HISTIDYL-tRNA SYNTHETASE-Fc CONJUGATES

Abstract

To provide histidyl-tRNA synthetase-Fc conjugates.SOLUTION: The present invention provides histidyl-tRNA synthetase and Fc region conjugate polypeptides (HRS-Fc conjugates), such as HRS-Fc fusion polypeptides, compositions comprising the same, and methods of using such conjugates and compositions for treating or diagnosing a variety of conditions. The HRS-Fc conjugates of the invention have improved controlled release properties, stability, half-life, and other pharmacokinetic and biological properties relative to corresponding, unmodified HRS polypeptides.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under U.S. Patent Act 119(e) to U.S. Provisional Application No. 61 / 789,011, filed March 15, 2013, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Statement The sequence listing accompanying this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence listing is named ATYR_116_01WO_ST25.txt. This text file is approximately 399 KB and was created on March 14, 2014, and has been submitted electronically via EFS-Web.

[0003] The present invention relates generally to conjugates, such as fusion polypeptides, of one or more histidyl-tRNA synthetase (HRS) polypeptide(s) and immunoglobulin Fc region(s), compositions comprising them, and methods of using such polypeptides and compositions to treat or diagnose various conditions. [Background technology]

[0004] Physiocrine domains are small, naturally occurring protein domains generally found in the aminoacyl-tRNA synthetase (AARS) gene family in higher organisms, but are not required for the well-established role of aminoacyl-tRNA synthetases in protein synthesis. Until the discovery of the physiocrine paradigm, aminoacyl-tRNA synthetases, a family of approximately 20 enzymes, were known only for their ubiquitous expression in all living cells and their essential role in the process of protein synthesis. However, more recent scientific findings now suggest that aminoacyl-tRNA synthetases have additional roles beyond protein synthesis and, in fact, have evolved in multicellular organisms to play important homeostatic roles in tissue physiology and disease.

[0005] Evidence for the existence of non-canonical functions of AARS includes well-defined sequence comparisons establishing that during evolution from simple unicellular organisms to more complex life forms, AARS evolved to become more structurally complex through the addition of accessory domains without losing their ability to facilitate protein synthesis.

[0006] Consistent with this hypothesis, a rich and diverse set of expanded functions for AARSs has been discovered in tRNA synthetases of higher eukaryotes, particularly humans. This data, based both on direct analysis of individual domains and on the discovery of mutations in tRNA synthetase genes that are causally associated with disease but that do not affect aminoacylation or protein synthesis activity, suggests that these newly appended domains, or physiocrines, are central to newly acquired non-canonical functions of AARSs.

[0007] Furthermore, there is increasing recognition that specific tRNA synthetases, such as histidyl-tRNA synthetase (HRS), can be released or secreted from living cells and provide important locally acting signals with immunomodulatory, chemotactic, and angiogenic properties. Direct confirmation of the role of AARS as extracellular signaling molecules has been obtained through studies demonstrating the secretion and extracellular release of specific tRNA synthetases, as well as through direct demonstration that the addition of fragments of tRNA synthetases containing newly added domains (Physiocrines) is active in a range of extracellular signaling pathways, while the addition of other fragments lacking these domains is not. These Physiocrines, such as HRS, present new, previously untapped opportunities to develop new classes of therapeutic proteins for treating human diseases.

[0008] To best utilize these and other activities in therapeutic or diagnostic settings, there is a need in the art for HRS polypeptides with improved pharmacokinetic properties. These improved therapeutic forms of HRS polypeptides would enable the development of more effective therapeutic regimens for the treatment of various diseases and disorders, requiring significantly less frequent administration than the unmodified protein. Summary of the Invention [Means for solving the problem]

[0009] Embodiments of the present invention relate generally to histidyl-tRNA synthetase (HRS) polypeptide conjugates having one or more immunoglobulin Fc regions covalently linked thereto, pharmaceutical compositions comprising such molecules, methods of manufacture, and methods for their therapeutic use. Among other advantages, the HRS-Fc conjugates of the present invention may possess improved pharmacokinetic properties and / or improved therapeutically relevant biological activity compared to the corresponding unmodified HRS polypeptide.

[0010] Thus, certain embodiments include an HRS polypeptide comprising an amino acid sequence at least 80% identical to any of SEQ ID NOs: 1-106, 170-181, or 185-191, or any of the sequences in Table D1, D3-D6, or D8, and an HRS fusion polypeptide comprising at least one Fc region fused to the C-terminus, N-terminus, or both, of the HRS polypeptide. In some embodiments, the HRS polypeptide comprises, consists of, or consists essentially of an amino acid sequence at least 90% identical to any of SEQ ID NOs: 1-106, 170-181, or 185-191, or any of the sequences in Table D1, D3-D6, or D8. In specific embodiments, the HRS polypeptide comprises, consists of, or consists essentially of an amino acid sequence of any one of SEQ ID NOs: 1-106, 170-181, or 185-191, or any of the sequences in Table D1, D3-D6, or D8.

[0011] In certain embodiments, the HRS polypeptide comprises amino acid residues 2-40, 2-45, 2-50, 2-55, 2-60, 2-66, or 1-506 of SEQ ID NO: 1, or an amino acid sequence at least 90% identical to residues 2-40, 2-45, 2-50, 2-55, 2-60, 2-66, or 1-506 of SEQ ID NO: 1. In some embodiments, the HRS polypeptide is up to about 40-80 amino acids in length and comprises residues 2-45 of SEQ ID NO: 1. In specific embodiments, the HRS polypeptide consists of or consists essentially of amino acid residues 2-40, 2-45, 2-50, 2-55, 2-60, 2-66, or 1-506 of SEQ ID NO: 1.

[0012] In some embodiments, at least one endogenous cysteine ​​residue of the HRS polypeptide is substituted with another amino acid or deleted. In certain embodiments, the at least one endogenous cysteine ​​residue is selected from Cys174, Cys191, Cys224, Cys235, Cys507, and Cys509. In certain embodiments, the at least one endogenous cysteine ​​residue is selected from Cys224, Cys235, Cys507, and Cys509. In specific embodiments, the endogenous cysteine ​​residues are Cys507 and Cys509. In some embodiments, all endogenous surface-exposed cysteine ​​residues are substituted with another amino acid or deleted.

[0013] In certain embodiments, the HRS polypeptide is tandemly repeated. In certain embodiments, the HRS polypeptide comprises a WHEP domain. In specific embodiments, the HRS polypeptide lacks a functional aminoacylation domain. In some embodiments, the HRS polypeptide consists essentially of a WHEP domain. In specific aspects, the WHEP domain of the HRS polypeptide, or a variant or fragment thereof, has a consensus sequence in Table D5.

[0014] In some embodiments, the Fc region and the HRS polypeptide are separated by a peptide linker, which in certain embodiments is about 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acids in length. In certain embodiments, the peptide linker is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100 amino acids in length. In certain embodiments, the peptide linker consists of Gly and / or Ser residues. In some embodiments, the peptide linker is a physiologically stable linker. In other embodiments, the peptide linker is a releasable linker, optionally an enzymatically cleavable linker. In specific embodiments, the peptide linker comprises the sequence of any one of SEQ ID NOs: 200-260, or any other peptide linker described herein.

[0015] In some embodiments, the Fc region is fused to the C-terminus of the HRS polypeptide. In certain embodiments, the Fc region is fused to the N-terminus of the HRS polypeptide.

[0016] In certain embodiments, the Fc region comprises one or more of the hinge, CH2, CH3 and / or CH4 domains from mammalian IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and / or IgM. In some embodiments, the Fc region comprises the hinge, CH2 and CH3 domains of IgG1. In some embodiments, the Fc region comprises the hinge, CH2 and CH3 domains of IgG2. In some embodiments, the Fc region comprises the hinge, CH2 and CH3 domains of IgG3. In certain embodiments, the HRS fusion polypeptide comprises the CH1, CH2 and CH3 domains of immunoglobulins. L , V L and V H Does not include the area.

[0017] In specific embodiments, the Fc region comprises any one of SEQ ID NOs: 128-163 or 339-342, or a variant, fragment, or combination thereof. In certain embodiments, the hinge domain is a modified IgG1 hinge domain comprising SEQ ID NO: 341.

[0018] In a specific embodiment, the Fc region is MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 339) or S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 340).

[0019] In certain embodiments, the HRS-Fc fusion polypeptide is Fc-HRS(2-60) (SEQ ID NO: 337), or HRS(1-60)-Fc (SEQ ID NO: 338), or Fc-HRS(2-40) (SEQ ID NO: 381), or HRS(1-40)-Fc (SEQ ID NO: 386), or Fc-HRS(2-45) (SEQ ID NO: 382), or HRS(1-45)-Fc (SEQ ID NO: 387), or Fc-HRS(2-50) (SEQ ID NO: 383), or HRS(1-50)-Fc (SEQ ID NO: 388), or Fc-HRS(2-55) (SEQ ID NO: 384), or HRS(1-55)-Fc (SEQ ID NO: 389), or Fc-HRS(2-66) (SEQ ID NO: 385), or HRS(1-66)-Fc (SEQ ID NO: 390), or Fc-HRS(2-60)HRS(2-60) (SEQ ID NO: 396).

[0020] In certain instances, the HRS fusion polypeptide has altered pharmacokinetics compared to the corresponding HRS polypeptide. Examples of altered pharmacokinetics include increased serum half-life, increased bioavailability, increased exposure, and / or decreased clearance. In certain instances, the exposure is increased by at least 100-fold. In some instances, the HRS fusion polypeptide has a half-life of at least 30 hours in mice. In certain instances, the bioavailability is increased subcutaneously by at least about 30%. In some instances, the HRS fusion polypeptide has altered immune effector activity compared to the corresponding HRS polypeptide. Examples of such immune effector activity include one or more of complement activation, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or antibody-dependent cell-mediated phagocytosis (ADCP).

[0021] In certain embodiments, the Fc region comprises a variant Fc region compared to the wild-type Fc region. In some embodiments, the variant Fc region comprises a sequence at least 90% identical to any one of SEQ ID NOs: 128-163 or 341, or a combination of said sequences. In certain embodiments, the variant Fc region comprises a hybrid of one or more Fc regions from different species, different Ig classes, or different Ig subclasses. In certain embodiments, the variant Fc region comprises a hybrid of one or more hinge, CH2, CH3, and / or CH4 domains of Fc regions from different species, different Ig classes, and / or different Ig subclasses.

[0022] In certain embodiments, the variant Fc region has an altered glycoform compared to the corresponding wild-type Fc region. In certain embodiments, the variant Fc region has altered pharmacokinetics compared to the corresponding wild-type Fc region. Examples of such altered pharmacokinetics include serum half-life, bioavailability, and / or clearance. In some embodiments, the variant Fc region has altered effector activity compared to the corresponding wild-type Fc region. Examples of such effector activity include one or more of complement activation, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or antibody-dependent cell-mediated phagocytosis (ADCP).

[0023] In certain embodiments, the variant Fc region has altered binding to one or more Fcγ receptors compared to the corresponding wild-type Fc region. Exemplary Fcγ receptors are described herein and known in the art.

[0024] In certain embodiments, the variant Fc region has altered binding to one or more FcRn receptors compared to the corresponding wild-type Fc region. Exemplary FcRn receptors are described herein and known in the art.

[0025] In some embodiments, the variant Fc region has altered (e.g., increased) solubility compared to the corresponding wild-type Fc region, and the HRS-Fc fusion polypeptide has altered solubility compared to the corresponding unmodified HRS polypeptide.

[0026] In a specific embodiment, the HRS-Fc fusion polypeptide is substantially in a dimeric form in physiological solution or under other physiological conditions, such as in vivo conditions. In a specific embodiment, the HRS-Fc fusion polypeptide has substantially the same secondary structure as the corresponding unmodified or differentially modified HRS polypeptide, as determined by UV circular dichroism analysis.

[0027] In some embodiments, the HRS-Fc fusion polypeptide has a plasma or serum pharmacokinetic AUC profile when administered to a mammal that is at least 5-fold greater than the corresponding unmodified HRS polypeptide.

[0028] In certain embodiments, the HRS-Fc fusion polypeptide has substantially the same activity in an assay for anti-inflammatory activity as the corresponding unmodified or differentially modified HRS polypeptide.

[0029] In certain embodiments, the HRS-Fc fusion polypeptide has more than twice the activity of the corresponding unmodified or differentially modified HRS polypeptide in an assay for anti-inflammatory activity.

[0030] In certain embodiments, the HRS-Fc fusion polypeptide has at least 30% greater stability than the corresponding unmodified or differentially modified HRS polypeptide when compared under similar conditions in PBS at pH 7.4 at room temperature for 7 days.

[0031] Specific examples of HRS-Fc fusion polypeptides may contain at least one of SEQ ID NOs: 107-110, 337-338, 349-350, 381-390, or 396, or an amino acid sequence that is at least 80%, 90%, 95%, or 98% identical to SEQ ID NOs: 107-110, 337-338, 349-350, 381-390, or 396. SEQ ID NOs: 107 and 338 are the amino acid sequences of exemplary C-terminal Fc fusion polypeptides to residues 1 to 60 of SEQ ID NO: 1 (HRS(1-60)_Fc); SEQ ID NOs: 108 and 337 are the amino acid sequences of exemplary N-terminal Fc fusion polypeptides to residues 1 to 60 of SEQ ID NO: 1 (Fc_HRS(1-60)); SEQ ID NO: 109 is the amino acid sequence of an exemplary C-terminal Fc fusion polypeptide to residues 1 to 506 of SEQ ID NO: 1 (HRS(1-506)_Fc); and SEQ ID NO: 110 is the amino acid sequence of an exemplary N-terminal Fc fusion polypeptide to residues 1 to 506 of SEQ ID NO: 1 (Fc_HRS(1-506)).

[0032] In some embodiments, the HRS-Fc fusion polypeptide has anti-inflammatory activity, for example, in a cell-based assay or upon administration to a subject.

[0033] Also included are compositions, such as pharmaceutical or therapeutic compositions, comprising the HRS-Fc fusion polypeptide described herein and a pharmaceutically acceptable or pharmaceutical-grade carrier or excipient. In some compositions, the polypeptide is at least about 95% pure and less than about 5% aggregated. In some embodiments, the composition is formulated for delivery via oral, subcutaneous, intranasal, pulmonary, or parenteral administration. In certain embodiments, the composition comprises a delivery vehicle selected from the group consisting of liposomes, micelles, emulsions, and cells.

[0034] In some embodiments, the composition is for use in a) treating an inflammatory or autoimmune disease, b) reducing muscle or lung inflammation optionally associated with an autoimmune or inflammatory disease, c) inducing tolerance to histidyl-tRNA synthetase (HRS) autoantigen, d) eliminating a set or subset of T cells involved in the autoimmune response to the HRS autoantigen, e) reducing tissue inflammation in a subject, optionally in muscle, lung and / or skin tissue, f) treating muscular dystrophy, g) treating rhabdomyolysis, muscle wasting, cachexia, muscle inflammation or muscle injury, and / or h) treating a disease associated with an autoantibody.

[0035] Also included is a dosing regimen that maintains an average steady-state concentration of histidyl-tRNA synthetase (HRS)-Fc fusion polypeptide in the plasma of a subject between about 300 pM and about 1,000 nM when a dosing interval of 3 days or more is used, the dosing regimen comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described herein.

[0036] Some embodiments include a method of maintaining histidyl-tRNA synthetase (HRS)-Fc fusion polypeptide levels above the minimum effective therapeutic level in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described herein.

[0037] Also included are methods of treating an inflammatory or autoimmune disease or condition in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described herein.

[0038] Some embodiments include a method of reducing muscle or lung inflammation associated with an autoimmune or inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described herein.

[0039] Certain embodiments include a method of inducing tolerance to a histidyl-tRNA synthetase (HRS) autoantigen in a subject in need thereof, comprising administering to the subject a therapeutic composition or an HRS-Fc fusion polypeptide described herein.

[0040] Some embodiments include a method of eliminating a set or subset of T cells involved in an autoimmune response to a histidyl-tRNA synthetase (HRS) autoantigen in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described herein.

[0041] Also included are methods for reducing tissue inflammation in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described herein. In certain embodiments, the tissue is selected from muscle, intestine, brain, lung, and skin.

[0042] Some embodiments include a method of treating muscular dystrophy in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described herein. In certain embodiments, the muscular dystrophy is selected from Duchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, and congenital muscular dystrophy.

[0043] Certain embodiments include methods of treating rhabdomyolysis, muscle wasting, cachexia, muscle inflammation, or muscle injury in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described herein.

[0044] Some embodiments include a method of treating a disease associated with an autoantibody, comprising administering a composition or AARS / HRS polypeptide described herein to a subject in need thereof. In some embodiments, the disease is selected from the group consisting of inflammatory myopathies, inflammatory myopathies including polymyositis, dermatomyositis and related disorders, polymyositis-scleroderma overlap, inclusion body myositis (IBM), antisynthetase syndrome, interstitial lung disease, arthritis, and Raynaud's phenomenon. In some embodiments, the composition is administered to the subject before the onset of disease symptoms. In some embodiments, the autoantibody is specific for histidyl-tRNA synthetase. In some embodiments, the HRS polypeptide comprises at least one epitope of histidyl-tRNA synthetase recognized by the disease-specific autoantibody. In some embodiments, the epitope is an immunodominant epitope recognized by antibodies in serum from the subject. In some embodiments, the HRS polypeptide blocks the binding of autoantibodies to native histidyl-tRNA synthetase. In some embodiments, the HRS polypeptide causes clonal deletion of autoreactive T cells. In some embodiments, the HRS polypeptide causes functional inactivation of T cells involved in autoimmune responses. In some embodiments, administration of the HRS polypeptide results in reduced muscle or lung inflammation. In some embodiments, the HRS polypeptide induces tolerance to autoantigens.

[0045] In certain embodiments, the composition is formulated for delivery via oral, intranasal, pulmonary, intramuscular, or parenteral administration.

[0046] Also included are isolated polynucleotides comprising a nucleotide sequence encoding the HRS-Fc conjugates or fusion polypeptides described herein, including vectors comprising such polynucleotides, and host cells comprising said polynucleotides and / or vectors.

[0047] Some embodiments include a method of producing the HRS-Fc fusion polypeptide described herein, comprising: a) culturing a host cell (e.g., an E. coli K-12 host cell) to express the HRS-Fc fusion polypeptide, wherein the host cell comprises a polynucleotide encoding the HRS-Fc fusion polypeptide described herein operably linked to a regulatory element; and b) isolating the HRS-Fc fusion polypeptide from the host cell. In a specific embodiment, the E. coli K-12 strain is selected from W3110 and UT5600. In certain embodiments, for example, the following are provided: (Item 1) (a) a histidyl-tRNA synthetase (HRS) polypeptide comprising an amino acid sequence at least 80% identical to any of SEQ ID NOs: 1-106, 170-181, or 185-191, or any of the sequences in Table D1, D3-D6, or D8; and (b) an HRS fusion polypeptide comprising at least one Fc region fused to the C-terminus, the N-terminus, or both, of the HRS polypeptide. (Item 2) 2. The HRS fusion polypeptide according to item 1, wherein the HRS polypeptide comprises an amino acid sequence at least 90% identical to any of SEQ ID NOs: 1 to 106, 170 to 181, or 185 to 191, or any of the sequences of Table D1, D3 to D6, or D8. (Item 3) 2. The HRS fusion polypeptide according to item 1, wherein the HRS polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 106, 170 to 181, or 185 to 191, or any one of the sequences of Table D1, D3 to D6, or D8. (Item 4) 2. The HRS fusion polypeptide of item 1, wherein the HRS polypeptide comprises an amino acid sequence that is at least 90% identical to residues 2 to 40, 2 to 45, 2 to 50, 2 to 55, 2 to 60, 2 to 66, or 1 to 506 of SEQ ID NO:1. (Item 5) 5. The HRS fusion polypeptide according to item 4, wherein the HRS polypeptide comprises amino acid residues 2 to 40, 2 to 45, 2 to 50, 2 to 55, 2 to 60, 2 to 66, or 1 to 506 of SEQ ID NO: 1. (Item 6) 6. The HRS fusion polypeptide of item 5, wherein the HRS polypeptide consists essentially of amino acid residues 2 to 40, 2 to 45, 2 to 50, 2 to 55, 2 to 60, 2 to 66, or 1 to 506 of SEQ ID NO:1. (Item 7) 7. The HRS fusion polypeptide according to item 6, wherein the HRS polypeptide consists of residues 2 to 40, 2 to 45, 2 to 50, 2 to 55, 2 to 60, 2 to 66, or 1 to 506 of SEQ ID NO: 1. (Item 8) 2. The HRS fusion polypeptide of item 1, wherein the HRS polypeptide is at most about 40 to 80 amino acids in length and comprises residues 2 to 45 of SEQ ID NO:1. (Item 9) 9. The HRS fusion polypeptide according to any one of items 1 to 8, wherein at least one endogenous cysteine ​​residue is substituted with another amino acid or deleted. (Item 10) 10. The HRS fusion polypeptide of item 9, wherein the at least one endogenous cysteine ​​residue is selected from Cys174, Cys191, Cys224, Cys235, Cys507 and Cys509. (Item 11) 10. The HRS fusion polypeptide of item 9, wherein the at least one endogenous cysteine ​​residue is selected from Cys224, Cys235, Cys507 and Cys509. (Item 12) 9. The HRS fusion polypeptide according to any one of items 1 to 8, wherein all endogenous surface-exposed cysteine ​​residues are substituted with another amino acid or deleted. (Item 13) 13. The HRS fusion polypeptide according to any one of items 1 to 12, wherein the HRS polypeptide is tandemly repeated. (Item 14) 14. The HRS fusion polypeptide according to any one of items 1 to 13, wherein the HRS polypeptide is selected from the sequences in Table D5. (Item 15) 15. The HRS fusion polypeptide of any one of items 1 to 14, wherein the HRS polypeptide comprises a WHEP domain. (Item 16) 16. The HRS fusion polypeptide of any one of items 1 to 15, wherein the Fc region and the HRS polypeptide are separated by a peptide linker. (Item 17) 17. The HRS fusion polypeptide of item 16, wherein the peptide linker is about 1 to 20 amino acids in length, 1 to 10 amino acids in length, or 1 to 5 amino acids in length. (Item 18) 17. The HRS fusion polypeptide of item 16, wherein the peptide linker is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. (Item 19) 19. The HRS fusion polypeptide according to any one of items 16 to 18, wherein the peptide linker consists of Gly and / or Ser residues. (Item 20) 19. The HRS fusion polypeptide according to any one of items 16 to 18, wherein the peptide linker is a physiologically stable linker. (Item 21) 19. The HRS fusion polypeptide of any one of items 16 to 18, wherein the peptide linker is a releasable linker, optionally an enzymatically cleavable linker. (Item 22) 22. The HRS fusion polypeptide according to any one of items 16 to 21, wherein the peptide linker comprises any one of the sequences of SEQ ID NOs: 200 to 260. (Item 23) 23. The HRS fusion polypeptide of any one of items 1 to 22, wherein the Fc region is fused to the C-terminus of the HRS polypeptide. (Item 24) 23. The HRS fusion polypeptide of any one of items 1 to 22, wherein the Fc region is fused to the N-terminus of the HRS polypeptide. (Item 25) 25. The HRS fusion polypeptide of any one of items 1 to 24, wherein the Fc region comprises one or more of the hinge, CH2, CH3 and / or CH4 domains derived from mammalian IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and / or IgM. (Item 26) 25. The HRS fusion polypeptide of any one of items 1 to 24, wherein the Fc region comprises the hinge, CH2 and CH3 domains of IgG1. (Item 27) 25. The HRS fusion polypeptide of any one of items 1 to 24, wherein the Fc region comprises the hinge, CH2 and CH3 domains of IgG2. (Item 28) 25. The HRS fusion polypeptide of any one of items 1 to 24, wherein the Fc region comprises the hinge, CH2 and CH3 domains of IgG3. (Item 29) The Fc region comprises the hinge, CH2 and CH3 domains of IgG2. 25. The HRS fusion polypeptide of any one of claims 24. (Item 30) Immunoglobulin CH1, C L , V L and V H 30. The HRS fusion polypeptide of any one of items 1 to 29, which does not contain a region. (Item 31) 2. The HRS fusion polypeptide of any of the preceding items, wherein the Fc region comprises any one of SEQ ID NOs: 128-163 or 339-342, or a variant, or fragment, or combination thereof. (Item 32) 2. The HRS fusion polypeptide of any preceding item, wherein the hinge region comprises SEQ ID NO: 341. (Item 33) The Fc region is selected from the group consisting of MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 339) and SDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 340). (Item 34) 33. The HRS fusion polypeptide according to any one of items 1 to 32, wherein the HRS fusion polypeptide comprises an amino acid sequence at least 90% identical to Fc-HRS(2-60) (SEQ ID NO: 337) or HRS(1-60)-Fc (SEQ ID NO: 338). (Item 35) 33. The HRS fusion polypeptide according to any one of items 1 to 32, wherein the HRS fusion polypeptide comprises an amino acid sequence at least 90% identical to Fc-HRS(2-40) (SEQ ID NO: 381) or HRS(1-40)-Fc (SEQ ID NO: 386). (Item 36) 33. The HRS fusion polypeptide according to any one of items 1 to 32, wherein the HRS fusion polypeptide comprises an amino acid sequence at least 90% identical to Fc-HRS(2-45) (SEQ ID NO: 382) or HRS(1-45)-Fc (SEQ ID NO: 387). (Item 37) 33. The HRS fusion polypeptide of any of items 1 to 32, wherein the HRS fusion polypeptide comprises an amino acid sequence at least 90% identical to Fc-HRS(2-50) (SEQ ID NO: 383) or HRS(1-50)-Fc (SEQ ID NO: 388). (Item 38) 33. The HRS fusion polypeptide according to any one of items 1 to 32, wherein the HRS fusion polypeptide comprises an amino acid sequence at least 90% identical to Fc-HRS(2-55) (SEQ ID NO: 384) or HRS(1-55)-Fc (SEQ ID NO: 389). (Item 39) 33. The HRS fusion polypeptide according to any one of items 1 to 32, wherein the HRS-Fc fusion polypeptide comprises an amino acid sequence at least 90% identical to Fc-HRS(2-66) (SEQ ID NO: 385) or HRS(1-66)-Fc (SEQ ID NO: 390). (Item 40) 33. The HRS fusion polypeptide according to any one of items 1 to 32, wherein the HRS-Fc fusion polypeptide comprises an amino acid sequence that is at least 90% identical to Fc-HRS(2-60)HRS(2-60) (SEQ ID NO: 396). (Item 41) 2. The HRS fusion polypeptide of any preceding item, having altered pharmacokinetics compared to the corresponding unmodified HRS polypeptide. (Item 42) 42. The HRS fusion polypeptide of item 41, wherein the altered pharmacokinetics is increased serum half-life, increased bioavailability, exposure and / or decreased clearance. (Item 43) 43. The HRS fusion polypeptide of claim 42, wherein the exposure is increased by at least 100-fold. (Item 44) 43. The HRS fusion polypeptide of claim 42, wherein the HRS fusion polypeptide has a half-life in mice of at least 30 hours. (Item 45) 43. The HRS fusion polypeptide of claim 42, wherein the bioavailability is at least about 30% increased subcutaneous bioavailability. (Item 46) 2. The HRS fusion polypeptide of any preceding item, having altered immune effector activity compared to a corresponding HRS polypeptide. (Item 47) 37. The HRS fusion polypeptide of claim 36, wherein the immune effector activity is one or more of complement activation, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or antibody-dependent cell-mediated phagocytosis (ADCP). (Item 48) 2. The HRS fusion polypeptide of any preceding item, wherein the Fc region comprises a variant Fc region compared to a wild-type Fc region. (Item 49) 49. The HRS fusion polypeptide of item 48, wherein the variant Fc region comprises a sequence at least 90% identical to any one of SEQ ID NOs: 128-163 or 341, or a combination of said sequences. (Item 50) 50. The HRS fusion polypeptide of item 48 or 49, wherein the variant Fc region comprises a hybrid of one or more Fc regions from different species, different Ig classes or different Ig subclasses. (Item 51) 51. The HRS fusion polypeptide of any one of items 48 to 50, wherein the variant Fc region comprises a hybrid of one or more hinge, CH2, CH3 and / or CH4 domains of an Fc region from a different species, a different Ig class and / or a different Ig subclass. (Item 52) 52. The HRS fusion polypeptide of any one of items 48 to 51, wherein the variant Fc region is an altered glycoform compared to the corresponding wild-type Fc region. (Item 53) 53. The HRS fusion polypeptide of any one of items 48 to 52, wherein the variant Fc region has altered pharmacokinetics compared to the corresponding wild-type Fc region. (Item 54) 54. The HRS fusion polypeptide of item 53, wherein the altered pharmacokinetics include serum half-life, bioavailability and / or clearance. (Item 55) 55. The HRS fusion polypeptide of any one of items 48 to 54, wherein the variant Fc region has altered effector activity compared to the corresponding wild-type Fc region. (Item 56) 56. The HRS fusion polypeptide of claim 55, wherein the effector activity is one or more of complement activation, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or antibody-dependent cell-mediated phagocytosis (ADCP). (Item 57) 57. The HRS fusion polypeptide of any one of items 48 to 56, wherein the variant Fc region has altered binding to one or more Fcγ receptors compared to the corresponding wild-type Fc region. (Item 58) 57. The HRS fusion polypeptide of any one of items 48 to 56, wherein the variant Fc region has altered binding to one or more FcRn receptors compared to the corresponding wild-type Fc region. (Item 59) 59. The HRS fusion polypeptide of any one of items 48 to 58, wherein the variant Fc region has altered solubility compared to the corresponding wild-type Fc region. (Item 60) 2. The HRS fusion polypeptide of any preceding item, which is substantially in a dimeric form in physiological solution. (Item 61) 2. The HRS fusion polypeptide of any preceding item, having substantially the same secondary structure as the corresponding unmodified HRS polypeptide, as determined via UV circular dichroism analysis. (Item 62) 2. The HRS fusion polypeptide of any preceding item, which, when administered to a mammal, has a plasma or serum pharmacokinetic AUC profile that is at least 5-fold greater than the corresponding unmodified HRS polypeptide. (Item 63) A histidyl-tRNA synthetase (HRS)-Fc fusion polypeptide comprising an amino acid sequence at least 80% identical to any one or more of SEQ ID NOs: 107-110, or 337-338, or 349-350, or 381-390, or 396. (Item 64) 2. The histidyl-tRNA synthetase (HRS)-Fc fusion polypeptide according to any one of the preceding items, which has anti-inflammatory activity. (Item 65) A therapeutic composition comprising an HRS-Fc fusion polypeptide and a pharmaceutically acceptable carrier or excipient. (Item 66) 66. The therapeutic composition of claim 65, wherein the polypeptide is at least about 95% pure and less than about 5% aggregated. (Item 67) The therapeutic composition of any preceding item, formulated for delivery via oral, subcutaneous, intranasal, pulmonary or parenteral administration. (Item 68) 20. The therapeutic composition of any preceding item, comprising a delivery vehicle selected from the group consisting of liposomes, micelles, emulsions, and cells. (Item 69) The therapeutic composition of any of the preceding items for use in a) treating an inflammatory or autoimmune disease, b) reducing muscle or lung inflammation optionally associated with an autoimmune or inflammatory disease, c) inducing tolerance to the histidyl-tRNA synthetase (HRS) autoantigen, d) eliminating a set or subset of T cells involved in the autoimmune response to the HRS autoantigen, e) reducing tissue inflammation in a subject, optionally in muscle, lung and / or skin tissue, f) treating muscular dystrophy, g) treating rhabdomyolysis, muscle wasting, cachexia, muscle inflammation or muscle injury, and / or h) treating a disease associated with an autoantibody. (Item 70) A dosing regimen that maintains an average steady-state concentration of histidyl-tRNA synthetase (HRS)-Fc fusion polypeptide in the plasma of a subject of between about 300 pM and about 1,000 nM when a dosing interval of 3 days or more is used, said dosing regimen comprising administering to said subject a therapeutic composition or HRS-Fc fusion polypeptide described in any preceding item. (Item 71) 1. A method for maintaining histidyl-tRNA synthetase (HRS)-Fc fusion polypeptide levels above the minimum effective therapeutic level in a subject in need thereof, the method comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described in any of the preceding items. (Item 72) A method for treating an inflammatory or autoimmune disease or condition in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described in any preceding item. (Item 73) A method for reducing muscle or lung inflammation associated with an autoimmune or inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described in any of the preceding items. (Item 74) A method for inducing tolerance to a histidyl-tRNA synthetase (HRS) autoantigen in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described in any of the preceding items. (Item 75) A method for eliminating a set or subset of T cells involved in an autoimmune response to a histidyl-tRNA synthetase (HRS) autoantigen in a subject in need thereof, the method comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described in any of the preceding items. (Item 76) A method of reducing tissue inflammation in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described in any preceding item. (Item 77) 77. The method of claim 76, wherein the tissue is selected from muscle, intestine, brain, lung, and skin. (Item 78) A method of treating muscular dystrophy in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described in any of the preceding items. (Item 79) 79. The method of claim 78, wherein the muscular dystrophy is selected from Duchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, and congenital muscular dystrophy. (Item 80) A method for treating rhabdomyolysis, muscle wasting, cachexia, muscle inflammation, or muscle injury in a subject in need thereof, comprising administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described in any of the preceding items. (Item 81) The method of any preceding item, wherein the composition is formulated for delivery via oral, intranasal, pulmonary, intramuscular, or parenteral administration. (Item 82) An isolated polynucleotide comprising a nucleotide sequence encoding the HRS-Fc fusion polypeptide according to any of the preceding items. (Item 83) 83. A vector comprising the isolated polynucleotide of item 82. (Item 84) A host cell comprising the vector described in Item 83. (Item 85) 78. A method for producing the histidyl-tRNA synthetase (HRS)-Fc fusion polypeptide of any preceding item, comprising: a) culturing E. coli K-12 host cells to express the HRS-Fc fusion polypeptide, wherein the host cells comprise the polynucleotide of 77 operably linked to a regulatory element; and b) isolating the HRS-Fc fusion polypeptide from the host cells. (Item 86) 86. The method of item 85, wherein the E. coli K-12 strain is selected from W3110 and UT5600. [Brief explanation of the drawings]

[0048] [Figure 1] FIG. 1 is a diagram showing the structural organization of exemplary immunoglobulins and provides an overview of antibody classes and subclasses. [Figure 2] Figure 2 shows an alignment of the Fc regions from human IgA1 (SEQ ID NO: 156), IgA2 (SEQ ID NO: 157), IgM (SEQ ID NO: 158), IgG1 (SEQ ID NO: 159), IgG2 (SEQ ID NO: 160), IgG3 (SEQ ID NO: 161), IgG4 (SEQ ID NO: 162), and IgE (SEQ ID NO: 163). The secondary structure of the Fcα is shown above the sequences. Carets (^) and asterisks (*) indicate residues contributing to 0-4% and 5-12% of the binding surface, respectively. [Figure 3] Figure 3 shows the results of SDS-PAGE analysis of full-length HRS and HRS(1-506) under reducing and non-reducing conditions. The results demonstrate that HRS(1-506) dramatically reduces disulfide-mediated interchain bond formation compared to the full-length protein. Samples (10 μg) were loaded onto a 4-12% Bis-Tris gel using MOPS-SDS running buffer. [Figure 4] Figure 4 shows the anti-inflammatory properties of exemplary HRS-derived polypeptides in a TNBS-induced mouse model of colitis. Studies were conducted on male BDF-1 mice, 12 mice per group. TNBS and budesonide were added to water at 5 mg / kg. HRS(1-60) (Resokine, (HisRSN4)) was administered daily at a concentration of 1 mg / kg or 5 mg / kg by intravenous injection starting 3 days before TNBS treatment. The figure shows the percent (%) survival of treated and untreated mice over approximately 80 hours. [Figure 5]Figure 5A shows the dosing regimen used to evaluate the therapeutic utility of HRS(1-506) in a statin myopathy model. Treatment groups included vehicle (n=11), 0.3 mpk HRS(1-506) (n=8), 1.0 mpk HRS(1-506) (n=8), and 3.0 mpk HRS(1-506) (n=8). Figure 5B shows the results of troponin C measurements after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg, and 3.0 mg / Kg HRS(1-506). This figure demonstrates the positive effect of HRS(1-506) in reducing statin-induced troponin C induction. [Figure 6] Figure 6A shows the results of CK measurements after 12 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg, and 3.0 mg / Kg of HRS(1-506). Figure 6B shows the same data after 15 days of treatment. This figure demonstrates the positive effect of HRS(1-506) in reducing statin-induced CK levels. [Figure 7] Figure 7 shows the levels of circulating HARS after 15 days of treatment with statins compared to vehicle controls, demonstrating that statins induce the release of extracellular HARS. [Figure 8] FIG. 8 shows representative H&E images of thigh flexor muscle sections at 10× magnification after 15 days of treatment with statin + / −0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 9] Figure 9 shows the results of gene expression profiling of statin-treated rat femoral flexor muscles. The data show changes in the expression of 137 genes selected to track muscle markers, immune cell function, inflammation, metabolic status, tissue repair, muscle growth, and atrophy. Gene expression values ​​were normalized to reference genes and presented as fold changes relative to the vehicle-treated group. [Figure 10]Figure 10A shows the results of gene expression profiling of statin-treated rat femoral flexor muscles. The data show the change in expression of 137 genes (as in Figure 7) to compare the relative change in gene expression in statin-treated animals compared to vehicle-treated animals. Figure 10B shows the relative change in gene expression in statin-treated animals also treated with HRS(1-506) compared to animals treated with statin alone. [Figure 11] FIG. 11 shows the results of gene expression profiling of 10 diabetes / metabolic syndrome-related genes in flexor muscle of statin-treated rats after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 12] FIG. 12 shows the results of gene expression profiling of 26 immune cell marker genes in flexor muscles of statin-treated rats after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 13-1] Figures 13A-D show the results of gene expression profiling of CD11a, CD11b, CD8a and CD8b genes in femoral flexor muscles of statin-treated rats after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 13-2] Figures 13A-D show the results of gene expression profiling of CD11a, CD11b, CD8a and CD8b genes in femoral flexor muscles of statin-treated rats after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 14-1] 14A-C show the results of gene expression profiling of CD18, CCR5 and CD45R genes in femoral flexor muscles of statin-treated rats after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 14-2]14A-C show the results of gene expression profiling of CD18, CCR5 and CD45R genes in femoral flexor muscles of statin-treated rats after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 15] FIG. 15 shows the results of gene expression profiling of 17 inflammation marker genes in femoral flexor muscles of statin-treated rats after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 16-1] Figures 16A-D show the results of gene expression profiling of the inflammatory cytokines IL-6, MCP1, IL-10 and interferon-gamma (IFN-γ) in the thigh flexor muscles of statin-treated rats after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 16-2] Figures 16A-D show the results of gene expression profiling of the inflammatory cytokines IL-6, MCP1, IL-10 and interferon-gamma (IFN-γ) in the thigh flexor muscles of statin-treated rats after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 17] FIG. 17 shows the results of gene expression profiling of 14 adhesion, development and fibrosis related genes in statin-treated rat femoral flexor muscle after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 18] FIG. 18 shows the results of gene expression profiling of 14 muscle wasting / atrophy-related genes in flexor muscles of statin-treated rats after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 19-1]Figure 19A shows the results of gene expression profiling of 14 muscle wasting / atrophy-related genes in flexor muscles of statin-treated rats after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg, and 3.0 mg / Kg of HRS(1-506). Figure 19B shows specific changes in MMP-3 and Figure 19C shows specific changes in MMP-9 gene expression under the same conditions. [Figure 19-2] Figure 19A shows the results of gene expression profiling of 14 muscle wasting / atrophy-related genes in flexor muscles of statin-treated rats after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg, and 3.0 mg / Kg of HRS(1-506). Figure 19B shows specific changes in MMP-3 and Figure 19C shows specific changes in MMP-9 gene expression under the same conditions. [Figure 20] FIG. 20 shows the results of gene expression profiling of statin-treated rat femoral flexor muscles of 29 myogenesis-related genes after 15 days of treatment with statin + / - 0.3 mg / Kg, 1.0 mg / Kg and 3.0 mg / Kg of HRS(1-506). [Figure 21] Figure 21 shows the results of SDS-PAGE analysis of purified Fc fusion proteins. Lane 1: See Blue Plus2 protein ladder (Life Technologies). Lanes 2 and 6: Fc-HRS(2-60) lot number 472. Lanes 3 and 7: HRS(1-60)-Fc lot number 473. Lanes 4 and 8: Fc-HRS(2-60) lot number 480. Lanes 5 and 9: HRS(1-60)-Fc lot number 482. Lanes 2 to 5 were run under non-reducing conditions, and lanes 6 to 9 were run under reducing conditions. [Figure 22] Figure 22 shows analytical size-exclusion HPLC analysis of a representative purified Fc-HRS(2-60) fusion after Protein A, cation exchange, and hydroxyapatite chromatography (overlay of duplicate injections). Purity is 99.2% major peak and 0.8% high molecular weight (HMW) species. [Figure 23-1]Figure 23A shows the concentration versus time of HRS(1-60) after either intravenous or subcutaneous injection into mice. Figure 23B shows the concentration versus time of Fc-HRS(2-60) and HRS(1-60)-Fc after intravenous injection into mice. Figure 23C shows the concentration versus time of Fc-HRS(2-60) and HRS(1-60)-Fc after subcutaneous injection into mice. [Figure 23-2] Figure 23A shows the concentration versus time of HRS(1-60) after either intravenous or subcutaneous injection into mice. Figure 23B shows the concentration versus time of Fc-HRS(2-60) and HRS(1-60)-Fc after intravenous injection into mice. Figure 23C shows the concentration versus time of Fc-HRS(2-60) and HRS(1-60)-Fc after subcutaneous injection into mice. [Figure 24] Figure 24A shows the Disease Activity Index (DAI) scores at termination in mice treated with different HRS-Fc fusion proteins. Bars indicate the mean DAI (±SEM) for each treatment group. DAI incorporates information on bleeding and diarrhea along with weight loss scores. Figure 24B shows the colon weight:length ratio at termination in compound-treated mice. Bars indicate the mean ratio (±SEM) for each treatment group. [Figure 25] Figure 25 shows an overview of transcriptional changes in the TNBS study. After normalization to naive animals (group 1), relative transcriptional changes are shown in TNBS-treated animals (group 2), TNBS and budesonide-treated animals (group 3), TNBS and test article A-treated animals (HRS(1-60); group 4), and TNBS and test article B-treated animals (Fc-HRS(2-60); groups 5 and 6). Each dot in the scatter plot represents a measured gene. In group 2, seven genes were upregulated by more than 10-fold (IL6, IL1b, MCP-1, MMP3, MMP9, CD11b, and IL10). [Figure 26-1]Figures 26A-26H show immune and inflammation-related genes upregulated by TNBS. Shown are the relative transcriptional changes of individual genes in TNBS-treated animals (Group 2), TNBS and budesonide-treated animals (Group 3), TNBS and test article A-treated animals (HRS(1-60); Group 4), and TNBS and test article B-treated animals (Fc-HRS(2-60); Groups 5 and 6) after normalization to naive animals (Group 1). Each dot in the scatter plot indicates the abundance of the gene of interest in each animal within the group. Significance was calculated using a Student's t-test, with * = p-value < 0.05 and ** = p-value < 0.01. [Figure 26-2] Figures 26A-26H show immune and inflammation-related genes upregulated by TNBS. Shown are the relative transcriptional changes of individual genes in TNBS-treated animals (Group 2), TNBS and budesonide-treated animals (Group 3), TNBS and test article A-treated animals (HRS(1-60); Group 4), and TNBS and test article B-treated animals (Fc-HRS(2-60); Groups 5 and 6) after normalization to naive animals (Group 1). Each dot in the scatter plot indicates the abundance of the gene of interest in each animal within the group. Significance was calculated using a Student's t-test, with * = p-value < 0.05 and ** = p-value < 0.01. [Figure 26-3] Figures 26A-26H show immune and inflammation-related genes upregulated by TNBS. Shown are the relative transcriptional changes of individual genes in TNBS-treated animals (Group 2), TNBS and budesonide-treated animals (Group 3), TNBS and test article A-treated animals (HRS(1-60); Group 4), and TNBS and test article B-treated animals (Fc-HRS(2-60); Groups 5 and 6) after normalization to naive animals (Group 1). Each dot in the scatter plot indicates the abundance of the gene of interest in each animal within the group. Significance was calculated using a Student's t-test, with * = p-value < 0.05 and ** = p-value < 0.01. [Figure 27]Figures 27A-27D show the relative percentages of different T cell populations in the spleens of naive mice, mice treated intracolonally with TNBS to induce experimental colitis, and mice treated with TNBS ± 0.5 mg / kg Fc-HRS(2-60). The percentages of live lymphocytes stained for (27A) CD3, (27B) CD8, (27C) CD4, and (27D) CD25 and FoxP3 are shown. Treg cells were further gated on CD4+ cells. DETAILED DESCRIPTION OF THE INVENTION

[0049] The practice of the present invention will employ, unless specifically indicated to the contrary, conventional methods of molecular biology and recombinant DNA technology within the skill of those in the art, many of which are described below for purposes of illustration, and such techniques are fully explained in the literature. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2000); DNA Cloning: A Practical Approach, Vols. I and II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed., 2004); Nucleic Acid Hybridization (B. Hames and S. Higgins, eds., 1985); Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009); Transcription and Translation (B. Hames and 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 ed., 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd ed., 2005). Poly(ethylene glycol), Chemistry and Biological Applications, ACS, Washington, 1997; Veronese, F. and J.M.See Harris (ed.), Peptide and protein PEGylation, Advanced Drug Delivery Reviews, Vol. 54(4), pp. 453-609 (2002); Zalipsky, S. et al., "Use of functionalized Poly(Ethylene Glycols) for modification of polypeptides," Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications.

[0050] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0051] 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0052] 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, "an element" means one element or more than one element.

[0053] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0054] 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 the 20 (L)-amino acids utilized during protein biosynthesis, as well as other amino acids, such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids known to those skilled in the art, such as norleucine, norvaline, p-fluorophenylalanine, and ethionine. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications can include, for example, substitutions or replacements of chemical groups and moieties on amino acids or derivatization of amino acids. Amino acid mimetics include organic structures that exhibit functionally similar properties, such as charge and charge-spatial characteristics, of the reference amino acids. For example, an organic structure that mimics arginine (Arg or R) has a positively charged moiety that is positioned in a similar molecular space and has the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures that maintain optimal space 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.

[0055] As used herein, a subject "at risk" of developing a disease or adverse reaction may or may not have detectable disease or disease symptoms, and may or may not exhibit detectable disease or disease symptoms prior to the treatment methods described herein. "At risk" indicates that the subject has one or more risk factors, which are measurable parameters that correlate with the development of a disease, as described herein and known in the art. A subject with one or more of these risk factors has a higher likelihood of developing a disease or adverse reaction than a subject without one or more of these risk factor(s).

[0056] An "autoimmune disease," as used herein, is a disease or disorder arising from and directed against an individual's own tissues. Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory responses, e.g., inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); systemic sclerosis and sclerosis; responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions, e.g., eczema and asthma, and other conditions involving T-cell infiltration and a chronic inflammatory response; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome syndrome); juvenile-onset diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes typically found in tuberculosis, sarcoidosis, polymyositis, inflammatory myopathy, interstitial lung disease, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases involving leukocyte extravasation; central nervous system (CNS) inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (cryoglobinemia or Coombs positive) anemia); myasthenia gravis; antigen-antibody complex-mediated disease; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; stiff man syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia, etc.

[0057] Throughout this specification, unless the context otherwise requires, the words "comprise," "comprises," and "comprising" are understood to mean the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to whatever precedes the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that other elements may not be present. "Consisting essentially of" means including any elements listed before the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited 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 action of the recited elements.

[0058] The term "clonal deletion" refers to the elimination (e.g., loss or death) of autoreactive T cells. Clonal deletion can be achieved centrally in the thymus, or in the periphery, or both.

[0059] The term "conjugate" is intended to refer to an entity formed as a result of the covalent attachment of a molecule, such as a biologically active molecule (e.g., an HRS polypeptide), to an immunoglobulin Fc region. One example of a conjugated polypeptide is a "fusion protein" or "fusion polypeptide," i.e., a polypeptide created by the joining of two or more coding sequences that originally encoded separate polypeptides; transcription of the joined coding sequences results in a single fusion polypeptide that typically possesses the functional properties derived from each of the separate polypeptides.

[0060] The phrases "endotoxin-free" or "substantially endotoxin-free" generally refer to compositions, solvents, and / or containers that contain at most trace amounts of endotoxin (e.g., amounts that have no clinically adverse physiological effects on a subject), preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain 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 lipooligosaccharides (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 cause fever, a drop in blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

[0061] Therefore, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxin from drug products and / or drug containers, since even small amounts can cause adverse effects in humans. Because temperatures exceeding 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 holding time is often sufficient to achieve a 3-log reduction in endotoxin levels. Other methods for removing endotoxin are contemplated, including, for example, chromatography and filtration methods, as described herein and known in the art. To reduce or otherwise eliminate the risk of endotoxin being present in the compositions of the present invention, methods of producing HRS-Fc conjugates in eukaryotic cells, such as mammalian cells, and isolating the HRS-Fc conjugates from such cells are also included. Methods of producing HRS-Fc conjugates in serum-free cells and isolating the HRS-Fc conjugates from such cells are preferred.

[0062] Endotoxin can be detected using conventional techniques known in the art. For example, the Limulus amebocyte lysate assay, which utilizes the 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 coagulation of Limulus lysate due to a powerful enzyme cascade that amplifies this reaction. Endotoxin can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially free of endotoxin, the endotoxin level can be less than about 0.001 EU / ml, less than 0.005 EU / ml, less than 0.01 EU / ml, less than 0.02 EU / ml, less than 0.03 EU / ml, less than 0.04 EU / ml, less than 0.05 EU / ml, less than 0.06 EU / ml, less than 0.08 EU / ml, less than 0.09 EU / ml, less than 0.1 EU / ml, less than 0.5 EU / ml, less than 1.0 EU / ml, less than 1.5 EU / ml, less than 2 EU / ml, less than 2.5 EU / ml, less than 3 EU / ml, less than 4 EU / ml, less than 5 EU / ml, less than 6 EU / ml, less than 7 EU / ml, less than 8 EU / ml, less than 9 EU / ml, or less than 10 EU / ml. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to about 1-10 EU / ml.

[0063] As used herein, the terms "function" and "functional" and the like refer to a biological function, an enzymatic function, or a therapeutic function.

[0064] "Homology" refers to the percentage of amino acids that are identical or that constitute conservative substitutions. Homology can be determined using a sequence comparison program such as GAP (Deveraux et al., Nucleic Acids Research. Vol. 12, pp. 387-395, 1984), which is incorporated herein by reference. In this method, sequences of similar or substantially different lengths to the sequences cited herein can be compared by inserting gaps into the alignment, such as gaps determined by the comparison algorithm used by GAP.

[0065] A "physiologically stable" linker refers to a linker that is substantially stable in water or under physiological conditions (e.g., in vivo, in vitro culture conditions, e.g., in the presence of one or more proteases), i.e., a linker that does not undergo degradation reactions (e.g., enzymatically degradable reactions) under physiological conditions to any appreciable extent over an extended period of time. Generally, a physiologically stable linker is one that exhibits a degradation rate of less than about 0.5%, less than about 1%, less than about 2%, less than about 3%, less than about 4%, or less than about 5% per day under physiological conditions.

[0066] "Isolated" refers to material that is substantially or essentially free from components that normally accompany it in its native context. For example, "isolated peptide" or "isolated polypeptide," etc., as used herein, include the in vitro isolation and / or purification of a peptide or polypeptide molecule from its natural cellular environment and from association with other cellular components; i.e., an isolated molecule is not significantly associated with in vivo materials.

[0067] The term "median effective concentration" or "EC 50 " refers to the concentration of a HRS-Fc conjugate described herein that induces a response intermediate between baseline and maximum after some specified exposure time; thus, the EC 50 indicates the concentration of a compound at which 50% of its maximal effect is observed. In certain embodiments, the EC 50 is indicated for "non-canonical" activity, as described above. EC 50 The EC also indicates the plasma concentration required to obtain 50% of the maximum effect in vivo. 90 "EC" refers to the concentration of a drug or composition at which 90% of its maximum effect is observed. 90 " is "EC 50 and Hill slope, or can be determined directly from the data using routine knowledge in the art. In some embodiments, the EC50 is less than about 0.01 nM, less than 0.05 nM, less than 0.1 nM, less than 0.2 nM, less than 0.3 nM, less than 0.4 nM, less than 0.5 nM, less than 0.6 nM, less than 0.7 nM, less than 0.8 nM, less than 0.9 nM, less than 1 nM, less than 2 nM, less than 3 nM, less than 4 nM, less than 5 nM, less than 6 nM, less than 7 nM, less than 8 nM, less than 9 nM , less than 10 nM, less than 11 nM, less than 12 nM, less than 13 nM, less than 14 nM, less than 15 nM, less than 16 nM, less than 17 nM, less than 18 nM, less than 19 nM, less than 20 nM, less than 25 nM, less than 30 nM, less than 40 nM, less than 50 nM, less than 60 nM, less than 70 nM, less than 80 nM, less than 90 nM or less than 100 nM. Preferably, the biotherapeutic composition has an EC of about 1 nM or less. 50 It has a value.

[0068] The "half-life" of an HRS-Fc conjugate can refer to the time it takes for the conjugate to lose half of its pharmacological, physiological, or other activity, compared to such activity at the time of administration into the serum or tissues of an organism, or compared to any other defined time point. "Half-life" can also refer to the time it takes for the amount or concentration of the HRS-Fc conjugate to decrease to half of the starting amount administered in the serum or tissues of an organism, compared to such amount or concentration at the time of administration into the serum or tissues of an organism, or compared to any other defined time point. Half-life can be measured in serum and / or any one or more selected tissues.

[0069] The terms "linkage," "linker," "linker moiety," or "L" are used herein to refer to a linker that can be used to separate an HRS polypeptide from another HRS polypeptide and / or from one or more Fc regions. The linker can be physiologically stable or can include a releasable linker, e.g., an enzymatically degradable linker (e.g., a proteolytically cleavable linker). In certain embodiments, the linker can be a peptide linker, e.g., as part of an HRS-Fc fusion protein. In some embodiments, the linker can be a non-peptide linker.

[0070] The terms "modulating" and "altering" typically include "increasing," "enhancing," or "stimulating," as well as "decreasing" or "reducing," by a statistically significant or physiologically significant amount or degree compared to a control. An "increased," "stimulated," or "enhanced" amount is typically a "statistically significant" amount and can include an increase that is 1.1-fold, 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points therebetween and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the amount produced by a composition, sample, or test subject without the composition (e.g., in the absence of any of the HRS-Fc conjugates of the present invention) or a control. A "decreased" or "reduced" amount is typically a "statistically significant" amount 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% decrease in the amount produced by no composition (in the absence of an agent or compound) or a control composition, including all integers therebetween. As one non-limiting example, when comparing canonical and non-canonical activity, the control may include the HRS-Fc conjugate of interest compared to a corresponding unmodified or differently modified (in sequence) HRS polypeptide. Other examples of comparisons and "statistically significant" amounts are described herein.

[0071] "Non-canonical" activity, as used herein, generally refers to either i) a new non-aminoacylating activity possessed by an HRS polypeptide of the present invention that is not possessed to any significant degree by the intact, native, full-length parent protein, or ii) an activity that was possessed by the intact, native, full-length parent protein, where, e.g., by isolating that activity from other activities possessed by the intact, native, full-length parent protein, the HRS polypeptide either exhibits a significantly higher (e.g., at least 20% higher) specific activity with respect to the non-canonical activity compared to the intact, native, full-length parent protein, or exhibits activity in a new context. For HRS polypeptides, non-limiting examples of non-canonical activities include extracellular signaling, including modulation of cell proliferation, modulation of cell migration, modulation of cell differentiation (e.g., hematopoiesis, neurogenesis, myogenesis, osteogenesis, and adipogenesis), modulation of gene transcription, modulation of apoptosis or other forms of cell death, modulation of cell signaling, modulation of cellular uptake or secretion, modulation of angiogenesis, modulation of cell binding, modulation of cellular metabolism, modulation of cytokine production or activity, modulation of cytokine receptor activity, inflammation, modulation of immunogenicity, and the like.

[0072] In certain embodiments, the "purity" of any given agent (e.g., an HRS-Fc conjugate, such as a fusion protein) in a composition can be specifically defined. For example, a particular composition can contain an agent that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimal points therebetween, as measured by, for example, but in no way limited to, high-pressure liquid chromatography (HPLC), a well-known form of column chromatography frequently used in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0073] Without wishing to be bound by any particular theory, an "enzymatically degradable linker" refers to a linker, e.g., an amino acid sequence, that is subject to degradation by one or more enzymes, e.g., peptidases or proteases.

[0074] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues and variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic, non-naturally occurring amino acids, e.g., chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers.

[0075] "Releasable linkers" include, but are not limited to, physiologically cleavable linkers and enzymatically degradable linkers. Thus, a "releasable linker" is a linker that can undergo either spontaneous hydrolysis or cleavage by some other mechanism (e.g., enzyme-catalyzed, acid-catalyzed, base-catalyzed, etc.) under physiological conditions. For example, a "releasable linker" can involve an elimination reaction with base abstraction of a proton (e.g., an ionizable hydrogen atom, Hα) as the driving force. For purposes herein, "releasable linker" is synonymous with "degradable linker." In certain embodiments, a releasable linker has a half-life of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours or more at pH 7.4, 25°C, e.g., physiological pH, human body temperature (e.g., in vivo).

[0076] " 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.

[0077] The term "solubility" refers to the ability of the HRS-Fc conjugate polypeptide provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as 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.), molality, 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 solution, such as PBS or NaCl (with or without NaP). In specific 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 HRS-Fc conjugate polypeptide 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, or 30 mg / ml at room temperature or 37°C.

[0078] As used herein, the term "subject" includes any animal that exhibits a condition that can be treated or diagnosed using the HRS-Fc conjugate polypeptide of the present invention, or that is at risk of exhibiting such a condition. Suitable subjects (patients) include experimental animals (e.g., mice, rats, rabbits, or guinea pigs), livestock and domestic animals, or pets (e.g., cats or dogs). Non-human primates and preferably human patients are also included.

[0079] "Substantially" or "essentially" means nearly wholly or completely, for example, 95%, 96%, 97%, 98%, 99% or more of a given amount.

[0080] "Treatment" or "treating" as used herein includes any desired effect on the symptoms or pathology of a disease or condition, and may even include minimal changes or improvements in one or more measurable markers of the disease or condition being treated. "Treatment" or "treating" does not necessarily indicate complete eradication or cure of the disease or condition or its associated symptoms. The subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to those skilled in the art.

[0081] Histidyl-tRNA synthetase-derived polypeptide Embodiments of the present invention relate to histidyl-tRNA synthetase polypeptide ("HRS or HisRS polypeptide")-Fc conjugates, including HRS-Fc conjugates comprising wild-type HRS sequences, naturally occurring sequences, non-naturally occurring sequences, and / or variants and fragments thereof. Specific examples of HRS-derived polypeptides include those with altered cysteine ​​content. Histidyl-tRNA synthetase belongs 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 attachment 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 synthetase typically exists as either a cytosolic homodimer or an alternatively spliced ​​mitochondrial form.

[0082] More recently, it has been established that some biological fragments or alternatively spliced ​​isoforms of eukaryotic histidyl-tRNA synthetase (physiocrine or HRS polypeptide), or in some contexts, the intact synthetase, modulate specific cell signaling pathways or have anti-inflammatory properties. These activities, which differ from the classical role of tRNA synthetase in protein synthesis, are collectively referred to herein as "non-canonical activities." These physiocrines can be naturally generated by either alternative splicing or proteolysis and can act in a cell-autonomous manner (i.e., within the host cell) or a non-cell-autonomous manner (i.e., outside the host cell) to regulate various homeostatic mechanisms. For example, as provided herein, HRS polypeptides, such as N-terminal fragments of histidyl-tRNA synthetase (e.g., HRS 1-48, HRS 1-60), can exert anti-inflammatory signals by, inter alia, blocking the migration, activation, or differentiation in vivo of inflammatory cells (e.g., monocytes, macrophages, T cells, B cells) associated with sites of active inflammation. Furthermore, certain mutations or deletions (e.g., HRS 1-506, HRS 1-60) compared to the full-length HRS polypeptide sequence confer increased activity and / or improved pharmacological properties. The sequences of certain exemplary HRS polypeptides are provided in Table D1.

[0083] [Table 1-1]

[0084] [Table 1-2]

[0085] [Table 1-3]

[0086] Several naturally occurring histidyl-tRNA synthetase single nucleotide polymorphisms (SNPs) and naturally occurring variants of the human gene have been sequenced and are known in the art to be at least partially functionally interchangeable. Some such variants of histidyl-tRNA synthetase (i.e., representative histidyl-tRNA synthetase SNPs) are shown in Table D2.

[0087] [Table 2-1]

[0088] [Table 2-2]

[0089] [Table 2-3]

[0090] Additionally, homologs and orthologs of the human gene exist in other species, as listed in Table D3, and therefore it is a matter of routine to select naturally occurring amino acids or nucleotide variants present in SNPs, or other naturally occurring homologs, in place of any of the human HRS polypeptide sequences listed in Tables D1, D4-D6, or D8.

[0091] [Table 3-1]

[0092] [Table 3-2]

[0093] Thus, in any of the methods, therapeutic compositions, and kits of the invention, the terms "HRS polypeptide," "HRS protein," or "HRS protein fragment" include all naturally occurring and synthetic forms of histidyl-tRNA synthetase that possess non-canonical activity, such as anti-inflammatory activity, and / or retain at least one epitope that specifically cross-reacts with autoantibodies or autoreactive T cells from subjects with a disease associated with autoantibodies to histidyl-tRNA synthetase. Such HRS polypeptides include the full-length human protein and HRS peptides derived from the full-length proteins listed in Tables D1, D3-D6, or D8. In some embodiments, the term HRS polypeptide refers to a polypeptide sequence derived from human histidyl-tRNA synthetase (SEQ ID NO: 1 in Table D1) that is about 45 or 50 to about 250 amino acids in length. In any of the HRS-Fc conjugates described herein, it is understood that the N-terminal amino acid of the HRS polypeptide (e.g., the N-terminal Met) may be deleted from any of the sequences listed in Tables D1, D3-D6, or D8 when creating a fusion protein or conjugate.

[0094] In some embodiments, the HRS polypeptide is between about 20-509, 20-508, 20-507, 50-506, 20-505, 50-504, 20-503, 20-502, 20-501, 20-500, 20-400, 20-300, 20-250, 20-200, or 20-100 amino acids in length. For example, in specific embodiments, the polypeptide is between about 20-25, 20-35, 20-40, 20-45, 20-55, 20-60, 20-65, 20-70, 20-75, 20-80, 20-85, 20-90, 20-95, or 20-100 amino acids in length, or between about 30-35, 30-40, 30-45, 30-55, 30-60, 30-65, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, or 30-100 amino acids in length, or between about 40-45, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90 , 40-95, or 40-100 amino acids in length, or between about 45-50, 45-55, 50-55, 50-60, 50-65, 50-70, 50-75, 50-80, 50-85, 50-90, 50-95, or 50-100 amino acids in length, or between about 60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, or 60-100 amino acids in length, or between about 70-75, 70-80, 70-85, 70-90, 70-95, or 70-100 amino acids in length, or between about 80-85, 80-90, 80-95, or 80-100 amino acids in length.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 9, 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, 150, 200, 250, 300, 350, 400, 450, 500, 501, 502, 503, 504, 505, 506, 507, 508, or 509 amino acids in length.

[0095] In some embodiments, the HRS polypeptide comprises up to about 1-5 x 10 -7 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 up to a concentration of M or greater. 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).

[0096] Thus, all such homologs, orthologs, and naturally occurring or synthetic isoforms of histidyl-tRNA synthetase (e.g., any of the proteins listed in Tables D1, D3-D6, or D8) are included in any of the methods, HRS-Fc conjugates, kits, and compositions of the invention, so long as they retain at least one epitope that specifically cross-reacts with autoantibodies or autoreactive T cells from a subject with a disease associated with autoantibodies to histidyl-tRNA synthetase or possess non-canonical activity. These HRS polypeptides may exist in their native form, i.e., as different variants that would be considered functionally equivalent variants of human histidyl-tRNA synthetase if they naturally occur in different species, or they may be functionally equivalent naturally occurring derivatives thereof that may differ in their amino acid sequence, for example, by truncation (e.g., from the N-terminus or C-terminus, or both) or deletion, addition, insertion, substitution, or post-translational modification of other amino acids. Naturally occurring chemical derivatives, including post-translational modifications and degradation products of any HRS polypeptide, including, for example, pyroglutamyl, isoaspartyl, proteolytic, phosphorylated, glycosylated, oxidized, isomerized, and deamidated variants of the HRS polypeptide or HRS-Fc conjugate, are also specifically included in any of the methods and compositions of the invention. HRS polypeptides and HRS-Fc conjugates may also be composed of naturally occurring and / or non-naturally occurring amino acids, as described herein.

[0097] As noted above, embodiments of the present invention include all homologs, orthologs, and naturally occurring isoforms of histidyl-tRNA synthetase (e.g., proteins listed in a table or sequence listing, or proteins derivable from such a table or sequence listing, or their corresponding nucleic acids listed in such a table or sequence listing), as well as "variants" of these HRS reference polypeptides. The described polypeptide "variant" refers to a polypeptide that is distinguished from a reference HRS polypeptide by the addition, deletion, and / or substitution of at least one amino acid residue and typically retains (e.g., mimics) or modulates (e.g., antagonizes) one or more non-canonical activities of the reference HRS polypeptide. Variants also include polypeptides that have been modified by the addition, deletion, and / or substitution of at least one amino acid residue to have improved stability or other pharmaceutical properties.

[0098] In certain embodiments, polypeptide variants are distinguished from reference polypeptides by one or more substitutions, which can be conservative or non-conservative, as described herein and known in the art.In certain embodiments, this polypeptide variant comprises conservative substitutions, and in this regard, it is well understood in the art that some amino acids can be changed to other amino acids with broadly similar properties without changing the nature of the activity of the polypeptide.In some embodiments, this variant comprises one or more conserved residues, including one or more of Leu7, Gln14, Gly15, Val18, Arg19, Leu21, Lys22, Lys25, Ala26, Val35, Leu38, Leu39, Leu41 and Lys42 (based on the numbering of SEQ ID NO: 1).

[0099] Specific examples of HRS polypeptide variants useful in any of the methods and compositions of the invention include full-length HRS polypeptides, or truncations or splice variants thereof (e.g., any of the proteins listed in the tables or sequence listings or derivable from such tables or sequence listings), that i) retain detectable non-canonical activity and / or retain at least one epitope that specifically cross-reacts with autoantibodies or autoreactive T cells from subjects with a disease associated with autoantibodies to histidyl-tRNA synthetase, and ii) have one or more additional amino acid insertions, substitutions, deletions, and / or truncations. In certain embodiments, a variant polypeptide comprises an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity or similarity to the corresponding sequence of an HRS reference polypeptide (e.g., any of the proteins listed in a table or sequence listing or derivable from such a table or sequence listing), as described herein, and substantially retains a non-canonical activity of that reference polypeptide. Also included are sequences that differ from a reference HRS sequence by the addition, deletion, 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, but retain the properties of the reference HRS polypeptide. In certain embodiments, the amino acid additions or deletions occur at the C-terminus and / or N-terminus of the HRS reference polypeptide. In certain embodiments, the amino acid additions include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more wild-type residues (i.e., from the corresponding full-length HRS polypeptide) that are proximal to the C-terminus and / or N-terminus of the HRS reference polypeptide.

[0100] In some embodiments, the HRS polypeptide comprises a polypeptide fragment of a full-length histidyl-tRNA synthetase of about 45-250 amino acids or about 50-250 amino acids, comprising, consisting of, or consisting essentially of the amino acids of the HRS polypeptide sequence set forth in one or more of SEQ ID NOs: 1-106, 170-181, or 185-191. In some embodiments, the HRS polypeptide comprises, consists of, or consists essentially of residues 1-141, 1-408, 1-113, or 1-60 of SEQ ID NO: 1. In some aspects, the HRS polypeptide is a splice variant comprising, consisting of, or consisting essentially of residues 1-60 + 175-509, 1-60 + 211-509, or 1-60 + 101-509 of SEQ ID NO: 1. In particular embodiments, the HRS polypeptide comprises, consists of, or consists essentially of residues 1-48 or 1-506 of SEQ ID NO:1.

[0101] In certain embodiments, the HRS polypeptides of the present invention comprise, consist of, or consist essentially of the smallest active fragment of a full-length HRS polypeptide capable of modulating anti-inflammatory activity in vivo or possessing antibody or autoreactive T cell blocking activity. In one aspect, such a smallest active fragment comprises, consists of, or consists essentially of the WHEP domain (i.e., about amino acids 1-43 of SEQ ID NO:1). In some aspects, this smallest active fragment comprises, consists of, or consists essentially of the aminoacylation domain (i.e., about amino acids 54-398 of SEQ ID NO:1). In some aspects, this smallest active fragment comprises, consists of, or consists essentially of the anticodon binding domain (i.e., about amino acids 406-501 of SEQ ID NO:1). Other exemplary active fragments are shown in Table D4 below.

[0102] [Table 4-1]

[0103] [Table 4-2]

[0104] [Table 4-3]

[0105] For some HRS polypeptides, about 20-40, 20-45, 20-50, 20-55, or 20-60, 20-65, or 20-67 contiguous or non-contiguous amino acids of the HRS polypeptide, or at least about 20-40, 20-45, 20-50, 20-55, or 20-60, 20-65, or 20-67 contiguous or non-contiguous amino acids of the HRS polypeptide, are derived from amino acids 1-67 of SEQ ID NO:1. In certain embodiments, 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, or 67 consecutive or non-consecutive amino acids of the HRS polypeptide are derived from amino acids 1-67 of SEQ ID NO: 1. The HRS polypeptide may comprise one or more of a WHEP domain, an aminoacylation domain, an anticodon-binding domain, or any combination thereof. In certain embodiments, the HRS polypeptide lacks a functional aminoacylation domain. In some embodiments, the polypeptide consists essentially of a WHEP domain derived from human HRS. Without wishing to be bound by any one theory, the unique orientation or conformation of the WHEP domain in certain HRS polypeptides may contribute to the enhanced non-canonical and / or antibody-blocking activity observed in these proteins.

[0106] Thus, in certain embodiments, the HRS polypeptide comprises, consists of, or consists essentially of a human HRS WHEP domain sequence. In some embodiments, the human HRS WHEP domain sequence is defined by specific conserved residues. For example, in some aspects, the HRS polypeptide comprises, consists of, or consists essentially of a human HRS WHEP domain consensus sequence in Table D5 below.

[0107] In certain embodiments, the HRS polypeptide may comprise 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 or all 29 amino acids of a flexible linker connecting the minimal domain to a heterologous protein (e.g., an Fc domain) or splice variant.

[0108] The expression "sequence identity" or, for example, "50% identical sequence to" as used herein refers to the degree to which their sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over the comparison window.Therefore, "percentage of sequence identity" 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 identical positions, and dividing this number of identical 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.

[0109] Terms used to describe the sequence relationship between two or more polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" is at least 12, but frequently 15-18, and often at least 25 monomer units in length, including nucleotides and amino acid residues. Because two polypeptides may each contain (1) sequences that are similar between the two polypeptides (i.e., only a portion of the complete polypeptide sequence) and (2) sequences that are diverse between the two polypeptides, sequence comparison between two (or more) polypeptides is typically performed by comparing the sequences of the two polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150, over which a sequence is compared to a reference sequence of the same number of contiguous positions after optimally aligning the two sequences. The comparison window may contain about 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning the comparison window may 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, WI, USA), or by inspecting and selecting the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of a variety of methods. Reference may also be made to the BLAST family of programs, as disclosed, for example, by Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Ausubel et al., "Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, Unit 19.3.

[0110] Calculation of sequence similarity or sequence identity (these terms are used interchangeably herein) between sequences can be performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, these sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In certain embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. Then, the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, these molecules are identical at that position.

[0111] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0112] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (1970, J. Mol. Biol. 48:444-453) incorporated into the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one to be used unless otherwise specified) is the Blossum62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences may also be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios 4:11-17) incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0113] The nucleic acid and protein sequences described herein can be used as "query sequences" to conduct searches against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990, J. Mol. Biol. 215:403-10). BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of representative programs (eg, XBLAST and NBLAST) can be used.

[0114] In certain embodiments, a variant polypeptide differs from the corresponding HRS reference sequence by at least 1%, but less than 20%, 15%, 10%, or 5% of the residues. If this comparison requires alignment, the sequences should be aligned to maximize similarity. Sequences "looped out" from deletions or insertions, or mismatches, are considered differences. These differences are suitably differences or changes in non-essential residues or conservative substitutions. In certain embodiments, the molecular weight of a variant HRS polypeptide differs from the molecular weight of an HRS reference polypeptide by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or more.

[0115] Also included are biologically active "fragments" of the HRS reference polypeptide, i.e., biologically active fragments of the HRS protein fragment. Exemplary biologically active fragments generally participate in an interaction, e.g., an intramolecular interaction or an intermolecular interaction. An intermolecular interaction can be a specific binding interaction or an enzymatic interaction. An intermolecular interaction can be an interaction between an HRS polypeptide and a cellular binding partner, e.g., a cellular receptor or other host molecule involved in a non-canonical activity of the HRS polypeptide.

[0116] Biologically active fragments of an HRS reference polypeptide include any of the following: e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 195, 200, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 2 0, 190, 200, 220, 240, 260, 280, 300, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, The polypeptide fragment may be 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 38, 359, 360, 361, 362, 363, 364, 365, 380, 400, 450, 500, 505 or more consecutive or non-consecutive amino acids. In certain embodiments, the biologically active fragment comprises a non-canonical activity-associated sequence, domain, or motif. In certain embodiments, the C-terminal or N-terminal region of any HRS reference polypeptide may be about 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, 60, 70, 75, 80, 85, 90, 95, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 210, 211, 212, 213, 214, 215, 216, 217, 218, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, It may be truncated by 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500 or more amino acids, or by about 10-50, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500 or more amino acids.Certain exemplary truncated HRS polypeptides and the consensus sequence of the WHEP domain of human HRS are shown in Table D5 below.

[0117] [Table 5-1]

[0118] [Table 5-2]

[0119] [Table 5-3]

[0120] It is understood that in any of the HRS-Fc conjugates of the present invention, the N-terminal amino acid of the HRS polypeptide (e.g., the N-terminal Met) may further be deleted from any of the exemplary truncated HRS polypeptides or other HRS sequences described herein.

[0121] Typically, a biologically active fragment has about 1%, 10%, 25%, or 50% or more of the activity of the biologically active HRS reference polypeptide from which it is derived (i.e., non-canonical activity). Exemplary methods for measuring such non-canonical activity are described in the Examples.

[0122] In some embodiments, HRS proteins, variants and biologically active fragments thereof bind to one or more cellular binding partners with an affinity 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, 50, 100 or 150 nM. In some embodiments, the binding affinity of an HRS protein fragment for selected cellular binding partners, particularly binding partners involved in non-canonical activities, may be at least about 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x or more (including all integers therebetween) stronger than that of the corresponding full-length HRS polypeptide or a particular alternatively spliced ​​HRS polypeptide variant.

[0123] As mentioned above, the HRS polypeptide can be altered in various ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are generally 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. Pat. No. 4,873,192, Watson, JD et al. (Molecular Biology of the Gene, 4th ed., Benjamin / Cummings, Menlo Park, NY, USA). See, e.g., "Synthetic Proteins," ...

[0124] 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, and such additional substitutions may further enhance the activity or stability of HRS polypeptides with altered cysteine ​​content. 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 containing the residue when the peptide is present in aqueous medium at physiological pH. Amino acids with acidic side chains include glutamic acid and aspartic acid.

[0125] 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.

[0126] 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).

[0127] Hydrophobic: These residues are uncharged at physiological pH and are repelled by aqueous solutions such that they 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 hydrophobic side chains include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine, and tryptophan.

[0128] Neutral / polar: These residues are not charged at physiological pH, but are not sufficiently repelled by aqueous solutions, so that they 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.

[0129] This description also characterizes certain amino acids as "small" because their side chains, even 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 carbons 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 as well as the α-carbon of the α-amino group. Several amino acid similarity matrices are known in the art (e.g., Dayhoff et al., 1978, A model of evolutionary change in (See, for example, the PAM120 and PAM250 matrices disclosed by the National Institute for Protein Sciences, Washington, DC). However, matrices for determining distance relationships by MO Dayhoff (ed.), Atlas of protein sequence and structure, Vol. 5, pp. 345-358, National Biomedical Research Foundation, Washington, DC; and Gonnet et al. (Science, Vol. 256:14430-1445, 1992) place proline in the same group as glycine, serine, alanine, and threonine. Therefore, for purposes of the present invention, proline is classified as a "small" amino acid.

[0130] 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 on the basis of known behavior.

[0131] 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. A residue is considered small if it contains a total of four or fewer carbon atoms, including the carboxyl carbon, provided that there is an additional polar substituent; otherwise, it is considered small if it contains three or fewer. Small residues are, of course, always non-aromatic. Depending on their structural properties, amino acid residues can fall into two or more classes. For naturally occurring protein amino acids, subclassification according to this scheme is presented in Table A.

[0132] [Table 5-4]

[0133] Conservative amino acid substitution also includes grouping based on side chain.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; the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine.For example, it is reasonable to expect that the replacement of leucine with isoleucine or valine, the replacement of aspartic acid with glutamic acid, the replacement of threonine with serine, or similar replacement of certain amino acids with structurally related amino acids will not have a significant effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional truncated and / or variant HRS polypeptide can be readily determined by assaying its non-canonical activity, as described herein. Conservative substitutions are shown in Table B under the heading of exemplary substitutions. Amino acid substitutions that fall within the scope of the present invention are generally achieved by selecting substitutions that do not significantly alter (a) the structure of the peptide backbone in the region of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, (c) the bulkiness of the side chain, or (d) their effect on maintaining biological function. After the substitutions are introduced, these variants are screened for biological activity.

[0134] [Table 5-5]

[0135] Alternatively, similar amino acids for conservative substitutions can be grouped into three categories based on the identity of their side chains. As described in Zubay, G., Biochemistry, 3rd 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.

[0136] The NMR structure of the WHEP domain of human HRS has been determined (see Nameki et al., Accession 1X59_A). Additionally, the crystal structures of full-length human HRS and an internal catalytic domain deletion mutant of HRS (HRSΔCD) have also been determined (see Xu et al., Structure. 20:1470-7, 2012; and U.S. Patent Application No. 61 / 674,639). These detailed physical descriptions of the protein, combined with the primary amino acid sequence of HRS, provide precise insight into the roles played by specific amino acids within the protein. Thus, one skilled in the art can use this information to identify structurally conserved domains, connecting regions, secondary structures such as alpha-helices, surface or solvent-exposed amino acids, unexposed or internal regions, catalytic sites, and ligand-interacting surfaces, among other structural features. Such a skilled artisan can then readily use that information, or other information, to engineer HRS variants that retain or improve the desired non-canonical activity, e.g., by preserving or altering characteristics of amino acid residues within or adjacent to these and other structural features, e.g., by preserving or altering the polarity, hydropathic index, charge, size, and / or positioning (i.e., inward, outward) of selected amino acid side chain(s) relative to the wild-type residue (e.g., Zaiwara et al., Mol Biotechnol. 51:67-102, 2012; Perona and Hadd, Biochemistry. 51:8705-29, 2012; Morin et al., Trends Biotechol. 29:159-66, 2011; Collins et al., Annu. Rev. Biophys. 40:81-98, 2011; and U.S. Patent Application No. 61 / 674,639).

[0137] Thus, predicted non-essential amino acid residues in truncated and / or variant HRS polypeptides are typically replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be randomly introduced along all or part of the HRS coding sequence, for example, by saturation mutagenesis, and the resulting mutants can be screened for the activity of the parent polypeptide to identify mutants that retain their activity. After mutagenesis of the coding sequence, the encoded peptide can be recombinantly expressed and the activity of the peptide can be determined. A "non-essential" amino acid residue is a residue that can be altered from the reference sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its non-canonical activities. Suitably, the alteration does not substantially abolish one of these activities, for example, the activity is at least 20%, 40%, 60%, 70%, or 80%, 100%, 500%, 1000% or more of the reference HRS sequence. An "essential" amino acid residue is one that, when altered from the reference sequence of an HRS polypeptide, results in abrogating the activity of the parent molecule such that less than 20% of the reference activity is present. For example, such essential amino acid residues include residues conserved in HRS polypeptides across different species, including sequences conserved in the active binding site(s) or motif(s) of HRS polypeptides from various sources.

[0138] Assays for determining anti-inflammatory activity, including those based on routine measurement of cytokine release from cells in vitro and in animal studies, are well established in the art (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), can be readily used to profile and optimize anti-inflammatory activity. Exemplary in vivo experimental systems are also described in the accompanying Examples.

[0139] In some embodiments, the HRS polypeptide may have one or more cysteine ​​substitutions, in which one or more naturally occurring (non-cysteine) residues are substituted with cysteine ​​(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 cysteine ​​substitutions are near the N-terminus and / or C-terminus of the HRS polypeptide (e.g., SEQ ID NOs: 1-106, 170-181, or 185-191), or other surface-exposed regions of the HRS polypeptide. Certain embodiments include those in which one or more of the residues within 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids of the N-terminus and / or C-terminus of any one of SEQ ID NOS: 1-106, 170-181, or 185-191 are substituted with a cysteine ​​residue. 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 1-5, or about 5-10, about 10-20, or about 20-30 amino acids in length. In some embodiments, fusion to the C-terminus is preferred.

[0140] Specific exemplary embodiments of such cysteine ​​engineered proteins based on the HRS polypeptide HRS(1-60) are shown in Table D6. This approach is directly applicable to the HRS polypeptides of Table D5 and other HRS polypeptides described herein.

[0141] [Table 6]

[0142] In some embodiments, the HRS polypeptide may include 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(s) into the HRS polypeptide may be combined with the elimination of other surface-exposed reactive cysteine ​​residues. Thus, in some embodiments, the HRS polypeptide may include one or more substitutions and / or deletions at Cys83, Cys174, Cys191, Cys196, Cys224, Cys235, Cys379, Cys455, Cys507, and / or Cys509 (as defined by SEQ ID NO: 1), for example, to remove naturally occurring cysteine ​​residues.

[0143] Specific embodiments include any one of SEQ ID NOS: 1-106, 170-181, or 185-191, or variants or fragments thereof, having 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 a 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 D7.

[0144] [Table 7]

[0145] In some embodiments, 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 further examples of desirable cysteine ​​mutations / substitutions (shown in bold and underlined) to reduce surface-exposed cysteine ​​residues include those listed below in Table D8.

[0147] [Table 8-1]

[0148] [Table 8-2]

[0149] [Table 8-3]

[0150] [Table 8-4]

[0151] [Table 8-5]

[0152] In some embodiments, such cysteine ​​substitution mutants are modified to engineer, insert, or otherwise introduce a new surface-exposed cysteine ​​residue at a defined surface-exposed position, where the introduced residue does not substantially interfere with the non-canonical activity of the HRS polypeptide. Specific examples include, for example, the insertion (or 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 an N- or C-terminal surface-exposed cysteine ​​insertion involves 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 HRS (SEQ ID NO: 1)) in any of the HRS polypeptides of SEQ ID NOs: 1-106, 170-181, or 185-191, or Table D1, D3-D6, or D8.

[0153] For some types of site-specific conjugation or attachment to an Fc region or a heterologous molecule, such as PEG or another 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, e.g., to facilitate transglutaminase-catalyzed attachment of a molecule(s) to the amide group of glutamine. In some embodiments, glutamine substitutions are introduced near the N-terminus and / or C-terminus of the HRS polypeptide (e.g., SEQ ID NOS: 1-106, 170-181, or 185-191, or the HRS polypeptides of Tables D1, D3-D6, or D8). Certain embodiments include those in which one or more of the residues within 0, 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 any one of SEQ ID NOs: 1-106, 170-181, or 185-191 are substituted with glutamine residues. These and related HRS polypeptides can 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.

[0154] For certain types of site-specific conjugation or attachment to an Fc region or a heterologous molecule, such as PEG or other heterologous molecule, the HRS polypeptide can have one or more lysine substitutions, where one or more naturally occurring (non-lysine) residues are substituted with lysine to facilitate, for example, acylation- or alkylation-based attachment of a molecule(s) to the amino group of the lysine. These methods also typically result in attachment of a molecule(s) to the N-terminal residue. In some embodiments, the lysine substitutions are near the N- and / or C-terminus of the HRS polypeptide (e.g., SEQ ID NOS: 1-106, 170-181, or 185-191, or an HRS polypeptide of Table D1, D3-D6, or D8). Certain embodiments include those in which one or more of the residues within 0, 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 to the N-terminus and / or C-terminus of any one of SEQ ID NOs: 1-106, 170-181, or 185-191 (or the HRS polypeptides of Tables D1, D3-D6, or D8) are substituted with lysine residues. These and related HRS polypeptides can 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.

[0155] 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.

[0156] 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.

[0157] In various embodiments, the present invention contemplates site-specific conjugation or attachment at any amino acid position in the HRS polypeptide by substituting a non-naturally occurring amino acid containing a functional group that forms a covalent bond with a functional group attached to the Fc region or a heterologous molecule, such as PEG or other heterologous molecule. Unnatural amino acids can be inserted or substituted, for example, at one or more of the residues within 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids of the N-terminus and / or C-terminus of any one of SEQ ID NOs: 1-106, 170-181, or 185-191 (or the HRS polypeptides of Tables D1, D3-D6, or D8); at the N-terminus and / or C-terminus of any one of SEQ ID NOs: 1-106, 170-181, or 185-191 (or the HRS polypeptides of Tables D1, D3-D6, or D8); or at the solvent-accessible surface amino acid residues described herein.

[0158] 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 exemplified by the following formula:

[0159] [ka]

[0160] 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 natural amino acids. For the structures of the 20 natural amino acids, see, for example, biochemistry textbooks, such as Biochemistry by L. Stryer, 3rd Edition, 1988, Freeman and Company, New York. It should be noted that the unnatural amino acids disclosed herein may be naturally occurring compounds other than the 20 alpha-amino acids. 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 with other amino acids, for example, 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, boronate, 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.

[0161] 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, These 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 invention, and the like.

[0162] Thus, non-naturally occurring amino acids can be selected that contain a functional group that forms a covalent bond 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.

[0163] 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 Fc regions or PEG to a given protein, thereby improving its pharmacokinetic properties.

[0164] Specific examples of amino acid analogs and mimetics can be found, for example, in Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, edited by Gross and Meinhofer, Vol. 5, p. 341, Academic Press, Inc., New York, NY (1983), the entire contents of which are incorporated herein by reference. Other examples include peralkylated amino acids, particularly permethylated amino acids. See, for example, Combinatorial Chemistry, edited by Wilson and Czarnik, Chapter 11, p. 235, John Wiley & Sons, Inc., the entire contents of which are incorporated herein by reference. & Sons Inc., New York, NY (1997). Still other examples include amino acids in which the amide moiety (and thus the amide backbone of the resulting peptide) has been replaced by, for example, a sugar ring, a steroid, a benzodiazepine, or a carbocycle. See, e.g., Burger's Medicinal Chemistry and Drug Discovery, edited by Manfred E. Wolff, Chapter 15, pp. 619-620, John Wiley & Sons Inc., New York, NY (1995), the entire contents of which are incorporated herein by reference. Methods for synthesizing peptides, polypeptides, peptidomimetics, and proteins are well known in the art (see, e.g., U.S. Pat. No. 5,420,109; M. Bodanzsky, Principles of Peptide Synthesis (1st ed. and revised 2nd ed.), Springer-Verlag, New York, NY (1984 and 1993), Chapter 7, each of which is incorporated herein by reference; Stewart and Young, Solid Phase Peptide Synthesis, (2nd ed.), Pierce Chemical Co., Rockford, Ill. (1984). Thus, the HRS polypeptides of the present invention can be composed of naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics.

[0165] Polynucleotides Certain embodiments relate to polynucleotides encoding HRS polypeptides or HRS-Fc fusion proteins. Also included are polynucleotides encoding any one or more of the Fc regions described herein, either alone or in combination with an HRS coding sequence. Among other uses, these embodiments can be utilized to recombinantly produce a desired HRS, Fc region, or HRS-Fc polypeptide or variant thereof, or to express HRS, Fc region, or HRS-Fc polypeptide in a selected cell or subject. Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many nucleotide sequences exist that encode the HRS polypeptides or HRS-Fc fusion proteins described herein. Some of these polynucleotides may bear minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage, such as polynucleotides optimized for human, yeast, or bacterial codon selection, are specifically contemplated by the present invention.

[0166] As will be appreciated by those skilled in the art, polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA, or synthetic) or RNA molecules. Additional coding or non-coding sequences can, but need not, be present within a polynucleotide of the invention, and polynucleotides can, but need not, be linked to other molecules and / or support materials.

[0167] The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an HRS-Fc fusion polypeptide or a portion thereof) or may comprise a variant or biologically functional equivalent of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions, and / or insertions, as described further below, such that, preferably, the activity of the encoded polypeptide is not substantially diminished compared to the unmodified polypeptide.

[0168] In a further embodiment, the present invention provides isolated polynucleotides comprising contiguous stretches of various lengths of sequence identical to or complementary to an HRS polypeptide or HRS-Fc fusion protein, wherein the isolated polynucleotide encodes a truncated HRS polypeptide described herein.

[0169] Thus, multiple polynucleotides can encode the HRS polypeptide, Fc region, and fusion protein of the present invention. Furthermore, the polynucleotide sequence can be manipulated for various reasons. Examples include, but are not limited to, the incorporation of preferred codons to enhance expression of the polynucleotide in various organisms (see generally Nakamura et al., Nucl. Acid. Res. 28:292, 2000). Furthermore, silent mutations can be incorporated to introduce or eliminate restriction sites, reduce the density of CpG dinucleotide motifs (see, e.g., Kameda et al., Biochem. Biophys. Res. Commun. 349:1269-1277, 2006), or reduce the ability of single-stranded sequences to form stem-loop structures (see, e.g., Zuker M., Nucl. Acid Res. 31:3406-3415, 2003). Additionally, mammalian expression can be further optimized by including a Kozak consensus sequence (i.e., (a / g)cc(a / g)ccATGg) (SEQ ID NO: 199) at the start codon. Kozak consensus sequences useful for this purpose are known in the art (Mantyh et al., PNAS 92:2662-2666, 1995; Mantyh et al., Prot. Exp. & Purif. 6:124, 1995). Exemplary wild-type and codon-optimized versions of various HRS polypeptides are provided below in Table D9.

[0170] [Table 9-1]

[0171] [Table 9-2]

[0172] [Table 9-3]

[0173] [Table 9-4]

[0174] [Table 9-5]

[0175] [Table 9-6]

[0176] Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the invention, and the polynucleotide may, but need not, be linked to other molecules and / or support materials. Thus, regardless of the length of the coding sequence itself, the polynucleotides of the invention may be combined with other DNA or RNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., so that their overall length may vary considerably.

[0177] It is therefore contemplated that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. Any portion or fragment (e.g., greater than about 6 nucleotides in length, greater than 7 nucleotides in length, greater than 8 nucleotides in length, greater than 9 nucleotides in length, or greater than 10 nucleotides in length) of an HRS reference polynucleotide (e.g., base numbers X through Y, where X is about 1 to 3000 or more and Y is about 10 to 3000 or more) or its complement, including 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, 40, 41, 41, 43, 44, 45, 46, 47, 48, 49, 50 , 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 270, 280, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 , 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 or more bases in length (including all integers in between).

[0178] Embodiments of the present invention also include "variants" of the HRS reference polynucleotide sequence. Polynucleotide "variants" may contain one or more substitutions, additions, deletions, and / or insertions compared to the reference polynucleotide. Generally, variants of the HRS reference polynucleotide sequence may have at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, typically at least about 75%, 80%, 85%, desirably about 90% to 95% or more, and more suitably about 98% or more sequence identity to a particular nucleotide sequence (e.g., SEQ ID NOS: 111-127, 182-184, 192-198, etc.; see also the Examples) as determined by a sequence alignment program described elsewhere herein using default parameters. In certain embodiments, variants may differ from reference sequences by 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, 41, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 (including all integers therebetween) bases or more.In certain embodiments, for example, when polynucleotide variants encode HRS polypeptides with non-canonical activity, the desired activity of the encoded HRS polypeptide is not substantially reduced compared to unmodified polypeptides.The effect on the activity of the encoded polypeptide can generally be evaluated as described herein. In some embodiments, these variants may alter the aggregation state of the HRS polypeptide, for example, to provide for an HRS polypeptide that exists predominantly as a monomer, dimer, or multimer in different embodiments.

[0179] Certain embodiments include polynucleotides that hybridize to reference HRS polynucleotide sequences (e.g., SEQ ID NOS: 111-127, 182-184, 192-198, etc.; see also the Examples) or their complements under stringency conditions described below. As used herein, the term "hybridizes under low stringency, moderate stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel et al. (1998, supra), sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference, and either can be used.

[0180] References herein to low stringency conditions include and encompass at least about 1% v / v to at least about 15% v / v formamide, and at least about 1 M to at least about 2 M salt for hybridization at 42° C., and at least about 1 M to at least about 2 M salt for washing at 42° C. Low stringency conditions can also include 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO (pH 7.2), 5% SDS for washing at room temperature. One embodiment of low stringency conditions involves hybridization in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2x SSC, 0.1% SDS at at least 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions).

[0181] Moderate stringency conditions include, but are not limited to, at least about 16% v / v to at least about 30% v / v formamide, and at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C., and at least about 0.1 M to at least about 0.2 M salt for washing at 55° C. Moderate stringency conditions may also include 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO (pH 7.2), 5% SDS for washing at 60-65° C. One embodiment of moderate stringency conditions includes hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C. High stringency conditions include and encompass from at least about 31% v / v to at least about 50% v / v formamide, and from about 0.01 M to about 0.15 M salt for hybridization at 42° C., and from about 0.01 M to about 0.02 M salt for washes at 55° C.

[0182] High stringency conditions may also include 1% BSA, 1 mM EDTA, 0.5 M NaHPO (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 0.2×SSC, 0.1% SDS for washes at temperatures above 65° C.; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO (pH 7.2), 1% SDS. One embodiment of high stringency conditions includes hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. One embodiment of very high stringency conditions includes hybridization in 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washes in 0.2×SSC, 1% SDS at 65° C.

[0183] Other stringency conditions are well known in the art, and those skilled in the art will recognize that various factors can be manipulated to optimize hybridization specificity. Optimizing the stringency of the final wash can serve to ensure a high degree of hybridization. For detailed examples, see Ausubel et al., supra, pp. 2.10.1-2.10.16, and Sambrook et al. (1989, supra), sections 1.101-1.104. Stringent washes are typically performed at temperatures between about 42°C and 68°C, although those skilled in the art will understand that other temperatures may be appropriate for stringent conditions. Maximum hybridization rates are typically determined by the T m It occurs below 20°C to 25°C. m It is well known in the art that T is the melting temperature or the temperature at which two complementary polynucleotide sequences dissociate. m Methods for estimating Λ are well known in the art (see Ausubel et al., supra, p. 2.10.8).

[0184] In general, the T of a perfectly matched DNA duplex m is the formula:T m =81.5+16.6(log 10 The total cleavage energy can be approximately estimated by (M) + 0.41(% G+C) - 0.63(% formamide) - (600 / length), where M is the amount of Na, preferably in the range of 0.01 molar to 0.4 molar. + %G+C is the sum of guanosine and cytosine bases as a percentage of the total number of bases within the G+C range between 30% and 75%; %formamide is the formamide concentration in volume percent; and length is the number of base pairs in the DNA duplex. m decreases by approximately 1°C for every 1% increase in the number of randomly mismatched base pairs. Washing is generally performed at T m -15°C, or T for moderate stringency m It is carried out at -30°C.

[0185] In one example of a hybridization procedure, a membrane (e.g., a nitrocellulose membrane or a nylon membrane) containing immobilized DNA is hybridized overnight at 42°C in a hybridization buffer containing labeled probes (50% deionized formamide, 5x SSC, 5x Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 0.1% bovine serum albumin), 0.1% SDS, and 200 mg / mL denatured salmon sperm DNA). The membrane is then subjected to two successive medium stringency washes (i.e., 2x SSC, 0.1% SDS for 15 minutes at 45°C, followed by 2x SSC, 0.1% SDS for 15 minutes at 50°C), followed by two successive higher stringency washes (i.e., 0.2x SSC, 0.1% SDS for 12 minutes at 55°C, followed by 0.2x SSC and 0.1% SDS for 12 minutes at 65-68°C).

[0186] Generation of HRS polypeptides and HRS-Fc conjugates HRS-Fc conjugate polypeptides can be prepared by any suitable procedure known in the art, for example, using standard solid-phase peptide synthesis (Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)) or by recombinant techniques using genetically engineered hosts. Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using an Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments can be chemically synthesized separately and combined using chemical methods to generate the desired molecule.

[0187] HRS-Fc conjugates can also be produced by expressing DNA or RNA sequences encoding the HRS polypeptide or HRS-Fc conjugate of interest in suitable host cells using well-known techniques. Polynucleotide sequences encoding HRS-Fc conjugates or HRS polypeptides can be prepared synthetically using established standard methods, such as the phosphoramidite method described by Beaucage et al., Tetrahedron Letters 22:1859-1869, 1981; or the method described by Matthes et al., EMBO Journal 3:801-805, 1984. According to the phosphoramidite method, oligonucleotides are synthesized, purified, double-stranded, and ligated to form a synthetic DNA construct, for example, in an automated DNA synthesizer. Alternatively, DNA or RNA constructs can be constructed using standard recombinant molecular biology techniques, including restriction enzyme-mediated cloning and PCR-based gene amplification. In some embodiments, for direct mRNA-mediated expression, the polynucleotide may be encapsulated in nanoparticles or liposomes to allow efficient delivery and uptake into cells, and optionally contain modified cap or tail structures to enhance stability and translation.

[0188] These polynucleotide sequences can also be of mixed genomic, cDNA, RNA, and synthetic origin. For example, a genomic or cDNA sequence encoding a leader peptide can be joined to a genomic or cDNA sequence encoding an HRS polypeptide or HRS-Fc conjugate, and then this DNA or RNA sequence can be modified at a single site by inserting a synthetic oligonucleotide encoding the desired amino acid sequence for homologous recombination according to well-known procedures, or preferably by generating the desired sequence by PCR using appropriate oligonucleotides. In some embodiments, a signal sequence can be included before the coding sequence. This sequence encodes a signal peptide N-terminal to the coding sequence that communicates with the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the medium. Typically, this signal peptide is cleaved off by the host cell before the protein leaves the cell. Signal peptides can be found in various proteins in prokaryotes and eukaryotes.

[0189] A variety of expression vector / host systems are known and can be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems, including mammalian cells and more specifically human cell lines, transformed with viral, plasmid, episomal, or integrative expression vectors.

[0190] "Control elements" or "regulatory sequences" present in an expression vector are untranslated regions of the vector—enhancers, promoters, 5' and 3' untranslated regions—that interact with host cellular proteins to effect transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of appropriate transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters can be used, such as the hybrid lacZ promoter of the pBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or the PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.). In mammalian cell systems, promoters derived from mammalian genes or mammalian viruses are generally preferred. If it is necessary to generate a cell line containing multiple copies of a polypeptide-encoding sequence, SV40- or EBV-based vectors can be advantageously used with an appropriate selectable marker.

[0191] Certain embodiments may use E. coli-based expression systems (see, e.g., Structural Genomics Consortium et al., Nature Methods. 5:135-146, 2008). These and related embodiments may rely in part or in whole on ligation-independent cloning (LIC) to generate suitable expression vectors. In specific embodiments, protein expression may be controlled by T7 RNA polymerase (e.g., the pET vector series) or modified pET vectors with alternative promoters, including, for example, the TAC promoter. These and related embodiments may utilize the λDE3 lysogen of BL21, expression host strain BL21(DE3), which supports T7-mediated expression and is deficient in lon and ompT proteases for improved target protein stability. Expression host strains harboring plasmids encoding tRNAs rarely used in E. coli, such as the ROSETTA™(DE3) and Rosetta 2(DE3) strains, are also included. In some embodiments, other E. coli K-12 strains, such as W3110(F), that may produce reduced levels of post-translational modifications during fermentation are also used. - lambda - IN(rrnD-rrnE)1 rph-1), and UT5600(F, araC14, leuB6(Am), secA206(aziR), lacY1, proC14, tsx67, Δ(ompTfepC)266, entA403, glnX44(AS), λ -Other E. coli strains, including those with the following genera (e.g., trpE38, rfbC1, rpsL109(strR), xylA5, mtl-1, thiE1), can be utilized. Cell lysis and sample handling can also be improved using reagents sold under the trademarks BENZONASE® nuclease and BUGBUSTER® Protein Extraction Reagent. For cell culture, autoinducing media can improve the efficiency of many expression systems, including high-throughput expression systems. This type of media (e.g., the OVERNIGHT EXPRESS™ Autoinduction System) initiates protein expression stepwise through metabolic shifts without the addition of artificial inducers such as IPTG.

[0192] Certain embodiments use a hexahistidine tag (e.g., one sold under the trademark HIS·TAG® fusion) followed by immobilized metal affinity chromatography (IMAC) purification or related techniques. However, in certain aspects, clinical-grade proteins can be isolated from E. coli inclusion bodies with or without the use of affinity tags (see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006). As a further example, certain embodiments can use a cold-shock-inducible E. coli high-yield production system, since overexpression of the protein in Escherichia coli at low temperatures improves its solubility and stability (see, e.g., Qing et al., Nature Biotechnology. 22:877-882, 2004).

[0193] High-density bacterial fermentation systems are also included. For example, high cell density cultivation of Ralstonia eutropha allows protein production at cell densities exceeding 150 g / L and expression of recombinant proteins at titers exceeding 10 g / L. In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, can be used. For reviews, see Ausubel et al. (supra) and Grant et al., Methods Enzymol. 153:516-544, 1987. Pichia pandoris expression systems (see, e.g., Li et al., Nature Biotechnology. 24:210-215, 2006; and Hamilton et al., Science 301:1244, 2003) are also included. Certain embodiments include yeast systems engineered to selectively glycosylate proteins, including, inter alia, yeast with humanized N-glycosylation pathways (see, e.g., Hamilton et al., Science. 313:1441-1443, 2006; Wildt et al., Nature Reviews Microbiol. 3:119-28, 2005; and Gerngross et al., Nature-Biotechnology. 22:1409-1414, 2004; U.S. Patent Nos. 7,629,163; 7,326,681; and 7,029,872). By way of example only, recombinant yeast cultures can be grown in Fernbach Flasks or 15 L, 50 L, 100 L, and 200 L fermentors, among others.

[0194] When a plant expression vector is used, expression of the polypeptide-encoding sequence can be driven by any of several promoters. For example, viral promoters, such as the 35S and 19S promoters of CaMV, can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6:307-311, 1987). Alternatively, plant promoters, such as the small subunit or heat shock promoter of RUBISCO, can be used (Coruzzi et al., EMBO J. 3:1671-1680, 1984; Broglie et al., Science. 224:838-843, 1984; and Winter et al., Results Probl. Cell Differ. 17:85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in several publicly available reviews (see, for example, Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191-196, 1992).

[0195] Insect systems can also be used to express a polypeptide of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda or Trichoplusia cells. A polypeptide-encoding sequence can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence inactivates the polyhedrin gene and generates recombinant virus lacking coat protein. These recombinant viruses can then be used to infect, for example, S. frugiperda or Trichoplusia cells in which the polypeptide of interest can be expressed (Engelhard et al., PNAS USA. 91:3224-3227, 1994). Also included are baculovirus expression systems, including expression systems utilizing SF9, SF21, and T. ni cells (see, e.g., Murphy and Piwnica-Worms, Curr Protoc Protein Sci. (See Chapter 5: Unit 5.4, 2001). Insect systems may offer post-translational modifications similar to those in mammalian systems.

[0196] For mammalian host cells, several expression systems are well known in the art and commercially available. Exemplary mammalian vector systems include, for example, Invitrogen's pCEP4, pREP4, and pREP7, Crucell's PerC6 system, and lentivirus-based systems such as Invitrogen's pLP1. For example, when adenovirus is used as an expression vector, a sequence encoding a polypeptide of interest can be ligated into an adenovirus transcription / translation complex consisting of a late promoter and a tripartite leader sequence. Insertion into the non-essential E1 or E3 region of the viral genome can be used to obtain a viable virus capable of expressing the polypeptide in infected host cells (Logan and Shenk, PNAS USA. 81:3655-3659, 1984). Additionally, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

[0197] Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals of NY Acad. Sci. 383:44-68, 1982); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA. 77:4216, 1980); and myeloma cell lines, such as NSO and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, Humana Press, Totowa, NJ, 2003), pp. 255-268. Certain preferred mammalian cell expression systems include CHO and HEK293 cell-based expression systems. Mammalian expression systems may utilize, for example, attached cell lines in T-flasks, roller bottles, or cell factories, or suspension cultures in, for example, 1 L and 5 L spinners, 5 L, 14 L, 40 L, 100 L, and 200 L stirred tank bioreactors, or 20 / 50 L and 100 / 200 L WAVE bioreactors, among others known in the art.

[0198] Also included are methods of cell-free protein expression. These and related embodiments typically utilize purified RNA polymerase, ribosomes, tRNA, and ribonucleotides. Such reagents can be produced, for example, by extraction from cells or cell-based expression systems.

[0199] Additionally, host cell strains can be selected for their ability to modulate the expression of the inserted sequences or process the expressed proteins in the desired manner. Such modifications of the polypeptide include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation, or the insertion of non-naturally occurring amino acids (see generally U.S. Patent Nos. 7,939,496; 7,816,320; 7,947,473; 7,883,866; 7,838,265; 7,829,310; 7,820,766; 7,820,766; 7,7737,226; 7,736,872; 7,638,299; 7,632,924; and 7,230,068). In some embodiments, such a non-naturally occurring amino acid can be inserted at position Cys130. Post-translational processing that cleaves a "prepro" form of the protein can also be used to facilitate correct insertion, folding, and / or function. In addition to bacterial cells, different host cells, such as yeast, CHO, HeLa, MDCK, HEK293, and W138, that possess or even lack specific cellular machinery and characteristic mechanisms for such post-translational activities can be selected to ensure correct modification and processing of the foreign protein.

[0200] HRS polypeptides or HRS-Fc conjugates produced by recombinant cells can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high-pressure liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary purification chemistries include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A / G), gel filtration, reversed-phase, ceramic HYPERD® ion exchange chromatography, and hydrophobic interaction columns (HIC), among others known in the art. Some exemplary methods are also disclosed in the Examples section.

[0201] HRS-Fc conjugate As described above, embodiments of the present invention relate to 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. A wide variety of Fc region sequences, including wild-type sequences from any number of species, as well as variants, fragments, hybrids, and chemically modified forms thereof, can be used in the HRS-Fc conjugates of the present invention. These HRS-Fc polypeptides may also (optionally) include one or more linkers that typically separate the Fc region(s) from the HRS polypeptide(s), including peptide linkers and chemical linkers, as described herein and known in the art. It is 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 acid(s), for example, to facilitate expression and / or cloning or to function as a linker sequence between the two proteins.

[0202] 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(s) 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.

[0203] Certain embodiments use HRS-Fc fusion proteins. "Fusion protein" is defined elsewhere herein, as are methods for making fusion proteins, and are well known in the art (e.g., for general disclosures and methods relating to Fc fusion proteins, see 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). 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), where the first HRS sequence is fused to the N-terminus of the Fc region and the second HRS sequence is fused to the C-terminus of the Fc region. In specific 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.

[0204] The term "HRS-Fc" can, but does not necessarily, refer to N-terminal or C-terminal attachment of the Fc region to the HRS polypeptide. For example, in certain instances, the term "Fc-HRS" refers to fusion of the Fc region to the N-terminus of the HRS polypeptide, and the term "HRS-Fc" refers to 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.

[0205] In some embodiments, the HRS-Fc fusion protein may comprise 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 D10. The preparation and sequences of specific tandem repeat HRS-Fc conjugates are illustrated in the Examples.

[0206] [Table 10]

[0207] Certain embodiments relate to, for example, HRS-Fc conjugates in which one or more Fc regions are chemically conjugated or crosslinked to an HRS polypeptide(s). In these and related aspects, the Fc region may be conjugated to the HRS polypeptide at the N-terminal region (e.g., within the first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc. amino acids), at an internal region (between the N-terminal region and the C-terminal region), and / or at 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), which covalently bonds 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. Yet 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 non-peptide polymers (e.g., PEG polymers; HRS-N-PEG-N-Fc conjugates) as cross-linkers between the Fc region(s) and the HRS polypeptide(s), 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.

[0208] 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(s), 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).

[0209] 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 is composed of two heavy chains and two light chains. These heavy chains can be divided into at least three functional regions: the Fd region, the Fc region (fragment crystallizable region), and the hinge region (see Figure 1), 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 ) domain and constant (CH1) domain, and the light chain variable (V L) domain and constant (C L ) domains together form the antigen-binding fragment or Fab region.

[0210] The Fc region of IgG, IgA, and IgD immunoglobulins contains heavy chain constant domains 2 and 3, termed the CH2 and CH3 regions, respectively; the Fc region of IgE and IgM immunoglobulins contains heavy chain constant domains 2, 3, and 4, termed the CH2, CH3, and CH4 regions, respectively. The Fc region is primarily responsible for the effector functions of the immunoglobulin, including, for example, complement fixation and binding to cognate Fc receptors on effector cells.

[0211] 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 site(s), 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(s) (see, e.g., Shin et al., Intern. Rev. Immunol. 10:177-186, 1993).

[0212] Thus, the term "Fc region" or "Fc fragment" or "Fc," as used herein, refers to a protein comprising one or more of the CH2, CH3, and / or CH4 regions from one or more selected immunoglobulin(s), including fragments and variants and combinations thereof. An "Fc region" may also comprise one or more hinge region(s) of the heavy chain constant region of the immunoglobulin. In certain embodiments, an Fc region comprises the CH1, CH2, CH3, and CH4 regions of an immunoglobulin. L , V L and / or V H Does not include one or more of the regions.

[0213] The Fc region may be derived from the CH2, CH3, CH4, and / or hinge region(s) 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. Figure 2 shows an alignment of the Fc regions from human IgA1 (SEQ ID NO: 156), IgA2 (SEQ ID NO: 157), IgM (SEQ ID NO: 158), IgG1 (SEQ ID NO: 159), IgG2 (SEQ ID NO: 160), IgG3 (SEQ ID NO: 161), IgG4 (SEQ ID NO: 162) and IgE (SEQ ID NO: 163).

[0214] 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 FcεR (e.g., FcαRI) compared to one or more FcγR, FcαR, and / or FcRn. In certain 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 of) the Fc region to one or more selected FcR(s) is increased, typically by about 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x or more (including all integers in between) compared to its binding to (or affinity for) one or more different FcR(s).

[0215] 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.

[0216] 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. Certain Fc regions have increased binding to FcγRIIIb relative to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, and / or FcγRIIIa.

[0217] 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 is composed of two extracellular Ig-like domains and is a member of both the immunoglobulin superfamily and the multichain immune recognition receptor (MIRR) family. It signals by associating with two FcRγ signaling chains.

[0218] 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 have increased binding to FcεRII compared to FcεRII. Other Fc regions have increased binding to FcεRII compared to FcεRI.

[0219] Table F1 below summarizes the characteristics of certain FcRs.

[0220] [Table 11]

[0221] The Fc region can be derived from immunoglobulin molecules of any animal, including vertebrates such as mammals, including 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 below (SEQ ID NOs: 128-154).

[0222] SEQ ID NO: 128 is the amino acid sequence of the human IgA1 hinge region (VPSTPPTPSPSTPPTPSPS).

[0223] SEQ ID NO: 129 is the amino acid sequence of the human IgA1 CH2 region (CCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKS).

[0224] Sequence number 130 is the amino acid sequence of the human IgA1 CH3 region (GNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY).

[0225] SEQ ID NO: 131 is the amino acid sequence of the human IgA2 hinge region (VPPPPP).

[0226] SEQ ID NO: 132 is the amino acid sequence of the human IgA2 CH2 region (CCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKS).

[0227] SEQ ID NO: 133 is the amino acid sequence of the human IgA2 CH3 region (GNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY).

[0228] SEQ ID NO: 134 is the amino acid sequence of the human IgD hinge region (ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTP).

[0229] SEQ ID NO: 135 is the amino acid sequence of the human IgD CH2 region (ECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREP).

[0230] SEQ ID NO: 136 is the amino acid sequence of the human IgD CH3 region (AAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPRSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK).

[0231] SEQ ID NO: 137 is the amino acid sequence of the human IgE CH2 region (VCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCA).

[0232] SEQ ID NO: 138 is the amino acid sequence of the human IgE CH3 region (DSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTS).

[0233] SEQ ID NO: 139 is the amino acid sequence of the human IgE CH4 region (GPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK).

[0234] SEQ ID NO: 140 is the amino acid sequence of the human IgG1 hinge region (EPKSCDKTHTCPPCP).

[0235] SEQ ID NO: 341 is the amino acid sequence of a modified human IgG1 hinge region-derived sequence (SDKTHTCPPCP).

[0236] SEQ ID NO: 141 is the amino acid sequence of the human IgG1 CH2 region (APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK).

[0237] SEQ ID NO: 142 is the amino acid sequence of the human IgG1 CH3 region (GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK).

[0238] SEQ ID NO: 342 is the amino acid sequence of the human IgG1 heavy chain sequence (MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK). It is understood that the Met residue in this human IgG1 heavy chain sequence may be deleted upon N-terminal fusion to, for example, an HRS polypeptide (see SEQ ID NO: 340).

[0239] SEQ ID NO: 143 is the amino acid sequence of the human IgG2 hinge region (ERKCCVECPPCP).

[0240] SEQ ID NO: 144 is the amino acid sequence of the human IgG2 CH2 region (APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK).

[0241] SEQ ID NO: 145 is the amino acid sequence of the human IgG2 CH3 region (GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK).

[0242] SEQ ID NO: 146 is the amino acid sequence of the human IgG3 hinge region (ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP).

[0243] SEQ ID NO: 147 is the amino acid sequence of the human IgG3 CH2 region (APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK).

[0244] SEQ ID NO: 148 is the amino acid sequence of the human IgG3 CH3 region (GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK).

[0245] SEQ ID NO: 149 is the amino acid sequence of the human IgG4 hinge region (ESKYGPPCPSCP).

[0246] SEQ ID NO: 150 is the amino acid sequence of the human IgG4 CH2 region (APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK).

[0247] SEQ ID NO: 151 is the amino acid sequence of the human IgG4 CH3 region (GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK).

[0248] SEQ ID NO: 152 is the amino acid sequence of the human IgM CH2 region (VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVP).

[0249] SEQ ID NO: 153 is the amino acid sequence of the human IgM CH3 region (DQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPK).

[0250] SEQ ID NO: 154 is the amino acid sequence of the human IgM CH4 region (GVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY).

[0251] Thus, the HRS-Fc conjugates of the present invention may comprise, consist of, or consist essentially of one or more of the human Fc region amino acid sequences of SEQ ID NOs: 128-163 or 339-342, 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, and optionally comprising, consisting of, or consisting essentially of any one or more of SEQ ID NOs: 128-154 or 341-342. 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 SEQ ID NOs: 128-154 or 339-342.

[0252] A particular Fc region comprises, consists of, or consists essentially of the human IgA1 sequence set forth in SEQ ID NOs: 128-130 or 156, in any order reading from N-terminus to C-terminus, including combinations thereof (e.g., SEQ ID NOs: 128 and 129 and 130; SEQ ID NOs: 128 and 129; SEQ ID NOs: 128 and 130; SEQ ID NOs: 129 and 130), and variants and fragments thereof. A particular Fc region comprises, consists of, or essentially consists of the human IgA1 sequence set forth in SEQ ID NO: 128. A particular Fc region comprises, consists of, or essentially consists of the human IgA1 sequence set forth in SEQ ID NO: 129. A particular Fc region comprises, consists of, or essentially consists of the human IgA1 sequence set forth in SEQ ID NO: 130.

[0253] Some Fc regions comprise, consist of, or consist essentially of the human IgA2 sequences set forth in SEQ ID NOs: 131-133 or 157, in any order reading from N-terminus to C-terminus, including combinations thereof (e.g., SEQ ID NOs: 131 and 132 and 133; SEQ ID NOs: 131 and 132; SEQ ID NOs: 131 and 133; SEQ ID NOs: 132 and 133), and variants and fragments thereof. A particular Fc region comprises, consists of, or essentially consists of the human IgA2 sequence set forth in SEQ ID NO: 131. A particular Fc region comprises, consists of, or essentially consists of the human IgA2 sequence set forth in SEQ ID NO: 132. A particular Fc region comprises, consists of, or essentially consists of the human IgA2 sequence set forth in SEQ ID NO: 133.

[0254] A particular Fc region comprises, consists of, or consists essentially of the human IgD sequences set forth in SEQ ID NOs: 134-136, in any order reading from N-terminus to C-terminus, including combinations thereof (e.g., SEQ ID NOs: 134 and 135 and 136; SEQ ID NOs: 134 and 135; SEQ ID NOs: 134 and 136; SEQ ID NOs: 135 and 136), as well as variants and fragments and combinations of these sequences. A particular Fc region comprises, consists of, or essentially consists of the human IgD sequence set forth in SEQ ID NO: 134. A particular Fc region comprises, consists of, or essentially consists of the human IgD sequence set forth in SEQ ID NO: 135. A particular Fc region comprises, consists of, or essentially consists of the human IgD sequence set forth in SEQ ID NO: 136.

[0255] A particular Fc region comprises, consists of, or consists essentially of the human IgE sequence set forth in SEQ ID NOs: 137-139 or 163, in any order reading from N-terminus to C-terminus, including combinations thereof (e.g., SEQ ID NOs: 137 and 138 and 139; SEQ ID NOs: 137 and 138; SEQ ID NOs: 137 and 139; SEQ ID NOs: 138 and 139), as well as variants and fragments and combinations of these sequences. A particular Fc region comprises, consists of, or essentially consists of the human IgE sequence set forth in SEQ ID NO: 137. A particular Fc region comprises, consists of, or essentially consists of the human IgE sequence set forth in SEQ ID NO: 138. A particular Fc region comprises, consists of, or essentially consists of the human IgE sequence set forth in SEQ ID NO: 139.

[0256] A particular Fc region comprises, consists of, or consists essentially of the human IgG1 sequence set forth in SEQ ID NOs: 140-142 or 159 or 339-342, in any order reading from N-terminus to C-terminus, including combinations thereof (e.g., SEQ ID NOs: 140 and 141 and 142; SEQ ID NOs: 140 and 141; SEQ ID NOs: 140 and 142; SEQ ID NOs: 141 and 142), as well as variants and fragments and combinations of these sequences. A particular Fc region comprises, consists of, or essentially consists of the human IgG1 sequence set forth in SEQ ID NO: 140. A particular Fc region comprises, consists of, or essentially consists of the human IgG1 sequence set forth in SEQ ID NO: 141. A particular Fc region comprises, consists of, or essentially consists of the human IgG1 sequence set forth in SEQ ID NO: 142. A particular Fc region comprises, consists of, or essentially consists of the human IgG1 sequence set forth in SEQ ID NO: 339. A particular Fc region comprises, consists of, or consists essentially of the human IgG1 sequence set forth in SEQ ID NO: 340. A particular Fc region comprises, consists of, or consists essentially of the human IgG1 sequence set forth in SEQ ID NO: 341. A particular Fc region comprises, consists of, or consists essentially of the human IgG1 sequence set forth in SEQ ID NO: 342.

[0257] A particular Fc region comprises, consists of, or consists essentially of the human IgG2 sequence set forth in SEQ ID NOs: 143-145 or 160, in any order read from N-terminus to C-terminus, including combinations thereof (e.g., SEQ ID NOs: 143 and 144 and 145; SEQ ID NOs: 143 and 144; SEQ ID NOs: 143 and 145; SEQ ID NOs: 144 and 145), as well as variants and fragments and combinations of these sequences. A particular Fc region comprises, consists of, or essentially consists of the human IgG2 sequence set forth in SEQ ID NO: 143. A particular Fc region comprises, consists of, or essentially consists of the human IgG2 sequence set forth in SEQ ID NO: 144. A particular Fc region comprises, consists of, or essentially consists of the human IgG2 sequence set forth in SEQ ID NO: 145.

[0258] A particular Fc region comprises, consists of, or consists essentially of the human IgG3 sequence set forth in SEQ ID NOs: 146-148 or 161, in any order reading from N-terminus to C-terminus, including combinations thereof (e.g., SEQ ID NOs: 146 and 147 and 148; SEQ ID NOs: 146 and 147; SEQ ID NOs: 146 and 148; SEQ ID NOs: 147 and 148), as well as variants and fragments and combinations of these sequences. A particular Fc region comprises, consists of, or essentially consists of the human IgG3 sequence set forth in SEQ ID NO: 146. A particular Fc region comprises, consists of, or essentially consists of the human IgG3 sequence set forth in SEQ ID NO: 147. A particular Fc region comprises, consists of, or essentially consists of the human IgG3 sequence set forth in SEQ ID NO: 148.

[0259] A particular Fc region comprises, consists of, or consists essentially of the human IgG4 sequence set forth in SEQ ID NOs: 149-151 or 162, in any order reading from N-terminus to C-terminus, including combinations thereof (e.g., SEQ ID NOs: 149 and 150 and 151; SEQ ID NOs: 149 and 150; SEQ ID NOs: 149 and 151; SEQ ID NOs: 150 and 151), as well as variants and fragments and combinations of these sequences. A particular Fc region comprises, consists of, or essentially consists of the human IgG4 sequence set forth in SEQ ID NO: 149. A particular Fc region comprises, consists of, or essentially consists of the human IgG4 sequence set forth in SEQ ID NO: 150. A particular Fc region comprises, consists of, or essentially consists of the human IgG4 sequence set forth in SEQ ID NO: 151.

[0260] A particular Fc region comprises, consists of, or consists essentially of the human IgM sequence set forth in SEQ ID NOs: 152-154 or 158, in any order reading from N-terminus to C-terminus, including combinations thereof (e.g., SEQ ID NOs: 152 and 153 and 154; SEQ ID NOs: 152 and 153; SEQ ID NOs: 152 and 154; SEQ ID NOs: 153 and 154), as well as variants and fragments and combinations of these sequences. A particular Fc region comprises, consists of, or essentially consists of the human IgM sequence set forth in SEQ ID NO: 152. A particular Fc region comprises, consists of, or essentially consists of the human IgM sequence set forth in SEQ ID NO: 153. A particular Fc region comprises, consists of, or essentially consists of the human IgM sequence set forth in SEQ ID NO: 154.

[0261] 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 (e.g., the human Ig sequences of SEQ ID NOs: 128-163).

[0262] 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 set forth in SEQ ID NOs: 128-163. In certain embodiments, the variant Fc region comprises an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity or similarity or homology to any one or more of SEQ ID NOs: 128-163. Also included are Fc regions that differ from one or more of SEQ ID NOs: 128-163 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. In certain embodiments, these amino acid additions or deletions are at the C-terminus and / or N-terminus of the Fc reference sequence.

[0263] In certain embodiments, the variant Fc region is optimally aligned with any one or more of SEQ ID NOs: 128-163, and is 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, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 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, 00, 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, 56 90, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more BLAST bit scores or sequence similarity scores, where the BLAST alignment used a BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

[0264] Also included are hybrid Fc regions, eg, 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 ... gD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgE / IgA1, IgE / IgA2, IgE / IgD, IgE / IgE, IgE / IgG1, IgE / IgG2, IgE / IgG3, IgE / IgG4, IgE / I gM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD I gG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM, IgM In a specific embodiment, the hybrid Fc region comprises, consists of, or consists essentially of 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 derived from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, or IgG4, and / or a CH4 domain derived from IgE and / or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from a human Ig.

[0265] 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 (or any combination thereof). In a specific embodiment, the hybrid Fc region comprises, consists of, or consists essentially of a human Ig (a fragment or variant thereof), and optionally includes a hinge derived from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4 and / or a CH3 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from a human Ig.

[0266] Particular 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 (or fragments thereof). In a specific embodiment, the hybrid Fc region comprises, consists of, or consists essentially of a human Ig (a fragment or variant thereof), and optionally includes a hinge derived from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4 and / or a CH2 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from a human Ig.

[0267] 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 , IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, I gG1 / 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 / IgG 1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, I In a specific embodiment, the Fc region comprises 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 comprises a CH3 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM and / or a CH4 domain derived from IgE and / or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from a human Ig.

[0268] Particular 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 / IgA2, I gG1 / 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 / IgG 1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, I In a specific embodiment, the Fc region comprises 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 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 a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from a human Ig.

[0269] 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.

[0270] Specific examples of hybrid Fc regions derived from combinations of IgG subclasses or combinations of human IgD and IgG can be found, for example, in WO 2008 / 147143.

[0271] 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 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., WO See US Patent Application No. 2005 / 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., US 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., US Patent Application No. 2010 / 0255013). Some embodiments can include an Fc region that has been optimized by substitution or deletion to reduce the level of fucosylation, e.g., to increase affinity for FcγRI, FcγRIa, or FcγRIIIa and / or to improve phagocytosis by FcγRIIa-expressing cells (see, e.g., US Patent Application No. 2010 / 0249382 and US Patent Application No. 2007 / 0148170).

[0272] As another example of an altered Fc glycoform, the Fc region may 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 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, which optionally has greater 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.

[0273] 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 of 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 of 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.

[0274] 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 WO 2000 / 42072 and WO 2004 / 016750. Specific 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 of Kabat et al. (see U.S. Patent Application Nos. 2009 / 0163699 and 20060173170).

[0275] Certain variant, fragment, hybrid, or modified Fc regions 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 compared to the corresponding wild-type Fc sequence. 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 contain 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 routine techniques in the art (see, e.g., 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 assessed in vivo by using animal models described, for example, in Clynes et al., PNAS. 95:652-656, 1998.

[0276] 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.

[0277] 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 a substitution at position 251 with leucine, a substitution at position 252 with tyrosine, tryptophan or phenylalanine, a substitution at position 254 with threonine or serine, a substitution at position 255 with arginine, a substitution at position 256 with glutamine, arginine, serine, threonine or glutamic acid, a substitution at position 308 with threonine, a substitution at position 309 with proline, a substitution at position 311 with serine, a substitution at position 312 with aspartic acid, a substitution at position 314 with leucine, a substitution at position 380 with leucine, a substitution at position 382 with leucine, a substitution at position 383 with leucine, a substitution at position 384 with leucine, a substitution at position 385 with leucine, a substitution at position 386 with leucine, a substitution at position 387 with leucine, a substitution at position 388 with leucine, a substitution at position 389 with proline, a substitution at position 390 with proline, a substitution at position 391 with serine, a substitution at position 392 with aspartic acid, a substitution at position 393 with leucine, a substitution at position 394 with leucine, a substitution at position 395 with leucine, a substitution at position 396 with leucine, a substitution at position 397 with leucine, a substitution at position 398 with leucine, a substitution at position 399 with proline, a substitution at position 399 with proline, a substitution at position 400 with proline, a substitution at position 401 with proline, a substitution at position 402 with proline, a substitution at position 403 with proline, a substitution at position 404 with proline, a substitution at position 405 These modifications include a substitution with arginine, aspartic acid, or serine at position 5, a substitution with threonine or proline at position 386, a substitution with arginine or proline at position 387, a substitution with proline, asparagine, or serine at position 389, a substitution with methionine or threonine at position 428, a substitution with tyrosine or phenylalanine at position 434, a substitution with histidine, arginine, lysine, or serine at position 433, and / or a substitution with 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.

[0278] 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.

[0279] 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 specific 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 this 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 (see Kabat et al., "Sequences of Proteins of Immunological Interest," 5th 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.

[0280] 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 Publication 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). The substitution(s) 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 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, 245R, 247A, 247D, 247E, 247F, 247M, 247N, 247Q, 247R, 247S, 247T, 247W, 247Y, 248F, 248P, 248Q, 248W, 249L, 24 9M, 249N, 249P, 249Y, 251H, 251I, 251W, 254D, 254E, 254F, 254G, 254H, 254I, 254K, 254L, 254M, 254N, 254P, 254Q, 254R, 254V, 254W, 254Y, 255K, 255N, 256H, 256I, 256K, 256L, 256V, 256W, 256Y, 257A, 257I, 257M, 257N, 257S, 25 8D, 260S, 262L, 264S, 265K, 265S, 267H, 267I, 267K, 268K, 269N, 269Q, 271T, 272H, 272K, 272L, 272R, 279A, 279D, 279F, 279G, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R, 279S, 279T, 279W, 279Y, 280T, 283F, 283G, 283H, 28 3I, 283K, 283L, 283M, 283P, 283R, 283T, 283W, 283Y, 285N, 286F, 288N, 288P, 292E, 292F, 292G, 292I, 292L, 293S,293V, 301W, 304E, 307E, 307M, 312P, 315F, 315K, 315L, 315P, 315R, 316F, 316K, 317P, 317T, 318N, 318P, 318T, 332F, 332G, 332L, 332M, 332S, 332V, 332W, 339D, 339E, 339F, 339G, 339H, 339I, 339K, 339L, 339M, 339N, 339Q, 339R, 339S, 339W, 339Y, 341D, 341E, 341F, 341H, 341I, 341K, 341L, 341M, 3 41N, 341P, 341Q, 341R, 341S, 341T, 341V, 341W, 341Y, 343A, 343D, 343E, 3 43F, 343G, 343H, 343I, 343K, 343L, 343M, 343N, 343Q, 343R, 343S, 343T, 34 3V, 343W, 343Y, 373D, 373E, 373F, 373G, 373H, 373I, 373K, 373L, 373M, 37 3N, 373Q, 373R, 373S, 373T, 373V, 373W, 375R, 376E, 376F, 376G, 376H, 376 I, 376L, 376M, 376N, 376P, 376Q, 376R, 376S, 376T, 376V, 376W, 376Y, 377 G, 377K, 377P, 378N, 379N, 379Q, 379S, 379T, 380D, 380N, 380S, 380T, 382D , 382F, 382H, 382I, 382K, 382L, 382M, 382N, 382P, 382Q, 382R, 382S, 382T , 382V, 382W, 382Y, 385E, 385P, 386K, 423N, 424H, 424M, 424V, 426D, 426L, 427N, 429A, 429F, 429M, 430A, 430D, 430F, 430G, 430H, 430I, 430K, 430L, 430M, 430N, 430P, 430Q, 430R, 430S, 430T, 430V, 430W, 430Y, 431H, 431K, 431P, 432R, 432S, 438G, 438K, 438L, 438T, 438W, 439E, 439H, 439Q, 440D, 440E, 440F, 440G, 440H, 440I, 440K, 440L, 440M, 440Q, 440T, 440V or 442K. As 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 substitution(s).

[0281] 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, 274, 275, 276, 278, 280, 281, 283, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 349, 350, 351, 3 Included are variant Fc regions comprising amino acid substitutions at one or more of positions 86, 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 a specific embodiment, 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, F275 and at least one amino acid substitution selected from the group consisting of W, 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 selected from the group consisting of 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 / I3 32Q, S239N / I332D, S239N / I332E, S239Q / I332D, A330Y / I332E, V264I / A330Y / I332E, A330L / I332E, V264I / A330L / I332E, L234E, L234Y, L234I , L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T, L32 8D / I332E, L328V / I332E, L328T / I332E, L328I / I332E, S239E / V264I / I 332E, S239Q / V264I / I332E, S239E / V264I / A330Y / I332E, S239D / A330Y / I332E, S239N / A330Y / I332E, S239D / A330L / I332E, S239N / A330L / I332 E. I332E, S239D / I332E / A330I, P230A, P230A / E233D / I332E, E272Y, K274 T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S32 In a more specific embodiment, the variant Fc region comprises at least one amino acid substitution selected from the group consisting of N297D / I332E, ...F241Y / F243Y / V262T / V264T / N297D / I332E, S239D / N297D / I332E, S239E / N297D / I 332E, S239D / D265Y / N297D / I332E, S239D / D265H / N297D / I332E, V264E / N297D / I3 In a specific embodiment, the variant Fc region comprises an amino acid substitution at position 332 (using the numbering of the EU index, 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.

[0282] Further examples include the following amino acid substitutions: 224N / Y, 225A, 228L, 230S, 239P, 240A, 241L, 243S / L / G / H / I, 244L, 246E, 247L / A, 252T, 254T / P, 258K, 261Y, 265V, 266A, 267G / N, 268N, 269K / G, 273A, 276D, 278H, 279M, 280N, 283G, 285R, 288R, 289A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 305A, 306I / F, 311R, 312N, 315D / K / S, 320R, 322E, 323A, 324T, 325S, 326E / R, 332T, 333D / G, 335I, 338R, 339T, 340Q, 341E, 342R, 344Q, 347R, 351S, 35 2A, 354A, 355W, 356G, 358T, 361D / Y, 362L, 364C, 365Q / P, 370R, 372L, 377V, 378T, 383N, 389S, 390D, 391C , 393A, 394A, 399G, 404S, 408G, 409R, 411I, 412A, 414M, 421S, 422I, 426F / P, 428T, 430K, 431S, 432P, 433 and variant Fc regions comprising one or more of the following substitutions: P, 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) F 243S, P247L, D265V, V266A, S383N, and T411I; (5) H224N, F243L, T393A, and H433P; (6) V240A, S267G, G341E, and E356G; (7) M252T, P291L, P352A, R355W, N390D, S408G, S426F, and A431S;(8) P228L, T289A, 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 L36 5P; (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, N31 and (23) variant Fc regions comprising or consisting of: 5S, F372L, A378T, N390D, Y391C, F404S, E430K, L432P, and K447E; and (24) 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).

[0283] Another specific example of an Fc variant comprises the sequence of SEQ ID NO: 155, 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 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.

[0284] 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.

[0285] 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 (apparent volume into which a drug distributes and reaches equilibrium between plasma and surrounding tissues immediately after intravenous injection), concentration (initial or steady-state concentration of drug in plasma), elimination rate constant (rate at which a drug is eliminated from the body), elimination rate (rate of infusion required to balance elimination), area under the curve (AUC or exposure; integral of the concentration-time curve after a single dose or at steady state), clearance (volume of plasma cleared 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.

[0286] 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 in 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.

[0287] 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 at least about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours, about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90 hours, about 96 hours, about 120 hours, or about 144 hours or more, 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 rat, mouse, monkey, or human).

[0288] 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.

[0289] 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.

[0290] Peptide Linker In certain embodiments, a peptide linker sequence may be used to separate the HRS polypeptide(s) and the Fc region(s) by a distance sufficient to ensure that each polypeptide folds into its desired secondary and tertiary structure. Such peptide linker sequences may be incorporated into the fusion protein using standard techniques well known in the art.

[0291] 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 can interact with functional epitopes on the first and second polypeptides; (3) physiological stability; and (4) the absence of hydrophobic or charged residues, or other characteristics, that can react with the functional epitopes of the polypeptides. See, e.g., George and Heringa, J. Protein Eng. 15:871-879, 2002.

[0292] Linker sequences generally can 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.

[0293] Peptide linkers can use any one or more naturally occurring amino acids, non-naturally occurring amino acids(s), amino acid analogs, and / or amino acid mimetics 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.

[0294] 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: 200), [GGSG] x (SEQ ID NO: 201), [GGGS] x (SEQ ID NO: 202), [GGGGS] x (SEQ ID NO: 203), [GN] x , [GGN] x , [GNN] x , [GNGN] x (SEQ ID NO: 204), [GGNG] x (SEQ ID NO: 205), [GGGN] x (SEQ ID NO: 206), [GGGGN] x (SEQ ID NO: 207) 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 skilled in the art.

[0295] 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-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 208); Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 209); Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly ly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 210); 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: 211); and Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Arg-(SEQ ID NO: 212).

[0296] Further non-limiting examples of linker peptides include DGGGS (SEQ ID NO: 213); TGEKP (SEQ ID NO: 214) (see, e.g., Liu et al., PNAS. 94:5525-5530, 1997); GGRR (SEQ ID NO: 215) (Pomerantz et al. 1995);(GGGGS) n(SEQ ID NO: 203) (Kim et al., PNAS. 93:1156-1160, 1996); EGKSSGSGSESKVD (SEQ ID NO: 216) (Chaudhary et al., PNAS. 87:1066-1070, 1990); KESGSVSSEQLAQFRSLD (SEQ ID NO: 217) (Bird et al., Science. 242:423-426, 1988), GGRRGGGS (SEQ ID NO: 218); LQRDGERP (SEQ ID NO: 219); LRQKDGGGSERP (SEQ ID NO: 220); LRQKd(GGGS)2ERP (SEQ ID NO: 221). In a specific embodiment, the linker sequence comprises a Gly3 linker sequence, which includes 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 and Berg, PNAS. 90:2256-2260, 1993; and PNAS. 91:11099-11103, 1994), or by phage display methods.

[0297] 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 Fc region(s) 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.

[0298] 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 (see Donnelly et al., J. Gen. Virol. 82:1027-1041, 2001). Exemplary 2A sites include the following sequences: LLNFDLLKLAGDVESNPGP (SEQ ID NO: 222); TLNFDLLKLAGDVESNPGP (SEQ ID NO: 223); LLKLAGDVESNPGP (SEQ ID NO: 224); NFDLLKLAGDVESNPGP (SEQ ID NO: 225); QLLNFDLLKLAGDVESNPGP (SEQ ID NO: 226); APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 227); VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT (SEQ ID NO: 228); LNFDLLKLAGDVESNPGP (SEQ ID NO: 229); LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 230); and EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 231). In one embodiment, 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.

[0299] Suitable protease cleavage sites and self-cleaving peptides are known to those of skill in the art (see, e.g., Ryan et al., J. Gener. Virol. 78:699-722, 1997; and Scymczak et al., Nature Biotech. 5:589-594, 2004). Exemplary protease cleavage sites include, but are not limited to, 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: 232), e.g., ENLYFQG (SEQ ID NO: 233) and ENLYFQS (SEQ ID NO: 234), where X represents any amino acid (TEV cleavage occurs between Q and G or between Q and S).

[0300] Further examples of enzymatically degradable linkers suitable for use in certain embodiments of the present invention 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:235), -Gly-Gly-Arg-, -Gly-Arg-Gly-Asp-Asn-Pro-(SEQ ID NO:236), -Gly-Arg-Gly-Asp-Ser-(SEQ ID NO:237), -Gly-Arg-Gly-Asp-Ser-Pro-Lys-(SEQ ID NO:238), -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: 239), -Ala-Ala-Pro-Leu- (SEQ ID NO: 240), -Ala-Ala-Pro-Phe- (SEQ ID NO: 241), -Ala-Ala-Pro-Ala- (SEQ ID NO: 242) and -Ala-Tyr-Leu-Val- (SEQ ID NO: 243).

[0301] 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: 244), -Gly-Pro-, Leu-Gly-Pro-Z- (SEQ ID NO: 245), -Gly-Pro-Ile-Gly-Pro-Z- (SEQ ID NO: 246), and -Ala-Pro-Gly-Leu-Z- (SEQ ID NO: 247), where Y and Z are amino acids. Illustrative examples of collagenase-cleavable amino acid sequences include, but are not limited to, -Pro-Leu-Gly-Pro-D-Arg-Z- (SEQ ID NO: 248), -Pro-Leu-Gly-Leu-Leu-Gly-Z- (SEQ ID NO: 249), -Pro-Gln-Gly-Ile-Ala-Gly-Trp- (SEQ ID NO: 250), -Pro-Leu-Gly-Cys(Me)-His- (SEQ ID NO: 251), -Pro-Leu-Gly-Leu-Tyr-Ala- (SEQ ID NO: 252), -Pro-Leu-Ala-Leu-Trp-Ala-Arg- (SEQ ID NO: 253), and -Pro-Leu-Ala-Tyr-Trp-Ala-Arg- (SEQ ID NO: 254), 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: 255); one example of a gelatinase-cleavable amino acid sequence is -Pro-Leu-Gly-Met-Tyr-Ser-Arg- (SEQ ID NO: 256).

[0302] Enzymatically degradable linkers suitable for use in certain embodiments of the present invention 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: 257) and -Gly-Ser-Asp-Lys-Pro- (SEQ ID NO: 258).

[0303] Enzymatically degradable linkers suitable for use in certain embodiments of the present invention also include amino acid sequences that can be degraded by cathepsin B, such as Val-Cit, Ala-Leu-Ala-Leu- (SEQ ID NO: 259), Gly-Phe-Leu-Gly- (SEQ ID NO: 260), and Phe-Lys.

[0304] 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.

[0305] 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.

[0306] Methods for Use Embodiments of the present invention relate to the discovery that Fc region-histidyl-tRNA synthetase (HRS-Fc) conjugate polypeptides, as well as fragments and variants thereof, provide improved methods for modulating inflammatory responses in a variety of useful ways, both in vitro and in vivo. Thus, the compositions of the present invention may be useful as immunomodulators for treating a wide range of pro-inflammatory, inflammatory, and / or autoimmune indications, including inflammatory responses, chronic inflammation, acute inflammation, and immune disorders, by modulating cells that mediate such inflammatory and / or autoimmune diseases, conditions, and disorders, either directly or indirectly. The usefulness of the compositions of the present invention as immunomodulators can be monitored using any of several art-known and available techniques, including, for example, migration assays (e.g., using leukocytes or lymphocytes), cytokine production assays, or cell viability or cell differentiation assays (e.g., using B cells, T cells, monocytes, or NK cells).

[0307] "Inflammation" generally refers to the biological response of tissues to harmful stimuli, such as pathogens, damaged cells (e.g., wounds), and irritants. The term "inflammatory response" refers to specific mechanisms by which inflammation is achieved and regulated, including, by way of example only, immune cell activation or migration, migration, autoimmunity and autoimmune diseases, cytokine production, kinin release, vasodilation, including fibrinolysis and coagulation, among others described herein and known in the art. Ideally, inflammation is a defensive attempt by the body to both remove the injurious stimulus and initiate the healing process for the affected tissue(s). In the absence of inflammation, wounds and infections would never heal, creating a situation in which the progressive destruction of tissue threatens survival. On the other hand, excessive or chronic inflammation can be associated with various diseases, such as hay fever, atherosclerosis, and rheumatoid arthritis, among others described herein and known in the art.

[0308] The clinical manifestations of chronic inflammation depend on the duration of the disease, the inflammatory lesion, the cause, and the affected anatomical area (see, e.g., Kumar et al., Robbins Basic Pathology - 8th Edition, 2009, Elsevier, London; Miller, LM, Pathology Lecture Notes, Atlantic Veterinary College, Charlottetown, PEI, Canada). Chronic inflammation is associated with a variety of pathological conditions or diseases, including, for example, allergies, Alzheimer's disease, anemia, aortic stenosis, arthritis, such as rheumatoid arthritis and osteoarthritis, cancer, congestive heart failure, fibromyalgia, fibrosis, heart attack, kidney failure, lupus, pancreatitis, stroke, surgical complications, inflammatory lung disease, inflammatory bowel disease, including Crohn's disease (CD) and ulcerative colitis (UC), atherosclerosis, neurological disorders, diabetes, metabolic disorders, obesity, and psoriasis, among others described herein and known in the art. Many other chronic diseases may also include an inflammatory component and thus may be treated with the HRS-Fc conjugates of the present invention, including, for example, muscular dystrophy and rhabdomyolysis. Thus, the HRS-Fc conjugates may be used to treat or manage chronic inflammation, to modulate any one or more of the individual chronic inflammatory responses, or to treat any one or more diseases or conditions associated with chronic inflammation.

[0309] Certain specific inflammatory responses include cytokine production and activity or related pathways. For example, certain exemplary embodiments relate to modulating cell signaling via nuclear factor-kB (NF-kB), for example, by increasing the downstream activity of this transcription factor. In certain cases, an increase in NF-kB activity can lead to an increase in cytokine signaling or activity, for example, pro-inflammatory cytokines (e.g., TNF-alpha or beta) and anti-inflammatory cytokines (e.g., IL-10).

[0310] Criteria for evaluating the signs and symptoms of inflammatory and other conditions, including for purposes of making differential diagnoses and also for monitoring treatment, such as determining whether a therapeutically effective dose has been administered over the course of treatment by determining improvement according to accepted clinical criteria, will be apparent to those skilled in the art and can be found, for example, in Berkow et al., eds., The Merck Manual, 16th ed., Merck and Co., Rahway, NJ, 1992; Goodman et al., eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., Pergamon Press, Inc., Elmsford, NY, (2001); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd ed., ADIS Press, Ltd., Williams and Wilkins, Baltimore, MD. (1987); Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985); Osolci et al., eds., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990); Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, CT (1992).

[0311] Also included are methods for modulating immune responses, such as innate or adaptive immune responses, by using any of the HRS-Fc conjugates described herein.As used herein, the term "immune response" includes measurable or observable reactions to antigens, vaccine compositions, or immunomodulatory molecules mediated by one or more cells of the immune system.Immune responses typically begin with the binding of antigens or immunomodulatory molecules to immune system cells.Reactions to antigens or immunomodulatory molecules can be mediated by many cell types, including the cells that initially bind to antigens or immunomodulatory molecules and cells involved in mediating innate immune responses, humoral immune responses, and cell-mediated immune responses.

[0312] Also included are methods of treating immune diseases. Exemplary immune system diseases, disorders, or conditions that can be treated according to the present invention include, but are not limited to, primary immunodeficiency, immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplantation (e.g., recent bone marrow transplant in an adult or child), chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic inflammatory demyelinating polyneuropathy, post-transfusion purpura, and the like.

[0313] Additionally, additional diseases, disorders, and conditions that may be treated with any of the HRS-Fc conjugates described herein include Guillain-Barré syndrome, anemia (e.g., anemia associated with parvovirus B19, patients with stable multiple myeloma who are at high risk for infection (e.g., recurrent infections), autoimmune hemolytic anemia (e.g., warm autoimmune hemolytic anemia), thrombocytopenia (e.g., neonatal thrombocytopenia) and immune-mediated neutropenia), transplantation (e.g., cytomegalovirus (CMV)-negative recipients of CMV-positive organs), hypogammaglobulinemia (e.g., hypogammaglobulinemic newborns with risk factors for infection or morbidity), epilepsy (e.g., refractory epilepsy), systemic vasculitis syndromes, myasthenia gravis (e.g., decompensation in myasthenia gravis), dermatomyositis, and polymyositis.

[0314] Additional autoimmune diseases, disorders, and conditions that may be treated with any of the HRS-Fc conjugates described herein include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, autoimmune cytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), multiple sclerosis, neuritis, uveitis ophthalmia, polyendocrinopathies, purpura (e.g., Henoch-Schoenlein purpura), Reiter's disease, stiff-man syndrome, autoimmune pulmonary inflammation, Guillain-Barré syndrome, insulin-dependent diabetes mellitus, and autoimmune inflammatory eye disease.

[0315] Hypothyroidism, including Hashimoto's thyroiditis and thyroiditis characterized by, for example, cell-mediated and humoral thyroid cytotoxicity; SLE (e.g., often characterized by circulating and locally generated immune complexes); Goodpasture's syndrome (e.g., often characterized by anti-basement membrane antibodies); pemphigus (e.g., often characterized by epithelial acantholytic antibodies); receptor autoimmunity, such as Graves' disease (e.g., often characterized by antibodies to the thyroid-stimulating hormone receptor; myasthenia gravis (e.g., often characterized by acetylcholine receptor antibodies); insulin resistance (e.g., often characterized by insulin receptor antibodies); autoimmune hemolytic anemia (e.g., often characterized by phagocytosis of antibody-sensitized red blood cells); and autoimmune thrombocytopenic purpura (e.g., often characterized by phagocytosis of antibody-sensitized platelets).

[0316] Additional autoimmune diseases, disorders, or conditions that may be treated with any of the HRS-Fc conjugates described herein include rheumatoid arthritis (e.g., often characterized by immune complexes in the joints); scleroderma with anti-collagen antibodies (e.g., often characterized by nucleolar and other nuclear antibodies); mixed connective tissue disease (e.g., often characterized by antibodies to extractable nuclear antigens, such as ribonucleoproteins); polymyositis / dermatomyositis (e.g., often characterized by non-histone antinuclear antibodies); pernicious anemia (e.g., often characterized by anti-parietal cell, anti-microsomal, and anti-intrinsic factor antibodies); idiopathic Addison's disease (e.g., often characterized by humoral and cell-mediated adrenal cytotoxicity); infertility (e.g., anti-sperm glomerulonephritis (e.g., often characterized by glomerular basement membrane antibodies or immune complexes); primary glomerulonephritis, due to IgA nephropathy; bullous pemphigoid (e.g., often characterized by IgG and complement at the basement membrane); Sjogren's syndrome (e.g., often characterized by multi-tissue antibodies and / or specific non-histone antinuclear antibodies (SS-B)); diabetes (e.g., often characterized by cell-mediated and humoral islet cell antibodies); and adrenergic drug resistance (e.g., often characterized by beta-adrenergic receptor antibodies), including adrenergic drug resistance associated with asthma or cystic fibrosis.

[0317] Still further autoimmune diseases, disorders, or conditions that may be treated with any of the HRS-Fc conjugates described herein include chronic active hepatitis (e.g., often characterized by smooth muscle antibodies); primary biliary cirrhosis (e.g., often characterized by antimitochondrial antibodies); other endocrine glandular deficiencies (e.g., in some cases characterized by specific tissue antibodies); vitiligo (e.g., often characterized by antimelanocyte antibodies); vasculitis (e.g., in some cases characterized by immunoglobulins and complement in the blood vessel walls and / or low serum complement); post-myocardial infarction state (e.g., often characterized by anti-myocardial antibodies); open heart surgery syndrome (e.g., often characterized by anti-myocardial antibodies); urticaria (e.g., often characterized by IgG and IgM antibodies to IgE); atopic dermatitis (e.g., often characterized by IgG and IgM antibodies to IgE); asthma (e.g., often characterized by IgG and IgM antibodies to IgE); inflammatory myopathies; and other inflammatory, granulomatous, degenerative and atrophic disorders.

[0318] Additional diseases and disorders that may be treated with any of the HRS-Fc conjugates described herein include those resulting from or associated with an imbalance of Th17 or other Th cell subtypes. Examples include psoriasis, psoriatic arthritis, atopic dermatitis (eczema), Barro concentric sclerosis, Schilder diffuse sclerosis, Marburg MS, IBD, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behçet's disease, indeterminate colitis, asthma, autoimmune myocarditis, endometriosis, adult-onset Still's syndrome (AOSD), Henoch-Schönlein purpura (HSP), Vogt-Koyanagi-Harada (VKH), periodontal disease, organ transplant failure, graft-versus-host disease, and Devic's disease (neuromyelitis optica).

[0319] In some aspects, the present invention includes a method for reducing muscle or lung inflammation associated with an autoimmune disease, comprising administering to a subject in need thereof a composition comprising any of the HRS-Fc conjugates described herein. Exemplary muscle inflammatory diseases and disorders include muscular dystrophy, exercise-induced muscle inflammation, inflammation associated with muscle injury or surgery, rhabdomyolysis, and related diseases and disorders described herein.

[0320] Methods for treating diseases associated with autoantibodies include administering to a subject in need thereof a therapeutic composition comprising any of the HRS-Fc conjugates described herein, wherein the HRS polypeptide comprises at least one epitope specifically recognized by the autoantibody.

[0321] Certain embodiments include a method of inducing tolerance to a histidyl-tRNA synthetase (HisRS) antigen, comprising administering to a subject a composition comprising any of the HRS-Fc conjugates described herein, wherein the HRS polypeptide comprises at least one epitope specifically recognized by an autoantibody, and wherein administration of the composition causes tolerization to the autoantigen.

[0322] Also included is a method for eliminating a set or subset of T cells involved in an autoimmune response to a histidyl-tRNA synthetase (HisRS) autoantigen, comprising administering to a subject a composition comprising any of the HRS-Fc conjugates described herein, wherein the HRS polypeptide comprises at least one epitope specifically recognized by an autoantibody or autoreactive T cells, and wherein administration of the composition causes clonal deletion of the autoreactive T cells.

[0323] In another embodiment, the invention includes a method of inducing anergy in T cells involved in an autoimmune response to a histidyl-tRNA synthetase (HRS) autoantigen, comprising administering to a subject a composition comprising any of the HRS-Fc conjugates described herein, wherein the HRS polypeptide comprises at least one epitope specifically recognized by an autoantibody or T cell, and wherein administration of the composition causes functional inactivation of the T cells involved in the autoimmune response.

[0324] In another embodiment, the invention provides a replacement therapy for treating a disease associated with histidyl-tRNA synthetase insufficiency, comprising administering to a subject in need thereof a therapeutic composition comprising any of the HRS-Fc conjugates described herein, wherein the HRS polypeptide functionally compensates for the histidyl-tRNA synthetase insufficiency.

[0325] In one embodiment of this replacement therapy, the histidyl-tRNA synthetase deficiency is caused by the presence of anti-Jo-1 antibodies. In one embodiment of this replacement therapy, the histidyl-tRNA synthetase deficiency is caused by a mutation in endogenous histidyl-tRNA synthetase that modulates the activity, expression, or cellular distribution of the endogenous histidyl-tRNA synthetase. In one embodiment, the histidyl-tRNA synthetase deficiency is associated with Perrault syndrome or Usher syndrome.

[0326] In any of these methods, the term "tolerance" refers to the persistent reduction or absence of immune responses to specific antigens in mammals, particularly humans. Tolerance is distinct from generalized immunosuppression, in which all immune responses or all specific classes of immune cells in the immune response, such as B cell-mediated immune responses, are reduced or eliminated. The development of tolerance can be routinely monitored by the absence or reduction of antibody concentrations against HRS polypeptides in the serum of a host subject after administration of a single or sequential dose of a therapeutic HRS-Fc conjugate. The development of tolerance is typically sufficient to reduce the symptoms of autoimmune disease in patients; for example, patients can be sufficiently improved to maintain normal activity in the absence or reduced amounts of common immunosuppressants such as corticosteroids.

[0327] In any of these methods and compositions, tolerance is typically long-lasting, meaning that tolerance has a duration of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months or more. Tolerance can result in selective B cell anergy or T cell anergy, or both.

[0328] In any of these methods, treatments, and therapeutic compositions, the term "disease associated with autoantibodies specific to histidyl-tRNA synthetase" refers to any disease or disorder in which antibodies to histidyl-tRNA synthetase are detected or detectable, regardless of whether other autoantibodies are also detected or whether they are thought to play a role in the progression or cause of the disease. Methods for detecting antibodies in patient samples can be performed by any standard procedure, including, for example, RIA, ELISA, immunoprecipitation, staining of tissues or cells (including transfected cells), antigen microarrays, mass spectrometry, specific neutralization assays, or one of several other methods known in the art for identifying desired antigen specificities. In some embodiments, antibody specificity can be further characterized by determining the antibody's ability to selectively bind to different splice variants and truncated or proteolytic forms of histidyl-tRNA synthetase. Relatively well-known human autoantibodies to histidyl-tRNA synthetase include, for example, antibodies against Jo-1.

[0329] In some embodiments of any of the claimed methods and compositions, the HRS polypeptide or HRS-Fc conjugate comprises an epitope derived from histidyl-tRNA synthetase that specifically cross-reacts with disease-associated autoantibodies against histidyl-tRNA synthetase. In some embodiments of any of the claimed methods and compositions, the HRS polypeptide or HRS-Fc conjugate comprises an epitope derived from histidyl-tRNA synthetase that specifically cross-reacts with disease-associated autoreactive T cells against histidyl-tRNA synthetase. In some embodiments of any of the claimed methods and compositions, the HRS polypeptide or HRS-Fc conjugate comprises an epitope that specifically cross-reacts with disease-associated autoantibodies against any other tRNA synthetase or non-tRNA synthetase autoantibodies.

[0330] In some embodiments of any of the claimed methods, the HRS polypeptide or HRS-Fc conjugate comprises an immunodominant epitope specifically recognized by a majority of antibodies from the sera of patients with a disease associated with autoantibodies to histidyl-tRNA synthetase. In some embodiments of any of the claimed methods, the HRS polypeptide or HRS-Fc conjugate comprises an immunodominant epitope specifically recognized by a majority of autoreactive T cells from the sera of patients with a disease associated with autoantibodies to histidyl-tRNA synthetase.

[0331] In some embodiments, the epitope is contained within the WHEP domain (approximately amino acids 1-43 of SEQ ID NO:1); the aminoacylation domain (approximately amino acids 54-398 of SEQ ID NO:1); or the anticodon binding domain (approximately amino acids 406-501 of SEQ ID NO:1) of the HRS polypeptide, or any combination thereof.

[0332] In some embodiments, the HRS polypeptide does not contain an epitope that specifically cross-reacts with disease-associated autoantibodies to histidyl-tRNA synthetase. In some embodiments, the HRS polypeptide is at most about 1 x 10 -7 In some embodiments, the HRS polypeptide does not significantly compete for binding of disease-associated autoantibodies to histidyl-tRNA synthetase in a competitive ELISA at concentrations up to about 5×10 -7 In some embodiments, the HRS polypeptide does not significantly compete for binding of disease-associated autoantibodies to histidyl-tRNA synthetase in a competitive ELISA at concentrations up to about 1×10 -6 Concentrations up to M do not significantly compete for binding of disease-associated autoantibodies to histidyl-tRNA synthetase in competitive ELISA.

[0333] Thus, in some embodiments, the HRS polypeptide has a lower affinity for disease-associated autoantibodies than wild-type histidyl-tRNA synthetase (SEQ ID NO: 1), as measured by competitive ELISA. In some embodiments, the HRS polypeptide has an apparent affinity for disease-associated autoantibodies that is at least about 10-fold less, or at least about 20-fold less, or at least about 50-fold less, or at least about 100-fold less than the affinity of disease-associated autoantibodies for wild-type human histidyl-tRNA synthetase (SEQ ID NO: 1). In one aspect, the autoantibody against histidyl-tRNA synthetase is an antibody against the Jo-1 antigen.

[0334] Examples of diseases associated with autoantibodies specific for histidyl-tRNA synthetase (as well as diseases associated with histidyl-tRNA synthetase insufficiency) include, but are not limited to, autoimmune diseases, inflammatory diseases, and inflammatory myopathies, including idiopathic inflammatory myopathy, polymyositis, statin-induced myopathy, dermatomyositis, interstitial lung disease (and other pulmonary fibrotic conditions) and related disorders, e.g., polymyositis-scleroderma overlap and inclusion body myositis (IBM), and conditions such as those found in antisynthetase syndromes, including interstitial lung disease, arthritis, esophageal hypoperistalsis, cardiovascular disease and other vascular findings, e.g., Raynaud's phenomenon; other examples of diseases associated with histidyl-tRNA synthetase insufficiency include genetic disorders resulting in insufficiency of active histidyl-tRNA synthetase, including Usher syndrome and Perot syndrome.

[0335] Polymyositis affects skeletal muscles (involved in movement) on both sides of the body. Polymyositis is rarely seen in people under 18 years of age; most cases occur in people between 31 and 60 years of age. In addition to the symptoms listed above, progressive muscle weakness leads to difficulty swallowing, speaking, rising from a seated position, climbing stairs, lifting objects, or lifting one's arms. People with polymyositis may also experience arthritis, shortness of breath, and cardiac arrhythmias. Polymyositis is often associated with antibodies against HisRS-containing synthetases, resulting in immune cell infiltration into damaged muscle cells. Therefore, HRS-Fc conjugates can be used to reduce immune cell activation and infiltration and treat polymyositis.

[0336] Dermatomyositis is characterized by a skin rash that precedes or accompanies progressive muscle weakness. This rash appears patchy, has purple and red discolorations, and characteristically occurs on the eyelids and on muscles used to extend or straighten joints, including the knuckles, elbows, knees, and toes. The red rash may also occur on the face, neck, shoulders, upper thorax, back, and other locations, and swelling may be present in the affected areas. The rash sometimes occurs without obvious muscle involvement. Adults with dermatomyositis may experience weight loss or low-grade fever, have inflamed lungs, and be sensitive to light. Unlike polymyositis, adult dermatomyositis may be associated with tumors of the breast, lungs, female reproductive tract, or intestine. Children and adults with dermatomyositis may develop calcium deposits (called calcinosis) that appear as hard bumps under the skin or in the muscles. Calcinosis most often occurs 1 to 3 years after disease onset, but may occur many years later. These deposits are more common in childhood dermatomyositis than in dermatomyositis that begins in adults. Dermatomyositis may be associated with collagen-vascular disease or autoimmune disorders.

[0337] In some cases of polymyositis and dermatomyositis, distal muscles (those located away from the trunk, such as those in the forearm and around the ankles and wrists) may be affected as the disease progresses. Polymyositis and dermatomyositis may be associated with collagen-vascular disease or autoimmune disease, which causes immune cell infiltration into damaged muscle cells. Therefore, HRS-Fc conjugates can be used to reduce immune cell activation and infiltration and treat dermatomyositis.

[0338] Inclusion body myositis (IBM) is characterized by progressive muscle weakness and wasting. The onset of muscle weakness is generally gradual (over months or years) and affects both proximal and distal muscles. Muscle weakness may affect only one side of the body. Small holes, called hollows, are occasionally seen in the cells of affected muscle fibers. Falling and stumbling are usually the first noticeable symptoms of IBM. For some individuals, the disorder begins with weakness in the wrists and fingers, causing difficulty pinching, buttoning, and gripping objects. Weakness of the wrist and finger muscles and atrophy (thinning or loss of muscle volume) of the forearm muscles and quadriceps muscles of the legs may be present. Difficulty swallowing occurs in approximately half of IBM cases. Symptoms of the disease usually begin after age 50, although the disease can occur earlier. Unlike polymyositis and dermatomyositis, IBM occurs more frequently in men than in women. Like other muscular dystrophies, IBM also causes progressive immune cell infiltration into damaged muscle cells. Therefore, HRS-Fc conjugates can be used to reduce immune cell activation and infiltration and treat IBM.

[0339] Juvenile myositis shares some similarities with adult dermatomyositis and polymyositis. Juvenile myositis typically affects children between the ages of 2 and 15, and its symptoms include proximal muscle weakness and inflammation, edema (abnormal collection of fluid in body tissues, causing swelling), muscle pain, fatigue, skin rash, abdominal pain, fever, and contractures (chronic shortening of muscles or tendons around joints, caused by inflammation in the tendons, preventing the joint from moving freely). Children with juvenile myositis may also have difficulty swallowing and breathing, and the heart may be affected. Approximately 20 to 30 percent of children with juvenile dermatomyositis develop calcinosis. Affected children may not have higher-than-normal levels of the muscle enzyme creatine kinase in their blood, but they do have higher-than-normal levels of other muscle enzymes. Juvenile myositis also results in progressive immune cell infiltration into damaged muscle cells. Therefore, HRS-Fc conjugates can be used to reduce immune cell activation and infiltration and treat juvenile myositis.

[0340] Statin-induced myopathy is associated with the long-term use of statins, which act through the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Although generally well tolerated, these medications have been described as inducers of muscle toxicity. More recently, there have been reports of patients in whom statin myopathy persisted even after drug cessation, which has been hypothesized to have an autoimmune etiology. The benefits of statins are undisputed in reducing the risk of coronary heart disease and the progression of coronary atherosclerosis. Nevertheless, associated complications can be life-threatening. It is estimated that more than 38 million people in the United States are currently taking statins; of these, up to 7% (>2.6 million) are predicted to develop muscle symptoms, and of these, up to 0.5% (>190,000) potentially progress to develop life-threatening myopathy.

[0341] All statins can cause muscle problems, and the risk increases with their lipophilicity, cholesterol-lowering capacity, and dosage. Cerivastatin, in particular, has been implicated as having a higher risk and has been withdrawn from the US market. Of the remaining statins, atorvastatin and simvastatin have higher muscle toxicity rates. Other non-statin lipid-lowering agents, such as niacin and fibrates, also carry a risk of muscle problems, especially when combined with statins. While it is impossible to predict which patients will develop statin-induced muscle problems, previous muscle problems may be a risk factor and should be considered when initiating statin treatment. A family history of myopathy is relevant to whether a patient may be a carrier of a genetic myopathy, as this may not be masked by the added stress of statin treatment. Other risk factors may include age over 80 years, underweight, female gender, hypothyroidism, certain genetic defects and Asian ancestry, and concomitant use of certain medications including calcium channel blockers, macrolide antibiotics, omeprazole, amiodarone, azole antifungals, histamine H2 receptor antagonists, nefazodone, cyclosporine, HIV protease inhibitors, warfarin, and grapefruit juice.

[0342] The most common muscle symptom caused by statins is muscle pain or muscle pain, which occurs in approximately 7% of statin users. Muscle pain can range from mild to severe and is often aggravated by muscle activity. For example, in patients with hypercholesterolemia and recent myocardial infarction, continued statin treatment may be appropriate if the symptoms are tolerable and there is an indication for intensive statin treatment.

[0343] Although baseline creatine kinase (CK) levels are not uniformly recommended by statin treatment authorities before initiation, CK levels can provide very useful information on whether muscle symptoms will subsequently develop. Muscle weakness may also occur, often fatiguing in quality and combined with pain and elevated CK levels. As with most myopathies, weakness is most pronounced proximal. Rare episodes of rhabdomyolysis have also occurred with statin treatment; these are much less frequent but can potentially be fatal. Muscle histological changes most typical of statin myopathy are cytochrome oxidase-negative fibers, increased lipid content, and ragged-red fibers. Autoimmune necrotizing myopathy is a rare form of statin myopathy. In these patients, discontinuing statin medication does not lead to recovery, even after several months of drug discontinuation. Patients have predominantly proximal, often painless weakness.

[0344] Diagnosis is based on the individual's medical history, physical examination, muscle strength test results, and blood samples showing elevated levels of various muscle enzymes and autoantibodies. Diagnostic tools include electromyography, which records the electrical activity that controls muscles during contraction and at rest, ultrasound, which searches for muscle inflammation, and magnetic resonance imaging, which reveals abnormal muscles and evaluates muscle disease. Muscle biopsies can be examined microscopically for signs of chronic inflammation, muscle fiber death, vascular deformation, or changes specific to the diagnosis of IBM. Thus, HRS-Fc conjugates can be used to reduce immune cell activation and infiltration into damaged muscles and treat statin-induced myopathy and rhabdomyolysis.

[0345] Interstitial lung disease (ILD) is a broad category of lung disease that includes more than 130 disorders characterized by lung scarring (i.e., "fibrosis") and / or inflammation. ILD accounts for 15 percent of cases seen by pulmonologists. ILD can arise from a variety of sources, ranging from other diseases to environmental factors. Some of the known causes of ILD include: connective tissue or autoimmune diseases, including, for example, scleroderma / progressive systemic sclerosis, lupus (systemic lupus erythematosus), rheumatoid arthritis, and polymyositis / dermatomyositis; and occupational and environmental exposures, including, for example, exposure to dust and certain gases, toxic substances, chemotherapy, and radiation therapy.

[0346] In ILD, tissue in the lungs becomes inflamed and / or scarred. The lung interstitium includes the areas in and around small blood vessels and alveoli (air sacs) where oxygen and carbon dioxide exchange occurs. Interstitial inflammation and scarring destroy this tissue, resulting in a decrease in the lungs' ability to extract oxygen from the air. Therefore, HRS-Fc conjugates can be used to reduce immune cell activation and infiltration into damaged lungs and treat ILD.

[0347] The progression of ILD varies from disease to disease and from person to person. Because interstitial lung disease disrupts the transport of oxygen and carbon dioxide in the lungs, its symptoms typically manifest as breathing problems. The two most common symptoms of ILD are shortness of breath with exertion and a non-productive cough.

[0348] Usher syndrome is the most common condition affecting both hearing and vision. The primary symptoms of Usher syndrome are hearing loss and retinitis pigmentosa (RP). RP causes night blindness and loss of peripheral vision (side vision) through progressive degeneration of the retina. As RP progresses, the visual field narrows until only central vision remains. Many people with Usher syndrome also have severe balance problems. Approximately 3-6 percent of all children with hearing loss and another 3-6 percent of children with hearing impairments have Usher syndrome. In developed countries such as the United States, approximately 4 babies per 100,000 births have Usher syndrome. Usher syndrome is inherited as an autosomal recessive trait. Several genetic loci, including histidyl t-RNA synthetase, have been associated with Usher syndrome (Puffenberger et al., (2012) PLoS ONE 7(1) e28936 doi:10.1371 / journal.pone.0028936).

[0349] There are three clinical types of Usher syndrome: types 1, 2, and 3. In the United States, types 1 and 2 are the most common types. Together, they account for approximately 90-95 percent of all cases in children with Usher syndrome.

[0350] Children with Usher syndrome type 1 are profoundly deaf from birth and have severe balance problems. Due to the balance problems associated with Usher syndrome type 1, children with the disorder have difficulty sitting without support and typically do not walk independently before the age of 18 months. These children usually begin to develop vision problems in early childhood, usually by the time they reach the age of 10. The vision problems most often begin with difficulty seeing at night but tend to progress quickly until the person is completely blind.

[0351] Children with Usher syndrome type 2 are born with moderate to severe hearing loss and normal balance. The severity of the hearing loss varies, but most of these children can benefit from hearing aids and are able to communicate verbally. Vision problems in Usher syndrome type 2 tend to progress more slowly than vision problems in type 1, and the onset of RP is often not apparent until the teenage years.

[0352] Children with Usher syndrome type 3 have normal hearing at birth. Most children with the disorder have normal to near-normal balance, although some may develop balance problems later in life. Hearing and vision deteriorate over time, but the rate at which they decline can vary from person to person, even within the same family. People with Usher syndrome type 3 may develop hearing loss by their teenage years, and they usually require hearing aids by mid- to late adulthood. Night blindness usually begins sometime during adolescence. Blind spots appear by late teenage years to early adulthood, and by mid-adulthood, the person is usually legally blind.

[0353] Perrault syndrome (PS) is characterized by ovarian dysgenesis in females with sensorineural hearing loss and, in some subjects, associated neurological abnormalities, including progressive cerebellar ataxia and intellectual disability. The exact prevalence of Perrault syndrome is unknown and is likely underdiagnosed, particularly in males, where hypogonadism is not a feature and symptoms remain undetected. The mean age at diagnosis is 22 years, following presentation of delayed puberty in females with sensorineural hearing loss. Hearing deficits were noted in all but one reported case (mean age at diagnosis: 8 years). Hearing loss is always sensorineural and bilateral, but the severity varies (from mild to severe), even among affected individuals from the same family. Ovarian dysgenesis has been reported in all female cases, whereas gonadal defects have not been detected in males. Amenorrhea is generally primary, although secondary amenorrhea has also been reported. Delayed growth (height below the 3rd percentile) has been reported in half of the reported cases. Although the exact frequency of neurological abnormalities is unknown, nine females and two males (aged 16–37 years) lacking neurological abnormalities have been reported. Neurological signs are progressive and generally manifest later in life, although delayed gait or frequent falls early in life have been noted in younger PS patients. Common neurological signs include ataxia, dyspraxia, limited extraocular movements, and polyneuropathy. Some cases with scoliosis have also been reported. PS is transmitted in an autosomal recessive manner, and mutations in mitochondrial histidyl-tRNA synthetase have recently been identified to cause ovarian dysgenesis and sensorineural hearing loss associated with Perot syndrome (Pierce et al., PNAS USA, Vol. 108(16), pp. 6543–6548, 2011).

[0354] Muscular dystrophies refer to a group of genetic disorders that result in a gradual decline in strength and muscle volume. All muscular dystrophies are characterized by muscle weakness driven by primary genetic defects in one or more muscle-specific genes. Furthermore, muscular dystrophies typically have a variable inflammatory component that drives muscle inflammation and ultimately enhances muscle tissue degeneration. Therefore, HRS-Fc conjugates can be used to reduce immune cell activation and infiltration into damaged muscles and treat muscular dystrophies. At least nine types of muscular dystrophies are generally recognized. In some embodiments, the muscular dystrophies are selected from Duchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, and congenital muscular dystrophy.

[0355] Duchenne muscular dystrophy (DMD): DMD affects young boys and causes progressive muscle weakness, usually beginning in the legs. It is the most severe form of muscular dystrophy. DMD occurs in approximately 1 in 3,500 male births, affecting approximately 8,000 boys and young men in the United States. A milder form occurs in a very small number of female carriers.

[0356] DMD is caused by mutations in the gene encoding the subsarcolemmal protein dystrophin, which functions within the dystrophin-associated glycoprotein complex (DGC), preventing the production of functional protein. The amount of dystrophin correlates with disease severity (i.e., the less dystrophin present, the more severe the phenotype). This DGC complex connects the intracellular cytoskeleton to the extracellular matrix. The DGC concentrates at the Z-lines of sarcomeres, providing force transmission across muscle fibers. Disruption of this connection creates membrane instability, ultimately leading to sarcolemmal rupture. Extracellular calcium influx alters molecular processes, such as muscle contraction, and activates proteolytic activity. Affected muscle fibers become necrotic or apoptotic, releasing mitogenic chemoattractants that initiate inflammatory processes. Cycles of degeneration and regeneration ultimately lead to irreversible muscle wasting and its replacement by fibrous and fatty tissue.

[0357] Boys with Duchenne muscular dystrophy usually begin showing symptoms during preschool. The legs are affected first, making walking difficult and causing balance problems. Most patients walk 3 to 6 months later than expected and have difficulty running. Contractures (permanent muscle tightness), most severe in the calf muscles, usually begin by age 5 or 6. Frequent falls and fractures commonly begin at this age. Climbing stairs and standing unaided can become impossible by age 9 or 10, and most boys use wheelchairs for mobility by age 12. Weakness of the trunk muscles around this age often leads to scoliosis (side-to-side curvature of the spine) and kyphosis (curvature of the front to back).

[0358] One of the most serious weaknesses of DMD is weakness of the diaphragm, a sheet of muscle in the upper abdomen that performs the primary functions of breathing and coughing. Diaphragm weakness leads to reduced energy and stamina, as well as increased lung infections due to the inability to cough effectively. Young men with DMD can survive into their 20s and beyond, provided they have mechanical ventilator support and good respiratory hygiene.

[0359] In some embodiments, subjects with DMD are characterized by one or more of the following, as measured, for example, by muscle biopsy: a positive Gower's sign, reflecting impairment of the lower limb muscles; high levels of creatine kinase (CPK-MM) in the blood; a genetic error in the Xp21 gene; or reduced levels of dystrophin.

[0360] HRS-Fc conjugates can be used alone or in combination with other therapies, such as antisense oligonucleotides (e.g., exon skipping therapies such as Eteplirsen), corticosteroids, beta2-agonists, physical therapy, respiratory support, stem cell therapy, and gene replacement therapy, to treat DMD. In some embodiments, administration of the HRS-Fc conjugate results in a statistically significant improvement in the 6-minute walk test.

[0361] Becker muscular dystrophy (BMD): BMD affects older boys and young men and follows a milder course than DMD. BMD occurs in approximately 1 in 30,000 male births. Becker muscular dystrophy is a less severe variant of Duchenne muscular dystrophy and is caused by the production of a truncated, but partially functional, form of dystrophin.

[0362] Symptoms of BMD usually appear from late childhood to early adulthood. Symptom progression may parallel that of DMD, but these symptoms are usually milder and their course is more variable. Scoliosis may occur, but is usually milder and progresses more slowly. Cardiac muscle disease (cardiomyopathy) is more common in BMD. Problems may include irregular heartbeat (arrhythmia) and congestive heart failure. Symptoms may include fatigue, shortness of breath, chest pain, and dizziness. Respiratory weakness may also occur, leading to the need for mechanical ventilation. HRS-Fc conjugates can be used alone or in combination with other therapies to treat BMD.

[0363] Emery-Dreifuss muscular dystrophy (EDMD): EDMD affects young boys and causes contractures and weakness in the calves, weakness in the shoulders and upper arms, and problems with the pathways that carry electrical impulses to the heart to pump (cardiac conduction defects). There are three subtypes of Emery-Dreifuss muscular dystrophy, distinguishable by their pattern of inheritance: X-linked, autosomal dominant, and autosomal recessive. The X-linked form is the most common. Each type varies in prevalence and symptoms. The disease is caused by mutations in the LMNA gene or, more commonly, by mutations in the EMD gene. Both genes encode protein components of the nuclear envelope.

[0364] EDMD usually begins in early childhood, often with contractures preceding muscle weakness. Weakness primarily affects the shoulders and upper arms, along with the calf muscles, resulting in foot drop. Most men with EDMD survive into middle age, but defects in heart rhythm (heart block) can be fatal if not treated with a pacemaker. HRS-Fc conjugates can be used alone or in combination with other therapies to treat EDMD.

[0365] Limb-girdle muscular dystrophy (LGMD): LGMD begins in late childhood and early adulthood, affects both men and women, and causes weakness in the muscles around the hips and shoulders. It is the most variable of the muscular dystrophies, and several different forms of the disease are currently recognized. Many people suspected of having LGMD have likely been misdiagnosed in the past, making it difficult to estimate the prevalence of the disease. The number of affected people in the United States may be in the thousands.

[0366] Although there are at least half a dozen genes that cause different types of LGMD, two major clinical forms of LGMD are generally recognized: the severe childhood form, which is similar in appearance to DMD but is inherited as an autosomal recessive trait;

[0367] Limb-girdle muscular dystrophy type 2B (LGMD2B) is caused by loss-of-function mutations in the dysferlin gene. Dysferlin is primarily expressed in skeletal and cardiac muscle, but also in monocytes, macrophages, and other tissues, where it localizes to cytoplasmic vesicles and cell membranes. Dysferlin appears to be involved in membrane fusion and membrane trafficking, as well as repair processes. LGMD2B is a late-onset (teenage / young adult) muscle disease characterized by progressive, symmetric muscle weakness and a predominantly aggressive immune / inflammatory pathology. Muscle biopsies typically reveal myofiber degeneration / regeneration along with macrophage / macrophage activation markers (HLA-DR, HLA-ABC, CD86), CD8 + Cytotoxic T cells and CD4 + They exhibit significant inflammatory cell infiltration, primarily consisting of T cells. Therefore, HRS-Fc conjugates can be used to reduce immune cell activation and infiltration into injured muscles and treat limb-girdle muscular dystrophies.

[0368] Symptoms of adult-onset LGMD usually appear in a person's teens or twenties and are characterized by progressive weakness and wasting of the muscles closest to the trunk. Contractures may occur, and the ability to walk is usually lost about 20 years after onset. Some people with LGMD develop respiratory weakness that requires the use of a ventilator. Lifespan may be somewhat shortened (autosomal dominant forms usually occur in later life and progress more slowly).

[0369] Facioscapulohumeral muscular dystrophy (FSH): FSH, also known as Landouzy-Dejerine disease, begins in late childhood to early adulthood, affects both men and women, and causes weakness in the muscles of the face, shoulders, and upper arms. The buttocks and legs may also be affected. FSH occurs in approximately 1 in 20,000 people, affecting approximately 13,000 people in the United States.

[0370] FSH varies in severity and age of onset, even among members of the same family. Symptoms most commonly begin in the teens or early twenties, but onset can occur in infancy or childhood. Symptoms tend to be more severe in individuals with earlier onset. The disease is named for the areas of the body most severely affected by it: the muscles of the face (facio-), shoulders (scapulo-), and upper arms (humeral). The buttocks and legs may be affected as well. Children with FSH often develop partial or complete hearing loss.

[0371] Two defects are required for FSHD: the first is a deletion of the D4Z4 repeat, and the second is a "toxic gain-of-function" of the DUX4 gene. The first symptom noted is often difficulty lifting objects above the shoulders. Weakness may be greater on one side than the other. Shoulder weakness also causes the scapula to protrude backward, a condition called scapular winging. FSHD is associated with inflammatory infiltration in specific muscle groups; therefore, HRS-Fc conjugates can be used to reduce immune cell activation and infiltration into damaged muscles and treat FSHD.

[0372] Myotonic dystrophy: Myotonic dystrophy, also known as Steinert's disease, affects both men and women and causes generalized weakness initially seen in the face, feet, and hands. This is accompanied by an inability to relax the affected muscles (myotonia). Symptoms can begin at birth through adulthood. Myotonic muscular dystrophy type 1 (DM1) is the most common form of muscular dystrophy, affecting more than 30,000 people in the United States. It results from an expansion of a short (CTG) repeat in the DNA sequence of the DMPK (myotonic dystrophy protein kinase) gene. Myotonic muscular dystrophy type 2 (DM2) is significantly rarer and is the result of an expansion of a CCTG repeat in the ZNF9 (zinc finger protein 9) gene.

[0373] Symptoms of myotonic dystrophy include facial weakness and a slack mouth. Symptoms include drooping of the jaw, drooping eyelids (ptosis), and muscle wasting in the forearms and calves. People with this dystrophy have difficulty relaxing their grip, especially when objects are cold. Myotonic dystrophy affects the heart muscle, causing arrhythmias and heart block, as well as the muscles of the digestive system, resulting in movement disorders and constipation. Other body systems are affected as well: myotonic dystrophy can cause cataracts, retinal degeneration, low IQ, frontal hair loss, skin disorders, testicular atrophy, sleep apnea, and insulin resistance. An increased need or desire for sleep is common, as is decreased motivation. Severe disability affects most people with this form of dystrophy within 20 years of onset, but most do not require a wheelchair, even in old age. Thus, HRS-Fc conjugates can be used to treat myotonic dystrophy, for example, by reducing inflammation associated with muscle tissue, including skeletal muscle (e.g., quadriceps) and / or cardiac tissue, among other tissues.

[0374] Oculopharyngeal muscular dystrophy (OPMD): OPMD affects adults of both genders and causes weakness in the eye muscles and pharynx. It is most common among French-Canadian families in Quebec and Spanish-American families in the southwestern United States.

[0375] OPMD usually begins in a person's 30s or 40s and is accompanied by weakness in the muscles that control the eyes and pharynx. Symptoms include ptosis, difficulty swallowing (dysphagia), and progression of weakness to other muscles in the face, neck, and sometimes upper limbs. Difficulty swallowing can lead to aspiration or introduction of food or saliva into the airway. Pneumonia may follow. Therefore, HRS-Fc conjugates can be used to treat OPMD, for example, by reducing inflammation associated with muscle tissue.

[0376] Distal muscular dystrophy (DD): DD begins in middle age or later and causes weakness in the muscles of the feet and hands. It is most common in Sweden and rare elsewhere in the world. DD usually begins in the 20s or 30s and involves weakness in the hands, forearms, and lower legs. Difficulty with fine movements such as typing or buttoning can be the first symptom. Symptoms progress slowly, and the disease usually does not affect lifespan. Therefore, HRS-Fc conjugates can be used to treat DD by reducing muscle tissue inflammation and associated inflammation.

[0377] Congenital muscular dystrophy (CMD): CMD is present from birth, causes generalized weakness, and usually progresses slowly. A subtype called Fukuyama CMD is also associated with mental retardation. Both are rare; Fukuyama CMD is more common in Japan.

[0378] CMD is characterized by severe muscle weakness from birth, with infants exhibiting "floppiness" and very little voluntary movement. Nevertheless, children with CMD can learn to walk, with or without some type of assistive device, and survive into young adulthood or beyond. In contrast, children with Fukuyama-type CMD are barely able to walk and have severe mental retardation. Most children with this type of CMD die during childhood. Therefore, similar to other muscular dystrophies, HRS-Fc conjugates can be used to treat CMD, for example, by reducing inflammation associated with muscle tissue inflammation.

[0379] Cachexia: Cachexia (or wasting syndrome) is typically characterized by weight loss, muscle atrophy, fatigue, weakness, and marked loss of appetite in individuals who are not actively attempting to lose weight. The official definition of cachexia is weight loss that cannot be nutritionally reversed. Even when affected patients consume more calories, lean body mass is lost, indicating the presence of a primary pathology.

[0380] Cachexia is experienced by patients with cancer, AIDS, chronic obstructive pulmonary disease, multiple sclerosis, congestive heart failure, tuberculosis, familial amyloid polyneuropathy, mercury poisoning (acromelodynia), and hormone deficiencies, among other diseases.

[0381] Cachexia can also be a symptom of a variety of underlying disorders, including cancer, metabolic acidosis (i.e., decreased protein synthesis and increased protein catabolism), certain infectious diseases (e.g., tuberculosis, AIDS), chronic pancreatitis, autoimmune disorders, or addiction to amphetamines. Cachexia physically debilitates patients to the point of immobility due to loss of appetite, asthenia, and anemia, and typically responds poorly to standard treatments.

[0382] Approximately 50% of all cancer patients suffer from cachexia. Patients with upper gastrointestinal and pancreatic cancers most frequently develop cachectic symptoms. In addition to increasing morbidity and mortality, exacerbating chemotherapy side effects, and reducing quality of life, cachexia is considered the direct cause of death in a large proportion of cancer patients, ranging from 22% to 40% of patients. Symptoms of cancer cachexia include progressive weight loss and depletion of host reserves of adipose tissue and skeletal muscle. Traditional treatment approaches include the use of appetite stimulants, 5-HT3 antagonists, nutrient supplementation, and COX-2 inhibitors.

[0383] The pathogenesis of cachexia is poorly understood but is predicted to involve multiple biological pathways, including proinflammatory cytokines such as TNF-α, neuroendocrine hormones, IGF-1, and tumor-specific factors such as proteolysis-inducing factor.

[0384] Thus, HRS-Fc conjugates can be used to treat cachexia and any of its associated underlying or secondary disorders or complications. HRS-Fc conjugates can be used alone or in combination with other therapies, such as dietary supplementation with a combination of high protein, leucine, and fish oil, antioxidants, progestogens (megestrol acetate, medroxyprogesterone acetate), and anti-cyclooxygenase-2 drugs, appetite stimulants, and 5-HT3 antagonists, among others.

[0385] Rhabdomyolysis: Rhabdomyolysis is the breakdown of muscle fibers in skeletal muscle tissue. Breakdown products are released into the bloodstream, and certain of these products, such as myoglobin, can be toxic to the kidneys and lead to kidney failure.

[0386] Symptoms include muscle pain, vomiting, confusion, coma, or abnormal heart rate and rhythm, the severity of which usually depends on the extent of muscle damage and whether kidney failure occurs. Kidney damage can cause reduced or absent urine production, usually approximately 12 to 24 hours after the initial muscle injury. Swelling of the injured muscle can cause compartment syndrome, or compression of surrounding tissues, such as nerves and blood vessels, in the same fascial compartment, resulting in blood loss and damage (e.g., loss of function) to the affected body part. Symptoms of this complication include pain or reduced sensation in the affected limb. Other complications include disseminated intravascular coagulation (DIC), a severe disruption of blood clotting that can lead to uncontrolled bleeding.

[0387] Initial muscle injury can be caused by, for example, physical factors (e.g., crush injury, strenuous exercise), altered blood supply (e.g., arterial thrombosis, embolism), altered metabolism (e.g., hyperglycemic hyperosmolar states, hyper- and hyponatremia, hypokalemia, hypocalcemia, hypophosphatemia, ketoacidosis, hypothyroidism), altered body temperature (hyperthermia, hypothermia), medications and toxins (e.g., statins, antipsychotic medications, neuromuscular blockers, diuretics, heavy metals, hemlock, insect or snake venom), drug abuse (e.g., alcohol, amphetamines, cocaine, heroin, ketamine, LDS, MDMA), infection (e.g., Coxsackievirus, influenza A virus, influenza B virus, Epstein-Barr virus, primary HIV infection, Plasmodium falciparum, herpesvirus, Legionella pneumophila, Salmonella) and autoimmune muscle damage (e.g., polymyositis, dermatomyositis). Certain genetic conditions also increase the risk of rhabdomyolysis, including glycolysis and glycogenolysis deficiencies (e.g., McArdle disease, phosphofructokinase deficiency, glycogen storage diseases VIII, IX, X, and XI), lipid metabolism deficiencies (e.g., carnitine palmitoyltransferase I and II deficiencies, acyl-CoA dehydrogenase subtype deficiencies (e.g., LCAD, SCAD, MCAD, VLCAD, 3-hydroxyacyl-coenzyme A dehydrogenase deficiency), thiolase deficiency), mitochondrial myopathies (e.g., succinate dehydrogenase, cytochrome c oxidase, and coenzyme Q10 deficiencies), and others, such as glucose-6-phosphate dehydrogenase deficiency, myoadenylate deaminase deficiency, and muscular dystrophies.

[0388] Rhabdomyolysis is usually diagnosed with blood and urine tests and may be indicated by abnormally elevated or gradually increasing creatinine and urea levels, decreased urine output, or a maroon discoloration of the urine. Primary treatments include intravenous fluids, dialysis, and hemofiltration.

[0389] Thus, HRS-Fc conjugates can be used to treat rhabdomyolysis and any of its associated secondary or underlying disorders or complications. HRS-Fc conjugates can be used alone or in combination with other therapies, including those intended to treat shock and preserve kidney function. Exemplary therapies include intravenous fluid infusion, usually isotonic saline (0.9% sodium chloride solution by volume), as well as renal replacement therapy. alternative These include the administration of renal renal therapy (RRT), such as hemodialysis, continuo...

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[Claim 1] A composition or method as described in the specification.

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