus, and Porcine teschovirus. In certain embodiments, the self-cleaving polypeptide site comprises a 2A or 2A-like site, sequence or domain (Donnelly et al., J. Gen. Virol. 82:1027-1041, 2001). Exemplary 2A sites include the following sequences: LLNFDLLKLAGDVESNPGP (SEQ ID NO: 195), TLNFDLLKLAGDVESNPGP (SEQ ID NO: 196), LLKLAGDVESNPGP (SEQ ID NO: 197), NFDLLKLAGDVESNPGP (SEQ ID NO: 198), QLLNFDLLKLAGDVESNPGP (SEQ ID NO: 199), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 200), VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT (SEQ ID NO: 201), LNFDLLKLAGDVESNPGP (SEQ ID NO: 202), LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 203), and EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 204). In some embodiments, this autocatalytic peptide cleavage site comprises a translational 2A signal sequence, such as, for example, the 2A region of the aphthovirus Foot-and-Mouth Disease Virus (FMDV) polyprotein, which is an 18 amino acid sequence. Further examples of 2A-like sequences that can be used include, for example, insect virus polyproteins, the NS34 protein of rotavirus C, and repetitive sequences in Trypanosoma spp., as described in Donnelly et al., Journal of General Virology. 82:1027-1041, 2001.

[0229] Suitable protease cleavage sites and self-cleaving peptides are known to those of skill in the art (see, e.g., Ryan et al., J. Gener. Virol. 78:699-722, 1997 and Scymczak et al., Nature Biotech. 5:589-594, 2004). Exemplary protease cleavage sites include, but are not limited to, potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, byovirus RNA-2 encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase cleavage sites. Due to its high cleavage stringency, in some embodiments, TEV (tobacco etch virus) protease cleavage sites such as EXXYXQ(G / S) (SEQ ID NO: 205), e.g., ENLYFQG (SEQ ID NO: 206) and ENLYFQS (SEQ ID NO: 207), are included, where X represents any amino acid (cleavage by TEV occurs between Q and G or between Q and S).

[0230] Additional examples of enzymatically cleavable linkers suitable for use in certain embodiments include, but are not limited to, amino acid sequences cleaved by serine proteases such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or subtilisin. Exemplary examples of thrombin-cleavable amino acid sequences include, but are not limited to, -Gly-Arg-Gly-Asp- (SEQ ID NO: 208), -Gly-Gly-Arg-, -Gly-Arg-Gly-Asp-Asn-Pro- (SEQ ID NO: 209), -Gly-Arg-Gly-Asp-Ser- (SEQ ID NO: 210), -Gly-Arg-Gly-Asp-Ser-Pro-Lys- (SEQ ID NO: 211), -Gly-Pro-Arg-, -Val-Pro-Arg-, and -Phe-Val-Arg-. Exemplary examples of elastase-cleavable amino acid sequences include, but are not limited to, -Ala-Ala-Ala-, -Ala-Ala-Pro-Val- (SEQ ID NO: 212), -Ala-Ala-Pro-Leu- (SEQ ID NO: 213), -Ala-Ala-Pro-Phe- (SEQ ID NO: 214), -Ala-Ala-Pro-Ala- (SEQ ID NO: 215), and -Ala-Tyr-Leu-Val- (SEQ ID NO: 216).

[0231] Enzymatically cleavable linkers also include amino acid sequences that can be cleaved by matrix metalloproteinases such as collagenase, stromelysin, and gelatinase. Exemplary examples of matrix metalloproteinase-cleavable amino acid sequences include, but are not limited to, -Gly-Pro-Y-Gly-Pro-Z- (SEQ ID NO: 217), -Gly-Pro-, Leu-Gly-Pro-Z- (SEQ ID NO: 218), -Gly-Pro-Ile-Gly-Pro-Z- (SEQ ID NO: 219), and -Ala-Pro-Gly-Leu-Z- (SEQ ID NO: 220), wherein Z is an amino acid. Exemplary examples of collagenase-cleavable amino acid sequences include, but are not limited to, -Pro-Leu-Gly-Pro-D-Arg-Z- (SEQ ID NO: 221), -Pro-Leu-Gly-Leu-Leu-Gly-Z- (SEQ ID NO: 222), -Pro-Gln-Gly-Ile-Ala-Gly-Trp- (SEQ ID NO: 223), -Pro-Leu-Gly-Cys(Me)-His- (SEQ ID NO: 224), -Pro-Leu-Gly-Leu-Tyr-Ala- (SEQ ID NO: 225), -Pro-Leu-Ala-Leu-Trp-Ala-Arg- (SEQ ID NO: 226), and -Pro-Leu-Ala-Tyr-Trp-Ala-Arg- (SEQ ID NO: 227), wherein Z is an amino acid. One exemplary example of a stromelysin-cleavable amino acid sequence is -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg- (SEQ ID NO: 228), and one example of a gelatinase-cleavable amino acid sequence is -Pro-Leu-Gly-Met-Tyr-Ser-Arg- (SEQ ID NO: 229).

[0232] Enzymatically cleavable linkers suitable for use in certain embodiments also include amino acid sequences that can be cleaved by angiotensin converting enzyme, such as, for example, -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro- (SEQ ID NO: 230) and -Gly-Ser-Asp-Lys-Pro- (SEQ ID NO: 231).

[0233] Enzymatically cleavable linkers suitable for use in certain embodiments also include amino acid sequences that can be cleaved by cathepsin B, such as Val-Cit, Ala-Leu-Ala-Leu-(SEQ ID NO: 232), Gly-Phe-Leu-Gly-(SEQ ID NO: 233), and Phe-Lys.

[0234] In certain embodiments, the releasable linker has a half-life of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours or more at pH 7.4, 25°C, e.g., physiological pH, human body temperature (e.g., in vivo, in serum, in a given tissue), or any intervening half-life. One of ordinary skill in the art can appreciate that the half-life of the HRS-Fc conjugate polypeptide can be precisely individualized by using a particular releasable linker.

[0235] However, in certain embodiments, any one or more of the peptide linkers are optional. For example, if the first and second polypeptides have non-essential N-terminal and / or C-terminal amino acid regions that can be used to separate functional domains and prevent steric hindrance, the linker sequence may not be required.

[0236] The HRS polypeptide and polynucleotide, e.g., an expressible polynucleotide, can be used in any of the compositions, methods, and / or kits described herein.

[0237] Immunomodulatory agent Certain embodiments use one or more immunomodulatory agents. Exemplary immunomodulatory agents include small molecules, polypeptides, e.g., antibodies and antigen-binding fragments thereof, ligands, small peptides, antisense agents, RNAi agents, and mixtures thereof.

[0238] In some embodiments, the immunomodulatory agent is selected from one or more of sphingosine-1-phosphate (S1P) and / or S1P receptor (S1PR) modulators, steroids, calcineurin inhibitors, mammalian target of rapamycin (mTOR) inhibitors, indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitors, inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors, cytokines and / or cytokine receptor inhibitors, B cell receptor inhibitors, kinase inhibitors, and cell growth inhibitors such as methotrexate.

[0239] In some embodiments, the immunomodulatory agent is pirfenidone, which is often used for the treatment of idiopathic pulmonary fibrosis (IPF). Pirfenidone has antifibrotic and anti-inflammatory properties in various in vitro systems and animal models of fibrosis. For example, cell-based studies have shown that pirfenidone decreases fibroblast proliferation, inhibits TGF-β-stimulated collagen production, and reduces the production of profibrotic mediators such as TGF-β. Pirfenidone has also been shown to decrease the production of inflammatory mediators such as TNF-α and IL-1β in both cultured cells and isolated human peripheral blood mononuclear cells. In the United States, pirfenidone is approved for the treatment of IPF as an oral dosage unit of 801 mg (three 267 mg capsules) taken orally three times a day for a total oral dose of 2403 mg / day. Further exemplary dosages of pirfenidone are described herein.

[0240] In some embodiments, the immunomodulatory agent is nintedanib, which is also used for the treatment of IPF. Nintedanib inhibits specific growth factor receptors involved in pulmonary fibrosis, including platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and vascular endothelial growth factor receptor (VEGFR). Nintedanib is thought to slow the progression of the IPF disease and delay the decline of lung function by blocking the signaling pathways involved in the fibrosis process. Nintedanib is formulated as a salt containing ethanesulfonic acid. In the United States, nintedanib is approved for the treatment of IPF as an oral dosage unit of 150 mg taken twice daily for a total of 300 mg / day, which can reduce the side effects associated with a dosage of approximately 100 mg taken twice daily for a total of 200 mg / day. Further exemplary dosages of nintedanib are described herein.

[0241] In some embodiments, the immunomodulatory agent is a sphingosine-1-phosphate (S1P) and / or S1P receptor (S1PR) modulator. General examples of modulators include S1P and / or S1PR antagonists or inhibitors, or S1P and / or S1PR agonists or activators. S1P is a bioactive lipid with various biological functions, including cell proliferation, differentiation, angiogenesis, chemotaxis, and trafficking. Many of the activities of S1P act through five closely related G protein-coupled receptors of the sphingosine-1-phosphate receptor family (S1PR), which play important roles in sphingolipid metabolism. S1PR includes S1PR 1 、S1PR 2 、S1PR 3 、S1PR 4 、and S1PR 5 . The expression of these receptors varies as follows: S1PR 1 、S1PR 2 、and S1PR 3 are expressed in a wide variety of cell types, mainly highly expressed on leukocytes, and S1PR 4 is mainly expressed in lymphocytes and hematopoietic tissues, and S1PR5 is mainly expressed in the spleen and the white matter of the central nervous system (CNS).

[0242] Certain non-limiting examples of S1P or S1PR modulators include amiselimod (also known as MT-1303; S1PR antagonist; see Kappos et al., Lancet Neurol 2016;15:1148-59, 2016), fingolimod (S1PR 1 functional antagonist), sonelixizumab (S1P-specific monoclonal antibody), KRP203 (S1PR 1 agonist), SEW2871 (S1PR 1 agonist), siponimod (S1PR 1 and S1PR 5 modulator), RPC1063 (S1PR 1 modulator), ONO-4641 (S1PR 1 and S1PR 5 agonist), JTE-013 (S1PR 2 antagonist), GSK2018682 (S1PR 1 agonist), ponesimod (S1PR 1 agonist), suramin (selective S1PR 3 and S1PR 5 antagonist), VPC23019 (aryl-amide analog; competitive S1PR 1 and S1PR 3 antagonist); and W146 (selective S1PR 1 antagonist). In certain embodiments, the S1P or S1PR modulator is amiselimod.

[0243] Certain S1P or S1PR modulators include an antibody or antigen-binding fragment or small molecule that specifically binds to S1P or S1PR (see, e.g., sonelixizumab that binds to S1P). In some embodiments, the antibody or antigen-binding fragment thereof is an S1P and / or S1PR antagonist. In certain embodiments, the antibody or antigen-binding fragment thereof is an S1P and / or S1PR agonist.

[0244] Certain S1PR antagonists or inhibitors include antisense agents and RNAi agents against the S1PR coding sequence (see, e.g., accession numbers NM_001400.4; NM_004230.3). Certain antisense agents specifically hybridize to a target region within the pre-mRNA or mRNA target sequence encoding S1PR, and the target region is selected from one or more of the AUG start codon of the mRNA, the region upstream of the AUG start codon, the region downstream of the AUG codon, the 3' or 5' splice site of the pre-processed mRNA, the branch point, the 3' untranslated region (UTR), and the polyadenylation signal sequence. Certain RNAi agents include a sense strand that is substantially identical to the mRNA target sequence encoding S1PR, and optionally, an antisense strand that is complementary or substantially complementary to the mRNA target sequence encoding S1PR.

[0245] In some embodiments, the immunomodulatory agent is a steroid or corticosteroid, e.g., a glucocorticoid. In certain embodiments, the steroid is an anti-inflammatory steroid. Examples of steroids include, among others, betamethasone, budesonide, cortisol (hydrocortisone), cortisone, deflazacort, deoxycorticosterone, dexamethasone, fludrocortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, and triamcinolone.

[0246] In some embodiments, the immunomodulatory agent is a calcineurin antagonist or inhibitor. Calcineurin is a calcium and calmodulin-dependent serine / threonine protein phosphatase that activates T cells. Specifically, calcineurin translocates to the nucleus, upregulates the expression of interleukin 2 (IL-2), and then activates the nuclear factor of activated T cells (NFATc), which stimulates the growth and differentiation of the T cell response. Specific examples of calcineurin antagonists or inhibitors include cyclosporine, pimecrolimus, and tacrolimus.

[0247] Certain calcineurin antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to calcineurin. Also included are antisense agents and RNAi agents against the calcineurin coding sequence or its subunits (see, e.g., accession numbers NM_000944; NM_021132; NM_005605; NM_000945; NM_147180). Certain antisense agents specifically hybridize to a target region within a pre-mRNA or mRNA target sequence encoding calcineurin or its subunits, and, for example, the target region is selected from one or more of the AUG start codon of the mRNA, the region upstream of the AUG start codon, the region downstream of the AUG codon, the 3' or 5' splice site of the pre-processed mRNA, the branch point, the 3' untranslated region (UTR), and the polyadenylation signal sequence. Certain RNAi agents include a sense strand that is substantially identical to an mRNA target sequence encoding calcineurin or its subunits, and optionally, an antisense strand that is complementary or substantially complementary to the mRNA target sequence encoding calcineurin or its subunits.

[0248] In some embodiments, the immunomodulatory agent is a mechanistic target of rapamycin (mTOR) antagonist or inhibitor. mTOR is a member of the phosphatidylinositol 3-kinase related kinase family of protein kinases and is localized in different intracellular compartments and is thus the catalytic subunit of two structurally distinct complexes, mTORC1 and mTORC2, that particularly affect their activation and function. As a core component of both complexes, mTOR acts as a serine / threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription. As a core component of mTORC2, mTOR also acts as a tyrosine protein kinase that promotes the activation of the insulin receptor and the insulin-like growth factor 1 receptor. mTORC2 is also involved in the control and maintenance of the actin cytoskeleton. mTOR plays a role in fibrosis and autoimmunity, and blockade of the mTORC pathway is under investigation as a treatment for such diseases.

[0249] Specific examples of mTOR inhibitors include everolimus, rapamycin, deforolimus, and temsirolimus. General examples of mTOR inhibitors include ATP-competitive mTOR kinase inhibitors, including mTORC1 / mTORC2 dual inhibitors, and mTOR / PI3K dual inhibitors that inhibit the catalytic isoforms of mTORC1, mTORC2, and PI3K. Specific examples include dactolisib, BGT226, SF1126, PKI-587, NVPBE235, sapitinib, AZD8055, and AZD2014.

[0250] Certain mTOR antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to mTOR or members of the mTOR complex. Also included are antisense agents and RNAi agents against the coding sequence of mTOR or members of the mTOR complex (see, e.g., Ravichandran et al., Hum Mol Genet. 23:4919-31, 2014). Certain antisense agents specifically hybridize to a target region within the pre-mRNA or mRNA target sequence encoding mTOR or a member of the mTOR complex, and, for example, the target region is selected from one or more of the AUG start codon of the mRNA, the region upstream of the AUG start codon, the region downstream of the AUG codon, the 3' or 5' splice site of the pre-processed mRNA, the branch point, the 3' untranslated region (UTR), and the polyadenylation signal sequence. Certain RNAi agents include a sense strand that is substantially identical to the mRNA target sequence encoding mTOR or a member of the mTOR complex, and optionally, an antisense strand that is complementary or substantially complementary to the mRNA target sequence encoding mTOR or a member of the mTOR complex.

[0251] In some embodiments, the immunomodulatory agent is an IDO antagonist or inhibitor. IDO is a tryptophan catabolic enzyme with immunosuppressive properties. For example, IDO is known to suppress T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. Specific examples of IDO antagonists or inhibitors include indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, e.g., Sheridan, Nature Biotechnology. 33:321-322, 2015). Certain IDO antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to IDO (see, e.g., Platten et al., Front Immunol. 5:673, 2014). Also included are antisense agents and RNAi agents against the IDO coding sequence (see, e.g., accession number AH002828.2). Certain antisense agents specifically hybridize to a target region within the pre-mRNA or mRNA target sequence encoding IDO, and for example, the target region is selected from one or more of the AUG start codon of the mRNA, the region upstream of the AUG start codon, the region downstream of the AUG codon, the 3' or 5' splice site of the pre-processed mRNA, the branch point, the 3' untranslated region (UTR), and the polyadenylation signal sequence. Certain RNAi agents include a sense strand that is substantially identical to the mRNA target sequence encoding IDO, and optionally, an antisense strand that is complementary or substantially complementary to the mRNA target sequence encoding IDO.

[0252] In some embodiments, the immunomodulatory agent is an inosine-5'-monophosphate dehydrogenase (IMPDH) antagonist or inhibitor. IMPDH is a purine biosynthetic enzyme that catalyzes the nicotinamide adenine dinucleotide (NAD+)-dependent oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), and is the rate-limiting step first identified for de novo synthesis of guanine nucleotides from IMP. Guanine nucleotide synthesis is essential for maintaining normal cell function and growth, and is also important for cell proliferation and maintenance of the immune response. In particular, B and T cells show dependence on IMPDH for normal activation and function, and show upregulated IMPDH expression.

[0253] Specific examples of IMPDH inhibitors include mycophenolic acid (mycophenolate mofetil), ribavirin, and 6TGMP (6-thioguanine monophosphate). Certain IMPDH antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to IMPDH. Also included are antisense agents and RNAi agents against the IMPDH coding sequence. Certain antisense agents specifically hybridize to a target region within the pre-mRNA or mRNA target sequence encoding IMPDH, for example, the target region is selected from one or more of the AUG start codon of the mRNA, the region upstream of the AUG start codon, the region downstream of the AUG codon, the 3' or 5' splice site of the pre-processed mRNA, the branch point, the 3' untranslated region (UTR), and the polyadenylation signal sequence. Certain RNAi agents include a sense strand that is substantially identical to the mRNA target sequence encoding IMPDH, and optionally, an antisense strand that is complementary or substantially complementary to the mRNA target sequence encoding IMPDH.

[0254] In some embodiments, the immunomodulatory agent is a cytokine and / or a cytokine receptor antagonist or inhibitor. A cytokine is a small (glyco)protein (having a molecular weight of 8-75 kDa) that affects hematopoiesis, immune response, and inflammation. Certain exemplary cytokine inhibitors reduce cytokine synthesis, reduce the concentration of cytokines in their free active form, block the interaction between cytokines and their cognate receptors, and / or prevent cytokine receptor signaling.

[0255] In some embodiments, the target cytokine or cytokine receptor is an inflammatory or pro-inflammatory cytokine or cytokine receptor. Examples of target cytokines include interleukin-1 (IL-1) including IL-1α and IL-1β, interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-20 (IL-20), interleukin-33 (IL-33), tumor necrosis factor (TNF), interferon gamma (IFN-γ), transforming growth factor-β (TGF-β), and granulocyte-macrophage colony-stimulating factor (GM-CSF), and their cognate cytokine receptors, such as IL-1R, IL-6R, IL-8R, IL-11R, IL-12R, IL-17R, IL-18R, IL-20R, ST2 (interleukin 1 receptor-like 1, IL1RL1), TNFR such as TNFR1, interferon-gamma receptor (IFNGR), TGF-β receptors such as TGFβR1 (ALK5) or TGFβR2, but are not limited thereto.

[0256] Specific examples of cytokines and / or cytokine receptor inhibitors include etanercept, a recombinant fusion protein of a soluble type II TNF receptor on a human IgG1 backbone, and TNF-alpha inhibitors such as infliximab, a chimeric anti-TNF-alpha monoclonal antibody containing a mouse TNF-alpha binding region and a human IgG1 backbone. Adalimumab, certolizumab, and golimumab are also included as TNF inhibitors.

[0257] Specific examples of interleukin inhibitors include, for example, IL-1R antagonists such as anakinra, IL-1 inhibitors such as rilonacept (a dimer fusion protein consisting of the ligand-binding domain of the extracellular portion of the IL-1R1 component and the interleukin-1 receptor accessory protein (IL-1RAcP) linked in-line to the fragment-crystallizable region (Fc region) of human IgG1 that binds and neutralizes IL-1), IL-2 competitive inhibitors such as basiliximab (a chimeric mouse-human monoclonal antibody against the alpha chain (CD25) of the IL-2 receptor of T cells) and daclizumab (a humanized monoclonal antibody that binds CD25), IL-1β-specific inhibitors such as canakinumab (a human monoclonal antibody), IL-17 antagonists such as ixekizumab (a humanized monoclonal antibody that binds IL-17) and secukinumab (a human IgG1κ monoclonal antibody that binds protein interleukin (IL)-17A), IL-5 inhibitors such as mepolizumab (a humanized monoclonal antibody that binds IL-5 and prevents binding to the alpha subunit of the IL-5 receptor) and reslizumab, IL-6 inhibitors such as siltuximab (an antibody that binds IL-6), sirukumab (an antibody that binds IL-6), sarilumab (an antibody that binds to the IL-6 receptor), tocilizumab (an antibody that binds to the IL-6 receptor), and IL-12 / IL-23 signaling inhibitors such as ustekinumab (an antibody that binds to the p-40 subunit of both IL-12 and IL-23).

[0258] Certain cytokines and / or cytokine receptor antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to a cytokine and / or cytokine receptor, such as one or more of the aforementioned cytokines and / or cytokine receptors. Also included are antisense agents and RNAi agents directed against cytokine and / or cytokine receptor coding sequences, such as one or more of the aforementioned cytokines and / or cytokine receptors. Certain antisense agents specifically hybridize to a target region within a pre-mRNA or mRNA target sequence encoding a cytokine or cytokine receptor, and for example, the target region is selected from one or more of the AUG start codon of the mRNA, the region upstream of the AUG start codon, the region downstream of the AUG codon, the 3' or 5' splice site of the pre-processed mRNA, the branch point, the 3' untranslated region (UTR), and the polyadenylation signal sequence. Certain RNAi agents include a sense strand that is substantially identical to an mRNA target sequence encoding a cytokine or cytokine receptor, and optionally, an antisense strand that is complementary or substantially complementary to the mRNA target sequence encoding a cytokine or cytokine receptor.

[0259] In some embodiments, the immunomodulatory agent is a kinase antagonist or inhibitor, i.e., an inhibitor that targets or is targeted to one or more kinases. Common examples include tyrosine kinase inhibitors (TKIs). Examples of target kinases include, among others, Janus kinases (JAK, including JAK1, JAK2, JAK3, TYK2), epidermal growth factor receptor (EGFR), receptor tyrosine-protein kinase erbB-2 (Her2 / neu or ERBB2), Bcr-Abl, c-SRC, mitogen-activated protein kinase (MAP) kinase, anaplastic lymphoma kinase (ALK), spleen tyrosine kinase (SYK), Bruton tyrosine kinase (BTK), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR, including VEGFR1, VEGFR2, VEGFR3), fibroblast growth factor receptor (FGFR), B-Raf, RET oncogene, platelet-derived growth factor receptor (PDGF-R), tropomyosin receptor kinase (Trk, including TrkA, TrkB, TrkC), and c-Met, but are not limited thereto. Thus, in certain embodiments, the kinase inhibitor is an inhibitor or antagonist of one or more of the aforementioned kinases.

[0260] Specific examples of kinase inhibitors include JAK inhibitors such as baricitinib, fedratinib, filgotinib, gandotinib, lestaurtinib, momelotinib, pacritinib, peficitinib, ruxolitinib, tofacitinib, and padasitinib. Further examples of kinase inhibitors include nintedanib, afatinib, axitinib, bosutinib, cetuximab, cobimetinib, crizotinib, cabozantinib, dasatinib, entrectinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, neratinib, nilotinib, pazopanib, pegaptanib, sorafenib, sunitinib, SU6656, toceranib, vandetanib, batatinib, and vemurafenib, but are not limited thereto.

[0261] Certain kinase antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to a kinase, such as one or more of the aforementioned kinases. Also included are antisense agents and RNAi agents against kinase coding sequences, such as one or more of the aforementioned kinases. Certain antisense agents specifically hybridize to a target region within a pre-mRNA or mRNA target sequence encoding a kinase, and for example, the target region is selected from one or more of the AUG start codon of the mRNA, the region upstream of the AUG start codon, the region downstream of the AUG codon, the 3' or 5' splice site of the pre-processed mRNA, the branch point, the 3' untranslated region (UTR), and the polyadenylation signal sequence. Certain RNAi agents include a sense strand that is substantially identical to the mRNA target sequence encoding a kinase, and optionally, an antisense strand that is complementary or substantially complementary to the mRNA target sequence encoding a kinase.

[0262] In some embodiments, the immunomodulatory agent is a B cell receptor inhibitor, such as an agent that targets CD20. B lymphocyte antigen CD20 or CD20 is an activation-glycosylated lymprotein (CD45R+, CD117+) expressed on the surface of all B cells starting from the pro-B stage, with its concentration increasing gradually until maturation. The protein has no known natural ligand, and its function is specifically to enable an optimal B cell immune response against T-independent antigens. Exemplary immunomodulatory agents against CD20 include the monoclonal antibodies ibritumomab tiuxetan, obinutuzumab, ofatumumab, ocrelizumab, rituximab, tositumomab, and belimumab.

[0263] In some embodiments, the immunomodulatory agent is a cytostatic or cytotoxic agent. Examples of cytostatic or cytotoxic agents include, among others, azathioprine, chlorambucil, cyclophosphamide, cyclosporine A, methotrexate, and nitrogen mustard.

[0264] In some embodiments, as described above, the immunomodulatory agent is a "small molecule" that refers to an organic compound that is of synthetic or biological origin (biomolecule) but is typically not a polymer. Organic compounds refer to a large class of compounds whose molecules contain carbon, and typically those containing only carbonates, simple oxides of carbon, or cyanides are excluded. "Biomolecule" generally refers to organic molecules produced by living organisms, including large polymeric molecules (biopolymers) such as peptides, polysaccharides, and nucleic acids, as well as small molecules such as primary metabolites, lipids, phospholipids, glycolipids, sterols, glycerolipids, vitamins, and hormones. "Polymer" generally refers to a large molecule or macromolecule composed of repeating structural units, which are usually linked by covalent chemical bonds.

[0265] In certain embodiments, the small molecule has a molecular weight of about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 500, 650, 600, 750, 700, 850, 800, 950, 1000, or 2000 daltons, or less than about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 500, 650, 600, 750, 700, 850, 800, 950, 1000, or 2000 daltons, typically about 300 - 700 daltons, including about 1000 - 2000 daltons or less than about 1000 - 2000 daltons.

[0266] Certain small molecules may have the characteristic of "specific binding" as described herein. For example, in some embodiments, the small molecule has a binding affinity (Kd) of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM, at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM, or less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM and specifically binds to a target (e.g., S1P, S1PR, calcineurin, mTOR, IDO, IMPDH, cytokine and / or cytokine receptor, B cell receptor, kinase).

[0267] In certain embodiments, the immunomodulatory agent is a polypeptide or a peptide. The terms "peptide" and "polypeptide" are used interchangeably herein, although in certain cases, "peptide" may refer to a shorter polypeptide, e.g., a polypeptide consisting of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids, including all integers and ranges in between (e.g., 5 - 10, 8 - 12, 10 - 15). Polypeptides and peptides can consist of naturally occurring amino acids and / or non-naturally occurring amino acids, as described herein. Antibodies are also included as polypeptides.

[0268] The binding properties of the polypeptide can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, the polypeptide specifically binds to a target molecule (e.g., S1P, S1PR, calcineurin, mTOR, IDO, IMPDH, cytokine and / or cytokine receptor, B cell receptor, kinase, or an epitope thereof) having an equilibrium dissociation constant ranging from about 10-7 to about 10-8 M. In some embodiments, the equilibrium dissociation constant ranges from about 10-9 M or less to about 10-10 M. In certain exemplary embodiments, the polypeptide has an affinity (Kd) for a target (specifically binding ones described herein, e.g., including S1P, S1PR, calcineurin, mTOR, IDO, IMPDH, cytokine and / or cytokine receptor, B cell receptor, or kinase) of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM, at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM, or less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM.

[0269] In some embodiments, the immunomodulatory agent is an antibody or "antigen-binding fragment thereof" that specifically binds to the targets described herein. The antibody or antigen-binding fragment can be essentially any type of antibody or antigen-binding fragment. As is well known in the art, an antibody is an immunoglobulin molecule that can specifically bind to a target (e.g., S1P, S1PR, calcineurin, mTOR, IDO, IMPDH, cytokine and / or cytokine receptor, B cell receptor, kinase) via at least one epitope recognition site located within the variable region of the immunoglobulin molecule.

[0270] As used herein, the term "antibody" includes not only intact polyclonal or monoclonal antibodies, but also their fragments (dAb, Fab, Fab’, F(ab’)2, Fv, etc.), single-chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins containing antibody moieties having the required specificity, humanized antibodies, chimeric antibodies, and any other modified form of immunoglobulin molecule containing the antigen-binding site or fragment (epitope recognition site) of the required specificity. Certain properties and characterizations of antibodies (and antigen-binding fragments thereof) are described in more detail herein.

[0271] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy and / or light chain that binds to a target antigen. In this regard, the antigen-binding fragments of the antibodies described herein can include one, two, three, four, five, or all six CDRs of the VH and VL sequences from an antibody that binds to a target molecule.

[0272] The term "antigen" is a molecule or part of a molecule that can be bound by a selective binding agent such as an antibody and can further be used to produce an antibody in an animal that can bind to the epitope of that antigen. An antigen can have one or more epitopes.

[0273] The term "epitope" includes any determinant, e.g., a polypeptide determinant, that can specifically bind to an immunoglobulin or T cell receptor. An epitope is a region of an antigen or target protein that is bound by an antibody. In certain embodiments, an epitope determinant includes a chemically active surface moiety of a molecule such as an amino acid, sugar side chain, phosphoryl, or sulfonyl, and in certain embodiments can have specific three-dimensional structural features and / or specific charge characteristics. An epitope can be continuous or discontinuous with respect to the primary structure of the antigen.

[0274] A molecule such as a polypeptide or antibody is said to exhibit "specific binding" or "selective binding" if it reacts or associates more frequently, rapidly, for a longer duration, and / or with greater affinity with a particular cell or substance than it does with an alternative cell or substance. An antibody "specifically binds" or "selectively binds" to a target if it binds to the target with greater affinity, binding activity, more readily, and / or for a longer duration than it binds to other substances, e.g., in a statistically significant amount. For example, an antibody that specifically or selectively binds to a particular epitope is an antibody that binds its specific epitope with greater affinity, binding activity, more readily, and / or for a longer duration than it binds to other epitopes. By reading this definition, it is understood that, for example, an antibody (or portion or epitope) that specifically or selectively binds to a first target may or may not specifically or selectively bind to a second target. Thus, "specific binding" or "selective binding" does not necessarily require exclusive binding (although it can include exclusive binding). Generally, but not necessarily, a reference to binding means preferential binding.

[0275] Immunological binding refers to the non-covalent interaction between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific, for example, by way of illustration and not limitation, electrostatic, ionic, hydrophilic, and / or hydrophobic attractive or repulsive forces, steric hindrance forces, hydrogen bonds, van der Waals forces, and other types of interactions that occur as a result of such interactions. The strength or affinity of an immunological binding interaction can be expressed by the dissociation constant (Kd) of the interaction, with a smaller Kd representing a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires the measurement of the rates of antigen-binding site / antigen complex formation and dissociation, which rates depend on the concentrations of the complex partners, the affinity of the interaction, and the geometric parameters that equally affect the rates in both directions. Thus, both the "on-rate constant" (K on ) and the "off-rate constant" (K off ) can be determined by calculation of the concentrations as well as the actual association and dissociation rates. The ratio of K off / K on allows cancellation of all parameters not related to affinity and is thus equal to the dissociation constant Kd.

[0276] Antibodies can be prepared by any of a variety of techniques known to those of skill in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies specific for a target polypeptide can be prepared, for example, using the techniques of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976 and improvements thereto. Methods for expressing human antibodies using transgenic animals such as mice are also included. See, for example, Neuberger et al., Nature Biotechnology 14:826, 1996, Lonberg et al., Handbook of Experimental Pharmacology 113:49-101, 1994, and Lonberg et al., Internal Review of Immunology 13:65-93, 1995. Specific examples include the VELOCIMMUNE® platform by REGENEREX® (see, for example, U.S. Patent No. 6,596,541).

[0277] Antibodies can also be made or identified by use of phage display or yeast display libraries (see, e.g., U.S. Patent No. 7,244,592, Chao et al., Nature Protocols. 1:755-768, 2006). Non-limiting examples of available libraries include cloned libraries or synthetic libraries such as the Human Combinatorial Antibody Library (HuCAL) in which the structural diversity of the human antibody repertoire is represented by seven heavy chain and seven light chain variable region genes. The combination of these genes results in 49 frameworks in the master library. By overlaying highly variable gene cassettes (CDR = Complementary Determining Region) on these frameworks, an enormous human antibody repertoire can be reproduced. Also included are human libraries designed using fragments of human donor origin encoding the light chain variable region, heavy chain CDR-3, synthetic DNA encoding the diversity of heavy chain CDR-1, and synthetic DNA encoding the diversity of heavy chain CDR-2. Other libraries suitable for use will be apparent to those skilled in the art.

[0278] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein include sets of heavy and light chain complementarity determining regions (CDRs) inserted respectively between sets of heavy and light chain framework regions (FRs) that provide support to the CDRs and define the spatial relationship of the CDRs to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of the heavy or light chain V region. Proceeding from the N-terminus of the heavy or light chain, these regions are designated "CDR1", "CDR2", and "CDR3", respectively. Thus, the antigen-binding site includes six CDRs, including a CDR set from each of the heavy or light chain V regions. A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit". X-ray crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, and that the most extensive antigen contacts are with the heavy chain CDR3. Thus, the molecular recognition site is primarily responsible for the specificity of the antigen-binding site.

[0279] As used herein, the term "FR set" refers to the four contiguous amino acid sequences that frame the CDRs of the CDR set of a heavy or light chain V region. Although some FR residues may contact the bound antigen, the FR residues, particularly those immediately adjacent to the CDRs, primarily serve to fold the V region into the antigen-binding site. Within the FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are presented as protruding loop motifs that form the antigen-binding surface. It is generally recognized that there are conserved structural regions of the FRs that influence the CDR loop shape folded into a particular "canonical" structure, regardless of the exact CDR amino acid sequence. Furthermore, it is known that certain FR residues are involved in non-covalent domain-to-domain contacts that stabilize the interaction between the antibody heavy and light chains.

[0280] The structure and location of immunoglobulin variable domains can be determined by reference to Kabat, E.A. et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987 and its most recent version.

[0281] “Monoclonal” antibodies are also included and refer to a homogeneous antibody population, and the monoclonal antibodies consist of amino acids (naturally occurring or not) involved in the selective binding of epitopes. Monoclonal antibodies are highly specific for a single epitope. The term “monoclonal antibody” includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (Fab, Fab’, F(ab’)2, Fv), single-chain (ScFv), variants thereof, fusion proteins containing antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified form of immunoglobulin molecule containing an antigen-binding fragment (epitope recognition site) with the required specificity and ability to bind the epitope. It is not intended to be limited with respect to the antibody source or the technique for making the antibody (e.g., hybridoma, phage selection, recombinant expression, by transgenic animals). The term includes whole immunoglobulins, as well as fragments such as those described above under the definition of “antibody”.

[0282] The proteolytic enzyme papain selectively cleaves the IgG molecule to yield several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer containing an intact antigen-binding site. The enzyme pepsin can cleave the IgG molecule to yield several fragments, including an F(ab’)2 fragment containing both antigen-binding sites. Fv fragments for use according to certain embodiments of the present invention can be generated by selective proteolytic cleavage of IgM and rarely IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly obtained using recombinant techniques known in the art. An Fv fragment contains a non-covalently linked VH::VL heterodimer containing an antigen-binding site that retains most of the antigen recognition and binding ability of the native antibody molecule. See Inbar et al., PNAS USA. 69:2659-2662, 1972, Hochman et al., Biochem. 15:2706-2710, 1976, and Ehrlich et al., Biochem. 19:4091-4096, 1980.

[0283] In certain embodiments, single-chain Fv or scFV antibodies are contemplated. For example, Kappa bodies (Ill et al., Prot. Eng. 10:949-57, 1997), minibodies (Martin et al., EMBO J 13:5305-9, 1994), diabodies (Holliger et al., PNAS 90:6444-8, 1993), or Janusins (Traunecker et al., EMBO J 10:3655-59, 1991 and Traunecker et al., Int. J. Cancer Suppl. 7:51-52, 1992) can be prepared according to the teachings of this application with respect to the selection of antibodies having the desired specificity using standard molecular biology techniques.

[0284] A single-chain Fv (sFv) polypeptide is a covalently linked VH::VL heterodimer expressed from a gene fusion containing a gene encoding VH and a gene encoding VL linked by a peptide-encoding linker. Huston et al. (PNAS USA. 85(16):5879-5883, 1988). A number of methods have been described for identifying chemical structures that convert the naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into sFv molecules that will fold into a three-dimensional structure substantially similar to that of the antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 by Huston et al., and U.S. Patent No. 4,946,778 by Ladner et al.

[0285] In certain embodiments, the antibodies described herein are in the form of "diabodies." Diabodies are multimers of polypeptides, each polypeptide comprising a first domain that includes a binding region of an immunoglobulin light chain and a second domain that includes a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site: the antigen-binding site is formed by the association of a first domain of one polypeptide within the multimer with a second domain of another polypeptide within the multimer (WO94 / 13804). The dAb fragment of an antibody consists of a VH domain (Ward et al., Nature 341:544-546, 1989). Diabodies and other multivalent or multispecific fragments can be constructed, for example, by gene fusion (see WO94 / 13804, and Holliger et al., PNAS USA. 90:6444-6448, 1993).

[0286] Also included are minibodies that include an scFv bound to a CH3 domain (see Hu et al., Cancer Res. 56:3055-3061, 1996). See also Ward et al., Nature. 341:544-546, 1989, Bird et al., Science. 242:423-426, 1988, Huston et al., PNAS USA. 85:5879-5883, 1988), PCT / US92 / 09965, WO94 / 13804, and Reiter et al., Nature Biotech. 14:1239-1245, 1996.

[0287] When attempting to use bispecific antibodies, these may be conventional bispecific antibodies, which can be produced in a variety of ways (Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993), for example, they may be prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments described above. Diabodies and scFvs can be constructed without an Fc region, potentially reducing the effect of anti-idiotypic reactions, using only variable domains.

[0288] In contrast to bispecific whole antibodies, bispecific diabodies can be particularly useful because they can be easily constructed and expressed in Escherichia coli. Diabodies (and many other polypeptides such as antibody fragments) with appropriate binding specificities can be readily selected using phage display from a library (WO94 / 13804). If one arm of the diabody is kept constant, for example, to have specificity for antigen X, a library with a diversified other arm can be created to select an antibody with appropriate specificity. Bispecific whole antibodies can be produced by the "knobs-into-holes" maneuver (Ridgeway et al., Protein Eng., 9:616-621, 1996).

[0289] In certain embodiments, the antibodies described herein may be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands and, for example, US2009 / 0226421). This antibody technology creates a stable, smaller antibody format that is expected to have a longer therapeutic window than current small antibody formats. IgG4 antibodies are thought to be inert and do not interact with the immune system. Full human IgG4 antibodies are modified by removing the hinge region of the antibody, thereby obtaining a half-molecule fragment with different stability characteristics compared to the corresponding intact IgG4 (GenMab, Utrecht). By splitting the IgG4 molecule in half, only one region that can bind to a cognate antigen (e.g., a disease target) remains in the UniBody®, and thus the UniBody® binds monovalently to only one site on the target cell.

[0290] In certain embodiments, the antibodies described herein may take the form of nanobodies. Nanobodies are encoded by a single gene and can be efficiently produced in most prokaryotic and eukaryotic hosts, such as Escherichia coli (see US Patent No. 6,765,087), fungi (e.g., Aspergillus or Trichoderma), and yeast (e.g., Saccharomyces, Kluyvermyces, Hansenula, or Pichia; see, for example, US Patent No. 6,838,254). The production process can be scaled up, and nanogram quantities of nanobodies have been produced. Nanobodies can be formulated as ready-to-use solutions with a long shelf life. The Nanoclone® method (see, for example, WO06 / 079372) is a proprietary method for generating nanobodies against a desired target based on the automated high-throughput selection of B cells.

[0291] In certain embodiments, the antibody or antigen-binding fragment thereof is humanized. These embodiments generally refer to chimeric molecules prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin of a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site can include either a complete variable domain fused onto a constant domain or just the CDRs transplanted into a suitable framework region of the variable domain. The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in a human individual, but leaves the potential for an immune response to the foreign variable region (LoBuglio et al., PNAS USA 86:4220-4224, 1989, Queen et al., PNAS USA. 86:10029-10033, 1988, Riechmann et al., Nature. 332:323-327, 1988). Exemplary methods for humanizing an antibody include those described in U.S. Patent No. 7,462,697.

[0292] Another approach focuses not only on providing human-derived constant regions, but also on modifying the variable regions to reshape them as closely as possible to the human form. The variable regions of both the heavy and light chains are known to contain three complementarity-determining regions (CDRs) adjacent to four framework regions (FRs) that are relatively conserved in a given species and presumably provide a scaffold for the CDRs (which vary depending on the epitope in question and determine binding ability). When non-human antibodies are prepared against a particular epitope, the variable regions can be "reshaped" or "humanized" by transplanting the CDRs derived from the non-human antibody into the FRs present in the human antibody to be modified. The application of this approach to various antibodies has been reported by Sato et al., Cancer Res. 53:851-856, 1993, Riechmann et al., Nature 332:323-327, 1988, Verhoeyen et al., Science 239:1534-1536, 1988, Kettleborough et al., Protein Engineering. 4:773-3783, 1991, Maeda et al., Human Antibodies Hybridoma 2:124-134, 1991, Gorman et al., PNAS USA. 88:4181-4185, 1991, Tempest et al., Bio / Technology 9:266-271, 1991, Co et al., PNAS USA. 88:2869-2873, 1991, Carter et al., PNAS USA. 89:4285-4289, 1992, and Co et al., J Immunol. 148:1149-1154, 1992. In some embodiments, the humanized antibody preserves all of the CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (1, 2, 3, 4, 5, 6) that have been altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs of the original antibody.

[0293] In certain embodiments, the antibody can be a chimeric antibody. In this regard, a chimeric antibody consists of an antigen-binding fragment of an antibody that is operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain can be from a different Ig class than the parental antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain can consist of CH2 and CH3 domains from one or more of different Ig classes. As described above for humanized antibodies, the antigen-binding fragment of a chimeric antibody can contain only one or more (e.g., 1, 2, 3, 4, 5, 6 CDRs of the antibodies described herein) of the CDRs of the antibodies described herein, or can contain the entire variable domain (VL, VH, or both).

[0294] In some embodiments, the immunomodulatory agent is or includes a "ligand" of the target molecule, e.g., a native ligand. A "ligand" generally refers to a substance or molecule that forms a complex with a target molecule (e.g., a biomolecule) to achieve a biological purpose, and generally includes a "protein ligand" that generates a signal by binding to a site on the target molecule or target protein. Thus, certain agents are in fact protein ligands that bind to the target molecule and generate a signal. Also included are "modified ligands", e.g., pharmacokinetic modifying factors, e.g., protein ligands fused to an Fc region derived from an immunoglobulin.

[0295] In some embodiments, the immunomodulatory agent or inhibitor is an antisense agent. Thus, in some embodiments, the target proteins, target sequences, and / or target genes (e.g., S1PR, calcineurin, mTOR, IDO, IMPDH, cytokines and / or cytokine receptors, B cell receptors, kinases) described herein are targeted by any of a variety of antisense agents, including oligonucleotide-based agents or methods. The antisense agent or oligonucleotide typically comprises a nucleotide sequence that targets a region within the target sequence (e.g., is sufficiently complementary to the region or specifically hybridizes to the region), and optionally comprises one or more of the following: a region that includes or surrounds the AUG start codon of the mRNA (e.g., a region upstream of the start codon, a region downstream of the start codon, a region that includes the start codon), the 3' or 5' splice site of the pre-processed mRNA, a pyrimidine-rich or polypyrimidine tract upstream of the splice acceptor site, exon-intron boundaries, intron-exon boundaries, branch sites, exon splicing enhancer elements, 5' and 3' untranslated regions, and polyadenylation signal sequences.

[0296] In certain embodiments, the antisense agent is capable of effectively modifying the expression of the target gene (e.g., reducing expression, altering splicing) upon administration to a subject in need thereof or upon contact with cells, e.g., muscle cells. This requirement is typically met when the antisense agent has the ability to be actively taken up by mammalian cells (e.g., muscle cells) and, when taken up, forms a duplex with the target RNA at a Tm above about 45°C.

[0297] Certain "antisense agents" include "antisense oligonucleotides", "antisense oligomers", and "oligonucleotides", and refer to linear sequences of nucleotides or nucleotide analogs, where the nucleobases can hybridize to a target sequence in RNA by Watson-Crick base pairing to form an oligonucleotide:RNA heteroduplex within the target sequence. The terms "antisense oligonucleotide", "antisense oligomer", "oligomer", and "compound" may be used interchangeably and can refer to oligonucleotides. Cyclic subunits can be based on ribose or another pentose sugar, or in certain embodiments, a morpholino group (see the description of morpholino oligonucleotides below). Among other antisense agents well known in the art, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), tricyclo-DNA oligomers, tricyclo-phosphorothioate oligonucleotides, and 2'-O-methyl oligonucleotides are also contemplated.

[0298] In certain embodiments, a "target sequence" includes a region that contains or surrounds the AUG start codon of an mRNA (e.g., a region upstream of the start codon, a region downstream of the start codon, a region containing the start codon), a 3' or 5' splice site of a pre-processed mRNA, a branch point, or a 3' non-coding mRNA region such as a 3'-UTR or a polyadenylation signal. The target sequence can be within an exon or an intron. A target sequence for a splice site can include an mRNA sequence having its 5' end 1 to about 25 base pairs downstream of the normal splice acceptor junction in a pre-processed mRNA (pre-mRNA). Exemplary target sequences for a splice region are any region of a pre-processed mRNA that includes the splice site, or is completely contained within an exon coding sequence, or spans a splice acceptor site or donor site. An antisense agent is more generally said to be "targeting" a biologically relevant target when it targets a nucleic acid of interest in a manner described herein and well known in the art, e.g., when it specifically hybridizes to it or is complementary thereto. Other examples of target regions or target sequences are described herein.

[0299] The term "targeting sequence" is the sequence in an oligonucleotide that is complementary (and, in further meaning, substantially complementary) to a "target sequence" in an RNA. Only all or a part of the antisense agent's sequence can be complementary to the target sequence. For example, in an antisense agent having 20 to 30 bases, about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 can be a targeting sequence that is complementary to the target region. Typically, the target sequence is formed by contiguous bases in the oligonucleotide, but alternatively, it can be formed by non-contiguous sequences that, when placed together from opposite ends of the oligonucleotide, constitute a sequence that spans the target sequence.

[0300] The target array may have "substantial" or "substantially" complementarity to the targeted array and still be functional for the purposes of this disclosure, i.e., the targeted array can still be "complementary". Preferably, the oligonucleotides used in this disclosure have at most one mismatch with the target array out of 10 nucleotides, preferably at most one mismatch out of 20 nucleotides. Alternatively, the antisense oligonucleotides used have at least 90% sequence homology or identity, at least 95% sequence homology or identity, or at least 98% sequence homology or identity with an exemplary antisense targeted array.

[0301] (i) Modified backbone structures, such as backbones other than the standard phosphodiester linkages found in naturally occurring oligonucleotides and polynucleotides, and / or (ii) modified sugar moieties, such as oligonucleotides having a morpholino moiety rather than a ribose or deoxyribose moiety, are included in non-naturally occurring oligonucleotides or "oligonucleotide analogs". Oligonucleotide analogs support bases that can hydrogen bond to standard polynucleotide bases by Watson-Crick base pairing, and the analog backbone presents the bases in a manner that allows such hydrogen bonding between the oligonucleotide analog molecule and the bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA) in a sequence-specific manner. Specific examples of analogs include those having a phosphorus-containing backbone that is substantially uncharged.

[0302] A "nuclease-resistant" oligonucleotide refers to one whose backbone is substantially resistant to nuclease cleavage by normal extracellular and intracellular nucleases in the body (e.g., by exonucleases such as 3'-exonuclease, endonuclease, RNase H, etc.), whether in a non-hybridized or hybridized form. That is, under normal nuclease conditions in the body where the oligonucleotide is exposed, the oligonucleotide shows little or no nuclease cleavage. A "nuclease-resistant heteroduplex" refers to a heteroduplex formed by an antisense oligonucleotide binding to its complementary target such that the heteroduplex is substantially resistant to in vivo degradation by intracellular and extracellular nucleases that can cleave double-stranded RNA / RNA or RNA / DNA complexes. A "heteroduplex" refers to a double-strand between an antisense oligonucleotide and a complementary portion of a target RNA.

[0303] In certain embodiments, the antisense oligonucleotide is recognized as a substrate for active or facilitated transport across a cell membrane, such as a muscle cell membrane. The ability of an oligonucleotide to form a stable double-strand with a target RNA can also relate to other properties of the oligonucleotide backbone, including the length and degree of complementarity of the antisense oligonucleotide to the target, the ratio of G:C base pairs to A:T base pairs, and the position of any mismatched bases. The ability of an antisense oligonucleotide to resist cellular nucleases can promote survival and the ultimate delivery of the agent to the cytoplasm of the cell. Accordingly, certain embodiments include non-naturally occurring antisense oligonucleotides that are nuclease-resistant or substantially nuclease-resistant.

[0304] In certain embodiments, the antisense oligonucleotide comprises an unnatural chemical backbone selected from phosphoramidate or phosphorodiamidate morpholino oligonucleotides (PMOs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs), phosphorothioate oligonucleotides, tricyclo-DNA oligonucleotides, tricyclo-phosphorothioate oligonucleotides, 2’O-Me-modified oligonucleotides (e.g., 2’O-methyl phosphorothioate oligonucleotides), or any combination of the foregoing.

[0305] When the oligonucleotide hybridizes to the target under physiological conditions, the antisense oligonucleotide has a Tm of substantially greater than 40°C or 45°C, preferably at least 50°C, typically 60°C - 80°C or higher, and "specifically hybridizes" to the target sequence or polynucleotide (e.g., pre-mRNA, mRNA). Such hybridization preferably corresponds to stringent hybridization conditions. At a given ionic strength and pH, the Tm is the temperature at which 50% of the target sequence hybridizes to the complementary polynucleotide. Such hybridization can occur by "substantial" or "substantive" complementarity of the antisense oligonucleotide to the target sequence, as well as by exact complementarity.

[0306] As used herein, "sufficient length" refers to an antisense oligonucleotide that is complementary to at least 8, more typically 8 - 40, contiguous nucleobases in the target sequence or gene described herein. An antisense oligonucleotide of sufficient length has at least a minimum number of nucleotides so as to be able to hybridize to a region of the target sequence or gene. Preferably, an oligonucleotide of sufficient length is 8 - 30 nucleotides in length. More preferably, an oligonucleotide of sufficient length is 9 - 27 nucleotides in length.

[0307] Antisense oligonucleotides generally contain multiple nucleotide subunits, each of which, taken together, has nucleobases that form or include a target sequence. Thus, in some embodiments, the antisense oligonucleotide ranges in length from about 10 to about 40 subunits, or about 10 to 30 subunits, and typically from 15 to 25 subunits. For example, in some embodiments, the antisense oligonucleotide is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 subunits in length, or ranges from about 10 to 40 subunits, 10 to 30 subunits, 14 to 25 subunits, 15 to 30 subunits, 17 to 30 subunits, 17 to 27 subunits, 10 to 27 subunits, 10 to 25 subunits, and 10 to 20 subunits. In certain embodiments, the antisense oligonucleotide is about 10 to about 40 or about 5 to about 30 nucleotides in length. In some embodiments, the antisense oligonucleotide is about 14 to about 25 or about 17 to about 27 nucleotides in length.

[0308] In some embodiments, the backbone of the antisense oligonucleotide is substantially uncharged and optionally recognized as a substrate for active or facilitated transport across the cell membrane. In some embodiments, all internucleotide linkages are uncharged. The ability of an oligonucleotide to form a stable duplex with a target RNA can also relate to other properties of its backbone, including the length and degree of complementarity of the antisense oligonucleotide to the target, the ratio of G:C base pairs to A:T base pairs, and the position of any mismatched bases. The ability of an antisense oligonucleotide to resist cellular nucleases can promote survival and the ultimate delivery of the agent to the cytoplasm of the cell.

[0309] In certain embodiments, the antisense oligonucleotide has at least one internucleotide linkage that is positively charged or cationic at physiological pH. In some embodiments, the antisense oligonucleotide has at least one internucleotide linkage that exhibits a pKa of from about 5.5 to about 12. Optionally, the antisense oligonucleotide has at least one inter-nucleoside linkage that contains both a basic nitrogen and an alkyl, aryl, or aralkyl group. In certain embodiments, the cationic internucleoside linkage comprises a 4-aminopiperidin-1-yl (APN) group or a derivative thereof. Without being bound by any particular theory, the presence of a cationic linkage (e.g., an APN group or an APN derivative) in the oligonucleotide is thought to facilitate binding to the negatively charged phosphate within the target nucleotide. Thus, the formation of a heteroduplex between the mutant RNA and the cationic linkage-containing oligonucleotide may be held together by both ionic attraction and Watson-Crick base pairing.

[0310] In some embodiments, the number of cationic linkages is at least 2 and about half or less of all inter-nucleotide linkages, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 cationic linkages, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or fewer cationic linkages. In some embodiments, however, up to all inter-nucleotide linkages are cationic linkages, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 of all inter-nucleotide linkages, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 are cationic linkages. In certain embodiments, an oligonucleotide of about 19 - 20 subunits has 2 - 10, for example, 4 - 8 cationic linkages, and the remainder may be uncharged linkages. In other certain embodiments, an oligonucleotide of 14 - 15 subunits has 2 - 7, for example, 2, 3, 4, 5, 6, or 7 cationic linkages, and the remainder may be uncharged linkages. Thus, the total number of cationic linkages within an oligonucleotide can vary from about 1 - 10 - 15 - 20 - 30 or more (including all integers therebetween) and can be dispersed throughout the oligonucleotide.

[0311] In some embodiments, an antisense oligonucleotide can have about 1 or up to about 1 cationic linkage per every 2 - 5, or 2, 3, 4, or 5 uncharged linkages, for example, about 4 - 5 or 4 or 5 per every 10 uncharged linkages.

[0312] Certain embodiments include antisense oligonucleotides comprising 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% cationic linkages. In certain embodiments, optimal improvement in antisense activity can be seen when about 25% of the backbone linkages are cationic. In certain embodiments, enhancement can be seen with a minority, e.g., 10 - 20% cationic linkages, or when the number of cationic linkages is in the range of 50 - 80%, e.g., about 60%.

[0313] In some embodiments, the cationic linkages are scattered along the backbone. Such oligonucleotides optionally contain at least two consecutive uncharged linkages, i.e., the oligonucleotide optionally does not have a pattern that is strictly alternating throughout its length. In certain examples, each one or two cationic linkages are separated by at least 1, 2, 3, 4, or 5 uncharged linkages along the backbone.

[0314] Also included are oligonucleotides having blocks of cationic linkages and blocks of uncharged linkages. For example, a central block of uncharged linkages may be adjacent to a block of cationic linkages, or vice versa. In some embodiments, the oligonucleotide has 5', 3' and central regions of approximately equal length, and the percentage of cationic linkages in the central region is greater than about 50%, 60%, 70%, or 80% of the total number of cationic linkages.

[0315] In certain antisense oligonucleotides, a substantial amount of cationic linkages (e.g., 70, 75%, 80%, 90% of the cationic linkages) are distributed near the "central region" backbone linkage, e.g., at the 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 central linkages. For example, a 16, 17, 18, 19, 20, 21, 22, 23, or 24-mer oligonucleotide may have at least 50%, 60%, 70%, or 80% of all cationic linkages localized at the 8, 9, 10, 11, or 12 central linkages.

[0316] As noted above, antisense oligonucleotides can use a variety of antisense chemistries. Examples of oligonucleotide chemistries include, but are not limited to, peptide nucleic acid (PNA), locked nucleic acid (LNA), phosphorothioate, 2’O-Me-modified oligonucleotides, morpholino, PMO, PPMO, PMOplus, and PMO-X chemistries, including any combination of the foregoing. Generally, PNA and LNA chemistries can utilize shorter targeting sequences due to their relatively high target binding strength compared to PMO and 2’O-Me oligonucleotides. Phosphorothioate and 2’O-Me-modified chemistries are often combined to generate a 2’O-Me-phosphorothioate backbone. See, for example, PCT Publications WO / 2013 / 112053 and WO / 2009 / 008725, which are hereby incorporated by reference in their entireties.

[0317] Peptide nucleic acid (PNA) is an analogue of DNA that is structurally homologous to the deoxyribose backbone consisting of N-(2-aminoethyl)glycine units to which pyrimidine or purine bases are attached. PNA containing natural pyrimidine and purine bases hybridizes to complementary oligonucleotides according to the Watson-Crick base pairing rules and mimics DNA with respect to base pair recognition (Egholm, Buchardt et al. 1993). The backbone of PNA is formed by peptide bonds rather than phosphodiester bonds, making PNA well-suited for antisense applications (see the following structure). The backbone is uncharged, resulting in PNA / DNA or PNA / RNA duplexes that exhibit greater than normal thermal stability. PNA is not recognized by nucleases or proteases.

[0318] Despite radical structural changes to the native structure, PNA can bind sequence specifically to DNA or RNA in a helical form. The characteristics of PNA include high binding affinity to complementary DNA or RNA, destabilization effects caused by single-base mismatches, resistance to nucleases and proteases, hybridization with DNA or RNA independent of salt concentration and triple-strand formation with homopurine DNA. PANAGENE™ has developed the Bts PNA monomer (Bts; benzothiazole-2-sulfonyl group) and the oligomerization process. PNA oligomerization using the Bts PNA monomer consists of repetitive cycles of deprotection, coupling, and capping. PNA can be synthetically generated using any technique known in the art. See, for example, U.S. Patent Nos. 6,969,766, 7,211,668, 7,022,851, 7,125,994, 7,145,006, and 7,179,896. See also U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262 for the preparation of PNA. Further teachings of PNA compounds can be found in Nielsen et al., Science, 254:1497-1500, 1991. Each of the foregoing is hereby incorporated by reference in its entirety.

[0319] Antisense oligonucleotides may also contain "locked nucleic acid" subunits (LNA). "LNA" is a member of a class of modifications called bridged nucleic acids (BNA). BNA is characterized by a covalent bond that locks the conformation of the ribose ring of the C3'-endo (northern) sugar puckering. In LNA, the bridge consists of a methylene between the 2'-O position and the 4'-C position. LNA enhances backbone preorganization and base stacking, and increases hybridization and thermal stability. The structure of LNA can be found, for example, in Wengel, et al., Chemical Communications (1998) 455, Tetrahedron (1998) 54:3607, and Accounts of Chem. Research (1999) 32:301), Obika, et al., Tetrahedron Letters (1997) 38:8735; (1998) 39:5401, and Bioorganic Medicinal Chemistry (2008) 16:9230.

[0320] The compounds of the present disclosure may incorporate one or more LNAs, and in some cases, the compounds may consist entirely of LNAs. Methods for the synthesis of individual LNA nucleoside subunits and their incorporation into oligonucleotides are described, for example, in U.S. Patent Nos. 7,572,582, 7,569,575, 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and 6,670,461, each of which is incorporated herein by reference in its entirety. Typical inter-subunit linkers include phosphodiester and phosphorothioate moieties, and alternatively, non-phosphorus-containing linkers may be used. One embodiment is an LNA-containing compound, wherein each LNA subunit is separated by a DNA subunit. Certain compounds consist of alternating LNA and DNA subunits where the inter-subunit linker is a phosphorothioate.

[0321] "Phosphorothioate" (or S-oligo) is a variant of normal DNA in which one of the non-bridging oxygens is replaced by sulfur. Sulfurization of the internucleotide bond reduces the action of endonucleases and exonucleases, including 5'- to 3'- and 3'- to 5'- DNA POL 1 exonucleases, nuclease S1 and P1, RNases, serum nucleases, and snake venom phosphodiesterase. Phosphorothioates are made by two main routes: by the action of a solution of elemental sulfur in carbon disulfide on hydrogen phosphonate, or by sulfuring triester phosphite with either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD) (see, for example, Iyer et al., J. Org. Chem. 55, 4693-4699, 1990). The latter method avoids the problems of the insolubility of elemental sulfur in most organic solvents and the toxicity of carbon disulfide. The TETD method and the BDTD method also result in higher purity phosphorothioates.

[0322] Tricyclo-DNA (tc-DNA) is a class of constrained DNA analogs in which each nucleotide is modified by the introduction of a cyclopropane ring to limit the conformational flexibility of the backbone and to optimize the backbone configuration of the twist angle γ. Homobasic adenine- and thymine-containing tc-DNA forms very stable A-T base pairs with complementary RNA. Tricyclo-DNA and their synthesis are described in International Patent Application Publication No. WO2010 / 115993. The compounds of the present disclosure may incorporate one or more tricyclic-DNA nucleotides, and in some cases, the compounds may consist entirely of tricyclic-DNA nucleotides.

[0323] Tricyclo-phosphorothioate nucleotides are tricyclo-DNA nucleotides having phosphorothioate subunit linkages. Tricyclo-phosphorothioate nucleotides and their synthesis are described in International Patent Application Publication No. WO2013 / 053928. The compounds of the present disclosure may incorporate one or more tricyclic-DNA nucleotides and, in some cases, the compounds may consist entirely of tricyclic-DNA nucleotides.

[0324] "2’O-Me oligonucleotide" molecules carry a methyl group at the 2’-OH residue of the ribose molecule. 2’-O-Me-RNA exhibits the same (or similar) behavior as DNA but is protected from nuclease degradation. 2’-O-Me-RNA can also be combined with phosphorothioate oligonucleotides (PTO) to further stabilize it. 2’O-Me oligonucleotides (phosphodiester or phosphorothioate) can be synthesized according to routine techniques in the art (see, for example, Yoo et al., Nucleic Acids Res. 32:2008-16, 2004). In some cases, 2’O-Me oligonucleotides contain phosphorothioate linkages (2’O-Me phosphorothioate oligonucleotides).

[0325] "Morpholino oligonucleotide" or "PMO" refers to an oligonucleotide having a backbone that supports nucleobases capable of hydrogen bonding to a typical polynucleotide, where the polymer lacks a pentose sugar backbone moiety and instead contains a morpholino ring. Thus, in a PMO, the morpholino ring structure supports a base pairing moiety and typically forms a sequence of a base pairing moiety designed to hybridize to an antisense target selected in a cell or subject being treated. Exemplary "morpholino" oligonucleotides include a morpholino subunit structure linked together by phosphoramidate or phosphorodiamidate linkages that attach the morpholino nitrogen of one subunit to the 4'-exocyclic carbon of an adjacent subunit, with each subunit containing a purine or pyrimidine nucleobase effective to bind to a base in a polynucleotide by base-specific hydrogen bonding. Morpholino oligonucleotides (including antisense oligonucleotides) are described, for example, in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, 5,506,337, and co-pending U.S. Patent Applications Nos. 12 / 271,036, 12 / 271,040, as well as PCT Publications WO / 2009 / 064471 and WO / 2012 / 043730, all of which are hereby incorporated by reference in their entirety.

[0326] Within an oligonucleotide structure, the phosphate groups are generally referred to as forming the "internucleoside linkages" of the oligonucleotide. The naturally occurring internucleoside linkages of RNA and DNA are 3'-to-5' phosphodiester linkages. A "phosphoramidate" group contains phosphorus with three attached oxygen atoms and one attached nitrogen atom, while a "phosphorodiamidate" group contains phosphorus with two attached oxygen atoms and two attached nitrogen atoms. In the uncharged or cationic subunit linkages of PMO and / or PMO-X oligonucleotides described herein, one nitrogen is always pendant to the backbone chain. The second nitrogen in a phosphorodiamidate linkage is typically the ring nitrogen in the morpholino ring structure.

[0327] "PMO-X" refers to a phosphorodiamidate morpholino oligonucleotide (PMO) having a phosphorus atom with a (i) covalent bond to a nitrogen atom of a morpholino ring and a (ii) second covalent bond to a ring nitrogen of 4-aminopiperidin-1-yl (i.e., APN) or a derivative of 4-aminopiperidin-1-yl. Exemplary PMO-X oligonucleotides are disclosed in PCT Application No. PCT / US2011 / 38459 and PCT Publication No. WO / 2013 / 074834, each of which is hereby incorporated by reference in its entirety. "PMO-apn" or "APN" refers to a PMO-X oligonucleotide comprising at least one internucleotide linkage in which the phosphorus atom is linked to the ring nitrogen of a morpholino group and 4-aminopiperidin-1-yl (i.e., APN). In certain embodiments, the antisense oligonucleotides comprising the targeting sequences described herein comprise at least one APN-containing linkage or APN-derivative-containing linkage. Certain embodiments include PMOs having from about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% APN / APN-derivative-containing linkages, with the remaining linkages (if less than 100%) being uncharged linkages, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 of all internucleotide linkages being APN / APN-derivative-containing linkages.

[0328] Additional antisense oligonucleotides / chemicals that can be used in accordance with the methods and compositions provided herein include those described in the following patents and patent applications, the contents of which are incorporated herein by reference: PCT Publication Nos. WO / 2007 / 002390, WO / 2010 / 120820, and WO / 2010 / 148249, U.S. Patent No. 7,838,657, and U.S. Patent Application No. 2011 / 0269820.

[0329] In some embodiments, the immunomodulatory agent or inhibitor is an RNA interference (RNAi) agent. Thus, in some embodiments, the target proteins, target sequences, and / or target genes (e.g., S1PR, calcineurin, mTOR, IDO, IMPDH, cytokines and / or cytokine receptors, B cell receptors, kinases) described herein are targeted by any of a variety of RNA-based agents or methods. RNA interference (RNAi) is an evolutionarily conserved gene silencing mechanism originally discovered in studies of the nematode Caenorhabditis elegans (Lee et al, Cell 75:843, 1993; Reinhart et al., Nature 403:901, 2000). Introduction of dsRNA into cells expressing the appropriate molecular machinery triggers RNA interference, which degrades the corresponding endogenous mRNA. This mechanism involves conversion of dsRNA into short RNAs that direct ribonucleases to homologous mRNA targets (see Ruvkun, Science 2294:797, 2001).

[0330] In certain embodiments, an RNA agent is said to "target" a biologically relevant target (gene) more generally when it comprises a sense strand corresponding to the "target sequence" of a target gene, as described herein and by methods well known in the art.

[0331] RNAi agents include RNAi nucleic acid molecules and RNAi nucleic acid analog molecules, such as small interfering nucleic acids and small interfering nucleic acid analogs (siNA), which include small interfering RNAs and small interfering RNA nucleic acid analogs (siRNA), for example, double-stranded RNAs and double-stranded RNA analogs (dsRNA), micro-RNAs and micro-RNA analogs (miRNA), as well as small hairpin RNAs and small hairpin RNA analogs (shRNA).

[0332] Certain embodiments use double-stranded ribonucleic acid (dsRNA) molecules as RNAi agents. dsRNA generally comprises two single strands. One strand of the dsRNA (the "sense" strand) comprises a nucleotide sequence that is substantially identical to a portion of the target gene or target sequence, and the other strand (the "complementary" or "antisense" strand) comprises a sequence that is complementary or substantially complementary to a portion of the target region. Substantially identical sequences include those that are at least about 80, 85, 90, 95, 97, 98, 99% identical to the target sequence. These strands are complementary enough to hybridize and form a duplex structure. In certain embodiments, the complementary RNA strand can be less than 30 nucleotides, less than 25 nucleotides in length, or more specifically 19-24 nucleotides in length. In certain aspects, the complementary nucleotide sequence can be 20-23 nucleotides in length, or 22 nucleotides in length. In certain embodiments, the sense strand of the RNAi agent is substantially identical to a portion of the target sequence described herein. In some embodiments, this antisense strand is complementary or substantially complementary to a portion of the target sequence described herein. In certain embodiments, the portion comprises, consists of, or consists essentially of about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or fewer adjacent nucleotides of the target sequence described herein.

[0333] Suitable siRNA sequences can be identified using any means known in the art. In some cases, using the exemplary target sequences described herein, the methods described in Elbashir et al., Nature, 411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) are combined with the rational design rules described in Reynolds et al., Nature Biotech., 22:326-330 (2004).

[0334] Generally, the 3' nucleotide sequence of the transcript from the target gene's AUG start codon can be scanned for dinucleotide sequences (e.g., AA, NA, CC, GG, or UU, where N is C, G, or U) (see, e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). The nucleotide immediately 3' of the dinucleotide sequence is identified as the potential siRNA sequence (i.e., the target sequence or sense strand sequence). In some cases, 19, 21, 23, 25, 27, 29, 31, 33, 35 or more nucleotides immediately 3' of the dinucleotide sequence are identified as potential siRNA sequences. In some embodiments, the dinucleotide sequence is an AA or NA sequence, and 19 nucleotides immediately 3' of the AA or NA dinucleotide are identified as the potential siRNA sequence. siRNA sequences are usually spaced at different positions along the length of the target gene. To further enhance the silencing efficiency of the siRNA sequence, potential siRNA sequences can be analyzed to identify, for example, sites in the target cell or organism that do not contain regions of homology with other coding sequences. For example, a suitable siRNA sequence of about 21 base pairs will typically not have more than 16-17 contiguous base pairs of homology with coding sequences in the target cell or organism. When the siRNA sequence is to be expressed from an RNA Pol III promoter, an siRNA sequence lacking more than 4 contiguous A's or T's is selected.

[0335] Once potential siRNA sequences are identified, the sequences can be analyzed using various criteria known in the art. For example, to enhance their silencing efficiency, siRNA sequences are analyzed by rational design algorithms for the following characteristics: (1) a G / C content of about 25% to about 60% G / C, (2) at least three A / U at positions 15 to 19 of the sense strand, (3) no internal repeats, (4) an A at position 19 of the sense strand, (5) an A at position 3 of the sense strand, (6) a U at position 10 of the sense strand, (7) no G / C at position 19 of the sense strand, and (8) no G at position 13 of the sense strand, and sequences having one or more of these characteristics can be identified. siRNA design tools that incorporate algorithms that specify appropriate values for each of these characteristics and are useful for the selection of siRNAs are known. Those skilled in the art will appreciate that sequences having one or more of the foregoing features can be selected as potential siRNA sequences for further analysis and testing.

[0336] Furthermore, potential siRNA target sequences having one or more of the following criteria can often be excluded as siRNAs: (1) sequences containing stretches of four or more identical bases in a row, (2) sequences containing G homopolymers (i.e., to reduce the likelihood of non-specific effects due to the structural features of these polymers), (3) sequences containing triple-base motifs (e.g., GGG, CCC, AAA, or TTT), (4) sequences containing stretches of seven or more G / C in a row, and (5) sequences containing tandem repeats of four or more bases in candidates that result in internal folded structures. However, those skilled in the art will appreciate that sequences having one or more of the foregoing features can still be selected for further analysis and testing as potential siRNA sequences.

[0337] In some embodiments, potential siRNA target sequences can be further analyzed based on, for example, the asymmetry of the siRNA duplex as described in Khvorova et al., Cell, 115:209-216 (2003), and Schwarz et al., Cell, 115:199-208 (2003). In certain embodiments, potential siRNA target sequences can be further analyzed based on, for example, the secondary structure at the mRNA target site as described in Luo et al., Biophys. Res. Commun., 318:303-310 (2004). For example, the secondary structure in the mRNA can be modeled using the Mfold algorithm to select siRNA sequences that are favorable for accessibility at mRNA target sites with less secondary structure in the form of base pairing and stem-loops.

[0338] Potential siRNA sequences can also be analyzed for the presence of any immunostimulatory properties, for example, using in vitro cytokine assays or in vivo animal models. Motifs in the sense and / or antisense strands of siRNA sequences such as GU-rich motifs (e.g., 5'-GU-3', 5'-UGU-3', 5'-GUGU-3', 5'-UGUGU-3', etc.) can also provide an indication as to whether the sequence is immunostimulatory. If an siRNA molecule is found to be immunostimulatory, it can then be modified to reduce its immunostimulatory properties as described herein. As a non-limiting example, under conditions such that the cells elicit a detectable immune response, the siRNA sequence can be contacted with mammalian responder cells to determine whether the siRNA is immunostimulatory or non-immunostimulatory. The mammalian responder cells can be from naive mammals (i.e., mammals that have not previously been exposed to the gene product of the siRNA sequence). The mammalian responder cells can be, for example, peripheral blood mononuclear cells (PBMCs), macrophages, etc. Detectable immune responses can include, for example, the production of cytokines or growth factors such as TNF-alpha, IFN-alpha, IFN-beta, IFN-gamma, IL-6, IL-12, or combinations thereof. An siRNA molecule identified as being immunostimulatory can then be modified to reduce its immunostimulatory properties by replacing at least one of the nucleotides in the sense strand and / or the antisense strand with a modified nucleotide. For example, less than about 30% (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) of the nucleotides within the duplex region of the siRNA duplex can be replaced with a modified nucleotide such as a 2'-OMe nucleotide. The modified siRNA can then be contacted with mammalian responder cells as described above to confirm that its immunostimulatory properties have been reduced or abrogated.

[0339] RNAi agents typically include a double-stranded portion (regardless of the presence of any and potentially preferred single-stranded overhangs), where the silencing is desired and thus the mRNA targeted by the RNAi agent is identical or nearly identical (e.g., showing 90% or more, e.g., at least 95% sequence identity, or showing a maximum of two and optionally only one mismatch) to the mRNA, and includes at least 16 bases, optionally at least 17 bases, more optionally at least 18 bases, even more optionally at least 19 bases, and typically 18 - 35 bases, optionally 19 - 30 bases, more optionally 20 - 25 bases, and even more optionally 21 - 23 bases.

[0340] In certain embodiments, at least one of the RNA strands includes a nucleotide overhang that is 1 - 4 nucleotides in length. In some embodiments, the dsRNA includes at least one chemically modified nucleotide. In certain aspects, a dsRNA including a single-stranded overhang of 1 - 4 nucleotides can include molecules where the unpaired nucleotide of the single-stranded overhang that is directly adjacent to the terminal nucleotide pair includes a purine base. In some embodiments, the last complementary nucleotide pair at both ends of the dsRNA is a G - C pair, or at least two of the last four terminal nucleotide pairs are G - C pairs.

[0341] In certain embodiments, the RNAi agent comprises a microRNA. MicroRNAs represent a large group of small RNAs that are naturally produced in organisms, and some of these regulate the expression of target genes. MicroRNAs are formed from approximately 70 nucleotide single-stranded hairpin precursor transcripts by Dicer. (See V. Ambros et al., Current Biology 13:807, 2003). MicroRNAs are not translated into protein; rather, they bind to specific messenger RNAs and thereby block translation. MicroRNAs are thought to inhibit translation by binding imperfectly to the target. Certain microRNAs can be transcribed as hairpin RNA precursors and processed to their mature form by the Dicer enzyme.

[0342] In certain embodiments, the RNAi agent or oligonucleotide is single-stranded. In some embodiments, the RNAi agent or oligonucleotide is double-stranded. Certain embodiments include small interfering RNAs (siRNAs). In certain embodiments, the first strand of the double-stranded oligonucleotide contains two or more nucleoside residues more than the second strand. In other embodiments, the first and second strands have the same number of nucleosides, but the first and second strands are offset such that the two terminal nucleosides on the first and second strands do not pair with residues on the complementary strand. In certain cases, the two unpaired nucleosides are thymidine residues.

[0343] In some cases where the modulator comprises siRNA, the agent comprises a region of sufficient homology to the target region and is of sufficient length with respect to nucleotides such that the siRNA agent or a fragment thereof is capable of mediating down-regulation of the target gene or RNA. It will be understood that the term "ribonucleotide" or "nucleotide" can refer to modified nucleotides or surrogate replacement moieties at one or more positions in the case of modified RNA or nucleotide surrogates. Thus, the siRNA agent is or comprises a region that is at least partially complementary to the target sequence. There need not be perfect complementarity between the siRNA agent and the target sequence, but the correspondence must be sufficient, for example, to enable the siRNA agent or a cleavage product thereof to effect sequence-specific silencing, for example, by RNAi cleavage of the target RNA. Complementarity or degree of homology to the target strand is most important in the antisense strand. Perfect complementarity is often desired, particularly in the antisense strand, although some embodiments include one or more, but preferably 10, 8, 6, 5, 4, 3, 2 or fewer mismatches to the target sequence. Mismatches are most tolerated in the terminal regions and, when present, are preferably within the terminal regions, for example, within 6, 5, 4, or 3 nucleotides of the 5' and / or 3' termini. The sense strand need only be sufficiently complementary to the antisense strand to maintain the overall duplex character of the molecule.

[0344] In some embodiments, the RNAi agent or oligonucleotide, e.g., siRNA oligonucleotide, is modified or contains nucleoside surrogates. The single-stranded regions of the siRNA agent may be modified or contain nucleoside surrogates, e.g., the unpaired regions of the hairpin structure, e.g., the region linking two complementary regions, may have modifications or nucleoside surrogates. For example, modifications for stabilizing one or more 3’ or 5’ ends of the siRNA agent against exonucleases or for facilitating entry of the antisense siRNA agent into RISC are also included. Exemplary modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), and special biotin or fluorescein reagents that occur as phosphoramidites and have another DMT-protected hydroxyl group to allow multiple couplings during RNA synthesis.

[0345] Certain siRNA agents include, for example, molecules that are long enough to cause an interferon response (cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363 - 366) and capable of entering the RISC (RNAi - induced silencing complex)). Also included are molecules that are not long enough to cause an interferon response (which can also be cleaved by Dicer and / or enter the RISC), for example, molecules of a size that allows entry into the RISC, such as molecules similar to Dicer cleavage products. Molecules that are short enough not to cause an interferon response are herein referred to as siRNA agents or shorter RNAi agents. The "siRNA agent or shorter RNAi agent" used refers to an siRNA agent that is short enough not to induce a harmful interferon response in human cells, for example, it has a double - stranded region of less than 60, preferably less than 50, 40, or 30 nucleotide pairs. The siRNA regulator or its cleavage products can down - regulate a target gene, for example, by inducing RNAi against the target RNA.

[0346] In some cases, each strand of the siRNA agent is about 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 or less than about 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides in length. For example, each strand can be about 21 - 25 nucleotides in length. Certain siRNA agents have a double - stranded region of about 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, for example, one or two 3' overhangs of 1 - 3 nucleotides.

[0347] In addition to homology to the target RNA and the ability to down-regulate the target gene, the siRNA agent may have one or more of the following characteristics: it can present bases (or modified bases) in a suitable three-dimensional framework such that, despite being modified for a very large number or even all of the nucleosides, accurate base pairing and the formation of a duplex structure with a homologous target RNA sufficient to allow down-regulation of the target, for example, by cleavage of the target RNA, can occur, and it may have an antisense strand that, despite being modified for a very large number or even all of the nucleosides, still has "RNA-like" properties, i.e., it can have the overall structural, chemical, and physical properties of an RNA molecule, not limited only to ribonucleotide-based content or even if there is only a partial ribonucleotide-based content. For example, the siRNA agent can contain a sense and / or antisense strand in which all nucleotide sugars contain, for example, 2'-fluoro instead of 2'-hydroxyl. This deoxyribonucleotide-containing agent may still be expected to exhibit RNA-like properties. Without wishing to be bound by theory, electronegative fluorine, when attached to the C2' position of ribose, prefers an axial orientation. This spatial preference of fluorine can similarly force the sugar to take a C3' end-packer. This is the same packing pattern observed in RNA molecules and results in an A-family type helix characteristic of RNA. Furthermore, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bond interactions as the water molecules known to stabilize the RNA structure. Generally, the modified moiety at the 2'-sugar position can enter into H-bonds that are more characteristic of the OH moiety of ribonucleotides than the H moiety of deoxyribonucleotides.

[0348] As used herein, the “single-stranded RNAi agent” is an RNAi agent composed of a single molecule. The single-stranded RNAi agent may contain a double-stranded region formed by intrastrand pairing. For example, the single-stranded RNAi agent can be or contain a hairpin structure or a pan-handle structure. The agent that regulates single-stranded RNAi is preferably antisense with respect to the target molecule. The single-stranded RNAi agent may be long enough to enter RISC and be involved in the cleavage of target mRNA via RISC. The single-stranded RNAi agent has a length of at least 14 nucleotides, more preferably at least 15, 20, 25, 29, 35, 40, or 50 nucleotides. It preferably has a length of less than 200, 100, or 60 nucleotides.

[0349] The hairpin RNAi agent can be equal to or have a double-stranded region of at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The double-stranded region can preferably be equal to or less than 200, 100, or 50 in length. Certain ranges of the double-stranded region are 15 - 30, 17 - 23, 19 - 23, and 19 - 21 nucleotide pairs. The hairpin can preferably have a single-stranded overhang or a terminal unpaired region at the 3' end and preferably on the antisense side of the hairpin. In certain embodiments, the overhang is 2 - 3 nucleotides in length.

[0350] Certain modulators used in accordance with the methods provided herein include RNAi oligonucleotides such as chimeric oligonucleotides or "chimeras" that contain two or more chemically distinct regions, each of which consists of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region that is modified to enhance resistance to nuclease degradation, enhance cellular uptake, and / or enhance binding affinity to a target nucleic acid. As a result, when using chimeric oligonucleotides, results similar to those obtained using shorter oligonucleotides as compared to phosphorothioate oligonucleotides can often be obtained. Chimeric oligonucleotides can be formed as a complex structure consisting of two or more oligonucleotides, modified oligonucleotides, oligonucleotides, and / or oligonucleotide mimetics, as described above. Such oligonucleotides are also referred to in the art as hybrids or gapmers. Representative U.S. patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Patent Nos. 5,013,830, 5,149,797, 5,220,007, 5,256,775, 5,366,878, 5,403,711, 5,491,133, 5,565,350, 5,623,065, 5,652,355, 5,652,356, 5,700,922, and 5,955,589, each of which is incorporated herein by reference. In certain embodiments, the chimeric oligonucleotide is RNA-DNA, DNA-RNA, RNA-DNA-RNA, DNA-RNA-DNA, or RNA-DNA-RNA-DNA, where the oligonucleotide is 5 to 60 nucleotides in length.

[0351] In some embodiments, the RNAi agent comprises an oligonucleotide comprising at least one ligand linked to a modified or unnatural nucleobase. A number of compounds can function as modified bases. The structure of the modified base is important to the extent that the modified base should not substantially interfere with the binding of the oligonucleotide to its target, e.g., mRNA. In certain embodiments, the modified base is a divalent radical of any one of difluorotolyl, nitropyrrolyl, nitroimidazolyl, nitroindolyl, naphthalenyl, anthracenyl, pyridinyl, quinolinyl, pyrenyl, or an unnatural nucleobase described herein. In certain embodiments, the unnatural nucleobase is difluorotolyl, nitropyrrolyl, or nitroimidazolyl. In certain embodiments, the unnatural nucleobase is difluorotolyl. A wide variety of ligands are well known in the art. For example, the ligand can be a steroid, bile acid, lipid, folic acid, pyridoxal, B12, riboflavin, biotin, aromatic compound, polycyclic compound, crown ether, intercalator, cleaving agent molecule, protein binder, or carbohydrate. In certain embodiments, the ligand is a steroid or an aromatic compound. In certain instances, the ligand is cholesteryl.

[0352] In some embodiments, the RNAi agent is an oligonucleotide linked to a ligand for the purpose of improving cellular targeting and uptake. For example, the RNAi agent can be linked to an antibody or an antigen-binding fragment thereof. As a further example, the RNAi agent can be linked to a specific ligand-binding molecule, such as a polypeptide or polypeptide fragment, that specifically binds to a particular cell surface receptor.

[0353] In certain embodiments, the RNAi agent comprises unnatural nucleobases. In some embodiments, the unnatural nucleobases are difluorotolyl, nitroimidazolyl, nitroindolyl, or nitropyrrolyl. In certain embodiments, the modulator provided to the aromatic compound relates to a double-stranded oligonucleotide sequence, and only one of the strands of the double-stranded contains unnatural nucleobases. In certain embodiments, the modulator used herein relates to a double-stranded oligonucleotide sequence, and both of these strands independently contain at least one unnatural nucleobase.

[0354] In certain cases, the ribose sugar moiety naturally present in the nucleoside is replaced with a hexose sugar. In certain embodiments, the hexose sugar is allose, altrose, glucose, mannose, gulose, idose, galactose, talose, or a derivative thereof. In preferred embodiments, the hexose is a D-hexose. In certain cases, the ribose sugar moiety naturally present in the nucleoside is replaced with a polycyclic heteroalkyl ring or a cyclohexenyl group. In certain cases, the polycyclic heteroalkyl group is a bicyclic ring containing one oxygen atom in the ring. In certain cases, the polycyclic heteroalkyl group is bicyclo[2.2.1]heptane, bicyclo[3.2.1]octane, or bicyclo[3.3.1]nonane. In certain embodiments, the backbone of the oligonucleotide is modified to improve the therapeutic or diagnostic properties of the oligonucleotide compound. In certain embodiments, at least one of the bases or at least one of the sugars of the oligonucleotide is modified to improve the therapeutic or diagnostic properties of the oligonucleotide compound. When the oligonucleotide is double-stranded, in certain cases, the two strands are complementary, partially complementary, or chimeric oligonucleotides.

[0355] Examples of modified RNAi agents include oligonucleotides that contain a modified backbone or unnatural internucleotide linkages. As defined herein, oligonucleotides having a modified backbone or unnatural internucleotide linkages include those that retain a phosphorus atom within the backbone and those that do not retain a phosphorus atom within the backbone. Modified oligonucleotides that do not have a phosphorus atom within their sugar backbone can also be considered oligonucleotides. Specific oligonucleotide chemical modifications are described below. It is not necessary for all positions within a given compound to be uniformly modified, and in fact, one or more of the following modifications can be incorporated even in a single oligonucleotide compound, or even in a single nucleotide thereof.

[0356] Examples of modified internucleotide linkages or backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates (those having a normal 3'-5' linkage, 2'-5'-linked analogs thereof, as well as those with reversed polarity, where the adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' and 2'-5' to 5'-2'). Various salts, mixed salts, and free acid forms are also included.

[0357] Representative U.S. patents that teach the preparation of the above phosphorus atom-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, 5,625,050, and 5,697,248, each of which is incorporated herein by reference.

[0358] Examples of modified internucleotide linkages or backbones (i.e., oligonucleotides) that do not contain a phosphorus atom therein have a backbone formed by short chain alkyl or cycloalkyl sugar linkages, sugar linkages incorporating heteroatoms and alkyl or cycloalkyl, or one or more short chain heteroatom or heterocyclic sugar linkages. These include morpholino linkages (partially formed from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones, methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones incorporating components of N, O, S, and CH 2 and the like.

[0359] Representative U.S. patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, each of which is incorporated herein by reference.

[0360] In other examples of oligonucleotide mimetics, both the sugar and the internucleoside linkages, i.e., the backbone of the nucleoside units, can be replaced with other groups. The nucleoside units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligonucleotide mimetic, which has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone, specifically, an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly attached to the atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, each of which is incorporated herein by reference. Further teachings of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497.

[0361] Also included are oligonucleotides using ribozymes. Synthetic RNA molecules and their derivatives that catalyze highly specific endoribonuclease activity are known as ribozymes. (See generally, U.S. Patent No. 5,543,508 by Haseloff et al. and U.S. Patent No. 5,545,729 by Goodchild et al.). The cleavage reaction is catalyzed by the RNA molecule itself. In naturally occurring RNA molecules, the self-cleaving site is present within a highly conserved region of the RNA secondary structure (Buzayan et al., PNAS USA. 83:8859, 1986). Modify naturally occurring autocatalytic RNA molecules to generate ribozymes that can target specific cellular or pathogenic RNA molecules with a high degree of specificity. Thus, ribozymes serve the same general purpose as antisense oligonucleotides (i.e., regulation of the expression of a specific gene) and, like oligonucleotides, are nucleic acids having a substantial portion in single-stranded, chain form. That is, ribozymes have a substantial chemical and functional identity with oligonucleotides and are therefore considered equivalents for the purposes described herein.

[0362] In certain cases, the RNAi agent can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to enhance the activity, cellular distribution, cell targeting, or cellular uptake of oligonucleotides, and procedures for performing such conjugation are available from the literature.Examples of non-ligand moieties include lipid moieties such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306, Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocolesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111, Kabanov et al., FEBS Lett., 1990, 259:327, Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651, Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).Typical conjugation protocols involve synthesizing oligonucleotides having amino linkers at one or more positions in the array. The amino groups are then reacted with the conjugated molecule using an appropriate coupling or activating reagent. The conjugation reaction can be carried out either while the oligonucleotide remains attached to the solid support or after cleaving the oligonucleotide into the liquid phase. Purification of the oligonucleotide conjugate by HPLC typically yields a pure conjugate.

[0363] Certain exemplary RNAi agents provide delivery in a vector. The term "vector" generally refers to a nucleic acid molecule, typically DNA, into which a nucleic acid segment can be inserted and cloned, i.e., propagated. Thus, a vector typically contains one or more unique restriction enzyme recognition sites and can be self-replicating in a defined host or vehicle organism because the cloned sequence is reproducible. Vectors can include plasmids, phagemids, bacteriophages, bacteriophage-derived vectors, PACs, BACs, linear nucleic acids such as linear DNA, viral vectors, and the like. Expression vectors are generally constructed to enable and / or effect the expression of a nucleic acid or ORF introduced therein in a desired expression system, e.g., in vitro, in a host cell, host organ, and / or host organism. For example, an expression vector may advantageously contain suitable regulatory sequences.

[0364] Exemplary vectors for use herein include viral vectors, which are known and include, for example, without limitation, vectors derived from retroviruses, vaccinia virus, pox virus, adenovirus, adeno-associated virus (AAV). Such viral vectors can be engineered by recombinant techniques known per se to introduce herein a nucleic acid sequence encoding any one of the antisense or RNAi agents disclosed herein.

[0365] For example, retroviral vectors can be used herein to deliver RNAi agents. Generally, a retroviral vector can contain a retroviral genomic sequence encoding the components necessary for (random) integration of a recombinant viral genome into a target host cell genome and nucleic acid sequence, e.g., in particular, a nucleic acid sequence encoding one of the antisense or RNAi agents disclosed herein. Such retroviral vectors can be readily constructed from various retroviruses using standard recombinant techniques, e.g., including retroviruses of types B, C, and D, as well as spumaviruses and lentiviruses (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989) (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985).

[0366] Recombinant adenoviral vectors may also be contemplated for the delivery and expression of RNAi agents, as disclosed herein, in host cells. Adenovirus-based viral vectors have the advantage of being able to infect non-dividing host cells, but the recombinant viral genome is not integrated into the host cell genome. For example, suitable adenoviral vectors, methods for constructing such recombinant adenoviral vectors, and methods for delivering recombinant vectors to host cells are described in Xia H et al. (2002) (Nat. Biotech. 20:1006-1010). The use of recombinant AAV (rAAV) vectors is also contemplated herein. rAAV vectors can infect both dividing and non-dividing cells and may integrate their recombinant viral genome into the host's recombinant viral genome. rAAV vectors can be generated from a variety of adeno-associated viruses, including, for example, serotypes 1-6. Generally, an rAAV vector can include, in order, a 5' adeno-associated virus inverted terminal repeat (ITR), a nucleic acid of interest, for example, a nucleic acid sequence encoding any one of the antisense or RNAi agents disclosed herein, operably linked to a sequence that regulates its expression in a host cell or host organism, and a 3' adeno-associated virus ITR. In addition, the rAAV vector may preferably have a polyadenylation signal. Suitable rAAV vectors are described, inter alia, in WO1994 / 13788, WO1993 / 24641, Goyenvalle et al. 2004 (Science 306:1796-1799), where the antisense sequence is linked to a modified U7 small nuclear RNA.

[0367] Other exemplary viral vectors for use herein are poxviruses such as vaccinia virus, for example, attenuated vaccinia virus, such as modified vaccinia virus Ankara (MVA) or NYVAC, avipoxviruses, for example, vectors derived from fowlpox virus or canarypox virus.

[0368] Further examples of modulators such as RNAi oligonucleotides, including siRNA oligonucleotides, can be found in U.S. Patent Application Publication Nos. 2009 / 0312531, 2009 / 0318676, 2011 / 0117125, 2011 / 0269814, 2007 / 0275465, 2007 / 0054279, 2006 / 0287260, 2006 / 0035254, and 2006 / 0008822, which are incorporated by reference.

[0369] Method of Use Certain embodiments include the use of the HRS polypeptide / expressible polynucleotide and compositions described herein, alone or in combination with an immunotherapeutic agent, to treat lung inflammation. Treatment of interstitial lung disease (ILD) and related disorders is also included. In some embodiments, the HRS polypeptide / expressible polynucleotide and compositions, methods, and / or combination therapies are used to reduce lung inflammation, treat one or more ILDs, and / or improve clinical symptoms or parameters of the disease in a subject in need of treatment of lung inflammation.

[0370] Accordingly, some embodiments are methods of doing so in a subject in need of treatment of lung inflammation, the method comprising administering to the subject a histidyl-tRNA synthetase (HRS) polypeptide (e.g., an HRS-Fc fusion polypeptide), or an expressible polynucleotide encoding an HRS polypeptide.

[0371] A particular embodiment is a method of doing so in a subject in need of treatment of lung inflammation, the method comprising administering to the subject (a) a histidyl-tRNA synthetase (HRS) polypeptide or an expressible polynucleotide encoding an HRS polypeptide, and (b) an immunomodulatory agent, such as described herein. In some embodiments, (a) and (b) are administered separately and optionally as defined herein. In a particular embodiment, (a) and (b) are administered together, optionally as a therapeutic composition described herein.

[0372] In certain embodiments, the immunomodulatory agent is pirfenidone or nintedanib. In some methods or compositions, pirfenidone is in individual dosage units in the range of about 50 to about 1000 mg, or about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg, about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg or less, or at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg in individual dosage units, for example, administered in about one, two, or three capsules for oral administration.,

[0373] In some embodiments, pirfenidone is in a daily dosage unit in the range of about 100 to about 4000 mg / day, or about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day, about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500,2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day or less, or at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day in a daily dosage unit, for example, administered in about 1, 2, 3, 4, 5, 6, 7, 8, 9 capsules for oral administration.,

[0374] In certain embodiments, pirfenidone is taken as about 800 mg (e.g., 801 mg), for example, administered as 3 capsules of about 267 mg each for individual dosages, or pirfenidone is administered in a daily dosage unit of about 2400 mg / day (e.g., 2403 mg / day), for example, taken as 3 capsules for individual dosages, and administered as 9 capsules of about 267 mg each for three times a day oral administration.,

[0375] In some methods or compositions, nintedanib is administered in individual dosage units that range from about 10 to about 500 mg, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg or less, or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg of individual dosage units, for example, in about one, two, or three capsules.

[0376] In some embodiments, nintedanib is in a daily dosage unit in the range of about 20 to about 1000 mg / day, or about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg / day, about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mg / day or less, or at least about 20, 30, 40, 50, 60, 70, 80, 90,...

Claims

1. (a) a histidyl-tRNA synthetase (HRS)-Fc fusion polypeptide, or an expressible polynucleotide encoding said HRS-Fc fusion polypeptide, wherein said HRS-Fc fusion polypeptide comprises an amino acid sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 157, 158, and 163-166; and (b) pirfenidone, which increases the serum concentration of the HRS-Fc fusion polypeptide compared to the HRS-Fc fusion polypeptide alone; 23. A therapeutic composition for use in treating pulmonary inflammation in a subject in need thereof, comprising:

2. The HRS-Fc fusion polypeptide has the structure of SEQ ID NO: 157 (Fc-HRS(2-60) or HRS ＦＣ１ 2. The therapeutic composition of claim 1, comprising, consisting of, or consisting essentially of

3. The therapeutic composition of any one of claims 1 to 2, wherein the HRS-Fc fusion polypeptide is at least about 80%, 85%, 90%, or 95% pure on a protein basis and is less than about 5% aggregated.

4. The therapeutic composition of any one of claims 1 to 3, wherein (a) is an expressible polynucleotide encoding said HRS-Fc fusion polypeptide, optionally a modified mRNA polynucleotide, said modified mRNA polynucleotide optionally comprising one or more non-natural bases and / or non-natural internucleotide linkages.

5. The therapeutic composition of any one of claims 1 to 4, wherein said HRS-Fc fusion polypeptide has a non-canonical activity, optionally an anti-inflammatory activity.

6. 6. The therapeutic composition of any one of claims 1-5, wherein the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or weight-by-weight basis and is substantially free of aggregates.

7. The therapeutic composition of any one of claims 1 to 5, which is substantially free of endotoxins.

8. A therapeutic composition according to any one of claims 1 to 5, comprising lipid nanoparticles.

9. The therapeutic composition of any one of claims 1 to 5, wherein the composition is in a syringe, optionally an injectable syringe, or wherein the composition is a capsule, optionally an oral capsule.

10. 10. Use of a therapeutic composition according to any one of claims 1 to 8 in the manufacture of a medicament for the treatment of pulmonary inflammation in a subject in need thereof.

11. 10. The composition of any one of claims 1-9, wherein the pirfenidone increases the serum concentration of the HRS-Fc fusion polypeptide in the subject by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200% or more compared to the HRS-Fc fusion polypeptide alone.

12. The pirfenidone is administered in individual dosage units ranging from about 50 to about 1000 mg, or in individual dosage units ranging from about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570 , 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg, about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 23 0, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 54 0, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850 , 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg or less, or at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 84 The composition according to any one of claims 1 to 9 and 11, administered in individual dosage units of 0, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg, optionally in one, two, or three capsules for oral administration.

13. The pirfenidone is administered in a daily dosage unit ranging from about 100 to about 4000 mg / day, or in a daily dosage unit ranging from about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 9 20, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 , 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day, about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day 60, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 57 0, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880 , 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700,2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day or less, or at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 31 0, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2 The composition of any one of claims 1 to 9 and 11, administered in daily dosage units of 500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day, optionally in about 1, 2, 3, 4, 5, 6, 7, 8, 9 capsules for oral administration.

14. 12. The composition of any one of claims 1-9 and 11, wherein the pirfenidone is administered in individual dosage units of about 800 mg (e.g., 801 mg) as three about 267 mg capsules for oral administration, optionally taken as three capsules per individual dosage.

15. 12. The composition of any one of claims 1-9 and 11, wherein the pirfenidone is administered in daily dosage units of about 2400 mg / day (e.g., 2403 mg / day) as nine about 267 mg capsules for oral administration three times a day, optionally taken as three capsules per individual dosage.

16. The composition of any one of claims 1-9 and 11-15, wherein the subject has or is at risk of having an interstitial lung disease (ILD).

17. 17. The composition of claim 16, wherein the ILD is idiopathic or associated with a connective tissue disease, an autoimmune disease, exposure to an inhaled substance or drug, an infectious disease, or malignancy.

18. 18. The composition of claim 17, wherein the ILD is selected from or associated with one or more of idiopathic interstitial pneumonia, idiopathic pulmonary fibrosis, sarcoidosis, Hammann-Rich syndrome, antisynthetase syndrome, idiopathic eosinophilic pneumonia, alveolar hemorrhage syndrome, pulmonary alveolar proteinosis, asbestosis, silicosis, beryllium disease, rheumatoid arthritis, lupus erythematosus, chronic graft-versus-host disease with pulmonary involvement, sclerosis (systemic) or scleroderma, polymyositis, dermatomyositis, chronic lung disease, asthma, bronchitis (respiratory bronchitis), pneumonia, hypersensitivity pneumonitis, chronic hypersensitivity pneumonitis, respiratory distress syndrome, Still's disease, acute lung injury, microscopic polyangiitis, pulmonary edema, pulmonary Langerhans cell histiocytosis, acute inhalation exposure, drug-induced pulmonary disease, desquamative interstitial pneumonia, and / or cystic fibrosis.

19. 19. The composition of claim 17 or 18, wherein the ILD is associated with one or more of surfactant protein B deficiency (mutations in SFTPB), surfactant protein C deficiency (mutations in SFTPC), ABCA3 deficiency (mutations in ABCA3), brain-pulmonary-thyroid syndrome (mutations in TTF1), or congenital pulmonary alveolar proteinosis (mutations in CSFR2A, CSFR2B), alveolar-capillary dysplasia (mutations in FoxF1), mutations in telomerase reverse transcriptase (TERT), mutations in telomerase RNA component (TERC), mutations in regulator of telomere elongation helicase 1 (RTEL1), and / or mutations in poly(A)-specific ribonuclease (PARN).

20. 20. The composition of claim 17, wherein the drug is selected from one or more of an antibiotic, a chemotherapeutic agent, an antiarrhythmic agent, and a statin drug.

21. 18. The composition of claim 17, wherein the infection is selected from one or more of atypical pneumonia, Pneumocystis pneumonia (PCP), tuberculosis, Chlamydia trachomatis, and respiratory syncytial virus (RSV), cryptogenic organizing pneumonia.

22. The composition of claim 17, wherein the malignant tumor is lymphangitis carcinomatosis or lymphoma.

23. 16. The composition of any one of claims 1-9 and 11-15, wherein the subject in need of treatment of the pulmonary inflammation has a condition selected from one or more of atopic asthma, non-atopic asthma, allergic asthma, atopic bronchial IgE-mediated asthma, bronchial asthma, essential asthma, true asthma, intrinsic asthma caused by pathophysiological perturbations, extrinsic asthma caused by environmental factors, essential asthma of unknown or unclear etiology, non-atopic asthma, bronchitis-like asthma, emphysematous asthma, exercise-induced asthma, allergen-induced asthma, cold-induced asthma, occupational asthma, infectious asthma caused by bacterial, fungal, protozoal, or viral infections, non-allergic asthma, incipient asthma, wheezing infant syndrome and bronchiolitis, chronic or acute bronchoconstriction, chronic bronchitis, small airway obstruction, and emphysema.

24. The composition of any one of claims 1 to 9 and 11 to 15, wherein the subject in need of treatment for pulmonary inflammation has an obstructive or inflammatory airways disease.

25. 25. The composition of claim 24, wherein the obstructive or inflammatory airways disease is selected from one or more of chronic eosinophilic pneumonia, chronic obstructive pulmonary disease (COPD), chronic bronchitis, COPD including emphysema or dyspnea, COPD characterized by irreversible progressive airway obstruction, and acute respiratory distress syndrome (ARDS).

26. 16. The composition of any one of claims 1 to 9 and 11 to 15, wherein the subject in need of treatment for lung inflammation has a condition associated with exacerbation of airway hyperresponsiveness resulting from other drug therapies, airway disease with pulmonary hypertension, bronchitis or acute bronchitis, acute laryngotracheobronchitis, arachidic bronchitis, catarrhal bronchitis, croupus bronchitis, dry bronchitis, infectious asthmatic bronchitis, productive bronchitis, staphylococcal or streptococcal bronchitis, vesicular bronchitis, acute lung injury, bronchiectasis, or a condition associated with cylindrical bronchiectasis, saccular bronchiectasis, fusiform bronchiectasis, capillary bronchiectasis, cystic bronchiectasis, dry bronchiectasis, or follicular bronchiectasis.

27. 27. The composition of any one of claims 1-9 and 11-26, wherein the subject in need of treatment for pulmonary inflammation has an Ashcroft score of 1, 2, 3, 4, 5, 6, 7, or 8.

28. 28. The composition of any one of claims 1-9 and 11-27, optionally increasing life expectancy of a subject in need of said pulmonary inflammation treatment by about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 years or more.

29. 29. The composition of any one of claims 1-9 and 11-28, which improves one or more clinical symptoms or parameters of pulmonary inflammation in a subject in need of treatment of said pulmonary inflammation.

30. 30. The composition of claim 29, wherein the one or more clinical symptoms or parameters are selected from one or more of pulmonary fibrosis, inflammatory cell infiltration in the lungs, respiratory function, and body weight.

31. 31. The composition of claim 30, wherein the composition ameliorates pulmonary fibrosis in a subject in need of treatment for said pulmonary inflammation, optionally as measured by a reduction in Ashcroft score of 1, 2, 3, 4, 5, 6, 7, or 8 grades lower in Ashcroft score as compared to the initial score.

32. (a) a histidyl-tRNA synthetase (HRS)-Fc fusion polypeptide, or an expressible polynucleotide encoding said HRS-Fc fusion polypeptide, wherein said HRS-Fc fusion polypeptide comprises, consists of, or consists essentially of an amino acid sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 157, 158, and 163-166; and (b) pirfenidone, which increases the serum concentration of the HRS-Fc fusion polypeptide compared to the HRS-Fc fusion polypeptide alone; 23. A patient care kit for use in treating pulmonary inflammation in a subject in need thereof, comprising:

33. 33. The patient care kit of claim 32, wherein (a) and (b) are in separate compositions.

34. 33. The patient care kit of claim 32, wherein (a) and (b) are present in the same composition.

35. The pirfenidone is administered in individual dosage units ranging from about 50 to about 1000 mg, or in individual dosage units ranging from about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570 , 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg, about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 23 0, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 54 0, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850 , 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg or less, or at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,33. The patient care kit of claim 32, wherein the individual dosage units are 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg, optionally in one, two, or three capsules for oral administration. ,

36. The pirfenidone is administered in a daily dosage unit ranging from about 100 to about 4000 mg / day, or in a daily dosage unit ranging from about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 9 20, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 , 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day, about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day 60, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 57 0, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880 , 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700,2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day or less, or at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 , 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 7 20, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 33. The patient care kit of claim 32, wherein the patient is administered 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day, optionally in about 1, 2, 3, 4, 5, 6, 7, 8, 9 capsules for oral administration.

37. 33. The patient care kit of claim 32, wherein the pirfenidone is in individual dosage units of about 800 mg (e.g., 801 mg), optionally as three about 267 mg capsules for oral administration taken as three capsules per individual dosage.

38. 38. The patient care kit of claim 37, wherein the pirfenidone is in a daily dosage unit of about 2400 mg / day (e.g., 2403 mg / day), optionally as nine about 267 mg capsules for oral administration three times per day, taken as three capsules per individual dosage.

39. 1. A composition for use in altering one or more pharmacokinetic characteristics of an HRS-Fc fusion polypeptide in a subject, comprising said HRS-Fc fusion polypeptide, or an expressible polynucleotide encoding said HRS-Fc fusion polypeptide, in combination with pirfenidone, wherein said HRS-Fc fusion polypeptide comprises an amino acid sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 157, 158, and 163-166, and wherein the one or more altered pharmacokinetic characteristics of the HRS-Fc fusion polypeptide is increased serum concentration.

40. The HRS-Fc fusion polypeptide has the structure of SEQ ID NO: 157 (Fc-HRS(2-60) or HRS ＦＣ１ 40. The composition of claim 39 comprising, consisting of, or consisting essentially of.

Images