TOMMUNOMUS009822353B2 U DITARTI MITTE (12 ) United States Patent (10 ) Patent No. : US 9 ,822 , 353 B2 Buechler et al. ( 45 ) Date of Patent: Nov . 21 , 2017 (54 ) PEGYLATED ASPARTYL - TRNA 5 ,753 , 480 A 5 / 1998 Lawlor SYNTHETASE POLYPEPTIDES 5 , 756 , 327 A 5 / 1998 Sassanfar et al . 5 ,759 ,833 A 6 / 1998 Shiba et al. 5 , 776 ,749 A 7 / 1998 Hodgson et al. (71 ) Applicant: a Tyr Pharma, Inc ., San Diego , CA 5 , 795 ,757 A 8 / 1998 Hodgson et al . (US ) 5 , 798 , 240 A 8 / 1998 Martinis et al . 5 , 801, 013 A 9 / 1998 Tao et al . (72 ) Inventors : Ying Ji Buechler, Carlsbad , CA (US ); 5 , 866 , 390 A 2 / 1999 Lawlor 5 , 885 , 815 A 3 / 1999 Sassanfar et al . Chi- Fang Wu , San Diego , CA (US ) ; 5 , 928 , 920 A 7 / 1999 Hodgson et al. Jeffrey Greve , Berkeley , CA (US ); 5 , 939 , 298 A 8 / 1999 Brown et al. John D . Mendlein , Encinitas, CA (US ) 6 , 225, 060 B1 5 / 2001 Clark et al. 6 , 255, 090 B1 7 / 2001 Famodu et al. ( 73 ) Assignee: aTyr Pharma, Inc ., San Diego , CA 6 , 265 ,188 B1 7 / 2001 Brown et al. (US ) 6 , 428 , 960 B1 8 / 2002 Clark et al. 6 , 548 , 060 B1 4 / 2003 Kim 6 , 696, 619 B1 2 / 2004 Famodu et al. ( * ) Notice: Subject to any disclaimer, the term of this 6 ,852 , 512 B2 2 / 2005 Choi et al. patent is extended or adjusted under 35 6 , 903 , 189 B2 6 /2005 Schimmel et al. U .S .C . 154 (b ) by 257 days . 7 , 037 , 505 B2 5 /2006 Kim et al . 7 , 067 , 126 B2 6 / 2006 Schimmel et al. 7 , 144 , 984 B2 12/ 2006 Schimmel et al. ( 21) Appl. No .: 14 / 360 , 573 7 ,196 , 068 B2 3 / 2007 Kim et al. 7 , 273 , 844 B2 9 / 2007 Schimmel et al . ( 22 ) PCT Filed : Dec. 6 , 2012 7 , 282 , 208 B2 | 10 / 2007 Kim 7 , 413 , 885 B2 8/ 2008 Schimmel et al. ( 86 ) PCT No. : PCT/ US2012 / 068296 7 ,459 , 529 B2 12 / 2008 Kim 7 ,476 , 651 B2 1 / 2009 Schimmel et al. $ 371 ( c ) ( 1 ) , 7 , 521 , 215 B2 4 /2009 Schimmel et al. ( 2 ) Date : May 23 , 2014 7 ,528 , 106 B2 5 /2009 Friedlander et al . (87 ) PCT Pub. No. : W02013 /086228 (Continued ) PCT Pub . Date : Jun . 13, 2013 FOREIGN PATENT DOCUMENTS CA 2531146 3 / 2005 (65 ) Prior Publication Data CN 1341725 3 / 2002 US 2015 /0159148 A1 Jun . 11, 2015 (Continued ) Related U . S . Application Data OTHER PUBLICATIONS (60 ) Provisional application No. 61/ 567 , 602 , filed on Dec . International Preliminary Report on Patentability for International 6 , 2011 . Application No. PCT/ US2009 / 048915 , dated Jan . 5 , 2011 . International Search Report and Written Opinion for International (51 ) Int. Ci. Application No. PCT/ US2009 / 048915 , dated Nov . 2 , 2009 . C12N 9 / 96 ( 2006 .01 ) Office Action for U . S . Appl. No . 12 /482 , 151 , dated Oct . 11 , 2011 , A61K 38 /43 ( 2006 .01 ) 43 pages . ( 2006 .01 ) Office Action for U . S . Appl. No. 12 /482 , 151, dated Mar. 18 , 2011, C12N 9 / 00 11 pages. A61K 47 / 48 ( 2006 .01 ) International Preliminary Report on Patentability for International A61K 38 /00 ( 2006 .01 ) Application No. PCT/ US2009 /046910 , dated Dec. 13 , 2010 . A61K 38 /53 ( 2006 .01 ) (52 ) U .S . CI. (Continued ) CPC ...... C12N 9 / 93 ( 2013 .01 ); A61K 47/ 48215 ( 2013 .01 ) ; C12N 9 / 96 (2013 . 01 ) ; C12Y Primary Examiner — Kagnew H Gebreyesus 601/ 01023 (2013 .01 ) ; A61K 38 / 00 (2013 .01 ) ; A61K 38 /53 (2013 .01 ) ; C12Y 601/ 01012 (74 ) Attorney , Agent, or Firm — Cooley LLP ( 2013 .01 ) (58 ) Field of Classification Search (57 ) ABSTRACT CPC ...... C12N 9 / 96 ; A61K 38 /43 ; CO7K 2319 /30 ; C12Y 601 / 01012 ; C12Y 601 / 01023 The present invention provides PEGylated aspartyl- tRNA See application file for complete search history. synthetase ( DRS ) polypeptides , compositions comprising the same, and methods of using such polypeptides and (56 ) References Cited compositions for treating or diagnosing a variety of condi tions. The PEGylated DRS polypeptides of the invention U .S . PATENT DOCUMENTS have improved controlled release properties , stability , half 5 ,370 , 995 A 12 / 1994 Hennecke et al. life , and other pharmacokinetic properties compared to 5 , 484 , 703 A 1/ 1996 Raben et al . non -PEGylated DRS polypeptides . 5 ,663 , 066 A 9 / 1997 Raben et al. 5 , 747 , 315 A 5 / 1998 Lawlor 5 , 750 , 387 A 5 / 1998 Hodgson et al. 9 Claims, 12 Drawing Sheets US 9, 822 , 353 B2 Page 2
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tu Tip Homeokine FIG . 12BDexamethasone US 9 ,822 , 353 B2 PEGYLATED ASPARTYL - TRNA manner, or where TLRs may be involved in the breakdown SYNTHETASE POLYPEPTIDES of immune tolerance . TLRs have been implicated in the pathogenesis of chronic inflammatory diseases such as CROSS -REFERENCE TO RELATED inflammatory bowel disease , rheumatoid arthritis , psoriasis , APPLICATIONS 5 and multiple sclerosis . It is now increasingly recognized that the successful This application claims benefit under 35 U . S . C . $ 119 ( e ) treatment of some autoimmune and inflammatory conditions of U . S . Provisional Application No . 61/ 567 ,602 , filed Dec . of tissues requires effective control of the inflammatory 6 , 2011 , which is incorporated by reference in its entirety . reaction in order to preserve tissue integrity and function , 10 without immune - compromising the patient . Recent experi STATEMENT REGARDING SEQUENCE mental evidence has shown that specific modulation of TLR LISTING pathways induces an improvement in several inflammatory conditions, without comprising tissue function , or enhancing The Sequence Listing associated with this application is bacterial or viral infections, suggesting the potential for new provided in text format in lieu of a paper copy, and is hereby 15 therapeutic anti- inflammatory strategies with significantly incorporated by reference into the specification . The name of improved side effect profiles. Moreover TLR agonists have the text file containing the Sequence Listing is already proved useful in clinical trials in allergic, infectious ATYR _ 108 _ 01 WO _ ST25 . txt. The text file is about 203 KB , and autoimmune diseases and are under development for a was created on Dec . 6 , 2012 , and is being submitted elec - broad range of other diseases including cancer , arthritis , tronically via EFS -Web . 20 multiple sclerosis , inflammatory bowel disease , see gener ally Zhu and Mohan ( 2010 ) Mediators of Inflammation BACKGROUND doi: 10 . 1155 / 2010 / 781235 ; Hennessy et al . , Nat. Rev . 9 : 293 307 , 2010 ). Therefore TLRs are becoming increasingly Technical Field recognized as novel potential therapeutic targets for the The present invention relates generally to PEGylated 25 modulation of a broad variety of diseases and disorders . aspartyl -tRNA synthetase (DRS ) polypeptides , composi - To best exploit these and other activities in therapeutic or tions comprising the same, and methods of using such diagnostic settings, there is a need in the art for DRS polypeptides and compositions for treating or diagnosing a polypeptides having improved pharmacokinetic properties . variety of conditions . The present invention is focused on the development of DRS Description of the Related Art 30 polypeptides that have been modified through the addition of Aspartyl -TRNA synthetases (DRS ) , and fragments and water soluble polymers , which retain the biological activity variants thereof, ( collectively DRS polypeptides ) have of the native DRS polypeptides and exhibit superior phar recently been shown to possess a variety of non - canonical macokinetic and other properties . activities of therapeutic and diagnostic relevance . In par- Although many suitable water soluble polymers exist, ticular it has been established that certain aspartyl- tRNA 35 polyethylene glycol (PEG ) is typically preferred as a water synthetase fragments are highly potent, endogenously pro - soluble polymer for attachment because it has good solu duced , Toll - like receptor modulators . Without being bound bility in both water and in many organic solvents, lacks of to any one specific theory of operation , it is believed that toxicity, and immunogenicity , and is also clear, colorless , such DRS polypeptides are released from macrophage cells odorless , and chemically stable . upon proteolytic cleavage , or through alternative splicing of 40 The term “ PEGylation ” refers to the modification of the full length AspRS tRNA synthetase and are capable of biological molecules by covalent conjugation with polyeth binding to and modulating the activity of immunomodula ylene glycol (PEG ) . PEGylation can change the physical and tory , and other cell types . Such DRS polypeptides when chemical properties of a biological molecule , such as its administered , provide for a novelmechanism of selectively conformation , electrostatic binding , hydrophobicity , and modulating inflammatory responses , without the side effect 45 pharmacokinetic profile . In general, PEGylation improves profiles typically associated with traditional anti - inflamma - drug solubility and decreases immunogenicity . PEGylation tory agents such as steroids also increases drug stability and the retention time of the Toll - like receptors ( TLRs) are a family of pattern recog - conjugates in blood , and reduces proteolysis and renal nition receptors that play a key role in initiating the rapid0 excretion , thereby allowing a reduced dosing frequency . In innate immune response in an organism . TLRs recognize 50 order to benefit from these favorable pharmacokinetic con certain pathogen or host derived cellular components which sequences , a variety of therapeutic proteins, peptides , and can be generally characterized as being either pathogen antibody fragments , as well as small molecule drugs, have associated molecular patterns, (PAMPs ) , or damage -associ - been PEGylated . ated molecular pattern molecules , (DAMPS ) respectively . These improved therapeutic forms of the DRS polypep PAMPS are typically unique to a given class of pathogen , 55 tides enable the development of more effective therapeutic and include for example bacterial components such as the regimens for the treatment of various diseases and disorders, lipopolysaccharide of Gram negative bacteria , and viral and require significantly less frequent administration than specific nucleic acid motifs or viral specific modifications of the unmodified proteins . RNA or DNA . By contrast DAMPS are typically endog enousmolecules released from dying host cells upon cellular 60 BRIEF DESCRIPTION OF THE DRAWINGS stress or tissue damage . TLRs are implicated in several chronic inflammatory and FIG . 1 shows an SDS - PAGE analysis of the purified immune mediated disorders by various potential mecha proteins AspRSIM (C76S ) (DRS ( 1 - 154 ) (C76S ) and the cor nisms, including those in which infectious agents have been responding non mutated protein AspRSIM (DRS ( 1 - 154 )) . proposed to initiate disease progression . For example in 65 Lanes 1 - 3 were run under reduced conditions, and lanes 4 - 6 scenarios in which endogenous damage signals or self - were run under non - reduced conditions. Lanes 1 and 4 : antigens cause chronic inflammation in a TLR dependent AspRS1M DRS ( 1 - 154 ) lot # D -N1 -V5H -046 , lanes 2 and 5 US 9 ,822 ,353 B2 AspRSIM (DRS (1 -154 )) lot # D -N1 - V5H -047 , lanes 3 and thereof. In some embodiments , at least one endogenous 6 : AspRS1M (C768 ) lot # D -N1 : 1 - V5H -048 . cysteine residue has been modified to another amino acid FIG . 2 shows a direct comparison of AspRS1M1 (grey residue . squares) and AspRS1M (C76S ) (black circles ) on their In some embodiments of the PEGylated DRS polypep ability to stimulate reporter gene activity mediated by the 5 tide , the DRS polypeptide is about 50 - 200 amino acids in TLR2 receptor in HEK — Blue 2 cells . Grey triangles length and comprises amino acid residues 1- 154 , 11 - 146 , Pam3C SK4. 13 - 146 , 23 - 154 , 1 - 171, or 1 - 174 of SEQ ID NO : 1 . In one FIG . 3 shows a direct comparison of AspRS1M1 ( grey aspect the PEGylated DRS polypeptide comprises amino squares ) and AspRSIM (C76S ) ( Black circles ) on their acid residues 1 - 154 . In one aspect the PEGylated DRS ability to stimulate reporter gene activity mediated by the polypeptide comprises amino acid residues 13 - 146 . In some TLR4 receptor in HEK - Blue 4 cells . embodiments , the Cys76 residue has been selectively FIG . 4 shows the SDS - PAGE analysis of samples of mutated to another amino acid . In some embodiments , the AspRS1M C76S ) PEGylated with different PEGylation Cy $ 130 residue has been selectively mutated to anoti amino acid . In some embodiments both the Cys76 residue reagents . A Lane 1, AspRSIM C76S ); Lane 2 , reaction mix 15 and the Cys130 residue have been mutated to another amino after PEGylation with SUNBRIGHT 40 K PEG (NOF acid . ME- 400MA ); Lane 3 purified reaction product from ( 2 ); In some embodiments the PEGylated DRS polypeptide Lane 4 Pool of purified product from lane 2 . FIG . 4B comprises a PEG moiety which has a molecular weight of PEGylated and unPEGylated samples of AspRS1M C768 ) between about 1 KDa and about 80 KDa. In some embodi after PEGylated with JemKem 40K PEG ( Cat # A3042 - 113 ) . 20 ments the PEG moiety has a molecular weight of between FIG . 5 shows the activity of the PEGylated and unPE about 20 KDa and about 60 KDa. In some embodiments the Gylated AspRS1 ^ (C76S ) in a TLR2 reporter gene assay. PEG moiety has a molecular weight of between about 30 Diamonds and triangles = UnPEGylated AspRS1M 076S ) ; KDa and about 50 KDa . In some embodiments the PEG Squares = PEGylated AspRSIM C76S ) . moiety has a molecular weight has a molecular weight of FIG . 6 shows the results of PK analysis of the PEGylated 25 about 40 KDa. In some embodiments the PEG moiety has a and unPEGylated samples of AspRS1M C76S ) in rats . molecular weight has a molecular weight of about 20 KDa. Diamonds = unPEGylated and Squares = PEGylated . In some embodiments the PEG moiety has a molecular FIG . 7 shows the WinNonLin analysis of the unPEGY weight has a molecular weight of about 10 KDa . In some lated AspRS1M ( C76S ) rat PK data . embodiments the PEG moiety has a molecular weight has a FIG . 8 shows the WinNonLin analysis of the PEGylated 30 molecular weight of about 5 KDa. In some embodiments the AspRS1M ( C76S ) rat PK data . PEG moiety has a molecular weight has a molecular weight FIG . 9 shows the results of PD analysis with respect to of about 2 KDa . In some embodiments the PEG moiety has TNF -alpha secretion in response to the injection of PEGY a molecular weight has a molecular weight of about 2 KDa . lated and unPEGylated AspRS1M 076S ) in rats . In some embodiments the PEG moiety has a molecular Diamonds = unPEGylated AspRS1 “ (C76S ) ; 35 weight has a molecular weight of less than 2 KDa. Squares = PEGylated AspRS1 “ (C76S ) ; triangles = vehicle In some embodiments of the PEGylated DRS polypeptide control. the PEG moiety is attached to an amino acid residue within FIG . 10 shows the results of PD analysis with respect to about 10 amino acid residues of the N - terminus of the DRS IL - 10 secretion in response to the injection of the PEGylated polypeptide . In some embodiments the PEG moiety is AspRS1M (C76S ) and unPEGylated AspRS1M1 (C76S ) in 40 attached to the N -terminal amino acid of the DRS polypep rats . Diamonds = unPEGylated ; Squares = PEGylated ; tide . In some embodiments the PEG moiety is attached to an triangles = vehicle control. amino acid residue within about 10 amino acid residues of FIG . 11 shows the results of the administration of the C - terminus . In some embodiments the PEG moiety is AspRS1M (C76S ) in a partial body irradiation survival attached to the C - terminal amino acid of the DRS polypep 45 tide . model; AspRS1M (C76S ) shown in squares and the PBS 45 In some embodiments of the PEGylated DRS polypeptide control shown as diamonds. the PEG moiety is attached to a cysteine ( C ) residue . In some FIG . 12 shows the results of the administration of embodiments the cysteine residue is naturally occurring . In AspRS1M (C76S ) in an MSU induced model of gout inflam one aspect, the naturally occurring cysteine residue is C76 or mation (squares ) , compared to vehicle control (PBS ) dia C130 . In one aspect it is attached to C130 . monds, and a positive control (dexamethasone ( triangles ) 50 In some embodiments the cysteine residue is introduced The insert shows the statistical significance for AspRS1M1 into , or appended onto the DRS polypeptide . In some ( C76S ) (“ Homeokine” ) compared to the vehicle control. embodiments of the PEGylated DRS polypeptide, the PEGylated DRS polypeptide comprises an inserted cysteine BRIEF SUMMARY OF THE INVENTION residue within about 10 amino acids of the N terminus, the 55 C -terminus , a solvent accessible surface amino acid of the Embodiments of the present invention relate generally to DRS polypeptide or any combination thereof. In some PEGylated aspartyl- tRNA synthetase (DRS ) polypeptides, embodiments of the PEGylated DRS polypeptide , the sol pharmaceutical compositions comprising such molecules, vent accessible surface amino acids of the DRS polypeptide used for the insertion of the cysteine residue are selected and methods for their therapeutic use . In one embodiment from the group consisting of: 5130 , G129, A107 , A72 , or the invention includes a PEGylated aspartyl- tRNA syn - 60 G95 . In some embodiments of the PEGylated DRS poly thetase (DRS ) polypeptide, comprising an amino acid peptide , the PEGylated DRS polypeptide comprises a sub sequence at least 80 % identical as that set forth in any of stituted cysteine residue wherein the substituted amino acid SEQ ID NOS : 1 , 3 - 24 , 29 , 31, or 74 - 117 , comprising at least is introduced at an amino acid position corresponding to any one PEG moiety covalently attached to an amino acid amino acid selected from 154 - 184 of SEO ID NO : 1 . In some residue within about 10 amino acid residues of the C -ter - 65 embodiments of the PEGylated DRS polypeptide at least minus or the N -terminus , or a solvent accessible surface one endogenous cysteine residue has been substituted with amino acid of the DRS polypeptide or any combination another amino acid to block PEGylation at that position . In US 9 ,822 ,353 B2 some embodiments the endogenous cysteine residue has CHO CH - CH2– CH2CH2 - O CH2 - , beenembodiments substituted the with endogenous a conservative cysteine amino residue acid is . Inselected some CHCH , °, CH°CH , CH, Ro , ? CH– , CH20°CH, CH? , CH?, CH?CH , from C76 and C130 . | CHO CH?CH , , CH , CH , CH , ? – In some embodiments of the PEGylated DRS polypeptide CH , — — CH , CH , CH , CH , O C ( O ) the PEG moiety is attached to a non - naturally occurring NH - CH2 – C (O ) - NH CH , CH , CH , C CH2 - CH2 - C ( O ) - NH - , aminoring amino acid . acidIn some comprises embodiments a side chain the nonhaving - naturally a functional occur - (0 )C -( ONH ) -NH CH2 CH – , CH , CH , , CH, C ( O) group selected from the group consisting of: an alkyl, aryl, NH - CH , CH , CH , CH - C ( O ) - NH - CH , — , aryl halide, vinyl halide , alkyl halide , acetyl, ketone , aziri - CH , CH , CH , - C ( O ) - NH — - C ( O ) - NH dine , nitrile , nitro , halide , acyl, keto , azido , hydroxyl, hydra - 10 CH , CH , CH , CH , CH , C (O ) - NH - CH , — zine, cyano , halo , hydrazide, alkenyl, alkynyl , ether , thio CH2 - CH , — , - CH2- CH2 - C (O ) - NH - CH , CH , — , ether, epoxide , sulfone , boronic acid , boronate ester, borane , CH2 - CH2 - CH2 - C ( O ) - NH - CH2 – CH , — phenylboronic acid , thiol, seleno , sulfonyl, borate , boronate , CH2 CH2 = C( O ) _ NH _ CH , CH2, CH3CH2 phospho, phosphono, phosphine , heterocyclic -, pyridyl, CH2 - CH2 - C ( O ) — NH - NH - C (O ) _ CH , naphthyl , benzophenone , a constrained ring such as a 15 CH2 - NH - C ( O ) - CH , — — CH , CH , — NH - C cyclooctyne, thio ester, enone , imine , aldehyde , ester, thio - ( ) CH2 , - NH - C ( O ) - CH2 - CH2 – CH , acid , hydroxylamine, amino , carboxylic acid , alpha -keto NH C ( O ) CH2 CH2, CHA CHANH C ( O ) carboxylic acid , alpha or beta unsaturated acids and amides , CH2 - CH , C ( O ) - NH - CH , — , - C ( O ) - NH — glyoxylamide , and organosilane group . In some embodi - CH2 - CH2 - , - O - C ( O ) - NH - CH , — , - 0 _ C (O ) ments the non - naturally occurring amino acid is selected NH - CH2 - CH2 - , - NH - CH , — — NH?CH , — from the group consisting of: p - acetyl- L - phenylalanine , - CH2 – CH2 - NH - CH , — — CH , CH , — NH O -methyl - L -tyrosine , L -3 - (2 -naphthyealanine , 3 -methyl CH2 – C ( O ) - CH2 C (O ) - CH2 - CH2 phenylalanine , 0 - 4 -allyl - L -tyrosine , 4 -propyl - L -tyrosine , | _ CH , C ( 0) CHÚ , CH , CH , C ( 0 ) CH tri- O -acetyl -GlcNAcß - serine, B - O -GlcNAC - L - serine , tri- O CH, CH , C ( O ) CH , CH?, Ý CH, CH , C acetyl- GalNAc - a - threonine , a -GalNAc - L - threonine , ( 0 ) , CH , CH , CH , C ( O ) NH?CH, CH , L -Dopa , a fluorinated phenylalanine, isopropyl- L - phenyl - 25 NH , CH2 CH2 CH2- C ( O ) - NH - CH2 CH2 alanine, p - azido - L - phenylalanine , p -acyl - L -phenylalanine , NH - C ( O ) - , - CH2 - CH2 - CH , - C ( O ) - NH - CH , — p -benzoyl - L - phenylalanine, L - phosphoserine , phosphonos - CH2 - NH - C ( O ) - CH , — bivalent cycloalkyl group , erine, phosphonotyrosine, p -iodo -phenylalanine , p -bro - N ( R ) — , Ris H or an organic radical selected from the mophenylalanine , p -amino - L -phenylalanine , and isopropyl - group consisting of alkyl, substituted alkyl, alkenyl, substi L -phenylalanine . 30 tuted alkenyl, alkynyl, substituted alkynyl, aryl and substi In some embodiments of the PEGylated DRS polypep - tuted aryl. tide, the PEGylated DRS polypeptide has the following In some embodiments , L in formula (I ) comprises a structure ( 1 ) : releasable linkage . In some embodiments , the releasable linkage is selected from the group consisting of : carboxylate X - L - Y -DRS ester , phosphate ester , anhydride , acetal, ketal, acyloxyalkyl wherein : 35 ether, imine , orthoester, thio ester, thiol ester, carbonate , and X is the PEG moiety ; hydrazone . L is an optional linker ; In some embodiments, L in formula ( I ) comprises a stable Y is a covalent linkage ; and linkage . In some embodiments , the stable linkage is selected DRS is the DRS polypeptide. from the group consisting of: succinimide , propionic acid , In some embodiments , X , in formula (1 ) is R , - 40 carboxymethylate linkages, ethers, carbamates, amides , ( CH2CH2O ) n or R , OCH2CH2) n , amines , carbamides, imides , aliphatic C — C bonds, and thio wherein Ri = alkyl, alkoxy , aryl , glucose , or galactose ; and ethers . n is 20 to 800 . In some embodiments , Y in formula ( I ) is selected from In some embodiments , R is an alkoxy selected from the the group consisting of: amide , secondary amine, carbonyl , group consisting of: methoxy , ethoxy , and benzyloxy . carboxylate , carbamate , carbamide , ester , formyl, acyl, thio In some embodiments , L in formula ( I ) comprises a chain carbonyl, thio ester, thioacetate , thioformate, thio ether, of 1 to 20 atoms selected from the group consisting of: C , S , alkoxyl, phosphoryl , phosphonate , phosphinate , amino , N , P , and O . amido , amidine, imine, cyano , nitro , azido , disulfide, sult In some embodiments , L in formula (1 ) comprises one or hydryl, sulfate , sulfonate , sulfamoyl, sulfonamido, sulfonyl , more of the following linkages: 0 , - NH - , - S — , heterocyclyl, aralkyl, aromatic moiety, hydrazone , het - C ( O ) - , C ( O ) - NH , NHC ( O ) - NH , O - C ( O ) - NH , 50 eroaromatic moiety , imino , sulfamoyl, sulfonate , silyl , ether, - CS) , CH , , CH , CH? , CH , CH , and alkylthio . CH2 – CH2CH2CH2CH2 - , In some embodiments , the PEGylated DRS polypeptides CH , O , CO CH , CH , — - CH , - 0 CH112 , — of formula ( I ) comprise a structure selected from the group CH , CH , 0 , 0 CH2= CH - CH2 consisting of :
H?c – ( OCH2CH2) = 0 = CH - CH2 = C _ NH _ DRS, H2COJ ( CH2CH2O ) = C _ NH _ DRS,
H?c – ( OCH2CH2) = 0 – CH2 – C _ NH _ DRS, H , CO – CH2CH2O ) = CH- CHI - SH – CH2CH2 – – NH DRS,
H?c – ( OCH2CH2) , — — C _ NH _ DRS, H , CO - (CH2CH2O ) – CH2CH2 – & — NH?N = c _ DRS, H3C0 — ( CH2CH20 )n — CH2CH2 – 0 – CH2 — — NH — N = C — DRS, US 9, 822 ,353 B2
- continued H3CO (CH3CH2O ) = CH- CHU - NH?c - NH N = c - DRS, O H3CO - ( CH2CH2O) = CH - CH2 - NH – NH – C _ NH?N = c _ DRS,
w
=
H?c – ( CH2CH2O ) = CH - CHU - NH –C 0 NH?N = c _ DRS, H3CO - ( CH2CH2O) = CH - CH2 - NH – NH – – NH – N = C – DRS,
S - DRS H3CO – CH2CH2O ) – CH2 – C - NH - CH - CHU - NH – – CH2CH2 - N
S — DRS H3CO — (CH2CH20 )n - CH2CH2 — N
- S - DRS H?CO - ( CH3CH2O ) = CH - CH2 - NH – & — CH - CH2 - N
- S — DRS H3C0 — (CH2CH20 )n - CH2CH2CH2 - NH - C - CH2CH2 - N
- S — DRS H?CO - ( CH3CH2O ) – 0 – CH2CH2 – – NH = CH - CH2 - NH – & — CH - CH2 - N
=
H3C — (OCH2CH )n - O - CH2CH2 - =5 - CH2CH2 - S — DRS
O
H3C — (OCH2CH2 ) n - O - CH2CH2 - O - NH - CH2CH2 - S - S — DRS , and H3C0 — (CH2CH20 )n - CH2CH2CH2CH2 - S — S - DRS ,
55 wherein n = 20 - 800 . In one aspect , the PEGylated DRS polypeptides of for mula ( 1 ) have the structure :
- S -DRS H3CO - ( CH2CH2) n – 0 – CH2CH2 – – NH – CH2CH2 - NH - C – CH2CH2 - N . US 9 ,822 ,353 B2 10 In some embodiments of the PEGylated DRS polypep connecting the lysine molecule to the DRS polypeptide tide, the PEGylated DRS polypeptide comprises a branched is attached via the C - terminal carboxylate group of the PEG polymer. In some embodiments of the PEGylated DRS lysine molecule ; polypeptide , the PEGylated DRS polypeptide has the fol Y is a covalent linkage between the DRS polypeptide and lowing structure ( II) : the remainder of the conjugate ; and DRS refers to a ( X -L1 ) m -B -L2 - Y- DRS DRS polypeptide as disclosed herein . wherein : In some embodiments of the PEGylated DRS polypep X is an independently selected PEG moiety for each m ; tide, the PEGylated DRS polypeptide has the following L , and L , are independently selected optional linkers , in structure (ID ) : wherein L , is also independently selected for each m ; m is 2 , 3 , 4 , or 5 ; B is a branching moiety ; X - L1 Y is a covalent linkage; and N — Lz - Y — DRS DRS is the DRS polypeptide. In some embodiments of the PEGylated DRS polypep 15 X - L2 tide, the PEGylated DRS polypeptide has the following structure ( IIA ) : wherein : X is an independently selected water soluble polymer moiety ; X - L . - CH2 20 L1, L2 and Lz are independently selected optional linkers ; Y is a covalent linkage between the DRS polypeptide and X - L2 — CH the remainder of the conjugate ; and DRS refers to a CH2 - L3 - Y - DRS DRS polypeptide as disclosed herein . 25 In some embodiments in any of formulae ( II ), (IIA ), ( IIB ) , wherein : ( IIC ) , or (IID ) each X is independently R , ( CH2CH2O ) , or X is an independently selected water soluble polymer R1 - ( OCH2CH2) n ., moiety ; wherein R , Falkyl, alkoxy , aryl, glucose , or galactose ; and I I ñ are independently selected ontional linkers . n is 20 to 800 . In some embodiments , R is an alkoxy Y is a covalent linkage between the DRS polypeptide and 30 selected from the group consisting of: methoxy , ethoxy, and the remainder of the conjugate ; and benzyloxy. DRS refers to a DRS polypeptide as disclosed herein . In some embodiments in any of formulae ( II ), ( IIA ), ( IIB ) , In some embodiments of the PEGylated DRS polypep - ( IICTIC ) , or ( IID ) L , and each of L , independently comprise a tide, the PEGylated DRS polypeptide has the following chain of 1 to 20 atoms selected from the group consisting of: 35 C , S , N , P , and 0 . structure ( IIB ) : In some embodiments in any of formulae ( II ), ( IIA ), ( IIB ) , (IIC ) , or ( IID ) L2 and each of L , independently comprise one or more of the following linkages : - 0 - - NH , - S — , X - L . - CH2 C ( O ) , C ( O ) _ NH, NHC ( O ) _ NH, 0 C( 0 ) _ NH, HC - L3 - Y - DRS 40 C ( S ) , CH , — , - CH2CH2 - , CH , CH , CH2– CH2CH2CH2CH2 — , O CH2 X - L2 - CH2 CH , 02 , 0 CH, CH, - , CH , 0 – CH , - CH2 CH2- 04 , 0 CH2CH2CH2 - , wherein : X is an independently selected water soluble polymer 45 - CHCH20 - CH2 CH2 - CH2 CH2 - 6 - , O CH2CH- CH20 CH2 - CH2CH , — moiety ; CH , CH20 - CH2CH2CH2 – CH – L1 , L2 and Lz are independently selected optional linkers ; CH , O CH , CH , — , — CH , CH , CH , O Y is a covalent linkage between the DRS polypeptide and CH , — , - CH2 - CH , CH , CH , 0 , - C (O ) the remainder of the conjugate ; and NHCH2 - , C ( O ) NH CH?CH , CHEC DRS refers to a DRS polypeptide as disclosed herein . 50 ( 0 ) - NH - CH , CH , CH - C ( O ) - NH - , In some embodiments of the PEGylated DRS polypep - C ( O ) - NH - CH , CH , CH , CH - C ( O ) tide, the PEGylated DRS polypeptide has the following NH - CH2 - CH , — , - CH2CH2 - C ( O ) - NH - CH2- , structure ( IIC ) : CH , CH?CH, C ( 0 ) NH?, C ( O ) NH? CH2 – CH2 – CH2 – CH2 – , CH , C ( O) NHCH2 55 CH3CH2 - , CH2 CH2 = C ( O ) NH – CH2 – CH2 – XJL CH , CH?CH , C ( O ) NH CH? , CH? Lysine- L3 - Y - DRS CH, CH3C( O ) _ NH _ CH2 = CH2, CH3CH2 CH , CH, C( 0 ) NH?, — NH C ( 0 ) CH X - L2 CH, NHC( O ) CHE , > CH, CH – NH2 60 ( 0 ) CHE , > NH C ( O ) CH2 CH2, CH3 wherein : NHC( 0 ) CH?CH , CH?CH, NH – C( O ) X is an independently selected water soluble polymer CH2 = CH2, C ( 0 ) NHCH , C ( O ) NH moiety ; CH , CH2 = , 0 C ( O ) _ NH _ CH, , 20 C ( O ) L1, L2 and Lz are independently selected optional linkers , NH - CH2 - CH2- , - NH - CH2 - , - NH - CH2 and wherein the linkers connecting the lysine residue to 65 CH2– CH2- NH - CH2 – CH2- CH2 - NH the water soluble polymer moiety are connected via the CH , — , C ( O ) - CH2 – C ( O ) - CH2 - CH2 - , amino groups of the lysine molecule , and the linker - CH2 - C ( O ) - CH2 - CH2 CH2 C ( O ) - CH2 - , US 9 ,822 , 353 B2 12 _ CH2CH2- C ( O ) _ CH2- CH2 - CH2CH2C carbonyl, carboxylate , carbamate , carbamide , ester , formyl, ( O ) - , – CH2 – CH2 – CH2 – CO) - NH - CH2 – CH2 – acyl, thiocarbonyl, thio ester , thioacetate , thioformate , thio NH?, – CH2 – CH2 – CH , C ( O ) NHCH , CH?A ether, alkoxyl, phosphoryl, phosphonate , phosphinate , amino , amido , amidine , imine , cyano , nitro , azido , disulfide , NHCH _ CNH ( 0 )_ C, ( O ) _CH CH , . _CH , bivalentCH , C (cycloalkyl 0 ) NHCH group , — 5 sulthydryl, sulfate , sulfonate , sulfamoyl, sulfonamido, sul fonyl, heterocyclyl, aralkyl, aromatic moiety , hydrazone , - N ( R ) — , Rºis H or an organic radical selected from the heteroaromatic moiety , imino , sulfamoyl, sulfonate , silyl, group consisting of alkyl, substituted alkyl, alkenyl, substi emether , and alkylthio . tuted alkenyl, alkynyl, substituted alkynyl, aryl and substi In some embodiments of the PEGylated DRS polypeptide tuted aryl. o of formulae ( II ), (IIA ), ( IIB ), ( IIC ), or (IID ) , and B is In some embodiments of the PEGylated DRS polypeptide selected from the group consisting of: an amino acid linkage of formulae (II ) , (IIA ) , ( IIB ) , ( IIC ), or (IID ) L , and each of or an aliphatic hydrocarbon chain of 3 to 6 carbons . L independently comprise a releasable linkage or a stable In some embodiments of the PEGylated DRS polypeptide linkage . of formula ( II ) , B is selected from arginine, histidine , lysine , In some embodiments of the PEGylated DRS polypeptide 15 glutamine , serine , threonine, asparagine , aspartic acid , glu of formulae (II ) , ( IIA ) , ( IIB ), ( IIC ), or ( IID ) L2 and each of tamic acid , cysteine , and seleno cysteine. In one aspect , B is L? independently comprise a releasable linkage . lysine . In some embodiments of the PEGylated DRS poly In some embodiments of the PEGylated DRS polypeptide peptide of formula ( II ), B is an aliphatic hydrocarbon chain of formulae ( II ) , (IIA ) , ( IIB ) , ( IIC ) , or ( IID ) L , and each of is derived from propane , butane , pentane , or hexane . In L , independently comprise a stable linkage . some embodiments of the PEGylated DRS polypeptide of In some embodiments of the PEGylated DRS polypeptide formula (II ) , B is an aliphatic hydrocarbon chain derived of formulae (II ) , (IIA ) , (IIB ), ( IIC ), or (IID ) the stable from a polyol selected from the groups consisting of : linkage is selected from the group consisting of: succinim - glycerol, erythritol, xylitol, and sorbitol. In some embodi ide, propionic acid , carboxymethylate linkages , ethers , car - ments of the PEGylated DRS polypeptide of formula ( II ) , B bamates , amides, amines, carbamides , imides , aliphatic 25 is an aliphatic hydrocarbon chain is derived from glycerol or C — C bonds, and thio ethers. propane. In some embodiments of the PEGylated DRS polypeptide In some embodiments of the PEGylated DRS polypeptide of formulae ( II ), (IIA ), (IIB ), ( IIC ) , or (IID ) Y is selected of formulae ( II ) , ( IIA ), ( IIB ) , ( IIC ), or (IID ) comprise a from the group consisting of: amide , secondary amine, structure selected from the group consisting of:
HC - ( OCH2CH2) n – 0 – C - NH 2_8
CH CH2 CH2 S - DRS HC?c NH - CH - CH2 – NH – C – CH2CH2 _ N H3C — (OCH CH2) n — 0 - 0C - NH ©
O= H?CO — (CH2CH20 – CH, HzC0 — (CH2CH2O ). — CH - S — DRS H?c – 0 – CH2CH2CH2 – NH?? – ( CH2) 2 -5 _ N
H ;CO — ( CH2CH20 ), — ÇH2 - S - DRS HC - 0 – CH2CH2CH – NH?º - ( CH2) 2 - 5 _ N H2C0och — ( CH2CH20 hos ) n - CH2Lo - enenen =xazio
H?CO (OCH3CH2 ) N – – 0 – CH, S - DRS H? – 0 = CH - CH - CH = « — NH = CH- CH2 – NH – & — ( CH2 )2 - 5 _ N H3C0 — (OCH ,CH ) n - N - C - 0 - CH2
o= US 9, 822 ,353 B2 13 14 - continued 14 H3C0 — (CH2CH20 )n - CH2 H3C0 — ( CH2CH20 ). — CH2 HzC0 — ( CH2CH20 ). — CH - S — DRS H?C _ 0 – CH2CH2CH – NH – & — ( CH2) 2 - 5 – N
and H3C0 — (CH2CH20 )n - CH2 H3C0 — ( CH2CH20 ), — CH2 H3C0 — ( CH2CH20 )n - CH2
H3C0 — (CH2CH2O )n — CH - S - DRS HC - 0 – CH2CH2CH2 – NH — — ( CH2) 2 - 5 - N
wherein n is independently any integer from 20 to 800 . monomethoxy polyethylene glycol (mPEG ) derivative of In some embodiments of the PEGylated DRS polypep about 40 ,000 Daltons is covalently attached to an amino acid tide, the PEGylated DRS polypeptide, is about 50 -200 25 residue within about 1, 2, 3, 4, 5, 6 , 7 , 8 , 9 , or 10 amino acid amino acids in length and comprises amino acid residues residues of the C -terminus or the N - terminus of the DRS 1 - 154 , 11 - 146 , 13 - 146 , 23 - 154 , 1 - 171, or 1- 174 of SEQ ID polypeptide . NO : 1 which is modified by C76S , and a maleimide monomethoxy polyethylene glycol (MPEG ) derivative is 30 , In particular embodiments , the amino acid residue to covalently attached via a thio ether linkage to C130 . 30 which themPEG derivative is attached is introduced into the In some embodiments of the PEGylated DRS polypep DRS polypeptide. In specific embodiments , the amino acid tide, the PEGylated DRS polypeptide, is about 50 -200 to which the mPEG derivative is attached is a cysteine amino acids in length and comprises amino acid residues ? residue . 1 - 154 , 11- 146 , 13 - 146 , 23 - 154 , 1 - 171 , or 1 - 174 of SEQ ID In some of the PEGylated DRS polypeptides, the DRS NO : 1 which is modified by C76S , and C130S and a maleim - polypeptide comprises a mutation of Cys203 , where a ide monomethoxy polyethylene glycol (MPEG ) derivative is maleimide monomethoxy polyethylene glycol (mPEG ) covalently attached via a thio ether linkage to an inserted derivative of about 40 ,000 Daltons is covalently attached to cysteine residue inserted within about 10 amino acids of the * a cysteine residue selected from the group consisting of C -terminus Cys76 , Cys130 , Cys259 , Cys334, and Cys349 . In certain In some embodiments of the PEGylated DRS polypep embodiments , the DRS polypeptide comprises cysteine tide , the PEGylated DRS polypeptide is about 50 -200 amino modifications at positions Cys203 , Cys334 and Cys349. In acids in length and comprises amino acid residues 1 - 154 , specific embodiments , the mPEG derivative is attached to 11- 146 , 13 - 146 , 23 - 154 , 1 - 171, or 1 - 174 of SEQ ID NO : 1 Cys130 . which is modified by C76S , and C130S and a maleimide monomethoxy polyethylene glycol (MPEG ) derivative is In some aspects , the full- length PEGylated DRS polypep covalently attached via a thio ether linkage to an inserted s 50 tide is modified by at least one cysteine modification cysteine residue inserted into a surface exposed amino acid selected from the group consisting of C76S , C203S , C259S , listed in Table D9 . C334S , and C349S substitution (s ), where a maleimide In some embodiments of the PEGylated DRS polypep monomethoxy polyethylene glycol (MPEG ) derivative of tide, the PEGylated DRS polypeptide is a full -length noth DRS 55 aboutabou 40 ,000 Daltons is covalently attached via a thio ether polypeptide comprising the sequence set forth in SEQ ID linkage to C130 . In specific aspects , the full - length PEGy. NO : 1 , which is modified by at least one cysteine modifica lated DRS polypeptide ismodified by C76S , C203S , C259S , tion selected from the group consisting of C76 , C203 , C259 , C334S , and C349S substitutions, where a maleimide C334 , and C349, where a maleimide monomethoxy poly - 60 monomenoxymonomethoxy polyethylenepory glycol (MPEG ) derivative of ethylene glycol (MPEG ) derivative of about 40, 000 Daltons about 40 ,000 Daltons is covalently attached via a thio ether is covalently attached to a solvent exposed cysteine residue . linkage to C130 . In some aspects , the PEGylated polypep In certain embodiments , the DRS polypeptide further com - tide exhibits a higher stability compared to a corresponding prises at least one cysteinecysteine modification selected fromfrom Cys76 non - PEGylated polypeptide . and Cys130 . In some embodiments , the DRS polypeptide In one aspect the PEGylated DRS polypeptide comprises comprises a mutation of Cys203 , where a maleimide the structure : US 9, 822 ,353 B2 15 16
- S - DRS HCO - ( CH2CH2) n > 0 - CH2CH2 –C NH - CH - CH2 - NH - C – CH2CH2 - N .
In one aspect the PEGylated DRS polypeptide comprises when compared under similar conditions at room tempera the structure : ture, for 7 days in PBS at pH 7 . 4 . In one embodiment the invention includes a dosing regi men which maintains an average steady - state concentration 15 of DRS polypeptide in the subjects ' plasma of between about 0 . 3 ug /ml and about 3 ug /ml when using a dosing S - DRS interval of 3 days or longer , comprising administering to the H3C0 — ( CH2CH20 )n - CH2CH2 - N patient a therapeutic dose of any of the PEGylated DRS polypeptides described herein . 20 In one embodiment the invention includes a method for maintaining DRS polypeptide levels above the minimum In one aspect the PEGylated DRS polypeptide comprises effective therapeutic level in a subject in need thereof, the structure : comprising administering to the subject a therapeutic dose of any of the PEGylated DRS polypeptides described herein . 25 In another aspect, the invention includes a method for treating an inflammatory response in a subject, comprising administering any of the herein disclosed PEGylated DRS H3CO - ( CH2CH2O) – CH2CH2 – NH – C – CH2CH2 – polypeptides to a subject in need thereof. In another aspect, the invention includes a method for S — DRS 30 treating a TLR associated disease in a subject in need thereof , comprising administering to the subject a therapeu tic dose of any of the herein disclosed PEGylated DRS polypeptides. In another aspect , the invention includes a method for 35 method formodulating TLR activity in a subject, comprising In one aspect the PEGylated DRS polypeptide comprises administering to the subject a therapeutic dose of any of the the structure : herein disclosed PEGylated DRS polypeptides .
- S — DRS H3CO - ( CH2CH2O) – CH2CH2CH2 - NH - C – CH2CH2 - N .
In one aspect of any of these PEGylated DRS polypep - In another aspect, the invention includes a method for tides , the PEGylated DRS polypeptide has substantially the method for killing cancer cells , comprising administering a same secondary structure as unmodified DRS polypeptide , 50 vaccine or immunogenic composition comprising any of the as determined via UV circular dichroism analysis . previously disclosed PEGylated DRS polypeptides to a In one aspectt of any of these PEGylated DRS polynenpolypep subject in need thereof. tides , the PEGylated DRS polypeptide has a plasma or sera In another aspect, the invention includes a method for pharmacokinetic AUC profile at least 5 - fold greater than treating a subject with cancer , or preventing the develop unmodified DRS polypeptide when administered to rats . 55 ment of cancer in a subject , comprising administering a In one aspect of any of these PEGylated DRS polypep vaccine or immunogenic composition comprising any of the tides, the PEGylated DRS polypeptide has substantially the previously disclosed PEGylated DRS polypeptides to a same activity of the unPEGylated protein in a TLR 2 or TLR subject in need thereof. 4 based assay. DETAILED DESCRIPTION OF THE In one aspect of any of these PEGylated DRS polypep INVENTION tides , the PEGylated DRS polypeptide has greater than 2 fold the activity of the unPEGylated protein in a TLR 2 or The practice of the present invention will employ, unless TLR 4 . indicated specifically to the contrary , conventional methods In one aspect of any of these PEGylated DRS polypep - 65 of molecular biology and recombinant DNA techniques tides, the PEGylated DRS polypeptide has a stability which within the skill of the art, many of which are described is at least 30 % greater than unmodified DRS polypeptide below for the purpose of illustration . Such techniques are US 9 ,822 ,353 B2 17 18 explained fully in the literature . See, e . g . , Sambrook , et al ., containing at least one double bond , such as ethenyl, n -pro Molecular Cloning : A Laboratory Manual ( 3rd Edition , penyl, isopropenyl, n -butenyl , isobutenyl, octenyl , decenyl , 2000 ) ; DNA Cloning : A Practical Approach , vol. I & II ( D . tetradecenyl, and the like . Glover, ed . ) ; Oligonucleotide Synthesis ( N . Gait, ed . , 1984 ) ; The term “ alkynyl” as used herein refers to a branched or Oligonucleotide Synthesis : Methods and Applications ( P . 5 unbranched hydrocarbon group of 2 to 15 atoms in length , Herdewijn , ed ., 2004 ); Nucleic Acid Hybridization (B . containing at least one triple bond , ethynyl , n - propynyl, Hames & S . Higgins, eds. , 1985 ) ; Nucleic Acid Hybridiza isopropynyl, n -butynyl , isobutynyl, octynyl, decynyl , and so tion : Modern Applications (Buzdin and Lukyanov , eds. , forth . As used herein , the term “ amino acid ” is intended to mean 2009 ) ; Transcription and Translation ( B . Hames & S . Hig 10 both naturally occurring and non -naturally occurring amino gins, eds ., 1984 ) ; Animal Cell Culture (R . Freshney, ed ., acids as well as amino acid analogs and mimetics . Naturally 1986 ) ; Freshney , R . I . (2005 ) Culture of Animal Cells , a occurring amino acids include the 20 ( L ) - amino acids uti Manual of Basic Technique, 5th Ed . Hoboken N . J ., John lized during protein biosynthesis as well as others such as Wiley & Sons ; B . Perbal, A Practical Guide to Molecular Cloning (3rd Edition 2010 ); Farrell, R ., RNA Methodologies: 15 homocysteine4 -hydroxyproline , citrulline , hydroxylysine and ornithine , desmosine , for ,example isodesmosine . Non , A Laboratory Guide for Isolation and Characterization (3rd naturally occurring amino acids include , for example , ( D ) Edition 2005 ) . Poly ( ethylene glycol) , Chemistry and Bio - amino acids, norleucine , norvaline , p - fluorophenylalanine , logical Applications , ACS , Washington , 1997 ; Veronese , F ., ethionine and the like , which are known to a person skilled and J . M . Harris, Eds. , Peptide and protein PEGylation , in the art . Amino acid analogs include modified forms of Advanced Drug Delivery Reviews, 54 (4 ) 453 -609 (2002 ) ; 20 naturally and non -naturally occurring amino acids. Such Zalipsky , S ., et al. , “ Use of functionalized Poly ( Ethylene modifications can include, for example, substitution or Glycols) for modification of polypeptides” in Polyethylene replacement of chemical groups and moieties on the amino Glycol Chemistry : Biotechnical and Biomedical Applica acid or by derivitization of the amino acid Amino acid tions. mimetics include, for example , organic structures which All publications , patents and patent applications cited 25 exhibit functionally similar properties such as charge and herein are hereby incorporated by reference in their entirety. charge spacing characteristic of the reference amino acid . Definitions For example , an organic structure which mimics Arginine Unless defined otherwise , all technical and scientific (Arg or R ) would have a positive charge moiety located in terms used herein have the same meaning as commonly similar molecular space and having the same degree of understood by those of ordinary skill in the art to which the 30 mobility as the e - amino group of the side chain of the invention belongs. Although any methods and materials naturally occurring Arg amino acid . Mimetics also include similar or equivalent to those described herein can be used constrained structures so as to maintain optimal spacing and in the practice or testing of the present invention , preferred charge interactions of the amino acid or of the amino acid methods and materials are described . For the purposes of the 35 functionaldetermine whatgroups structures. Those constituteskilled in functionallythe art know equivalent or can present invention , the following terms are defined below . amino acid analogs and amino acid mimetics . The articles “ a ” and “ an ” are used herein to refer to one “ Aryl” means one or more aromatic rings , each of 5 or 6 or to more than one ( i . e . , to at least one ) of the grammatical core carbon atoms. Aryl includes multiple aryl rings that object of the article . By way of example , " an element" may be fused , as in naphthyl or unfused , as in biphenyl. Aryl means one element or more than one element. 40 rings may also be fused or unfused with one or more cyclic By “ about” is meant a quantity , level, value , number , hydrocarbon , heteroaryl, or heterocyclic rings . As used frequency, percentage, dimension , size , amount, weight or herein , " aryl” includes heteroaryl. length that varies by as much as 30 , 25 , 20 , 15 , 10 , 9, 8 , 7 , “ Atom length ” or “ chain length ” refers to the number of 6 , 5 , 4 , 3 , 2 or 1 % to a reference quantity , level , value, atoms making up a particular fragment, spacer, linker or the number, frequency , percentage , dimension , size , amount, 45 like . By chain length is meant the number of atoms in a weight or length . single chain , not counting substituents . For instance , An “ alkyl” or “ alkylene ” group , depending upon its CH — counts as one atom with respect to chain length , position in a molecule and the number of points of attach - CH ,CH ,CH , — counts as 3 atoms with respect to chain ment of the group to atoms other than hydrogen , refers to a length , and so on . hydrocarbon chain or moiety , typically ranging from about 50 “ Bifunctional” in the context of a polymer of the inven 1 to 50 atoms in length . Such hydrocarbon chains are tion refers to a PEG polymer possessing two reactive preferably but not necessarily saturated unless so indicated functional groups which may be the same or different . and may be branched or straight chain , although typically “ Branched” in reference to the geometry or overall struc straight chain is preferred in particular embodiments . Exem - ture of a PEG polymer refers to polymer having 2 or more plary alkyl groups include methyl, ethyl, propyl, butyl, 55 PEG polymer “ arms. ” A branched polymer may possess 2 , pentyl, 1 -methylbutyl , 1 - ethylpropyl , 3 -methylpentyl , and 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 or more PEG polymer arms. the like . “ Branch moiety ” refers to a moiety comprising one or " Alicyclic ” refers to any aliphatic compound that contains more atoms at which a PEG polymer splits or branches from a ring of carbon atoms. An alicyclic group is one that a linear structure into one or more additional PEG polymer contains a “ cycloalkyl” or “ cycloalkylene” group as defined 60 arms. above that is substituted with one or more alkyl or alkylenes. Throughout this specification , unless the context requires " Alkoxy ” refers to an 0 - R group , wherein R is alkyl otherwise , the words " comprise , " " comprises, " and " com or substituted alkyl, preferably C , - C20 alkyl ( e . g . , methoxy , prising ” will be understood to imply the inclusion of a stated ethoxy, propyloxy, benzyl, etc . ), and in some embodiments , step or element or group of steps or elements but not the preferably C ,- C5. 65 exclusion of any other step or element or group of steps or As used herein , “ alkenyl” refers to a branched or elements . By “ consisting of" is meant including , and limited unbranched hydrocarbon group of 1 to 15 atoms in length , to , whatever follows the phrase " consisting of. ” Thus , the US 9 ,822 ,353 B2 19 20 phrase “ consisting of” indicates that the listed elements are levels may be less than about 0 .001 , 0 .005 , 0 .01 , 0 .02 , 0 .03 , required or mandatory , and that no other elements may be 0 .04 , 0 .05 , 0 .06 , 0 .08 , 0 .09 , 0 . 1 , 0 . 5, 1 . 0 , 1. 5 , 2 , 2 . 5 , 3, 4 , 5 , present. By “ consisting essentially of ” is meant including 6 , 7 , 8 , 9 , or 10 EU /ml . Typically , 1 ng lipopolysaccharide any elements listed after the phrase , and limited to other (LPS ) corresponds to about 1 - 10 EU . elements that do not interfere with or contribute to the 5 “ Electrophile ” refers to an ion , atom , or collection of activity or action specified in the disclosure for the listed atoms that may be ionic , having an electrophilic center, i. e ., elements . Thus, the phrase “ consisting essentially of indi a center that is electron seeking , capable of reacting with a cates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect 10 The terms " end -capped ” and “ terminally capped ” are the activity or action of the listed elements . interchangeably used herein to refer to a terminal or end The term " conjugate ” is intended to refer to the entity point of a polymer having an end - capping moiety . Typically , formed as a result of covalent attachment of a molecule , e . g ., although not necessarily , the end - capping moiety comprises a biologically active molecule , to a reactive polymer mol a hydroxy or an alkoxy group , more preferably a C1- 10 ecule , preferably a branched reactive polymer of the inven - 15 alkoxy group , and still more preferably a C1 -5 alkoxy group . tion . Thus , examples of end - capping moieties include alkoxy “ Cycloalkyl"” or “" cycloalkylene ”” , dependingdepending uponupon its ( e . g ., methoxy, ethoxy and benzyloxy ) , as well as aryl, position in a molecule and the number of points of attach - heteroaryl, cycloalkyl , heterocyclo , and the like . In particu ment to atoms other than hydrogen , refers to a saturated or lar embodiments , the end -capping moiety may include one unsaturated cyclic hydrocarbon chain , including polycyclics 20 or more atoms of the terminalmonomer in the polymer ( e. g. , such as bridged , fused , or spiro cyclic compounds, prefer the end -capping moiety “ methoxy ” in CH2O (CH2CH2O ) n ably made up of 3 to about 12 carbon atoms, more preferably and CH (OCH CH ) , – ]. In addition , saturated , unsatu 3 to about 8 . rated , substituted and unsubstituted forms of each of the The recitation " endotoxin free ” or “ substantially endo - foregoing are envisioned . Moreover, the end -capping group toxin free " relates generally to compositions , solvents , and 25 can also be a silane or acrylate . In certain embodiments , the or vessels that contain at most trace amounts ( e . g ., amounts end -capping group can also comprise a phospholipid . When having no clinically adverse physiological effects to a sub the polymer has an end -capping group comprising a phos ject) of endotoxin , and preferably undetectable amounts of pholipid , unique properties can be imparted to the polymer endotoxin . Endotoxins are toxins associated with certain and the resulting conjugate , e . g ., DRS polypeptide . Exem bacteria , typically gram -negative bacteria , although endo - 30 plary phospholipids include, without limitation , phosphati toxins may be found in gram -positive bacteria , such as dylcholines, such as, for example , dilauroylphosphatidyl Listeria monocytogenes. The most prevalent endotoxins are choline , dioleylphosphatidylcholine , lipopolysaccharides (LPS ) or lipo -oligo - saccharides (LOS ) dipalmitoylphosphatidylcholine, disteroylphosphatidylcho found in the outer membrane of various Gram -negative line , behenoylphosphatidylcholine , arachidoylphosphatidyl bacteria , and which represent a central pathogenic feature in 35 choline , and lecithin . the ability of these bacteria to cause disease . Small amounts As used herein , the terms " function ” and “ functional” and of endotoxin in humansmay produce fever, a lowering of the the like refer to a biological , enzymatic , or therapeutic blood pressure, and activation of inflammation and coagu - function . lation , among other adverse physiological effects . “ Homology ” refers to the percentage number of amino Therefore , in pharmaceutical production , it is often desir - 40 acids that are identical or constitute conservative substitu able to remove most or all traces of endotoxin from drug tions. Homology may be determined using sequence com products and/ or drug containers, because even small parison programs such as GAP (Deveraux et al. , 1984 , amounts may cause adverse effects in humans. A depyroge - Nucleic Acids Research 12 , 387 - 395 ) , which is incorporated nation oven may be used for this purpose , as temperatures in herein by reference . In this way sequences of a similar or excess of 300° C . are typically required to break down most 45 substantially different length to those cited herein could be endotoxins. For instance, based on primary packaging mate compared by insertion of gaps into the alignment, such gaps rial such as syringes or vials , the combination of a glass being determined , for example, by the comparison algorithm temperature of 250° C . and a holding time of 30 minutes is used by GAP. often sufficient to achieve a 3 log reduction in endotoxin A “ hydrolytically stable ” linkage or bond refers to a levels . Other methods of removing endotoxins are contem - 50 linker, or chemical bond , that is substantially stable in water, plated , including , for example , chromatography and filtra - that is to say, does not undergo hydrolysis under physiologi tion methods, as described herein and known in the art . Also cal conditions to any appreciable extent over an extended included are methods of producing DRS polypeptides in and period of time. Examples of hydrolytically stable linkages isolating them from eukaryotic cells such as mammalian include, but are not limited to , the following : succinimide, cells to reduce , if not eliminate , the risk of endotoxins being 55 propionic acid , carboxymethylate linkages , ethers, carbam present in a composition of the invention . Preferred are ates, amides, amines , carbamides , imides , aliphatic C - C methods of producing DRS polypeptides in and isolating bonds, thio ethers, thiocarbamates, thiocarbamides , and the them from serum free cells . like . Generally , a hydrolytically stable linkage is one that Endotoxins can be detected using routine techniques exhibits a rate of hydrolysis of less than about 0 . 5 % , about known in the art . For example , the Limulus Ameobocyte 60 1 % , about 2 % , about 3 % , about 4 % , or about 5 % per day Lysate assay, which utilizes blood from the horseshoe crab , under physiological conditions . is a very sensitive assay for detecting presence of endotoxin . By “ isolated ” is meant material that is substantially or In this test, very low levels of LPS can cause detectable essentially free from components that normally accompany coagulation of the limulus lysate due a powerful enzymatic it in its native state . For example , an “ isolated peptide ” or an cascade that amplifies this reaction . Endotoxins can also be 65 “ isolated polypeptide” and the like , as used herein , includes quantitated by enzyme - linked immunosorbent assay the in vitro isolation and / or purification of a peptide or ( ELISA ) . To be substantially endotoxin free , endotoxin polypeptide molecule from its natural cellular environment, US 9 ,822 ,353 B2 21 22 and from association with other components of the cell; i. e ., “ stimulated ” or “ enhanced ” amount is typically a “ statisti it is not significantly associated with in vivo substances . cally significant” amount, and may include an increase that The term " half maximal effective concentration ” or is 1. 1, 1. 2 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 15 , 20 , 30 or more times “ EC50 ” refers to the concentration of a PEGylated DRS ( e . g ., 500 , 1000 times ) ( including all integers and decimal polypeptide agent described herein at which it induces a 5 points in between and above 1 , e . g . , 1 . 5 , 1 . 6 , 1 . 7 . 1 . 8 , etc . ) response halfway between the baseline and maximum after the amount produced by no composition ( e . g ., in the absence some specified exposure time; the EC 50 of a graded dose of any of the PEGylated DRS polypeptides of the invention ) response curve therefore represents the concentration of a or a control composition , sample or test subject. A compound at which 50 % of its maximal effect is observed . “ decreased ” or “ reduced ” amount is typically a " statistically In certain embodiments , the EC50 of an agent provided 10 significant” amount, and may include a 1 % , 2 % , 3 % , 4 % , herein is indicated in relation to a " non -canonical ” activity , 5 % , 6 % , 7 % , 8 % , 9 % , 10 % , 11 % , 12 % , 13 % , 14 % , 15 % , as noted above. EC50 also represents the plasma concentra 16 % , 17 % , 18 % , 19 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , tion required for obtaining 50 % of a maximum effect in 50 % , 55 % , 60 % ,65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , or vivo . Similarly , the " EC90 ” refers to the concentration of an 100 % decrease in the amount produced by no composition agent or composition at which 90 % of its maximal effect is 15 (the absence of an agent or compound ) or a control com observed . The “ EC . .” can be calculated from the “ EC50 ” position , including all integers in between . As one non and the Hill slope , or it can be determined from the data limiting example, a control in comparing canonical and directly , using routine knowledge in the art . In some embodi non -canonical activities could include the PEGylated DRS ments , the EC o of a PEGylated DRS protein is less than polypeptide of interest compared to a corresponding un about 0 .01 , 0 .05 , 0 . 1 , 0 . 2 , 0 .3 , 0 . 4 , 0 . 5 , 0 . 6 , 0 . 7 , 0 . 8 , 0 . 9 , 1 , 20 PEGylated DRS polypeptide . Other examples of " statisti 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , cally significant” amounts are described herein . 20 , 25 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , or 100 nM . Preferably , “ Monofunctional” in the context of a polymer of the biotherapeutic composition will have an EC50 value of about invention refers to a PEG polymer possessing a single 1 nM or less. reactive functional group . “ Heteroaryl” is an aryl group containing from one to four 25 “ Multifunctional” in the context of a polymer of the heteroatoms, preferably N , O , or S , or a combination thereof. invention means a PEG polymer having 3 or more functional Heteroaryl rings may also be fused with one or more cyclic groups attached thereto , where the functional groups may be hydrocarbon , heterocyclic , aryl, or heteroaryl rings. the same or different . Multifunctional polymers of the inven “ Heterocycle ” or " heterocyclic ” means one or more rings tion will typically comprise from about 3 to 100 functional of 5 - 12 atoms, preferably 5 - 7 atoms, with or without unsatu - 30 groups, or from 3 to 50 functional groups, or from 3 to 25 ration or aromatic character and having at least one ring functional groups , or from 3 to 15 functional groups , or from atom which is not a carbon . Preferred heteroatoms include 3 to 10 functional groups, or will contain 3 , 4 , 5 , 6 , 7 , 8 , 9 sulfur, oxygen , and nitrogen . or 10 functional groups attached to the polymer backbone. The terms “ functional group , ” “ active moiety, ” “ reactive “ Non - canonical” activity as used herein , refers generally site , " " chemically reactive group , ” and “ chemically reactive 35 to either i ) a new activity possessed by DRS polypeptide of moiety ” are used in the art and herein to refer to distinct, the invention that is not possessed to any significant degree definable portions or units of a molecule . The terms are by the intact native full length parental protein , or ii ) an somewhat synonymous in the chemical arts and are used activity that was possessed by the by the intact native full herein to indicate the portions of molecules that perform l ength parental protein , where the DRS polypeptide either some function or activity and are reactive with other mol- 40 exhibits a significantly higher ( i . e . , at least 20 % greater ) ecules . The term “ active ," when used in conjunction with a specific activity with respect to the non - canonical activity functional group , is intended to include those functional compared to the intact native full length parental protein , or groups that react readily with electrophilic or nucleophilic exhibits the activity in a new context; for example by groups on other molecules, in contrast to those groups that isolating the activity from other activities possessed by the require strong catalysts or highly impractical reaction con - 45 intact native full length parental protein . In the case of DRS ditions in order to react (i . e ., " non - reactive” or “ inert ” polypeptides, non - limiting examples of non - canonical groups) . activities include extracellular signaling including the The term " linkage ,” “ linker, " " linker moiety , ” or “ L ” is modulation of TLRs, modulation of cell proliferation , modu used herein to refer to an atom or a collection of atoms used lation of cell migration , modulation of cell differentiation to link , preferably by one or more covalent bonds, intercon - 50 ( e . g . , hematopoiesis , neurogenesis , myogenesis , osteogen necting moieties such as two polymer segments or a termi- esis , and adipogenesis ) , modulation of gene transcription , nus of a polymer and a reactive functional group present on modulation of apoptosis or other forms of cell death , modu a polypeptide, e . g . , a DRS polypeptide . The linker may be lation of cell signaling, modulation of cellular uptake , or hydrolytically stable or may include a releasable linkage secretion , modulation of angiogenesis , modulation of cell such as a physiologically hydrolyzable or enzymatically 55 binding , modulation of cellular metabolism , modulation of degradable linkage . cytokine production or activity , modulation of cytokine " Lower alkyl” or " lower alkylene ” refers to an alkyl or receptor activity , modulation of inflammation , immunoge alkylene group as defined above containing from 1 to 6 nicity , and the like. carbon atoms, and may be straight chain or branched , as " Non - interfering substituents ” are those groups that, exemplified by methyl, ethyl, n - butyl, i - butyl, t - butyl. 60 when present in a molecule , are typically non - reactive with “ Lower cycloalkyl" or " lower cycloalkylene ” refers to a other functional groups contained within the molecule . cycloalkyl group or cycloalkylene group containing from 1 “ Nucleophile ” refers to an ion or atom or collection of to 6 carbon atoms. atoms that may be ionic , having a nucleophilic center, i . e . , The term “modulating ” includes “ increasing, " " enhanc a center that is seeking an electrophilic center , and capable ing ” or “ stimulating, " as well as “ decreasing ” or “ reducing, " 65 of reacting with an electrophile . typically in a statistically significant or a physiologically As used herein , the term " polyalkylene glycol” or “ poly significant amountas compared to a control. An “ increased, ” (alkene glycol) ” refers to polyethylene glycol (poly ( ethylene US 9 ,822 , 353 B2 23 24 glycol) ) , polypropylene glycol, polybutylene glycol, and peutics ' Advanced PEGylation also offers diverse PEG derivatives thereof. The term “ polyalkylene glycol” encom coupling technologies to improve the safety and efficacy of passes both linear and branched polymers and average therapeutics . Additional PEGs for use in forming a DRS molecular weights of between 0 . 1 kDa and 100 kDa. Other polypeptide conjugate of the invention include those avail exemplary embodiments are listed , for example , in commer - 5 able from Polypure (Norway ) , from QuantaBioDesign LTD cial supplier catalogs , such as Shearwater Corporation ' s (Ohio ) JenKem Technology , Nanocs Corporation , and Sun catalog “ Polyethylene Glycol and Derivatives for Biomedi- bio , Inc (South Korea ) . Further PEG reagents suitable for cal Applications” (2001 ) . use in forming a conjugate of the invention , and methods of As used herein , the terms “ PEG ,” “ polyethylene glycol” conjugation are described in the Pasut. G ., et al. , Expert and “ poly (ethylene glycol) ” as used herein , are interchange - 10 Opin . Ther. Patents (2004 ), 14 ( 6 ) 859 - 893 . able and meant to encompass any water -soluble poly ( ethyl- A number of investigators have disclosed the preparation ene oxide ) derivative . PEG is a well -known polymer with of linear or branched PEG polymers and derivatives or good solubility in many aqueous and organic solvents , conjugates thereof (see , e . g . , U . S . Pat. Nos. 4 , 904 , 584 ; which exhibits low toxicity , lack of immunogenicity , and is 5 ,428 , 128 ; 5 ,621 ,039 ; 5 ,622 , 986 ; 5 ,643 , 575 ; 5 ,728 ,560 ; clear , colorless , odorless, and stable . For these reasons and 15 5 ,730 ,990 ; 5 ,738 ,846 ; 5 ,811 , 076 ; 5 ,824 ,701 ; 5 ,840 ,900 ; others , PEG has been selected as the preferred polymer for 5 , 880 , 131; 5 , 900 , 402 ; 5 , 902, 588 ; 5 ,919 ,455 ; 5 . 951 . 974 : attachment, but it has been employed solely for purposes of 5 ,965 , 119 ; 5 , 965 ,566 ; 5 , 969 ,040 ; 5 , 981, 709 ; 6 ,011 ,042 ; illustration and not limitation . Similar products may be 6 ,042 , 822 ; 6 , 113 , 906 ; 6 , 127 , 355 ; 6 , 132 ,713 ; 6 , 177 ,087 : obtained with other water- soluble polymers , as described 6 , 180 , 095 ; 6 , 448 , 369; 6 ,495 ,659 ; 6 ,602 , 498 ; 6 , 858 ,736 ; herein , including without limitation ; polyvinyl alcohol, 20 6 ,828 ,401 ; 7 , 026 , 440 ; 7 ,608 ,678 ; 7 ,655 ,747 ; 7 ,786 ,221 ; other poly (alkylene oxides ) such as poly ( propylene glycol) 7 , 872 , 072 ; and 7 , 910 ,661 , each of which is incorporated and the like , poly ( oxyethylated polyols ) such as poly ( oxy - herein by reference in its entirety . ethylated glycerol) and the like , carboxymethylcellulose , In certain embodiments , the “ purity ” of any given agent dextran , polyvinyl alcohol, polyvinyl purrolidone, poly - 1, 3 - (e . g ., PEGylated DRS polypeptide ) in a composition may be dioxolane , poly - 1 , 3 , 6 - trioxane , ethylene /maleic anhydride , 25 specifically defined . For instance , certain compositions may and polyaminoacids. One skilled in the art will be able to comprise an agent that is at least 80, 85 , 90 , 91 , 92 , 93, 94 , select the desired polymer based on the desired dosage , 95 , 96 , 97 , 98 , 99 , or 100 % pure , including all decimals in circulation time, resistance to proteolysis , and other consid between , as measured , for example and by no means limit erations. ing , by high pressure liquid chromatography (HPLC ) , a Typically , PEGs for use in accordance with the invention 30 well- known form of column chromatography used fre comprise the following structure “ _ OCH , CH ) n - ” where q uently in biochemistry and analytical chemistry to separate , ( n ) is about 1 to 4000 , alternatively from about 20 to 1400 , identify , and quantify compounds. or about 20 -800 . In particular embodiments , PEG also " physiologically cleavable ” or “ hydrolyzable” or includes “ _ 0 — (CH , CH , O ) . - CH , CH , ” and “ degradable ” bond is a bond that reacts with water ( i .e ., is “ _ OCH CH2) n 0 ” depending upon whether or not the 35 hydrolyzed ) under physiological conditions. The tendency terminal oxygens have been displaced . Throughout the of a bond to hydrolyze in water will depend not only on the specification and claims, it should be understood that in general type of linkage connecting two central atoms but certain embodiments , the term “ PEG " includes structures also on the substituents attached to these central atoms. having various terminal or " end capping ” groups and so Appropriate hydrolytically unstable or weak linkages forth . The term “ PEG ” also means a polymer that contains 40 include, but are not limited to : carboxylate ester , phosphate a majority , that is to say , greater than 50 % , of ester , anhydride , acetal , ketal, acyloxyalkyl ether , imine , - OCH , CH , - repeating subunits . With respect to specific orthoester, thio ester , thiol ester, carbonate , and hydrazone , forms, the PEG can take any number of a variety of peptides and oligonucleotides . Without wishing to be bound molecular weights , as well as structures or geometries such to any particular theory, an “ enzymatically degradable link as “ branched ,” “ linear, ” “ forked, " “ multifunctional, ” and the 45 age ” means a linkage , e. g. , amino acid sequence , that is like, to be described in greater detail below . subject to degradation by one or more enzymes , e . g ., pep Representative polymeric reagents and methods for con - tidases or proteases. jugating such polymers to an active moiety are described in The terms " polypeptide ” and “ protein ” are used inter Harris , J . M . and Zalipsky, S . , Eds , Poly ( ethylene glycol) , changeably herein to refer to a polymer of amino acid Chemistry and Biological Applications, ACS , Washington , 50 residues and to variants and synthetic analogues of the same. 1997 ; Veronese , F . , and J . M . Harris , Eds. , Peptide and Thus, these terms apply to amino acid polymers in which Protein PEGylation , Advanced Drug Delivery Reviews , one or more amino acid residues are synthetic non - naturally 54 ( 4 ); 453 -609 (2002 ) ; Zalipsky, S . , et al. , “ Use of Func occurring amino acids, such as a chemical analogue of a tionalized Poly Ethylene Glycols ) for Modification of Poly corresponding naturally occurring amino acid , as well as to peptides ” in Polyethylene Glycol Chemistry : Biotechnical 55 naturally -occurring amino acid polymers . and Biomedical Applications, J . M . Harris, ed . , Plenus Press , A “ releasable linkage ” includes , but is not limited to , a New York ( 1992 ) ; Zalipsky (1995 ) Advanced Drug Reviews physiologically cleavable bond , a hydrolyzable bond , and an 16 :157 -182 ; and in Roberts et al. , Adv. Drug Delivery enzymatically degradable linkage . Thus, a “ releasable link Reviews, 54 , 459 - 476 (2002 ) . age ” is a linkage that may undergo either spontaneous A wide variety of PEG derivatives are both commercially 60 hydrolysis , or cleavage by some other mechanism (e . g. , available and suitable for use in the preparation of the enzyme- catalyzed , acid - catalyzed , base - catalyzed , and so PEG -conjugates of the invention . For example , NOF Corp . 's forth ) under physiological conditions. For example , a SUNBRIGHT® Series provides numerous PEG derivatives , " releasable linkage” can involve an elimination reaction that including methoxypolyethylene glycols and activated PEG has a base abstraction of a proton , ( e . g ., an ionizable derivatives such as succinimidyl ester , methoxy - PEG 65 hydrogen atom , Ha ), as the driving force . For purposes amines , maleimides , and carboxylic acids, for coupling by herein , a " releasable linkage ” is synonymous with a various methods to DRS polypeptides and Nektar Thera “ degradable linkage .” In particular embodiments , a releas US 9 , 822 ,353 B2 25 26 able linkage has a half life at pH 7 . 4 , 25° C ., e . g ., a or condition , and may include even minimal changes or physiological pH , human body temperature , of about 30 improvements in one or more measurable markers of the min ., about 1 hour, about 2 hour, about 3 hours , about 4 disease or condition being treated . “ Treatment” or “ treating ” hours, about 5 hours , about 6 hours , about 12 hours, about does not necessarily indicate complete eradication or cure of 18 hours , about 24 hours, about 36 hours, about 48 hours, 5 the disease or condition , or associated symptoms thereof. about 72 hours , or about 96 hours or more . The subject receiving this treatment is any subject in need By “ statistically significant” , it is meant that the result was thereof. Exemplary markers of clinical improvement will be unlikely to have occurred by chance. Statistical significance apparent to persons skilled in the art . can be determined by any method known in the art . Com As used herein , the term " water soluble polymer” refers monly used measures of significance include the p - value , 10 to any polymer that is soluble in aqueous solvents . Linkage which is the frequency or probability with which the of water soluble polymers to DRS polypeptides can result in observed event would occur, if the null hypothesis were true . changes including , but not limited to , increased or modu If the obtained p -value is smaller than the significance level , lated serum half- life , or increased or modulated therapeutic then the null hypothesis is rejected . In simple cases , the half - life relative to the unmodified form , modulated immu significance level is defined at a p - value of 0 . 05 or less . 15 nogenicity , modulated physical association characteristics The term “ substituted ” as in , for example , " substituted such as aggregation and multimer formation , altered recep alkyl, ” refers to a moiety ( e . g . , an alkyl group ) substituted tor binding, altered receptor dimerization or multimeriza with one or more non - interfering substituents , such as , but tion , modulated toxicity , and modulation of one or more the not limited to : C3 - C , cycloalkyl, e. g ., cyclopropyl, cyclobu - biological activities of DRS polypeptides including side tyl , and the like ; halo , e . g . , fluoro , chloro , bromo, and iodo ; 20 effects found with current DRS therapeutics . The water cyano ; alkoxy, lower phenyl; substituted phenyl; and the soluble polymer may or may not have its own biological like . For substitutions on a phenyl ring, the substituents may activity , and may be utilized as a linker for attaching DRS be in any orientation (i . e . , ortho , meta , or par polypeptides to other substances , including but not limited “ Substituted heteroaryl” is heteroaryl having one or more to one or more DRS polypeptides , and / or one or more non - interfering groups as substituents . 25 biologically active molecules . Suitable polymers include , “ Substituted heterocycle ” is a heterocycle having one or but are not limited to , polyethylene glycol, polyethylene more side chains formed from non - interfering substituents. glycol propionaldehyde, mono C , -C10 alkoxy or aryloxy The term “ solubility ” refers to the property of a PEGy - derivatives thereof (described in U . S . Pat. No . 5 , 252, 714 lated DRS polypeptide provided herein to dissolve in a which is incorporated by reference ), monomethoxy -polyeth liquid solvent and form a homogeneous solution . Solubility 30 ylene glycol, polyvinyl pyrrolidone , polyvinyl alcohol, is typically expressed as a concentration , either by mass of polyamino acids , divinylether maleic anhydride, N - ( 2 -Hy solute per unit volume of solvent (g of solute per kg of droxypropyl) - methacrylamide , dextran , dextran derivatives solvent, g per dL ( 100 mL ) , mg/ml , etc . ) , molarity , molality, including dextran sulfate , polypropylene glycol, polypropyl mole fraction or other similar descriptions of concentration . ene oxide / ethylene oxide copolymer, polyoxyethylated The maximum equilibrium amount of solute that can dis - 35 polyol, heparin , heparin fragments , polysaccharides, oli solve per amount of solvent is the solubility of that solute in gosaccharides , glycans , cellulose and cellulose derivatives , that solvent under the specified conditions, including tem including but not limited to methylcellulose and carboxym perature , pressure , pH , and the nature of the solvent. In ethyl cellulose, starch and starch derivatives , polypeptides, certain embodiments , solubility is measured at physiological polyalkylene glycol and derivatives thereof, copolymers of pH , or other pH , for example , at pH 5 . 0, pH 6 . 0, pH 7. 0 , or 40 polyalkylene glycols and derivatives thereof, polyvinyl ethyl pH 7 . 4 . In certain embodiments , solubility is measured in ethers , and alpha -beta -poly [ ( 2 -hydroxyethyl ) -DL -aspart water or a physiological buffer such as PBS or NaCl (with amide, and the like , or mixtures thereof. or without NaP ) . In specific embodiments , solubility is Specific examples of such water soluble polymers measured at relatively lower pH ( e . g ., pH 6 . 0 ) and relatively include, but are not limited to , polyalkyl ethers and alkoxy higher salt ( e . g ., 500 mM NaCl and 10 mM NaP ) . In certain 45 capped analogs thereof ( e . g ., polyoxyethylene glycol, poly embodiments , solubility is measured in a biological fluid oxyethylene /propylene glycol, and methoxy or ethoxy (solvent ) such as blood or serum . In certain embodiments , capped analogs thereof, especially polyoxyethylene glycol , the temperature can be about room temperature ( e . g . , about the latter is also known as polyethyleneglycol or PEG ) ; 20 , 21, 22 , 23 , 24 , 25° C . ) or about body temperature (37° polyvinylpyrrolidones ; polyvinylalkyl ethers; polyoxazo C . ) . In certain embodiments , a PEGylated DRS polypeptide 50 lines , polyalkyl oxazolines and polyhydroxyalkyl oxazo has a solubility of at least about 0 . 1 , 0 . 2 , 0 . 3 , 0 . 4 , 0 .5 , 0 .6 , lines ; polyacrylamides, polyalkyl acrylamides , and polyhy 0 .7 , 0. 8 , 0 .9 , 1, 2 , 3, 4 , 5 , 6 , 7 , 8, 9 , 10 , 11 , 12 , 13 , 14 , 15 , droxyalkyl acrylamides ( e . g . , 16 , 17 , 18 , 19, 20 , 25 , or 30 mg/ ml at room temperature or polyhydroxypropylmethacrylamide and derivatives at 37° C . thereof) ; polyhydroxyalkyl acrylates ; polysialic acids and A “ subject, " as used herein , includes any animal that 55 analogs thereof; hydrophilic peptide sequences; polysaccha exhibits a symptom , or is at risk for exhibiting a symptom , rides and their derivatives , including dextran and dextran which can be treated or diagnosed with a PEGylated DRS derivatives, e. g ., carboxymethyldextran , dextran sulfates , polypeptide of the invention . Suitable subjects (patients ) aminodextran ; cellulose and its derivatives , e . g ., carboxym include laboratory animals (such as mouse , rat , rabbit , or ethyl cellulose , hydroxyalkyl celluloses ; chitin and its guinea pig ) , farm animals , and domestic animals or pets 60 derivatives, e . g . , chitosan , succinyl chitosan , carboxymeth (such as a cat or dog ). Non -human primates and , preferably , ylchitin , carboxymethylchitosan ; hyaluronic acid and its human patients , are included . derivatives ; starches ; alginates ; chondroitin sulfate ; albu “ Substantially ” or “ essentially ” means nearly totally or min ; pullulan and carboxymethyl pullulan ; polyaminoacids completely , for instance , 95 % or greater of some given and derivatives thereof, e . g ., polyglutamic acids, polyly quantity . 65 sines , polyaspartic acids, polyaspartamides; maleic anhy “ Treatment ” or “ treating , " as used herein , includes any dride copolymers such as : styrene maleic anhydride copo desirable effect on the symptoms or pathology of a disease lymer, divinylethyl ether maleic anhydride copolymer; US 9 ,822 , 353 B2 27 28 polyvinyl alcohols ; copolymers thereof; terpolymers aspartyl- tRNA synthetases , or in some contexts the intact thereof; mixtures thereof; and derivatives of the foregoing . synthetase , can dissociate from the multisubunit complex , Aspartyl- tRNA Synthetase Derived Polypeptides and activate certain cell - signaling pathways , or act within Embodiments of the present invention relate to the use of non -naturally occurring Aspartyl- tRNA synthetase derived 5 thewhi nucleus to modulate transcription . These activities , polypeptides with altered cysteine content (“ DRS polypepderived 5 which are distinct from the classical role of tRNA syn tides ” ) . Aspartyl - tRNA synthetases belong to the class I thetases in protein synthesis , are collectively referred to tRNA synthetase family , which has two highly conserved herein as “ non canonical activities " . These DRS polypep sequence motifs at the active site , HIGH (SEQ ID NO : 42 ) tides may be produced naturally by either alternative splic and KMSKS ( SEQ ID NO : 43 ) . Class I tRNA synthetases are ing or proteolysis , and can act in a cell autonomous ( i. e . , widely recognized as being responsible the specific attach - " within the host cell) , or non - cell autonomous fashion (i . e ., ment of an amino acid to its cognate tRNA in a 2 step outside the host cell ) to regulate a variety of homeostatic reaction : the amino acid ( AA ) is first activated by ATP to mechanisms. For example, as provided in the present inven form AA - AMP and then transferred to the acceptor end of tion , the N -terminal fragment of Aspartyl- tRNA synthetase , the tRNA . The full length Aspartyl- tRNA synthetases typi cally exists as a homodimer ; and also forms part of a 1515 DRS ( 1 - 154 ), is capable of modulating the activity of certain multisubunit complex that typically includes the proteins TLRs in vivo . In addition , certain mutations or deletions AIMP1, AIMP2, EEF1A1 and the tRNA synthetases for Arg, relative to the full -length DRS polypeptide sequence confer Asp , Glu , Gin , Ile , Leu , Lys, Met and Pro . increased TLR binding or other non - canonical activities. More recently it has been established that some biological The sequences of various exemplary DRS polypeptides are fragments , or alternatively spliced isoforms of eukaryotic provided in Tables D1 to D8 . TABLE D1 Amino Acid Residue Range of SEO ID SEQ ID Name NO : 1 Amino acid and nucleic acid sequence NO : Exemplary DRS Polypeptides Full length Protein / MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQS AspRS Human OEKPDRVLVRVRDLTIOKADEVVWVRARVHTSRAK sequence 1 - 501 GKQCFLVLRQQQFNVQALVAVGDHAS KOMVKFAA NINKESIVDVEGVVRKVNOKIGSCTQQDVELHVQKI YVISLAEPRLPLOLDDAVRPEAEGEEEGRATVNODT RLDNRVIDLRTSTSOAVFRLOSGICHLFRETLINKGFV EIOTPKIISAASEGGANVFTVSYFKNNAYLAOSPOLY KOMCICADFEKVFSIGPVFRAEDSNTHRHLTEFVGLD IEMAFNYHYHEVMEEIADTMVQIFKGLQERFOTEIQ TVNKOFPCEPFKFLEPTLRLEYCEALAMLREAGVEM GDEDDLSTPNEKLLGHLVKEKYDTDFYILDKYPLAV RPFYTMPDPRNPKOSNSYDMFMRGEEILSGAORIHD POLLTERALHHGIDLEKIKAYIDSFRFGAPPHAGGGI GLERVTMLFLGLHNVROTSMFPRDPKRLTP Exemplary AspRS nucleic Acids Full length DNA / Human / ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 2 AspRS 1 - 1506 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT sequence AAAGAGAGATATGGAATATCTTCAATGATACAATCACA Human codon AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT usage TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA TACAAGATTAGACAACAGAGTCATTGATCTTAGGACAT CAACTAGTCAGGCAGTC??ccGT c?ccAGT ??GGCATCT GCCATCTCTTCCGAGAAACTTTAATTAACAAAGGTTTTG ?GGAAATCCAAACTccTAAAATTATTICAGCTGccAGT GAAGGAGGAGCCAATGTTTTTACTGTGTCATATTTTAA AAATAATGCATACCTGGCTCAGTCCCCACAGCTATATA AGCAAATGTGCATTTGTGCTGATTTTGAGAAGGTTTTCT CTATTGGACCAGTATTCAGAGCGGAAGACTCTAATACC CATAGACATCTAACTGAGTTTGTTGGTTTGGACATTGAA ATGGCTTTTAATTACCATTACCACGAAGTTATGGAAGA AATTGCTGACACCATGGTACAAATATTCAAAGGACTTC AAGAAAGGTTTCAGACTGAAATTCAAACAGTGAATAAA CAGTTCCCATGTGAGCCATTCAAATTTTTGGAGCCAACT CTAAGACTAGAATATTGTGAAGCATTGGCTATGCTTAG GGAAGCTGGAGTCGAAATGGGAGATGAAGACGATCTG AGCACACCAAATGAAAAGCTGTTGGGTCATTTGGTAAA US 9 ,822 , 353 B2 29 30 TABLE D1 - continued Amino Acid Residue Range of SEO ID SEQ ID Name NO : 1 Amino acid and nucleic acid sequence NO : GGAAAAGTATGATACAGATTTTTATATTCTTGATAAAT ??ccATTGGCTGTAAGAccTTTC?A?AccATGccTGAcc CAAGAAATCCCAAACAGTCCAACTCTTACGATATGTTC ATGAGAGGAGAAGAAATATTGTCAGGAGCTCAAAGAA TACATGATcc??????GCTAACAGAGAGAGc??????? CATGGAATTGATTTGGAGAAAATTAAGGCTTACATTGA TTCCTTCCGCTTTGGAGCCCCTCCTCATGCTGGTGGAGG CATTGGATTGGAACGAGTTACTATGCTGTTTCTGGGATT GCATAATGTTcGTCAGACCTccATGTTccc?cGTGATcc CAAACGACTCACTCCTTAG
TABLE D2 Exemplary N - terminal DRS polypeptide Fragments Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Amino acid sequence NO : AspRsiNi Protein / MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEK 3 Human / 1 - 154 PDRVLVRVRDLTICKADEVVWVRARVHTSRAKGKQCFL VLRQQQFNVQALVAVGDHASKQMVKFAANINKESIVDV EGVRKVNOKIGSCTQQDVELHVQKIYVISLAEPRLPL AspRS1N11 Protein / MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK Human / 1 - 171 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQCFL VLROOOFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPLQL DDAVRPEAEGEEEGR AspRS1N12 Protein / MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSCEK 5 Human / 1 - 174 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQCFL VLROQOFNVQALVAVGDHASKOMVKFAANINKESIVDV EGWRKVNOKIGSCTQQDVELHVOKIYVISLAEPRLPLQL DDAVRPEAEGEEEGRATV ASPRS1N13 Protein / MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK Human / 1 - 182 PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL VLROOOFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPLQL DDAVRPEAEGEEEGRATVNODTRLDN ASPRS1N4 Protein MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEK Human / 1 - 184 PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHAS KQMVKFAANINKESIVDV EGWRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPLQL DDAVRPEAEGEEEGRATVNQDTRLDNRV AspRS1N2 Protein / MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK Human 1 - 274 PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNOKIGSCTOODVELHVOKIYVISLAEPRLPLOL DDAVRPEAFGEEEGRATVNQDTRLDNRVIDLRTSTSQAVF RLQSGICHLFRETLINKGFVEIQTPKIISAASEGGANVFTVS YFKNNAYLAOS POLYKOMCI CADFEKVFSIGPVFRA ASPRS1N3 Protein MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSCEK Human / 1 - 224 PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHASKOMVKFAANINKESIVDV EGVVRKKVNCKIGSCTOQDVELHVOKIYVISLAEPRLPLQL DDAVRPEAFGEEEGRATVNQDTRLDNRVIDLRTSTSQAVF RLOSGICHLFRETLINKGFVEIQTPKII DRS 1 - 182 1 - 182 MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEK 74 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQCFL VLRQQQFNVQALVAVGDHASKQMVKFAANINKESIVDV EGWRKVNOKIGSCTQQDVELHVQKIYVISLAEPRLPLQL DDAVRPEAFGEEEGRATVNQDTRLDN DRS 1 - 1801 - 180 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIOSOEK 7575 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKOCFL US 9 ,822 ,353 B2 31 32 TABLE D2 - continued Exemplary N - terminal DRS polypeptide Fragments
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Amino acid sequence NO : VLROOOFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNOKIGSCTOQDVELHVOKIYVISLAEPRLPLQL DDAVRPEAPGEEKGRATVNQDTRL DRS 1 - 178 1 - 178 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL VLROOOFNVQALVAVGDHAS KOMVKFAANINKEISIVDV EGWRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPLQL DDAVRPEAEGEEEGRATVNODT DRS 1 - 176 1 - 176 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK 77 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHAS KQMVKFAANINKESIVDV EGVVRKVNOKIGSCTOODVELHVOKIYVISLAEPRLPLQL DDAVRPEAEGEEEGRATVNQ DRS 1 - 174 1 - 174 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIOSOEK 78 PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNOKIGSCTOODVELHVOKIYVISLAEPRLPLOL DDAVRPEAEGEEEGRATV DRS 1 - 1721 - 172 MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEK 79 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNQKIGSCTOQDVELHVQKIYVISLAEPRLPLQL DDAVRPEAEGEEEGRA
DRS 1 - 170 1 - 170 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIOSOEK 80 PDRVLRVRDLTIQKADEWVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPLQL DDAVRPEAEGEEEG DRS 1 - 168 1 - 168 MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEK 81 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGWRKVNOKIGSCTQQDVELHVOKIYVISLAEPRLPLQL DDAVRPEAEGEE DRS 1 - 166 1 - 166 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK 82 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQCFL VLROQQFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNOKIGSCTOODVELHVOKIYVISLAEPRLPLOL DDAVRPEAEG DRS 1 - 164 1 - 164 MPSASASRKSOEKPREIMDAAFDYAKERYGISSMIQSQEK 83 PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPLQL DDAVRPEA DRS 1 - 162 1 - 162 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK 84 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQCFL VLRQQQFNVQALVAVGDHASKQMVKFAANINKESIVDV EGVVRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPLQL DDAVRP DRS 1 - 160 1 - 160 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIOSOEK 85 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQCFL VLRQQQFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNQKKIGCTQQDVELHVQKIYVISLAEPRLPLQL DDAV DRS 1 - 158 1 - 158 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK 86 PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHASKQMVKFAANINKESIVDV EGVVRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPLQL DD
DRS 1 - 156 1 - 156 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK 87 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKOCFL US 9 ,822 ,353 B2 33 34 TABLE D2 - continued Exemplary N - terminal DRS polypeptide Fragments
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Amino acid sequence NO : VLROOOFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNQKIGSCTOQDVELHVQKIYVISLAEPRLPLQL DRS 1 - 154 1 - 154 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK 88 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNOKIGSCTOODVELHVOKIYVISLAEPRLPL DRS 1 - 152 1 - 152 MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEK 89 PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHASKOMVKFAANINKESIVDV EGVVRKVNOKIGSCTQQDVELHVQKIYVISLAEPRL DRS 1 - 1501 - 150 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK 90 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQCFL FLRQQQFNVQALVAVGDHASKOMVKFAANINKESIVDV EGVVRKVNOKIGSCTQQDVELHVOKIYVISLAEP DRS 1 - 148 148 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIOSOEK 91 PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL VLRQQQFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGVVRKVNQKIGSCTQQDVELHVQKIYVISLA daseDRS 1 - 146 1 - 146 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK 92 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQCFL VLRQQQFNVQALVAVGDHAS KOMVKFAANINKESIVDV EGWRKVNOKIGSCTQQDVELHVQKIYVIS sakDRS 3 - 154 3 - 154 ASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEKPDR 93 VLVRVRDLTIQKADEVVWVRARVHTSRAKGKOCFLVLR QOOFNVOALVAVGDHAS KOMVKFAANINKESIVDVEGV VRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPL DRS 5 - 154 5 - 154 ASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEKPDRVL 94 VRVRDLTIQKADEVVWVRARVHTSRAKGKOCFLVLROQ QFNVQALVAVGDHASKQMVKFAANINKESIVDVEGVR KVNQKIGSCTQQDVELHVQKIYVISLAE PRLPL DRS 7 - 154 7 - 154 TKSQEKPREIMDAAEDYAKERYGISSMI QSQEKPDRVLVR 95 VRDLTIQKADEVVWVRARVHTSRAKGKQCFLVLRQQQF NVOALVAVGDHAS KOMVKFAANINKESIVDVEGVVRKV NOKIGSCTQQDVELHVQKIYVISLAEPRLPL DRS 9 - 154 9 - 154 SOEKPREIMDAAEDYAKERYGISSMIQSQEKPDRVLVRVR 96 DLTIQKADEVVWVRARVHTSRAKGKQCFLVERQQQFNV QALVAVGDHASKQMVKFAANINKESIVDVEGVVRKVNO KIGSCTQQDVELHVQKIYVISLAEPRLPL EKPREIMDAAEDYAKERYGISSMIQSQEKPDRVLVRVRDL 97 DRS 11 - 154 11- 154 TICKADEVVWVRARVHTSRAKGKOCFLVLRQOQFNVOA LVAVGDHASKOMVKFAANINKESIVDVEGVVRKVNOKIG SCTQQDVELHVQKIYVISLAEPRLPL DRS 13 - 154 13 - 154 PREIMDAAEDYAKERYGISSMIQSQEKPDRVLVRVRDLTI 98 QKADEVVWVRARVHTSRAKGKOCFLVLROQQFNVQALV AVGDHASKQMVKFAANINKESIVDVEGVVRKVNOKIGSC per TOODVELHVOKIYVISLAEPRLPL DRS15 - 154 15 - 154 EIMDAAEDYAKERYGISSMIQSQEKPDRVLVRVRDLTIQK 99 ADEVVWVRARVHTSRAKGKOCFLVLRQQQFNVQALVAV GDHASKQMVKFAANINKESIVDVEGVVRKVNOKIGSCTO QDVELHVOKIYVISLAEPRLPL DRS 17 - 154 17 - 154 MDAAEDYAKERYGISSMIQSQEKPDRVLVRVRDLTIQKA 100 DEVVWVRARVHTSRAKGKOCFLVLRQQQFNVQALVAVG DHASKOMVKFAANINKESIVDVEGVVRKVNQKIGSCTQQ DVELHVQKIYVISLAEPRLPL DRS 19 - 154 19 - 154 MDAAEDYAKERYGISSMIQSQEKPDRVLVRVRDLTIQKA 101 DEVVWVRARVHTSRAKGKOCFLVLRQQQFNVQALVAVG DHASKOMVKFAANINKESIVDVEGVVRKVNQKIGSCTQQ DVELHVOKIYVISLAEPRL US 9 ,822 ,353 B2 35 36 TABLE D2 - continued Exemplary N - terminal DRS polypeptide Fragments
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Amino acid sequence NO : DRS 21 - 154 21 - 154 MDAAEDYAKERYGISSMIQSQEKPDRVLVRVRDLTIQKA 102 DEVVWVRARVHTSRAKGKOCFLVLRQQQFNVQALVAVG DHASKOMVKFAANINKESIVDVEGVVRKVNQKIGSCTQQ DVELHVQKIYVISLAEPRL DRS 23 - 154 23 - 154 AAEDYAKERYGISSMIQSQEKPDRVLVRVRDLTIQKADEV 103 VWVRARVHTSRAKGKOCFLVLROOOFNVOALVAVGDH AS KOMVKFAANINKESIVDVEGVVRKVNQKIGSCTQQDV ELHVOKIYVISLAEPRL DRS 11 - 146 11 - 146 MOEKPREIMDAAEDYAKERYGISSMIOSOEKPDRVLVRV 104 RDLTIQKADEVVWVRARVHTSRAKGKOCFLVLROQQFN VQALVAVGDHASKQMVKFACNINKESIVDVEGVVRKVN QKIGSCTQQDVELHVQKIYVIS DRS 13 - 146 13 - 146 MKPREIMDAAEDYAKERYGISSMIQSQEKPDRVLVRVRD 105 LTIOKADEVVWVRARVHTSRAKGKOCFLVEROOOFNVO ALVAVGDHASKOMVKFACNINKESIVDVEGVVRKVNOKI GSCTQQDVELHVQKIYVIS DRS 13 13 - 146 MKPREIMDAAEDYAKERYGISSMIQSQEKPDRVLVRVRD 106 146 / A1060 LTIOKADEVVWVRARVHTSRAKGKOCFLVEROOOFNVO ALVAVGDHASKOMVKFACNINKESIVDVEGVVRKVNOKI GSCTQQDVELHVQKIYVIS DRS 17 - 146 17 - 146 MIMDAAEDYAKERYGISSMIQSQEKPDRVLVRVRDLTIQK 107 ADEVVWVRARVHTSRAKGKQCFLVLRQQQFNVQALVAV GDHASKQMVKFACNINKESIVDVEGVVRKVNQKIGSCTO ODVELHVQKIYVIS DRS 21 - 146 21 - 146 MAEDYAKERYGISSMIQSQEKPDRVLVRVRDLTICKADE 108 WWVRARVHTSRAKGKQCFLVLRQQQFNVQALVAVGD HASKQMVKFACNINKES IVDVEGVVRKVNQKIGSCTQQD VELHVQKIYVIS
TABLE D3 Exemplary Internal DRS polypeptide Fragments
Amino Acid Residue Range SEQ ID Name of SEQ ID NO : Amino acid sequence NO : AspRsil Protein / QEKPDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQ 10 Human / CFLVLRQQQFNVQALVAVGDHASKOMVKFAANINKESIV 38 - 292 DVEGVVRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPL QLDDAVRPEAEGEEEGRATVNQDTRLDNRVIDLRTSTSQ AVFRLQSGICHLFRETLINKGFVEIQTPKIISAASEGGANVF TVSYFKNNAYLAOSPOLYKOMCICADFEKVFSIGPVFRAE DSNTHRHLTEFVGLDIE AspRS12 Protein / DYAKERYGISSMIQSQEKPDRVLVRVRDLTIQKADEVVW 11 Human / VRARVHTSRAKGKQCFLVLRQQQFNVQALVAVGDHASK 23 - 154 OMVKFAANINKESIVDVEGVVRKVNOKIGSCTOODVELH VOKIYVISLAEPRLPL
AspRs13 Protein / SMIQSQEKPDRVLVRVRDLTIQKADEVVWVRARVHTSRA 12 Human / KGKQCFLVLRQQQFNVQALVAVGDHASKQMVKFAANIN 33 - 154 KESIVDVEGVVRKVNQKIGSCTQQDVELHVQKIYVISLAE PRLPL US 9 ,822 , 353 B2 37 38 TABLE 14 Exemplary C - Terminal DRS polypeptide Fragments Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Amino acid sequence NO : ASPRS1C Protein / YHYHEVMEEIADTMVQIFKGLQERFQTEIQTVNKQFPCEP 13 Human / FKFLEPTLRLEYCEALAMLREAGVEMGDEDDLSTPNEKLL 297 - 501 GHLVKEKYDTDFYILDKYPLAVRPFYTMPDPRNPKQSNS YDMFMRGEEILSGAQRIHDPQLLTERALHHGIDLEKIKAYI DSFRFGAPPHAGGGIGLERVTMLFLGLHNVRQTSMFPRDP KRLTP
ASPRS1C2 Protein / MVKFAANINKESIVDVEGVVRKVNOKIGSCTOODVELHV 14 Human / QKIYVISLAEPRLPLQLDDAVRPEAEGEEEGRATVNQDTR 101 - 501 LDNRVIDLRTSTSQAVFRLOSGICHLFRETLINKGFVEIQTP KIISAASEGGANVFTVSYFKNNAYLAQSPOLYKQMCICAD FEKVFSIGPVFRAEDSNTHRHLTEFVGLDIEMAFNYHYHE VMEEIADTMVQIFKGLQERFQTEIQTVNKOFPCEPFKFLEP TLRLEYCEALAMLREAGVEMGD EDDLSTPNEKLLGHLVK EKYDTDFYILDKY PLAVRPFYTMPDPRNPKOSNSYDMFM RGEEILSGAQRIHDPOLLTERALHHGIDLEKIKAYIDSFRFG APPHAGGGIGLERVTMLFLGLHNVRQTSMFPRDPKRLTP
TABLE D5 Exemplary Alternatively Spliced DRS polypeptide Variants Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Amino acid sequence NO : ASPRS1N6 Protein / MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEK 15 Human / 1 PGKOCFLVLROQQFNVQALVAVGDHASKOMVKFAANIN 41 + 73 - 501 KESIVDVEGWRKVNOKIGSCTOODVELHVOKIYVISLAE PRLPLOLDDAVRPEAEGEEEGRATVNODTRLDNRVIDLRT STSOAVFRLOSGICHLFRETLINKGFVEIQTPKIISAASEGG ANVFTVSYFKNNAYLAOSPOLYKOMCICADFEKVFSIGPV FRAEDSNTHRHLTEFVGLDIEMAFNYHYHEVMEEIADTM VOIFKGLOERFOTEIQTVNKOFPCEPFKFLEPTLRLEYCEA LAMLREAGVEMGDEDDLSTPNEKLLGHLVKEKYDTDFYI LDKYPLAVRPFYTMPDPRNPKQSNSYDMFMRGEEILSGA QRIHDPOLLTERALHHGIDLEKIKAYIDSFRFGAPPHAGGG IGLERVTMLFLGLHNVRQTSMFPRDPKRLTP ASPRS1N7 Protein / MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEK 16 Human / 1 - 141 + PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQCFL 189 - 501 VLRQQQFNVQALVAVGDHASKOMVKFAANINKESIVDV EGVVRKVNOKIGSCTOODVELHVOKTSTSOAVFRLOSGIC HLFRETLINKGFVEIQTPKIISAASEGGANVFTVSYFKNNA YLAQSPOLYKOMCICADFEKVFSIGPVFRAEDSNTHRHLT EFVGLDIEMAFNYHYHEVMEEIADTMVQIFKGLQERFQTE IOTVNKOFPCEPFKFLEPTLRLEYCEALAMLREAGVEMGD EDDLSTPNEKLLGHLVKEKYDTDFYILDKYPLAVRPFYTM PDPRNP KOSNSYDMFMRGEEILSGAORIHD POLLTERALH HGIDLEKIKAYIDSFRFGAPPHAGGGIGLERVTMLFLGLHN VROTSMFPRDPKRLTP ASPRS1N8 Protein / MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK 17 Human PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKOCFL 1 - 319 + 369 -501 VLRQQQFNVQALVAVGDHASKOMVKFAANINKES IVDV EGVVRKVNOKIGSCTOODVELHVOKIYVISLAEPRLPLOL DDAVRPEAEGEEEGRATVNQDTRLDNRVIDLRTSTSQAVF RLOSGICHLFRETLINKGFVEIQTPKIISAASEGGANVFTVS YFKNNAYLAQSPOLYKOMCICADFEKVFSIGPVFRAEDSN THRHLTEFVGLDI EMAFNYHYHEVMEEIADTMVOIFKGL OESTPNEKLLGHLVKEKYDTDFYILDKYPLAVRPFYTMPD PRNPKQSNSYDMFMRGEEILSGAQRIHDPQLLTERALHHG IDLEKIKAYIDSFRFGAPPHAGGGIGLERVTMLFLGLHNVR QTSMFPRDPKRLTP ASPRS1N9 Protein / MPSASASRKSQEKPREIMDAAEDWNELLCCFWDCIMFVR 18 DRS ( 1 - 22 + Human / PPCSLVIPNDSLLKFTLCHLTPVWMTERDPASKKKKKKES 63 aa ) 1 - 22 + 63 aa HTYSFO US 9 ,822 , 353 B2 39 40 TABLE D5 - continued Exemplary Alternatively Spliced DRS polypeptide Variants Amino Acid Residue Range of SEQ ID SEO ID Name NO : 1 Amino acid sequence NO : ASPRSINO Protein / MPSASASRKSQEKPREIMDAAEGNSAS 19 DRS ( 1 - 22 + Human 5 aa ) 1 - 22 + 5 aa Asprsi C2 Protein / MVKFAANINKESIVDVEGVVRKVNOKIGSCTOQDVELHV 20 Human OKIYVISLAEPRLPLOLDDAVRPEAEGEEEGRATVNODTR 101 - 501 LDNRVIDLRTSTSOAVFRLOSGICHLFRETLINKGFVEIOTP KIISAASEGGANVFTVSYFKNNAYLAQS POLYKOMCICAD FEKVFSIGPVFRAEDSNTHRHLTEFVGLDIEMAFNYHYHE VMEEIADTMVQIFKGLQERFQTEIQTVNKOFPCEPFKFLEP TLRLEYCEALAMLREAGVEMGDEDDLSTPNEKLLGHLVK EKYDTDFYILDKYPLAVRPFYTMPDPRNPKOSNSYDMFM RGEEILSGAQRIHDPQLLTERALHHGIDLEKIKAYIDSFRFG APPHAGGGIGLERVTMLFLGLHNVRQTSMFPRDPKRLTP ASPRS1C3 Protein / MLFLGLHNVRQTSMFPRDPKRLTP 21 DRS ( 478 Human 501 ) 478 - 501
A number of naturally occurring aspartyl- tRNA syn - 25 thetase single nucleotide polymorphisms (SNPs ) and natu TABLE D6 - continued rally occurring variants of the human gene have been Human Aspartyl - tRNA synthetase SNPs sequenced , and are known in the art to be at least partially Gene Bank Gene Bank functionally interchangeable . Additionally homologs and Accession Nucleotide Accession Nucleotide orthologs of the human gene exist in other species, and it 30 Number Change Number Change would thus be a routine matter to select a naturally occurring variant such as a DRS polypeptide encoded by a SNP, or rs116755289 rs567363 other naturally occurring variant in place of any of the DRS polypeptide sequences listed in Tables D1- D5 . Several such rs116723553 A / G rs561980 A / G variants of aspartyl- tRNA synthetase (i .e . , representative 35 rs116719241 C / T rs522086 C / T aspartyl- tRNA synthetase SNPs) are shown in Table D6 . rs 116626412 C / T rs309172 C / T TABLE D6 rs116599033 A / G rs309171 Human Aspartyl - tRNA synthetase SNPs 40 rs116528963 C / T rs309170 C / T Gene Bank Gene Bank rs116504104 A / G rs309169 C / T Accession Nucleotide Accession Nucleotide Number Change Number Change rs116503734 AT rs309168 C / T rs2164332 c / G rs309167 C / T rs118100102 c / T 45 rs116471228 G / T rs117859527 c / G rs2164331 C / T rs 116460118 AT rs309166 C / T rs117847055 A / G rs1867632 A / G HHrs116376572 A / G rs309165 C / T rs117843158 A / C rs1803167 c / T rs116373537 G / T rs309164 A / G 50 rs117754321 A / C rs1803166 C / T rs116190965 C / T rs309163 C / T rs117605910 c / G rs1803165 G / T rs116114585 AT rs309162 A / T rs117587018 AG rs1347442 C / T rs116069651 C / T rs309161 c / T 55 rs117448010 A / C rs895285 A / G rs116013288 C / T rs309160 A /G rs117438984 A / G rs834734 C / T rs115947325 C / T rs309159 A /G rs117395206 GT rs689002 A / G rs115876148 C / T rs309158 C / T rs117045416 C / T rs687670 60 C / T rs115771261 C / T rs309157 A /G rs116899241 C / T rs 661562 A /C rs115749352 A / G rs309156 C / G rs116807764 C / T rs660002 CT rs115704588 C / T rs309155 A / G rs116756668 C / T rs 640727 A / T 65 rs115691888 AC rs309154 C / T US 9 ,822 , 353 B2 41 42 TABLE D6 - continued TABLE D6 - continued Human Aspartyl - tRNA synthetase SNPs Human Aspartyl - tRNA Synthetase SNPs
Gene Bank Gene Bank Gene Bank Gene Bank Accession Nucleotide Accession Nucleotide Accession Nucleotide Accession Nucleotide Number Change Number Change Number Change Number Change rs115651129 C / G rs309153 A / G rs113853485 G / T rs2305101 G / T rs115572299 C / T rs309150 A / T 10 rs113759327 C / G rs2278683 A / C rs115553816 AG rs309149 C / T rs113676252 C / T rs2278682 C / G rs115530645 C / T rs7587285 C / T rs113641203 G / T rs2278681 C / T rs115475999 C / T rs7585928 c / G rs113342018 G / T rs2164333 A / T rs115469964 A / C rs7573555 C / T rs113328159 - / C rs13397074 A / C rs115332530 AG rs6760465 A / T rs113316632 AT rs13392680 A / T rs115330084 C / G rs6757965 A / G 20 rs113200654 A / T rs13388887 c / T rs115316382 AG rs 6754311 C / T rs113155677 A / G rs13034773 A / C rs115306423 C / T rs6752967 A / G rs113148022 AAAAAAAAAAAAA rs13025460 A / T AAAAAAATCCAA rs115253602 A / G rs6750549 A / G 25 rs113012086 A / G rs13007697 G / T rs115249754 C / T rs6743537 A / G rs112923773 A / G rs13004546 C / T rs115248017 C / G rs 6742701 rs112910626 C / T rs12999871 A / C rs114986027 C / T rs 6740254 30 rs112868187 C / T rs12990346 G / T rs114977327 c / T rs 6738266 C / T rs112849402 A / G rs12990316 C / T rs114851922 C / T rs 6733398 A / G rs112848056 C / T rs 12624144 C / T rs114841878 AG rs6724595 A / G 35 rs112835147 C / T rs12623506 A / G rs114832662 A / G rs6711493 A / G rs112767522 C / T rs12617586 C / T rs114830940 A / G rs6430594 A / G rs112396243 C / T rs12615624 A /G rs114489290 C / T rs5834455 - / T rs112369881 A / T rs12613540 C / T rs114428384 C / T rs5834454 - / AA 40 rs112319042 C / T rs12613074 G / T rs114422751 C / T rs5834453 - /AAAAT rs112300736 G / T rs12477103 A / C rs114414669 A / C rs4954551 A / G ??? rs112205661 C / T rs12474975 A / T rs114412783 C / T rs4597591 A / T 45 rs112205423 G / T rs12471430 A / T rs114399267 C / T rs4538260 A / G rs112138368 C / T rs11895669 G / T rs114398361 A / G rs4278979 C / T rs112136466 C / T rs11895436 A / G rs114345514 C / T rs3820789 C /G 50 rs111956746 AG rs11892136 G / T rs114337780 A / C rs3768999 rs111909933 C / T rs11889473 A / C rs114164361 C / G rs3768998 A / C rs111766943 A / G rs11548872 rs114162105 A / T rs3768997 AG 55 rs111731189 C / T rs11548870 A / G rs114126158 AG rs 3768996 c / G rs111716305 C / T rs11375996 - / A rs114110228 AC rs3112496 C / T . rs111670530 C / T rs11345750 - / A rs114058841 G / T rs3098104 A / T rs111613855 A / G rs11340194 rs113998842 GT rs2839741 AT rs111608134 C / T rs11319623 - / A rs113995718 A / C rs2556175 C / T rs111600480 AG rs11297201 - / T rs113884130 C / T rs2322725 C / T 65 rs111578911 A / C rs10610928 - / CTCT rs113882668 A / C rs2307720 - / TTAG US 9 ,822 , 353 B2 43 44 TABLE D6 - continued TABLE D6 - continued Human Aspartyl - tRNA synthetase SNPs Human Aspartyl - tRNA Synthetase SNPs
Gene Bank Gene Bank Gene Bank Gene Bank Accession Nucleotide Accession Nucleotide Accession Nucleotide Accession Nucleotide Number Change Number Change Number Change Number Change rs111533002 - / T rs10606646 - / AAAA rs79018926 C / T rs16832221 C / T rs111432741 C / T rs10598545 - / AAAA rs78993580 A / G rs 16832205 A / G 10 rs111346414 C / T rs10566195 - / TGA rs78943662 - / A rs16832200 c / T rs111261866 C / T rs 10546948 rs78919277 G / T rs16832172 C / T rs80342688 A / C rs10205844 C / G rs78915112 A / C rs16832162 A / T 15 rs80296238 A / C rs35332762 - / C rs 78898735 AT rs13404551 C / T rs80290607 G / T rs35323281 - / A rs78793088 A / G rs13399128 A / G rs80201497 A / C rs35250856 - / C rs78784878 G / T rs71417582 C / T rs80160510 c / T rs35207721 - / C rs78770570 c / T rs71417581 C / G rs80095420 C / T rs35180509 - / A rs78700806 C / G rs71400535 - / A rs79933222 A / C rs35066766 - / T rs78638278 C / T rs67636722 - / A rs79908186 G / T rs34855029 - / A 25 rs78629157 A / G rs67591467 - / A rs79826902 A / G rs 34818704 - / G rs78628013 C / T rs66527494 - / A rs79811988 G / T rs34764820 - / T rs78577601 A / T rs66508408 - / AA rs79778906 c / T rs 34762161 - / T rs78537103 c / T rs62159056 A / C rs79745746 c / T rs34744196 - ?KT/ A rs78518056 A / C rs62159055 A / T rs79719188 C / T rs34739918 rs78512447 A / T rs61569739 - / AA rs79715594 C / T rs34719779 - / T 35 rs78497838 - / TTT rs61297566 - / AAATA rs79685879 - / TT rs34713850 - / A rs78383997 A / T rs61222539 C / T rs79613305 A / C rs34698626 - / AA rs 78283445 C / G rs61133344 C / T rs79513920 C / T rs34675243 - / A 40 rs78275586 G / T rs60878223 - / T rs79507949 AG rs34613097 - / A rs78274583 C / T rs 60538468 A / C rs79494100 A / T rs34442772 rs78258066 A / G rs60485095 - / TT rs79478181 A / T rs34398897 - rs60318326 C / T 45 rs78168253 C / T rs79327246 C / G rs34215176 - / G rs78143716 A /G rs59584448 - / A rs79301888 C / T rs 34180776 - / G rs78130363 A / G rs59505882 - / A rs79274257 A / G rs 34142242 - / T rs78083497 A / C rs59464486 G / T 50 rs79268627 A / T rs34050823 - / T rs78081965 G / T rs59199326 - / TT rs79238496 A / G rs17718194 rs78076875 C / T rs58805013 A / C rs79231002 C / T rs16832417 C / T rs78026280 A / G rs58799551 - / G rs79227800 C / T rs16832413 A / C 55 rs78015725 G / T rs58666594 G / T rs79173488 A / G rs16832394 A / C rs77987440 C / T rs57046249 - / A rs79161420 - / A rs16832326 A / G rs77972711 A / G rs56721192 - / AA rs79139071 A / G rs16832275 C /G rs77930020 A / C rs56100046 A / T rs79137850 C / T rs 16832274 C / T rs77902883 c / T rs55951873 A / G rs79121686 C / T rs16832248 C / G rs 77883526 A / T rs55815289 - / A rs79078468 G / T rs16832243 C / T 65 rs77862927 - / TT rs55759471 G / T US 9 ,822 , 353 B2 45 46 TABLE D6 - continued TABLE D6 - continued Human Aspartyl - tRNA synthetase SNPs Human Aspartyl - tRNA Synthetase SNPs
Gene Bank Gene Bank Gene Bank Gene Bank Accession Nucleotide Accession Nucleotide Accession Nucleotide Accession Nucleotide Number Change Number Change Number Change Number Change rs77837755 A / C rs55641281 A / G rs76677887 C / T rs71930676 - / A rs77793053 C / T rs41269823 A /G rs76672039 C / T rs71746189 - / A 10 rs77774340 AC rs41269821 AG rs76496496 A / G rs71701797 - / AAAA rs77753457 c / T rs36023868 rs76460134 A / C rs71697066 - / A rs77752694 A / T rs35921927 A / G rs76456107 A / G rs 71535212 A / T 15 rs77743403 G / T rs35814998 - rs76448970 A / G rs71535211 C / T rs77707512 C / T rs35760856 - / C rs76433055 C / T rs 71417587 AC rs77697045 C / T rs35460584 rs76392392 A /G rs71417586 A / C rs77694994 A / G rs35363362 c / G rs76357426 C / T rs 71417585 rs77654242 G / T rs74661004 C / T ? $ 76350348 A / G rs71417584 C / G rs77546304 C / T rs74527665 C / T rs76337990 C / T rs71417583 A / C rs77516029 C / T rs 74479926 C / T 25 rs76306255 G / T rs309148 C / T rs77511888 A / C rs 74462337 G / T rs76302219 C / T rs309147 rs77507602 A / T rs74399174 A / C rs76296777 A / G rs309146 A / G rs77390314 A / G rs74398392 C / T 30 rs76285313 A / T rs309145 A / G rs77341293 A / C rs 74266318 G / T rs76189476 A / G rs309144 C/ T rs77340433 C / T rs73957079 C / T rs76089705 G / T rs309143 A / G rs77244692 A / G rs73957078 C / T 35 rs76047098 c / T rs309142 C/ T rs77241600 c / T rs73957074 A / C rs75999734 C / T rs309141 A /C rs77194466 A / T rs73957073 A /G rs75990169 A / G rs309140 A / C rs77182879 A / G rs73957072 A / G rs75935955 C / T rs309120 C / G rs77177301 G / T rs73957071 A / G rs75874749 C / T rs309119 A / G rs77147958 AG rs73957070 A / G rs75843843 C / G rs309115 C /T rs77144439 A / T rs73957069 C / G C / T A / T 45 rs75843510 rs309114 rs77113180 A /G rs73957068 C / T rs75842188 A /G rs309113 rs77092452 AG rs72974121 AG rs75800473 G / T rs309112 G / T rs77052188 G / T rs72974120 c / G rs75794936 A / C rs192822 A / T 50 rs77051588 c / T rs72974119 A / G rs75753154 C / T rs177917 C / T rs76986930 A / C rs72974109 A / G rs75732042 c / G rs 167442 G / T rs76946722 - / AA rs72423998 - / A rs75683158 G / T rs 71518151 ACTTTTTGATGG 55 GGTTGT ( SEQ rs76862952 A / C rs72366475 - / T ID NO : 44 ) / ccTTTTTCATG rs76856516 G / T rs72355283 - / A GGCTTGTTTTT TTCTTGTAAAT rs76798249 A / C rs72313616 - / TT TTGTTT ( SEQ ID NO : 45 ) rs76793136 A /G rs72270342 - / A 60 rs75667274 C / T rs75123144 - / AG rs76792531 A / G rs72268157 - / A rs 75657010 A / T rs 75071131 A / T rs76732000 G / T rs72097458 - / A rs75647121 C / T rs74959174 C / T rs76729798 C / T rs71937749 - / AA rs75572938 A / T rs74833182 A / T US 9 ,822 , 353 B2 47 48 tial T cell epitopes , and directed evolution approaches to TABLE D6 - continued identify less immunogenic forms. Human Aspartyl - tRNA synthetase SNPs As noted above , embodiments of the present invention Gene Bank Gene Bank include all homologues , orthologs , and naturally -occurring Accession Nucleotide Accession NucleotideNucleotide 5 isoforms of aspartyl -tRNA synthetase ( e . g . , any of the Number Change Number Change proteins , or their corresponding nucleic acids listed in Tables D1 to D8 which i ) retain detectable non canonical activity . Also included are “ variants ” of these DRS reference rs75560320 A / G rs74777619 C / T polypeptides. The recitation polypeptide " variant” refers to rs75524146 C / T rs74771413 C /G 10 polypeptides that are distinguished from a reference DRS polypeptide by the addition , deletion , and / or substitution of rs75437018 C / G rs74674565 C / T at least one amino acid residue, and which typically retain rs75402079 A / C rs75346069 c / T ( e . g ., mimic ) or modulate ( e . g . , antagonize ) one or more non -canonical activities of a reference DRS polypeptide . rs75394224 C / G rs75298650 A /G The structure of human aspartyl- tRNA synthetase has been determined to a resolution of 1 .7A . (WO2010 / 120509 ) pro rs75365510 A / G rs75214175 A / G viding a detailed physical description of the protein , which in conjunction with the primary amino acid sequence pro Accordingly , the terms “ DRS polypeptide” “ DRS pro vides precise insights into the roles played by specific amino tein ” or “ DRS protein fragment” as used herein includes all 20 acids within the protein . Accordingly it is within the skill of naturally occurring and synthetic forms of the aspartyl- those in the art to identify amino acids suitable for substi tRNA synthetase that retain non canonical activity . Such tution and to design variants with substantially unaltered , DRS polypeptides include the full length human protein , as improved , or decreased activity with no more than routine well as the DRS peptides derived from the full length protein experimentation . listed in Tables D1- D5 , as well as naturally occurring 25 In certain embodiments, a polypeptide variant is distin variants , for example as disclosed in Table D6 , exemplary guished from a reference polypeptide by one or more cysteine mutants listed in Table D7, and synthetic codon substitutions , which may be conservative or non - conserva optimized forms and other coding sequences as exemplified tive , as described herein and known in the art. In certain by the nucleic acid sequences in Table D8. In some embodi- embodiments , the polypeptide variant comprises conserva ments, the term DRS polypeptide refers to a polypeptide 30 tive substitutions and, in this regard , it is well understood in sequence derived from human aspartyl- tRNA synthetase the art that some amino acids may be changed to others with (SEQ ID NO : 1 in Table D1 ) comprising at least one muta - broadly similar properties without changing the nature of the tion at either Cys76 or Cys130 . activity of the polypeptide . DRS Variants Specific examples of DRS polypeptide variants useful in Thus all such homologues, orthologs , and naturally - oc - 35 any of the methods and compositions of the invention curring , or synthetic isoforms of aspartyl- tRNA synthetase include full - length DRS polypeptides , or truncations or ( e . g . , any of the proteins or nucleic acids listed in Tables Di s plice variants thereof ( e . g ., any of the proteins or nucleic to D8) are included in any of the methods, kits and phar acids listed in Tables D1 to D8 which i) retain detectable non maceutical compositions of the invention , as long as they canonical activity and ii ) have one or more additional amino retain detectable non canonical activity . 40 acid substitutions. In certain embodiments , a variant poly The DRS polypeptides may be in their native form , i .e . , as peptide includes an amino acid sequence having at least different variants as they appear in nature in different species about 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , which may be viewed as functionally equivalent variants of 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % or more human aspartyl- tRNA synthetase , or they may be function - sequence identity or similarity to a corresponding sequence ally equivalent natural derivatives thereof, which may differ 45 of a DRS reference polypeptide, as described herein , ( e . g . , in their amino acid sequence , e . g . , by truncation ( e . g . , from any of the proteins or nucleic acids listed in Tables D1 to D8 the N - or C -terminus or both ) or other amino acid deletions, and substantially retains the non -canonical activity of that additions , insertions , substitutions , or post - translational reference polypeptide . Also included are sequences differing modifications . Naturally -occurring chemical derivatives , from the reference DRS sequences by the addition , deletion , including post - translational modifications and degradation 50 or substitution of 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11, 12 , 13 , 14 , products of any DRS polypeptide , are also specifically 15 , 16 , 17 , 18 , 19 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 110 , included in any of the methods and pharmaceutical compo - 120 , 130 , 140 , 150 or more amino acids but which retain the sitions of the invention including , e . g . , pyroglutamyl, iso - properties of the reference DRS polypeptide . In certain aspartyl, proteolytic , phosphorylated , glycosylated , oxida embodiments , the amino acid additions or deletions occur at tized , isomerized , and deaminated variants of a DRS 55 the C - terminal end and / or the N - terminal end of the DRS polypeptide. reference polypeptide . In certain embodiments , the amino It is known in the art to synthetically modify the acid additions include 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , sequences of proteins or peptides , while retaining their 14 , 15 , 16 , 17 , 18 , 19 , 20 , 30 , 40 , 50 or more wild -type useful activity , and this may be achieved using techniques residues ( i . e . , from the corresponding full - length DRS poly which are standard in the art and widely described in the 60 peptide ) that are proximal to the C - terminal end and /or the literature , e . g . , random or site - directed mutagenesis , cleav - N -terminal end of the DRS reference polypeptide . age, and ligation of nucleic acids , or via the chemical In some embodiments , the DRS polypeptides comprise a synthesis or modification of amino acids or polypeptide polypeptide fragment of the full length Aspartyl- tRNA syn chains . Similarly it is within the skill in the art to address thetase of about 50 to 250 amino acids, which comprises, or and / or mitigate immunogenicity concerns if they arise using 65 consists essentially of the amino acids 1 -224 , 1 - 184, 1 - 174 , a DRS polypeptide or variant thereof, e . g . , by the use of 1 - 171, 11- 146 , 13 - 146 , 1 - 154 or 23 - 154 of the DRS poly automated computer recognition programs to identify poten - peptide sequence set forth in SEQ ID NO : 1 , comprising at US 9 ,822 ,353 B2 49 50 least one mutation at either Cys76 or Cys130 ( using the window ” to identify and compare local regions of sequence numbering of SEQ ID NO : 1 ) , and variants thereof. similarity. A “ comparison window ” refers to a conceptual In certain embodiments , a DRS polypeptide of the inven - segment of at least 6 contiguous positions , usually about 50 tion comprises the minimal active fragment of a full- length to about 100 , more usually about 100 to about 150 in which DRS polypeptide capable of modulating TLR activity etc . , 5 a sequence is compared to a reference sequence of the same in vivo or having other desirable non - canonical aspartyl- number of contiguous positions after the two sequences are tRNA synthetase activities. In one aspect, such a minimal optimally aligned . The comparison window may comprise active fragment consists essentially of the anticodon binding additions or deletions ( i . e . , gaps ) of about 20 % or less as domain , ( i. e ., about amino acids 23 - 154 of SEO ID NO : 1 ) . compared to the reference sequence (which does not com In some aspects , the minimal active fragment consists essen - 10 prise additions or deletions ) for optimal alignment of the two tially of the anticodon binding domain anticodon binding sequences. Optimal alignment of sequences for aligning a domain , and N -terminal amphiphilic helix ( i . e ., about amino comparison window may be conducted by computerized acids 1 - 154 of SEQ ID NO : 1 . In some aspects , of either of implementations of algorithms (GAP , BESTFIT , FASTA , these embodiments , the minimal active fragment consists and TFASTA in the Wisconsin Genetics Software Package essentially of the anticodon binding domain anticodon bind - 15 Release 7 .0 , Genetics Computer Group , 575 Science Drive ing domain , and N -terminal amphiphilic helix and a variable Madison , Wis . , USA ) or by inspection and the best align amount of the flexible 29 amino acid linker (amino acids 154 ment ( i. e ., resulting in the highest percentage homology over to 182 of SEQ ID NO : 1 ). In different embodiments , such the comparison window ) generated by any of the various minimal active fragments may comprise 1 , 2 , 3 , 4 , 5 , 6 , 7 , methods selected . Reference also may be made to the 8 , 9 , 10 , 11, 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 20 BLAST family of programs as for example disclosed by 25 , 26 , 27 , 28 , or all 29 amino acids of the flexible linker. Altschul et al ., 1997 , Nucl . Acids Res. 25 : 3389 . A detailed Without wishing to be bound by any one theory , the discussion of sequence analysis can be found in Unit 19 .3 of unique orientation , or conformation , of the anticodon -rec - Ausubel et al. , " Current Protocols in Molecular Biology , " ognition domain in certain DRS polypeptides may contrib - John Wiley & Sons Inc , 1994 - 1998 , Chapter 15 . ute to the enhanced non canonical activities observed in 25 Calculations of sequence similarity or sequence identity these proteins . In certain embodiments, non -canonical activ - between sequences ( the terms are used interchangeably ity may be modulated by the selective deletion , in whole or herein ) can be performed as follows. To determine the part of the Amphiphilic helix domain , anticodon - recognition percent identity of two amino acid sequences , or of two domain , or the aminoacylation domain . Specific examples of nucleic acid sequences, the sequences can be aligned for splice variants that accomplish such embodiments include 30 optimal comparison purposes ( e . g . , gaps can be introduced for example AspRS1 ^ and AspRS1C2 ( partial deletion of in one or both of a first and a second amino acid or nucleic the anticodon binding domain ), AspRS1 ^ 7 (partial deletion acid sequence for optimal alignment and non -homologous of both the anticodon binding domain and aminoacylation sequences can be disregarded for comparison purposes ) . In domain ) , AspRS1M (partial deletion of the aminoacylation certain embodiments , the length of a reference sequence domain ) . In some embodiments of the present invention , all 35 aligned for comparison purposes is at least 30 % , preferably such DRS polypeptides comprise at least one mutation at at least 40 % , more preferably at least 50 % , 60 % , and even either Cys76 or Cys130 (using the numbering of SEQ ID more preferably at least 70 % , 80 % , 90 % , 100 % of the length NO : 1) . of the reference sequence . The amino acid residues or The recitations “ sequence identity ” or, for example, com - nucleotides at corresponding amino acid positions or nucleo prising a " sequence 50 % identical to ,” as used herein , refer 40 tide positions are then compared . When a position in the first to the extent that sequences are identical on a nucleotide sequence is occupied by the same amino acid residue or by -nucleotide basis or an amino acid -by -amino acid basis nucleotide as the corresponding position in the second over a window of comparison . Thus , a " percentage of sequence , then the molecules are identical at that position . sequence identity ” may be calculated by comparing two The percent identity between the two sequences is a optimally aligned sequences over the window of compari- 45 function of the number of identical positions shared by the son , determining the number of positions at which the sequences , taking into account the number of gaps , and the identical nucleic acid base ( e . g . , A , T , C , G , I ) or the identical length of each gap , which need to be introduced for optimal amino acid residue ( e . g . , Ala , Pro , Ser , Thr, Gly , Val, Leu , alignment of the two sequences . Ile, Phe , Tyr, Trp , Lys, Arg , H is , Asp , Glu , Asn , Gin , Cys The comparison of sequences and determination of per and Met ) occurs in both sequences to yield the number of 50 cent identity between two sequences can be accomplished matched positions, dividing the number of matched posi - using a mathematical algorithm . In a preferred embodiment, tions by the total number of positions in the window of the percent identity between two amino acid sequences is comparison ( i. e ., the window size ) , and multiplying the determined using the Needleman and Wunsch , ( 1970 , J . result by 100 to yield the percentage of sequence identity . Mol. Biol. 48 : 444 -453 ) algorithm which has been incorpo Terms used to describe sequence relationships between 55 rated into the GAP program in the GCG software package , two or more polypeptides include " reference sequence , " using either a Blossum 62 matrix or a PAM250 matrix , and " comparison window ," " sequence identity , " " percentage of a gap weight of 16 , 14 , 12 , 10 , 8 , 6 , or 4 and a length weight sequence identity ” and “ substantial identity .” A “ reference of 1 , 2 , 3 , 4 , 5 , or 6 . In yet another preferred embodiment, sequence ” is at least 12 but frequently 15 to 18 and often at the percent identity between two nucleotide sequences is least 25 monomer units , inclusive of nucleotides and amino 60 determined using the GAP program in the GCG software acid residues, in length . Because two polypeptides may each package , using a NWSgapdna. CMP matrix and a gap weight comprise ( 1 ) a sequence (i .e ., only a portion of the complete of 40 , 50 , 60 , 70 , or 80 and a length weight of 1 , 2 , 3 , 4 , 5 , polypeptides sequence ) that is similar between the two or 6 . A particularly preferred set of parameters ( and the one polypeptides, and ( 2 ) a sequence that is divergent between that should be used unless otherwise specified ) are a Blos the two polypeptides , sequence comparisons between two 65 sum 62 scoring matrix with a gap penalty of 12 , a gap extend (or more ) polypeptides are typically performed by compar- penalty of 4 , and a frameshift gap penalty of 5 . The percent ing sequences of the two polypeptides over a “ comparison identity between two amino acid or nucleotide sequences US 9 ,822 ,353 B2 52 can also be determined using the algorithm of E . Meyers and The E - Value , or expected value, describes the likelihood W . Miller ( 1989, Cabios, 4 : 11 - 17 ) which has been incor that a sequence with a similar score will occur in the porated into the ALIGN program (version 2 .0 ), using a database by chance. It is a prediction of the number of PAM120 weight residue table , a gap length penalty of 12 and different alignments with scores equivalent to or better than a gap penalty of 4 . 5 S that are expected to occur in a database search by chance . The nucleic acid and protein sequences described herein The smaller the E - Value , the more significant the alignment . can be used as a “ query sequence” to perform a search For example , an alignment having an E value of e - 117 means that a sequence with a similar score is very unlikely to occur against public databases to , for example , identify other simply by chance . Additionally , the expected score for family members or related sequences. Such searches can beDe 10 aligning a random pair of amino acids is required to be performed using the NBLAST and XBLAST programs negative, otherwise long alignments would tend to have high ( version 2 . 0 ) of Altschul, et al. , ( 1990 , J . Mol. Biol, 215 : score independently of whether the segments aligned were 403- 10 ) . BLAST nucleotide searches can be performed with related . Additionally , the BLAST algorithm uses an appro the NBLAST program , score = 100 , wordlength = 12 to obtain priate substitution matrix , nucleotide or amino acid and for nucleotide sequences homologous to nucleic acid molecules 15 gapped alignments uses gap creation and extension penal of the invention . BLAST protein searches can be performed ties . For example , BLAST alignment and comparison of with the XBLAST program , score = 50 , wordlength = 3 to polypeptide sequences are typically done using the BLO obtain amino acid sequences homologous to protein mol SUM62 matrix , a gap existence penalty of 11 and a gap ecules of the invention . To obtain gapped alignments for extension penalty of 1 . comparison purposes, Gapped BLAST can be utilized as 20 In one embodiment, sequence similarity scores are described in Altschul et al. , ( 1997 , Nucleic Acids Res, 25 : reported from BLAST analyses done using the BLOSUM62 3389 - 3402 ). When utilizing BLAST and Gapped BLAST matrix , a gap existence penalty of 11 and a gap extension programs, the default parameters of the respective programs penalty of 1 . ( e . g . , XBLAST and NBLAST ) can be used . In a particular embodiment, sequence identity / similarity In certain embodiments , variant polypeptides differ from 25 scores provided herein refer to the value obtained using the corresponding DRS reference sequences by at least 1 % GAP Version 10 (GCG , Accelrys, San Diego , Calif. ) using but less than 20 % , 15 % , 10 % or 5 % of the residues. ( If this the following parameters : % identity and % similarity for a comparison requires alignment, the sequences should be nucleotide sequence using GAP Weight of 50 and Length aligned for maximum similarity . “ Looped ” out sequences Weight of 3 , and the nwsgapdna .cmp scoring matrix ; % from deletions or insertions , or mismatches, are considered 30 identity and % similarity for an amino acid sequence using differences. ) The differences are, suitably , differences or GAP Weight of 8 and Length Weight of 2 , and the BLO changes at a non -essential residue or a conservative substi- SUM62 scoring matrix (Henikoff and Henikoff (1992 ) Proc tution . In certain embodiments , the molecular weight of a Natl Acad Sci USA 89 : 10915 - 10919 ) . GAP uses the algo variant DRS polypeptide differs from that of the DRS rithm of Needleman and Wunsch ( 1970 ) J Mol Biol 48 : 443 reference polypeptide by about 1 % , 2 % , 3 % , 4 % , 5 % , 6 % , 35 453 , to find the alignment of two complete sequences that 7 % , 8 % , 9 % , 10 % , 11 % , 12 % , 13 % , 14 % , 15 % , 16 % , 17 % , maximizes the number ofmatches and minimizes the num 18 % , 19 % , 20 % , or more . ber of gaps. In one embodiment, as noted above , polynucleotides In one particular embodiment, the DRS polypeptides and / or polypeptides can be evaluated using a BLAST align - comprise an amino acid sequence that can be optimally ment tool. A local alignment consists simply of a pair of 40 aligned with a DRS reference polypeptide sequence ( e . g . , sequence segments , one from each of the sequences being amino acids 1 - 224 , 1 - 184 , 1 - 174 , 1 - 171, 1 - 154 or 23 -154 of compared . A modification of Smith - Waterman or Sellers the DRS polypeptide sequence set forth in SEQ ID NO : 1 , algorithms will find all segment pairs whose scores cannot optionally comprising at least one mutation at either Cys76 be improved by extension or trimming , called high -scoring o r Cys130 (using the numbering of SEQ ID NO : 1 ) ; any one segment pairs (HSPs ) . The results of the BLAST alignments 45 of SEQ ID NOs: 1 , 3 - 24 , 29 , 31, or 74 - 117 ) to generate a include statistical measures to indicate the likelihood that the BLAST bit scores or sequence similarity scores of at least BLAST score can be expected from chance alone . about 50 , 60 , 70 , 80 , 90 , 100 , 100 , 110 , 120 , 130 , 140 , 150 , The raw score, S , is calculated from the number of gaps 160 , 170 , 180 , 190 , 200 , 210 , 220 , 230 , 240 , 250 , 260 , 270 , and substitutions associated with each aligned sequence 280 , 290 , 300 , 310 , 320 , 330 , 340 , 350 , 360 , 370 , 380 , 390 , wherein higher similarity scores indicate a more significant 50 400 , 410 , 420 , 430 , 440 , 450 , 460 , 470 , 480 , 490 , 500 , 510 , alignment . Substitution scores are given by a look -up table 520 , 530 , 540 , 550 , 560 , 570 , 580 , 590 , 600 , 610 , 620 , 630 , ( see PAM , BLOSUM ). 640 , 650 , 660 , 670 , 680 , 690 , 700 , 710 , 720 , 730 , 740 , 750 , Gap scores are typically calculated as the sum of G , the 760 , 770 , 780 , 790 , 800 , 810 , 820 , 830 , 840 , 850 , 860 , 870 , gap opening penalty and L , the gap extension penalty . For a 880 , 890 , 900 , 910 , 920 , 930 , 940 , 950 , 960 , 970 , 980 , 990 , gap of length n , the gap cost would be G + Ln . The choice of 55 1000 , or more , including all integers and ranges in between , gap costs , G and L is empirical , but it is customary to choose wherein the BLAST alignment used the BLOSUM62 a high value for G ( 10 - 15 ) , e . g ., 11, and a low value for L matrix , a gap existence penalty of 11 , and a gap extension ( 1 - 2 ) e .g ., 1 . penalty of 1 . The bit score , S ', is derived from the raw alignment score Also included are biologically active “ fragments " of the S in which the statistical properties of the scoring system 60 DRS reference polypeptides , i .e . , biologically active frag used have been taken into account. Bit scores are normalized ments of the DRS protein fragments . Representative bio with respect to the scoring system , therefore they can be logically active fragments generally participate in an inter used to compare alignment scores from different searches . action , e . g . , an intramolecular or an inter -molecular The terms “ bit score” and “ similarity score ” are used inter - interaction . An inter- molecular interaction can be a specific changeably . The bit score gives an indication of how good 65 binding interaction or an enzymatic interaction . An inter the alignment is ; the higher the score , the better the align - molecular interaction can be between a DRS polypeptide ment. and a cellular binding partner, such as a cellular receptor or US 9 , 822, 353 B2 53 54 other host molecule that participates in the non -canonical al. , ( 1978 ) Atlas of Protein Sequence and Structure (Natl . activity of the DRS polypeptide . Biomed . Res . Found. , Washington , D . C . ) . A biologically active fragment of an DRS reference Biologically active truncated and / or variant DRS poly polypeptide can be a polypeptide fragment which is , for peptides may contain conservative amino acid substitutions example , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 5 at various locations along their sequence , as compared to a 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 35 , 40 , 45 , 50 , 55 , 60 , 65 , 70 . reference DRS amino acid residue, and such additional 75 , 80 , 85 , 90 , 95 , 100 , 110 , 120 , 130 , 140 , 150 , 160 , 170 , substitutions may further enhance the activity or stability of the DRS polypeptides with altered cysteine content. A 180, 190 , 200 , 220 , 240 , 260 , 280 , 300 , 320 , 321, 322 , 323 , " conservative amino acid substitution " is one in which the 324 , 325 , 326 , 327 , 328 , 329 , 330 , 331 , 332 , 333 , 334 , 335? , 10 amino acid residue is replaced with an amino acid residue 336 , 337, 338 , 339 , 340 , 341, 342, 343 , 344 , 345, 346 , 347, having a similar side chain . Families of amino acid residues 348 , 349 , 350 , 351 , 352 , 353 , 354 , 355 , 356 , 357 , 38 , 359 , having similar side chains have been defined in the art, 360 , 361, 362, 363, 364, 365 , 380 , 400 , 450 , 500 or more which can be generally sub -classified as follows: contiguous or non -contiguous amino acids, including all Acidic : The residue has a negative charge due to loss of integers ( e . g ., 101, 102 , 103 ) and ranges ( e . g . , 50 - 100 , 15 H ion at physiological pH and the residue is attracted by 50 - 150 , 50 - 200 ) in between , of the amino acid sequences set aqueous solution so as to seek the surface positions in the forth in any one of the DRS reference polypeptides conformation of a peptide in which it is contained when the described herein . In certain embodiments , a biologically peptide is in aqueous medium at physiological pH . Amino active fragment comprises a non -canonical activity - related acids having an acidic side chain include glutamic acid and sequence , domain , or motif. In certain embodiments , the 20 aspartic acid . C - terminal or N - terminal region of any DRS reference Basic : The residue has a positive charge due to association polypeptide may be truncated by about 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , with H ion at physiological pH or within one or two pH units 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 25 , 30 , 35 , 40 , thereof ( e . g ., histidine ) and the residue is attracted by 45 , 50 , 60 , 70 , 80 , 90 , 100 , 110 , 120 , 130 , 140 , 150 , 160 , aqueous solution so as to seek the surface positions in the 170 , 180 , 190 , 200 , 250 , 300 , 350 , 400 , 450 , 500 or more 25 conformation of a peptide in which it is contained when the amino acids, or by about 10 - 50 , 20 - 50 , 50 - 100 , 100 - 150 , peptide is in aqueous medium at physiological pH . Amino 150 - 200 , 200 - 250 , 250 - 300 , 300 -350 , 350 -400 , 400 -450 , acids having a basic side chain include arginine, lysine and 450 - 500 or more amino acids , including all integers and histidine . ranges in between ( e . g ., 101, 102 , 103 , 104 , 105 ) , so long as Charged : The residues are charged at physiological pH the truncated DRS polypeptide retains the non - canonical 30 and , therefore , include amino acids having acidic or basic activity of the reference polypeptide . Typically , the biologi - side chains ( i. e . , glutamic acid , aspartic acid , arginine, lysine cally -active fragment has no less than about 1 % , 10 % , 25 % , and histidine ) . or 50 % of an activity of the biologically - active ( i. e ., non - Hydrophobic : The residues are not charged at physiologi canonical activity ) DRS reference polypeptide from which it cal pH and the residue is repelled by aqueous solution so as is derived . Exemplary methods for measuring such non - 35 to seek the inner positions in the conformation of a peptide canonical activities are described in the Examples in which it is contained when the peptide is in aqueous In some embodiments , DRS proteins , variants , and bio - medium . Amino acids having a hydrophobic side chain logically active fragments thereof, bind to one or more include tyrosine , valine , isoleucine , leucine , methionine , cellular binding partners with an affinity of at least about phenylalanine and tryptophan . 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 , 40 Neutral/ polar : The residues are not charged at physiologi 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , cal pH , but the residue is not sufficiently repelled by aqueous 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 40 , 50 , 100 , or 150 nM . solutions so that it would seek inner positions in the con In some embodiments , the binding affinity of a DRS protein formation of a peptide in which it is contained when the fragment for a selected cellular binding partner , particularly peptide is in aqueousmedium . Amino acids having a neutral/ a binding partner that participates in a non - canonical activ - 45 polar side chain include asparagine , glutamine, cysteine, ity , can be stronger than that of the corresponding full length histidine , serine and threonine . DRS polypeptide or a specific alternatively spliced DRS This description also characterizes certain amino acids as polypeptide variant, by at least about 1 .5x , 2x , 2 .5x , 3x , “ small” since their side chains are not sufficiently large , even 3 .5x , 4x , 4 . 5x , 5x , 6x, 7x , 8X , 9X , 10X, 15x, 20x , 25x , 30x , if polar groups are lacking , to confer hydrophobicity . With 40x , 50x , 60x , 70x , 80x , 90x , 100x , 200x , 300m , 400m , 50 the exception of proline, “ small ” amino acids are those with 500x , 600x , 700x , 800x , 900x, 1000x or more ( including all four carbons or less when at least one polar group is on the integers in between ). side chain and three carbons or less when not. Amino acids As noted above , a DRS polypeptide may be altered in having a small side chain include glycine , serine , alanine various ways including amino acid substitutions, deletions, and threonine. The gene - encoded secondary amino acid truncations, and insertions . Methods for such manipulations 55 proline is a special case due to its known effects on the are generally known in the art. For example , amino acid secondary conformation of peptide chains. The structure of sequence variants of a DRS reference polypeptide can be proline differs from all the other naturally occurring amino prepared by mutations in the DNA . Methods for mutagen - acids in that its side chain is bonded to the nitrogen of the esis and nucleotide sequence alterations are well known in a -amino group , as well as the a - carbon . Several amino acid the art. See , for example , Kunkel (1985 , Proc. Natl. Acad . 60 similarity matrices are known in the art ( see e . g ., PAM120 Sci. USA . 82 : 488 -492 ) , Kunkel et al. , ( 1987 , Methods in matrix and PAM250 matrix as disclosed for example by Enzymol, 154 : 367 - 382 ) , U . S . Pat . No. 4 , 873 , 192 , Watson , J . Dayhoff et al . , 1978 , A model of evolutionary change in D . et al ., (“ Molecular Biology of the Gene " , Fourth Edition , proteins ) . Matrices for determining distance relationships In Benjamin /Cummings , Menlo Park , Calif. , 1987 ) and the M . O . Dayhoff , ( ed . ) , Atlas of protein sequence and struc references cited therein . Guidance as to appropriate amino 65 ture , Vol. 5 , pp . 345 -358 , National Biomedical Research acid substitutions that do not affect biological activity of the Foundation , Washington D . C . , and by Gonnet et al. , ( Sci protein of interestmay be found in the model of Dayhoff et e nce, 256 : 14430 - 1445 , 1992 ) , however , include proline in US 9 ,822 ,353 B2 55 56 the same group as glycine, serine, alanine and threonine . biological function . After the substitutions are introduced , Accordingly , for the purposes of the present invention , the variants are screened for biological activity . proline is classified as a “ small ” amino acid . The degree of attraction or repulsion required for classi TABLE B fication as polar or nonpolar is arbitrary and , therefore , 5 amino acids specifically contemplated by the invention have Exemplary Amino Acid Substitutions been classified as one or the other . Most amino acids not Original Preferred specifically named can be classified on the basis of known Residue Exemplary Substitutions Substitutions behavior. Ala Val, Leu , Ile Val Amino acid residues can be further sub -classified as 10 Arg Lys, Gin , Asn Lys cyclic or non -cyclic , and aromatic or non -aromatic , self Asn Gln , His , Lys , Arg Gin explanatory classifications with respect to the side -chain Asp Glu Glu Cys Ser Ser substituent groups of the residues , and as small or large . The Gln Asn , His , Lys, Asn residue is considered small if it contains a total of four Glu Asp , Lys Asp carbon atoms or less , inclusive of the carboxyl carbon , 15 Gly Pro Pro provided an additional polar substituent is present; three or His Asn , Gln , Lys , Arg Arg Ile Leu , Val, Met, Ala , Phe , Norleu Leu less if not. Small residues are , of course , always non Leu Norleu , Ile , Val, Met , Ala , Phe Ile aromatic . Dependent on their structural properties , amino Lys Arg , Gln , Asn Arg acid residues may fall in two or more classes. For the Met Leu , Ile , Phe Leu naturally - occurring protein amino acids , sub -classification 20 Phe Leu , Val, Ile , Ala Leu Pro Gly Gly according to this scheme is presented in Table A . Ser Thr Thr Thr Ser Ser TABLE A Trp Tyr Tyr Tyr Tip, Phe , Thr , Ser Phe Amino acid sub - classification 25 Val Ile , Leu , Met, Phe , Ala , Norleu Leu Sub - classes Amino acids Alternatively , similar amino acids for making conserva Acidic Aspartic acid , Glutamic acid Basic Noncyclic : Arginine , Lysine; Cyclic : Histidine tive substitutions can be grouped into three categories based Charged Aspartic acid , Glutamic acid , Arginine, Lysine , on the identity of the side chains . The first group includes Histidine 30 glutamic acid , aspartic acid , arginine , lysine , histidine , Small Glycine , Serine, Alanine, Threonine, Proline Polar/ neutral Asparagine , Histidine, Glutamine , Cysteine , which all have charged side chains; the second group Serine , Threonine includes glycine , serine , threonine , cysteine, tyrosine , glu Polar / large Asparagine , Glutamine tamine , asparagine ; and the third group includes leucine , Hydrophobic Tyrosine, Valine , Isoleucine, Leucine , Methionine, Phenylalanine, Tryptophan 25 isoleucine , valine , alanine , proline, phenylalanine , trypto Aromatic Tryptophan , Tyrosine, Phenylalanine phan , methionine , as described in Zubay, G . , Biochemistry , Residues that influence Glycine and Proline third edition , Wm . C . Brown Publishers ( 1993 ). chain orientation Thus , a predicted non - essential amino acid residue in a truncated and/ or variant DRS polypeptide is typically Conservative amino acid substitution also includes group - 40 replaced with another amino acid residue from the same side ings based on side chains. For example , a group of amino chain family . Alternatively , mutations can be introduced acids having aliphatic side chains is glycine , alanine , valine , randomly along all or part of a DRS coding sequence , such leucine , and isoleucine ; a group of amino acids having as by saturation mutagenesis , and the resultant mutants can aliphatic -hydroxyl side chains is serine and threonine ; a be screened for an activity of the parent polypeptide to group of amino acids having amide - containing side chains is 45 identify mutants which retain that activity . Following muta asparagine and glutamine ; a group of amino acids having genesis of the coding sequences, the encoded peptide can be aromatic side chains is phenylalanine , tyrosine , and trypto - expressed recombinantly and the activity of the peptide can phan ; a group of amino acids having basic side chains is be determined A “ non - essential” amino acid residue is a lysine , arginine , and histidine ; and a group of amino acids residue that can be altered from the reference sequence of an having sulphur- containing side chains is cysteine and 50 embodiment polypeptide without abolishing or substantially methionine . For example, it is reasonable to expect that altering one or more of its non canonical activities . Suitably , replacement of a leucine with an isoleucine or valine, an the alteration does not substantially abolish one of these aspartate with a glutamate , a threonine with a serine , or a activities, for example , the activity is at least 20 % , 40 % , similar replacement of an amino acid with a structurally 60 % , 70 % or 80 % 100 % , 500 % , 1000 % or more of the related amino acid will not have a major effect on the 55 reference DRS sequence . An " essential” amino acid residue properties of the resulting variant polypeptide . Whether an is a residue that , when altered from the reference sequence amino acid change results in a functional truncated and/ or of a DRS polypeptide , results in abolition of an activity of variant DRS polypeptide can readily be determined by the parent molecule such that less than 20 % of the reference assaying its non - canonical activity, as described herein . activity is present. For example, such essential amino acid Conservative substitutions are shown in Table B under the 60 residues include those that are conserved in DRS polypep heading of exemplary substitutions . Amino acid substitu t ides across different species , including those sequences that tions falling within the scope of the invention , are , in are conserved in the active binding site ( s ) or motif (s ) of DRS general, accomplished by selecting substitutions that do not polypeptides from various sources . differ significantly in their effect on maintaining ( a ) the For certain types of site -specific PEGylation , described structure of the peptide backbone in the area of the substi - 65 below , DRS polypeptides may have one or more cysteine tution , ( b ) the charge or hydrophobicity of the molecule at substitutions , where one or more naturally occurring (non the target site , ( c ) the bulk of the side chain , or (d ) the cysteine ) residues are substituted with cysteine , for example , US 9 ,822 ,353 B2 57 58 to facilitate thiol- based attachment of PEG molecules. In with a cysteine substitution within the first 23 amino acids, some embodiments , cysteine substitutions are near the including the DRS polypeptides of any of SEQ ID NOs: 1 , N - terminus and / or C -terminus of the DRS polypeptide ( e. g. , 3 -24 , 29, 31 , or 74 - 117 . Specific embodiments of such DRS polypeptides with a C - terminal cysteine substitution include SEQ ID NOS: 1 , 3 - 24 , 29, 31, or 74 - 117 ) . Particular embodi01 5 for example , those with a cysteine substitution with the last ments include where one or more of residues within 0 , 1 , 2 , 5 20 amino acids, including the DRS polypeptides of any of 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11, 12 , 13, 14 , 15 , 16 , 17 , 18 , 19 , 20 , SEQ ID NOs: 1 , 3 - 24 , 29 , 31, or 74 - 117 . 21 , 22 , 23 , 24 , or 25 amino acids relative to the N - terminus These and related DRS polypeptides may also have and /or C - terminus of any one of SEQ ID NOS : 1, 3 -24 , 29 , additional substitutions at C76 and / or C130 , to remove 31 , or 74 - 117 are substituted with a cysteine residue . In o naturally occurring cysteine residues , and to facilitate site some embodiments , cysteine residues may be added to the specific pegylation at the selectively introduced cysteine DRS polypeptide through the creation of N , or C - terminal residue ( s ) . Specific embodiments include any one of SEQ fusion proteins. Such fusion proteins may be of any length , IDID NOS :1 , 3 -24 , 29 , 31, or 74 - 117 , or variants thereof, but will typically be about 1 - 5 , or about 5 - 10 , about 10 to 20 , having at mutation at C76 and /or C130 . Exemplary muta or about 20 to 30 amino acids in length . In some embodi- 15 tions at these positions include for example the mutation of ments , fusion to the C - terminus is preferred . cysteine to serine , alanine , leucine , or glycine . Various Specific embodiments of such DRS polypeptides with an exenexemplary proteins with reduced cysteine content are listed N -terminal cysteine substitution , include for example , those in Table D7 . TABLE D7 Exemplary Variants with reduced cysteine content
Amino Acid Residue Range of SEO ID SEQ ID Name NO : 1 Amino acid sequence NO : ASPRS1N1 1 - 154 MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSOEK 22 ( C76S ) PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOSFLV LROQOFNVQALVAVGDHASKOMVKFAANINKESIVDVE GVVRKVNOKIGSCTQQDVELHVQKIYVISLAEPRLPL ASPRSINI 1 - 154 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK ( C130S ) PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL 23 VLROOOFNVOALVAVGDHASKOMVKFAANINKESIVDV EGWRKVNOKIGSSTOQDVELHVOKIYVISLAEPRLPL AspRsiNi 1 - 154 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIOSOEK ( 0769 , C130S ) PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOSFLV 24 LRQQQFNVQALVAVGDHAS KOMVKFAANINKESIVDVE GVVRKVNQKIGSSTOQDVELHVQKIYVISLAEPRLPL DRS C3345 1 - 501 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL 109 VLRQQQFNVQALVAVGDHASKQMVKFAANINKESIVDV EGVVRKVNOKIGSCTOODVELHVOKIYVISLAEPRLPLOL DDAVRPEAEGEEEGRATVNODTRLDNRVIDLRTSTSOAVF RLOSGICHLFRETLINKGFVEIOTPKIISAASEGGANVFTVS YFKNNAYLAOS POLYKOMCICADFEKVFSIGPVFRAEDSN THRHLTEFVGLDI EMAFNYHYHEVMEEIADTMVOIFKGL QERFOTEIQTVNKOFPSEPFKFLEPTLRLEYCEALAMLREA GVEMGDEDDLSTPNEKLLGHLVKEKYDTDFYILDKYPLA VRPFYTMPDPRNPKOSNSYDMFMRGEEILSGAORIHDPOL LTERALHHGIDLEKI KAYIDSFRFGAPPHAGGGIGLERVTM LFLGLHNVROTSMFPRDPKRLTP DRS C3495 1 - 501 MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEK PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL 110 VLROQQFNVQALVAVGDHASKOMVKFAANINKESIVDV EGVVRKVNOKIGSCTOODVELHVOKIYVISLAEPRLPLOL DDAVRPEAEGEEEGRATVNQDTRLDNRVIDLRTSTSQAVF RLOSGICHLFRETLINKGFVEIQTPKIISAASEGGANVFTVS YFKNNAYLAQSPOLYKOMCICADFEKVFSIGPVFRAEDSN THRHLTEFVGLDI EMAFNYHYHEVMEEIADTMVQIFKGL QERFOTEIQTVNKOFPCEPFKFLEPTLRLEYSEALAMLREA GVEMGDEDDLSTPNEKLLGHLVKEKYDTDFYILDKYPLA VRPFYTMPDPRNP KOSNSYDMFMRGEEILSGAORIHDPOL LTERALHHGIDLEKIKAYIDSFRFGAPPHAGGGIGLERVTM LFLGLHNVROTSMFPRDPKRLTP DRS 1 - 501 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK C334S / C3495 PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQCFL 111 VLRQQQFNVQALVAVGDHASKQMVKFAANINKESIVDV EGVVRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPLQL DDAVRPEAEGEEEGRATVNODTRLDNRVIDLRTSTSQAVF RLOSGICHLFRETLINKGFVEIQTPKIISAASEGGANVFTVS YFKNNAYLAOS POLYKOMCICADFEKVFSIGPVFRAEDSN US 9 ,822 ,353 B2 59 TABLE D7 - continued Exemplary Variants with reduced cysteine content Amino Acid Residue Range of SEQ ID SEO ID Name NO : 1 Amino acid sequence NO : THRHLTEFVGLDI EMAFNYHYHEVMEEIADTMVOIFKGL QERFOTEIQTVNKOFPSEPFKFLEPTLRLEYSEALAMLREA GVEMGDEDDLSTPNEKLLGHLVKEKYDTDFYILDKYPLA VRPFYTMPDPRNPKOSNSYDMFMRGEEILSGAORIHDPOL LTERALHHGIDLEKI KAYIDSFRFGAPPHAGGGIGLERVTM LFLGLHNVROTSMFPRDPKRLTP DRS C203A 1 - 501 MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEK PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQCFL 112 VLRQQQFNVQALVAVGDHASKOMVKFAANINKESIVDV EGVVRKVNOKIGSCTOQDVELHVOKIYVISLAEPRLPLQL DDAVRPEAEGEEEGRATVNQDTRLDNRVIDLRTSTSQAVF RLQSGIAHLFRETLINKGFVEIQTPKIISAASEGGANVFTVS YFKNNAYLAQSPOLYKOMCICADFEKVFSIGPVFRAEDSN THRHLTEFVGLDI EMAFNYHYHEVMEEIADTMVOIFKGL QERFOTEIQTVNKOFPCEPFKFLEPTLRLEYCEALAMLREA GVEMGDEDDLSTPNEKLLGHLVKEKYDTDFYILDKYPLA VRPFYTMPDPRNP KOSNSYDMFMRGEEILSGAORIHDPOL LTERALHHGIDLEKI KAYIDSFRFGAPPHAGGGIGLERVTM LFLGLHNVROTSMFPRDPKRLTP DRS C203V 1 - 501 MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEK PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL 113 VLRQQQFNVQALVAVGDHASKQMVKFAANINKESIVDV EGVVRKVNOKIGSCTOODVELHVOKIYVISLAEPRLPLOL DDAVRPEAEGEEEGRATYNODTRLDNRVIDLRTSTSOAVF RLOSGIVHLFRETLINKGFVEIQTPKIISAASEGGANVFTVS YFKNNAYLAOSPOLYKOMCICADFEKVFSIGPVFRAEDSN THRHLTEFVGLDI EMAFNYHYHEVMEEIADTMVOIFKGL QERFOTEIQTVNKOFPCEPFKFLEPTLRLEYCEALAMLREA GVEMGDEDDLSTPNEKLLGHLVKEKYDTDFYILDKYPLA VRPFYTMPDPRNPKQSNSYDMFMRGEEILSGAQRIHDPOL LTERALHHGIDLEKI KAYIDSFRFGAPPHAGGGIGLERVTM LFLGLHNVROTSMFPRDPKRLTP DRS 1 - 501 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK 114 C344S / C3495 / PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL C203A VLROQOFNVQALVAVGDHASKOMVKFAANINKESIVDV EGVVRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPLQL DDAVRPEAEGEEEGRATVNQDTRLDNRVIDLRTSTSQAVF RLOSGIAHLFRETLINKGFVEIOTPKIISAASEGGANVFTVS YFKNNAYLAQSPOLYKOMCICADFEKVFSIGPVFRAEDSN THRHLTEFVGLDI EMAFNYHYHEVMEEIADTMVOIFKGL OERFOTEIOTYNKOFPSEPFKFLEPTLRLEYSEALAMLREA GVEMGDEDDLSTPNEKLLGHLVKEKYDTDFYILDKYPLA VRPFYTMPDPRNP KOSNSYDMFMRGEEILSGAORIHDPOL LTERALHHGIDLEKI KAYIDSFRFGAPPHAGGGIGLERVTM LFLGLHNVRQTSMFPRDPKRLTP DRS 1 - 501 MPSASARKSQEKPREIMDAAEDYAKERYGISSMIQSQEK 115 C334S / C3495 / PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL C203V VLRQQQFNVQALVAVGDHASKOMVKFAANINKESIVDV EGVVRKVNOKIGSCTOODVELHVOKIYVISLAEPRLPLOL DDAVRPEAEGEEEGRATVNODTRLDNRVIDLRTSTOAVF RLOSGIVHLFRETLINKGFVEIOTPKIISAASEGGANVFTVS YF KNNAYLAOSPOLYKOMCIAADFEKVFSIGPVFRAEDSN THRHLTEFVGLDI EMAFNYHYHEVMEEIADTMVOIFKGL QERFQTEIQTVNKOFPSEPFKFLEPTLRLEYSEALAMLREA GVEMGDEDDLSTPNEKLLGHLVKEKYDTDFYILDKYPLA VRPFYTMPDPRNP KOSNSYDMFMRGEEILSGAORIHDPOL LTERALHHGIDLEKIKAYIDSFRFGAPPHAGGGIGLERVTM LFLGLHNVROTSMFPRDPKRLTP DRS 1 - 501 MPSASASRKSQEKPREIMDAAEDYAKERYGISSMIQSQEK 116 C334S / C349S / PDRVLVRVRDLTIOKADEVVWVRARVHTSRAKGKOCFL C259A / C203A VLRQQQFNVQALVAVGDHASKQMVKFAANINKESIVDV EGVVRKVNOKIGSCTOODVELHVOKIYVISLAEPRLPLOL DDAVRPEAEGEEEGRATVNQDTRLDNRVIDLRTSTSQAVF RLQSGIAHLFRETLINKGFVEIQTPKIISAASEGGANVFTVS YFKNNAYLAOSPOLYKOMCIAADFEKVFSIGPVFRAEDSN THRHLTEFVGLDIEMAFNYHYHEVMEEIADTMVQIFKGL QERFQTEIQTVNKOFPSEPFKFLEPTLRLEYSEALAMLREA TTT GVEMGDEDDLSTPNEKLLGHLVKEKYDTDFYILDKYPLA US 9 ,822 ,353 B2 61 TABLE D7 - continued Exemplary Variants with reduced cysteine content Amino Acid Residue Range of SEQ ID SEO ID Name NO : 1 Amino acid sequence NO : VRPFYTMPDPRNPKQSNSYDMFMRGEEILSGAQRIHDPOL LTERALHHGIDLEKIKAYIDSFRFGAPPHAGGGIGLERVTM LFLGLHNVRQTSMFPRDPKRLTP DRS 1 - 501 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEK 117 C334S / C3495 / PDRVLVRVRDLTIQKADEVVWVRARVHTSRAKGKQCFL C259A / C203V VLROOOFNVQALVAVGDHASKOMVKFAANINKESIVDV EGWRKVNQKIGSCTQQDVELHVQKIYVISLAEPRLPLQL DDAVRPEAEGEEEGRATVNODTRLDNRVIDLRTSTSQAVF RLOSGIVHLFRETLINKGFVEIOTPKIISAASEGGANVFTVS YFKNNAYLAQSPOLYKOMCIAADFEKVFSIGPVFRAEDNS THRHLTEFVGLDI EMAFNYHYHEVMEEIADTMVOIFKGL QERFQTEIQTVNKQFPSEPFKFLEPTLRLEYSEALAMLREA GVEMGDEDDLSTPNEKLLGHLVKEKYDTDFYILDKYPLA VRPFYTMPDPRNPKQSNSYDMFMRGEEILSGAQRIHDPOL LTERALHHGIDLEKIKAYIDSFRFGAPPHAGGGIGLERVTM LFLGLHNVRQTSMFPRDPKRLTP
For some types of site - specific pegylation , DRS polypep - crystal structure of an exemplary DRS polypeptide tides may have one or more glutamine substitutions, where - (WO2010 /120509 ) . Based on this analysis several amino one or more naturally -occurring ( non - glutamine ) residues acids on the surface may potentially be used as mutation are substituted with glutamine , for example , to facilitate sites to introduce functional groups suitable for PEGylation . transglutaminase - catalyzed attachment of PEG molecules to The following Table D9 lists the surface accessibility score the glutamine ’ s amide group . In some embodiments , gluta - 30 of amino acids based on the crystal structure above . In this mine substitutions are introduced near the N - terminus and /or table , the higher scores represent better accessibility . C -terminus of the DRS polypeptide (e .g ., SEQ ID NOS: 1, Accordingly , higher scores (for example , > 40 ) are preferred 3 -24 , 29 , 31 , or 74 - 117 ) . Particular embodiments include for better PEG - coupling efficiency . Accordingly in some where one or more of residues within 0 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , embodiments an amino acid position selected from Table D9 8 , 9 , 10 , 11, 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21, 22 , 23 , 24 35 may used to introduce a cysteine, lysine , glutamine , or non or 25 amino acids relative to the N - terminus and / or C - ter naturally occurring amino acid . minus of any one of SEQ ID NOS : 1 , 3 - 24 , 29 , 31 , or 74 - 117 are substituted with a glutamine residue . These and related TABLE D9 DRS polypeptides can also include substitutions ( e . g . , con Surface Exposed amino acids servative substitutions ) to remove any naturally -occurring 40 glutamine residues, if desired , and thereby regulate the Position Amino Acid Score degree of site -specific pegylation . 125 63 For other types of site -specific pegylation , DRS polypep tides may have one or more lysine substitutions , where one 51 or more naturally - occurring (non - lysine ) residues are sub - 45 stituted with lysine , for example , to facilitate acylation or alkylation -based attachment of PEG molecules to the OvauAWNA lysine ’ s amino group . These methods also typically result in 43 attachment of PEG to the N -terminal residue . In some 104 uuuuuuu99 10 108 embodiments , lysine substations are near the N - terminus 50 130 and / or C - terminus of the DRS polypeptide ( e . g ., SEQ ID 132 NOS : 1 , 3 - 24 , 29 , 31 , or 74 - 117 ) . Particular embodiments 151 include where one or more of residues within 0 , 1 , 2 , 3 , 4 , 152 u 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 91 ZOAHOAAAZOEAAAIOCOXO 22 , 23 , 24 or 25 amino acids to the N - terminus and/ or 55 127 C - terminus of any one of SEQ ID NOS : 1 , 3 - 24 , 29 , 31 , or 129 74 - 117 are substituted with a lysine residue . These and 50 TioNa 107 related DRS polypeptides can also include substitutions 72 ( e . g . , conservative substitutions ) to remove any naturally 39 occurring lysine residues, if desired , and thereby regulate the 60 100 #Ob degree of site - specific pegylation . ARRARAHAN 95 Site - specific PEGylation of DRS polypeptides may also be performed by substituting one or more solvent accessible In particular embodiments , a solvent accessible surface surface amino acids of a DRS polypeptide . For example , amino acid from Table D9 is selected from the group suitable solvent accessible amino acids may be determined 65 consisting of: alanine, glycine, and serine , and can be based on the predicted solvent accessibility using the SPID - substituted with naturally occurring amino acids including , DER server (http :/ / sppider .cchmc . org /) using the published but not limited to , cysteine, glutamine, or lysine, or a US 9 ,822 ,353 B2 64 non -naturally occurring amino acid that is optimized for site Specific examples of unnatural amino acids include , but specific PEGylation . In certain embodiments, one or more are not limited to , p -acetyl - L -phenylalanine , O -methyl - L solvent accessible surface amino acids of the DRS polypep tyrosine , an L - 3 - ( 2 - naphthyl) alanine , a 3 -methyl - phenylala tide are selected from the group consisting of: C130 , G129 , nine , an 0 - 4 - allyl- L - tyrosine, a 4 -propyl - L - tyrosine , a tri A107, A72 and G95 are , substituted with cysteine, gluta - 5 O - acetyl- GlcNAcß - serine , B - O -GlcNAc - L - serine, a tri - 0 mine , lysine , or a non -naturally occurring amino acid . acetyl- GalNAc - a -threonine , an a -GalNAc - L - threonine, an In various embodiments , the present invention contem - L - Dopa , a fluorinated phenylalanine , an isopropyl - L - phe plates site - specific pegylation at any amino acid position in nylalanine, a p -azido - L -phenylalanine , a p - acyl- L - phenyl a DRS polypeptide by virtue of substituting a non -naturally alanine , a p -benzoyl - L -phenylalanine , an L -phosphoserine , occurring amino acid comprising a functional group that will a phosphonoserine, a phosphonotyrosine , a p - iodo -phenyl form a covalent bond with the functional group attached to alanine, a p -bromophenylalanine , a p - amino - L -phenylala a PEG moiety . Non -natural amino acids can be inserted or nine , an isopropyl- L - phenylalanine, those listed below , or substituted at, for example , one or more of residues within elsewhere herein , and the like . 0 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 16 Accordingly , one may select a non -naturally occurring 19 , 20 , 21 , 22 , 23 , 24 or 25 amino acids relative to the amino acid comprising a functional group that forms a N -terminus and /or C -terminus of any one of SEQ ID NOS: covalent bond with any preferred functional group of a PEG 1 , 3 - 24 , 29, 31, or 74 - 117 ; at the N - terminus and / or C - ter - moiety . Non -natural amino acids , once selected , can either minus of any one of SEQ ID NOS : 1, 3 -24 , 29 , 31, or be purchased from vendors , or chemically synthesized . Any 74 - 117 ; or a solvent accessible surface amino acid residue as 20 number ofnon -natural amino acids may be incorporated into described in Table D9. the target molecule and may vary according to the number In particular embodiments , non -naturally occurring of desired water soluble polymers , e . g . , PEG moieties , that amino acids include , without limitation , any amino acid , are to be attached . The PEG moieties may be attached to all modified amino acid , or amino acid analogue other than or only some of the non - natural amino acids. Further, the selenocysteine and the following twenty genetically 25 same or different non -natural amino acids may be incorpo encoded alpha -amino acids: alanine , arginine , asparagine , rated into a DRS polypeptide, depending on the desired aspartic acid , cysteine, glutamine , glutamic acid , glycine , outcome. In certain embodiments , about 1, 2 , 3 , 4 , 5 , 6 , 7 , histidine, isoleucine, leucine, lysine, methionine, phenylala 8 , 9 , 10 or more non -natural amino acids are incorporated nine , proline , serine, threonine, tryptophan , tyrosine , valine . into a DRS polypeptide any or all of which may be conju The generic structure of an alpha - amino acid is illustrated by 30 gated to a PEG comprising a desired functional group . the following formula : In certain aspects , the use of non - natural amino acids can be utilized to modify ( e . g ., increase ) a selected non - canoni cal 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 , as described elsewhere H2N CO2H herein . For instance , certain non - natural amino acids allow selective attachment of polymers such as PEG to a given A non -natural amino acid is typically any structure having protein , and thereby improve their pharmacokinetic proper the foregoing formula wherein the R group is any substituent 40 ties . other than one used in the twenty natural amino acids . See , Specific examples of amino acid analogs and mimetics e .g . , any biochemistry text such as Biochemistry by L . can be found described in , for example , Roberts and Vel Stryer, 3rd ed . 1988 , Freeman and Company, New York , for laccio , The Peptides: Analysis , Synthesis , Biology, Eds . structures of the twenty natural amino acids . Note that the Gross and Meinhofer, Vol. 5 , p . 341 , Academic Press, Inc. , non - natural amino acids disclosed herein may be naturally 45 New York , N . Y . ( 1983 ) , the entire volume of which is occurring compounds other than the twenty alpha -amino incorporated herein by reference . Other examples include acids above. Because the non -natural amino acids disclosed peralkylated amino acids , particularly permethylated amino herein typically differ from the natural amino acids in side acids. See , for example , Combinatorial Chemistry , Eds. chain only, the non -natural amino acids form amide bonds Wilson and Czarnik , Ch . 11 , p . 235 , John Wiley & Sons Inc . , with other amino acids, e. g ., natural or non -natural , in the 50 New York , N . Y. ( 1997 ), the entire book of which is incor same manner in which they are formed in naturally occur - porated herein by reference . Yet other examples include ring proteins . However , the non -natural amino acids have amino acids whose amide portion (and , therefore , the amide side chain groups that distinguish them from the natural backbone of the resulting peptide ) has been replaced , for amino acids . For example , R in foregoing formula optionally example , by a sugar ring , steroid , benzodiazepine or carbo comprises an alkyl- , aryl- , aryl halide , vinyl halide , alkyl 55 cycle . See , for instance , Burger ' s Medicinal Chemistry and halide, acetyl, ketone , aziridine, nitrile , nitro , halide, acyl- , Drug Discovery , Ed . Manfred E . Wolff , Ch . 15 , pp . 619 -620 , keto - , azido - , hydroxyl- , hydrazine , cyano - , halo -, hydraz - John Wiley & Sons Inc ., New York , N . Y . ( 1995 ) , the entire ide, alkenyl, alkynyl , ether, thio ether , epoxide, sulfone , book of which is incorporated herein by reference .Methods boronic acid , boronate ester, borane , phenylboronic acid , for synthesizing peptides, polypeptides, peptidomimetics thiol, seleno - , sulfonyl- , borate , boronate , phospho , 60 and proteins are well known in the art ( see, for example , phosphono , phosphine, heterocyclic -, pyridyl, naphthyl, U .S . Pat . No . 5 ,420 , 109 ; M . Bodanzsky, Principles of Pep benzophenone , a constrained ring such as a cyclooctyne , tide Synthesis ( 1st ed . & 2d rev. ed . ), Springer - Verlag , New thio ester , enone , imine , aldehyde, ester , thioacid , hydrox York , N . Y . (1984 & 1993 ) , see Chapter 7 ; Stewart and ylamine , amino , carboxylic acid , alpha -keto carboxylic acid , Young , Solid Phase Peptide Synthesis , (2d ed . ) , Pierce alpha or beta unsaturated acids and amides , glyoxylamide , 65 Chemical Co ., Rockford , ill . ( 1984 ), each of which is or organosilane group , or the like or any combination incorporated herein by reference ) . Accordingly , the DRS thereof. polypeptides of the present invention may be composed of US 9 ,822 ,353 B2 65 66 naturally occurring and non -naturally occurring amino acids modified , and then the remaining free cysteine at position as well as amino acid analogs and mimetics . 130 used to selectively chemically couple another molecule . In one embodiment of any of these methods , composi - Polynucleotides tions and kits , the DRS polypeptide is AspRS1M /DRS ( 1 - Certain embodiments relate to polynucleotides that 154 ) comprising at least one mutation at either Cys76 or 5 encode a DRS polypeptide . Among other uses , these Cys130 . embodiments may be utilized to recombinantly produce a In some embodiments , the DRS polypeptide may com desired DRS polypeptide or variant thereof, or to express the prise at mutation at Cys76 and /or Cys130 , wherein the DRS polypeptide in a selected cell or subject. It will be substituted amino acid is independently selected from the appreciated by those of ordinary skill in the art that, as a group consisting of all 19 alternative naturally occurring 10 result of the degeneracy of the genetic code , there are many amino acids except Cys, or a non -naturally occurring amino nucleotide sequences that encode a DRS polypeptide as acid . described herein . Some of these polynucleotides may bear In some embodiments , the DRS polypeptide may com - minimal homology to the nucleotide sequence of any native prise at mutation at Cys76 and / or Cys130 , wherein the gene . Nonetheless , polynucleotides that vary due to differ substituted amino acid is independently selected from the 15 ences in codon usage are specifically contemplated by the group consisting of Ser, Ala , Gly , Met , Leu , Val; Ile and Thr. present invention , for example polynucleotides that are In some embodiments , the DRS polypeptide may com - optimized for human , yeast or bacterial codon selection . prise at mutation at Cys76 and / or Cys130 wherein the Therefore , multiple polynucleotides can encode the DRS substituted amino acid is independently selected from the polypeptides of the invention . Moreover, the polynucleotide group consisting of Ser and Ala . 20 sequence can be manipulated for various reasons. Examples In some embodiments , the DRS polypeptide may com - include but are not limited to the incorporation of preferred prise at mutation at Cys76 and / or Cys130 wherein the codons to enhance the expression of the polynucleotide in substituted amino acid is independently selected from the various organisms (see generally Nakamura et al. , Nuc . group consisting of Asp , Glu , Arg , Lys, Gln , and Asn . Acid . Res . (2000 ) 28 ( 1 ) : 292 ). In addition , silent mutations In some embodiments the DRS polypeptide may com - 25 can be incorporated in order to introduce , or eliminate prise at mutation at Cys76 and / or Cys130 wherein the restriction sites, decrease the density of CpG dinucleotide substituted amino acid is independently selected from the motifs (see for example, Kameda et al ., Biochem . Biophys. group consisting of H is , Pro , Tyr, Trp and Phe . Res. Commun ( 2006 ) 349 (4 ) : 1269 - 1277 ) or reduce the In some embodiments , the DRS polypeptide may com - ability of single stranded sequences to form stem - loop prise at mutation at Cys76 and /or Cys130 wherein the 30 structures: ( see , e . g . , Zuker M . , Nucl. Acid Res. (2003 ) ; substitution is a independently selected from Ser, Ala , Gly, 31 ( 13 ) : 3406 - 3415 ) . In addition , mammalian expression can Met, Leu , Val ; Ile and Thr, and a non -naturally occurring be further optimized by including a Kozak consensus amino acid . sequence [ i. e . , ( a / g ) cc ( a / g /ccATGgl at the start codon . In any of these various embodiments , Cys76 may be Kozak consensus sequences useful for this purpose are selectively modified , while Cys130 remains unmodified . 35 known in the art (Mantyh et al . PNAS 92 : 2662 - 2666 Conversely , in some embodiments , Cys130 may be selec - ( 1995 ) ; Mantyh et al . Prot. Exp . & Purif. 6 , 124 (1995 ) ) . tively modified , while Cys76 remains unmodified . In some Exemplary codon optimized versions of the wild type full embodiments both Cys76 and Cys130 may be independently length DRS polypeptide and AspRS1M are provided in modified using any combination of the sub - groupings listed Table D8 , below , as are other exemplary DRS coding above. In some embodiments , Cys76 may be selectively sequences . TABLE D8 DRS DNA Sequences Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Nucleic acid sequence NO : AspRS1N1 DNA / ATGCCGAGCGCGAGCGCCAGCCGTAAGAGCCAGGAAA 25 Synthetic / AACCACGTGAGATTATGGATGCCGCAGAGGACTATGCG Codon AAAGAACGTTACGGTATTTCCAGCATGATCCAATCTCA optimized 1 GGAGAAACCGGACCGCGTTCTGGTTCGTGTTCGCGATC 462 TGACCATTCAGAAGGCGGACGAGGTGGTTTGGGTGCGT GCGCGCGTGCACACCAGCCGTGCAAAAGGCAAACAGT GCTTTCTGGTCCTGCGTCAGCAGCAATTCAACGTCCAG GCGCTGGTGGCAGTGGGTGACCACGCCAGCAAACAAAT GGTGAAGTTCGCTGCTAACATCAATAAAGAATCCATTG TTGATGTTGAAGGCGTCGTTCGCAAGGTCAATCAAAAG ATCGGCTCGTGTACGCAACAAGATGTCGAGCTGCATGT GCAGAAGATTTACGTCATCAGCCTGGCGGAGCCGCGTT ???????? AspRS1N1 DNA / ATGCCGAGCGCGAGCGCCAGCCGTAAGAGCCAGGAAA ( C765 ) Synthetic / AACCACGTGAGATTATGGATGCCGCAGAGGACTATGCG Codon AAAGAACGTTACGGTATTTCCAGCATGATCCAATCTCA optimized 1 GGAGAAACCGGACCGCGTTCTGGTTCGTGTTCGCGATC 462 TGACCATTCAGAAGGCGGACGAGGTGGTTTGGGTGCGT GCGCGCGTGCACACCAGCCGTGCAAAAGGCAAACAGA GCTTTCTGGTCCTGCGTCAGCAGCAATTCAACGTCCAG GCGCTGGTGGCAGTGGGTGACCACGCCAGCAAACAAAT US 9 ,822 ,353 B2 67 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Nucleic acid sequence NO : GGTGAAGTTCGCTGCTAACATCAATAAAGAATCCATTG TTGATGTTGAAGGCGTCGTTCGCAAGGTCAATCAAAAG ATCGGCTCGTGTACGCAACAAGATGTCGAGCTGCATGT GCAGAAGATTTACGTCATCAGCCTGGCGGAGCCGCGTT TGCCGCTGGGTAAGCCGATCCCTAACCCGCTGTTGGGT CTGGACAGCACGCATCACCATCACCACCACTAA Full length DNA / ATGccATCAGcc?cAGCATCTcGTAAAAGccAGGAAAA 27 AspRS Synthetic / ACCGCGCGAAATCATGGACGCTGCCGAAGATTATGCCA sequence Codon AAGAGCGCTATGGTATCAGTTCGATGATCCAGTCACAA optimized 1 GAGAAACCAGATCGTGTGCTGGTCCGTGTTCGTGACCT 1503 GACCATCCAGAAAGCGGATGAAGTTGTTTGGGTCCGTG CTCGTGTTCATACAAGCCGTGCCAAAGGCAAACAGTGC ??ccTGGT???GCGTCAACAGCAGTTTAAcGTICAGGcc ??GGTAGccGTTGGTGATCAcGccTCAAAACAAATGGT GAAATTcGcccccAACATCAACAAAGAGAGCATCGTCG ACGTTGAAGGTGTCGTCCGTAAAGTGAATCAGAAAATC GGCTCCTGTACACAGCAAGATGTGGAGCTGCATGTCCA AAAAATCTATGTCATCTCACTGGccGAAcc?cGTCTGcc TCTGCAACTGGATGATGCTGTACGCCCTGAAGCTGAAG GCGAAGAAGAAGGTCGTGCTACGGTTAATCAGGATACT CGCCTGGACAACCGTGTCATTGATCTGCGCACCTCAAC CTCTCAAGCGGTATTCCGCCTGCAATCCGGCATCTGTCA CCTGTTCCGTGAAACGCTGATCAACAAAGGGTTTGTGG AGATTCAGACCCCGAAAATCATTAGTGCCGCCAGCGAA GGTGGAGCAAATGTGTTTACCGTGTCCTATTTCAAAAA CAATGccTATCTGGCACAGTCTccTCAGCTGTATAAAC AAATGTGTATCTGTGCTG????cGAGAAAGTGTTCTCA ATCGGGCCGGTATTCCGTGCAGAGGATAGCAACACACA CCGCCATCTGACCGAATTTGTAGGCCTGGACATCGAAA TGGCCTTCAACTATCATTATCACGAGGTGATGGAAGAA ATCGCTGATACAATGGTACAGATCTTTAAAGGGCTGCA AGAACGCTTTCAAACAGAGATTCAAACCGTCAATAAAC AGTTCCCGTGTGAACCGTTCAAATTTCTGGAACCGACC CTGCGTCTGGAATATTGTGAAGCACTGGCTATGCTGCG CGAAGCTGGTGTCGAAATGGGTGATGAGGATGACCTGT CTACCCCTAACGAAAAACTGCTGGGCCACCTGGTAAAA GAAAAATATGACACAGACTTCTATATCCTGGACAAATA ?ccGCTGGCAGTTcGTccGTTTTATACGATGccTGATcc TCGTAATCCGAAACAAAGCAACTCCTATGACATGTTCA TGCGTGGTGAAGAGATCCTGTCTGGTGCTCAACGTATC CATGATCCACAGCTGCTGACAGAACGTGCACTGCATCA CGGTATTGATCTGGAGAAAATCAAAGCCTATATCGACT CCTTTCGCTTTGGTGCCCCTCCACATGCCGGTGGTGGAA TTGGGCTGGAGCGTGTAACAATGCTGTTCCTGGGACTG CACAAcGTccGTCAAAccTCAATGTTTccACGTGAccCT AAACGTCTGACACCT DRS - C3345 1 - 1503 / ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 118 Reduced AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT cysteine content AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA TACAAGATTAGACAACAGAGTCATTGATCTTAGGACAT CAACTAGTCAGGCAGTc??ccGTCTccAGTCTGGCATCT GCCATCTCTTCCGAGAAACTTTAATTAACAAAGGTTTTG TGGAAATCCAAACTCCTAAAATTATTTCAGCTGCCAGT GAAGGAGGAGCCAATGTTTTTACTGTGTCATATTTTAA AAATAATGCATAccTGGCTCAGTccccACAG??????? AGCAAATGTGCATTTGTGCTGATTTTGAGAAGGTTTTCT CTATTGGACCAGTATTCAGAGCGGAAGACTCTAATACC CATAGACATCTAACTGAGTTTGTTGGTTTGGACATTGAA ATGGCTTTTAATTACCATTACCACGAAGTTATGGAAGA AATTGCTGACACCATGGTACAAATATTCAAAGGACTTC US 9 ,822 ,353 B2 69 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Nucleic acid sequence NO : AAGAAAGGTTTCAGACTGAAATTCAAACAGTGAATAAA CAGTTCCCATCTGAGCCATTCAAATTTTTGGAGCCAACT CTAAGACTAGAATATTGTGAAGCATTGGCTATGCTTAG GGAAGCTGGAGTCGAAATGGGAGATGAAGACGATCTG AGCACACCAAATGAAAAGCTGTTGGGTCATTTGGTAAA GGAAAAGTATGATACAGATTTTTATATTCTTGATAAAT ??ccATTGGCTGTAAGAccTTTC?A?AccATGccTGAcc CAAGAAATC?CAAACAGTCCAACTCTTACGATATGTTC ATGAGAGGAGAAGAAATATTGTCAGGAGCTCAAAGAA TACATGATCCTCAACTGCTAACAGAGAGAGCTTTACAT CATGGAATTGATTTGGAGAAAATTAAGGCTTACATTGA TTCCTTCCGCTTTGGAGCCCCTCCTCATGCTGGTGGAGG CATTGGATTGGAACGAGTTACTATGCTGTTTCTGGGATT GCATAATGTTCGTCAGAccTCCATGTTccc?cGTGATcc CAAACGACTCACTCCT DRS - C3495 1 - 1503 / ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 119 Reduced AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT cysteine content AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA TACAAGATTAGACAACAGAGTCATTGATCTTAGGACAT CAACTAGT CAGGCAGTC??ccGT c?ccAGTCTGGCATCT GCCATCTCTTCCGAGAAACTTTAATTAACAAAGGTTTTG TGGAAATCCAAACTCCTAAAATTATTTCAGCTGCCAGT GAAGGAGGAGCCAATGTTTTTACTGTGTCATATTTTAA AAATAATGCATACCTGGCTCAGTCCCCACAGCTATATA AGCAAATGTGCATTTGTGCTGATTTTGAGAAGGTTTTCT CTATTGGACCAGTATTCAGAGCGGAAGACTCTAATACC CATAGACATCTAACTGAGTTTGTTGGTTTGGACATTGAA ATGGCTTTTAATTACCATTACCACGAAGTTATGGAAGA AATTGCTGACACCATGGTACAAATATTCAAAGGACTTC AAGAAAGGTTTCAGACTGAAATTCAAACAGTGAATAAA CAGTTCCCATGTGAGCCATTCAAATTTTTGGAGCCAACT CTAAGACTAGAATATTCTGAAGCATTGGCTATGCTTAG GGAAGCTGGAGTCGAAATGGGAGATGAAGACGATCTG AGCACACCAAATGAAAAGCTGTTGGGTCATTTGGTAAA GGAAAAGTATGATACAGATTTTTATATTCTTGATAAAT ATCCATTGGCTGTAAGACCTTTCTATACCATGCCTGACC CAAGAAATCCCAAACAGTCCAACTCTTACGATATGTTC ATGAGAGGAGAAGAAATATTGTCAGGAGCTCAAAGAA TACATGATCCTCAACTGCTAACAGAGAGAGCTTTACAT CATGGAATTGATTTGGAGAAAATTAAGGCTTACATTGA TTCCTTCCGCTTTGGAGCCCCTCCTCATGCTGGTGGAGG CATTGGATTGGAACGAGTTACTATGCTGTTTCTGGGATT GCATAATGTTCGTCAGACCTCCATGTTCCCTCGTGATCC CAAAcGACTCACTcc? DRS 1 - 1503 / ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 120 C334S / C349S Reduced AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT cysteine content AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA TACAAGATTAGACAACAGAGTCATTGATCTTAGGACAT CAACTAGTCAGGCAGTCTTCCGTCTCCAGTCTGGCATCT US 9 ,822 ,353 B2 71 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Nucleic acid sequence NO : GCCATCTCTTCCGAGAAACTTTAATTAACAAAGGTTTTG TGGAAATCCAAACTCCTAAAATTATTTCAGCTGCCAGT GAAGGAGGAGCCAATGTTTTTACTGTGTCATATTTTAA AAATAATGCATACCTGGCTCAGTCCCCACAGCTATATA AGCAAATGTGCATTTGTGCTGATTTTGAGAAGGTTTTCT CTATTGGACCAGTATTCAGAGCGGAAGACTCTAATACC CATAGACATCTAACTGAGTTTGTTGGTTTGGACATTGAA ATGGCTTTTAATTACCATTACCACGAAGTTATGGAAGA AATTGCTGACACCATGGTACAAATATTCAAAGGACTTC AAGAAAGGTTTCAGACTGAAATTCAAACAGTGAATAAA CAGTTCCCATCTGAGCCATTCAAATTTTTGGAGCCAACT CTAAGACTAGAATATTCTGAAGCATTGGCTATGCTTAG GGAAGCTGGAGTCGAAATGGGAGATGAAGACGATCTG AGCACACCAAATGAAAAGCTGTTGGGTCATTTGGTAAA GGAAAAGTATGATACAATTTTTATATTCTTGATAAAT ATCCATTGGCTGTAAGACCTTTCTATACCATGCCTGACC CAAGAAATCCCAAACAGTCCAACTCTTACGATATGTTC ATGAGAGGAGAAGAAATATTGTCAGGAGCTCAAAGAA TACATGATCCTCAACTGCTAACAGAGAGAGCTTTACAT CATGGAATTGATTTGGAGAAAATTAAGGCTTACATTGA TTCCTTCCGCTTTGGAGCCCCTCCTCATGCTGGTGGAGG CATTGGATTGGAACGAGTTACTATGCTGTTTCTGGGATT GCATAATGTTCGTCAGACCTCCATGTTCCCTCGTGATCC CAAACGAc???????? DRS C203A 1 - 501 / ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 121 Reduced AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT cysteine content AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA TACAAGATTAGACAACAGAGTCATTGATCTTAGGACAT CAACTAGTCAGGCAGTCTTCCGTCTCCAGTCTGGCATCG CCCATCTCTTCCGAGAAACTTTAATTAACAAAGGTTTTG TGGAAATCCAAACTCCTAAAATTATTTCAGCTGCCAGT GAAGGAGGAGCCAATGTTTTTACTGTGTCATATTTTAA AAATAATGCATAccTGccTCAGTccccACAG??????? AGCAAATGTGCATTTGTGCTGATTTTGAGAAGGTTTTCT CTATTGGACCAGTATTCAGAGCGGAAGACTCTAATACC CATAGACATCTAACTGAGTTTGTTGGTTTGGACATTGAA ATGGCTTTTAATTACCATTACCACGAAGTTATGGAAGA AATTGCTGACACCATGGTACAAATATTCAAAGGACTTC AAGAAAGGTTTCAGACTGAAATTCAAACAGTGAATAAA CAGTTCCCATGTGAGCCATTCAAATTTTTGGAGCCAACT CTAAGACTAGAATATTGTGAAGCATTGGCTATGCTTAG GGAAGCTGGAGTCGAAATGGGAGATGAAGACGATCTG AGCACACCAAATGAAAAGCTGTTGGGTCATTTGGTAAA GGAAAAGTATGATACAGATTTTTATATTCTTGATAAAT ??ccATTGGCTGTAAGAccTTTC?A?AccATGccTGAcc CAAGAAATCCCAAACAGTCCAACTCTTACGATATGTTC ATGAGAGGAGAAGAAATATTGTCAGGAGCTCAAAGAA TACATGATCCTCAACTGCTAACAGAGAGAGCTTTACAT CATGGAATTGATTTGGAGAAAATTAAGGCTTACATTGA TTCCTTCCGCTTTGGAGCCCCTCCTCATGCTGGTGGAGG CATTGGATTGGAACGAGTTACTATGCTGTTTCTGGGATT GCATAATGTTcGTCAGAcc?ccATGTTccc?cGTGATcc CAAACGACTCACTcc? DRS C203V 1 - 1503 / ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 122 Reduced AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT cysteine content AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG US 9 ,822 ,353 B2 73 74 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Nucleic acid sequence NO : CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA TACAAGATTAGACAACAGAGTCATTGATCTTAGGACAT CAACTAGTCAGGCAGTCTTCCGTCTCCAGTCTGGCATCG TCCATCTCTTCCGAGAAACTTTAATTAACAAAGGTTTTG TGGAAATCCAAACTCCTAAAATTATTTCAGCTGCCAGT GAAGGAGGAGCCAATGTTTTTACTGTGTCATATTTTAA AAATAATGCATACCTGGCTCAGTCCCCACAGCTATATA AGCAAATGTGCATTTGTGCTGATTTTGAGAAGGTTTTCT ?????GGACCAGTATTCAGAGCGGAAGACTc??????? CATAGACATCTAACTGAGTTTGTTGGTTTGGACATTGAA ATGGCTTTTAATTACCATTACCACGAAGTTATGGAAGA AATTGCTGACACCATGGTACAAATATTCAAAGGACTTC AAGAAAGGTTTCAGACTGAAATTCAAACAGTGAATAAA CAGTTCCCATGTGAGCCATTCAAATTTTTGGAGCCAACT CTAAGACTAGAATATTGTGAAGCATTGGCTATGCTTAG GGAAGCTGGAGTCGAAATGGGAGATGAAGACGATCTG AGCACACCAAATGAAAAGCTGTTGGGTCATTTGGTAAA GGAAAAGTATGATACAGATTTTTATATTCTTGATAAAT ATCCATTGGCTGTAAGACCTTTCTATACCATGCCTGACC CAAGAAATCCCAAACAGTCCAACTCTTACGATATGTTC ATGAGAGGAGAAGAAATATTGTCAGGAGCTCAAAGAA TACATGATCCTCAACTGCTAACAGAGAGAGCTTTACAT CATGGAATTGATTTGGAGAAAATTAAGGCTTACATTGA TTCCTTCCGCTTTGGAGCCCCTCCTCATGCTGGTGGAGG CATTGGATTGGAACGAGTTACTATGCTGTTTCTGGGATT GCATAATGTTcGTCAGAccccATGT ?????cGTGATcc CAAAcGACTCACTcc? DRS 1 - 1503 / ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 123 C3343 / C3495 / Reduced AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT C203A cysteine content AAAGAGAGATATGGAATATCTTCAATGATACAATCACA en esos montes de content AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACTis TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA TACAAGATTAGACAACAGAGTCATTGATCTTAGGACAT CAACTAGTCAGGCAGTCTTCCGTCTCCAGTCTGGCATCG ???????c??ccGAGAAACTTTAATTAACAAAGGTTTTG TGGAAATCCAAACTCCTAAAATTATTTCAGCTGCCAGT GAAGGAGGAGCCAATGTTTTTACTGTGTCATATTTTAA AAATAATGCATACCTGGCTCAGTCCCCACAGCTATATA AGCAAATGTGCATTTGTGCTGATTTTGAGAAGGTTTTCT CTATTGGACCAGTATTCAGAGCGGAAGACTCTAATACC CATAGACATCTAACTGAGTTTGTTGGTTTGGACATTGAA ATGGCTTTTAATTACCATTACCACGAAGTTATGGAAGA AATTGCTGACACCATGGTACAAATATTCAAAGGACTTC AAGAAAGGTTTCAGACTGAAATTCAAACAGTGAATAAA CAGTTcccATCTGAGCCATICAAATTTTTGGAGCCAACT CTAAGACTAGAATATTCTGAAGCATTGGCTATGCTTAG GGAAGCTGGAGTCGAAATGGGAGATGAAGACGATCTG AGCACACCAAATGAAAAGCTGTTGGGTCATTTGGTAAA GGAAAAGTATGATACAGATTTTTATATTCTTGATAAAT ATCCATTGGCTGTAAGACCTTTCTATACCATGCCTGACC CAAGAAATCCCAAACAGTCCAACTCTTACGATATGTTC ATGAGAGGAGAAGAAATATTGTCAGGAGCTCAAAGAA TACATGATCCTCAACTGCTAACAGAGAGAGCTTTACAT CATGGAATTGATTTGGAGAAAATTAAGGCTTACATTGA TTCCTTCCGCTTTGGAGCCCCTCCTCATGCTGGTGGAGG CATTGGATTGGAACGAGTTACTATGCTGTTTCTGGGATT US 9 ,822 ,353 B2 75 76 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Nucleic acid sequence NO : GCATAATGTTcGTCAGAcc?cCATEGTTccc?cGTGATcc CAAACGACTCACTcc? DRS 1 - 501 / ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 124 C334S / C3495 / Reduced AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT C203V cysteine content AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA TACAAGATTAGACAACAGAGTCATTGATCTTAGGACAT CAACTAGTCAGGCAGTCTTCCGTCTCCAGTCTGGCATCG ??????????ccGAGAAACTTTAATTAACAAAGGTTTTG TGGAAATccAAACTccTAAAATTATTICAGCTGccAGT GAAGGAGGAGCCAATGTTTTTACTGTGTCATATTTTAA AAATAATGCATACCTGGCTCAGTCCCCACAGCTATATA AGCAAATGTGCATTTGTGCTGATTTTGAGAAGGTTTTCT CTATTGGACCAGTATTCAGAGCGGAAGACTCTAATACC CATAGACATCTAACTGAGTTTGTTGGTTTGGACATTGAA ATGGCTTTTAATTACCATTACCACGAAGTTATGGAAGA AATTGCTGACACCATGGTACAAATATTCAAAGGACTTC AAGAAAGGTTTCAGACTGAAATTCAAACAGTGAATAAA CAGTTCCCATCTGAGCCATTCAAATTTTTGGAGCCAACT CTAAGACTAGAATATTCTGAAGCATTGGCTATGCTTAG GGAAGCTGGAGTCGAAATGGGAGATGAAGACGATCTG AGCACACCAAATGAAAAGCTGTTGGGTCATTTGGTAAA GGAAAAGTATGATACAGATTTTTATATTCTTGATAAAT ATCCATTGGCTGTAAGACCTTTCTATACCATGCCTGACC CAAGAAATCCCAAACAGTCCAACTCTTACGATATGTTC ATGAGAGGAGAAGAAATATTGTCAGGAGCTCAAAGAA TACATGATccTCAACTGCTAACAGAGAGAGCTTTACAT CATGGAATTGATTTGGAGAAAATTAAGGCTTACATTGA TTCCTTCCGCTTTGGAGCCCCTCCTCATGCTGGTGGAGG CATTGGATTGGAACGAGTTACTATGCTGTTTCTGGGATT GCATAATGTTCGTCAGACCTCCATGTTCCCTCGTGATCC CAAACGACTCACTcCT DRS 1 - 1503 / ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 125 C334S / C349S / Reduced AGCCGCGGGAGAT CATGGACGCGGCGGAAGATTATGCT C259A / C203A cysteine content AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT G???cTTAGTccTAcGTCAGCAGCAGTTTAATGTccAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGccAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA TACAAGATTAGACAACAGAGTCATTGATCTTAGGACAT CAACTAGTCAGGCAGTCTTCCGTCTCCAGTCTGGCATCG CCCATCTCTTCCGAGAAACTTTAATTAACAAAGGTTTTG TGGAAATCCAAACTCCTAAAATTATTTCAGCTGCCAGT GAAGGAGGAGCCAATGTTTTTACTGTGTCATATTTTAA AAATAATGCATAccTGGCTCAGTccccACAG??????? AGCAAATGTGCATTGCGGCTGATTTTGAGAAGGTTTTCT CTATTGGACCAGTATTCAGAGCGGAAGACTCTAATACC CATAGACATCTAACTGAGTTTGTTGGTTTGGACATTGAA ATGGCTTTTAATTACCATTACCACGAAGTTATGGAAGA AATTGCTGACACCATGGTACAAATATTCAAAGGACTTC AAGAAAGGTTTCAGACTGAAATTCAAACAGTGAATAAA CAGTTcccATCTGAGCCATICAAATTTTTGGAGCCAACT CTAAGACTAGAATATTCTGAAGCATTGGCTATGCTTAG GGAAGCTGGAGTCGAAATGGGAGATGAAGACGATCTG US 9 ,822 ,353 B2 77 78 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Nucleic acid sequence NO : AGCACACCAAATGAAAAGCTGTTGGGTCATTTGGTAAA GGAAAAGTATGATACAGATTTTTATATTCTTGATAAAT ATCCATTGGCTGTAAGACCTTTCTATACCATGCCTGACC CAAGAAATCCCAAACAGTCCAACTCTTACGATATGTTC ATGAGAGGAGAAGAAATATTGTCAGGAGCTCAAAGAA TACATGATCCTCAACTGCTAACAGAGAGAGCTTTACAT CATGGAATTGATTTGGAGAAAATTAAGGCTTACATTGA ??cc??ccGcTTTGGAGcccc?ccTCATGCTGGTGGAGG CATTGGATTGGAACGAGTTACTATGCTGTTTCTGGGATT GCATAATGTTcGTCAGAcc?ccATGTTccc?cGTGATcc CAAACGAc???????? DRS 1 - 1503 / ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 126 C334S / C3495 / Reduced AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT C259A / C203V cysteine content AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGccAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA TACAAGATTAGACAACAGAGTCATTGATCTTAGGACAT CAACTAGTCAGGCAGTc??ccGTc?ccAGTCTGGCATCG TCCATCTCTTCCGAGAAACTTTAATTAACAAAGGTTTTG TGGAAATCCAAACTCCTAAAATTATTTCAGCTGCCAGT GAAGGAGGAGCCAATGTTTTTACTGTGTCATATTTTAA AAATAATGCATAccTGGCTCAGTccccACAG??????? AGCAAATGTGCATTGCGGCTGATTTTGAGAAGGTTTTCT CTATTGGACCAGTATTCAGAGCGGAAGACTCTAATACC CATAGACATCTAACTGAGTTTGTTGGTTTGGACATTGAA ATGGCTTTAATTACCATTACCACGAAGTTATGGAAGA AATTGCTGACACCATGGTACAAATATTCAAAGGACTTC AAGAAAGGTTTCAGACTGAAATTCAAACAGTGAATAAA CAGTTcccATCTGAGCCATICAAATTTTTGGAGCCAACT CTAAGACTAGAATATTCTGAAGCATTGGCTATGCTTAG GGAAGCTGGAGTCGAAATGGGAGATGAAGACGATCTG AGCACACCAAATGAAAAGCTGTTGGGTCATTTGGTAAA GGAAAAGTATGATACAGATTTTTATATTCTTGATAAAT ATCCATTGGCTGTAAGACCTTTCTATACCATGCCTGACC CAAGAAATCCCAAACAGTCCAACTCTTACGATATGTTC ATGAGAGGAGAAGAAATATTGTCAGGAGCTCAAAGAA TACATGATCCTCAACTGCTAACAGAGAGAGCTTTACAT CATGGAATTGATTTGGAGAAAATTAAGGCTTACATTGA ??cc??ccccTTTGGAGcccc?ccTCATGCTGGTGGAGG CATTGGATTGGAACGAGTTACTATGCTGTTTCTGGGATT GCATAATGTTcGTCAGAcc?ccATGTTccc?cGTGATcc CAAACGACTCACTCCT DRS 1 - 182 ATGCCCAGCGCCAGCGCCAGCCGCAAAGTCAGGAGA 127 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA TACAAGATTAGACAAC DRS 1 - 180 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 128 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT US 9 ,822 ,353 B2 79 80 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Nucleic acid sequence NO : TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA TACAAGATTA DRS 1 - 178 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 129 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTICAATGAT????????? AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGccAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAGGA ???? DRS 1 - 176 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 130 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTTAACCAG DRS 1 - 174 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 131 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCTACTGTT DRS 1 - 172 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 132 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT G???cTTAGTccTAcGTCAGCAGCAGTTTAATGTccAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGAAGAGCT US 9 ,822 ,353 B2 81 82 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Nucleic acid sequence NO : DRS 1 - 170 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 133 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAGGAAGGA DRS 1 - 168 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 134 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGAGAAGAG DRS 1 - 166 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 135 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGccAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCAG AAGGA DRS 1 - 164 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 136 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAT G???cTTAGTccTAcGTCAGCAGCAGTTTAATGTccAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGATGCTGTTCGGCCTGAGGCA DRS 1 - 162 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 137 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GccccTGCAGCTGGATGATGCTGTTcGGcc? US 9 ,822 ,353 B2 83 84 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEO ID Name NO : 1 Nucleic acid sequence NO : DRS 1 - 160 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 138 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GccccTGCAGCTGGATGATGCTGTT DRS 1 - 158 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 139 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTGGATGAT DRS 1 - 156 ATGCCCAGCGCCAGCGCCAGCCGCAAAGTCAGGAGA 140 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GCCCCTGCAGCTG DRS 1 - 154 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 141 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT GccccTG DRS 1 - 152 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 142 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCT
DRS 1 - 150 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 143 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA US 9 ,822 ,353 B2 85 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEO ID Name NO : 1 Nucleic acid sequence NO : AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCTGAACCC DRS 1 - 148 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 144 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGTTTGGCT DRS 1 - 146 ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGA 145 AGCCGCGGGAGATCATGGACGCGGCGGAAGATTATGCT AAAGAGAGATATGGAATATCTTCAATGATACAATCACA AGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACT TGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGT GCAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGT GCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGG CTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATG GTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGT GGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAA ATTGGAAGCTGTACACAGCAAGACGTTGAGTTACATGT TCAGAAGATTTATGTGATCAGT
DRS 3 - 154 GCCAGCGCCAGCCGCAAGAGTCAGGAGAAGCCGCGGG 146 AGATCATGGACGCGGCGGAAGATTATGCTAAAGAGAG ATATGGAATATCTTCAATGATACAATCACAAGAAAAAC CAGATCGAGTTTTGGTTCGGGTTAGAGACTTGACAATA CAAAAAGCTGATGAAGTTGTTTGGGTACGTGCAAGAGT TCATACAAGCAGAGCTAAAGGGAAACAGTGCTTCTTAG TCCTACGTCAGCAGCAGTTTAATGTCCAGGCTCTTGTGG CGGTGGGAGACCATGCAAGCAAGCAGATGGTTAAATTT GCTGCCAACATCAACAAAGAGAGCATTGTGGATGTAGA AGGTGTTGTGAGAAAAGTGAATCAGAAAATTGGAAGCT GTACACAGCAAGACGTTGAGTTACATGTTCAGAAGATT TATGTGATCAGTTTGGCTGAAccccGT????????? DRS 5 - 154 GCCAGCCGCAAGAGTCAGGAGAAGCCGCGGGAGATCA 147 TGGACGCGGCGGAAGATTATGCTAAAGAGAGATATGG AATATCTTCAATGATACAATCACAAGAAAAACCAGATC GAGTTTTGGTTCGGGTTAGAGACTTGACAATACAAAAA GCTGATGAAGTTGTTTGGGTACGTGCAAGAGTTCATAC AAGCAGAGCTAAAGGGAAACAGTGCTTCTTAGTCCTAC GTCAGCAGCAGTTTAATGTCCAGGCTCTTGTGGCGGTG GGAGACCATGCAAGCAAGCAGATGGTTAAATTTGCTGC CAACATCAACAAAGAGAGCATTGTGGATGTAGAAGGT GTTGTGAGAAAAGTGAATCAGAAAATTGGAAGCTGTAC ACAGCAAGACGTTGAGTTACATGTTCAGAAGATTTATG TGATCAGTTTGGCTGAACCCCGTCTGCCCCTG DRS 7 - 154 CGCAAGAGTCAGGAGAAGCCGCGGGAGATCATGGACG 148 CGGCGGAAGATTATGCTAAAGAGAGATATGGAATATCT TCAATGATACAATCACAAGAAAAACCAGATCGAGTTTT GGTTCGGGTTAGAGACTTGACAATACAAAAAGCTGATG AAGTTGTTTGGGTACGTGCAAGAGTTCATACAAGCAGA GCTAAAGGGAAACAGTGCTICTTAGTccTACGTCAGCA GCAGTTTAATGTCCAGGCTCTTGTGGCGGTGGGAGACC ATGCAAGCAAGCAGATGGTTAAATTTGCTGCCAACATC AACAAAGAGAGCATTGTGGATGTAGAAGGTGTTGTGAG AAAAGTGAATCAGAAAATTGGAAGCTGTACACAGCAA US 9 ,822 ,353 B2 87 88 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Nucleic acid sequence NO : GACGTTGAGTTACATGTTCAGAAGATTTATGTGATCAG TTTGGCTGAACCCCGTCTGCCCCTG DRS 9 - 154 AGTCAGGAGAAGCCGCGGGAGATCATGGACGCGGCGG 149 AAGATTATGCTAAAGAGAGATATGGAATATCTTCAATG ATACAATCACAAGAAAAACCAGATCGAGTTTTGGTTCG GGTTAGAGACTTGACAATACAAAAAGCTGATGAAGTTG TTTGGGTACGTGCAAGAGTTCATACAAGCAGAGCTAAA GGGAAACAGTGCTTCTTAGTCCTACGTCAGCAGCAGTT TAATGTCCAGGCTCTTGTGGCGGTGGGAGACCATGCAA GCAAGCAGATGGTTAAATTTGCTGCCAACATCAACAAA GAGAGCATTGTGGATGTAGAAGGTGTTGTGAGAAAAGT GAATCAGAAAATTGGAAGCTGTACACAGCAAGACGTTG AGTTACATGTTCAGAAGATTTATGTGATCAGTTTGGCTG AAccccGTCTGccccTG DRS 11 - 154 GAGAAGCCGCGGGAGATCATGGACGCGGCGGAAGATT 150 ATGCTAAAGAGAGATATGGAATATCTTCAATGATACAA TCACAAGAAAAACCAGATCGAGTTTTGGTTCGGGTTAG AGACTTGACAATACAAAAAGCTGATGAAGTTGTTTGGG TACGTGCAAGAGTTCATACAAGCAGAGCTAAAGGGAA ACAGTGCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGT CCAGGCTCTTGTGGCGGTGGGAGACCATGCAAGCAAGC AGATGGTTAAATTTGCTGCCAACATCAACAAAGAGAGC ATTGTGGATGTAGAAGGTGTTGTGAGAAAAGTGAATCA GAAAATTGGAAGCTGTACACAGCAAGACGTTGAGTTAC ATGTTCAGAAGATTTATGTGATCAGTTTGGCTGAACCCC GTCTGCCCCTG DRS 13 - 154 CCGCGGGAGATCATGGACGCGGCGGAAGATTATGCTAA 151 AGAGAGATATGGAATATCTTCAATGATACAATCACAAG AAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGACTTG ACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGTGC AAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGTGC TTCTTAGTCCTACGTCAGCAGCAGTTTAATGTCCAGGCT CTTGTGGCGGTGGGAGACCATGCAAGCAAGCAGATGGT TAAATTTGCTGCCAACATCAACAAAGAGAGCATTGTGG ATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAAATT GGAAGCTGTACACAGCAAGACGTTGAGTTACATGTTCA GAAGATTTATGTGATCAGTTTGGCTGAACCCCGTCTGCC ???? DRS15 - 154 GAGATCATGGACGCGGCGGAAGATTATGCTAAAGAGA 152 GATATGGAATATCTTCAATGATACAATCACAAGAAAAA CCAGATCGAGTTTTGGTTCGGGTTAGAGACTTGACAAT ACAAAAAGCTGATGAAGTTGTTTGGGTACGTGCAAGAG TTCATACAAGCAGAGCTAAAGGGAAACAGTGCTTCTTA GTCCTACGTCAGCAGCAGTTTAATGTCCAGGCTCTTGTG GCGGTGGGAGACCATGCAAGCAAGCAGATGGTTAAATT TGCTGCCAACATCAACAAAGAGAGCATTGTGGATGTAG AAGGTGTTGTGAGAAAAGTGAATCAGAAAATTGGAAG CTGTACACAGCAAGACGTTGAGTTACATGTTCAGAAGA TTTATGTGATCAGTTTGGCTGAACCCCGTCTGCCCCTG DRS 17 - 154 ATGGACGCGGCGGAAGATTATGCTAAAGAGAGATATG 153 GAATATCTTCAATGATACAATCACAAGAAAAACCAGAT CGAGTTTTGGTTCGGGTTAGAGACTTGACAATACAAAA AGCTGATGAAGTTGTTTGGGTACGTGCAAGAGTTCATA CAAGCAGAGCTAAAGGGAAACAGTGCTTCTTAGTCCTA CGTCAGCAGCAGTTTAATGTCCAGGCTCTTGTGGCGGT GGGAGACCATGCAAGCAAGCAGATGGTTAAATTTGCTG CCAACATCAACAAAGAGAGCATTGTGGATGTAGAAGGT GTTGTGAGAAAAGTGAATCAGAAAATTGGAAGCTGTAC ACAGCAAGACGTTGAGTTACATGTTCAGAAGATTTATG TGATCAGTTTGGCTGAACCCCGTCTGCCCCTG DRS 19 - 154 GCGGCGGAAGATTATGCTAAAGAGAGATATGGAATATC 154 TTCAATGATACAATCACAAGAAAAACCAGATCGAGTTT TGGTTCGGGTTAGAGACTTGACAATACAAAAAGCTGAT GAAGTTGTTTGGGTACGTGCAAGAGTTCATACAAGCAG AGCTAAAGGGAAACAGTGCTICTTAGTccTACGTCAGC AGCAGTTTAATGTCCAGGCTCTTGTGGCGGTGGGAGAC CATGCAAGCAAGCAGATGGTTAAATTTGCTGCCAACAT US 9 ,822 ,353 B2 89 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEO ID Name NO : 1 Nucleic acid sequence NO : CAACAAAGAGAGCATTGTGGATGTAGAAGGTGTTGTGA GAAAAGTGAATCAGAAAATTGGAAGCTGTACACAGCA AGACGTTGAGTTACATGTTCAGAAGATTTATGTGATCA GTTTGGCTGAAccccGTCTGccccTG DRS 21 - 154 GCGGCGGAAGATTATGCTAAAGAGAGATATGGAATATC 155 TTCAATGATACAATCACAAGAAAAACCAGATCGAGTTT TGGTTCGGGTTAGAGACTTGACAATACAAAAAGCTGAT GAAGTTGTTTGGGTACGTGCAAGAGTTCATACAAGCAG AGCTAAAGGGAAACAGTGCTTCTTAGTCCTACGTCAGC AGCAGTTTAATGTCCAGGCTCTTGTGGCGGTGGGAGAC CATGCAAGCAAGCAGATGGTTAAATTTGCTGCCAACAT CAACAAAGAGAGCATTGTGGATGTAGAAGGTGTTGTGA GAAAAGTGAATCAGAAAATTGGAAGCTGTACACAGCA AGACGTTGAGTTACATGTTCAGAAGATTTATGTGATCA GTTTGGCTGAACCCCGTCTG DRS 23 - 154 GCGGCGGAAGATTATGCTAAAGAGAGATATGGAATATC 156 TTCAATGATACAATCACAAGAAAAACCAGATCGAGTTT TGGTTCGGGTTAGAGACTTGACAATACAAAAAGCTGAT GAAGTTGTTTGGGTACGTGCAAGAGTTCATACAAGCAG AGCTAAAGGGAAACAGTGCTTCTTAGTCCTACGTCAGC AGCAGTTTAATGTCCAGGCTCTTGTGGCGGTGGGAGAC CATGCAAGCAAGCAGATGGTTAAATTTGCTGCCAACAT CAACAAAGAGAGCATTGTGGATGTAGAAGGTGTTGTGA GAAAAGTGAATCAGAAAATTGGAAGCTGTACACAGCA AGACGTTGAGTTACATGTTCAGAAGATTTATGTGATCA GTTTGGCTGAACCC DRS 11 - 146 ATGCAGGAGAAGCCGCGGGAGATCATGGACGCGGCGG 157 AAGATTATGCTAAAGAGAGATATGGAATATCTTCAATG ATACAATCACAAGAAAAACCAGATCGAGTTTTGGTTCG GGTTAGAGACTTGACAATACAAAAAGCTGATGAAGTTG TTTGGGTACGTGCAAGAGTTCATACAAGCAGAGCTAAA GGGAAACAGTGCTTCTTAGTCCTACGTCAGCAGCAGTT TAATGTCCAGGCTCTTGTGGCGGTGGGAGACCATGCAA GCAAGCAGATGGTTAAATTTGCTTGCAACATCAACAAA GAGAGCATTGTGGATGTAGAAGGTGTTGTGAGAAAAGT GAATCAGAAAATTGGAAGCTGTACACAGCAAGACGTTG AGTTACATGTTCAGAAGATTTATGTGATCAGT DRS 13 - 146 ATGAAGCCGCGGGAGATCATGGACGCGGCGGAAGATT 158 ATGCTAAAGAGAGATATGGAATATCTTCAATGATACAA TCACAAGAAAAACCAGATCGAGTTTTGGTTCGGGTTAG AGACTTGACAATACAAAAAGCTGATGAAGTTGTTTGGG TACGTGCAAGAGTTCATACAAGCAGAGCTAAAGGGAA ACAGTGCTTCTTAGTCCTACGTCAGCAGCAGTTTAATGT CCAGGCTCTTGTGGCGGTGGGAGACCATGCAAGCAAGC AGATGGTTAAATTTGCTTGCAACATCAACAAAGAGAGC ATTGTGGATGTAGAAGGTGTTGTGAGAAAAGTGAATCA GAAAATTGGAAGCTGTACACAGCAAGACGTTGAGTTAC ATGTTCAGAAGATTTATGTGATCAGT DRS 13 ATGAAGCCGCGGGAGATCATGGACGCGGCGGAAGATT 159 146 / A1060 ATGCTAAAGAGAGATATGGAATATCTTCAATGATACAA TCACAAGAAAAACCAGATCGAGTTTTGGTTCGGGTTAG AGACTTGACAATACAAAAAGCTGATGAAGTTGTTTGGG TACGTGCAAGAGTTCATACAAGCAGAGCTAAAGGGAA ACAGTGC??????GTccTACGTCAGCAGCAGTTTAATGT CCAGGCTCTTGTGGCGGTGGGAGACCATGCAAGCAAGC AGATGGTTAAATTTGCTTGCAACATCAACAAAGAGAGC ATTGTGGATGTAGAAGGTGTTGTGAGAAAAGTGAATCA GAAAATTGGAAGCTGTACACAGCAAGACGTTGAGTTAC ATGTTCAGAAGATTTATGTGATCAGT DRS 17 - 146 ATGATCATGGACGCGGCGGAAGATTATGCTAAAGAGA 160 GATATGGAATATCTTCAATGATACAATCACAAGAAAAA CCAGATCGAGTTTTGGTTCGGGTTAGAGACTTGACAAT ACAAAAAGCTGATGAAGTTGTTTGGGTACGTGCAAGAG TTCATACAAGCAGAGCTAAAGGGAAACAGTGCTTCTTA GTccTACGTCAGCAGCAGTTTAATGTccAGGC????GTG GCGGTGGGAGACCATGCAAGCAAGCAGATGGTTAAATT TGCTTGCAACATCAACAAAGAGAGCATTGTGGATGTAG US 9 ,822 ,353 B2 91 TABLE D8 - continued DRS DNA Sequences
Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Nucleic acid sequence NO : AAGGTGTTGTGAGAAAAGTGAATCAGAAAATTGGAAG CTGTACACAGCAAGACGTTGAGTTACATGTTCAGAAGA TTTATGTGATCAGT
DRS 21 - 146 ATGGCGGAAGATTATGCTAAAGAGAGATATGGAATATC 161 TTCAATGATACAATCACAAGAAAAACCAGATCGAGTTT TGGTTCGGGTTAGAGACTTGACAATACAAAAAGCTGAT GAAGTTGTTTGGGTACGTGCAAGAGTTCATACAAGCAG AGCTAAAGGGAAACAGTGCTTCTTAGTCCTACGTCAGC AGCAGTTTAATGTCCAGGCTCTTGTGGCGGTGGGAGAC CATGCAAGCAAGCAGATGGTTAAATTTGCTTGCAACAT CAACAAAGAGAGCATTGTGGATGTAGAAGGTGTTGTGA GAAAAGTGAATCAGAAAATTGGAAGCTGTACACAGCA AGACGTTGAGTTACATGTTCAGAAGATTTATGTGATCA GT
Additional coding or non -coding sequencesmay , but need 100 (including all integers in between ) or more bases. In not, be present within a polynucleotide of the present certain embodiments , such as when the polynucleotide vari invention , and a polynucleotide may , but need not, be linked 25 ant encodes a DRS polypeptide having a non -canonical to other molecules and /or support materials . Hence , the activity , the desired activity of the encoded DRS polypeptide polynucleotides of the present invention , regardless of the is not substantially diminished relative to the unmodified length of the coding sequence itself , may be combined with polypeptide . The effect on the activity of the encoded other DNA sequences, such as promoters , polyadenylation polypeptide may generally be assessed as described herein . signals , additional restriction enzyme sites, multiple cloning 30 Certain embodiments include polynucleotides that sites , other coding segments, and the like , such that their hybridize to a reference DRS polynucleotide sequence , overall length may vary considerably . ( such as for example , SEQ ID NO : 2 , 25 - 28 , 30 , 36 - 37 , or It is therefore contemplated that a polynucleotide frag - 118 - 161 ) or to their complements, under stringency condi ment of almost any length may be employed ; with the total tions described below . As used herein , the term “ hybridizes length preferably being limited by the ease of preparation 35 under low stringency, medium stringency, high stringency , and use in the intended recombinant DNA protocol. or very high stringency conditions” describes conditions for Included are polynucleotides of about 10 , 11, 12 , 13 , 14 , 15 , hybridization and washing .Guidance for performing hybrid 16 , 17 , 18 , 19 , 20 , 21, 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , ization reactions can be found in Ausubel et al. , (1998 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41, 41 , 43 , 44 , 45 , 46 , 47 , supra ) , Sections 6 . 3 . 1 - 6 . 3 . 6 . Aqueous and non - aqueous 48 , 49, 50 , 60 , 70 , 80 , 90 , 100 , 110 , 120 , 130 , 140 , 150 , 160 , 40 methods are described in that reference and either can be 170 , 180 , 190 , 200 , 220 , 240 , 260 , 270 , 280 , 300 , 350 , 400 , used . 450 , 500 , 550 , 600 , 650 , 700 , 750 , 800 , 850 , 900 , 950 , 1000 , Reference herein to low stringency conditions include and 1100 , 1200 , 1300 , 1400 , 1500 , 1600 , 1700 , 1800 , 1900 , encompass from at least about 1 % v /v to at least about 15 % 2000 , 2100 , 2200 , 2300 , 2400 , 2500 , 2600 , 2700 , 2800 , v / v formamide and from at least about 1 M to at least about 2900 , 3000 or more ( including all integers in between ) bases 45 2 M salt for hybridization at 42° C ., and at least about 1 M in length , including any portion or fragment ( e . g ., greater to at least about 2 M salt for washing at 42° C . Low than about 6 , 7 , 8 , 9 , or 10 nucleotides in length ) of an AARS stringency conditions also may include 1 % Bovine Serum reference polynucleotide ( e . g . , base number X - Y , in which X Albumin (BSA ) , 1 mM EDTA , 0 . 5 M NaHPO , ( pH 7 . 2 ) , 7 % is about 1 - 3000 or more and Y is about 10 - 3000 or more ), SDS for hybridization at 65° C ., and ( i ) 2xSSC , 0 . 1 % SDS ; or its complement. 50 or ( ii ) 0 . 5 % BSA , 1 mM EDTA , 40 mM NaHPO (pH 7 . 2 ) , Embodiments of the present invention also include " vari - 5 % SDS for washing at room temperature . One embodiment ants ” of the AARS reference polynucleotide sequences . of low stringency conditions includes hybridization in 6x Polynucleotide " variants " may contain one or more substi - sodium chloride / sodium citrate (SSC ) at about 45° C . , tutions , additions , deletions and / or insertions in relation to a followed by two washes in 0 . 2xSSC , 0 . 1 % SDS at least at reference polynucleotide . Generally , variants of an AARS 55 50° C . ( the temperature of the washes can be increased to reference polynucleotide sequence may have at least about 55° C . for low stringency conditions) . 30 % , 40 % 50 % , 55 % , 60 % , 65 % , 70 % , generally at least Medium stringency conditions include and encompass about 75 % , 80 % , 85 % , desirably about 90 % to 95 % or more , from at least about 16 % v / v to at least about 30 % v / v and more suitably about 98 % or more sequence identity to formamide and from at least about 0 . 5 M to at least about 0 . 9 that particular nucleotide sequence (Such as for example , 60 M salt for hybridization at 42° C ., and at least about 0 . 1 M SEQ ID NO : 2 , 25 - 28 , 30 , 36 - 37 , or 118 - 161 ) as determined to at least about 0 . 2 M salt for washing at 55° C . Medium by sequence alignment programs described elsewhere herein stringency conditions also may include 1 % Bovine Serum using default parameters . In certain embodiments , variants Albumin (BSA ) , 1 mM EDTA , 0 . 5 M NaHPO4 (pH 7 . 2 ) , 7 % may differ from a reference sequence by about 1 , 2 , 3 , 4 , 5 , SDS for hybridization at 65° C ., and ( i) 2xSSC , 0 . 1 % SDS ; 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 65 or (ii ) 0 . 5 % BSA , 1 mM EDTA , 40 mM NaHPO4 (pH 7 . 2 ) , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 5 % SDS for washing at 60 -65° C . One embodiment of 39 , 40 , 41, 41, 43 , 44 , 45 , 46 , 47 , 48 , 49, 50 , 60 , 70 , 80 , 90 , medium stringency conditions includes hybridizing in US 9 ,822 ,353 B2 93 94 6xSSC at about 45° C ., followed by one or more washes in Am . Chem . Soc. 85 :2149 -2154 (1963 ) ) , or by recombinant 0 .2xSSC , 0 . 1 % SDS at 60° C . High stringency conditions technology using a genetically modified host. Protein syn include and encompass from at least about 31 % v / v to at thesis may be performed using manual techniques or by least about 50 % v / v formamide and from about 0 .01 M to automation . Automated synthesis may be achieved , for about 0 . 15 M salt for hybridization at 42° C ., and about 0 .01 5 example , using Applied Biosystems 431A Peptide Synthe M to about 0 .02 M salt for washing at 55° C . sizer (Perkin Elmer) . Alternatively, various fragments may High stringency conditions also may include 1 % BSA , 1 be chemically synthesized separately and combined using mM EDTA , 0 . 5 M NaHPO4 (pH 7 . 2 ) , 7 % SDS for hybrid chemical methods to produce the desired molecule . ization at 65° C . , and (i ) 0 .2xSSC , 0 . 1 % SDS; or ( ii ) 0 . 5 % DRS polypeptides can also be produced by expressing a BSA , 1 mM EDTA , 40 mM NaHPO4 (pH 7 . 2 ), 1 % SDS for 10 DNA sequence encoding the DRS polypeptide in question ) washing at a temperature in excess of 65° C . One embodi- in a suitable host cell by well -known techniques . The ment of high stringency conditions includes hybridizing in polynucleotide sequence coding for the DRS polypeptide 6xSSC at about 45° C ., followed by one or more washes in may be prepared synthetically by established standard meth 0 .2xSSC , 0 . 1 % SDS at 65° C . One embodiment of very high ods, e .g ., the phosphoamidite method described by stringency conditions includes hybridizing in 0 . 5 M sodium 15 Beaucage et al. ( 1981 ) Tetrahedron Letters 22 : 1859 - 1869 , or phosphate , 7 % SDS at 65° C . , followed by one or more the method described by Matthes et al. ( 1984 ) EMBO washes in 0 .2xSSC , 1 % SDS at 65° C . Journal 3 : 801 -805 . According to the phosphoramidite Other stringency conditions are well known in the art and method , oligonucleotides are synthesized , e . g . , in an auto a skilled artisan will recognize that various factors can be matic DNA synthesizer , purified , duplexed and ligated to manipulated to optimize the specificity of the hybridization . 20 form the synthetic DNA construct. Alternatively the DNA Optimization of the stringency of the final washes can serve construct can be constructed using standard recombinant to ensure a high degree of hybridization . For detailed molecular biological techniques including restriction examples, see Ausubel et al . , supra at pages 2 . 10 . 1 to 2 . 10 . 16 enzyme mediated cloning and PCR based gene amplifica and Sambrook et al . ( 1989 , supra ) at sections 1 .101 to 1 . 104 . tion . While stringent washes are typically carried out at tempera - 25 The polynucleotide sequences may also be of mixed tures from about 42° C . to 68° C ., one skilled in the art will genomic , cDNA , and synthetic origin . For example , a appreciate that other temperatures may be suitable for strin - genomic or cDNA sequence encoding a leader peptide may gent conditions . Maximum hybridization rate typically be joined to a genomic or cDNA sequence encoding the DRS occurs at about 20° C . to 25° C . below the Tm for formation polypeptide, after which the DNA sequence may be modi of a DNA -DNA hybrid . It is well known in the art that the 30 fied at a site by inserting synthetic oligonucleotides encod Tis the melting temperature , or temperature at which two ing the desired amino acid sequence for homologous recom complementary polynucleotide sequences dissociate . Meth bination in accordance with well -known procedures or ods for estimating Tm are well known in the art ( see Ausubel preferably generating the desired sequence by PCR using et al. , supra at page 2 . 10 . 8 ) . suitable oligonucleotides . In some embodiments a signal In general, the T , of a perfectly matched duplex of DNA 35 sequence can be included before the coding sequence . This may be predicted as an approximation by the formula : sequence encodes a signal peptide N - terminal to the coding Ty = 81. 5 + 16 .6 ( log10 M ) + 0 .41 % G + C ) - 0 .63 % forma- sequence which communicates to the host cell to direct the mide ) - (600 / length ) wherein : M is the concentration of Nat, polypeptide to the cell surface or secrete the polypeptide into preferably in the range of 0 .01 molar to 0 . 4 molar ; % G + C the media . Typically the signal peptide is clipped off by the is the sum of guanosine and cytosine bases as a percentage 40 host cell before the protein leaves the cell. Signal peptides of the total number of bases , within the range between 30 % can be found in variety of proteins in prokaryotes and and 75 % G + C ; % formamide is the percent formamide eukaryotes. concentration by volume; length is the number of base pairs A variety of expression vector/ host systems are known in the DNA duplex . The Tm of a duplex DNA decreases by and may be utilized to contain and express polynucleotide approximately 1° C . with every increase of 1 % in the 45 sequences . These include , but are not limited to , microor number of randomly mismatched base pairs . Washing is ganisms such as bacteria transformed with recombinant generally carried out at T - 15° C . for high stringency , or bacteriophage , plasmid , or cosmid DNA expression vectors ; Tm - 30° C . for moderate stringency. yeast transformed with yeast expression vectors ; insect cell In one example of a hybridization procedure , a membrane systems infected with virus expression vectors ( e . g . , bacu ( e . g ., a nitrocellulose membrane or a nylon membrane ) 50 lovirus ) ; plant cell systems transformed with virus expres containing immobilized DNA is hybridized overnight at 42° sion vectors ( e . g ., cauliflower mosaic virus , CaMV ; tobacco C . in a hybridization buffer (50 % deionized formamide , mosaic virus, TMV) or with bacterial expression vectors 5xSSC , 5xDenhardt' s solution ( 0 .1 % ficoll, 0 . 1 % polyvi (e . g ., Ti or PBR322 plasmids) ; or animal cell systems, nylpyrollidone and 0 . 1 % bovine serum albumin ) , 0 . 1 % SDS including mammalian cell and more specifically human cell and 200 mg /mL denatured salmon sperm DNA ) containing 55 systems transformed with viral , plasmid , episomal or inte a labeled probe. The membrane is then subjected to two grating expression vectors . sequential medium stringency washes (i .e . , 2xSSC , 0 . 1 % The " control elements ” or “ regulatory sequences” present SDS for 15 min at 45° C ., followed by 2xSSC , 0 . 1 % SDS in an expression vector are non - translated regions of the for 15 min at 50° C . ), followed by two sequential higher vector enhancers , promoters , 5 ' and 3 ' untranslated stringency washes ( i .e . , 0 .2xSSC , 0 . 1 % SDS for 12 min at 60 regions — which interact with host cellular proteins to carry 55° C . followed by 0 .2xSSC and 0 . 1 % SDS solution for 12 out transcription and translation . Such elements may vary in min at 65 -68° C . their strength and specificity . Depending on the vector Production of DRS Polypeptides and PEGylated DRS Poly system and host utilized , any number of suitable transcrip peptides tion and translation elements, including constitutive and DRS polypeptide may be prepared by any suitable pro - 65 inducible promoters, may be used . For example , when cedure known to those of skill in the art for example , by cloning in bacterial systems, inducible promoters such as the using standard solid - phase peptide synthesis (Merrifield , J. hybrid lacZ promoter of the PBLUESCRIPT phagemid US 9 ,822 , 353 B2 95 96 (Stratagene , La Jolla , Calif . ) or PSPORT1 plasmid (Gibco In cases where plant expression vectors are used , the BRL , Gaithersburg , Md .) and the like may be used . In expression of sequences encoding polypeptides may be mammalian cell systems, promoters from mammalian genes driven by any of a number of promoters . For example , viral or from mammalian viruses are generally preferred . If it is promoters such as the 35S and 19S promoters of CaMV may necessary to generate a cell line that contains multiple copies 5 be used alone or in combination with the omega leader of the sequence encoding a polypeptide , vectors based on sequence from TMV ( Takamatsu , EMBO J. 6 : 307 -311 SV40 or EBV may be advantageously used with an appro ( 1987 ) ) . Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used priate selectable marker. ( Coruzzi et al . , EMBO J . 3 : 1671 - 1680 ( 1984 ) ; Broglie et al. , Certain embodiments may employ E . coli -based expres 10 Science 224 :838 - 843 (1984 ) ; and Winter et al. , Results sion systems (see , e. g. , Structural Genomics Consortium et Probl. Cell Differ. 17 :85 - 105 ( 1991 ) ) . These constructs can al. , Nature Methods. 5 : 135 - 146 , 2008 ) . These and related be introduced into plant cells by direct DNA transformation embodiments may rely partially or totally on ligation -inde or pathogen -mediated transfection . Such techniques are pendent cloning (LIC ) to produce a suitable expression described in a number of generally available reviews (see , vector. In specific embodiments , protein expression may be 15 e . g .. Hobbs in McGraw Hill. Yearbook of Science and controlled by a 17 RNA polymerase (e . g. , PET vector Technology, pp . 191 - 196 ( 1992 ) ). series ) . These and related embodiments may utilize the An insect system may also be used to express a polypep expression host strain BL21 (DE3 ) , a ADE3 lysogen of BL21 tide of interest. For example , in one such system , that supports T7 -mediated expression and is deficient in lon Autographa californica nuclear polyhedrosis virus and ompT proteases for improved target protein stability . 20 ( AcNPV ) is used as a vector to express foreign genes in Also included are expression host strains carrying plasmids Spodoptera frugiperda cells or in Trichoplusia cells . The encoding tRNAs rarely used in E . coli , such as ROSETTATM sequences encoding the polypeptide may be cloned into a ( DE3 ) and Rosetta 2 (DE3 ) strains . Cell lysis and sample non -essential region of the virus, such as the polyhedrin handling may also be improved using reagents sold under gene , and placed under control of the polyhedrin promoter . the trademarks BENZONASE® nuclease and BUG - 25 Successful insertion of the polypeptide -encoding sequence BUSTER® Protein Extraction Reagent. For cell culture , will render the polyhedrin gene inactive and produce recom auto - inducing media can improve the efficiency of many binant virus lacking coat protein . The recombinant viruses expression systems, including high - throughput expression may then be used to infect, for example, S . frugiperda cells systems. Media of this type ( e . g ., OVERNIGHT or Trichoplusia cells in which the polypeptide of interest EXPRESSTM Autoinduction System ) gradually elicit protein 30 may be expressed ( Engelhard et al ., Proc . Natl. Acad . Sci. expression through metabolic shift without the addition of U . S. A . 91: 3224 - 3227 ( 1994 ) ). Also included are baculovirus artificial inducing agents such as IPTG . expression systems, including those that utilize SF9 , SF21 , Particular embodiments employ hexahistidine tags ( such and T . ni cells (see , e . g ., Murphy and Piwnica -Worms , Curr as those sold under the trademark HIS•TAG® fusions) , Protoc Protein Sci . Chapter 5 :Unit5 . 4 , 2001 ) . Insect systems followed by immobilized metal affinity chromatography 35 can provide post - translation modifications that are similar to ( IMAC ) purification , or related techniques . In certain mammalian systems. aspects , however , clinical grade proteins can be isolated In mammalian host cells , a number of expression systems from E . coli inclusion bodies, without or without the use of are well known in the art and commercially available . affinity tags ( see, e . g . , Shimp et al. , Protein Expr Purif . Exemplary mammalian vector systems include for example , 50 : 58 -67 , 2006 ) . As a further example , certain embodiments 40 pCEP4 , PREP4, and PREP7 from Invitrogen , the PerC6 may employ a cold - shock induced E . coli high -yield pro - system from Crucell, and Lentiviral based systems such as duction system , because over- expression of proteins in PLP1 from Invitrogen , and others . For example , in cases Escherichia coli at low temperature improves their solubility where an adenovirus is used as an expression vector, and stability ( see , e . g ., Qing et al. , Nature Biotechnology. sequences encoding a polypeptide of interest may be ligated 22 :877 - 882 , 2004 ) . 45 into an adenovirus transcription / translation complex con Also included are high - density bacterial fermentation sisting of the late promoter and tripartite leader sequence . systems. For example , high cell density cultivation of Ral- Insertion in a non -essential El or E3 region of the viral stonia eutropha allows protein production at cell densities of genome may be used to obtain a viable virus which is over 150 g / L , and the expression of recombinant proteins at capable of expressing the polypeptide in infected host cells titers exceeding 10 g /L . In the yeast Saccharomyces cerevi- 50 (Logan & Shenk , Proc. Natl . Acad. Sci. U .S . A . 81: 3655 siae , a number of vectors containing constitutive or induc - 3659 ( 1984 ) ) . In addition , transcription enhancers , such as ible promoters such as alpha factor, alcohol oxidase , and the Rous sarcoma virus (RSV ) enhancer , may be used to PGH may be used . For reviews, see Ausubel et al. ( supra ) increase expression in mammalian host cells . and Grant et al . , Methods Enzymol. 153 : 516 - 544 ( 1987 ) . Examples of useful mammalian host cell lines include Also included are Pichia pandoris expression systems (see , 55 monkey kidney CV1 line transformed by SV40 (COS - 7 , e . g ., Li et al. , Nature Biotechnology . 24 , 210 - 215 , 2006 ; and ATCC CRL 1651 ) ; human embryonic kidney line (293 or Hamilton et al ., Science , 301 : 1244 , 2003 ). Certain embodi 293 cells sub - cloned for growth in suspension culture , ments include yeast systems that are engineered to selec Graham et al. , J . Gen Vivol. 36 :59 (1977 ) ); baby hamster tively glycosylate proteins, including yeast that have human - kidney cells (BHK , ATCC CCL 10 ) ; mouse sertoli cells ized N - glycosylation pathways , among others ( see , e . g ., 60 ( TM4, Mather, Biol. Reprod . 23 :243 - 251 (1980 ) ) ; monkey Hamilton et al. , Science . 313 : 1441- 1443 , 2006 ; Wildt et al. , kidney cells (CV1 ATCC CCL 70 ); African green monkey Nature Reviews Microbiol. 3 : 119 - 28 , 2005 ; and Gerngross kidney cells (VERO - 76 , ATCC CRL - 1587 ) ; human cervical et al. , Nature - Biotechnology . 22 : 1409 - 1414 , 2004 ; U . S . Pat . carcinoma cells (HELA , ATCC CCL 2 ) ; canine kidney cells Nos. 7 ,629 , 163 ; 7 ,326 ,681 ; and 7 ,029 ,872 ) . Merely by way (MDCK , ATCC CCL 34 ) ; buffalo rat liver cells (BRL 3A , of example , recombinant yeast cultures can be grown in 65 ATCC CRL 1442 ) ; human lung cells (W138 , ATCC CCL Fernbach Flasks or 15 L , 50 L , 100 L , and 200 L fermentors , 75 ) ; human liver cells (Hep G2, HB 8065 ) ;mouse mammary among others . tumor (MMT 060562 , ATCC CCL51) ; TR1 cells (Mather et US 9 ,822 , 353 B2 97 98 al. , Annals N . Y . Acad . Sci. 383 :44 -68 ( 1982 ) ); MRC 5 cells ; PEGylated polypeptides can also be designed so as to FS4 cells ; and a human hepatoma line (Hep G2) . Other control the rate of release of the bioactive polypeptide into useful mammalian host cell lines include Chinese hamster a patient' s bloodstream , and thus, PEGylation of therapeutic ovary (CHO ) cells , including DHFR -CHO cells (Urlaub et polypeptides is a useful and attractive strategy for designing al ., PNAS USA 77 :4216 ( 1980 )) ; and myeloma cell lines 5 next generation polypeptide- based drugs. Moreover , site such as NSO and Sp2 / 0 . For a review of certain mammalian specific PEGylation also minimizes undesirable collateral host cell lines suitable for antibody production , see , e . g ., effects on the therapeutic activities of the PEGylated poly Yazaki and Wu ,Methods in Molecular Biology , Vol. 248 ( B . peptide . PEGylation of polypeptide -based drugs can change K . C Lo , ed . , Humana Press , Totowa, N . J ., 2003 ) , pp . their physical and chemical properties, such as conforma 255 - 268 . Certain preferred mammalian cell expression sys - 10 tion , electrostatic binding, hydrophobicity , and pharmacoki tems include CHO and HEK293 -cell based expression sys - netic profile . PEGylation also improves drug solubility , tems. Mammalian expression systems can utilize attached stability , and the retention time of the conjugates in blood cell lines , for example , in T- flasks, roller bottles , or cell and decreases immunogenicity , proteolysis and renal excre factories, or suspension cultures, for example , in 1 L and 5 tion , thereby allowing a reduced dosing frequency . L spinners, 5 L , 14 L , 40 L , 100 L and 200 L stir tank 15 In various embodiments , the present invention contem bioreactors , or 20 /50 L and 100 / 200 L WAVE bioreactors , plates, in part, PEGylated DRS polypeptides , compositions among others known in the art . comprising such PEGylated polypeptides , and methods of Also included is cell - free expression of proteins. These use thereof. In certain embodiments , the PEGylated DRS and related embodiments typically utilize purified RNA polypeptides described herein have improved pharmacoki polymerase , ribosomes, tRNA and ribonucleotides ; these 20 netic properties compared to non -PEGylated DRS polypep reagents may be produced by extraction from cells or from tides . a cell- based expression system . PEG polymers suitable for conjugation to DRS polypep In addition , a host cell strain may be chosen for its ability tides of the invention are not limited to a particular structure to modulate the expression of the inserted sequences or to and can be linear ( e . g . , monofunctional PEG or bifunctional process the expressed protein in the desired fashion . Such 25 PEG ), branched or multi- armed ( e . g ., PEG attached to a modifications of the polypeptide include, but are not limited polyol core or forked PEG ) , dendritic . In one embodiment, to , post - translationalmodifications such as acetylation , car the internal structure of a polyalkylene glycol polymer can boxylation , glycosylation , phosphorylation , lipidation , and be organized in any number of different patterns and can be acylation , or the insertion of non - naturally occurring amino selected from the group consisting of homopolymer , alter acids ( see generally U . S . Pat . Nos. ; 7 , 939 , 496 ; 7 ,816 ,320 ; 30 nating copolymer, random copolymer, block copolymer, 7 ,947 ,473 ; 7 ,883 ,866 ; 7 ,838 ,265 ; 7, 829 ,310 ; 7 ,820 ,766 ; alternating tripolymer , random tripolymer, and block trip 7 ,820 ,766 ; 7 ,7737 ,226 ; 7 ,736 ,872 ; 7 ,638 , 299 ; 7 ,632 , 924 ; olymer. and 7 ,230 ,068 ). In some embodiments , such non -naturally In particular embodiments , the total average molecular occurring amino acids may be inserted at position Cys130 . weight of the PEG polymers polymer in the conjugate is Post- translational processing which cleaves a " prepro ” form 35 typically from about 1 , 000 Daltons to about 150 ,000 Dal of the protein may also be used to facilitate correct insertion , tons. Exemplary ranges of total average molecular weights folding and / or function . Different host cells such as yeast, of PEG polymers conjugated to a DRS polypeptide of the CHO , HeLa , MDCK , HEK293 , and W138 , in addition to invention include , but are not limited to : about 1 , 000 Dal bacterial cells , which have or even lack specific cellular tons to about 120 ,000 Daltons , about 10 ,000 Daltons to machinery and characteristic mechanisms for such post- 40 about 100 , 000 Daltons , about 10 , 000 Daltons to about translational activities, may be chosen to ensure the correct 80 , 000 Daltons , about 10 ,000 Daltons to about 60 , 000 modification and processing of the foreign protein . Daltons, about 10 ,000 Daltons to about 40 , 000 Daltons , The DRS polypeptides produced by a recombinant cell about 20 ,000 Daltons to about 100 ,000 Daltons , about can be purified and characterized according to a variety of 20 ,000 Daltons to about 80 , 000 Daltons , about 20 ,000 techniques known in the art . Exemplary systems for per- 45 Daltons to about 60 ,000 Daltons, about 20 ,000 Daltons to forming protein purification and analyzing protein purity about 40 ,000 Daltons, about 40, 000 Daltons to about 120 , include fast protein liquid chromatography (FPLC ) ( e . g ., 000 Daltons, about 40 ,000 Daltons to about 100 ,000 Dal AKTA and Bio -Rad FPLC systems) , high -pressure liquid tons, about 40 ,000 Daltons to about 80 ,000 Daltons, or about chromatography (HPLC ) ( e . g ., Beckman and Waters 40 , 000 Daltons to about 60 ,000 Daltons, or any intervening HPLC ) . Exemplary chemistries for purification include ion 50 range . exchange chromatography (e .g ., Q , S ), size exclusion chro In some embodiments , low molecular weight PEG poly matography, salt gradients , affinity purification ( e. g ., Ni, Co , mers may be preferred , and these may range from about 200 FLAG , maltose , glutathione , protein A / G ) , gel filtration , Daltons to about 5 ,000 Daltons. Exemplary ranges of low reverse - phase , ceramic HYPERD® ion exchange chroma - molecular weight PEG polymers conjugated to a DRS tography , and hydrophobic interaction columns (HIC ) , 55 polypeptide of the invention include, but are not limited to : among others known in the art. Several exemplary methods about 200 Daltons to about 2 , 000 Daltons , about 500 Dal are also disclosed in the Examples sections . tons to about 2 , 000 Daltons, about 800 Daltons to about PEGylated DRS Polypeptides 2000 Daltons , about 1000 Daltons to about 2000 Daltons . A wide variety of PEG polymers can be linked to DRS Exemplary ranges of total average molecular weights of polypeptides of the present invention to modulate biological 60 PEG polymers conjugated to a DRS polypeptide of the properties of the DRS polypeptide, and / or provide new invention include, but are not limited to : about 500 Daltons, biological properties to the DRS polypeptide . PEG polymers about 1 , 000 Daltons, about 5000 Daltons, about 10 ,000 can be linked to the DRS polypeptide via a naturally Daltons, about 15 ,000 Daltons, about 20 ,000 Daltons, about encoded amino acid , via a non -naturally encoded amino 25 , 000 Daltons, about 30 ,000 Daltons , about 35 , 000 Dal acid , or any functional substituent of a natural or non -natural 65 tons, about 40 ,000 Daltons, about 45 ,000 Daltons, about amino acid , or any substituent or functional group added to 50 , 000 Daltons, about 55 , 000 Daltons, about 60 ,000 Dal a natural or non - natural amino acid . tons, about 65 ,000 Daltons, about 70 ,000 Daltons, about US 9 ,822 ,353 B2 99 100 75 ,000 Daltons, about 80, 000 Daltons, about 85, 000 Dal - (1992 ), Zalipsky (1995 ) Advanced Drug Reviews 16 :157 tons, about 90 ,000 Daltons, about 100 , 000 Daltons, about 182 ; and Roberts et al. , Advanced Drug Delivery Reviews 54 110 ,000 Daltons, about 120 ,000 Daltons, about 130 ,000 (2002 ): 459 -476 . Daltons, about 140 , 000 Daltons, or about 150 , 000 Daltons In general , PEG functional groups suitable for conjugat any intervening molecular weight. 5 ing PEG to a polypeptide of the invention include , but are In particular embodiments comprising 1 , 2 , 3 , 4 , 5 , 6 , 7 , not limited to a carboxylic acid , ester, aldehyde , aldehyde 8 , 9 , or 10 or more PEG polymers conjugated to a DRS hydrate , acetal, hydroxy, protected hydroxy, carbonate , alk polypeptide of the present invention , the average molecular enyl, acrylate , methacrylate , acrylamide, substituted or weight of each PEG polymer conjugated to the DRS poly - unsubstituted thiol, halogen , substituted or unsubstituted peptide includes , but is not limited to : about 1000 Daltons, 10 amine , protected amine , hydrazide , protected hydrazide , about 5000 Daltons , about 10 .000 Daltons, about 15 ,000 succinimidyl, isocyanate , isothiocyanate , dithiopyridine , Daltons , about 20 , 000 Daltons, about 25 , 000 Daltons , about vinylpyridine, iodoacetamide , epoxide, hydroxysuccinim 30 .000 Daltons , about 35 . 000 Daltons , about 40 . 000 Dal- idyl, azole , maleimide , sulfone , allyl, vinylsulfone , tresyl, sulfo - N - succinimidyl, dione , mesyl, tosyl, or glyoxal. tons, about 45 ,000 Daltons, about 50 ,000 Daltons, about 15 In one embodiment , the active functional group of a PEG 55 ,000 Daltons, about 60 , 000 Daltons, about 65 ,000 Dal moiety selected from the group consisting of: a carboxylic tons, about 70 ,000 Daltons, about 75 ,000 Daltons, about acid , ester , aldehyde , aldehyde hydrate , acetal, hydroxy , 80 , 000 Daltons, about 85, 000 Daltons , about 90 , 000 Dal protected hydroxy, carbonate , alkenyl , acrylate, methacry tons , or about 100 ,000 Daltons, or any intervening molecu late , acrylamide , substituted or unsubstituted thiol, halogen , lar weight. 20 substituted or unsubstituted amine , protected amine , hydraz It will be appreciated that because virtually all PEG ide , protected hydrazide , succinimidyl, isocyanate , isothio polymers exist as mixtures of diverse high molecular mass, cyanate , dithiopyridine , vinylpyridine , iodoacetamide , the PEG polymer molecular weights (MW ) above represent epoxide, hydroxysuccinimidyl , azole , maleimide, sulfone , the average MWs of different sized chains within the poly allyl, vinylsulfone, tresyl, sulfo - N - succinimidyl, dione , mer . The PEG polymers of the invention will for a given 25 mesyl, tosyl , and glyoxal forms a covalent linkage with a molecular weight typically consist of a range of ethylene non -natural amino acid having the formula : glycol (or ethyleneoxide; OCH2CH2) monomers . For example , a PEG polymer of molecular weight 2000 Da will typically consist of 43 10 monomers, the average being 30 around 43 -44 monomers . The PEG polymers of the present invention will typically H2NC02H comprise a number of subunits, e . g ., each “ n ” , “ n , ” or “ n , " in any of the claimed compounds may each independently wherein the sidechain , R , of the non -naturally occurring be from about 1 to about 1000 , from about 1 to about 800 , 35 amino acid comprises a functional group selected from the from about 1 to about 600 , from about 1 to about 400 , from group consisting of: alkyl- , aryl- , aryl halide , vinyl halide , about 1 to about 300 , from about 1 to about 200 . Well- suited alkyl halide, acetyl, ketone , aziridine, nitrile , nitro , halide , PEG groups are such wherein the number of subunits ( i . e . , acyl- , keto - , azido - , hydroxyl- , hydrazine, cyano - , halo - , n , n , or ny) are independently selected from the group hydrazide , alkenyl, alkynyl, ether , thio ether , epoxide, sul consisting of from about 800 to about 1000 ; from about 800 40 fone , boronic acid , boronate ester , borane , phenylboronic to about 950 ; from about 600 to about 850 ; from about 400 acid , thiol, seleno -, sulfonyl- , borate , boronate , phospho , to about 650 ; from about 200 to about 450 , from about 180 phosphono , phosphine , heterocyclic - , pyridyl, naphthyl , to about 350 ; from about 100 to about 150 ; from about 35 benzophenone , a constrained ring such as a cyclooctyne , to about 55 ; from about 42 to about 62 ; from about 12 to thio ester , enone , imine , aldehyde , ester, thioacid , hydrox about 25 subunits, from about 1 to 10 subunits . In certain 45 ylamine , amino , carboxylic acid , alpha -keto carboxylic acid , embodiments the PEGylated DRS polypeptide will have a alpha or beta unsaturated acids and amides, glyoxylamide , molecular weight of about 40 kDa, and thus n for each PEG or organosilane group , and the like and any combination chain in the branch chain PEGs will be within the range of thereof. about 440 to about 550 , or about 450 to about 520 . In another embodiment, the active functional group of a Branched versions of the PEG polymer ( e . g . , a branched 50 PEG moiety selected from the group consisting of: a car 40 ,000 Da PEG polymer comprised of two or more 10 ,000 boxylic acid , ester, aldehyde , aldehyde hydrate , acetal , Da to 20 ,000 Da PEG polymers or the like ) having a total hydroxy, protected hydroxy, carbonate , alkenyl, acrylate , molecular weight of any of the foregoing can also be used . methacrylate , acrylamide, substituted or unsubstituted thiol, Typically , PEG polymers are activated with a suitable halogen , substituted or unsubstituted amine , protected activating group appropriate for coupling to a desired site on 55 amine, hydrazide, protected hydrazide , succinimidyl, iso a DRS polypeptide . Thus, a polymeric reagent will possess cyanate , isothiocyanate , dithiopyridine , vinylpyridine , iodo a functional group for reaction with a corresponding func - acetamide , epoxide, hydroxysuccinimidyl, azole , maleim tional group on a DRS polypeptide, e . g . , lysine , cysteine , ide , sulfone, allyl, vinylsulfone , tresyl, sulfo - N histidine , arginine , aspartic acid , glutamic acid , serine , succinimidyl, dione, mesyl , tosyl , and glyoxal forms a threonine, tyrosine , and the N - terminal amino and C -termi - 60 covalent linkage with a non - natural amino acid selected nal carboxylic acid group of amino acids . Representative from the group consisting of: p - acetyl- L - phenylalanine , polymeric reagents and methods for conjugating these poly - O -methyl - L - tyrosine , L - 3 - ( 2 - naphthyl) alanine , 3 -methyl mers to an active moiety are known in the art and further phenylalanine , 0 - 4 -allyl - L - tyrosine , 4 -propyl - L - tyrosine , described in Zalipsky, S . , et al. , “ Use of Functionalized Poly tri - O - acetyl- GlcNAcß - serine, B - O -GlcNAc - L - serine, tri - 0 ( Ethylene Glycols ) for Modification of Polypeptides” in 65 acetyl- GalNAc - a - threonine , a -GalNAc - L - threonine , Polyethylene Glycol Chemistry Biotechnical and Biomedi - L -Dopa , fluorinated phenylalanine , isopropyl- L -phenylala cal Applications, J . M . Harris , Plenus Press , New York nine , p -azido - L -phenylalanine , p - acyl- L -phenylalanine , US 9 ,822 ,353 B2 101 102 p -benzoyl - L - phenylalanine , L - phosphoserine , phosphonos PEGylation of a DRS polypeptide via amino group , with erine , phosphonotyrosine, p - iodo - phenylalanine , p -bro a PEG reagent bearing an N -hydroxysuccinimide ester mophenylalanine, p - amino - L -phenylalanine , and isopropyl - (NHS group ) , is typically carried out at room temperature , or L - phenylalanine. 4° C ., in a polar aprotic solvent such as dimethylformamide In particular embodiments , the active functional group of 5 (DMF ) or acetonitrile , or a combination thereof (with small a PEG polymer is conjugated to an amino group of a DRS amounts of water to solubilize the peptide ) under slightly polypeptide . In this approach , the PEG bearing the active basic pH conditions, e . g ., from pHs ranging from about 7 . 5 functional group is reacted with the DRS polypeptide in to about 9 . Reaction times are typically in the range of 1 to aqueous media under appropriate pH conditions, at room 24 hours , depending upon the pH and temperature of the temperature or 4° C . , for a few hours to overnight. Typically 10 reaction . the polymeric reagent is coupled to the activated functional PEGylation of a DRS polypeptide via amino group , with group via a linker as described herein . a PEG reagent bearing an aldehyde group , is typically Suitable active functional groups to conjugate PEG to an conducted under mild conditions, in the presence of sodium amino group of the polypeptide , such as those found in cyanoborohydride ( 10 equiv . ) , 4° C . , at pHs from about 5 to lysine , arginine , or histidine residues or an N - terminal 15 10 , for about 20 to 36 hours . PEGylation may be conducted , residue of a DRS polypeptide, include, but are not limited to : for example , in 100 mM sodium acetate or 100 mM sodium succinimidyl esters (NHS ), e . g . , succinimidyl carbonate , biphosphate buffer at pH 5 .06 .0 . The buffer may additionally succinimidyl carboxylmethyl, succinimidyl glutarate , suc contain 20 mM sodium cyanoborahydride. The molar ratio cinimidyl valerate , succinimidyl succinate , and the like ; of compound to mPEG - aldehyde may be 1: 5 - 1 : 10 . The p -nitrophenyl esters , e .g ., p -nitrophenyl carbonate , p -nitro - 20 PEGylation reaction is then stirred overnight at ambient or phenyl carboxymethyl, p - nitrophenyl glutarate , p -nitrophe - refrigeration temperature . nyl valerate , p - nitrophenyl succinate , and the like; succin - PEGylation of a DRS polypeptide via amino group , with imidyl carbamate (NSC ); dichlorotriazines ; tresylates; a PEG reagent bearing p -Nitrophenyloxycarbonyl group , is benzotriazole carbonates ; trichlorophenyl carbonates ; iso - typically conducted with borate or phosphate buffer at pHs cyanates ; isothiocyanates; acyl azides ; sulfonyl chloride ; 25 from about pH 8 to 8 . 3 pH , at room temperature overnight. aldehydes , e . g ., proprionaldehyde , acetalaldehyde , butyral- For all the coupling reactions, varying ratios of polymeric dehyde, and the like ; carboxylic acid derivatives , e . g . , pro - reagent to the DRS polypeptide may be employed , e . g ., from pionic acid , butanoic acid , and the like ; imidioesters , e . g ., an equimolar ratio up to a 10 - fold molar excess of polymer carbonylimidazole , oxycarbonylimidazoles , and the like ; reagent. Typically , up to a 2 - fold molar excess of polymer cyclic imide thiones ; epoxides ; acrylates ; and anhydrides . 30 reagent will suffice . In the following Table D10 , selected Exemplary activated PEGs capable of reacting with amino PEGylation reagents are listed . Obviously other active groups of the DRS polypeptide include , e . g ., those listed in groups and linkers may be employed , and are known to Table D10 . those skilled in the art . TABLE D10
Abbreviation & Molecular Weight Range Structure /Functionality ( in Da ) Exemplary Activated Linear PEGs Capable of Reacting With Amino Groups
SUNBRIGHT ME -020CS MW = 2 ,000 SUNBRIGHT ME- 050CS MW = 5 ,000 CH3 – 0 – ( CH2CH2O) – C – CH2CH2C _ 0 _ N SUNBRIGHT ME- 100CS MW = 10 ,000 SUNBRIGHT ME - 200CS MW = 20 , 000 N -hydroxysuccinimide ester SUNBRIGHT ME - 300CS MW = 30 ,000 SUNBRIGHT ME -400CS MW = 40 ,000
SUNBRIGHT ME- 050GS MW = 5 ,000 SUNBRIGHTME - 200GS MW = 20 , 000 CH3 — O - (CH2CH2O )n1 - 0 - 0 - N SUNBRIGHT ME- 300GS MW = 30 , 000 SUNBRIGHT ME- 400GS MW = 40 , 000 aureonlineN - hydroxysuccinimide ester US 9 ,822 ,353 B2 103 104 103 TABLE D10 -continued Abbreviation & Molecular Weight Range Structure /Functionality ( in Da ) SUNBRIGHT ME - 050TS MW = 5 ,000 SUNBRIGHT ME - 200TS MW = 20 ,000 CH3 — 0 — ( CH2CH2O ) , 1 - 0 - 0 - N SUNBRIGHT ME- 300TS MW = 30 , 000 SUNBRIGHT ME - 400TS MW = 40 ,000 N - hydroxysuccinimide ester
SUNBRIGHT ME - 020AS MW = 2 ,000 SUNBRIGHT ME- 050AS CH3 — O - (CH2CH2O ) 1 - CH2 - 0 - 0 N MW = 5 ,000
N -hydroxysuccinimide ester SUNBRIGHTME - 050HS MW = 5 ,000 SUNBRIGHTME - 200HS MW = 20 ,000 CH3 — 0 - ( CH2CH2O ) n1 - ( CH2) 50 — 0 - N SUNBRIGHT ME- 300HS MW = 30 ,000 SUNBRIGHT ME- 400HS MW = 40 ,000 N -hydroxysuccinimide ester SUNBRIGHT MENP -020H MW = 2 ,000 CH3 — 0 — (CH2CH20 )n1 - 0 - 0 -NO2 SUNBRIGHT MENP -050H MW = 5 ,000 SUNBRIGHTMENP - 10T p - Nitrophenyl MW = 10 ,000 SUNBRIGHTMENP - 20T MW = 20 ,000 SUNBRIGHT MENP - 30T MW = 30 ,000 SUNBRIGHT MENP - 40T MW = 40 ,000 CH , — — ( CH2CH2O ) – N = C = 0 Isocyanate
SUNBRIGHT ME - 050AL MW = 5 . 000 CH3 — 0 - ( CH2CH2O ) n - CH2 - C - 0 - CH2CH2CH SUNBRIGHT ME- 100AL MW = 10 ,000 Aldehyde SUNBRIGHTME - 200AL MW = 20 ,000 SUNBRIGHT ME -300AL MW = 30 ,000 SUNBRIGHT ME - 400AL MW = 40 ,000 SUNBIO P1PAL - 5 MW = 5 ,000 CH3 — 0 — ( CH2CH20 + n CH2CH2CH SUNBIO P1PAL - 10 MW = 10 ,000 Aldehyde SUNBIO P1PAL - 20 MW = 20 ,000 SUNBIO P1PAL - 30 MW = 30 ,000 SUNBIO P1APAL - 5 MW = 5 ,000 CH3 — O - (CH2CH20 +n CH2CNHCH2CH2CH SUNBIO P1APAL - 10 MW = 10 ,000 Amide Aldehyde SUNBIO P1APAL - 20 MW = 20 ,000 SUNBIO P1APAL - 30 MW = 30 , 000 US 9 ,822 ,353 B2 105 106 TABLE D10 -continued Abbreviation & Molecular Weight Range Structure / Functionality ( in Da )
SUNBIO P1TPAL - 5 MW = 5 ,000 CH3 — O - ( CH2CH20 + n CNHCH2CH2CH Urethane Aldehyde SUNBIO PIBAL - 5 MW = 5 ,000 CH3 — 0 — (CH2CH20tn CH2CH2CH2CH SUNBIO P1BAL - 10 MW = 10 , 000 Aldehyde SUNBIO P1BAL - 20 MW = 20 ,000 SUNBIO P1BAL - 30 MW = 30 ,000 SUNBIO P1ABAL - 5 MW = 5 ,000 CH3 — O - (CH2CH20 +n CH2CNHCH2CH2CH2CH SUNBIO P1ABAL - 10 MW = 10 ,000 Amide Aldehyde SUNBIO P1ABAL - 20 MW = 20 ,000 SUNBIO PLABAL - 30 MW = 30 ,000 SUNBIO P1TBAL - 5 MW = 5 ,000 CH3 — O - (CH2CH2O ) n - CNHCH2CH2CH2CH Urethane Aldehyde
X |= 0 , y = 1 SUNBRIGHT- AS X = 0 , y = 5 SUNBRIGHT- HS ? = 2 SUNBRIGHT- CS | ROCH3CH2Ow = C( Ox = (CH2 ) y - c( O) = 0 < N XXXX = 1 , ?y = 3 SUNBRIGHT- GS
N -hydroxysuccinimide ester
z = 2 SUNBRIGHT-MA z = 5 SUNBRIGHT-MA3 RjO (CH2CH2O ) n — ( CH2)2NH — ( CH2) 2 — N
Maleimide Exemplary Activated Branched PEGs Capable of Reacting With Amino Groups JENKEM A0001- 1 Y -NHS - 40K CH3 – 0 < (CH3CHO 1 CH?CH, N - CH2 - 0 - 0 - N CH3 — 0 — (CH2CH207n2 - CH2CH2 N -hydroxysuccinimide ester R1 - O - ( CH2CH20 )n? - CH2 SUNBRIGHT GL2 - 200GS2 MW = 20 ,000 R1 - O - (CH2CH20 ) n2 — CH SUNBRIGHT GL2- 400GS2 MW = 40 ,000 H2C - OCH2CH2CH2NHC ( CH2) 3C0 — N SUNBRIGHT GL2 - 400GS2 MW = 60 ,000 SUNBRIGHT GL2 - 800GS2 MW = 80 ,000 N -hydroxysuccinimide ester US 9 ,822 ,353 B2 107 108 TABLE D10 - continued Abbreviation & Molecular Weight Range Structure /Functionality ( in Da ) SUNBRIGHT GL2 - 100NP R1 - 0 — ( CH2CH20 ) n1 CH2 MW = 10 ,000 R = 0 < ( CH2CH2O) ng – CH 0 SUNBRIGHT GL2- 200NP MW = 20 ,000 H2C = OCO - NO2 SUNBRIGHT GL2- 400NP MW = 40 ,000 SUNBRIGHT GL2 -600NP p - Nitrophenyl MW = 60 , 000 SUNBRIGHT GL2- 800NP MW = 80 ,000 R1 - O - (CH2CH20 ) n1 CH2 SUNBRIGHT GL2 - 200TS MW = 20, 000 R = 0 < ( CH2CH2O) 2 – CH 0 SUNBRIGHT GL2 - 400TS MW = 40 ,000 H2C - O0O - N SUNBRIGHT GL2 - 600TS MW = 60 ,000 SUNBRIGHT GL2- 800TS MW = 80 ,000 N -hydroxysuccinimide ester
SUNBRIGHT GL2- 200AL3 R ; — o — ( CH2CH20) , 1 - CH2 MW = 20 ,000 R - 0 – ( CH2CH2O ) 2 – CH 0 SUNBRIGHT GL2 - 400AL3 MW = 40 ,000 H2C - OÖNHCH2CH2CH SUNBRIGHT GL2 - 600AL3 MW = 60 ,000 Aldehyde SUNBRIGHT GL2 - 800AL3 MW = 80 ,000
SUNBRIGHT GL3 R ; — 0 — (CH2CH20 ), 1 - CH2 400AL1000 R1 - O - (CH2CH20 ) ,2 — CH 0 MW = 50 ,000 H2C - OËNH (CH2 ) 30 ( CH2CH2O )nz - CH2CH2CH Aldehyde
SUNBRIGHT GL3 R1 - O - ( CH2CH20 ) , 1 CH2 400NP100U RR1 - OO -— (CHCH20 CH2CH20 )-n2 — CHCI 0 MW = 50 ,000 H2C - OËNH (CH2 ) 30 ( CH2CH2OACILO )nz - 00 A NO2No. p - Nitrophenyl R1 - 0 — (CH2CH20 ), 1 - CH2 SUNBRIGHT GL3 400GS100U R = 0 < ( CH3CH2O) 2 – CH ? MW = 50 ,000 HC - OCNH (CH2 ) 30 (CH2CH2O )n3C (CH2 ) 300 — N
N -hydroxysuccinimide ester
R1 - O - (CH2CH20 )n1 — CH2 SUNBRIGHT GL3 400HS100U R1 - 0 — (CH2CH20 )- 2 — CH O MW = 50 ,000 kannH2C - OCNH (CH2 ) 30 (CH2CH2O )nz ( CH2) 5CO — N N -hydroxysuccinimide ester US 9 ,822 ,353 B2 109 110 TABLE D10 -continued Abbreviation & Molecular Weight Range Structure/ Functionality ( in Da ) R = 0 < ( CH2CH2O) n = 0 < c( O) - NH SUNBRIGHT LY -400NS HC - CH2 MW = 40 ,000 H?C – CH2 CH - C R = 0 ( CH2CH2O ) 2 – 0 – COO – NH 0 _ N
R1 - 0 — (CH2CH20 )n1 CH2 MW = 40 ,000 HC - OCH2CH2CH2NHC( CH2) 3C R - O - (CH2CH20 ) n2 - CH2
In certain embodiments , the active functional group of a os amides , orthopyridyl disulfides, haloacetyls , alkyl halide PEG polymer is conjugated to a thiol group of a DRS - derivatives , aziridines , acrylol derivatives arylating agents , polypeptide. Suitable active functional groups to conjugate and the like . PEG to a thiol group of the polypeptide, such as those found Exemplary activated PEGs capable of reacting with in a cysteine residue of a DRS polypeptide, include, but are amino groups of the DRS polypeptide include , e. g ., those not limited to : thiols , maleimides, vinylsulfones , iodoacet listed in Table D11 . TABLE D11 Abbreviation & Molecular Structure / Functionality Weight Range (in Da) Exemplary Activated Linear PEGs Capable of Reacting With Thiol Groups NOCS PEG2- 0001 MW = 5000 NOCS PEG2 - 0002 MW = 2000 NANOCS PEG2 - 0003 MW = 1000 NANOCS PEG2 - 0004 MW = 10000 CH3 — 0 – (CH2CH20 ). — CH2CH2 — N NANOCS PEG2 - 0005 MW = 20000 NANOCS PEG2 -0006 MW = 30000 NANOCS PEG2- 0006 MW = 40000 Maleimide
SUNBRIGHT ME - 020MA MW = 2 ,000 SUNBRIGHT ME - 050MA MW = 5 . 000 SUNBRIGHT ME - 120MA MW = 12 ,000 SUNBRIGHT ME- 200MA MW = 20 , 000 CH3 — 0 - ( CH2CH20 +n - CH2CH2CH2NHC (CH2 ) 2 — N SUNBRIGHT ME- 300MA MW = 30 ,000 SUNBRIGHT ME- 400MA MW = 40 ,000
Maleimide SUNBRIGHT ME- 2001A MW = 20 ,000 SUNBRIGHT ME- 300IA MW = 30 ,000 CH3 — 0 - (CH2CH2O )n - CH2CH2CH2NHC - CH2I SUNBRIGHT ME- 400IA MW = 40 ,000 Iodoacetamide
ENKEM A3073 M -MAL - 2000 ENKEM A3014 M -MAL - 5000 42 - N ENKEM A3045 M -MAL - 10K CH3 - 0 - ( CH - CHO - CH2 - ~ - NH - CH - CH2 - NH - - CH , ENKEM A3002 M -MAL - 20K ENKEM A3046 M -MAL - 30K Maleimide ENKEM A3042 M -MAL - 40K US 9, 822 , 353 B2 111 112 TABLE D11 - continued Abbreviation & Molecular Structure/ Functionality Weight Range (in Da ) JENKEM A3034 M - VS - 5000 JENKEM A3006 M - VS - 20K CH3 — 0 – ( CH2CH2O + n CH2CH2 - 5
Vinyl sulfone NANOCS PEG2- 0011 MW = 5000 NANOCS PEG2- 0012 MW = 2000 CH3 — 0 – ( CH2CH2O + n - S - S — NANOCS PEG2- 0014 MW = 10000 Orthopyridyldisufide Exemplary Activated Branched PEGs Capable of Reacting With Thiol Groups ENKEM A0002 - 1 Y -MAL - 40K CH3 - 0 – (CH2CH2OTnT CH2CH2 N - CH2 - CH2 - N CH , — o - (CH3CH20 ,2 - CH2 – CH
Maleimide
SUNBRIGHT GL2 - 200GS MW = 20 ,000 CH – 0 - (CH , CH, oni - CH , SUNBRIGHT GL2 -400GS MW = 40 , 000 SUNBRIGHT GL2 -600GS MW = 60 ,000 CH3 – 0 – ( CH2CH2O42 - CH SUNBRIGHT GL2 -800GS MW = 80 ,000 H2C - OCH2CH2CH2NHC (CH2 ) — N
Maleimide
SUNBRIGHT LY - 400MA MW = 40 , 000 CH3 – 0 – ( CH2CH2O ) = 0 – – NH CH2 CH2 _ CH2
= CH2 CH3 – 0 - ( CH2CH2O ) , 0 - C - HN - HC - C - N
PEGylation of a DRS polypeptide via amino group , with wild - type DRS cysteines residues may be first substituted a PEG reagent bearing a maleimide group , iodoacetamide or with another amino acid to prevent attachment of the PEG vinyl sulfone is typically carried out in phosphate buffer 55 polymer to these wild - type cysteines ( e . g . , C76 , C130 ) , for 50 - 100 mM under mild conditions around pH 6 . 5 - 7 .5 and at example , to prevent the PEG molecule ( s ) from disrupting an 4° C . for 4 to 24 hours . otherwise desirable biological activity . In particular embodiments , PEG polymers may be In other embodiments , the active functional group of a attached to wild - type cysteine residues ( i . e . , cysteine resi - PEG polymer is conjugated to a carboxylic acid group of a dues present in the wild -type DRS sequence ) , or to " substi - 60 DRS polypeptide , e . g . , at the C - terminus. Suitable active tuted ” or “ inserted ” cysteine residues ( e . g ., cysteine residues functional groups to conjugate the PEG to the carboxylic introduced into the wild -type sequence by replacing a natu - acid group of the DRS polypeptide include, but are not rally occurring residue with a cysteine , or by inserting a limited to : primary amines, hydrazines, and hydrazides , e . g . , cysteine into the sequence without necessarily altering or carbazates , semicarbazates , thiocarbazates , and the like . removing the nearby residues , e . g ., by appending an N - or 65 Exemplary activated PEGs capable of reacting with car C - terminal fusion protein ; ), so as to target the PEG to a boxylic acid groups of the DRS polypeptide include , e . g . , desired location . In certain embodiments, certain of the those listed in Table D12 . US 9 ,822 , 353 B2 113 114 TABLE D12 Exemplary Activated Linear PEGs Capable of Reacting With Carboxylate Groups Structure / Functionality Abbreviation & Molecular Weight Range ( in Da ) CH3 – 0 – (CH2CH2 ) n CH2CH2CH2NH2 SUNBRIGHT MEPA - 20H MW = 2 ,000 Primary Amine SUNBRIGHT MEPA - 50H MW = 5 , 000 SUNBRIGHT MEPA - 12T MW = 12 , 000 SUNBRIGHT MEPA - 20T MW = 20 ,000 SUNBRIGHT MEPA - 30T MW = 30 ,000 SUNBRIGHT MEPA -40T MW = 40 , 000 CH , O ( CH - CH2O) , CHACHANH , SUNBRIGHTMEPA - 20H MW = 2 ,000 Primary Amine SUNBRIGHT ME - 050EA MW = 5 , 000 SUNBRIGHT ME- 100EA MW = 12 , 000 SUNBRIGHT ME - 200EA MW = 20 ,000 SUNBRIGHT ME - 300EA MW = 30 . 000 SUNBRIGHT ME - 400EA MW = 40 , 000 JENKEM A3060 M - HZ MW = 5 ,000 JENKEM A3096 M - HZ MW = 10 ,000 CH3 – 0 - ( CH2CH2O) – CH2C - NH – NH JENKEM A3059 M -HZ MW = 20 ,000 JENKEM A3065 M -HZ MW = 30 ,000
PEGylation of a DRS polypeptide via carboxyl group , wherein : with a PEG reagent bearing a primary amine can be carried X is a water soluble polymer moiety ; out in 50 mM Phosphate buffer (pH 7 . 2 ) , in the presence of L is an optional linker ; WSC ( 2 eq ) , 4C , for 10 to 24 hours . PEGylation of a DRS 25 Y is a covalent linkage ; and polypeptide via carboxyl group , with a PEG reagent bearing DRS is a DRS polypeptide . a hydrazide group can be carried out in the presence of In another embodiment, the present invention , contem N , N - dicyclohexylcarbodiimide (DCC ), or in presence of a plates, in part, DRS polypeptides conjugated to branch water soluble coupling agent such as N - ( - 3 -dimethylamino chain water soluble polymers comprising two or more, e . g . , propyl) - N '- ethylcarbodiimide hydrochloride (EDC ) under 30 two , three , four, five , six , seven , eight, nine , ten , or more water soluble moieties mildly acid conditions (pH 6 . 0 - 6 . 5 ) . Illustrative multi- armed water soluble polymers having 2 In further embodiments , the active functional group of a arms, 3 arms, 4 arms, and 8 arms are known in the art , and PEG polymer is conjugated to a hydroxyl group of a DRS are available commercially e . g ., from Nanocs , NOF, Nektar , polypeptide. Suitable active functional groups to conjugate 35 SunBio and Jenkem . In particular embodiments , any com the PEG to the hydroxyl group of the polypeptide , such as mercially available branched water soluble polymer, such as those found in a serine, threonine , or tyrosine residue of a any branched chain PEG is suitable for PEGylation of a DRS polypeptide , include, but are not limited to : amines, hydraz polypeptide . Additional branched -water soluble polymers ides , epoxides , p -nitrophenylcarbonates , and isocyanates. for use in forming a DRS polypeptide conjugate of the In various embodiments , the present invention provides 40 present invention can be prepared following techniques PEGylated aspartyl- tRNA synthetase (DRS ) polypeptides , known to those skilled in the art. (See generally Pasut et al ., comprising at least one PEG moiety covalently attached to (2004 ) Protein , peptide and non -peptide drug PEGylation an amino acid residue within about 5 , about 10 , about 15 , for therapeutic application Expert Opinin . Ther. Patents about 20 , or about 25 amino acid residues of the C - terminus, 146 ) 859 -894 ) and are also described in U . S . Patent Appli the N -terminus , or a solvent accessible surface amino acid of 45 cation Publication Nos. 20050009988 , 20060194940 , the DRS polypeptide or any combination thereof. In certain 20090234070 , 20070031371 , U . S . Pat. Nos . 6 ,664 ,331 ; preferred embodiments , the PEG moieties comprise linear or 6 , 362, 254 ; 6 ,437 ,025 ; 6 ,541 , 543 ; 6 ,664 , 331; 6 ,730 ,334 ; branched PEG polymers . 6 , 774 , 180 ; 6 , 838 ,528 ; 7 ,030 , 278 ; 7 ,026 , 440 ; 7 , 053 , 150 ; In one embodiment, the present invention , contemplates , 7 ,157 , 546 ; 7 ,223 ,803 ; 7 , 265 , 186 ; 7 ,419 ,600 ; 7 ,432 ,330 ; in part , DRS polypeptides conjugated to a linear water 50 7 ,432 , 331 ; 7 ,511 , 094 ; 7 ,528 , 202 ; 7 ,589 , 157; and PCT pub soluble polymer, e .g ., a PEG moiety . A wide variety of linear lication numbers WO2005000360 , WO2005108463, water soluble polymers , comprising functional groups suit - WO2005107815 , WO2005028539 and WO200605108463 . able for conjugation to amino , thiol , hydroxyl, and carbox The branching moiety ( i. e . , central core molecule ) can be ylic acid groups of a DRS polypeptide are commercially an aliphatic hydrocarbon having a carbon chain length of at available in the art , e . g ., from Nanocs Corporation , NOF 55 least three carbon atoms ( i. e . , propane, butane, pentane, Corporation , SunBio , Nektar, and Jenkem Technology . In hexane , heptane , octane , nonane , decane , and the like ) or an particular embodiments , any commercially available water appropriate amino acid backbone , e . g . , lysine, arginine , soluble polymer is suitable for conjugation to a DRS poly - histidine , glutamine, serine , threonine , asparagine , aspartic peptide . In various embodiments, a linear , water soluble acid , glutamic acid , cysteine , and seleno cysteine . polymer conjugated to a DRS polypeptide of the invention 60 Other suitable core molecules include polyols , which are comprises a water soluble polymer moiety , optionally bound then further functionalized . Such polyols include aliphatic to a linker, and a covalent linkage that binds the DRS polyols having from 1 to 10 carbon atoms and from 1 to 10 polypeptide to the remainder of the conjugate . A generalized hydroxyl groups, including ethylene glycol, alkane diols , structure (I ) of a water soluble polymer conjugated to a DRS alkyl glycols , alkylidene alkyl diols , alkyl cycloalkane diols , polypeptide of the invention has the following structure : 65 1 , 5 - decalindiol, 4 , 8 - bis (hydroxymethyl ) tricyclodecane , cycloalkylidene diols, dihydroxyalkanes, trihydroxyalkanes, X - L - Y -DRS (I ) and the like . Cycloaliphatic polyols may also be employed , US 9 , 822, 353 B2 115 116 including straight chained or closed -ring sugars and sugar In certain embodiments the branched DRS polypeptide alcohols , such as mannitol, sorbitol, inositol, xylitol, que polymer conjugates of the invention may have a generalized brachitol, threitol, arabitol, erythritol, adonitol, ducitol, facose , ribose , arabinose , xylose , lyxose , rhamnose, galac tose , glucose , fructose , sorbose , mannose , pyranose , altrose , 5 talose , tagitose , pyranosides , sucrose , lactose , maltose , and X - L1 - CH2 the like . Additional aliphatic polyols include derivatives of X - L2 — CH glyceraldehyde , glucose , ribose , mannose , galactose , and related stereoisomers . Other core polyols that may be used CH2 - L3 - Y — DRS include crown ether, cyclodextrins, dextrins and other car - 10 bohydrates such as starches and amylose . Typical polyols wherein : include glycerol, pentaerythritol, sorbitol, and trimethylol X is an independently selected water soluble polymer propane . Other suitable cores include polyamines , and PEG moiety ; moieties comprising multiple functional terminal end L1, L2 and L? are independently selected optional linkers ; groups. In one embodiment, the branching moiety comprises 15 Y is a covalent linkage between the DRS polypeptide and a lysine residue . the remainder of the conjugate ; and Since the branched polymers of the invention combine at DRS refers to a DRS polypeptide as disclosed herein . least two polymer arms in a single molecule , a polymer with In certain embodiments the branched DRS polypeptide sufficient molecular weight to impart beneficial properties to polymer conjugates of the invention may have a generalized a DRS polypeptide can be formed using shorter , easier to 20 formula ( IIB ) : prepare polymer chains. The branched polymers of the invention are preferably monofunctional, meaning the poly mer molecule contains only a single reactive site for con X - L1 - CH2 jugation to a DRS polypeptide . HC - L3 - Y - DRS Although the carbon atoms of the branching moiety can 25 have PEG polymers extending from any of the aforemen X - L2CH2 tioned carbons, in particular embodiments, the overall branched conjugate is symmetrical . For example , for a three wherein : carbon branching moiety , the PEG polymers extend from X is an independently selected water soluble polymer positions 1 and 3 , with a site suitable for covalent attachment 30 moiety ; to a DRS polypeptide extending from the central carbon or L1, L2 and Lz are independently selected optional linkers ; the carbon at position 2 . Similarly , for a five carbon branch Y is a covalent linkage between the DRS polypeptide and ing moiety , the PEG polymers can extend from positions 1 the remainder of the conjugate ; and DRS refers to a and 5 , with a site suitable for covalent attachment to a DRS DRS polypeptide as disclosed herein . polypeptide extending from position 3 , or PEG polymers 35 In certain embodiments the branched DRS polypeptide extending from positions 2 and 4 , or , if a highly branched polymer conjugates of the invention may have a generalized structure is desired , with PEG polymers extending from formula ( IIC ) : each of positions 1 , 2 , 4 , and 5 . In certain embodiments, the overall branched conjugate is asymmetrical, for example , in anan embodiment comprising a four carbon branching moiety . 40 X - L1 For example , for a four carbon branching moiety , the PEG Lysine - L3 - Y - DRS polymers extend from positions 1 , 2 , and 3 , with a site suitable for covalent attachment to a DRS polypeptide X - L2 extending from the central carbon or the carbon at position 45 wherein : A DRS polypeptide comprising branched chain water X is an independently selected water soluble polymer soluble polymer conjugate of the invention will typically moiety ; comprise at least two water soluble moieties , each optionally L1 , L2 and Lz are independently selected optional linkers , bound to a linker, covalently attached to a branching moiety , and wherein the linkers connecting the lysine residue to also optionally bound to a linker , covalently attached to a 50 the water soluble polymer moiety are connected via the covalent linkage that binds the DRS polypeptide to the amino groups of the lysine molecule , and the linker connecting the lysine molecule to the DRS polypeptide remainder of the conjugate . A generalized structure ( II) of is attached via the C - terminal carboxylate group of the the branched DRS polypeptide polymer conjugates of the lysine molecule ; invention is shown below : Y is a covalent linkage between the DRS polypeptide and (X -L 1) m -B - L2- Y -DRS (II ) S5 the remainder of the conjugate ; and DRS refers to a wherein : DRS polypeptide as disclosed herein . X is an independently selected water soluble polymer In certain embodiments the branched DRS polypeptide moiety, for each m ; polymer conjugates of the invention may have a generalized L? and Ly are independently selected optional linkers , 60 formula" (IID ) : wherein L , is also independently selected for each m ; m is 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , or 10 , preferably 2 to about 5 X - L ( e . g ., 2 , 3 , 4 , or 5 ) ; B is a branching moiety ; N - L3 - Y - DRSDRS Y is a covalent linkage between the DRS polypeptide and 65 X - L2 the remainder of the conjugate ; and DRS is a DRS polypeptide as disclosed elsewhere herein . US 9 ,822 ,353 B2 117 118 wherein : can be the same linker or each linker can be different from X is an independently selected water soluble polymer each other linker. In certain embodiments any one or more moiety ; of the linkers are optional. L1, L2 and Lz are independently selected optional linkers ; The particular linkage between the DRS polypeptide and Y is a covalent linkage between the DRS polypeptide and 5 the water -soluble polymer or branch moiety depends on a number of factors , including the desired stability of the the remainder of the conjugate ; and DRS refers to a linkage, its hydrophobicity , the particular linkage chemistry DRS polypeptide as disclosed herein . employed , and impact on the aqueous solubility, and aggre In different embodiments of any of the generalized struc gation state of the PEGylated DRS polypeptide . Exemplary tures ( I ) , ( II ) , ( IIA ) , (IIB ) , ( IIC ) or (IID ) each water soluble 10 linkages are hydrolytically stable , and water soluble , repre polymer moiety , X , is independently selected and is repre sentative suitable linker can comprise any combination of sented by the formula R , ( CH2CH2O ) n or R , - amide, a urethane (also known as carbamate ), amine, thio ( OCH2CH2) m ; wherein R , is selected from alkyl, alkoxy, and ether (also known as sulfide ), or urea (also known as aryl groups . carbamide ) groups . In different embodiments n is from about 1 to about 1 , 200 , 15 There are many commercially available examples of from about 10 to about 1 ,000 , from about 20 to about 800 , suitable water -soluble linker moieties and / or these can be from about 50 to about 600 , or from about 100 to about 500 . prepared following techniques known to those skilled in the In one embodiment, n is about 5 , about 10 , about 20 , about art. Certain illustrative exemplary linker moieties are 50 , about 100 , about 200 , about 300 , about 400 , about 500 , described below . The corresponding activated intermediates about 600 , about 700 , about 800 , about 900 , about 1 , 000 , 20 are provided in Tables D10 -D12 . about 1 , 100 , about 1 , 200 , about 1 , 300 , about 1 , 400 , or about Suitable linkers can have an overall chain length of about 1 ,500 or any intervening integer . In some embodiments , nis 1 - 100 atoms, 1 - 80 atoms, 1 -60 atoms, 1 -40 atoms, 1 - 30 from about 200 to about 800 . atoms, 1 - 20 atoms, or 1, 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11, 12 , 13 , Typically , branched PEGylated DRS polypeptides of gen 14 , 15 , 16 , 17 , 18 , 19 , or 20 atoms, wherein the atoms in the eral formula ( II ) comprise two or more, three or more , four, 25 chain comprise C , S , N , P , and O . In certain embodiments , or more , or five or more of the same PEG polymer. That is a linker is optional, e .g ., a PEG conjugated polypeptide does to say , the polymer arms are each PEG polymers composed not comprises a linker. In further embodiments a PEG of the same type of subunits, which have similar geometries comprising a functional group is directly conjugated to a and similar molecular weights . Typically in the PEGylated polypeptide . 30 Illustrative examples of linkers or linkages useful in DRS polypeptides of general formula ( I ) , ( II ), ( IIA ), ( IIB ) , particular embodiments of the present invention include , but ( IIC ) or ( IID ) , each PEG moiety , X , may be end -capped , are not limited to one or more of the following: O - , having at least one terminus capped with a relatively inert ~ NH , S , C ( 0 ) , C ( O ) _ NH, NHC ( O ) NH, group , R1. Suitable inert groups for R , include, but are not | 0 C ( O ) _ NH , C ( S ) , CH? , – CH2 – CH , , limited to alkyl groups, alkoxy groups , aryl groups , and 35 CH , CH2CH2– CH2CH2 CH2 - CH2 - , sugars, such as, for example glucose , galactose , fructose , or 0 _ CH2 – CH2 - 0 , 0 - CH2- CH2 - , sucrose . In particular embodiments , R , is an alkoxy group _ CHO CH , — , CH , CH , 0 , -O CH , — including, but not limited to methoxy, ethoxy , propoxy, CH2 - CH2 – CH2 – 0 CH2– CH2 – CH2 – butoxy , pentoxy, hexoxy , or benzyloxy . In some embodi- CH . _ 0 _ CH , CH2CH2CH20 - , ments , R , is methoxy, and the PEG moiety is a methoxy - 40 0 CH , CH , CH , CH , CH , - 0 _ CH , PEG or mPEG . CH , CH?, CH , CH2- O CH2CH2 , Those of ordinary skill in the art will recognize that the CH , CH2CH2 - O - CH2 - , CH2CH2CH2 foregoing discussion describing linear and branched chain CH - 04 , C ( O ) - NH - CH2 - , - C ( O ) - NH - CH2 PEGs for use in forming a DRS polypeptide conjugate is by CH - , - CH2 - C ( O ) - NH - CH2 - CH2 CH2- C no means exhaustive and is merely illustrative, and that all 45 (0 ) — NH — , C ( O ) - NH - CH2- CH2- CH2 - , water soluble polymers , and PEG structures having the CH2- C ( O ) - NH - CH2 - CH2 – CH2CH2 - C qualities described herein are contemplated . Moreover, ( 0 ) NH CH? ” , CH?CH, CH, C ( 0 ) NH?, based on the instant invention , one of ordinary skill in the art C ( 0 ) NH CH2 CH2 CH2 CH2, CHÚC can readily determine the appropriate size and optimal ( 0 ) - NH - CH2 - CH2 - CH , — — CH , CH - C ( O ) structure of alternative PEGylated DRS polypeptides using 50 NH - CH2CH2 – CH2CH2CH2- C (O )- NH routine experimentation , for example , by obtaining the CH? , CH2 CH2 CH2C( ) NH – CH2 – CH2 clearance profile for each conjugate by administering the CH2CH2CH2- CH2- C ( O ) - NH - NH - C ( 0 ) conjugate to a patient and taking periodic blood and /or urine CH2– CH2 - NH - C ( O ) CH2– CH2 CH2 – samples, as described herein . Once a series of clearance NHC ( 0 ) CH , — , - NH - C ( O ) - CH2CH , — profiles has been obtained for each tested conjugate , a 55 CH2- NH C ( O ) CH2CH2, CH - CH2- NH - C conjugate or mixture of conjugates , having the desired ( O ) CHA CHA, C ( 0 ) NH CH?, C ( O ) NH clearance profile ( s) can be determined CH = CH2 - , OC( O ) _ NH _ CH2 , OC( O ) Linkers NHCH2 CH2 = , > NH CH?, – NH2CH2 In particular embodiments , the conjugates of the inven - CH2– CH2 - NH - CH2 - , - CH2CH2 - NH tion comprise one or more linkers , e . g . , L , L1, L2. In a linear 60 CH2– C ( O ) CH2 – C ( O ) CH2CH2 - , PEGylated DRS conjugate , linkers separate the PEG poly - CH2- C ( O ) - CH2 – CH2CH2C(O ) CH2 mers from the covalent linkage to a DRS polypeptide. In a CH2 CH2- C ( O ) CH2CH2– CH2– CH2- C branched PEGylated DRS conjugate , linkers separate the (0 ) — , - CH2 - CH2CH2 - C (O ) - NH - CH2CH2 – PEG polymers from the branch moiety and/ or the branch NH - CH2CH2CH2 - C ( O ) - NH - CH2 - CH2 moiety from the covalent bond that links the conjugate to a 65 NH - C ( O ) - , - CH2CH2CH2- C ( O ) - NH - CH2 DRS polypeptide of the invention . Each linker can be CH2- NH - C ( O ) - CH2- , bivalent cycloalkyl group , independently selected . Each linker in a branched conjugate — N (R ) , Rºis H or an organic radical selected from the US 9 ,822 ,353 B2 119 120 group consisting of alkyl , substituted alkyl, alkenyl, substi and acid labile linkers . Among the acid labile linkers can be tuted alkenyl, alkynyl, substituted alkynyl, aryl and substi disulfide bond , hydrazone - containing linkers and thiopropi tuted aryl. Additionally , any of the above linker moieties onate - containing linkers . may further include an ethylene oxide oligomer chain com Enzymatically degradable linkages suitable for use in prising 1 to 20 ethylene oxide monomer units fi. e . , 5 particular embodiments of the present invention include , but - (CH _ CH 01- 2014 That is , the ethylene oxide oligomer are not limited to : an amino acid sequence cleaved by a chain can occur before or after the linker , and optionally in serine protease such as thrombin , chymotrypsin , trypsin , between any two atomsof a linker moiety comprised of two elastase , kallikrein , or substilisin . Illustrative examples of or more atoms. Also , the oligomer chain would not be thrombin -cleavable amino acid sequences include, but are considered part of the linker moiety if the oligomer is 10 not limited to : -Gly - Arg -Gly - Asp - (SEQ ID NO :46 ) , -Gly adjacent to a polymer segment and merely represent an Gly - Arg - , -Gly - Arg -Gly -Asp -Asn -Pro - (SEQ ID NO :47 ) , extension of the polymer segment. -Gly - Arg -Gly - Asp -Ser - (SEQ ID NO :48 ) , -Gly - Arg -Gly Each linker moiety may be hydrolytically stable or may Asp - Ser- Pro - Lys - (SEQ ID NO :49 ) , -Gly - Pro - Arg - , - Val include a releasable linkage such as a physiologically hydro Pro -Arg -, and Phe - Val - Arg -. Illustrative examples of lyzable or enzymatically degradable linkage. 15 elastase -cleavable amino acid sequences include , but are not Releasable Linkers limited to : - Ala - Ala - Ala - , - Ala - Ala -Pro - Val - (SEQ ID In particular embodiments , the PEG and related polymer NO :50 ), - Ala - Ala - Pro -Leu - (SEQ ID NO : 51) , - Ala - Ala -Pro derivatives of the invention are capable of imparting Phe - (SEQ ID NO :52 ), - Ala - Ala -Pro - Ala - (SEQ ID NO :53 ) , improved water solubility , increased size , a slower rate of and - Ala - Tyr -Leu - Val- (SEQ ID NO : 54 ) . kidney clearance , and reduced immunogenicity to a conju - 20 Enzymatically degradable linkages suitable for use in gate formed by covalent attachment thereto , while also particular embodiments of the present invention also include providing for controllable hydrolytic release of a DRS amino acid sequences that can be cleaved by a matrix polypeptide into an aqueous environment — by virtue of the metalloproteinase such as collagenase , stromelysin , and design of the linkages provided herein . The invention can be gelatinase . Illustrative examples of matrix metalloprotein used to enhance the solubility and blood circulation lifetime 25 ase -cleavable amino acid sequences include, but are not of DRS polypeptides in the bloodstream , while also deliv - limited to : -Gly - Pro - Y -Gly -Pro - Z - (SEQ ID NO :55 ) , -Gly ering a DRS polypeptide into the bloodstream that, subse - Pro - , Leu -Gly - Pro - Z - ( SEQ ID NO :56 ), -Gly - Pro - Ile -Gly quent to hydrolysis , is substantially free of PEG . The inven - Pro - Z - (SEQ ID NO :57 ) , and - Ala -Pro -Gly -Leu - Z - (SEQ tion is especially useful in those cases where DRS ID NO : 58 ) , where Y and Z are amino acids . Illustrative polypeptides, when permanently conjugated to PEG , dem - 30 examples of collagenase - cleavable amino acid sequences onstrate reduced activity . By using the linkages as provided include, but are not limited to : -Pro -Leu - Gly -Pro - D - Arg - Z herein , such DRS polypeptides can maintain their therapeu - (SEQ ID NO : 59 ) , - Pro -Leu -Gly -Leu -Leu -Gly - Z - (SEQ ID tic activity when in conjugated form . NO : 60 ), - Pro -Gln - Gly - Ile - Ala -Gly - Trp - (SEQ ID NO :61 ) , Representative , but non - limiting , examples of releasable - Pro -Leu -Gly - Cys (Me ) -His - (SEQ ID NO :62 ) , -Pro - Leu linkages include physiologically cleavable bonds, hydrolyz - 35 Gly - Leu - Tyr- Ala - (SEQ ID NO :63 ) , -Pro -Leu - Ala -Leu - Trp able bonds , and enzymatically degradable linkages . In par- - Ala -Arg - (SEQ ID NO :64 ) , and - Pro - Leu - Ala - Tyr - Trp - Ala ticular embodiments , a releasable linkage has a half life at Arg - (SEQ ID NO : 65 ) , where Z is an amino acid . An pH 7 . 4 , 25° C ., e . g . , a physiological pH , human body illustrative example of a stromelysin - cleavable amino acid temperature , of about 30 min ., about 1 hour , about 2 hour, sequence is - Pro - Tyr -Ala - Tyr- Tyr -Met - Arg - (SEQ ID about 3 hours , about 4 hours , about 5 hours , about 6 hours , 40 NO : 66 ); and an example of a gelatinase - cleavable amino about 12 hours , about 18 hours , about 24 hours, about 36 acid sequence is - Pro -Leu -Gly -Met - Tyr - Ser - Arg - (SEQ ID hours , about 48 hours , about 72 hours , or about 96 hours or NO :67 ) . more or any intervening half - life . One having skill in the art Enzymatically degradable linkages suitable for use in would appreciate that the half life of a PEGylated DRS particular embodiments of the present invention also include polypeptide can be finely tailored by using a particular 45 amino acid sequences that can be cleaved by an angiotensin releasable linkage . converting enzyme , such as, for example , -Asp -Lys -Pro -, Appropriate hydrolytically unstable or weak linkages -Gly - Asp -Lys - Pro - (SEQ ID NO :68 ) , and -Gly -Ser - Asp include , but are not limited to : carboxylate ester , phosphate Lys -Pro - (SEQ ID NO :69 ) . ester, anhydride , acetal , ketal , acyloxyalkyl ether , imine , Enzymatically degradable linkages suitable for use in orthoester , thio ester , thiol ester , carbonate , and hydrazone , 50 particular embodiments of the present invention also include peptides and oligonucleotides . amino acid sequences that can be degraded by cathepsin B , Additional illustrative embodiments of hydrolytically such as , for example , Val- Cit , Ala -Leu - Ala -Leu (SEQ ID unstable or weak linkages include, but are not limited to : NO :70 ), Gly - Phe -Leu -Gly (SEQ ID NO :71 ) and Phe- Lys . - 02C – (CH2 ) - 0 , where b is from 1 to 5 , - 0 Examples of hydrolytically stable linkages include , but (CH2 ) 5 - C02 - ( CH2) . — , where b is from 1 to 5 and c is 55 are not limited to , the following: succinimide, propionic from 2 -5 , - 0 - (CH ) n - C02 - ( CH2) . - 04 , where b is acid , carboxymethylate linkages, ethers , carbamates , from 1 to 5 and c is from 2 -5 , (CH2 ) . OPO3 — amides , amines , carbamides, imides, aliphatic C — C bonds , ( CH2) - , where b is 1 - 5 and b ' is 1 - 5, - C (O ) - (NH thio ethers , thiocarbamates , thiocarbamides, and the like . CHR - CO ) . - NH - CHR — , where a is 2 to 20 and R is a Generally, a hydrolytically stable linkage is one that exhibits substituent found on an a - amino acid , O (CH2 ) . — 60 a rate of hydrolysis of less than about 1 - 2 % per day under CO , CHCH , CH , — , where b is from 1- 5 , physiological conditions . 0 C6H4- CH = N (CH2 ) - 0 , where b is from 1 -5 , In certain embodiments , the half- life of the PEGylated and – O (CH2 ) – CH2CH = N — ( CH2) . - 0 , where DRS polypeptide conjugate is regulated by incorporating each b is independently from 1 - 5 . one or more linkers of various stability into the conjugate . Other illustrative examples of releasable linkers can be 65 For example, if a relatively stable PEGylated DRS conjugate benzyl elimination - based linkers , trialkyl lock -based linkers is desired , the conjugate can comprise one or more linkers (or trialkyl lock lactonization based ), bicine- based linkers , that are hydrolytically stable or resistant to degradation . US 9 , 822 ,353 B2 121 122 Hydrolytically stable linkers are known in the art and are idizaole functional group and a DRS polypeptide comprising described elsewhere herein , and generally result in a rate of an amino group results in an carbamate linkage ; a reaction hydrolysis of about 0 . 5 % , about 1 % , about 2 % , about 3 % , between a PEG comprising a p -nitrophenyl carbonate func about 4 % , or about 5 % per day . Illustrative examples of tional group and a DRS polypeptide comprising an amino hydrolytically stable linkers that can be used in PEGylated 5 group results in an carbamate linkage ; a reaction between a PEG comprising a trichlorophenyl carbonate functional DRS conjugates of the invention include, but are not limited group and a DRS polypeptide comprising an amino group to : succinimide, propionic acid , carboxymethylate linkages , results in an carbamate linkage ; a reaction between a PEG ethers , carbamates , amides , amines , carbamides , imides , comprising a thio ester functional group and a DRS poly aliphatic C — C bonds, thio ethers , thiocarbamates , thiocar peptide comprising an n - terminal amino group results in an bamides, and the like. amide linkage ; a reaction between a PEG comprising a In other embodiments , a PEGylated DRS conjugate com proprionaldehyde functional group and a DRS polypeptide prises one or more releasable linkages that result in a shorter comprising an amino group results in a secondary amine half - life and more rapid clearance of the conjugate . For linkage ; a reaction between a PEG comprising a butyralde example , PEG can be prepared with ester linkages in the hyde functional group and a DRS polypeptide comprising an polymer backbone that are subject to hydrolysis. This hydro - 15 amino group results in a secondary amine linkage ; a reaction lysis results in cleavage of the polymer into fragments of between a PEG comprising a acetal functional group and a lower molecular weight . Other hydrolytically degradable DRS polypeptide comprising an amino group results in a linkages are known in the art and described elsewhere secondary amine linkage ; a reaction between a PEG com herein , and include carbonate linkages ; imine linkages prising a piperidone functional group and a DRS polypep resulting , for example , from reaction of an amine and an 20 tide comprising an amino group results in a secondary amine aldehyde; phosphate ester linkages formed , for example , by linkage ; a reaction between a PEG comprising a methylke reacting an alcohol with a phosphate group ; hydrazone tone functional group and a DRS polypeptide comprising an linkages which are typically formed by reaction of a hydraz - amino group results in a secondary amine linkage; a reaction ide and an aldehyde ; acetal linkages that are typically between a PEG comprising a tresylate functional group and formed by reaction between an aldehyde and an alcohol; and 25 a DRS polypeptide comprising an amino group results in a ortho ester linkages that are , for example , formed by reac - secondary amine linkage ; a reaction between a PEG com tion between a formate and an alcohol. Other suitable prising a maleimide functional group and a DRS polypeptide releasable linkers for use in branched conjugates of the comprising an amino group results in a secondary amine invention include enzymatically degradable linkages and linkage ; a reaction between a PEG comprising a aldehyde discussed elsewhere herein . 30 functional group and a DRS polypeptide comprising an Such optional features of the conjugate , i. e ., the introduc amino group results in a secondary amine linkage ; and a tion of one or more degradable linkages into the polymer reaction between a PEG comprising a hydrazine functional chain , may provide for additional control over the final group and a DRS polypeptide comprising an carboxylic acid desired pharmacological properties of the conjugate upon group results in a secondary amine linkage . administration . For example , a large and relatively inert 35 In another non -limiting example , a reaction between a PEGylated DRS polypeptide conjugate ( i. e ., having one or PEG comprising a maleimide functional group and a DRS more high molecular weight PEG chains attached thereto , polypeptide comprising a thiol group results in a thio ether for example , one or more PEG chains having a molecular linkage ; a reaction between a PEG comprising a vinyl weight greater than about 10 ,000 ) may be administered , sulfone functional group and a DRS polypeptide comprising which is then hydrolyzed in vivo to generate a bioactive 40 a thiol group results in a thio ether linkage; a reaction DRS polypeptide conjugate possessing a portion of the between a PEG comprising a thiol functional group and a original PEG chain or lacking PEG entirely . In this way, the DRS polypeptide comprising a thiol group results in a properties of the PEGylated DRS polypeptide conjugate can di- sulfide linkage; a reaction between a PEG comprising a be more effectively tailored to balance the bioactivity and orthopyridyl disulfide functional group and a DRS polypep circulating half -life of the conjugate over time. 45 tide comprising a thiol group results in a di -sulfide linkage ; Covalent Linkages (“ Y ” ) and a reaction between a PEG comprising an iodoacetamide In forming the PEGylated DRS polypeptide conjugates of functional group and a DRS polypeptide comprising a thiol the invention , the branching moiety or a linker comprises a group results in a thio ether linkage . functional group that forms a covalent bond or linkage , Y , The particular coupling chemistry employed will depend with a functional group on a DRS polypeptide , thereby 50 upon the structure of the biologically active agent, the forming a conjugate . Exemplary functional groups of linkers potential presence of multiple functional groups within the and DRS polypeptides are disclosed elsewhere herein , supra . biologically active molecule , the need for protection /depro Illustrative examples of covalent linkages, Y , in any of the tection steps , chemical stability of the molecule , and the like, PEGylated DRS polypeptide conjugates of the invention and will be readily determined by one skilled in the art . include , but are not limited to : amide , secondary amine , 55 Illustrative linking chemistry useful for preparing the carbonyl, carboxylate , carbamate , carbamide , ester, formyl, branched polymer conjugates of the invention can be found , acyl, thiocarbonyl, thio ester, thioacetate , thioformate , thio for example , in Wong, S . H ., (1991 ) , “ Chemistry of Protein ether, alkoxyl, phosphoryl, phosphonate , phosphinate , Conjugation and Crosslinking " , CRC Press , Boca Raton , amino , amido , amidine , imine , cyano , nitro , azido , disulfide , Fla . and in Brinkley, M . ( 1992 ) “ A Brief Survey of Methods sulthydryl, sulfate , sulfonate , sulfamoyl, sulfonamido , sul- 60 for Preparing Protein Conjugates with Dyes , Haptens, and fonyl, heterocyclyl , aralkyl, aromatic moiety , hydrazone , Crosslinking Reagents ” , in Bioconjug . Chem . , 3 , 2013 . heteroaromatic moiety , imino , sulfamoyl, sulfonate , silyl , More specific structural embodiments of the conjugates of ether , or alkylthio . the invention will now be described , all of which are For example , a reaction between a PEG comprising a intended to be encompassed by the structure above . The succinimidyl ester functional group and a DRS polypeptide 65 specific structures shown below are presented as exemplary comprising an amino group results in an amide linkage ; a structures only, and are not intended to limit the scope of the reaction between a PEG comprising a oxycarbonylim - invention . US 9 ,822 ,353 B2 123 124 In one embodiment, a PEGylated DRS polypeptide com -continued prises any of the structures 1 - 5 : Ê (( 1 ) 5 H3CO - (CH2CH20 ) n - CH2CH2 - SH - CH2CH2 - C - NH - DRS, or HzC — (OCH2CH2 )n - O - CH2CH2 - C — NH — DRS, Û HzC — (OCH2CH2 ) n - 0 — C — NH — DRS , H3CO - ( CH CH2O) – – NH - DRS, (3) 10 wherein “ NH ” of NH -DRS refers to an amino group of a DRS polypeptide and n is any integer from 1 to 800 . H?c – ( OCH2CH2) n – 0 – CH2 – – NH - DRS, In another embodiment, a PEGylated DRS polypeptide comprises the structure :
© H3CO ( CH2CH2O) n – CH2CH2 = C _ NH?N = C – DRS, © H3CO - ( CH2CH2O) = CH - CH2 – 0 – CH2 – C?NH?N = c _ DRS, @ H3CO - ( CH2CH2O) = CH - CHU - NH - C?NH?N = c - DRS, @ H?CO – CH2CH2O) , = CH -CHU - NH – NH?c – NH?N = c - DRS, @ v
= H4C - ( CH2CH2O ) = CH -CH2 - NH – 0 – NH?N = c - DRS, or @ H3CO - ( CH2CH2O) – CH2CH2 - NH – NH – C?NH?N =c DRS,
wherein “ C ” of C -DRS refers to carboxyl group of a DRS 40 polypeptide and n is any integer from 1 to 800 . In another embodiment, a PEGylated DRS polypeptide comprises the structure :
( 12 )
- S - DRS H3COcomo - ( CH - CH2O) - –e CH2 - –L C - NH -. CH- CH - NH – & — CH - CH2 - N . ( 13 )
- S — DRS H3CO — ( CH2CH2O )n - CH2CH2 - N
samenstenment ( 14 ) S — DRS noH3CO- - ( CH2CH2Oamatovaciu) = CH - CH2 - NH – C – CH2CH2 - N . US 9 ,822 ,353 B2 125 126 125 -continued ( 15 )
- S - DRS H?CO < ( CH2CH2O) = CH - CH - CHU - NH = C = CH- CHU - N .
( 16 )
- S — DRS H3CO - ( CH2CH2) = 0 = CH - CH2 – & — NH = CH - CH2 - NH CH= CH - CH - N
( 17 ) ( 17 ) H3C — (OCH2CH2 )n - O - CH2CH2 - S - CH2CH2 - S — DRS
1. 0 - c01c15- 0 – anas. LainO =- - (18 )
=O H3C — (OCH2CH2 ) n - O - CH2CH2 — C — NH - CH2CH2 — s — s — DRS, or ( 19 ) H3C0 — ( CH2CH20 )n - CH2CH2CH2CH2 — 5 — — DRS ,
wherein “ S ” of S -DRS refers to thiol group of a DRS polypeptide and n is any integer from 1 to 800 . In one embodiment, a branched PEGylated DRS poly peptide of the invention comprises the structure:
(20 ) H?C ( OCH2CH2 – 0 – – NH CH2 7-8CH2 CH2 CH2 - S — DRS HC - c - NH - CH - CHU - NH?? – CH2CH2 - N HzC — (OCH , CH ), — 0 - 0 — NH
=
O H3CO — (CH2CH20 ) , — CH H3C0 — ( CH2CH2O ) n - CH - S - DRS H?c – 0 – CH2CH2CH2 - NH – & — ( CH2) 2- 5 - N
H3C0 — (CH2CH20 ). — CH2 S - DRS HC _ 0 – CH2CH2CH2 - NH — — ( CH2) 2 - 5 - N H3CO - (CH2CH20 )n - CH2 US 9 ,822 , 353 B2 127 128 -continued ( 23 )
H3C — (OCH2CH2 ) n - N - O - O - CH2 DE FS- DRS HC _ 0 – CH2CH2CH2 – – NH – CH2CH2 – NH — — ( CH2CH2) 2 - 5 - N H3C — (OCH2CH2 ) - N - C - 0 - CH2
= H?CO -( CH -CHO - CH, ( 24 ) H3C0 — ( CH2CH20 )n - CH2 H3C0 — ( CH2CH20 )n - CH S - DRS or H , C — 0 — CH2 - CH2CH2CH2 - NH - C - (CH2 ) 2 -5 — N
H3C0 — ( CH2CH20 ) . — CH2 (25 ) H3C0 — ( CH2CH20 )n - CH2 H3C0 — ( CH2CH20 )n - CH2 HzC0 — (CH2CH20 ) n - CH S - DRS H?C = 0 = CH - CH- CH - NH - C = ( CH2) 2 - 5 - N
wherein “ S ” of S -DRS refers to thiol group of a DRS comprising the sequence set forth in SEQ ID NO : 4 ( 1- 171) , polypeptide and n is independently selected any integer from and which differs from SEQ ID NO : 4 by at least the 1 to 800 . 35 mutation C76S , and further comprises a maleimide In different embodiments of any of the disclosed DRS monomethoxy polyethylene glycol (mPEG ) derivative of conjugates, the DRS polypeptide is a full -length DRS poly general formula ( 1) having a molecular weight of about peptide, or a truncated , or splice variant thereof , ( see 1 ,000 to 60, 000 Daltons that is covalently attached via a thio Sequence Listing or Tables ) which comprises a linear or branched chain polyethylene glycol (mPEG ) derivative of 401ether linkage to C130 . general formula ( I) , ( II ), ( IIA ) , ( IIB ), ( IIC ) or ( IID ) of about In some embodiments , the present invention provides 1, 000 to 60 ,000 Daltons. In one aspect of any of these DRS PEGylated aspartyl- tRNA synthetase (DRS ) polypeptides, conjugates , the conjugated polymer has a structure selected comprising the sequence set forth in SEQ ID NO : 5 ( 1 - 174 ), from any of compounds ( 1 ) to (25 ) . and which differs from SEO ID NO : 5 by at least the In some embodiments , the DRS polypeptide differs from 45smutation " C76S , and further comprises a maleimide any DRS polypeptides described herein (see Sequence List monomethoxy polyethylene glycol (mPEG ) derivative of ing or Tables ) by at least one amino acid selected from C76S general formula (1 ) having a molecular weight of about and C130S . In some embodiments the conjugate comprises 1 , 000 to 60 ,000 Daltons that is covalently attached via a thio a linear or branched chain polyethylene glycol (mPEG ) ether linkage to C130 . derivative of general formula ( I ), ( II) , ( IIA ), (IIB ) , (IIC ) or 50 In some embodiments , the present invention provides ( IID ) of about 1 , 000 to 60 , 000 Daltons that is covalently PEGylated aspartyl- tRNA synthetase (DRS ) polypeptides , attached to the DRS polypeptide via a thio ether linkage . In comprising the sequence set forth in SEQ ID NO :6 ( 1 - 182 ) , one aspect of any of these DRS conjugates , the conjugated and which differs from SEQ ID NO : 6 by at least the polymer has a structure selected from any of compounds mutation C76S , and further comprises a maleimide ( 12 ) to (25 ) . In one aspect the conjugated DRS polypeptide 55 monomethoxy polyethylene glycol (mPEG ) derivative of is coupled via amino acid C130 general formula (I ) having a molecular weight of about In some embodiments , the present invention provides 1 ,000 to 60 , 000 Daltons that is covalently attached via a thio PEGylated aspartyl- tRNA synthetase (DRS ) polypeptides , ether linkage to C130 . comprising the sequence set forth in SEQ ID NO : 3 (1 - 154 ), In some embodiments , the present invention provides and which differs from SEQ ID NO : 3 by at least the 60 PEGylated aspartyl- tRNA synthetase (DRS ) polypeptides, mutation C76S, and further comprises a maleimide comprising the sequence set forth in SEQ ID NO : 7 ( 1 - 184 ) , monomethoxy polyethylene glycol (mPEG ) derivative of and which differs from SEQ ID NO : 7 by at least the general formula (1 ) having a molecular weight of about mutation C76S , and further comprises a maleimide 1 , 000 to 60 ,000 Daltons that is covalently attached via a thio monomethoxy polyethylene glycol (mPEG ) derivative of ether linkage to C130 . 65 general formula ( 1 ) having a molecular weight of about In some embodiments , the present invention provides 1 , 000 to 60 , 000 Daltons that is covalently attached via a thio PEGylated aspartyl- tRNA synthetase (DRS ) polypeptides , ether linkage to C130 . US 9 ,822 ,353 B2 129 130 In some embodiments , the present invention provides chronic inflammatory responses, or treat any one or more PEGylated aspartyl -tRNA synthetase (DRS ) polypeptides , diseases or conditions associated with chronic inflammation . comprising the sequence set forth in SEQ ID NO : 11 (23 - Certain specific inflammatory responses include cytokine 154 ) , and which differs from SEQ ID NO : 11 by at least the production and activity , and related pathways. For instance , mutation C76S , and further comprises a maleimide 5 certain exemplary embodiments relate to modulating cell monomethoxy polyethylene glycol (mPEG ) derivative of signaling through nuclear factor -kB (NF -kB ) , such as by general formula ( 1 ) having a molecular weight of about increasing the downstream activities of this transcription factor. In certain instances, increases in NF -kB activity can 1 ,000 to 60 ,000 Daltons that is covalently attached via a thio lead to increases in cytokine signaling or activity , such as ether linkage to C130 . 10 pro - inflammatory cytokines ( e . g ., TNFalpha or beta ), and Methods for Use anti - inflammatory cytokines ( e. g ., IL - 10 ). Embodiments of the present invention relate to the dis Criteria for assessing the signs and symptoms of inflam covery that aspartyl- tRNA synthetase (DRS ) polypeptides , matory and other conditions , including for purposes of and fragments and variants thereof, with altered cysteine making differential diagnosis and also for monitoring treat content offer improved methods of modulating Toll like 15 ments such as determining whether a therapeutically effec receptors ( TLRs) in a variety of useful ways , both in vitro tive dose has been administered in the course of treatment, and in vivo . The compositions of the invention may thus be e . g ., by determining improvement according to accepted useful as immunomodulators for treating anti- or pro - in clinical criteria , will be apparent to those skilled in the art flammatory indications by modulating the cells that mediate , and are exemplified by the teachings of e . g ., Berkow et al. , either directly or indirectly , autoimmune and /or inflamma - 20 eds ., The Merck Manual, 16th edition , Merck and Co . , tory disease , conditions and disorders . The utility of the Rahway, N . J . , 1992 ; Goodman et al. , eds ., Goodman and compositions of the invention as immunomodulators can be Gilman ’s The Pharmacological Basis of Therapeutics, 10th monitored using any of a number of known and available edition , Pergamon Press , Inc ., Elmsford , N . Y . , ( 2001 ) ; techniques in the art including , for example , migration Avery ' s Drug Treatment : Principles and Practice of Clinical assays ( e .g ., using leukocytes or lymphocytes) , cytokine 25 Pharmacology and Therapeutics , 3rd edition , ADIS Press , production assays , or cell viability assays ( e . g ., using Ltd ., Williams and Wilkins, Baltimore, Md . ( 1987 ) ; Ebadi, B - cells , T -cells , monocytes or NK cells ). Pharmacology , Little , Brown and Co . , Boston , ( 1985 ) ; " Inflammation ” refers generally to the biological response Osolci al. , eds. , Remington ' s Pharmaceutical Sciences , 18th of tissues to harmful stimuli , such as pathogens, damaged edition , Mack Publishing Co . , Easton , Pa . ( 1990 ) ; Katzung , cells ( e . g ., wounds ), and irritants . The term “ inflammatory 30 Basic and Clinical Pharmacology, Appleton and Lange , response ” refers to the specific mechanisms by which Norwalk , Conn . ( 1992 ) . inflammation is achieved and regulated , including , merely Also included are methods of modulating an immune by way of illustration , immune cell activation or migration , response , such as an innate immune response . As used cytokine production , vasodilation , including kinin release , herein , the term “ immune response” includes a measurable fibrinolysis , and coagulation , among others described herein 35 or observable reaction to an antigen , vaccine composition , or and known in the art . Ideally , inflammation is a protective immunomodulatory molecule mediated by one or more cells attempt by the body to both remove the injurious stimuli and of the immune system . An immune response typically initiate the healing process for the affected tissue or tissues begins with an antigen or immunomodulatory molecule In the absence of inflammation , wounds and infections binding to an immune system cell. A reaction to an antigen would never heal , creating a situation in which progressive 40 or immunomodulatory molecule may be mediated by many destruction of the tissue would threaten survival . On the cell types, including a cell that initially binds to an antigen other hand , excessive or chronic inflammation may associate or immunomodulatory molecule and cells that participate in with a variety of diseases, such as hay fever, atherosclerosis , mediating an innate, humoral, cell- mediated immune and rheumatoid arthritis , among others described herein and response . known in the art . 45 An “ innate immune response , ” as used herein , may Clinical signs of chronic inflammation are dependent involve binding of pathogen - associated molecular patterns upon duration ofthe illness , inflammatory lesions , cause and (PAMPs ) or damage - associated molecular pattern mol anatomical area affected , ( see , e . g . , Kumar et al. , Robbins ecules , (DAMPS ) or a DRS polypeptide to cell surface Basic Pathology - 8 ft Ed ., 2009 Elsevier , London ; Miller , L receptors, such as toll- like receptors . Activation of toll - like M , Pathology Lecture Notes , Atlantic Veterinary College , 50 receptors and Ipaf -signaling pathways in response to PAMPs Charlottetown , PEI, Canada ) . Chronic inflammation is asso or other signals leads to the production of immunomodula ciated with a variety of pathological conditions or diseases, tory molecules, such as cytokines and co -stimulatory mol including , for example , allergies , Alzheimer ' s disease , ane - ecules , which induce and /or enhance an immune response . mia , aortic valve stenosis , arthritis such as rheumatoid Cells involved in the innate immune response include , for arthritis and osteoarthritis , cancer , congestive heart failure , 55 example , dendritic cells , macrophages , natural killer cells , fibromyalgia , fibrosis , heart attack , kidney failure , lupus, and neutrophils , among others. gout and gout flares , pancreatitis, hepatitis , stroke, surgical Certain embodiments relate to increasing an innate complications, acetaminophen - induced liver toxicity , immune response . Other embodiments relate to decreasing inflammatory lung disease , inflammatory bowel diseases an innate immune response . In certain aspects , an innate including Crohn ' s disease (CD ) , necrotizing enterocolitis, 60 immune response is mediated by one or more toll - like and ulcerative colitis (UC ) , atherosclerosis , neurological receptors ( TLRs ) , such as TLR2 and /or TLR4 . Certain DRS disorders, (neuro ) inflammatory disorders, diabetes , meta - polypeptides of the invention bind to TLRS such as TLR2 bolic disorders, obesity , graft versus host disease , myositis , and /or TLR4. More generally , DRS polypeptides are capable emphysema/ COPD and psoriasis , among others described of selectively modulating host immune responses via spe herein and known in the art . Hence, DRS polypeptide 65 cific interactions with Toll like receptors , and may therefore compositions may be used to treat or manage chronic be used to modulate host immune responses and thereby to inflammation ,modulate any of one ormore of the individual manage diseases and conditions associated with the same, as US 9 ,822 ,353 B2 131 132 described herein and known in the art . Exemplary uses for aureus, S . epidermidis, S . saprophytics , Streptococcus aga the DRS polypeptides of the invention therefore include lactiae , S. pneumoniae , S. pyogenes, Treponema pallidum , both methods for the treatment and prevention of TLR Vibrio cholera , Yersinia pestis, Bordatella pertussis , and associated diseases, as well as for use in the breakdown of otitis media ( e . g . , often caused by Streptococcus pneumo immune tolerance , for example for the development of 5 niae , Haemophilus influenzae , or Moraxella catarrhalis ) , vaccines , and in the development of immune therapies . among others described elsewhere herein . Exemplary " TLR associated diseases” include for Examples of parasitic infectious diseases include , but are example , inflammatory conditions , and diseases and disor - not limited to , Amoebiasis ( e . g ., Entemoeba histolytica ) , ders associated with the dysfunction of the innate immune Hookworm Disease ( e . g . , nematode parasites such as Neca response , including for example , autoimmunity , cancer, 10 tor americanus and Ancylostoma duodenale ) , Leishmania allergy , autoimmunity , radiation induced toxicity, and the sis , Malaria ( four species of the protozoan parasite Plasmo treatment and prevention of bacterial and viral infections. dium ; P . falciparum , P . vivax , P . ovale , and P . malariae ) , Accordingly in one embodiment the present invention Schistosomiasis (parasitic Schistosoma ; S . mansoni, S . hae includes a method for treating a TLR associated disease in matobium , and S . japonicum ) , Onchocerca volvulus (River a subject in need thereof, comprising administering to the 15 blindness ) , Trypanosoma cruzi (Chagas disease American subject a therapeutic dose of a DRS polypeptide ( e . g . , any of sleeping sickness ) , and Dracunculus medinensis , lymphatic the proteins or nucleic acids listed in Tables D1 to D8 which filariasis . Certain DRS polypeptide compositions may be i ) retain detectable non canonical activity , and ii ) which useful in the treatment or reduction of endotoxic shock , comprise , or have been modified to comprise at least one which often results from exposure to foreign antigens , such mutation at either Cys76 or Cys130 (using the numbering of 20 as lipopolysacchande (LPS ) . Because endotoxic shock can SEQ ID NO : 1 ) which replaces the native cysteine with be mediated by TLR signaling , and naturally -occurring another naturally , or non - naturally , occurring amino acid . endogenous DRS polypeptide fragments may stimulate Exemplary uses associated with the breakdown of TLRs , certain of the binding agents, antisense agents , or immune tolerance include for example the development of RNAi agents provided herein may render a subject more vaccines and adjutants comprising DRS polypeptides mixed 25 resistant to endotoxic shock by antagonizing or otherwise with antigens, or comprising DRS fusion proteins with reducing the endogenous DRS polypeptide fragment- medi antigens , which exhibit enhanced immunogenicity . In some ated stimulation of TLR2 and / or TLR4 . embodiments the antigen is a self - antigen . DRS polypeptide Also included are methods of treating immune diseases. compositions that stimulate innate immunity ( e . g ., via TLR2 Illustrative immune system diseases, disorders or conditions and / r TLR4) can be useful in the treatment of a wide variety 30 that may be treated according to the present invention of conditions, either alone or in combination with other include, but are not limited to , primary immunodeficiencies , therapies . Specific examples of such conditions include immune -mediated thrombocytopenia , Kawasaki syndrome, infectious diseases , such as bacterial, viral, and parasitic bone marrow transplant ( for example , recent bone marrow infectious diseases . DRS polypeptide compositions that transplant in adults or children ) , chronic B cell lymphocytic stimulate innate immunity can also be useful as vaccine 35 leukemia , HIV infection ( for example , adult or pediatric adjuvants , to enhance a subject 's immune response to the HIV infection ), chronic inflammatory demyelinating poly primary antigen , whether in a live , attenuated , or other type neuropathy , post- transfusion purpura , and the like . of vaccine . Additionally, further diseases , disorders and conditions Examples of viral infectious diseases or agents ( and their include Guillain -Barre syndrome, anemia ( for example , ane corresponding vaccines ) include , but are not limited to , 40 mia associated with parvovirus B19 , patients with stable Hepatitis A , Hepatitis B , Hepatitis C , Hepatitis E , Calicivi- multiple myeloma who are at high risk for infection ( for ruses associated diarrhoea , Rotavirus diarrhoea , Haemophi- example , recurrent infection ) , autoimmune hemolytic ane lus influenzae B pneumonia and invasive disease , influenza , mia (for example, warm -type autoimmune hemolytic ane measles, mumps, rubella , Parainfluenza associated pneumo - mia ) , thrombocytopenia ( for example, neonatal thrombocy nia , Respiratory syncytial virus (RSV ) pneumonia , Severe 45 topenia ) , and immune -mediated neutropenia ) , Acute Respiratory Syndrome (SARS ) , Human papillomavi- transplantation ( for example , cytomegalovirus ( CMV ) rus , Herpes simplex type 2 genital ulcers , HIV /AIDS , Den negative recipients of CMV -positive organs) , hypogamma gue Fever, Japanese encephalitis , Tick -borne encephalitis , globulinemia ( for example , hypogammaglobulinemic neo West- Nile virus associated disease , Yellow Fever , Epstein - nates with risk factor for infection or morbidity ) , epilepsy Barr virus, Lassa fever, Crimean -Congo haemorrhagic fever, 50 ( for example , intractable epilepsy ), systemic vasculitic syn Ebola haemorrhagic fever, Marburg haemorrhagic fever, dromes, myasthenia gravis ( for example , decompensation in Rabies , Rift Valley fever, Smallpox , leprosy, upper and myasthenia gravis ) , dermatomyositis, and polymyositis . lower respiratory infections , poliomyelitis , among others. Further autoimmune diseases , disorders and conditions described elsewhere herein . include but are not limited to , autoimmune hemolytic ane Examples of bacterial infections disease or agents 55 mia , autoimmune neonatal thrombocytopenia , idiopathic include, but are not limited to , Bacillus antracis, Borellia thrombocytopenia purpura , autoimmunocytopenia , hemo burgdorferi , Brucella abortus, Brucella canus, Brucella lytic anemia , antiphospholipid syndrome, dermatitis , aller melitensis , Brucella suis, Campylobacter jejuni, Chlamydia gic encephalomyelitis , myocarditis , relapsing polychondri pneumoniae, Chlamydia psitacci, Chlamydia trachomatis , tis , rheumatic heart disease , glomerulonephritis ( for Clostridium botulinum , C . difficile , C . perfringens, C . tetani, 60 example , IgA nephropathy ) , multiple sclerosis , neuritis , Corynebacterium diphtheriae ( i. e . , diphtheria ), Enterococ uveitis ophthalmia , polyendochnopathies, purpura ( for cus, Escherichia coli, Haemophilus influenza , Helicobacter example , Henloch - Scoenlein purpura ), Reiter' s disease , pylori, Legionella pneumophila , Leptospira , Listeria mono stiff -man syndrome, autoimmune pulmonary inflammation , cytogenes , Mycobacterium leprae , M . tuberculosis , Myco - Guillain - Barre Syndrome , insulin dependent diabetes mel plasma pneumoniae, Neisseria gonorrhea , N . meningitidis , 65 litus, and autoimmune inflammatory eye disease . Pseudomonas aeruginosa , Rickettsia recketisii , Salmonella Additional autoimmune diseases , disorders or conditions typhi, S. typhimurium , Shigella sonnei, Staphylococcus include , but are not limited to , autoimmune thyroiditis ; US 9 ,822 ,353 B2 133 134 hypothyroidism , including Hashimoto ' s thyroiditis and thy - IgE ) ; inflammatory myopathies ; and other inflammatory , roiditis characterized , for example , by cell- mediated and granulomatous, degenerative , and atrophic disorders . humoral thyroid cytotoxicity ; SLE (which is often charac - Further embodiments the present invention include meth terized , for example , by circulating and locally generated ods for killing cancer cells, comprising administering a immune complexes ) ; Goodpasture ' s syndrome (which is 5 vaccine or immunogenic composition comprising a DRS often characterized , for example , by anti -basement mem - polypeptide of the invention fused to , or associated with an brane antibodies ) ; pemphigus (which is often characterized , antigen , or vector comprising a nucleic acid encoding a DRS for example , by epidermal acantholytic antibodies ) ; receptor polypeptide fused to an antigen , to a subject in need thereof. autoimmunities such as , for example, Graves ' disease Such DRS polypeptides may comprise any of the proteins or (which is often characterized , for example, by antibodies to 10 nucleic acids listed in Tables D1 to D8 which i) retain a thyroid stimulating hormone receptor ; myasthenia gravis , detectable non canonical activity , and ii ) which comprise , or which is often characterized , for example , by acetylcholine have been modified to comprise at least one mutation at receptor antibodies ) ; insulin resistance (which is often char - either Cys76 or Cys130 (using the numbering of SEQ ID acterized , for example, by insulin receptor antibodies ) ; auto - NO : 1 ) which replaces the native cysteine with another immune hemolytic anemia (which is often characterized , for 15 naturally , or non - naturally , occurring amino acid ) . In some example , by phagocytosis of antibody - sensitized red blood embodiments the antigen is a self- antigen , in some embodi cells ); and autoimmune thrombocytopenic purpura ( which is moments the antigen is a tumor derived antigen . In some often characterized , for example , by phagocytosis of anti embodiments, the antigen is a pathogen derived antigen . In body -sensitized platelets ) . some embodiments the pathogen derived antigen is derived Further autoimmune diseases , disorders or conditions 20 from a virus , bacteria or prion . In some embodiments , the include , but are not limited to , rheumatoid arthritis (which is antigen is fused to the DRS polypeptide through conjugation often characterized , for example , by immune complexes in at Cys130 . In some embodiments the antigen and DRS joints ) ; scleroderma with anti - collagen antibodies (which is polypeptide are mixed together . often characterized , for example , by nucleolar and other In some embodiments the present invention includes a nuclear antibodies ) ; mixed connective tissue disease , (which 25 method for treating a subject with cancer, or preventing the is often characterized , for example , by antibodies to extract development of cancer in a subject , comprising administer able nuclear antigens , for example , ribonucleoprotein ) ; ing a vaccine or immunogenic composition comprising a polymyositis /dermatomyositis (which is often characterized , DRS polypeptide of the invention fused to an antigen , or for example , by nonhistone anti -nuclear antibodies ); perni- vector comprising a nucleic acid encoding a DRS polypep cious anemia (which is often characterized , for example , by 30 tide fused to an antigen , wherein the vaccine elicits an antiparietal cell , antimicrosome, and anti -intrinsic factor immune response to the cancer. Such DRS polypeptides may antibodies ) ; idiopathic Addison ' s disease (which is often comprise any of the proteins or nucleic acids listed in Tables characterized , for example , by humoral and cell -mediated D1 to D8 which i ) retain detectable non canonical activity , adrenal cytotoxicity ) ; infertility (which is often character and ii ) which comprise , or have been modified to comprise ized , for example , by antispennatozoal antibodies ) ; glom - 35 at least one mutation at either Cys76 or Cys130 (using the erulonephritis (which is often characterized , for example, by numbering of SEQ ID NO : 1 ) which replaces the native glomerular basementmembrane antibodies or immune com - cysteine with another naturally , or non - naturally, occurring plexes ) ; by primary glomerulonephritis , by IgA nephropa - amino acid ) . In some embodiments the antigen is a self thy ; bullous pemphigoid ( which is often characterized , for antigen , in some embodiments the antigen is a tumor derived example , by IgG and complement in the basement mem - 40 antigen . In some embodiments , the antigen is a pathogen brane) ; Sjogren ' s syndrome (which is often characterized , derived antigen . In some embodiments the pathogen derived for example , by multiple tissue antibodies and/ or the specific a ntigen is derived from a virus , bacteria or prion . In some nonhistone antinuclear antibody (SS - B ) ) ; diabetes mellitus embodiments , the antigen is fused to the DRS polypeptide (which is often characterized , for example , by cell -mediated through conjugation at Cys130 . and humoral islet cell antibodies ) ; and adrenergic drug 45 In some embodiments the present invention includes a resistance , including adrenergic drug resistance with asthma method for overcoming tolerance of a subject to an antigen , or cystic fibrosis (which is often characterized , for example , comprising administering a vaccine or immunogenic com by beta -adrenergic receptor antibodies ) . position comprising a DRS polypeptide of the invention Still further autoimmune diseases , disorders or conditions fused to the antigen , or vector comprising a nucleic acid include, but are not limited to chronic active hepatitis (which 50 encoding a DRS polypeptide fused to the antigen . In differ is often characterized , for example by smooth muscle anti - ent embodiments, the antigen may be selected self- antigens , bodies ) ; primary biliary cirrhosis (which is often character - tumor derived antigens , pathogen derived antigens . In some ized, for example , by anti- mitochondrial antibodies ); other embodiments the pathogen derived antigen is derived from endocrine gland failure (which is characterized , for example , a virus, bacteria or prion . In some embodiments , the antigen by specific tissue antibodies in some cases ) ; vitiligo (which 55 is fused to the DRS polypeptide through conjugation at is often characterized , for example , by anti -melanocyte Cys130 . antibodies ) ; vasculitis (which is often characterized , for Pharmaceutical Formulations, Administration , and Kits example , by immunoglobulin and complement in vessel Embodiments of the present invention include composi walls and / or low serum complement ) ; post -myocardial tions comprising PEGylated DRS polypeptides formulated infarction conditions which are often characterized , for 60 in pharmaceutically -acceptable or physiologically -accept example, by anti -myocardial antibodies ) ; cardiotomy syn - able solutions for administration to a cell, subject, or an drome (which is often characterized , for example , by anti - animal, either alone, or in combination with one or more myocardial antibodies) ; urticaria (which is often character other modalities of therapy . It will also be understood that, ized , for example , by IgG and IgM antibodies to IgE ); atopic if desired , the compositions of the invention may be admin dermatitis (which is often characterized , for example , by 65 istered in combination with other agents as well, such as, IgG and IgM antibodies to IgE ) ; asthma (which is often e . g ., other proteins or polypeptides or various pharmaceu characterized , for example , by IgG and IgM antibodies to tically -active agents . There is virtually no limit to other US 9 , 822 ,353 B2 135 136 components that may also be included in the compositions, least about 95 % purity , or in some embodiments, at least provided that the additional agents do not adversely affect 98 % purity . Purity may be determined via any routine the modulatory or other effects desired to be achieved . analytical method as known in the art. For pharmaceutical production , DRS polypeptide thera - In another aspect , such compositions have a high molecu peutic compositions will typically be substantially endo - 5 lar weight aggregate content of less than about 10 % , com toxin free . Endotoxins are toxins associated with certain pared to the total amount of protein present, or in some bacteria , typically gram -negative bacteria , although endo - embodiments such compositions have a high molecular toxins may be found in gram -positive bacteria , such as weight aggregate content of less than about 5 % , or in some Listeria monocytogenes . The most prevalent endotoxins are aspects such compositions have a high molecular weight lipopolysaccharides (LPS ) or lipo - oligo -saccharides (LOS ) 10 aggregate content of less than about 3 % , or in some embodi found in the outer membrane of various Gram -negative ments a high molecular weight aggregate content of less bacteria , and which represent a central pathogenic feature in than about 1 % . High molecular weight aggregate content the ability of these bacteria to cause disease . Small amounts may be determined via a variety of analytical techniques of endotoxin in humansmay produce fever , a lowering of the including for example , by size exclusion chromatography , blood pressure , and activation of inflammation and coagu - 15 dynamic light scattering , or analytical ultracentrifugation . lation , among other adverse physiological effects . Pharmaceutical compositions may include pharmaceuti Endotoxins can be detected using routine techniques cally acceptable salts of a DRS polypeptide . For a review on known in the art . For example , the Limulus Ameobocyte suitable salts , see Handbook of Pharmaceutical Salts: Prop Lysate assay, which utilizes blood from the horseshoe crab , erties, Selection , and Use by Stahl and Wermuth (Wiley is a very sensitive assay for detecting presence of endotoxin . 20 VCH , 2002 ) . Suitable base salts are formed from bases In this test, very low levels of LPS can cause detectable which form non - toxic salts . Representative examples coagulation of the limulus lysate due a powerful enzymatic include the aluminum , arginine , benzathine , calcium , cho cascade that amplifies this reaction . Endotoxins can also be line , diethylamine , diolamine , glycine , lysine , magnesium , quantitated by enzyme- linked immunosorbent assay meglumine, olamine , potassium , sodium , tromethamine , and ( ELISA ) . 25 zinc salts . Hemisalts of acids and bases may also be formed , To be substantially endotoxin free , endotoxin levels may e . g . , hemisulphate and hemicalcium salts . Compositions to be less than about 0 .001 , 0 .005 , 0 . 01, 0 .02 , 0 . 03 , 0 .04 , 0 .05 , be used in the invention suitable for parenteral administra 0 . 06 , 0 . 08 , 0 .09 , 0 . 1 , 0 . 5 , 1 .0 , 1 . 5 , 2 , 2 . 5 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , tion may comprise sterile aqueous solutions and /or suspen or 10 EU /mg of protein . Typically , 1 ng lipopolysaccharide sions of the pharmaceutically active ingredients preferably ( LPS ) corresponds to about 1- 10 EU . 30 made isotonic with the blood of the recipient, generally In certain embodiments , as noted herein , the DRS poly - using sodium chloride , glycerin , glucose , mannitol, sorbitol, peptide compositions have an endotoxin content of less than and the like . Organic acids suitable for forming pharmaceu about 10 EU /mg of DRS polypeptide, or less than about 5 tically acceptable acid addition salts include , by way of EU /mg of DRS polypeptide , less than about 3 EU /mg of example and not limitation , acetic acid , trifluoroacetic acid , DRS polypeptide, or less than about 1 EU /mg of DRS 35 propionic acid , hexanoic acid , cyclopentanepropionic acid , polypeptide , or less than about 0 . 1 EU /mg of DRS polypep - glycolic acid , oxalic acid , pyruvic acid , lactic acid , malonic tide, or less than about 0 .01 EU /mg of DRS polypeptide . In acid , succinic acid , malic acid , maleic acid , fumaric acid , certain embodiments , as noted above , the DRS polypeptide tartaric acid , citric acid , palmitic acid , benzoic acid , 3 - ( 4 pharmaceutical compositions are about 95 % endotoxin free , hydroxybenzoyl) benzoic acid , cinnamic acid , mandelic preferably about 99 % endotoxin free , and more preferably 40 acid , alkylsulfonic acids ( e . g ., methanesulfonic acid , ethane about 99 .99 % endotoxin free on wt/ wt protein basis . sulfonic acid , 1 , 2 -ethane -disulfonic acid , 2 -hydroxyethane Pharmaceutical compositions comprising a therapeutic sulfonic acid , etc .) , arylsulfonic acids ( e . g . , benzenesulfonic dose of a PEGylated DRS polypeptide include all homo- acid , 4 -chlorobenzenesulfonic acid , 2 -naphthalenesulfonic logues , orthologs, and naturally -occurring isoformsof aspar - acid , 4 - toluenesulfonic acid , camphorsulfonic acid , etc . ) , tyl -tRNA synthetase ( e . g . , any of the proteins or nucleic 45 4 -methylbicyclo ( 2 . 2 . 2 ) - oct - 2 - ene - 1 - carboxylic acid , gluco acids listed in Tables D1 to D8 which i ) retain detectable non heptonic acid , 3 - phenylpropionic acid , trimethylacetic acid , canonical activity, and ii ) which comprise , or have been tertiary butylacetic acid , lauryl sulfuric acid , gluconic acid , modified to comprise , at least one mutation at either Cys76 glutamic acid , hydroxynaphthoic acid , salicylic acid , stearic or Cys130 (using the numbering of SEQ ID NO : 1 ) which acid , muconic acid , and the like. replaces the native cysteine with another amino acid . 50 In particular embodiments , the carriermay include water. In some embodiments such pharmaceutical compositions In some embodiments , the carrier may be an aqueous may comprise an arginine buffer , which may be present in solution of saline , for example , water containing physiologi any of the pharmaceutical compositions within the range of cal concentrations of sodium , potassium , calcium , magne about 1 mM to about 100 mM . In different embodiments, the sium , and chloride at a physiological pH . In some embodi arginine buffer may be present at a concentration of about 1 55 ments , the carrier may be water and the formulation may mM , about 10 mM , about 20 mM , about 40 mM , about 50 further include NaCl. In some embodiments , the formulation mM , about 60 mM , about 70 mM , about 80 mM , about 90 may be isotonic . In some embodiments , the formulation may mM , or about 100 mM . be hypotonic . In other embodiments, the formulation may be In one aspect such compositions may comprises PEGy . hypertonic . In some embodiments , the formulation may be lated DRS polypeptides that are substantially monodisperse , 60 isomostic . In some embodiments , the formulation is sub meaning that the DRS polypeptide compositions exist pri - stantially free of polymers ( e . g ., gel -forming polymers , marily ( i. e. , at least about 90 % , or greater ) in one apparent polymeric viscosity - enhancing agents , etc . ) . In some molecular weight form when assessed for example , by size embodiments , the formulation is substantially free of vis exclusion chromatography , dynamic light scattering , or ana - cosity - increasing agents ( e . g ., carboxymethylcellulose , lytical ultracentrifugation . 65 polyanionic polymers, etc . ) . In some embodiments , the In another aspect , such compositions have a purity (on a formulation is substantially free of gel- forming polymers . In protein basis ) of at least about 90 % , or in some aspects at some embodiments, the viscosity of the formulation is about US 9 ,822 ,353 B2 137 138 the same as the viscosity of a saline solution containing the Inc . , and SABER (Sucrose Acetate Isobutyrate Extended same concentration of a DRS polypeptide ( or a pharmaceu - Release ) developed by Durect Corporation , and lipid -based tically acceptable salt thereof ). systems such as DepoFoam developed by SkyePharma. In the pharmaceutical compositions of the invention , Solutions of the active compounds as free base or phar formulation of pharmaceutically - acceptable excipients and 5 macologically acceptable salts may be prepared in water carrier solutions is well -known to those of skill in the art , as suitably mixed with a surfactant, such as hydroxypropylcel is the development of suitable dosing and treatment regi- lulose . Dispersions may also be prepared in glycerol, liquid mens for using the particular compositions described herein polyethylene glycols , and mixtures thereof and in oils . in a variety of treatment regimens , including e . g . , oral, Under ordinary conditions of storage and use , these prepa parenteral, intravenous , intranasal, and intramuscular 10 rations contain a preservative to prevent the growth of administration and formulation . microorganisms. In certain embodiments , the PEGylated DRS polypeptide The pharmaceutical forms suitable for injectable use have a solubility that is desirable for the particular mode of include sterile aqueous solutions or dispersions and sterile administration , such intravenous administration . Examples powders for the extemporaneous preparation of sterile of desirable solubility ' s include at least about 1 mg/ ml , at 15 injectable solutions or dispersions ( U . S . Pat. No . 5 , 466 , 468 , least about 10 mg/ml , at least about 25 mg/ ml , and at least specifically incorporated herein by reference in its entirety ) . about 50 mg/ ml . In all cases the form should be sterile and should be fluid to In certain applications , the pharmaceutical compositions the extent that easy syringability exists . It should be stable disclosed herein may be delivered via oral administration to under the conditions of manufacture and storage and should a subject . As such , these compositions may be formulated 20 be preserved against the contaminating action of microor with an inert diluent or with an edible carrier, or they may ganisms, such as bacteria and fungi. The carrier can be a be enclosed in hard - or soft- shell gelatin capsule , or they solvent or dispersion medium containing , for example , may be compressed into tablets , or they may be incorporated water, ethanol, polyol (e . g ., glycerol, propylene glycol, and directly with the food of the diet. liquid polyethylene glycol, and the like ), suitable mixtures Pharmaceutical compositions suitable for the delivery of 25 thereof, and /or vegetable oils . Proper fluidity may be main DRS polypeptides and methods for their preparation will be tained , for example , by the use of a coating , such as lecithin , readily apparent to those skilled in the art. Such composi- by the maintenance of the required particle size in the case tions and methods for their preparation may be found , e . g . , of dispersion and by the use of surfactants . The prevention in Remington 's Pharmaceutical Sciences, 19th Edition of the action ofmicroorganisms can be facilitated by various (Mack Publishing Company, 1995 ) . 30 antibacterial and antifungal agents , for example , parabens , Administration of a therapeutic dose of a DRS polypep - chlorobutanol, phenol, sorbic acid , thimerosal , and the like . tide may be by any suitable method known in the medicinal In many cases, it will be preferable to include isotonic arts , including for example , oral, intranasal , parenteral agents , for example , sugars or sodium chloride . Prolonged administration include intravitreal, subconjuctival, sub - absorption of the injectable compositions can be brought tenon , retrobulbar, suprachoroidal intravenous, intra - arte - 35 about by the use in the compositions of agents delaying rial, intraperitoneal, intrathecal, intraventricular, intraure absorption , for example, aluminum monostearate and gela thral, intrasternal , intracranial, intramuscular , intrasynovial, tin . intraocular, topical and subcutaneous. Suitable devices for For parenteral administration in an aqueous solution , for parenteral administration include needle ( including example , the solution should be suitably buffered if neces microneedle ) injectors , needle - free injectors, and infusion 40 sary and the liquid diluent first rendered isotonic with techniques . sufficient saline or glucose . These particular aqueous solu Parenteral formulations are typically aqueous solutions tions are especially suitable for intravenous, intramuscular, which may contain excipients such as salts , carbohydrates , subcutaneous and intraperitoneal administration . In this con and buffering agents (preferably to a pH of from 3 to 9 ), but, nection , a sterile aqueous medium that can be employed will for some applications , they may be more suitably formulated 45 be known to those of skill in the art in light of the present as a sterile non -aqueous solution or as a dried form to be disclosure . For example , one dosage may be dissolved in 1 used in conjunction with a suitable vehicle such as sterile , ml of isotonic NaCl solution and either added to 1000 ml of pyrogen - free water. The preparation of parenteral formula - hypodermoclysis fluid or injected at the proposed site of tions under sterile conditions, e . g . , by lyophilization , may infusion (see , e . g . , Remington ' s Pharmaceutical Sciences , readily be accomplished using standard pharmaceutical 50 15th Edition , pp . 1035 - 1038 and 1570 - 1580 ) . Some varia techniques well -known to those skilled in the art . tion in dosage will necessarily occur depending on the Formulations for parenteral administration may be for - condition of the subject being treated . The person respon mulated to be immediate and /or sustained release . Sustained sible for administration will , in any event, determine the release compositions include delayed , modified , pulsed , appropriate dose for the individual subject. Moreover , for controlled , targeted and programmed release . Thus a DRS 55 human administration , preparations should meet sterility , polypeptide may be formulated as a suspension or as a solid , pyrogenicity , and the general safety and purity standards as semi- solid , or thixotropic liquid for administration as an required by FDA Office of Biologics standards . implanted depot providing sustained release of DRS poly - Sterile injectable solutions can be prepared by incorpo peptides . Examples of such formulations include without rating the active compounds in the required amount in the limitation , drug - coated stents and semi- solids and suspen - 60 appropriate solvent with the various other ingredients enu sions comprising drug -loaded poly (DL - lactic - co - glycolic ) merated above , as required , followed by filtered steriliza acid (PGLA ) , poly (DL - lactide -co - glycolide ) (PLG ) or poly tion . Generally , dispersions are prepared by incorporating ( lactide ) ( PLA ) lamellar vesicles or microparticles, the various sterilized active ingredients into a sterile vehicle hydrogels (Hoffman A S : Ann . N . Y. Acad . Sci. 944 : 62 - 73 which contains the basic dispersion medium and the ( 2001) ) , poly - amino acid nanoparticles systems, such as the 65 required other ingredients from those enumerated above . In Medusa system developed by Flamel Technologies Inc ., non the case of sterile powders for the preparation of sterile aequous gel systems such as Atrigel developed by Atrix , injectable solutions , the preferred methods of preparation US 9 ,822 ,353 B2 139 140 are vacuum -drying and freeze -drying techniques which Examples of antihistamines include, but are not limited to , yield a powder of the active ingredient plus any additional loradatine, hydroxyzine , diphenhydramine, chlorpheni desired ingredient from a previously sterile - filtered solution ramine , brompheniramine , cyproheptadine , terfenadine, cle thereof. mastine, triprolidine, carbinoxamine, diphenylpyraline , The compositions disclosed herein may be formulated in 5 phenindamine , azatadine , tripelennamine , dexchlorpheni a neutral or salt form . Pharmaceutically -acceptable salts , ramine , dexbrompheniramine , methdilazine , and trimpra zine doxylamine , pheniramine , pyrilamine, chiorcyclizine , include the acid addition salts ( formed with the free amino thonzylamine , and derivatives thereof. groups of the protein ) and which are formed with inorganic Examples of antibiotics include , but are not limited to : acids such as, for example , hydrochloric or phosphoric 10 Aminoglycosides ( e. g ., amikacin , apramycin , arbekacin , acids, or such organic acids as acetic , oxalic , tartaric ,man bambermycins, butirosin , dibekacin , dihydrostreptomycin , delic , and the like . Salts formed with the free carboxyl fortimicin ( s ) , gentamicin , isepamicin , kanamycin , micron groups can also be derived from inorganic bases such as, for omicin , neomycin , neomycin undecylenate , netilmicin , example , sodium , potassium , ammonium , calcium , or ferric paromomycin , ribostamycin , sisomicin , spectinomycin , hydroxides , and such organic bases as isopropylamineYamine ,, 15 streptomycin , tobramycin , trospectomycin ), amphenicols trimethylamine , histidine , procaine and the like . Upon for ( e . g . , azidamfenicol, chloramphenicol, florfenicol , thiam mulation , solutions will be administered in a manner com phenicol ), ansamycins ( e . g . , rifamide, rifampin , rifamycin patible with the dosage formulation and in such amount as sv, rifapentine , rifaximin ), lactams ( e . g ., carbacephems ( e . g . , is therapeutically effective. The formulations are easily loracarbef) , carbapenems (e . g ., biapenem , imipenem , mero administered in a variety of dosage forms such as injectable 20 penem , panipenem ), cephalosporins ( e. g ., cefaclor , solutions, drug - release capsules , and the like . cefadroxil , cefamandole , cefatrizine , cefazedone , cefazolin , As used herein , " carrier ” includes any and all solvents , cefcapene pivoxil , cefclidin , cefdinir , cefditoren , cefepime, dispersion media , vehicles, coatings, diluents , antibacterial cefetamet, cefixime, cefinenoxime, cefodizime, cefonicid , and antifungal agents, isotonic and absorption delaying cefoperazone , ceforanide , cefotaxime, cefotiam , cefozopran , agents , buffers , carrier solutions , suspensions , colloids , and 25 cefpimizole , cefpiramide, cefpirome, cefpodoxime proxetil , the like . The use of such media and agents for pharmaceu - cefprozil , cefroxadine , cefsulodin , ceftazidime, cefteram , tical active substances is well known in the art. Except ceftezole, ceftibuten , ceftizoxime, ceftriaxone, cefuroxime, insofar as any conventionalmedia or agent is incompatible cefuzonam , cephacetrile sodium , cephalexin , cephaloglycin , with the active ingredient, its use in the therapeutic compo - cephaloridine, cephalosporin , cephalothin , cephapirin sitions is contemplated . Supplementary active ingredients 30 sodium , cephradine, pivcefalexin ), cephamycins ( e .g ., cef can also be incorporated into the compositions. buperazone, cefinetazole , cefminox, cefotetan , cefoxitin ) , The phrase " pharmaceutically -acceptable ” refers to monobactams (e . g. , aztreonam , carumonam , tigemonam ), molecular entities and compositions that do not produce an oxacephems, flomoxef, moxalactam ) , penicillins ( e . g . , allergic or similar untoward reaction when administered to amdinocillin , amdinocillin pivoxil, amoxicillin , ampicillin , a human . The preparation of an aqueous composition that 35 apalcillin , aspoxicillin , azidocillin , azlocillin , bacampicillin , contains a protein as an active ingredient is well understood benzylpenicillinic acid , benzylpenicillin sodium , carbenicil in the art. Typically , such compositions are prepared as lin , carindacillin , clometocillin , cloxacillin , cyclacillin , injectables, either as liquid solutions or suspensions ; solid dicloxacillin , epicillin , fenbenicillin , floxacillin , hetacillin , forms suitable for solution in , or suspension in , liquid prior lenampicillin , metampicillin , methicillin sodium , mezlocil to injection can also be prepared . The preparation can also 40 lin , nafcillin sodium , oxacillin , penamecillin , penethamate be emulsified . hydriodide , penicillin g benethamine , penicillin g benzath PEGylated DRS polypeptides for use in the present inven - ine , penicillin g benzhydrylamine, penicillin g calcium , tion may also be administered topically , ( intra dermally , or penicillin g hydrabamine , penicillin g potassium , penicillin transdermally to the skin , mucosa , or surface of the eye , g procaine , penicillin n , penicillin o , penicillin v , penicillin either alone or in combination with one or more antihista - 45 v benzathine , penicillin v hydrabamine, penimepicycline , mines, one or more antibiotics , one or more antifungal phenethicillin potassium , piperacillin , pivampicillin , propi agents , one or more beta blockers , one or more anti -inflam - cillin , quinacillin , sulbenicillin , sultamicillin , talampicillin , matory Agents , one or more antineoplastic agents , one or temocillin , ticarcillin ), other ( e . g ., ritipenem ), lincosamides more immunosuppressive agents , one or more antiviral (e . g ., clindamycin , lincomycin ), macrolides ( e . g ., azithro agents , one or more antioxidant agents , or other active 50 mycin , carbomycin , clarithromycin , dirithromycin , erythro agents . Formulations for topical and ocular administration mycin , erythromycin acistrate , erythromycin estolate , eryth may be formulated to be immediate and / or modified release . romycin glucoheptonate , erythromycin lactobionate , Modified release formulations include delayed , sustained , erythromycin propionate , erythromycin stearate , josamycin , pulsed , controlled , targeted and programmed release . leucomycins, midecamycins, miokamycin , oleandomycin , Typical formulations for this purpose include gels , hydro - 55 primycin , rokitamycin , rosaramicin , roxithromycin , spi gels , lotions , solutions, eye drops, creams, ointments , dust ramycin , troleandomycin ) , polypeptides ( e . g . , amphomycin , ing powders, dressings, foams, films, skin patches, wafers , bacitracin , capreomycin , colistin , enduracidin , enviomycin , implants , sponges, fibers , bandages, and microemulsions . fusafungine , gramicidin s , gramicidin ( s ) , mikamycin , poly Liposomes may also be used . Typical carriers include alco - myxin , pristinamycin , ristocetin , teicoplanin , thiostrepton , hol, water , mineral oil , liquid petrolatum , white petrolatum , 60 tuberactinomycin , tyrocidine, tyrothricin , vancomycin , vio glycerin , polyethylene glycol, and propylene glycol. Pen - mycin , virginiamycin , zinc bacitracin ), tetracyclines ( e . g . , etration enhancers may be incorporated — see , e . g . , Finnin apicycline , chlortetracycline , clomocycline , demeclocy and Morgan : J. Pharm . Sci. 88 ( 10 ): 955 -958 , ( 1999 ). Other cline , doxycycline , guamecycline , lymecycline , meclocy means of topical administration include delivery by elec - cline , methacycline, minocycline , oxytetracycline , pen troporation , iontophoresis, phonophoresis , sonophoresis , 65 imepicycline , pipacycline , rolitetracycline , sancycline , and microneedle or needle -free injection ( e. g . , the systems tetracycline ) , and others ( e . g ., cycloserine , mupirocin , sold under the trademarks POWDERJECTTM , BIOJECTTM ) . tuberin ). 2 .4 -Diaminopyrimidines ( e . g . , brodimoprim , tet US 9 ,822 ,353 B2 141 142 roxoprim , trimethoprim ), nitrofurans ( e .g ., furaltadone, fura streptonigrin , streptozocin , tubercidin , zinostatin , zorubi zolium chloride , nifuradene , nifuratel, nifurfoline , nifurpiri cin ) , antimetabolites ( e . g ., folic acid analogs ( e . g ., denop nol, nifurprazine , nifurtoinol, nitrofurantoin ), quinolones terin , edatrexate , methotrexate , piritrexim , pteropterin , and analogs ( e . g ., cinoxacin , ciprofloxacin , clinafloxacin , Tomudex® , trimetrexate ), purine analogs ( e . g ., cladribine , difloxacin , enoxacin , fleroxacin , flumequine, grepafloxacin , 5 fludarabine , 6 -mercaptopurine , thiamiprine , thioguanine ), lomefloxacin , miloxacin , nadifloxacin , nalidixic acid , nor pyrimidine analogs ( e . g . , ancitabine, azacitidine , 6 - azauri floxacin , ofloxacin , oxolinic acid , pazufloxacin , pefloxacin , dine , carmofur , cytarabine , doxifluridine, emitefur , enocit pipemidic acid , piromidic acid , rosoxacin , rufloxacin , spar - abine . floxuridine . fluorouracil, gemcitabine , tagafur ) . floxacin , temafloxacin , tosufloxacin , trovafloxacin ), sulfo namides (e . g. , acetyl sulfamethoxypyrazine, benzylsulf - 10 , Examples of Antiinflammatory Agents Include but are not amide, chloramine - b , chloramine - t, dichloraminet , Limited to Steroidal Antiinflammatory Agents and Non n2- formylsulfisomidine, mafenide, 4 '- ( methylsulfamoyl ) sul Steroidal Antiinflammatory Agents. Exemplary Steroidal fanilanilide , noprylsulfamide , phthalylsulfacetamide, phth Antiinflammatory include acetoxypregnenolone , alclometa alylsulfathiazole , salazosulfadimidine, succinylsulfathiaz sone , algestone , amcinonide , beclomethasone , betametha ole . sulfabenzamide. sulfacetamide , sulfachlorpyridazine. 15 sone , budesonide , chloroprednisone , clobetasol, clobeta sulfachrysoidine , sulfacytine , sulfadiazine , sulfadicramide, sone, clocortolone , cloprednol, corticosterone , cortisone , sulfadimethoxine , sulfadoxine , sulfaethidole , sulfaguani cortivazol, deflazacort , desonide , desoximetasone , dexam dine , sulfaguanol, sulfalene , sulfaloxic acid , sulfamerazine, ethasone, diflorasone , diflucortolone, difluprednate , enox sulfameter , sulfamethazine , sulfamethizole , sulfamethomi olone , fluazacort, flucloronide , flumethasone , flunisolide , dine , sulfamethoxazole, sulfamethoxypyridazine , sulfame - 20 fluocinolone acetonide, fluocinonide , fluocortin butyl, fluo trole , sulfamidochrysoidine, sulfamoxole , sulfanilamide, cortolone, fluorometholone , fluperolone acetate , flupred 4 - sulfanilamidosalicylic acid , n4 -sulfanilylsulfanilamide , nidene acetate , fluprednisolone, flurandrenolide , fluticasone sulfanilylurea , n - sulfanilyl - 3 , 4 - xylamide, sulfanitran , sulfa propionate , formocortal, halcinonide , halobetasol propi perine , sulfaphenazole , sulfaproxyline, sulfapyrazine , sul onate , halometasone , halopredone acetate , hydrocortamate , fapyridine , sulfasomizole , sulfasymazine , sulfathiazole , sul- 25 hydrocortisone, loteprednol etabonate , mazipredone , fathiourea , sulfatolamide , sulfisomidine , sulfisoxazole ) medrysone , meprednisone , methylprednisolone , mometa sulfones ( e . g ., acedapsone , acediasulfone , acetosulfone sone furoate , paramethasone , prednicarbate , prednisolone, sodium , dapsone, diathymosulfone , glucosulfone sodium , prednisolone 25 - diethylamino - acetate , prednisolone sodium solasulfone, succisulfone , sulfanilic acid , p - sulfanilylben - phosphate , prednisone , prednival, prednylidene , rimex zylamine , sulfoxone sodium , thiazolsulfone ) , and others 30 olone , tixocortol, triamcinolone , triamcinolone acetonide , ( e . g . , clofoctol, hexedine , methenamine , methenamine anhy triamcinolone benetonide , and triamcinolone hexacetonide . dromethylene - citrate ,methenamine hippurate ,methenamine Exemplary Non - Steroidal Antiinflammatory Agents mandelate , methenamine sulfosalicylate , nitroxoline , tauro - include Aminoarylcarboxylic acid derivatives ( e . g ., enfe lidine, xibornol) . namic acid , etofenamate , flufenamic acid , isonixin , meclofe Examples of antifungal agents include, but are not limited 35 namic acid , mefenamic acid , niflumic acid , talniflumate , to Polyenes ( e .g ., amphotericin b , candicidin , dermostatin , terofenamate , tolfenamic acid ) , arylacetic acid derivatives filipin , fungichromin , hachimycin , hamycin , lucensomycin , ( e .g ., aceclofenac, acemetacin , alclofenac, amfenac , amtol mepartricin , natamycin , nystatin , pecilocin , perimycin ) , oth - metin guacil , bromfenac , bufexamac , cinmetacin , clopirac , ers ( e . g . , azaserine , griseofulvin , oligomycins, neomycin diclofenac sodium , etodolac , felbinac , fenclozic acid , fenti undecylenate , pyrrolnitrin , siccanin , tubercidin , viridin ) , 40 azac , glucametacin , ibufenac , indomethacin , isofezolac , Allylamines ( e . g ., butenafine, naftifine , terbinafine ) , imida isoxepac, lonazolac, metiazinic acid , mofezolac , oxameta zoles ( e . g ., bifonazole , butoconazole , chlordantoin , chlormi- cine , pirazolac , proglumetacin , sulindac, tiaramide , tolme dazole , cloconazole , clotrimazole , econazole , enilconazole , tin , tropesin , zomepirac ) , arylbutyric acid derivatives ( e . g . , fenticonazole , flutrimazole , isoconazole, ketoconazole , bumadizon , butibufen , fenbufen , xenbucin ), arylcarboxylic lanoconazole , miconazole , omoconazole , oxiconazole 45 acids ( e . g . , clidanac , ketorolac , tinoridine ) , arylpropionic nitrate , sertaconazole , sulconazole , tioconazole ), thiocar - acid derivatives ( e . g . , alminoprofen , benoxaprofen , bermo bamates ( e . g ., tolciclate , tolindate , tolnaftate ) , triazoles ( e . g ., profen , bucloxic acid , carprofen , fenoprofen , flunoxaprofen , fluconazole , itraconazole , saperconazole , terconazole ) oth - flurbiprofen , ibuprofen , ibuproxam , indoprofen , ketoprofen , ers ( e . g ., acrisorcin , amorolfine , biphenamine , bromosalicyl- loxoprofen , naproxen , oxaprozin , piketoprolen , pirprofen , chloranilide , buclosamide , calcium propionate , chlorphen - 50 pranoprofen , protizinic acid , suprofen , tiaprofenic acid , esin , ciclopirox , cloxyquin , coparaffinate , diamthazole ximoprofen , zaltoprofen ), pyrazoles ( e . g . , difenamizole , epi dihydrochloride, exalamide , flucytosine, halethazole , hexe - rizole ) , pyrazolones (e . g. , apazone , benzpiperylon , fepra tidine , loflucarban , nifuratel , potassium iodide, propionic zone, mofebutazone , morazone , oxyphenbutazone , phenylb acid , pyrithione , salicylanilide , sodium propionate , sulben utazone , pipebuzone , propyphenazone , ramifenazone , tine , tenonitrozole , triacetin , ujothion , undecylenic acid , zinc 55 suxibuzone , thiazolinobutazone ) , salicylic acid derivatives propionate ) . ( e . g . , acetaminosalol, aspirin , benorylate , bromosaligenin , Examples of beta blockers include but are not limited to calcium acetylsalicylate , diflunisal, etersalate, fendosal , gen acebutolol, atenolol, labetalol, metoprolol, propranolol, tisic acid , glycol salicylate, imidazole salicylate, lysine timolol, and derivatives thereof. acetylsalicylate , mesalamine, morpholine salicylate , 1 -naph Examples of antineoplastic agents include , but are not 60 thyl salicylate , olsalazine , parsalmide, phenyl acetylsalicy limited to Antibiotics and analogs ( e . g ., aclacinomycins , late , phenyl salicylate , salacetamide , salicylamide o -acetic actinomycin f? , anthramycin , azaserine, bleomycins, cac - acid , salicylsulfuric acid , salsalate , sulfasalazine ), thiazin tinomycin , carubicin , carzinophilin , chromomycins, dac - ecarboxamides ( e . g . , ampiroxicam , droxicam , isoxicam , lor tinomycin , daunorubicin , 6 - diazo - 5 -oxo - L -norleucine , noxicam , piroxicam , tenoxicam ), g -acetamidocaproic acid , doxorubicin , epirubicin , idarubicin , menogaril, mitomycins, 65 s - adenosylmethionine, 3 - amino - 4 - hydroxybutyric acid , mycophenolic acid , nogalamycin , olivomycines , peplomy - amixetrine , bendazac , benzydamine , bucolome, difenpir cin , pirarubicin , plicamycin , porfiromycin , puromycin , amide, ditazol , emorfazone , fepradinol, guaiazulene, nabu US 9 ,822 ,353 B2 143 144 metone, nimesulide , oxaceprol, paranyline , perisoxal, pro from about 0 . 6 % to about 0 . 9 % , from about 0 . 7 % to about quazone , superoxide dismutase , tenidap , and zileuton . 0 . 9 % , from about 0 . 8 % to about 0 . 9 % , about 0 . 9 % , about Examples of antiviral agents include interferon gamma, 0 % , about 0 .05 % , about 0 .01 % , about 0 .09 % , about 0 . 1 % , zidovudine , amantadine hydrochloride, ribavirin , acyclovir, about 0 . 2 % , about 0 . 3 % , about 0 . 4 % , about 0 . 5 % , about valciclovir , dideoxycytidine , phosphonoformic acid , ganci - 5 0 . 6 % , about 0 . 7 % , or about 0 . 8 % . In certain embodiments , clovir , and derivatives thereof . the aqueous saline solution will be isotonic ( e . g . , NaCl Examples of antioxidant agents include ascorbate , alpha concentration of about 0 . 9 % NaCl ( w / v ) ) . In certain embodi tocopherol, mannitol, reduced glutathione , various carote - ments, the aqueous solution will contain a NaCl concentra noids, cysteine , uric acid , taurine , tyrosine , superoxide dis - tion of about 0 . 5 % , about 0 . 7 % , about 0 . 8 % , about 0 . 85 , or mutase , lutein , zeaxanthin , cryotpxanthin , astazanthin , 10 about 0 . 75 % . As will be appreciated the skilled artisan , lycopene , N - acetyl- cysteine , carnosine , gamma- glutamyl- depending on the concentrations of other components , for cysteine , quercitin , lactoferrin , dihydrolipoic acid , citrate , example where the DRS polypeptides are present as salts of, Ginkgo Biloba extract , tea catechins , bilberry extract, vita - the concentration of NaCl or other salt needed to achieve an mins E or esters of vitamin E , retinyl palmitate , and deriva - formulation suitable for administration may vary . tives thereof. Other therapeutic agents include squalamine , 15 In some embodiments , where the ocular formulation is carbonic anhydrase inhibitors , alpha - 2 adrenergic receptor substantially free of viscosity - increasing agents , the formu agonists , antiparasitics , antifungals , and derivatives thereof. lation may be substantially free of viscosity - increasing The exact dose of each component administered will , of agents such as, but not limited to polyanionic polymers , course , differ depending on the specific components pre - water soluble cellulose derivatives ( e . g . , hypromellose (also scribed , on the subject being treated , on the severity of the 20 known as HPMC , hydroxypropylmethyl cellulose , and disease , e . g ., severity of the inflammatory reaction , on the hydroxypropylcellulose) , hydroxyethylcellulose , carboxm manner of administration and on the judgment of the pre - ethylcellulose , etc .) , polyvinyl alcohol , polyvinyl pyrroli scribing physician . Thus , because of patient- to -patient vari - done , chondroitin sulfate , hyaluronic acid , soluble starches, ability , the dosages given above are a guideline and the etc . In some variations , the formulation does not incorporate physician may adjust doses of the compounds to achieve the 25 a hydrogel or other retention agent ( e . g ., such as those treatment that the physician considers appropriate . disclosed in U . S . Pat. Pub . No. 2005 /0255144 ( incorporated As will be understood by the skilled artisan , for DRS by reference herein in its entirety ) ) , e . g ., where they hydro polypeptide ocular formulations where the carrier includes a gel may include , hydrogels incorporating homopolymers ; gel -forming polymer , in certain formulations the inclusion copolymers ( e . g . , tetrapolymers of hydroxymethylmethacry of salt ( s ), in particular saline solution , is contraindicated as 30 late , ethylene glycol, dimethylmethacrylate, and methacrylic inclusion of salt may either cause the solution to gel prior to acid ) , copolymers of trimethylene carbonate and polygly topical administration , as with certain in situ gel - forming colicacid , polyglactin 910 , glyconate , poly - p - dioxanone , polymers ( e .g ., gellan gel) , or the inclusion of salts may polyglycolic acid , polyglycolic acid felt , poly - 4 -hydroxybu inhibit the gelling properties of the gel- forming polymer. tyrate , a combination of poly ( L - lactide ) and poly ( L - lactide The skilled artisan will be able to select appropriate com - 35 co - glycolide ), glycol methacrylate , poly -DL - lactide , or Pri binations based on the desired properties of the formulation macryl) ; composites of oxidized regenerated cellulose , and characteristics of gel - forming polymers known in the polypropylene, and polydioxanone or a composite of poly art. propylene and poligelcaprone ; etc . In some variations, the Suitable aqueous saline solutions will be understood by formulations do not include one or more of polyvinyl those of skill in the art and may include, for example , 40 alcohol, hydroxypropyl methylcellulose , polyethylene gly solutions at a pH of from about pH 4 . 5 to about pH 8 . 0 . In col 400 castor oil emulsion , carboxymethylcellulose further variations of aqueous solutions ( where water is sodium , propylene glycol, hydroxypropyl guar, carboxym included in the carrier ), the pH of the formulation is between ethylcelluose sodium , white petrolatum ,mineral oil, dextran any of about 6 and about 8 . 0 ; between about 6 and about 7 . 5 ; 70 , glycerin , hypromellose , flaxseed oil, fish oils, omega 3 between about 6 and about 7 . 0 ; between about 6 . 2 and about 45 and omega 6 fatty acids, lutein , or primrose oil. In some 8 ; between about 6 .2 and about 7 . 5 ; between about 7 and variations , the formulations do not include one or more of about 8 ; between about 6 . 2 and about 7 . 2 , between about 5 . 0 the carriers described in U . S . Pat. No . 4 ,888 ,354 ( incorpo and about 8 . 0 ; between about 5 and about 7 .5 ; between about rated by reference herein in its entirety ) , e. g . , such as one or 5 . 5 and about 8 . 0 ; between about 6 . 1 and about 7 . 7 ; between more of oleic acid , ethanol, isopropanol, glycerol about 6 . 2 and about 7 . 6 ; between about 7 . 3 and about 7 . 4 ; 50 monooleate , glycerol diooleate , methyl laurate , propylene about 6 . 0 ; about 7 . 1 ; about 6 . 2 ; about 7 . 3 ; about 6 . 4 ; about glycol, propanol or dimethyl sulfoxide . In some variations , 6 . 5 ; about 6 . 6 ; about 6 . 7 ; about 6 . 8 ; about 6 . 9 ; about 7 . 0 ; the formulations are substantially free of glycerol diooleate about 7 . 1 ; about 7 . 2 ; about 7 . 3 ; about 7 . 4 ; about 7 . 5 ; about and isopropanol. 7 . 6 ; or about 8 .0 . In some variations, the DRS polypeptide In particular embodiments, the gel- forming polymer may formulation has a pH of about 6 . 0 to about 7 . 0 . In some 55 be , for example , a polysaccharide . In certain embodiments , variations, the formulation has a pH of about 7 . 4 . In par - the polysaccharide is gellan gum . Gellan gum refers to a ticular variations, the formulation has a pH of about 6 . 2 to heteropolysaccharide elaborated by the bacterium about 7 . 5 . Pseudomonas elodea , though the name " gellan gum ” is In certain embodiments the concentration of the salt ( e . g . , more commonly used in the field . Gellan gum , in particular NaCl) will be, for example , from about 0 % to about 0 . 9 % 60 the formulation GELRITE® is described in detail in U . S . ( w / v ) . For example , the concentration of salt may be from Pat. No . 4 , 861, 760 ( hereby incorporated by reference in its about 0 .01 to about 0 . 9 % , from about 0 .02 % to about 0 . 9 % , entirety ) , in particular in its use in formulation of timolol. from about 0 .03 % to about 9 % , from about 0 . 05 % to about GELRITE® , a low acetyl clarified grade of gellan gum , is 0 . 9 % from about 0 . 07 % to about 0 . 9 % , from about 0 .09 % to commercially available from Merck & Co (Rahway , N . J . ) about 0 . 9 % , from about 0 . 1 % to about 0 . 9 % from about 65 and gellan gum can be commercially obtained from , among 0 . 2 % to about 0 . 9 % , from about 0 . 3 % to about 0 . 9 % , from others CPKelco ( Atlanta , Ga. ) . The preparation of polysac about 0 . 4 % to about 0 . 9 % from about 0 . 5 % to about 0 . 9 % , charides such as gellan gum is described in , for example , US 9 ,822 ,353 B2 145 146 U . S . Pat. Nos . 4, 326 ,053 and 4 ,326 ,052 , which are hereby preservatives (e .g ., benzalkonium chloride, benzethonium incorporated by reference in their entirety . chloride , chlorhexidine , chlorobutanol, methylparaben , phe In certain embodiments , the gel- forming polymer is pres nylethyl alcohol, propylparaben , thimerosal, phenylmercu ent at a concentration of from about 0 . 03 % to about 2 % ric nitrate , phenylmercuric borate , phenylmercuric acetate , ( w / v ) . In some embodiments , the gel - forming polymer is 5 or combinations of two or more of the foregoing ) , salt ( e . g . , present at a concentration from about 0 . 03 % to about 1 .75 % ; NaCl) and one or more buffering agents (e . g. , one or more from about 0 .03 % to about 1. 5 % , from about 0 .03 % to about phosphate buffers ( e. g. , dibasic sodium phosphate , monoba 1 .25 % , from about 0 .03 % to about 1 % , from about 0 .03 % to sic sodium phosphate, combinations thereof, etc . ) , citrate about 0 . 9 % , from about 0 .03 % to about 0 . 8 % , from about buffers, maleate buffers , borate buffers , and combination of 0 .03 % to about 0 . 7 % , from about 0 .03 % to about 0 .6 % , from 10 two or more of the foregoing .) . about 0 . 03 % to about 0 . 5 % , from about 0 .05 % to about 2 % , In particular embodiments, the chelating agent is EDTA from about 0 .05 % to about 1. 75 % ; from about 0 .05 % to disodium , the preservative is benzalkonium chloride , the salt about 1 . 5 % , from about 0 .05 % to about 1 . 25 % , from about is NaCl, and the buffering agents are dibasic sodium phos 0 .05 % to about 1 % , from about 0 .05 % to about 0 . 9 % , from phate and monobasic sodium phosphate . In certain of these about 0 .05 % to about 0 .8 % , from about 0 .05 % to about 15 embodiments , the formulation is substantially free of poly 0 . 7 % , from about 0 . 05 % to about 0 .6 % , from about 0 .05 % mer . In some embodiments , the formulation is substantially to about 0 . 5 % , from about 0 . 1 % to about 2 % , from about free of substantially viscosity - increasing agent( s ) ( e . g . , car 0 . 1 % to about 1 . 75 % ; from about 0 . 1 % to about 1 . 5 % , from boxymethylcellulose , polyanionic polymers , etc . ) . In some about 0 . 1 % to about 1 . 25 % , from about 0 . 1 % to about 1 % , embodiments , the viscosity of the formulation is about the from about 0 . 1 % to about 0 . 9 % , from about 0 . 1 % to about 20 same as the viscosity of a saline solution containing the same 0 . 8 % , from about 0 . 1 % to about 0 . 7 % , from about 0 . 1 % to concentration of a DRS polypeptide (or a pharmaceutically about 0 . 6 % , from about 0 . 1 % to about 0 . 5 % , from about acceptable salt thereof) . In some of these embodiments, the 0 .2 % to about 2 % , from about 0 . 2 % to about 1. 75 % ; from concentration of a DRS polypeptide (or a pharmaceutically about 0 . 2 % to about 1 . 5 % , from about 0 . 2 % to about 1 .25 % , acceptable salt thereof) if from about 0 . 02 % to about 3 % , from about 0 . 2 % to about 1 % , from about 0 . 2 % to about 25 from about 0 . 02 % to about 2 % , from about 0 . 02 % to about 0 . 9 % , from about 0 . 2 % to about 0 . 8 % , from about 0 . 2 % to 1 % ( w / v ) . In certain embodiments, the concentration of a about 0 . 7 % , from about 0 . 2 % to , about 0 . 6 % , from about DRS polypeptide (or a pharmaceutically acceptable salt 0 . 2 % to about 0 . 5 % , or from about 0 . 5 % to about 1 . 5 % . In thereof) , is about 0 .01 % , about 0 .02 % , about 0 .03 % , about some embodiments , the concentration of gel - forming poly . 0 .05 % , about 0 .07 % , about 0 . 1 % , about 0 . 3 % , about 0 . 4 % , mer is about 0 . 1 % , about 0 . 2 % , about 0 . 4 % , about 0 .6 % , 30 about 0 . 5 % , about 0 . 6 % , about 0 . 8 % or about 1 % ( w / v ) . about 0 . 8 % , about 1 % . In certain embodiments , where the carrier includes water, In particular embodiments , the gel- forming polymer is a viscosity - increasing agent may also be included in the gellan gum at a concentration of from about 0 .05 % to about formulation . The skilled artisan will be familiar with vis 2 % ( w / v ), from about 0 . 1 % to about 2 % ( w / v ) , from about cosity - increasing agents that are suitable ( e . g ., water- soluble 0 . 1 % to about 1 % (w /v ) , from about 0 .05 % to about 1 % 35 cellulose derivatives ( e. g ., hypromellose (also known as ( w / v ) or from about 0 . 1 % to about 0 . 6 % ( w / v ) . In some HPMC , hydroxypropylmethyl cellulose, and hydroxypropy embodiments , the concentration of gellan gum is about lcellulose ), hydroxyethylcellulose , carboxmethylcellulose , 0 . 1 % , about 0 . 2 % , about 0 . 4 % , about 0 . 6 % , about 0 . 8 % , etc . ) , polyvinyl alcohol, polyvinyl pyrrolidone , chondroitin about 1 % . sulfate , hyaluronic acid , and soluble starches . It is intended In some embodiments of the ocular formulations, the 40 that when viscosity - increasing agents are used , they are not formulation may include additional components such as one included in high enough concentrations such that the for or more preservatives , one or more surfactants , or one or mulation would form a gel prior to or after administration more pharmaceutical agents . In particular embodiments , the ( e . g . , wherein the concentration of the viscosity - increasing formulation may include additional components such as one agent is not sufficient to induce gel formation ) . or more preservatives, one or more surfactants , one or more 45 While exact concentrations of viscosity - increasing agents tonicity agents, one or more buffering agents , one or more will depend upon the selection and concentration of other chelating agents , one or more viscosity - increasing agents , components in the formulation as well as the particular one or more salts , or one or more pharmaceutical agents . In viscosity -increasing agent( s ) selected , in general, viscosity certain of these embodiments , the formulation may include increasing agents may be present in a concentration such ( in addition to a DRS polypeptide ( or a pharmaceutically 50 that the viscosity of the resulting solution is less than about acceptable salt thereof) and carrier ) : one or more preserva - 1000 centipoise . In certain embodiments , the viscosity of the tives , one or more buffering agents ( e .g ., one, two , three , formulation is less than about 900 , less than about 800 , less etc .) , one ormore chelating agents , and one ormore salts . In than about 700 , less than about 600 , less than about 500 , less some embodiments , the formulation may include ( in addi- than about 400 , less than about 300 , less than about 200 , less tion to a DRS polypeptide ( or a pharmaceutically acceptable 55 than about 150 , less than about 100 , less than about 50 salt thereof ) and carrier ): one or more preservatives , one or centipoise . In some embodiments , the viscosity of the for more tonicity agents , one or more buffering agents , one or mulation is about 200 , about 150 , about 100 , about 50 more chelating agents , and one or more viscosity - increasing centipoise . In particular embodiments , the viscosity is less agents . than about 200 centipoise . In others , less than about 120 In some embodiments , the viscosity of the formulation is 60 centipoise or less than about 100 centipoise . In some about the same as the viscosity of a saline solution contain - embodiments , the viscosity is about 100 centipoise . In others ing the same concentration of a DRS polypeptide ( or a about 50 centipoise . In still other embodiments the viscosity pharmaceutically acceptable salt thereof) . In some embodi- is about 200 centipoise . Methods for measuring viscosity are ments , the formulation is substantially free of gel- forming well known to the skilled artisan . For example , as described polymers . In certain embodiments , where the carrier is 65 in United States Pharmacopoeia 29 ( Chapter 911 ) Viscosity , water , the formulation may additionally include one or more page 2785 (which is herein incorporated by reference in its chelating agents ( e . g ., EDTA disodium ( EDTA ) , one or more entirety ) . As is well known to the skilled artisan , formula US 9 ,822 , 353 B2 147 148 tions commonly considered “ gels ” will have viscosity sig - more of the foregoing preservatives . In certain embodi nificantly greater than 1000 centipoise , for example , greater ments , the preservative is benzalkonium chloride . than about 2000 centipoise , greater than about 5000 centi As will be appreciated by the skilled artisan , preservatives poise . may be present in concentrations of from about 0 .001 % to In some embodiments , including (but not limited to ) 5 about 0 . 7 % ( w / v ) . In particular embodiments , the preserva where the use of salts is contraindicated as described above , tive ( s ) may be present in a concentration of from about the ocular formulation may further include one or more tonicity agents . As used herein , the term “ tonicity agent” and 0. 001 % to about 0 . 5 % ( w /v ); from about 0 .001 % to about its cognates refers to agents that adjust the tonicity of the 0 .05 % ( w / v ), from about 0 .001 % to about 0 .02 % ( w / v ) , from formulation , but are not salts ( e . g . , not NaCl) , which , as will 10 about 0 .001 % to about 0 .015 % ( w / v ) , from about 0 . 001 % to be appreciated by the skill artisan in view of the teaching about 0 .005 % ( w /v ), from about 0 .01 % to about 0 .02 % , provided herein , are contraindicated for some formulations from about 0 .002 % to about 0 .01 % , from about 0 . 015 % to due to the presence of certain of the gel - forming polymers about 0 .05 % , less than about < 0 . 5 % , from about 0 . 005 % to or viscosity - increasing agents . These agents may be used to about 0 .01 % , from about 0 .001 % to about 0 . 15 % , from prepare formulations that are isotonic or near isotonic ( e . g . , 15 about 0 .002 % to about 0 .004 % , from about 0 .001 % to about somewhat hyper - or hypo - isotonic ; e . g . , within about + 20 % , 0 .002 % . In some embodiments the concentration of the about + 15 % , about + 10 % , about 15 % of being isotonic ) . preservative may be, for example , about 0 .001 % , about Tonicity agent( s ) may also be used in formulations where the 0 .005 % , about 0 .01 % , about 0 .02 % , about 0 . 03 % , about use of salts is not contraindicated . 0 .05 % , about 0 . 1 % , about 0 . 2 % , about 0 . 5 % , or about 0 . 7 % Tonicity agents that may be used to adjust the tonicity of 2020 ( w / v ) . Typical concentrations ( w / v ) for various commonly formulation the formulations described herein and are used preservatives are listed in Table C below . known to the skilled artisan and can be selected based on the teaching provided herein . For example , tonicity agents TABLE C include polyols ( e . g ., sugar alcohols ( e . g ., mannitol, etc .) , Approximate Concentration trihydroxy alcohols ( e .g ., glycerin , etc . ) , propylene glycol or 25 Preservative Range ( w / v ) polyethylene glycol, etc . ) , or combinations of two or more Benzalkonium chloride 0 .01 - 0 .02 % polyols . Likewise, the concentration of the tonicity agent( s ) Benzethonium chloride 0 . 01- 0 .02 % will depend upon the identity and concentrations of the other Chlorhexidine 0 . 002 - 0 .01 % components in the formulation and can be readily deter Chlorobutanol < 0 . 5 % Methylparaben 0 . 015 - 0 . 05 % mined by the skilled artisan in view of the teaching provided 30 Phenylethyl alcohol < 0 . 5 % herein . Propylparaben 0 .005 - 0 .01 % In certain embodiments , the tonicity agent is glycerin or Thimerosal 0 .001 - 0 . 15 % mannitol. In some embodiments, the tonicity agent is glyc Phenylmercuric nitrate 0 . 002 - 0 . 004 % erin . In other embodiments it is , mannitol. In still others a Phenylmercuric borate 0 .002 - 0 .004 combination of mannitol and glycerin may be used . Exem - 35 Phenylmercuric acetate 0 . 001 - 0 . 002 plary concentrations of tonicity agents include , for example from about 0 . 001 to about 3 % . In some embodiments , the In certain embodiments , the formulation may additionally concentration of the tonicity agent ( e . g . , mannitol or glyc - include a surfactant, or combinations of two or more sur erin ) is , for example , about 0 .001 % to about 2 . 7 % , about factants. In particular embodiments, the formulation is sub 0 .001 % to about 2 . 5 % , about 0 . 001 % to about 2 % , about 40 stantially free of surfactant. As used herein , the term “ sub 0 .001 % to about 1 . 5 % , about 0 .001 % to about 1 % , about stantially free ” is intended to refer to levels of a particular 0 .01 % to about 3 % , about 0 .01 % to about 2 . 7 % , about component that are undetectable using routine detection 0 .01 % to about 2 . 5 % , about 0 . 01 % to about 2 % , about methods and protocols known to the skilled artisan . For 0 .01 % to about 1 . 5 % , about 0 . 01 % to about 1 % , about 0 . 1 % example , HPLC ( including chiral HPLC , chiral HPLC /MS , to about 3 % , about 0. 1 % to about 2. 7 % , about 0 . 1 % to about 45 LC /MS / MS etc .) , thin layer chromatography, mass spec 2 .5 % , about 0 . 1 % to about 2 % , about 0 .1 % to about 1 . 5 % , trometry , polarimetry measurements , Gas - chromatography about 1 % , about 0 .01 % about 1 % to about mass spectrometry , or others . 3 % ; about 1 % to about 2 . 5 % ; about 1 % to about 2 % ; about I n particular embodiments , the ocular formulation may 1 % to about 1 . 8 % ; about 1 % to about 1 . 5 % ; or about further include a chelating agent ( e . g ., EDTA disodium 0 . 001 % , about 0 .01 % , about 0 . 05 % , about 0 . 08 % , about 50 (EDTA ) ( e . g . , EDTA disodium ( dihydrate ), etc .) citrates , 0 . 1 % , about 0 . 2 % , about 0 . 5 % , about 0 . 8 % , about 1 % , about etc .) . In some embodiments , a combination of chelating 1 . 5 % , about 1 . 8 % , about 2 % , about 2 . 2 % , about 2 . 5 % , about agents may be present. As will be appreciated by those of 2 . 8 % , or about 3 % ( w / v ). In certain embodiments , the skill in the field , chelating agents can be used to hinder tonicity agent is mannitol. In some of these embodiments , degradation of the formulation components and thereby the carrier includes a gel - forming agent ( e. g ., gellan gum ). 55 increase the shelf life of ocular formulations. As will be In some embodiments , the tonicity agent is mannitol. In appreciated by the skilled artisan , use of EDTA in combi certain of these embodiments , the carrier includes a viscos - nation with gellan gum formulation may be contraindicated ity - increasing agent ( e . g . , water soluble cellulose derivatives as the EDTA can cause gel formation prior to administration ( e. g ., hypromellose ), polyvinyl alcohol, polyvinyl pyrroli - of the gellan gum formulation . done , chondroitin sulfate , hyaluronic acid , or soluble 60 Typical concentrations for chelating agents are from about starches ) . 0 . 005 % to 0 . 1 % ( w / v ) . For example , from about 0 . 005 % to In some embodiments , the ocular formulation may addi - about 0 .09 % , from about 0 . 005 % to about 0 . 08 % , from tionally include a preservative ( e . g . , benzalkonium chloride, about 0 . 005 % to about 07 % , from about 0 . 005 % , to about benzethonium chloride , chlorhexidine, chlorobutanol, meth - 0 .06 % , from about 0 . 005 % to about 0 .05 % , from about ylparaben , Phenylethyl alcohol, propylparaben , thimerosal, 65 0 . 005 to about 0 . 04 % , from about 0 . 005 % to about 0 .03 % , phenylmercuric nitrate , phenylmercuric borate , or phenyl from about 0 .01 % to about 0 . 1 % , from about 0 .01 % to about mercuric acetate, peroxides ), or a combination of two or 0 . 09 % , from about 0 .01 % to about 0 .08 % , from about 0 .01 % US 9 ,822 ,353 B2 149 150 to about 0 .07 % , from about 0 .01 % to about 0 .06 % , from but will depend on such factors as weight, diet , concurrent about 0 .01 % to about 0 .05 % , from about 0 .01 % to about medication and other factors which those skilled in the 0 .04 % , etc . In certain embodiments , the concentration of medical arts will recognize . chelating agent ( s ) is about 0 . 005 % , about 0 .01 % , about In certain embodiments , the pharmaceutical compositions 0 .02 % , about 0 .03 % , about 0 .05 % , about 0 . 06 % , about 5 may be delivered by intranasal sprays, inhalation , and /or 0 .07 % , about 0 .08 % , about 0 . 09 % , or about 0 . 1 % . other aerosol delivery vehicles. Methods for delivering In particular embodiments , the chelating agent is EDTA genes, polynucleotides , and peptide compositions directly to disodium . In certain embodiments , the chelating agent is the lungs via nasal aerosol sprays have been described e . g . , EDTA disodium (dihydrate ). In some of these embodiments , in U . S . Pat. Nos. 5 ,756 , 353 and 5 , 804 , 212 (each specifically the EDTA disodium dihydrate is present at a concentration incorporated herein by reference in its entirety ) . Likewise , of about 0 .01 % ( w / v ) . thele delivery of drugs using intranasal microparticle resins In some embodiments , the ocular formulation may addi- ( Takenaga et al. , 1998 ) and lysophosphatidyl- glycerol com tionally include one or more buffering agents ( e . g . , phos - pounds ( U . S . Pat. No . 5 , 725 ,871 , specifically incorporated phate buffer( s ) ( e . g . , sodium phosphate buffers ( e . g . , dibasic 1 herein by reference in its entirety ) are also well -known in the sodium phosphate , monobasic sodium phosphate , etc . ) , cit - pharmaceutical arts . Likewise , transmucosal drug delivery rate buffers , maleate buffers , borate buffers , etc . ). As will be in the form of a polytetrafluoroetheylene support matrix is appreciated by the skilled artisan , the one or more buffering described in U . S . Pat. No. 5 , 780 ,045 (specifically incorpo agent( s ) should be selected in combination with the other rated herein by reference in its entirety ) . components of a given formulation to achieve a pH suitable 20 In certain embodiments , the delivery may occur by use of for use ( e . g . , pH of about 4 . 5 to about 8 ). liposomliposomes , nanocapsules, microparticles , microspheres , In certain embodiments , the buffering agent is a phosphate lipid particles, vesicles , and the like, for the introduction of buffer or combination of two or more phosphate buffers . In the compositions of the present invention into suitable host certain embodiments , the buffering agents are dibasic cells . In particular, the compositions of the present invention sodium phosphate and monobasic sodium phosphate . 25 may be formulated for delivery either encapsulated in a lipid Typical concentrations for buffering agent( s ) for example , particle , a liposome, a vesicle , a nanosphere , a nanoparticle phosphate buffering agent( s ) may be from about 0 .005 molar or the like . The formulation and use of such delivery to 0 . 1 molar. In some embodiments, the buffering agent ( s ) vehicles can be carried out using known and conventional may be at a concentration of about 0 .01 to about 0 . 1 , from techniques . about 0 .01 to about 0 . 08 , from about 0 .01 to about 0 .05 , 30 from about 0 .01 to about 0 . 04 , from about 0 .02 to about 0 . 1 , In certain embodiments , the agents provided herein may from about 0 .02 to about 0 .08 , from about 0 .02 to about be attached to a pharmaceutically acceptable solid substrate , 0 .06 , from about 0 .02 to about 0 . 05 , from about 0 .02 to including biocompatible and biodegradable substrates such about 0 .04 molar , etc. In particular embodiments , there are as polymers and matrices. Examples of such solid substrates two buffering agents . Exemplary buffering agents include a 35 include , without limitation , polyesters , hydrogels ( for combination of dibasic sodium phosphate ( e . g ., dibasic example , poly ( 2 -hydroxyethyl - methacrylate ), or poly (vinyl sodium phosphate .7H . O ) and monobasic sodium phosphate alcohol) ) , polylactides ( U . S . Pat. No . 3 , 773 , 919 ) , copoly ( e . g ., monobasic sodium phosphate anhydrous) . In some mers of L - glutamic acid and y - ethyl- L - glutamate , non -de embodiments , the concentration of the buffering agent( s ) is gradable ethylene - vinyl acetate , degradable lactic acid about 0 .005 molar, about 0 . 01 molar, about 0 . 02 molar, 40 glycolic acid copolymers such as poly ( lactic -co - glycolic about 0 . 03 molar, about 0 .04 molar, about 0 . 05 molar , about acid ) (PLGA ) and the LUPRON DEPOTTM ( injectable 0 .06 molar , about 0 .07 molar, or about 0 . 1 molar. microspheres composed of lactic acid - glycolic acid copoly An additional aspect of the invention includes use of the mer and leuprolide acetate ), poly - D -( - )- 3 -hydroxybutyric formulations as described herein in the manufacture of a acid , collagen , metal, hydroxyapatite , bioglass , aluminate , medicament. Particularly , the manufacture of a medicament 45 bioceramic materials , and purified proteins . for use in the treatment and / or prevention of conditions as In one particular embodiment, the solid substrate com described herein . Further , the formulations, variously prises AtrigelTM ( QLT, Inc . , Vancouver, B . C . ). The Atrigel® described herein , are also intended for use in the manufac drug delivery system consists of biodegradable polymers ture of a medicament for use in treatment and / or prevention dissolved in biocompatible carriers. Pharmaceuticals may be of the conditions and , in accordance with the methods , 50 blended into this liquid delivery system at the time of described herein , unless otherwise noted . manufacturing or, depending upon the product , may be Methods of formulation are well known in the art and are added later by the physician at the time of use . When the disclosed , for example , in Remington : The Science and liquid product is injected into the subcutaneous space Practice of Pharmacy, Mack Publishing Company, Easton , through a small gauge needle or placed into accessible tissue Pa ., 19th Edition ( 1995 ) . The compositions and agents 55 sites through a cannula , water in the tissue fluids causes the provided herein may be administered according to the polymer to precipitate and trap the drug in a solid implant . methods of the present invention in any therapeutically The drug encapsulated within the implant is then released in effective dosing regime . The dosage amount and frequency a controlled manner as the polymer matrix biodegrades with are selected to create an effective level of the agent without time. harmful effects . The effective amount of a compound of the 60 In particular embodiments , the amount of a PEGylated present invention will depend on the route of administration , DRS composition the agent administered will generally the type of warm -blooded animal being treated , and the range from a dosage of from about 0 . 1 to about 100 physical characteristics of the specific warm -blooded animal mg/ kg / day, and typically from about 0 . 1 to 10 mg/ kg where under consideration . These factors and their relationship to administered orally or intravenously . In particular embodi determining this amount are well known to skilled practi - 65 ments , a dosage is 5 mg/ kg or 7 . 5 mg/ kg . For humans, the tioners in the medical arts . This amount and the method of daily dosage used may range from , about 0 . 1 mg/ kg to 0 . 5 administration can be tailored to achieve optimal efficacy mg/ kg , about 1 mg/ kg to 5 mg/ kg , about 5 mg/kg to 10 US 9 ,822 ,353 B2 151 152 mg/ kg , about 10 mg/ kg to 20 mg/ kg , about 20 mg/ kg to 30 or between about 0 . 3 ug/ ml to about 3 ug /ml . In certain mg/ kg , about 30 mg/ kg to 50 mg/ kg , and about 50 mg/ kg to embodiments , an intravenous dosage is an amount sufficient 100 mg/ kg / 24 hours . to achieve a blood plasma concentration (Cmor ) of between In certain embodiments , a composition or agent is admin about 1 ug /ml to about 10 ug/ ml or between about 2 ug /ml istered in a single dosage of 0 . 1 to 10 mg/ kg or 0 . 5 to 5 5 and about 6 ug /ml . In a related embodiment, the total mg/ kg . In other embodiments , a composition or agent is concentration of an agent in the blood plasma of the subject administered in a dosage of 0 . 1 to 50 mg/ kg/ day , 0 . 5 to 20 has a mean trough concentration of less than about 20 ug /ml mg/ kg /day , or 5 to 20 mg/ kg / day. and / or a steady state concentration of less than about 20 In various embodiments , the dosage is about 50 - 2500 mg ug/ ml . In a further embodiment, the total concentration of an per day, 100 -2500 mg/ day , 300 - 1800 mg/ day , or 500 - 1800 10 agent in the blood plasma of the subject has a mean trough mg/ day . In one embodiment , the dosage is between about concentration of less than about 10 ug /ml and / or a steady 100 to 600 mg/ day. In another embodiment, the dosage is state concentration of less than about 10 ug /ml . between about 300 and 1200 mg/ day . In particular embodi - In yet another embodiment, the total concentration of an ments , the composition or agent is administered at a dosage agent in the blood plasma of the subject has a mean trough of 100 mg/ day , 240 mg/ day 300 mg/ day , 600 mg/ day , 1000 15 concentration of between about 1 ng /ml and about 10 ug/ ml mg/ day , 1200 mg/ day , or 1800 mg/ day, in one or more doses and /or a steady state concentration of between about 1 ng /ml per day ( i . e . , where the combined doses achieve the desired and about 10 ug /ml . In one embodiment, the total concen daily dosage ) . In related embodiments , a dosage is 100 mg tration of an agent in the blood plasma of the subject has a bid , 150 mg bid , 240 mg bid , 300 mgbid , 500 mg bid , or 600 mean trough concentration of between about 0 . 3 ug /ml and mg bid . In various embodiments , the composition or agent 20 about 3 ug /ml and / or a steady state concentration of between is administered in single or repeat dosing . The initial dosage about 0 . 3 ug /ml and about 3 ug /ml . and subsequent dosages may be the same or different. In particular embodiments , a composition or agent is In some embodiments , total daily dose may be about administered in an amount sufficient to achieve in the 0 .001 mg, about 0 . 005 mg, about 0 .01 mg, about 0 . 05 mg, mammal a blood plasma concentration having a mean about 0 . 1 mg, 0 .5 mg, 1 mg, about 2 mg, about 3 mg, about 25 trough concentration of between about 1 ng /ml and about 10 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, ug /ml and / or a steady state concentration of between about about 9 mg, about 10 mg, about 20 mg, about 30 mg, about 1 ng /ml and about 10 ug /ml . In related embodiments , the 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 total concentration of the agent in the blood plasma of the mg, about 90 mg or about 100 mg/ 24 hours . For repeated mammal has a mean trough concentration of between about administrations over several days or longer , depending on 30 0 . 3 ug/ ml and about 3 ug /ml and / or a steady state concen the condition , the treatment is sustained until a desired tration of between about 0 . 3 ug /ml and about 3 ug /ml . suppression of disease symptoms occurs. The progress of In particular embodiments of the present invention , the these and other therapies ( e . g . , ex vivo therapies ) can be effective amount of a composition or agent, or the blood readily monitored by conventional methods and assays and plasma concentration of composition or agent is achieved or based on criteria known to the physician or other persons of 35 maintained , e . g . , for at least 15 minutes , at least 30 minutes , skill in the art. at least 45 minutes , at least 60 minutes, at least 90 minutes , It will be further appreciated that for sustained delivery at least 2 hours, at least 3 hours , at least 4 hours , at least 8 devices and compositions the total dose of DRS contained in hours, at least 12 hours , at least 24 hours , at least 48 hours , such delivery system will be correspondingly larger depend at least 3 days , at least 4 days , at least 5 days , at least 6 days, ing upon the release profile of the sustained release system . 40 at least one week , at least 2 weeks, at least one month , at Thus, a sustained release composition or device that is least 2 months , at least 4 months, at least 6 months, at least intended to deliver DRS polypeptide over a period of 5 days one year , at least 2 years , or greater than 2 years . will typically comprise at least about 5 to 10 times the daily In certain DRS polypeptide -based embodiments , the dose of DRS polypeptide ; a sustained release composition or amount of polypeptide administered will typically be in the device that is intended to deliver a DRS peptide over a 45 range of about 0 . 1 ug/ kg to about 0 . 1 mg/ kg to about 50 period of 365 days will typically comprise at least about 400 mg/ kg of patient body weight. Depending on the type and to 800 times the daily dose of the DRS polypeptide ( depend severity of the disease , about 0 . 1 ug/ kg to about 0 . 1 mg/ kg ing upon the stability and bioavailability of the DRS poly - to about 50 mg/ kg body weight ( e. g ., about 0 . 1 - 15 mg/ kg / peptide when administered using the sustained release sys - dose ) of polypeptide can be an initial candidate dosage for tem ) . 50 administration to the patient, whether, for example , by one In certain embodiments , a composition or agent is admin - or more separate administrations, or by continuous infusion . istered orally or intravenously , e . g . , by infusion over a period For example , a dosing regimen may comprise administering of time of about, e . g ., 10 minutes to 90 minutes . In other an initial loading dose of about 4 mg/ kg , followed by a related embodiments , a composition or agent is administered weekly maintenance dose of about 2 mg/ kg of the polypep by continuous infusion , e . g . , at a dosage of between about 55 tide , or about half of the loading dose . However, other 0 . 1 to about 10 mg/ kg /hr over a time period . While the time dosage regimens may be useful. A typical daily dosage period can vary , in certain embodiments the time period may might range from about 0 . 1 ug/ kg to about 1 ug /kg to 100 be between about 10 minutes to about 24 hours or between mg/ kg or more , depending on the factors mentioned above . about 10 minutes to about three days. For repeated administrations over several days or longer, In particular embodiments , an effective amount or thera - 60 depending on the condition , the treatment is sustained until peutically effective amount is an amount sufficient to a desired suppression of disease symptoms occurs . achieve a total concentration of the composition or agent in In particular embodiments, the effective dosage achieves the blood plasma of a subject with a Cmar of between about the blood plasma levels or mean trough concentration of a 0 . 1 ug /ml and about 20 ug /ml or between about 0 . 3 ug /ml composition or agent described herein . These may be readily and about 20 ug /ml . In certain embodiments , an oral dosage 65 determined using routine procedures . is an amount sufficient to achieve a blood plasma concen - Embodiments of the present invention , in other aspects , tration (Cmax ) of between about 0 . 1 ug /ml to about 5 ug/ ml provide kits comprising one or more containers filled with US 9 , 822 , 353 B2 153 154 one or more of the polypeptides, polynucleotides , antibod The corresponding translated protein sequence is: ies, multiunit complexes, compositions thereof, etc ., of the invention , as described herein . The kits can include written instructions on how to use such compositions ( e . g . , to ( SEO ID NO : 29 ) modulate cellular signaling , angiogenesis , cancer, inflam - 5- MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIQSQEKPDRVLVRVRD matory conditions, diagnosis etc . ) . LTIQKADEVVWVRARVHTSRAKGKOSFLVLROQQFNVQALVAVGDHASKO The kits herein may also include a one or more additional MVKFAANINKESIVDVEGVVRKVNQKIGSCTQQDVELHVQKIYVISLAEP therapeutic agents or other components suitable or desired for the indication being treated , or for the desired diagnostic i RLPLGKPIPNPLLGLDSTHHHHHH application . An additional therapeutic agent may be con - As a control, the non -mutated AspRS1N1 protein was also tained in a second container, if desired . Examples of addi tional therapeutic agents include , but are not limited to prepared , using wild type (human codon usage ) , and cloned anti - neoplastic agents , anti - inflammatory agents , antibacte into the identical expression cassette . The nucleic acid rial agents , antiviral agents , angiogenic agents , etc . 15 sequence of the native AspRS1M1 is as follows: The kits herein can also include one or more syringes or other components necessary or desired to facilitate an ( SEQ ID NO : 30 ) intended mode of delivery ( e . g . , stents, implantable depots , ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGAAGCCGCGGGAGAT etc . ) . CATGGACGCGGCGGAAGATTATGCTAAAGAGAGATATGGAATATCTTCAA The present invention now will be described more fully 20 by the following examples . This invention may , however, be TGATACAATCACAAGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGAC embodied in many different forms and should not be con TTGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGTGCAAGAGTTCA strued as limited to the embodiments set forth herein ; rather, these embodiments are provided so that this disclosure will TACAAGCAGAGCTAAAGGGAAACAGTGCTTCTTAGTCCTACGTCAGCAGC be thorough and complete , and will fully convey the scope of the invention to those skilled in the art. AGTTTAATGTCCAGGCTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAG ATGGTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGTGGATGTAGA EXAMPLES AGGTGTTGTGAGAAAAGTGAATCAGAAAATTGGAAGCTGTACACAGCAAG 30 Example 1 ACGTTGAGTTACATGTTCAGAAGATTTATGTGATCAGTTTGGCTGAACCC CGTCTGCCCCTGGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTC Production of DRS Polypeptides TACGCACCACCACCACCACCACTGA 353 The encoding protein , containing the identical C - terminal Codon Optimization and Gene Synthesis : An E . coli tag, but the wild type Cys76 is shown below : codon optimized nucleic acid sequence encoding the DRS polypeptide AspRS1M (C76S ) (comprising amino acids 1 - 154 , and a cysteine - > serine mutation at position 76 ) was ( SEQ ID NO : 31 ) designed for optimal E . coli expression using the algorithm 40 MPSASASRKSOEKPREIMDAAEDYAKERYGISSMIOSOEKPDRVLVRVRD developed by DNA2 .0 (Menlo Park , Calif. ) . The gene was synthesized with a C -terminal V5H is tag and subcloned into LTIQKADEVVWVRARVHTSRAKGKOCFLVLRQQQFNVQALVAVGDHASKO PJExpress411 vector where the T7 promoter was used to MVKFAANINKESIVDVEGVVRKVNQKIGSCTQQDVELHVQKIYVISLAEP drive the transcription and the kanamycin resistance was used for antibiotic selection . The codon -optimized DNA 45 RLPLGKPIPNPLLGLDSTHHHHHH sequence is as follows: Expression Strains : BL21 -CodonPlus (DE3 )- RIPL com petent cells (Agilent cat. no . 230280 ) were transformed with ( SEO ID NO : 28 ) the non -mutated AspRS1M expression construct . BL21 ATGCCGAGCGCGAGCGCCAGCCGTAAGAGCCAGGAAAAACCACGTGAGAT 50 (DE3 ) competent cells (Novagen , cat. no . 69450 ) were transformed with the AspRS1M (C76S ) expression con TATGGATGCCGCAGAGGACTATGCGAAAGAACGTTACGGTATTTCCAGCA struct. Briefly, the plasmid ( 1 uL ) was added into 50 uL of the competent cells . The reaction was mixed and incubated TGATCCAATCTCAGGAGAAACCGGACCGCGT???GGTTCGTGTTCGCGAT on ice for 30 minutes . The reaction was heat - shocked for at CTGACCATTCAGAAGGCGGACGAGGTGGTTTGGGTGCGTGCGCGCGTGCA 55 42° C . for 30 sec followed by a cold - shock on ice for 2 minutes . Then the SOC medium (500 UL ) was added and the CAccAGccGTGCAAAAGGCAAACAGAGC?????GGTccTGCGTCAGCAGC tube was incubated at 37° C ., 250 rpm for 1 hour. Finally, an AATTCAACGTCCAGGCGCTGGTGGCAGTGGGTGACCACGCCAGCAAACAA aliquot of the culture (50 UL ) was spread on the Kanamycin plate ( Teknova S9641 ) and incubated at 37° C . overnight . ATGGTGAAGTTCGCTGCTAACATCAATAAAGAATCCATTGTTGATGTTGA 60 Single colony was picked and used for expression scale - up . Fed -Batch Fermentation Production of Proteins: MOYE AGGCGTCGTTCGCAAGGTCAATCAAAAGATCGGCTCGTGTACGCAACAAG medium was prepared by mixing 200 mL sterile M9minimal ATGTCGAGCTGCATGTGCAGAAGATTTACGTCATCAGCCTGGCGGAGCCG salt 5x (BD248510 ) , 778 mL 30 g / L yeast extract in sterile purified water (BD212750 ) , 20 mL sterilized 20 % glucose CGTTTcccccTGGGTAAC?CGATcccTAAcccccTGTTGGGTCTGGACAG 65 (Sigma G7021) and 2 mL sterile 1 .0 M MgSO4 (Sigma CACGCATCACCATCACCACCACTAA M7506 ) . The feeding solution contains 5 % yeast extract , 50 % glucose , trace elements and 2 g /L magnesium sulfate . US 9 ,822 ,353 B2 155 156 Kanamycin sulfate ( Invitrogen 15160 ) was added to a final TABLE E1 concentration of 100 ug/ mL in both M9YE and feeding solution . Production yields for different AspRS1N1 variants A 4 L fermentor (Sartorius Biostat B plus ) with MFCS/ DA software was used for the fed - batch fermentation of both 5 Purified protein yield proteins. The agitation was set at 1000 rpm . The pH value DRS polypeptide form (mg / g cell pellet ) was controlled at 7 .0 automatically by the addition of 30 % ammonium hydroxide (Sigma 221228 ) and 30 % phosphoric AspRS1M1 (C765 ) 1 .72 = 0 . 25 ( n = 8 ) acid (Sigma P5811) . The air was provided at a flow rate of AspRsiNi 1 . 38 + 0 .57 ( n = 7 ) 4 L /min with an oil - free diaphragm air compressor ( Cole Parmer ). The air was passed through a 0 . 2 um Midisart 2000 filter (Sartorius 17805 ). The pure oxygen (West Air ) was An analysis of representative proteins by SDS- gel is supplied automatically to control the dissolved oxygen level shown in FIG . 1 . The gel demonstrates that the purified at 70 % . The temperature was controlled at 30° C . with a AspRSIM (C76S ) has less low molecular weight impurities, Neslab RTE7 circulator ( Thermo Scientific ). The foaming4 15 and contains less disulfide cross- linked dimer species , com was controlled by addition of the antifoam 204 (Sigma pared to comparable batches of AspRS1M prepared under A8311 ) . The initial volume of MOYE medium in the fer identical conditions. mentor was 3 L . The fermentor was inoculated with 150 mL Moreover an analysis of the proteins endotoxin content of the seed culture grown overnight at 30° C . and 250 rpm . 20 reveals that the AspRS1 ^ (C76S ) proteins exhibited a When the glucose was depleted in the vessel, the concen significantly reduced endotoxin content compared to the trated feeding solution was introduced into the vessel by a non -mutated AspRS1M . ( Table E2 ) . peristaltic pump set at 0 . 9 ml/ min . When the optical density of the cells at 600 nm reached about 30 , the culture was TABLE E2 induced with 0 .5 mM IPTG ( Fisher Scientific BP1755 ). The 25 Endotoxin Content culture was run overnight ( about 18 -hour fed - batch phase ) and harvested by centrifugation at 6 , 000 g for 1 hour. The Average Endotoxin level cell pellet was stored at - 20° C . until purification . The DRS polypeptide form in purified protein ( EU /mg ) expression of each protein was confirmed by SDS- PAGE AspRS1N1 (C76S ) 7 . 3 ( n = 8 ) analysis ( data not shown) . 30 AspRS1N1 43. 5 ( n = 7 ) Purification of Proteins: Frozen cell pellets from each production run were resuspended in 4 volumes (i . e. , 4 mL /g Accordingly it is concluded that the DRS polypeptides cell pellet ) of Lysis Buffer ( 50 mM Tris , 300 mM NaC1, 25 comprising a reduced a cysteine content, specifically mM Imidazole , 14 mM 13 -ME , pH 8 . 0 ). Complete EDTA FREE protease inhibitor cocktail tablets (Roche Cat . # 05 improved production yields and significantly less endotoxin 056 489 001 ) were added to the suspension at a ratio of 1 contamination compared to the corresponding non mutated tablet/ 50 mL . The suspension was passed through a micro protein . fluidizer (Microfluidics ) twice at 14 ,000 psi with cooling by ice . The lysate was centrifuged at 35, 000xg for 45 min at 4° 40 C . The supernatant was filtered through 0 .45 + 0 .22 um Example 2 Sartobran capsule filters (Sartorius ) . The clarified lysate was bound to the Ni- NTA resin (Qiagen ), pre -equilibrated with Ni-NTA Binding Buffer (50 Production of DRS Polypeptides in Mammalian mM Tris , 300 mM NaCl, 25 mM Imidazole , pH 8 . 0 ) . The 45 Cells column was washed with 300 column volumes of Ni-NTA Binding Buffer + 0 . 1 % Triton X - 114 followed by 33 column As an alternative production system , exemplary DRS volumes of the Ni- NTA Binding Buffer. The bound protein , polypeptides were prepared using a mammalian expression D1 - C76S , was eluted with 5 column volumes of Ni- NTA system . This approach has the potential advantage of elimi Elution Buffer ( 50 mM Tris , 300 mM NaCl, 300 mM 50 nating any potential contamination of the DRS polypeptides Imidazole , pH 8 . 0 ) . with E . coli derived endotoxins . The purified proteins were dialyzed into a buffer contain ing 20 mM sodium phosphate , 200 mM Arginine, at pH 7 . 3 . Cloning : The AspRS1M1 fragment ( amino acid 1- 154 of The dialyzed protein was passed through a Q membrane human cytoplasmic Aspartyl- tRNA synthetase ) was ampli filter (Sartobind - Q from Sartorius or Mustang - Q from Pall ) 55 fied by polymerase chain reaction (PCR ) using the following or a Q - Sepharose column (GE Healthcare ) for further endo primer pairs synthesized at Integrated DNA Technologies to toxin removal, and then filtered through a 0 . 22 um sterile create either cytoplasmic , or secreted versions of the filter. AspRS1M1 Comparison of Production Yield , Purity and Endotoxin Content of AspRS1M (C76S ) with AspRS1M1. Primer Pair 1 A direct comparison of the yields of soluble proteins from ( SEO ID NO : 32 ) the AspRS1M1 (C76S ) and non -mutated AspRS1M1 con AGTCTTGCACTTGTCACGAATTCGATGCCCAGCGCCAGCGCCAGC structs , over several independent production runs, ( Table E1 ) reveals that the AspRS1M (C76S ) variant has a consis ( SEQ ID NO : 33 ) tently higher yield compared to the non -mutated parent 65 CGGTGGGCATGTGTGAGTTTTGTCTCACTTGTCGTCATCGTCTTTGTAGT protein . Table E1 lists the average purification yield of CCGTAGAATCGAGACCGAGGAGAGG AspRS1M (C76S ) and non -mutated AspRS1M . US 9 ,822 , 353 B2 157 158 - continued The intracellular AspRS1M sequence is as follows : Primer Pair 2 ( SEO ID NO : 34 ) ( SEO ID NO : 37 ) GATCACCGGCGAAGGAGGGCCACCATGCCCAGCGCCAGCGCCAGC ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGAAGCCGCGGGAGAT ( SEQ ID NO : 35 ) cGGT GGGCATGTGTGAGTTTTGTCTCACTTGTCGTCATCGTCTTTGTAGT CATGGACGCGGCGGAAGATTATGCTAAAGAGAGATATGGAATATCTTCAA CCGTAGAATCGAGACCGAGGAGAGG TGATACAATCACAAGAAAAACCAGATCGAGTTTTGGTTCGGGTTAGAGAC The primers were mixed with the template (AspRS1N1 10 TTGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGTGCAAGAGTTCA nucleic acid fragment in the pET28 vector ) ( see above ), TACAAGCAGAGCTAAAGGGAAACAGTGCTTCTTAGTCCTACGTCAGCAGC Accuprime pfx supermix ( Invitrogen cat. no . 12344 -040 ) AGTTTAATGTCCAGGCTCTTGTGGCGGTGGGAGACCATGCAAGCAAGCAG and denatured for 5 minutes at 95° C . The amplification was done in the Eppendorf thermal cycler for 35 cycles of 95° C . ATGGTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTGTGGATGTAGA for 30 seconds, 52° C . for 30 seconds and 68° C . for 40 AGGTGTTGTGAGAAAAGTGAATCAGAAAATTGGAAGCTGTACACAGCAAG seconds. The amplified fragments were purified with QIA quick PCR Purification Kit ( Qiagen cat . no . 28104 ) . The ACGTTGAGTTACATGTTCAGAAGATTTATGTGATCAGTTTGGCTGAACCC fragment size , quantity and purity were confirmed on the 1 % agarose gel in the TAE buffer ( Invitrogen cat. no . 15558 ) . 20 CGTCTGCCCCTGGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTC The fragment was inserted into the pFUSE -hIgG1 - Fc2 ( Invi- TACGGACTACAAAGACGATGACGACAAGTGA vogen cat. no . pfuse -hg1fc2 ) by mutagenesis using the The hEF1- HTLV promoter comprising the Elongation QuikChange Lightning Site -Directed Mutagenesis Kit (Agi - Factor -la (EF - 1a ) core promoter and the R segment and lent, cat. no . 210518 ) . Eighteen thermal cycles were per part of the U5 sequence of the Human T -Cell Leukemia formed at 95° C . for 30 seconds, 52° C . for 30 seconds and 25 Virus (HTLV ) Type 1 Long Terminal Repeat was used to 68° C . for 4 minutes . After mutagenesis , the sample was drive the transcription . The V5 (GKPIPNPLLGLDST ) treated with Dpn I enzyme at 37° C . and transformed into (SEO ID NO : 72 ) and Flag (DYKDDDDK ) (SEO ID NO :73 ) tags were added to the C - terminus of the D1 fragments for XL10 gold competent cells . The heat shock was done at 42° detection and purification purpose . The Sh ble gene from C . for 30 seconds followed by 2 minutes on ice . The XL 10 30 Streptoalloteichus hindustanus was used for antibiotic resis gold transformants were resuspended in SOC medium and 30 tance . The Simian Virus 40 late polyadenylation signal incubated at 37° C . for 1 hour and then were spread onto enables the cleavage and polyadenylation resulting in stable zeocin agar and incubated at 37° C . overnight. Multiple mRNA . colonies were grown in terrific broth overnight at 37° C . and Expression . The FREESTYLETM MAX CHO Expression the plasmids were purified with QIAprep Spin Miniprep Kit - System ( Invitrogen cat. no . K9000 - 20 ) was used for expres ( Qiagen cat . no . 27106 ). The plasmids were sequenced to sion of the secretory form of AspRS1M . The CHO - S cells confirm the DNA identity . The correct clones were trans were thawed from liquid nitrogen and grown in the serum formed into NovaBlue competent cells (Novagen cat. no . free medium ( FREESTYLETM CHO Expression Medium ) 70181) and grown in 250 ml MOYE medium at 37° C . supplemented with 8 mM L -Glutamine in a 37° C . incubator overnight. The maxiprep was performed using the HiSpeed containing a humidified atmosphere of 8 % CO , in air on an 40 orbital shaker platform rotating at 125 rpm . The cells were Plasmid Maxi Kit ( Qiagen cat. no . 12663) . The concentra diluted to 2 - 3x10 cells /ml when the density reached about tion and purity were determined by measuring A260 , A280 109 cells /ml and were repeated a few passages . The DNA and A230 . The purified plasmids were stored at - 20° C . was mixed 1 : 1 with the Freestyle Max reagent in the Optipro before transfection . SFM and incubated 10 minutes at room temperature . The The secretory AspRS1M sequence is as follows: 45 complex was added slowly into the cells at the density about 106 cells/ ml . The cell density and viability were monitored daily until harvest. ( SEO ID NO : 36 ) The FREESTYLETM 293 Expression ( Invitrogen cat. no . ATGTACAGGATGCAACTccTGTCTTGCATTGCACTAAGTCTIGCACTIGT K9000 -01 ) was used for expression of the intracellular form CACGAATTCGATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGAAGC 50 of AspRS1" . The 293 - F cells were thawed from liquid nitrogen and grown in the serum - free medium ( FREE CGCGGGAGATCATGGACGCGGCGGAAGATTATGCTAAAGAGAGATATGGA STYLETM 293 Expression Medium ) supplemented with ATATCTTCAATGATACAATCACAAGAAAAACCAGATCGAGTTTTGGTTCG Glutamax - I in a 37° C . incubator containing a humidified atmosphere of 8 % CO , in air on an orbital shaker platform GGTTAGAGACTTGACAATACAAAAAGCTGATGAAGTTGTTTGGGTACGTG 55 rotating at 125 rpm . The cells were diluted to 2 - 3x10 cells /ml when the density reached about 10 cells/ ml and CAAGAGTTCATACAAGCAGAGCTAAAGGGAAACAGTGCTTCTTAGTCCTA were repeated for a few passages. The DNA was mixed 1 : 2 CGTCAGCAGCAGTTTAATGTCCAGGCTCTTGTGGCGGTGGGAGACCATGC with the 293transfectin reagent in the Opti -MEM I and incubated 20 -30 minutes at room temperature. The complex AAGCAAGCAGATGGTTAAATTTGCTGCCAACATCAACAAAGAGAGCATTG 60 was added slowly into the cells at the density about 106 TGGATGTAGAAGGTGTTGTGAGAAAAGTGAATCAGAAAATTGGAAGCTGT cells /ml . The cell density and viability were monitored daily until harvest. ACACAGCAAGACGTTGAGTTACATGTTCAGAAGATTTATGTGATCAGTTT Purification . In the case of secretory form of AspRS1M , the supernatant of the cell culture was separated from the GGCTGAAccccGTCTGccccTGGGTAAGCC???ccCTAAccc????c??? 65 cells by centrifugation . The clarified sample was loaded onto GTCTCGATTCTACGGACTACAAAGACGATGACGACAAGTGA M2 agarose ( Sigma cat. no . A2220 ) in a gravity column . The resin was then washed with TBS (50 mM Tris HC1, with 150 US 9 ,822 ,353 B2 159 160 mM NaC1, pH 7 . 4 ) . The bound protein was eluted with 0 . 1 minutes at 56° C . ) , 100 mg/mL ZEOCINTM , 2 mM L - glu M glycine HC1, pH 3 . 0 and neutralized immediately with tamine ) . Cells were plated at a concentration of 50 , 000 1M Tris buffer at pH8 .0 . cells/ well in a 96 well plate in a total volume of 100 ul , and In the case of intracellular form of AspRSIMI, the cells DRS polypeptides , (AspRS1M or AspRS1M (C76S ) ) , were were recovered by centrifugation . The cells were lysed using 5 added to each well at the concentrations shown for 16 hours . On the next day, SEAP detection medium ( QUANTI M - PER Mammalian Protein Extraction Reagent (Pierce cat. BLUETM ) ( Invivogen Catalog code : rep - qb1 ) was prepared no . 78501 ) and then centrifuged to remove the insoluble following the manufacturer ' s instructions and 120 uL was debris . The clarified lysate was loaded onto M2 agarose added per well to a clear flat- bottom 96 -well plate, followed (Sigma cat. no . A2220 ) in a gravity column . The resin was by ( 20 uL ) of cell supernatant. Samples were incubated at then washed with TBS (50 mM Tris HC1, with 150 mM " 37° C . for 24 hours . SEAP levels were determined using a NaCl, pH 7 . 4 ) . The bound protein was eluted with 0 . 1 M spectrophotometer and reading absorbance at 650 nM . glycine HCl, pH 3 . 0 and neutralized immediately with 1M Results . The results shown in FIGS. 2 and 3, demonstrate Tris buffer at pH8. 0 . The purified protein was analyzed by that the DRS polypeptide AspRS1 " (C76S ) exhibited sig SDS - PAGE and Western blot. Purified proteins may be nificantly more activity , and displayed an apparent EC50 evaluated for binding to TLRs as described in Example 3 13 which was significantly higher compared to the non -mutated AspRS1M parent molecule with respect to both TLR2 and below . TLR4 receptor binding ( Table E3 ). Example 3 TABLE E3 20 - Evaluation of Biological Activity Activity of AspRS1N1 variant C76S on TLR2 and TLR4 receptors To evaluate the binding of the DRS polypeptides to Fold increase in activity human toll like receptors a series of studies were conducted DRS polypeptide form over AspRS1M1 with commercially available reporter HEK 293 and THP - 1 TLR2 Activity cell lines over expressing the TLR 2 and TLR 4 receptors . Genetically modified Human HEK293 cells sold under AspRsimi (C768 ) 3. 2 + 0. 14 (n = 2 ) the trademark HEK - BlueTM TLR cells ( Invivogen ) selec TLR4 Activity tively express the TLR2 or TLR4 receptors and include a AspRS1N1 ( C765) secreted embryonic alkaline phosphatase (SEAP )reporter 3 . 6 + 0 .17 (n = 2 ) gene under the control of an IFN -beta minimal promoter 30 which is fused to five NF - kB and AP - 1 transcription factors These results demonstrate the DRS polypeptides with binding sites . With the use of specific TLR 2 or 4 agonists altered cysteine content, and in particular DRS mutants (respectively ), HEK -BLUETM TLR2 and HEK -BLUETM comprising the mutation of cysteine 76 to another amino TLR4 cells activate NF - kB and / or AP - 1 leading to the acid , result in the creation of new product forms which secretion of SEAP which is measurable when using SEAP5 35» surprisingly exhibit enhanced activities , improved produc detection reagent . The HEK - BLUETM TLR2 cells are co tion yields and further surprisingly demonstrate reduced transfected with the LPS co - receptor protein CD14 to endotoxin content . enhance TLR2 responsiveness and improve signal quality . Example 4 The parent cell expresses endogenous levels of TLR1, 3 , 5 ,9 6 and also NOD1. The THP- 1 monocyte reporter cells to Mutation of C76 and 0130 to Other Amino Acids ( Invivogen THP1- XBlueTM cells ) . Stably express CD14 , MD - 2 , & and also include a secreted embryonic alkaline To determine whether other favorable mutations in addi phosphatase (SEAP ) reporter gene under the control of tion to Cys76 - > Ser could be identified , both cysteine resi NF -kB and AP - 1 promoter elements as described above . dues ( i. e ., those at either Cys76 or Cys130 ) were mutated to Methods . HEK - BLUETM - TLR2 or HEK - BLUETM - TLR4 43 all 19 alternative naturally occurring amino acid residues . To cells were washed twice with PBS, trypsinized and resus accomplish this in either the native human codon usage DRS pended in fresh Growth Medium (Growth Medium : DMEM , polypeptides, or the E . coli optimized DRS polypeptides, the 4 .5 g /L glucose , 10 % heat - inactivated fetalbovine serum (30 following primers were used : TABLE E4 Mutagenesis Primer Sequences Amino Acid Residue Range of SEQ ID SEQ ID Name NO : 1 Nucleic acid sequence NO : Human C76X 211 - 247 GCTAAAGGGAAACAGNNNTTCTTAGTCCTACGTCAG 3838 Primer C ( NNN = AGC ) Human C13 OX 367 - 403 GTGAATCAGAAAATTGGAAGCNNNACACAGCAAGA 39 Primer CG ( NNN = AGC ) E . coli codon 208 - 247 CGTGCAAAAGGCAAACAGNNNTTTCTGGTCCTGCGT 40 optimized 076X CAGC ( NNN = AGC ) Primer US 9 ,822 ,353 B2 161 162 TABLE E4 - continued Mutagenesis Primer Sequences Amino Acid Residue Range of SEQ ID SEO ID Name NO : 1 Nucleic acid sequence NO : E . coli codon 369 - 409 CAATCAAAAGATCGGCTCGNNNACGCAACAAGATGT 41 optimized CGAGC ( NNN = AGC ) C130X Primer
Mutations at either position were introduced by mutagen Example 5 esis using the QuikChange Lightning Site -Directed Muta - 15 genesis Kit (Agilent , cat. no . 210518 ) as described above . After mutagenesis , the sample was treated with Dpn I Production of DRS Cysteine Mutants enzyme at 37° C . and transformed into XL10 gold compe tent cells as described in Example 2 . Multiple colonies were Creation of DRS Cysteine Mutants : To improve the grown in terrific broth overnight at 37° C . and the resulting stability of full length DRS and reduce the impact of plasmids were purified with QIAprep Spin Miniprep Kit 20 non -specific disulfide bond mediated aggregation formation , (Oiagen cat. no . 27106 ) . The plasmids were sequenced to potential problematic cysteines were identified based on the confirm the identity of the amino acid substitution of each crystal structure (see , e . g . , commonly owned U . S . applica clone . The representative clones were transformed into tion Ser. No . 12 /751 , 358 ) , and mutated into Ser or Ala or NovaBlue competent cells (Novagen cat. no . 70181) and Val. In particular cysteines C334 , C349 , C203 and C259 in grown in 250 ml MIYE medium at 37° C . overnight. A 25 wild type DRS were initially targeted for mutagenesis . To maxiprep was performed using the HiSpeed Plasmid Maxi systematically assess the impact of each cysteine in medi Kit ( Qiagen cat . no . 12663 ) to create a plasmid stock of ating protein aggregation , mini libraries were created in mutant for further analysis. The concentration and purity which each DRS cysteine mutant could contain either a were determined by measuring A260 , A280 and A230 . The mutation on one cysteine position or multiple positions . To purified plasmids were stored at - 20° C . before transfection 30 make DRS mutants C334S , C349S , C334S /C349S , C334S / into E . coli or mammalian cells using the methods described C349S /C259A /C203A , C334S /C349S /C259A /C203V , above in Examples 1 and 2 . C334S / C349S /C203A , C334S / C349S /C203V , C203A and To assess the impact of the mutation of Cys76 or Cys130 , C203V , the following primers were used as listed in Table representative clones were transformed into E . coli, or 76 mammalian cells , and the production yields, endotoxin contents were compared . Also , the relative1 Yerus activity, Colomm of the 35 purified proteins are compared in the HEK293 - TLR2 and TABLE E6 HEK293 - TLR4 expressing cell lines as described above . Mutation Oligo sequence SEQ ID NO : The optimal substitutions are identified based on the results C3345 CAGTTCCCATCTGAGCCATTC 162 obtained . Representative results are shown in Table E5. 40 C3495 GACTAGAATATTCTGAAGCATTGGC 163 TABLE E5 C203A CCAGTCTGGCATCGCCCATCTCTTCC 164 EU /mg + < 1 EU /mg , + + < 5 EU /mg , C203V CCAGTCTGGCATCGTCCATCTCTTCC 165 Yield + + + < 10 EU /mg , 45 + < 1 . 2 mg, + + > 1 . 2 mg, + + + > 1 . 4 mg, + + + + < 20 EU /mg , C259A CCACAGCTATATAAGCAAATGTGCAT 166 Variant + + + + > 2 . 0 mg + + + + + > 20 EU /mg TGCGGCTGATTTTGAG
C76A + + + + + + + + + C761 + + + + + + Mutations at cysteine positions were introduced by muta C76L +I + + + 50 genesis using the QuickChange Lightning Site -Directed C76T + + + + + C76V Mutagenesis Kit (Agilent , cat. no . 210518 ) following the C130F + + manufacturer ' s instructions . After mutagenesis, the sample C130L + + + + + + + was treated with Dpn I enzyme at 37° C . and transformed C130T +I+ + + + into XL10 gold competent cells using routine procedures . C130V + + + + + + 55 Multiple colonies were grown in LB media overnight at 37° C . and the resulting plasmids are purified with QIAprep Spin The results show that C76V , C76L , and C76T show Miniprep Kit ( Qiagen cat. no . 27106 ) . The plasmids were enhanced yields and reduced endotoxin content. Addition sequenced to confirm the identity of the amino acid substi ally the results show that C130T and C130V demonstrate tution of each clone . enhanced yields and reduced endotoxin content. The DRS cysteine mutant DNA sequences are as follows:
1 . DRS - C334S : ( SEQ ID NO : 118 ) ATGCCCAGCGCCAGCGCCAGCCGCAAGAGTCAGGAGAAGCCGCGGGAGATCATGGACGCG GCGGAAGATTATGCTAAAGAGAGATATGGAATATCTTCAATGATACAATCACAAGAAAAAC