Compositions and methods for treating lung inflammation

Abstract

To provide compositions and methods for the treatment of lung inflammation, including interstitial lung diseases (ILDs).SOLUTION: Embodiments of the present disclosure relate to therapies, including combination therapies for the treatment of lung inflammation, including interstitial lung diseases (ILDs), which include the use of at least one histidyl-tRNA synthetase (HRS) polypeptide or an expressible polynucleotide that encodes the HRS polypeptide, alone or in combination with at least one immunomodulatory agent. Embodiments of the present disclosure relate, in appropriate portions, to a therapeutic composition, comprising (a) an HRS polypeptide, or an expressible polynucleotide that encodes the HRS polypeptide and (b) an immunomodulatory agent.SELECTED DRAWING: Figure 3

Description

Technical Field

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

[0002] Statement Regarding Sequence Listing The Sequence Listing related to the present application is provided in text format in lieu of a paper copy, and is incorporated herein by reference. The name of the text file containing the Sequence Listing is ATYR_131_01WO_ST25.txt. The text file is approximately 276 KB, created on April 18, 2018, and has been electronically submitted via EFS-Web.

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

[0004] Interstitial lung disease (ILD) is a group of heterogeneous disorders affecting the pulmonary interstitium, in which inflammation of heterogeneous disorders is the main pathogenesis. Among ILD designations, there are many fibrotic pulmonary conditions generally recognized to have a measurable inflammatory component involving both innate and adaptive immune mechanisms that contribute to pathogenesis at several levels.

[0005] Patients suffering from ILD often suffer from progressive debilitating respiratory symptoms, and significantly higher and increased mortality is observed compared to the general population. As a group, these conditions represent a large unmet medical need, for which there are few effective treatments without significant adverse side effects. Summary of the Invention Means for Solving the Problem

[0006] Embodiments of the present disclosure, in appropriate aspects, relate to a therapeutic composition, wherein (a) a histidyl-tRNA synthetase (HRS) polypeptide, or an expressible polynucleotide encoding an HRS polypeptide, and (b) an immunomodulator; the present invention relates to said therapeutic composition comprising the above.

[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 to 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 to 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 fused 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 contains, consists of, or essentially consists 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 is at least about 80%, 85%, 90%, or 95% pure on a protein basis and less than about 5% aggregated.

[0011] In some embodiments, (a) is an expressible polynucleotide encoding an HRS polypeptide, optionally a modified mRNA polynucleotide, which optionally comprises one or more non-natural bases and / or non-natural nucleotide bonds.

[0012] In some embodiments, the HRS polypeptide has non-canonical activity and, optionally, anti-inflammatory activity.

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

[0014] In some embodiments, the S1P and / or S1PR modulator is amicelimod (S1PR antagonist), fingolimod (S1PR1 functional antagonist), sonepcizumab (S1P-specific monoclonal antibody), KRP203 (S1PR1 agonist), SEW2871 (S1PR1 agonist), siponimod (S1PR1 and S1PR5 modulator), RPC1063 (S1PR1 modulator), ONO-4641 (S1PR1 and S1PR5 agonist), JTE-013 (S1PR2 antagonist), GS Amiserimod is optionally selected from K2018682 (S1PR1 agonist), Ponesimod (S1PR1 agonist), Suramin (selective S1PR3 and S1PR5 antagonist), VPC23019 (aryl-amide analog; competitive S1PR1 and S1PR3 antagonist), and W146 (selective S1PR1 antagonist), antisense or RNAi agents targeting S1PR, and antibodies or antigen-binding fragments or small molecules that specifically bind to S1P and / or S1PR, in a range of approximately 0.1 mg to approximately 10 mg. The dosage units are approximately 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 than or equal to approximately 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 at least approximately 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 arbitrarily, amicelimod is approximately 0 The dosage units are in the range of 0.1 mg / kg to approximately 10 mg / kg, or approximately 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, approximately 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 approximately 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, antisense or RNAi agents targeting calcineurin or its subunits, as well as antibodies or antigen-binding fragments or small molecules that specifically bind to calcineurin or its subunits.

[0017] In some embodiments, the mTOR inhibitor is an ATP-competitive mTOR kinase inhibitor, an mTORC1 / mTORC2 bipolar inhibitor, and / or an mTOR / PI3K bipolar inhibitor, or the mTOR inhibitor is selected from one or more of the following: everolimus, rapamycin, deforolimus, temsirolimus, dactricib, BGT226, SF1126, PKI-587, NVPBE235, sapanicertib, AZD8055, AZD2014, mTOR-targeting antisense or RNAi agents, and antibodies or antigen-binding fragments or small molecules that specifically bind to mTOR.

[0018] In some embodiments, the IDO inhibitor is selected from indoximod (NLG-8189), 1-methyltryptophan (1MT), β-carbolin (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat, antisense or RNAi agents targeting IDO, as well as antibodies or antigen-binding fragments or small molecules that specifically bind to IDO.

[0019] In some embodiments, the IMPDH inhibitor is selected from one or more of the following: mycophenolic acid (mycophenolate mofetil), ribavirin, and 6TGMP (6-thioguanine monophosphate), antisense or RNAi agents that target IMPDH, and antibodies or antigen-binding fragments or small molecules that specifically bind to IMPDH.

[0020] In some embodiments, the cytokine inhibitors include interleukin-1 (IL-1), 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-gamma), transforming growth factor-β (TGF-β), and granulocyte-macrophage colony-stimulating factor (GM-CSF), and / or IL-1R, IL-6R, I The cytokine inhibitors are selected from one or more cytokine receptors selected from L-8R, IL-11R, IL-12R, IL-17R, IL-18R, and IL-20R; TNFRs such as ST2 (interleukin-1 receptor-like 1, IL1RL1) and TNFR1; interferon-gamma receptors (IFNGRs); and TGF-β receptors such as TGFβR1 (ALK5) or TGFβR2. Furthermore, the cytokine and / or cytokine receptor inhibitors are selected from antisense or RNAi agents that target cytokines and / or cytokine receptors, as well as antibodies or antigen-binding fragments or small molecules that specifically bind to cytokines or cytokine receptors.

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

[0022] In some embodiments, the kinase inhibitors include Janus kinase (JAK1, JAK2, JAK3, TYK2, and other JAKs), 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's tyrosine kinase (BTK), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor (VE). The kinase inhibitors are selected from one or more of the following: GFR1, VEGFR2, VEGFR3 (including VEGFRs), fibroblast growth factor receptor (FGFR), B-Raf, RET proto-oncogene, platelet-derived growth factor receptor (PDGF-R), tropomyosin receptor kinases (TrkA, TrkB, TrkC (including Trk)), and c-Met. Furthermore, the kinase inhibitors are selected from antisense or RNAi agents that target kinases, as well as antibodies or antigen-binding fragments or small molecules that specifically bind to kinases.

[0023] In some embodiments, the kinase inhibitor is nintedanib, baricitinib, fedratinib, filgotinib, gandotinib, restaurtinib, momerotinib, pacritinib, peficitinib, ruxolitinib, tofacitinib, padacitinib, afatinib, axitinib, bosutinib, cetuximab, cobimetinib, crizotinib, cabozantinib, dasatinib, Select one or more of the following: entrectinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mbritinib, neratinib, nilotinib, pazopanib, pegaptanib, sorafenib, sunitinib, SU6656, toceranib, vandetanib, batalanib, and vemurafenib.

[0024] In some embodiments, the B cell receptor inhibitor is selected from CD20-targeting antisense or RNAi agents, as well as antibodies or antigen-binding fragments or small molecules that specifically bind to CD20, or the B cell receptor inhibitor is optionally selected from one or more of ibritumomab / tiuxetan, obinutuzumab, okalatuzumab, ocrelizumab, rituximab, tocitumomab, and bertuzumab.

[0025] In some embodiments, the antisense agent is about 10 to 40 base pairs long and is optionally selected from morpholino oligonucleotides (PMOs), peptide nucleic acids (PNAs), 2'O-methylphosphorothioate oligonucleotides, tricyclophosphorothioate oligonucleotides, and locked nucleic acids (LNAs).

[0026] In some embodiments, the antisense agent specifically hybridizes to a target region within a pre-mRNA encoding a target protein or an mRNA target sequence, the target region being selected from one or more of the following: 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-treated mRNA, a branch point, the 3' untranslated region (UTR), and a polyadenylation signal sequence.

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

[0028] In some embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody, optionally a humanized antibody, or optionally an Fv fragment or a 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-based or weight-to-weight basis, and is substantially free of aggregates.

[0030] In some embodiments, the composition is substantially free of endotoxins.

[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 in a capsule, for example, an oral capsule.

[0033] A method for treating lung inflammation in a subject requiring treatment, wherein the subject (a) a histidyl-tRNA synthetase (HRS) polypeptide, or an expressible polynucleotide encoding an HRS polypeptide, (b) Methods also include administering an immunomodulator.

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

[0035] In some embodiments, the HRS polypeptide includes an Fc region to form an HRS-Fc fusion polypeptide, for example, this HRS-Fc fusion polypeptide contains, consists of, or essentially consists 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 is SEQ ID NO: 157 (Fc-HRS(2-60) or HRS FC1 ) containing, consisting of, or essentially consisting of.

[0036] In some embodiments, immunomodulators alter one or more pharmacokinetic characteristics of HRS polypeptides compared to HRS polypeptides alone. In certain embodiments, the altered pharmacokinetic characteristics of HRS polypeptides are increased serum concentration, increased serum half-life, increased bioavailability, increased exposure (AUC), increased serum concentration, and / or decreased clearance.

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

[0038] In some embodiments, the HRS polypeptide is sequence number 157 (Fc-HRS(2-60) or HRSF C1 An immunomodulator containing, consisting of, or essentially consisting of, is pirfenidone. In some embodiments, pirfenidone increases the serum concentration of HRS polypeptide in a subject by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200% or more compared to HRS polypeptide alone.

[0039] In some embodiments, pirfenidone is administered in individual dose units ranging from approximately 50 to approximately 1000 mg, or approximately 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, 8 70, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000mg, approximately 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210 ,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,10,520,5 30, 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 0, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or less than 1000 mg, or at least approximately 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,It is administered orally in individual dose 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, in one, two, or three capsules.

[0040] In some embodiments, the dose of pirfenidone is in daily dose units ranging from approximately 100 to approximately 4000 mg / day, or approximately 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, 8 90, 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, 28 00, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day, approximately 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 2 30, 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 approximately 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, The daily dose is 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day, administered in units of approximately 1, 2, 3, 4, 5, 6, 7, 8, or 9 oral capsules.

[0041] In some embodiments, pirfenidone is administered in individual dose units of approximately 800 mg (e.g., 801 mg), optionally as three capsules of approximately 267 mg each for oral administration. In some embodiments, pirfenidone is administered in daily dose units of approximately 2400 mg / day (e.g., 2403 mg / day), optionally as nine capsules of approximately 267 mg each for oral administration three times a day, optionally as three capsules each for individual doses.

[0042] In some embodiments, nintedanib is administered in individual dose units ranging from about 10 to about 500 mg, or in doses of 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, approximately 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, 27 0, 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 approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 It is administered in individual dose units of 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, either in approximately one, two, or three capsules.

[0043] In some embodiments, nintedanib is administered in daily dose units ranging from approximately 20 to approximately 1000 mg / day, or approximately 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, 8 40, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 , 1000mg / day, approximately 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, 4 80, 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, 79 0, 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 approximately 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, 7 The daily dose is 70, 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 / day, administered in units of approximately 1, 2, 3, 4, 5, or 6 capsules.

[0044] In some embodiments, nintedanib is administered in a daily dose unit of approximately 100–150 mg or approximately 200–300 mg / day, either once or twice daily.

[0045] In some embodiments, subjects have or are at risk of developing 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, ILD is selected from or associated with one or more of the following: idiopathic interstitial pneumonia, idiopathic pulmonary fibrosis, sarcoidosis, Hanmann-Ricci syndrome, antisynthesis syndrome, idiopathic eosinophilic pneumonia, alveolar hemorrhage syndrome, alveolar proteinosis, asbestosis, silicosis, beryllium disease, rheumatoid arthritis, lupus erythematosus, chronic graft-versus-host disease with lung injury, 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 lung disease, desquamative interstitial pneumonia, and / or cystic fibrosis.

[0047] In some embodiments, ILD is associated with one or more of the following: surfactant protein B deficiency (mutation in SFTPB), surfactant protein C deficiency (mutation in SFTPC), ABCA3 deficiency (mutation in ABCA3), cerebropulmonary thyroid syndrome (mutation in TTF1), or congenital alveolar proteinosis (mutation 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 the following: antibiotics, chemotherapeutic agents, antiarrhythmic agents, and statins. In some embodiments, the infection is selected from one or more of the following: Pneumocystis pneumonia (PCP), tuberculosis, Chlamydia trachomatis, and respiratory syncytial virus (RSV), and idiopathic organizing pneumonia. In some embodiments, the malignant tumor is carcinomatous lymphangitis or lymphoma.

[0049] In some embodiments, the subjects requiring it have one or more conditions selected from atopic asthma, non-atopic asthma, allergic asthma, atopic bronchial IgE-mediated asthma, bronchial asthma, essential asthma, true asthma, endogenous asthma caused by pathophysiological disturbance, exogenous asthma caused by environmental factors, essential asthma of unknown or unclear causes, 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, protozoan, or viral infections, non-allergic asthma, first-onset asthma, wheezing infant syndrome and bronchiolitis, chronic or acute bronchoconstriction, chronic bronchitis, peripheral airway obstruction, and pulmonary emphysema.

[0050] In some embodiments, the subjects requiring it have an obstructive or inflammatory airway disease. In some embodiments, the obstructive or inflammatory airway disease is selected from one or more of the following: chronic eosinophilic pneumonia, chronic obstructive pulmonary disease (COPD), COPD encompassing chronic bronchitis, COPD characterized by irreversible progressive airway obstruction, emphysema or dyspnea, and acute respiratory distress syndrome (ARDS).

[0051] In some embodiments, subjects requiring treatment for lung inflammation have conditions associated with worsening airway hyperresponsiveness resulting from other drug therapies, airway diseases with pulmonary hypertension, bronchitis or acute bronchitis, acute laryngotracheobronchitis, arachidic bronchitis, catarrhal bronchitis, croup bronchitis, dry bronchitis, infectious asthmatic bronchitis, productive bronchitis, staphylococcal or streptococcal bronchitis, vesicular bronchitis, acute lung injury, bronchiectasis, or conditions associated with columnar bronchiectasis, saccular bronchiectasis, fusiform bronchiectasis, capillary bronchiectasis, cystic bronchiectasis, dry bronchitis, or follicular bronchiectasis.

[0052] In some embodiments, the object requiring it has 1, 2, 3, 4, 5, 6, 7, or 8 Ashcroft scores.

[0053] A particular embodiment increases the average life expectancy of the subject requiring it, 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] A particular embodiment improves one or more clinical symptoms or parameters of lung inflammation in a subject requiring it.

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

[0056] Certain embodiments optionally improve pulmonary fibrosis in subjects requiring it by approximately or at least approximately 10, 20, 30, 40, 50, 60, 70, 80, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months or longer, as measured over a period of approximately 10, 20, 30, 40, 50, 60, 700, 800, 900, 1000%, or more, when measured over a period of approximately 1, 2, 3, 4, 5, 6, 7, 8, 90, 1000%, or more.

[0057] A particular embodiment optionally improves pulmonary fibrosis in a subject requiring it, as measured by a decrease in the Ashcroft score, where the Ashcroft score decreases by 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 approximately or at least approximately 10, 20, 30, 40, 50, 60, 70, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months or longer, as measured over a period of approximately 10, 20, 30, 40, 50, 600, 700, 800, 900, 1000%, or more.

[0059] Certain embodiments optionally improve respiratory function by approximately or at least approximately 10, 20, 30, 40, 50, 60, 70, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months or longer, when measured over a period of approximately 10, 20, 30, 40, 50, 60, 70, 800, 900, 1000%, or more. In some embodiments, the improved respiratory function is selected from one or more of the following: increased expiratory time, increased inspiratory time, decreased maximum expiratory flow rate, decreased maximum inspiratory flow rate, decreased respiratory volume per minute (RMV), and decreased respiratory rate.

[0060] (a) a histidyl-tRNA synthetase (HRS) polypeptide, or an expressible polynucleotide encoding the HRS polypeptide, (b) Patient care kits, including the administration of immunomodulators, are also included.

[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 immunomodulator is pirfenidone or nintedanib.

[0063] In some embodiments, pirfenidone is administered in individual dose units ranging from approximately 50 to approximately 1000 mg (optionally in approximately one, two, or three capsules for oral administration), or in doses of approximately 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, 70 0, 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 100 0mg, approximately 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, 3 60, 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 1000mg or less, or at least approximately 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, 68 These are individual dosage units of 0, 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.

[0064] In some embodiments, pirfenidone is administered in daily dose units ranging from approximately 100 to approximately 4000 mg / day (optionally in approximately 3, 4, 5, 6, 7, 8, or 9 capsules for oral administration), or in doses of approximately 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 ,370,380,390,400,410,420,430,440,450,460,470,480,490,500,10,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,6 80, 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, 99 0, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 36 00, 3700, 3800, 3900, or 4000 mg / day, approximately 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 3 30, 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 less At least approximately 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, 53 0, 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, 97 These are individual dose units of 0, 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.

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

[0066] In some embodiments, nintedanib is administered in individual dose units ranging from approximately 10 to approximately 500 mg (optionally in approximately one, two, or three capsules for oral administration), or in doses of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 mg (optionally in approximately one, two, or three capsules for oral administration). , 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 500mg, approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 1 60, 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 approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, These are individual dose units of 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.

[0067] In some embodiments, nintedanib is administered in daily dose units ranging from approximately 20 to approximately 1000 mg / day (in any one, two, three, four, five, or six capsules), or in doses of approximately 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, 6 60, 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, 97 0, 980, 990, 1000mg / day, approximately 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 14 0, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 3 00, 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, 61 0, 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 approximately 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, 6 These are daily dose units of 40, 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, and 1000 mg / day.

[0068] In some embodiments, nintedanib is administered in daily dose units ranging from approximately 100 to 150 mg, or from approximately 200 to 300 mg / day, either once or twice daily.

[0069] Methods for altering one or more pharmacokinetic characteristics of an HRS-Fc fusion polypeptide in a subject also include administering to the subject an expressible polynucleotide encoding the HRS-Fc fusion polypeptide, either in combination with the HRS-Fc fusion polypeptide or pirfenidone. In some embodiments, the HRS-Fc fusion polypeptide contains, consists of, or essentially consists 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 is SEQ ID NO: 157 (Fc-HRS(2-60) or HRS FC1 ) containing, consisting of, or essentially consisting of.

[0070] Methods for treating lung inflammation (as described herein) in subjects requiring such treatment also include administering to a subject an HRS-Fc fusion polypeptide, or an expressible polynucleotide encoding an HRS-Fc fusion polypeptide. In some embodiments, the HRS-Fc fusion polypeptide comprises, consists of, or essentially consists 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 is SEQ ID NO: 157 (Fc-HRS(2-60) or HRS FC1 ) containing, consisting of, or essentially consisting of. [Brief explanation of the drawing]

[0071] [Figure 1] The BAL fluid cell count is shown. Individual cell counts are shown using a line indicating the group mean: "no tx" indicates no treatment, "Veh" indicates treatment with the vehicle, "Dex." indicates treatment with dexamethasone, "Ninte." indicates treatment with nintedanib, and "TA" indicates treatment with the test substance. Statistical comparisons were performed within the PO or IV treatment group by one-way ANOVA, followed by Dunnett's post-hoc test. *p<0.05, **p<0.01 vs IV vehicle. [Figure 2] The histological fibrosis (Ashcroft) score is shown. Mean data from all areas scored within each group are shown (+SEM): "no tx" indicates no treatment, "Veh" indicates treatment with vehicle, "Dex." indicates treatment with dexamethasone, "Ninte." indicates treatment with nintedanib, and "TA" indicates treatment with the test substance. Statistical comparisons were performed within the PO or IV treatment groups by one-way ANOVA followed by Dunnett's post-hoc test. *p<0.05, **p<0.01 vs. each vehicle. [Figure 3]The respiratory rate (RMV) on day 15 is shown. Individual RMVs are shown using a line indicating the group mean: "Veh" indicates treatment with the vehicle, "Ninte." indicates treatment with nintedanib, and "TA" indicates treatment with the test substance. The dose of the test substance is shown in parentheses. Group IV was compared using one-way ANOVA, and the PO group was compared using a t-test. *p<0.05, **p<0.01, ***p<0.001. [Figure 4] The histological fibrosis (Ashcroft) score is shown. Mean data from all areas scored within each group are shown (+SEM): "Veh" indicates treatment with the vehicle, "Ninte." indicates treatment with nintedanib, and "TA" indicates treatment with the test substance. The dose of the test substance is shown in parentheses. Statistical comparisons were performed by t-tests within the PO or IV treatment group. *p<0.05 vs IV vehicle. [Figure 5] Interstitial / alveolar inflammatory cell infiltration scores are shown. Individual mean scores within each group are shown: "Veh" indicates treatment with the vehicle, "Ninte." indicates treatment with nintedanib, and "TA" indicates treatment with the test substance. The dose of the test substance is shown in parentheses. Statistical comparisons were performed by t-tests within the PO or IV treatment groups. *p<0.05 vs IV vehicle. [Figure 6] Mean serum HRSFC1 levels in pMol (+SEM): In each group that completed the study on day 22, statistical comparisons were performed by t-tests with p<0.01, p<0.001, and p<0.0005 between the group treated with the test substance in combination with the vehicle (Veh), the group treated with the test substance in combination with nintedanib, or the group treated with the test substance in combination with pirfenidone. [Modes for carrying out the invention]

[0072] In the practice of this 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 described 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, RI (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 JMHarris, Eds., 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] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this disclosure belongs. Any method, material, composition, reagent, cell, or similar or equivalent to those described herein may be used in the practice or testing of the subject matter of this disclosure, but preferred methods and materials are described herein. All publications and references, including but not limited to patent and patent application documents cited herein, are incorporated herein by reference in their entirety, so that each individual publication or reference is specifically and individually indicated as being incorporated herein by reference as being fully described herein. Any patent application for which this application claims priority is also incorporated herein by reference in their entirety in the manner described above for publications and references.

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

[0075] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical purposes of the article. For example, "component" means one or more components.

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

[0077] An "antagonist" or "inhibitor" refers to a biological structure or chemical agent that interferes with or otherwise reduces the physiological effects of another drug or molecule. In some cases, antagonists bind specifically to the other drug or molecule. Full and partial antagonists are included.

[0078] An "agonist" refers to a biological structure or chemical agent that increases or enhances the physiological effects of another drug or molecule. In some cases, agonists specifically bind to other drugs or molecules. This includes complete and partial agonists.

[0079] The term "anergy" refers to the functional inactivation of the T-cell or B-cell response to antigen-induced restimulation.

[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 mimics. 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 known to those skilled in the art, norleucine, norvaline, p-fluorophenylalanine, and ethionine. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications may include, for example, substitution or replacement of chemical groups and parts on an amino acid, or derivatization of an amino acid. Amino acid mimics include, for example, organic structures that exhibit functionally similar properties, such as the charge and charge spacing characteristic of a reference amino acid. For example, an organic structure that mimics arginine (Arg or R) may have a positively charged moiety located in a similar molecular space and possess mobility comparable to the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimics also include structures constrained to maintain optimal spacing and charge interactions between amino acids or amino acid functional groups. Those skilled in the art know, or can determine, which structures constitute functionally equivalent amino acid analogs and amino acid mimics.

[0081] As used herein, a “at-risk” subject for developing 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 method described herein. “At-risk” means that the subject has one or more risk factors, which are measurable parameters associated with developing a disease, as described herein and known in the art. Subjects having one or more of these risk factors are more likely to develop a disease or adverse reaction than subjects not having one or more of these risk factors.

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

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

[0084] The term "clonal removal" refers to the removal (e.g., loss or death) of autoreactive T cells. Clonal removal can primarily occur within or around the thymus, or both.

[0085] Throughout this disclosure, unless otherwise required by context, the words “comprise,” “comprises,” and “comprising” shall be understood to imply the inclusion of the step or element, or group of steps or elements, described herein, but not to imply the exclusion of any other step or element, or group of steps or elements.

[0086] "Consists of" means that what follows the phrase "consists of" is included and limited to it. Therefore, the phrase "consists of" indicates that the enumerated elements are required or essential, and that other elements cannot exist at all. "Essentially consists of" means that it includes any elements enumerated after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the enumerated elements. Therefore, the phrase "essentially consists of" indicates that the enumerated elements are required or essential, but other elements may or may not exist, depending on whether they substantially affect the activity or action of the enumerated elements.

[0087] The terms “endotoxin-free” or “substantially endotoxin-free” generally refer to compositions, solvents, and / or containers containing at most trace amounts of endotoxins (e.g., amounts that do not have clinically harmful physiological effects on the subject), preferably undetectable amounts of endotoxins. Endotoxins, while endotoxins can also be found in Gram-positive bacteria such as Listeria monocytogenes, are toxins associated with certain microorganisms, e.g., bacteria, typically Gram-negative bacteria. The most common endotoxins are lipopolysaccharides (LPS) or lipo-oligosaccharides (LOS) found in the outer membranes of various Gram-negative bacteria, which are central to the pathogenicity of these bacteria in their ability to cause disease. Small amounts of endotoxins in humans can produce fever, decreased blood pressure, and activation of inflammation and coagulation, among other harmful physiological effects.

[0088] Therefore, since even small amounts of endotoxins in pharmaceutical products can cause harmful effects in humans, it is often desirable to remove most or all traces of endotoxins from drug products and / or drug containers. Since temperatures above 300°C are typically required to decompose most endotoxins, depyrogenation ovens can be used for this purpose. For example, based 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 for removing endotoxins, including, for example, chromatography and filtration methods, described herein and known in the art, are also considered.

[0089] Endotoxins can be detected using conventional techniques known in the art. For example, the Limulus amebosite lysate assay, which utilizes blood from horseshoe crabs, is a highly sensitive assay for detecting the presence of endotoxins. In this test, very low levels of LPS can cause detectable coagulation of Limulus lysates due to a potent enzyme cascade that amplifies the reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). Endotoxin levels may be less than approximately 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 free of endotoxins. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to approximately 1–10 EU.

[0090] As used herein, the terms “contacting cells,” “introducing,” or “delivering” include the delivery of the agents described herein (e.g., polypeptide agents, polynucleotide agents) to cells by methods customary 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-permeable peptide” (CPP) or “cell uptake-enhancing peptide moiety” are used interchangeably and refer to cationic cell-permeable peptides, also called “transport peptides,” “carrier peptides,” or “peptide delivery domains.” In some embodiments, when systemically administered, the peptide induces cell permeability (e.g., muscle cells) within about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells in a given cell culture population, or at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells, or has the ability to enable the translocation of macromolecules within multiple tissues (e.g., muscle tissue) in vivo or in other forms of administration. In some embodiments, the CPP is derived from the formula -[(C(O)CHR'NH) m The CPP is of ]R'' (wherein R' is a naturally occurring amino acid or a side chain of a 1- or 2-carbon analog thereof, 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 in whole by reference). In some embodiments, m is an integer selected from 1 to 50, and when m is 1, the portion is a single amino acid or a derivative thereof. Any of the polynucleotide agents described herein (e.g., antisense, RNAi agents) can be conjugated to a CPP to improve uptake into target cells, for example, muscle cells.

[0092] The terms “median effective concentration” or “EC50” refer to the concentration of a drug described herein (e.g., HRS polypeptide or other drug) that induces an intermediate response between baseline and maximum after some specified exposure time; therefore, the EC50 of a stepwise dose-response curve represents the concentration of the compound at which 50% of its maximum effect is observed. EC50 also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. Similarly, “EC90” refers to the concentration of a drug or composition at which 90% of its maximum effect is observed. “EC90” can be calculated from “EC50” and the Hill gradient, or it can be determined directly from the data using conventional knowledge in the art. In some embodiments, the EC50 of the drug is 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 less than 500 nM. In some embodiments, the biotherapeutic composition will have an EC50 value of about 1 nM or less.

[0093] "Homologousity" refers to the percentage number of identical or constituting conserved substitutions of amino acids. Homologousity can be determined using sequence comparison programs such as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). In this way, sequences of similar or substantially different lengths to the sequences cited herein can be compared by inserting gaps during 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 related mechanisms that regulate cytokine expression and release (e.g., interferons and interferon-signaling), induce cell death, and inhibit protein synthesis.

[0095] "Isolated" means material that substantially or essentially does not contain components that would normally accompany it in its natural state. For example, as used herein, "isolated polynucleotide," "isolated oligonucleotide," or "isolated oligonucleotide" may refer to a polynucleotide that has been purified or extracted from sequences adjacent to it in its natural state, for example, a DNA fragment extracted from sequences adjacent to that fragment in a genome. When the term "isolate" refers to cells, it refers to the purification of cells (e.g., fibroblasts, lymphoblasts) from a source subject (e.g., a subject suffering from polynucleotide recurrent disease). In the context of mRNA or proteins, "isolate" refers to the recovery of mRNA or protein from a source, for example, cells.

[0096] The term “modulate” optionally includes “increasing” or “decreasing” one or more quantifiable parameters by a defined and / or statistically significant amount. “Increasing” or “enhancing,” “boosting” or “enhancing,” or “stimulating” or “stimulating” generally refers to the ability of one or more agents or compositions to produce or induce a greater physiological response (i.e., downstream effect) in a cell or subject compared to the response caused by the absence of the agent / compound or a control compound. The relevant physiological or cellular response (in vivo or in vitro) is obvious to those skilled in the art and may include an increase in skeletal muscle mass in a tissue or subject requiring it. The “increased” or “enhanced” amount is typically a “statistically significant” amount and may include increases of 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), including any integers and decimals between and above 1 (e.g., 1.5, 1.6, 1.7, 1.8), the amount produced without the drug / compound (absence of the drug) or by the control compound. The terms “decreased” or “inhibited” may generally refer to the ability of one or more drugs 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, as described herein, when measured by routine techniques of diagnostic technology. The relevant physiological or cellular response (in vivo or in vitro) may be obvious to those skilled in the art and may include a reduction or improvement in the symptoms or pathology of lung inflammation or ILD, as described herein. A “decrease” in response may be “statistically significant” compared to the response produced without the drug or composition, or by the control drug or composition, and may include decreases 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 a composition can be specifically defined. For example, a particular composition 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 intermediate minorities, 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.

[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 lipid-soluble molecules. The lipid core is stabilized by a surfactant (e.g., an emulsifier) ​​and may contain one or more of the following, or combinations thereof: triglycerides (e.g., tristearin), diglycerides (e.g., glycerol behenate), monoglycerides (e.g., glycerol monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate). Lipid nanoparticles are, for example, described by Petrilli et al., Curr Pharm. This is described in Biotechnol. 15:847-55, 2014, and in U.S. Patents No. 6,217,912, No. 6,881,421, No. 7,402,573, No. 7,404,969, No. 7,550,441, No. 7,727,969, No. 8,003,621, No. 8,691,750, No. 8,871,509, No. 9,017,726, No. 9,173,853, No. 9,220,779, No. 9,227,917, and No. 9,278,130 (these are incorporated in their entirety by reference).

[0099] As used herein, “nucleic acid base” (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 naturally occurring analogs of purines and pyrimidines that confer improved properties, such as binding affinity to oligonucleotides. Exemplary analogs include hypoxanthine (a base component of nucleoside inosine), 2,6-diaminopurine, 5-methylcytosine, C5-propynyl-modified pyrimidine, and 9-(aminoethoxy)phenoxazine (G-clamp).

[0100] Further examples of base-pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, guanine, and pyrimidine analogs such as hypoxanthine, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pseudoisocytosine, and pseudouracil, in which their respective amino groups are protected with an acyl protecting group, as well as other modified nucleic acid bases such as 8-substituted purines, 8-substituted xanthines, or 8-substituted hypoxanthines (the latter two being natural degradation products). 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 considered.

[0101] Further examples of base pairing moieties include, but are not limited to, size-expanded nucleic acid bases with one or more benzene rings added. Nuclear base 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 SA, et al., Nat. Rev. Genet., 2005, 6, 553-543, Romesberg, FE, et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733, and Hirao, I., Curr. Opin. Chem. Biol., 2006, 10, 622-627 are intended to be useful for the synthesis of oligonucleotides described herein. Examples of size-enlarged nucleic acid bases are shown below. [ka]

[0102] Nucleic acid bases covalently linked to ribose, sugar analogs, or morpholino contain nucleosides. A "nucleotide" consists of a nucleoside along with one phosphate group. The phosphate group covalently links to adjacent nucleotides, forming an oligomer.

[0103] The terms “polypeptide” and “protein” are used interchangeably herein and refer to polymers of amino acid residues, as well as their variants and synthetic analogs. Therefore, 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. Typically, these terms refer to nucleotides, ribonucleotides, or deoxynucleotides in polymeric form, or modified forms of any of these types, that are at least 10 nucleotides long. These terms include single-stranded and double-stranded forms of DNA. The terms “isolated DNA,” “isolated polynucleotide,” and “isolated nucleic acid” refer to isolated molecules that do not contain the whole-genome DNA of a particular species. Therefore, an isolated DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences but is isolated substantially apart from or purified from the whole-genome DNA of the species from which the DNA segment is obtained. Non-coding polynucleotides that do not encode polypeptides (e.g., primers, probes, oligonucleotides) are also included. Recombinant vectors are also included, such as expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, and viruses.

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

[0106] An "expression control sequence" includes a nucleic acid or a corresponding amino acid regulatory sequence, such as a promoter, leader, enhancer, intron, recognition motif for RNA, or DNA-binding protein, polyadenylation signal, terminator, internal ribosome entry site (IRES), secretory signal, intracellular localization signal, or intracellular or cellular region of a coding sequence in a host cell, all of which have the ability to influence transcription or translation. Exemplary expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

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

[0108] Numerous promoters, including structural, inducible, and repressive promoters, are well known in the art from a variety of different sources. 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 synthesized based on sequences publicly available from depositary institutions such as ATCC, as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiating transcription in one direction) or bidirectional (i.e., initiating 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, and the RSV promoter. Inducible promoters include the Tet system (US Patent Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci. (1996) 93(8):3346-3351, T-REx™ 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, US Patent No. 7,112,715), and Kramer & Fussenegger Methods. This includes Mol. Biol. (2005) 308:123-144) or any promoter known in the art that is suitable for desired intracellular expression.

[0109] "Expressable polynucleotides include cDNA, RNA, mRNA, or other polynucleotides comprising at least one coding sequence and optionally at least one expression control sequence, e.g., transcription and / or translation regulatory elements, which can express the encoded polypeptide (e.g., HRS polypeptide) upon introduction into cells, e.g., in a subject."

[0110] In some embodiments, the expressible polynucleotide is a modified RNA or modified mRNA polynucleotide, e.g., an RNA analog not found in nature. In certain embodiments, the modified RNA or mRNA polypeptide comprises one or more modified or unnatural bases, e.g., nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T), and / or uracil (U). In some embodiments, the modified mRNA comprises one or more modified or unnatural nucleotide linkages. Expressible RNA polynucleotides for delivering encoded therapeutic polypeptides are described, for example, in Kormann et al., Nat Biotechnol. 29:154-7, 2011, and U.S. Patent Applications 2015 / 0111248, 2014 / 0243399, 2014 / 0147454, and 2013 / 0245104, which are incorporated in their entirety by reference.

[0111] In some embodiments, various viral vectors that can be used to deliver expressible polynucleotides include adenovirus vectors, herpesvirus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, and retrovirus vectors. In some cases, the retrovirus vector is a derivative of a mouse or avian retrovirus vector, or a lentivirus vector. Examples of retrovirus vectors into which a single foreign gene may be inserted include, but are not limited to, Moloney mouse leukemia virus (MoMuLV), Harvey mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous sarcoma virus (RSV). Many further retrovirus vectors can incorporate multiple genes. All of these vectors can move or incorporate genes for selectable markers so that transduced cells can be identified and generated. For example, by inserting a target polypeptide sequence into the viral vector together with another gene encoding a ligand for a receptor on a specific target cell, the vector can achieve target specificity. Retroviral vectors can achieve target specificity, for example, by inserting polynucleotides that encode proteins. Exemplary targeting can be achieved by using antibodies that target retroviral vectors. Those skilled in the art know, or can easily identify, specific polynucleotide sequences that can be delivered to the retroviral genome, enabling target-specific delivery of retroviral vectors, without excessive experimentation.

[0112] In certain cases, the expressible polynucleotides described herein may be engineered to localize within a cell, potentially within a specific compartment such as the nucleus, or to be secreted from the cell or translocated to the cell's plasma membrane. In exemplary embodiments, the expressible polynucleotide is engineered for nuclear localization.

[0113] This specification also includes biologically active “variants” and “fragments” of polypeptides, as well as the polynucleotides encoding them. A “variant” contains one or more substitutions, additions, deletions, and / or insertions compared to a reference polypeptide or polynucleotide (see, e.g., Table and Sequence Listing). A variant polypeptide or polynucleotide comprises 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, similarity, or homology to a reference sequence as described herein, and substantially retains the activity of the reference sequence. The following also include sequences that consist of, or differ from, a 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, or substantially retain the activity of the 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” means, as used herein, the degree to which the sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis across a comparison window. Thus, “percentage of sequence identity” can be calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in both sequences where 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) are present, obtaining the number of matching positions, dividing this number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The optimal alignment of sequences for aligning a comparison window can be achieved by computerized execution of algorithms (GAP, BESTFIT, FASTA, and TFASTA in 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 produces the highest percentage homology across the comparison window) generated by any of the various methods of inspection and selection. References can also be made to programs of the BLAST family, such as those disclosed by 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. A commonly used measure of significance is the p-value, which is the frequency or probability of the observed event occurring if the null hypothesis were true. If the resulting p-value is smaller than the significance level, the null hypothesis is rejected. In a simple example, this significance level is defined by a p-value of 0.05 or less.

[0116] The term "solubility" refers to the property of a drug provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as concentration, by any of the following: mass of solute per unit volume of solvent (e.g., g, g / dL (100 mL), mg / ml of solute per 1 kg of solvent), molar concentration, gravimetric concentration, mole fraction, or any other similar description of concentration. The maximum equilibrium amount of solute that can dissolve per unit volume of solvent is the solubility of that solute in that solvent under specific conditions, including temperature, pressure, pH, and the properties of the solvent. In certain embodiments, solubility is measured at physiological pH or other pH levels, e.g., pH 5.0, pH 6.0, pH 7.0, or pH 7.4. In certain embodiments, solubility is measured in water or physiological buffer, e.g., PBS or NaCl (with or without NaP). In certain embodiments, solubility is measured at relatively low pH (e.g., pH 6.0) and relatively high salt (e.g., 500 mM NaCl and 10 mM NaP). In certain embodiments, solubility is measured in biological fluids (solvents), such as blood or serum. In certain embodiments, the temperature may be around room temperature (e.g., around 20, 21, 22, 23, 24, 25°C) or around body temperature (37°C). In certain embodiments, the drug 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] "Target" or "target that requires it" includes mammalian targets such as humans.

[0118] "Substantially" or "essentially" means almost entirely or completely, for example, 95% or more of a given quantity.

[0119] "Therapeutic response" refers to the improvement in symptoms (whether or not it is sustained) resulting from the 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 as described herein. A target may include coding and non-coding sequences, a 5' upstream sequence, a 3' downstream sequence, and other RNA sequences as described herein.

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

[0122] As used herein, “quantify,” “quantify,” or other related terms refer to determining the amount, mass, or concentration of nucleic acids, polynucleotides, oligonucleotides, peptides, polypeptides, or proteins in a unit volume.

[0123] As used herein, the terms “therapeutic effective dose,” “therapeutic dose,” “prophylactic effective dose,” or “diagnostic effective dose” refer to the amount of 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 the antisense or RNAi agent in the patient’s plasma or other tissue compartments (e.g., muscle tissue) above a minimum effective therapeutic level.

[0124] As used herein, “treatment” of an object (e.g., a mammal, e.g., a human) or 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, the administration of pharmaceutical compositions, and may be performed either prophylactically or after the onset of a pathological event or contact with a pathogen. “Prophylactic” treatment also includes, which may be aimed at slowing the rate of progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. “Treatment” or “prevention” does not necessarily imply the complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

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

[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, and expressible polynucleotides encoding the HRS polypeptide. Histidyl-tRNA synthetases belong to the class II tRNA synthetase family, which have three highly conserved sequence motifs. Class I and II tRNA synthetases are widely recognized as being responsible for the specific binding of amino acids to their homologous tRNAs in a two-step reaction: the amino acids (AA) are first activated by ATP to form AA-AMP, which is then transferred to the receptor end of the tRNA. Full-length histidyl-tRNA synthetases typically exist either as cytosolic homodimers or as alternatively spliced ​​mitochondrial forms.

[0127] Certain biological fragments or alternatively spliced ​​isoforms of eukaryotic histidyl-tRNA synthetases, or in some contexts, intact full-length synthetases, modulate certain therapeutically relevant cellular signaling pathways and / or possess 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 synthetases provided herein (e.g., HRS1-48, HRS1-60), can exert anti-inflammatory signals, among other things, by in vivo blocking the migration, activation, or differentiation of inflammatory cells (e.g., monocytes, macrophages, T cells, B cells, NK cells, dendritic cells) associated with the site of active inflammation. In addition, certain mutations or deletions (e.g., HRS1-506, HRS1-60) confer increased activity and / or improved pharmacological properties compared to the full-length HRS polypeptide sequence. Some specific exemplary HRS polypeptide sequences 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

[0128] Therefore, in certain embodiments, the HRS polypeptide comprises, consists of, or essentially comprises mammalian HRS amino acid sequences in Table H1 (e.g., SEQ ID NOs: 1-117) or their active variants or fragments. In some embodiments, the HRS polypeptide comprises, consists of, or essentially comprises human HRS amino acid sequences in Table H1 (e.g., SEQ ID NOs: 1-109) or their active variants or fragments. In some embodiments, the expressible polynucleotide encodes an HRS polypeptide comprising, consisting of, or essentially comprising amino acid sequences in Table H1 (e.g., SEQ ID NOs: 1-117), e.g., human HRS sequences in Table H1 (SEQ ID NOs: 1-109) or their active variants or fragments.

[0129] As described above, HRS polypeptides can be modified in various ways, including amino acid substitution, deletion, shortening, addition, and insertion. Methods for such operations are generally well known in the art. For example, amino acid sequence variants of HRS reference polypeptides can be prepared by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification 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, JD et al. ("Molecular Biology of the Gene", Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and the references cited therein. Guidance for selecting amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

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

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

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

[0133] Hydrophobicity: These residues are uncharged at physiological pH, and when the peptide is in an aqueous medium, they are repelled by the aqueous solution, determining the internal position of the peptide conformation containing these residues. 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 are not sufficiently repelled by aqueous solutions to determine the internal position of the peptide conformation containing these residues 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 explanation also characterizes certain amino acids as "small" because the side chains of those amino acids are not large enough to confer hydrophobicity even without a polar group. With the exception of proline, "small" amino acids are those with four or fewer carbon atoms if at least one polar group is present on the side chain, and three or fewer carbon atoms if none is present. Amino acids with small side chains include glycine, serine, alanine, and threonine. The gene-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 all other naturally occurring amino acids in that its side chain is bonded to the nitrogen and α-carbon of the α-amino group. Several amino acid similarity matrices are well known in the art (see, for example, the PAM120 matrix and the PAM250 matrix, disclosed, for example, in Dayhoff et al., 1978, A model of evolutionary change in proteins). However, the matrix for determining distance relationships, as presented by MODayhoff, (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), includes proline in the same group as glycine, serine, alanine, and threonine. Therefore, proline is classified as a "small" amino acid.

[0136] The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary, and therefore, the amino acids specifically intended by this invention are classified as either one or the other. Most amino acids that are not specifically named can be classified based on known behavior.

[0137] Amino acid residues can be further subdivided into cyclic or acyclic, aromatic or non-aromatic, trivial classifications with respect to the side-chain substituents of the residue, and 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 further polar substituents are present; otherwise, it is considered small if it contains three or fewer carbon atoms. Small residues are, of course, always non-aromatic. Depending on their structural characteristics, amino acid residues may belong to two or more classes. For naturally occurring protein amino acids, the subclassifications following this scheme are presented in Table A. [Table A-1] [Table A-2]

[0138] Conservative amino acid substitutions also include classifications based on side chains. For example, the group of amino acids with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains are serine and threonine; the group of amino acids with amide-containing side chains are asparagine and glutamine; the group of amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains are lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains are cysteine ​​and methionine. For example, substitutions of leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or similar substitutions of amino acids with structurally related amino acids will 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. Conservative substitutions are shown in Table B under the heading "Exemplary Substitutions." Amino acid substitutions that fall 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 bulkiness of the side chain, or (d) their effect on maintaining 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 conservative substitution can be grouped into three categories based on the nature of their side chains. (Zubay, G., Biochemistry, third) As described in 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, for example, one or more non-cysteine ​​residues being substituted with cysteine ​​residues (e.g., to alter stability, to facilitate thiol-based conjugate of Fc fragments, to facilitate thiol-based binding of PEG or other molecules). In some embodiments, one or more cysteine ​​substitutions are at the N-terminus and / or C-terminus of the HRS polypeptide, or in the vicinity of other surface-exposed regions of the HRS polypeptide. Certain embodiments include cases where one or more residues within amino acids 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 are substituted with cysteine ​​residues at the N-terminus and / or C-terminus of the HRS polypeptide. In some embodiments, cysteine ​​residues may be added to the HRS polypeptide via the creation of an N-terminal or C-terminal fusion protein. Such fusion proteins can be of any length, but are typically about 1–5, 5–10, 10–20, or 20–30 amino acids long.

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

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

[0143] In some embodiments, the HRS polypeptide may have variants in which endogenous or naturally occurring cysteine ​​residues are mutated to or deleted by alternative amino acids. 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 includes, for example, one or more substitutions and / or deletions in any one or more of 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, including combinations thereof.

[0144] Certain embodiments include HRS polypeptides from Table H1 having any one or more mutations or deletions from Cys83, Cys174, Cys191, Cys196, Cys224, Cys235, Cys379, and Cys455, or deletions of Cys507 and Cys509 due to the deletion of the three C-terminal amino acids (Δ507-509), for example. Exemplary mutations at these positions include, for example, mutations from cysteine ​​to serine, alanine, leucine, valine, or glycine. In certain embodiments, the amino acid residue for the specific cysteine ​​substitution may 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 embodiment, the cysteine ​​residues selected for substitution are selected from Cys224, Cys235, Cys507, and Cys509. In some embodiments, the last three (C-terminal) residues of SEQ ID NO: 1 are deleted to remove residues 507-509. In some embodiments, these cysteines are selected for mutation or deletion to exclude intramolecular cysteine ​​pairs, such as Cys174 and Cys191.

[0146] Specific examples of cysteine ​​mutations / substitutions (shown in bold and underlined) to reduce surface-exposed cysteine ​​residues include those listed below in Table H4. [Table H4-1] [Table H4-2]

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

[0148] In some embodiments, such cysteine ​​substitution variants are modified to manipulate, insert, or otherwise introduce a new surface-exposed cysteine ​​residue at a defined surface-exposed site, 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 a further cysteine ​​residue at the N-terminus or C-terminus of any of the reduced-cysteine ​​HRS polypeptides described above. In some embodiments, such N-terminus or C-terminus surface-exposed cysteine ​​insertions into the reduced-cysteine ​​variant of the HRS polypeptide include the re-insertion of the last one, last two, or last three naturally occurring C-terminal amino acids of the full-length human HRS, e.g., all or part of the sequence CIC (Cys Ile Cys). Exemplary reduced cysteine ​​variants include, for example, any combination of mutations (or deletions thereof) in residues Cys174, Cys191, Cys224, and Cys235 in any of the HRS polypeptides in Table H1, as well as deletions or substitutions of Cys507 and Cys509 (based on the numbering of full-length human cytoplasmic HRS (SEQ ID NO: 1)).

[0149] For several types of site-specific conjugates or binding to heterologous molecules such as the Fc region or PEG or other heterologous molecules, the HRS polypeptide may have one or more glutamine substitutions, where one or more naturally occurring (non-glutamine) residues are substituted with glutamine, for example, to facilitate the transglutaminase-catalyzed binding of the molecule to the glutamine amide group. In some embodiments, the glutamine substitution is introduced near the N-terminus and / or C-terminus of the HRS polypeptide. Certain embodiments include cases where one or more residues within amino acids 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 are substituted with glutamine residues near the N-terminus and / or C-terminus of the HRS polypeptide. These and related HRS polypeptides may also include substitutions (e.g., conservative substitutions) to remove any naturally occurring glutamine residues and, if desired, to modulate the degree of site-specific conjugation or binding.

[0150] For several types of site-specific conjugates or binding to heterologous molecules such as the Fc region or PEG or other heterologous molecules, the HRS polypeptide may have one or more lysine substitutions, where one or more naturally occurring (non-lysine) residues are substituted with lysine to facilitate binding, for example, based on acylation or alkylation of the molecule to the amide group of lysine. These methods also typically result in binding of the molecule to the N-terminal residue. In some embodiments, the lysine substitution is in the vicinity of the N-terminus and / or C-terminus of the HRS polypeptide. Certain embodiments include cases where one or more residues within amino acids 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 are substituted with lysine residues relative to the N-terminus and / or C-terminus of the HRS polypeptide. These and related HRS polypeptides may also include substitutions (e.g., conservative substitutions) to remove any naturally occurring lysine residues and, if desired, to modulate the degree of site-specific conjugation or binding.

[0151] Site-specific conjugation to HRS polypeptides 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 predicted solvent accessibility using the SPIDDER server (http: / / sppider.cchmc.org / ) with the published crystal structure of an exemplary HRS polypeptide (see Xu et al., Structure. 20:1470-7, 2012 and U.S. Patent Application No. 61 / 674,639). Based on this analysis, several amino acids on the surface can potentially be used as mutation sites to introduce 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. Therefore, in some embodiments, amino acid positions with a surface accessibility score greater than 40 can be used to introduce cysteine, lysine, glutamine, or other amino acids that do not exist naturally.

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

[0153] Certain embodiments involve site-specific conjugation or binding to an HRS polypeptide at any amino acid position by substituting a non-naturally occurring amino acid containing 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 may be inserted or substituted, for example, at the N-terminus and / or C-terminus of the HRS polypeptide as described herein, or at a solvent-accessible surface amino acid residue, in one or more residues within amino acids 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.

[0154] In certain embodiments, amino acids that do not exist in nature include, but are not limited to, any amino acid, modified amino acids, or amino acid analogs other than selenocysteine, and 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, and valine. The general structure of an alpha-amino acid is illustrated by the following formula: [ka]

[0155] Non-natural amino acids are typically any structure having the formula described above, where the R group is any substituent other than those used in the 20 natural amino acids. For the structures of the 20 natural amino acids, see, for example, biochemistry textbooks such as Biochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York. Note that the non-natural amino acids disclosed herein may be naturally occurring compounds other than the 20 alpha-amino acids described above. The non-natural amino acids disclosed herein typically differ from natural amino acids only in their side chains, so these non-natural amino acids form amide bonds with other natural or non-natural amino acids in the same manner as they are formed in naturally occurring proteins, for example. However, these non-natural amino acids have side chain groups that distinguish them from natural amino acids. For example, R in the above formula may optionally include 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, selen-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic-, pyridyl, naphthyl, benzophenone, cyclooctin, bound rings such as thioester, enone, imine, aldehyde, ester, thio acid, hydroxylamine, amino, carboxylic acid, alpha-ketocarboxylic acid, alpha- or beta-unsaturated acid and amide, glyoxylamide, or 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-methylphenylalanine, 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, Examples of such substances include, but are not limited to, 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, and those listed below or elsewhere in the present invention.

[0157] Therefore, it is possible to select naturally occurring amino acids that contain functional groups that form a covalent bond with any preferred functional group of the desired molecule (e.g., Fc region, PEG). Once selected, the non-natural amino acids can either be purchased from suppliers or chemically synthesized. Any number of non-natural amino acids can be incorporated into the target molecule, which may vary depending on the number of desired molecules to be conjugated. These molecules may be conjugated to all or some of the non-natural amino acids. Furthermore, the same or different non-natural amino acids may 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 may be incorporated into the HRS polypeptide, and any or all of them may be conjugated to molecules containing the desired functional groups.

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

[0159] Specific examples of amino acid analogs and mimetic compounds 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, NY (1983), the entire contents of which are incorporated herein by reference. Other examples include peralkylated amino acids, particularly permethylated amino acids. For example, see Combinatorial Chemistry, Eds. Wilson and Czarnik, Ch. 11, p. 235, John Wiley & Sons Inc., New York, NY (1997), the entire contents of which are incorporated herein by reference. Further examples include amino acids in which their amide portion (and therefore the amide backbone of the resulting peptide) is replaced, for example, by a sugar ring, steroid, benzodiazepine, or carbocycle. For example, see Burger's Medicinal Chemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620, John Wiley & Sons Inc., New York, NY (1995), the entire contents of which are incorporated herein by reference. Methods for synthesizing peptides, polypeptides, peptide mimes, and proteins are well known in the art (for example, U.S. Patent No. 5,420,109; M. Bodanzsky, Principles of Peptide Synthesis (1st ed. & 2d rev. ed.), Springer-Verlag, New York, NY (1984 & 1993), see Chapter 7; see Stewart and Young, Solid Phase Peptide Synthesis, (2d ed.), Pierce Chemical Co., Rockford, Ill. (1984), each of which is incorporated herein by reference).Therefore, HRS polypeptides can be composed of naturally occurring amino acids, naturally occurring amino acids, as well as amino acid analogs and mimetic compounds.

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

[0161] In a particular embodiment, this HRS polypeptide is approximately 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, 9 4, 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 acid lengths, at least approximately 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 acid lengths, and / or up to approximately 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 acid lengths, including all integers within that range, and including, consisting of, or essentially consisting of, amino acid sequences in Table H1, Table H2, or Table H4.

[0162] In certain embodiments, the HRS polypeptide has at least one non-canonical activity, such as anti-inflammatory activity, or cross-reactivity with autoantibodies or autoreactive T cells from subjects having a disease associated with an autoantibody against histidyl-tRNA synthetase (e.g., Jo-1 antibody). Assays for determining anti-inflammatory activity, including conventional measurement-based assays of cytokine release from in vitro cells and animal studies, are well-established in the art (see, for example, 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. An exemplary in vivo experimental setup is also described in the attached examples.

[0163] In some embodiments, the HRS polypeptide does not significantly compete for the binding of disease-related autoantibodies (e.g., Jo-1 antibody) to wild-type histidyl-tRNA synthetase in competitive ELISA up to concentrations of approximately 1–5 × 10⁻⁷ M or higher. Therefore, in some embodiments, the HRS polypeptide has a lower affinity for disease-related autoantibodies than wild-type histidyl-tRNA synthetase (SEQ ID NO: 1) when measured in competitive ELISA. In some embodiments, the HRS polypeptide has an apparent affinity for disease-related autoantibodies (e.g., Jo-1 antibody) that is at least approximately 10 times, at least approximately 20 times, at least approximately 50 times, or at least approximately 100 times lower than the affinity for disease-related autoantibodies to wild-type human (SEQ ID NO: 1).

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

[0165] In other embodiments, fusion proteins of HRS polypeptides 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, but are not limited to, fusions to human albumin (Osborn et al.: Eur.J.Pharmacol. 456(1-3):149-158, (2002)), antibody Fc domains, polyGlu or polyAsp sequences, and transferrin. Furthermore, fusions with conformationally disordered polypeptide sequences consisting of amino acids Pro, Ala, and Ser ("PAS-modified"), or with hydroxyethyl starch (marketed under the trademark HESYLATION®), provide a simple method for increasing the hydrodynamic volume of the HRS polypeptide. This additional elongation results in a bulky, random structure, which significantly increases the size of the resulting fusion protein. This means that the generally rapid clearance of smaller HRS polypeptides via renal filtration is delayed by several orders of magnitude. Furthermore, it has been shown that the use of IgG fusion proteins allows 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 HRS polypeptides include, for example, fusions to T cell-binding ligands, including MHC class I and II proteins, β-2 microglobulin, LFA-3 moieties, heavy chain Fc region moieties, and conjugates and derivatives thereof. Examples of such fusion proteins are described, for example, in EP1964854, U.S. Patents 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 synthetic or naturally occurring secretory signal sequences derived from other well-characterized secretory proteins. In some embodiments, such proteins can be processed by proteolytic cleavage to form the HRS polypeptide in situ. In some embodiments, the HRS polypeptide may include heterologous proteolytic cleavage sites to enable situ expression and production of the HRS polypeptide at intracellular or extracellular locations. Other fusion proteins may include, for example, the fusion of the HRS polypeptide to ubiquitin to provide a new N-terminal amino acid, or the use of secretory signals to mediate high-level secretion of the HRS polypeptide into an extracellular medium, or N-terminal or C-terminal epitope tags and fusion to cell-permeable peptides to improve purification or detection.

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

[0169] Certain embodiments include HRS-Fc conjugates comprising at least one Fc region covalently bonded to one or more HRS polypeptides. Examples of HRS-Fc conjugates include fusion proteins and various forms of chemically crosslinked proteins. Wild-type sequences from any number of species, as well as their variants, fragments, hybrids, and chemically modified forms, may be used in HRS-Fc conjugates. HRS-Fc polypeptides 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. It will be understood that in any of these HRS-Fc conjugates, native N or C-terminal amino acids of the HRS polypeptide, or 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 two proteins.

[0170] HRS-Fc conjugated polypeptides may offer various advantages compared to unconjugated or unmodified HRS polypeptides, such as the corresponding HRS polypeptide with the same or similar sequence but without the conjugated Fc region. For example, covalent conjugation of one or more Fc regions may alter (e.g., increase, decrease) the solubility, half-life (e.g., in serum, in selected tissue, in vitro under storage conditions, e.g., at room temperature or under refrigeration), dimerization or multimerization properties, and biological activity (one or multiple) of HRS polypeptides compared to unmodified HRS polypeptides with the same or similar sequences, by providing, for example, Fc region-related effector function (e.g., activation of the classical complement cascade, interaction with immunoeffector cells via Fc receptors (FcR), compartmentalization of immunoglobulins), cellular uptake, intracellular transport, tissue distribution, and / or bioavailability. In certain embodiments, the Fc region can confer effector functions related to complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or antibody-dependent cell-mediated phagocytosis (ADCP), which are thought to play a role in clearing specific target cells such as tumor cells and infected cells.

[0171] Certain embodiments utilize HRS-Fc fusion proteins. "Fusion protein," as well as methods for constructing fusion proteins, are defined elsewhere herein and are well known in the art (see, for example, U.S. Patents 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 relating to Fc fusion proteins). In HRS-Fc fusion proteins, the Fc region may be fused to the N-terminus, C-terminus, or both of the HRS polypeptide. In some embodiments, one or more Fc regions may be fused internally to an HRS sequence by, for example, positioning the Fc region between a first HRS sequence (e.g., a domain) and a second HRS sequence (e.g., a 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 contain different functional domains of the HRS polypeptide). A particular HRS-Fc fusion protein may also include further heterogeneous protein sequences, namely non-Fc regions and non-HRS polypeptide sequences.

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

[0173] In some embodiments, the HRS-Fc fusion protein may optionally comprise a tandem repeat copy 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 sequencing of specific tandem repeat HRS-Fc conjugates are illustrated in the examples. [Table H5]

[0174] Certain embodiments relate to HRS-Fc conjugates, for example, HRS-Fc conjugates in which one or more Fc regions are chemically conjugated or crosslinked to an HRS polypeptide. In these and related embodiments, the Fc regions can be conjugated to the HRS polypeptide in the N-terminal region (e.g., the first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 amino acids), the internal region (between the N-terminal and C-terminal regions), and / or the C-terminal region (e.g., within the last 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 amino acids). Polypeptides can be conjugated or crosslinked to other polypeptides according to various conventional techniques of the art. For example, certain techniques use carboxyl-reactive carbodiimide crosslinking agents EDC (or EDAC) that are covalently bonded via D, E, and C-terminal carboxyl groups. Other techniques use activated EDC that is covalently bonded via K and N-terminal amino groups. Other techniques use m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or sulfo-MBS that is covalently bonded via thiol groups of cysteine ​​residues (see also U.S. Patent Application No. 2007 / 0092940 for cysteine-operated Ig regions that may be used for thiol conjugates). Such crosslinked proteins may also include linkers that are cleavable or otherwise releaseable (e.g., enzymatically cleavable linkers, hydrolyzable linkers) and incleavable linkers (i.e., physiologically stable linkers). Certain embodiments may use non-peptide polymers (e.g., PEG polymers; HRS-N-PEG-N-Fc conjugate) as crosslinkers between the Fc region and the HRS polypeptide, as described, for example, in U.S. Patent Application No. 2006 / 0269553. For an exemplary description of the Fc region conjugate, see also U.S. Patent Application No. 2007 / 0269369.

[0175] In certain specific embodiments, as discussed in more detail below, variant or otherwise modified Fc regions may be used, including those having altered properties or biological activities compared to a wild-type Fc region. Examples of modified Fc regions include, for example, those having sequences mutated by one or more amino acid substitutions, insertions, deletions, or truncations compared to a wild-type sequence, hybrid Fc polypeptides consisting of domains from different immunoglobulin classes / subclasses, Fc polypeptides having altered glycosylation / sialylation patterns, and Fc polypeptides modified or derivatized by, for example, biotinylation (see, e.g., U.S. Patent Application No. 2010 / 0209424), phosphorylation, sulfation, or the like, or any combination of the foregoing. Such modifications can alter (e.g., increase or 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 , fluctuation), its immunogenicity, its complement fixation or activation, and / or the CDC / ADCC / ADCP-related activity 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 comprises the variable (V H ) and constant (CH1) domains of the heavy chain, and the variable (V L ) and constant (C LTogether with the domain, it forms an antigen-binding fragment or Fab region.

[0177] The Fc regions of IgG, IgA, and IgD immunoglobulins contain heavy chain constant domains 2 and 3, designated as CH2 and CH3 regions, respectively, while the Fc regions of IgE and IgM immunoglobulins contain heavy chain constant domains 2, 3, and 4, designated as CH2, CH3, and CH4 regions, respectively. The Fc regions are primarily responsible for immunoglobulin effector functions, including, for example, complement binding and binding to homologous Fc receptors on effector cells.

[0178] The hinge region (found in IgG, IgA, and IgD) acts as a flexible spacer, allowing the Fab portion to move freely in space relative to the Fc region. In contrast to the constant region, the hinge region is structurally diverse, varying in both sequence and length across immunoglobulin classes and subclasses. 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 its hinge region, conferring significant resistance of the hinge region polypeptide to intestinal proteases. Residues in the hinge proximal region of the CH2 domain can also influence the specificity of the interaction between immunoglobulins and their respective Fc receptors (see, e.g., Shin et al., Intern. Rev. Immunol. 10:177-186, 1993).

[0179] Accordingly, the terms “Fc region,” “Fc fragment,” or “Fc,” as used herein, refer to a protein comprising one or more CH2, CH3, and / or CH4 regions derived from one or more selected immunoglobulins, including those fragments, variants, and combinations. “Fc region” may also include one or more hinge regions of the heavy chain constant region of the immunoglobulin. In certain embodiments, the Fc region is the CH1, C of the immunoglobulin. L , V L, and / or V H It does not include one or more of the regions.

[0180] The Fc region may be derived from the CH2, CH3, CH4, and / or hinge regions of any one or more immunoglobulin classes, including but not limited to IgA, IgD, IgE, IgG, and IgM, including their subclasses and combinations. In some embodiments, the Fc region is derived from IgA immunoglobulin, including subclass IgA1 and / or IgA2. In certain embodiments, the Fc region is derived from IgD immunoglobulin. In certain embodiments, the Fc region is derived from IgE immunoglobulin. In some embodiments, the Fc region is derived from IgG immunoglobulin, including subclass IgG1, IgG2, IgG3, and / or IgG4. In certain embodiments, the Fc region is derived from IgM immunoglobulin.

[0181] A particular Fc region demonstrates specific binding to one or more Fc receptors (FcRs). Examples of the Fc receptor class include the Fcγ receptor (FcγR), Fcα receptor (FcαR), Fcε receptor (FcεR), and neonatal Fc receptor (FcRn). For example, a particular Fc region increases binding (or affinity for) to one or more FcγRs compared to FcαR, FcεR, and / or FcRn. In some embodiments, the Fc region increases binding to FcαR compared to one or more FcγR, FcεR, and / or FcRn. In other embodiments, the Fc region increases binding to FcαR (e.g., FcαRI) compared to one or more FcγR, FcαR, and / or FcRn. In certain embodiments, the Fc region increases binding to FcRn compared to one or more FcγR, FcαR, and / or FcεR. In a particular embodiment, the binding (or affinity) of an Fc region to one or more selected FcRs increases by typically about 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times or more (including all integers in between).

[0182] Examples of FcγR include FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. FcγRI(CD64) is expressed on macrophages and dendritic cells and plays a role in phagocytosis, respiratory bursts, cytokine stimulation, and dendritic cell endocytotic transport. FcγRI expression is upregulated by both GM-CSF and γ-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 bursts, and cytokine stimulation. FcγRIIa expression 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 the immune receptor tyrosine-based activation motif (ITAM)-mediated response and is therefore an inhibitory receptor. FcγRIIc expression 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 bursts, cytokine stimulation, platelet aggregation and degranulation, as well as NK-mediated ADCC. FcγRIII expression is upregulated by C5a, TGF-β, and γ-IFN and downregulated by IL-4. FcγRIIIb is a GPI-bound receptor expressed on PMNs.

[0183] Certain Fc regions increase binding to FcγRI compared to FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb. Some embodiments increase binding to FcγRIIa compared to FcγRI, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb. Certain Fc regions increase binding to FcγRIIb compared to FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb. Certain Fc regions increase binding to FcγRIIc compared to FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and / or FcγRIIIb. Some Fc regions increase binding to FcγRIIIa compared to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, and / or FcγRIIIb. Certain Fc regions increase binding to FcγRIIIb 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 multi-chain 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 and is expressed on epithelial Langerhans cells, eosinophils, mast cells, and basophils, playing a major role in regulating 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 type C lectin that can function as a membrane-bound or soluble receptor. FcεRII regulates B cell proliferation and differentiation and blocks IgE binding on eosinophils, monocytes, and basophils. Certain Fc regions increase binding to FcεRI compared to FcεRII. Other Fc regions increase binding to FcεRII compared to FcεRI. Table H6 below summarizes the characterization of a particular FcR. [Table H6-1] [Table H6-2]

[0186] The Fc region can be derived from immunoglobulin molecules of any animal, including vertebrates such as cattle, goats, pigs, dogs, mice, rabbits, hamsters, rats, guinea pigs, non-human primates, and mammals such as humans. The amino acid sequences of the CH2, CH3, CH4, and hinge regions 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] Therefore, the Fc region of the HRS-Fc conjugate may contain, consist of, or essentially consist of one or more of the human Fc region amino acid sequences in Table H7, including their variants, fragments, homologs, orthologues, paralogs, and combinations. Certain exemplary embodiments include an Fc region whose size is in the range of approximately 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 acid lengths, and optionally include, consist of, or essentially consist of, any one or more of the sequences in Table H7. Certain embodiments include Fc regions of up to approximately 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, and optionally include, consist of, or essentially consist of, any one or more sequences in Table H7.

[0188] A particular Fc region contains, consists of, or essentially consists of, the human IgA1 sequences from Table H7, including their combinations, variants, and fragments, in any order read from the N-terminus to the C-terminus. A particular Fc region contains, consists of, or essentially consists of, the human IgA1 sequences from Table H7. A particular Fc region contains, consists of, or essentially consists of, the human IgA1 sequences from Table H7. A particular Fc region contains, consists of, or essentially consists of, the human IgA1 sequences from Table H7.

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

[0190] A particular Fc region contains, consists of, or essentially consists of, the human IgD sequences from Table H7, including their combinations, as well as variants and fragments of these sequences and combinations, in any order read from N-terminus to C-terminus. A particular Fc region contains, consists of, or essentially consists of, the human IgE sequences from Table H7, including their combinations, as well as variants and fragments of these sequences and combinations, in any order read from N-terminus to C-terminus. A particular Fc region contains, consists of, or essentially consists of, the human IgG1 sequences from Table H7, including their combinations, as well as variants and fragments of these sequences and combinations, in any order read from N-terminus to C-terminus. A particular Fc region contains, consists of, or essentially consists of, the human IgG2 sequences from Table H7, including their combinations, in any order read from N-terminus to C-terminus. A particular Fc region contains, consists of, or essentially consists of, the human IgG3 sequences from Table H7, including their combinations, in any order read from N-terminus to C-terminus. A particular Fc region contains, consists of, or essentially consists of, human IgG4 sequences from Table H7, including combinations thereof, in any order read from N-terminus to C-terminus. A particular Fc region contains, consists of, or essentially consists of, human IgM sequences from Table H7, including combinations thereof, as well as variants and fragments of these sequences and combinations, in any order read from N-terminus to C-terminus.

[0191] An exemplary HRS-Fc fusion conjugate is provided in Table H8 below. [Table H8-1] [Table H8-2] [Table H8-3] [Table H8-4] [Table H8-5] [Table H8-6]

[0192] Accordingly, in certain specific 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 set forth in Table H8 (SEQ ID NOs: 157 to 172) 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 set forth in Table H8 (e.g., SEQ ID NOs: 157 to 172) or active variants or fragments thereof.

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

[0194] A hybrid Fc region is an Fc region that includes a combination of Fc domains (e.g., hinge, CH2, CH3, CH4) from immunoglobulins of different species, different Ig classes, and / or different Ig subclasses.Common examples include the following combinations of CH2 / CH3 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 / Ig E, 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, I gE / 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 / I gG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / Ig The hybrid Fc region includes, consists of, or essentially consists of M, IgM / IgA1, IgM / IgA2, IgM / IgD, IgM / IgE, IgM / IgG1, IgM / IgG2, IgM / IgG3, IgM / IgG4, IgM / IgM (or fragments or variants thereof), and optionally includes a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, or IgG4, and / or a CH4 domain from IgE and / or IgM. In certain embodiments, this hinge, CH2, CH3, and CH4 domains are from human Ig.

[0195] Further examples include the following combinations of CH2 / CH4 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 (and The hybrid Fc region includes, consists of, or essentially consists of (fragments or variants thereof), and optionally includes a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4, and / or a CH3 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In certain embodiments, the hinge, CH2, CH3, and CH4 domains are from human Ig.

[0196] In certain specific examples, the following combinations of CH3 / CH4 domains are: 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 (and The hybrid Fc region includes, consists of, or essentially consists of (fragments or variants thereof), and optionally includes a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4, and / or a CH2 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In certain embodiments, the hinge, CH2, CH3, and CH4 domains are from human Ig.

[0197] Specific examples include the following combinations of hinge / CH2 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 / I gM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA 2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, Ig G2 / 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 / IgG 2. A hybrid Fc region comprising, consisting of, or essentially consisting of, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), optionally comprising a CH3 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or a CH4 domain from IgE and / or IgM. In certain embodiments, the hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0198] In certain specific cases, the following combinations of hinge / CH3 domains are possible: 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 / Ig A2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, I gG2 / 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 / Ig The hybrid Fc region includes, consists of, or essentially consists of G2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), and optionally includes a CH2 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or a CH4 domain from IgE and / or IgM. In certain embodiments, the hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0199] Some examples include hybrid Fc regions containing, consisting of, or essentially consisting of, the following hinge / CH4 domain combinations: 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), and optionally containing a CH2 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or a CH3 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM.

[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] Derivatized or otherwise modified Fc regions are also included. In certain embodiments, the Fc region may be modified, for example, by phosphorylation, sulfation, acrylication, glycosylation, methylation, farnesylation, acetylation, amidation, etc., compared to the wild-type or naturally occurring Fc region. In certain embodiments, the Fc region may include the wild-type or naturally occurring glycosylation pattern, or may include increased glycosylation compared to the natural form, decreased glycosylation compared to the natural form, or may be completely deglycosylated. As an example of a modified Fc glycoform, decreased glycosylation of the Fc region reduces the binding of the first complement component C1 to the C1q region, the decrease in ADCC-related activity, and / or the decrease in CDC-related activity. Therefore, certain embodiments use deglycosylated or nonglycosylated Fc regions. For example, see WO2005 / 047337 for the generation of an exemplary nonglycosylated Fc region. Another example of an Fc region glycoform can be generated by substituting a cysteine ​​residue at position Q295 according to the numbering system of Kabat et al. (see, for example, U.S. Patent Application No. 2010 / 0080794). Certain embodiments may include an Fc region in which approximately 80–100% of the glycoprotein in the Fc region contains a mature core carbohydrate structure lacking fructose (see, for example, U.S. Patent Application No. 2010 / 0255013). Some embodiments may 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, for example, U.S. Patent Application No. 2010 / 0249382 and U.S. Patent Application No. 2007 / 0148170).

[0202] Another example of a modified Fc glycoform is an Fc region which may contain an oligomannose-type N-glycan and optionally have one or more of the following: increased ADCC activity compared to the corresponding Fc region or HRS-Fc conjugate containing a complex-type N-glycan; increased binding affinity to FcγRIIIA (and certain other FcR); similar or increased binding specificity to targets of HRS polypeptides; similar or higher binding affinity to targets of HRS polypeptides; and / or similar or lower binding affinity to mannose receptors (see, for example, U.S. Patent Application No. 2007 / 0092521 and U.S. Patent No. 7,700,321). As another example, enhanced affinity of the Fc region to FcγR has been achieved using manipulated glycoforms generated by antibody expression in manipulated or variant cell lines (see, for example, Umana et al., Nat Biotechnol. 17:176-180, 1999; Davies et al., Biotechnol Bioeng. 74:288-294, 2001; Shields et al., J BiolChem. 277:26733-26740, 2002; Shinkawa et al., 2003; J BiolChem. 278:3466-3473, 2003; and U.S. Patent Application No. 2007 / 0111281). Certain Fc region glycoforms include an increased proportion of N-glycosidic complex glycans that lack the fucose at position 1 of N-acetylglucosamine at position 6 at the reducing end of the glycan (see, for example, U.S. Patent Application No. 2010 / 0092997). Certain embodiments may include an Fc region of IgG glycosylated by at least one galactose moiety linked 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 of these and related modified glycosylation approaches result in a substantial enhancement of the Fc domain's ability to selectively bind to FcR such as FcγRIII, its ability to mediate ADCC, and its ability to modify other properties of the Fc domain, as described herein.

[0203] A particular variant, fragment, hybrid, or otherwise modified Fc region may 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 an Fc region may 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, a variant, fragment, hybrid, or modified Fc region may 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 in this specification.

[0204] Specific examples of Fc variants with altered (e.g., increased, decreased) FcR binding can be found, for example, in U.S. Patents Nos. 5,624,821 and 7,425,619, U.S. Patent Applications Nos. 2009 / 0017023, 2009 / 0010921, and 2010 / 0203046, as well as WO2000 / 42072 and WO2004 / 016750. Certain examples include human Fc regions having one or more substitutions at positions 298, 333, and / or 334, e.g., S298A, E333A, and / or K334A (based on the EU index numbering by Kabat et al.), which have been shown to increase binding to the activating receptor FcγRIIIa and decrease binding to the inhibitory receptor FcγRIIb. These mutations can be combined to obtain double and triple mutant variants with further improvements in 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 BiolChem. 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, such as those disclosed above by Umana et al. and in U.S. Patent No. 7,662,925. Some embodiments include an Fc region that includes one or more substitutions selected from 434S, 252Y / 428L, 252Y / 434S, and 428L / 434S (see U.S. Patent Applications No. 2009 / 0163699 and No. 2006 / 0173170) based on the EU index numbering by Kabat et al.

[0205] A particular variant, fragment, hybrid, or modified Fc region may have altered effector function compared to the corresponding wild-type Fc sequence. For example, such an Fc region 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 an Fc region 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 compared to the corresponding wild-type Fc sequence. As merely illustrative examples, an Fc region may include deletions or substitutions at complement binding sites, such as C1q binding sites, and / or deletions or substitutions at ADCC sites. 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, for example, Zuckerman et al., CRC Crit Rev Microbiol. 7:1-26, 1978). Useful effector cells for such assays include, but are not limited to, natural killer (NK) cells, macrophages, and other peripheral blood mononuclear cells (PBMCs). Alternatively, certain Fc effector functions can be evaluated in vivo by using animal models, for example, 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 an Fc region may have an increased half-life compared to the corresponding wild-type Fc sequence. In other embodiments, a variant hybrid or modified Fc region may have a decreased half-life compared to the corresponding wild-type Fc sequence. 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 measured in one or more bodily fluids, including blood, serum, plasma, urine, or cerebrospinal fluid, or in a given tissue, such as the liver, kidney, muscle, central nervous system tissue, or bone. As an example, modifications to an Fc region that alter its ability to bind to FcRn may alter its half-life in vivo. Assays for measuring in vivo pharmacokinetic properties (e.g., mean in vivo elimination half-life), and non-limiting examples of Fc modifications that alter their binding to FcRn are described, for example, in U.S. Patent Nos. 7,217,797 and 7,732,570; and U.S. Patent Applications Nos. 2010 / 0143254 and 2010 / 0143254.

[0207] Further non-limiting examples of modifications to alter stability or half-life include substitutions / deletions in one or more amino acid residues selected from 251–256, 285–290, and 308–314 in the CH2 domain, and 385–389 and 428–436 in the CH3 domain, according to the numbering system of Kabat et al. See U.S. Patent Application No. 2003 / 0190311. Specific examples include any combination thereof, substitution by leucine at position 251, substitution by tyrosine, tryptophan, or phenylalanine at position 252, substitution by threonine or serine at position 254, substitution by arginine at position 255, substitution by glutamine, arginine, serine, threonine, or glutamate at position 256, substitution by threonine at position 308, substitution by proline at position 309, substitution by serine at position 311, substitution by aspartate at position 312, substitution by leucine at position 314, and 38 This includes substitutions at position 5 with arginine, aspartate, or serine; substitutions at position 386 with threonine or proline; substitutions at position 387 with arginine or proline; substitutions at position 389 with proline, asparagine, or serine; substitutions at position 428 with methionine or threonine; substitutions at position 434 with tyrosine or phenylalanine; substitutions at position 433 with histidine, arginine, lysine, or serine; and / or substitutions at position 436 with histidine, tyrosine, arginine, or threonine. Such modifications optionally increase the affinity of the Fc region to 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, variant hybrids or modified Fc regions may have decreased solubility compared to the corresponding wild-type Fc sequence. Solubility may be measured, for example, in vitro (e.g., under physiological conditions) according to conventional techniques in the art. Exemplary solubility measurements are described elsewhere in this specification.

[0209] Further examples of variants include Fc regions of IgG having conserved or non-conserved substitutions (as described elsewhere herein) at one or more of the 250, 314, or 428 positions of the heavy chain, or any combination thereof, for example, at positions 250 and 428, or 250 and 314, or 314 and 428, or 250, 314, and 428 (see, for example, 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 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., conserved or non-conserved substitutions, deletions). In certain embodiments, the CH2 domain of an IgG Fc variant includes amino acid substitutions at positions 228, 234, 235, and / or 331 to attenuate the effector function of the Fc region (e.g., human IgG4 with 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 (Kabat et al., “Sequences of Proteins of Immunological Interest,” 5 thSee Ed., National Institutes of Health, Bethesda, Md. (1991). Certain embodiments of these and related embodiments optionally alter (e.g., increase, decrease) FcRn binding and / or serum half-life without reducing effector activity such as ADCC-related activity or CDC-related activity.

[0210] Further examples include variant Fc regions containing one or more amino acid substitutions at positions 279, 341, 343, or 373 of the wild-type Fc region, or any combination thereof (see, for example, U.S. Patent Application No. 2007 / 0224188). For human IgG, the wild-type amino acid residues at these positions are valine (279), glycine (341), proline (343), and tyrosine (373). The substitutions may be conserved or non-conserved, or may include amino acids or mimics that do not exist naturally, as described herein. In addition to these substitutions, certain embodiments may also use variant Fc regions comprising 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, 2 45R, 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, 25 4R, 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, 279 F, 279G, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R, 279S, 279T, 279W, 279Y, 280T, 283F, 283G, 283H, 283I, 2 83K, 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, 3 76L, 376M, 376N, 376P, 376Q, 376R, 376S, 376T, 376V, 376W, 376Y, 377G, 3 77K, 377P, 378N, 379N, 379Q, 379S, 379T, 380D, 380N, 380S, 380T, 382D, 3 82F, 382H, 382I, 382K, 382L, 382M, 382N, 382P, 382Q, 382R, 382S, 382T, 3 82V, 382W, 382Y, 385E, 385P, 386K, 423N, 424H, 424M, 424V, 426D, 426L, 42 7N, 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 is as follows:This is a numbering system for EU indicators (see Kabat et al., 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 bound. Preferably, the altered effector function is an increase or decrease in ADCC, an increase or decrease in CDC, an increase or decrease in Clq binding affinity, an increase or decrease in FcR (preferably FcRn) binding affinity, or an increase or decrease in FcR (preferably FcRn) binding affinity compared to the corresponding Fc region lacking such amino acid substitution.

[0211] Further examples include 221st, 222nd, 224th, 227th, 228th, 230th, 231st, 223rd, 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 241st, 243rd, 244th, 245th, 246th, 247th, 249th, 250th, 258th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272nd, 273rd, The variant Fc region includes an amino acid substitution at one or more of the following positions: 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, for example, U.S. Patent No. 7,662,925). In certain embodiments, the variant Fc region is 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 The molecule contains at least one amino acid substitution selected from the group consisting of 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 specific embodiments, variant Fc region is 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 / I 332D, S239N / I332E, S239Q / I332D, A330Y / I332E, V264I / A330Y / I332E, A330L / I332E, V264I / A330L / I332E, L234E, L234Y, L234I, L235D, L2 35S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T, L328D / I332E, L328V / I332E, L328T / I332E, L328I / I332E, S239E / V264I / I332E, S23 9Q / V264I / I332E, S239E / V264I / A330Y / I332E, S239D / A330Y / I332E, S239N / A330Y / I332E, S239D / A330L / I332E, S239N / A330L / I332E, V264 I / 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, It contains at least one amino acid substitution selected from the group consisting of 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. Furthermore, in certain embodiments, this variant Fc region is N297D / I332E, F241Y / F243Y / V262T / V264T / N297D / I332E,This includes 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 contains an amino acid substitution at position 332 (using the EU index, Kabat et al., numbering described 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 the EU index numbering by 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, 320 R, 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, 3 91C, 393A, 394A, 399G, 404S, 408G, 409R, 411I, 412A, 414M, 421S, 422I, 426F / P, 428T, 430K, 431S, 432P The variant Fc region includes one or more of 433P, 438L, 439E / R, 440G, 441F, 442T, 445R, 446A, and 447E, where optionally, this variant has altered recognition and / or altered effector function of the Fc ligand compared to the parent Fc polypeptide, and the residue numbering is the EU index numbering by Kabat et al. Specific examples of these and related embodiments include the following set 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, P24 7L, 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,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, (14) L306I, K326R, and Q362L, (15) H224Y, P230S, V323A, E333D, K338R, and S364C, (16) T335I, K414M, and P445R, (17) T335I and K414M, (18) P247A, E258K, D280N, K288R, N297D, T299A, K322E, Q342R, S354A, and L365P 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, The variant Fc region includes F372L, A378T, N390D, Y391C, F404S, E430K, L432P, and K447E, as well as (23)E269G, Y278H, N325S, and K370R, where the residue numbering is the EU index numbering by Kabat et al. (see, for example, U.S. Patent Application No. 2010 / 0184959).

[0213] Another specific example of Fc variants comprises the Fc sequence of Table H7, wherein Xaa at position 1 is Ala or is 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 is absent (see, for example, U.S. Patent Application No. 2007 / 0253966). Certain of these Fc regions and related HRS-Fc conjugates have increased half-life, reduced effector activity, and / or significantly lower immunogenicity than wild-type Fc sequences.

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

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

[0216] Therefore, 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 larger than the corresponding unmodified or differently modified HRS polypeptide when administered to mammals under the same or comparable conditions. In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide exhibits at least 10%, 20%, 30%, 40%, 500%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% greater stability (e.g., measured by half-life) compared to the corresponding unmodified or differently modified HRS polypeptide when compared in PBS at pH 7.4 under similar conditions at room temperature, for example, over approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or 1, 2, 3, 4 weeks.

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

[0218] In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has greater bioavailability after subcutaneous (SC) administration compared with the corresponding unmodified HRS polypeptide. In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has bioavailability of 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 compared with 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 by 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 anti-inflammatory activity assay. In other embodiments, the HRS-Fc fusion polypeptide has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more than 20 times the activity of the corresponding unmodified or differently modified HRS polypeptide in an anti-inflammatory activity assay.

[0220] In certain embodiments, a peptide linker sequence may 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 a peptide linker sequence may be incorporated into a conjugate or fusion protein using standard techniques well known in the art.

[0221] A particular peptide linker sequence may 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 and second polypeptides; (3) physiological stability; and (4) the absence of hydrophobic or charged residues that can react with functional epitopes on the polypeptides, or other characteristics. See, for example, George and Heringa, J Protein Eng. 15:871-879, 2002.

[0222] Linker sequences can generally be 1 to about 200 amino acids long. Specific linkers can be about 1-200 amino acids, 1-150 amino acids, 1-100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, It may have the total amino acid length of 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] Peptide linkers may be any one or more naturally occurring amino acids, non-naturally occurring amino acids, amino acid analogs, and / or amino acid mimes described elsewhere in this specification and known in the art. Certain amino acid sequences that may be useful 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 Nos. 4,935,233 and 4,751,180. Certain peptide linker sequences include Gly residues, Ser residues, and / or Asn residues. Other near-neutral amino acids such as Thr and Ala may also be used in peptide linker sequences if desired.

[0224] Certain exemplary linkers include Gly, Ser, and / or Asn-containing linkers such as [G] x [S] x [N] x [GS] x [GGS] x [GSS] x [GSGS] x (Sequence No. 173), [GGSG] x (Sequence No. 174), [GGGS] x (Sequence ID 175), [GGGGS] x (Sequence ID 176), [GN] x [GGN] x [GNN] x [GNGN] x (Sequence No. 177), [GGNG] x (Sequence No. 178), [GGGN] x (Sequence No. 179), [GGGGN] x (Sequence ID 180) contains a linker, in the formula, x These are 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 are obvious 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-Gly-Ser-Gly-Gly-Gly-(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-(Sequence ID 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-(Sequence ID 184), and Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Arg-(Sequence ID 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). nThis includes (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), LQRRDGERP (SEQ ID NO: 192), LRQKDGGGSERP (SEQ ID NO: 193), and LRQKd(GGGS)2ERP (SEQ ID NO: 194). In certain embodiments, this linker sequence includes a Gly3 linker sequence containing three glycine residues. In certain embodiments, flexible linkers can be rationally designed using computer programs 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.

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

[0228] In certain embodiments, the linker peptide includes an autocatalytic or autocleavable peptide cleavage site. In a particular embodiment, the autocleavable peptide includes polypeptide sequences obtained from potivirus and cardiovirus 2A peptide, FMDV (foot-and-mouth disease virus), equine rhinitis A virus, Thosea asigna virus, and porcine teishouvirus. In a particular embodiment, the autocleavable polypeptide site includes a 2A or 2A-like site, sequence, or domain (Donnelly et al., J. Gen. Virol. 82:1027-1041, 2001). The example 2A site includes 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, the autocatalytic peptide cleavage site includes a translation 2A signal sequence, such as the 2A region of the aft-virus 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 insect virus polyproteins, the NS34 protein of rotavirus type C, and repeat sequences in Trypanosoma spp., as described, for example, by Donnelly et al., Journal of General Virology. 82:1027-1041, 2001.

[0229] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., J.Gener.Virol.78:699-722, 1997 and Scymczak et al., Nature Biotech.5:589-594, 2004). Examples of protease cleavage sites include, but are not limited to, those of potivirus NIa protease (e.g., tobacco eczema virus protease), potivirus HC protease, potivirus P1 (P35) protease, biovirus NIa protease, biovirus RNA-2 encoding protease, aftovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungrosspheric virus) 3C-like protease, PYVF (parsnip's macular virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to its high cleavage stringency, some embodiments include TEV (tobacco eczema 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), where X represents any amino acid (cleavage by TEV occurs between Q and G or between Q and S).

[0230] Further examples of enzymatically cleavable linkers suitable for use in specific 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 degradable 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), where Z is an amino acid. 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), where Z is an amino acid. One 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 specific embodiments also include amino acid sequences that can be cleaved by angiotensin-converting enzyme, such as -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro- (SEQ ID NO: 230), and -Gly-Ser-Asp-Lys-Pro- (SEQ ID NO: 231).

[0233] Enzymatically degradable linkers suitable for use in specific embodiments also include amino acid sequences that can be degraded 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 releaseable 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 longer, or any intervening 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). Those skilled in the art will understand that the half-life of the HRS-Fc conjugate polypeptide can be precisely individualized by using a specific releaseable 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, then linker sequences may not be required.

[0236] HRS polypeptides and polynucleotides, such as expressible polynucleotides, may be used in any of the compositions, methods, and / or kits described herein.

[0237] Immunomodulators Certain embodiments utilize one or more immunomodulators. Exemplary immunomodulators include small molecules, polypeptides, such as antibodies and their antigen-binding fragments, ligands, small amounts of peptides, antisense agents, RNAi agents, and mixtures thereof.

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

[0239] In some embodiments, the immunomodulator is pirfenidone, which is often used for the treatment of idiopathic pulmonary fibrosis (IPF). Pirfenidone has anti-fibrotic and anti-inflammatory properties in various in vitro and animal models of fibrosis. For example, cell-based studies have shown that pirfenidone reduces fibroblast proliferation, inhibits TGF-β-stimulated collagen production, and reduces the production of fibrosis mediators such as TGF-β. Pirfenidone has also been shown to reduce 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 in 801 mg oral dose units (three 267 mg capsules), taken orally three times daily, for a total oral dose of 2403 mg / day. Further exemplary doses of pirfenidone are described herein.

[0240] In some embodiments, the immunomodulator 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 IPF disease and delay the decline of lung function by blocking signaling pathways involved in the fibrotic process. Nintedanib is formulated as a salt containing ethanesulfonic acid. In the United States, nintedanib is approved for the treatment of IPF in oral dose units of 150 mg taken twice daily for a total of 300 mg / day, which can reduce side effects compared to a dose of approximately 100 mg taken twice daily for a total of 200 mg / day. Further exemplary doses of nintedanib are described herein.

[0241] In some embodiments, the immunomodulator is a sphingosine-1-phosphate (S1P) and / or S1P receptor (S1PR) modulator. Common 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 a variety of biological functions, including cell proliferation, differentiation, angiogenesis, chemotaxis, and transport. Many of the activities of S1P act via five closely related G protein-coupled receptors of the sphingosine-1-phosphate receptor family (S1PR), which play important roles in sphingolipid metabolism. S1PR includes S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5. The expression of these receptors differs as follows: S1PR1, S1PR2, and S1PR3 are expressed in a wide variety of cell types, but are mainly expressed in large quantities on leukocytes; S1PR4 is mainly expressed in lymphocytes and hematopoietic tissues; and S1PR5 is mainly expressed in the white matter of the spleen and central nervous system (CNS).

[0242] Specific non-limiting examples of S1P or S1PR modulators include amicelimod (also known as MT-1303; S1PR antagonist; see Kappos et al., Lancet Neurol 2016;15:1148-59, 2016), fingolimod (S1PR1 functional antagonist), sonepcizumab (S1P-specific monoclonal antibody), KRP203 (S1PR1 agonist), SEW2871 (S1PR1 agonist), siponimod (S1PR1 and S1PR5 modulator), RPC1063 (S1PR1 modulator), and ONO-4641 (S1PR1 modulator). This includes S1P (S1PR5 agonist), JTE-013 (S1PR2 antagonist), GSK2018682 (S1PR1 agonist), Ponesimod (S1PR1 agonist), Slamin (selective S1PR3 and S1PR5 antagonist), VPC23019 (aryl-amide analog; competitive S1PR1 and S1PR3 antagonist); and W146 (selective S1PR1 antagonist). In certain embodiments, the S1P or S1PR modulator is an amicerimod.

[0243] A particular S1P or S1PR modulator includes an antibody or antigen-binding fragment or small molecule that specifically binds to S1P or S1PR (see, for example, sonepcizumab that binds to S1P). In some embodiments, the antibody or its antigen-binding fragment is an S1P and / or S1PR antagonist. In certain embodiments, the antibody or its antigen-binding fragment is an S1P and / or S1PR agonist.

[0244] Certain S1PR antagonists or inhibitors include antisense and RNAi agents against the S1PR coding sequence (see, e.g., acceptance 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, the target region being selected from one or more of the following: 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-treated mRNA, a branch point, the 3' untranslated region (UTR), and a polyadenylation signal sequence. Certain RNAi agents include a sense strand substantially identical to the mRNA target sequence encoding S1PR, and optionally, an antisense strand complementary or substantially complementary to the mRNA target sequence encoding S1PR.

[0245] In some embodiments, the immunomodulator is a steroid or corticosteroid, such as 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 immunomodulator 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 activated T cell nuclear factor (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, antigen-binding fragments, or small molecules that specifically bind to calcineurin. Antisense and RNAi agents against calcineurin coding sequences or their subunits are also included (see, for example, acceptance 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, for example, the target region being selected from one or more of the following: 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-treated mRNA, a branch point, the 3' untranslated region (UTR), and a polyadenylation signal sequence. A particular RNAi agent comprises a sense strand substantially identical to an mRNA target sequence encoding calcineurin or a subunit thereof, and optionally an antisense strand complementary to or substantially complementary to the mRNA target sequence encoding calcineurin or a subunit thereof.

[0248] In some embodiments, immunomodulators are mechanistic target (mTOR) antagonists or inhibitors of rapamycin. mTOR is a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases and is a catalytic subunit of two structurally distinct complexes: mTORC1 and mTORC2, which localize to different intracellular compartments and therefore particularly influence 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 insulin receptors and insulin-like growth factor 1 receptors. mTORC2 is also involved in the regulation and maintenance of the actin cytoskeleton. mTOR plays a role in fibrosis and autoimmunity, and blocking the mTORC pathway is being studied as a treatment for such diseases.

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

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

[0251] In some embodiments, the immunomodulator 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, induce and activate Treg and bone marrow-derived suppressor cells, and promote tumor angiogenesis. Specific examples of IDO antagonists or inhibitors include indoximod (NLG-8189), 1-methyltryptophan (1MT), β-carbolin (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epcadostat (e.g., Sheridan, Nature). See Biotechnology.33:321-322, 2015. Certain IDO antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to IDO (see, e.g., Platten et al., Front Immunol.5:673, 2014). Antisense agents and RNAi agents against IDO-coding sequences are also included (see, e.g., acceptance number AH002828.2). Certain antisense agents specifically hybridize to a target region within a pre-mRNA or mRNA target sequence encoding IDO, for example, the target region being selected from one or more of the following: the AUG start codon of mRNA, the region upstream of the AUG start codon, the region downstream of the AUG codon, the 3' or 5' splice site of pre-treated mRNA, a branch point, the 3' untranslated region (UTR), and a polyadenylation signal sequence. A particular RNAi agent comprises a sense strand substantially identical to the mRNA target sequence encoding IDO, and optionally an antisense strand complementary to or substantially complementary to the mRNA target sequence encoding IDO.

[0252] In some embodiments, the immunomodulator is an inosine-5'-monophosphate dehydrogenase (IMPDH) antagonist or inhibitor. IMPDH is a purine biosynthesis 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 recognized for the 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 maintaining cell proliferation and immune responses. In particular, B and T cells show dependence on IMPDH in normal activation and function and exhibit 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. Antisense agents and RNAi agents against IMPDH coding sequences are also included. Certain antisense agents specifically hybridize to a target region within a pre-mRNA or mRNA target sequence encoding IMPDH, for example, the target region being selected from one or more of the following: the AUG start codon of mRNA, the region upstream of the AUG start codon, the region downstream of the AUG codon, the 3' or 5' splice site of pre-treated mRNA, a branch point, the 3' untranslated region (UTR), and a polyadenylation signal sequence. A particular RNAi agent comprises a sense strand substantially identical to the mRNA target sequence encoding IMPDH, and optionally an antisense strand complementary to or substantially complementary to the mRNA target sequence encoding IMPDH.

[0254] In some embodiments, immunomodulators are cytokines and / or cytokine receptor antagonists or inhibitors. Cytokines are small (glycoproteins) (with molecular weights of 8-75 kDa) that influence hematopoiesis, immune responses, and inflammation. Certain exemplary cytokine inhibitors reduce cytokine synthesis, decrease cytokine concentrations in their free, active form, block interactions between cytokines and their homogeneous receptors, and / or interfere with 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), 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), and interferon-gamma (IFN-gamma). Examples include, but are not limited to, transforming growth factor-β (TGF-β), granulocyte-macrophage colony-stimulating factor (GM-CSF), and their allocytokine 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), TNFRs such as TNFR1, interferon-gamma receptor (IFNGR), and TGF-β receptors such as TGFβR1 (ALK5) or TGFβR2.

[0256] Specific examples of cytokine and / or cytokine receptor inhibitors include etanercept, a recombinant fusion protein of a soluble type II TNF receptor on the human IgG1 backbone, and TNF-alpha inhibitors such as infliximab, a chimeric anti-TNF-alpha monoclonal antibody containing a mouse TNF-alpha binding domain and the 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 lilonacept (a dimeric fusion protein consisting of the ligand-binding domain of the extracellular portion of the IL-1R1 component and an IL-1 receptor accessory protein (IL-1RAcP) inline linked to the fragment-crystallized portion (Fc region) of human IgG1 that binds to and neutralizes IL-1), IL-2 competitive inhibitors such as basiliximab (a chimeric mouse-human monoclonal antibody against the α chain (CD25) of the IL-2 receptor on T cells) and daclizumab (a humanized monoclonal antibody that binds to CD25), IL-1β specific inhibitors such as canakinumab (a human monoclonal antibody), and ixekizumab (which binds to IL-17). This includes IL-17 antagonists such as secukinumab (a humanized monoclonal antibody that binds to the protein interleukin (IL)-17A) and IL-5 inhibitors such as mepolizumab (a humanized monoclonal antibody that binds to 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 to IL-6), sirukmab (an antibody that binds to IL-6), cerilumab (an antibody that binds to the IL-6 receptor), and tocilizumab (an antibody that binds to the IL-6 receptor), as well as 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] A particular cytokine and / or cytokine receptor antagonist or inhibitor includes an antibody or antigen-binding fragment or small molecule that specifically binds to one or more of the cytokines and / or cytokine receptors described above. Also included are cytokine and / or cytokine receptor coding sequences, such as antisense agents and RNAi agents against one or more of the cytokines and / or cytokine receptors described above. A particular antisense agent specifically hybridizes to a target region within a premRNA or mRNA target sequence encoding a cytokine or cytokine receptor, for example, the target region being selected from one or more of the AUG start codon of mRNA, the region upstream of the AUG start codon, the region downstream of the AUG codon, the 3' or 5' splice site of pre-treated mRNA, a branch point, the 3' untranslated region (UTR), and a polyadenylation signal sequence. A particular RNAi agent includes a sense strand that is substantially identical to the 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 immunomodulator is a kinase antagonist or inhibitor, that is, an inhibitor that targets or is targeted against one or more kinases. A common example is a tyrosine kinase inhibitor (TKI). Examples of target kinases include, but are not limited to, Janus kinase (JAK1, JAK2, JAK3, and 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's tyrosine kinase (BTK), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR1, VEGFR2, and VEGFR3), fibroblast growth factor receptor (FGFR), B-Raf, RET proto-oncogene, platelet-derived growth factor receptor (PDGF-R), tropomyosin receptor kinase (TrkA, TrkB, and TrkC), and c-Met. Therefore, in certain embodiments, the kinase inhibitor is one or more inhibitors or antagonists of the aforementioned kinases.

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

[0261] A particular kinase antagonist or inhibitor includes an antibody or antigen-binding fragment or small molecule that specifically binds to a kinase, e.g., one or more of the aforementioned kinases. Kinase-coding sequences, e.g., antisense agents and RNAi agents against one or more of the aforementioned kinases, are also included. A particular antisense agent specifically hybridizes to a target region within a pre-mRNA or mRNA target sequence encoding a kinase, e.g., the target region is selected from one or more of the following: 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-treated mRNA, a branching point, the 3' untranslated region (UTR), and a polyadenylation signal sequence. A particular RNAi agent includes a sense strand that is substantially identical to the mRNA target sequence encoding the kinase, and optionally, an antisense strand that is complementary or substantially complementary to the mRNA target sequence encoding the kinase.

[0262] In some embodiments, the immunomodulator is a B cell receptor inhibitor, such as a drug targeting CD20. The B lymphocyte antigen CD20, or CD20, is an activated-glycosylated phosphoprotein (CD45R+, CD117+) expressed on the surface of all B cells starting from the pro-B phase, and its concentration gradually increases until maturation. The protein has no known intrinsic ligand, and its function is specifically to enable an optimal B cell immune response to T-independent antigens. Exemplary immunomodulators targeting CD20 include the monoclonal antibodies ibritumomab / tiuxetan, obinutuzumab, okalatuzumab, ocrelizumab, rituximab, tositumomab, and bertuzumab.

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

[0264] In some embodiments, as described above, immunomodulators are “small molecules” referring to organic compounds that are synthetic or of biological origin (biomolecules) but are not typically polymers. Organic compounds refer to a larger class of compounds whose molecules contain carbon, typically excluding those containing only carbonates, simple carbon oxides, or cyanides. “Biomolecules” generally refer to organic molecules produced by living organisms, including large polymer molecules (biopolymers) such as peptides, polysaccharides, and nucleic acids, as well as small molecules such as major secondary metabolites, lipids, phospholipids, glycolipids, sterols, glycerolipids, vitamins, and hormones. “Polymers” generally refer to large molecules or macromolecules consisting of repeating structural units, which are usually linked by covalent chemical bonds.

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

[0266] Certain small molecules may have the "specific binding" characteristic described herein. For example, in some embodiments, the small molecules may be approximately 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 approximately 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 It specifically binds to targets (e.g., S1P, S1PR, calcineurin, mTOR, IDO, IMPDH, cytokines and / or cytokine receptors, B cell receptors, kinases) with binding affinity (Kd) of 25, 26, 27, 28, 29, 30, 40, or 50 nM, or approximately 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 less than 50 nM.

[0267] In certain embodiments, immunomodulators are polypeptides or peptides. The terms “peptide” and “polypeptide” are used interchangeably herein, but in certain cases, “peptide” may refer to shorter polypeptides, e.g., polypeptides 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 may consist of naturally occurring and / or non-naturally occurring amino acids as described herein. Antibodies also include polypeptides.

[0268] The binding properties of polypeptides can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, polypeptides specifically bind to target molecules (e.g., S1P, S1PR, calcineurin, mTOR, IDO, IMPDH, cytokines and / or cytokine receptors, B cell receptors, kinases, or their epitopes) having equilibrium dissociation constants ranging from about 10⁻⁷ to about 10⁻⁸ M. In some embodiments, the equilibrium dissociation constants range from about 10⁻⁹ M or less to about 10⁻¹⁰ M. In certain exemplary embodiments, the polypeptide is bound to the targets described herein (including those that specifically bind, e.g., S1P, S1PR, calcineurin, mTOR, IDO, IMPDH, cytokines and / or cytokine receptors, B cell receptors, or kinases) in concentrations 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 approximately 0.01, 0.05, It has an affinity (Kd) of 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 less than 50 nM.

[0269] In some embodiments, the immunomodulator is an antibody or "antigen-binding fragment thereof" that specifically binds to a target as 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, cytokines and / or cytokine receptors, B cell receptors, kinases) via at least one epitope recognition site located within the variable region of the immunoglobulin molecule.

[0270] As used herein, the term “antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also their fragments (dAb, Fab, Fab', F(ab')2, Fv, etc.), single-chain (ScFv), their synthetic variants, naturally occurring variants, fusion proteins containing an antibody moiety with an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and immunoglobulin molecules in any other modified form containing an antigen-binding site or fragment (epitope recognition site) of the required specificity. Certain properties and characterizations of antibodies (and their antigen-binding fragments) 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 the immunoglobulin heavy chain and / or light chain that binds to the antigen of interest. In this regard, the antigen-binding fragments of antibodies described herein may contain one, two, three, four, five, or all six CDRs of the VH and VL sequences from the antibody that binds to the target molecule.

[0272] The term "antigen" refers to a molecule or a part of a molecule that can be conjugated by a selective binder, such as an antibody, and can be used in animals to produce an antibody that can bind to the epitope of that antigen. An antigen may have one or more epitopes.

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

[0274] Molecules such as polypeptides or antibodies are said to exhibit "specific binding" or "selective binding" if they react or associate with a particular cell or substance more frequently, rapidly, for a longer duration, and / or with greater affinity than they would with alternative cells or substances. An antibody "specifically binds" or "selectively binds" if it binds to a target more readily and / or for a longer duration with greater affinity, binding activity, and / or longer duration than it would with other substances, for example, in statistically significant amounts. For example, an antibody that specifically or selectively binds to a particular epitope is an antibody that binds to that specific epitope more readily and / or for a longer duration with greater affinity, binding activity, and / or longer duration than it would with other epitopes. By reading this definition, it can also be understood that, for example, an antibody (or partial or epitope) that specifically or selectively binds to a first target may or may not specifically or selectively bind to a second target. Therefore, "specific coupling" or "selective coupling" does not necessarily require exclusive coupling (although it can include exclusive coupling). Generally, but not always, a reference to coupling implies preferential coupling.

[0275] Immunological binding refers to non-covalent interactions of a type that occur between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific, for example and not limited to electrostatic, ionic, hydrophilic, and / or hydrophobic attractive or repulsive forces, steric hindrance forces, hydrogen bonds, van der Waals forces, and other interactions. The strength or affinity of an immunological binding interaction can be expressed by the dissociation constant (Kd) of the interaction, where a smaller Kd represents 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 measuring the rates of antigen-binding site / antigen complex formation and dissociation, where these rates depend on geometric parameters that equally affect the concentration of the complex partner, the affinity of the interaction, and the rates in both directions. Thus, the "on-rate constant" (Kd) is called on ) and "off-speed constant" (K off Both of these can be determined by calculating the concentration as well as the actual association and dissociation rates. off / K on The ratio allows for the cancellation of all parameters unrelated to affinity, and is therefore equal to the dissociation constant Kd.

[0276] Antibodies can be prepared by any of the various techniques known to those skilled in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies specific to the target polypeptide can be prepared, for example, using the technique described in Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976 and its modifications. 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 constructed or identified using 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 clonal or synthetic libraries, such as the Human Combinatorial Antibody Library (HuCAL), where the structural diversity of the human antibody repertoire is represented by seven heavy-chain and seven light-chain variable region genes. Combinations of these genes result in 49 frameworks within the master library. By superimposing highly variable gene cassettes (CDRs = Complementarity Determining Regions) onto these frameworks, a vast human antibody repertoire can be reproduced. Human libraries designed using human donor-derived fragments encoding light-chain variable regions, 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 are also included. Other libraries suitable for use will be apparent to those skilled in the art.

[0278] In certain embodiments, the antibody and its antigen-binding fragment described herein include heavy-chain and light-chain CDR sets inserted between heavy-chain and light-chain framework region (FR) sets, respectively, providing support to the CDRs and defining the spatial relationships of the CDRs to each other. As used herein, the term “CDR set” refers to 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 represented as “CDR1,” “CDR2,” and “CDR3,” respectively. Thus, the antigen-binding site contains six CDRs, each containing a CDR set from the heavy or light-chain V region. A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) is referred herein to as a “molecular recognition unit.” Crystallographic analysis of numerous antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contact with the bound antigen, with the most extensive antigenic contact being with 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 adjacent amino acid sequences that frame the CDR of a CDR set in a heavy or light chain V region. While some FR residues can come into contact with the binding antigen, FRs, particularly those directly adjacent to the CDR, are primarily responsible for the folding of the V region into the antigen-binding site. Within FRs, certain amino acid residues and specific 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 CDR is presented as a protruding loop motif that forms the antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs that influence the shape of the CDR loop folded into a certain “canonical” structure, regardless of its exact CDR amino acid sequence. Furthermore, it is known that certain FR residues are involved in non-covalent interdomain contacts that stabilize the interaction between antibody heavy and light chains.

[0280] The structure and location of the immunoglobulin variable domain can be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, 1987, and its latest edition.

[0281] The term "monoclonal" antibodies also include, referring to a homogeneous population of antibodies, which consist of amino acids (naturally occurring or not naturally occurring) involved in the selective binding of epitopes. Monoclonal antibodies are highly specific to a single epitope. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (Fab, Fab', F(ab')2, Fv), single-chain (ScFv), their variants, fusion proteins containing antigen-binding moieties, humanized monoclonal antibodies, chimeric monoclonal antibodies, and immunoglobulin molecules in any other modified form containing antigen-binding fragments (epitope-recognition sites) with the required specificity and ability to bind to epitopes. It is not intended to be limited in terms of antibody sources or methods for producing antibodies (e.g., by hybridomas, phage selection, recombinant expression, or transgenic animals). The term includes all immunoglobulins, as well as the aforementioned fragments, etc., under the definition of "antibody."

[0282] The proteolytic enzyme papain selectively cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer containing an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to produce 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 produced 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. Fv fragments contain a non-covalent 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-stranded Fv or scFV antibodies are intended. For example, Kappa antibodies (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 using standard molecular biology techniques, and in regard to the selection of antibodies with desired specificity, follow the teachings of this application.

[0284] Single-stranded Fv(sFv) polypeptides are covalently linked VH::VL heterodimers expressed from a gene fusion containing a VH-encoding gene and a VL-encoding gene linked by a peptide-encoding linker. (Huston et al., PNAS USA. 85(16):5879-5883, 1988). Numerous methods for identifying the chemical structure to convert naturally aggregated but chemically separated light and heavy polypeptide chains into sFv molecules that fold from the antibody V region into a three-dimensional structure substantially similar to that of the antigen-binding site have been described. See, for example, U.S. Patents 5,091,513 and 5,132,405 by Huston et al., and U.S. Patent 4,946,778 by Ladner et al.

[0285] In certain embodiments, the antibodies described herein are in the form of a “diabody.” A diabody is a polypeptide polymer in which each polypeptide comprises a first domain containing a binding region for an immunoglobulin light chain and a second domain containing a binding region for an immunoglobulin heavy chain, and these two domains are linked (e.g., by a peptide linker) but cannot associate with each other to form an antigen-binding site: the antigen-binding site is formed by the association of the first domain of one polypeptide in the polymer with the second domain of another polypeptide in the polymer (WO94 / 13804). The dAb fragment of the antibody consists of a VH domain (Ward et al., Nature 341:544-546, 1989). Diabodies and other polyvalent 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] Minibodies containing scFv bound to the CH3 domain are also included (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 bispecific antibodies are to be used, these may be conventional bispecific antibodies and can be manufactured by various methods (Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993), for example, chemically or from hybrid hybridomas, or any of the bispecific antibody fragments described above. The diabody and scFv can be constructed without the Fc region using only the variable domain, potentially reducing the effect of the anti-idiotype reaction.

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

[0289] In certain embodiments, the antibodies described herein may be provided in the form of UniBody®. UniBody® is an IgG4 antibody from which the hinge region has been removed (see GenMab Utrecht, The Netherlands, also see, e.g., US2009 / 0226421). This antibody technology produces a stable, smaller antibody format that is expected to have a longer therapeutic window than the current smaller antibody format. Since IgG4 antibodies are considered inactive, they do not interact with the immune system. A whole human IgG4 antibody can be modified by removing the hinge region of that antibody, thereby yielding a halving fragment with different stability properties compared to the corresponding intact IgG4 (GenMab, Utrecht). By halving the IgG4 molecule, only one region capable of binding to a congener antigen (e.g., a disease target) remains in UniBody®, and therefore, 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. These nanobodies are encoded by a single gene and are efficiently produced in almost all prokaryotic and eukaryotic hosts, e.g., Escherichia coli (see U.S. Patent No. 6,765,087), fungi (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyvermyces, Hansenula, or Pichia (see U.S. Patent No. 6,838,254)). The production process can be scaled up, producing nanobodies in quantities of several kilograms. The nanobodies can be formulated as ready-to-use solutions with long shelf lives. The nanoclone method (see, for example, WO06 / 079372) is a proprietary method for producing nanobodies against desired targets based on automated, high-throughput selection of B cells.

[0291] In certain embodiments, the antibody or its antigen-binding fragment is humanized. These embodiments refer to chimeric molecules having an antigen-binding site derived from a non-human immunoglobulin species, typically prepared using recombinant techniques, as well as the rest of the immunoglobulin structure of that molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may consist of either a complete variable domain fused to a constant domain or just a CDR 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 domain as an immunogen in a human organism, but leaves the possibility of an immune response to the exogenous variable domain (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). An exemplary method for humanizing antibodies is the method described in U.S. Patent No. 7,462,697.

[0292] Another approach focuses not only on providing a constant region derived from humans, but also on modifying the variable region to reshape it as closely as possible to human form. Both the heavy and light chain variable regions are known to be relatively conserved in a given species and contain three complementarity-determining regions (CDRs) adjacent to four framework regions (FRs) that putatively provide a scaffold for CDRs (which vary depending on the epitope in question and determine binding ability). When a non-human antibody is prepared for a specific epitope, the variable region can be "reshaped" or "humanized" by transplanting 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 is documented in: 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 This has been reported by USA.89:4285-4289,1992 and Co et al., J Immunol.148:1149-1154,1992. In some embodiments, the humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from the mouse antibody). In other embodiments, the humanized antibody has one or more modified CDRs (one, two, three, four, five, or six) 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 may be a chimeric antibody. In this respect, a chimeric antibody consists of an antigen-binding fragment of an antibody 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 may be from a different Ig class than the parent 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 may consist of CH2 and CH3 domains from one or more of the different Ig classes. As described above with respect to humanized antibodies, the antigen-binding fragment of a chimeric antibody may consist of only one or more CDRs of the antibodies described herein (e.g., one, two, three, four, five, or six CDRs of the antibodies described herein), or it may consist of the entire variable domain (VL, VH, or both).

[0294] In some embodiments, immunomodulators are or contain a “ligand” of a 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 perform a biological purpose, and generally includes “protein ligands” that generate a signal by binding to a site on the target molecule or target protein. Thus, a particular drug is, in fact, a protein ligand that binds to a target molecule and generates a signal. “Modified ligands,” e.g., pharmacokinetic modifiers, e.g., protein ligands fused to an Fc region derived from immunoglobulins are also included.

[0295] In some embodiments, the immunomodulator or inhibitor is an antisense agent. Thus, in some embodiments, the target protein, target sequence, and / or target gene (e.g., S1PR, calcineurin, mTOR, IDO, IMPDH, cytokines and / or cytokine receptors, B cell receptors, kinases) described herein are targeted by any variety of antisense agents, including oligonucleotide-based drugs or methods. Antisense agents or oligonucleotides typically include a nucleotide sequence that targets a region within a target sequence (e.g., is sufficiently complementary to the region or specifically hybridizes to the region), and optionally include one or more of the following: a region containing or surrounding the AUG start codon of mRNA (e.g., a region upstream of the start codon, a region downstream of the start codon, or a region containing the start codon), a 3' or 5' splice site of pre-treated mRNA, a pyrimidine-rich or polypyrimidine tract upstream of a splice receptor site, an exon-intron boundary, an intron-exon boundary, a branching site, an exon splicing enhancer component, 5' and 3' untranslated regions, and a polyadenylation signal sequence.

[0296] In certain embodiments, an antisense agent can effectively modify the expression of a target gene (e.g., reduce expression, alter splicing) upon administration to a target requiring administration, or upon contact with cells, such as muscle cells. This requirement is typically met when the antisense agent (a) has the ability to be actively taken up by mammalian cells (e.g., muscle cells), and (b) upon takeup, forms a double helix with the target RNA at a Tm greater than approximately 45°C.

[0297] Certain “antisense agents” include “antisense oligonucleotides,” “antisense oligomers,” and “oligonucleotides,” referring to linear sequences of nucleotides or nucleotide analogs in which nucleic acid bases can hybridize to a target sequence in RNA via Watson-Crick base pairs to form an oligonucleotide:RNA heteroduplex within the target sequence. The terms “antisense oligonucleotide,” “antisense oligomer,” “oligomer,” and “compound” are used interchangeably and may refer to oligonucleotides. The cyclic subunit may 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 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 intended.

[0298] In certain embodiments, the “target sequence” includes a region containing or surrounding the AUG start codon of mRNA (e.g., a region upstream of the start codon, a region downstream of the start codon, or a region containing the start codon), a 3' or 5' splice site of pre-treated mRNA, a branch point, or a 3' non-coding mRNA region such as a 3'-UTR or polyadenylation signal. The target sequence may be within an exon or an intron. A target sequence for a splice site may include an mRNA sequence whose 5' end is 1 to about 25 base pairs downstream of a typical splice receptor junction in pre-treated mRNA (premRNA). An exemplary target sequence for a splice region may be any region of pre-treated mRNA containing the splice site, or it may be entirely contained within an exon-coding sequence, or it may span a splice receptor site or a donor site. An antisense agent is said to “target” a biologically relevant target when it targets a target nucleic acid in a manner described herein and known in the art, for example, when it is specifically hybridized to or complementary to it. Other examples of target regions or target sequences are described herein.

[0299] The term "targeting sequence" refers to a sequence in an oligonucleotide that is complementary (and moreover, substantially complementary) to the "target sequence" in RNA. Only the entire sequence or a portion of the antisense agent may be complementary to the target sequence. For example, in an antisense agent with 20–30 bases, approximately 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 may be a targeting sequence complementary to the target region. Typically, the target sequence is formed by a sequence of bases in the oligonucleotide, but alternatively, it may be formed by a discontinuous sequence that, for example, forms a sequence spanning the target sequence when placed together from opposite ends of the oligonucleotide.

[0300] The target sequence may have “approximate” or “substantial” complementarity with the targeting sequence and may still function for the purposes of this disclosure; that is, the targeting sequence may still be “complementary.” Preferably, the oligonucleotide used in this disclosure has at most one mismatch with the target sequence per 10 nucleotides, preferably at most one mismatch per 20 nucleotides. Alternatively, the antisense oligonucleotide used has 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 targeting sequence.

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

[0302] "Nuclease-resistant" oligonucleotides refer to oligonucleotides whose backbone, in non-hybridized or hybridized forms, is substantially resistant to nuclease cleavage by normal extracellular and intracellular nucleases in the body (e.g., by exonucleases such as 3'-exonuclease, endonuclease, and RNase H). That is, under normal nuclease conditions in the body to which the oligonucleotide is exposed, the oligonucleotide exhibits little to no nuclease cleavage. "Nuclease-resistant heteroduplex" refers to a heteroduplex formed by the binding of an antisense oligonucleotide to its complementary target such that the heteroduplex is substantially resistant to in vivo degradation by intracellular and extracellular nucleases capable of cleaving double-stranded RNA / RNA or RNA / DNA complexes. "Heteroduplex" refers to the double helix between the antisense oligonucleotide and the complementary portion of the target RNA.

[0303] In certain embodiments, antisense oligonucleotides are recognized as substrates for active or facilitated transport across cell membranes, e.g., muscle cell membranes. The ability of an oligonucleotide to form a stable double helix with target RNA may also relate to other properties of the oligonucleotide backbone, including the length and degree of complementarity of the antisense oligonucleotide with respect to the target, the ratio of G:C base matches to A:T base matches, and the position of any mismatched bases. The ability of an antisense oligonucleotide to resist cellular nucleases may promote survival and the final delivery of drugs to the cell's cytoplasm. Therefore, certain embodiments may include antisense oligonucleotides that are not naturally occurring and are nuclease-resistant or substantially nuclease-resistant.

[0304] In certain embodiments, the antisense oligonucleotide comprises a non-natural chemical skeleton 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-methylphosphorothioate oligonucleotides), or any combination thereof.

[0305] When an oligonucleotide hybridizes to a target under physiological conditions, the antisense oligonucleotide "specifically hybridizes" to the target sequence or polynucleotide (e.g., premRNA, mRNA) with a Tm substantially above 40°C or 45°C, preferably at least 50°C, typically 60°C to 80°C or higher. Such hybridization preferably corresponds to stringent hybridization conditions. Given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes to complementary polynucleotides. Such hybridization can occur due to the "approximate" or "substantial" complementarity of the antisense oligonucleotide to the target sequence, as well as due to precise complementarity.

[0306] As used herein, “sufficient length” refers to an antisense oligonucleotide that is complementary to at least 8, more typically 8 to 40, consecutive nucleic acid bases in the target sequence or gene described herein. A sufficient length antisense oligonucleotide has at least a minimum number of nucleotides so that it can hybridize to a region of the target sequence or gene. Preferably, a sufficient length oligonucleotide has a length of 8 to 30 nucleotides. More preferably, a sufficient length oligonucleotide has a length of 9 to 27 nucleotides.

[0307] Antisense oligonucleotides generally contain multiple nucleotide subunits, each of which, when combined, forms or contains a target sequence. Therefore, in some embodiments, antisense oligonucleotides range 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 nucleotides or about 5 to about 30 nucleotides in length. In some embodiments, the antisense oligonucleotide has a nucleotide length of about 14 to about 25 or about 17 to about 27 nucleotides.

[0308] In some embodiments, the antisense oligonucleotide backbone is substantially uncharged and optionally recognized as a substrate for active or facilitated transport across the cell membrane. In some embodiments, all nucleotide-nucleotide bonds are uncharged. The ability of an oligonucleotide to form a stable double helix with target RNA may also relate to other properties of its backbone, including the length and degree of complementarity of the antisense oligonucleotide with respect to the target, the ratio of G:C base matches to A:T base matches, and the position of any mismatched bases. The ability of an antisense oligonucleotide to resist cellular nucleases may promote survival and the ultimate delivery of drugs to the cell's cytoplasm.

[0309] In certain embodiments, the antisense oligonucleotide has at least one internucleotide bond that is positively charged or cationic at physiological pH. In some embodiments, the antisense oligonucleotide has at least one internucleotide bond exhibiting a pKa of about 5.5 to about 12. Optionally, the antisense oligonucleotide has at least one internucleoside bond containing both a basic nitrogen and an alkyl, aryl, or aralkyl group. In certain embodiments, the cationic internucleoside bond contains a 4-aminopiperzin-1-yl (APN) group or a derivative thereof. Without being constrained by any particular theory, the presence of a cationic linkage (e.g., an APN group or APN derivative) in the oligonucleotide is thought to facilitate binding to a negatively charged phosphate within the target nucleotide. Therefore, 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 two and about half of all internucleotide links, 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 internucleotide bonds are cationic links, 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, 3 5, 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, are cationic linkages. In certain embodiments, oligonucleotides of about 19-20 subunits may have 2-10, for example, 4-8 cationic linkages and the remainder being uncharged linkages. In other certain embodiments, oligonucleotides of 14-15 subunits may have 2-7, for example, 2, 3, 4, 5, 6, or 7 cationic linkages and the remainder being uncharged linkages. Therefore, the total number of cationic linkages within an oligonucleotide can vary from approximately 1 to 10 to 15 to 20 to 30 or more (including all integers in between), and can be scattered throughout the oligonucleotide.

[0311] In some embodiments, the antisense oligonucleotide may have about one or at most about one cationic linkage for every 2-5, or 2, 3, 4, or 5 uncharged bonds, for example, about 4-5 or 4 or 5 for every 10 uncharged bonds.

[0312] Certain embodiments include antisense oligonucleotides containing 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 may be observed when approximately 25% of the skeletal linkages are cationic. In certain embodiments, enhancement may be observed with a small number of cationic linkages, e.g., 10–20%, or when the number of cationic linkages is in the range of 50–80%, e.g., approximately 60%.

[0313] In some embodiments, the cationic linkages are scattered along the backbone. Such oligonucleotides optionally contain at least two consecutive uncharged bonds; that is, the oligonucleotides optionally do not have a strictly alternating pattern along their entire length. In certain examples, each one or two cationic linkages are separated along the backbone by at least one, two, three, four, or five uncharged bonds.

[0314] Oligonucleotides having blocks of cationic linkages and blocks of uncharged linkages are also included. 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 about 50%, 60%, 70%, or more than 80% of the total number of cationic linkages.

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

[0316] As described above, antisense oligonucleotides can utilize a variety of antisense chemicals. Examples of oligonucleotide chemicals include, but are not limited to, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), phosphorothioates, 2'O-Me-modified oligonucleotides, morpholinos, PMOs, PPMOs, PMOplus, and PMO-X chemicals, and any combination thereof. Generally, PNA and LNA chemicals can utilize shorter targeting sequences due to their relatively higher target binding strength compared to PMOs and 2'O-Me oligonucleotides. Phosphothioates and 2'O-Me-modified chemicals are often combined to produce a 2'O-Me-phosphorothioate skeleton. See, for example, PCT Publications WO / 2013 / 112053 and WO / 2009 / 008725, which are incorporated herein by reference in their entirety.

[0317] Peptide nucleic acids (PNAs) are DNA analogs structurally isomorphic to a deoxyribose backbone consisting of N-(2-aminoethyl)glycine units to which pyrimidines or purine bases are attached. PNAs containing natural pyrimidines and purine bases hybridize to complementary oligonucleotides according to the Watson-Crick base pairing rule, mimicking DNA in terms of base pair recognition (Egholm, Buchardt et al. 1993). The PNA backbone is formed by peptide bonds rather than phosphodiester bonds, making PNA well-suited for antisense applications (see structure below). The backbone is uncharged, resulting in PNA / DNA or PNA / RNA double helixes that exhibit greater thermal stability than usual. PNAs are not recognized by nucleases or proteases.

[0318] Regardless of radical structural changes to its native structure, PNA is capable of sequence-specific binding to DNA or RNA in a helical form. Features of PNA include high binding affinity to complementary DNA or RNA, destabilization effects induced by single-nucleotide mismatches, resistance to nucleases and proteases, and hybridization with DNA or RNA independent of salt concentration and triple-strand formation with homopurine DNA. PANAGENE® has developed Bts PNA monomers (Bts; benzothiazole-2-sulfonyl group) and oligomerization processes. PNA oligomerization using Bts PNA monomers consists of iterative cycles of deprotection, coupling, and capping. PNA can be synthesized using any technique known in the art. See, for example, U.S. Patents 6,969,766, 7,211,668, 7,022,851, 7,125,994, 7,145,006, and 7,179,896. See also U.S. Patents 5,539,082, 5,714,331, and 5,719,262 for the preparation of PNA. Further teachings on PNA compounds can be found in Nielsen et al., Science, 254:1497-1500, 1991. Each of the foregoing is incorporated herein by reference in whole.

[0319] Antisense oligonucleotides may also contain a “locked nucleic acid” subunit (LNA). LNA is a member of a class of modifications called crosslinked nucleic acids (BNA). BNA is characterized by a covalent bond that locks the conformation of the ribose ring of a C30-endo (northern) sugar packer. In LNA, the crosslink consists of a methylene group between the 2'-O and 4'-C positions. LNA enhances the pre-organization and base stacking of the skeleton, increasing 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 This can be found in Chemistry (2008) 16:9230.

[0320] The compounds of this 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. Patents 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 incorporates the whole by reference. Typical intersubunit linkers include phosphodiester and phosphorothioate moieties, and alternatively, non-phosphorus-containing linkers may be used. One embodiment is an LNA-containing compound in which 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] Phosphothioates (or S-oligonucleotides) are variants of normal DNA in which one of the non-crosslinking oxygen atoms is replaced by sulfur. Sulfurization of internucleotide bonds reduces the action of endonucleases and exonucleases, including 5'-to-3' and 3'-to-5' DNA POL 1 exonuclease, nucleases S1 and P1, RNases, serum nucleases, and snake venom phosphodiesterase. Phosphothioates are produced by two main pathways: by the action of a solution of elemental sulfur in carbon disulfide on hydrogen phosphonate, or by sulfurizing triester phosphite with either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD) (see, e.g., Iyer et al., J. Org. Chem. 55, 4693-4699, 1990). The latter method avoids the problems of elemental sulfur being insoluble in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also yield phosphorothioates of higher purity.

[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 skeleton and optimize the arrangement of the skeleton at a twist angle γ. Homobasic adenine and thymine-containing tc-DNA form highly stable AT base pairs with complementary RNA. Tricyclo-DNA and their synthesis are described in International Patent Application Publication WO2010 / 115993. The compounds of this disclosure may incorporate one or more tricyclyl-DNA nucleotides, and in some cases, the compounds may consist entirely of tricyclyl-DNA nucleotides.

[0323] Tricyclophosphorothioate nucleotides are tricyclo-DNA nucleotides having inter-phosphorothioate subunit links. Tricyclophosphorothioate nucleotides and their synthesis are described in International Patent Application Publication WO2013 / 053928. The compounds of this disclosure may incorporate one or more tricyclyl-DNA nucleotides, and in some cases, the compounds may consist entirely of tricyclyl-DNA nucleotides.

[0324] "2'O-Me oligonucleotide" molecules support a methyl group at the 2'-OH residue of a ribose molecule. 2'-O-Me RNA behaves the same as (or similar to) DNA but is protected from nuclease degradation. 2'-O-Me RNA can also be combined with phosphorothioate oligonucleotides (PTOs) for further stabilization. 2'O-Me oligonucleotides (phosphodiesters or phosphothioates) 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 include phosphorothioate linkages (2'O-Me phosphorothioate oligonucleotides).

[0325] A “morpholino oligonucleotide” or “PMO” refers to an oligonucleotide having a backbone that supports nucleic acid bases that can hydrogen-bond to a typical polynucleotide, where the polymer lacks a pentose sugar backbone but instead contains a morpholino ring. Thus, in a PMO, the morpholino ring structure supports the base-pairing moiety and typically forms a sequence of base-pairing moieties designed to hybridize to a selected antisense target in a cell or subject being treated. An exemplary “morpholino” oligonucleotide comprises a morpholino subunit structure linked together by a phosphoramidate or phosphorodiamidate linkage, where the morpholino nitrogen of one subunit is bonded to the 4' outer ring carbon of an adjacent subunit, and each subunit contains a purine or pyrimidine nucleic acid base that is effective in binding to a base in a polynucleotide by base-specific hydrogen bonding. Morpholino oligonucleotides (including antisense oligonucleotides) are described, for example, in U.S. Patents 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 pending U.S. Patent Applications Nos. 12 / 271,036 and 12 / 271,040, as well as PCT Publications WO / 2009 / 064471 and WO / 2012 / 043730, all of which are incorporated herein by reference in their entirety.

[0326] Within oligonucleotide structures, phosphate groups are generally referred to as forming “nucleoside linkages” of the oligonucleotide. Naturally occurring nucleoside linkages in RNA and DNA are 3'-5' phosphodiester linkages. A “phosphoamideate” group contains phosphorus with three attached oxygen atoms and one attached nitrogen atom, while a “phosphodiamidate” 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 skeletal chain. The second nitrogen in a phosphorodiamidate linkage is typically the ring nitrogen in a morpholino ring structure.

[0327] "PMO-X" refers to a phosphorodiamidate morpholino oligonucleotide (PMO) having a phosphorus atom having a covalent bond with (i) the nitrogen atom of a morpholino ring, and (ii) a second covalent bond with the ring nitrogen of 4-aminopiperzin-1-yl (i.e., APN) or a derivative of 4-aminopiperzin-1-yl. Exemplary PMO-X oligonucleotides are disclosed in PCT application PCT / US2011 / 38459 and PCT publication WO / 2013 / 074834, each of which is incorporated herein by reference in whole. "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 morpholino group and the ring nitrogen of 4-aminopiperzin-1-yl (i.e., APN). In certain embodiments, an antisense oligonucleotide comprising a targeted sequence described herein comprises at least one APN-containing linkage or APN derivative-containing linkage. Certain embodiments include a PMO having approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% APN / APN derivative-containing links, with the remaining links (if less than 100%) being uncharged links, for example, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 of all internucleotide links. 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 are APN / APN derivative-containing links.

[0328] Further antisense oligonucleotides / chemicals that may 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 Publications 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 immunomodulator or inhibitor is an RNA interference (RNAi) agent. Thus, in some embodiments, the target protein, target sequence, and / or target gene (e.g., S1PR, calcineurin, mTOR, IDO, IMPDH, cytokines and / or cytokine receptors, B cell receptors, kinases) described herein are targeted by any variety of RNA-based agents or methods. RNA interference (RNAi) is an evolutionarily conserved gene silencing mechanism originally discovered in the study of the nematode Caenorhabditis elegans (Lee et al., Cell 75:843, 1993; Reinhart et al., Nature 403:901, 2000). RNA interference is triggered by introducing dsRNA into a cell expressing the appropriate molecular mechanism, which degrades the corresponding endogenous mRNA. This mechanism involves the conversion of dsRNA into shorter RNAs, which 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) when it contains a sense strand corresponding to a "target sequence" of a target gene, in a manner described herein and known in the art.

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

[0332] In certain embodiments, a double-stranded ribonucleic acid (dsRNA) molecule is used as the RNAi agent. A dsRNA generally consists of two single strands. One strand of the dsRNA ("sense" strand) contains a nucleotide sequence that is substantially identical to a portion of the target gene or target sequence, and the other strand ("complementary" or "antisense" strand) contains 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, or 99% identical to the target sequence. These strands are sufficiently complementary to hybridize and form a double structure. In certain embodiments, the complementary RNA strand may have fewer than 30 nucleotides, a nucleotide length of less than 25 nucleotides, or even 19–24 nucleotides. In certain embodiments, the complementary nucleotide sequence may have a nucleotide length of 20–23 nucleotides, or 22 nucleotides. 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, the antisense strand is complementary to or substantially complementary to a portion of the target sequence described herein. In certain embodiments, the portion comprises, consists of, or essentially comprises about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 adjacent nucleotides of the target sequence described herein, 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 fewer adjacent nucleotides.

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

[0334] Generally, the nucleotide sequence 3' of the AUG start codon of the transcript from the target gene 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 nucleotides immediately 3' to the dinucleotide sequence are 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' to the dinucleotide sequence are identified as the potential siRNA sequence. In some embodiments, the dinucleotide sequence is an AA or NA sequence, and 19 nucleotides immediately 3' to the AA or NA dinucleotide are identified as the potential siRNA sequence. siRNA sequences are typically spaced at different positions along the length of the target gene. To further enhance the silencing efficiency of siRNA sequences, potential siRNA sequences can be analyzed to identify regions in the target cell or organism that do not contain areas homologous to other coding sequences. For example, a suitable siRNA sequence of approximately 21 base pairs will typically not have more than 16-17 adjacent base pairs homologous to coding sequences in the target cell or organism. If the siRNA sequence is to be expressed from an RNA Pol III promoter, siRNA sequences lacking more than four adjacent A' or T' base pairs are selected.

[0335] Once potential siRNA sequences are identified, they can be analyzed using various criteria known in the art. For example, to enhance their silencing efficiency, siRNA sequences can be analyzed using rational design algorithms to identify sequences having one or more of the following characteristics: (1) a G / C content of approximately 25% to 60% G / C, (2) at least three A / U at positions 15-19 of the sense strand, (3) no internal repeats, (4) A at position 19 of the sense strand, (5) A at position 3 of the sense strand, (6) 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. siRNA design tools are known that incorporate algorithms useful for siRNA selection, specifying appropriate values ​​for each of these characteristics. Those skilled in the art will understand that sequences having one or more of the aforementioned characteristics 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 siRNA: (1) sequences containing stretches of four or more identical bases in a column; (2) sequences containing homopolymers of G (i.e., to reduce the possibility of nonspecific 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 column; and (5) sequences containing serial repeats of four or more bases in the candidate that result in an internal folding structure. However, those skilled in the art will understand that sequences having one or more of the aforementioned features can still be selected for further analysis and testing as potential siRNA sequences.

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

[0338] Potential siRNA sequences can also be analyzed for the presence of any immunostimulant 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 indicator of whether the sequence is immunostimulant. If an siRNA molecule is found to be immunostimulant, it can then be modified to reduce its immunostimulant properties, as described herein. As a non-limiting example, an siRNA sequence can be brought into contact with mammalian responder cells under conditions that elicit a detectable immune response to determine whether the siRNA is immunostimulant or non-immunostimulant. Mammalian responder cells may be from naive mammals (i.e., mammals that have not previously come into contact with the gene products of the siRNA sequence). Mammalian responder cells may be, for example, peripheral blood mononuclear cells (PBMCs), macrophages, etc. Detectable immune responses may include the production of cytokines or growth factors such as TNF-alpha, IFN-alpha, IFN-beta, IFN-gamma, IL-6, IL-12, or combinations thereof. siRNA molecules identified as immunostimulant may then be modified to reduce their immunostimulant properties by replacing at least one nucleotide in the sense and / or antisense strands with a modified nucleotide. For example, less than approximately 30% (e.g., less than 30%, 25%, 20%, 15%, 10%, or 5%) of the nucleotides in the double-strand region of the siRNA double-strand may be replaced with a modified nucleotide such as a 2'OMe nucleotide. The modified siRNA may then be brought into contact with mammalian responder cells, as described above, to confirm that its immunostimulant properties have been reduced or suppressed.

[0339] The RNAi agent typically includes a double-stranded portion (despite any and potentially preferred presence of any single-stranded overhand) that is identical or nearly identical to the mRNA targeted by the RNAi agent (e.g., exhibiting 90% or more sequence identity, e.g., at least 95%, or showing up to two and optionally only one mismatch), comprising 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 RNA strand contains a nucleotide overhang of 1 to 4 nucleotides in length. In some embodiments, the dsRNA contains at least one chemically modified nucleotide. In certain embodiments, the dsRNA containing a single-stranded overhang of 1 to 4 nucleotides may contain a molecule in which the unpaired nucleotide of the single-stranded overhang directly adjacent to the terminal nucleotide pair contains a purine base. In some embodiments, the last complementary nucleotide pairs on both ends of the dsRNA are GC pairs, or at least two of the last four terminal nucleotide pairs are GC pairs.

[0341] In certain embodiments, RNAi agents include microRNAs. MicroRNAs represent a large group of small RNAs naturally produced in organisms, some of which regulate the expression of target genes. MicroRNAs are formed by Dicer from single-stranded hairpin precursor transcripts of approximately 70 nucleotides (see V. Ambros et al., Current Biology 13:807, 2003). MicroRNAs are not translated into proteins, but rather bind to specific messenger RNAs, thereby blocking translation. MicroRNAs are thought to inhibit translation by improperly base-pairing with their targets. Certain microRNAs may be transcribed as hairpin RNA precursors and processed into their mature forms by Dicer enzymes.

[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 RNA (siRNA). In certain embodiments, the first strand of a double-stranded oligonucleotide contains two or more nucleoside residues than the second strand. In other embodiments, the first and second strands have the same number of nucleosides, but the first and second strands cancel each other out so that 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] Where the regulator includes siRNA, the agent contains a region of sufficient homology to the target region and is of sufficient length with respect to nucleotides, so that the siRNA agent or a fragment thereof can mediate the downregulation of the target gene or RNA. It will also be understood that the terms “ribonucleotide” or “nucleotide” may refer to a modified nucleotide or surrogate substitution region at one or more positions in the case of modified RNA or nucleotide surrogates. Thus, the siRNA agent is or contains a region that is at least partially complementary to the target sequence. While complete complementarity between the siRNA agent and the target sequence is not required, the correspondence must be sufficient, for example, to allow the siRNA agent or its cleavage product to lead to sequence-specific silencing by RNAi cleavage of the target RNA, for example. The degree of complementarity or homology with the target strand is most important in the antisense strand. Complete complementarity is often desired, especially in the antisense strand, but some embodiments contain one or more, but preferably 10, 8, 6, 5, 4, 3, 2 or fewer, mismatches with respect to the target sequence. Mismatches are most tolerant in the terminal regions and, if present, preferably within the terminal regions, for example, within the terminal regions of 6, 5, 4, or 3 nucleotides at the 5' and / or 3' ends. The sense strand only needs to be sufficiently complementary to the antisense strand to maintain the overall double-stranded properties of the molecule.

[0344] In some embodiments, the RNAi agent or oligonucleotide, such as siRNA oligonucleotide, may be modified or contain a nucleoside surrogate. The single-stranded region of the siRNA agent may be modified or contain a nucleoside surrogate, for example, the unpaired region of a hairpin structure, such as the region linking two complementary regions, may have the modification or nucleoside surrogate. Modifications for stabilizing one or more 3' or 5' ends of the siRNA agent with respect to an exonuclease, or for facilitating the entry of an antisense siRNA agent into RISC, are also included. Exemplary modifications may include C3 (or C6, C7, C12) aminolinkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (C3, C6, C9, C12, debase, triethylene glycol, hexaethylene glycol), and special biotin or fluorescein reagents that result as phosphoramidites and have another DMT-protected hydroxyl group to enable multiple couplings during RNA synthesis.

[0345] Certain siRNA agents include, for example, molecules long enough to induce an interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter RISC (RNAi-induced silencing complex)). Also included are molecules long enough not to induce an interferon response (which can also be cleaved by Dicer and / or enter RISC), for example, molecules of a size that allows entry into RISC, such as molecules similar to Dicer cleavage products. Molecules short enough not to induce an interferon response are referred to herein as siRNA agents or shorter RNAi agents. As used, “siRNA agents or shorter RNAi agents” refers to siRNA agents short enough not to induce a harmful interferon response in human cells, for example, having a double-stranded region of less than 60, preferably less than 50, 40, or 30 nucleotide pairs. siRNA modifiers or their cleavage products can downregulate target genes, for example, by inducing RNAi against target RNA.

[0346] In some cases, each strand of an siRNA agent has a nucleotide length of approximately 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides, or less than approximately 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides. For example, each strand may have a nucleotide length of approximately 21 to 25 nucleotides. A particular siRNA agent has a double-stranded region of approximately 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleoti pairs, and one or more overhangs, e.g., one or two 3' overhangs of 1 to 3 nucleotides.

[0347] In addition to homology to target RNA and the ability to downregulate target genes, siRNA agents may have one or more of the following properties: they may have antisense strands that can present bases (or modified bases) in a suitable three-dimensional framework to enable accurate base pairing and the formation of a double-stranded structure with homologous target RNA sufficient to enable downregulation of the target by, for example, cleavage of target RNA, despite modifications to a very large number or even all nucleosides; and they may still have "RNA-like" properties despite modifications to a very large number or even all nucleosides, i.e., they may have the overall structural, chemical, and physical properties of an RNA molecule, not limited to, or even partially based on, ribonucleotide-based contents. For example, an siRNA agent may contain sense and / or antisense strands in which all nucleotide sugars contain, for example, a 2'-fluoro instead of a 2'-hydroxyl. This deoxyribonucleotide-containing agent may still be expected to exhibit RNA-like properties. While not wishing to be constrained by theory, electronegative fluorine, when bound to the C2' position of ribose, prefers axial orientation. This spatial preference of fluorine can also compel the sugar to take a C3' end packer. This is the same puckering pattern observed in RNA molecules, resulting in the A-family helix characteristic of RNA. Furthermore, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bond interactions as water molecules, which are known to stabilize RNA structures. Generally speaking, modification moieties at the 2' sugar position can enter hydrogen bonds that are more characteristic of the OH portion of ribonucleotides than of the H portion of deoxyribonucleotides.

[0348] As used herein, “single-stranded RNAi agent” is an RNAi agent composed of a single molecule. A single-stranded RNAi agent may include a double-stranded region formed by intra-strand pairs, for example, a single-stranded RNAi agent may be or may include a hairpin structure or a pan-handle structure. The agent modulating 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 participate in RISC-mediated cleavage of the target mRNA. The single-stranded RNAi agent has a nucleotide length of at least 14, more preferably at least 15, 20, 25, 29, 35, 40, or 50 nucleotides. It is preferably less than 200, 100, or 60 nucleotides.

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

[0350] Certain modifiers used according to the methods provided herein include RNAi oligonucleotides in which the chimeric oligonucleotide or "chimera" contains 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, which is modified to enhance resistance to nuclease degradation, enhance cellular uptake, and / or enhance binding affinity to target nucleic acids. As a result, when using chimeric oligonucleotides, results similar to those obtained when using shorter oligonucleotides can often be obtained compared to phosphorothioate oligonucleotides. Chimeric oligonucleotides can be formed as complex structures consisting of two or more oligonucleotides, modified oligonucleotides, oligonucleotides, and / or oligonucleotide mimetic compounds, as described above. Such oligonucleotides are also referred to in the art as hybrids or gapmers. Representative U.S. patents teaching the preparation of such hybrid structures include, but are not limited to, U.S. Patents 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 has a length of 5 to 60 nucleotides.

[0351] In some embodiments, RNAi agents include oligonucleotides comprising at least one ligand linked to a modified or unnatural nucleic acid base. Numerous compounds can function as modified bases. The structure of the modified base is important to the extent that it should not substantially hinder 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 the following unnatural nucleic acid bases: difluorotolyl, nitropyrrolyl, nitroimidazolyl, nitroindolyl, naphthalenyl, anthracenyl, pyridinyl, quinolinyl, pyrenyl, or any of the unnatural nucleic acid bases described herein. In certain embodiments, the unnatural nucleic acid base is difluorotolyl, nitropyrrolyl, or nitroimidazolyl. In certain embodiments, the unnatural nucleic acid base is difluorotolyl. A wide variety of ligands are well known in the art. For example, ligands can be steroids, bile acids, lipids, folic acid, pyridoxal, B12, riboflavin, biotin, aromatic compounds, polycyclic compounds, crown ethers, interfering substances, cleavage molecules, protein binders, or carbohydrates. In certain embodiments, the ligand is a steroid or an aromatic compound. In certain cases, the ligand is cholesteryl.

[0352] In some embodiments, RNAi agents are oligonucleotides that are linked to ligands for the purpose of improving cell targeting and uptake. For example, RNAi agents may be linked to antibodies or their antigen-binding fragments. As a further example, RNAi agents may be linked to specific ligand-binding molecules, such as polypeptides or polypeptide fragments, that specifically bind to particular cell surface receptors.

[0353] In certain embodiments, the RNAi agent contains a non-natural nucleic acid base. In some embodiments, the non-natural nucleic acid base is difluorotolyl, nitroimidazolyl, nitroindolyl, or nitropyrrolyl. In certain embodiments, the modifier provided for an aromatic compound relates to a double-stranded oligonucleotide sequence, where only one of the two strands contains a non-natural nucleic acid base. In certain embodiments, the modifier used herein relates to a double-stranded oligonucleotide sequence, where both of these strands independently contain at least one non-natural nucleic acid base.

[0354] In certain cases, the naturally occurring ribose sugar moiety in the nucleoside is replaced with a hexose sugar. In certain embodiments, the hexose sugar is allose, altrose, glucose, mannose, gross, idose, galactose, talose, or derivatives thereof. In preferred embodiments, the hexose is D-hexose. In certain cases, the naturally occurring ribose sugar moiety 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 within 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 oligonucleotide skeleton is modified to improve the therapeutic or diagnostic properties of the oligonucleotide compound. In certain embodiments, at least one base or at least one sugar of the oligonucleotide is modified to improve the therapeutic or diagnostic properties of the oligonucleotide compound. In some cases, when the oligonucleotide is double-stranded, the two strands are complementary, partially complementary, or chimeric oligonucleotides.

[0355] Examples of modified RNAi agents include oligonucleotides containing modified skeletons or unnatural internucleotide linkages. Oligonucleotides having modified skeletons or unnatural internucleotide linkages, as defined herein, include those that retain a phosphorus atom in their skeleton and those that do not. Modified oligonucleotides that do not have a phosphorus atom in their intersugar skeleton can also be considered oligonucleotides. Specific oligonucleotide chemical modifications are described below. The modifications do not need to be uniform at all positions in a given compound; in fact, one or more of the following modifications may be incorporated into a single oligonucleotide compound, or even in a single nucleotide of that compound.

[0356] Examples of modified nucleotide linkages or backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotryesters, and boranophosphates (those with normal 3'-5' linkages, their 2'-5' linked analogues, and those with reversed polarity, where adjacent nucleoside unit pairs are linked from 3'-5' to 5'-3' and from 2'-5' to 5'-2'). Various salts, mixed salts, and free acid forms are also included.

[0357] Representative U.S. patents teaching the preparation of the phosphorus atom-containing linkages described above include U.S. Patents 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, and 5,45 Examples include, but are not limited to, Nos. 3,496, 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 nucleotide linkages or skeletons (i.e., oligonucleotides) that do not contain a phosphorus atom include short-chain alkyl or cycloalkyl sugar linkages, mixed heteroatom and alkyl or cycloalkyl sugar linkages, or skeletons formed by one or more short-chain heteroatoms or heterocyclic sugar linkages. These include morpholino linkages (partially formed from the sugar portion of a nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamate, methyleneimino, and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and other skeletons with mixed N, O, S, and CH2 components.

[0359] Representative U.S. patents teaching the preparation of the above oligonucleotides include U.S. Patents No. 5,034,506, No. 5,166,315, No. 5,185,444, No. 5,214,134, No. 5,216,141, No. 5,235,033, No. 5,264,562, No. 5,264,564, No. 5,405,938, No. 5,434,257, No. 5,466,677, No. 5,470,967, No. 5,489,677, and No. 5,541,307. Including, but not limited to, Patent Nos. 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 mimetic compounds, both the sugar and nucleoside linkages, i.e., the nucleoside unit backbone, may be substituted with other groups. The nucleoside unit is maintained for hybridization with a suitable nucleic acid target compound. One such oligonucleotide, oligonucleotide mimetic compound, that has been shown to have outstanding hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is substituted with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleic acid base is retained and is directly or indirectly bonded to the atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patents 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] Oligonucleotides that utilize ribozymes are also included. 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 autocatalytic cleavage site is located within a highly conserved region of the RNA secondary structure (Buzayan et al., PNAS USA. 83:8859, 1986). Modifying naturally occurring autocatalytic RNA molecules produces ribozymes that can target specific cellular or pathogenic RNA molecules with high specificity. Therefore, ribozymes serve the same general purposes as antisense oligonucleotides (i.e., regulating the expression of specific genes) and, like oligonucleotides, are nucleic acids that have a substantial portion of their stranded state. In other words, ribozymes have substantial chemical and functional identity with oligonucleotides and are therefore considered equivalent for the purposes described herein.

[0362] In certain cases, RNAi agents can be modified with non-ligand groups. Numerous non-ligand molecules have been conjugated to enhance the activity, cellular distribution, cellular targeting, or cellular uptake of oligonucleotides, and procedures for performing such conjugations are available in the references. Examples of non-ligand moieties include lipid moieties, e.g., cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), and thioethers, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765; thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533); aliphatic chains, e.g., 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, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan This includes polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). A typical conjugation protocol involves synthesizing oligonucleotides having aminolinkers at one or more positions in their sequence. The conjugated molecule is then reacted with the amino group using an appropriate coupling or activating reagent. The conjugation reaction can be carried out either while the oligonucleotide remains bound to a solid support or after cleavage in the liquid phase.Purification of oligonucleotide conjugates by HPLC typically yields a pure conjugate.

[0363] Certain exemplary RNAi agents offer delivery via vectors. The term “vector” generally refers to a nucleic acid molecule, typically DNA, in which nucleic acid segments can be inserted and cloned, i.e., propagated. Thus, a vector typically contains one or more unique restriction enzyme recognition sites, and the cloned sequence is reproducible, thus enabling self-replication in a defined host or vehicle organism. Vectors may include plasmids, phagemids, bacteriophages, bacteriophage-derived vectors, PACs, BACs, linear nucleic acids, e.g., linear DNA, viral vectors, etc. Expression vectors are generally configured to enable and / or perform the expression of the nucleic acid or ORF introduced therein in a desired expression system, e.g., in vitro, in host cells, host organs, and / or host organisms. For example, an expression vector may advantageously contain preferred regulatory sequences.

[0364] Exemplary vectors for use herein include viral vectors, which are known and, for example, but not limited to, vectors derived from retroviruses, vaccinia viruses, poxviruses, adenoviruses, and adeno-associated viruses (AAVs). Such viral vectors may be manipulated by recombinant techniques known to themselves to introduce herein a nucleic acid sequence encoding either an antisense or RNAi agent disclosed herein.

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

[0366] Recombinant adenovirus vectors may also be intended for the delivery and expression of RNAi agents in host cells, as disclosed herein. Adenovirus-based viral vectors have the advantage of being able to infect non-dividing host cells, although the recombinant viral genome is not integrated into the host cell genome. For example, suitable adenovirus vectors, methods for constructing such recombinant adenovirus 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 intended herein. RAAV vectors can infect both dividing and non-dividing cells, and their recombinant viral genome may be integrated into the host recombinant viral genome. RAAV vectors may be produced from a variety of adeno-associated viruses, including, for example, serotypes 1-6. Generally, RAAV vectors may, in order, comprise a 5' adeno-associated virus reverse terminal repeat (ITR), a nucleic acid sequence encoding either an antisense or RNAi agent as disclosed herein, which is operably linked to a target nucleic acid, for example, a sequence that regulates its expression in a host cell or host organism, and a 3' adeno-associated virus ITR. In addition, rAAV vectors may preferably have a polyadenylation signal. Suitable RAAV vectors include, among others, WO1994 / 13788, WO1993 / 24641, Goyenvalle et al. This was described in al.2004 (Science 306:1796-1799), where the antisense sequence is ligated to a modified U7 small nuclear RNA.

[0367] Other exemplary viral vectors for use herein include poxviruses such as vaccinia virus, e.g., attenuated vaccinia virus, e.g., modified virus Ankara (MVA) or NYVAC, avipoxvirus, e.g., vectors derived from avian diphtheria virus or canarypox virus.

[0368] Further examples of regulators, including RNAi oligonucleotides such as siRNA oligonucleotides, can be found in U.S. Patent Publications 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] How to use Certain embodiments involve the use of HRS polypeptides / expressable polynucleotides and compositions described herein to treat lung inflammation alone or in combination with immunotherapeutic agents. This also includes the treatment of interstitial lung disease (ILD) and related disorders. In some embodiments, HRS polypeptides / expressable polynucleotides and compositions, methods, and / or combination therapies are used to reduce lung inflammation in subjects requiring treatment of lung inflammation, to treat one or more ILDs, and / or to improve the clinical symptoms or parameters of the disease.

[0370] Accordingly, some embodiments include methods for treating lung inflammation in subjects requiring treatment, 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 for treating lung inflammation in a subject requiring treatment, comprising administering to the subject (a) a histidyl-tRNA synthetase (HRS) polypeptide or an expressible polynucleotide encoding an HRS polypeptide, and (b) an immunomodulator, for example, as described herein. In some embodiments, (a) and (b) are administered separately and optionally as described herein. In certain embodiments, (a) and (b) are administered together and optionally as a therapeutic composition as described herein.

[0372] In certain embodiments, the immunomodulator is pirfenidone or nintedanib. In some methods or compositions, pirfenidone is used in individual dose units ranging from about 50 to about 1000 mg, or in amounts of 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, 52 0, 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 1000mg, approximately 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 less than 1000 mg, 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, 75 Individual dose units of 0, 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 are administered, for example, in approximately one, two, or three capsules for oral administration.

[0373] In some embodiments, pirfenidone is administered in daily dose units ranging from approximately 100 to approximately 4000 mg / day, or approximately 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, approximately 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, 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, 8 60, 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 approximately 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, The daily dose is 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day, administered in units of approximately 1, 2, 3, 4, 5, 6, 7, 8, or 9 oral capsules.

[0374] In certain embodiments, pirfenidone is administered in the form of three capsules of approximately 267 mg each for oral administration, for example, three capsules per individual dose, in a daily dose unit of approximately 2400 mg / day (e.g., 2403 mg / day), for example, nine capsules of approximately 267 mg each for oral administration, three times a day, in a daily dose unit of approximately 2400 mg / day (e.g., 2403 mg / day).

[0375] In some methods or compositions, nintedanib is administered in individual dose units ranging from approximately 10 to approximately 500 mg, or in doses of approximately 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, approximately 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 approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 18 Individual dose units of 0, 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 are administered, for example, in approximately one, two, or three capsules.

[0376] In some embodiments, nintedanib is administered in daily dose units ranging from approximately 20 to approximately 1000 mg / day, or approximately 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, 8 40, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 , 1000mg / day, approximately 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, 4 80, 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, 79 0, 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 approximately 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, 7 The daily dose is 70, 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 / day, administered in units of approximately 1, 2, 3, 4, 5, ...

Claims

1. (a) A histidyl-tRNA synthetase (HRS)-Fc fusion polypeptide, or an expressible polynucleotide encoding the HRS-Fc fusion polypeptide, wherein the 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, (b) Nintedanib, which increases the serum concentration of the HRS-Fc fusion polypeptide compared to the HRS-Fc fusion polypeptide alone, A therapeutic composition for use in the treatment of lung inflammation in subjects requiring treatment of lung inflammation, comprising [a specific compound / component].

2. The HRS-Fc fusion polypeptide is sequence number 157 (Fc-HRS(2-60) or HRS ＦＣ１ The therapeutic composition according to claim 1, comprising, consisting of, or essentially consisting of )

3. The therapeutic composition according to 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 less than about 5% aggregated.

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

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

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

7. A therapeutic composition according to 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 according to any one of claims 1 to 5, wherein the composition is in a syringe, optionally in an injectable syringe, or the composition is in the form of a capsule, optionally an oral capsule.

10. Use of the therapeutic composition according to any one of claims 1 to 8 in the manufacture of a pharmaceutical for the treatment of lung inflammation in a subject requiring treatment of lung inflammation.

11. The composition according to any one of claims 1 to 9, wherein the nintedanib 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 nintedanib is administered in individual dose units ranging from approximately 10 to approximately 500 mg, or in doses of approximately 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, 3 70, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg, approximately 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 approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 2 The composition according to any one of claims 1 to 9 and 11, administered in individual dose units of 40, 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, in any one of approximately one, two, or three capsules.

13. The nintedanib is administered in daily dose units ranging from approximately 20 to approximately 1000 mg / day, or in units of approximately 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, approx. 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, 8 20, 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 approximately 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, The composition according to any one of claims 1 to 9 and 11, administered in a daily dose unit of 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg / day, in any one of approximately one, two, three, four, five, or six capsules.

14. The composition according to any one of claims 1 to 9 and 11, wherein the nintedanib is administered in a daily dose unit of approximately 100 to 150 mg, or approximately 200 to 300 mg / day, either once or twice daily.

15. The composition according to any one of claims 1 to 9 and 11, wherein the nintedanib is administered in the form of nine capsules containing approximately 267 mg each, taken three times a day, in a daily dose unit of approximately 2400 mg / day (e.g., 2403 mg / day), which can be optionally taken as three capsules per individual dose.

16. The composition according to any one of claims 1 to 9 and 11 to 15, wherein the subject is suffering from or at risk of suffering from interstitial lung disease (ILD).

17. The composition according to claim 16, wherein the ILD is idiopathic or related to connective tissue disease, autoimmune disease, exposure to inhaled substances or drugs, infection, or malignant tumor.

18. The composition according to claim 17, wherein the ILD is selected from or associated with one or more of the following: idiopathic interstitial pneumonia, idiopathic pulmonary fibrosis, sarcoidosis, Hanmann-Ricci syndrome, antisynthesis syndrome, idiopathic eosinophilic pneumonia, alveolar hemorrhage syndrome, alveolar proteinosis, asbestosis, silicosis, beryllium disease, rheumatoid arthritis, lupus erythematosus, chronic graft-versus-host disease with lung injury, 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 lung disease, desquamative interstitial pneumonia, and / or cystic fibrosis.

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

20. The composition according to claim 17, wherein the drug is selected from one or more of antibiotics, chemotherapeutic agents, antiarrhythmic agents, and statins.

21. The composition according to claim 17, wherein the infectious disease is selected from one or more of atypical pneumonia, Pneumocystis pneumonia (PCP), tuberculosis, Chlamydia trachomatis, respiratory syncytial virus (RSV), and idiopathic organizing pneumonia.

22. The composition according to claim 17, wherein the malignant tumor is carcinomatous lymphangitis or lymphoma.

23. The composition according to any one of claims 1 to 9 and 11 to 15, wherein the subject requiring treatment for the aforementioned lung inflammation has one or more conditions selected from atopic asthma, non-atopic asthma, allergic asthma, atopic bronchial IgE-mediated asthma, bronchial asthma, essential asthma, true asthma, endogenous asthma caused by pathophysiological disturbance, exogenous asthma caused by environmental factors, essential asthma of unknown or unclear causes, bronchitis-like asthma, emphysematous asthma, exercise-induced asthma, allergen-induced asthma, cold-induced asthma, occupational asthma, as well as infectious asthma caused by bacterial, fungal, protozoan, or viral infections, non-allergic asthma, first-onset asthma, wheezing infant syndrome and bronchiolitis, chronic or acute bronchoconstriction, chronic bronchitis, peripheral airway obstruction, and pulmonary emphysema.

24. The composition according to any one of claims 1 to 9 and 11 to 15, wherein the subject requiring treatment for the lung inflammation has an obstructive or inflammatory airway disease.

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

26. The composition according to any one of claims 1 to 9 and 11 to 15, wherein the subject requiring treatment for the aforementioned lung inflammation has a condition related to worsening airway hyperresponsiveness resulting from other drug therapies, airway disease with pulmonary hypertension, bronchitis or acute bronchitis, acute laryngotracheobronchiitis, arachidate bronchitis, catarrhal bronchitis, croup bronchitis, dry bronchitis, infectious asthmatic bronchitis, proliferative bronchitis, staphylococcal or streptococcal bronchitis, vesicular bronchitis, acute lung injury, bronchiectasis, or columnar bronchiectasis, saccular bronchiectasis, fusiform bronchiectasis, capillary bronchiectasis, cystic bronchiectasis, dry bronchitis, or follicular bronchiectasis.

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

28. The composition according to any one of claims 1 to 9 and 11 to 27, which optionally increases the average life expectancy of a subject requiring treatment for the lung inflammation 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. The composition according to any one of claims 1 to 9 and 11 to 28, which improves one or more clinical symptoms or parameters of lung inflammation in a subject requiring treatment for the lung inflammation.

30. The composition according to claim 29, wherein 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. The composition according to claim 30, which optionally improves pulmonary fibrosis in a subject requiring treatment for inflammation of the lung, as measured by a decrease in the Ashcroft score, which is a decrease of 1, 2, 3, 4, 5, 6, 7, or 8 grades in the Ashcroft score compared to an initial score.

32. (a) a histidyl-tRNA synthetase (HRS)-Fc fusion polypeptide, or an expressible polynucleotide encoding the HRS-Fc fusion polypeptide, wherein the HRS-Fc fusion polypeptide contains, consists of, or essentially consists of an amino acid sequence that is at least 95% identical to a sequence selected from SEQ ID NOs. 157, 158 and 163-166, (b) Nintedanib, which increases the serum concentration of the HRS-Fc fusion polypeptide compared to the HRS-Fc fusion polypeptide alone, A patient care kit for use in the treatment of lung inflammation in subjects requiring treatment of lung inflammation, including [mention specific components / items].

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

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

35. The nintedanib is administered in individual dose units ranging from approximately 10 to approximately 500 mg, or in doses of approximately 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, 36 0, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg, approximately 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, 2 The patient care kit according to claim 32, comprising, arbitrarily, approximately one, two, or three capsules, each containing an individual dose unit of 20, 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.

36. The nintedanib is administered in daily dose units ranging from approximately 20 to approximately 1000 mg / day, or in units of approximately 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, approx. 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, 8 20, 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 approximately 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 The patient care kit according to claim 32, comprising, arbitrarily, approximately one, two, three, four, five, or six capsules in daily dose units of 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, and 1000 mg / day.

37. The patient care kit according to claim 32, wherein the nintedanib is administered in a daily dose unit of approximately 100 to 150 mg, or approximately 200 to 300 mg / day, and is optionally administered once or twice daily.

38. The patient care kit according to claim 37, wherein the nintedanib is administered in a daily dose unit of about 100 or 150 mg, or in a dose of about 200 to 300 mg / day, either once or twice daily.

39. A composition for use in altering one or more pharmacokinetic characteristics of an HRS-Fc fusion polypeptide in a subject, comprising the HRS-Fc fusion polypeptide, or an expressible polynucleotide encoding the HRS-Fc fusion polypeptide, in combination with nintedanib, wherein the 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 the altered pharmacokinetic characteristic of the HRS-Fc fusion polypeptide is an increase in serum concentration.

40. The HRS-Fc fusion polypeptide is sequence number 157 (Fc-HRS(2-60) or HRS ＦＣ１ The composition according to claim 39, comprising, consisting of, or essentially consisting of )

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