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 APPLICATIONS This application claims the benefit of priority under 35 USC § 119(e) to U.S. patent application Ser. No. 62 / 487,812, filed April 20, 2017, the disclosure of which is incorporated by reference in its entirety.

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

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

[0004] Interstitial lung diseases (ILDs) are a heterogeneous group of disorders affecting the pulmonary interstitium in which inflammation is a primary pathogenesis. Within the ILD designation, there are many fibrotic lung conditions that are generally recognized as having a measurable inflammatory component, involving both innate and adaptive immune mechanisms that contribute to pathogenesis at several levels.

[0005] Patients with ILDs often suffer from progressively debilitating respiratory symptoms and experience significantly higher and increased mortality rates compared to the general population. As a group, these conditions represent a high unmet medical need for which there are few effective treatments that do not have significant undesirable side effects. Summary of the Invention [Means for solving the problem]

[0006] Embodiments of the present disclosure include, in appropriate part, a therapeutic composition comprising: (a) a histidyl-tRNA synthetase (HRS) polypeptide, or an expressible polynucleotide encoding an HRS polypeptide; (b) an immunomodulatory agent.

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

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

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

[0010] In some embodiments, the HRS-Fc fusion polypeptide comprises, consists of, or consists essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to 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 is less than about 5% aggregated.

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

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

[0013] In some embodiments, the immunomodulatory agent is selected from one or more of pirfenidone, nintedanib, sphingosine-1-phosphate (S1P) and / or S1P receptor (S1PR) modulators, steroids, optionally glucocorticoids, calcineurin inhibitors, 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 cytostatic agents, optionally methotrexate.

[0014] In some embodiments, the S1P and / or S1PR modulator is selected from the group consisting of amiselimod (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 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, and optionally, amicelimod is in the range of about 0.1 mg to about 10 mg. or about 0.1, 0.2, 0.3, 0.337, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg, or about 0.1, 0.2, 0.3, 0.337, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg or less, or at least about 0.1, 0.2, 0.3, 0.337, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg, or optionally, wherein amiserimod is about 0 In dosage units ranging from about 0.1 mg / kg to about 10 mg / kg, or about 0.1, 0.2, 0.3, 0.337, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg, or less than about 0.1, 0.2, 0.3, 0.337, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / kg.

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

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

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

[0018] In some embodiments, the IDO inhibitor is selected from indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat, antisense or RNAi agents that target IDO, and 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 mycophenolic acid (mycophenolate mofetil), ribavirin, and 6TGMP (6-thioguanine monophosphate), an antisense or RNAi agent that targets IMPDH, and an antibody or antigen-binding fragment or small molecule that specifically binds to IMPDH.

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

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

[0022] In some embodiments, the kinase inhibitor is selected from the group consisting of Janus kinases (JAKs, including JAK1, JAK2, JAK3, TYK2), epidermal growth factor receptor (EGFR), receptor tyrosine-protein kinase erbB-2 (Her2 / neu or ERBB2), Bcr-Abl, c-SRC, mitogen-activated protein kinase (MAP) kinases, anaplastic lymphoma kinase (ALK), spleen tyrosine kinase (SYK), Bruton's tyrosine kinase (BTK), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGF), and vascular endothelial growth factor receptor (VEGF). and an inhibitor of a kinase selected from one or more of VEGFRs (including VEGFR1, VEGFR2, VEGFR3), fibroblast growth factor receptors (FGFRs), B-Raf, RET proto-oncogene, platelet-derived growth factor receptor (PDGF-R), tropomyosin receptor kinases (Trks including TrkA, TrkB, TrkC), and c-Met, further wherein the kinase inhibitor is selected from an antisense or RNAi agent that targets the kinase, and an antibody or antigen-binding fragment or small molecule that specifically binds to the kinase.

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

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

[0025] In some embodiments, the antisense agent is about 10-40 bases in length and is optionally selected from morpholino oligonucleotides (PMOs), peptide nucleic acids (PNAs), 2'O-methyl phosphorothioate oligonucleotides, tricyclo-phosphorothioate oligonucleotides, and locked nucleic acids (LNAs).

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

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

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

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

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

[0031] Certain compositions include lipid nanoparticles.

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

[0033] 1. A method of treating pulmonary inflammation in a subject in need thereof, comprising administering to the subject: (a) a histidyl-tRNA synthetase (HRS) polypeptide, or an expressible polynucleotide encoding an HRS polypeptide; (b) an immunomodulatory agent.

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

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

[0036] In some embodiments, the immunomodulator alters one or more pharmacokinetic characteristics of the HRS polypeptide compared to the HRS polypeptide alone. In certain embodiments, the one or more altered pharmacokinetic characteristics of the HRS polypeptide 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 immunomodulatory agent is pirfenidone or nintedanib.

[0038] In some embodiments, the HRS polypeptide is SEQ ID NO: 157 (Fc-HRS(2-60) or HRSF C1 and the immunomodulatory agent is pirfenidone. In some embodiments, pirfenidone increases the serum concentration of 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 the HRS polypeptide alone.

[0039] In some embodiments, pirfenidone is provided in individual dosage units ranging from about 50 to about 1000 mg, or about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1 560, 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 1000 mg, 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, 510, 520, 530 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 1000 mg or less, or at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,and optionally in one, two, or three capsules for oral administration in individual dosage units of 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg.

[0040] In some embodiments, the dosage of pirfenidone is in daily dosage units ranging from about 100 to about 4000 mg / day, or about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 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, 1010, 1020, 1030 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, 230, 240, 250, 260, 270, 280, 290, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day 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 about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690 , 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 2000, In daily dosage units of 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mg / day, optionally administered in about 1, 2, 3, 4, 5, 6, 7, 8, or 9 capsules for oral administration.

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

[0042] In some embodiments, nintedanib is administered in individual dosage units ranging from about 10 to about 500 mg, or in individual dosage units ranging from 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, 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, 97 , 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 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 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 individual dosage units, optionally administered in about one, two, or three capsules.

[0043] In some embodiments, nintedanib is administered in a daily dosage unit ranging from about 20 to about 1000 mg / day, or about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 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, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 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 about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 7 Daily dosage units of 70, 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, optionally administered in about 1, 2, 3, 4, 5, or 6 capsules.

[0044] In some embodiments, nintedanib is administered in daily dosage units ranging from about 100-150 mg, or from about 200-300 mg / day, optionally in once or twice daily doses. In some embodiments, nintedanib is administered in daily dosage units ranging from about 100-150 mg, or from about 200-300 mg / day, optionally in once or twice daily doses.

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

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

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

[0048] In some embodiments, the drug is selected from one or more of an antibiotic, a chemotherapeutic agent, an antiarrhythmic agent, and a statin drug. In some embodiments, the infectious disease is selected from one or more of Pneumocystis pneumonia (PCP), tuberculosis, Chlamydia trachomatis, and respiratory syncytial virus (RSV), and cryptogenic organizing pneumonia. In some embodiments, the malignancy is lymphangitic carcinoma or lymphoma.

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

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

[0051] In some embodiments, the subject in need of treatment for pulmonary inflammation has a condition associated with worsening airway hyperresponsiveness resulting from other medications, airway disease with pulmonary hypertension, bronchitis or acute bronchitis, acute laryngotracheobronchitis, arachidonic acid bronchitis, catarrhal bronchitis, croupus bronchitis, bronchitis sicca, infectious asthmatic bronchitis, productive bronchitis, staphylococcal or streptococcal bronchitis, vesicular bronchitis, acute lung injury, bronchiectasis, or a condition associated with cylindrical bronchiectasis, saccular bronchiectasis, fusiform bronchiectasis, capillary bronchiectasis, cystic bronchiectasis, sicca bronchiectasis, or follicular bronchiectasis.

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

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

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

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

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

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

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

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

[0060] (a) a histidyl-tRNA synthetase (HRS) polypeptide, or an expressible polynucleotide encoding said HRS polypeptide; (b) an immunomodulatory agent; and a patient care kit comprising administering the immunomodulatory agent.

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

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

[0063] In some embodiments, pirfenidone is provided in individual dosage units ranging from about 50 to about 1000 mg (optionally in about 1, 2, or 3 capsules for oral administration), or in individual dosage units ranging from about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 96 390, 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, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 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 1000 mg or less, or at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 68 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 a daily dosage unit ranging from about 100 to about 4000 mg / day (optionally in about 3, 4, 5, 6, 7, 8, or 9 capsules for oral administration), or in a daily dosage unit ranging from about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, , 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, 330, 340, 350, 360, 3700, 3800, 3900, or 4000 mg / day 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 At most 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, 520, 530 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 In individual dosage 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 in individual dosage units of about 800 mg (e.g., 801 mg), for example, as three capsules of about 267 mg for oral administration, each of which is taken as three capsules per dosage. In certain embodiments, pirfenidone is in daily dosage units of about 2400 mg / day (e.g., 2403 mg / day), for example, as nine capsules of about 267 mg for oral administration three times a day, each of which is taken as three capsules per dosage.

[0066] In some embodiments, nintedanib is administered in individual dosage units ranging from about 10 to about 500 mg (optionally in about 1, 2, or 3 capsules for oral administration), or in individual dosage units ranging from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 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, 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 , 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, 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 about 10, 20, 30, 40, 50, 60, 70, 80, 90, In individual dosage 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 a daily dosage unit ranging from about 20 to about 1000 mg / day (optionally in about 1, 2, 3, 4, 5, or 6 capsules), or in a daily dosage unit ranging from about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 9 350, 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 about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, In daily dosage 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 dosage units ranging from about 100-150 mg, or from about 200-300 mg / day, optionally in once or twice daily doses. In some embodiments, nintedanib is administered in daily dosage units ranging from about 100-150 mg, or from about 200-300 mg / day, optionally in once or twice daily doses.

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

[0070] Also included are methods of treating pulmonary inflammation (as described herein) in a subject in need thereof, comprising administering to the 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 consists essentially of an amino acid sequence 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 ) comprising, consisting of, or consisting essentially of. [Brief explanation of the drawings]

[0071] [Figure 1] BAL fluid cell counts are shown. Individual cell counts are shown with lines indicating group means: "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 test article. Statistical comparisons were performed by one-way ANOVA within PO or IV treatment groups followed by Dunnett's post-hoc test. *p<0.05, **p<0.01 vs. IV vehicle. [Figure 2] Histological fibrosis (Ashcroft) scores are shown. Mean data from all fields 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 test article. Statistical comparisons were performed by one-way ANOVA followed by Dunnett's post-hoc test within PO or IV treatment groups. *p<0.05, **p<0.01 vs. respective vehicle. [Figure 3]Respiratory minute volumes (RMVs) on day 15 are shown. Individual RMVs are shown with lines indicating group means: "Veh" indicates treatment with vehicle, "Ninte." indicates treatment with nintedanib, and "TA" indicates treatment with test article. Test article doses are indicated in parentheses. IV groups were compared by one-way ANOVA, and PO groups were compared by t-test. *p<0.05, **p<0.01, ***p<0.001. [Figure 4] Histological fibrosis (Ashcroft) scores are shown. Mean data from all fields scored within each group are shown (+SEM): "Veh" indicates treatment with vehicle, "Ninte." indicates treatment with nintedanib, and "TA" indicates treatment with test article. Test article doses are indicated in parentheses. Statistical comparisons were performed by t-test within PO or IV treatment groups. *p<0.05 vs. IV vehicle. [Figure 5] Interstitial / alveolar inflammatory cell infiltration scores are shown. The mean individual scores within each group are shown: "Veh" indicates treatment with vehicle, "Ninte." indicates treatment with nintedanib, and "TA" indicates treatment with test article. The dose of test article is indicated in parentheses. Statistical comparisons were performed by t-test within PO or IV treatment groups. *p<0.05 vs. IV vehicle. [Figure 6] Mean serum HRSFC1 levels in pMol are shown (+SEM): In each group, which ended on day 22 of the study: Statistical comparisons were made by t-test between groups treated with vehicle (Veh) in combination with test article, nintedanib in combination with test article, or pirfenidone in combination with test article, respectively, with **p<0.01, ***p<0.001, ****p<0.0005. DETAILED DESCRIPTION OF THE INVENTION

[0072] The practice of the present invention will employ, unless specifically indicated to the contrary, conventional methods of molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below by way of example, and such techniques are explained fully in the literature. 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 meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any method, material, composition, reagent, cell, similar or similar to those described herein, similar or similar to those described herein, can be used in the practice or testing of the subject matter of this disclosure, preferred methods and materials are described herein. All publications and references, including but not limited to patent documents and patent application documents cited herein, are incorporated herein by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated herein by reference as if fully set forth. Any patent application to which this application claims priority is also incorporated herein by reference in its entirety in the manner described above for publications and references.

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

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

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

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

[0078] "Agonist" refers to a biological structure or chemical agent that increases or enhances the physiological effect of another drug or molecule. In some cases, agonists specifically bind to the other drug or molecule. Full and partial agonists are included.

[0079] The term "anergy" refers to the functional inactivation of T cell or B cell responses to restimulation with an antigen.

[0080] As used herein, the term "amino acid" is intended to refer to both naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics. Naturally occurring amino acids include, for example, the 20 (L)-amino acids utilized during protein biosynthesis, as well as others, such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, and the like, which are known to those skilled in the art. Amino acid analogs include modified versions of naturally occurring and non-naturally occurring amino acids. Such modifications can include, for example, substitutions or replacements of chemical groups and moieties on the amino acid or derivatization of the amino acid. Amino acid mimetics include organic structures that exhibit functionally similar properties, such as the charge and charge spacing characteristic of the 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 have the same mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include structures that are constrained to maintain optimal spacing and charge interactions of amino acids or amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0081] As used herein, a subject that is "at risk" of developing disease or adverse reaction may or may not have detectable disease or disease symptoms, and may or may not show detectable disease or disease symptoms before the treatment method described herein. "At risk" refers to the subject having one or more risk factors, which are measurable parameters that correlate with developing disease, as described herein and known in the art.Subjects that have one or more of these risk factors have a higher probability of developing disease or adverse reaction than subjects that do not have one or more of these risk factors.

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

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

[0084] The term "clonal depletion" refers to the elimination (e.g., loss or death) of autoreactive T cells. Clonal depletion can be achieved primarily within the thymus or periphery, or both.

[0085] Throughout this disclosure, unless the context requires otherwise, the words "comprise", "comprises", and "comprising" will be understood to imply the inclusion of stated steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements.

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

[0087] The terms "endotoxin-free" or "substantially endotoxin-free" generally refer to compositions, solvents, and / or containers containing at most trace amounts of endotoxin (e.g., amounts that have no clinically adverse physiological effects in a subject), preferably undetectable amounts. Endotoxins are toxins associated with certain microorganisms, e.g., bacteria, typically gram-negative bacteria, although endotoxins can also be found in gram-positive bacteria such as Listeria monocytogenes. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligosaccharides (LOS), found in the outer membrane of various gram-negative bacteria and represent a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans can produce fever, a drop in blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

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

[0089] Endotoxin can be detected using conventional techniques known in the art. For example, the Limulus amebocyte lysate assay, which utilizes blood from horseshoe crabs, is a highly sensitive assay for detecting the presence of endotoxin. In this test, very low levels of LPS can cause detectable clotting of the Limulus lysate due to a powerful enzyme cascade that amplifies this reaction. Endotoxin can also be quantified by enzyme-linked immunosorbent assay (ELISA). To ensure that endotoxin is substantially free, endotoxin levels can 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 active compound. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to approximately 1-10 EU.

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

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

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

[0093] " Homology " refers to the percentage of amino acids that are identical or that constitute conservative substitutions. Homology can be determined using 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 in the alignment, and such gaps are 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 protect the host from infection by pathogens, and the associated mechanisms that regulate cytokine expression and release (e.g., interferon and interferon-signaling), induce cell death, and inhibit protein synthesis.

[0095] "Isolated" refers to a material that is substantially or essentially free from components that normally accompany it in its natural state. For example, as used herein, "isolated polynucleotide," "isolated oligonucleotide," or "isolated oligonucleotide" can refer to a polynucleotide that has been purified or removed from the sequences adjacent to the polynucleotide in its naturally occurring state, e.g., a DNA fragment that has been removed from the sequences adjacent to the fragment in the genome. When referring to cells, the term "isolate" refers to the purification of cells (e.g., fibroblasts, lymphoblasts) from a source subject (e.g., a subject suffering from a polynucleotide repeat disease). In the context of mRNA or protein, "isolate" refers to the recovery of mRNA or protein from a source, e.g., a cell.

[0096] The term "modulate" optionally includes "increasing" or "decreasing" one or more quantifiable parameters by a defined and / or statistically significant amount. "Increase" or "increasing," "enhance" or "enhancing," or "stimulate" or "stimulating" generally refer to the ability of one or more agents or compositions to produce or cause a greater physiological response (i.e., downstream effect) in a cell or subject compared to the response caused by no agent / compound or a control compound. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include an increase in skeletal muscle mass in a tissue or a subject in need thereof. An "increased" or "enhanced" amount is typically a "statistically significant" amount and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more (e.g., 500, 1000-fold), including all integers and decimal points between and above 1 (e.g., 1.5, 1.6, 1.7, 1.8), over the amount produced by no agent / compound (absence of agent) or a control compound. The terms "reduce" or "inhibit" generally refer to the ability of one or more agents or compositions to "lower" a target gene or related physiological or cellular response, such as the expression of a symptom of a disease or condition, as described herein, as measured by routine techniques in the diagnostic arts. Related physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art and may include a reduction or amelioration of pulmonary inflammation or ILD symptoms or pathology, as described herein. A "reduction" in response may be "statistically significant" compared to the response produced by no agent or composition or a control agent or composition and may include a 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% reduction, including all integers in between.

[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 fractions, as measured, for example, and in no way limiting, 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 comprising 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 comprise one or more of the following: 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), including combinations thereof. Lipid nanoparticles are described, for example, in Petrilli et al., Curr Pharm. Biotechnol. 15:847-55, 2014, and U.S. Patent Nos. 6,217,912, 6,881,421, 7,402,573, 7,404,969, 7,550,441, 7,727,969, 8,003,621, 8,691,750, 8,871,509, 9,017,726, 9,173,853, 9,220,779, 9,227,917, and 9,278,130, which are incorporated by reference in their entireties.

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

[0100] Further examples of base-pairing moieties include, 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, which have their respective amino groups protected with acyl protecting groups, as well as other modified nucleobases such as octa-substituted purines, octa-substituted xanthines, or octa-substituted hypoxanthines (the latter two being naturally occurring 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 contemplated.

[0101] Further examples of base pairing moiety include, but are not limited to, the size-expanded nucleobase that adds one or more benzene rings.The nucleobase substitutions described in Glen Research catalogue (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, Hirao, I., Curr.Opin.Chem.Biol.,2006,10,622-627 are contemplated to be useful for synthesizing the oligonucleotide described herein. Examples of size-expanded nucleobases are shown below. [ka]

[0102] A nucleobase covalently linked to a ribose, sugar analog, or morpholino comprises a nucleoside. A "nucleotide" consists of a nucleoside with one phosphate group. The phosphate group covalently links adjacent nucleotides to each other to form an oligomer.

[0103] The terms "polypeptide" and "protein" are used interchangeably herein and refer to polymers of amino acid residues and their variants and synthetic analogs. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic, non-naturally occurring amino acids, such as chemical analogs of 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. The terms typically refer to polymeric forms of nucleotides, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide, typically at least 10 bases in length. The terms include single- and double-stranded forms of DNA. The terms "isolated DNA," "isolated polynucleotide," and "isolated nucleic acid" refer to molecules isolated free of total genomic DNA of a particular species. Thus, an isolated DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences but that has been isolated substantially free of, or purified free of, total genomic DNA of the species from which the DNA segment is obtained. Non-coding polynucleotides (e.g., primers, probes, oligonucleotides) that do not encode polypeptides are also included. Recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, viruses, etc., are also included.

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

[0106] "Expression control sequences" include nucleic acid or corresponding amino acid regulatory sequences, such as promoters, leaders, enhancers, introns, recognition motifs for RNA or DNA binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES), secretion signals, subcellular localization signals, and the like, which are capable of influencing transcription or translation, or the intracellular or cellular location of a coding sequence in a host cell. 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 capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. As used herein, a promoter sequence is bounded at its 3' end by a transcription initiation site, extends upstream (5' direction), and includes the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. Within the promoter sequence, a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for binding RNA polymerase may be found. Eukaryotic promoters do not necessarily contain "TATA" and "CAT" boxes, but often do. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

[0108] Numerous promoters, including constitutive, inducible, and repressible promoters from a variety of different sources, are well known in the art. Representative sources include, for example, viral, mammalian, insect, plant, yeast, and bacterial cell types, and suitable promoters from these sources are readily available or can be synthetically produced based on publicly available sequences online or from depositories such as the ATCC, as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci. (1996) 93(8):3346-3351, the T-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, U.S. Pat. No. 7,112,715, and Kramer & Fussenegger, Methods Mol. Biol. (2005) 308:123-144) or any promoter known in the art suitable for expression in the desired cells.

[0109] "An expressible polynucleotide comprises a cDNA, RNA, mRNA, or other polynucleotide that comprises at least one coding sequence and, optionally, at least one expression control sequence, e.g., a transcriptional and / or translational regulatory element, and is capable of expressing an encoded polypeptide (e.g., an HRS polypeptide) upon introduction into a cell, e.g., a cell in a subject."

[0110] In some embodiments, the expressible polynucleotide is a modified RNA or modified mRNA polynucleotide, such as a non-naturally occurring RNA analog. In certain embodiments, the modified RNA or mRNA polypeptide contains one or more modified or unnatural bases, such as nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T), and / or uracil (U). In some embodiments, the modified mRNA contains one or more modified or unnatural internucleotide 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 Application Nos. 2015 / 0111248, 2014 / 0243399, 2014 / 0147454, and 2013 / 0245104, which are incorporated by reference in their entireties.

[0111] In some embodiments, various viral vectors that can be used to deliver expressible polynucleotides include adenoviral vectors, herpesvirus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, and retroviral vectors. In some cases, the retroviral vector is a derivative of a murine or avian retroviral vector, or is a lentiviral vector. Examples of retroviral vectors into which a single foreign gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous sarcoma virus (RSV). Many additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate genes for selectable markers so that transduced cells can be identified and generated. For example, by inserting a polypeptide sequence of interest into the viral vector along 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 a polynucleotide encoding a protein.An exemplary targeting can be achieved by using an antibody to target retroviral vectors.Those skilled in the art will know or can easily identify the specific polynucleotide sequence that can be delivered to retroviral genome, which allows target-specific delivery of retroviral vectors, without undue experimentation.

[0112] In certain instances, the expressible polynucleotides described herein are 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 plasma membrane of the cell. In exemplary embodiments, the expressible polynucleotides are engineered for nuclear localization.

[0113] Also included herein are biologically active "variants" and "fragments" of the polypeptides described herein, as well as the polynucleotides encoding them. "Variants" contain one or more substitutions, additions, deletions, and / or insertions compared to a reference polypeptide or polynucleotide (see, for example, the tables and sequence listings). A variant polypeptide or polynucleotide comprises an amino acid or polynucleotide sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity or homology with the reference sequence described herein, and substantially retains the activity of the reference sequence. Also included are 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, while substantially retaining the activity of the reference sequence. In certain embodiments, the additions or deletions include C-terminal and / or N-terminal additions and / or deletions.

[0114] The term " sequence identity " or, for example, " sequence identity of 50% to " as used herein refers to the degree to which their sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis over the comparison window.Therefore, " sequence identity percentage " can be calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions where the same nucleic acid base (for example, A, T, C, G, I) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) exists in both sequences to obtain the number of matched positions, and dividing this number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for aligning a comparison window can be performed by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA), or by inspecting and selecting the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by any of a variety of methods. Reference can also be made to the BLAST family of programs, e.g., as 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. Commonly used measures of significance include p-value, which is the frequency or probability that the observed event occurs if the null hypothesis is true. If the obtained p-value is less than significance level, the null hypothesis is rejected. In a simple example, this significance level is defined as a p-value of 0.05 or less.

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

[0117] A "subject" or "subject in need thereof" includes a mammalian subject, such as a human subject.

[0118] "Substantially" or "essentially" means nearly wholly or completely, for example, 95% or more of a given amount.

[0119] "Therapeutic response" refers to an improvement in symptoms (whether sustained or not) upon administration of the therapeutic response.

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

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

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

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

[0124] As used herein, "treatment" of a subject (e.g., a mammal, e.g., a human) or cell is any type of intervention used to attempt to alter the natural course of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition, and may be performed either prophylactically or after the onset of a pathological event or contact with a pathogen. "Prophylactic" treatment is also included, which may be directed to reducing 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 indicate 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., a polypeptide) that is most frequently observed in a population and is thus arbitrarily designed the "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), including conjugates (e.g., Fc conjugates), variants, and fragments thereof, as well as expressible polynucleotides encoding the HRS polypeptides. Histidyl-tRNA synthetases belong to the class II tRNA synthetase family, which has three highly conserved sequence motifs. Class I and II tRNA synthetases are widely recognized as responsible for the specific binding of an amino acid to its cognate tRNA in a two-step reaction: an amino acid (AA) is first activated by ATP to form AA-AMP, which is then transferred to the acceptor end of the tRNA. Full-length histidyl-tRNA synthetases typically exist as either cytosolic homodimers or alternatively spliced ​​mitochondrial forms.

[0127] Certain biological fragments or alternatively spliced ​​isoforms of eukaryotic histidyl-tRNA synthetase, or in some contexts, the intact full-length synthetase, regulate certain therapeutically relevant cell signaling pathways and / or have anti-inflammatory properties. These activities, which differ 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 by, inter alia, blocking the migration, activation, or differentiation of inflammatory cells (e.g., monocytes, macrophages, T cells, B cells, NK cells, dendritic cells) associated with sites of active inflammation in vivo. 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. The sequences of certain exemplary HRS polypeptides are provided in Table H1 below. [Table H1-1] [Table H1-2] [Table H1-3] [Table H1-4] [Table H1-5] [Table H1-6] [Table H1-7] [Table H1-8] [Table H1-9] Table H1-10 Table H1-11 Table H1-12 Table H1-13 Table H1-14

Table H1-15

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

[0129] As mentioned above, HRS polypeptides can be modified in various ways, including amino acid substitutions, deletions, truncations, additions, and insertions. Methods for such manipulations are generally well known in the art. For example, amino acid sequence variants of the HRS reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations 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 applying amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

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

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

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

[0133] Hydrophobic: These residues are uncharged at physiological pH and are repelled by aqueous solution such that when the peptide is in aqueous medium, the residue seeks an interior position in the conformation of the peptide in which it is contained. 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 solution so as to seek an interior position in the conformation of the peptide in which they are contained when the peptide is in aqueous medium. Amino acids with neutral / polar side chains include asparagine, glutamine, cysteine, histidine, serine, and threonine.

[0135] This description also characterizes certain amino acids as "small" because their side chains, even when lacking polar groups, are not large enough to confer hydrophobicity. With the exception of proline, "small" amino acids are those with four or fewer carbons if at least one polar group is present on the side chain, and three or fewer if not. Amino acids with small side chains include glycine, serine, alanine, and threonine. The genetically encoded secondary amino acid proline is a special case due to its known effect on the secondary conformation of peptide chains. The structure of proline differs from all other naturally occurring amino acids in that its side chain is attached to the nitrogen and α-carbon of the α-amino group. Several amino acid similarity matrices are known in the art (see, e.g., the PAM120 matrix and the PAM250 matrix, e.g., as disclosed by Dayhoff et al., 1978, "A model of evolutionary change in proteins"). However, the matrix for determining distance relationships by M O Dayhoff, (ed.), Atlas of protein sequence and structure, Vol. 5, pp. 345-358, National Biomedical Research Foundation, Washington DC, and Gonnet et al. (Science, 256:14430-1445, 1992) 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, amino acids specifically contemplated by the present invention have been classified as one or the other. Most amino acids not specifically named can be classified based on known behavior.

[0137] Amino acid residues can be further subclassified as cyclic or acyclic, and aromatic or non-aromatic, self-evident classifications based on the residue's side-chain substituents, and as small or large. Residues are considered small if they contain a total of four or fewer carbon atoms, including the carboxyl carbon, provided that additional polar substituents are present; otherwise, they are considered small if they contain three or fewer. Small residues are, of course, always non-aromatic. Depending on their structural characteristics, amino acid residues may fall into more than one class. For naturally occurring protein amino acids, subclassification according to this scheme is 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 is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, substitutions of leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or similar substitutions of an amino acid with a structurally related amino acid will not significantly affect the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional truncated and / or variant HRS polypeptide can be easily determined by assaying its non-canonical activity, as described herein. Conservative substitutions are shown in Table B under the heading of exemplary substitutions. Amino acid substitutions that fall within the scope of the invention are generally achieved by selecting substitutions that do not significantly alter (a) the structure of the peptide backbone in the area of ​​the substitution, (b) the charge or hydrophobicity of the molecule at the target site, (c) the bulk of the side chain, or (d) its effect on maintaining 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 identity of their side chains. As described in the "Ammonists' Guide to the Ammonium Amino Acids," edition, Wm. C. Brown Publishers (1993), the first group includes glutamic acid, aspartic acid, arginine, lysine, and histidine, all of which have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, and asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, and methionine.

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

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

[0142] Thus, in certain embodiments, the HRS polypeptide comprises, consists of, or consists essentially of an amino acid sequence in Table H2 (SEQ ID NOS:118-120), or an active variant or fragment thereof. In some embodiments, the expressible polynucleotide encodes an HRS polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence in Table H2 (e.g., SEQ ID NOS:118-120), or an active variant or fragment thereof.

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

[0144] Certain embodiments include HRS polypeptides of Table H1 with a mutation or deletion of any one or more of Cys83, Cys174, Cys191, Cys196, Cys224, Cys235, Cys379, Cys455, or a deletion of Cys507 and Cys509, e.g., by deletion of the C-terminal three amino acids (Δ507-Δ509). Exemplary mutations at these positions include, for example, a cysteine ​​to serine, alanine, leucine, valine, or glycine mutation. In certain embodiments, the amino acid residue for the specific cysteine ​​substitution can be selected from naturally occurring substitutions found in HRS orthologs from other species and organisms. Exemplary substitutions of this type are shown in Table H3. [Table H3]

[0145] In some embodiments, the naturally occurring cysteines selected for mutagenesis are selected based on their surface exposure. Thus, in one aspect, the cysteine ​​residues selected for substitution are selected from Cys224, Cys235, Cys507, and Cys509. In some embodiments, the last three (C-terminal) residues of SEQ ID NO:1 are deleted, such that residues 507-509 are deleted. In some embodiments, these cysteines are selected for mutation or deletion to eliminate an intramolecular cysteine ​​pair, 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] Thus, in certain embodiments, the HRS polypeptide comprises, consists of, or consists essentially of an amino acid sequence in Table H4 (SEQ ID NOS:121-127), or an active variant or fragment thereof. In some embodiments, the expressible polynucleotide encodes an HRS polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence in Table H4 (e.g., SEQ ID NOS:121-127), or an active variant or fragment thereof.

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

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

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

[0151] Site-specific conjugation to an HRS polypeptide can also be achieved 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 / ) using published crystal structures of exemplary HRS polypeptides (see Xu et al., Structure. 20:1470-7, 2012 and U.S. Patent Application No. 61 / 674,639). Based on this analysis, some 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 the amino acid based on the crystal structure can be calculated, with a higher score indicating better accessibility. In certain embodiments, a higher score (e.g., >40) is preferred. Thus, in some embodiments, amino acid positions with a surface accessibility score greater than 40 can be used to introduce cysteine, lysine, glutamine, or other non-naturally occurring amino acids.

[0152] In certain embodiments, the solvent-accessible surface amino acids 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 non-naturally occurring amino acids optimized for site-specific conjugation or attachment.

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

[0154] In certain embodiments, non-naturally occurring amino acids include, but are not limited to, any amino acid, modified amino acid, or amino acid analog other than selenocysteine, and the 20 genetically encoded alpha-amino acids: 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] Unnatural 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 naturally occurring amino acids. For the structures of the 20 naturally occurring amino acids, see, for example, a biochemistry textbook such as Biochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York. It should be noted that the unnatural amino acids disclosed herein can be naturally occurring compounds other than the 20 alpha-amino acids described above. Because the unnatural amino acids disclosed herein typically differ from natural amino acids only in their side chains, these unnatural amino acids form amide bonds, for example, with other amino acids, natural or unnatural, in the same manner as they are formed in naturally occurring proteins. However, these unnatural amino acids have side chain groups that distinguish them from natural amino acids. For example, R in the above formula optionally includes 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 ester, borane, phenylboronic acid, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic-, pyridyl, naphthyl, benzophenone, constrained ring such as cyclooctyne, thioester, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, carboxylic acid, alpha-ketocarboxylic acid, alpha or beta unsaturated acids and amides, glyoxylamide, or organosilane group, or the like, or any combination thereof.

[0156] Specific examples of unnatural amino acids include p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, β-O-GlcNAc-L-serine, tri-O-acetyl-GalNAc-α-threonine, α-GalNAc-L-threonine, L-Dopa, fluorinated phenylalanine, Examples of suitable phenylalanine 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, those listed below or listed elsewhere in this specification, and the like.

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

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

[0159] Specific examples of amino acid analogs and mimetics can be found, for example, in Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341, Academic Press, Inc., New York, NY (1983), the entire contents of which are incorporated herein by reference. Other examples include per-alkylated amino acids, particularly per-methylated amino acids. See, for example, 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. Still other examples include amino acids whose amide moiety (and therefore the amide backbone of the resulting peptide) is replaced by, for example, a sugar ring, a steroid, a benzodiazepine, or a 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 peptide, polypeptide, peptidomimetic and protein are well known in the art (for example, see 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), Chapter 7; Stewart and Young, Solid Phase Peptide Synthesis, (2d ed.), Pierce Chemical Co., Rockford, Ill. (1984), each of which is incorporated herein by reference).Thus, HRS polypeptides can be composed of naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics.

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

[0161] In certain embodiments, the HRS polypeptide has a length of about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 13 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 acids in length, at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 501, 502, 503, 504, 505, 506, 507, 508, or 509 amino acids in length, and or up to about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 , 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 501, 502, 503, 504, 505, 506, 507, 508, or 509 amino acids in length, including all integer ranges therebetween, and comprising, consisting of, or consisting essentially of an amino acid sequence in Table H1, Table H2, or Table H4.

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

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

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

[0165] In other embodiments, fusion proteins of HRS polypeptides with other (non-HARS) proteins (e.g., heterologous proteins or polypeptides) are also included; these fusion proteins can modulate the biological activity, secretion, antigenicity, targeting, biological lifespan, ability to penetrate cell membranes or the 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 the amino acids Pro, Ala, and Ser ("PASylated") or hydroxyethyl starch (sold under the trademark HESYLATION®) provide a simple method for increasing the hydrodynamic volume of HRS polypeptides. This additional extension results in a bulky, random structure that significantly increases the size of the resulting fusion protein. This approach delays the generally rapid clearance of smaller HRS polypeptides via renal filtration by several orders of magnitude. Furthermore, the use of IgG fusion proteins has been shown to enable some fusion proteins to penetrate the blood-brain barrier (Fu et al., (2010) Brain Res. 1352:208-13).

[0166] Examples of fusion proteins that modulate the antigenic or immunomodulatory properties of HRS polypeptides include fusions to T cell-binding ligands, including, for example, MHC class I and II proteins, b-2 microglobulin, portions of LFA-3, portions of the Fc region of the heavy chain, and conjugates and derivatives thereof; examples of such fusion proteins are described, for example, in EP 1964854, U.S. Patent Nos. 5,468,481, 5,130,297, 5,635,363, 6,451,314, and US 2009 / 0280135.

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

[0168] In certain embodiments, the use of unnatural amino acids can be utilized to modify (e.g., increase) selected non-canonical activities of HRS polypeptides or to alter the in vivo or in vitro half-life of the protein. Unnatural amino acids can also be used to facilitate (selective) chemical modification (e.g., pegylation) of HRS proteins, as described elsewhere herein. For example, certain unnatural amino acids allow for the selective attachment 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 linked to one or more HRS polypeptides. Examples of HRS-Fc conjugates include fusion proteins and various forms of chemically cross-linked proteins. Wild-type sequences from any number of species, as well as variants, fragments, hybrids, and chemically modified forms thereof, may be used in the HRS-Fc conjugates. The HRS-Fc polypeptide may also (optionally) comprise one or more linkers, including peptide linkers and chemical linkers, that typically separate the Fc region from the HRS polypeptide, as described herein and known in the art. It will be understood that in any of these HRS-Fc conjugates, the native N- or C-terminal amino acids of the HRS polypeptide, or the native N- or C-terminal amino acids in the Fc domain, may be deleted and / or replaced with non-native amino acids, for example, to facilitate expression and / or cloning or to function as a linker sequence between the two proteins.

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

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

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

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

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

[0175] In certain embodiments, variant or otherwise modified Fc regions may be used, including those with altered properties or biological activity compared to the wild-type Fc region, as discussed in more detail below. Examples of modified Fc regions include those with sequences that are mutated, e.g., by one or more amino acid substitutions, insertions, deletions, or truncations compared to the wild-type sequence, hybrid Fc polypeptides composed of domains from different immunoglobulin classes / subclasses, Fc polypeptides with altered glycosylation / sialylation patterns, and Fc polypeptides that have been modified or derivatized, e.g., by biotinylation (see, e.g., U.S. Patent Application Publication No. 2010 / 0209424), phosphorylation, sulfation, etc., or any combination of the above. Such modifications may improve or alter 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, among other properties described herein, compared to the corresponding wild-type Fc sequence. max , t max , C min , variation), its immunogenicity, its complement fixation or activation, and / or CDC / ADCC / ADCP-related activity of the Fc region can be used to alter (e.g., increase, decrease).

[0176] The "Fc region" of the HRS-Fc conjugates provided herein is usually 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 of which is found only in IgG, IgA, and IgD immunoglobulins. The Fd region is the variable (V) region of the heavy chain. H ) and constant (CH1) domains of the light chain, L ) and steady-state (C L) domains together form the 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 the 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 the CH2, CH3, and CH4 regions, respectively. The Fc regions are primarily responsible for immunoglobulin effector functions, including, for example, complement fixation and binding to cognate 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 relative to the Fc region. In contrast to the constant region, the hinge region is structurally diverse, varying in both sequence and length among 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 the 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 an immunoglobulin and its respective Fc receptor (see, e.g., Shin et al., Intern. Rev. Immunol. 10:177-186, 1993).

[0179] Thus, the terms "Fc region" or "Fc fragment" or "Fc," as used herein, refer to a protein comprising one or more CH2, CH3, and / or CH4 regions from one or more selected immunoglobulins, including fragments, variants, and combinations thereof. An "Fc region" may also comprise one or more hinge regions of an immunoglobulin heavy chain constant region. In certain embodiments, an Fc region comprises the CH1, CH2, CH3, and / or CH4 regions of an immunoglobulin. L , V L, and / or V H Does not include one or more of the regions.

[0180] The Fc region can 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 subclasses and combinations thereof. In some embodiments, the Fc region is derived from an IgA immunoglobulin, including subclasses IgA1 and / or IgA2. In certain embodiments, the Fc region is derived from an IgD immunoglobulin. In certain embodiments, the Fc region is derived from an IgE immunoglobulin. In some embodiments, the Fc region is derived from an IgG immunoglobulin, including subclasses IgG1, IgG2, IgG2, IgG3, and / or IgG4. In certain embodiments, the Fc region is derived from an IgM immunoglobulin.

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

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

[0183] Certain Fc regions have increased binding to FcγRI compared to FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb. Some embodiments have increased binding to FcγRIIa compared to FcγRI, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb. Certain Fc regions have increased binding to FcγRIIb compared to FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb. Certain Fc regions have increased binding to FcγRIIc compared to FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and / or FcγRIIIb. Some Fc regions have increased binding to FcγRIIIa relative to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, and / or FcγRIIIb, while certain Fc regions have increased binding to FcγRIIIb relative 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 and is a member of both the immunoglobulin superfamily and the multichain immune recognition receptor (MIRR) family, and signals by associating with two FcRγ signaling chains.

[0185] FcεR includes FcεRI and FcεRII. The high-affinity receptor FcεRI is a member of the immunoglobulin superfamily and is expressed on epithelial Langerhans cells, eosinophils, mast cells, and basophils, where it plays 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 C-type lectin that can function as a membrane-bound or soluble receptor. FcεRII regulates B cell proliferation and differentiation and blocks IgE binding from eosinophils, monocytes, and basophils. Certain Fc regions enhance binding to FcεRII compared to FcεRII. Other Fc regions enhance binding to FcεRII compared to FcεRI. Table H6 below summarizes the characterization of certain FcRs. [Table H6-1] [Table H6-2]

[0186] The Fc region can be derived from immunoglobulin molecules of any animal, including vertebrates such as mammals, such as cows, goats, pigs, dogs, mice, rabbits, hamsters, rats, guinea pigs, non-human primates, and humans. The amino acid sequences of 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] Thus, the Fc region of the HRS-Fc conjugate can comprise, consist of, or consist essentially of one or more of the human Fc region amino acid sequences of Table H7, including variants, fragments, homologs, orthologs, paralogs, and combinations thereof. Certain exemplary embodiments comprise an Fc region ranging in size from about 20-50, 20-100, 20-150, 20-200, 20-250, 20-300, 20-400, 50-100, 50-150, 50-200, 50-250, 50-300, 50-400, 100-150, 100-200, 100-250, 100-300, 100-350, 100-400, 200-250, 200-300, 200-350, or 200-400 amino acids in length, optionally comprising, consisting of, or consisting essentially of any one or more of the sequences in Table H7. Certain embodiments comprise an Fc region of up to about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400 or more amino acids, optionally comprising, consisting of, or consisting essentially of any one or more of the sequences in Table H7.

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

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

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

[0191] Exemplary HRS-Fc fusion conjugates are provided below in Table H8. [Table H8-1] [Table H8-2] [Table H8-3] [Table H8-4] [Table H8-5] [Table H8-6]

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

[0193] As noted above, certain embodiments utilize variants, fragments, hybrids, and / or otherwise modified forms of the Fc regions described herein and known in the art. Included are variants having 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 in Table H7 or Table H8. Polypeptide and polynucleotide variants are described elsewhere herein.

[0194] Hybrid Fc regions are, for example, Fc regions that comprise a combination of Fc domains (eg, 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 In certain embodiments, the hybrid Fc region comprises, consists of, or consists essentially of IgM, IgM / IgA1, IgM / IgA2, IgM / IgD, IgM / IgE, IgM / IgG1, IgM / IgG2, IgM / IgG3, IgM / IgG4, IgM / IgM (or a fragment or variant 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, the 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 (also or a fragment or variant thereof), and optionally includes a hybrid Fc region comprising 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 a human Ig.

[0196] Certain examples include the following combinations of CH3 / 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 (also or a fragment or variant thereof), and optionally includes a hybrid Fc region comprising 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 a human Ig.

[0197] Particular 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 / IgM 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 consisting essentially of IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or a fragment or variant thereof), and 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 a specific embodiment, the hinge, CH2, CH3, and CH4 domains are from a human Ig.

[0198] Certain examples include the following combinations of hinge / 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 / 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 In certain embodiments, the hybrid Fc region comprises, consists of, or consists essentially of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM (or a fragment or variant thereof), and optionally comprises 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 from a human Ig.

[0199] Some examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of hinge / CH4 domains: IgA1 / IgE, IgA1 / IgM, IgA2 / IgE, IgA2 / IgM, IgD / IgE, IgD / IgM, IgG1 / IgE, IgG1 / IgM, IgG2 / IgE, IgG2 / IgM, IgG3 / IgE, IgG3 / IgM, IgG4 / IgE, IgG4 / IgM (or fragments or variants thereof), and optionally comprising 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 IgG subclass combinations 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 aspects, the Fc region may be modified, e.g., by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc., compared to a wild-type or naturally occurring Fc region. In certain embodiments, the Fc region may comprise a wild-type or native glycosylation pattern, or alternatively, may comprise increased glycosylation compared to the native form, decreased glycosylation compared to the native form, or may be completely deglycosylated. As an example of an altered Fc glycoform, decreased glycosylation of the Fc region reduces binding to the C1q region of the first complement component C1, a decrease in ADCC-related activity, and / or a decrease in CDC-related activity. Accordingly, certain embodiments utilize deglycosylated or aglycosylated Fc regions. For the generation of exemplary aglycosylated Fc regions, see, e.g., WO2005 / 047337. Another example of an Fc region glycoform can be generated by substituting position Q295 with a cysteine ​​residue according to the numbering system of Kabat et al. (See, e.g., U.S. Patent Application No. 2010 / 0080794). Certain embodiments can include an Fc region in which approximately 80-100% of the glycoproteins in the Fc region comprise a mature core carbohydrate structure lacking fructose (See, e.g., U.S. Patent Application No. 2010 / 0255013). Some embodiments can include an Fc region optimized by substitution or deletion to reduce the level of fucosylation, e.g., to increase affinity for FcγRI, FcγRIa, or FcγRIIIa and / or improve phagocytosis by FcγRIIa-expressing cells (See, e.g., U.S. Patent Application No. 2010 / 0249382 and U.S. Patent Application No. 2007 / 0148170).

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

[0203] Certain variant, fragment, hybrid, or otherwise modified Fc regions 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 Fc regions may have increased binding to one or more Fcγ receptors, Fcα receptors, Fcε receptors, and / or neonatal Fc receptors compared to the corresponding wild-type Fc sequence. In other embodiments, variant, fragment, hybrid, or modified Fc regions may have decreased binding to one or more Fcγ receptors, Fcα receptors, Fcε receptors, and / or neonatal Fc receptors compared to the corresponding wild-type Fc sequence. Specific FcRs are described elsewhere herein.

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

[0205] Certain variant, fragment, hybrid, or modified Fc regions may have altered effector functions compared to the corresponding wild-type Fc sequence. For example, such Fc regions may have increased complement binding or activation, increased Clq binding affinity, increased CDC-related activity, increased ADCC-related activity, and / or increased ADCP-related activity. In other embodiments, such Fc regions may have decreased complement binding or activation, decreased Clq binding affinity, decreased CDC-related activity, decreased ADCC-related activity, and / or decreased ADCP-related activity compared to the corresponding wild-type Fc sequence. By way of illustrative example only, the Fc region may include deletions or substitutions in complement binding sites, such as the Clq binding site, and / or deletions or substitutions in 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, or in addition, certain Fc effector functions can be evaluated in vivo by using animal models described, for example, in Clynes et al. PNAS. 95:652-656, 1998.

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

[0207] Further non-limiting examples of modifications to alter stability or half-life include substitutions / deletions at one or more of 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 Publication No. 2003 / 0190311. Specific examples include substitutions at position 251 with leucine, substitutions at position 252 with tyrosine, tryptophan, or phenylalanine, substitutions at position 254 with threonine or serine, substitutions at position 255 with arginine, substitutions at position 256 with glutamine, arginine, serine, threonine, or glutamate, substitutions at position 308 with threonine, substitutions at position 309 with proline, substitutions at position 311 with serine, substitutions at position 312 with aspartate, substitutions at position 314 with leucine, substitutions at position 380 with leucine, substitutions at position 382 with leucine, substitutions at position 383 with leucine, substitutions at position 384 with leucine, substitutions at position 385 with leucine, substitutions at position 386 with leucine, substitutions at position 387 with leucine, substitutions at position 388 with leucine, substitutions at position 389 with leucine, substitutions at position 389 with leucine, substitutions at position 390 with leucine, substitutions at position 391 with leucine, substitutions at position 392 with leucine, substitutions at position 393 with leucine, substitutions at position 394 with leucine, substitutions at position 395 with leucine, substitutions at position 396 with leucine, substitutions at position 397 with leucine, substitutions at position 398 with leucine, substitutions at position 399 with leucine, substitutions at position 400 with leucine, substitutions at position 401 with leucine, substitutions at position 402 with leucine, substitutions at position 403 with leucine, substitutions These modifications include substitutions with arginine, aspartate, or serine at position 5, threonine or proline at position 386, arginine or proline at position 387, proline, asparagine, or serine at position 389, methionine or threonine at position 428, tyrosine or phenylalanine at position 434, histidine, arginine, lysine, or serine at position 433, and / or histidine, tyrosine, arginine, or threonine at position 436. Such modifications optionally increase the affinity of the Fc region for FcRn, thereby increasing half-life, compared to the corresponding wild-type Fc region.

[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 can be measured according to routine techniques in the art, for example, in vitro (e.g., under physiological conditions). Exemplary solubility measurements are described elsewhere herein.

[0209] Further examples of variants include IgG Fc regions with conservative or non-conservative substitutions (as described elsewhere herein) at one or more of positions 250, 314, or 428 of the heavy chain, or any combination thereof, e.g., 250 and 428, or 250 and 314, or 314 and 428, or 250, 314, and 428 (see, e.g., U.S. Patent Application No. 2011 / 0183412). In certain embodiments, the residue at position 250 is substituted with glutamic acid or glutamine, and / or the residue at position 428 is substituted with leucine or phenylalanine. As another illustrative example of an IgG Fc variant, any one or more of the amino acid residues at positions 214-238, 297-299, 318-322, and / or 327-331 may be used as suitable targets for modification (e.g., conservative or non-conservative substitution, deletion). In certain embodiments, the CH2 domain of the IgG Fc variant contains 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 that of the EU index (Kabat et al., "Sequences of Proteins of Immunological Interest," 5 th(See, Ed., National Institutes of Health, Bethesda, Md. (1991)). Certain of these and related embodiments have altered (e.g., increased, decreased) FcRn binding and / or serum half-life, optionally without a concomitant reduction in effector function, such as ADCC- or CDC-related activity.

[0210] Further examples include variant Fc regions comprising one or more amino acid substitutions at positions 279, 341, 343, or 373 of the wild-type Fc region, or any combination thereof (see, e.g., U.S. Patent Application No. 2007 / 0224188). The wild-type amino acid residues at these positions for human IgG are valine (279), glycine (341), proline (343), and tyrosine (373). Substitutions may be conservative or non-conservative, or may include non-naturally occurring amino acids or mimetics as described herein. Alone or in combination with these substitutions, certain embodiments may also use variant Fc regions comprising at least one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions selected from the following: 235G, 235R, 236F, 236R, 236Y, 237K, 237N, 237R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 244L, 244H, 244I ... 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 noted above, the numbering of residues in the heavy chain isThe numbering is EU index (see Kabat et al., supra). Such variant Fc regions typically confer an altered effector function or an altered serum half-life to the HRS polypeptide to which the variant Fc region is operably linked. Preferably, the altered effector function is an increase in ADCC, a decrease in ADCC, an increase in CDC, a decrease in CDC, an increase in Clq binding affinity, a decrease in Clq binding affinity, an increase in FcR (preferably FcRn) binding affinity, or a decrease in FcR (preferably FcRn) binding affinity, compared to a corresponding Fc region lacking such amino acid substitutions.

[0211] Further examples include positions 221, 222, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, Included are variant Fc regions comprising amino acid substitutions at one or more of 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, e.g., U.S. Pat. No. 7,662,925). In certain embodiments, the variant Fc region is selected from the group consisting of P230A, E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W , N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V, L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R, and T335Y. In other specific embodiments, the 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, and 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. In a more specific embodiment, the variant Fc region is N297D / I332E, F241Y / F243Y / V262T / V264T / N297D / I332E,and N297D / S298A / A330Y / I332E. In certain embodiments, the variant Fc region comprises an amino acid substitution at position 332 (using the EU index numbering of Kabat et al., supra). Exemplary 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 that of the EU index of Kabat et al. Among other properties described herein, such variant Fc regions may have increased affinity for FcγRs, 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 , 433P, 438L, 439E / R, 440G, 441F, 442T, 445R, 446A, 447E, where optionally the variant has altered recognition of an Fc ligand and / or altered effector function compared to the parent Fc polypeptide, and the residue numbering is that of the EU index of Kabat et al. Specific examples of these and related embodiments include the following sets of substitutions: (1) N276D, R292Q, V305A, I377V, T394A, V412A, and K439E, (2) P244L, K246E, D399G, and K409R, (3) S304G, K320R, S324T, K326E, and M358T, (4) F243S, P244L, P244R, D399G, and K409R, (5) S304G, K320R, S324T, K326E, and M358T, (6) F243S, P244R, D399G, and K409R, (7) S304G, K320R, S324T, K326E, and M358T, (8) F243S, P244R, D399G, and K409R, (9) S304G, K320R, S324T, K326E, and M358T, (10) S304G, K320R, S324T, K326E, and M358T, (11) S304G, K320R, S324T, K326E, and M358T, (12) S304G, K320R, S324T 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, L306I, K326R, and Q362L, (14) H224Y, P230S, V323A, E333D, K338R, and S364C, (15) T335I, K414M, and P445R, (16) T335I and K414M, (17) P247A, E258K, D280N, K288R, N297D, T299A, K322E, Q342R, S354A, and L365P, (18) H268N, V279M, A339T, N361D, and S426P, (19) C261Y, K290E, L306F, Q311R, E333G, and Q438L, (20) E283G, N315K, E333G, R344Q, L365P, and S442T, (21) Q347R, N361Y, and K439R, (22) S239P, S254P, S267N, H285R, N315S, Included are variant Fc regions comprising or consisting of F372L, A378T, N390D, Y391C, F404S, E430K, L432P, and K447E, and (23) E269G, Y278H, N325S, and K370R, where the residue numbering is that of the EU index of Kabat et al. (see, e.g., U.S. Patent Application No. 2010 / 0184959).

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

[0214] The variant Fc region may also have one or more mutated hinge regions, for example, as described in U.S. Patent Application Publication No. 2003 / 0118592. For example, one or more cysteines in the hinge region may be deleted or substituted with different amino acids. 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 reduced dimerization ability compared to a wild-type Ig hinge region.

[0215] As described above, HRS-Fc conjugates, e.g., HRS-Fc fusion proteins, typically have altered (e.g., improved, increased, or decreased) pharmacokinetic properties compared to the corresponding HRS polypeptide. Examples of pharmacokinetic properties include stability or half-life, bioavailability (fraction of drug absorbed), tissue distribution, volume of distribution (the apparent volume into which a drug distributes immediately after intravenous injection and reaches equilibrium between plasma and surrounding tissues), concentration (initial or steady-state concentration of drug in plasma), elimination rate constant (the rate at which a drug is eliminated from the body), elimination rate (the rate of infusion required to balance elimination), area under the curve (AUC or exposure; the integral of the concentration-time curve after a single dose or at steady state), clearance (the volume of plasma cleared of drug per unit time), C max (peak plasma concentration of drug after oral administration), t max (C max time to reach C min These include the peak-to-trough fluctuation (the lowest concentration the drug reaches before the next dose is administered) and the fluctuation (the peak-to-trough fluctuation within one dosing interval at 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. Indeed, some HRS-Fc conjugates have increased non-canonical biological activity.

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

[0217] In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide is incubated for about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours, about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90 hours, about 96 hours, about 120 hours, or about 144 hours or more, or for at least about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or more. , about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours, about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90 hours, about 96 hours, about 120 hours, or about 144 hours or more, or 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, in a given species such as a rat, mouse, monkey, or human).

[0218] In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has greater bioavailability after subcutaneous (SC) administration than the corresponding unmodified HRS polypeptide. In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has a 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 than 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 in an anti-inflammatory activity assay as the corresponding unmodified or differently modified HRS polypeptide. 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 20 times more activity in an anti-inflammatory activity assay than the corresponding unmodified or differently modified HRS polypeptide.

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

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

[0222] Linker sequences can generally be from 1 to about 200 amino acids in length. Particular linkers can be from about 1 to 200 amino acids, 1 to 150 amino acids, 1 to 100 amino acids, 1 to 90 amino acids, 1 to 80 amino acids, 1 to 70 amino acids, 1 to 60 amino acids, 1 to 50 amino acids, 1 to 40 amino acids, 1 to 30 amino acids, 1 to 20 amino acids, 1 to 10 amino acids, 1 to 5 amino acids, 1 to 4 amino acids, 1 to 3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 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 It may have an overall 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 can be any one or more naturally occurring amino acids, non-naturally occurring amino acids, amino acid analogs, and / or amino acid mimetics as described elsewhere herein and known in the art. Specific amino acid sequences that can be usefully used as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., PNAS USA.83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. Specific peptide linker sequences include Gly, Ser, and / or Asn residues. Other near-neutral amino acids, such as Thr and Ala, can 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 (SEQ ID NO: 173), [GGSG] x (SEQ ID NO: 174), [GGGS] x (SEQ ID NO: 175), [GGGGS] x (SEQ ID NO: 176), [GN] x , [GGN] x , [GNN] x , [GNGN] x (SEQ ID NO: 177), [GGNG] x (SEQ ID NO: 178), [GGGN] x (SEQ ID NO: 179), [GGGGN] x (SEQ ID NO: 180) linker, wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. Other combinations of these and related amino acids will be apparent to those of skill in the art.

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

[0226] Further non-limiting examples of linker peptides include DGGGS (SEQ ID NO: 186), TGEKP (SEQ ID NO: 187) (see, e.g., Liu et al., PNAS. 94:5525-5530, 1997), GGRR (SEQ ID NO: 188) (Pomerantz et al. 1995), (GGGGS) n(SEQ ID NO: 176) (Kim et al., PNAS. 93: 1156-1160, 1996), EGKSSGSGSESKVD (SEQ ID NO: 189) (Chaudhary et al., PNAS. 87: 1066-1070, 1990), KESGSVSSEQLAQFRSLD (SEQ ID NO: 190) (Bird et al., Science. 242: 423-426, 1988), GGRRGGGS (SEQ ID NO: 191), LQRDGERP (SEQ ID NO: 192), LRQKDGGGSERP (SEQ ID NO: 193), LRQKd(GGGS)2ERP (SEQ ID NO: 194). In certain embodiments, the linker sequence comprises a Gly3 linker sequence, which contains 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 methods.

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

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

[0229] Suitable protease cleavage sites and self-cleaving peptides are known to those of skill in the art (see, for example, Ryan et al., J. Gener. Virol. 78:699-722, 1997 and Scymczak et al., Nature Biotech. 5:589-594, 2004). Exemplary protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2 encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to its high cleavage stringency, some embodiments include a TEV (tobacco etch virus) protease cleavage site 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 (TEV cleavage occurs between Q and G or between Q and S).

[0230] Further examples of enzymatically degradable linkers suitable for use in certain embodiments include, but are not limited to, amino acid sequences that are cleaved by serine proteases, such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or subtilisin. Illustrative 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-. Illustrative 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. Illustrative 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. Illustrative 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 illustrative 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 degradable linkers suitable for use in certain 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 certain 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 releasable linker has a half-life of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours or more, 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 tailored by using a particular releasable linker.

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

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

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

[0238] In some embodiments, the immunomodulatory agent is selected from one or more of sphingosine-1-phosphate (S1P) and / or S1P receptor (S1PR) modulators, steroids, calcineurin inhibitors, 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 cytostatic agents 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 systems 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 fibrogenic 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 as an 801 mg oral dosage unit (three 267 mg capsules) taken orally three times a day for a total oral administration of 2403 mg / day. Further exemplary dosages of pirfenidone are described herein.

[0240] In some embodiments, the immunomodulatory agent is nintedanib, which is also used for the treatment of IPF. Nintedanib inhibits certain 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). It is believed that nintedanib slows the progression of IPF and slows 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 as an oral dosage unit of 150 mg taken twice daily for a total of 300 mg / day, which can reduce side effects compared to a dosage of approximately 100 mg taken twice daily for a total of 200 mg / day. Further exemplary dosages of nintedanib are described herein.

[0241] In some embodiments, the 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 various biological functions, including cell proliferation, differentiation, angiogenesis, chemotaxis, and transport. Many of the activities of S1P are exerted through five closely related G protein-coupled receptors of the sphingosine-1-phosphate receptor family (S1PR), which play an important role in sphingolipid metabolism. S1PRs include 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 primarily expressed in large amounts on leukocytes, S1PR4 is primarily expressed in lymphocytes and hematopoietic tissues, and S1PR5 is primarily expressed in the spleen and white matter of the central nervous system (CNS).

[0242] Specific non-limiting examples of S1P or S1PR modulators include amiselimod (also known as MT-1303; S1PR antagonist; see Kappos et al., Lancet Neurol 2016;15:1148-59, 2016), fingolimod (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 and S1PR5 agonist), JTE-013 (S1PR2 antagonist), GSK2018682 (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). In certain embodiments, the S1P or S1PR modulator is amiselimod.

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

[0244] Certain S1PR antagonists or inhibitors include antisense agents and RNAi agents for S1PR coding sequences (see, for example, accession numbers NM_001400.4; NM_004230.3). Certain antisense agents specifically hybridize to target regions within the pre-mRNA or mRNA target sequence encoding S1PR, and the target region is 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-processed mRNA, branch point, 3' untranslated region (UTR), and polyadenylation signal sequence. Certain RNAi agents include a sense strand that is substantially identical to the mRNA target sequence encoding S1PR, and optionally an antisense strand that is complementary or substantially complementary to the mRNA target sequence encoding S1PR.

[0245] In some embodiments, the immunomodulatory agent is a steroid or corticosteroid, 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 immunomodulatory agent is a calcineurin antagonist or inhibitor. Calcineurin is a calcium and calmodulin-dependent serine / threonine protein phosphatase that activates T cells. Specifically, calcineurin translocates to the nucleus, upregulating the expression of interleukin 2 (IL-2), and then activates the nuclear factor of activated T cells (NFATc), which stimulates the growth and differentiation of T cell responses. Specific examples of calcineurin antagonists or inhibitors include cyclosporine, pimecrolimus, and tacrolimus.

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

[0248] In some embodiments, the immunomodulatory agent is a mechanistic target of rapamycin (mTOR) antagonist or inhibitor. mTOR is a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases and is the catalytic subunit of two structurally distinct complexes: mTORC1 and mTORC2, which are localized in different intracellular compartments, thereby specifically affecting 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 activation of the insulin receptor and insulin-like growth factor 1 receptor. mTORC2 is also involved in the regulation and maintenance of the actin cytoskeleton. mTOR plays a role in fibrosis and autoimmunity, and blockade of the mTORC pathway is under investigation as a treatment for such diseases.

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

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

[0251] In some embodiments, the immunomodulatory agent is an IDO antagonist or inhibitor. IDO is a tryptophan catabolic enzyme with immunoinhibitory properties. For example, IDO is known to suppress T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. Specific examples of IDO antagonists or inhibitors include indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epcadostat (see, e.g., Sheridan, Nature 2000, 14, 144-145). Biotechnology. 33:321-322, 2015). Certain IDO antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to IDO (see, e.g., Platten et al., Front Immunol. 5:673, 2014). Antisense agents and RNAi agents against the IDO coding sequence are also included (see, e.g., Accession No. 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 is selected from one or more of the AUG start codon of the mRNA, the region upstream of the AUG start codon, the region downstream of the AUG codon, the 3' or 5' splice site of a pre-processed mRNA, the branch point, the 3' untranslated region (UTR), and the polyadenylation signal sequence. Certain RNAi agents include a sense strand that is substantially identical to an mRNA target sequence encoding IDO, and optionally an antisense strand that is complementary or substantially complementary to an mRNA target sequence encoding IDO.

[0252] In some embodiments, the immunomodulatory agent is an inosine-5'-monophosphate dehydrogenase (IMPDH) antagonist or inhibitor. IMPDH is a purine biosynthetic enzyme that catalyzes the nicotinamide adenine dinucleotide (NAD+)-dependent oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), and is the first identified rate-limiting step 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 response. In particular, B and T cells exhibit a dependency on IMPDH for 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, 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 target regions within the pre-mRNA or mRNA target sequence encoding IMPDH, for example, the target region is 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-processed mRNA, branch point, 3' untranslated region (UTR), and polyadenylation signal sequence. Certain RNAi agents include a sense strand that is substantially identical to an mRNA target sequence encoding IMPDH, and optionally, an antisense strand that is complementary or substantially complementary to an mRNA target sequence encoding IMPDH.

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

[0255] In some embodiments, the target cytokine or cytokine receptor is an inflammatory or pro-inflammatory cytokine or cytokine receptor. Examples of target cytokines include interleukin-1 (IL-1), including IL-1α and IL-1β, interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-20 (IL-20), interleukin-33 (IL-33), tumor necrosis factor (TNF), interferon gamma (IFN-γ), and IL-1β. IL-1R, IL-6R, IL-8R, IL-11R, IL-12R, IL-17R, IL-18R, IL-20R, ST2 (interleukin 1 receptor-like 1, IL1RL1), TNFR such as TNFR1, interferon-gamma receptor (IFNGR), TGF-β receptor such as TGFβR1 (ALK5) or TGFβR2.

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

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

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

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

[0260] Specific examples of kinase inhibitors include JAK inhibitors such as baricitinib, fedratinib, filgotinib, gandotinib, lestaurtinib, momelotinib, pacritinib, peficitinib, ruxolitinib, tofacitinib, and 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, mubritinib, neratinib, nilotinib, pazopanib, pegaptanib, sorafenib, sunitinib, SU6656, toceranib, vandetanib, vatalanib, and vemurafenib.

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

[0262] In some embodiments, the immunomodulatory agent is a B cell receptor inhibitor, e.g., an agent targeting CD20. B lymphocyte antigen CD20, or CD20, is an activation-glycosylated phosphoprotein (CD45R+, CD117+) expressed on the surface of all B cells beginning at the pro-B stage and gradually increasing in concentration until maturation. The protein has no known natural ligand, and its function is to specifically enable optimal B cell immune responses to T-independent antigens. Exemplary immunomodulatory agents directed against CD20 include the monoclonal antibodies ibritumomab tiuxetan, obinutuzumab, ocralizumab, rituximab, tositumomab, and veltuzumab.

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

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

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

[0266] Certain small molecules may have the "specific binding" characteristic described herein. For example, in some embodiments, the small molecule may have a specific binding affinity of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM, at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 40, or 50 nM, or less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM.

[0267] In certain embodiments, the immunomodulatory agent is a polypeptide or peptide. The terms "peptide" and "polypeptide" are used interchangeably herein, although in certain instances, "peptide" can refer to shorter polypeptides, such as 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 can be composed of naturally occurring and / or non-naturally occurring amino acids, as described herein. Antibodies are also included as polypeptides.

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

[0269] In some embodiments, the immunomodulatory agent is an antibody or "antigen-binding fragment thereof" that specifically binds to a target 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 fragments thereof (such as dAb, Fab, Fab', F(ab')2, Fv), single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion having an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and any other modified form of an immunoglobulin molecule that contains an antigen-binding site or fragment (epitope-recognition site) of the required specificity. Certain properties and characterizations of antibodies (and antigen-binding fragments thereof) are described in more detail herein.

[0271] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy and / or light chain that binds to an antigen of interest. In this regard, the antigen-binding fragment of an antibody described herein can 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 portion of a molecule that can be bound by a selective binding agent, such as an antibody, and that can be used to produce antibodies in an animal that can bind to an epitope of that antigen. An antigen can have one or more epitopes.

[0273] The term "epitope" includes any determinant, e.g., a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. An epitope is a region of an antigen or target protein that is bound by an antibody. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryls, or sulfonyls, and in certain embodiments, can have specific three-dimensional structural characteristics and / or specific charge characteristics. Epitopes can be contiguous or discontinuous with respect to the primary structure of the antigen.

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

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

[0276] Antibodies can be prepared by any of a variety of 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 a polypeptide of interest can be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, or modifications thereof. Methods of 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. A particular example includes the VELOCIMMUNE® platform by REGENEREX® (see, eg, US Pat. No. 6,596,541).

[0277] Antibodies can also be generated 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 clone or synthetic libraries, such as the Human Combinatorial Antibody Library (HuCAL), in which the structural diversity of the human antibody repertoire is represented by seven heavy chain and seven light chain variable region genes. The combination of these genes generates 49 frameworks within the master library. By overlaying these frameworks with highly variable gene cassettes (CDRs = complementarity-determining regions), a vast human antibody repertoire can be reproduced. Also included are human libraries designed using fragments of human donor origin encoding the light chain variable region and heavy chain CDR-3, synthetic DNA encoding diversity for heavy chain CDR-1, and synthetic DNA encoding diversity for heavy chain CDR-2. Other libraries suitable for use will be apparent to those skilled in the art.

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

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

[0280] The structure and location of immunoglobulin variable domains 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 editions.

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

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

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

[0284] Single-chain 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 have been described for identifying chemical structures for converting the naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into sFv molecules that fold into a three-dimensional structure substantially similar to that of an antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 by Huston et al. and U.S. Patent No. 4,946,778 by Ladner et al.

[0285] In certain embodiments, the antibodies described herein are in the form of a "diabody." A diabody is a multimer of polypeptides, each polypeptide comprising a first domain containing an immunoglobulin light chain binding region and a second domain containing an immunoglobulin heavy chain binding region, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site: the antigen-binding site is formed by the association of a first domain of one polypeptide in the multimer with a second domain of another polypeptide in the multimer (WO94 / 13804). An antibody dAb fragment consists of a VH domain (Ward et al., Nature 341:544-546, 1989). Diabodies and other multivalent or multispecific fragments can be constructed, for example, by gene fusion (see WO 94 / 13804, and Holliger et al., PNAS USA. 90:6444-6448, 1993).

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

[0287] If bispecific antibodies are to be used, they may be conventional bispecific antibodies and may be produced in a variety of ways (Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993), for example, prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments described above. Diabodies and scFvs can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic 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 specificities can be readily selected from libraries using phage display (WO 94 / 13804). If one arm of the diabody is consistently maintained to have specificity for, for example, antigen X, a library can be generated in which the other arm is diversified, and antibodies with the appropriate specificity can be selected. Bispecific whole antibodies can be generated by "knobs-into-holes" engineering (Ridgeway et al., Protein Eng., 9:616-621, 1996).

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

[0290] In certain embodiments, the antibodies described herein may take the form of nanobodies. Minibodies are encoded by a single gene and are efficiently produced in almost all prokaryotic and eukaryotic hosts, such as Escherichia coli (see U.S. Pat. No. 6,765,087), molds (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyvermyces, Hansenula, or Pichia (see, e.g., U.S. Pat. No. 6,838,254). The production process is scalable, and multi-kilogram quantities of nanobodies have been produced. Nanobodies can be formulated as ready-to-use solutions with long shelf lives. The Nanoclone method (see, e.g., WO 06 / 079372) is a proprietary method for generating nanobodies against desired targets based on automated high-throughput selection of B cells.

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

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

[0293] In certain embodiments, the antibody may be a chimeric antibody. In this regard, 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 an Ig class different from that of 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 include 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 may include the entire variable domain (VL, VH, or both).

[0294] In some embodiments, an immunomodulatory agent is or comprises a "ligand," e.g., a natural ligand, of a target molecule. "Ligand" generally refers to a substance or molecule that forms a complex with a target molecule (e.g., a biomolecule) to fulfill 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, certain agents are actually protein ligands that bind to a target molecule and generate a signal. Also included are "modified ligands," e.g., pharmacokinetic modifiers, e.g., protein ligands fused to an Fc region from an immunoglobulin.

[0295] In some embodiments, the immunomodulatory or inhibitory agent is an antisense agent.Thus, in some embodiments, the target protein, target sequence, and / or target gene described herein (for example, S1PR, calcineurin, mTOR, IDO, IMPDH, cytokine and / or cytokine receptor, B cell receptor, kinase) is targeted by any of various antisense agents, including oligonucleotide-based agents or methods. Antisense agents or oligonucleotides typically comprise a base sequence that targets (e.g., is sufficiently complementary to or specifically hybridizes to) a region within a target sequence, and optionally includes one or more of the following: the region including or surrounding the AUG start codon of an mRNA (e.g., the region upstream of the start codon, the region downstream of the start codon, the region including the start codon), the 3' or 5' splice site of a preprocessed mRNA, a pyrimidine-rich or polypyrimidine tract upstream of the splice acceptor site, an exon-intron boundary, an intron-exon boundary, a branch site, an exon splicing enhancer element, a 5' and 3' untranslated region, and a polyadenylation signal sequence.

[0296] In certain embodiments, an antisense agent can effectively modify (e.g., reduce expression or alter splicing) target gene expression upon administration to a subject in need thereof or upon contact with a cell, e.g., a muscle cell. 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 uptake, forms a duplex with the target RNA at a Tm of greater than about 45°C.

[0297] Certain "antisense agents" include "antisense oligonucleotides," "antisense oligomers," and "oligonucleotides," which refer to linear sequences of nucleotides or nucleotide analogs in which the nucleobases can hybridize to a target sequence in RNA through Watson-Crick base pairing 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 subunits may be based on ribose or another pentose sugar, or, in certain embodiments, morpholino groups (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 contemplated.

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

[0299] The term "targeting sequence" refers to a sequence in an oligonucleotide that is complementary (and by extension, substantially complementary) to a "target sequence" in RNA. The entire sequence of an antisense agent or only a portion thereof may be complementary to a target sequence. For example, in an antisense agent having 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 that is complementary to the target region. Typically, the target sequence is formed by contiguous bases in the oligonucleotide, but alternatively, it may be formed by non-contiguous sequences that, when placed together, for example, from opposite ends of the oligonucleotide, constitute a sequence spanning the target sequence.

[0300] Target sequence can have "approximate" or "substantial" complementarity with target sequence and still function for the purpose of the present disclosure, that is, target sequence can still be "complementary".Preferably, the oligonucleotide used in the present disclosure has at most one mismatch with target sequence in 10 nucleotides, preferably at most one mismatch in 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 exemplary antisense target sequence.

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

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

[0303] In certain embodiments, antisense oligonucleotides are recognized as substrates for active or facilitated transport across cell membranes, for example, muscle cell membranes.The ability of oligonucleotides to form stable duplexes with target RNA can also be related to other properties of oligonucleotide backbone, including the length and degree of complementarity of antisense oligonucleotides with respect to target, the ratio of G:C base match and A:T base match, and the position of any mismatched base.The ability of antisense oligonucleotides to resist cellular nuclease can promote survival and the ultimate delivery of drugs to cell cytoplasm.Therefore, certain embodiments include non-naturally occurring antisense oligonucleotides that are nuclease-resistant or substantially nuclease-resistant.

[0304] In certain embodiments, the antisense oligonucleotide comprises a non-natural chemical backbone selected from a phosphoramidate or phosphorodiamidate morpholino oligonucleotide (PMO), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a phosphorothioate oligonucleotide, a tricyclo-DNA oligonucleotide, a tricyclo-phosphorothioate oligonucleotide, a 2'O-Me-modified oligonucleotide (e.g., a 2'O-methyl phosphorothioate oligonucleotide), or any combination of the foregoing.

[0305] When an oligonucleotide hybridizes to a target under physiological conditions, the antisense oligonucleotide "specifically hybridizes" to a target sequence or polynucleotide (e.g., pre-mRNA, 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. At a given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes to a complementary polynucleotide. Such hybridization can occur with "approximate" or "substantial" complementarity of the antisense oligonucleotide to the target sequence, as well as with exact complementarity.

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

[0307] Antisense oligonucleotides generally comprise multiple nucleotide subunits, each having a nucleobase that, when taken together, form or comprise a targeting sequence. Thus, in some embodiments, antisense oligonucleotides range in length from about 10 to about 40 subunits, or from about 10 to 30 subunits, and typically from 15 to 25 subunits. For example, in some embodiments, antisense oligonucleotides are 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 ranging 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. In certain embodiments, antisense oligonucleotides are about 10 to about 40 or about 5 to about 30 nucleotides in length. In some embodiments, the antisense oligonucleotides are about 14 to about 25 or about 17 to about 27 nucleotides in length.

[0308] In some embodiments, the backbone of antisense oligonucleotide is substantially uncharged, and optionally recognized as a substrate for active or facilitated transport across cell membrane.In some embodiments, all internucleotide bonds are uncharged.The ability of an oligonucleotide to form a stable duplex with target RNA can also be related 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 match and A:T base match, and the position of any mismatched base.The ability of antisense oligonucleotide to resist cellular nuclease can promote survival and the ultimate delivery of drugs to the cytoplasm of cells.

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

[0310] In some embodiments, the number of cationic linkages is at least 2 and no more than about half the total internucleotide linkages, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 cationic linkages, or no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 cationic linkages. In some embodiments, however, up to all internucleotide linkages are cationic linkages, e.g., up to 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, 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 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, cationic linkages. In certain embodiments, oligonucleotides of about 19-20 subunits may have 2-10, e.g., 4-8, cationic linkages, with the remainder being uncharged. In other certain embodiments, oligonucleotides of 14-15 subunits may have 2-7, e.g., 2, 3, 4, 5, 6, or 7, cationic linkages, with the remainder being uncharged. Thus, the total number of cationic linkages within an oligonucleotide can vary from about 1 to 10 to 15 to 20 to 30 or more (including all integers in between), and can be dispersed throughout the oligonucleotide.

[0311] In some embodiments, the antisense oligonucleotide may have about 1 or up to about 1 cationic linkage for every 2-5, or 2, 3, 4, or 5 uncharged linkages, e.g., about 4-5 or 4 or 5 for every 10 uncharged linkages.

[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 seen when about 25% of the backbone linkages are cationic. In certain embodiments, enhancement may be seen with a low number of cationic linkages, e.g., 10-20%, or when the number of cationic linkages is in the range of 50-80%, e.g., about 60%.

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

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

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

[0316] As mentioned above, antisense oligonucleotides can use various antisense chemicals.Examples of oligonucleotide chemicals include, but are not limited to, peptide nucleic acid (PNA), locked nucleic acid (LNA), phosphorothioate, 2'O-Me-modified oligonucleotide, morpholino, PMO, PPMO, PMOplus and PMO-X chemicals, including any combination of the above.Generally, PNA and LNA chemicals can utilize shorter targeting sequences due to their relatively high target binding strength compared with PMO and 2'O-Me oligonucleotide.Phosphorothioate and 2'O-Me-modified chemicals are often combined to produce 2'O-Me-phosphorothioate backbone.See, for example, PCT Publication No. WO / 2013 / 112053 and PCT Publication No. WO / 2009 / 008725, which are incorporated herein by reference in their entirety.

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

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

[0319] Antisense oligonucleotides may also contain "locked nucleic acid" subunits (LNAs). "LNAs" are members of a class of modifications called bridged nucleic acids (BNAs). BNAs are characterized by a covalent bond that locks the conformation of the ribose ring of the C30-endo (northern) sugar pucker. In LNAs, the bridge consists of a methylene between the 2'-O and 4'-C positions. LNAs enhance backbone preorganization and base stacking, increasing hybridization and thermal stability. The structure of LNA is described, for example, in Wengel, et al., Chemical Communications (1998) 455; Tetrahedron (1998) 54:3607; and Accounts of Chem. Research (1999) 32:301; Obika, et al., Tetrahedron Letters (1997) 38:8735; (1998) 39:5401; and Bioorganic Medicinal Chemistry (2008) 16:9230.

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

[0321] "Phosphorothioates" (or S-oligos) are variants of normal DNA in which one of the non-bridging oxygens is replaced by sulfur. Sulfuration of the internucleotide bond 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 phosphodiesterases. Phosphorothioates are generated by two major routes: by the action of a solution of elemental sulfur in carbon disulfide on hydrogen phosphonate, or by sulfurization of a phosphite triester with either tetraethylthiuram disulfide (TETD) or 3H-1,2-bensodithiol-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 insolubility 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 analogues in which each nucleotide is modified by the introduction of a cyclopropane ring, with the backbone configuration optimized to limit conformational flexibility and the torsion angle γ. Homobasic adenine- and thymine-containing tc-DNA forms highly stable AT base pairs with complementary RNA. Tricyclo-DNAs and their synthesis are described in International Patent Application Publication No. WO2010 / 115993. Compounds of the present disclosure may incorporate one or more tricyclyl-DNA nucleotides, and in some cases, the compounds may consist entirely of tricyclyl-DNA nucleotides.

[0323] Tricyclo-phosphorothioate nucleotides are tricyclo-DNA nucleotides with phosphorothioate intersubunit linkages.Tricyclo-phosphorothioate nucleotides and their synthesis are described in International Patent Application Publication No. WO2013 / 053928.The compounds of the present disclosure can incorporate one or more tricyclyl-DNA nucleotides, and in some cases, the compounds can be entirely composed of tricyclyl-DNA nucleotides.

[0324] A "2'O-Me oligonucleotide" molecule carries a methyl group at the 2'-OH residue of the 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 (PTO) for further stabilization. 2'O-Me oligonucleotides (phosphodiester or phosphothioate) can be synthesized according to routine techniques in the art (see, for example, Yoo et al., Nucleic Acids Res. 32:2008-16, 2004). In some cases, 2'O-Me oligonucleotides contain phosphorothioate linkages (2'O-Me phosphorothioate oligonucleotides).

[0325] " Morpholino oligonucleotide " or " PMO " refers to an oligonucleotide having a backbone that supports nucleobases that can hydrogen bond with typical polynucleotides, and the polymer lacks a pentose sugar backbone, but instead contains a morpholino ring.Therefore, in PMO, the morpholino ring structure supports base-pairing moieties, typically forming a sequence of base-pairing moieties designed to hybridize with the selected antisense target in cells or the subject being treated.Exemplary " morpholino " oligonucleotides comprise morpholino subunit structures linked together by phosphoramidate or phosphorodiamidate linkages, which connect the morpholino nitrogen of one subunit to the 4' exocyclic carbon of the adjacent subunit, and each subunit comprises a purine or pyrimidine nucleobase that is effective for binding to a base in a polynucleotide through base-specific hydrogen bonding. Morpholino oligonucleotides (including antisense oligonucleotides) are described, for example, in U.S. Pat. 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 Ser. Nos. 12 / 271,036 and 12 / 271,040, and PCT publications WO / 2009 / 064471 and WO / 2012 / 043730, all of which are incorporated by reference herein in their entireties.

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

[0327] "PMO-X" refers to a phosphorodiamidate morpholino oligonucleotide (PMO) having (i) a covalent bond to the nitrogen atom of the morpholino ring, and (ii) a phosphorus atom with a second covalent bond to 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 No. PCT / US2011 / 38459 and PCT Publication No. WO / 2013 / 074834, each of which is incorporated herein by reference in its entirety. "PMO-apn" or "APN" refers to a PMO-X oligonucleotide containing 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, antisense oligonucleotides containing the targeting sequences described herein contain at least one APN-containing linkage or APN derivative-containing linkage. Certain embodiments include PMOs having about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% APN / APN derivative-containing linkages, with the remaining linkages (if less than 100%) being uncharged linkages, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 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, 95, 96, 97, 98, 99, 100, 101, 10 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 linkages.

[0328] Additional 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 Publication Nos. WO / 2007 / 002390, WO / 2010 / 120820, and WO / 2010 / 148249, U.S. Patent No. 7,838,657, and U.S. Patent Application No. 2011 / 0269820.

[0329] In some embodiments, the immunomodulatory or inhibitory agent 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, cytokine and / or cytokine receptor, B cell receptor, kinase) described herein is targeted by any of various 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 cells that express the appropriate molecular machinery, which degrades the corresponding endogenous mRNA. This mechanism involves the conversion of dsRNA into short RNAs that target 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 comprises a sense strand that corresponds to the "target sequence" of the target gene, more generally, in a manner described herein and well known in the art.

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

[0332] Certain embodiments use double-stranded ribonucleic acid (dsRNA) molecules as RNAi agents. dsRNA generally comprises two single strands. One strand of the dsRNA (the "sense" strand) contains a nucleotide sequence that is substantially identical to a portion of a target gene or target sequence, while the other strand (the "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 duplex structure. In certain embodiments, the complementary RNA strand can be less than 30 nucleotides, less than 25 nucleotides in length, or even 19-24 nucleotides in length. In certain aspects, the complementary nucleotide sequence can be 20-23 nucleotides in length, or 22 nucleotides in length. In certain embodiments, the sense strand of the RNAi agent is substantially identical to a portion of the target sequence described herein.In some embodiments, the antisense strand is complementary or substantially complementary to a portion of the target sequence described herein. In certain embodiments, the portion comprises, consists of, or consists essentially of about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or no more than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a target sequence described herein.

[0333] Suitable siRNA sequence can be identified by any means known in the art.In some cases, by using the exemplary target sequence described herein, the method described in Elbashir et al., Nature, 411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) is combined with the rational design rule 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 of interest can be scanned for dinucleotide sequences (e.g., AA, NA, CC, GG, or UU, where N is C, G, or U) (see, e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). The nucleotide immediately 3' of the dinucleotide sequence is identified as a potential siRNA sequence (i.e., target sequence or sense strand sequence). In some cases, 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotides immediately 3' of the dinucleotide sequence are identified as a potential siRNA sequence. In some embodiments, the dinucleotide sequence is an AA or NA sequence, and the 19 nucleotides immediately 3' of the AA or NA dinucleotide are identified as a potential siRNA sequence. siRNA sequences are usually spaced at different positions along the length of the target gene. To further enhance the silencing efficiency of siRNA sequences, potential siRNA sequences can be analyzed to identify sites that do not contain regions of homology with other coding sequences in the target cell or organism, for example. For example, a suitable siRNA sequence of about 21 base pairs will typically not have more than 16-17 adjacent base pairs of homology with coding sequences in the target cell or organism. If the siRNA sequence is to be expressed from an RNA Pol III promoter, an siRNA sequence lacking more than four adjacent A's or T's is selected.

[0335] Once potential siRNA sequences are identified, the sequences can be analyzed using various criteria known in the art.For example, to improve their silencing efficiency, rational design algorithms can be used to analyze siRNA sequences to identify sequences with one or more of the following characteristics: (1) G / C content of about 25% to about 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.The siRNA design tool that incorporates an algorithm that specifies the appropriate value of each of these characteristics and is useful for selecting siRNA is known.Those skilled in the art will understand that sequences with one or more of the above-mentioned characteristics can be selected as potential siRNA sequences for further analysis and testing.

[0336] Furthermore, potential siRNA target sequences with one or more of the following criteria can often be eliminated as siRNAs: (1) sequences containing a stretch of four or more identical bases in a row; (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 a stretch of seven or more G / Cs in a row; and (5) sequences containing tandem repeats of four or more bases in a candidate that result in an internal fold structure. However, one of skill in the art will understand that sequences with one or more of the aforementioned characteristics can still be selected for further analysis and testing as potential siRNA sequences.

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

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

[0339] An RNAi agent typically comprises a double-stranded portion (notwithstanding the optional and potentially preferred presence of any single-stranded overhand) comprising at least 16 bases, optionally at least 17 bases, more optionally at least 18 bases, even more optionally at least 19 bases, and usually 18-35 bases, optionally 19-30 bases, more optionally 20-25 bases, and even more optionally 21-23 bases, that is identical or nearly identical (e.g., exhibiting 90% or more, e.g., at least 95% sequence identity with, or exhibiting up to two and optionally only one mismatch) to the mRNA whose silencing is desired and therefore targeted by the RNAi agent.

[0340] In certain embodiments, at least one of the RNA strands comprises a nucleotide overhang of 1 to 4 nucleotides in length. In some embodiments, the dsRNA comprises at least one chemically modified nucleotide. In certain aspects, a dsRNA comprising a 1 to 4 nucleotide single-stranded overhang may comprise a molecule in which the unpaired nucleotide of the single-stranded overhang directly adjacent to the terminal nucleotide pair comprises a purine base. In some embodiments, the last complementary nucleotide pair on both ends of the dsRNA is a GC pair, or at least two of the last four terminal nucleotide pairs are GC pairs.

[0341] In certain embodiments, the RNAi agent comprises a microRNA. 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 about 70 nucleotides. (See V. Ambros et al., Current Biology 13:807, 2003). MicroRNAs are not translated into proteins, but instead bind to specific messenger RNAs, thereby blocking translation. It is believed that microRNAs inhibit translation by imprecise base pairing with targets. Certain microRNAs can be transcribed as hairpin RNA precursors and processed into 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 the double-stranded oligonucleotide contains two or more nucleoside residues than the second strand. In other embodiments, the first strand and the second strand have the same number of nucleosides, but the first and second strands are offset so that the two terminal nucleosides on the first strand and the second strand are not paired with residues on the complementary strand. In certain cases, the two unpaired nucleosides are thymidine residues.

[0343] In some cases, when the modulating agent comprises siRNA, the agent comprises a region of sufficient homology with the target region and is of sufficient length in terms of nucleotides so that the siRNA agent or its fragment can mediate the down-regulation of target gene or RNA.It will also be understood that the term "ribonucleotide" or "nucleotide" can refer to the modified nucleotide or surrogate replacement portion at one or more positions in the case of modified RNA or nucleotide surrogate.Therefore, the siRNA agent is or comprises a region that is at least partially complementary to the target sequence.Although there is no need for perfect complementarity between the siRNA agent and the target sequence, the correspondence must be sufficient to allow the siRNA agent or its cleavage product to induce sequence-specific silencing, for example, by RNAi cleavage of the target RNA.The degree of complementarity or homology with the target strand is most important in the antisense strand.Perfect complementarity is often desired, especially in the antisense strand, but some embodiments comprise one or more, but preferably 10, 8, 6, 5, 4, 3, 2 or less mismatches with the target sequence. Mismatches are most tolerated in the terminal regions, and if present, are preferably within the terminal regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5' and / or 3' termini. The sense strand need only be sufficiently complementary to the antisense strand to maintain the overall double-stranded character of the molecule.

[0344] In some embodiments, RNAi agents or oligonucleotides, such as siRNA oligonucleotides, are modified or contain nucleoside surrogates. The single-stranded region of an siRNA agent can be modified or contain nucleoside surrogates; for example, the unpaired region of a hairpin structure, such as the region connecting two complementary regions, can have modifications or nucleoside surrogates. Modifications are also included, for example, to stabilize one or more 3' or 5' ends of the siRNA agent against exonucleases or to facilitate the antisense siRNA agent entering RISC. Exemplary modifications include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), and special biotin or fluorescein reagents that are generated as phosphoramidites and have another DMT-protected hydroxyl group to allow multiple coupling 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 them to enter 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 herein, "siRNA agents or shorter RNAi agents" refers to siRNA agents that are short enough not to induce a harmful interferon response in human cells, for example, they have a duplex region of less than 60, but preferably less than 50, 40, or 30 nucleotide pairs. An siRNA modulator or its cleavage product can, for example, downregulate a target gene by inducing RNAi against the target RNA.

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

[0347] In addition to the homology with target RNA and the ability to down-regulate target gene, siRNA agent may have one or more of the following properties: it may have an antisense strand that can present bases (or modified bases) in a suitable three-dimensional framework, so that it can form a duplex structure with homologous target RNA, even if a large number or even all nucleosides are modified, and can form accurate base pairs and, for example, by cleaving target RNA, enough to enable down-regulation of the target; even if a large number or even all nucleosides are modified, it may still have " RNA-like " properties, that is, it may have the overall structural, chemical and physical properties of RNA molecules, even if it is not limited to ribonucleotide-based content, or even if it is only partially ribonucleotide-based content.For example, siRNA agent may contain, for example, sense and / or antisense strands in which all nucleotide sugars contain, for example, 2' fluoro instead of 2' hydroxyl.This deoxyribonucleotide-containing agent may still be expected to show RNA-like properties. Without wishing to be bound by theory, electronegative fluorine prefers an axial orientation when attached to the C2' position of ribose. This spatial preference of fluorine can also force the sugar to adopt a C3' end pucker. This is the same puckering mode observed in RNA molecules, resulting in the A-family helix characteristic of RNA. Furthermore, because fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen-bonding interactions with water molecules known to stabilize RNA structure. Generally, modified moieties at the 2' sugar position can enter into H-bonds, which are more characteristic of the OH moiety of ribonucleotides than the H moiety of deoxyribonucleotides.

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

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

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

[0351] In some embodiments, RNAi agents include oligonucleotides that contain at least one ligand linked to modified or unnatural nucleobases.Many compounds can function as modified bases.The structure of modified bases is important to the extent that modified bases should not substantially interfere with the binding of oligonucleotides to their target, for example, mRNA.In certain embodiments, modified bases are difluorotolyl, nitropyrrolyl, nitroimidazolyl, nitroindolyl, naphthalenyl, anthracenyl, pyridinyl, quinolinyl, pyrenyl, or any one of the divalent radicals of unnatural nucleobases described herein.In certain embodiments, unnatural nucleobases are difluorotolyl, nitropyrrolyl, or nitroimidazolyl.In certain embodiments, unnatural nucleobases are difluorotolyl.A wide variety of ligands are well known in the art. For example, the ligand may be a steroid, bile acid, lipid, folic acid, pyridoxal, B12, riboflavin, biotin, aromatic compounds, polycyclic compounds, crown ethers, interfering substances, cleavage agent molecules, protein binders, or carbohydrates. In certain embodiments, the ligand is a steroid or aromatic compound. In certain cases, the ligand is cholesteryl.

[0352] In some embodiments, RNAi agent is the oligonucleotide that is connected to ligand for the purpose of improving cell targeting and uptake.For example, RNAi agent can be connected to antibody or its antigen binding fragment.As another example, RNAi agent can be connected to specific ligand binding molecule, such as polypeptide or polypeptide fragment, that specifically binds to specific cell surface receptor.

[0353] In certain embodiments, the RNAi agent comprises an unnatural nucleobase.In some embodiments, the unnatural nucleobase is difluorotolyl, nitroimidazolyl, nitroindolyl or nitropyrrolyl.In certain embodiments, the regulator provided in the aromatic compound is related to a double-stranded oligonucleotide sequence, and only one of the two strands comprises an unnatural nucleobase.In certain embodiments, the regulator used herein is related to a double-stranded oligonucleotide sequence, and both strands independently comprise at least one unnatural nucleobase.

[0354] In certain instances, the ribose sugar moiety naturally present in a nucleoside is replaced with a hexose sugar. In certain aspects, the hexose sugar is allose, altrose, glucose, mannose, gulose, idose, galactose, talose, or a derivative thereof. In a preferred embodiment, the hexose is D-hexose. In certain instances, the ribose sugar moiety naturally present in a nucleoside is replaced with a polycyclic heteroalkyl ring or a cyclohexenyl group. In certain instances, the polycyclic heteroalkyl group is a bicyclic ring containing one oxygen atom in the ring. In certain instances, the polycyclic heteroalkyl group is bicyclo[2.2.1]heptane, bicyclo[3.2.1]octane, or bicyclo[3.3.1]nonane. In certain embodiments, the backbone of an oligonucleotide is modified to improve the therapeutic or diagnostic properties of the oligonucleotide compound. In certain embodiments, at least one of the bases or at least one of the sugars of the oligonucleotide is modified to improve the therapeutic or diagnostic properties of the oligonucleotide compound. 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 the oligonucleotides that contain modified backbones or non-natural internucleotide linkages.As defined herein, the oligonucleotides that have modified backbones or non-natural internucleotide linkages include those that retain a phosphorus atom in the backbone and those that do not retain a phosphorus atom in the backbone.The modified oligonucleotides that do not have a phosphorus atom in their intersugar backbone can also be considered as oligonucleotides.Specific oligonucleotide chemical modifications are described below.Not all positions in a given compound need to be uniformly modified, and in fact, one or more of the following modifications can be incorporated into a single oligonucleotide compound, or even in a single nucleotide thereof.

[0356] Examples of modified internucleotide linkages or backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates (including those with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reverse polarity, where adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' and 2'-5' to 5'-2'). Various salts, mixed salts, and free acid forms are also included.

[0357] Representative U.S. patents that teach the preparation of the above phosphorus atom-containing linkages include U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,450,945, and the like. 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 internucleotide linkages or backbones (i.e., oligonucleotides) that do not contain a phosphorus atom therein include backbones formed by short alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short heteroatom or heterocyclic intersugar linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate, methyleneimino, and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones containing mixed N, O, S, and CH moieties.

[0359] Representative U.S. patents that teach the preparation of the above oligonucleotides include U.S. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, and 5,541,307. , 5,561,225, 5,596,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, each of which is incorporated herein by reference.

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

[0361] Also included are oligonucleotides that use ribozymes. Synthetic RNA molecules and their derivatives that catalyze highly specific endoribonuclease activity are known as ribozymes. (See generally U.S. Patent No. 5,543,508 to Haseloff et al. and U.S. Patent No. 5,545,729 to 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). Naturally occurring autocatalytic RNA molecules can be modified to generate ribozymes that can target specific cellular or pathogenic RNA molecules with high specificity. Thus, ribozymes serve the same general purpose as antisense oligonucleotides (i.e., controlling the expression of specific genes), and, like oligonucleotides, are nucleic acids that have a significant portion of a single-stranded chain state. That is, ribozymes have substantial chemical and functional identity to oligonucleotides and are therefore considered equivalents for the purposes described herein.

[0362] In certain cases, RNAi agents can be modified by non-ligand groups.Many non-ligand molecules have been conjugated to enhance the activity, cellular distribution, cell targeting or cell uptake of oligonucleotides, and the procedures for carrying out such conjugation are available in references.Examples of non-ligand moieties include lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, such as hexyl-5-tritylthiol (Manoharan et al., Ann. NY Acad. 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 such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), 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). Typical conjugation protocol involves synthesizing an oligonucleotide with an amino linker at one or more positions of the sequence.Then, the conjugated molecule is reacted with the amino group using a suitable coupling or activation reagent.The conjugation reaction can be carried out while the oligonucleotide is still attached to the solid support, or after the oligonucleotide is cleaved in the liquid phase.Purification of the oligonucleotide conjugate by HPLC typically yields the pure conjugate.

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

[0364] Exemplary vectors for use herein include viral vectors, which are known and include, for example, but not limited to, vectors derived from retroviruses, vaccinia viruses, poxviruses, adenoviruses, and adeno-associated viruses (AAVs). Such viral vectors can be engineered by recombinant techniques known per se to introduce therein a nucleic acid sequence encoding any one of the antisense or RNAi agents disclosed herein.

[0365] For example, retroviral vectors can be used herein to deliver RNAi agents.Generally, retroviral vectors can include retroviral genome sequences that encode the components necessary for the integration of recombinant viral genomes (randomly) into the target host cell genome and nucleic acid sequences, for example, particularly the nucleic acid sequences encoding one of the antisense or RNAi agents disclosed herein.Such retroviral vectors can be easily constructed using standard recombinant techniques from various 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 Laboratory Press, 1990). Laboratory, 1985).

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

[0367] Other exemplary viral vectors for use herein are vectors derived from poxviruses such as vaccinia viruses, e.g., attenuated vaccinia viruses, e.g., Modified Virus Ankara (MVA) or NYVAC, avipox viruses, e.g., fowl diphtheria viruses, or canarypox viruses.

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

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

[0370] Accordingly, some embodiments include a method of treating pulmonary inflammation in a subject in need thereof, 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] Certain embodiments include methods of treating pulmonary inflammation in a subject in need thereof, comprising administering to the subject (a) a histidyl-tRNA synthetase (HRS) polypeptide or an expressible polynucleotide encoding an HRS polypeptide, and (b) an immunomodulatory agent, e.g., as described herein. In some embodiments, (a) and (b) are administered separately, optionally as described herein. In certain embodiments, (a) and (b) are administered together, optionally as a therapeutic composition described herein.

[0372] In certain embodiments, the immunomodulatory agent is pirfenidone or nintedanib. In some methods or compositions, pirfenidone is provided in individual dosage units ranging from about 50 to about 1000 mg, or in individual dosage units ranging from about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 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 1000 mg, 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 1000 mg or less, or at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 10, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 75 In individual dosage 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, for example, in about one, two, or three capsules for oral administration.

[0373] In some embodiments, pirfenidone is administered in a daily dosage unit ranging from about 100 to about 4000 mg / day, or about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 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, 1010, 1020, 1030, , 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, 9...

Claims

[Claim 1] The invention as described in the drawings.

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