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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/93—Ligases (6)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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  • C12Y—ENZYMES
  • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
  • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
  • C12Y601/01012—Aspartate-tRNA ligase (6.1.1.12)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
  • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/50—Fusion polypeptide containing protease site
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  • C12Y—ENZYMES
  • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
  • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
  • C12Y601/01002—Tryptophan-tRNA ligase (6.1.1.2)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates generally to conjugates, such as fusion proteins, of one or more aspartyl-tRNA synthetase (DRS) polypeptide(s) and immunoglobulin Fc region(s), compositions comprising the same, and methods of using such polypeptides and compositions for treating or diagnosing a variety of conditions.
• DRS aspartyl-tRNA synthetase
• DRS Aspartyl-tRNA synthetases
• AspRS polypeptides have recently been shown to possess a variety of non-canonical activities of therapeutic and diagnostic relevance. In particular it has been established that certain aspartyl-tRNA synthetase fragments are highly potent, endogenously produced, Toll-like receptor modulators. Without being bound to any one specific theory of operation, it is believed that such DRS polypeptides are released from macrophage cells upon proteolytic cleavage, or through alternative splicing of the full length DRS tRNA synthetase and are capable of binding to and modulating the activity of immunomodulatory, and other cell types. Such DRS polypeptides when administered, provide for a novel mechanism of selectively modulating inflammatory responses, without the side effect profiles typically associated with traditional anti-inflammatory agents such as steroids.
• TLRs Toll-like receptors
• PAMPs pathogen associated molecular patterns
• DAMPS damage-associated molecular pattern molecules
• PAMPS are typically unique to a given class of pathogen, and include for example bacterial components such as the lipopolysaccharide of Gram negative bacteria, and viral specific nucleic acid motifs or viral specific modifications of RNA or DNA.
• DAMPS are typically endogenous molecules released from dying host cells upon cellular stress or tissue damage.
• TLRs are implicated in several chronic inflammatory and immune mediated disorders by various potential mechanisms, including those in which infectious agents have been proposed to initiate disease progression. For example in scenarios in which endogenous damage signals or self-antigens cause chronic inflammation in a TLR dependent manner, or where TLRs may be involved in the breakdown of immune tolerance. TLRs have been implicated in the pathogenesis of chronic inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis, psoriasis, and multiple sclerosis.
• TLR agonists have already proved useful in clinical trials in allergic, infectious and autoimmune diseases and are under development for a broad range of other diseases including cancer, arthritis, multiple sclerosis, inflammatory bowel disease, see generally Zhu and Mohan (2010) Mediators of Inflammation doi:10.1155/2010/781235; Hennessy et al, Nat. Rev. 9:293-307, 2010). Therefore TLRs are becoming increasingly recognized as novel potential therapeutic targets for the modulation of a broad variety of diseases and disorders.
• DRS polypeptides having improved pharmacokinetic properties. These improved therapeutic forms of the DRS polypeptides enable the development of more effective therapeutic regimens for the treatment of various diseases and disorders, and require significantly less frequent administration than the unmodified proteins.
• FIG 1 shows an SDS-PAGE analysis of the purified proteins AspRSl N1 (C76S) (DRS(1-
• Lanes 1-3 were run under reduced conditions, and lanes 4-6 were run under non-reduced conditions. Lanes 1 and 4: AspRSl N1 DRS(1-154) lot # D-N1-V5H-046, lanes 2 and 5 AspRSl N1 (DRS(1- 154))lot # D-N1-V5H-047, lanes 3 and 6: AspRSl N1 (C76S) (SEQ ID NO:29) lot # D-Nl : l-V5H-048.
• Figure 2 shows a direct comparison of AspRSl (SEQ ID NO:31) (grey squares) and AspRSl (C76S) (SEQ ID NO:29) (black circles) on their ability to stimulate reporter gene activity mediated by the TLR2 receptor in HEK- Blue 2 cells. Grey triangles - Pam3C SK4.
• Figure 3 shows a direct comparison of AspRSl N1 (SEQ ID NO:31) (grey squares) and AspRSl N1 (C76S) (SEQ ID NO:29) (Black circles) on their ability to stimulate reporter gene activity mediated by the TLR4 receptor in HEK-Blue 4 cells.
• Figure 4 shows SDS-PAGE analysis of a DRS-Fc (SEQ ID NO:37) purification.
• Lane 1 clarified lysate; lane 2, MabSelect flow-through; lane 3, MabSelect wash; lane 4, purified DRS-Fc.
• Figure 5 shows an SEC analysis of a DRS-Fc fusion protein (SEQ ID NO:37).
• the upper trace is 280nm absorbance, and the lower trace is 260nm absorbance.
• Figure 6 illustrates the structural make-up of an exemplary immunoglobulin, and provides an overview of antibody classes and subclasses.
• Figure 7 shows an alignment of Fc regions from human IgAl (SEQ ID NO:66), IgA2 (SEQ ID NO:67), IgM (SEQ ID NO:68), IgGl (SEQ ID NO:69), IgG2 (SEQ ID NO:70), IgG3 (SEQ ID NO:71), IgG4 (SEQ ID NO:72), and IgE (SEQ ID NO:73).
• the secondary structure of Fca is shown above the sequences. Carets ( ⁇ ) and asterisks (*) show residues that contribute respectively to 0-4% and 5-12%> of the binding surface.
• Figure 8 shows the results of the administration of AspRSl N1 ( C76S) in a partial body irradiation survival model; AspRSl N1 ( C76S) shown in squares and the PBS control shown as diamonds.
• Figures 9A and 9B show the results of the administration of AspRSl N1 ( C76S) in an MSU induced model of gout inflammation (squares), compared to vehicle control (PBS) diamonds, and a positive control (dexamethasone (triangles)
• the insert shows the statistical significance for AspRSl N1 ( C76S) ("Homeokine") compared to the vehicle control.
• Embodiments of the present invention relate generally to aspartyl-tRNA synthetase (DRS) polypeptide conjugates having one or more immunoglobulin Fc regions covalently attached thereto, pharmaceutical compositions comprising such molecules, methods of manufacture, and methods for their therapeutic use.
• DRS-Fc conjugates of the present invention can possess improved pharmacokinetic properties and/ or improved therapeutically relevant biological activities, relative to corresponding, un-modified DRS polypeptides.
• DRS fusion polypeptides comprising a DRS amino acid sequence at least 80% identical to any one of SEQ ID NOS: l, 3-24, 29, 31, or 154-197, and at least one Fc region fused to the C-terminus, the N-terminus, or both of the DRS polypeptide.
• the DRS polypeptide comprises an amino acid sequence at least 90% identical to any of SEQ ID NOS: 1, 3-24, 29, 31, or 154-197.
• the DRS polypeptide comprises an amino acid sequence of any one of SEQ ID NOS: 1, 3-24, 29, 31, or 154-197.
• the DRS polypeptide is about 130-300 amino acids in length and comprises amino acid residues 1-154, 11-146, 13-146, 23-154, 1-171, or 1-174, 1-182, 1-184, 1-224, or 1- 274 of SEQ ID NO:l, or an amino acid sequence at least 90% identical to residues 1-154, 11-146, 13-146, 23-154, 1-171, or 1-174, 1-182, 1-184, 1-224, or 1-274 of SEQ ID NO :1.
• the DRS polypeptide is about 130-200 amino acids in length and comprises amino acid residues 1-154, 11-146, 13- 146, 23-154, 1-171, 1-174, 1-182, or 1-184 of SEQ ID NO: l, or an amino acid sequence at least 90% identical to residues 1-154, 11-146, 13-146, 23-154, 1-171, 1-174, 1-182, or 1-184 of SEQ ID NO:l.
• the DRS polypeptide is about 130-175 amino acids in length and comprises amino acid residues 1-154, 23-154, 1-171, or 1-174 of SEQ ID NO:l, or an amino acid sequence at least 90% identical to residues 1-154, 23-154, 1-171, or 1-174 of SEQ ID NO: 1.
• the DRS polypeptide comprises amino acid residues 1-154, 11-146, 13-146, 23-154, 1-171, or 1-174 of SEQ ID NO:l .
• the DRS polypeptide consists essentially of amino acid residues 1-154 of SEQ ID NO:l .
• the DRS polypeptide consists essentially of amino acid residues 13-146 of SEQ ID NO: l.
• the DRS polypeptide comprises an OB fold domain, an N-terminal amphiphilic helix, or both.
• the Fc region and the DRS polypeptide are separated by a peptide linker.
• the peptide linker is about 1-200 amino acids, about 1-150 amino acids, about 1- 100 amino acids, about 1-90 amino acids, about 1-80 amino acids, about 1-70 amino acids, about 1-60 amino acids, about 1-50 amino acids, about 1-40 amino acids, about 1-30 amino acids, about 1-20 amino acids, about 1-10 amino acids, or about 1-5 amino acids in length.
• peptide linker is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100 amino acids in length.
• the peptide linker consists or consists essentially of Gly and/or Ser residues.
• the peptide linker is a physiologically stable linker.
• the peptide linker is a releasable linker, optionally an enzymatically-cleavable linker.
• the peptide linker comprises a sequence of any one of SEQ ID NOS:80-139.
• the Fc region is fused to the C-terminus of the DRS polypeptide. In certain embodiments, the Fc region is fused to the N-terminus of the DRS polypeptide.
• the Fc region comprises one or more of a hinge, CH 2 , CH 3 , and/or CH 4 domain from a mammalian IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, and/or IgM.
• the DRS fusion polypeptide does not comprise the CHi, C L , V L , and V H regions of an immunoglobulin.
• the Fc region comprises any one of SEQ ID NOS:38-64, or a variant, or a fragment, or a combination thereof.
• the DRS fusion polypeptide has altered pharmacokinetics relative to a corresponding DRS polypeptide. Examples of said altered pharmacokinetics include increased serum half-life, increased bioavailability, and/or decreased clearance.
• the DRS fusion polypeptide has altered immune effector activity relative to a corresponding DRS polypeptide. Examples of such immune effector activities include one or more of complement activation, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or antibody-dependent cell- mediated phagocytosis (ADCP).
• the Fc region comprises a variant Fc region, relative to a wild-type Fc region.
• the variant Fc region comprises a sequence that is at least 90% identical to any one of SEQ ID NOS:38-64, or a combination of said sequences.
• the variant Fc region comprises a hybrid of one or more Fc regions from different species, different Ig classes, or different Ig subclasses.
• the variant Fc region comprises a hybrid of one or more hinge, CH 2 , CH 3 , and/or CH 4 domains of Fc regions from different species, different Ig classes, and/or different Ig subclasses.
• the variant Fc region is a modified glycoform, relative to a corresponding, wild-type Fc region.
• the variant Fc region has altered pharmacokinetics relative to a corresponding, wild-type Fc region. Examples of such altered pharmacokinetics include serum half-life, bioavailability, and/or clearance.
• the variant Fc region has altered effector activity relative to a corresponding, wild-type Fc region. Examples of such effector activities include one or more of complement activation, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or antibody-dependent cell- mediated phagocytosis (ADCP).
• CDC complement-dependent cytotoxicity
• ADCC antibody-dependent cell-mediated cytotoxicity
• ADCP antibody-dependent cell- mediated phagocytosis
• the variant Fc region has altered binding to one or more Fey receptors, relative to a corresponding, wild-type Fc region.
• Fey receptors are described herein and known in the art.
• the variant Fc region has altered ⁇ e.g., increased) solubility, relative to a corresponding, wild-type Fc region, and the DRS-Fc fusion protein has altered solubility, relative to a corresponding, unmodified DRS polypeptide.
• the DRS-Fc fusion polypeptide is substantially in dimeric form in a physiological solution, or under other physiological conditions, such as in vivo conditions.
• the DRS-Fc fusion polypeptide has substantially the same secondary structure a corresponding unmodified or differently modified DRS polypeptide, as determined via UV circular dichroism analysis.
• the DRS-Fc fusion polypeptide has a plasma or sera pharmacokinetic AUC profile at least 5-fold greater than a corresponding, unmodified DRS polypeptide when administered to a mammal.
• the DRS-Fc fusion polypeptide has substantially the same activity of a corresponding unmodified or differently modified DRS polypeptide in a TLR 2 or TLR 4 based assay.
• the DRS-Fc fusion polypeptide has greater than 2 fold the activity of a corresponding unmodified or differently modified DRS polypeptide in a TLR 2 or TLR 4 based assay.
• the DRS-Fc fusion polyptide has a stability which is at least 30% greater than a corresponding unmodified or differently modified DRS polypeptide when compared under similar conditions at room temperature, for 7 days in PBS at pH 7.4.
• DRS-Fc fusion polypeptides comprise SEQ ID NO:36 or 37, or an amino acid sequence at least 80%, 90%, 95%, 98% identical to SEQ ID NO:36 or 37.
• the invention includes a dosing regimen which maintains an average steady- state concentration of DRS polypeptide in the subjects' plasma of between about 0.3 ⁇ g/ml and about 3 ⁇ g/ml when using a dosing interval of 3 days or longer, comprising administering to the patient a therapeutic dose of any of the DRS-Fc fusion polypeptides described herein.
• the invention includes a method for maintaining DRS polypeptide levels above the minimum effective therapeutic level in a subject in need thereof, comprising administering to the subject a therapeutic dose of any of the DRS-Fc fusion polypeptides described herein.
• the invention includes a method for treating an inflammatory response in a subject, comprising administering any of the previously disclosed DRS-Fc fusion polypeptides described herein to a subject in need thereof.
• the invention includes a method for treating a TLR associated disease in a subject in need thereof, comprising administering to the subject a therapeutic dose of any of the DRS-Fc fusion polypeptides described herein.
• the invention includes a method for method for modulating TLR activity in a subject, comprising administering to the subject a therapeutic dose of any of the DRS-Fc fusion polypeptides described herein.
• the invention includes a method for method for killing cancer cells, comprising administering a vaccine or immunogenic composition comprising any of the DRS-Fc fusion polypeptides described herein to a subject in need thereof.
• the invention includes a method for treating a subject with cancer, or preventing the development of cancer in a subject, comprising administering a vaccine or immunogenic composition comprising any of the DRS-Fc fusion polypeptides described herein to a subject in need thereof.
• the invention includes a method for overcoming tolerance to an antigen in a subject, comprising administering a vaccine or immunogenic composition comprising any of the DRS-Fc fusion polypeptides described herein to a subject in need thereof.
• isolated polynucleotides comprising a nucleotide sequence that encodes a DRS-Fc fusion polypeptide described herein, including vectors that comprise such polynucleotides, and host cells that comprise said polynucleotides and/or vectors.
• Some embodiments include methods for manufacturing a DRS-Fc fusion polypeptide described herein, comprising a) culturing a host cell to express a DRS-Fc fusion polypeptide, wherein the host cell comprises a polynucleotide that encodes a DRS-Fc fusion polypeptide described herein, which is operably linked to a regulatory element; and b) isolating the DRS-Fc fusion polypeptide from the host cell.
• an element means one element or more than one element.
• amino acid is intended to mean both naturally occurring and non- naturally occurring amino acids as well as amino acid analogs and mimetics.
• Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4- hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example.
• Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art.
• Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids.
• Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivitization of the amino acid.
• Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid.
• Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.
• conjugate is intended to refer to the entity formed as a result of covalent attachment of a molecule, e.g., a biologically active molecule, to an immunoglobulin Fc region.
• a conjugate polypeptide is a "fusion protein " or "fusion polypeptide, " that is, a polypeptide that is created through the joining of two or more coding sequences, which originally coded for separate polypeptides; translation of the joined coding sequences results in a single, fusion polypeptide, typically with functional properties derived from each of the separate polypeptides.
• endotoxin free or “substantially endotoxin free” relates generally to compositions, solvents, and/or vessels that contain at most trace amounts (e.g., amounts having no clinically adverse physiological effects to a subject) of endotoxin, and preferably undetectable amounts of endotoxin.
• Endotoxins are toxins associated with certain bacteria, typically gram-negative bacteria, although endotoxins may be found in gram-positive bacteria, such as Listeria monocytogenes.
• the most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligo-saccharides (LOS) found in the outer membrane of various Gram-negative bacteria, and which represent a central pathogenic feature in the ability of these bacteria to cause disease.
• LPS lipopolysaccharides
• LOS lipo-oligo-saccharides
• a depyrogenation oven may be used for this purpose, as temperatures in excess of 300°C are typically required to break down most endotoxins.
• a glass temperature of 250°C and a holding time of 30 minutes is often sufficient to achieve a 3 log reduction in endotoxin levels.
• Other methods of removing endotoxins are contemplated, including, for example, chromatography and filtration methods, as described herein and known in the art.
• DRS polypeptides in and isolating them from eukaryotic cells such as mammalian cells to reduce, if not eliminate, the risk of endotoxins being present in a composition of the invention.
• Endotoxins can be detected using routine techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes blood from the horseshoe crab, is a very sensitive assay for detecting presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the limulus lysate due a powerful enzymatic cascade that amplifies this reaction.
• Endotoxins can also be quantitated by enzyme-linked immunosorbent assay (ELISA).
• ELISA enzyme-linked immunosorbent assay
• endotoxin levels may be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU/ml.
• 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.
• Homology refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al, Nucleic Acids Research. 12, 387-395, 1984), which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
• 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, for example, in the presence of one or more proteases), that is to say, it does not undergo a degradation reaction (e.g., enzymatically degradable reaction) under physiological conditions to any appreciable extent over an extended period of time.
• a physiologically stable linker is one that exhibits a rate of degradation of less than about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5% per day under physiological conditions.
• isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
• an “isolated peptide” or an “isolated polypeptide “ and the like, as used herein, includes the in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell; i.e., it is not significantly associated with in vivo substances.
• EC 50 half maximal effective concentration
• EC 50 of an agent provided herein is indicated in relation to a “non-canonical " activity, as noted above.
• EC 50 also represents the plasma concentration required for obtaining 50% of a maximum effect in vivo.
• EC 90 refers to the concentration of an agent or composition at which 90% of its maximal effect is observed.
• the "EC90” can be calculated from the "EC50 " and the Hill slope, or it can be determined from the data directly, using routine knowledge in the art.
• the EC50 of a DRS-Fc conjugate is less than about 0.01, 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nM.
• biotherapeutic composition will have an EC50 value of about InM or less.
• half-life of a DRS-Fc conjugate can refer to the time it takes for the conjugate to lose half of its pharmacologic, physiologic, or other activity, relative to such activity at the time of administration into the serum or tissue of an organism, or relative to any other defined time -point.
• Hydrof-life can also refer to the time it takes for the amount or concentration of a DRS-Fc conjugate to be reduced by half of a starting amount administered into the serum or tissue of an organism, relative to such amount or concentration at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point.
• the half-life can be measured in serum and/or any one or more selected tissues.
• linkage is used herein to refer to a linker that can be used to separate a DRS polypeptides from another DRS polypeptide and/or from one or more Fc regions.
• the linker may be physiologically stable or may include a releasable linker such as an enzymatically degradable linker (e.g. , proteolytically cleavable linkers).
• the linker may be a peptide linker, for instance, as part of a DRS-Fc fusion protein.
• the linker may be a non-peptide linker.
• modulating " and “altering” include “increasing, “ “enhancing “ or “stimulating, “ as well as “decreasing” or “reducing, “ typically in a statistically significant or a physiologically significant amount or degree relative to a control.
• An “increased, " “stimulated “ or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.
• a “decreased " or “reduced” amount is typically a "statistically significant " amount, and may include a 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 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%o decrease in the amount produced by no composition (the absence of an agent or compound) or a control composition, including all integers in between.
• a control in comparing canonical and non-canonical activities could include the DRS-Fc conjugate of interest compared to a corresponding (sequence-wise), unmodified or differently modified DRS polypeptide.
• Other examples of comparisons and "statistically significant" amounts are described herein.
• Non-canonical activity refers generally to either i) a new activity possessed by DRS polypeptide of the invention that is not possessed to any significant degree by the intact native full length parental protein, or ii) an activity that was possessed by the by the intact native full length parental protein, where the DRS polypeptide either exhibits a significantly higher (i.e., at least 20% greater) specific activity with respect to the non-canonical activity compared to the intact native full length parental protein, or exhibits the activity in a new context; for example by isolating the activity from other activities possessed by the intact native full length parental protein.
• non-limiting examples of non-canonical activities include extracellular signaling including the modulation of TLRs, , modulation of cell proliferation, modulation of cell migration, modulation of cell differentiation (e.g., hematopoiesis, neurogenesis, myogenesis, osteogenesis, and adipogenesis), modulation of gene transcription, modulation of apoptosis or other forms of cell death, modulation of cell signaling, modulation of cellular uptake, or secretion, modulation of angiogenesis, modulation of cell binding, modulation of cellular metabolism, modulation of cytokine production or activity, modulation of cytokine receptor activity, modulation of inflammation, immunogenicity, and the like.
• extracellular signaling including the modulation of TLRs, , modulation of cell proliferation, modulation of cell migration, modulation of cell differentiation (e.g., hematopoiesis, neurogenesis, myogenesis, osteogenesis, and adipogenesis), modulation of gene transcription, modulation of apoptosis or other forms
• the "purity" of any given agent (e.g., DRS-Fc conjugate such as a fusion protein) in a composition may be specifically defined.
• certain compositions may comprise an agent that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between, as measured, for example and by no means limiting, by high pressure liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.
• HPLC high pressure liquid chromatography
• an "enzymatically degradable linker” means a linker, e.g., amino acid sequence, which is subject to degradation by one or more enzymes, e.g., peptidases or proteases.
• polypeptide and protein are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
• a “releasable linker” includes, but is not limited to, a physiologically cleavable linker and an enzymatically degradable linker.
• a “releasable linker” is a linker that may undergo either spontaneous hydrolysis, or cleavage by some other mechanism (e.g., enzyme-catalyzed, acid-catalyzed, base-catalyzed, and so forth) under physiological conditions.
• a “releasable linker” can involve an elimination reaction that has a base abstraction of a proton, (e.g. , an ionizable hydrogen atom, Ha), as the driving force.
• a “releasable linker” is synonymous with a “degradable linker. "
• a releasable linker has a half life at pH 7.4, 25°C, e.g., a physiological pH, human body temperature (e.g., in vivo), of about 30 minutes, about 1 hour, about 2 hour, 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.
• Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.
• solubility refers to the property of a DRS-Fc conjugate polypeptide provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL), mg/ml, etc.), molarity, molality, mole fraction or other similar descriptions of concentration.
• the maximum equilibrium amount of solute that can dissolve per amount of solvent is the solubility of that solute in that solvent under the specified conditions, including temperature, pressure, pH, and the nature of the solvent.
• solubility is measured at physiological pH, or other pH, for example, at pH 5.0, pH 6.0, pH 7.0, or pH 7.4. In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaP). In specific embodiments, solubility is measured at relatively lower pH (e.g., pH 6.0) and relatively higher salt (e.g., 500mM NaCl and lOmM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25°C) or about body temperature (37°C).
• a DRS-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 at 37°C.
• a “subject, " as used herein, includes any animal that exhibits a symptom, or is at risk for exhibiting a symptom, which can be treated or diagnosed with a DRS-Fc conjugate polypeptide of the invention.
• Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog).
• Non-human primates and, preferably, human patients, are included.
• “Substantially” or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%o, 98%o, 99%o or greater of some given quantity.
• therapeutically effective amount refers to the level or amount of agent such, as a DRS-Fc conjugate polypeptide or derivative thereof, needed to treat or improve a condition, or reduce injury or damage without causing significant negative or adverse side effects.
• Treatment or “treating, " as used herein, includes any desirable effect on the symptoms or pathology of a disease or condition, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. "Treatment " or “treating” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. The subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art. Aspartyl-tRNA synthetase derived polypeptides
• Embodiments of the present invention relate to the use of aspartyl-tRNA synthetase polypeptides (DRS or AspRS polypeptides), including wild-type sequences, naturally-occurring sequences, and non- naturally occurring sequences, and also include variants and fragments thereof.
• DRS or AspRS polypeptides include those with altered cysteine content.
• Aspartyl-tRNA synthetases belong to the class I tRNA synthetase family, which has two highly conserved sequence motifs at the active site, HIGH (SEQ ID NO:152) and KMSKS (SEQ ID NO: 153).
• Class I tRNA synthetases are widely recognized as being responsible the specific attachment of an amino acid to its cognate tRNA in a 2-step reaction: the amino acid (AA) is first activated by ATP to form AA- AMP and then transferred to the acceptor end of the tRNA.
• the full length Aspartyl-tRNA synthetases typically exists as a homodimer; and also forms part of a multisubunit complex that typically includes the proteins AIMPl, AIMP2, EEFlAl and the tRNA synthetases for Arg, Asp, Glu, Gin, lie, Leu, Lys, Met and Pro.
• DRS polypeptides may be produced naturally by either alternative splicing or proteolysis, and can act in a cell autonomous (i.e., within the host cell), or non-cell autonomous fashion (i.e., outside the host cell) to regulate a variety of homeostatic mechanisms.
• the N- terminal fragment of aspartyl-tRNA synthetase, DRS (1-154) is capable of modulating the activity of certain TLRs in vivo.
• certain mutations or deletions relative to the full-length DRS polypeptide sequence confer increased TLR binding or other non-canonical activities.
• the sequences of various exemplary DRS polypeptides are provided in Tables Dl to D5 and D7.
• DRS 1-170 1-170 MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEKPDRVL 160
• DRS 1-162 1-162 MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEKPDRVL 164
• DRS 21- 21-154 MDAAEDYAKERYGISSMIQSQEKPDRVLVRVRDLTIQ ADEWW 182 154 VRARVHTSRAKGKQCFLVLRQQQF VQALVAVGDHASKQMVK
• DRS 21- 21-146 MAEDYAKERYGISSMIQSQEKPDRVLVRVRDLTIQ ADEVVWVR 188 146 ARVHTSRAKGKQCFLVLRQQQF VQALVAVGDHASKQMVKFA
• SNPs single nucleotide polymorphisms
• naturally occurring variants of the human gene have been sequenced, and are known in the art to be at least partially functionally interchangeable. Additionally homologs and orthologs of the human gene exist in other species, and it would thus be a routine matter to select a naturally occurring variant such as a DRS polypeptide encoded by a SNP, or other naturally occurring variant in place of any of the DRS polypeptide sequences listed in Tables D1-D5 or D7.
• SNPs naturally occurring variant such as a DRS polypeptide encoded by a SNP
• Table D6 Several such variants of aspartyl-tRNA synthetase ⁇ i.e., representative aspartyl-tRNA synthetase SNPs are shown in Table D6.
• DRS polypeptide “DRS protein” or “DRS protein fragment” as used herein includes all naturally-occurring and synthetic forms of the aspartyl-tRNA synthetase that optionally retain at least one non canonical activity.
• DRS polypeptides include the full length human protein, the DRS peptides derived from the full length protein listed in Tables D1-D5, naturally occurring variants, for example as disclosed in Table D6, the exemplary cysteine mutants listed in Table D7, and synthetic codon optimized forms and other coding sequences as exemplified by the nucleic acid sequences in Table D9, among others.
• DRS polypeptide refers to a polypeptide sequence derived from human aspartyl-tRNA synthetase (SEQ ID NO:l in Table Dl) comprising at least one mutation at either Cys76 or Cysl30.
• the DRS polypeptides may be in their native form, i.e., as different variants as they appear in nature in different species which may be viewed as functionally equivalent variants of human aspartyl - tRNA synthetase, or they may be functionally equivalent natural derivatives thereof, which may differ in their amino acid sequence, for example, by truncation (e.g., from the N- or C-terminus or both) or other amino acid deletions, additions, insertions, substitutions, or post-translational modifications.
• Naturally- occurring chemical derivatives including post-translational modifications and degradation products of any DRS polypeptide, are also specifically included in any of the methods and pharmaceutical compositions of the invention including, e.g., pyroglutamyl, iso-aspartyl, proteolytic, phosphorylated, glycosylated, oxidatized, isomerized, and deaminated variants of a DRS polypeptide.
• embodiments of the present invention include all homologues, orthologs, and naturally-occurring isoforms of aspartyl-tRNA synthetase (e.g., any of the proteins, or their corresponding nucleic acids listed in or derivable from Tables Dl to D9, which optionally retain at least one detectable non canonical activity). Also included are “variants " of these DRS reference polypeptides.
• the recitation polypeptide "variant" refers to polypeptides that are distinguished from a reference DRS polypeptide by the addition, deletion, and/or substitution of at least one amino acid residue, and which typically retain (e.g., mimic) or modulate (e.g., antagonize) one or more non-canonical activities of a reference DRS polypeptide.
• the structure of human aspartyl-tRNA synthetase has been determined to a resolution of 1.7A. ⁇ See WO2010/120509) providing a detailed physical description of the protein, which in conjunction with the primary amino acid sequence provides precise insights into the roles played by specific amino acids within the protein. Accordingly it is within the skill of those in the art to identify amino acids suitable for substitution and to design variants with substantially unaltered, improved, or decreased activity with no more than routine experimentation.
• a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative, as described herein and known in the art.
• the polypeptide variant comprises conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide.
• DRS polypeptide variants useful in any of the methods and compositions of the invention include full-length DRS polypeptides, or truncations or splice variants thereof ⁇ e.g., any of the proteins or nucleic acids listed in or derivable from Tables Dl to D9 which i) optionally retain detectable non canonical activity and ii) have one or more additional amino acid substitutions, insertions, or deletions).
• a variant polypeptide 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%o or more sequence identity or similarity to a corresponding sequence of a DRS reference polypeptide, as described herein ⁇ e.g., any of the proteins or nucleic acids listed in or derivable from Tables Dl to D9 and substantially retains the non-canonical activity of that reference polypeptide).
• the amino acid additions or deletions occur at the C-terminal end and/or the N-terminal end of the DRS reference polypeptide.
• the amino acid additions include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more wild-type residues ⁇ e.g., from the corresponding full-length DRS polypeptide or a polypeptide listed in or derivable from Tables Dl to D9) that are proximal to the C-terminal end and/or the N-terminal end of the DRS reference polypeptide.
• Certain illustrative embodiments comprise a DRS polypeptide fragment that ranges in size from about 20-50, 20-100, 20-150, 20-200, 20-250, 20-300, 20-400, or 20-500 amino acids in length. In other embodiments, the DRS polypeptide fragment ranges in size from about 50-100, 50-150, 50-200, 50-250, 50-300, 50-400, or 50-500 amino acids in length.
• the DRS polypeptide fragment ranges in size from about 100-120, 100-130, 100-140, 100-150, 100-200, 100-250, 100-300, 100-400, or 100-500 amino acids in length, or from about 130-150, 150-175, 150-200, 150-250, 150-300, 150-400, or 150-500 amino acids in length. In still other illustrative embodiments, the DRS polypeptide fragment ranges in size from about 200-300, 200-250, 200-400, or 200-500 amino acids in length.
• the DRS polypeptide or fragment will comprise or consists essentially of the amino acids 1-224, 1-184, 1-174, 1-171, 1-154, 11-146, 13-146, or 23-154 of the DRS polypeptide sequence set forth in SEQ ID NO:l, optionally comprising at least one mutation at either Cys76 or Cysl30 (using the numbering of SEQ ID NO:l), and variants thereof.
• Certain embodiments comprise a polypeptide fragment of the full-length aspartyl-tRNA synthetase of up to about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids, which comprises, or consists essentially of the amino acids 1-224, 1-184, 1-174, 1-171, 1-154, 11-146, 13-146, or 23-154 of the DRS polypeptide sequence set forth in SEQ ID NO:l, optionally comprising at least one mutation at either Cys76 or Cysl30 (using the numbering of SEQ ID NO:l), and variants thereof.
• a DRS polypeptide of the invention comprises the minimal active fragment of a full-length DRS polypeptide capable of modulating TLR activity etc., in vivo or having other desirable non-canonical aspartyl-tRNA synthetase activities.
• a minimal active fragment consists essentially of the anticodon binding domain (e.g., about amino acids 23-154 or 13-146 of SEQ ID NO:l).
• the DRS polypeptide comprises an amphiphilic helix, such as the N-terminal amphiphilic helix of about residues 1-22 of SEQ ID NO:l, and/or an OB fold domain.
• the minimal active fragment consists essentially of the anticodon binding domain anticodon binding domain, and N-terminal amphiphilic helix ⁇ e.g., about amino acids 1-154 of SEQ ID NO:l). In some aspects, of either of these embodiments, the minimal active fragment consists essentially of the anticodon binding domain anticodon binding domain, and N-terminal amphiphilic helix and a variable amount of the flexible 29 amino acid linker ⁇ e.g., amino acids 154 to 182 of SEQ ID NO: l). In different embodiments, such minimal active fragments 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 the flexible linker.
• non-canonical activity may be modulated by the selective deletion, in whole or part of the Amphiphilic helix domain, anticodon-recognition domain, or the aminoacylation domain.
• Specific examples of splice variants that accomplish such embodiments include for example AspRSl N6 and AspRSl C2 (partial deletion of the anticodon binding domain), AspRSl N? (partial deletion of both the anticodon binding domain and aminoacylation domain), AspRSl N? (partial deletion of the aminoacylation domain).
• all such DRS polypeptides comprise at least one mutation at Cys76, Cysl30, Cys 203, Cys259, Cys334, or Cys349 (using the numbering of SEQ ID NO:l).
• sequence identity or, for example, comprising a "sequence 50% identical to, " as used herein, refer to the extent that sequences are identical on a nucleotide -by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
• a “percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. , A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• the identical nucleic acid base e.g. , A, T, C, G, I
• the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys,
• reference sequence " “comparison window, " “sequence identity, “ “percentage of sequence identity” and “substantial identity. "
• a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polypeptides may each comprise (1) a sequence (i.e., only a portion of the complete polypeptides sequence) that is similar between the two polypeptides, and (2) a sequence that is divergent between the two polypeptides, sequence comparisons between two (or more) polypeptides are typically performed by comparing sequences of the two polypeptides over a "comparison window " to identify and compare local regions of sequence similarity.
• a “comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• sequence similarity or sequence identity between sequences can be performed as follows.
• the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
• the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%>, 60%>, and even more preferably at least 70%, 80%>, 90%, 100%> of the length of the reference sequence.
• amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When 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 the molecules are identical at that position.
• the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
• the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
• the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
• a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penally of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
• the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4: 1 1-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penally of 12 and a gap penally of 4.
• nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
• Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al , ⁇ J. Mol. Biol, 215: 403-10, 1990).
• Gapped BLAST can be utilized as described in Altschul et al, (Nucleic Acids Res, 25: 3389-3402, 1997).
• the default parameters of the respective programs e.g., XBLAST and NBLAST.
• variant polypeptides differ from the corresponding DRS reference sequences by at least 1% but less than 20%, 15%, 10%> or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, suitably, differences or changes at a non-essential residue or a conservative substitution.
• the molecular weight of a variant DRS polypeptide differs from that of the DRS reference polypeptide by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or more.
• polynucleotides and/or polypeptides can be evaluated using a BLAST alignment tool.
• a local alignment consists simply of a pair of sequence segments, one from each of the sequences being compared.
• a modification of Smith- Waterman or Sellers algorithms will find all segment pairs whose scores cannot be improved by extension or trimming, called high-scoring segment pairs (HSPs).
• HSPs high-scoring segment pairs
• the results of the BLAST alignments include statistical measures to indicate the likelihood that the BLAST score can be expected from chance alone.
• the raw score, S is calculated from the number of gaps and substitutions associated with each aligned sequence wherein higher similarity scores indicate a more significant alignment. Substitution scores are given by a look-up table (see PAM, BLOSUM).
• Gap scores are typically calculated as the sum of G, the gap opening penally and L, the gap extension penally.
• the gap cost would be G+Ln.
• the choice of gap costs, G and L is empirical, but it is customary to choose a high value for G (10-15), e.g., 11, and a low value for L (1-2) e.g., 1.
• bit score, S' is derived from the raw alignment score S in which the statistical properties of the scoring system used have been taken into account. Bit scores are normalized with respect to the scoring system, therefore they can be used to compare alignment scores from different searches. The terms “bit score " and “similarity score " are used interchangeably. The bit score gives an indication of how good the alignment is; the higher the score, the better the alignment.
• the E-Value describes the likelihood that a sequence with a similar score will occur in the database by chance. It is a prediction of the number of different alignments with scores equivalent to or better than S that are expected to occur in a database search by chance. The smaller the E- Value, the more significant the alignment. For example, an alignment having an E value of e "117 means that a sequence with a similar score is very unlikely to occur simply by chance. Additionally, the expected score for aligning a random pair of amino acids is required to be negative, otherwise long alignments would tend to have high score independently of whether the segments aligned were related. Additionally, the BLAST algorithm uses an appropriate substitution matrix, nucleotide or amino acid and for gapped alignments uses gap creation and extension penalties. For example, BLAST alignment and comparison of polypeptide sequences are typically done using the BLOSUM62 matrix, a gap existence penally of 11 and a gap extension penalty of 1.
• sequence similarity scores are reported from BLAST analyses done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penally of 1.
• sequence identity/similarity scores provided herein refer to the value obtained using GAP Version 10 (GCG, Accelrys, San Diego, Calif.) using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA. 89: 10915-10919, 1992).
• GAP uses the algorithm of Needleman and Wunsch (1970) J Mol Biol 48:443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
• the DRS polypeptides comprise an amino acid sequence that can be optimally aligned with a DRS reference polypeptide sequence described herein (e.g., amino acid residues 1-224, 1-184, 1-174, 1-171, 1-154, 11-146, 13-146, or 23-154 of the DRS polypeptide sequence set forth in SEQ ID NO:l, optionally comprising at least one mutation at either Cys76 or Cysl30 (using the numbering of SEQ ID NO:l); or any one of SEQ ID NOS: l, 3-24, 29, 31, or 154-197) to generate a BLAST bit scores or sequence similarity scores of at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,
• biologically active fragments of the DRS reference polypeptides, i.e., biologically active fragments of the DRS protein fragments.
• Representative biologically active fragments generally participate in an interaction, e.g., an intramolecular or an inter-molecular interaction.
• An inter- molecular interaction can be a specific binding interaction or an enzymatic interaction.
• An inter-molecular interaction can be between a DRS polypeptide and a cellular binding partner, such as a cellular receptor or other host molecule that participates in the non-canonical activity of the DRS polypeptide.
• a biologically active fragment of a DRS reference polypeptide can be a polypeptide fragment which is, 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, 180, 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, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 38, 359, 360, 361, 362, 363, 364, 365, 380, 400, 450, 500 or more contiguous or non-contiguous amino acids, including
• a biologically active fragment comprises a non-canonical activity-related sequence, domain, or motif.
• the C-terminal or N-terminal region of any DRS reference polypeptide may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500 or more amino acids, or by about 10-50, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500 or more amino acids, including all integers and ranges in between (e.g., 101, 102, 103, 104, 105), so long as the truncated DRS polypeptide retains the non- canonical activity of the reference polypeptide.
• the biologically-active fragment has no less than about 1%, 10%, 25%, or 50% of an activity of the biologically-active (i.e., non-canonical activity) DRS reference polypeptide from which it is derived. Exemplary methods for measuring such non- canonical activities are described in the Examples.
• DRS proteins, variants, and biologically active fragments thereof bind to one or more cellular binding partners with an affinity of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 100, or 150 nM.
• the binding affinity of a DRS protein fragment for a selected cellular binding partner can be stronger than that of the corresponding full length DRS polypeptide or a specific alternatively spliced DRS polypeptide variant, by at least about 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, lOx, 15x, 20x, 25x, 3 Ox, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, lOOOx or more (including all integers in between).
• a DRS polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
• amino acid sequence variants of a DRS reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (Proc. Natl. Acad. Sci. USA. 82: 488-492, 1985), Kunkel et al, (Methods in Enzymol. 154: 367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D.
• Biologically active truncated and/or variant DRS polypeptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to a reference DRS amino acid residue, and such additional substitutions may further enhance the activity or stability of the DRS polypeptides with altered cysteine content.
• a "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub- classified as follows:
• Acidic The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
• Amino acids having an acidic side chain include glutamic acid and aspartic acid.
• the residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
• Amino acids having a basic side chain include arginine, lysine and histidine.
• the residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).
• amino acids having acidic or basic side chains i.e., glutamic acid, aspartic acid, arginine, lysine and histidine.
• Hydrophobic The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
• Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.
• Neutral/polar The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
• Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.
• proline This description also characterizes certain amino acids as “small " since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity.
• small amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not.
• Amino acids having a small side chain include glycine, serine, alanine and threonine.
• the gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains.
• the structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the a-amino group, as well as the a-carbon.
• the degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behavior.
• Amino acid residues can be further sub-classified as cyclic or non-cyclic, and aromatic or non- aromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large.
• the residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not.
• Small residues are, of course, always non-aromatic.
• amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub- classification according to this scheme is presented in Table A.
• Conservative amino acid substitution also includes groupings based on side chains.
• a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic -hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
• Amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, (c) the bulk of the side chain, or (d) the biological function. After the substitutions are introduced, the variants are screened for biological activity.
• similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains.
• the first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains;
• the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine;
• the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm.C. Brown Publishers (1993).
• a predicted non-essential amino acid residue in a truncated and/or variant DRS polypeptide is typically replaced with another amino acid residue from the same side chain family.
• mutations can be introduced randomly along all or part of a DRS coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity.
• the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined.
• a "nonessential " amino acid residue is a residue that can be altered from the reference sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its non canonical activities.
• the alteration does not substantially abolish one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% 100%, 500%, 1000% or more of the reference DRS sequence.
• An "essential " amino acid residue is a residue that, when altered from the reference sequence of a DRS polypeptide, results in abolition of an activity of the parent molecule such that less than 20% of the reference activity is present.
• such essential amino acid residues include those that are conserved in DRS polypeptides across different species, including those sequences that are conserved in the active binding site(s) or motif(s) of DRS polypeptides from various sources.
• DRS polypeptides may have one or more cysteine substitutions, where one or more naturally- occurring (non-cysteine) residues are substituted with cysteine, for example, to facilitate thiol-based attachment of other molecules.
• cysteine substitutions are near the N-terminus and/or C-terminus of the DRS polypeptide (e.g., SEQ ID NOS: 1 , 3-24, 29, 31 , or 154-197).
• Particular embodiments include where one or more of residues within 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 amino acids relative to the N-terminus and/or C-terminus of any one of SEQ ID NOS:l, 3-24, 29, 31 , or 154-197 are substituted with a cysteine residue.
• cysteine residues may be added to the DRS polypeptide through the creation of N-terminal, or C-terminal fusion proteins.
• Such fusion proteins may be of any length, but will typically be about 1 -5, or about 5-10, about 10 to 20, or about 20 to 30 amino acids in length.
• fusion to the C-terminus is preferred.
• DRS polypeptides with an N-terminal cysteine substitution include for example, those with a cysteine substitution within the first 23 amino acids, including the DRS polypeptides of any of SEQ ID NOS: 1 , 3-24, 29, 31 , or 154-197.
• DRS polypeptides with a C-terminal cysteine substitution include for example, those with a cysteine substitution with the last 20 amino acids, including the DRS polypeptides of any of SEQ ID NOs: 1, 3-24, 29, 31, or 154-197.
• DRS polypeptides may also have additional substitutions at C76 and/or CI 30, to remove naturally-occurring cysteine residues.
• Specific embodiments include any one of SEQ ID NOS: l , 3-24, 29, 31, or 154-197, or variants thereof, having at mutation at C76 and/or C130. Exemplary mutations at these positions include for example the mutation of cysteine to serine, alanine, leucine, or glycine.
• Various exemplary proteins with reduced cysteine content are listed in Table D7.
• DRS polypeptides may have one or more glutamine substitutions, where one or more naturally- occurring (non-glutamine) residues are substituted with glutamine.
• glutamine substitutions are introduced near the N-terminus and/or C-terminus of the DRS polypeptide (e.g., SEQ ID NOS: l, 3-24, 29, 31, or 154-197).
• Particular embodiments include where one or more of residues within 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids relative to the N-terminus and/or C-terminus of any one of SEQ ID NOS: l, 3-24, 29, 31, or 154-197 are substituted with a glutamine residue.
• These and related DRS polypeptides can also include substitutions (e.g., conservative substitutions) to remove any naturally-occurring glutamine residues.
• DRS polypeptides may have one or more lysine substitutions, where one or more naturally- occurring (non-lysine) residues are substituted with lysine.
• lysine substations are near the N-terminus and/or C-terminus of the DRS polypeptide (e.g., SEQ ID NOS: l, 3-24, 29, 31, or 154-197).
• Particular embodiments include where one or more of residues within 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids to the N-terminus and/or C- terminus of any one of SEQ ID NOS: l , 3-24, 29, 31, or 154-197 are substituted with a lysine residue.
• DRS polypeptides can also include substitutions (e.g., conservative substitutions) to remove any naturally-occurring lysine residues, if desired.
• DRS variants may also be created by substituting one or more solvent accessible surface amino acids of a DRS polypeptide, or alternatively, by avoiding substitution of solvent accessible surface amino acids.
• Suitable solvent accessible amino acids may be determined based on the predicted solvent accessibility using the SPPIDER server (http://sppider.cchmc.org/) using the published crystal structure of an exemplary DRS polypeptide (WO2010/120509). Based on this analysis several amino acids on the surface may potentially be used as mutation sites, for instance, by conservative substitution to minimize effects on surface interactions, or by non-conservative substitution to interfere with certain surface interactions.
• Table D8 lists the surface accessibility score of amino acids based on the crystal structure above. In this table, the higher scores represent better accessibility.
• an amino acid position selected from Table D8 may be used to introduce a cysteine, lysine, glutamine, or non-naturally occurring amino acid.
• an amino acid position selected from Table D8 may be used to introduce a conservative or non-conservative substitution.
• a DRS variant may retain one or more or all of the amino acid residues from Table D8.
• a DRS variant my retain an amino acid residue from Table D8 having a score of greater than about 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63.
• a solvent accessible surface amino acid from Table D8 is selected from the group consisting of: alanine, glycine, and serine, and can be substituted with naturally occurring amino acids including, but not limited to, cysteine, glutamine, or lysine, or a non-naturally occurring amino acid.
• one or more solvent accessible surface amino acids of the DRS polypeptide are selected from the group consisting of: C130, G129, A107, A72 and G95 are, substituted with cysteine, glutamine, lysine, or a non-naturally occurring amino acid.
• certain DRS polypeptides may contain one or more non-naturally occurring amino acids.
• non-naturally occurring amino acids include, without limitation, any amino acid, modified amino acid, or amino acid analogue other than selenocysteine and the following twenty genetically encoded alpha-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.
• the generic structure of an alpha-amino acid is illustrated by the following formula:
• a non-natural amino acid is typically any structure having the foregoing formula wherein the R group is any substituent other than one used in the twenty natural amino acids. See, e.g., any biochemistry text such as Biochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York, for structures of the twenty natural amino acids. Note that the non-natural amino acids disclosed herein may be naturally occurring compounds other than the twenty alpha-amino acids above. Because the non-natural amino acids disclosed herein typically differ from the natural amino acids in side chain only, the non-natural amino acids form amide bonds with other amino acids, e.g., natural or non -natural, in the same manner in which they are formed in naturally occurring proteins.
• R in foregoing formula optionally comprises an 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, thio ether, epoxide, sulfone, boronic acid, boronate ester, borane, phenylboronic acid, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic-, pyridyl, naphthyl, benzophenone, a constrained
• unnatural amino acids include, but are not limited to, p-acetyl-L- phenylalanine, 0-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3 -methyl -phenylalanine, an O-4-allyl- L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAc -serine, ⁇ - ⁇ -GlcNAc-L-serine, a tri-O-acetyl- GalNAc-a-threonine, an a-GalNAc-L-threonine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L- phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-
• non-natural amino acids can be utilized to modify ⁇ e.g., increase) a selected non-canonical activity of a DRS polypeptide, or to alter the in vivo or in vitro half-life of the protein.
• Non-natural amino acids can also be used to facilitate (selective) chemical modifications ⁇ e.g., PEGylation) of a DRS protein.
• certain non-natural amino acids allow selective, protein- protein attachment of Fc regions to a DRS polypeptide, and thereby improve their pharmacokinetic properties.
• amino acid analogs and mimetics can be found described in, for example, Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341, Academic Press, Inc., New York, N.Y. (1983), the entire volume of which is incorporated herein by reference.
• Other examples include peralkylated amino acids, particularly permethylated amino acids. See, for example, Combinatorial Chemistry, Eds. Wilson and Czarnik, Ch. 11, p. 235, John Wiley & Sons Inc., New York, N.Y. (1997), the entire book of which is incorporated herein by reference.
• Yet other examples include amino acids whose amide portion (and, therefore, the amide backbone of the resulting peptide) has been replaced, for example, by a sugar ring, steroid, benzodiazepine or carbo cycle. See, for instance, Burger's Medicinal Chemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620, John Wiley & Sons Inc., New York, N.Y. (1995), the entire book of which is incorporated herein by reference. Methods for synthesizing peptides, polypeptides, peptidomimetics and proteins are well known in the art (see, for example, U.S. Pat. No. 5,420,109; M.
• DRS polypeptides of the present invention may be composed of naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetic s.
• the DRS polypeptide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe
• AspRSl N1 /DRS(l-154) comprising at least one mutation at Cys76 and/or Cysl30.
• the DRS polypeptide is DRS (11-146) comprising at least one mutation at Cys76 and/or Cysl30.
• the DRS polypeptide is DRS (13-146) comprising at least one mutation at Cys76 and/or Cysl30.
• the DRS polypeptide is full- length DRS (SEQ ID NO:l) comprising at least one mutation at Cys76, Cysl30, Cys203, Cys259, Cys334, and/or Cys339.
• the DRS polypeptide may comprise at mutation at Cys76, Cysl30, Cys203, Cys259, Cys334, and/or Cys339, wherein the substituted amino acid is independently selected from the group consisting of all 19 alternative naturally occurring amino acids except Cys, or a non- naturally occurring amino acid.
• the DRS polypeptide may comprise at mutation at Cys76, Cys 130, Cys203, Cys259, Cys334, and/or Cys339, wherein the substituted amino acid is independently selected from the group consisting of Ser, Ala, Gly, Met, Leu, Val; He and Thr.
• the DRS polypeptide may comprise at mutation at Cys76, Cys 130, Cys203, Cys259, Cys334, and/or Cys339, wherein the substituted amino acid is independently selected from the group consisting of Ser and Ala.
• the DRS polypeptide may comprise at mutation at Cys76, Cys 130, Cys203, Cys259, Cys334, and/or Cys339, wherein the substituted amino acid is independently selected from the group consisting of Asp, Glu, Arg, Lys, Gin, and Asn.
• the DRS polypeptide may comprise at mutation at Cys76, Cysl30, Cys203, Cys259, Cys334, and/or Cys339, wherein the substituted amino acid is independently selected from the group consisting of His, Pro, Tyr, Trp and Phe.
• the DRS polypeptide may comprise at mutation at Cys76, Cys 130,
• Cys203, Cys259, Cys334, and/or Cys339 wherein the substitution is a independently selected from Ser, Ala, Gly, Met, Leu, Val; He and Thr, and a non-naturally occurring amino acid.
• Cys76 and/or Cys203 may be selectively modified, while Cysl30 remains unmodified. Conversely, in some embodiments, Cysl30 and/or Cys203 may be selectively modified, while Cys76 remains unmodified. In some embodiments Cys76, Cysl30, and/or Cys203 may be independently modified using any combination of the sub-groupings listed above.
• Cys203, Cys259, Cys334, and Cys349 may be selectively modified, while Cysl30 remains unmodified.
• Cys76, Cys203, Cys259, Cys334, and Cys349 may be selectively modified, while Cysl30 remains unmodified.
• Cys76 may be selectively modified, where the cysteine at position 130 is used to selectively chemically couple another molecule, such as an Fc region.
• Certain embodiments relate to polynucleotides that encode a DRS polypeptide, such as a DRS-Fc fusion protein. Also included are polynucleotides that encode any one or more of the Fc regions described herein, alone or in combination with a DRS coding sequence. Among other uses, these embodiments may be utilized to recombinantly produce a desired DRS, Fc region, or DRS-Fc polypeptide or variant thereof, or to express the DRS, Fc region, or DRS-Fc polypeptide in a selected cell or subject.
• polynucleotides can encode the DRS polypeptides, Fc regions, and fusion proteins of the invention.
• the polynucleotide sequence can be manipulated for various reasons. Examples include but are not limited to the incorporation of preferred codons to enhance the expression of the polynucleotide in various organisms (see generally Nakamura et al, Nuc. Acid. Res. 28:292, 2000).
• silent mutations can be incorporated in order to introduce, or eliminate restriction sites, decrease the density of CpG dinucleotide motifs (see for example, Kameda et al. , Biochem. Biophys. Res. Commun.
• mammalian expression can be further optimized by including a Kozak consensus sequence (i.e., (a/g)cc(a/g)ccATGg) (SEQ ID NO:79) at the start codon.
• Kozak consensus sequences useful for this purpose are known in the art (Mantyh et al, PNAS 92: 2662-2666, 1995; Mantyh et al,. Prot. Exp. & Purif. 6: 124, 1995).
• Exemplary codon optimized versions of the wild type full length DRS polypeptide and AspRSl N1 are provided in Table D9, below.

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