Histidyl-tRNA synthetase Fc conjugate

Abstract

To provide histidyl-tRNA synthetase Fc conjugate. [Solution] The present invention provides a conjugate polypeptide of histidyl-tRNA synthetase and an Fc region (HRS-Fc conjugate), for example, an HRS-Fc fusion polypeptide, a composition comprising the same, and a method of using such conjugates and compositions to treat or diagnose various conditions. The HRS-Fc conjugate of the present invention has improved controlled release properties, stability, half-life, and other pharmacokinetic and biological properties compared to the corresponding unmodified HRS polypeptide.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 61 / 789,011, filed on 15 March 2013 under 119(e) of the U.S. Patent Act, which is incorporated herein by reference in its entirety.

[0002] Statement regarding sequence listings The sequence listing accompanying this application is provided in text format instead of being a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is ATYR_116_01WO_ST25.txt. This text file is approximately 399KB in size, was created on March 14, 2014, and submitted electronically via EFS-Web.

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

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

[0005] Evidence for the existence of non-canonical function in AARS includes well-defined sequence comparisons that establish that during the evolution from simple single-celled organisms to more complex life forms, AARS evolved to become structurally more complex through the addition of attached domains without losing its ability to facilitate protein synthesis.

[0006] Consistent with this hypothesis, a rich and diverse set of extended functions related to AARS has been found in higher eukaryotes, particularly human tRNA synthetases. Based on both direct analysis of individual domains and the discovery of mutations in tRNA synthetase genes that are causally associated with disease but do not affect aminoacylation or protein synthesis activity, these data suggest that these newly attached domains or physiocrines are central to the newly acquired non-canonical functions of AARS.

[0007] Furthermore, there is a growing recognition that specific tRNA synthesizers, 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 an extracellular signaling molecule has been obtained through studies demonstrating the secretion and extracellular release of specific tRNA synthesizers, as well as direct demonstration that the addition of tRNA synthetase fragments containing newly attached domains (physiocrines) is active within the extracellular signaling pathway, while the addition of other fragments lacking these domains is not. These physiocrines, e.g., HRS, present previously untapped new opportunities to develop a new primary position in the therapeutic protein class for treating human diseases.

[0008] To best utilize these and other activities in therapeutic or diagnostic settings, HRS polypeptides with improved pharmacokinetic properties are needed in the field. These improved therapeutic forms of HRS polypeptides enable the development of more effective therapeutic regimens for the treatment of various diseases and disorders and require significantly lower administration frequencies than unmodified proteins. [Overview of the Initiative] [Means for solving the problem]

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

[0010] Accordingly, certain embodiments include an HRS polypeptide comprising an amino acid sequence or any sequence from Table D1, D3-D6, or D8 that is at least 80% identical to any of SEQ ID NOs: 1-106, 170-181, or 185-191, 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 essentially consists of, an amino acid sequence or any sequence from Table D1, D3-D6, or D8 that is at least 90% identical to any of SEQ ID NOs: 1-106, 170-181, or 185-191. In certain embodiments, the HRS polypeptide comprises, consists of, or essentially consists of, one amino acid sequence or any sequence from Table D1, D3-D6, or D8 that is at least 90% identical to any of the SEQ ID NOs: 1-106, 170-181, or 185-191.

[0011] In certain embodiments, the HRS polypeptide contains an amino acid sequence that is at least 90% identical to amino acid residues 2-40, 2-45, 2-50, 2-55, 2-60, 2-66, or 1-506 of SEQ ID NO: 1, or 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 approximately 40-80 amino acid lengths and contains residues 2-45 of SEQ ID NO: 1. In specific embodiments, the HRS polypeptide consists of, or essentially consists 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 for or deleted by another amino acid. In certain embodiments, this at least one endogenous cysteine ​​residue is selected from Cys174, Cys191, Cys224, Cys235, Cys507, and Cys509. In certain embodiments, this at least one endogenous cysteine ​​residue is selected from Cys224, Cys235, Cys507, and Cys509. In specific embodiments, this endogenous cysteine ​​residue is Cys507 and Cys509. In some embodiments, all endogenous surface-exposed cysteine ​​residues are substituted for or deleted by another amino acid.

[0013] In certain embodiments, the HRS polypeptide is repeated in tandem. In certain embodiments, the HRS polypeptide contains 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 embodiments, the WHEP domain or a variant or fragment of the HRS polypeptide has the consensus sequence in Table D5.

[0014] In some embodiments, the Fc region and HRS polypeptide are separated by a peptide linker. In certain embodiments, this peptide linker is approximately 1-200 amino acid length, 1-150 amino acid length, 1-100 amino acid length, 1-90 amino acid length, 1-80 amino acid length, 1-70 amino acid length, 1-60 amino acid length, 1-50 amino acid length, 1-40 amino acid length, 1-30 amino acid length, 1-20 amino acid length, 1-10 amino acid length, or 1-5 amino acid length. In certain embodiments, the peptide linker is approximately 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 acid lengths. In certain embodiments, this peptide linker consists of Gly residues and / or Ser residues. In some embodiments, this peptide linker is a physiologically stable linker. In other embodiments, this peptide linker is a releaseable linker, optionally an enzymatically cleavable linker. In specific embodiments, the peptide linker comprises any one of the sequences of SEQ ID NOs. 200 to 260, or other peptide linkers described herein.

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

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

[0017] In specific embodiments, this Fc region includes one of sequence numbers 128-163 or 339-342, or variants, fragments, or combinations thereof. In certain embodiments, this hinge domain is a modified IgG1 hinge domain including sequence number 341.

[0018] In certain embodiments, this Fc region is MSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 339) or S Contains at least 90% identical amino acid sequence to DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 340).

[0019] In certain embodiments, this 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) It contains at least 90% identical amino acid sequences to (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 cases, this HRS fusion polypeptide has modified pharmacokinetics compared to the corresponding HRS polypeptide. Examples of such modified pharmacokinetics include increased serum half-life, increased bioavailability, increased exposure, and / or decreased clearance. In certain cases, this exposure is increased by at least 100 times. In some cases, this HRS fusion polypeptide has a half-life of at least 30 hours in mice. In certain cases, this bioavailability is increased subcutaneous bioavailability by at least about 30%. In some cases, this HRS fusion polypeptide has modified immunoeffector activity compared to the corresponding HRS polypeptide. Examples of such immunoeffector activity include one or more of complement activation, complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or antibody-dependent cell-mediated phagocytosis (ADCP).

[0021] In certain embodiments, this Fc region includes a variant Fc region compared to a wild-type Fc region. In some embodiments, this variant Fc region includes a sequence, or a combination thereof, that is at least 90% identical to one of sequence numbers 128-163 or 341. In certain embodiments, this variant Fc region includes a hybrid of one or more Fc regions derived from different species, different Ig classes, or different Ig subclasses. In certain embodiments, this variant Fc region includes a hybrid of one or more hinges, CH2, CH3, and / or CH4 domains of Fc regions derived from different species, different Ig classes, and / or different Ig subclasses.

[0022] In certain embodiments, this variant Fc region is a modified glycoform compared to the corresponding wild-type Fc region. In certain embodiments, this 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, this 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 cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or antibody-dependent cell-mediated phagocytosis (ADCP).

[0023] In certain embodiments, this variant Fc region has modified 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, this variant Fc region has modified 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, this variant Fc region has modified (e.g., increased) solubility compared to the corresponding wild-type Fc region, and this HRS-Fc fusion polypeptide has modified solubility compared to the corresponding unmodified HRS polypeptide.

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

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

[0028] In certain embodiments, this HRS-Fc fusion polypeptide exhibits substantially the same activity as the corresponding unmodified HRS polypeptide or a differently modified HRS polypeptide in an anti-inflammatory activity assay.

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

[0030] In certain embodiments, this HRS-Fc fusion polypeptide exhibits at least 30% higher stability than the corresponding unmodified HRS polypeptide or a differently modified HRS polypeptide when compared under similar conditions over 7 days at room temperature in PBS at pH 7.4.

[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, or 381-390, or 396. SEQ ID NOs: 107 and 338 are the amino acid sequences of an exemplary C-terminal Fc fusion polypeptide to residues 1-60 of SEQ ID NO: 1 (HRS(1-60)_Fc); SEQ ID NOs: 108 and 337 are the amino acid sequences of an exemplary N-terminal Fc fusion polypeptide to residues 1-60 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 1-506 of SEQ ID NO: 1 (HRS(1-506)_Fc); SEQ ID NO: 110 is the amino acid sequence of an exemplary N-terminal Fc fusion polypeptide to residues 1-506 of SEQ ID NO: 1 (Fc_HRS(1-506)).

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

[0033] This specification also includes compositions comprising the HRS-Fc fusion polypeptide described herein and pharmaceutically acceptable or pharmaceutical-grade carriers or excipients, such as pharmaceutical compositions or therapeutic compositions. 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 intended for use in: a) treating inflammatory or autoimmune diseases; b) reducing muscle or lung inflammation optionally associated with autoimmune or inflammatory diseases; c) in inducing tolerance to histidyl-tRNA synthetase (HRS) autoantigens; d) eliminating sets or subsets of T cells involved in the autoimmune response to HRS autoantigens; e) reducing tissue inflammation in subjects, optionally in muscle, lung, and / or skin tissues; f) treating muscular dystrophy; g) treating rhabdomyolysis, muscle wasting, cachexia, myoinflammatory or myocardial injury; and / or h) treating autoantibody-associated diseases.

[0035] A drug regimen that maintains an average steady-state concentration of histidyl-tRNA synthetase (HRS)-Fc fusion polypeptide in the subject's plasma between approximately 300 pM and approximately 1,000 nM, when using a drug interval of three days or more, also includes a drug regimen that comprises the step of administering the therapeutic composition or HRS-Fc fusion polypeptide described herein to the subject.

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

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

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

[0039] A particular embodiment includes a method for inducing tolerance to a histidyl-tRNA synthetase (HRS) autoantigen in a subject requiring such tolerance, comprising the step of administering to the subject a therapeutic composition or HRS-Fc fusion polypeptide described herein.

[0040] Some embodiments include 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 requiring such elimination, the method comprising the step of administering a therapeutic composition or HRS-Fc fusion polypeptide described herein to the subject.

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

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

[0043] A particular embodiment includes a method for treating rhabdomyolysis, muscle wasting, cachexia, myositis, or muscle injury in a subject requiring such treatment, comprising the step of administering a therapeutic composition or HRS-Fc fusion polypeptide described herein to the subject.

[0044] Some embodiments include a method for treating an autoantibody-associated disease, comprising the step of 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 myopathy, polymyositis, dermatomyositis and related disorders, polymyositis-scleroderma duplication, inclusion body myositis (IBM), anti-synthetase 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 to 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 an antibody in the serum of the subject. In some embodiments, this HRS polypeptide blocks the binding of autoantibodies to native histidyl-tRNA synthetase. In some embodiments, this HRS polypeptide induces clonal elimination of autoreactive T cells. In some embodiments, this HRS polypeptide induces functional inactivation of T cells involved in autoimmune responses. In some embodiments, administration of this HRS polypeptide results in reduced muscle or lung inflammation. In some embodiments, this 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 an HRS-Fc conjugate or fusion polypeptide as described herein, including a vector comprising such polynucleotides, as well as host cells comprising the polynucleotides and / or vectors.

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

[0048] [Figure 1] Figure 1 is a diagram illustrating the structural composition of exemplary immunoglobulins, providing an overview of antibody classes and subclasses. [Figure 2] Figure 2 shows the alignment of the Fc region derived 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 Fcα is shown above the sequence. Carets (^) and asterisks (*) indicate residues contributing 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. These results demonstrate that HRS(1-506) dramatically reduces the formation of disulfide-mediated interchain bonds compared to the full-length protein. Samples (10 μg) were loaded onto 4–12% Bis-Tris gels using MOPS-SDS running buffer. [Figure 4] Figure 4 shows the anti-inflammatory properties of exemplary HRS-derived polypeptides in a TNBS-inducible mouse model of colitis. The study was conducted on male BDF-1 mice in groups of 12 mice / group. TNBS and budesonide were added to water at 5 mg / kg. HRS(1-60)(Resokine, (HisRSN4)) was administered daily at concentrations of 1 mg / kg or 5 mg / kg by intravenous injection, starting 3 days before TNBS treatment. This figure shows the percentage survival (%) of treated and untreated mice over approximately 80 hours. [Figure 5]Figure 5A shows the drug regimens used to evaluate the therapeutic usefulness of HRS(1-506) in a statin myopathy model. The treatment regimens 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 in HRS(1-506) with statins + / - 0.3 mg / Kg, 1.0 mg / Kg, and 3.0 mg / Kg. This figure shows 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 statins + / - 0.3 mg / kg, 1.0 mg / kg, and 3.0 mg / kg in HRS(1-506). Figure 6B shows the same data after 15 days of treatment. This figure demonstrates the positive effect of HRS(1-506) on reducing statin-induced CK levels. [Figure 7] Figure 7 shows the levels of circulating HARS after 15 days of statin treatment compared to the vehicle control. This figure shows that statins (stains) induce the release of extracellular HARS. [Figure 8] Figure 8 shows representative H&E images of flexor thigh muscle sections at 10x magnification after 15 days of treatment with statins + / - 0.3 mg / kg, 1.0 mg / kg, and 3.0 mg / kg at HRS(1-506). [Figure 9] Figure 9 shows the results of gene expression profiling of the thigh flexor muscle of statin-treated rats. This data shows changes in muscle markers, as well as in the expression of 137 genes selected to track immune cell function, inflammation, metabolic status, tissue recovery, muscle growth, and atrophy. Gene expression values ​​were standardized against a reference gene and are shown as a percentage change relative to the vehicle-treated group. [Figure 10]Figure 10A shows the results of gene expression profiling of the thigh flexor muscle of statin-treated rats. This data shows changes in the expression of 137 genes (same as in Figure 7) to compare the relative changes in gene expression in statin-treated animals with those treated with vehicle-treated animals. Figure 10B shows the relative changes in gene expression in statin-treated animals that were also treated with HRS(1-506) compared with animals treated with statin alone. [Figure 11] Figure 11 shows the results of gene expression profiling of 10 diabetes / metabolic syndrome-related genes in the thigh 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 HRS(1-506). [Figure 12] Figure 12 shows the results of gene expression profiling of 26 immune cell marker genes in the thigh 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 HRS(1-506). [Figure 13-1] Figures 13A-D show the results of gene expression profiling of the CD11a, CD11b, CD8a, and CD8b genes in the thigh 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 HRS(1-506). [Figure 13-2] Figures 13A-D show the results of gene expression profiling of the CD11a, CD11b, CD8a, and CD8b genes in the thigh 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 HRS(1-506). [Figure 14-1] Figures 14A-C show the results of gene expression profiling of the CD18, CCR5, and CD45R genes in the thigh 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 HRS(1-506). [Figure 14-2]Figures 14A-C show the results of gene expression profiling of the CD18, CCR5, and CD45R genes in the thigh 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 HRS(1-506). [Figure 15] Figure 15 shows the results of gene expression profiling of 17 inflammatory marker genes in the thigh 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 HRS(1-506). [Figure 16-1] Figures 16A-D show the results of gene expression profiling of inflammatory cytokines IL-6, MCP1, IL-10, and interferon-gamma (IFN-γ) in the thigh 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 HRS(1-506). [Figure 16-2] Figures 16A-D show the results of gene expression profiling of inflammatory cytokines IL-6, MCP1, IL-10, and interferon-gamma (IFN-γ) in the thigh 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 HRS(1-506). [Figure 17] Figure 17 shows the results of gene expression profiling of 14 adhesion, development, and fibrosis-related genes in the thigh 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 HRS(1-506). [Figure 18] Figure 18 shows the results of gene expression profiling of 14 muscle wasting / atrophy-related genes in the thigh 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 HRS(1-506). [Figure 19-1]Figure 19A shows the results of gene expression profiling of 14 muscle wasting / atrophy-related genes in the thigh 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 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 the thigh 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 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] Figure 20 shows the results of gene expression profiling of 29 myogenesis-related genes in the thigh 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 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 2-5 were run under non-reducing conditions, and lanes 6-9 were run under reducing conditions. [Figure 22] Figure 22 shows the analytical size exclusion HPLC analysis of a representative purified Fc-HRS(2-60) fusion after protein A, cation exchange, and hydroxyapatite chromatography (double injection overlay). Purity is 99.2% for the major peak and 0.8% for the high molecular weight (HMW) species. [Figure 23-1]Figure 23A shows the time-to-time concentration of HRS(1-60) after either intravenous or subcutaneous injection into mice. Figure 23B shows the time-to-time concentration of Fc-HRS(2-60) and HRS(1-60)-Fc after intravenous injection into mice. Figure 23C shows the time-to-time concentration of Fc-HRS(2-60) and HRS(1-60)-Fc after subcutaneous injection into mice. [Figure 23-2] Figure 23A shows the time-to-time concentration of HRS(1-60) after either intravenous or subcutaneous injection into mice. Figure 23B shows the time-to-time concentration of Fc-HRS(2-60) and HRS(1-60)-Fc after intravenous injection into mice. Figure 23C shows the time-to-time concentration 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 the end of the study in mice treated with different HRS-Fc fusion proteins. The bars represent 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 the end of the study in mice treated with the compound. The bars represent the mean ratio (±SEM) for each treatment group. [Figure 25] Figure 25 shows an overview of transcriptional changes in the TNBS study. It shows relative transcriptional changes in TNBS-treated animals (Group 2), animals treated with TNBS and budesonide (Group 3), animals treated with TNBS and test item A (HRS(1-60); Group 4), and animals treated with TNBS and test item B (Fc-HRS(2-60); Groups 5 and 6) after standardization against naive animals (Group 1). Each dot in the scatter plot represents the 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. Relative transcriptional changes of individual genes are shown in TNBS-treated animals (Group 2), animals treated with TNBS and budesonide (Group 3), animals treated with TNBS and test item A (HRS(1–60); Group 4), and animals treated with TNBS and test item B (Fc-HRS(2–60); Groups 5 and 6) after standardization against 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 Student's t-test, where * = p-value < 0.05 and ** = p-value < 0.01. [Figure 26-2] Figures 26A–26H show immune and inflammation-related genes upregulated by TNBS. Relative transcriptional changes of individual genes are shown in TNBS-treated animals (Group 2), animals treated with TNBS and budesonide (Group 3), animals treated with TNBS and test item A (HRS(1–60); Group 4), and animals treated with TNBS and test item B (Fc-HRS(2–60); Groups 5 and 6) after standardization against 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 Student's t-test, where * = p-value < 0.05 and ** = p-value < 0.01. [Figure 26-3] Figures 26A–26H show immune and inflammation-related genes upregulated by TNBS. Relative transcriptional changes of individual genes are shown in TNBS-treated animals (Group 2), animals treated with TNBS and budesonide (Group 3), animals treated with TNBS and test item A (HRS(1–60); Group 4), and animals treated with TNBS and test item B (Fc-HRS(2–60); Groups 5 and 6) after standardization against 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 Student's t-test, where * = 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 spleen of naive mice, mice treated intracolonically with TNBS to induce experimental colitis, and mice treated with TNBS ± 0.5 mg / kg Fc-HRS(2-60). (27A) shows the percentage of viable lymphocytes stained for CD3, (27B) CD8, (27C) CD4, and (27D) CD25 and FoxP3. Treg cells were further gated against CD4+ cells. [Modes for carrying out the invention]

[0049] The implementation of this invention will utilize conventional methods of molecular biology and recombinant DNA techniques within the scope of the art of those skilled in the art, unless otherwise specifically indicated, many of which are described below for illustrative purposes. Such techniques are well described in the literature. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2000); DNA Cloning: A Practical Approach, Volumes I and II (edited by D. Glover); Oligonucleotide Synthesis (edited by N. Gait, 1984); Oligonucleotide Synthesis: Methods and Applications (edited by P. Herdewijn, 2004); Nucleic Acid Hybridization (edited by B. Hames and S. Higgins, 1985); Nucleic Acid Hybridization: Modern Applications (edited by Buzdin and Lukyanov, 2009); Transcription and Translation (edited by B. Hames and S. Higgins, 1984); Animal Cell Culture (edited by R. Freshney, 1986); Freshney, RI (2005), Culture of Animal Cells, a Manual of Basic Technique, 5th edition, Hoboken NJ, John Wiley & Sons; B. Perbal, A Practical Guide to Molecular Cloning (3rd edition, 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd edition, 2005). Poly(ethylene glycol), Chemistry and Biological Applications, ACS, Washington, 1997; Veronese, F. and JMSee Harris (ed.), Peptide and protein PEGylation, Advanced Drug Delivery Reviews, Vol. 54 (No. 4), pp. 453-609 (2002); and 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 incorporated herein by reference in their entirety.

[0051] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described. For the 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 more (i.e., at least one) of the grammatical objects of the article. For example, “an element” means one element or more than one element.

[0053] "Approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by approximately 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 mean both naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimics. 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, norleucine, norvaline, p-fluorophenylalanine, and ethionine. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications may include, for example, substitution or replacement of chemical groups and parts on an amino acid, or derivatization of an amino acid. Amino acid mimics include, for example, organic structures that exhibit functionally similar properties to a reference amino acid, such as charge and charge space characteristics. For example, an organic structure mimicking arginine (Arg or R) has a positively charged moiety that is positioned in a molecular space similar to the e-amino group of the side chain of the naturally occurring Arg amino acid and has the same degree of mobility. Mimics also include constrained structures that maintain the optimal spatial and charge interactions of the amino acid or amino acid functional group. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimics.

[0055] As used herein, a subject “at risk” of developing a disease or adverse reaction may or may not have a detectable disease or symptoms of a disease, and may or may not have exhibited a detectable disease or symptoms of a disease prior to the treatment method described herein. “At risk” means that a 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 having one or more of these risk factors is more likely to develop a disease or adverse reaction than a subject having one or more of these risk factors.

[0056] As used herein, "autoimmune disease" refers to a disease or disorder that originates from and affects the tissues of an individual. Examples of autoimmune diseases or autoimmune disorders include, but are not limited to, inflammatory responses, e.g., inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis) and associated responses; respiratory distress syndromes (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions, e.g., eczema and asthma, as well as other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion disorders; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes mellitus (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome Syndrome; juvenile-onset diabetes; and immune responses associated with cytokine and T lymphocyte-mediated acute and delayed hypersensitivity, typically found in tuberculosis, sarcoidosis, polymyositis, inflammatory myopathy, interstitial lung disease, granulomatous diseases, and vasculitis; pernicious anemia (Addison's disease); diseases involving extravasation of leukocytes; central nervous system (CNS) inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (cryoglobinemia or Coombs positive). Anemia is included, but not limited to; myasthenia gravis; antigen-antibody complex-mediated diseases; anti-glomerular basement membrane antibody disease; antiphospholipid antibody syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenia gravis; bullous pemphigoid; pemphigus; autoimmune polyglandular endocrine disorder; Reiter's disease; Stiff-Mann 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 specifically requires otherwise, the words “comprise,” “comprises,” and “comprising” are understood to mean that they include the steps or elements or groups of steps or elements described, but do not exclude any other steps or elements or groups of steps or elements. “Consists of” means that it includes and is limited to any of the things that precede the phrase “consists of.” Thus, the phrase “consists of” indicates that the enumerated elements are required or essential, and other elements may or may not be present. “Essentially consists of” means that it includes any elements enumerated before this phrase, and is limited to other elements that do not interfere with or contribute to the activity or action of the enumerated elements as identified in this disclosure. Thus, the phrase “essentially consists of” indicates that the enumerated elements are required or essential, but other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the enumerated elements.

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

[0059] The term “conjugate” is intended to refer to entities formed as a result of the covalent bonding of a biologically active molecule (e.g., an HRS polypeptide) to an immunoglobulin Fc region. An example of a conjugated polypeptide is a “fusion protein” or “fusion polypeptide,” i.e., a polypeptide created by the linking of two or more coding sequences that originally encode separate polypeptides; the transcription of the linked coding sequences yields a single fusion polypeptide that typically possesses functional properties derived from each of the separate polypeptides.

[0060] The descriptions “endotoxin-free” or “substantially endotoxin-free” generally refer to compositions, solvents, and / or containers containing at most trace amounts of endotoxin (e.g., amounts that do not have clinically harmful physiological effects on the subject), preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain bacteria, typically Gram-negative bacteria, although they can also be found in Gram-positive bacteria such as Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS) found in the outer membranes of various Gram-negative bacteria, which are central to the pathogenicity of these bacteria in their ability to cause disease. Small amounts of endotoxin in humans can cause fever, decreased blood pressure, and activation of inflammation and coagulation, among other harmful physiological effects.

[0061] Therefore, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxins from drug products and / or drug containers, because even small amounts can cause harmful effects in humans. Since temperatures above 300°C are typically required to decompose most endotoxins, depyrogenation ovens can be used for this purpose. For example, based on the main packaging material such as syringes or vials, a combination of a glass temperature of 250°C and a holding time of 30 minutes is often sufficient to achieve a 3-log reduction in endotoxin levels. Other methods for removing endotoxins are considered, including, for example, chromatography and filtration methods, which are described herein and known in the art. Methods for generating HRS-Fc conjugates in eukaryotic cells such as mammalian cells and isolating HRS-Fc conjugates from such cells are also included to reduce, or otherwise eliminate, the risk of endotoxin presence in the compositions of the present invention. Methods for generating HRS-Fc conjugates in serum-free cells and isolating HRS-Fc conjugates from such cells are preferred.

[0062] Endotoxins can be detected using conventional techniques known in the field. For example, the Limulus amebosite lysate assay, which utilizes horseshoe crab blood, is a highly sensitive assay for detecting the presence of endotoxins. In this test, very low levels of LPS can cause detectable coagulation of Limulus lysates due to a potent enzyme cascade that amplifies the reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). To ensure that the product is virtually endotoxin-free, endotoxin levels may be less than approximately 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 approximately 1 to 10 EU.

[0063] As used herein, the terms “function” and “functional” refer to biological, enzymatic, or therapeutic functions.

[0064] "Homologousity" refers to the percentage of identical or constituting conserved substitutions of amino acids. Homologousity can be determined using sequence comparison programs such as GAP (Deveraux et al., Nucleic Acids Research, Vol. 12, pp. 387-395, 1984), which is incorporated herein by reference. In this way, sequences of similar or substantially different lengths to sequences cited herein can be compared by inserting gaps into the alignment, such as gaps determined, for example, 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, such as in the presence of one or more proteases), i.e., a linker that does not undergo a degradation reaction (e.g., an enzymatically degradable reaction) under physiological conditions to any appreciable extent over an extended period. Generally, a physiologically stable linker is a linker that exhibits a rate of degradation 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" means a material that is substantially or essentially free of the components that are normally associated with it in its native situation. For example, an "isolated peptide" or "isolated polypeptide" as used herein includes the in vitro isolation and / or purification of the peptide or polypeptide molecule from its natural cellular environment and from its association with other components of the cell; i.e., the isolated molecule is not significantly associated with substances in vivo.

[0067] The term "half-maximal effective concentration" or "EC 50 " refers to the concentration of the HRS-Fc conjugate described herein that induces a response intermediate between baseline and maximum after some specified exposure time; thus, the EC 50 of a stepwise dose-response curve indicates the concentration of the compound at which 50% of its maximum effect is observed. In certain embodiments, the EC 50 of the agents provided herein is shown with respect to "off-target" activity. The EC 50 also indicates the plasma concentration required to obtain 50% of the maximum effect in vivo. Similarly, "EC 90 " refers to the concentration of an agent or composition at which 90% of its maximum effect is observed. The "EC 90 " can be calculated from the "EC 50 " and the Hill slope or can be determined directly from the data using conventional knowledge in the art. In some embodiments, the EC of the HRS-Fc conjugate50 This includes values ​​less than approximately 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, and less than 9 nM. The EC is 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 biotherapy composition has an EC of about 1 nM or less. 50 It has a value.

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

[0069] The terms “linker,” “linker moiety,” or “L” are used herein to refer to a linker that may be used to separate an HRS polypeptide from another HRS polypeptide and / or from one or more Fc regions. This linker may be physiologically stable or may include a releaseable linker, such as an enzymatically degradable linker (e.g., a proteolytically cleavable linker). In certain embodiments, this linker may be a peptide linker, for example, as part of an HRS-Fc fusion protein. In some embodiments, this linker may be a non-peptide linker.

[0070] The terms “modulate” and “modulate” typically include “increase,” “enhance,” or “stimulate,” as well as “decrease” or “reduce,” 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 may include increases of 1.1 times, 1.2 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times or more (e.g., 500 times, 1000 times) (including all integers and decimals between them and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) in the absence of the composition (e.g., in the absence of any of the HRS-Fc conjugates of the present invention) or the control composition, sample, or test subject. The amount "reduced" or "reduced" is typically a "statistically significant" amount and may include reductions of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the amount produced without the composition (in the absence of the drug or compound) or by the control composition. As one non-limiting example, the control when comparing canonical activity with non-canonical activity may include the HRS-Fc conjugate of interest compared to the corresponding unmodified or differently modified HRS polypeptide (in sequence). Other examples of comparisons and "statistically significant" quantities are described herein.

[0071] "Non-canonical" activity, as used herein, generally refers to either i) a novel non-aminoacylation activity possessed by the 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, wherein, for example, by isolating that activity from other activities possessed by the intact native full-length parent protein, the HRS polypeptide exhibits either a significantly higher specific activity (e.g., at least 20% higher) with respect to non-canonical activity compared to the intact native full-length parent protein, or exhibits activity in a novel context. In the case of HRS polypeptides, non-canonical activity is not limited to extracellular signaling, including modulation of cell proliferation, cell migration, cell differentiation (e.g., hematopoiesis, neurogenesis, myogenesis, osteogenicity, and adipogenesis), gene transcription, apoptosis or other forms of cell death, cellular signaling, cellular uptake or secretion, angiogenesis, cell binding, cellular metabolism, cytokine production or activity, cytokine receptor activity, inflammation, and immunogenicity.

[0072] In certain embodiments, the "purity" of any given agent in the composition (e.g., an HRS-Fc conjugate such as a fusion protein) can be specifically defined. For example, a particular composition may include agents that are at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between, as measured by 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] While we do not wish to be bound by any particular theory, "enzymatically degradable linker" means a linker, such as an amino acid sequence, that is subject to degradation by one or more enzymes, such as peptidases or proteases.

[0074] The terms “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues, as well as their variants and synthetic analogs. Accordingly, 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 and enzymatically degradable linkers. Therefore, a "releasable linker" is a linker that, under physiological conditions, can undergo either spontaneous hydrolysis or cleavage by several other mechanisms (e.g., enzyme-catalyzed, acid-catalyzed, base-catalyzed, etc.). For example, a "releasable linker" may involve an elimination reaction having a base abstraction of a proton (e.g., an ionizable hydrogen atom, Hα) as the driving force. For the purposes of this specification, "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 field. A commonly used measure of significance is the p-value, which is the frequency or probability of the observed event occurring if the null hypothesis were true. If the resulting p-value is smaller than the significance level, the null hypothesis is rejected. In a simple example, this significance level is defined by a p-value of 0.05 or less.

[0077] The term "solubility" refers to the property 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, by any of the following: mass of solute per unit volume of solvent (g of solute per 1 kg of solvent, g / dL (100 mL), mg / ml, etc.), molar concentration by volume, molar concentration by weight, mole fraction, or other similar descriptions of concentration. The maximum equilibrium amount of solute that can dissolve per unit volume of solvent is the solubility of that solute in that solvent under specific conditions, including temperature, pressure, pH, and the properties of the solvent. In certain embodiments, solubility is measured at physiological pH or other pH levels, e.g., pH 5.0, pH 6.0, pH 7.0, or pH 7.4. In certain embodiments, solubility is measured in water or physiological buffer, e.g., PBS or NaCl (with or without NaP). In specific embodiments, solubility is measured at relatively low pH (e.g., pH 6.0) and relatively high salt (e.g., 500 mM NaCl and 10 mM NaP). In certain embodiments, solubility is measured in biological fluids (solvents), such as blood or serum. In certain embodiments, the temperature may be 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] "Subject" as used herein includes any animal exhibiting, or at risk of exhibiting, a condition that can be treated or diagnosed with the HRS-Fc conjugate polypeptide of the present invention. Suitable subjects (patients) include laboratory 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 almost entirely or completely, for example, 95%, 96%, 97%, 98%, 99%, or more of a given quantity.

[0080] "Treatment" or "doing treatment," as used herein, includes any desired effect on the symptoms or pathology of a disease or condition, and may include even the smallest change or improvement in one or more measurable markers of the disease or condition being treated. "Treatment" or "doing treatment" does not necessarily indicate the complete eradication or cure of the disease or condition or its associated symptoms. The subject receiving this treatment is any subject who needs it. 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 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 having modified cysteine ​​content. Histidyl-tRNA synthetases belong to the class II tRNA synthetase family, which have three highly conserved sequence motifs. Class I and II tRNA synthetases are widely recognized as being responsible for the specific binding of amino acids to their homologous tRNAs in a two-step reaction: the amino acid (AA) is first activated by ATP to form AA-AMP, which is then transferred to the acceptor terminus of the tRNA. Full-length histidyl-tRNA synthetases typically exist either as cytosolic homodimers or as alternatively spliced ​​mitochondrial forms.

[0082] More recently, several biological fragments or alternatively spliced ​​isoforms (Physiocrines or HRS polypeptides) of eukaryotic histidyl-tRNA synthetases, or in some contexts, intact synthetases, have been established to modulate specific cellular signaling pathways or possess anti-inflammatory properties. These activities, distinct from the classical role of tRNA synthetases in protein synthesis, are collectively referred to herein as “non-canonical activities.” These Physiocrines may be spontaneously produced by either alternative splicing or proteolysis and may 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 in the present invention, HRS polypeptides, such as the N-terminal fragments of histidyl-tRNA synthetase (e.g., HRS 1-48, HRS 1-60), can exert anti-inflammatory signals, among other things, by blocking the migration, activation, or differentiation of inflammatory cells (e.g., monocytes, macrophages, T cells, B cells) associated with the site of active inflammation in vivo. Furthermore, specific 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. Sequences of specific 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 human genes have been sequenced and are known in the art to be at least partially functionally interchangeable. Some such variants of histidyl-tRNA synthetases (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] Furthermore, homologs and orthologues of human genes exist in other species, as listed in Table D3, and therefore it is customary to select naturally occurring amino acids, nucleotide variants present in SNPs, or other naturally occurring homologs instead 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] Accordingly, in any of the methods, therapeutic compositions, and kits of the present 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 activities such as anti-inflammatory activity and / or harbor at least one epitope that specifically cross-reacts with autoantibodies or autoreactive T cells derived from subjects having autoantibodies against histidyl-tRNA synthetase and associated diseases. Such HRS polypeptides include full-length human proteins, as well as HRS peptides derived from 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 long. It is understood that in any of the HRS-Fc conjugates described herein, the N-terminal amino acid of the HRS polypeptide (e.g., 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 approximately 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 acid lengths. For example, in a specific embodiment, this polypeptide is between approximately 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 acid lengths, or between approximately 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 acid lengths, or between approximately 40-45, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90 , between 40 and 95, or between 40 and 100 amino acid lengths, or approximately between 45 and 50, 45 and 55, 50 and 55, 50 and 60, 50 and 65, 50 and 70, 50 and 75, 50 and 80, 50 and 85, 50 and 90, 50 and 95, or between 50 and 100 amino acid lengths, or approximately between 60 and 65, 60 and 70, 60 and 75, 60 and 80, 60 and 85, 60 and 90, 60 and 95, or between 60 and 100 amino acid lengths, or approximately between 70 and 75, 70 and 80, 70 and 85, 70 and 90, 70 and 95, or between 70 and 100 amino acid lengths, or approximately between 80 and 85, 80 and 90, 80 and 95, or between 80 and 100 amino acid lengths.In a particular embodiment, this HRS polypeptide is approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 6 The amino acid lengths are 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.

[0095] In some embodiments, this HRS polypeptide is up to approximately 1 to 5 × 10 -7 Up to M or higher concentrations, the HRS polypeptide does not significantly compete for the binding of disease-related autoantibodies (e.g., Jo-1 antibody) to wild-type histidyl-tRNA synthetase in competitive ELISA. Therefore, in some embodiments, the HRS polypeptide has a lower affinity for disease-related autoantibodies than wild-type histidyl-tRNA synthetase (SEQ ID NO: 1) when measured in competitive ELISA. In some embodiments, the HRS polypeptide has an apparent affinity for disease-related autoantibodies (e.g., Jo-1 antibody) that is at least about 10 times, at least about 20 times, at least about 50 times, or at least about 100 times lower than the affinity for disease-related autoantibodies to wild-type human (SEQ ID NO: 1).

[0096] Accordingly, all such homologs, orthologues, 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 present invention, insofar as they possess at least one epitope that specifically cross-reacts with autoantibodies or autoreactive T cells derived from subjects having autoantibodies against histidyl-tRNA synthetase and associated diseases, or possess non-canonical activity. These HRS polypeptides may exist in their native form, i.e., as different variants that can be considered functionally equivalent variants of human histidyl-tRNA synthetase when they occur naturally in different species, or they may be functionally equivalent natural derivatives whose amino acid sequences may differ, for example, by shortening (e.g., from the N-terminus or C-terminus or both) or by deletion, addition, insertion, substitution, or post-translational modification of other amino acids. For example, naturally occurring chemical derivatives of any HRS polypeptide, including pyroglutamyl, isoaspartyl, proteolytic, phosphorylated, glycosylated, oxidized, isomerized, and deamination variants of the HRS polypeptide or HRS-Fc conjugate, as well as post-translational modifications and degradation products of the HRS polypeptide or HRS-Fc conjugate, are also specifically included in any of the methods and compositions of the present invention. The HRS polypeptide and HRS-Fc conjugate may also consist of naturally occurring amino acids and / or amino acids not naturally occurring, as described herein.

[0097] As described above, embodiments of the present invention include all homologs, orthologues, and naturally occurring isoforms of histidyl-tRNA synthetases (e.g., proteins listed in the table or sequence listing, or proteins derivable from such table or sequence listing, or any of their corresponding nucleic acids listed in such table or sequence listing) as well as “variants” of these HRS reference polypeptides. A “variant” of a described polypeptide means a polypeptide that is identified from the 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 modified by the addition, deletion, and / or substitution of at least one amino acid residue to have improved stability or other pharmaceutically acceptable properties.

[0098] In certain embodiments, the polypeptide variant is identified from the reference polypeptide by one or more substitutions, which may be conserved or non-conservative, as described herein and known in the art. In certain embodiments, the polypeptide variant comprises a conserved substitution, and in this regard, it is well understood in the art that some amino acids may be replaced with other amino acids having broadly similar properties without altering the activity properties of the polypeptide. In some embodiments, the 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 in Sequence ID No. 1).

[0099] Specific examples of HRS polypeptide variants useful in any of the methods and compositions of the present invention include full-length HRS polypeptides, or shortened or spliced ​​variants thereof (e.g., any proteins listed in the table or sequence listing, or proteins derivable from such table or sequence listing), which 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 having autoantibodies against histidyl-tRNA synthetase and associated diseases, and ii) have one or more further amino acid insertions, substitutions, deletions and / or shortenings. In certain embodiments, the 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 the HRS reference polypeptide (e.g., any of the proteins listed in the table or sequence listing or any protein derivable from such table or sequence listing), as described herein, and substantially retains the non-canonical activity of the reference polypeptide. The following also include sequences that differ from the reference HRS sequence due to 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 that retain the characteristics of the reference HRS polypeptide. In certain embodiments, this addition or deletion of amino acids occurs at the C-terminus and / or N-terminus of the HRS reference polypeptide. In certain embodiments, this amino acid addition includes 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., derived 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 to 250 amino acids or about 50 to 250 amino acids, which includes, consists of, or are essentially composed of, the amino acids of the HRS polypeptide sequence shown in one or more of SEQ ID NOs: 1-106, 170-181, or 185-191. In some embodiments, the HRS polypeptide includes, consists of, or is essentially composed of, residues 1-141, 1-408, 1-113, or 1-60 of SEQ ID NO: 1. In some embodiments, the HRS polypeptide is a splice variant which includes, consists of, or is essentially composed of, residues 1-60+175-509, 1-60+211-509, or 1-60+101-509 of SEQ ID NO: 1. In certain embodiments, the HRS polypeptide contains, consists of, or is essentially composed of residues 1-48 or 1-506 of SEQ ID NO: 1.

[0101] In certain embodiments, the HRS polypeptide of the present invention comprises, consists of, or is essentially composed of, a minimal 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 embodiment, such minimal active fragment comprises, consists of, or is essentially composed of, a WHEP domain (i.e., approximately amino acids 1-43 of SEQ ID NO: 1). In some embodiments, this minimal active fragment comprises, consists of, or is essentially composed of, an aminoacylation domain (i.e., approximately amino acids 54-398 of SEQ ID NO: 1). In some embodiments, this minimal active fragment comprises, consists of, or is essentially composed of, an anticodon-binding domain (i.e., approximately 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, approximately 20-40, 20-45, 20-50, 20-55, or 20-60, 20-65, or 20-67 consecutive or discontinuous amino acids of the HRS polypeptide, or at least approximately 20-40, 20-45, 20-50, 20-55, or 20-60, 20-65, or 20-67 consecutive or discontinuous amino acids, are derived from amino acids 1-67 of SEQ ID NO: 1. In certain embodiments, approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67 consecutive or discontinuous amino acids of the HRS polypeptide are derived from amino acids 1-67 of SEQ ID NO: 1. This HRS polypeptide may contain one or more of the WHEP domain, aminoacylation domain, anticodon-binding domain, or any combination thereof. In certain embodiments, this HRS polypeptide lacks a functional aminoacylation domain. In some embodiments, this polypeptide is essentially derived from a human HRS-derived WHEP domain. While we do not wish to be bound to any one theory, the unique orientation or conformation of the WHEP domain in certain HRS polypeptides may contribute to the enhanced non-canonical activity and / or antibody-blocking activity observed in these proteins.

[0106] Accordingly, in certain embodiments, the HRS polypeptide contains, consists of, or is essentially derived from the human HRS WHEP domain sequence. In some embodiments, the human HRS WHEP domain sequence is defined by specific conserved residues. For example, in some embodiments, the HRS polypeptide contains, consists of, or is essentially derived from the 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 that connects a minimal domain to a heterologous protein (e.g., an Fc domain) or a splice variant.

[0108] The term “sequence identity” or, for example, “sequences 50% identical to ~” means, as used herein, the degree to which the sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis across a comparison window. Thus, “percentage of sequence identity” can be calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in both sequences where identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are present, obtaining the number of matching positions, dividing this number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0109] Terms used to describe the sequence relationships between two or more polypeptides include “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.” A “reference sequence” is a monomer unit of length, including nucleotides and amino acid residues, of at least 12, but frequently 15–18 and often at least 25. Since each of two polypeptides may 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 comparisons between two (or more) polypeptides are typically performed by comparing the sequences of the two polypeptides across a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of at least six consecutive positions, usually about 50–100, more typically about 100–150, where, after the two sequences have been optimally aligned, the sequences are compared to the reference sequence at the same number of consecutive positions. The comparison window may contain approximately 20% or less of additions or deletions (i.e., gaps) compared to the reference sequence (which contains neither additions nor deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning the comparison window can be achieved by computerized execution of algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by the best alignment (i.e., producing the highest percentage homology across the comparison window) generated by any of the various methods of inspection and selection. References may also be made to programs of the BLAST family, such as those disclosed by Altschul et al., 1997, Nucl. Acids Res. Vol. 25:3389. A detailed discussion of sequence analysis can be found in Ausubel et al., "Current Protocols in This can be found in "Molecular Biology," John Wiley & Sons Inc., 1994–1998, in Chapter 15, Unit 19.3.

[0110] The calculation of sequence similarity or sequence identity between sequences (these terms are used interchangeably herein) may be carried out as follows: To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, these sequences may be aligned for the purpose of best comparison (for example, gaps may be introduced into one or both of the first and second amino acid sequences or nucleic acid sequences for best alignment, and non-homologous sequences may be ignored for the purpose of comparison). In certain embodiments, the length of the reference sequence aligned for the purpose of comparison is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. Then, amino acid residues or nucleotides at the corresponding amino acid 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, then these molecules are identical at that position.

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

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

[0113] The nucleic acid and protein sequences described herein may be used as “query sequences” for performing searches against public databases, for example, to identify other family members or related sequences. Such searches may be performed using the NBLAST and XBLAST programs (version 2.0) by Altschul et al. (1990, J. Mol. Biol, Vol. 215: pp. 403-4010). A BLAST nucleotide search may be performed using the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. A BLAST protein search may be performed using the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparative purposes, Gapped BLAST may be used as described by Altschul et al. (Nucleic Acids Res. Vol. 25: pp. 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of representative programs (e.g., XBLAST and NBLAST) may be used.

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

[0115] This also includes biologically active "fragments" of the HRS reference polypeptide, i.e., biologically active fragments of the HRS protein fragment. Representative biologically active fragments are generally involved in interactions, such as intramolecular or intermolecular interactions. Intermolecular interactions can be specific binding interactions or enzymatic interactions. Intermolecular interactions can be interactions between the HRS polypeptide and its cellular binding partner, such as cellular receptors or other host molecules involved in the non-canonical activity of the HRS polypeptide.

[0116] The biologically active fragments of the HRS reference polypeptide include all integers (e.g., 101, 102, 103) and ranges (e.g., 50-100, 50-150, 50-200) between them, and the amino acid sequences shown in any one of the HRS reference polypeptides described herein, for example, 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, 18 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 discontinuous amino acids. In certain embodiments, the biologically active fragment includes a non-canonical activity-related sequence, domain, or motif. In certain embodiments, the C-terminal or N-terminal region of any HRS reference polypeptide is approximately 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, including all integers and ranges between them (e.g., 101, 102, 103, 104, 105), as long as the truncated HRS polypeptide retains the non-canonical activity of the reference polypeptide. Only amino acids 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500 or more may be shortened, or only amino acids approximately 10-50, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500 or more may be shortened.The consensus sequences for specific exemplary truncated HRS polypeptides and 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., N-terminal Met) may be further deleted from any of the exemplary truncated HRS polypeptides described herein or other HRS sequences.

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

[0122] In some embodiments, HRS proteins, variants, and their biologically active fragments bind to one or more cellular binding partners with affinities 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 to a selected cellular binding partner, particularly a binding partner involved in non-canonical activity, may be at least about 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, 300×, 400×, 500×, 600×, 700×, 800×, 900×, 1000× or greater (including all integers in between), compared to that of the corresponding full-length HRS polypeptide or certain alternatively spliced ​​HRS polypeptide variants.

[0123] As described above, HRS polypeptides can be modified in various ways, including amino acid substitution, deletion, shortening, and insertion. Methods for such operations are generally known in the art. For example, amino acid sequence variants of HRS reference polypeptides can be prepared by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are well known in the art. For example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. Vol. 82: pp. 488-492), Kunkel et al. (1987, Methods in Enzymol, Vol. 154: 367-382), U.S. Patent No. 4,873,192, Watson, JD et al. ("Molecular Biology of the Gene", 4th edition, Benjamin / Cummings, Menlo) See Park, Calif., 1987 and the references cited therein. Guidance for selecting amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0124] Biologically active truncated and / or variant HRS polypeptides may contain conserved amino acid substitutions at various positions along their sequence compared to a reference HRS amino acid residue, and such further substitutions may further enhance the activity or stability of the HRS polypeptide with the altered cysteine ​​content. A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art, and can generally be subdivided as follows: Acidic: This residue has a negative charge due to the loss of H ions at physiological pH, and is attracted by aqueous solutions to determine the surface position in the conformation of the peptide containing this residue when the peptide is present in an aqueous medium at physiological pH. Amino acids with acidic side chains include glutamic acid and aspartic acid.

[0125] Basicity: This residue has a positive charge due to association with H ions at or within one or two pH units of physiological pH (e.g., histidine), and when the peptide is present in an aqueous medium at physiological pH, this residue is attracted by the aqueous solution to determine the surface position in the conformation of the peptide containing this residue. Amino acids with basic side chains include arginine, lysine, and histidine.

[0126] Charge: These residues are charged at physiological pH, and therefore include amino acids with acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine, and histidine).

[0127] Hydrophobicity: These residues are uncharged at physiological pH, and when the peptide is in an aqueous medium, they are repelled by the aqueous solution, determining the internal position of the peptide conformation containing these residues. Amino acids with hydrophobic side chains include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine, and tryptophan.

[0128] Neutral / Polar: These residues are uncharged at physiological pH, but they are not sufficiently repelled by aqueous solutions, and as a result, these residues determine the internal position of the conformation of the peptide containing them when the peptide is in an aqueous medium. Amino acids with neutral / polar side chains include asparagine, glutamine, cysteine, histidine, serine, and threonine.

[0129] This explanation also characterizes certain amino acids as "small" because their side chains are not large enough to confer hydrophobicity, even without polar groups. With the exception of proline, "small" amino acids are those with four or fewer carbon atoms if at least one polar group is present on the side chain, and those with three or fewer carbon atoms if none is present. Amino acids with small side chains include glycine, serine, alanine, and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effect on the secondary conformation of the peptide chain. The structure of proline differs from all other naturally occurring amino acids in that its side chain is bonded to the nitrogen and α-carbon of the α-amino group. Several amino acid similarity matrices are known in the field (e.g., Dayhoff et al., 1978, A model of evolutionary change in See, for example, the PAM120 matrix and the PAM250 matrix disclosed by proteins. However, the matrix 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: pp. 14430-1445, 1992) includes proline in the same group as glycine, serine, alanine, and threonine. Therefore, for the purposes of this 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, the amino acids specifically intended by this invention are classified as either one or the other. Most amino acids that are not specifically named can be classified based on their known behavior.

[0131] Amino acid residues can be further subdivided into cyclic or acyclic, aromatic or non-aromatic, trivial classifications with respect to the side-chain substituents of the residue, and small or large. A residue is considered small if it contains a total of four or fewer carbon atoms, including the carboxyl carbon, provided that further polar substituents are present; otherwise, it is considered small if it contains three or fewer. Small residues are, of course, always non-aromatic. Depending on their structural characteristics, amino acid residues can fall into two or more classes. For naturally occurring protein amino acids, the subclassifications following this scheme are presented in Table A.

[0132] [Table 5-4]

[0133] Conservative amino acid substitutions also include groupings based on side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, it is reasonable to predict that substitution of leucine with isoleucine or valine, substitution of aspartic acid with glutamic acid, substitution of threonine with serine, or similar substitutions of some amino acid with structurally related amino acids will not have a significant impact on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional truncated and / or variant HRS polypeptide can be readily determined by assaying its non-canonical activity, as described herein. Conservative substitutions are shown in Table B under the heading "Exemplary Substitutions." Amino acid substitutions that fall within the scope of the present invention are generally achieved by selecting substitutions that do not significantly differ in (a) the structure of the peptide backbone in the region of substitution, (b) the charge or hydrophobicity of the molecule at the target site, (c) the bulkiness of the side chain, or (d) their effect on maintaining biological function. After the substitution is introduced, these variants are screened for biological activity.

[0134] [Table 5-5]

[0135] Alternatively, similar amino acids for conservative substitution can be grouped into three categories based on the nature 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). Furthermore, the crystal structures of full-length human HRS and the internal catalytic domain deletion mutant of HRS (HRSΔCD) have also been determined (see Xu et al., Structure. Vol. 20: pp. 1470-1477, 2012; and U.S. Patent Application No. 61 / 674,639). Together with the primary amino acid sequence of HRS, these detailed physical descriptions of the protein provide accurate insights into the roles played by specific amino acids within the protein. Thus, those skilled in the art can use this information to identify structurally conserved domains, linking regions, secondary structures such as alpha-helices, surface or solvent-exposed amino acids, unexposed or internal regions, catalytic sites, and ligand interaction surfaces, among other structural features. A person skilled in the art can then use that information or other information to easily manipulate an HRS variant that retains or improves the desired non-canonical activity, for example, by preserving or modifying the characteristics of amino acid residues in or adjacent to these and other structural features, for example, by preserving or modifying the polarity, hydroxyl index, charge, size and / or positioning (i.e., inward, outward) of selected amino acid side chains compared to wild-type residues (e.g., Zaiwara et al., Mol Biotechnol. Vol. 51: pp. 67-102, 2012; Perona and Hadd, Biochemistry. Vol. 51: pp. 8705-29, 2012; Morin et al., Trends Biotechol. Vol. 29: pp. 159-66, 2011; Collins et al., Annu. Rev. See Biophys, Vol. 40: pp. 81–98, 2011; and U.S. Patent Application No. 61 / 674,639.

[0137] Therefore, predicted non-essential amino acid residues in the truncated and / or variant HRS polypeptide are typically replaced by other amino acid residues from the same side-chain family. Alternatively, mutations may be introduced randomly along all or part of the HRS coding sequence, for example by saturation mutagenesis, and the resulting mutants may 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 may be recombinantly expressed, and the peptide's activity may be determined. "Non-essential" amino acid residues are those that can be modified from the reference sequence of the embodiment polypeptide without invalidating or substantially altering one or more of their non-canonical activities. Appropriately, this modification does not substantially invalidate one of these activities, for example, this activity is at least 20%, 40%, 60%, 70%, or 80%, 100%, 500%, 1000%, or more than%, or 1000% of the activity of the reference HRS sequence. "Essential" amino acid residues are those that, when altered from the reference sequence of an HRS polypeptide, result in the inactivation of the parent molecule's activity to such an extent that less than 20% of the reference activity remains. For example, such essential amino acid residues include residues conserved across different species of HRS polypeptides, 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 conventional measurement-based assays of cytokine release from in vitro cells and animal studies, are well established in the field (e.g., Wittmann et al., J Vis Exp. (vol. 65):e4203.doi:10.3791 / 4203, 2012; Feldman et al., Mol Cell. vol. 47:pp. 585-595, 2012; Clutterbuck et al., J Proteomics. vol. 74:pp. 704-715, 2011; Giddings and Maitra, J See Biomol Screen, Vol. 15: pp. 1204-1210, 2010; Wijnhoven et al., Glycoconj J. Vol. 25: pp. 177-1785, 2008; and Frow et al., Med Res Rev. Vol. 24: pp. 276-2798, 2004. These can be readily used to profile and optimize anti-inflammatory activity. Exemplary in vivo experimental systems are also described in the attached examples.

[0139] In some embodiments, the HRS polypeptide may have one or more cysteine ​​substitutions, where one or more naturally occurring (non-cysteine) residues are substituted with cysteine ​​(for example, to alter stability, to facilitate thiol-based conjugation of Fc fragments, or to facilitate thiol-based binding of PEG or other molecules). In some embodiments, the cysteine ​​substitution is located at the N-terminus and / or C-terminus of the HRS polypeptide (e.g., SEQ ID NOs: 1-106, 170-181, or 185-191), or in the vicinity of other surface-exposed regions of the HRS polypeptide. Certain embodiments include cases where one or more 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 are substituted with cysteine ​​residues at any one of the N-terminus and / or C-terminus of SEQ ID NOs. In some embodiments, the cysteine ​​residues may be added to the HRS polypeptide via the creation of an N-terminal or C-terminal fusion protein. Such fusion proteins may be of any length, but are typically about 1–5, or about 5–10, about 10–20, or about 20–30 amino acids long. In some embodiments, fusion to the C-terminus is preferred.

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

[0141] [Table 6]

[0142] In some embodiments, the HRS polypeptide may include variants in which endogenous or naturally occurring cysteine ​​residues are mutated or deleted to alternative amino acids. In some embodiments, the insertion or substitution of cysteine ​​residues in the HRS polypeptide may be combined with the exclusion of other surface-exposed reactive cysteine ​​residues. Thus, in some embodiments, the HRS polypeptide may include one or more substitutions and / or deletions in Cys83, Cys174, Cys191, Cys196, Cys224, Cys235, Cys379, Cys455, Cys507 and / or Cys509 (as defined by SEQ ID NO: 1) to remove naturally occurring cysteine ​​residues, for example.

[0143] Specific embodiments include one of sequence numbers 1-106, 170-181, or 185-191, or variants or fragments thereof, having one or more mutations or deletions of Cys83, Cys174, Cys191, Cys196, Cys224, Cys235, Cys379, or Cys455, or deletions of Cys507 and Cys509 due to, for example, a deletion of the three C-terminal amino acids (Δ507-509). Exemplary mutations at these positions include, for example, mutations from cysteine ​​to serine, alanine, leucine, valine, or glycine. In certain embodiments, the amino acid residue for the specific cysteine ​​substitution may be selected from naturally occurring substitutions found in HRS orthologs of other species and organisms. Exemplary substitutions of this type are shown in Table D7.

[0144] [Table 7]

[0145] In some embodiments, the naturally occurring cysteine selected for mutagenesis is selected based on its surface exposure. Thus, in one aspect, the cysteine residue selected for substitution is 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 missing. In some embodiments, these cysteines are selected for mutation or deletion so as to exclude an intramolecular cysteine pair, such as Cys174 and Cys191.

[0146] Specific further examples of desired cysteine mutations / substitutions (indicated in bold underline) to reduce surface-exposed cysteine residues are included in 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 variants are modified to manipulate, insert, or otherwise introduce a new surface-exposed cysteine ​​residue at a defined surface-exposed location, where the introduced residue does not substantially interfere with the non-canonical activity of the HRS polypeptide. Specific examples include, for example, the insertion (or re-insertion) of a further cysteine ​​residue at the N-terminus or C-terminus of any of the reduced cysteine ​​HRS polypeptides. In some embodiments, such N-terminus or C-terminus surface-exposed cysteine ​​insertions include the re-insertion of the last one, last two, or last three naturally occurring C-terminal amino acids of full-length human HRS into the reduced cysteine ​​variant of the HRS polypeptide, e.g., the re-insertion of all or part of the sequence CIC (Cys Ile Cys). Exemplary reduced cysteine ​​variants include, for example, any combination of mutations (or deletions thereof) at residues Cys174, Cys191, Cys224, and Cys235 in any of the HRS polypeptides of SEQ ID NOs. 1-106, 170-181, or 185-191 or Table D1, D3-D6, or D8, as well as / or deletions or substitutions of Cys507 and Cys509 (based on the numbering of full-length human HRS (SEQ ID NO: 1)).

[0153] For some types of site-specific conjugate or binding to heterologous molecules such as the Fc region or PEG or other heterologous molecules, the HRS polypeptide may have one or more glutamine substitutions, where one or more naturally occurring (non-glutamine) residues are substituted with glutamine, for example, to facilitate the transglutaminase-catalyzed binding of the molecule(s) to the glutamine amide group. In some embodiments, the glutamine substitution is introduced near the N-terminus and / or C-terminus of the HRS polypeptide (e.g., SEQ ID NOs: 1-106, 170-181 or 185-191, or HRS polypeptides in Tables D1, D3-D6 or D8). Certain embodiments include cases where one or more 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 are substituted with glutamine residues at the N-terminus and / or C-terminus of any one of SEQ ID NOs: 1-106, 170-181, or 185-191. These and related HRS polypeptides may also include substitutions (e.g., conservative substitutions) to remove any naturally occurring glutamine residues and, if desired, to modulate the degree of site-specific conjugation or binding.

[0154] For certain types of site-specific conjugates or binding to heterologous molecules such as the Fc region or PEG or other heterologous molecules, HRS polypeptides may have one or more lysine substitutions, where one or more naturally occurring (non-lysine) residues are substituted with lysine to facilitate binding, for example, based on the acylation or alkylation of the molecule(s) to the amino group of lysine. These methods also typically result in the binding of the molecule(s) to the N-terminal residue. In some embodiments, the lysine substitution is 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 HRS polypeptides in Tables D1, D3-D6 or D8). Certain embodiments include cases where one or more 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 are substituted with lysine residues at the N-terminus and / or C-terminus of any one of the HRS polypeptides in SEQ ID NOs: 1-106, 170-181, or 185-191 (or Table D1, D3-D6, or D8). These and related HRS polypeptides may also include substitutions (e.g., conservative substitutions) to remove any naturally occurring lysine residues and, if desired, to modulate the degree of site-specific conjugation or binding.

[0155] Site-specific conjugation to HRS polypeptides can also be carried out by substituting one or more solvent-accessible surface amino acids of the HRS polypeptide. For example, suitable solvent-accessible amino acids can be determined using the published crystal structure of an exemplary HRS polypeptide and based on predicted solvent accessibility using the SPIDDER server (http: / / sppider.cchmc.org / ) (see Xu et al., Structure. Vol. 20: pp. 1470-1477, 2012; and U.S. Patent Application No. 61 / 674,639). Based on this analysis, several amino acids on the surface can potentially be used as mutation sites for introducing functional groups suitable for conjugation or binding. A surface accessibility score for amino acids based on the crystal structure can be calculated, where a higher score indicates better accessibility. In certain embodiments, a higher score (e.g., >40) is preferred. Therefore, in some embodiments, amino acid positions with a surface accessibility score greater than 40 can be used to introduce cysteine, lysine, glutamine, or other amino acids that do not exist naturally.

[0156] In certain embodiments, the solvent-accessible surface amino acids may be selected from the group consisting of alanine, glycine, and serine, and may be substituted with naturally occurring amino acids including but not limited to cysteine, glutamine, or lysine, or with naturally occurring non-natural amino acids optimized for site-specific conjugation or binding.

[0157] In various embodiments, the present invention aims to achieve site-specific conjugation or binding at any amino acid position in an HRS polypeptide by substituting an amino acid that does not exist in nature with a functional group that forms a covalent bond with a functional group bound to an Fc region or other heterologous molecule such as PEG or other heterologous molecules. Non-natural amino acids may be inserted or substituted, for example, in one or more residues of up to 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 at the N-terminus and / or C-terminus of any one of the HRS polypeptides in SEQ ID NOs: 1-106, 170-181, or 185-191 (or HRS polypeptides in Table D1, D3-D6, or D8); or in solvent-accessible surface amino acid residues as described herein.

[0158] In certain embodiments, amino acids not found in nature include, but are not limited to, any amino acid, modified amino acids, or amino acid analogs other than selenocysteine, and alpha-amino acids encoded by the following 20 genes: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The general structure of an alpha-amino acid is illustrated by the following formula:

[0159] [ka]

[0160] Non-natural amino acids are typically any structure having the formula described above, where the R group is any substituent other than those used in the 20 natural amino acids. For the structures of the 20 natural amino acids, see, for example, a biochemistry textbook, e.g., Biochemistry by L. Stryer, 3rd edition, 1988, Freeman and Company, New York. Note that the non-natural amino acids disclosed herein may be naturally occurring compounds other than the 20 alpha-amino acids described above. The non-natural amino acids disclosed herein typically differ from natural amino acids only in their side chains, so these non-natural amino acids form amide bonds with other natural or non-natural amino acids in the same manner as they are formed in naturally occurring proteins, for example. However, these non-natural amino acids have side chain groups that distinguish them from natural amino acids. For example, R in the above formulas may be of any choice, including alkyl-, aryl-, aryl halide, vinyl halide, alkyl halide, acetyl, ketone, aziridine, nitrile, nitro, halide, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynyl, ether, thioether, epoxide, sulfone, boronic acid, boronic acid ester, borane, phenylboronic acid, thiol, selen-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic-, pyridyl, naphthyl, benzophenone, cyclooctin, bound rings such as thioester, enone, imine, aldehyde, ester, thio acid, hydroxylamine, amino, carboxylic acid, alpha-ketocarboxylic acid, alpha- or beta-unsaturated acid and amide, glyoxylamide, or organosilane group, or any combination thereof.

[0161] Specific examples of non-natural amino acids include p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, β-O-GlcNAc-L-serine, tri-O-acetyl-GalNAc-α-threonine, α-GalNAc-L-threonine, L-Dopa, fluorinated phenylalanine, This includes, but is not limited to, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and those listed below or elsewhere in the present invention.

[0162] Therefore, it is possible to select non-natural amino acids that contain functional groups that form a covalent bond with any preferred functional group of the desired molecule (e.g., Fc region, PEG). Once selected, the non-natural amino acids can either be purchased from suppliers or chemically synthesized. Any number of non-natural amino acids can be incorporated into the target molecule, which may vary depending on the number of desired molecules to be conjugated. These molecules may be conjugated to all or some of the non-natural amino acids. Furthermore, the same or different non-natural amino acids may be incorporated into the HRS polypeptide, depending on the desired outcome. In certain embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-natural amino acids may be incorporated into the HRS polypeptide, and any or all of them may be conjugated to molecules containing the desired functional groups.

[0163] In certain embodiments, the use of non-natural amino acids may be utilized to modify (e.g., increase) the selective non-canonical activity of HRS polypeptides or to alter the in vivo or in vitro half-life of proteins. Non-natural amino acids may also be used to facilitate (selective) chemical modification (e.g., pegylation) of HRS proteins, as described elsewhere in this specification. For example, certain non-natural amino acids enable the selective binding of Fc domains or polymers such as PEG to a given protein, thereby improving its pharmacokinetic properties.

[0164] Specific examples of amino acid analogs and mimics 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. For example, in *Combinatorial Chemistry*, edited by Wilson and Czarnik, Chapter 11, p. 235, John Wiley, the entire contents of which are incorporated herein by reference. See John Wiley & Sons Inc., New York, NY (1997). Other examples include amino acids in which the amide portion (and therefore the amide backbone of the resulting peptide) is replaced by, for example, a sugar ring, steroid, benzodiazepine, or carbocycle. See, for example, 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 content of which is incorporated herein by reference. Methods for synthesizing peptides, polypeptides, peptide mimes, and proteins are well known in the art (for example, U.S. Patent No. 5,420,109, each of which is incorporated herein by reference; M. Bodanzsky, Principles of Peptide Synthesis (1st and 2nd revised editions), Springer-Verlag, New York, NY (1984 and 1993), see Chapter 7; Stewart and Young, Solid Phase Peptide Synthesis (2nd edition), Pierce See Chemical Co., Rockford, Ill. (1984). Accordingly, the HRS polypeptides of the present invention may consist of naturally occurring amino acids and non-naturally occurring amino acids, as well as amino acid analogs and mimics.

[0165] Polynucleotides Certain embodiments relate to polynucleotides encoding an HRS polypeptide or an HRS-Fc fusion protein. Alone or in combination with an HRS coding sequence, they also include polynucleotides encoding any one or more of the Fc regions described herein. 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 an HRS, Fc region or HRS-Fc polypeptide in a selected cell or subject. As a result of the degeneracy of the genetic code, those skilled in the art will understand that there are many nucleotide sequences encoding the HRS polypeptides and HRS-Fc fusion proteins described herein. Some of these polynucleotides may have minimal homology to the nucleotide sequences of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage frequency, for example, polynucleotides optimized for human, yeast or bacterial codon selection, are specifically contemplated by the present invention.

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

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

[0168] In further embodiments, the present invention provides isolated polynucleotides comprising continuous stretches of various lengths of sequences identical or complementary to an HRS polypeptide or HRS-Fc fusion protein, wherein the isolated polynucleotides encode the truncated HRS polypeptide described herein.

[0169] Therefore, multiple polynucleotides may encode the HRS polypeptide, the Fc region, and the fusion protein of the present invention. Furthermore, this polynucleotide sequence may be manipulated for a variety of reasons. Examples include, but are not limited to, the incorporation of preferred codons to enhance the expression of polynucleotides in various organisms (see, in general, Nakamura et al., Nuc. Acid. Res. vol. 28: p. 292, 2000). In addition, silent mutations may be incorporated to introduce or eliminate restriction sites, to reduce the density of CpG dinucleotide motifs (see, for example, Kameda et al., Biochem. Biophys. Res. Commun. vol. 349: pp. 1269-1277, 2006), or to reduce the ability of single-stranded sequences to form stem-loop structures (see, for example, Zuker M., Nucl. Acid Res. vol. 31: pp. 3406-3415, 2003). Furthermore, mammalian expression can be further optimized by including the Kozak consensus sequence (i.e., (a / g)cc(a / g)ccATGg) (Sequence ID 199) in the start codon. A Kozak consensus sequence useful for this purpose is known in the field (Mantyh et al., PNAS Vol. 92: pp. 2662-2666, 1995; Mantyh et al., Prot. Exp. & Purif. Vol. 6: p. 124, 1995). Various exemplary wild-type and codon-optimized versions of HRS polypeptides are provided in Table D9 below.

[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] Further coding or non-coding sequences may, but not necessarily, be present within the polynucleotide of the present invention, and the polynucleotide may, but not necessarily, be linked to other molecules and / or support materials. Thus, regardless of the length of the coding sequence itself, the polynucleotide of the present invention may be combined with other DNA or RNA sequences, such as promoters, polyadenylation signals, further restriction enzyme sites, multicloning sites, other coding segments, etc., and as a result, their overall lengths may vary considerably.

[0177] Therefore, polynucleotide fragments of almost any length can be used, and their total length is preferably intended to be limited by the ease of preparation and use in the intended recombinant DNA protocol. HRS reference polynucleotides (e.g., base numbers X-Y, where X is approximately 1-3000 or greater, and Y is approximately 10-3000 or greater) or any part or fragment of their complement (e.g., longer than approximately 6 nucleotides, longer than 7 nucleotides, longer than 8 nucleotides, longer than 9 nucleotides, or longer than 10 nucleotides), including approximately 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 This includes polynucleotides with base lengths of 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 or more (including all integers in between).

[0178] Embodiments of the present invention also include “variants” of the HRS reference polynucleotide sequence. A polynucleotide “variant” may include one or more substitutions, additions, deletions and / or insertions compared to the reference polynucleotide. Generally, a variant of the HRS reference polynucleotide sequence may have at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more sequence identity with respect to a particular nucleotide sequence (e.g., SEQ ID NOs. 111-127, 182-184, 192-198, etc.; see also Examples), as determined by a sequence alignment program described elsewhere herein using default parameters. In certain embodiments, the variant may differ from the reference sequence by approximately 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 in between) bases or more. In certain embodiments, for example, if the polynucleotide variant encodes an HRS polypeptide having non-canonical activity, the desired activity of the encoded HRS polypeptide is not substantially diminished compared to the unmodified polypeptide. The effect on the activity of the encoded polypeptide can generally be evaluated as described herein. In some embodiments, these variants may modify the aggregation state of the HRS polypeptide to provide, for example, HRS polypeptides that primarily exist as monomers, dimers, or multimers 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 Examples) or their complements under the stringency conditions described below. As used herein, the terms “hybridize under low stringency, moderate stringency, high stringency, or very high stringency conditions” describe the conditions for hybridization and washing. Guidance for carrying out hybridization reactions can be found in Ausubel et al. (1998, above), sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in the references and may be used.

[0180] The reference herein to low stringency conditions includes, and encompasses, at least about 1% v / v to at least about 15% v / v formamide and at least about 1M to at least about 2M salt for hybridization at 42°C, and at least about 1M to at least about 2M salt for washing at 42°C. Low stringency conditions may also include (i) 2×SSC, 0.1% SDS for hybridization at 65°C, and (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at room temperature. One embodiment of low stringency conditions includes hybridization in 6 × sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2 × SSC, 0.1% SDS at at least 50°C (the washing temperature can be increased up to 55°C for low stringency conditions).

[0181] Moderate stringency conditions include, and encompass, at least about 16% v / v to at least about 30% v / v formamide and at least about 0.5M to at least about 0.9M salt for hybridization at 42°C, and at least about 0.1M to at least about 0.2M salt for washing at 55°C. Moderate stringency conditions may also include 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5M NaHPO4 (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 NaHPO4 (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 and 0.1% SDS at 60°C. High stringency conditions include, and encompass, at least about 31% v / v to at least about 50% v / v formamide and about 0.01M to about 0.15M salt for hybridization at 42°C, and about 0.01M to about 0.02M salt for washing at 55°C.

[0182] High stringency conditions may also include (i) 0.2 × SSC, 0.1% SDS for hybridization at 65°C; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS for washing at temperatures above 65°C. 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 recognize that various factors can be manipulated to optimize the specificity of hybridization. Optimizing the stringency of the final wash can help ensure a high degree of hybridization. For detailed examples, see Ausubel et al., pp. 2.10.1-2.10.16 above, and Sambrook et al. (1989, above), sections 1.101-1.104. Stringent washing is typically performed at temperatures of approximately 42°C to 68°C, but those skilled in the art will understand that other temperatures may be suitable for stringent conditions. The maximum hybridization rate is typically T for the formation of DNA-DNA hybrids. m It occurs below that temperature, around 20°C to 25°C. m It is well known in the field that this is the melting temperature or the temperature at which two complementary polynucleotide sequences dissociate. m Methods for estimating this are well known in the field (see Ausubel et al., page 2.10.8 above).

[0184] Generally, the T of a double helix of perfectly matched DNA m is, formula: T m =81.5+16.6(log 10 The approximate value can be predicted by the formula M) + 0.41(%G+C) - 0.63(%formamide) - (600 / length), where M is preferably in the range of 0.01 molar concentration to 0.4 molar concentration, Na + %G+C is the concentration; %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 volume percentage of formamide concentration; and length is the number of base pairs in the DNA double helix. m For every 1% increase in the number of randomly mismatched base pairs, the temperature decreases by approximately 1°C. Washing is generally recommended for high stringency. m -15℃ or for moderate stringency m The test will be conducted at -30°C.

[0185] In one example of a hybridization procedure, a membrane containing immobilized DNA (e.g., a nitrocellulose membrane or nylon membrane) is hybridized overnight at 42°C in a hybridization buffer containing a labeled probe (50% deionized formamide, 5×SSC, 5×Denhardt solution (0.1% Ficol, 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 consecutive washes of moderate stringency (i.e., 2×SSC, 0.1%SDS for 15 minutes at 45°C, followed by 2×SSC, 0.1%SDS for 15 minutes at 50°C), and then two subsequent consecutive washes of higher stringency (i.e., 0.2×SSC, 0.1%SDS solution for 12 minutes at 55°C, followed by 0.2×SSC and 0.1%SDS solution for 12 minutes at 65-68°C).

[0186] Generation of HRS polypeptide and HRS-Fc conjugate 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. Vol. 85: pp. 2149-2154 (1963)), or by recombinant techniques using genetically modified hosts. Protein synthesis can be carried out using manual techniques or by automation. Automated synthesis can be achieved, for example, using the Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments can be chemically synthesized separately to produce the desired molecule and then combined using chemical methods.

[0187] HRS-Fc conjugates can also be generated by expressing the HRS polypeptide or HRS-Fc conjugate in question in a suitable host cell using well-known techniques. Polynucleotide sequences encoding the HRS-Fc conjugate or HRS polypeptide can be prepared by synthesis using established standard methods, e.g., the phosphoamidite method described by Beaucage et al., Tetrahedron Letters Vol. 22: pp. 1859-1869, 1981; or the method described by Matthes et al., EMBO Journal Vol. 3: pp. 801-805, 1984. According to the phosphoamidite method, oligonucleotides are synthesized, purified, double-stranded, and ligated, for example, in an automated DNA synthesizer, to form a synthetic DNA construct. Alternatively, the DNA or RNA construct 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 enable efficient delivery and uptake into cells, and optionally include a modified cap or tail structure to enhance stability and translation.

[0188] These polynucleotide sequences can also be of mixed genome, cDNA, RNA, and synthetic origin. For example, a genome or cDNA sequence encoding a leader peptide may be conjugated to a genome or cDNA sequence encoding an HRS polypeptide or HRS-Fc conjugate, and this DNA or RNA sequence may then be modified at one site by inserting a synthetic oligonucleotide encoding the desired amino acid sequence for homologous recombination according to a well-known procedure, or preferably by PCR using a suitable oligonucleotide to generate the desired sequence. In some embodiments, a signal sequence may be included before the coding sequence. This sequence encodes an N-terminal signal peptide relative to the coding sequence, communicating with the host cell to direct the polypeptide toward the cell surface or to secrete the polypeptide into the culture medium. Typically, this signal peptide is cleaved by the host cell before the protein leaves the cell. Signal peptides can be found in a variety of proteins in prokaryotes and eukaryotes.

[0189] Various expression vectors / host systems are known and can be used for expression because they contain polynucleotide sequences. These include microorganisms, e.g., bacteria transformed with recombinant bacteriophages, plasmids or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell lines infected with viral expression vectors (e.g., baculovirus); plant cell lines 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 lines, including mammalian cells and more specifically human cell lines, transformed with viral, plasmid, episomal or integrated expression vectors.

[0190] The “regulatory elements” or “regulatory sequences” present in an expression vector are the untranslated regions of the vector—enhancers, promoters, and the 5' and 3' untranslated regions—that interact with host cellular proteins to carry out transcription and translation. The intensity and specificity of such elements can vary. Depending on the vector system and host used, any number of appropriate transcription and translation elements, including constitutive and inductive promoters, may be used. For example, when cloning in bacterial systems, inductive promoters, such as the hybrid lacZ promoter of the PBLUESCRIPT phagemide (Stratagene, La Jolla, Calif.) or the PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.), may be used. In mammalian cell systems, promoters derived from mammalian genes or mammalian viruses are generally preferred. When it is necessary to generate cell lines containing multiple copies of the polypeptide-encoding sequence, SV40 or EBV-based vectors, along with appropriate selectable markers, may be advantageously used.

[0191] Certain embodiments may utilize an E. coli-based expression system (see, e.g., Structural Genomics Consortium et al., Nature Methods. Vol. 5: 135-146, 2008). These and related embodiments may partially or entirely rely on ligation-independent cloning (LIC) to generate appropriate expression vectors. In specific embodiments, protein expression may be controlled by T7 RNA polymerase (e.g., pET vector series) or modified pET vectors having alternative promoters, such as the TAC promoter. These and related embodiments may utilize the BL21 λDE3 lysogen, 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 possessing plasmids encoding tRNAs rarely used in E. coli, e.g., Rosetta®(DE3) strain and Rosetta 2(DE3) strain are also included. In some embodiments, other E. coli K-12 strains, such as W3110(F), may produce reduced levels of post-translational modification during fermentation. - 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 may be used, including trpE38, rfbC1, rpsL109(strR), xylA5, mtl-1, thiE1). Cell lysis and sample handling may also be improved by using reagents sold under the trademarks BENZONASE® nuclease and BUGBUSTER® Protein Extraction Reagent. For cell culture, autoinduction media can improve the efficiency of many expression systems, including high-throughput expression systems. This type of medium (e.g., OVERNIGHT EXPRESS® Autoinduction System) induces stepwise protein expression via metabolic shifts without the addition of artificial inducers such as IPTG.

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

[0193] High-density bacterial fermentation systems are also included. For example, cultivation of Ralstonia eutropha at high cell densities enables protein production at cell densities exceeding 150 g / L and recombinant protein expression at titers exceeding 10 g / L. In the yeast Saccharomyces cerevisiae, several vectors can be used, including constitutive or inductive promoters, such as alpha factor, alcohol oxidase, and PGH. For a review, see Ausubel et al. (above) and Grant et al., Methods Enzymol. Vol. 153: pp. 516-544, 1987. Pichia pandoris expression systems are also included (e.g., Li et al., Nature Biotechnology. Vol. 24: pp. 210-215, 2006; and Hamilton et al., Science, Vol. 301: p. 1244, 2003). Certain embodiments include, among other things, yeast systems engineered to selectively glycosylate proteins, including yeast having a humanized N-glycosylation pathway (see, for example, Hamilton et al., Science. 313: pp. 1441-1443, 2006; Wildt et al., Nature Reviews Microbiol. 3: pp. 119-128, 2005; and Gerngross et al., Nature-Biotechnology. 22: pp. 1409-1414, 2004; U.S. Patent No. 7,629,163; U.S. Patent No. 7,326,681; and U.S. Patent No. 7,029,872). Simply as an example, recombinant yeast cultures can be grown, among other things, in Fernbach Flasks or 15 L, 50 L, 100 L, and 200 L fermenters.

[0194] When plant expression vectors are used, the expression of the polypeptide-encoding sequence can be driven by one of several promoters. For example, viral promoters, such as the 35S and 19S promoters of CaMV, can be used alone or in combination with omega-leader sequences derived from TMV (Takamatsu, EMBO J. 6: pp. 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: pp. 1671-1680, 1984; Broglie et al., Science. 224: pp. 838-843, 1984; and Winter et al., Results Probl. Cell Differ. 17: pp. 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-based systems can also be used to express target polypeptides. For example, in one such system, the Autographa californica nuclear polyhedron disease virus (AcNPV) is used as a vector to express an exogenous gene in Spodoptera frugiperda or Trichoplusia cells. The polypeptide-coding 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-coding sequence inactivates the polyhedrin gene and produces a recombinant virus lacking the coat protein. These recombinant viruses can then be used, for example, to infect S. frugiperda or Trichoplusia cells that can express the target polypeptide (Engelhard et al., PNAS USA. 91: pp. 3224-3227, 1994). This also includes baculovirus expression systems, including those utilizing SF9, SF21, and T.ni cells (e.g., Murphy and Piwnica-Worms, Curr Protoc Protein Sci.). See Chapter 5: Unit 5.4, 2001. Insect systems may provide post-translational modifications similar to those found in mammals.

[0196] In mammalian host cells, several expression systems are well-known and commercially available in this field. 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, the sequence encoding the target polypeptide can be ligated into an adenovirus transcription / translation complex consisting of a late promoter and a tripartite leader sequence. Insertions in non-essential E1 or E3 regions of the viral genome can be used to obtain viable viruses capable of expressing polypeptides in infected host cells (Logan and Shenk, PNAS USA. Vol. 81: pp. 3655-3659, 1984). Furthermore, transcriptional enhancers, such as the Roussarcoma 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 cell line (COS-7, ATCC CRL 1651); and the human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen). Virol. Vol. 36: p. 59, 1977); Baby hamster kidney cells (BHK, ATCC CCL 10); Mouse Sertoli cells (TM4, Mather, Biol. Reprod. Vol. 23: pp. 243-251, 1980); Monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); Human cervical tumor 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 CCL 51); TR1 cells (Mather et al., Annals NY Acad. Sci. Volume 383: pp. 44-68, 1982); these include MRC 5 cells; FS4 cells; and human hepatoma cells (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA. Volume 77: p. 4216, 1980); and myeloma cell lines, e.g., NSO and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Volume 248 (edited by BKC Lo, Humana Press, Totowa, NJ, 2003), pp. 255-268. Specific preferred mammalian cell expression systems include CHO and HEK293 cell-based expression systems. Mammalian expression systems can utilize, for example, cell lines attached to T-flasks, roller bottles, or cell factories, which are known in the art, 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.

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

[0199] Furthermore, host cell lines may be selected for their ability to modulate the expression of the inserted sequence or to process the expressed protein in a desired manner. Such modifications of polypeptides include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation, or insertion of amino acids not naturally occurring (see generally U.S. Patents 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 naturally occurring amino acids may be inserted at position Cys130. Post-translational processing that cleaves the "prepro" form of the protein may also be used to facilitate precise insertion, folding, and / or function. In addition to bacterial cells, different host cells having or lacking specific cellular machinery and characteristic mechanisms for such post-translational activity, such as yeast, CHO, HeLa, MDCK, HEK293, and W138, may be selected to ensure precise modification and processing of foreign proteins.

[0200] HRS polypeptides or HRS-Fc conjugates produced by recombinant cells can be purified and characterized according to various 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) and high-pressure liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistry for purification, among others known in the art, includes ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradient, 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). Several 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 bonded to one or more HRS polypeptides. Examples of HRS-Fc conjugates include fusion proteins and various forms of chemically crosslinked proteins. A wide range of Fc region sequences, including wild-type sequences from any number of species, as well as their variants, fragments, hybrids, and chemically modified forms, 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. In any of these HRS-Fc conjugates, the native N-terminal or C-terminal amino acids of the HRS polypeptide, or the native N-terminal or C-terminal amino acids in the Fc domain, may be deleted and / or replaced with non-native amino acids(s), for example, to facilitate expression and / or cloning, or to function as a linker sequence between two proteins.

[0202] HRS-Fc conjugated polypeptides may offer various advantages compared to unconjugated or unmodified HRS polypeptides, such as the corresponding HRS polypeptide with the same or similar sequence but without the conjugated Fc region(s). For example, covalent conjugation of one or more Fc regions may alter (e.g., increase, decrease) the solubility, half-life (e.g., in serum, selected tissue, in vitro under storage conditions, e.g., at room temperature or under refrigeration), dimerization or multimerization properties, and biological activity(single or multiple) of HRS polypeptides compared to unmodified HRS polypeptides with the same or similar sequences, by providing, for example, Fc region-related effector function (e.g., activation of the classical complement cascade, interaction with immunoeffector cells via Fc receptors (FcRs), immunoglobulin compartmentalization), cellular uptake, intracellular transport, tissue distribution, and / or bioavailability. In certain embodiments, the Fc region can confer effector functions related to complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or antibody-dependent cell-mediated phagocytosis (ADCP), which are thought to play a role in clearing specific target cells such as tumor cells and infected cells.

[0203] Certain embodiments utilize HRS-Fc fusion proteins. "Fusion protein," as well as methods for constructing fusion proteins, are defined elsewhere herein and are well known in the art (see, for example, U.S. Patents 5,116,964; 5,428,130; 5,455,165; 5,514,582; 6,406,697; 6,291,212; and 6,300,099 for general disclosures and methods relating to Fc fusion proteins). In HRS-Fc fusion proteins, the Fc region may be fused to the N-terminus, C-terminus, or both of the HRS polypeptide. In some embodiments, one or more Fc regions may be fused internally to an HRS sequence by, for example, positioning the Fc region between a first HRS sequence (e.g., a domain) and a second HRS sequence (e.g., a domain), where the first HRS sequence is fused to the N-terminus of the Fc region and the second HRS sequence is fused to the C-terminus of the Fc region. In specific embodiments, these first and second HRS sequences are identical. In other embodiments, these first and second HRS sequences are different (e.g., they contain different functional domains of the HRS polypeptide). A particular HRS-Fc fusion protein may also include further heterogeneous protein sequences, namely non-Fc regions and non-HRS polypeptide sequences.

[0204] The term "HRS-Fc" may, but not necessarily, indicate the N-terminal or C-terminal fusion of an Fc region to an HRS polypeptide. For example, in certain cases, the term "Fc-HRS" indicates the fusion of an Fc region to the N-terminus of an HRS polypeptide, while the term "HRS-Fc" indicates the fusion of an Fc region to the C-terminus of an HRS polypeptide. However, either term may be used more generally to refer to any fusion protein or conjugate of an Fc region and an HRS polypeptide.

[0205] In some embodiments, the HRS-Fc fusion protein may comprise a tandem repeat copy 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 sequencing of specific tandem repeat HRS-Fc conjugates are illustrated in the examples.

[0206] [Table 10]

[0207] Certain embodiments relate to HRS-Fc conjugates in which one or more Fc regions are chemically conjugated or crosslinked to an HRS polypeptide(s). In these and related embodiments, the Fc region may be conjugated to an HRS polypeptide in the N-terminal region (e.g., within the first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 amino acids), the internal region (between the N-terminal and C-terminal regions), and / or the C-terminal region (e.g., within the last 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 amino acids). Polypeptides may be conjugated or crosslinked to other polypeptides according to various conventional techniques of the art. For example, certain techniques use carboxyl-reactive carbodiimide crosslinkers EDC (or EDAC) covalently linked via D, E, and C-terminal carboxyl groups. Other techniques use activated EDC that is covalently bonded via K and N-terminal amino groups. Other techniques use m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or sulfo-MBS that is covalently bonded via thiol groups of cysteine ​​residues (see also U.S. Patent Application No. 2007 / 0092940 for cysteine-operated Ig regions that can be used for thiol conjugates). Such crosslinked proteins may also include linkers that are cleavable or otherwise releaseable (e.g., enzymatically cleavable linkers, hydrolyzable linkers) and linkers that are not cleavable (i.e., physiologically stable linkers). Certain embodiments may use non-peptide polymers (e.g., PEG polymers; HRS-N-PEG-N-Fc conjugates) as crosslinkers between Fc regions and HRS polypeptides, as described, for example, U.S. Patent Application No. 2006 / 0269553. For an exemplary description of the Fc region conjugate, see also U.S. Patent Application No. 2007 / 0269369.

[0208] In certain embodiments, variant or otherwise modified Fc regions may be used, including those having altered properties or biological activity compared to the wild-type Fc region(s), as will be discussed in more detail below. Examples of modified Fc regions include, for example, those having sequences mutated by one or more amino acid substitutions, insertions, deletions or shortenings compared to the wild-type region; hybrid Fc polypeptides composed of domains from different immunoglobulin classes / subclasses; Fc polypeptides having altered glycosylation / sialic acid addition patterns; and Fc polypeptides modified or derivatized by, for example, biotinylation (see, for example, U.S. Patent Application No. 2010 / 0209424), phosphorylation, sulfation, etc., or any combination thereof. Such modifications, compared to the corresponding wild-type Fc sequence, affect the binding properties of the Fc region to one or more specific FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcRn), and their pharmacokinetic properties (e.g., stability or half-life, bioavailability, tissue distribution, distribution volume, concentration, efflux rate constant, efflux rate, area under the curve (AUC), clearance, C12 max t max , C min It can be used to alter (e.g., increase, decrease) its immunogenicity, its complement fixation or activation, and / or its CDC / ADCC / ADCP-related activity in the Fc region.

[0209] The “Fc region” of the HRS-Fc conjugate provided herein is typically derived from the heavy chain of an immunoglobulin (Ig) molecule. A typical Ig molecule consists of two heavy chains and two light chains. These heavy chains can be divided into at least three functional regions: an Fd region, an Fc region (a fragment crystallizable region), and a hinge region (see Figure 1), the latter of which is found only in IgG, IgA, and IgD immunoglobulins. The Fd region is a variable (V) region of the heavy chain. H ) domain and constant (CH1) domain, with variable (V) light chain L) domain and constant (C L Together with the domain, it forms an antigen-binding fragment or Fab region.

[0210] The Fc regions of IgG, IgA, and IgD immunoglobulins contain heavy chain constant domains 2 and 3, referred to as the CH2 and CH3 regions, respectively; the Fc regions of IgE and IgM immunoglobulins contain heavy chain constant domains 2, 3, and 4, referred to as the CH2, CH3, and CH4 regions, respectively. These Fc regions are primarily responsible for the effector functions of immunoglobulins, including complement binding to and from homologous 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 in space relative to the Fc region. In contrast to the constant region, the hinge region is structurally diverse, varying in both sequence and length across immunoglobulin classes and subclasses. The hinge region may also contain one or more glycosylation sites, each containing several structurally distinct types of sites for carbohydrate binding. For example, IgA1 contains five glycosylation sites within a 17-amino acid segment of its hinge region, conferring significant resistance of the hinge region polypeptide to intestinal proteases. Residues in the hinge proximal region of the CH2 domain can also influence the specificity of interactions between immunoglobulins and their respective Fc receptors (see, e.g., Shin et al., Intern. Rev. Immunol. Vol. 10: pp. 177-186, 1993).

[0212] Accordingly, the terms “Fc region,” “Fc fragment,” or “Fc,” as used herein, refer to a protein comprising one or more CH2, CH3, and / or CH4 regions derived from one or more selected immunoglobulins, including those fragments, variants, and combinations. “Fc region” may also include one or more hinge regions of the heavy chain constant region of the immunoglobulin. In certain embodiments, the Fc region is the CH1, C of the immunoglobulin. L , V L and / or V H It does not include one or more of the regions.

[0213] This Fc region can 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 their subclasses and combinations. In some embodiments, this Fc region is derived from IgA immunoglobulin containing subclass IgA1 and / or IgA2. In certain embodiments, this Fc region is derived from IgD immunoglobulin. In certain embodiments, this Fc region is derived from IgE immunoglobulin. In some embodiments, this Fc region is derived from IgG immunoglobulin containing subclass IgG1, IgG2, IgG3, and / or IgG4. In certain embodiments, this Fc region is derived from IgM immunoglobulin. Figure 2 shows the alignment of Fc regions derived 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] A specific Fc region demonstrates specific binding to one or more Fc receptors (FcRs). Examples of the Fc receptor class include the Fcγ receptor (FcγR), Fcα receptor (FcαR), Fcε receptor (FcεR), and neonatal Fc receptor (FcRn). For example, a specific Fc region has increased binding (or affinity for) to one or more FcγRs compared to FcαR, FcεR, and / or FcRn. In some embodiments, the Fc region has increased binding to FcαR compared to one or more FcγR, FcεR, and / or FcRn. In other embodiments, the Fc region has increased binding to FcεR (e.g., FcαRI) compared to one or more FcγR, FcαR, and / or FcRn. In certain embodiments, the Fc region has increased binding to FcRn compared to one or more FcγR, FcαR, and / or FcεR. In certain embodiments, the binding (or affinity) of an Fc region to one or more selected FcRs increases, typically around 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, 300×, 400×, 500×, 600×, 700×, 800×, 900×, 1000× or greater (including all integers in between), compared to its binding (or affinity for it) to one or more different FcRs.

[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 bursts, cytokine stimulation, and dendritic cell endocytotic transport. FcγRI expression is upregulated by both GM-CSF and γ-interferon (γ-IFN) and downregulated by interleukin-4 (IL-4). FcγRIIa is expressed on polymorphonuclear leukocytes (PMNs), macrophages, dendritic cells, and mast cells. FcγRIIa plays a role in phagocytosis, respiratory bursts, and cytokine stimulation. FcγRIIa expression is upregulated by GM-CSF and γ-IFN and decreased by IL-4. FcγIIb is expressed on B cells, PMNs, macrophages, and mast cells. FcγIIb inhibits the immune receptor tyrosine-based activation motif (ITAM)-mediated response and is therefore an inhibitory receptor. FcγRIIc expression is upregulated by intravenous immunoglobulin (IVIG) and IL-4, and decreased by γ-IFN. FcγRIIc is expressed on NK cells. FcγRIIIa is expressed on natural killer (NK) cells, macrophages, mast cells, and platelets. This receptor is involved in phagocytosis, respiratory bursts, cytokine stimulation, platelet aggregation and degranulation, and NK-mediated ADCC. FcγRIII expression is upregulated by C5a, TGF-β, and γ-IFN, and downregulated by IL-4. FcγRIIIb is a GPI-binding 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 compared to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc and / or FcγRIIIb. Certain Fc regions have increased binding to FcγRIIIb compared 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 consists of two extracellular Ig-like domains and is a member of both the immunoglobulin superfamily and the multi-chain immune recognition receptor (MIRR) family, and signals by associating with two FcRγ signaling chains.

[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, playing a major role in regulating allergic responses. FcεRI is also expressed on antigen-presenting cells and regulates the production of pro-inflammatory cytokines. The low-affinity receptor FcεRII (CD23) is a type C lectin that can function as a membrane-bound or soluble receptor. FcεRII regulates B cell proliferation and differentiation and blocks IgE binding on eosinophils, monocytes, and basophils. Certain Fc regions have increased binding to FcεRI compared to FcεRII. Other Fc regions have increased binding to FcεRII compared to FcεRI.

[0219] Table F1 below summarizes the characteristics of a specific FcR.

[0220] [Table 11]

[0221] The Fc region can be derived from immunoglobulin molecules of any animal, including vertebrates such as cattle, goats, pigs, dogs, mice, rabbits, hamsters, rats, guinea pigs, non-human primates, and mammals such as humans. The amino acid sequences of the CH2, CH3, CH4 and hinge regions from exemplary wild-type human IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM immunoglobulins are shown below (SEQ ID NOs: 128-154).

[0222] Sequence ID 128 is the amino acid sequence of the human IgA1 hinge region (VPSTPPTPSPSTPPTPSPS).

[0223] Sequence ID No. 129 is the amino acid sequence of the human IgA1 CH2 region (CCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKS).

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

[0225] Sequence ID 131 is the amino acid sequence (VPPPPP) of the human IgA2 hinge region.

[0226] Sequence ID 132 is the amino acid sequence of the human IgA2 CH2 region (CCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKS).

[0227] Sequence ID 133 is the amino acid sequence of the human IgA2 CH3 region (GNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY).

[0228] Sequence ID 134 is the amino acid sequence of the human IgD hinge region (ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTP).

[0229] Sequence ID 135 is the amino acid sequence of the human IgD CH2 region (ECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREP).

[0230] Sequence ID 136 is the amino acid sequence of the human IgD CH3 region (AAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPRSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK).

[0231] Sequence ID 137 is the amino acid sequence of the human IgE CH2 region (VCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCA).

[0232] Sequence ID 138 is the amino acid sequence of the human IgE CH3 region (DSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTS).

[0233] Sequence ID 139 is the amino acid sequence of the human IgE CH4 region (GPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK).

[0234] Sequence ID 140 is the amino acid sequence of the human IgG1 hinge region (EPKSCDKTHTCPPCP).

[0235] Sequence ID 341 is the amino acid sequence (SDKTHTCPPCP) of a modified human IgG1 hinge region.

[0236] Sequence ID 141 is the amino acid sequence of the human IgG1 CH2 region (APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK).

[0237] Sequence ID 142 is the amino acid sequence of the human IgG1 CH3 region (GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK).

[0238] Sequence ID 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, for example, during N-terminal fusion to the HRS polypeptide (see Sequence ID 340).

[0239] Sequence ID 143 is the amino acid sequence of the human IgG2 hinge region (ERKCCVECPPCP).

[0240] Sequence ID 144 is the amino acid sequence of the human IgG2 CH2 region (APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK).

[0241] Sequence ID 145 is the amino acid sequence of the human IgG2 CH3 region (GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK).

[0242] Sequence ID 146 is the amino acid sequence of the human IgG3 hinge region (ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP).

[0243] Sequence ID 147 is the amino acid sequence of the human IgG3 CH2 region (APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK).

[0244] Sequence ID 148 is the amino acid sequence of the human IgG3 CH3 region (GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK).

[0245] Sequence ID 149 is the amino acid sequence of the human IgG4 hinge region (ESKYGPPCPSCP).

[0246] Sequence ID 150 is the amino acid sequence of the human IgG4 CH2 region (APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK).

[0247] Sequence ID 151 is the amino acid sequence of the human IgG4 CH3 region (GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK).

[0248] Sequence ID 152 is the amino acid sequence of the human IgM CH2 region (VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVP).

[0249] Sequence ID 153 is the amino acid sequence of the human IgM CH3 region (DQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPK).

[0250] Sequence ID 154 is the amino acid sequence of the human IgM CH4 region (GVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY).

[0251] Accordingly, the HRS-Fc conjugate of the present invention may include, consist of, or essentially consist of, one or more of the human Fc region amino acid sequences of SEQ ID NOs. 128-163 or 339-342, including their variants, fragments, homologs, orthologs, paralogs, and combinations. Certain exemplary embodiments include an Fc region whose size is in the range of approximately 20-50, 20-100, 20-150, 20-200, 20-250, 20-300, 20-400, 50-100, 50-150, 50-200, 50-250, 50-300, 50-400, 100-150, 100-200, 100-250, 100-300, 100-350, 100-400, 200-250, 200-300, 200-350, or 200-400 amino acid lengths, and optionally include, consist of, or essentially consist of, any one or more of SEQ ID NOs: 128-154 or 341-342. Certain embodiments include an Fc region of up to approximately 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400 or more amino acids, and optionally include, consist of, or essentially consist of, any one or more of SEQ ID NOs. 128-154 or 339-342.

[0252] A particular Fc region contains, consists of, or essentially consists of, the human IgA1 sequences shown in sequence numbers 128-130 or 156, including their combinations (e.g., sequence numbers 128 and 129 and 130, sequence numbers 128 and 129; sequence numbers 128 and 130; sequence numbers 129 and 130), as well as their variants and fragments, in any order read from N-terminus to C-terminus. A particular Fc region contains, consists of, or essentially consists of, the human IgA1 sequence shown in sequence number 128. A particular Fc region contains, consists of, or essentially consists of, the human IgA1 sequence shown in sequence number 129. A particular Fc region contains, consists of, or essentially consists of, the human IgA1 sequence shown in sequence number 130.

[0253] Some Fc regions contain, consist of, or essentially consist of, the human IgA2 sequences shown in SEQ ID NOs. 131-133 or 157, in any order read from N-terminus to C-terminus, including combinations thereof (e.g., SEQ ID NOs. 131, 132, and 133; SEQ ID NOs. 131 and 132; SEQ ID NOs. 131 and 133; SEQ ID NOs. 132, 133, and 133.

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

[0255] A particular Fc region contains, consists of, or essentially consists of, the human IgE sequences shown in sequence numbers 137-139 or 163, in any order read from N-terminus to C-terminus, including their combinations (e.g., sequence numbers 137, 138, and 139; sequence numbers 137 and 138; sequence numbers 137 and 139; sequence numbers 138 and 139), as well as variants, fragments, and combinations of these sequences. A particular Fc region contains, consists of, or essentially consists of, the human IgE sequence shown in sequence number 137. A particular Fc region contains, consists of, or essentially consists of, the human IgE sequence shown in sequence number 138. A particular Fc region contains, consists of, or essentially consists of, the human IgE sequence shown in sequence number 139.

[0256] A particular Fc region contains, consists of, or essentially consists of, the human IgG1 sequences shown in SEQ ID NOs. 140-142 or 159 or 339-342, in any order read from N-terminus to C-terminus, including their combinations (e.g., SEQ ID NOs. 140, 141, and 142; SEQ ID NOs. 140 and 141; SEQ ID NOs. 141, 142, and 142). A particular Fc region contains, consists of, or essentially consists of, the human IgG1 sequences shown in SEQ ID NOs. 140. 141. 142. 339. A specific Fc region contains, is derived from, or is essentially derived from, the human IgG1 sequence shown in SEQ ID NO: 340. A specific Fc region contains, is derived from, or is essentially derived from, the human IgG1 sequence shown in SEQ ID NO: 341. A specific Fc region contains, is derived from, or is essentially derived from, the human IgG1 sequence shown in SEQ ID NO: 342.

[0257] A particular Fc region contains, consists of, or essentially consists of, the human IgG2 sequences shown in SEQ ID NOs. 143-145 or 160, in any order read from N-terminus to C-terminus, including their combinations (e.g., SEQ ID NOs. 143, 144, and 145; SEQ ID NOs. 143 and 144; SEQ ID NOs. 143 and 145; SEQ ID NOs. 144. 145.

[0258] A particular Fc region contains, consists of, or essentially consists of, the human IgG3 sequences shown in SEQ ID NOs. 146-148 or 161, in any order read from N-terminus to C-terminus, including their combinations (e.g., SEQ ID NOs. 146, 147, and 148; SEQ ID NOs. 146 and 147; SEQ ID NOs. 146 and 148; SEQ ID NOs. 147. 148.

[0259] A particular Fc region contains, consists of, or essentially consists of, the human IgG4 sequences shown in SEQ ID NOs. 149-151 or 162, in any order read from N-terminus to C-terminus, including their combinations (e.g., SEQ ID NOs. 149, 150, and 151; SEQ ID NOs. 149 and 150; SEQ ID NOs. 149 and 150; SEQ ID NOs. 151.

[0260] A particular Fc region contains, consists of, or essentially consists of, the human IgM sequences shown in SEQ ID NOs. 152-154 or 158, in any order read from N-terminus to C-terminus, including their combinations (e.g., SEQ ID NOs. 152, 153, and 154; SEQ ID NOs. 152 and 153; SEQ ID NOs. 152 and 154; SEQ ID NOs. 153; 154; 154; 153; 154.

[0261] As described above, certain embodiments utilize variants, fragments, hybrids, and / or other modified forms of Fc regions described herein and known in the art (e.g., human Ig sequences of sequence numbers 128-163).

[0262] The variants include those having one or more amino acid substitutions, insertions, deletions, and / or shortenings compared to any one or more of the reference sequences shown in SEQ ID NOs: 128-163. In certain embodiments, the variant Fc region includes 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, similarity, or homology with any one or more of the reference sequences shown in SEQ ID NOs: 128-163. The Fc region also includes one or more of sequence numbers 128-163 that are different from those of sequence numbers 128-163 due to the addition, deletion, insertion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 110, 120, 130, 140, 150 or more amino acids. In certain embodiments, these additions or deletions of amino acids are located 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 sequence numbers 128-163, and includes all integers and ranges between them, at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 2 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 The amino acid sequences included are capable of generating a BLAST bit score or sequence similarity score of 0, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more, where the BLAST alignment used a BLOSUM62 matrix, a gap presence penalty of 11, and a gap extension penalty of 1.

[0264] Hybrid Fc regions also include Fc regions that contain combinations of Fc domains (e.g., hinge, CH2, CH3, CH4) derived 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 / IgE, I 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 / IgA1, IgM / IgA2, IgM / IgD, IgM / IgE, IgM / IgG1, IgM / IgG2, IgM / IgG3, IgM / IgG4, IgM / IgM (or fragments or variants thereof), or essentially consisting thereof, and optionally including a hybrid Fc region containing 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 specific embodiments, this hinge, CH2, CH3, and CH4 domains are derived from 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 those combinations). The hybrid Fc region includes, consists of, or essentially consists of, 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 specific embodiments, this hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0266] Specific 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 their cross-sections). The hybrid Fc region includes, consists of, or essentially consists of (a fragment or variant) 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 specific embodiments, this hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0267] Specific examples include the following combinations of hinge / CH2 domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / 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 The hybrid Fc region includes, consists of, or essentially consists of gG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), and optionally includes 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 specific embodiments, this hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0268] Specific 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 The hybrid Fc region includes, consists of, or essentially consists of gG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), and optionally includes a CH2 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 specific embodiments, this hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0269] In some examples, the following combinations of hinge / CH4 domains are included, consist of, or essentially consist of IgG3 / IgE, IgG4 / IgM, IgG4 / IgE, IgG4 / IgM, IgG1 / IgE, IgG1 / IgM, IgG2 / IgE, IgG2 / IgM, IgG3 / IgE, IgG3 / IgM, IgG4 / IgE, IgG4 / IgM (or fragments or variants thereof), and the hybrid Fc region includes, optionally, a CH2 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM and / or a CH3 domain derived 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] The Fc region may also be modified by derivatization or other means. In certain embodiments, this Fc region may be modified, for example, by phosphorylation, sulfation, acrylication, glycosylation, methylation, farnesylation, acetylation, amidation, etc., compared to a wild-type or naturally occurring Fc region. In certain embodiments, this Fc region may include a wild-type or native glycosylation pattern, or may include increased glycosylation compared to the native form, decreased glycosylation compared to the native form, or may be completely deglycosylated. As an example of a modified Fc glycoform, decreased glycosylation of the Fc region reduces the binding of the first complement component C1 to the C1q region, the decrease in ADCC-related activity, and / or the decrease in CDC-related activity. Thus, certain embodiments use a deglycosylated or nonglycosylated Fc region. For example, WO See 2005 / 047337. Another example of an Fc region glycoform can be generated by substituting the Q295 position with a cysteine ​​residue according to the numbering system of Kabat et al. (see, for example, U.S. Patent Application No. 2010 / 0080794). Certain embodiments may include an Fc region containing a mature core carbohydrate structure in which approximately 80–100% of the glycoprotein in the Fc region lacks fructose (see, for example, U.S. Patent Application No. 2010 / 0255013). Some embodiments may include an Fc region optimized by substitution or deletion to reduce the level of fucosylation, for example, to increase affinity for FcγRI, FcγRIa, or FcγRIIIa and / or to improve phagocytosis by FcγRIIa-expressing cells (see U.S. Patent Application Nos. 2010 / 0249382 and U.S. Patent Application No. 2007 / 0148170).

[0272] Another example of a modified Fc glycoform is an Fc region which may contain an oligomannose-type N-glycan and optionally have one or more of the following: increased ADCC activity compared to the corresponding Fc region or HRS-Fc conjugate containing a complex-type N-glycan; increased binding affinity to FcγRIIIA (and certain other FcR); similar or increased binding specificity to HRS polypeptide targets; similar or higher binding affinity to HRS polypeptide targets; and / or similar or lower binding affinity to mannose receptors (see, for example, U.S. Patent Application No. 2007 / 0092521 and U.S. Patent No. 7,700,321). As another example, enhanced affinity of the Fc region to FcγR has been achieved using manipulated glycoforms generated by antibody expression in manipulated or variant cell lines (see, e.g., Umana et al., Nat Biotechnol. 17: pp. 176-180, 1999; Davies et al., Biotechnol Bioeng. 74: pp. 288-294, 2001; Shields et al., J Biol Chem. 277: pp. 26733-26740, 2002; Shinkawa et al., 2003, J Biol Chem. 278: pp. 3466-3473, 2003; and U.S. Patent Application No. 2007 / 0111281). Certain Fc region glycoforms include an increased proportion of N-glycosidic complex glycans that lack the fucose at position 1 of N-acetylglucosamine at position 6 of the glycan's reducing end (see, for example, U.S. Patent Application No. 2010 / 0092997). Certain embodiments may include an Fc region of IgG glycosylated by at least one galactose moiety linked to each terminal sialic acid moiety by an α-2,6 linkage, which optionally has higher anti-inflammatory activity compared to the corresponding wild-type Fc region (see U.S. Patent Application No. 2008 / 0206246).Certain of these and related modified glycosylation approaches, as described herein, result in a substantial enhancement of the Fc domain's ability to selectively bind to FcR such as FcγRIII, its ability to mediate ADCC, and its ability to modify other properties of the Fc domain.

[0273] Certain variants, fragments, hybrids, 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 an Fc region may have increased binding to one or more of the Fcγ receptor, Fcα receptor, Fcε receptor, and / or neonatal Fc receptor compared to the corresponding wild-type Fc sequence. In other embodiments, a variant, fragment, hybrid, or modified Fc region may have decreased binding to one or more of the Fcγ receptor, Fcα receptor, Fcε receptor, and / or neonatal Fc receptor compared to the corresponding wild-type Fc sequence. Specific FcRs are described elsewhere in this specification.

[0274] Specific examples of Fc variants having 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 in WO 2000 / 42072 and WO 2004 / 016750. Specific examples include human Fc regions having one or more substitutions at positions 298, 333, and / or 334, e.g., S298A, E333A, and / or K334A (based on the EU index numbering by Kabat et al.), which have been shown to increase binding to the activating receptor FcγRIIIa and decrease binding to the inhibitory receptor FcγRIIb. These mutations can be combined to obtain double and triple mutant variants with further improvements in binding to FcR. Certain embodiments include the S298A / E333A / K334A triple mutant having increased binding to FcγRIIIa, decreased binding to FcγRIIb, and increased ADCC (see, e.g., Shields et al., J Biol Chem. vol. 276: pp. 6591-6604, 2001; and Presta et al., Biochem Soc Trans. vol. 30: pp. 487-490, 2002). See also Umana et al., above; and engineered Fc glycoforms having increased binding to FcR, as disclosed in U.S. Patent No. 7,662,925. Some embodiments include an Fc region that includes one or more substitutions selected from 434S, 252Y / 428L, 252Y / 434S, and 428L / 434S (see U.S. Patent Application No. 2009 / 0163699 and U.S. Patent Application No. 20060173170) based on the EU index of Kabat et al.

[0275] Certain variants, fragments, hybrids, or modified Fc regions have altered effector functions compared to the corresponding wild-type Fc sequence. For example, such an Fc region may have increased complement binding or activation, increased Clq binding affinity, increased CDC-related activity, increased ADCC-related activity, and / or increased ADCP-related activity compared to the corresponding wild-type Fc sequence. In other embodiments, such an Fc region may have decreased complement binding or activation, decreased Clq binding affinity, decreased CDC-related activity, decreased ADCC-related activity, and / or decreased ADCP-related activity compared to the corresponding wild-type Fc sequence. As merely illustrative examples, an Fc region may include deletions or substitutions at complement binding sites, such as C1q binding sites, and / or deletions or substitutions at ADCC sites. Examples of such deletions / substitutions are described, for example, in U.S. Patent No. 7,030,226. Many Fc effector functions, such as ADCC, can be assayed according to conventional techniques in the field (see, e.g., Zuckerman et al., CRC Crit Rev Microbiol. 7: pp. 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 furthermore, specific Fc effector functions can be evaluated in vivo by using animal models, for example, Clynes et al., PNAS. 95: pp. 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 an Fc region may have an increased half-life compared to the corresponding wild-type Fc sequence. In other embodiments, a variant hybrid or modified Fc region may have a decreased half-life compared to the corresponding wild-type Fc sequence. Half-life can be measured in vitro (e.g., under physiological conditions) or in vivo according to conventional techniques in the art, such as radiolabeling, ELISA, or other methods. In vivo measurements of stability or half-life can be performed in one or more bodily fluids, including blood, serum, plasma, urine, or cerebrospinal fluid, or in a given tissue, such as the liver, kidney, muscle, central nervous system tissue, or bone. As an example, modifications to an Fc region that alter its ability to bind to FcRn may alter its half-life in vivo. Assays for measuring in vivo pharmacokinetic properties (e.g., mean efflux half-life in vivo) and non-limiting examples of Fc modifications that alter their binding to FcRn are described, for example, in U.S. Patent Nos. 7,217,797 and 7,732,570; and U.S. Patent Application Nos. 2010 / 0143254.

[0277] Further non-limiting examples of modifications to alter stability or half-life include substitutions / deletions in one or more amino acid residues selected from 251–256, 285–290, and 308–314 in the CH2 domain, and 385–389 and 428–436 in the CH3 domain, according to the numbering system of Kabat et al. See U.S. Patent Application No. 2003 / 0190311. Specific examples include any combination thereof, substitution by leucine at position 251, substitution by tyrosine, tryptophan, or phenylalanine at position 252, substitution by threonine or serine at position 254, substitution by arginine at position 255, substitution by glutamine, arginine, serine, threonine, or glutamic acid at position 256, substitution by threonine at position 308, substitution by proline at position 309, substitution by serine at position 311, substitution by aspartic acid at position 312, substitution by leucine at position 314, and 38 This includes substitutions with arginine, aspartic acid, or serine at position 5; threonine or proline at position 386; arginine or proline at position 387; proline, asparagine, or serine at position 389; methionine or threonine at position 428; tyrosine or phenylalanine at position 434; histidine, arginine, lysine, or serine at position 433; and / or histidine, tyrosine, arginine, or threonine at position 436. Such modifications optionally increase the affinity of the Fc region to FcRn compared to the corresponding wild-type Fc region, thereby increasing the half-life.

[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 may be measured, for example, in vitro (e.g., under physiological conditions) according to conventional techniques in the art. Exemplary solubility measurements are described elsewhere in this specification.

[0279] Further examples of variants include the Fc region of IgG having conserved or non-conserved substitutions (as described elsewhere herein) at one or more of the 250, 314, or 428 positions of the heavy chain, or any combination thereof, for example, at positions 250 and 428, or 250 and 314, or 314 and 428, or 250, 314 and 428 (see, for example, U.S. Patent Application No. 2011 / 0183412). In 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 exemplary example of an IgG Fc variant, any one or more amino acid residues at positions 214–238, 297–299, 318–322, and / or 327–331 can be used as suitable targets for modification (e.g., conserved or non-conserved substitutions, deletions). In certain embodiments, the CH2 domain of this IgG Fc variant includes amino acid substitutions at positions 228, 234, 235, and / or 331 to attenuate the effector function of the Fc region (e.g., human IgG4 with Ser228Pro and Leu235Ala mutations) (see U.S. Patent No. 7,030,226). Here, the numbering of residues in the heavy chain is EU index numbering (see Kabat et al., "Sequences of Proteins of Immunological Interest," 5th edition, National Institutes of Health, Bethesda, Md. (1991)). Certain of these and related embodiments optionally alter (e.g., increase, decrease) FcRn binding and / or serum half-life without reducing effector activity such as ADCC-related activity or CDC-related activity.

[0280] Further examples include variant Fc regions containing one or more amino acid substitutions at positions 279, 341, 343, or 373 of the wild-type Fc region, or any combination thereof (see, for example, U.S. Patent Application No. 2007 / 0224188). For human IgG, the wild-type amino acid residues at these positions are valine (279), glycine (341), proline (343), and tyrosine (373). The substitution(s) may be conserved or non-conserved, or may include amino acids or mimics that do not exist naturally, as described herein. In addition to these substitutions, certain embodiments may also use variant Fc regions comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions selected from: 235G, 235R, 236F, 236R, 236Y, 237K, 237N, 237R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 244L, 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 described above, the numbering of residues in the heavy chain is as follows:This is a numbering system for EU indicators (see Kabat et al., above). Such variant Fc regions typically confer altered effector function or altered serum half-life to the HRS polypeptide to which the variant Fc region is operably bound. Preferably, this altered effector function is an increase or decrease in ADCC, an increase or decrease in CDC, an increase or decrease in Clq binding affinity, an increase or decrease in FcR (preferably FcRn) binding affinity, or an increase or decrease in FcR (preferably FcRn) binding affinity compared to the corresponding Fc region lacking such amino acid substitution(s).

[0281] Further examples include 221st, 222nd, 224th, 227th, 228th, 230th, 231st, 223rd, 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 241st, 243rd, 244th, 245th, 246th, 247th, 249th, 250th, 258th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272nd, 273rd, 274th, 275th, 276th, 278th, 280th, 281st, 283rd, 285th, 2 The variant Fc region includes an amino acid substitution at one or more of the following 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, for example, U.S. Patent No. 7,662,925). In a specific embodiment, this variant Fc region is P230A, E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T, K274E, K274R, K274L, K274Y, F275 The substitution includes 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, this variant Fc region is V264I, F243L / V264I, L328M, I332E, L328M / I332E, V264I / I332E, S298A / I332E, S239E / I332E, S239Q / I332E, S239E, A330Y, I332D, L328I / I332E,L328Q / I332E, V264T, V240I, V266I, S239D, S239D / I332D, S239D / I332E, S239D / I332N, S239D / I332Q, S239E / I332D, S239E / I332N, S239E / 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 The variant comprises at least one amino acid substitution selected from the group consisting of 4D, S324I, S324V, K326I, K326T, T335D, T335R, T335Y, V240I / V266I, S239D / A330Y / I332E / L234I, S239D / A330Y / I332E / L235D, S239D / A330Y / I332E / V240I, S239D / A330Y / I332E / V264T, S239D / A330Y / I332E / K326E, and S239D / A330Y / I332E / K326T. In a more specific embodiment, the variant Fc region is N297D / I332E,F241Y / F243Y / V262T / V264T / N297D / I332E, S239D / N297D / I332E, S239E / N297D / I 332E, S239D / D265Y / N297D / I332E, S239D / D265H / N297D / I332E, V264E / N297D / I3 The variant includes a series of substitutions selected from the group consisting of 32E, Y296N / N297D / I332E, N297D / A330Y / I332E, S239D / D265V / N297D / I332E, S239D / D265I / N297D / I332E, and N297D / S298A / A330Y / I332E. In specific embodiments, this variant Fc region includes an amino acid substitution at position 332 (using EU index, Kabat et al., the numbering above). Examples of substitutions include 332A, 332D, 332E, 332F, 332G, 332H, 332K, 332L, 332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, and 332Y. The numbering of residues in the Fc region follows the EU index numbering by Kabat et al. Among other properties described herein, such variant Fc regions may have increased affinity for FcγR, increased stability, and / or increased solubility compared to the corresponding wild-type Fc region.

[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 The variant Fc region includes one or more of P, 438L, 439E / R, 440G, 441F, 442T, 445R, 446A, and 447E, where optionally, this variant has altered recognition and / or altered effector function of the Fc ligand compared to the parent Fc polypeptide, and the residue numbering is the EU index numbering by Kabat et al. Specific examples of these and related embodiments include the following 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 The variant Fc region includes or comprises 5S, F372L, A378T, N390D, Y391C, F404S, E430K, L432P, and K447E; and (23)E269G, Y278H, N325S, and K370R, where the residue numbering is the EU index numbering by Kabat et al. (see, for example, U.S. Patent Application No. 2010 / 0184959).

[0283] Another specific example of an Fc variant is the sequence of Sequence ID No. 155, in which Xaa at position 1 is Ala or absent; Xaa at position 16 is Pro or Glu; Xaa at position 17 is Phe, Val or Ala; Xaa at position 18 is Leu, Glu or Ala; Xaa at position 80 is Asn or Ala; and / or Xaa at position 230 is Lys or absent (see, for example, U.S. Patent Application No. 2007 / 0253966). These specific Fc regions and associated HRS-Fc conjugates have increased half-life, reduced effector activity, and / or significantly lower immunogenicity than wild-type Fc sequences.

[0284] The variant Fc region may also have one or more mutated hinge regions, as described, for example, in U.S. Patent Application No. 2003 / 0118592. For example, one or more cysteines in the hinge region may be deleted or substituted with different amino acids. The mutated hinge region may contain no cysteine ​​residues or contain one, two, or three fewer cysteine ​​residues than the corresponding wild-type hinge region. In some embodiments, the Fc region having this type of mutated hinge region exhibits reduced dimerization ability compared to the wild-type Ig hinge region.

[0285] As described above, HRS-Fc conjugates, such as HRS-Fc fusion proteins, typically have modified (e.g., improved, increased, or decreased) pharmacokinetic properties compared to the corresponding HRS polypeptides. Examples of pharmacokinetic properties include stability or half-life, bioavailability (fraction of drug absorbed), tissue distribution, volume of distribution (apparent volume at which the drug is distributed immediately after intravenous injection and reaches equilibrium between plasma and surrounding tissue), concentration (initial or steady-state concentration of the drug in plasma), efflux rate constant (rate at which the drug is removed from the body), efflux rate (rate of infusion required to balance efflux), area under the curve (AUC or exposure; integral of the concentration-time curve after a single dose or at steady state), clearance (volume of plasma cleared per unit time of the drug), and C max (Peak plasma concentration of the drug after oral administration), t max (C max (Time until it reaches), C min This includes the lowest concentration the drug reaches before the next dose is administered, and variability (peak-trough variation within a single dosing interval in a steady state). In some embodiments, these improved properties are achieved without significant alteration of the secondary structure and / or reduction of the non-canonical biological activity of the HRS polypeptide. In fact, some HRS-Fc conjugates have increased non-canonical biological activity.

[0286] Accordingly, in some embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has a plasma or serum pharmacokinetic AUC profile that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 200, 300, 400, or 500 times larger than the corresponding unmodified or differently modified HRS polypeptide when administered to mammals under the same or comparable conditions. In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide exhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% greater stability (e.g., measured by half-life) compared to the corresponding unmodified or differently modified HRS polypeptide when compared in PBS at pH 7.4 for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or 1, 2, 3, 4 weeks under similar conditions at room temperature.

[0287] In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide is used for approximately 30 minutes, approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 12 hours, approximately 18 hours, approximately 20 hours, approximately 24 hours, approximately 30 hours, approximately 36 hours, approximately 40 hours, approximately 48 hours, approximately 50 hours, approximately 60 hours, approximately 70 hours, approximately 72 hours, approximately 80 hours, approximately 84 hours, approximately 90 hours, approximately 96 hours, approximately 120 hours, or approximately 144 hours or longer. , or having a biological half-life of at least approximately 30 minutes, approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 12 hours, approximately 18 hours, approximately 20 hours, approximately 24 hours, approximately 30 hours, approximately 36 hours, approximately 40 hours, approximately 48 hours, approximately 50 hours, approximately 60 hours, approximately 70 hours, approximately 72 hours, approximately 80 hours, approximately 84 hours, approximately 90 hours, approximately 96 hours, approximately 120 hours or approximately 144 hours or more, at pH 7.4, 25°C, for example, physiological pH, human body temperature (e.g., in vivo, in serum, in a given tissue, in a given species such as a rat, mouse, monkey or human), or any intervening half-life.

[0288] In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has greater bioavailability after subcutaneous (SC) administration compared with the corresponding unmodified HRS polypeptide. In certain embodiments, the HRS-Fc conjugate or HRS-Fc fusion polypeptide has at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more bioavailability compared with 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 as the corresponding unmodified or differently modified HRS polypeptide in an anti-inflammatory activity assay. In other embodiments, the HRS-Fc fusion polypeptide has more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times the activity of the corresponding unmodified or differently modified HRS polypeptide in an anti-inflammatory activity assay.

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

[0291] A specific peptide linker sequence may be selected based on the following exemplary factors: (1) the ability to adopt a flexible, extended conformation; (2) the inability to adopt a secondary structure that can interact with functional epitopes on the first and second polypeptides; (3) physiological stability; and (4) the absence of hydrophobic or charged residues that can react with functional epitopes on the polypeptides, or other characteristics. See, for example, George and Heringa, J Protein Eng. Vol. 15: pp. 871-879, 2002.

[0292] Linker sequences can generally be 1 to about 200 amino acids long. Specific linkers can be about 1-200 amino acids, 1-150 amino acids, 1-100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, It may have the total amino acid length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more amino acids.

[0293] The peptide linker may be any one or more naturally occurring amino acids, non-naturally occurring amino acids, amino acid analogs, and / or amino acid mimes, as described elsewhere in this specification and known in the art. Specific amino acid sequences that may be useful as linkers include those disclosed in Maratea et al., Gene 40: pp. 39–46, 1985; Murphy et al., PNAS USA. 83: pp. 8258–8262, 1986; and U.S. Patents 4,935,233 and 4,751,180. Specific peptide linker sequences include Gly residues, Ser residues, and / or Asn residues. Other nearly neutral amino acids, such as Thr and Ala, may also be used in the peptide linker sequence, 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 (Sequence ID 200), [GGSG] x (Sequence No. 201), [GGGS] x (Sequence ID 202), [GGGGS] x (Sequence ID 203), [GN] x [GGN] x [GNN] x [GNGN] x (Sequence No. 204), [GGNG] x (Sequence No. 205), [GGGN] x (Sequence No. 206), [GGGGN] x (Sequence No. 207) contains a linker, in the formula, x The values ​​are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. Other combinations of these and related amino acids 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-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 208); Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-(SEQ ID NO: 209); Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-G ly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(Sequence ID 210); Asp-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Lys-Asp-Ala-Ala-Ala-Arg-Glu-Ala-Ala-Ala-Arg-Asp-Ala-Ala-Ala-Lys-(Sequence ID 211); and Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Arg-(Sequence ID 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. Vol. 94: pp. 5525-5530, 1997); and GGRR (SEQ ID NO: 215) (Pomerantz et al.). 1995);(GGGGS) nThis includes (SEQ ID NO: 203) (Kim et al., PNAS. Vol. 93: pp. 1156-1160, 1996); EGKSSGSGSESKVD (SEQ ID NO: 216) (Chaudhary et al., PNAS. Vol. 87: pp. 1066-1070, 1990); KESGSVSSEQLAQFRSLD (SEQ ID NO: 217) (Bird et al., Science. Vol. 242: pp. 423-426, 1988), GGRRGGGS (SEQ ID NO: 218); LRQRDGERP (SEQ ID NO: 219); LRQKDGGGSERP (SEQ ID NO: 220); LRQKd(GGGS)2ERP (SEQ ID NO: 221). In specific embodiments, this linker sequence includes a Gly3 linker sequence containing three glycine residues. In certain embodiments, the flexible linker may be rationally designed using a computer program capable of modeling both the DNA binding site and the peptide itself (Desjarlais and Berg, PNAS. 90: pp. 2256-2260, 1993; and PNAS. 91: pp. 11099-11103, 1994), or by phage display.

[0297] The peptide linker may be physiologically stable or may include a releaseable linker, such as a physiologically degradable linker or an enzymatically cleavable linker (e.g., a proteolytically cleavable linker). In certain embodiments, one or more releaseable linkers may result in a shorter half-life and faster clearance of the conjugate. These and related embodiments may be used, for example, to enhance the solubility and circulating lifetime of HRS polypeptides in the bloodstream, while also delivering HRS polypeptides substantially free of Fc regions into the bloodstream following linker degradation. These embodiments are particularly useful when demonstrating reduced activity when HRS polypeptides are permanently conjugated to Fc regions. By using the linkers provided herein, such HRS polypeptides may maintain their therapeutic activity when in the conjugated form. As another example, a large, relatively inactive HRS-Fc conjugate polypeptide may be administered, which is then degraded in vivo (via a degradable linker) to produce a bioactive HRS polypeptide that possesses a portion of the Fc region or lacks the Fc region entirely. In these and other ways, the properties of the HRS-Fc conjugate polypeptide can be more efficiently individualized to balance the bioactivity and circulating half-life of the HRS polypeptide over time.

[0298] In certain embodiments, the linker peptide includes an autocatalytic or autocleavable peptide cleavage site. In a particular embodiment, the autocleavable peptide includes polypeptide sequences obtained from potivirus and cardiovirus 2A peptide, FMDV (foot-and-mouth disease virus), equine rhinitis A virus, Thosea asigna virus, and porcine teishouvirus. In a particular embodiment, this autocleavable polypeptide site includes a 2A or 2A-like site, sequence, or domain (see Donnelly et al., J. Gen. Virol. 82: pp. 1027-1041, 2001). The example 2A site includes the following sequences: LLNFDLLKLAGDVESNPGP(sequence code 222);TLNFDLLKLAGDVESNPGP(sequence code 223);LLKLAGDVESNPGP(sequence code 224);NFDLLKLAGDVESNPGP(sequence code 225);QLLNFDLLKLAGDVESNPGP(sequence code 226);APVKQTLNFDLLKLAGDVESNPGP(sequence code 227);VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT(sequence code 228);LNFDLLKLAGDVESNPGP(sequence code 229);LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP(sequence code 230); and EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP(sequence code 231). In one embodiment, the autocatalytic peptide cleavage site includes a translation 2A signal sequence, such as the 2A region of the aft-virus foot-and-mouth disease virus (FMDV) polyprotein, which is a sequence of 18 amino acids. Further examples of 2A-like sequences that can be used include insect virus polyproteins, the NS34 protein of rotavirus type C, and repeat sequences in Trypanosoma spp., as described by Donnelly et al., Journal of General Virology, Vol. 82: pp. 1027-1041, 2001.

[0299] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., J. Gener. Virol. Vol. 78: pp. 699-722, 1997; and Scymczak et al., Nature Biotech. Vol. 5: pp. 589-594, 2004). Exemplary protease cleavage sites include, but are not limited to, those of potivirus NIa protease (e.g., tobacco eczema virus protease), potivirus HC protease, potivirus P1 (P35) protease, biovirus NIa protease, biovirus RNA-2 encoding protease, aftovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungrosspheric virus) 3C-like protease, PYVF (parsnip's macular virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to its high cleavage stringency, some embodiments include TEV (tobacco eczema virus) protease cleavage sites such as EXXYXQ(G / S) (SEQ ID NO: 232), for example, ENLYFQG (SEQ ID NO: 233) and ENLYFQS (SEQ ID NO: 234), where X represents any amino acid (cleavage by TEV occurs between Q and G or between Q and S).

[0300] Further examples of enzymatically cleavable linkers suitable for use in specific embodiments of the present invention include, but are not limited to, amino acid sequences cleaved by serine proteases, such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or subtilisin. Exemplary examples of thrombin-cleavable amino acid sequences include, but are not limited to, -Gly-Arg-Gly-Asp-(SEQ ID NO: 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-. Exemplary 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. Exemplary examples of amino acid sequences cleavable by matrix metalloproteinases 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. Exemplary 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 example of a stromelysin-cleavable amino acid sequence is -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg-(SEQ ID NO: 255); an 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 specific 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 specific 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 releaseable linker has a half-life of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours or 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 individualized by using a specific releaseable 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-terminal and / or C-terminal amino acid regions that can be used to separate functional domains and prevent steric hindrance, the linker sequence may not be required.

[0306] Instructions for use Embodiments of the present invention relate to the discovery that Fc-domain-histidyl-tRNA synthetase (HRS-Fc) conjugate polypeptides, as well as their fragments and variants, provide improved methods for modulating inflammatory responses in a variety of useful ways, both in vitro and in vivo. Accordingly, 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 diseases, by modulating cells that directly or indirectly mediate such inflammatory and / or autoimmune diseases, conditions, and disorders. The usefulness of the compositions of the present invention as immunomodulators may be monitored using any of several available techniques known in the art, 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] In general, "inflammation" refers to the biological response of tissue to harmful stimuli, such as pathogens, damaged cells (e.g., wounds), and irritants. The term "inflammatory response" refers, by example only, to the specific mechanisms by which inflammation is achieved and regulated, including, among others described herein and known in the art, activation or migration of immune cells, autoimmune and autoimmune diseases, cytokine production, kinin release, fibrinolysis, and coagulation and vasodilation. Ideally, inflammation is the body's attempt at defense, both to remove the damaging stimulus and to initiate the healing process for the affected tissue(s). In the absence of inflammation, wounds and infections never heal, creating a situation where progressive destruction of tissue threatens survival. On the other hand, excessive or chronic inflammation may be associated with various diseases, among others described herein and known in the art, such as hay fever, atherosclerosis, and rheumatoid arthritis.

[0308] The clinical signs of chronic inflammation depend on the duration of the disease, the inflammatory lesion, the cause, and the anatomical region affected (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, among others described herein and known in the art, allergies, Alzheimer's disease, anemia, aortic stenosis, arthritis, e.g., rheumatoid arthritis and osteoarthritis, cancer, congestive heart failure, fibromyalgia, fibrosis, heart attack, renal 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 mellitus, metabolic disorders, obesity, and psoriasis. Many other chronic diseases may also include inflammatory components and, therefore, may be treated with the HRS-Fc conjugate of the present invention, including, for example, muscular dystrophy and rhabdomyolysis. Accordingly, the HRS-Fc conjugate may be used to treat or manage chronic inflammation, to modulate one or more of the individual chronic inflammatory responses, or to treat any one or more diseases or conditions associated with chronic inflammation.

[0309] Specific inflammatory responses include cytokine production and activity, or related pathways. For example, certain exemplary embodiments relate to modulating nuclear factor-κB (NF-κB)-mediated cellular signaling, for instance, by increasing the downstream activity of this transcription factor. In certain cases, an increase in NF-κB activity may result in an increase in cytokine signaling or activity, such as 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 differential diagnosis and monitoring of treatment, such as determining whether a therapeutically effective dose was administered during the course of treatment by determining improvement according to accepted clinical criteria, are evident to those skilled in the art, e.g., Berkow et al., The Merck Manual, 16th edition, Merck and Co., Rahway, NJ, 1992; Goodman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th edition, Pergamon Press, Inc., Elmsford, NY, (2001); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, Ltd., Williams and Wilkins, Baltimore, MD. (1987); Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985); Osolci et al., Remington's This is illustrated by the teachings in Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, PA (1990); and Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, CT (1992).

[0311] Methods for modulating immune responses, such as innate or adaptive immune responses, through the use of any of the HRS-Fc conjugates described herein are also included. As used herein, the term “immune response” includes measurable or observable responses 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 an antigen or immunomodulatory molecule to immune system cells. Responses to antigens or immunomodulatory molecules can be mediated by many cell types, including cells that initially bind to antigens or immunomodulatory molecules and cells involved in mediating innate, humoral, and cell-mediated immune responses.

[0312] Methods for treating immune disorders are also included. Exemplary immune system disorders, conditions, 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 transplantation in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic inflammatory demyelinating polyneuropathy, and post-transfusion purpura.

[0313] Furthermore, additional diseases, disorders, and conditions that can 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 at high risk of 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., hypogammaglobulinemia in 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] Further autoimmune diseases, disorders, and conditions that can 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, polyendochnopathy, purpura (e.g., Henoch-Schoenlein purpura), Reiter's disease, Stiff-Mann syndrome, autoimmune pneumonia, Guillain-Barré syndrome, insulin-dependent diabetes mellitus, and autoimmune inflammatory eye diseases.

[0315] Further autoimmune diseases, disorders, or conditions that can be treated with any of the HRS-Fc conjugates described herein include, but are not limited to, autoimmune thyroiditis; hypothyroidism, including Hashimoto's thyroiditis and thyroiditis characterized by, for example, cell-mediated and humoral thyroid cytotoxicity; SLE (often characterized by, for example, circulating and locally generated immune complexes); Goodpasture syndrome (often characterized by, for example, anti-basement membrane antibodies); pemphigus (often characterized by, for example, epithelial acantholytic antibodies); receptor autoimmunity such as Graves' disease (often characterized by, for example, antibodies against thyroid-stimulating hormone receptors; and myasthenia gravis (often characterized by, for example, acetylcholine receptor antibodies); insulin resistance (often characterized by, for example, insulin receptor antibodies); autoimmune hemolytic anemia (often characterized by, for example, phagocytosis of antibody-sensitized red blood cells); and autoimmune thrombocytopenic purpura (often characterized by, for example, phagocytosis of antibody-sensitized platelets).

[0316] Further autoimmune diseases, disorders, or conditions that can 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 against extractable nuclear antigens, e.g., ribonucleoproteins); polymyositis / dermatomyositis (e.g., often characterized by non-histone antinuclear antibodies); pernicious anemia (e.g., often characterized by antiparietal, anti-microsome, 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 antibodies) Adrenergic drug resistance (often characterized by beta-adrenergic receptor antibodies), including ad which is often characterized by (antispennatozoal) antibodies; glomerulonephritis (often characterized by, e.g., glomerular basement membrane antibodies or immune complexes); IgA nephropathy due to primary glomerulonephritis; bullous pemphigoid (often characterized by, e.g., IgG and complement in the basement membrane); Sjögren's syndrome (often characterized by, e.g., multitissue antibodies and / or specific nonhistone antinuclear antibodies (SS-B)); diabetes mellitus (often characterized by, e.g., cell-mediated and humoral islet cell antibodies); and adrenergic drug resistance (often characterized by, e.g., beta-adrenergic receptor antibodies), which is often characterized by, adrenergic drug resistance, including adrenergic drug resistance with asthma or cystic fibrosis.

[0317] Further autoimmune diseases, disorders, or conditions that can 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 anti-mitochondrial antibodies); other endocrine gland failures (e.g., sometimes characterized by specific tissue antibodies); vitiligo (e.g., often characterized by anti-melanocyte antibodies); and vasculitis (e.g., characterized by immunoglobulins and complement in the vascular wall and / or low serum complement). This includes, but is not limited to, other inflammatory, granulomatous, degenerative, and atrophic disorders; post-myocardial infarction conditions (e.g., often characterized by anti-myocardial antibodies); open-heart surgery syndromes (e.g., often characterized by anti-myocardial antibodies); urticaria (e.g., often characterized by IgG and IgM antibodies against IgE); atopic dermatitis (e.g., often characterized by IgG and IgM antibodies against IgE); asthma (e.g., often characterized by IgG and IgM antibodies against IgE); inflammatory myopathy; and other inflammatory, granulomatous, degenerative, and atrophic disorders.

[0318] Further 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), Barlow concentric sclerosis, Schilder diffuse sclerosis, Marburg-type 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 disorder (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 embodiments, the present invention includes a method for reducing muscle or lung inflammation associated with autoimmune diseases, comprising the step of administering a composition comprising one of the HRS-Fc conjugates described herein to a subject in need thereof. Exemplary inflammatory diseases and disorders of muscle include muscular dystrophy, exercise-induced myositis, inflammation associated with muscle injury or surgery, rhabdomyolysis, and related diseases and disorders described herein.

[0320] A method for treating a disease associated with an autoantibody, comprising the step of administering a therapeutic composition comprising one of the HRS-Fc conjugates described herein to a subject in need thereof, wherein the HRS polypeptide comprises at least one epitope specifically recognized by the autoantibody.

[0321] A particular embodiment of the method for inducing tolerance to a histidyl-tRNA synthetase (HisRS) antigen comprises the step of administering to a subject a composition comprising one of the HRS-Fc conjugates described herein, wherein the HRS polypeptide comprises at least one epitope that is specifically recognized by an autoantibody, and the administration of the composition causes tolerance to the autoantigen.

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

[0323] In another embodiment, the present invention provides a method for inducing anergy in T cells involved in an autoimmune response to a histidyl-tRNA synthetase (HRS) autoantigen, comprising the step of administering to a subject a composition comprising one 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 the administration of the composition causes functional inactivation of the T cells involved in the autoimmune response.

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

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

[0326] In any of these methods, the term “tolerance” refers to a sustained reduction or absence of the immune response to a specific antigen in mammals, particularly humans. Tolerance is distinct from general immunosuppression, where all of the immune response, or all of a particular class of immune cells in the immune response, e.g., the B-cell mediated immune response, is reduced or eliminated. The onset of tolerance can be conventionally monitored by the absence or reduction of the concentration of antibodies against HRS polypeptides in the serum of a host subject after administration of a single or sequential dose of a treatment HRS-Fc conjugate. The onset of tolerance is typically sufficient to reduce the symptoms of the autoimmune disease in the patient, for example, the patient may be sufficiently improved to maintain normal activity in the absence or reduced doses of common immunosuppressants such as corticosteroids.

[0327] In any of these methods and compositions, tolerance is typically persistent, meaning that tolerance has a duration of approximately one month, two months, three months, four months, five months, or six months or longer. Tolerance may result in selective B-cell anergy, 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 against histidyl-tRNA synthetase are detected or detectable, regardless of whether other autoantibodies are also detected or are thought to play a role in the progression or cause of the disease. Methods for detecting antibodies in patient samples may be carried out by any standard procedure, including, for example, by 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 specificity. In some embodiments, antibody specificity may be further characterized by determining the ability of antibodies to selectively bind to different splice variants and truncated or proteolytic forms of histidyl-tRNA synthetase. Relatively well-known human autoantibodies against histidyl-tRNA synthetase include, for example, antibodies against Jo-1.

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

[0330] In some embodiments of the claimed method, the HRS polypeptide or HRS-Fc conjugate comprises an immunodominant epitope that is specifically recognized by a majority of antibodies derived from the serum of patients having autoantibodies against histidyl-tRNA synthetase and associated diseases.

[0331] In some embodiments, this 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); or any combination thereof of the HRS polypeptide.

[0332] In some embodiments, the HRS polypeptide does not contain an epitope that specifically cross-reacts with disease-associated autoantibodies against histidyl-tRNA synthetase. In some embodiments, the HRS polypeptide has up to approximately 1 × 10⁻⁶ -7 Up to concentration M, there is no significant competition for the binding of disease-associated autoantibodies to histidyl-tRNA synthetase in competitive ELISA. In some embodiments, this HRS polypeptide binds up to approximately 5 × 10⁶ times. -7 Up to concentration M, there is no significant competition for the binding of disease-associated autoantibodies to histidyl-tRNA synthetase in competitive ELISA. In some embodiments, this HRS polypeptide can bind up to approximately 1 × 10⁶ times. -6 Up to concentration M, there is no significant competition for the binding of disease-related autoantibodies to histidyl-tRNA synthetase in competitive ELISA.

[0333] Accordingly, in some embodiments, the HRS polypeptide has a lower affinity for disease-related 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-related autoantibodies that is at least about 10 times, at least about 20 times, at least about 50 times, or at least about 100 times lower than the affinity for disease-related autoantibodies against wild-type human (SEQ ID NO: 1). In one embodiment, the autoantibody against histidyl-tRNA synthetase is an antibody against the Jo-1 antigen.

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

[0335] Polymyositis affects the skeletal muscles (involved in movement) on both sides of the body. Polymyositis is rare 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, standing up from a seated position, climbing stairs, lifting objects, or raising the arms. People with polymyositis may also experience arthritis, shortness of breath, and cardiac arrhythmias. Polymyositis is often associated with antibodies against synthetases, including HisRS, resulting in immune cell infiltration into damaged muscle cells. Therefore, HRS-Fc conjugates may be used to treat polymyositis by reducing immune cell activation and infiltration.

[0336] Dermatomyositis is characterized by a skin rash that precedes or accompanies progressive muscle weakness. This rash appears patchy, has purple and red discoloration, and typically occurs on the eyelids and on the muscles used to extend or straighten joints, including the knuckles, elbows, knees, and toes. Red rashes may also occur on the face, neck, shoulders, upper chest, back, and other locations, and swelling may be present in the affected area. The rash occasionally occurs without apparent muscle involvement. Adults with dermatomyositis may experience weight loss or low-grade fever, may have inflamed lungs, and may be sensitive to light. Unlike polymyositis, adult dermatomyositis may be associated with tumors of the breast, lungs, female genitalia, or intestines. Children and adults with dermatomyositis may develop calcium deposits (called calcifications) that appear as firm bumps under the skin or in the muscles. Calcifications most commonly occur 1 to 3 years after the onset of the disease, but can also occur many years later. These deposits are more frequently seen in childhood dermatomyositis than in adult-onset dermatomyositis. Dermatomyositis may be associated with collagen-vascular disease or autoimmune disease.

[0337] In some cases of polymyositis and dermatomyositis, distal muscles (muscles away from the trunk, e.g., in the forearms and around the ankles and wrists) may be affected as the disease progresses. Polymyositis and dermatomyositis may be associated with collagen-vascular diseases or autoimmune diseases that result in immune cell infiltration into damaged muscle cells. Therefore, HRS-Fc conjugates may be used to treat dermatomyositis by reducing immune cell activation and infiltration.

[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 voids are sometimes found in the cells of the affected muscle fibers. Falls and stumbling are usually the first noticeable symptoms of IBM. In some individuals, the disorder begins with weakness in the wrists and fingers, causing difficulty in pinching, buttoning, and gripping objects. Weakness of the wrist and finger muscles, as well as atrophy (thinning or loss of muscle volume) of the forearm muscles and quadriceps femoris muscles of the legs, may be present. Dysphagia occurs in about half of IBM cases. Symptoms of the disease usually begin after age 50, but the disease can occur earlier. Unlike polymyositis and dermatomyositis, IBM occurs more frequently in men than in women. Like other muscular dystrophy, IBM also involves progressive immune cell infiltration into damaged muscle cells. Therefore, HRS-Fc conjugates may be used to treat IBM by reducing immune cell activation and infiltration.

[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 accumulation 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 muscles and tendons, which restricts the free movement of the joints). Children with juvenile myositis may also experience dysphagia and dyspnea, and the heart may be affected. Approximately 20–30 percent of children with juvenile dermatomyositis develop calcification. Affected children may not have elevated levels of the muscle enzyme creatine kinase in their blood, but may have elevated levels of other muscle enzymes. Juvenile myositis also involves progressive immune cell infiltration into damaged muscle cells. Therefore, HRS-Fc conjugates may be used to reduce immune cell activation and infiltration, and to treat juvenile myositis.

[0340] Statin-induced myopathy is associated with long-term use of statins, which act through the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Although generally well-tolerated, these drug therapies have been described as inducers of myotoxicity. More recently, there have been reports of patients whose statin myopathy persists even after drug discontinuation, which is hypothesized to have an autoimmune cause. The benefits of statins are undeniable in reducing the risk of progression of coronary heart disease and 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 which up to 7% (>2.6 million) are projected to develop muscular symptoms, and of these up to 0.5% (>190,000) are projected to potentially progress to life-threatening myopathy.

[0341] All statins can cause muscle problems, and the risk increases with their lipophilicity, cholesterol-lowering ability, and dosage. Cerivastatin, in particular, is implied to have a higher risk and has been withdrawn from the US market. Of the remaining statins, atorvastatin and simvastatin have higher rates of myotoxicity. 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 have statin-induced muscle problems, a history of muscle problems can be a risk factor and should be considered when initiating statin treatment. A family history of myopathy is relevant because it may indicate that the patient is a carrier of genetic myopathy, which may not be masked by the added stress of statin treatment. Other risk factors may include age over 80 years, low body weight, female sex, hypothyroidism, certain genetic defects and Asian ancestry, as well as the conjunctivitis 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 soreness, which occurs in about 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 a recent myocardial infarction, continued statin treatment may be appropriate if the symptoms are tolerable and the patient is an indication for strong statin treatment.

[0343] While baseline creatine kinase (CK) levels are not uniformly recommended by the institutions guiding statin treatment before initiating statin therapy, CK levels can provide very useful information about whether muscle symptoms will develop later. Muscle weakness can also occur, often being qualitatively fatigued and combined with pain and elevated CK. As with most myopathy, weakness is most pronounced proximally. Rare episodes of rhabdomyolysis have also occurred with statin treatment; these are far less frequent but can be potentially fatal. The most typical muscle histological changes that can be seen in statin myopathy are cytochrome oxidase-negative fibers, increased lipid content, and red, ragged fibers. Autoimmune necrotizing myopathy is a rare form of statin myopathy. In these patients, discontinuation of statin medication does not lead to recovery, even after several months of drug withdrawal. Patients have predominantly proximal, often painless weakness.

[0344] Diagnosis is based on the individual's medical history, the results of physical examination and muscle strength tests, and blood samples showing elevated levels of various muscle enzymes and autoantibodies. Diagnostic tools include electromyography to record the electrical activity controlling muscles during contraction and at rest, ultrasound to look for muscle inflammation, and magnetic resonance imaging to reveal abnormal muscles and assess muscle disease. Muscle biopsies may 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 may be used to treat statin-induced myopathy and rhabdomyolysis by reducing the activation and infiltration of immune cells into damaged muscle.

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

[0346] In ILD, the tissues in the lungs become inflamed and / or scarred. The lung interstitium includes the small blood vessels and alveoli (air sacs) where oxygen and carbon dioxide exchange takes place, as well as the surrounding areas. Inflammation and scarring of the interstitium 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 treat ILD by activating immune cells and reducing their infiltration into damaged lung tissue.

[0347] The progression of ILD varies depending on the disease and from person to person. Interstitial lung disease disrupts the transfer of oxygen and carbon dioxide in the lungs, so its symptoms typically manifest as breathing problems. The two most common symptoms of ILD are shortness of breath during exercise and a dry cough.

[0348] Usher syndrome is the most common condition affecting both hearing and vision. The main 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 field of vision narrows to the point where 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 impairment have Usher syndrome. In developed countries such as the United States, about 4 babies per 100,000 births have Usher syndrome. Usher syndrome is inherited as an autosomal recessive trait. Several gene loci, including histidyl tRNA synthetase, have been associated with Usher syndrome (Puffenberger et al., (2012) PLoS ONE vol. 7(1) e28936 doi:10.1371 / journal.pone.0028936).

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

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

[0351] Children with type 2 Usher syndrome are born with moderate to severe hearing loss and normal balance. While the severity of hearing loss varies, most of these children can benefit from hearing aids and communicate verbally. Visual problems in type 2 Usher syndrome tend to progress more slowly than those in type 1, and the onset of RP (reactive brain function) is often not apparent until adolescence.

[0352] Children with Usher syndrome type 3 have normal hearing at birth. Most children with this disorder have normal to near-normal balance, but some may develop balance problems later. 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 teens, and they usually require hearing aids by mid-adulthood or late adulthood. Night blindness usually begins at some point during adolescence. A blind spot appears by late teens or early adulthood, and by mid-adulthood, the person is usually legally blind.

[0353] Perot syndrome (PS) is characterized by ovarian dysplasia in females with sensorineural hearing loss, and, in some subjects, associated neurological abnormalities including progressive cerebellar ataxia and intellectual disability. The exact prevalence of Perot syndrome is unknown, and it is likely to be underdiagnosed, particularly in males where hypogonadism is not characteristic and symptoms remain undetected. The mean age at diagnosis is 22 years after the presentation of delayed puberty in females with sensorineural hearing loss. Hearing loss was 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 dysplasia has been reported in all female cases, but gonadal deficiency has not been detected in males. Amenorrhea is generally primary, but secondary amenorrhea has also been reported. Delayed growth (height below the 3rd percentile) was reported in half of the reported cases. The exact frequency of neurological abnormalities is unknown, but nine females and two males (16–37 years old) without neurological abnormalities have been reported. Neurological signs are progressive and generally appear in old age, but delayed gait or frequent falls in the early stages have been noted in younger PS patients. Common neurological signs include ataxia, impaired coordination, limited extraocular movement, and polyneuropathy. Some cases with scoliosis have also been reported. PS transmission is autosomal recessive, and mutations in mitochondrial histidyl tRNA synthetase have recently been identified as causing ovarian dysplasia and sensorineural hearing loss associated with Perot syndrome (Pierce et al., PNAS USA. vol. 108 (no. 16) pp. 6543–6548, 2011).

[0354] Muscular dystrophy refers to a group of hereditary disorders characterized by progressive decline in muscle strength and volume. All muscular dystrophys are characterized by muscle weakness driven by a primary genetic defect in one or more muscle-specific genes. Furthermore, muscular dystrophy typically has variable inflammatory components that drive muscle inflammation and ultimately enhance muscle tissue degeneration. Therefore, HRS-Fc conjugates can be used to treat muscular dystrophy by reducing the activation and infiltration of immune cells into damaged muscle. At least nine types of muscular dystrophy are generally recognized. In some embodiments, this muscular dystrophy is selected from Duchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreyfus 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 about 1 in 3,500 male births and affects 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 dystrophin, a submembrane muscle protein that functions within the dystrophin-associated glycoprotein complex (DGC), which prevents the production of functional proteins. The amount of dystrophin correlates with the severity of the disease (i.e., the less dystrophin present, the more severe the phenotype). This DGC complex connects the intracellular cytoskeleton to the extracellular matrix. DGCs are concentrated at the Z-line of the sarcomere, conferring force transmission to the muscle fibers. Disruption of this connection results in membrane instability, ultimately leading to muscle cell membrane rupture. The influx of extracellular calcium alters molecular processes such as muscle contraction and activates proteolytic activity. Affected muscle fibers become necrotic or apoptotic, releasing pro-mitotic chemoattractants, which initiate inflammatory processes. The degeneration and regeneration cycle ultimately leads to irreversible muscle wasting, as well as replacement with fibrous and adipose tissue.

[0357] Boys with Duchenne muscular dystrophy typically begin showing symptoms in preschool. The legs are the first to be affected, making walking difficult and causing balance problems. Most patients walk 3 to 6 months later than predicted and have difficulty running. Contractures (permanent muscle tension) usually begin by age 5 or 6, most severely in the calf muscles. Frequent falls and fractures commonly begin at this age. Climbing stairs and standing up without assistance may become impossible by age 9 or 10, and most boys use wheelchairs for mobility by age 12. Weakening of the trunk muscles around this age often leads to scoliosis (lateral curvature of the spine) and kyphosis (curvature from front to back).

[0358] One of the most severe forms of weakness in DMD is weakness of the diaphragm, the sheet of muscles in the upper abdomen that performs the primary functions of breathing and coughing. Diaphragmatic 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, however, with the assistance of mechanical ventilation and good respiratory hygiene.

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

[0360] HRS-Fc conjugates can be used in the treatment of DMD either alone or in combination with other therapies, such as antisense oligonucleotides (e.g., exon skipping therapies such as Eteplirsen), corticosteroids, beta-2 agonists, physiotherapy, respiratory support, stem cell therapy, and gene replacement therapy. In some embodiments, administration of HRS-Fc conjugates results in a statistically significant improvement in the 6-minute walk test.

[0361] Becker muscular dystrophy (BMD): BMD affects older boys and young males 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 shortened but partially functional form of dystrophin.

[0362] Symptoms of BMD typically appear in late childhood or early adulthood. While the progression of symptoms may parallel that of DMD, these symptoms are usually milder and the process is more variable. Scoliosis may occur, but is usually milder and progresses more slowly. 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, potentially requiring mechanical ventilation. HRS-Fc conjugates can be used in the treatment of BMD, either alone or in combination with other therapies.

[0363] Emery-Dreyfus muscular dystrophy (EDMD): EDMD affects young boys and causes contractures and weakness in the calves, weakness in the shoulders and upper arms, and problems in the pathway by which electrical impulses travel to the heart to make it beat (cardiac conduction defects). There are three subtypes of Emery-Dreyfus muscular dystrophy that can be identified by the pattern of inheritance: X-linked, autosomal dominant, and autosomal recessive. The X-linked form is the most common. Each type has different prevalence and symptoms. The disease is caused by mutations in the LMNA gene, or more commonly, mutations in the EMD gene. Both genes encode protein components of the nuclear membrane.

[0364] EDMD typically begins in early childhood, often accompanied by contractures preceding muscle weakness. The weakness initially affects the shoulders and upper arms, along with the calf muscles, leading to foot drop. While most men with EDMD survive into middle age, a deficit in cardiac rhythm (heart block) can be fatal if not treated with a pacemaker. HRS-Fc conjugates can be used in the treatment of EDMD, either alone or in combination with other therapies.

[0365] Limb-girdle muscular dystrophy (LGMD): LGMD begins in late childhood or early adulthood, affects both males and females, and causes weakness in the muscles around the buttocks and shoulders. It is the most variable of the muscular dystrophy, with several different types of the disease 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 individuals in the United States may be in the thousands.

[0366] While there are at least half a dozen genes that cause various types of LGMD, two major clinical forms of LGMD are usually recognized. The severe childhood form resembles DMD in appearance 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 dyspherin gene. Dyspherin is primarily expressed in skeletal and cardiac muscle, but is also expressed in monocytes, macrophages, and other tissues, where it localizes to cytoplasmic vesicles and cell membranes. Dyspherin appears to be involved in membrane fusion and membrane transport, as well as repair processes. LGMD2B is a late-onset (teenage / young adult) muscle disease characterized by progressive symmetrical muscle weakness and markedly malignant immune / inflammatory pathology. Muscle biopsies typically reveal macrophage / macrophage activation markers (HLA-DR, HLA-ABC, CD86), CD8, along with muscle fiber degeneration / regeneration. + Cytotoxic T cells and CD4 + It exhibits significant inflammatory cell infiltration, primarily from T cells. Therefore, HRS-Fc conjugates may be used to treat limb-girdle muscular dystrophy by reducing the activation and infiltration of immune cells into damaged muscle.

[0368] Adult-onset LGMD typically manifests in a person's teens or twenties and is characterized by progressive weakness and wasting of the muscles closest to the trunk. Contractures may develop, and the ability to walk is usually lost about 20 years after onset. Some people with LGMD develop respiratory failure requiring the use of a ventilator. Life expectancy may be somewhat shortened (the autosomal dominant form usually develops in old age and progresses relatively slowly).

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

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

[0371] Two deletions are required for FSHD: the first is a deletion of the D4Z4 repeat, and the second is a “toxic gain-of-function” form of the DUX4 gene. The first noticeable symptom 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, which is called scapular winging. FSHD is associated with inflammatory infiltration in certain muscle groups, and therefore, the HRS-Fc conjugate may be used to treat FSHD by reducing the activation of immune cells and infiltration into damaged muscles.

[0372] Myotonic Dystrophy: Myotonic dystrophy, also known as Steinert's disease, affects both men and women and causes generalized weakness, initially appearing in the face, feet, and hands. This is accompanied by the inability to relax the affected muscles (myotonia). Symptoms can begin from birth or throughout 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 increase in short (CTG) repeats in the DNA sequence of the DMPK (myotonic dystrophy protein kinase) gene. Myotonic muscular dystrophy type 2 (DM2) is considerably rarer and results from an increase in CCTG repeats in the ZNF9 (zinc finger protein 9) gene.

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

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

[0375] OPMD typically begins in a person's 30s or 40s and involves weakness in the muscles that control the eyes and pharynx. Symptoms include ptosis (drooping eyelids), dysphagia (difficulty swallowing), and progressive weakness in the muscles of the face, neck, and occasionally other muscles of the upper limbs. Dysphagia can lead to aspiration or introduction of food or saliva into the airway. Pneumonia may follow. Therefore, HRS-Fc conjugate may 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 legs and hands. It is most common in Sweden and rare elsewhere in the world. DD usually begins in the 20s or 30s and is accompanied by weakness in the hands, forearms, and lower legs. Difficulty with fine motor skills such as typing or buttoning clothes may be the first symptom. The symptoms progress slowly, and the disease usually does not affect life expectancy. Therefore, HRS-Fc conjugate may 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 intellectual disability. 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 form of assistive device, and survive into early adulthood or beyond. In contrast, children with Fukuyama-type CMD are barely able to walk and have severe intellectual disability. Most children with this type of CMD die in childhood. Therefore, as with other muscular dystrophy, the HRS-Fc conjugate 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 trying to lose weight. The formal definition of cachexia is weight loss that cannot be reversed nutritionally. Lean body mass is lost even when the affected patient is consuming more calories, indicating the presence of a primary pathology.

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

[0381] Cachexia can also be a sign of a variety of underlying conditions, 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 amphetamine addiction. Physically, cachexia can lead to immobility due to loss of appetite, asthenia, and anemia, and the response to standard care is usually poor.

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

[0383] The pathogenesis of cachexia is not well understood, but it is predicted that multiple biological pathways are involved, including pro-inflammatory cytokines such as TNF-α, neuroendocrine hormones, IGF-1, and tumor-specific factors such as proteolytic factors.

[0384] Therefore, HRS-Fc conjugates may be used to treat cachexia and its associated underlying or secondary disorders or complications. HRS-Fc conjugates may be used alone or, among other things, in combination with other treatments, such as dietary supplements with high protein, leucine, and fish oil combinations, antioxidants, progestogens (megestrol acetate, medroxyprogesterone acetate), as well as anticyclooxygenase-2 drugs, appetite stimulants, and 5-HT3 antagonists.

[0385] Rhabdomyolysis: Rhabdomyolysis is the breakdown of muscle fibers in skeletal muscle tissue. The breakdown products are released into the bloodstream, and certain parts of these products, such as myoglobin, are harmful to the kidneys and can 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 injury and whether renal failure develops. Kidney damage can cause reduced or absent urine production, usually about 12–24 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, which can lead to blood loss and damage to the affected body part (e.g., loss of function). Symptoms of this complication include pain or reduced sensation in the affected limb. Other complications include disseminated intravascular coagulation (DIC), severe disruption of blood clotting, which can lead to uncontrolled bleeding.

[0387] Initial muscle injury can be caused by, for example, physical factors (e.g., crush injuries, strenuous exercise), altered blood supply (e.g., arterial thrombosis, embolism), altered metabolism (e.g., hyperglycemic hyperosmolarity, hypernatremia and hyponatremia, hypokalemia, hypocalcemia, hypophosphatemia, ketoacidosis, hypothyroidism), altered body temperature (hypertension, hypothermia), drug therapy and toxins (e.g., statins, antipsychotic drugs, 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). It can be caused by pneumophila (Salmonella) and autoimmune muscle injuries (e.g., polymyositis, dermatomyositis). In addition, certain genetic conditions increase the risk of rhabdomyolysis, including glycolysis and glycogenolysis deficiencies (e.g., McArdle's disease, phosphofructokinase deficiency, glycogen storage diseases VIII, IX, X, and XI), lipid metabolism deficiencies (e.g., carnitine palmitoyltransferase I and II deficiencies, deficiencies of acyl-CoA dehydrogenase subtypes (e.g., LCAD, SCAD, MCAD, VLCAD, 3-hydroxyacyl-coenzyme A dehydrogenase deficiency), thiolase deficiencies), mitochondrial myopathy (e.g., succinate dehydrogenase, cytochrome c oxidase, and coenzyme Q10 deficiencies), as well as others, including glucose-6-phosphate dehydrogenase deficiency, myoadenylate deaminase deficiency, and muscular dystrophy.

[0388] Rhabdomyolysis is usually diagnosed by blood and urine tests and may be indicated by abnormally elevated or gradually increasing creatinine and urea levels, decreased urine volume, or a reddish-brown discoloration of the urine. Primary treatment includes intravenous fluid resuscitation, dialysis, and hemofiltration.

[0389] Therefore, HRS-Fc conjugates may be used to treat rhabdomyolysis and any associated secondary or underlying disorders or complications. HRS-Fc conjugates may be used alone or in combination with other treatments, including those intended to treat shock and preserve renal function. Exemplary treatments include intravenous fluids, usually isotonic saline (0.9 wt% sodium chloride solution by volume), and kidney alternative This includes the administration of therapeutic interventions (RRT), such as hemodialysis, continuous hemofiltration, and peritoneal dialysis.

[0390] More generally, the HRS-Fc conjugates described herein can reduce inflammatory responses, for example, by reducing the activation, differentiation, migration, or infiltration of immune cells into selected tissues, increasing the production of anti-inflammatory cytokines, or reducing the production or activity of pro-inflammatory cytokines, among other mechanisms. Furthermore, specific methods of the present invention, which involve blocking the binding, action, or production of anti-histidyl-tRNA synthetase antibodies or autoreactive T cells, have utility for treating a wide range of autoimmune and inflammatory diseases and disorders associated with anti-histidyl-tRNA synthetase antibodies, other autoantibodies, and other causes of histidyl-tRNA synthetase deficiency.

[0391] Pharmaceutical formulations, administrations, and kits Embodiments of the present invention include compositions comprising an HRS-Fc conjugate polypeptide formulated ...

Claims

[Claim 1] The kit or method described in the specification.

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