US 2010.0041872A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0041872 A1 DeFrees et al. (43) Pub. Date: Feb. 18, 2010

(54) GLYCEROL LINKED PEGYLATED SUGARS (86) PCT NO.: PCT/US07/80471 AND GLYCOPEPTDES S371 (c)(1), (76) Inventors: Shawn DeFrees, North Wales, PA (2), (4) Date: Jul. 28, 2009 (US); Xiao Zeng, Warrington, PA Related U.S. Application Data (US) (60) Provisional application No. 60/828,208, filed on Oct. Correspondence Address: 4, 2006. LEYDIG VOIT & MAYER, LTD Publication Classification TWO PRUDENTIAL PLAZA, SUITE 4900, 180 (51) Int. C. NORTH STETSONAVENUE C07K I4/745 (2006.01) CHICAGO, IL 60601-6731 (US) (52) U.S. Cl...... S30/381 (21) Appl. No.: 12/443,428 (57) ABSTRACT The present invention provides conjugates between peptides (22) PCT Fled: Oct. 4, 2007 and PEG moieties through glycerol linkers. Preparation of CMP-SA-Glycerol-PEG-40kDa

--nor-oYokn- 3D. oo-()-No,O rA50

mPEG-40 kDa nitrophenyl carbonate CMP-SA-Glycine 1.2g 30.0 g

1)THF/HO (3:1)rt, 5 days HO OH WP O ONC. 2) NaOH, pH 9.5, 1 hr --norO O O &ay 3) TFF, 1 kDa MWCO O HN CO2 Hó 6 4) Q-sepharose anion exchange Yokn- -1o O N HO *N 3. OH 5) TFF, 1 kDa MWCO O nr-450 CMP-SA-Glycerol-PEG-40 kDa 10.11 g, 36% isolated yield 100% purity by UV Patent Application Publication Feb. 18, 2010 Sheet 1 of 47 US 2010/0041872 A1

FIGURE 1

: eqXOy-?Ed-IoueoÁIÐ-VS-dWOjouo??euedeud Patent Application Publication Feb. 18, 2010 Sheet 2 of 47 US 2010/0041872 A1

FIGURE 2

(6z'))"be'Joucºz }IeSuun?poS (60£)"be'Jou! suoppuoouomoeoseqMOv-03d-IoleoÁIÐ-vs-awo %98p???Á6!L’OLp??glund

(aujoÁIÐ-VS-dWo"sauuuyzzAn)Aq?und Patent Application Publication Feb. 18, 2010 Sheet 3 of 47 US 2010/0041872 A1

FIGURE 3

sseoouduo?eo???undeqXOV-9Bd-IoueoÁIÐ-VS-dWO ?eseo(TEHIJ º1, (speeg615)

?sOue?d?S-O Á?deu6O?euol?O Patent Application Publication Feb. 18, 2010 Sheet 4 of 47 US 2010/0041872 A1

FIGURE 4

009 s SQL

eQX07-9Bd-IoueOÁIÐ-VS-dWO?ouo?eo??undesoue?des-o

'(zuL'OAedKLueelos?euelqeueW eso|n||30p??eJeue6ex;eq>||Oy-l)xz) Patent Application Publication Feb. 18, 2010 Sheets of 47 US 2010/0041872 A1

FIGURE 5

o

Tri -1 N

T --9. gPage6E E GvR.' r SSP's 938649eA. o of 1 site.BESE-R. S w O s'. wr B7778 E s 999 it (D OSSEF.6'sEaseeviv- N t; (6:1 ? E7WES987 l? s

O mism O) -ris a'a () 0. 2. D CD D a a.

C el O) ? fl. SS N. e S -1 O w l c Y v O e s s 2 w D N 4. used w s S.O ver ld Patent Application Publication Feb. 18, 2010 Sheet 6 of 47 US 2010/0041872 A1

FIGURE 6A Protein Organism GenBank I GenPept PDB 3D At 1g08280 Arabidopsis thaliana ACO11438 AAF 18241.1 Q84WOO BTOO4583 AAO42829.1 (9SGD2 NC003070|NP 1723.05.1 At1g08660/F22O13.14 n.d.

At3.g48820/T21J1890

(X-2,3-sialyltransferase ST3GA-IV N-2,3-sialyltransferase A585768 St3Ga Y-2,6-sialyltransferase AJ620651 CAFO5850.1

Siato (X-2,8-sialyltransferase SIAT8A OC-2,8-Sialyltransferase CAG27883 Siat8D (x-2,8-sialyltransferase ST8Si- (Siat8C CMP X-2,6-

sialyltransferase NM 177517|NP 803483.1 ST6Ga

fragment --

ST3Ga- (Siat4B --- sialyltransferase BOS taurus n.d. AJ748842 CAG44451.1 ST3Gal-lil (Siates ST3Gal-VI (Siat10

SEVEEEEEEEEStóGalNAc-V Bos taurus n.d. AJ620949 CAF06586.1 Q9BEG4 Branchiostonna n.d. AF391289 AAM18873.1 Q8T771 floridae polysialyltransferase Cercopithecus 24.99.- AF210729 AAF17105.1 Q9TTO9 (PST) (fragment) aethiops ST8Sia IV polysialyltransferase Cercopithecus 2.499.- AF210318 AAF17104.1 Q9TT10 (STX) (fragment) aethiops ST8Sial ST3Gall (Siat4 Ciona Savignyi n.d. Aj626814 CAF25172.1

ST3Gal Siata C-2,8- AAE28634 Q64690 polysialyltransferase CAA86822.1 ST8Sia IV Gal (1,314-GlcNAc C AAP22942.1 2,3-sialyltransferase St3Gal Gall1,314-GlcNAc cy AAP22943.1 2,3-sialyltransferase St3Galil (fragment (X-2,3-sialyltransferase CAHO4017.1 ST3Gal (Siat4 na. Issa. C-2,3-sialyltransferase Danio rerio ST3Galil (Siat5) a first CAHO4018.1 Patent Application Publication Feb. 18, 2010 Sheet 7 of 47 US 2010/0041872 A1

FIGURE 6B Organism EC# GenBank 1 GenPept 3D (X-2,3-sialyltransferase Danio rerio n.d. AJ626821 CAF25,179.1 ST3Gal (Siates C-2,3-sialyltransferase ST3Gal IV (Siat4C cC-2,3-sialyltransferase ST3Gal W-r (Siat5 CC-2,6-sialyltransferase ST6Gal Siat 1 EE-2,6-sialyltransferase A634.459 CAG2.568.0.1 ST6GalNAc Siat/B CC-2,6-sialyltransferase ST6GalNAc V (Siat7E) fragment DC-2,6-sialyltransferase AJ646883 ICAG26712.1 ST6GalNAc VI (Siat7F) fragment X-2,8-sialyltransferase AJ715535 CAG29374.1 ST8Sia I (Siat 8A) (fragment) X-2,8-sialyltransferase AJ715543 CAG2938.2.1 ST8Sia Il (Siat 8C) fragment cC-2,8-sialyltransferase CAG29384.1 ST8Sia IV (Siat 8D) cC-2,8-sialyltransferase AJ715546 CAG29385.1 ST8SiaV (Siat 8E) O-2,8-sialyltransferase AJ715551 CAG29390.1 ST8Sia VI (Siat 8F) fragment galactosamide (X-2,6- sialyltransferase ll ST6Gall N-glycan CX-2,8- n.d. sialyltransferase - ST3Gal-related (siatér) St3Gal-V st6GalNAc-V X-2,6-sialyltransferase Drosophila (CG4871) ST6Gall melanogaster

(X-2,3-sialyltransferase ST3Ga-V Gaius gains ind AJ627.204it; CAF25503.1SAE C-2,3-sialyitransferase ST3Gall NM 205217NP 990548.1 (C-2,3-sialyltransferase Gallus gallus 2.4.99.- AFO35250 AAC 14163.1 O73724 ST3Gal IV (fragment (X-2,3-sialytransferase Gallus gallus AJ585761 ICAE51385.2 ST3GAL (X-2,6-sialyltransferase Gallus gallus w CAFO5852.1 Siat/b AJ620653 X-2,6-sialyltransferase Gallus gallus X75558 CAA53235.1 Q92182 ST60sal NM 205241NP 990572.1 co-2,6-sialyltransferase Gallus gallus AAE68028.1 O921.83 Patent Application Publication Feb. 18, 2010 Sheet 8 of 47 US 2010/0041872 A1

FIGURE 6C Protein Organism EC GenBank I GenPept Swiss Prot PDB 3D ST6CalNAc - AAE68029.1 74946 CAA52902. NM 20524ONP 990571.1 C-2,6-sialyltransferase Gallus gallus 2.4.99.- X77775 AAE68030.1 Q921.84 ST6GalNAc NM 205233CAA54813.1 NP 990564.1 C-2,6-sialyltransferase Gallus gallus n.d. AJ634455 CAG2.5677.1 ST6GalNAc III (SIAT7C) fragment (C-2,6-sialyltransferase Gallus gallus AJ646877 ICAG26706.1 ST6GalNAc V (SIAT7E) fragment X-2,8-sialyltransferase Gallus gallus 24.99.- U73176 AAC28888.1 P79783 (GD3 Synthase) ST8Sia X-2,8-sialyltransferase Gallus gallus AJ 6994.19 (CAG27881.1 SAT8B OC-2,8-sialyltransferase Gallus gallus AJ699420 CAG27882.1 SAT8C (X-2,8-sialyltransferase Gallus gallus AJ699424 CAG27886.1 SIAT8F O-2,8-syalyltransferase Gallus gallus AJ704564 CAG28697.1 ST8Six-WSAT8C galactosamide X-2,6- Gallus gallus n.d. A627629 CAF29497.1 sialyltransferase ll ST6Galil Gallus qallus AY515.255 AAS83519.1 polysialyltransferase Gallus gallus ST8Sia IV

(X-2,3-sialyltransferase ST3Ga AF059321 L13972 AF155238 AF1861.91

O-2,3-sialyltransferase Q16842 ST3 Ga. OOO654

23768

C-2,3-sialyltransferase BC050380

ST3Galil (SiaT6) AF425851 AF425852 AF425853 AF425854 AAO13862.1 AAO13863.1

AAO13870.1 AAO1387.1.1

Patent Application Publication Feb. 18, 2010 Sheet 10 of 47 US 2010/0041872 A1

FIGURE 6E Protein Organism GenBank I GenPept swapo PDB 3D NM O18414 NP 06.0884.1 F O-2,8- 41680 polysialyltransferase BCO27866 ST8Sia IV BCO53657 AAH53657.1 NM OO5668 NP OO5659.1 X-2,8-sialyltransferase L32867 (GD3 synthase) ST8Sia L43494 BCO46158 AAH46158.1 Q93064 AACR53140.1 AAS75783.1 BAA05391.1 CAA548911 NP 003025.1 (X-2,8-sialyltransferase AAA36613.1 Q92186 ST8Sial B51242.1 Q92470 AAC24458.1 Q92746 BCO69584 AAH69584.1 NM OO6011 NP OO60.02.1 O-2,8-sialyltransferase AFOO4668 AAB87642.1 ST8Sial AFOO3092 AAC 15901.2 NM 015879 NP 056963. cC-2,8-sialyltransferase AAC51727.1 O15466

ENSPOOOOOO20221 (fragment) lactosylceramide X-2,3 24.99.9 AF05026 IAAD 14634.1 Q9UNP4 sialyltransferase AF1194.15 AAF66146.1 O94902 BCO65936 AAH65936.1 (ST3Gal W) AY152815 AAO16866.1 AAP65066 AAP65066.1 AY359105 AAC89463.1 ABO18356 BAA33950.1 CAE893.20.1

N- Homo sapiens BCOO6564 BCOO7802

acetylgalactosaminide BCO 16299

C-2,6-sialyltransferase AY358.672 (ST6GalNAc VI) ABO35173 AKO23900 AU507293 AX880950

N- Homo sapiens AF127142 AAFOO102.1 acetylgalactosaminide BCO36705 AAH36705.1 C-2,6-sialyltransferase AAP63349. IV (ST6GalNAc IV) AB035172 BAA87034.1

BAA91281.1 CAB44354.1 CACO7404.1 CAC24981.1 CAC27250.1 CAF 14360.1

NM_014403 NP 055218.3 NM 175039 NP 7782O4.1 ST8SIA-V1 (fragment) Aj621.583 CAF21722.1 homosen a M 291725 XP 291725.2 unnamed protein Homo sapiens AKO21929 BAB13940.1 Q9HAA9 Oroduct AX881696 CAE91353.1 Gali-1,314-GlcNAc C Mesocricetus 2.499.6 A.J245699 CAB53394.1 O90XF6 Patent Application Publication Feb. 18, 2010 Sheet 11 of 47 US 2010/0041872 A1

FIGURE 6F

1 3D auratus MeSOCricetus auratus pope CAB53395.1 Q9QXF5

MeSOCricetus a Fiates AAD33879.1 Ch9WUL1

auratuS MeSOCricetus 24.99.- AJ2457.01 CAB53396.1 Q90XF4 auratus X-2,3-Sialyltransferase Mus musculus 24.994 AF214028 AAF60973.1 P54751 ST3Gal BAC27356.1 Q11202 AKO78469 BAC37.290.1 73523 CAA51919.1 NM OO9177NP 033203.1 cC-2,3-sialyltransferase St3gal2 MuS nuSCulus AAH15264.1 Q11204 ST3Gal AAH66064.1 BAC28752.1 BAC28859.1

C-2,3-sialyltransferase St3gal3 MuS musculus ST3Gail s 3-Sialyltransferase ST3Gal V

(X-2,3-sialyltransferase MuS musculus ST3Gal VI ABO63326 BAB79494.1 AKO33562 BAC2836O1

X-2,6-sialyltransferase Stögalnac2 Mus musculus - P70277 ST6GalNAc BCO10208 AAH102081 Q9DC24

AB027198 BABOO637.1 Q9JJM5 AKOO4613 BAB23410.1 X93999 CAA63821.1 X94000 CAA63822.1 NM OO918ONP 033206.2 or-2,6-sialyltransferase Stögal1 MuS musculus 2.499.1 AAE680311 Q64685 ST6Gall BCO27833 AAH27833.1 Q8BM62 D16106 BAA03680.1 O8K1 L1 AKO34768 BAC288281 AKO84124 BAC39120.1 NM 145933NP 666045.1 CE-2,6-sialyltransferase Stögal2 MuS musculus AKO82566 BAC38534.1 O8BUU4 ST6Gal ABO95093 BAC87752.1 AK1294-62 BAC9827.2.1 NM 172829NP 766417.1 ox-2,6-sialyltransferase Stögalnac1 Mus musculus 24.99.3 Y11274 CAA72137.1 Q9QZ39 NMO 11371 INP 0355011 Q9JJP5 ST6GalNAc (X-2,6-sialyltransferase Stögalnac3 Mus musculus ST6GalNAc

Patent Application Publication Feb. 18, 2010 Sheet 13 of 47 US 2010/0041872 A1

FIGURE 6H

3D NM 016973NP 058669.1 M138 Myxoma virus AADOOO69.1 AF17O726 IAAE61323.1 NC 001132 AAE61326.1 AAF15026.1 NP 051852.1 (X-2,3-sialyltransferase n.d. AJ585760 CAE51384.1 St3Gal mykiss (X-2,6-sialyltransferase A.J82O649 CAFO5848.1 nvkiss OC-2,8- Oncorhynchus ABO94402 BAC77411.1 mykiss GalNAc (x-2,6- Oncorhynchus n.d. ABO97943 BAC77520.1 sialyltransferase mykiss RtST6GalNAc (Y-2,3-sialyltransferase Oryctolagus 24.99.- AF121967 AAF28871.1 Q9N257 ST3GaV Cuniculus OJ1217 FO2.7 Oryza sativa BA D O 76 1 6 . (japonica cultivar OSJNBa0043L24.2 or Oryza sativa n.d. AL731626 CAD41185.1 OSJNBbOOO2J11.9 (japonica cultivar AL662969 CAE04714.1 PO683fo2.18 or Oryza sativa n.d. APOO3289 BAB63715.1 PO489B03.1 (japonica cultivar APOO3794 BAB90552.1 CAG26705.

C-2,3-sialyltransferase ST3Gal (SiatA) (Y-2,3-sialyltransferase AJ744804 CAG3284.0.1 ST3Gal (Siat5 cC-2,3-sialyltransferase ST3Gal (Siats Alias carasin. ST3Gal IV (SiatAc Asia: Arasia. (C-2,3-sialyltransferase ST3GaW Siat10 Arace assau. (X-2,6-sialyltransferase Sia/A Atario Ages 5.1 O-2,6-sialyltransferase AJ748741 ICAG386.16.1 SiafB (X-2,6-sialyltransferase Pan troglodytes - - - as34454 CAG25676.1 ST6GalNAc (Siat/C cC-2,6-sialyltransferase Pan troglodytes d. A646870 CAG26699.1 ST6GalNAc IV (Siat7D) fragment X-2,6-sialyltransferase Pan troglodytes ST6GalNAc V (Siat/E cx-2,6-sialyltransferase Pan troglodytes ST6GalNAc VI (Siat7F) fragment cC-2,8-sialyltransferase Pan troglodytes

8A Siat 8. A (X-2,8-sialyltransferase 8 B Siat 8 B CC-2,8-sialyltransferase 8 C Si at 8 C C-2,8-sialyltransferase Pan troglodytes AJ697661 8 D Si at8 D C-2,8-sialyltransferase Pan troglodytes A697662 Patent Application Publication Feb. 18, 2010 Sheet 14 of 47 US 2010/0041872 A1

FIGURE 6 Organism EC GenBank I GenPept Swiss Prot 3D 8E (Siat8E X-2,8-sialyltransferase 8F (Sat3F galactosamide O-2,6- sialyltransferase I ST6Gall; Siat 1 galactosamide (X-2,6- Pan troglodytes - - - A627.625 CAF294.93.1 sialyltransferase il ST6Gall GM3 synthase ST3Gal Pan troglodytes n.d. AJ744807 CAG32843.1 V Si a t9

S138L. Rabbit fibroma virus n.d. NC 001266NP 052025 Kasza C-2,3-Sialyltransferase Rattus norvegicus M97754 AAA42146.1 CO2734 ST 3 al NM 03.1697|NP 113885.1 C-2,3-sialyltransferase Rattus norvegicus a AJ626825 CAF251 83.1 ST3Gal IV Siat4c C-2,3-sialyltransferase Rattus norvegicus AJ626743 CAF25053.1 ST3GaW (I-2,6-sialyltransferase Rattus norvegicus 2.4.99.- X76988 CAA54293.1 C 11205 ST3Gall NM 031695NP 113883.1 (Y-2,6-sialyltransferase Rattus norvegicus 24.99.1 M18769 AAA41196.1 P 13721 ST6GasG M83143 AABO7233.1 C-2,6-sialyltransferase Rattus norvegicus ST6GalNAc (Siat/A a less CAG25684.1 X-2,6-sialyltransferase Rattus norvegicus ST60alNAc II (Siat7B n.d. Jesus, CAG25679.1 ox-2,6-sialyltransferase Rattus norvegicus AAC42086.1 C64686 ST6GalNAc

cx-2,6-sialyltransferase Rattus norvegicus n.d. Aj646871 ICAG26700.1 ST6GalNAc IV (Siat7D) fragment cx-2,6-sialyltransferase Rattus norvegicus AJ646872 CAG26701.1 ST6GalNAc V (SiatfE X-2,6-sialyitransferase Rattus norvegicus AJ346881 ICAG267.10.1 ST6GalNAc VI (Siat7F) fragment (C-2,8-sialyltransferase Rattus norvegicus AAC27541.1 F70554 G D 3 Sy t h a S 8 ) S T 8 Si a BAAO8213.1 F97713 X-2,8-sialyltransferase Rattus norvegicus

SAT8E (X-2,8-sialyltransferase Rattus nonvegicus SIAT8F 2.499.- L13445 AAA42147.1 (0.7977 cC-2,8-sialyltransferase Rattus norvegicus ST8Sia II NM 057156NP 476497.1 (264688 oc-2,8-sialyltransferase Rattus norvegicus 2.4.99.- AAB5OO61.1 P97877 ST8Sial NMO 13029|NP 037161.1

D-2,8-sialyltransferase Rattus norvegicus 2.499.- U90215 AAB49989.1 OO8563 ST8Sia IV galactosamide ox-2,6- Rattus norvegicus AJS27626 CAF29494.1 sialyltransferasell (ST6Galll) GM3 synthase ST3Gal Rattus nonvegicus AB018049 BAA33492.1 O88830 V NM 031337NP 112627.1 Patent Application Publication Feb. 18, 2010 Sheet 15 of 47 US 2010/0041872 A1

FIGURE 6J PDB 1 3D sialyltransferase Rattus norvegicus ind AJ748840 CAG44449.1 ST3Gal- (Siat4A) cC-2,3-sialyltransferase Silurana tropicalis St3Gal na Asses CAE51387.1 — (X-2,6-sialyltransferase Silurana tropicalis CAF05849. Siat/b (x-2,6-sialyltransferase Strongylocentrotus AJ699.425 Stégalnac) purpuratus 0-2,3-sialyltransferase Sus scrofa nd AJ585765 ICAE51389.1 ST3GAL-III) X-2,3-Sialyltransferase Sus scrofa n.d.

ST3GAL-IV) X-2,3-sialyltransferase isSus scrofa AAA31125.1 CO2745

ST3Ga (X-2,6-sialyltransferase Sus scrofa AAD33059.1 Q9XSG8 fragment) ST6Cal galactosamide C-2,6- CAFO6585.2 sialyltransferase ST6GalNAc-V sialyltransferase SuS SCrofa AFO41031 AAC15633.1 O62717 fragment) ST6Call ST6GALNAC-V SuS SCrofa A620.948 CAF06585.1 (C-2,3-sialyltransferase Takifugu rubripes AJ744805 CAG3284.1.1 Siat5-r X-2,3-sialyltransferase Takifugu rubripes AJ626816 CAF25174.1 ST3Gal (Siat4 CC-2,3-sialyltransferase Takifugu rubripes ST3Galil (Siat5) fragment Takifugu rubripes ST3Gal (Siates Takifugu rubripes - - - ST6Gal Siat1 Takifugu rubripes ST6GalNAc (SiatfB (X-2,6-sialyltransferase Takifugu rubripes ST6GalNAc II B (Siat7B related (X-2,6-sialyltransferase Takifugu rubripes n.d. AJ634456 CAG25678.1

ST6GalNAc II (Siat7C) fragment (C-2,6-sialyltransferase Takifugu rubripes 2.499.3 Y17466 CAB44338.1 ST6GalNAc IV (siat7D) AJ64.6869 CAG26698.1 fragment (X-2,6-sialyltransferase Takifugu rubripes n.d. A646873 CAG26702.1 ST6GalNAc V (Siat7E) frament CX-2,6-sialyltransferase Takifugu rubripes n.d. AJ646880 CAG26709.1 ST6GalNAc VI (Siat7F) fragment cC-2,8-sialyitransferase Takifugu rubripes n.d. AJ715534 CAG29373.1 ST8Sia I (Siat 8A) fragment CC-2,8-Sialyltransferase Takifugu rubripes AJ715538 ICAG29377.1 ST8Sia II (Siat 8B) fragment (X-2,8-sialyltransferase Takifugu rubripes 5541 CAG29380.1 ST8Sia III (Siat 8C) fragment Takifugu rubripes C.d AJ715542 CAG29381.1 ST8Siar Siat 8Cr O-2,8-sialyltransferase |aligu rubripes n.d. AJ715547 ICAG29386.1 ST8Sia V (Siat 8E) Patent Application Publication Feb. 18, 2010 Sheet 16 of 47 US 2010/0041872 A1

FIGURE 6K Organism EC# GenBank 1 GenPept P f fragment (C-2,8-sialyltransferase Takifugu rubripes - AJ715549 ICAG29388.1 ST8Sia VI (Siat 8F) fragment (X-2,8-sialyltransferase Takifugu rubripes AJ715550 CAG29389.1 ST8Sia VI (Siat 8F (x-2,3-sialyltransferase AJ744806 CAG32842.1 nicroviridis ST3Ga(X-2,3-sialyltransferase (Siata Tetraodoniqroviridis - - Aria: AG38. (X-2,3-sialyltransferase Tetraodon AJ626822 CAF251 80.1 ST3Galil (Siates nigrowiridis C-2,6-sialyltransferase Tetraodon na Assas: CAG25683.1 S6GalNAc (Siat/B niqroviridis (X-2,6-sialyltransferase Tetraodon AJ646879 CAG26708.1 ST6GalNAc V (Siat7E) nigrowiridis fragment CC-2,8-sialyltransferase Tetraodor AJ715536 ICAG2.9375.1 ST8Sia I (Siat 8A) nigrowinidis fragment C-2,8-sialyltransferase Tetraodon AJ715537. CAG29376.1 ST8Sia II (Siat 8B) nigrowiridis fragment (X-2,8-sialyltransferase Tetraodon AJ715539 CAG29378.1 ST8Sia III (Siat 8C) nigrowinidis fragment (C-2,8-sialyltransferase Tefraodon AJ715540 CAG29379.1 ST8Sia Ilir (Siat 8Cr) nigrowiridis fragment EX-2,8-sialyltransferase Tetraodon AJ715548 CAG29387.1 ST8Sia V (Siat 8E) nigroviridis fragment C-2,3-sialyltransferase Xenopus laevis na Asesi AEsses. St3GalcC-2,3-sialyltransferase enopus laevis na Asses St3Ga-VI C-2,3-sialyltransferase Xenopus laevis ld A626823E: CAF25181.1 cC-2,8- Xenopus laevis 24.99.- ABOO7468 BAA3267.1 polysialyltransferase C-2,8-sialyltransferase enopus laevis AY272056 IAAQ16162.1 ST8Six-1 (Siat8A:GD3 AY272057 AAG16163.1 Synthase AJ704562 CAG28695.1 enopus laevis BCO68760 AAH68760.1 MGC:81265 XenopuS tropicalis AJ626744 CAF25054.1 3Gai-V (C-2,3-sialyltransferase Xenopus tropicalis AJ622908 CAF22058.1 Siat4C (C-2,6-sialyltransferase enopus tropicalis AJ 646878 CAG26.707.1 ST6GalNAc V (Siat7E) fragment (X-2,8-Sialyltransferase enopus tropicalis AJ715544 CAG29383.1 ST8Sia III (Siat 8C) fragment galactosamide ox-2,6- enopus tropicalis AJ627628 CAF29496.1 sialyltransferasell ST6Call

sialytransferase StaSial Xe Op S tT Op! Cal S AY652775 IAAT67042 poly-C-2,8-sialosyl C h eric hi8 COl K f 2 4.--4. - M76370 AAA242.13.1 Q57269 sialyltransferase (NeuS X60598 CAA43053.1 polysialyltransferase IScherichia Coli K92 2.4-- M884.79 AAA24215.1 Q474.04 Patent Application Publication Feb. 18, 2010 Sheet 17 of 47 US 2010/0041872 A1

FIGURE 6L Protein Organism ECi. GenBank I GenPept Swarolife (X-2,8 Neisseria 2.4.-- M95053 AAA20478.1 C51281 3D polysialyltransferase meningitidis B1940 X78068 CAA54985.1 (51145 SE Neisseria n.d. U75650 AAB53842.1 OO6435 neningitidis FAM18 polysialyltransferase Neisseria AY234.192 AAO85290.1 SiaD)(fragment meningitidis M1019 SiaD (fragment) Neisseria AY281046 IAAP34769.1 eningitidis M209 SiaD (fragment) AY281044 AAP34767.1 Asneringitidis M3045eas polysialyltransferase AY234,191 IAAO85289.1 SiaD) (fragment neningitidis M3315 SiaD (fragment) AY281047 AAP34770.1 neringitidis M3515 polysialyltransferase SiaD) (fragment neringitidis M4211 SiaD (fragment) Neisseria n.d. AY28.1048 AAP3477.1.1 meninqitidis M4642 polysialyltransferase Neisseria n.d. AY234193 SiaD) (fragment meningitidis M5.177 -- Siad Neisseria AY281043 meningitidis M5178 SiaD (fragment) Neisseria AY281045 nerinditiois M980 NMBOO67 Neisseria NC 003112 |NP 273131 tneringitidis MC58 Lst Aeromonas punctatan.d. AF126256 AAS666.24.1 Sch3 ORF2 Haemophilus M94.855 AAA24979.1 influenzae A2 H1699

X-2,3-sialyltransferase Neisseria donorrhoeae F62 X-2,3-sialyltransferase Neisseria 24.99.4 U60662 AAC44544.2 eningitidis 126E, NRCC 4010 X-2,3-sialyltransferase Neisseria 2.499.4 U60661 AAC44543.1 meningitidis 406Y, NRCC 4030 (X-2,3-sialyltransferase Neisseria (NMBO922) meningitidis MC58

NMA1118 Neisseria n.d. neringitidis Z2491 NC 003116 NP 283887.1

PMO508 Pasteurella AE006086 nutocida PM70 SE.5a, a 245445.1 Waah Salmonella enterica

SARB25 Salmonella enterica (8KS92 Waah SARB3 Waah Salmonella enterica n.d. SARB39 Waa Salmonella enterica n.d.

SARB53 Waah Salmonella enterica n.d. Q8KS91

SARB57 Waah Salmonella enterica n.d. AF519793 AAM82556.1Q8KS89 SARB71 Waah Salmonella enterica n.d. AF519792 AAM82555.1Q8KS90 Patent Application Publication Feb. 18, 2010 Sheet 18 of 47 US 2010/0041872 A1

FIGURE 6M EC# GenBank I GenPept SwissProtPDB 3D SARB8 Waa H Salmonella enterica AF519779 AAM88840.1 C8KS99 SARC1OW Waah (fragment) Salmonella enterica AF519781 AAM88842.1 SARC2 Waah (fragment) Salmonella enterica AAM88843.1 C8KS98 SARC 13 Waah (fragment) Salmonella enterica AF519783 IAAM88844.1 G8KS97 SARC4 Waa H SalmOnella enterica AF519784 SARC15II Waah SalmOnella enterica AF519785 AAM88846.1 Q8KS95 SARC16/ Waah (fragment) Salmonella enterica AF519772 AAM88834.1 Waa H (fragment) Salmonellaf i. enterica Waa SalmOnella enterica Astoria amass Waah Salmonella enterica SARC6lla Waah Salmonella enterica n.d. Waah SalmOnella enterica i if V UDP-glucose: D-1,2- SalmOnella enterica AF511116 AAM48166.1 glucosyltransferase Subsp. arizonae aa SARC 5 bifunctional CX-2,3-2,8- Campylobacter 3. e ra S e C St eiuni ATCC 43449 S al t Sf Cst Campylobacter ieiun'i 81-176 (X-2,3-sialyltransferase Campylobacter Cst-ill ieiuni ATCC 43429 (C-2,3-sialyltransferase Campylobacter AK854.19.1

Cst-III eiuni ATCC 43430 (X-2,3-sialyltransferase Campylobacter AAG43979.1 CSt-l feiuni ATCC 43432 ox-2,3/8- Campylobacter n.d. AAK91725.1 Ch93MQ0 S al t 2 sfe ra S e CS t eiuri ATCC 43438 cC-2,3-sialyltransferase 2.4.99.- AF167344 IAAF34137.1 CSt-l ieiuni ATCC 434.46 X-2,3-sialyltransferase 2 4.99.- AF401528 AA05990.1 (93D05 Cst-II eiuni ATCC 43456 C-2,3-lex-2,8- 2.4.99.- AY044868 AAK96001.1 Q938X6 S al tr3. Sfe a S e C St eiuni ATCC 4346O cy-2,318 AF216647 AAL36462.1 S al tr.a n sfe 8 S e C St feitini ATCC 700297 R F AY422197 AAR82875.1 eiuni GB11 ORFX-2,3-sialyltransferase 2 4.99. AF195055 AAG29922.1 cSt eiuni MSC57360 (X-2,3-sialyltransferase 2 499 - A139077 CAB73395.1 Q9PNF4 CSt C1140 eitri NCTC 11168 NC 002163 NP282288.1 CC-2,3/X-2,8- Campylobacter AAO96669.1 Se eiuni O1O CAFO4167. Si al t 3. Sfer a CSt C-2,31-2,8- Campylobacter sialyltransferasell iejuni O:19 Cst EX-2,3/X-2,8- Campylobacter AX934436 CAFO417.1.1 sialyltransferase II iejuni O:36 Cstill (X-2,3-2,8- Campylobacter AX934434 CAF041 70.1 Patent Application Publication Feb. 18, 2010 Sheet 19 of 47 US 2010/0041872 A1

FIGURE 6N

protein organism ECI GenBank Gonrep 3E.

CStillHansease I fun of (X-2,3/ox-2,8- Campylobacter AAO96670.1 sialyltransferase Il ejuni O:41 AAT17967.1 CStil AX934,429 CAFO4168.1 CSt- eiuni OH4384 AA S362611 sialyltransferase (Cst- eiuni OH4384 AX934.425 CAFO41661 1 RO8 HI0352 (fragment) Haemophilus U32720 AAC22013.1 P24324 influenzae Rd X57315 CAA40567.1 NC 000907 NP 438516.1

Pasteurella nutocida PM70 NC 002663 NP 246111.1 AAd96672. patent US 6503744 GE AAT17969.1 patent US 6699.705 unknown ind GS, Unknown. AAT17970.1 patent US 66997.05 Givisio Unknown. USSompaten 6709834 USSirom 6503744 paten EO 1 US 66997.05 Oate?t US 6503744 Sequence 35 from patent US 6503744 AAS36262.1 fragment Sequence 48 from at 7988.1 oatent US 6699.705 Sequence 5 from patent AAT17966.1

US 66997.05 Sequence 9 from patent AAO96671.1 US 6503744 Patent Application Publication Feb. 18, 2010 Sheet 20 of 47 US 2010/0041872 A1

FIGURE 7A 12AP1/E5 - Viventia Biotech AIDS vaccine - ANRS, CIBG, Hesed 1964 - Aventis Biomed, Hollis-Eden, Rome, United 20K growth hormone-AMUR Biomedical, American Home Products, 28P6/E6 - Viventia Biotech Maxygen 3-Hydroxyphthaloyl-beta-lactoglobulin airWay receptor ligand-IC Innovations 4-IBBligand gene therapy - AJW2 - Ajinomoto 64-CuMAb conjugate TETA-1A3 AK 30 NGF- Akermes Mallinckrodt Institute of Radiology Albuferon - Human Genome Sciences 64-Cu MAb conjugate TETA-CT84.66 albumin-Biogen, DSMAnti-Infectives, 64-Cu Trastuzumab TETA conjugate Genzyme Transgenics, PPL Therapeutics, Genentech TranXenoGen, Welfide Corp. A 200-Amgen aldesleukin -- Chiron A10255- Eli Lilly alefacept - Biogen A1PDX-Hedral Therapeutics Alemtuzumab A6- Angstrom Allergy therapy - ALK-Abello/Maxygen, aaAT-Ill-Genzyme ALK-Abello/RP Scherer AbCiximab - CentOCOr allergy vaccines - Allergy Therapeutics AB.001 - Atlantic BioPharmaceuticals Anidofibatide -- Aventis Pasteur ABT-828 - Abbott AnOrine - SRC VB VECTOR ACCutin ALP242 - Gruenenthal Actinohivin Alpha antitrypsin - Arrival Hyland activin - Biotech Australia, Human ImmunO/ProMetic/Protease Sciences Therapeutics, Curis Alpha-1 antitrypsin- Cutter, Bayer, PPL AD 439-TanOX Therapeutics, Profile, ZymoGenetics, AD 519-TanOX Arriva Adalimumab -- Cambridge Antibody Tech. Alpha-1 protease inhibitor-Genzyme Adenocarcinoma Vaccine-Biomira - NIS Transgenics, Welfide Corp. Adenosine deanimase -- Enzond Alpha-galactose fusion protein Adenosine A2B receptor antagonists - Immunomedics Adenosine Therapeutics Alpha-galactosidase A -- Research ADP-001 - Axis Genetics Corporation Technologies, Genzyme AF 13948-Affymax Alpha-glucosidase - Genzyme, Novazyme Afelimomab - Knoll Alpha-lactalbumin AFP-SCAN- Immunomedics Alpha-L-iduronidase-Transkaryotic AG 2195 - Corixa Therapies, BioMarin agalsidase alfa-Transkaryotic Therapies alteplase - Genentech agalsidase beta - Genzyme alvircept Sudotox-NIH AGENT-AntiSOma ALX1-11-SNPS Pharmaceuticals A 300 - Autommune Alzheimer's disease gene therapy A-101 - Teva AM-133. AMRAD A-102 - Teva Amb a 1 immunostim conj. - DynaVax Al-201 - Autoimmune AMD 3100 - AnorMED-NS Al-301 - Autoimmune AMD 3465- AnorMED- NIS Patent Application Publication Feb. 18, 2010 Sheet 21 of 47 US 2010/0041872 A1

FIGURE 7B

AMD 3465 - AnorMED -- NIS Anti-B7-2MAb GL-1 AMD Fab -- Genentech Anti-B7-2-gelonin immunotoxin Amediplase-Menarini, Novartis Antibacterialslantifungals -- AM-F9 DiverSa/IntraBiotics Amoebiasis vaccine Anti-beta-amyloid monoclonal antibodies - Amphiregulin - Octagene Cambridge Antibody Tech. Wyeth-Ayerst anakinra - Amgen Anti-BLySantibodies - Cambridge analgesic - NobeX Antibody Tech. /Human Genome Sciences ancestim - Amgen Antibody-drug Conjugates - Seattle AnergiX.RA - Corixa, Organon GeneticS/EOS Angiocidin-Inkine Anti-C5MAb BB5-1 - Alexion angiogenesis inhibitors--ILEX Anti-C5MAb N19-8 - Alexion AngioMab-Antisoma Anti-C8 MAb Angiopoietins - Regeneron/Procter & anticancer cytokines - BioPulse Gamble anticancer matrix-Telios Integra angiostatin - EntreMed Anticancer monoclonal antibodies - ARIUS, Angiostatinlendostatin gene therapy - ImmuneX Genetix Pharmaceuticals anticancer peptides - Maxygen, Micrologix angiotensin-Il, topical-Maret Anticancer prodrug Tech. --Alexion Anthrax-EluSys Therapeutics/US Army Antibody Technologies Medical Research Institute anticancer Troy-Bodies - Affite - Affitech Anthrax VaCCine anticancer Vaccine - NIH Antiplatelet-derived growth factor Dhuman anticancers - Epimmune monoclonal antibodies - CuraGen Anti-CCR5/CXCR4 sheep MAb - KS Anti-17-1AMAb 3622W94 Biomedix Holdings GlaxoSmithKline Anti-CD11a MAb KBA Anti-2C4MAb - Genentech Anti-CD11a MAb M17 anti-4-1BB monoclonal antibodies - Bristol Anti-CD11a MAb TA-3- Myers Squibb Anti-CD1 a MAb WT.1 - Anti-Adhesion Platform Tech. -Cytovax Anti-CD11b MAb -- Pharmacia Anti-adipocyte MAb - Cambridge Antibody Anti-CD11b MAb LM2 Tech/ObeSys Anti-CD154MAb - Biogen antiallergics - Maxygen Anti-CD16-anti-CD30 MAb - Biotest antiallergy vaccine - Acambis Anti-CD18 MAb - Pharmacia Anti-alpha-4-integrin MAb Anti-CD19 MAb B43 Anti-alphav33 integrin MAb-Applied Anti-CD19 MAb-liposomal sodium butyrate Molecular Evolution Conjugate Anti-angiogenesis monoclonal antibodies - Anti-CD 147 KSBiomedix/Schering AG Anti-CD19 MAb-Saporin Conjugate Anti-B4MAb-DC1 Conjugate - ImmunoGen Anti-CD 19-dSFW-PE38-immunotoxin Anti-B7 antibody PRIMATIZED-IDEC Anti-CD2 MAb 12-15 Anti-B7-1 MAb 16-10A1 Anti-CD2MAb B-E2 - Diaclone Anti-B7-1 MAb 1G10 Anti-CD2 MAb OX34 Patent Application Publication Feb. 18, 2010 Sheet 22 of 47 US 2010/0041872 A1

FIGURE 7C Anti-CD2 MAb OX54 Anti-CD4MAb YTS 177-9 Anti-CD2MAb OX55 Anti-CD40 ligand MAb 5c8-Biogen Anti-CD2 MAb RM2-1 Anti-CD40MAb Anti-CD2MAbRM2-2 Anti-CD40. MAb 5D12-TanOX Anti-CD2 MAb RM2-4 Anti-CD44 MAb A3D8 Anti-CD20 MAb BCAB20 Anti-CD44 MAb GKWA3 Anti-CD20-anti-Fc alpha Ribispecific MAb Anti-CD44 MAb M7 Medarex, TenOVUS Anti-CD44 MAb KM81 Anti-CD22MAb-Saporin-6 Complex Anti-CD44 variant monoclonal antibodies - Anti-CD3 immunotoxin Corixal Hebrew University Anti-CD3 MAb 145-2C11 - Pharming Anti-CD45MAb BC8--131 Anti-CD3 MAb CD4lgG conjugate Anti-CD45RBMAb Genentech Anti-CD48MAb Huly-m3 Anti-CD3 MAbhumanised-Protein Design, Anti-CD48 MAb WM-63 RW Johnson Anti-CD5 MAb -- Becton Dickinson Anti-CD3 MAb WT32 Anti-CD5 MAb OX19 Anti-CD3 MAb-ricin-chain-A conjugate Anti-CD6 MAb Anti-CD3 MAb-xanthine-Oxidase Conjugate Anti-CD7 MAb-PAP conjugate Anti-CD7 MAb-ricin-chain-A conjugate Anti-CD30 MAb BerH2-- Medac Anti-CD8MAb-Amerimmune, Cytodyn, Anti-CD30MAb-Saporin Conjugate Becton DickinSOn Anti-CD30-SCFV-ETA'-immunotoxin Anti-CD8 MAb 2-43 Anti-CD38 MAbAT13/5 Anti-CD8 MAb OX8 Anti-CD38 MAb-Saporin Conjugate Anti-CD80 MAb P16C 10 - DEC Anti-CD3-anti-CD19 bispecific MAb Anti-CD80 MAb P7C10-D Vaccine Anti-CD3-anti-EGFR MAb Anti-CD8-idarubicin conjugate Anti-CD3-anti-interleukin-2-receptor MAb Anti-CEAMAb CE-25 Anti-CD3-anti-MOV18 MAb -- CentOCOr Anti-CEAMAb MN 14 - Immunomedics Anti-CD3-anti-SCLC bispecific MAb Anti-CEAMAb MN14-PE40 conjugate Anti-CD4 idiotype vaccine ImmunOmedicS Anti-CD4MAb - CentOCOr, IDEC Anti-CEAMAb T84.66-interleukin-2 Pharmaceuticals, Xenova Group Conjugate Anti-CD4 MAb 16H5 Anti-CEA sheep MAb - KSBiomedix Anti-CD4. MAb 4162W94 - GlaxoSmithKline Holdings Anti-CD4 MAb B-F5 - Diaclone Anti-Cell Surface monoclonal antibodies - Anti-CD4MAb GK1-5 Cambridge Antibody Tech. IPharmacia Anti-CD4 MAb KT6 Anti-C-erbB2-anti-CD3 bifunctional MAb Anti-CD4. MAb OX38 Otsuka Anti-CD4MAb PAP conjugate - Bristol Anti-CMVMAb - Scotgen Myers Squibb Anti-Complement Anti-CD4 MAb RIB 5-2 Anti-CTLA-4MAb Anti-CD4. MAb W3/25 Anti-EGFR catalytic antibody-- Hesed Anti-CD4MAb YTA 3.1.2 Biomed Patent Application Publication Feb. 18, 2010 Sheet 23 of 47 US 2010/0041872 A1

FIGURE 7D anti-EGFR immunotoxin - VAX Anti-ICAM-1 MAb HA58 Anti-EGFR MAb-Abgenix Anti-ICAM-1 MAb YN1117.4 Anti-EGFR MAb 528 Anti-ICAM-3MAb ICM3. ICOS Anti-EGFR MAb KSB 107 - KSBiomedix Anti-idiotype breast cancer vaccine 11D10 Anti-EGFR MAb-DM1 Conjugate Anti-idiotype breast cancer vaccine ImmunOGen ACA14C5 Anti-EGFR MAb-LA1 - Anti-idiotype cancer vaccine - InClone Anti-EGFR sheep MAb - KSBiomedix Systems/Merck KGaA ImClone, Viventia Anti-FAP MAb F19-1-131 Biotech Anti-FaslgMMAb CH11 Anti-idiotype cancer vaccine 1A7 --Titan Anti-Fas MAb Jo2 Anti-idiotype cancer vaccine 3H1 - Titan Anti-Fas MAb RK-8 Anti-idiotype cancer vaccine TriAb-Titan Anti-Flt-1 monoclonal antibodies -- ImClone Anti-idiotype Chlamydia trachomatis Anti-fungal peptides -- State University of Vaccine New York Anti-idiotype Colorectal cancer vaccine antifungal tripeptides-BTG Novartis Anti-ganglioside GD2 antibody-interleukin-2 Anti-idiotype Colorectal cancer vaccine fusion protein-Lexigen OnyVax Anti-GM2MAb - Kyowa Anti-idiotype melanoma vaccine-IDEC Anti-GM-CSF receptor monoclonal Pharmaceuticals antibodies - AMRAD Anti-idiotype ovarian cancer vaccine ACA Anti-gp130MAb-Tosoh 125 Anti-HCA monoclonal antibodies - Anti-idiotype ovarian cancer Vaccine AR54 AltaRex/Epigen - AltaReX Anti-hCG antibodies - Abgenix/AV Anti-idiotype Ovarian cancer vaccine CA BioPharma 125-AltaRex, Biomira Anti-heparanase human monoclonal Anti-IgE catalytic antibody - Hesed Biomed antibodies - Oxford Anti-IgE MAb E26 - Genentech Glycosciences/Medarex Anti-IGF-1 MAb Anti-hepatitis C virus human monoclonal anti-inflammatory - GeneMax antibodies - XTL Biopharmaceuticals anti-inflammatory peptide - BTG Anti-HER-2 antibody gene therapy anti-integrin peptides - Burnha Anti-herpes antibody - Epicyte Anti-interferon-alpha-receptor MAb 64G12 Anti-HIV antibody - Epicyte Pharma Pacific Management anti-HIV catalytic antibody-- Hesed Biomed Anti-interferon-gamma MAb - Protein anti-HIV fusion protein-Idun Design Labs anti-HIV proteins - Cangene Anti-interferon-gamma polyclonal antibody Anti-HM1-24MAb - Chugai - Advanced Biotherapy Anti-hR3MAb Anti-interleukin-10 MAb Anti-Human-Carcinoma-Antigen MAb Anti-interleukin-12 MAb Epicyte Anti-interleukin-1-beta polyclonal antibody - Anti-ICAM-1 MAb -- Boehringer Ingelheim R&D Systems Anti-ICAM-1 MAb 1A-29 - Pharmacia Anti-interleukin-2 receptor MAb 2A3 Patent Application Publication Feb. 18, 2010 Sheet 24 of 47 US 2010/0041872 A1

FIGURE 7E Anti-interleukin-2 receptor MAb33B3-1- Anti-properdin monoclonal antibodies - Immunotech Abgenix/Gliatech Anti-interleukin-2 receptor MAbART-18 Anti-PSMA (prostrate specific membrane Anti-interleukin-2 receptor MAb LO-Tact-1 antigen) Anti-interleukin-2 receptor MAb Mikbeta1 Anti-PSMAMAb J591-BZLBiologics Anti-interleukin-2 receptor MAb NDS61 Anti-Rev MAbgene therapy Anti-interleukin-4 MAb 11B11 Anti-RSV antibodies - Epicyte, Intracell Anti-interleukin-5MAb - Wallace Anti-RSV monoclonal antibodies - Laboratories Medarex/MedImmune, Applied Molecular Anti-interleukin-6MAb - CentOCOr, Evolution/Medimmune Diaclone, Pharmadigm Anti-RSVMAb, inhalation Anti-interleukin-8MAb-Abgenix Akermes/Medlmmune Anti-interleukin-8MAb-Xenotech Anti-RT genetherapy Anti-JL1 MAb Antisense K-ras RNA genetherapy Anti-Klebsiella sheep MAb - KSBiomedix Anti-SF-25 MAb Holdings Anti-spermantibody - Epicyte Anti-Laminin receptor MAb-liposomal Anti-Tac(FV)-PE38 conjugate doxorubicin Conjugate Anti-TAPA/CD81 MAbAMP1 Anti-LCGMAb - Cytoclonal Anti-tat genetherapy Anti-lipopolysaccharide MAb - VitaResc Anti-TCR-alphabeta MAb H57-597 Anti-L-Selectin monoclonal antibodies - Anti-TCR-alphabeta MAb R73 Protein Design Labs, Abgenix, Stanford Anti-tenascin MAb BC-4-1-131 University Anti-TGF-beta human monoclonal Anti-MBL monoclonal antibodies - antibodies - Cambridge Antibody Tech., Alexion/Brigham and Women's Hospital Genzyme Anti-MHC monoclonal antibodies Anti-TGF-beta MAb 2.07 - Genentech Anti-MIF antibody humanised-IDEC, Antithrombin Ill - Genzyme Transgenics, Cytokine PharmaSciences Aventis, Bayer, Behringwerke, CSL, Anti-MRSAWRSA sheep MAb - KS Myriad Biomedix Holdings Anti-Thy1 MAb Anti-mu MAb - Novartis Anti-Thy1.1 MAb Anti-MUC-1 MAb Anti-tissue factor?factor VIIA sheep MAb Anti-MUC 18 KSBiomedix Anti-Nogo-A MAb IN1 Anti-TNF monoclonal antibodies - Anti-nuclear autoantibodies - Procyon Centocor, Chiron, Peptech, Pharacia, Anti-OVarian Cancer monoclonal antibodies - SerOno - Dompe Anti-TNF sheep MAb - KSBiomedix Anti-p185 monoclonal antibodies Holdings Anti-p43 MAb Anti-TNFalpha MAb - Genzyme Antiparasitic vaccines Anti-TNFalpha MAb B-C7 - Diaclone Anti-PDGF/bFGFsheep MAb - KS Anti-tooth decay MAb-Planet BioTech. Biomedix Anti-TRAIL receptor-1 MAb-Takeda Antitumour RNases - NIH Patent Application Publication Feb. 18, 2010 Sheet 25 of 47 US 2010/0041872 A1

FIGURE 7F Anti-VCAMMAb 2A2 - Alexion Aurintricarboxylic acid-high molecular Anti-VCAMMAb 3F4- Alexion Weight Anti-VCAM-1 MAb Autoimmune disorders - GPC Anti-VECMAb -- ImClone Biotech/MorphoSys Anti-VEGF MAb - Genentech Autoimmune disorders and transplant Anti-VEGF MAb2C3 rejection - Bristol-Myers Squibb/Genzyme Anti-VEGF sheep MAb -- KSBiomedix Tra Holdings Autoimmune disorderSlcancer Anti-VLA-4MAb HP1/2- Biogen Abgenix/Chiron, CuraGen Anti-VLA-4MAb PS/2 Autotaxin Anti-VLA-4MAb R1-2 Avicidin-NeoRX Anti-VA-4MAb TA-2 aXOgenesis factor-1 - Boston Life Sciences Anti-VAP-1 human MAb AXOkine - Regeneron Anti-VRE sheep MAb - KSBiomedix B cell lymphoma vaccine - Biomira Holdings B7-1 gene therapy - ANUP-TranXenOGen BABS proteins - Chiron ANUP-1.-- Pharis BAM-002- Novelos Therapeutics AOP-RANTES -- Senetek Basiliximab (anti CD25 MAb) - Novartis Apan-CH - Praecis Pharmaceuticals Bay-16-9996 - Bayer APC-8024-Demegen Bay-39-9437 - Bayer ApOA-1 - Milano, Pharmacia Bay-50-4798 - Bayer Apogen - Alexion BB-10153 - British Biotech apolipoprotein A1 - Avanir BBT-001 - Bolder BioTech. Apolipoprotein E-Bio-Tech. General BBT-002- Bolder BioTech. Applaggin - Biogen BBT-003 - BOlder BioTech. aprotinin-ProdiGene BBT-004 -- Bolder BioTech. APT-07OC - AdPOTech BBT-005-- Bolder BioTech. AR 177 - Aronex Pharmaceuticals BBT-006-- Bolder BioTech. AR 209 - Aronex Pharmaceuticals, BBT-007 - BOlder BioTech. Antigenics BCH-2763 - Shire AR545C BCSF - Millenium Biologix ARGENT gene delivery systems - ARIAD BDNF - Regeneron- Amgen Arresten Becaplermin - Johnson & Johnson, Chiron ART-123 - Asahi Kasei Bectumomab - Immunomedics arylsulfatase B - BioMarin Beriplast -- Aventis Arylsulfatase B, Recombinant human Beta-adrenergic receptor genetherapy - BioMarin University of Arkansas AS 1051 - Ajinomoto bFGF-- ScioS AS-BCL-Intracell B151013 - Behringwerke AG Asparaginase - Merck BBH 1 - Boehringer Ingelheim ATL-101 - Alizyme BIM-23190 - Beaufour-lpsen Atrial natriuretic peptide - Pharis birch pollen immunotherapy - Pharmacia bispecific fusion proteins - NIH Patent Application Publication Feb. 18, 2010 Sheet 26 of 47 US 2010/0041872 A1

FIGURE 7G Bispecific MAb 2B1 - Chiron Calcitonin - Oral-Nobex, Emisphere, Bitistatin Pharmaceutical Discovery BIWA 4 - Boehringer Ingelheim Calcitonin gene-related peptide -- Asahi blood Substitute - Northfield, Baxter Intl. Kasei-Unigene BLP-25 - Biomira Calcitonin, human - Suntory BLS-0597 -- Boston Life Sciences Calcitonin, nasal-Novartis, Unigene BLyS-Human Genome Sciences Calcitonin, PanOderm -- Elan BLyS radiolabelled - Human Genome calcitonin, Peptitrol - Shire Sciences calcitonin, salmon - Therapicon BM 06021 - Boehringer Mannheim calin-Biopharm BM-202 - BioMarin Calphobindin BM-301 - BioMarin Calphobindin - Kowa BM-301 - BioMarin Calreticulin - NYU BM-302- BioMarin Campath-1G BMP 2 - Genetics Institute/Medtronic Campath-1M Sofamor Danek, Genetics Institute? Cancer therapy - Cangene Collagenesis, Genetics Cancer Vaccine-Aixie, Aventis Pasteur, Institute/Yamanouch Center of Molecular immunology,YM BMP2 gene therapy BioSciences, Cytos, Genzyme, BMP52-- Aventis Pasteur, Biopharm Transgenics, Globelmmune, geneon, BMP-2 - Genetics Institute ImClone, Virogenetics, InterCell, lomai, BMS 182248 - Bristol-Myers Squibb Jenner Biotherapies, Memorial Sloan BMS 202448- Bristol-Myers Squibb Kettering Cancer Center, Sydney Kimmel bone growth factors--isoTis Cancer Center, NovaVax, Protein BPC-15 -- Pfizer Sciences, Argonex, SIGA brain natriuretic peptide Cancer Vaccine ALVAC-CEA B7.1- Breast Cancer - Oxford Aventis Pasteur/Therion Biologics GlycoSciences/Medarex Cancer Vaccine CEA-TRICOM-- Aventis Breast cancer Vaccine - Therion Biologics, Pasteur/Therion Biologics Oregon Cancer vaccine gene therapy - Cantab BSSL-PPL Therapeutics Pharmaceuticals BST-2001 - BioStratum Cancer Vaccine HER-2/neu - Corixa BST-3002- BioStratum Cancer Vaccine THERATOPE - Biomira BT 322 cancer vaccine, PolyMASC-Valentis butyrylcholinesterase -- Shire Candida Vaccine - Corixa, InhibiteX C 6822- COR Therapeutics CanStatin - LEX C1 esterase inhibitor - Pharming CAP-18 - PanOrama C3d adjuvant-AdProTech Cardiovascular genetherapy - Collateral CAB-2.1 - Millennium Therapeutics Calcitonin - Inhale Therapeutics Systems, Carperitide - Suntory Aventis, Genetronics, TranXenoGen, CaSOcidin-1 - Pharis Unigene, Rhone Poulenc Rohrer CAT152- Cambridge Antibody Tech. CAT192 - Cambridge Antibody Tech. Patent Application Publication Feb. 18, 2010 Sheet 27 of 47 US 2010/0041872 A1

FIGURE 7H CAT 213 - Cambridge Antibody Tech. Chlamydia pneumoniae vaccine - Antex Catalase-Enzon Biologics Cat-PAD - Circassia Chlamydia trachomatis vaccine - Antex CB0006 - Celltech Biologics CCK(27-32)- AkzoNobel Chlamydia vaccine - GlaxoSmithKline CCR2-64 - NIH Cholera vaccine CVD 103-HgR-Swiss CD, Procept - Paligent Serum and Vaccine institute Berne CD154 gene therapy Cholera vaccine CVD 112 - Swiss Serum CD39 - Immunex and Vaccine Institute Berne CD39-L2-Hyseq Cholera Vaccine inactivated Oral - SBL CD39-L4 - Hyseq Vaccin CD4 fusion toxin - Senetek Chrysalin - Chrysalis BioTech. CD4 gG - Genentech C-782 - Hitachi Kase CD4 receptor antagonists - Ciliary neurotrophic factor - Fidia, Roche Pharmacopeia/Progenics CIM project - Active Biotech CD4 soluble - Progenics CL 329753 - Wyeth-Ayerst CD4, Soluble -- Genzyme Transgenics CL22, Cobra - ML Laboratories CD40 ligand-Immunex Clenoliximab - DEC CD4-ricin chain A - Genentech Clostridium difficile antibodies - Epicyte CD59 genetherapy - Alexion clotting factors - Octagene CD8TIL cell therapy - Aventis Pasteur CMB401-- Celltech CD8, soluble - AvideX CNTF-Sigma-Tau CD95 ligand-Roche Cocaine abuSe Vaccine - Cantab, CDP 571 - Celltech Immulogic, Scripps CDP 850 - Celltech COccidiomycosis vaccine - Arizo CDP-860 (PEG-PDGFMAb) - Celltech Collagen.--Type - Pharming CDP870 - Celtech Collagen formation inhibitors - FibroGen CDS-1 - Ernest Orlando Collagen/hydroxyapatite/bone growth factor Cedelizumab --Ortho-McNeil - Aventis Pasteur, Biopharm, Orquest Cetermin -- inSmed Collagenase - BioSpecifics CETP Vaccine -- Avant Colorectal Cancer Vaccine -- Wistar Institute Cetrorelix Component B, Recombinant- Serono Cetuximab Connective tissue growth factor inhibitors - CGH 400 - NOWartis FibroGen Taisho CGP 42934 - NOWartis COntortrostatin CGP 51901 - TanOX Contraceptive vaccine - Zonagen CGRP-Unigene Contraceptive vaccine hCG CGS 27913-Novartis Contraceptive vaccine male reversible - CGS 32359 - Novartis MMUCON Chagas disease vaccine - Corixa Contraceptive vaccine Zona pellucida chemokines - Immune Response Zonagen CHH 380 - NOWartis Copper-64 labelled MAbTETA-1A3 - NCI Chitinase - Genzyme, ICOS Coralyne Patent Application Publication Feb. 18, 2010 Sheet 28 of 47 US 2010/0041872 A1

FIGURE 7. CorSevin M Daclizumab (anti-IL2R MAb) - Protein C-peptide analogues - Schwarz Design Labs CP-1500- Consensus DAMP - Incyte Genomics CRF - Neurobiological Tech. Daniplestim - Pharmacia CRGDfW pentapeptide darbepoetin alfa - Amgen CRL 1095- CytRx DB-3019 - Diabetogen CRL 1336 - CytRx DCC- Genzyme CRL 1605 - CytRx DDF - Hyseq CS-560 - Sankyo decorin-integra, Telios CSF-ZymoGenetics defensins - Large Scale Biology CSF-G-Hangzhou, Dong-A, Hanmi DEGR-Vila CSF-GM-Cangene, Hunan, LG Chem Delmmunised antibody 3B6/22 AGEN CSF-M -- Zarix Deimmunised anti-cancer antibodies - CT 1579 - Merck FroSSt Biovation/Viragen CT 1786 - Merck FroSSt Dendroamide A CT-112A - BTG Dengue vaccine - Bavarian Nordic, Merck CTB-134L-Xenova denileukin diffitoX - Ligand CTC-111 - KaketSuken DES-1101 - DeSmOS CTGF - FibroGen desirudin - Novartis CTLA4-g-Bristol-Myers Squibb desmopressin - Unigene CTLA4-g gene therapy - Desmoteplase-Merck, Schering AG CTP-37 - AV BioPharma Destabilase C-type natriuretic peptide - Suntory Diabetes gene therapy - DeveloGen, Pfizer CVS 995 - Corvas Intl. Diabetes therapy - Crucell CX397 - Nikko Kyodo Diabetes type 1 vaccine-Diamyd CY 1747 - Epimmune Therapeutics CY 1748 - Epimmune DiaCIM-YMBioSciences Cyanovirin-N dialytic oligopeptides - Research Corp Cystic fibrosis therapy - CBRIVAX Diamyd-Diamyd Therapeutics CYT 351 DiaPep227-Pepgen cytokine Traps - Regeneron DiaVax- Corixa Cytokines - Enzon, Cytoclonal Digoxin MAb - Glaxo Cytomegalovirus glycoprotein Vaccine Diphtheria tetanus pertussis-hepatitis B Chiron, Aquila Biopharmaceuticals, vaccine - GlaxoSmithKline Aventis Pasteur, Virogenetics DIR therapy - Solis Therapeutics - Cytomegalovirus vaccine live -- Aventis DNase - Genentech Pasteur Dornase alfa - Genentech Cytosine deaminase genetherapy - DOrnase alfa, inhalation - Genentech GlaxoSmithKline Doxorubicin-anti-CEAMAb Conjugate DA-3003-Dong-A Immunomedics DAB389interleukin-6 - Senetek DP-107 - TrimeriS DAB389interleukin-7 drotrecogin alfa-Eli Lilly DTctGMCSF Patent Application Publication Feb. 18, 2010 Sheet 29 of 47 US 2010/0041872 A1

FIGURE 7J DTP-polio vaccine -- Aventis Pasteur enzyme linked antibody nutrient depletion DU 257-KM231 antibody conjugate therapy - KSBiomedix Holdings Kyowa Eosinophil-derived neutralizing agent dural graft matrix-integra EP-51216 - Asta Medica Duteplase - Baxter Intl. EP-51389 - Asta Medica DWP-401 - Daewoong EPH family ligands - Regeneron DWP-404 - Daewoong Epidermal growth factor - Hitachi Kasei, DWP-408-Daewoong Johnson & Johnson DX 88 (Epi-KAL2)-Dyax Epidermal growth factor fusion toxin DX890 (elastin inhibitors) - Dyax Senetek E Coli O157 waCCine - NIH Epidermal growth factor-genistein E21-R-BresaGen EP-HNE-4-Dyax Eastern equine encephalitis virus vaccine EPI-KAL2-Dyax EchiCetin Epoetin-alfa-Amgen, Dragon Echinhibin 1 Pharmaceuticals, Nanjing Huaxin Echistatin - Merck Epratuzumab-Immunomedics Echitamine Epstein-Barr virus vaccine ECromeximab-Kyowa Hakko Aviron/SmithKline Beecham, Bioresearch EC-SOD - PPL Therapeutics Eptacog alfa-Novo Nordisk Eculizumab (5G1.1) - Alexion Eptifibatide - COR Therapeutics EDF - Ajinomoto erb-38 EDN derivative - NIH Erlizumab -- Genentech EDNA - NIH erythropoietin- Akermes, ProLease, Dong EdobaCOmab --XOMA A, Elanex, Genetics Institute, LG Chem, Edrecolomab -- CentOCOr Protein Sciences, Serono, Snow Brand, EF5077 SRC VB VECTOR, Transkaryotic Efalizumab - Genentech Therapies EGF fusion toxin- Seragen, Ligand Erythropoietin Beta-Hoffman La Roche EGF-P64k Vaccine - Center of Molecular Erythropoietin/Epoetin alfa - Chugai immunology Escherichia Colivaccine - North American EL246 - LigoCyte Vaccine, SBL Vaccin, Swiss Serum and elastase inhibitor - Synergen WaCCine Institute Berne elcatonin - Therapicon etanercept-Immunex EMD 72000 - Merck KGaA examOrelin - Mediolanum Emdogain - BIORA Exendin 4- Amylin emfiermin -- AMRAD exonuclease VI Emoctakin - Novartis F 105-- CentOCOr enamel matrix protein-BIORA F-992 - Fornix Endo III - NYU Factor IX-Alpha Therapeutics, Welfide endostatin - EntreMed, Pharis Corp., CSL, enetics InstitutelAHP, Enhancins - Micrologix Pharmacia, PPL Therapeutics Enlimomab -- isis Pharm. Factor IX gene therapy - Cell Genesys Enoxaparin sodium - Pharmuka Patent Application Publication Feb. 18, 2010 Sheet 30 of 47 US 2010/0041872 A1

FIGURE 7K Factor VII-Novo Nordisk, Bayer, Baxter follitropin alfa - Akermes, ProLease, int. POW(derJect, Serono, AkzoNobel Factor Vila-PPL Therapeutics, Follitropin Beta-Bayer, Organon ZymoGenetics FP59 Factor Vill-Bayer Genentech, Beaufour FSH-Ferring lpSen, CLB, inex, Octagen, Pharmacia, FSH + LH-Ferring Pharming F-spondin- CeNeS Factor WIll-PEGylated - Bayer fusion protein delivery system - UAB Factor WIll fragments - Pharmacia Research Foundation Factor WIll gene therapy - Targeted fusion toxins - Boston Life Sciences Genetics G5598 -- Genentech Factor VIII sucrose formulation-Bayer, GA-II - Transkaryotic Therapies Genentech Gamma-interferon analogues - SRC VB Factor VIII-2-Bayer VECTOR Factor Vill-3-Bayer Ganirelix-Roche Factor Xa inhibitors - Merck, Novo Nordisk, gastric lipase-Meristem Mochida Gavilimomab Factor XIII-ZymoGenetics G-CSF-Amgen, SRC VB VECTOR Factors VIlland IX gene therapy - Genetics GDF-1 - CeNeS Institute? Targeted Genetics GDF-5- Biopharm Famoxin - Genset GDNF (glial derived neurotrophic factor)- Fas (delta) TM protein-LXR BioTech. Amgen FaSTR - Human GenOme Sciences gelsolin - Biogen Felvizumab -- Scotgen Gemtuzumab OZOgamicin - Celltech FFR-Vila - NOVO Nordisk Gene-activated epoetin-alfa-Aventis FG-001-F-Gene Pharma-Transkaryotic Therapies FG-002-F-Gene Glanzmann thrombasthenia gene therapy FG-004-F-Gene Glatiramer acetate -- Yeda FG-005-F-Gene glial growth factor 2- CeNeS FGF + fibrin-Repair GLP-1 - Amylin, Suntory, TheraTech, Fibrimage - Bio-Tech. General Watson fibrin-binding peptides - ISIS Innovation GLP-1 peptide analogues - Zealand fibrinogen - PPL Therapeutics, Pharming Pharaceuticals fibroblast growth factor - Chiron, NYU, glucagon - Eli Lilly, ZymoGenetics Ramot, ZymoGenetics Glucagon-like peptide-17-36 amide fibrolase conjugate - Schering AG Suntory Filgrastim-Amgen Glucogen-like peptide - Amylin filgrastim - PDA modified-Xencor Glucocerebrosidase - Genzyme FLT-3 ligand- Immunex glutamate decarboxylase - Genzyme FN18 CRM9 Transgenics follistatin -- Biotech Australia, Human Glycoprotein S3-Kureha Therapeutics GM-CSF -- Immunex GM-CSF tumour vaccine - POWoderJect Patent Application Publication Feb. 18, 2010 Sheet 31 of 47 US 2010/0041872 A1

FIGURE 7L GnRH immunotherapeutic-Protherics Hemolink - HemOSol GOSerelin (LhrHantagonist) - AstraZeneca hepapoietin - Snow Brand gp75 antigen -- ImClone heparanase - InSight gp96- Antigenics heparinase - Ibex GP 0100 - Galenica heparinase ill- Ibex GR4991W93 - GlaxoSmithKline Hepatitis A vaccine - American Biogenetic Granulocyte Colony-stimulating factor Sciences Dong-A Hepatitis A vaccine inactivated Granulocyte Colony-stimulating factor Hepatitis A vaccine Nothav - Chiron Conjugate Hepatitis A-hepatitis B vaccine - graSS allergy therapy - DynaVax GlaxoSmithKline GRF1-44 - ICN hepatitis B therapy - Tripep Growth Factor - Chiron, Atrigel, Atrix, Hepatitis B vaccine - Amgen, Chiron SpA, Innogenetics, ZymoGenetics, Novo Meiji Milk, NIS, Prodeva, PowderJect, growth factor peptides - Biotherapeutics Rhein Biotech growth hormone - LG Chem Hepatitis B vaccine recombinant-Evans growth hormone, Recombinant human Vaccines, Epitec Combiotech, Genentech, Serono MedImmune, Merck Sharp & Dohme, GT4086 - Gliatech Rhein Biotech, Shantha Biotechnics, GW 353430 - GlaxoSmithKline Vector, Yeda GW-278884 - GlaxoSmithKline Hepatitis B vaccine recombinant TGP943 H 11 - Viventia Biotech Takeda H5N1 influenza A virus VaCCine - Protein Hepatitis C vaccine - Bavarian Nordic, Sciences Chiron, Innogenetics Acambis, haemoglobin - Biopure Hepatitis D vaccine - Chiron Vaccines haemoglobin 3011, Recombinant-Baxter Hepatitis Evaccine recombinant HealthCare GenelabS/GlaxoSmithKline, NovaVax haemoglobin Crosfumaril-Baxter Intl. hepatocyte growth factor-Panorama, haemoglobin stabilized- Ajinomoto SOSei haemoglobin, recombinant-Apex hepatocyte growth factor kringle fragments - HAF-Immune Response - EntreMed Hantavirus Vaccine Her-2/Neupeptides - Corixa HB 19 Herpes simplex glycoprotein DNA vaccine HBNF - Regeneron Merck, Wyeth-Lederle Vaccines-Malvern, HCC-1 - Pharis Genentech, GlaxoSmithKline, Chiron, hCG - Milkhaus Takeda hCG vaccine - Zonagen Herpes simplex vaccine - Cantab HE-317 - Hollis-Eden Pharmaceuticals Pharmaceuticals, CEL-SC, Henderson Heat shock protein cancer and influenza Morley Vaccines -- StressGen Herpes simplex vaccine live - ImClone Helicobacter pylori vaccine - Acambis, Systems/Wyeth-Lederle, Aventis Pasteur AstraZeneca/CSL, Chiron, Provalis HGF derivatives -- Dompe Helistat-G - GalaGen hAPP Vaccine -- CruCell Patent Application Publication Feb. 18, 2010 Sheet 32 of 47 US 2010/0041872 A1

FIGURE 7M Hib-hepatitis B vaccine - Aventis Pasteur host-vector vaccines - Henogen HIC 1 HPM.1 - Chugai HIP-- Altachem HPV Vaccine - MediGene Hirudins-Biopharma, Cangene, Dongkook, HSA - Meristem Japan Energy Corporation, Pharmacia HSF - StreSSGen Corporation, SIR International, Sanofi HSP carriers-Weizmann, Yeda, Peptor Synthelabo, Sotragene, Rhein Biotech HSPPC-70 - Antigenics HIV edible Vaccine - PrOdiGene HSPPC-96, pathogen-derived - Antigenics HIV gp120 vaccine - Chiron, Ajinomoto, HSV863 - NOWartis GlaxoSmithKline, ID Vaccine, Progenics, HTLV-DNA Vaccine VaxGen HTLV-I WaCCine HIV gp120 Vaccine genetherapy HTLV-II Vaccine - ACCeSS HIV gp160 DNA vaccine - PowderJect, HU901 - TanOX Aventis Pasteur, Oncogen, Hyland HU23F2G-ICOS Immuno, Protein Sciences HUHMFG1 HIV gp41 vaccine - Panacos Huma YM - Intracell HIV HGP-30W WaCCine - CEL-SC Human Krebs Statika - Yamanouchi HIV immune globulin-Abbott, Chiron human monoclonal antibodies HIV peptides - American Home Products Abgenix/Biogen, Abgenixl Corixa, HIV vaccine - Applied bioTech. Axis Abgenix Immunex, Abgenix/Lexicon, Genetics, Biogen, Bristol-Myers Squibb, Abgenix Pfizer, Athersys/Medarex, Genentech, Korea Green CrOSS, NS, Biogen/MorphoSys, CATISearle, Oncogen, Protein Sciences Corporation, CentOCOrlMedarex, CorixalKirin Brewery, Terumo, Tonen Corporation, Wyeth Corixa/Medarex, EOS BioTech./Medarex, Ayerst, Wyeth-Lederle Vaccines-Malvern, EOSIXenerex, Exelixis/Protein Design Advanced BioScience Laboratories, Labs, immunoGen Raven, Medarex/ Bavarian Nordic, Bavarian Nordic/Statens B.Twelve, MorphOSys/immunoGen, XTL Serum Institute, GeneCure, immune Biopharmaceuticals/Dyax, Response, Progenics, Therion Biologics, Human monoclonal antibodies - United Biomedical, Chiron Medarex/Northwest Biotherapeutics, HIV Vaccine VCP1433- AVentis Pasteur Medarex/Seattle Genetics HIV Vaccine VCP1452-- Aventis Pasteur humannetrin-1 - Exelixis HIV Vaccine VCP205 - AVentis Pasteur human papillomavirus antibodies - Epicyte HL-9 - American BioScience Human papillomavirus vaccine - Biotech HM-9239 - Cytran Australia, IDEC, StressGen HML-103 - HemoSol Human papillomavirus vaccine MED 501 - HML-104 -- HemOSol MedImmune/GlaxoSmithKline HML-105 - HemOSol Human papillomavirus vaccine MED HML-109-HemOSO 503/MED 504 HML-110 - HemOSol Medmmune/GlaxoSmithKline HML-121 - HemOSol Human papillomavirus vaccine TA-CIN hNLP-- Pharis Cantab Pharmaceuticals HookWOrm Vaccine Patent Application Publication Feb. 18, 2010 Sheet 33 of 47 US 2010/0041872 A1

FIGURE 7N Human papillomavirus vaccine TA-HPV IL-7-Dap 389 fusion toxin-Ligand Cantab Pharmaceuticals IM-862 - Cytran Human papillomavirus vaccine TH-GW MC-1 C 11 - ImClone CantablGlaxoSmithKline imigluCerase - Genzyme human polyclonal antibodies - Biosite/Eos Immune globulin intravenous (human) - BioTech.) Medarex Hoffman La ROChe human type II antifactor WIll monoclonal immune privilege factor - Proneuron antibodies - ThrombOGenicS immunoCal -- Immunotec humanised antiglycoprotein Ib murine Immunogenetherapy - Briana Bio-Tech mOnOClonal antibodies -- ThrombOGenics Immunoliposomal 5-fluorodeoxyuridine HumarAD - IntraCell dipalmitate HuMax EGFR - Genmab immunosuppressant vaccine-Aixie HuMax-CD4 - Medarex immunotoxin-Antisoma, NIH HuMax-IL15 - Genmab ImmurAIT-Re-188-mmunOmedics HYB 190 - Hybridon imreg-1 - Imreg HYB 676- Hybridon infertility - Johnson & Johnson, E-TRANS -125 MAb A33-- Celltech Infliximab - CentOCOr britumomab tiuxetan -- IDEC Influenza virus Vaccine - Avents Pasteur, BT-94.01 - Ibex Protein Sciences BT-94O2- Ibex inhibin - Biotech Australia, Human IC 14 - ICOS Therapeutics ldarubicin anti-Ly-2.1 - Inhibitory G protein gene therapy IDEC 114 - DEC NKP-2001 -- inkine IDEC 131 - DEC InOlimOmab - Diaclone IDEC 152 - DEC insulin - Autolimmune, Altea, Biobras, IDM 1 - DM BioSante, Bio-Tech. General, Chong Kun DPS - Hollis-Eden Pharmaceuticals Dang, Emisphere, Flamel, Provalis, Rhein iduronate-2-sulfatase - Transkaryotic Biotech, TranXenoGen Therapies insulin (bovine) - Novartis GFIBP-2-13 - Pharis insulin analogue - Eli Lilly IGN-101 - geneon Insulin Aspart-Novo Nordisk KHRO2-keton insulin detemir - Novo Nordisk IL-11 - Genetics Institute/AHP insulin glargine - Aventis IL-13-PE38 - NeOPharm insulin inhaled-inhale Therapeutics IL-17 receptor - Immunex Systems, Akermes IL-18BP -- Yeda insulin Oral -- noVax IL-1Hy1 - Hyseq insulin, AeroDOSe - AeroGen IL-1? - Celltech insulin, AERX - Aradigm IL-1 ($ adjuvant-Celltech insulin, BEODAS-Elan L-2-- Chiron insulin, Biphasix - Helix IL-2 + IL-12 - Hoffman La-ROche insulin, buCCal- Generex IL-6/SL-6R fusion - Hadasit insulin, I2R - Flemington IL-6R derivative - TOSOh insulin, intranasal - Bentley Patent Application Publication Feb. 18, 2010 Sheet 34 of 47 US 2010/0041872 A1

FIGURE 7O insulin, Oral-NobeX, Unigene Interferon Gamma-Boehringer Ingelheim, insulin, OraSome -- Endorex Sheffield, Rentschler, Hayashibara insulin, ProMaxx - Epic interferon receptor, Type - Serono insulin, Quadrant-Elan interferon(Gamma1B) - Genentech insulin, recombinant-Aventis Interferon-alpha-2b + ribavirin-Biogen, insulin, Spiros-Elan ICN insulin, TransferSOme -- IDEA Interferon-alpha-2b genetherapy - insulin, Zymo, recombinant - Novo Nordisk Schering-Plough insulinotropin - Scios Interferon-Con1 gene therapy - Insulysin gene therapy - interleukin-1 antagonists - Dompe integrin antagonists - Merck interleukin-1 receptor antagonist - Abbott interferon (Alpha2) - SRC VB VECTOR, Bioresearch, Pharmacia Viragen, Dong-A, Hoffman La-Roche, Interleukin-1 receptor type -- ImmuneX Genentech interleukin-1 receptor Type Il-ImmuneX interferon - BioMedicines, Human Genome Interleukin-1 trap - Regeneron Sciences Interleukin-1-alpha - Immunex/Roche interferon (Alfa-n3)-Interferon Sciences interleukin-2 - SRC VB VECTOR, Intl. Ajinomoto, Biomira, Chiron interferon (Alpha), Biphasix-Helix IL-2 diphtheria toxin-Ligand interferon (Alpha)-Amgen, BioNative, Interleukin-3- Cangene Novartis, Genzyme Transgenics, Interleukin-4 - Immunology Ventures, Hayashibara, Inhale Therapeutics Sanofi Winthrop, Schering-Plough, Systems, Medusa, Flamel, Dong-A, Immunexl Sanofi Winthrop, Bayer, Ono GeneTrol, Nastech, Shantha, interleukin-4+TNF-Alpha - NIH Wassermann, LG Chem, Sumitomo, interleukin-4 agonist - Bayer Aventis, Behring EGIS, Pepgen, Servier, interleukin-4 fusion toxin - Ligand Rhein Biotech, Interleukin-4 receptor - Immunex, Immun interferon (Alpha2A) Interleukin-6- Ajinomoto, Cangene, Yeda, interferon (Alpha2B)- Enzon, Schering Genetics Institute, Novartis Plough, Biogen, IDEA interleukin-6 fusion protein interferon (Alpha-N1)- GlaxoSmithKline interleukin-6 fusion toxin-Ligand, SerOno interferon (beta)- Rentschler, GeneTrol, interleukin-7 -IC Innovations Meristem, Rhein Biotech, Toray, Yeda, interleukin-7 receptor - ImmuneX Daiichi, Mochida interleukin-8 antagonists - Kyowa interferon (Beta1A) - Serono, Biogen HakkO/Millennium/Pfizer interferon (beta1A), inhale - Biogen interleukin-9 antagonists - Genaera interferon ($1b)- Chiron Interleukin-10-DNAX, Schering-Plough interferon (tau)- Pepgen Interleukin-10 gene therapy - Interferon alfacon-1-Amgen interleukin-12 - Genetics Institute, Hoffman Interferon alpha-2a vaccine La-ROche Interferon Beta 1b - Schering/Chiron, interleukin-13 - Sanofi InterMune interleukin-13 antagonists - AMRAD Interleukin-13-PE38OQR Patent Application Publication Feb. 18, 2010 Sheet 35 of 47 US 2010/0041872 A1

FIGURE 7P

interleukin-15 -- Immunex larOnidase - BioMarin interleukin-16 - Research Corp LaSSafever vaccine interleukin-18 - GlaxoSmithKline LCAT - NIH Interleukin-18 binding protein -- Serono LDP 01 - Millennium lor-P3 - Center of Molecular immunology LDPO2- Millennium IP-10 - NIH Lecithinized Superoxide dismutase - IPF - Metabolex Seikagaku IR-501 - Immune Response LelF adjuvant- Corixa ISIS 91.25-isis Pharmaceuticals leishmaniasis Vaccine - Corixa ISURF No. 1554 - Millennium lenercept - Hoffman La-Roche SURF No. 1866 - lowa State Univer. Lenograstim-Aventis, Chugal ITF-1697 - ItalfarmaCO lepirudin-Aventis XC 162 - lxion leptin-Amgen, IC Innovations J695 - Cambridge Antibody Tech., Leptingene therapy - Chiron Corporation Genetics Inst. Knoll leptin, 2nd-generation - Amgen Jagged + FGF- Repair leridistim - Pharmacia JKC-362 - Phoenix Pharmaceuticals leuprolide, ProMaxx-Epic JTP-2942-Japan Tobacce leuprorelin, oral - Unigene Juman monoclonal antibodies - LeuTech - Papatin Medarex/Raven LEXO32- SuperGen K02-AXys Pharmaceuticals LiDEPT - NOVartis Keliximab - DEC Lintuzumab (anti-CD33 MAb) - Protein Keyhole limpet haemocyanin Design Labs KGF-- Amgen lipase - Altus BiologicS KM871 - Kyowa lipid A vaccine - EntreMed KP 135 - SCiOS lipid-linked anchor Tech. -ICRT, ID KP-022 - ScioS Biomedical Kringle 5 liposome-CD4 Tech. -- Sheffield KSB 304 Listeria monocytogenes vaccine KSB-201 - KSBiomedix LMB 1 L 696418 - Merck LMB 7 L 703801 - Merck LMB 9 - Battelle Memorial Institute, NIH L1 - ACOrda LM-CD45-Cantab Pharmaceuticals -761191 - Merck lovastatin - Merck lactoferrin-Meristem, Pharming, Agennix LSA-3 lactoferrin cardio -- Pharming LT-? receptor - Biogen LAG-3 - Serono lung cancer vaccine - Corixa LAT - GEMMA luSupultide - Scios LAK cell cytotoxin-Arizona L-VaX-AVAX lamellarins - PharmaMar/University of LY355455- Eli Lilly Malaga LY366405- Eli Lilly laminin A peptides - NIH LY-355101- Eli Lilly lanoteplase - Genetics Institute Patent Application Publication Feb. 18, 2010 Sheet 36 of 47 US 2010/0041872 A1

FIGURE 7Q Lyme disease DNA vaccine-Vical/Aventis MDX240 - Medarex Pasteur MDX 33 Lyme disease vaccine - Aquila MDX 44 - Medarex Biopharmaceuticals, Aventis, Pasteur, MDX 447 - Medarex Symbicom, GlaxoSmithKline, Hyland MDXH210 - Medarex Immuno, MedImmune MDXRA - Houston BioTech., Medarex Lymphocytic choriomeningitis virus vaccine ME-104 -- Pharmexa lymphoma vaccine-Biomira, Genitope Measles Vaccine LYP18 Mecasermin - Cephalon/Chiron, Chiron lySplasminogen, recombinant MED 488 - MedImmune LySOSOmal storage disease gene therapy - MED 500 Avigen MED 507 - BioTransplant lysostaphin-Nutrition 21 melanin Concentrating hormone-- M 23- Gruenenthal NeurOCrine BioSciences M1 monoclonal antibodies - ACOrda melanoCortins -- OMRF Therapeutics Melanoma monoclonal antibodies - Viragen MA 16N7C2 - CorvaS Intl. melanoma vaccine - GlaxoSmithKline, malaria Vaccine -- GlaxoSmithKline, AkzoNobel, Avant, Aventis Pasteur, AdProTech, Antigenics, Apovia, Aventis Bavarian Nordic, BioVector, CancerVax, Pasteur, Axis Genetics, Behringwerke, Genzyme Molecular Oncology, Humbolt, CDCP, Chiron Vaccines, Genzyme ImClone Systems, Memorial, NYU, OXXOn Transgenics, Hawaii, MedImmune, NIH, Melanoma vaccine Magevac-Therion NYU, OXXOn, Roche/Saramane, Biotech memory enhancers - Scios Australia, RX Tech meningococcal B vaccine - Chiron Malaria Vaccine CDC/NIMALVAC-1 meningococcal vaccine - CAMR malaria vaccine, multicomponent Mening0COCCal Vaccine group B Conjugate mammaglobin - Corixa - North American Vaccine mammastatin - Biotherapeutics Meningococcal vaccine group B mannan-binding lectin-Natimmu recombinant - BioChem Vaccines, mannan-MUC1 - PSirOn MicroScience MAP 30 Mening0COCCal vaccine group Y COnjugate Marinovir - Phytera - North American WaCCine MARStem - Maret Mening0COccal vaccine groups A B and C MB-015 - MOChida Conjugate-North American Vaccine MBP-Immulogic Mepolizumab - GlaxoSmithKline MCI-028-Mitsubishi-Tokyo Metastatin - EntreMed, Takeda MCIF - Human GenOme Sciences Met-CkB7 - Human GenOme Sciences MDC - Advanced BioScience - Akzo met-enkephalin-TN Nobel, ICOS METH-1 - Human Genome Sciences MDX 11 - Medarex methioninase - AntiCancer MDX210 - Medarex Methionine lyase gene therapy - MDX 22 - Medarex AntiCancer MDX 22 Patent Application Publication Feb. 18, 2010 Sheet 37 of 47 US 2010/0041872 A1

FIGURE 7R Met-RANTES- Genexa Biomedical, MAb 323A3 - CentOCOr SerOno MAb 3C5 Metreleptin MAb 3F12 Microtubule inhibitor MAb MAb 3F8 Immunogen/Abgenix MAb 426 MGDF - Kirin MAb 425 - Merck KGaA MGV-Progenics MAb 447-52D-Merck Sharp & Dohme micrin - EndoCrine MAb 45-2D9-- haematoporphyrin microplasmin - ThromboGenics Conjugate MIF - GeneticS Institute MAb 4B4 migration inhibitory factor - NIH MAb 4E3-CPA Conjugate - BCM Oncologia Mim CD4.1-Xycte Therapies MAb 4E3-daunorubicin Conjugate mirostipen - Human Genome Sciences MAb 50-6 Mitumomab (BEC-2)-ImClone Systems, MAb 50-61A - Institut Pasteur Merck KGaA MAb 5A8- Biogen MK 852- Merck MAb 791TI36-methotrexate Conjugate MLN 1202 (Anti-CCR2 monoclonal MAb 7C11.e8 antibody)-Millenium Pharmaceuticals MAb 7E11 C5-selenocystamine Conjugate Mobenakin - NIS MAb 93KA9 - NOVartis mogramOstim- Genetics Institute, Novartis MAb A5B7-cisplatin conjugate monoclonal antibodies - Abgenix/Celltech, Biodynamics Research, Pharmacia ImmuSol Medarex, Viragen/Roslin MAbA5B7-1-131 Institute, Cambridge Antibody Tech/Elan MAb A7 MAb 108 MAbA717-- EXOCell MAb 10D5 MAb A7-zinostatin Conjugate MAb 14.18-interleukin-2 immunocytokine MAb ABX-RB2 - Abgenix Lexigen MAb ACA 11 MAb 14C2a MAb AFP-1-131 - ImmunomedicS MAb 15A10 MAb AP1 MAb 170 - Biomira MAb AZ1 MAb 177Lu CC49 MAb B3-LysPE40 conjugate MAb 17F9 MAbB4 - United Biomedical MAb 1D7 MAb B43 Genistein-conjugate MAb F7 - Immune NetWOrk MAb B43.13-TC-99m - Biomira MAb 1H10-doxorubicin Conjugate MAbB43-PAP conjugate MAb 26-2F MAb B4G7-gelonin Conjugate MAb 2A11 MAb BCM43-daunorubicin Conjugate MAb 2E1 - RWJohnson BCM Oncologia MAb 2F5 MAb BIS-1 MAb 31.1 - International Biolmmune MAb BMS 181170 - Bristol-Myers Squibb Systems MAb BR55-2 MAb32 - Cambridge Antibody Tech., MAb BW494 Peptech MAb C242-DM1 Conjugate -ImmunoGen Patent Application Publication Feb. 18, 2010 Sheet 38 of 47 US 2010/0041872 A1

FIGURE 7S MAb C242-PE conjugate MAb KS1-4-methotrexate conjugate MAb C30-6 MAb L6- Bristol-Myers Squibb, Oncogen MAb CA208-cytorhodin-S conjugate MAb LiCO 16-88 Hoechst Japan MAb LL2-1-131 - Immunomedics MAb CC49 - Enzon MAb LL2-Y-90 MAb Ch1418 MAb LS2D617-- Hybritech MAb CH14.18-GM-CSF fusion protein MAb LYM-1-gelonin conjugate Lexigen MAb LYM-1-1-131 MAb ChCE7 MAb LYM-1-Y-90 MAb C-137 - AMRAD MAb LYM-2- Peregrine MAb cisplatin Conjugate MAb M195 MAb CLB-CD19 MAb M195-bismuth 213 conjugate MAb CLB-CD19W Protein Design Labs MAb CLL-1 - Peregrine MAb M195-gelonin conjugate MAb CLL-1-GM-CSF conjugate MAb M195-1-131 MAb CLL-1-IL-2 Conjugate-Peregrine MAb M195-Y-90 MAb CLN gG-- doxorubicin Conjugates MAbMA 33H1 - Sanofi MAb Conjugates - Tanox MAbMAD11 MAb D612 MAb MGb2 MAb Da B02 MAbMINT5 MAb DC101 - ImClone MAb MK2-23 MAb EA 1 MAb MOC31 ETA(252-613) conjugate MAb EC708 -- BioVation MAb MOC-31-in-111 MAb EP-5C7-Protein Design Labs MAb MOC-31-PE Conjugate MAb ERC-1 - CRT MAb MR6 MAb F105 gene therapy MAb MRK-16 - Aventis Pasteur MAb FC 2.15 MAb MS11G6 MAb G250 - CentOCOr MAb MX-DTPA BrE-3 MAb GA6 MAb MY9 MAb GA733 MAb Nd2 - TOSOh MAb Gliomab-H - Viventia Biotech MAb NG-1 - Hygeia MAb HB2-Saporin Conjugate MAb NM01 - Nissin FOOd MAb HD 37 MAb OC 125 MAb HD37-ricin chain-A conjugate MAb OC 125-CMA conjugate MAb HNK20 - ACambis MAb OK-1 - Ortho-McNeil MAbhuN901-DM1 conjugate MAb OX52- Bioproducts for Science ImmunOGen MAb PMA5 MAb -131 CC49 -- COrixa MAb PR1 MAb CO25 MAb prost 30 MAb ICR12-CPG2 conjugate MAb R-24 MAb ICR-62 MAb R-24 C Human GD3 - Celltech MAb Rac-ricin A Conjugate MAb RFB4-ricin chain A conjugate MAb K1 MAb RFT5-ricin chain A Conjugate Patent Application Publication Feb. 18, 2010 Sheet 39 of 47 US 2010/0041872 A1

FIGURE 7T MAb SC 1 muCOSal tolerance - Aberdeen MAb SM-3 - CRT mullerian inhibiting subst MAb SMART 1D10- Protein Design Labs muplestim - Genetics Institute, Novartis, MAb SMART ABL 364 - NOVartis DSMAnti-infectives MAb SN6f murine MAb -- KSBiomedix MAb SN6f-deglycosylated ricin A chain Mutant somatropin - JCR Pharmaceutical Conjugate MV833.--ToagOsei MAb SN6 Mycoplasma pulmonis Vaccine MAb SN7-ricin chain A conjugate Mycoprex-XOMA MAb T101-Y-90 conjugate -- Hybritech myeloperOxidase - Henogen MAb T-88 - Chiron myostatin - Genetics Institute MAb TB94 - Cancer ImmunoBiology NaColomab tafenatoX -- Pharmacia MAb TEC 11 Nagrecor-Scios MAbTES-23- Chugai nagrestipen - British Biotech MAb TM31 - AWant NAP-5- COrvaS Intl. MAb TNT-1.-- Cambridge Antibody Tech., NAPC2 - COrvaS Intl. Peregrine nartograstim- Kyowa MAb TNT-3 Natalizumab - Protein Design Labs MAb TNT-3-IL2 fusion protein Nateplase-NIH, Nihon Schering MAb TP3-At-211 nateplase - Schering AG MAb TP3-PAP conjugate NB-3001 - NeurOCrine BioSci. MAb UJ13A - CRT NB-5788 - NeurOCrine BioSci. MAb UN3 NB-6024 - NeurOCrine BioSci. MAb ZME-018-gelonin conjugate Nef inhibitOrS - BR MAb-BC2 - GlaxoSmithKline Neisseria gonorrhoea vaccine. Antex MAb-DM1 Conjugate -ImmunoGen Biologics MAb-ricin-chain-A Conjugate-XOMA Neomycin B-arginine Conjugate MAb-temoportin Conjugates Nerelimomab -- Chiron Monopharm C-Viventia Biotech Nerve growth factor - Amgen - Chiron, monteplase - Eisai Genentech montirelin hydrate - Gruenenthal Nerve growth factor gene therapy morOctoCog alfa - Genetics Institute nesiritide citrate - ScioS MorOctoCog-alfa - Pharmacia neuregulin-2-CeNeS MP4 neuroCan - NYU MP-121 - Biopharm neuronal delivery system - CAMR MP-52 - Biopharm Neurophil inhibitory Factor - Corvas MRA - Chugai Neuroprotective vaccine - University of MS 28168 - Mitsui Chemicals, Nihon Auckland Schering neurotrophic chimaeras -- RegenerOn MSH fusion toxin-Ligand neurotrophic factor-NsGene, CereMedix MS-99 - Genaera NeuroVax - Immune Response MT 201 - Micromet neurturin - Genentech MuC-1 vaccine - Corixa neutral endopeptidase - Genentech Patent Application Publication Feb. 18, 2010 Sheet 40 of 47 US 2010/0041872 A1

FIGURE 7U NGF enhancers - NeuroSearch Onychomycosis vaccine - Boehringer NHL Vaccine - Large Scale Biology Ingelheim NIP45- BOston Life Sciences opebecan-XOMA NK-B20 opioids - Arizona NM01 - Nissin FOOd Oprelvekin - Genetics Institute NM-139 - NitroMed Oregovomab -- AltaRex NMMP - Genetics Institute Org-33408 b-AkzoNobel NN-2211 - NOVO NOrdisk Orolip DP-EpiCept Noggin - Regeneron OryzacyStatin Nonacog alfa OSA peptides - GenSciRegeneration Norelin - BioStar Osteoblast-cadherin GF-- Pharis Norwalk virus vaccine Osteocalcin-thymidine kinase gene therapy NRLU 10 - NeORX Osteogenic protein- Curis NRLU OPE - NeORX Osteopontin - Orapharma NT-3- Regeneron Osteoporosis peptides - Integra, Telios NT-4/5 - Genentech Osteoprotegerin-Amgen, SnowBrand NU 3056 otitis media vaccines - Antex Biologics NU 3076 ovarian cancer - University of Alabama NX 1838 - Gilead Sciences OX40-lgG fusion protein - Cantab, Xenova NY ESO-1/CAG-3 antigen - NIH P246 - Diatide NYWAC-7 - Aventis Pasteur P30-AlfaCell NZ-1002-Novazyme p1025 - Active Biotech obesity therapy - Nobex P-113 - Demegen OC 10426-- Ontogen P-16 peptide-Transition Therapeutics OC 144093-- Ontogen p43 - Ramot OCIF-Sankyo P-50 peptide-Transition Therapeutics Oct-43 - Otsuka p53 + RAS vaccine - NIH, NC Odulimomab - Immunotech PACAP(1-27) analogue OKPSA - liposomal paediatric vaccines - Chiron OKT3-gamma-1-ala-ala Pafase -- ICOS OM 991 PAGE-4 plasmid DNA-IDEC OM 992 PA-2 - Biotech Australia, Human Omalizumab - Genentech Therapeutics OnCoimmunin-L-NIH Palifermin (keratinocyte growth factor)- Oncolysin B- ImmunoGen Amgen Oncolysin CD6- ImmunoGen PaliwiZumab - MedImmune Oncolysin M-immunoGen PAM 4 - Merck Oncolysin S-- ImmunoGen pamiteplase - Yamanouchi OnCophage - Antigenics pancreatin, Minitabs - Eurand OnCostatin M-Bristol-Myers Squibb Pangen - Fournier OncoVax-CL-Jenner Biotherapies Pantarin - Selective Genetics OncoVax-P-Jenner Biotherapies Parainfluenza virus vaccine - Pharmacia, Onercept -- Yeda Pierre Fabre Patent Application Publication Feb. 18, 2010 Sheet 41 of 47 US 2010/0041872 A1

FIGURE 7W paraOxanase -- Esperion peptide vaccine - NIH, NC parathyroid hormone - Abiogen, Korea Pexelizumab Green CrOSS pexiganan acetate - Genaera Parathyroid hormone (1-34)- Pharmaprojects No. 3179 - NYU Chugai/Suntory Pharmaprojects No. 3390 - Ernest Orlando Parkinson's disease gene therapy - Cell Pharmaprojects No. 3417 - Sumitomo Genesysl Ceregene Pharmaprojects No. 3777 - Acambis Parvovirus vaccine - MedImmune Pharmaprojects No. 4209-XOMA PCP-SCan - ImmunOmedics Pharmaprojects No. 4349 - Baxter Intl. PDGF-- Chiron Pharmaprojects No. 4651 PDGF Cocktail - Theratechnologies Pharmaprojects No. 4915- Avanir peanut allergy therapy - DynaVax Pharmaprojects No. 5156 - Rhizogenics PEG anti-ICAMMAb-Boehringer Pharmaprojects No. 5200 - Pfizer Ingelheim Pharmaprojects No. 5215 - Origene PEGasparaginase-Enzon Pharmaprojects No. 5216 - Origene PEG glucocerebrosidase Pharmaprojects No. 5218 - Origene PEGhirudin-Knoll Pharmaprojects No. 5267 - ML PEG interferon-alpha-2a-Roche Laboratories PEG interferon-alpha-2b + ribavirin Pharmaprojects No. 5373-MorphoSys Biogen, Enzon, ICN Pharmaceuticals, Pharmaprojects No. 5493-Metabolex Schering-Plough Pharmaprojects No. 5707 - Genentech PEG MAbA5B7 Pharmaprojects No. 5728 - Autogen Pegacaristim-Amgen - Kirin Brewery - Pharmaprojects No. 5733 - BicMarin ZymoGenetics Pharmaprojects No. 5757 - NIH Pegaldesleukin-Research Corp Pharmaprojects No. 5765- Gryphon pegaSpargase -- EnZOn Pharmaprojects No. 5830 - AntiCancer pegfilgrastim-Amgen Pharmaprojects No. 5839 - Dyax PEG-interferon Alpha-Viragen Pharmaprojects No. 5849 - Johnson & PEG-interferon Alpha2A - Hoffman La Johnson ROChe Pharmaprojects No. 5860 - Mitsubishi PEG-interferon Alpha2B-Schering Tokyo Plough Pharmaprojects No. 5869 - Oxford PEG-r-hirudin-Abbott GlycoSciences PEG-rHuMGDF-Amgen Pharmaprojects No. 5883 - Asahi Brewery PEG-uricase -- Mountain View Pharmaprojects No. 5947 - StressGen Pegvisomant-Genentech Pharmaprojects No. 5961 -- PEGylated proteins, PolyMASC- Valentis Theratechnologies PEGylated recombinant native human leptin Pharmaprojects No. 5962 - NIH - ROChe Pharmaprojects No. 5966 - NIH Pemtumomab Pharmaprojects No. 5994 - Pharming Penetratin - Cyclace Pharmaprojects No. 5995 - Pharming Pepscan-Antisoma Pharmaprojects No. 6023 - IMMUCON peptide G-Peptech, ICRT Pharmaprojects No. 6063 - Cytoclonal Patent Application Publication Feb. 18, 2010 Sheet 42 of 47 US 2010/0041872 A1

FIGURE 7W Pharmaprojects No. 6073--SIDDCO PneumoCOCCal vaccine - Antex Biologics, Pharmaprojects No. 6115- Genzyme Aventis Pasteur Pharmaprojects No. 6227-NIH Pneum0COCCal Vaccine intranasal Pharmaprojects No. 6230 - NIH BioChem VaccineS/BioVector Pharmaprojects No. 6236 - NIH PR1A3 Pharmaprojects No. 6243 - NIH PR-39 Pharmaprojects No. 6244 - NIH prailmorelin - Kaken Pharmaprojects No. 6281 - Senetek Pretarget-Lymphoma - NeoRX Pharmaprojects No. 6365- NIH Priliximab -- CentOCOr Pharmaprojects No. 6368 - NIH PRO 140 - Progenics Pharmaprojects No. 6373 - NIH PRO 2000 - Procept Pharmaprojects No. 6408-Pan Pacific PRO 367-Progenics Pharmaprojects No. 6410 - Athersys PRO 542- Progenics Pharmaprojects No. 6421 - Oxford pro-Apo A-l-Esperion GlycoSciences prolactin - Genzyme Pharmaprojects No. 6522 - Maxygen Prosaptide TX14(A) - Bio-Tech. General Pharmaprojects No. 6523-- Pharis prostate cancer antbodies - Immunex, Pharmaprojects No. 6538 - Maxygen UrOCOr Pharmaprojects No. 6554 - APALEXO prostate cancer antibody therapy - Pharmaprojects No. 6560 - Ardana Genentech/UroGenesys, Pharmaprojects No. 6562- Bayer Genotherapeutics Pharmaprojects No. 6569-Eos prostate cancer immunotherapeutics -- The Phenoxazine PSMA Development Company Phenylase-Ibex prostate cancer vaccine -- Aventis Pasteur, Pigment epithelium derived factor Zonagen, Corixa, Dendreon, Jenner plasminogen activator inhibitor-1, Biotherapies, Therion Biologics recombinant- DuPont Pharmaceuticals prostate-specific antigen - EntreMed Plasminogen activators - Abbott protein A - RepliCen Laboratories, American Home Products, protein adhesives -- Enzon Boehringer Mannheim, Chiron protein C-Baxter Intl. PPL Therapeutics, Corporation, DuPont Pharmaceuticals, Eli ZymoGenetics Lilly, Shionogi, Genentech, Genetics protein C activator - Gilead Sciences Institute, GlaxoSmithKline, HemispherX protein kinase Rantags - NIH Biopharma, Merck & Co, Novartis, protirelin-Takeda Pharmacia Corporation, Wakamoto, Yeda protocadherin 2- Caprion plasminogen-related peptides - Bio-Tech. Pro-urokinase-Abbott, Bristol-Myers General/MGH Squibb, Dainippon, Tosoh-Welfide platelet factor 4 - RepliCen P-selectinglycoprotein ligand-1 - Genetics Platelet-derived growth factor-Amgen Institute ZymoGenetics pseudomonal infections - InterMune plusonermin-Hayashibara Pseudomonas Vaccine -CytoVax PMD-2850 - ProtheriCS PSGL-g-American Home Products PSP-94 - Procyon Patent Application Publication Feb. 18, 2010 Sheet 43 of 47 US 2010/0041872 A1

FIGURE 7X PTH 1-34 - Nobex Respiratory syncytial virus vaccine Quilimmune-M-Antigenics inactivated R744 - ROChe Respiratory syncytial virus-parainfluenza R101933 virus vaccine - Aventis Pasteur, R 125224 - Sankyo Pharmacia RA therapy - Cardion Retepiase - Boehringer Mannheim, Rabies Vaccine recombinant-Aventis Hoffman La-ROche Pasteur, BioChem Vaccines, Kaketsuken Retropep - RetroScreen Pharmaceuticals RFB4 (dsFW) PE38 RadioTheraCIM -- YMBioSciences RF 641 -- American Home Products Ramot project No. 1315- Ramot RFTS - UAB Research Foundation Ramot project No. K-734A-Ramot RG 12986 -- Aventis Pasteur Ramot project No. K-734B - Ramot RG83852 -- Aventis Pasteur Ranibizumab (Anti-VEGF fragment) - RG-1059- RepliCen Genentech rGCR - NIH RANK-- Immunex rGLP-1.-- Restoragen ranpinase - Alfacell rGRF - Restoragen ranpinase-anti-CD22MAb - Alfacell rh Insulin-Eli Lilly RANTES inhibitOr - Milan RHAMM targeting peptides - Cangene RAPID drug delivery systems - ARIAD rHb 1.1 - Baxter Intl. rasburicase - Sanofi rhCC10 - Claragen rBPI-21, topical-XOMA rhCG-SerOno RC 529 - Corixa Rheumatoid arthritis gene therapy rCFTR - Genzyme Transgenics Rheumatoid arthritis vaccine - Veterans RD 62198 Affairs Medical Center rDnase - Genentech rh H -- SerOno RDP-58 - SangStat Ribozyme genetherapy - Genset RecepTox-Foe - Keryx Rickettsial vaccine recombinant RecepTox-GnRH - Keryx, MTR RIGScan CR - Neoprobe Technologies RIP-3-Rigel RecepTox-MBP-Keryx, MTR Rituximab - Genentech Technologies RK-0202 - RXKinetix recFSH-AkzoNobel, Organon RLT peptide -- Esperion REGA 3G 12 rMINE - VAX Regavirumab -- Teijin rmCRP-mmtech relaxin- Connetics Corp RN-1001 - RenOVO Renal cancer vaccine - Macropharm RN-3 - RenOVO repifermin - Human Genome Sciences RNAse conjugate -Immunomedics Respiratory syncytial virus PFP-2 vaccine RO 631908 - ROChe Wyeth-Lederle Rotavirus Vaccine - Merck Respiratory syncytial virus Vaccine RP431 - DuPont Pharmaceuticals GlaxoSmithKline, Pharmacia, Pierre Fabre RP-128 - ReSolution RPE65 gene therapy - Patent Application Publication Feb. 18, 2010 Sheet 44 of 47 US 2010/0041872 A1

FIGURE 7Y

RPR 110173 - AVentis Pasteur SERP-1 - Viron RPR 115135 - AVentis Pasteur sertenef-Dainippon RPR 116258A-AVentis Pasteur Serum albumin, Recombinant human rPSGL-lg-American Home Products Aventis Behring r-SPC surfactant-Byk Gulden Serum-derived factor - Hadasit RSV antibody - Medimmune Sevirumab - Novartis Ruplizumab -- Biogen SGN 14 - Seatle Genetics rV-HER-2/neu - Therion Biologics SGN 15 - Seatle Genetics SA 1042 - Sankyo SGN 17119 - Seatle Genetics Sacrosidase - Orphan Medical SGN 30 - Seatle Genetics Sant 7 SGN-10 - Seatle Genetics SargramOstim - ImmuneX SGN-11 - Seatle Genetics Saruplase - Gruenenthal SH 306 - DuPont Pharmaceuticals Satumomab - Cytogen ShanVaC-B-Shantha SB 1 - COR Therapeutics Shigella flexnerivaccine - Avant, Acambis, SB207448- GlaxoSmithKline NovaVax SB208651 - GlaxoSmithKline Shigella Sonneivaccine SB240683 - GlaxoSmithKline sICAM-1 - Boehringer Ingelheim SB 24.9415 - GlaxoSmithKline Siltepiase - Genzyme SB 249417 - GlaxoSmithKline SIV Vaccine - EndoCon Institut Pasteur SB 6- COR Therapeutics SK896 - Sanwa Kagaku Kenkyusho SBRA 31012 SK-827 - Sanwa Kagaku Kenkyusho SC 56929. Pharmacia SkeleteX - CellFactorS SCA binding proteins - Curis, Enzon SKF 106160 - GlaxoSmithKline scFV(14E1)-ETA Berlex Laboratories, S-nitroSO-AR545C Schering AG SNTP -- Active Biotech ScFV(FRP5)-ETA Somatomedin-1 - GroPep, Mitsubishi SCFV6C6-PE40 Tokyo, NIH SCH 55700 - Celltech Somatomedin-1 carrier protein - inSmed Schistosomiasis vaccine - GlaxO Somatostatin -Ferring Wellcome/Medeva, Brazil Somatotropin/ SCPF - Advanced Tissue Sciences Human Growth Hormone - Bio-Tech. SCuPA-suPAR complex-Hadasit General, Eli Lilly SD-9427 - Pharmacia somatropin - Bio-Tech. General, Akermes, SDF-1 - OnO ProLease, Aventis Behring, BioVector, SDZ 215918 - NOVartis Cangene, Dong-A, Eli Lilly, Emisphere, SDZ 28.0125 - NOVartis Enact, Genentech, Genzyme Transgenics, SDZ 89104 - NOVartis Grandis/InfiMed, CSL InfiMed, MacroMed, SDZABL 364 - Novartis Novartis, NOVO Nordisk, Pharmacia SDZMMA 383 - NOVartis SerOno, TranXenoGen Secretin-Ferring, Repligen somatropin derivative - Schering AG serine protease inhibs -- Pharis somatropin, AIR-Eli Lilly Sermorelin acetate -- SerOno Somatropin, inhaled -- Eli LillylAkermes Patent Application Publication Feb. 18, 2010 Sheet 45 of 47 US 2010/0041872 A1

FIGURE 7Z Somatropin, Kabi-Pharmacia T cell receptor peptide vaccine Somatropin, OraSome - Novo Nordisk T4N5 liposomes - AGI Dermatics Sonermin-Dainippon Pharmaceutical TACl, soluble -- ZymoGenetics SP(V5.2)C - Supertek targeted apoptosis - AntiSOma SPf66 tasonermin - Boehringer Ingelheim sphingomyelinase - Genzyme TASP SR 29001- Sanofi TASP-V SR 41476 - Sanofi Tat peptide analogues - NIH SR-29001 - Sanofi TBP - Yeda SS1(dsFV)-PE38-NeoPharm TBPI ?32 microglobulin -- Avidex TBV25H-NH (32-microglobulin fusion proteins - NIH TC 99m ior Cea? - Center of Molecular ?3-amyloid peptides - CeNeS Immunology ?-defensin - Pharis TC 99m P748 - Diatide StaphyloCOCCus aureus infections - Tc 99m Votumumab -- intraCell InhibiteX/ZLB TC-99m rh-Annexin V-Theseus Imaging StaphyloCOCCUS aureus Vaccine Conjugate - teceleukin-Biogen Nabi tenecteplase - Genentech StaphyloCOCCuS therapy - Tripep Teriparatide - Armour Pharmaceuticals, Staphylokinase-Biovation, Prothera, Asahi Kasei, Eli Lilly Thrombogenetics terlipressin-Ferring Streptococcal A vaccine - M6 testisin -- AMRAD Pharmaceuticals, North American WaCCine Tetrafibricin -- ROChe StreptoCOccal B vaccine - MicroScience TFP -- EntreMed Streptococcal B vaccine recombinant tgD-IL-2-Takeda Biochem VaCCines TGF-Alpha-ZymoGenetics StreptoCOCCUS pyogenes vaccine TGF--- Kolon STRL-33 - NIH TGF-2-InSmed Subalin -- SRC VB VECTOR TGF-33 - OS SUIS - United Biomedical Thalassaemia gene therapy - Crucell SUS-LHRH - United Biomedical TheraCIM-h-R3 - Center of Molecular SUN-E3001 - Suntory Immunology, YMBioSciences super high affinity monoclonal antibodies - Theradigm-HBV-Epimmune YM BioSciences Theradigm-HPV - Epimmune Superoxide dismutase - Chiron, Enzon, Theradigm-malaria - Epimmune Ube industries, Bio-Tech, Yeda Theradigm-melanoma - Epimmune SuperOxide dismutase-2 - OXIS TheraFab - Antisoma suppressin - UAB Research Foundation ThGRF1-29 - Theratechnologies SY-161-P5 - ThrombOGenics ThGRF1-44 - Theratechnologies SY-162 - ThromboGenics Thrombin receptor activating peptide Systemic lupus erythematosus vaccine Abbott MedClone/VivORX thrombomodulin-lowa, NovOcastra T cell receptor peptides-Xoma Patent Application Publication Feb. 18, 2010 Sheet 46 of 47 US 2010/0041872 A1

FIGURE 7AA Thrombopoietin-Dragon Pharmaceuticals, Transforming growth factor-beta-1- Genentech Genentech thrombopoietin, Pliva -- Receptron transport protein - Genesis Thrombospondin 2 Trastuzumab -- Genetech thrombostatin - Thromgen TRH-Ferring thymalfasin -- SciClone Triabin - Schering AG thymocartin-Gedeon Richter TriCOnal thymosin Alpha1- NIH Triflavin thyroid stimulating hormone - Genzyme troponin - Boston Life Sciences tlCAM-1 - Bayer TRP-2A. NIH Tick anticoagulant peptide - Merck trypsin inhibitor - Mochida TIF-XOma TSP-1 genetherapy TifacOgin-Chiron, NIS, Pharmacia TT-232 Tissue factor-Genentech TTS-CD2 - Active Biotech Tissue factor pathway inhibitor Tuberculosis vaccine -- Aventis Pasteur, TJN-135 - TSumura Genesis TM27 - Avant Tumor Targeted Superantigens - Active TM 29 - AWant Biotech - Pharmacia TMC-151 - Tanabe Seiyaku tumour vaccines - PhotoCure TNF tumour necrosis factor - ASahi Kasei tumour-activated prodrug antibody TNF Alpha- Cytimmune Conjugates - Millennium/ImmunoGen TNF antibody - Johnson & Johnson tumstatin-LEX TNF binding protein-Amgen TuVirumab - Novartis TNF degradation product - Oncotech TV-4710-Teva TNF receptor - Immunex TWEAK receptor - Immunex TNF receptor 1, soluble-Amgen TXU-PAP TNFTumour necrosis factor-alpha-Asahi TY-10721-TOA Elyo Kasei, Genetech, Mochida Type I diabetes vaccine - Research Corp TNF-Alpha inhibitor-Tripep Typhoid vaccine CVD 908 TNFR:Fc gene therapy - Targeted Genetics U 143677 - Pharmacia TNF-SAM2 U81749 - Pharmacia ToleriMab-Innogenetics UA 1248 - Arizona Toxoplasma gondii vaccine UGIF - Sheffield GlaxoSmithKline UIC 2 TP 9201 - Telios UK 101 TP10 - AWant UK-279276 - CorvaS Intl. TP20 -- Avant urOdilatin - Pharis tPA - CentOCOr urofolitrophin - Serono trafermin -- ScioS Urokinase - Abbott TRAILIApo2L-Immunex uteroferrin-Pepgen TRAIL-R1 MAb - Cambridge Antibody V20- GLYCODesign Technologies V2 wasopressin receptor gene therapy transferrin-binding proteins - CAMR WaCCines - Active Biotech Patent Application Publication Feb. 18, 2010 Sheet 47 of 47 US 2010/0041872 A1

FIGURE 7AB Varicella ZOster glycoprotein vaccine WT1 vaccine - Corixa Research Corporation Technologies WX-293-- Wilex BioTech. Varicella ZOster virus Vaccine live -- Cantab WX-360 - Wilex BioTech. Pharmaceuticals WX-UK1 - Wilex BioTech. Vascular endothelial growth factor XMP-500 - XOMA Genentech, University of California XomaZyme-791-XOMA Vascular endothelial growth factors - R&D XTL 001-XTLBiopharmaceuticals Systems XTL 002--XTL Biopharmaceuticals VaSCular targeting agents -- Peregrine yeast delivery system - Globelmmune VaSOpermeation enhancement agents - Yersinia pestis vaccine Peregrine YIGSR-Stealth -- Johnson & Johnson VaSOStatin - NIH Yissum Project No. D-0460-Yissum WCL - Bio-Tech. General YM 207-Yamanouchi VEGF- Genentech, Scios YM337 - Protein Design Labs VEGF inhibitor - Chugai Yttrium-90 labelled biotin VEGF-2 - Human GenOme Sciences Yttrium-90-labeled anti-CEAMAb T84.66 VEGF-Trap-Regeneron ZD 0490 - AstraZeneca visCumin, recombinant-MadauS ZicOnotide-Elan Vitaxin ZK 157138 - Berlex Laboratories Vitrase -- ISTA Pharmaceuticals Zolimomab aritoX West Nile virus Vaccine - Bavarian Nordic Zorcell - Immune Response WP 652 ZRXL peptides - Novartis US 2010/0041872 A1 Feb. 18, 2010

GLYCEROL LINKED PEGYLATED SUGARS 0004. The polymeric modifying group can be attached at AND GLYCOPEPTIDES any position of a glycosyl moiety on a peptide. Moreover, the polymeric modifying group can be bound to a glycosyl resi CROSS-REFERENCE TO RELATED due at any position in the amino acid sequence of a wild type APPLICATIONS or mutant peptide. 0001. The present application is a U.S. National Phase of 0005. In an exemplary embodiment, the invention pro PCT Application No. PCT/US2007/080471 filed Oct. 4, 2007 vides a peptide that is conjugated through a glycosyl linking and claims priority under 35 USC 119(e) to The present group to a polymeric modifying group. Exemplary peptide application claims priority to U.S. Provisional Patent Appli conjugates include a glycosyl linking group having a formula cation No. 60/828,208, filed on Oct. 4, 2006, which is incor selected from: porated herein by reference in its entirety for all purposes.

SUMMARY OF THE INVENTION (R) 0002. In an exemplary embodiment, “glycopegylated molecules of the invention are produced by the enzyme medi R6 ated formation of a conjugate between a glycosylated or O non-glycosylated peptide and an enzymatically transferable saccharyl moiety that includes a modifying group. Such as a R5 R3 polymeric modifying group Such as poly(ethylene glycol), R4 within its structure. In an exemplary embodiment, the peptide II is a member selected from bone morphogenetic proteins (e.g., (R) BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, O O BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, R6 BMP-14, BMP-15), neurotrophins (e.g., NT3, NT-4, NT-5), growth differentiation factors (e.g., GDF-5), glial cell line derived neurotrophic factor (GDNF), brain derived neu R4 R3 rotrophic factor (BDNF), nerve growth factor (NGF), von Willebrand factor (vWF) protease, Factor VII, Factor Vla, Factor VIII, Factor IX, Factor X, Factor XI, B-domaindeleted Ia Factor VIII, vWF-Factor VIII fusion protein having full length Factor VIII, VWF-Factor VIII fusion protein having R B-domain deleted Factor VIII, erythropoietin (EPO), granu O ; and locyte colony stimulating factor (G-CSF), Granulocyte-Mac rophage Colony Stimulating Factor (GM-CSF)interferon R5 R3 alpha, interferon beta, interferon gamma, C.-antitrypsin R4 (ATT, or C-1 protease inhibitor), glucocerebrosidase, Tissue Type Plasminogen Activator (TPA), Interleukin-2 (IL-2), urokinase, human DNase, insulin, Hepatitis B surface protein IIa (HbsAg), human growth hormone, TNF Receptor-IgG Fc region fusion protein (EnbrelTM), anti-HER2 monoclonal antibody (HerceptinTM), monoclonal antibody to Protein F of Respiratory Syncytial Virus (SynagisTM), monoclonal anti r/R4 R3 body to TNF-C. (RemicadeTM), monoclonal antibody to gly coprotein IIb/IIIa (ReoproTM), monoclonal antibody to CD20 (RituxanTM), anti-thrombin III (AT III), human Chorionic Gonadotropin (hCG), alpha-galactosidase (FabrazymeTM), 0006. In Formulae I, Ia, II or IIa, R is H, CHOR7, alpha-iduronidase (AldurazymeTM), follicle stimulating hor COOR, COO or OR7, in which R7 represents H, substituted mone, beta-glucosidase, anti-TNF-alpha monoclonal anti or unsubstituted alkyl or substituted or unsubstituted het body (MLB 5075), glucagon-like peptide-1 (GLP-1), gluca eroalkyl. The symbols R. R. R. Rand R' independently gon-like peptide-2 (GLP-2), beta-glucosidase, alpha comprise H, substituted or unsubstituted alkyl, OR, NHC(O) galactosidase A and fibroblast growth factor. The polymeric Rand a saccaryl moiety. The index d is 0 or 1. RandR are modifying group is attached to the saccharyl moiety (i.e., independently selected from H, substituted or unsubstituted through a single group formed by the reaction of two reactive alkyl, substituted or unsubstituted heteroalkyl, sialic acid. At groups) or through a linker moiety, e.g., Substituted or unsub least one of R. R. R. R. or R' includes the polymeric stituted alkyl, substituted or unsubstituted heteroalkyl, etc. modifying group e.g., PEG. In an exemplary embodiment, R 0003. Thus, in one aspect, the present invention provides a and R, together with the carbon to which they are attached conjugate between a PEG moiety, e.g., PEG and a peptide that are components of the side chain of a sialyl moiety. In a has an in Vivo activity similar or otherwise analogous to further exemplary embodiment, this side chain is functional art-recognized therapeutic peptide. In the conjugate of the ized with the polymeric modifying group. invention, the PEG moiety is covalently attached to the pep 0007. In an exemplary embodiment, the polymeric modi tide via an intact glycosyl linking group. Exemplary intact fying group is bound to the glycosyl linking group, generally glycosyl linking groups include sialic acid moieties that are through a heteroatom on the glycosyl core (e.g., N, O), derivatized with PEG. through a linker, L, as shown below: US 2010/0041872 A1 Feb. 18, 2010

stituent that would be found in the carbohydrate from which its name is derived, e.g., deoxy Sugars. Still further exemplary unnatural Sugars include both oxidized (e.g., -onic and (R)-L -uronic acids) and reduced (Sugar alcohols) carbohydrates. The Sugar moiety can be a mono-, oligo- or poly-saccharide. 0013 Exemplary natural sugars of use as components of glycosyl linking groups in the present invention include glu R" is the polymeric modifying group and L is selected from a cose, glucosamine, galactose, galactosamine, fucose, man bond and a linking group. The index w represents an integer nose, mannosamine, Xylanose, ribose, N-acetyl glucose, selected from 1-6, preferably 1-3 and more preferably 1-2. N-acetyl glucosamine, N-acetyl galactose, N-acetyl galac Exemplary linking groups include Substituted or unsubsti tosamine, and Sialic acid. tuted alkyl, substituted or unsubstituted heteroalkyl moieties 0014. In one embodiment, the present invention provides a and sialic acid. An exemplary component of the linker is an peptide conjugate comprising the moiety: acyl moiety. Another exemplary linking group is an amino acid residue (e.g., cysteine, serine, lysine, and short oligopep tides, e.g., Lys-Lys, Lys-Lys-Lys, Cys-Lys, Ser-Lys, etc.). OH 0008. When L is a bond, it is formed by reaction of a reactive functional group on a precursor of R and a reactive O COOH functional group of complementary reactivity on a precursor HO of the glycosyl linking group. When L is a non-zero order linking group, L can be in place on the glycosyl moiety prior to reaction with the R" precursor. Alternatively, the precursors G-HN of R' and L can be incorporated into a preformed cassette that OH is Subsequently attached to the glycosyl moiety. As set forth herein, the selection and preparation of precursors with appropriate reactive functional groups is within the ability of wherein D is a member selected from OHand R'-L-HN : those skilled in the art. Moreover, coupling of the precursors G is a member selected from H and R-L- and —C(O)(C- proceeds by chemistry that is well understood in the art. C.)alkyl, R' is a moiety comprising a straight-chain or 0009. In an exemplary embodiment L is a linking group branched poly(ethylene glycol) residue; and L is a linker, e.g., that is formed from an amino acid, or Small peptide (e.g., 1-4 a bond (“Zero order'), substituted or unsubstituted alkyl and amino acid residues) providing a modified Sugar in which the substituted or unsubstituted heteroalkyl. In exemplary polymeric modifying moiety is attached through a substituted embodiments, when D is OH, G is R'-L-, and when G is alkyl linker. Exemplary linkers include glycine, lysine, serine —C(O)(C-C)alkyl, D is R'-L-NH-. and cysteine. Amino acid analogs, as defined herein, are also 0015. In another aspect, the invention provides a peptide ofuse as linker components. The amino acid may be modified conjugate comprising a glycosyl linking group, wherein the with an additional component of a linker, e.g., alkyl, het glycosyl linking group is attached to an amino acid residue of eroalkyl, covalently attached through an acyl linkage, for said peptide, and wherein said glycosyl linking group com example, an amide or urethane formed through an amine prises a sialyl linking group having a formula which is a moiety of the amino acid residue. member selected from: 0010. In an exemplary embodiment, the glycosyl linking group has a structure according to Formulae I or Ia and R' includes the polymeric modifying group. In another exem plary embodiment, Rincludes both the polymeric modifying group and a linker, L. joining the polymeric modifying group to the glycosyl core. L. can be a linear or branched structure. Similarly, the polymeric modifying group can be branched or linear. 0011. The polymeric modifying group comprises two or more repeating units that can be water-soluble or essentially insoluble in water. Exemplary water-soluble polymers of use in the compounds of the invention include PEG, e.g., m-PEG, PPG, e.g., m-PPG, polysialic acid, polyglutamate, polyaspar tate, polylysine, polyethyeleneimine, biodegradable poly CAA mers (e.g., polylactide, polyglyceride), and functionalized (CAA), OH PEG, e.g., terminal-functionalized PEG. 0012. The glycosyl core of the glycosyl linking groups of use in the peptide conjugates are selected from both natural Accor-HA(CAA) and unnatural furanoses and pyranoses. The unnatural sac charides optionally include an alkylated or acylated hydroxyl and/or amine moiety, e.g., ethers, esters and amide Substitu ents on the ring. Other unnatural saccharides include an H. hydroxyl, ether, ester oramide Substituent at a position on the ring at which Such a substituent is not present in the natural saccharide. Alternatively, the carbohydrate is missing a Sub US 2010/0041872 A1 Feb. 18, 2010

0017. In an exemplary embodiment, the polymeric modi -continued fying group has a structure including a moiety according to R16-X2 the following formulae:

(OCH2CH2)A CH are modifying groups. R is a member selected from H. CHOR, COOR, COO and OR'R' is a member selected A’(CH2CH2O) H from H. Substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl. R and Rare members indepen O O dently selected from H. Substituted or unsubstituted alkyl, OR, and NHC(O)R. Rand Rare independently selected r from H, substituted or unsubstituted alkyl, substituted or i-C,H - unsubstituted heteroalkyl and sialyl. La is a linker selected from a bond, substituted or unsubstituted alkyl and substi tuted or unsubstituted heteroalkyl. X, R'' and R'' are inde (OCH2CH2)A pendently selected from non-reactive group and polymeric moieties (e.g. poly(alkylene oxide), e.g., PEG). Non-reactive CH groups include groups that are considered to be essentially A’(CH2CH2O). H unreactive, neutral and/or stable at physiological pH, e.g., H. substituted or unsubstituted alkyl, substituted or unsubsti tuted heteroalkyl and the like. An exemplary polymeric moi ety includes the branched structures set forth in Formula IIIa and its exemplars, below. One of skill in the art will appreciate Ox that the PEG moiety in these formulae can be replaced with other polymers. Exemplary polymers include those of the poly(alkylene oxide) family. X and X’ are independently 0018. In another exemplary embodiment according to the selected linkage fragments joining polymeric moieties R' formula above, the polymeric modifying group has a struc and R7 to C. The index j is an integer selected from 1 to 15. ture according to the following formula: 0016. In another exemplary embodiment, the polymeric modifying group has a structure according to the following H. H. formula: (OCH2CH2)A

A’(CH2CH2O) H (IIIa) (OCH2CH2)A H La CA3 A4 H (CAA), A’(CH2CH2O)m A7 0019. In an exemplary embodiment, m and n are integers independently selected from about 1 to about 5000, prefer (CAA9) ably from about 100 to about 4000, more preferably from CA10All about 200 to about 3000, even more preferably from about 300 to about 2000 and still more preferably from about 400 to La about 1000. In an exemplary embodiment, m and n are inte gers independently selected from about 1 to about 500. In an exemplary embodiment, m and n are integers independently selected from about 1 to about 70, about 70 to about 150, in which the indices m and n are integers independently about 150 to about 250, about 250 to about 375 and about 375 selected from 0 to 5000.A.A.A.A.A.A.A.A.A.A.' to about 500. In an exemplary embodiment, m and n are and A' are members independently selected from H, substi integers independently selected from about 10 to about 35, tuted or unsubstituted alkyl, substituted or unsubstituted het about 45 to about 65, about 95 to about 130, about 210 to eroalkyl, substituted or unsubstituted cycloalkyl, substituted about 240, about 310 to about 370 and about 420 to about 480. or unsubstituted heterocycloalkyl, substituted or unsubsti In an exemplary embodiment, m and n are integers selected tuted aryl, substituted or unsubstituted heteroaryl, from about 15 to about 30. In an exemplary embodiment, m - NA'A'. - OA' and SiA'A'. A' and A'are mem and n are integers selected from about 50 to about 65. In an bers independently selected from substituted or unsubstituted exemplary embodiment, m and n are integers selected from alkyl, substituted or unsubstituted heteroalkyl, substituted or about 100 to about 130. In an exemplary embodiment, mand unsubstituted cycloalkyl, substituted or unsubstituted hetero in are integers selected from about 210 to about 240. In an cycloalkyl, substituted or unsubstituted aryl, and substituted exemplary embodiment, m and n are integers selected from or unsubstituted heteroaryl. about 310 to about 370. In an exemplary embodiment, mand US 2010/0041872 A1 Feb. 18, 2010

in are integers selected from about 430 to about 470. In an 0022. In an exemplary embodiment, the invention pro exemplary embodiment, A' and A are each members vides a modified Sugar having the following formula: selected from —OH and —OCH. 0020 Exemplary polymeric modifying groups according OH to this embodiment include the moiety: OH

H. H. O R2 || OH (OCH2CH2)OCH3 (R)-L-NHly - T - R 3 N-(

wherein R' is the polymeric moiety; L is selected from a bond and a linking group; R is a member selected from H. HN and CHOR7, COOR7 and OR7: R7 is a member selected from H, substituted or unsubstituted alkyl and substituted or unsub stituted heteroalkyl; R and R are members independently selected from H, substituted or unsubstituted alkyl, OR and NHC(O)R’; and R' and Rare independently selected from H, substituted or unsubstituted alkyl, substituted or unsubsti H. H. tuted heteroalkyl, sialic acid and polysialic acid. The index w (OCH2CH2)OCH represents an integer selected from 1-6, preferably 1-3 and more preferably 1-2. Exemplary linking groups include Sub CHO(CH2CH2O). H stituted or unsubstituted alkyl, substituted or unsubstituted O heteroalkyl moieties and sialic acid. An exemplary compo H nent of the linker is an acyl moiety. H O 0023 The present invention provides methods of forming conjugates of peptides. The methods include contacting a N peptide with a modified Sugar donor that bears a modifying H group covalently attached to a Sugar. The modified Sugar moiety is transferred from the donor onto an amino acid or glycosyl residue of the peptide by the action of an enzyme. 0021. The invention provides a peptide conjugate com Representative enzymes include, but are not limited to, gly prising a glycosyl linking group, wherein the glycosyl linking cosyltransferases, e.g., sialyltransferases. The method includes contacting the peptide with: a) a modified Sugar group is attached to an amino acid residue of the peptide, and donor, and b) an enzyme capable of transferring a modified wherein the glycosyl linking group comprises a sialyl linking Sugar moiety from the modified Sugar donor onto an amino group having the formula: acid or glycosyl residue of the peptide, under conditions appropriate to transfer a modified Sugar moiety from the donor to an amino acid or glycosyl residue of the peptide, HO thereby synthesizing said peptide conjugate. OH 0024. In a preferred embodiment, prior to step a), the R. O peptide is contacted with a sialidase, thereby removing at OH least a portion of the Sialic acid on the peptide. O O 0025. In another preferred embodiment, the peptide is R3 NH l O and contacted with a sialidase, a glycosyltransferase and a modi R4 N: o1a-k N-1No fied Sugar donor. In this embodiment, the peptide is in contact O with the sialidase, glycosyltransferase and modified Sugar donor essentially simultaneously, no matter the order of addi tion of the various components. The reaction is carried out under conditions appropriate for the sialidase to remove a sialic acid residue from the peptide; and the glycosyltrans ferase to transfer a modified sugar moiety from the modified Sugar donor to an amino acid or glycosyl residue of the ^n ----- peptide. 0026. In another preferred embodiment, the desialylation and conjugation are performed in the same vessel, and the is a modifying group. The index S is an integer selected from desialylated peptide is preferably not purified prior to the 1 to 20. The index fis an integer selected from 1 to 2500. Q is conjugation step. In another exemplary embodiment, the a member selected from H and substituted or unsubstituted method further comprises a ‘capping step involving Sialyla C-C alkyl. tion of the peptide conjugate. This step is performed in the US 2010/0041872 A1 Feb. 18, 2010

same reaction vessel that contains the sialidase, sialyltrans wherein a is an integer from 0 to 10, with a modified sugar ferase and modified Sugar donor without prior purification. having the formula: 0027. In another preferred embodiment, the desialylation of the peptide is performed, and the asialo peptide is purified. The purified asialo peptide is then Subjected to conjugation OH OH reaction conditions. In another exemplary embodiment, the OH method further comprises a ‘capping step involving Sialyla OH O tion of the peptide conjugate. This step is performed in the O R2 || OH same reaction vessel that contains the sialidase, sialyltrans o1 P-O ferase and modified Sugar donor without prior purification. 3 O O O 0028. In another exemplary embodiment, the capping (R)-L-NH R N ( step, sialylation of the peptide conjugate, is performed in the R 4 N same reaction vessel that contains the sialidase, sialyltrans \ / ferase and modified Sugar donor without prior purification. 0029. In an exemplary embodiment, the contacting is for a time less than 20 hours, preferably less than 16 hours, more preferably less than 12 hours, even more preferably less than and an appropriate transferase which transfers the glysocyl 8 hours, and still more preferably less than 4 hours. linking group onto a member selected from the GalNAc, Gal 0030. In a further aspect, the present invention provides a and the Sia of said glycosyl group, under conditions appro peptide conjugate reaction mixture. The reaction mixture priate for said transfer. An exemplary modified sugar is CMP comprises: a) a sialidase; b) an enzyme which is a member sialic acid modified, through a linker moiety, with a polymer, selected from glycosyltransferase, exoglycosidase and e.g., a straight chain or branched poly(ethylene glycol) moi endoglycosidase; c) a modified Sugar, and d) a peptide. ety. The radicals in the formula above are substantially the 0031. In another exemplary embodiment, the ratio of the same identity as those found in the identical formula herein sialidase to the peptide is selected from 0.1 U/L:2 mg/mL to above. 10 U/L:1 mg/mL, preferably 0.5U/L:2 mg/mL, more prefer 0037. The peptide can be acquired from essentially any ably 1.0 U/L:2 mg/mL, even more preferably 10 U/L:2 Source, however, in one embodiment, prior to being modified mg/mL, still more preferably 0.1 U/L: 1 mg/mL, more pref as discussed above, the peptide is expressed in a Suitable host. erably 0.5 U/L: 1 mg/mL, even more preferably 1.0 U/L: 1 Mammalian (e.g., BHK, CHO), bacteria (e.g., E. coli) and mg/mL, and still more preferably 10 U/L: 1 mg/mL. insect cells (e.g., Sf-9) are exemplary expression systems 0032. In an exemplary embodiment, at least 10%, 20%, providing a peptide of use in the compositions and methods 30%, 40%, 50%, 60%, 70% or 80% of said peptide conjugate set forth herein. includes at most two PEG moieties. The PEG moieties can be 0038. Other objects and advantages of the invention will added in a one-pot process, or they can be added after the be apparent to those of skill in the art from the detailed asialo is purified. description that follows. 0033. In another exemplary embodiment, at least 10%, DESCRIPTION OF THE DRAWINGS 20%, 30%, 40%, 50%, 60%, 70% or 80% of the peptide conjugate include at most one PEG moiety. The PEG moiety 0039 FIG. 1 illustrates the preparation of CMP-sialic can be added in a one-pot process, or it can be added after the acid-Glycerol PEG 40 kD. asialo peptide is purified. 0040 FIG. 2 illustrates reaction conditions for the prepa ration of CMP-sialic acid-Glycerol PEG 40 kD. 0034. In a further exemplary embodiment, the method 004.1 FIG. 3 illustrates the purification process for CMP further comprises “capping, or adding sialic acid to the sialic acid-Glycerol PEG 40 kD. peptide conjugate. In another exemplary embodiment, siali 0042 FIG. 4 illustrates the purification process involving dase is added, followed by a delay of 30 min, 1 hour, 1.5 Q-Sepharose for CMP-sialic acid-Glycerol PEG 40 kD. hours, or 2 hours, followed by the addition of the glycosyl 0043 FIG. 5 is an "H NMR spectra of CMP-sialic acid transferase, exoglycosidase, or endoglycosidase. Glycerol PEG 40 kD. 0035. In another exemplary embodiment, sialidase is 0044 FIG. 6 is a table providing exemplary sialyltrans added, followed by a delay of 30 min, 1 hour, 1.5 hours, or 2 ferases of use in forming the glycoconjugates of the inven hours, followed by the addition of the glycosyltransfase, tion, e.g., to glycoPEGylate peptides with a modified sialic exoglycosidase, or endoglycosidase. Other objects and acid. advantages of the invention will be apparent to those of skill 004.5 FIG. 7 is a table of the peptides to which one or more in the art from the detailed description that follows. glycosyl linking groups can be attached to order to provide 0036. In another exemplary embodiment, the method the peptide conjugates of the invention. includes: (a) contacting a peptide comprising a glycosyl group selected from: DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTS Abbreviations 0046 PEG, poly(ethyleneglycol); PPG, poly(propyleneg lycol); Ara, arabinosyl; Fru, fructosyl: Fuc, fucosyl; Gal, as and as galactosyl; GalNAc, N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc, N-acetylglucosaminyl; Man, mannosyl, Man Ac, mannosaminyl acetate; Xyl, Xylosyl; NeuAc, sialyl or US 2010/0041872 A1 Feb. 18, 2010

N-acetylneuraminyl; Sia, sialyl or N-acetylneuraminyl; and are not gene-encoded may also be used in the present inven derivatives and analogues thereof. tion. Furthermore, amino acids that have been modified to include reactive groups, glycosylation sites, polymers, thera Definitions peutic moieties, biomolecules and the like may also be used in 0047 Unless defined otherwise, all technical and scien the invention. All of the amino acids used in the present tific terms used herein generally have the same meaning as invention may be either the D- or L-isomer. The L-isomer is commonly understood by one of ordinary skill in the art to generally preferred. In addition, other peptidomimetics are which this invention belongs. Generally, the nomenclature also useful in the present invention. As used herein, "peptide' used herein and the laboratory procedures in cell culture, refers to both glycosylated and unglycosylated peptides. Also molecular genetics, organic chemistry and nucleic acid chem included are peptides that are incompletely glycosylated by a istry and hybridization are those well known and commonly system that expresses the peptide. For a general review, see, employed in the art. Standard techniques are used for nucleic Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO acid and peptide synthesis. The techniques and procedures ACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel are generally performed according to conventional methods Dekker, New York, p. 267 (1983). A listing of some of the in the art and various general references (see generally, Sam peptides of the invention is provided in FIG. 7. brooketal. MOLECULAR CLONING: A LABORATORY MANUAL, 0.052 The term "peptide conjugate.” refers to species of 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold the invention in which a peptide is conjugated with a modified Spring Harbor, N.Y., which is incorporated herein by refer Sugar as set forth herein. ence), which are provided throughout this document. The 0053. The term “amino acid refers to naturally occurring nomenclature used herein and the laboratory procedures in and synthetic amino acids, as well as amino acid analogs and analytical chemistry, and organic synthetic described below amino acid mimetics that function in a manner similar to the are those well known and commonly employed in the art. naturally occurring amino acids. Naturally occurring amino Standard techniques, or modifications thereof, are used for acids are those encoded by the genetic code, as well as those chemical syntheses and chemical analyses. amino acids that are later modified, e.g., hydroxyproline, 0048 All oligosaccharides described herein are described Y-carboxyglutamate, and O-phosphoserine. Amino acid ana with the name or abbreviation for the non-reducing saccha logs refers to compounds that have the same basic chemical ride (i.e., Gal), followed by the configuration of the glycosidic structure as a naturally occurring amino acid, i.e., an a carbon bond (C. or B), the ring bond (1 or 2), the ring position of the that is bound to a hydrogen, a carboxyl group, an amino reducing saccharide involved in the bond (2, 3, 4, 6 or 8), and group, and an R group, e.g., homoserine, norleucine, then the name or abbreviation of the reducing saccharide (i.e., methionine sulfoxide, methionine methyl sulfonium. Such GlcNAc). Each saccharide is preferably a pyranose. For a analogs have modified R groups (e.g., norleucine) or modi review of Standard glycobiology nomenclature, see, Essen fied peptide backbones, but retain the same basic chemical tials of Glycobiology Varki et al. eds. CSHL Press (1999). structure as a naturally occurring amino acid. Amino acid 0049. Oligosaccharides are considered to have a reducing mimetics refers to chemical compounds that have a structure end and a non-reducing end, whether or not the saccharide at that is different from the general chemical structure of an the reducing end is in fact a reducing Sugar. In accordance amino acid, but that functionina manner similar to a naturally with accepted nomenclature, oligosaccharides are depicted occurring amino acid. herein with the non-reducing end on the left and the reducing 0054 As used herein, the term “modified sugar,” or end on the right. "modified Sugar residue', refers to a naturally- or non-natu 0050. The term “sialic acid” or “sialyl refers to any mem rally-occurring carbohydrate that is enzymatically added ber of a family of nine-carbon carboxylated Sugars. The most onto an amino acid or a glycosyl residue of a peptide in a common member of the sialic acid family is N-acetyl process of the invention. The modified sugar is selected from neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glyc enzyme Substrates including, but not limited to Sugar nucle ero-D-galactononulopyranos-1-onic acid (often abbreviated otides (mono-, di-, and tri-phosphates), activated Sugars (e.g., as Neu5Ac, NeuAc, or NANA). A second member of the glycosyl halides, glycosyl mesylates) and Sugars that are nei family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in ther activated nor nucleotides. The “modified sugar is which the N-acetyl group of NeuAc is hydroxylated. A third covalently functionalized with a “modifying group. Useful sialic acid family member is 2-keto-3-deoxy-nonuloSonic modifying groups include, but are not limited to, PEG moi acid (KDN) (Nadano et al. (1986).J. Biol. Chem. 261: 11550 eties, therapeutic moieties, diagnostic moieties, biomolecules 11557; Kanamori et al., J. Biol. Chem. 265: 21811-21819 and the like. The modifying group is preferably not a naturally (1990)). Also included are 9-substituted sialic acids such as a occurring, oran unmodified carbohydrate. The locus of func 9-O C-C acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O- tionalization with the modifying group is selected Such that it acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9- does not prevent the “modified sugar from being added deoxy-Neu5Ac. For review of the sialic acid family, see, e.g., enzymatically to a peptide. Varki, Glycobiology 2: 25-40 (1992); Sialic Acids: Chemis 0055 As used herein, the term “polymeric moiety” refers try, Metabolism and Function, R. Schauer, Ed. (Springer to a water-soluble or water-insoluble polymer. The term Verlag, New York (1992)). The synthesis and use ofsialic acid “water-soluble' refers to moieties that have some detectable compounds in a sialylation procedure is disclosed in interna degree of solubility in water. Methods to detect and/or quan tional application WO92/16640, published Oct. 1, 1992. tify water solubility are well known in the art. Exemplary 0051. “Peptide” refers to a polymer in which the mono water-soluble polymers include peptides, saccharides, poly mers are amino acids and are joined together through amide (ethers), poly(amines), poly(carboxylic acids) and the like. bonds, alternatively referred to as a polypeptide. Addition Peptides can have mixed sequences of be composed of a ally, unnatural amino acids, for example, B-alanine, phenylg single amino acid, e.g., poly(lysine). An exemplary polysac lycine and homoarginine are also included. Amino acids that charide is poly(sialic acid). An exemplary poly(ether) is poly US 2010/0041872 A1 Feb. 18, 2010

(ethylene glycol). Poly(ethylene imine) is an exemplary No. 5,932.462, which is incorporated by reference herein in polyamine, and poly(acrylic) acid is a representative poly its entirety, can also be used as the polymer backbone. In an (carboxylic acid). Preferred water-soluble polymers are exemplary embodiment, the branched polymer is itself essentially non-fluorescent, or emit such a minimal amount of attached to a branching moiety (e.g., cysteine, serine, lysine, fluorescence that they are inappropriate for use as a fluores and oligomers of lysine). cent marker in an assay. Polymers that are not naturally occur 0.058 Many other polymers are also suitable for the inven ring Sugars may be used. In addition, the use of an otherwise tion. Polymer backbones that are non-peptidic and water naturally occurring Sugar that is modified by covalent attach ment of another entity (e.g., poly(ethylene glycol), poly(pro soluble, within about 2 to about 300 loci for attachment, are pylene glycol), poly(aspartate), biomolecule, therapeutic particularly useful in the invention. Examples of suitable moiety, diagnostic moiety, etc.) is also contemplated. The polymers include, but are not limited to, other poly(alkylene term water-soluble polymer also encompasses species Such glycols). Such as poly(propylene glycol) (“PPG'), copoly as saccharides (e.g., dextran, amylose, hyalouronic acid, poly mers of ethylene glycol and propylene glycol and the like, (sialic acid), heparans, heparins, etc.); poly (amino acids), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vi e.g., poly(glutamic acid); nucleic acids; synthetic polymers nylpyrrolidone), poly(hydroxypropylmethacrylamide), poly (e.g., poly(acrylic acid), poly(ethers), e.g., poly(ethylene gly (C.-hydroxy acid), poly(vinyl alcohol), polyphosphaZene, col); peptides, proteins, and the like. Representative water polyoxazoline, poly(N-acryloylmorpholine), such as insoluble polymers include, but are not limited to, polyphos described in U.S. Pat. No. 5,629,384, which is incorporated phazines, poly(vinyl alcohols), polyamides, polycarbonates, by reference herein in its entirety, and copolymers, terpoly polyalkylenes, polyacrylamides, polyalkylene glycols, poly mers, and mixtures thereof. Although the molecular weight of alkylene oxides, polyalkylene terephthalates, polyvinyl each chain of the polymer backbone can vary, it is typically in ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrroli the range of from about 100 Da to about 100,000 Da, often done, polyglycolides, polysiloxanes, polyurethanes, poly(m- from about 6,000 Da to about 80,000 Da. ethyl methacrylate), poly(ethyl methacrylate), poly(butyl 0059. The “area under the curve' or “AUC”, as used herein methacrylate), poly(isobutyl methacrylate), poly(hexyl in the context of administering a peptide drug to a patient, is methacrylate), poly(isodecyl methacrylate), poly(lauryl defined as total area under the curve that describes the con methacrylate), poly(phenyl methacrylate), poly(methyl acry centration of drug in Systemic circulation in the patient as a late), poly(isopropyl acrylate), poly(isobutyl acrylate), poly function of time from Zero to infinity. (octadecyl acrylate) polyethylene, polypropylene, poly(eth 0060. The term “half-life' or “t/3', as used herein in the ylene glycol), poly(ethylene oxide), poly (ethylene context of administering a peptide drug to a patient, is defined terephthalate), poly(vinyl acetate), polyvinyl chloride, poly as the time required for plasma concentration of a drug in a styrene, polyvinyl pyrrolidone, pluronics and polyvinylphe patient to be reduced by one half. There may be more than one nol and copolymers thereof. In addition, the use of an other half-life associated with the peptide drug depending on mul wise naturally occurring Sugar that is modified by covalent tiple clearance mechanisms, redistribution, and other mecha attachment of another entity (e.g., poly(ethylene glycol), poly nisms well known in the art. Usually, alpha and beta half-lives (propylene glycol), poly(aspartate), biomolecule, therapeutic are defined Such that the alpha phase is associated with redis moiety, diagnostic moiety, etc.) is also contemplated. Addi tribution, and the beta phase is associated with clearance. tional examples of water-soluble and water-insoluble poly However, with protein drugs that are, for the most part, con mers are described in the application. fined to the bloodstream, there can be at least two clearance 0056. The polymer backbone of the water-soluble poly half-lives. For some glycosylated peptides, rapid beta phase mer can be poly(ethylene glycol) (i.e. PEG). However, it clearance may be mediated via receptors on macrophages, or should be understood that other related polymers are also endothelial cells that recognize terminal galactose, N-acetyl suitable for use in the practice of this invention and that the galactosamine, N-acetylglucosamine, mannose, or fucose. use of the term PEG or poly(ethylene glycol) is intended to be Slower beta phase clearance may occur via renal glomerular inclusive and not exclusive in this respect. The term PEG filtration for molecules with an effective radius <2 nm (ap includes poly(ethylene glycol) in any of its forms, including proximately 68 kD) and/or specific or non-specific uptake alkoxy PEG, difunctional PEG, multiarmed PEG, forked and metabolism in tissues. GlycoPEGylation may cap termi PEG, branched PEG, pendent PEG (i.e. PEG or related poly nal Sugars (e.g., galactose or N-acetylgalactosamine) and mers having one or more functional groups pendent to the thereby block rapid alpha phase clearance via receptors that polymer backbone), or PEG with degradable linkages recognize these Sugars. It may also confer a larger effective therein. radius and thereby decrease the volume of distribution and 0057 The polymer can be linear or branched. Branched tissue uptake, thereby prolonging the late beta phase. Thus, polymers are generally known in the art. Typically, a the precise impact of glycoPEGylation on alpha phase and branched polymer has a central branch core moiety and a beta phase half-lives may vary depending upon the size, state plurality of linear or branched polymer chains linked to the of glycosylation, and other parameters, as is well known in central branch core. PEG is commonly used in branched the art. Further explanation of “half-life' is found in Pharma forms that can be prepared by addition of ethylene oxide to ceutical Biotechnology (1997, DFA Crommelin and RD Sin various polyols, such as glycerol, pentaerythritol and Sorbitol. delar, eds., Harwood Publishers, Amsterdam, pp 101-120). The central branch moiety can also be derived from several 0061 The term “glycoconjugation,” as used herein, refers amino acids. Such as lysine. The branched poly(ethylene gly to the enzymatically mediated conjugation of a modified col) can be represented in general form as R( PEG-OH), in Sugar species to an amino acid or glycosyl residue of a which R represents the core moiety, Such as glycerol or pen polypeptide, e.g., a G-CSF peptide of the present invention. A taerythritol, and m represents the number of arms. Multi Subgenus of glycoconjugation' is 'glyco-PEGylation in armed PEG molecules, such as those described in U.S. Pat. which the modifying group of the modified Sugar is poly US 2010/0041872 A1 Feb. 18, 2010

(ethylene glycol), and alkyl derivative (e.g., m-PEG) or reac 0067. As used herein, “pharmaceutically acceptable car tive derivative (e.g., H.N-PEG, HOOC-PEG) thereof. rier' includes any material, which when combined with the 0062. The terms “large-scale” and “industrial-scale” are conjugate retains the conjugates activity and is non-reactive used interchangeably and refer to a reaction cycle that pro with the Subject's immune systems. Examples include, but duces at least about 250 mg, preferably at least about 500 mg. are not limited to, any of the standard pharmaceutical carriers and more preferably at least about 1 gram of glycoconjugate Such as a phosphate buffered saline Solution, water, emulsions at the completion of a single reaction cycle. Such as oil/water emulsion, and various types of wetting 0063. The term, “glycosyl linking group, as used herein agents. Other carriers may also include Sterile solutions, tab refers to a glycosyl residue to which a modifying group (e.g., lets including coated tablets and capsules. Typically Such PEG moiety, therapeutic moiety, biomolecule) is covalently carriers contain excipients such as starch, milk, Sugar, certain attached; the glycosyl linking group joins the modifying types of clay, gelatin, Stearic acid or salts thereof, magnesium group to the remainder of the conjugate. In the methods of the or calcium Stearate, talc, vegetable fats or oils, gums, glycols, invention, the 'glycosyl linking group' becomes covalently or other known excipients. Such carriers may also include attached to a glycosylated or unglycosylated peptide, thereby flavor and color additives or other ingredients. Compositions linking the agent to an amino acid and/or glycosyl residue on comprising Such carriers are formulated by well known con the peptide. A glycosyl linking group' is generally derived ventional methods. from a “modified sugar by the enzymatic attachment of the 0068. As used herein, “administering, means oral admin "modified Sugar to an amino acid and/or glycosyl residue of istration, administration as a Suppository, topical contact, the peptide. The glycosyl linking group can be a saccharide intravenous, intraperitoneal, intramuscular, intralesional, derived structure that is degraded during formation of modi intranasal or subcutaneous administration, or the implanta fying group-modified Sugar cassette (e.g., oxidation->Schiff tion of a slow-release device e.g., a mini-osmotic pump, to the base formation->reduction), or the glycosyl linking group Subject. Administration is by any route including parenteral, may be intact. An "intact glycosyl linking group' refers to a and transmucosal (e.g., oral, nasal, vaginal, rectal, or trans linking group that is derived from a glycosyl moiety in which dermal). Parenteral administration includes, e.g., intrave the Saccharide monomer that links the modifying group and nous, intramuscular, intra-arteriole, intradermal, Subcutane to the remainder of the conjugate is not degraded, e.g., oxi ous, intraperitoneal, intraventricular, and intracranial. dized, e.g., by Sodium metaperiodate. “Intact glycosyl linking Moreover, where injection is to treat a tumor, e.g., induce groups of the invention may be derived from a naturally apoptosis, administration may be directly to the tumor and/or occurring oligosaccharide by addition of glycosyl unit(s) or into tissues surrounding the tumor. Other modes of delivery removal of one or more glycosyl unit from a parent saccharide include, but are not limited to, the use of liposomal formula Structure. tions, intravenous infusion, transdermal patches, etc. 0064. The term, “non-glycosidic modifying group', as 0069. The term “ameliorating” or “ameliorate” refers to used herein, refers to modifying groups which do not include any indicia of Success in the treatment of a pathology or a naturally occurring Sugar linked directly to the glycosyl condition, including any objective or subjective parameter linking group. Such as abatement, remission or diminishing of symptoms or 0065. The term “targeting moiety, as used herein, refers an improvement in a patient's physical or mental well-being. to species that will selectively localize in a particular tissue or Amelioration of symptoms can be based on objective or Sub region of the body. The localization is mediated by specific jective parameters; including the results of a physical exami recognition of molecular determinants, molecular size of the nation and/or a psychiatric evaluation. targeting agent or conjugate, ionic interactions, hydrophobic (0070. The term “therapy” refers to “treating” or “treat interactions and the like. Other mechanisms of targeting an ment of a disease or condition including preventing the agent to a particular tissue or region are known to those of disease or condition from occurring in an animal that may be skill in the art. Exemplary targeting moieties include antibod predisposed to the disease but does not yet experience or ies, antibody fragments, transferrin, HS-glycoprotein, coagu exhibit symptoms of the disease (prophylactic treatment), lation factors, serum proteins, B-glycoprotein, G-CSF, GM inhibiting the disease (slowing or arresting its development), CSF, M-CSF, EPO and the like. providing relief from the symptoms or side-effects of the 0066. As used herein, “therapeutic moiety” means any disease (including palliative treatment), and relieving the dis agent useful for therapy including, but not limited to, antibi ease (causing regression of the disease). otics, anti-inflammatory agents, anti-tumor drugs, cytotox (0071. The term “effective amount’ or “an amount effec ins, and radioactive agents. “Therapeutic moiety' includes tive to” or a “therapeutically effective amount’ or any gram prodrugs of bioactive agents, constructs in which more than matically equivalent term means the amount that, when one therapeutic moiety is bound to a carrier, e.g., multivalent administered to an animal for treating a disease, is sufficient agents. Therapeutic moiety also includes proteins and con to effect treatment for that disease. structs that include proteins. Exemplary proteins include, but 0072 The term "isolated’ refers to a material that is sub are not limited to, Granulocyte Colony Stimulating Factor stantially or essentially free from components, which are used (GCSF), Granulocyte Macrophage Colony Stimulating Fac to produce the material. For peptide conjugates of the inven tor (GMCSF), Interferon (e.g., Interferon-C., -f, -y), Interleu tion, the term "isolated’ refers to material that is substantially kin (e.g., Interleukin II), serum proteins (e.g., Factors VII, or essentially free from components which normally accom VIa, VIII, IX, and X), Human Chorionic Gonadotropin pany the material in the mixture used to prepare the peptide (HCG). Follicle Stimulating Hormone (FSH) and Lutenizing conjugate. “Isolated and “pure' are used interchangeably. Hormone (LH) and antibody fusion proteins (e.g. Tumor Typically, isolated peptide conjugates of the invention have a Necrosis Factor Receptor ((TNFR)/Fc domain fusion pro level of purity preferably expressed as a range. The lower end tein)). of the range of purity for the peptide conjugates is about 60%, US 2010/0041872 A1 Feb. 18, 2010

about 70% or about 80% and the upper end of the range of (0079. The term “substantially” in the above definitions of purity is about 70%, about 80%, about 90% or more than “substantially uniform' generally means at least about 40%, about 90%. at least about 70%, at least about 80%, or more preferably at 0073. When the peptide conjugates are more than about least about 90%, and still more preferably at least about 95% 90% pure, their purities are also preferably expressed as a of the acceptor moieties for a particular glycosyltransferase range. The lower end of the range of purity is about 90%, are glycosylated. about 92%, about 94%, about 96% or about 98%. The upper 0080 Where substituent groups are specified by their con end of the range of purity is about 92%, about 94%, about ventional chemical formulae, written from left to right, they 96%, about 98% or about 100% purity. equally encompass the chemically identical Substituents, 0074 Purity is determined by any art-recognized method which would result from writing the structure from right to of analysis (e.g., band intensity on a silver stained gel, poly left, e.g., -CHO— is intended to also recite —OCH2—. acrylamide gel electrophoresis, HPLC, or a similar means). I0081. The term “alkyl,” by itself or as part of another 0075 “Essentially each member of the population, as Substituent means, unless otherwise stated, a straight or used herein, describes a characteristic of a population of branched chain, or cyclic hydrocarbon radical, or combina peptide conjugates of the invention in which a selected per tion thereof, which may be fully saturated, mono- or polyun centage of the modified Sugars added to a peptide are added to saturated and can include di- and multivalent radicals, having multiple, identical acceptor sites on the peptide. “Essentially the number of carbon atoms designated (i.e. C-Co means each member of the population' speaks to the “homogeneity' one to ten carbons). Examples of saturated hydrocarbon radi of the sites on the peptide conjugated to a modified Sugar and cals include, but are not limited to, groups such as methyl, refers to conjugates of the invention, which are at least about ethyl, n-propyl, isopropyl. n-butyl, t-butyl, isobutyl, sec-bu 80%, preferably at least about 90% and more preferably at tyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, least about 95% homogenous. homologs and isomers of for example, n-pentyl, n-hexyl, 0076 “Homogeneity.” refers to the structural consistency n-heptyl, n-octyl, and the like. An unsaturated alkyl group is across a population of acceptor moieties to which the modi one having one or more double bonds or triple bonds. fied Sugars are conjugated. Thus, in a peptide conjugate of the Examples of unsaturated alkyl groups include, but are not invention in which each modified Sugar moiety is conjugated limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(buta to an acceptor site having the same structure as the acceptor dienyl), 2.4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and site to which every other modified Sugar is conjugated, the 3-propynyl, 3-butynyl, and the higher homologs and isomers. peptide conjugate is said to be about 100% homogeneous. The term “alkyl, unless otherwise noted, is also meant to Homogeneity is typically expressed as a range. The lower end include those derivatives of alkyl defined in more detail of the range of homogeneity for the peptide conjugates is below, such as "heteroalkyl. Alkyl groups that are limited to about 60%, about 70% or about 80% and the upper end of the hydrocarbon groups are termed “homoalkyl. range of purity is about 70%, about 80%, about 90% or more I0082. The term “alkylene' by itself or as part of another than about 90%. Substituent means a divalent radical derived from an alkane, 0077. When the peptide conjugates are more than or equal as exemplified, but not limited, by —CH2CH2CHCH . to about 90% homogeneous, their homogeneity is also pref and further includes those groups described below as “het erably expressed as a range. The lower end of the range of eroalkylene.” Typically, an alkyl (or alkylene) group will have homogeneity is about 90%, about 92%, about 94%, about from 1 to 24 carbon atoms, with those groups having 10 or 96% or about 98%. The upper end of the range of purity is fewer carbon atoms being preferred in the present invention. about 92%, about 94%, about 96%, about 98% or about 100% A “lower alkyl or “lower alkylene' is a shorter chain alkyl or homogeneity. The purity of the peptide conjugates is typically alkylene group, generally having eight or fewer carbon determined by one or more methods known to those of skill in atOmS. the art, e.g., liquid chromatography-mass spectrometry (LC I0083. The terms “alkoxy.” “alkylamino” and “alkylthio” MS), matrix assisted laser desorption mass time of flight (or thioalkoxy) are used in their conventional sense, and refer spectrometry (MALDITOF), capillary electrophoresis, and to those alkyl groups attached to the remainder of the mol the like. ecule via an oxygen atom, an amino group, or a Sulfur atom, 0078 “Substantially uniform glycoform” or a “substan respectively. tially uniform glycosylation pattern, when referring to a I0084. The term "heteroalkyl.” by itself or in combination glycopeptide species, refers to the percentage of acceptor with another term, means, unless otherwise stated, a stable moieties that are glycosylated by the glycosyltransferase of straight or branched chain, or cyclic hydrocarbon radical, or interest (e.g., fucosyltransferase). For example, in the case of combinations thereof, consisting of the stated number of car a C.1.2 fucosyltransferase, a substantially uniform fucosyla bon atoms and at least one heteroatom selected from the tion pattern exists if substantially all (as defined below) of the group consisting of O, N, Si and S, and wherein the nitrogen Galf 1.4-GlcNAc-R and sialylated analogues thereof are and Sulfur atoms may optionally be oxidized and the nitrogen fucosylated in a peptide conjugate of the invention. In the heteroatom may optionally be quaternized. The heteroatom fucosylated structures set forth herein, the Fuc-GlcNAc link (s) O, NandS and Si may be placed at any interior position of age is generally C.1.6 or C.1.3, with C.1.6 generally preferred. the heteroalkyl group or at the position at which the alkyl It will be understood by one of skill in the art, that the starting group is attached to the remainder of the molecule. Examples material may contain glycosylated acceptor moieties (e.g., include, but are not limited to. —CH2—CH2—O—CH, fucosylated Galf1.4-GlcNAc-R moieties). Thus, the calcu CH-CH NH-CH, CH, CH, N(CH)—CH, lated percent glycosylation will include acceptor moieties CH, S CH, CH, CH, CH, S(O)-CH, that are glycosylated by the methods of the invention, as well —CH2—CH2—S(O), CH, —CH=CH-O CH, —Si as those acceptor moieties already glycosylated in the starting (CH), —CH-CH=N OCH, and -CH=CH-N material. (CH)—CH. Up to two heteroatoms may be consecutive, US 2010/0041872 A1 Feb. 18, 2010

Such as, for example, —CH2 NH-OCH and —CH2— those alkyl groups in which a carbon atom (e.g., a methylene O Si (CH). Similarly, the term "heteroalkylene' by itself group) has been replaced by, for example, an oxygen atom or as part of another Substituent means a divalent radical (e.g., phenoxymethyl 2-pyridyloxymethyl, 3-(1-naphthy derived from heteroalkyl, as exemplified, but not limited by, loxy)propyl, and the like). CH2—CH2—S—CH2—CH2— and —CH2—S—CH2 I0089. Each of the above terms (e.g., “alkyl,”“heteroalkyl.” CH NH CH-. For heteroalkylene groups, heteroatoms “aryland "heteroaryl) is meant to include both substituted can also occupy either or both of the chain termini (e.g., and unsubstituted forms of the indicated radical. Preferred alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenedi substituents for each type of radical are provided below. amino, and the like). Still further, for alkylene and heteroalky 0090 Substituents for the alkyl and heteroalkyl radicals lene linking groups, no orientation of the linking group is (including those groups often referred to as alkylene, alkenyl, implied by the direction in which the formula of the linking heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocy group is written. For example, the formula—C(O).R— rep cloalkyl, cycloalkenyl, and heterocycloalkenyl) are generi resents both —C(O).R— and—R'C(O), . cally referred to as “alkyl group substituents.” and they can be I0085. The terms “cycloalkyl and “heterocycloalkyl, by one or more of a variety of groups selected from, but not themselves or in combination with other terms, represent, limited to: OR', -O, -NR', -N OR', NR'R'', SR', unless otherwise stated, cyclic versions of “alkyl and “het -halogen, -SiR'R"R", OC(O)R', -C(O)R', -COR', eroalkyl, respectively. Additionally, for heterocycloalkyl, a CONR'R", OC(O)NR'R", NR"C(O)R', NR C(O) heteroatom can occupy the position at which the heterocycle NR"R", NR"C(O).R', NR C(NR'R"R")=NR", is attached to the remainder of the molecule. Examples of NR-C(NR'R")=NR", S(O)R', S(O),R', S(O) cycloalkyl include, but are not limited to, cyclopentyl, cyclo NR'R'" —NRSOR', —CN and—NO in a number ranging hexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the from Zero to (2m'+1), where m' is the total number of carbon like. Examples of heterocycloalkyl include, but are not lim atoms in such radical. R', R", R" and R" each preferably ited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-pip independently refer to hydrogen, substituted or unsubstituted eridinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tet heteroalkyl, Substituted or unsubstituted aryl, e.g., aryl Sub rahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2- stituted with 1-3 halogens, substituted or unsubstituted alkyl, yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the alkoxy or thioalkoxy groups, or arylalkyl groups. When a like. compound of the invention includes more than one R group, I0086. The terms “halo' or “halogen.” by themselves or as for example, each of the R groups is independently selected as part of another substituent, mean, unless otherwise stated, a are each R', R", R" and R" groups when more than one of fluorine, chlorine, bromine, or iodine atom. Additionally, these groups is present. When R' and R" are attached to the terms such as "haloalkyl are meant to include monoha same nitrogen atom, they can be combined with the nitrogen loalkyl and polyhaloalkyl. For example, the term “halo(C- atom to form a 5-, 6-, or 7-membered ring. For example, C.)alkyl is mean to include, but not be limited to, trifluo —NR'R'" is meant to include, but not be limited to, 1-pyrro romethyl, 2.2.2-trifluoroethyl, 4-chlorobutyl, lidinyl and 4-morpholinyl. From the above discussion of sub 3-bromopropyl, and the like. stituents, one of skill in the art will understand that the term 0087. The term “aryl' means, unless otherwise stated, a “alkyl is meant to include groups including carbon atoms polyunsaturated, aromatic, Substituent that can be a single bound to groups other than hydrogen groups. Such as ring or multiple rings (preferably from 1 to 3 rings), which are haloalkyl (e.g., —CF and —CHCF) and acyl (e.g., -C(O) fused together or linked covalently. The term "heteroaryl CH, —C(O)CF, —C(O)CHOCH, and the like). refers to aryl groups (or rings) that contain from one to four (0091 Similar to the substituents described for the alkyl heteroatoms selected from N, O, and S, wherein the nitrogen radical, Substituents for the aryl and heteroaryl groups are and Sulfur atoms are optionally oxidized, and the nitrogen generically referred to as “aryl group substituents.” The sub atom(s) are optionally quaternized. A heteroaryl group can be stituents are selected from, for example: halogen, —OR', attached to the remainder of the molecule through a heteroa —O, —NR', —N OR', NR'R", SR', -halogen, tom. Non-limiting examples of aryl and heteroaryl groups SiR'R"R", OC(O)R', C(O)R', - COR', include phenyl, 1-naphthyl 2-naphthyl, 4-biphenyl, 1-pyrro CONR'R", OC(O)NR'R", NR"C(O)R', NR C(O) lyl. 2-pyrrolyl, 3-pyrrolyl 3-pyrazolyl, 2-imidazolyl, 4-imi NR"R", NR"C(O).R', NR C(NR'R"R") NR", dazolyl pyrazinyl, 2-oxazolyl, 4-oxazolyl 2-phenyl-4-OX NR-C(NR'R")=NR", S(O)R', S(O),R', S(O) azolyl, 5-oxazolyl 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, NR'R", NRSOR', CN and - NO, R', N, -CH 2-thiazolyl, 4-thiazolyl, 5-thiazolyl 2-furyl, 3-furyl, 2-thie (Ph), fluoro(C-C)alkoxy, and fluoro(C-C)alkyl, in a nyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, number ranging from Zero to the total number of open 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, valences on the aromatic ring system; and where R', R", R" 5-indolyl, 1-isoquinolyl, 5-isoquinolyl 2-quinoxalinyl, and R" are preferably independently selected from hydro 5-quinoxalinyl, 3-quinolyl, tetrazolyl, benzobfuranyl. gen, Substituted or unsubstituted alkyl, Substituted or unsub benzobthienyl, 2,3-dihydrobenzo 1,4-dioxin-6-yl, benzo stituted heteroalkyl, substituted or unsubstituted aryland sub 1.3dioxol-5-yl and 6-quinolyl. Substituents for each of the stituted or unsubstituted heteroaryl. When a compound of the above noted aryl and heteroaryl ring systems are selected invention includes more than one R group, for example, each from the group of acceptable substituents described below. of the R groups is independently selected as are each R', R", 0088 For brevity, the term “aryl' when used in combina R" and R" groups when more than one of these groups is tion with other terms (e.g., aryloxy, arylthioxy, arylalkyl) present. In the schemes that follow, the symbol X represents includes both aryl and heteroaryl rings as defined above. “R” as described above. Thus, the term “arylalkyl is meant to include those radicals 0092. Two of the substituents on adjacentatoms of the aryl in which an aryl group is attached to an alkyl group (e.g., or heteroaryl ring may optionally be replaced with a substitu benzyl, phenethyl, pyridylmethyl and the like) including ent of the formula -T-C(O)—(CRR)-U—, wherein T and U US 2010/0041872 A1 Feb. 18, 2010

are independently —NR— —O—, —CRR'— or a single 0097 “Salt counterion', as used herein, refers to posi bond, and u is an integer of from 0 to 3. Alternatively, two of tively charged ions that associate with a compound of the the Substituents on adjacent atoms of the aryl or heteroaryl invention when one of its moieties is negatively charged (e.g. ring may optionally be replaced with a Substituent of the COO ). Examples of salt counterions include H", HO". formula -A-(CH), B , wherein A and B are indepen ammonium, potassium, calcium, lithium, magnesium and dently —CRR' —O— —NR— —S —S(O)—, Sodium. —S(O) , —S(O)NR' - or a single bond, and r is an inte (0098. As used herein, the term “CMP-SA-PEG” is a cyti ger of from 1 to 4. One of the single bonds of the new ring so dine monophosphate molecule which is conjugated to a sialic formed may optionally be replaced with a double bond. Alter acid which comprises a polyethylene glycol moiety. If a natively, two of the Substituents on adjacent atoms of the aryl length of the polyethylene glycol chain is not specified, then or heteroaryl ring may optionally be replaced with a substitu any PEG chain length is possible (e.g. 1 kD, 2 kD. 5 kD, 10 ent of the formula—(CRR). X (CR"R") , where Zand kD, 20 kD, 30 kD, 40 kD). An exemplary CMP-SA-PEG is dare independently integers of from 0 to 3, and X is —O— compound 5 in Scheme 1. NR— —S —S(O)— —S(O)—, or —S(O)NR' . I. Introduction The substituents R. R', R" and R" are preferably indepen dently selected from hydrogen or substituted or unsubstituted 0099] To improve the effectiveness of recombinant pep (C-C)alkyl. tides used for therapeutic purposes, the present invention provides conjugates of glycosylated and unglycosylated pep 0093. As used herein, the term "heteroatom' is meant to tides with a modifying group. The modifying groups can be include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si). selected from polymeric modifying groups such as, e.g., PEG 0094. As used herein, Factor VII peptide refers to both (m-PEG), PPG (m-PPG), etc., therapeutic moieties, diagnos Factor VII and Factor VIa peptides. The terms generally refer tic moieties, targeting moieties and the like. Modification of to variants and mutants of these peptides, including addition, the peptides, e.g., with a water-soluble polymeric modifying deletion, substitution and fusion protein mutants. Where both group can improve the stability and retention time of the Factor VII and Factor VIa are used, the use is intended to be recombinant peptides in a patient's circulation, and/or reduce illustrative of two species of the genus “Factor VII peptide'. the antigenicity of recombinant peptides. 0.095 The invention is meant to include salts of the com 0100. The peptide conjugates of the invention can be pounds of the invention which are prepared with relatively formed by the enzymatic attachment of a modified Sugar to nontoxic acids or bases, depending on the particular substitu the glycosylated or unglycosylated peptide. A glycosylation ents found on the compounds described herein. When com site and/or a modified glycosyl group provides a locus for pounds of the present invention contain relatively acidic func conjugating a modified Sugar bearing a modifying group to tionalities, base addition salts can be obtained by contacting the peptide, e.g., by glycoconjugation. the neutral form of Such compounds with a sufficient amount 0101 The methods of the invention also make it possible of the desired base, either neat or in a suitable inert solvent. to assemble peptide conjugates and glycopeptide conjugates Examples of base addition salts include sodium, potassium, that have a Substantially homogeneous derivatization pattern. lithium, calcium, ammonium, organic amino, or magnesium The enzymes used in the invention are generally selective for salt, or a similar salt. When compounds of the present inven a particular amino acid residue, combination of amino acid tion contain relatively basic functionalities, acid addition residues, particular glycosyl residues, or combination of gly salts can be obtained by contacting the neutral form of Such cosyl residues of the peptide. The methods are also practical compounds with a Sufficient amount of the desired acid, for large-scale production of peptide conjugates. Thus, the either neat or in a suitable inert solvent. Examples of acid methods of the invention provide a practical means for large addition salts include those derived from inorganic acids like scale preparation of peptide conjugates having preselected hydrochloric, hydrobromic, nitric, carbonic, monohydrogen uniform derivatization patterns. The methods are particularly carbonic, phosphoric, monohydrogenphosphoric, dihydro well suited for modification of therapeutic peptides, includ genphosphoric, Sulfuric, monohydrogensulfuric, hydriodic, ing but not limited to, glycopeptides that are incompletely orphosphorous acids and the like, as well as the salts derived glycosylated during production in cell culture cells (e.g., from relatively nontoxic organic acids like acetic, propionic, mammalian cells, insect cells, plant cells, fungal cells, yeast isobutyric, maleic, malonic, benzoic, Succinic, Suberic, cells, or prokaryotic cells) or transgenic plants or animals. fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolyl 0102 The present invention also provides conjugates of Sulfonic, citric, tartaric, methanesulfonic, and the like. Also glycosylated and unglycosylated peptides with increased included are salts of amino acids such as arginate and the like, therapeutic half-life due to, for example, reduced clearance and salts of organic acids like glucuronic or galactunoric rate, or reduced rate of uptake by the immune or reticuloen acids and the like (see, for example, Berge et al., “Pharma dothelial system (RES). Moreover, the methods of the inven ceutical Salts', Journal of Pharmaceutical Science 66: 1-19 tion provide a means for masking antigenic determinants on (1977)). Certain specific compounds of the present invention peptides, thus reducing or eliminating a host immune contain both basic and acidic functionalities that allow the response against the peptide. Selective attachment of target compounds to be converted into either base or acid addition ing agents can also be used to target a peptide to a particular salts. tissue or cell surface receptor that is specific for the particular 0096. The neutral forms of the compounds are preferably targeting agent. regenerated by contacting the salt with a base or acid and 0103) Determining optimal conditions for the preparation isolating the parent compounds in the conventional manner. of peptide conjugates with water-soluble polymers, e.g., The parentform of the compound differs from the various salt involves the optimization of numerous parameters, which are forms in certain physical properties. Such as solubility in dependent on the identity of the peptide and of the water polar solvents. soluble polymer. For example, when the polymer is poly US 2010/0041872 A1 Feb. 18, 2010

(ethylene glycol), e.g., a branched poly(ethylene glycol), a in which the symbols a, b, c, d and S represent a positive, balance is preferably established between the amount of poly non-Zero integer, and t is either 0 or a positive integer. The mer utilized in the reaction and the viscosity of the reaction "agent’, or modifying group, can be a therapeutic agent, a mixture attributable to the presence of the polymer: if the bioactive agent, a detectable label, a polymeric modifying polymer is too highly concentrated, the reaction mixture group such as a water-soluble polymer (e.g., PEG, m-PEG, becomes Viscous, slowing the rate of mass transfer and reac tion. PPG, and m-PPG) or the like. The “agent’, or modifying 0104 Furthermore, though it is intuitively apparent to add group, can be a peptide, e.g., enzyme, antibody, antigen, etc. an excess of enzyme, the present inventors have recognized The linker can be any of a wide array of linking groups, infra. that, when the enzyme is present in too great of an excess, the Alternatively, the linker may be a single bond or a “Zero order excess enzyme becomes a contaminant whose removal linker. requires extra purification steps and material and unnecessar ily increases the cost of the final product. II. A. Peptide 0105 Moreover, it is generally desired to produce a pep 0109 The peptide in the peptide conjugate is a member tide with a controlled level of modification. In some selected from the peptides in FIG. 7. In these cases, the instances, it is desirable to add one modified Sugar preferen peptide in the peptide conjugate is a member selected from tially. In other instances, it is desirable to add two modified Sugars preferentially. Thus, the reaction conditions are pref bone morphogenetic proteins (e.g., BMP-1, BMP-2, BMP-3, erably controlled to influence the degree of conjugation of the BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, modifying groups to the peptide. BMP-11, BMP-12, BMP-13, BMP-14, BMP-15), neurotro 0106 The present invention provides conditions under phins (e.g., NT-3, NT-4, NT-5), growth differentiation factors which the yield of a peptide, having the desired level of (e.g., GDF-5), glial cell line-derived neurotrophic factor conjugation, is maximized. The conditions in the exemplary (GDNF), brain derived neurotrophic factor (BDNF), nerve embodiments of the inventions also recognize the expense of the various reagents and the materials and time necessary to growth factor (NGF), von Willebrand factor (VWF) protease, purify the product: the reaction conditions set forth herein are Factor VII, Factor VIIa, Factor VIII, Factor IX, Factor X, optimized to provide excellent yields of the desired product, Factor XI, B-domain deleted Factor VIII, v WF-Factor VIII while minimizing waste of costly reagents. fusion protein having full-length Factor VIII, VWF-Factor VIII fusion protein having B-domain deleted Factor VIII, II. The Compositions of Matter/Peptide Conjugates erythropoietin (EPO), granulocyte colony stimulating factor 0107. In a first aspect, the present invention provides a (G-CSF), Granulocyte-Macrophage Colony Stimulating Fac conjugate between a modified Sugar and a peptide. The tor (GM-CSF), interferon alpha, interferon beta, interferon present invention also provides a conjugate between a modi gamma, C.-antitrypsin (ATT, or C-1 protease inhibitor, glu fying group and a peptide. A peptide conjugate can have one cocerebrosidase, Tissue-Type Plasminogen Activator (TPA), of several forms. In an exemplary embodiment, a peptide Interleukin-2 (IL-2), urokinase, human DNase, insulin, conjugate can comprise a peptide and a modifying group Hepatitis B surface protein (HbsAg), human growth hor linked to an amino acid of the peptide through a glycosyl mone, TNF Receptor-IgG Fc region fusion protein (En linking group. In another exemplary embodiment, a peptide brelTM), anti-HER2 monoclonal antibody (HerceptinTM), conjugate can comprise a peptide and a modifying group monoclonal antibody to Protein F of Respiratory Syncytial linked to a glycosyl reside of the peptide through a glycosyl Virus (SynagisTM), monoclonal antibody to TNF-C. (Remi linking group. In another exemplary embodiment, the peptide conjugate can comprise a peptide and a glycosyl linking cadeTM), monoclonal antibody to glycoprotein IIb/IIIa (Re group which is bound to both a glycopeptide carbohydrate oproTM), monoclonal antibody to CD20 (RituxanTM), anti and directly to an amino acid residue of the peptide backbone. thrombin III (ATIII), human Chorionic Gonadotropin (hCG), In yet another exemplary embodiment, a peptide conjugate alpha-galactosidase (FabrazymeTM), alpha-iduronidase (Al can comprise a peptide and a modifying group linked directly durazymeTM), follicle stimulating hormone, beta-glucosi to an amino acid residue of the peptide. In this embodiment, dase, anti-TNF-alpha monoclonal antibody, glucagon-like the peptide conjugate may not comprise a glycosyl group. In peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), beta any of these embodiments, the peptide may or not be glyco glucosidase, alpha-galactosidase A and fibroblast growth fac Sylated. tor. In certain embodiments, the peptide in the peptide con 0108. The conjugates of the invention will typically cor jugate is Factor VIII. In other embodiments, the peptide in the respond to the general structure: peptide conjugate is interferon alpha.

Peptide US 2010/0041872 A1 Feb. 18, 2010

0110. In an exemplary embodiment, the polymeric modi 0112 Exemplary polymeric modifying groups according fying group has a structure including a moiety according to to this embodiment include the moiety: the following formulae: H H H (OCH2CH2)OCH H (OCH2CH2)A'; A’(CH2CH2O). H

A’(CH2CH2O) H H O

H La H and H O HN\- CH A’(CH2CH2O). H

NYCl, X. CHO(CH2CH2O).

CH A(CH2CH2O) H 0113. In an exemplary embodiment, in which the modify ing group is a branched water-soluble polymer, Such as those shown above, it is generally preferred that the concentration of sialidase is about 1.5 to about 2.5 U/L of reaction mixture. More preferably the amount of sialidase is about 2 U/L. Ox 0114. In another exemplary embodiment, about 5 to about 9 grams of peptide Substrate is contacted with the amounts of sialidase set forth above. 0111. In an exemplary embodiment, m and n are integers 0115 The modified sugar is present in the reaction mix independently selected from about 1 to about 5000, prefer ture in an amount from about 1 gram to about 6 grams, ably from about 100 to about 4000, more preferably from preferably from about 3 grams to about 4 grams. It is gener about 200 to about 3000, even more preferably from about ally preferred to maintain the concentration of a modified 300 to about 2000 and still more preferably from about 400 to Sugar having a branched water-soluble polymer modifying about 1000. In an exemplary embodiment, m and n are inte moiety, e.g., the moiety shown above, at less than about 0.5 mM. gers independently selected from about 1 to about 500. In an 0116. In certain embodiments, the modifying group is a exemplary embodiment, m and n are integers independently branched poly(alkylene oxide), e.g., poly(ethylene glycol), selected from about 1 to about 70, about 70 to about 150, having a molecular weight from about 20 kD to about 60 kD. about 150 to about 250, about 250 to about 375 and about 375 more preferably, from about 30 kD to about 50 kD, and even to about 500. In an exemplary embodiment, m and n are more preferably about 40 kD. In other embodiments, the integers independently selected from about 10 to about 35, modifying group is a branched poly(alkylene oxide), e.g., about 45 to about 65, about 95 to about 130, about 210 to poly(ethylene glycol), having a molecular weight of at least about 240, about 310 to about 370 and about 420 to about 480. about 80 kD. preferably at least about 100 kD, more prefer In an exemplary embodiment, m and n are integers selected ably at least about 120 kD. at least about 140 kD or at least about 160 kD. In yet another embodiment, the branched poly from about 15 to about 30. In an exemplary embodiment, m (alkylene oxide), e.g., poly(ethylene glycol) is at least about and n are integers selected from about 50 to about 65. In an 200kD, such as from at least about 80 kD to at least about 200 exemplary embodiment, m and n are integers selected from kD, including at least about 160 kD and at least about 180 kD. about 100 to about 130. In an exemplary embodiment, mand As those of skill will appreciate, the molecular weight of in are integers selected from about 210 to about 240. In an polymers is often polydisperse, thus, the phrase “about in the exemplary embodiment, m and n are integers selected from context of molecular weight preferably encompasses a range about 310 to about 370. In an exemplary embodiment, mand of values around the stated number. For example, a preferred in are integers selected from about 430 to about 470. In an modifying group having a molecular weight of about 40kDis exemplary embodiment, A' and A are each members one that has a molecular weight from about 35 kD to about 45 selected from —OH and —OCH. kD. Those of skill will appreciate that the reliance on US 2010/0041872 A1 Feb. 18, 2010

branched PEG structures set forth above is simply for clarity of illustration, the PEG can be replaced by substantially any -continued

polymeric moiety, including, without limitation those species R 14 set forth in the definition of “polymeric moiety’ found herein. 0117 Regarding the glycosyltransferase concentration, in a presently preferred embodiment, using the modifying group set forth above, the ratio of glycosyltransferase to peptide is R131. about 40 ug/mL transferase to about 200 uM peptide. R12 11 II. B. Modified Sugar 0118. In an exemplary embodiment, the peptides of the Other saccharyl moieties that are useful in forming the com invention, such as Factor VIII, interferon alpha, and the pep positions of the invention include, but are not limited to tides listed in FIG. 7, are reacted with a modified sugar, thus fucose and Sialic acid, as well as amino Sugars such as glu forming a peptide conjugate. A modified Sugar comprises a cosamine, galactosamine, mannosamine, the 5-amine ana "Sugar donor moiety' as well as a "sugar transfer moiety'. logue of Sialic acid and the like. The Saccharyl moiety can be The Sugar donor moiety is any portion of the modified Sugar a structure found in nature or it can be modified to provide a that will be attached to the peptide, either through a glycosyl site for conjugating the modifying group. For example, in one moiety or amino acid moiety, as a conjugate of the invention. embodiment, the modified Sugar provides a sialic acid deriva The Sugar donor moiety includes those atoms that are chemi tive in which the 9-hydroxy moiety is replaced with an amine. cally altered during their conversion from the modified Sugar The amine is readily derivatized with an activated analogue of to the glycosyl linking group of the peptide conjugate. The a selected modifying group. Sugar transfer moiety is any portion of the modified Sugar that I0121 Examples of modified sugars of use in the invention will be not be attached to the peptide as a conjugate of the are described in PCT Patent Application No. PCT/US05/ invention. For example, a modified Sugar of the invention is 002522, which is herein incorporated by reference. the PEGylated sugar nucleotide, PEG-sialic acid CMP. For I0122. In a further exemplary embodiment, the invention PEG-sialic acid CMP, the sugar donor moiety, or PEG-sialyl utilizes modified sugars in which the 6-hydroxyl position is donor moiety, comprises PEG-Sialic acid while the Sugar converted to the corresponding amine moiety, which bears a transfer moiety, or sialyl transfer moiety, comprises CMP. linker-modifying group cassette Such as those set forth above. 0119. In modified sugars of use in the invention, the sac Exemplary glycosyl groups that can be used as the core of charyl moiety is preferably a saccharide, a deoxy-saccharide, these modified sugars include Glu, Gal, GalNAc, Glc, an amino-saccharide, oran N-acyl saccharide. The term 'sac GlcNAc, Fuc, Xyl, Man, and the like. A representative modi charide' and its equivalents, "saccharyl. “Sugar,” and 'gly fied Sugar according to this embodiment has the formula: cosyl refer to monomers, dimers, oligomers and polymers. The Sugar moiety is also functionalized with a modifying group. The modifying group is conjugated to the saccharyl R 14 moiety, typically, through conjugation with an amine, Sulfhy dryl or hydroxyl, e.g., primary hydroxyl, moiety on the Sugar. R13 In an exemplary embodiment, the modifying group is attached through an amine moiety on the Sugar, e.g., through an amide, a urethane or a urea that is formed through the R12 R10 reaction of the amine with a reactive derivative of the modi fying group. 0120 Any saccharyl moiety can be utilized as the sugar donor moiety of the modified Sugar. The saccharyl moiety can be a known Sugar, such as mannose, galactose or glucose, or in which R'-R'' are members independently selected from a species having the stereochemistry of a known Sugar. The H, OH, C(O)CH, NH, and NHC(O)CH. R' is a link to general formulae of these modified Sugars are: another glycosyl residue (-O-glycosyl) or to an amino acid of the Factor VII/Factor VIIa peptide (-NH-(Factor VII/ Factor VIIa)). R'' is OR', NHR' or NH-L-R. R. and NH-L- R" are as described above. II. C. Glycosyl Linking Groups I0123. In an exemplary embodiment, the invention pro vides a peptide conjugate formed between a modified Sugar of the invention and a peptide. In another exemplary embodi ment, when the modifying group on the modified Sugar includes the moiety: US 2010/0041872 A1 Feb. 18, 2010

CAA CH (CAA),

Achcho-A(CAA) CA10Alll and the peptide in the peptide conjugate is a member selected CH from the peptides in FIG.7. In yet another exemplary embodi A’(CH2CH2O). H ment, the peptide in the peptide conjugate is a member O selected from bone morphogenetic proteins (e.g., BMP-1, O BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, N BMP-15), neurotrophins (e.g., NT-3, NT-4, NT-5), growth rulls’,H differentiation factors (e.g., GDF-5), glial cell line-derived neurotrophic factor (GDNF), brain derived neurotrophic fac tor (BDNF), nerve growth factor (NGF), von Willebrand fac tor (vWF) protease, Factor VII, Factor VIa, Factor VIII, Fac 0.125. In an exemplary embodiment, modifying group on tor IX, Factor X, Factor XI, B-domain deleted Factor VIII, the modified Sugar includes the moiety: VWF-Factor VIII fusion protein having full-length Factor VIII, VWF-Factor VIII fusion protein having B-domain deleted Factor VIII, erythropoietin (EPO), granulocyte H colony stimulating factor (G-CSF), Granulocyte-Macroph H (OCH2CH2)A'. age Colony Stimulating Factor (GM-CSF), interferon alpha, interferon beta, interferon gamma, C.-antitrypsin (ATT, or A'(CH2CH2O). H C-1 protease inhibitor), glucocerebrosidase, Tissue-Type H La Plasminogen Activator (TPA), Interleukin-2 (IL-2), uroki H nase, human DNase, insulin, Hepatitis B surface protein (Hb SAg), human growth hormone, TNF Receptor-IgGFc region fusion protein (EnbrelTM), anti-HER2 monoclonal antibody (HerceptinTM), monoclonal antibody to Protein F of Respira In an exemplary embodiment, A' and A are each members tory Syncytial Virus (SynagisTM), monoclonal antibody to selected from —OH and —OCH. TNF-C. (RemicadeTM), monoclonal antibody to glycoprotein IIb/IIIa (ReoproTM), monoclonal antibody to CD20 (Rit 0.126 Exemplary polymeric modifying groups according uxanTM), anti-thrombin III (ATIII), human Chorionic Gona to this embodiment include the moiety: dotropin (hCG), alpha-galactosidase (FabrazymeTM), alpha iduronidase (AldurazymeTM), follicle stimulating hormone, H beta-glucosidase, anti-TNF-alpha monoclonal antibody, glu H (OCH2CH2)OCH cagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), beta-glucosidase, alpha-galactosidase A and fibro CHO(CH2CH2O). H blast growth factor. In certain embodiments the peptide is Factor VIII or interferon alpha. In this embodiment, the sugar H O donor moiety (such as the Saccharyl moiety and the modify H and ing group) of the modified Sugar becomes a glycosyl linking O group'. The glycosyl linking group' can alternatively refer HN to the glycosyl moiety which is interposed between the pep tide and the modifying group. \- 0.124. In an exemplary embodiment, the polymeric modi HN fying group includes a moiety having a structure according to the following formulae: US 2010/0041872 A1 Feb. 18, 2010 16

-continued -continued IIa H (OCH2CH2)OCH R O y CHO(CH2CH2O). H O H H O

HNN-R N-S In Formulae I and Ia R is H, CHOR7, COOR7 or OR7, in H which R" represents H, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl. When COOR is a carboxylic acid or carboxylate, both forms are represented by 0127. As will be appreciated by those of skill in the art, the the designation of the single structure COO or COOH. In PEG moieties in each of the structures shown above can be Formulae I, Ia, II or Ila, the symbols R. R. R. R. and R' replaced by any other polymeric moiety, including, without independently represent H, substituted or unsubstituted alkyl, OR, NHC(O)R. The index d is 0 or 1. Rand Rare inde limitation, those species defined herein as "polymeric moi pendently selected from H. Substituted or unsubstituted alkyl, eties. substituted or unsubstituted heteroalkyl, sialic acid or poly 0128. Due to the versatility of the methods available for sialic acid. At least one of R. R. R. R. or R' includes a adding and/or modifying glycosyl residues on a peptide, the modifying group. This modifying group can be a polymeric glycosyl linking groups can have substantially any structure. modifying moiety e.g., PEG, linked through a bond or a In the discussion that follows, the invention is illustrated by linking group. In an exemplary embodiment, R and R', reference to the use of selected derivatives of furanose and together with the carbon to which they are attached are com pyranose. Those of skill in the art will recognize that the focus ponents of the pyruvyl side chain of sialic acid. In a further exemplary embodiment, the pyruvyl side chain is functional of the discussion is for clarity of illustration and that the ized with the polymeric modifying group. In another exem structures and compositions set forth are generally applicable plary embodiment, R and R', together with the carbon to across the genus of glycosyl linking groups and modified which they are attached are components of the side chain of Sugars. The glycosyl linking group can comprise virtually any sialic acid and the polymeric modifying group is a component mono- or oligo-Saccharide. The glycosyl linking groups can of R be attached to an amino acid either through the side chain or 0.130. In an exemplary embodiment, the invention utilizes through the peptide backbone. Alternatively the glycosyl a glycosyl linking group that has the formula: linking groups can be attached to the peptide through a sac charyl moiety. This saccharyl moiety can be a portion of an O-linked or N-linked glycan structure on the peptide. 0129. In an exemplary embodiment, the invention pro vides a peptide conjugate comprising an intact glycosyl link Ol NH-L-R ing group having a formula that is selected from: in which J is a glycosyl moiety, L is a bond or a linker and R' is a modifying group, e.g., a polymeric modifying group. (R) Exemplary bonds are those that are formed between an NH moiety on the glycosyl moiety and a group of complementary O R2 R6 reactivity on the modifying group. For example, when R' O includes a carboxylic acid moiety, this moiety may be acti vated and coupled with the NH, moiety on the glycosyl resi R5 R3 due affording a bond having the structure NHC(O)R. J is preferably a glycosyl moiety that is “intact, not having been II degraded by exposure to conditions that cleave the pyranose (R) or furanose structure, e.g. oxidative conditions, e.g., sodium periodate. I0131 Exemplary linkers include alkyl and heteroalkyl moieties. The linkers include linking groups, for example acyl-based linking groups, e.g., —C(O)NH , —OC(O) NH , and the like. The linking groups are bonds formed Ia between components of the species of the invention, e.g., between the glycosyl moiety and the linker (L), or between the linker and the modifying group (R'). Other exemplary linking groups are ethers, thioethers and amines. For example, in one embodiment, the linker is an amino acid residue. Such as a glycine residue. The carboxylic acid moiety of the glycine is converted to the corresponding amide by reaction with an amine on the glycosyl residue, and the amine US 2010/0041872 A1 Feb. 18, 2010 of the glycine is converted to the corresponding amide or 10137. In the formulae above, R' and L are as described urethane by reaction with an activated carboxylic acid or above. Further detail about the structure of exemplary R' carbonate of the modifying group. groups is provided below. (0132) An exemplary species of NH-L-R' has the formula: 0.138. In still a further exemplary embodiment, the conju gate is formed between a peptide and a modified Sugar in NH {C(O)(CH),NHS{C(O)(CH),(OCH2CH2)CO which the modifying group is attached through a linker at the (CH2)NHR', in which the indices sandt are independently 6-carbon position of the modified sugar. Thus, illustrative 0 or 1. The indices a, b and dare independently integers from glycosyl linking groups according to this embodiment have 0 to 20, and c is an integer from 1 to 2500. Other similar the formula: linkers are based on species in which an —NH moiety is replaced by another group, for example. —S. —O or —CH2. As those of skill will appreciate one or more of the bracketed O O moieties corresponding to indices Sandt can be replaced with R-L-N a substituted or unsubstituted alkyl or heteroalkyl moiety. H 0.133 More particularly, the invention utilizes compounds in which NH-L-R is: NHC(O)(CH)NHC(O)(CH), R13 R11 R12

in which the radicals areas discussed above. Glycosyl linking groups include, without limitation, glucose, glucosamine, NH(CH)NHR', and NHR'. In these formulae, the indices a, N-acetyl-glucosamine, galactose, galactosamine, N-acetyl band dare independently selected from the integers from 0 to galactosamine, mannose, mannosamine, N-acetyl-man 20, preferably from 1 to 5. The index c is an integer from 1 to nosamine, and the like. about 2500. 0.139. In one embodiment, the present invention provides a 0134. In an exemplary embodiment, c is selected such that peptide conjugate comprising the following glycosyl linking the PEG moiety is approximately 1 kD, 5 kD, 10, kD, 15 kD. group: 20 kD, 25 kD, 30 kD, 35 kD, 40 kD or 45 kD. 0135 For the purposes of convenience, the glycosyl link ing groups in the remainder of this section will be based on a OH sialyl moiety. However, one of skill in the art will recognize D that another glycosyl moiety, Such as mannosyl, galactosyl, O COOH glucosyl, or fucosyl, could be used in place of the Sialyl HO moiety. O 0136. In an exemplary embodiment, the glycosyl linking group is an intact glycosyl linking group, in which the gly G-HN cosyl moiety or moieties forming the linking group are not OH degraded by chemical (e.g., Sodium metaperiodate) or enzy matic (e.g., oxidase) processes. Selected conjugates of the wherein D is a member selected from OHand R'-L-HN : invention include a modifying group that is attached to the G is a member selected from H and R-L- and —C(O)(C- amine moiety of an amino-saccharide, e.g., mannosamine, C.)alkyl, R' is a moiety comprising a straight-chain or glucosamine, galactosamine, sialic acid etc. Exemplary branched poly(ethylene glycol) residue; and L is a linker, e.g., modifying group-intact glycosyl linking group cassettes a bond (“Zero order'), substituted or unsubstituted alkyl and according to this motif are based on a sialic acid structure, substituted or unsubstituted heteroalkyl. In exemplary Such as those having the formulae: embodiments, when D is OH, G is R'-L-, and when G is —C(O)(C-C)alkyl, D is R'-L-NH-. OH 0140. In one embodiment, the present invention provides a HO peptide conjugate comprising the following glycosyl linking group: O COOH HO and OH R-L-NH D O COOM OH HO OH RI-L-HN O G-N O COOH H HO OH D is a member selected from -OH and R'-L-HN : G is a OH member selected from R'-L-and-C(O)(C-C)alkyl-R'; R' is a moiety comprising a member selected from a straight chain poly(ethylene glycol) residue and branched poly(eth US 2010/0041872 A1 Feb. 18, 2010 18 ylene glycol) residue; and M is a member selected from H, a 0144. In a still further exemplary embodiment, the glyco salt counterion and a single negative charge; L is a linker syl linking group has the formula: which is a member selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted het eroalkyl. In an exemplary embodiment, when D is OH, G is R'-L-. In another exemplary embodiment, when G is C(O) OH (C-C)alkyl, D is R'-L-NH-. 0141. In any the compounds of the invention, a COOH group can alternatively be COOM, wherein M is a member O COOH (Sia), selected from H, a negative charge, and a salt counterion. HO 0142. The invention provides a peptide conjugate that O-GalNac includes a glycosyl linking group having the formula: OH OH D wherein D and Gareas described above and the indext is 0 or O COOH 1. HO O-Gal 0145. In yet another embodiment, the glycosyl linking group has the formula: G-HN

OH OH wherein D and G are as described above. 0143. In other embodiments, the glycosyl linking group has the formula: O COOH HO O-(Sia)-(Gal-GlcNAc); OH O cooH "F")F OH HO

G oals wherein D and G are as described above and the index p OH represents and integer from 1 to 10; and a is either 0 or 1. 0146 In another exemplary embodiment, the peptide con wherein D and G are as described above and the index t is jugate comprises a glycosyl moiety selected from the formu 0 or 1. lae:

CAA (CAA), OH

Accio-HA(CAA) R2

O s R3 R4 (OCH2CH2)A CH OH OH A'(CH2CH2O), H OH CH OH O R2 OH A’(CH2CH2O). H O O R2 O O HC N R3 La R4

US 2010/0041872 A1 Feb. 18, 2010 29 in which the index.a and the linker L'areas discussed above. The index p is an integer from 1 to 10. The indices tanda are independently selected from 0 or 1. Each of these groups can R6 R6 be included as components of the mono-, bi-, tri- and tetra R5 R5 antennary saccharide structures set forth above. AA is an O amino acid residue of the peptide. One of skill in the art will R R2 R 4 R2 appreciate that the PEG moiety in these formulae can be replaced with other non-reactive group and polymeric moi 3 NAc eties. Exemplary polymers include those of the poly(alkylene oxide) family. Non-reactive groups include groups that are R -- considered to be essentially unreactive, neutral and/or stable at physiological pH, e.g., H. Substituted or unsubstituted R6 R6 alkyl, substituted or unsubstituted heteroalkyland the like. An 5 O exemplary polymeric moiety includes the branched struc 5 O R 4 R2 tures set forth in Formula IIIa and its exemplars. R R4 R2 R 0147 In an exemplary embodiment, the PEG moiety has a molecular weight of about 20 kD. In another exemplary embodiment, the PEG moiety has a molecular weight of about 5 kD. In another exemplary embodiment, the PEG moiety has a molecular weight of about 10 kD. In another exemplary embodiment, the PEG moiety has a molecular weight of about 40 kD. In other embodiments, the modifying group is a branched poly(alkylene oxide), e.g., poly(ethylene glycol), having a molecular weight of at least about 80 kD. preferably at least about 100 kD, more preferably at least about 120kD, at least about 140 kD or at least about 160 kD. In yet another embodiment, the branched poly(alkylene oxide), e.g., poly(ethylene glycol) is at least about 200 kD. such as from at least about 80 kD to at least about 200 kD, wherein at least one of R. R. R, R or R has a structure including at least about 160 kD and at least about 180 kD. In which is a member selected from an exemplary embodiment, the branched polymer is itself attached to a branching moiety (e.g., cysteine, serine, lysine, and oligomers of lysine). 0148. In an exemplary embodiment, the glycosyl linking group is a branched SA-PEG-10 kD moiety based on a cys CAA teine residue, and one or two of these glycosyl linking groups are covalently attached to the peptide. In another exemplary (CAA), embodiment, the glycosyl linking group is a branched SA L PEG-10 kD moiety based on a lysine residue, and one or two x- SN and Accio-Hi of these glycosyl linking groups are covalently attached to the (CAA) peptide. In an exemplary embodiment, the glycosyl linking CA10All H group is a branched SA-PEG-10 kD moiety based on a cys N-N teine residue, and one or two of these glycosyl linking groups La are covalently attached to the peptide. In an exemplary embodiment, the glycosyl linking group is a branched SA PEG-10 kD moiety based on a lysine residue, and one or two in which the variables are as described above. Those of skill of these glycosyl linking groups are covalently attached to the will appreciate that the reliance on branched PEG structures peptide. In an exemplary embodiment, the glycosyl linking set forth above is simply for clarity of illustration, the PEG group is a branched SA-PEG-5 kD moiety based on a cysteine can be replaced by Substantially any polymeric moiety, residue, and one, two or three of these glycosyl linking groups including, without limitation those species set forth in the are covalently attached to the peptide. In an exemplary definition of “polymeric moiety’ found herein. embodiment, the glycosyl linking group is a branched SA I0150. In an exemplary embodiment, at least one of R. R. PEG-5 kD moiety based on a lysine residue, and one, two or R, R or R has a structure according to the following for three of these glycosyl linking groups are covalently attached mula: to the peptide. In an exemplary embodiment, the glycosyl linking group is a branched SA-PEG-40 kD moiety based on a cysteine residue, and one or two of these glycosyl linking H groups are covalently attached to the peptide. In an exemplary H (OCH2CH2)A embodiment, the glycosyl linking group is a branched SA PEG-40 kD moiety based on a lysine residue, and one or two A’(CH2CH2O). H of these glycosyl linking groups are covalently attached to the H peptide. H Le-N 0149. In another exemplary embodiment, the peptide con H jugate comprises a glycosyl moiety selected from the formu lae: US 2010/0041872 A1 Feb. 18, 2010 30

In an exemplary embodiment, A' and AZ are each selected from —OH and —OCH. -continued 0151 Exemplary polymeric modifying groups according R6 R6 to this embodiment include: R3 O R5 R4 R2 R5 -O H R4 H (OCH2CH2)OCH NAc O CHO(CH2CH2O). H

H H O O

> O and R6 HN O R2 O R3, R3 R'

wherein R. R. R. Ror Rare as described above. 0154) In another exemplary embodiment, the peptide con

jugate comprises a glycosyl moiety selected from the formu lae:

OH

OH

OH O R2

O (R)-L-NH R3 5: R4 0152. In an exemplary embodiment, only one of R. R.

R, R or R has a structure which includes the modifying 6 groups described above. (R) -L-N R 0153. In another exemplary embodiment, the peptide con- w jugate comprises a glycosyl moiety selected from the formu- R O R2 lae: 3 R O

OH R6 s OH R5 O R6 OH R 2 H O R2 R (R)-L-N. O R3 Y R O R2

5 3 R R NAc O R4

6 R5 R R6 R6

R4 O R2 R5 R4 R. R. (R)-L-N-ly - - \ R2

NAc 3 3 O- ; o- : R Y US 2010/0041872 A1 Feb. 18, 2010 31

0156 Exemplary polymeric modifying groups according -continued to this embodiment include:

R6 H H (OCH2CH2)OCH (R)-L-NHly - T - R O R2 CHO(CH2CH2O). H

NAc H O H and O HN R6 \- HN (R)-L-NH - O R2

H O (OCH2CH2)OCH3. ; and

CH3O(CH2CH2O)

O O HNN N R6 H R3 (R)-L-Ní R' In an exemplary embodiment, m and n are integers indepen dently selected from about 1 to about 1000. In an exemplary embodiment, m and n are integers independently selected in which L-(R') is a member selected from from about 1 to about 500. In an exemplary embodiment, m and n are integers independently selected from about 1 to about 70, about 70 to about 150, about 150 to about 250, about 250 to about 375 and about 375 to about 500. In an exemplary (OCH2CH2)A embodiment, m and n are integers independently selected from about 10 to about 35, about 45 to about 65, about 95 to As about 130, about 210 to about 240, about 310 to about 370 and R16-X2 (CAA), about 420 to about 480. In an exemplary embodiment, mand -L: in are integers selected from about 15 to about 30. In an XS-C and A'(CH2CH2O). A7 exemplary embodiment, m and n are integers selected from R17-X (CAA) about 50 to about 65. In an exemplary embodiment, m and n are integers selected from about 100 to about 130. In an AA. exemplary embodiment, m and n are integers selected from N. about 210 to about 240. In an exemplary embodiment, mand in are integers selected from about 310 to about 370. In an exemplary embodiment, m and n are integers selected from in which the variables are as described above. about 430 to about 470. I0155) In an exemplary embodiment, L-(R'), has a struc 0157. In another exemplary embodiment, the peptide con ture according to the following formula: jugate comprises a glycosyl moiety selected from the formu lae:

H OH

OH

OH CO Le

In an exemplary embodiment, A' and A are each selected OH from —OH and —OCH. US 2010/0041872 A1 Feb. 18, 2010

-continued -continued P" '" OH (R')-L-N. (R')-L-N. OH O O - O HO R 2 HO R 2 (R')R)-L-NH HO R2

OH O s NAc O s O ; and

OH

H O (R')R)-L-N HO R2 O OH O s HO O R2 OH OH (R)-L-NH HO O (R')R)-L-NH HO R 2

NAc O s wherein the variables are as described above. 0158. In another exemplary embodiment, species accord ing to this embodiment include:

O O HO OH 2,

HO "Na CCC - O 1. Cl O H OH Né---N-r O HO

1. N-nor- D- O OH No-fn- N-1- -r O HO R2 O

CCC O O OH US 2010/0041872 A1 Feb. 18, 2010

-continued OH

O R2

NAc O

OH OH O R2

O and

O

O R2 OH

wherein the variables are as discussed above. 0159. In an exemplary embodiment, a glycoPEGylated -continued peptide conjugate of the invention is selected from the for (GlcNAc-Gal)-R's mulae set forth below: (Fuc) Man-(GlcNAc-Gal)-R's AA-GlcNAc-GlcNAc-Man op (Fuc), yuanasa-R". olo scies are oloAA-GlcNAc-GlcNAc-Man l (GlcNAc-Gal)-R' wherein the variables are as described above. 0160. In the formulae above, the index t is an integer from 0 to 1 and the index p is an integer from 1 to 10. The symbol op (Fuc), Yu. R" represents H, OH (e.g., Gal-OH), a sialyl moiety, a sialyl linking group (i.e., sialyl linking group-polymeric modifying AA- Giescensin group (Sia-L-R), or a sialyl moiety to which is bound a olo Man-(GlcNAc-Gal)-R''. polymer modified sialyl moiety (e.g., Sia-Sia-L-R) ("Sia (Fuc), Man-(GlcNAc-Gal)-R' Sia?”)), a galactosyl moiety, a galactosyl linking group (i.e., galactosyl linking group-polymeric modifying group (Gal-L- AA- Giescensin R"), or a sialyl moiety to which is bound a polymer modified olo Man-(GlcNAc-Gal)-R': galactosyl moiety (e.g., Sia-Gal-L-R") ("Sia-Gal”)), a galac tosaminyl moiety, a galactosaminyl linking group (i.e., galac (GlcNAc-Gal)-R 5 tosaminyl linking group-polymeric modifying group (Gal NAc-L-R), or a sialyl moiety to which is bound a polymer op s yielease -R15 modified galactosaminyl moiety (e.g., Sia-GalNAc-L-R) AA-GlcNAc-GlcNAc-Man (“Sia-GalNAc)), a glucosyl moiety, a glucosyl linking group (i.e., glucosyl linking group-polymeric modifying olo Man-(GlcNAc-Gal)-R''. group (Glc-L-R), or a sialyl moiety to which is bound a (Fuc) Man-(GlcNAc-Gal)-R' polymer modified glucosyl moiety (e.g., Sia-Glc-L-R) ("Sia-Glc)), a glucosaminyl moiety, a glucosaminyl linking AA-GlcNAc-GlcNAc-Man group (i.e., glucosaminyl linking group-polymeric modifying group (GlcNAc-L-R), or a sialyl moiety to which is bound a olo nicknacay R15; and polymer modified glucosaminyl moiety (e.g., Sia-GlcNAc (GlcNAc-Gal)-R' L-R) ("Sia-GlcNAc)), a mannosyl moiety, a mannosyl linking group (i.e., mannosyl linking group-polymeric modi US 2010/0041872 A1 Feb. 18, 2010 34 fying group (Man-L-R), or a sialyl moiety to which is bound a polymer modified mannosyl moiety (e.g., Sia-Man-L-R) -continued ("Sia-Man)), a fucosyl moiety, a fucosyl linking group (i.e., fucosyl linking group-polymeric modifying group (Fuc-L- R"), or a sialyl moiety to which is bound a polymer modified fucosyl moiety (e.g., Sia-Fuc-L-R) ("Sia-Fuc”)). Exem op (Fuc), yaclescue plary polymer modified saccharyl moieties have a structure AA-GlcNAc-GlcNAc-Man according to Formulae I, Ia, II or IIa. An exemplary peptide olo Man-GlcNAc-Gal-R'; conjugate of the invention will include at least one glycan having a R' that includes a structure according to Formulae I, Ia, II and IIa. The oxygen, with the open valence, of For mulae I, Ia, II or IIa is preferably attached through a glyco and combinations thereof. sidic linkage to a carbon of a Gal or GalNAc moiety. In a further exemplary embodiment, the oxygen is attached to the (0163. In each of the formulae above, R' is as discussed carbon at position 3 of a galactose residue. In an exemplary above. Moreover, an exemplary peptide conjugate of the embodiment, the modified sialic acid is linked C-2,3-to the invention will include at least one glycan with an R' moiety galactose residue. In another exemplary embodiment, the having a structure according to Formulae I, Ia, II or IIa. sialic acid is linked C-2,6-to the galactose residue. 0164. In another exemplary embodiment, the glycosyl 0161 In an exemplary embodiment, the sialyl linking linking group comprises at least one glycosyl linking group group is a sialyl moiety to which is bound a polymer modified having the formula: sialyl moiety (e.g., Sia-Sia-L-R) ("Sia-Sia”). Here, the gly cosyl linking group is linked to a galactosyl moiety through a sialyl moiety: (GlcNAc-Gal)-R'; and (GlcNAc-Gal)-Sia-R'

Gal-Sia-Sia-L-R'. wherein R' is said sialyl linking group; and the index p is an integer selected from 1 to 10. An exemplary species according to this motif is prepared by 0.165. In an exemplary embodiment, the glycosyl linking conjugating Sia-L-R' to a terminal sialic acid of a glycan moiety has the formula:

OH HO

HO HOOC O O GlcNAc-i-Gal-O H NH ul-S r N-re O 1-1f OCH OH O using an enzyme that forms Sia-Sia bonds, e.g., CST-II, in which b is an integer from 0 to 1. The index s represents an ST8Sia-II, ST8Sia-III and ST8Sia-IV. integer from 1 to 10; and the index frepresents an integer from 0162. In another exemplary embodiment, the glycans on 1 to 2500. the peptide conjugates have a formula that is selected from the group: 0166 In an exemplary embodiment, the polymeric modi fying group is PEG. In another exemplary embodiment, the PEG moiety has a molecular weight of about 20 kD. In (Fuc), Man another exemplary embodiment, the PEG moiety has a molecular weight of about 5 kD. In another exemplary AA-GlcNAc-GlcNAc-Man embodiment, the PEG moiety has a molecular weight of olo Man-GlcNAc-Gal-R'': about 10 kD. In another exemplary embodiment, the PEG moiety has a molecular weight of about 40 kD. In other embodiments, the modifying group is a branched poly(alky lene oxide), e.g., poly(ethylene glycol), having a molecular op (Fuc), unaccur. and weight of at least about 80 kD. preferably at least about 100 AA-GlcNAc-GlcNAc-Man kD, more preferably at least about 120 kD. at least about 140 olo l kD or at least about 160 kD. In yet another embodiment, the branched poly(alkylene oxide), e.g., poly(ethylene glycol) is US 2010/0041872 A1 Feb. 18, 2010

at least about 200 kD, such as from at least about 80 kD to at least about 200 kD, including at least about 160 kD and at least about 180 kD. 0167. In an exemplary embodiment, the glycosyl linking group is a linear SA-PEG-10 kD moiety, and one or two of CAA these glycosyl linking groups are covalently attached to the (CAA),5. A 6 peptide. In another exemplary embodiment, the glycosyl linking group is a linear SA-PEG-20 kD moiety, and one or two of these glycosyl linking groups are covalently attached Accio-Hi to the peptide. In an exemplary embodiment, the glycosyl (CAA) linking group is a linear SA-PEG-5 kD moiety, and one, two CA10All or three of these glycosyl linking groups are covalently attached to the peptide. In an exemplary embodiment, the glycosyl linking group is a linear SA-PEG-40 kD moiety, and one or two of these glycosyl linking groups are covalently attached to the peptide. and the peptide in the peptide conjugate is a member selected 0.168. In another exemplary embodiment, the glycosyl from the peptides in FIG. 7. In another exemplary embodi linking group is a sialyl linking group having the formula: ment, the peptide in the peptide conjugate is a member selected from bone morphogenetic proteins (e.g., BMP-1, HO BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, OH BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, HOOC. O BMP-15), neurotrophins (e.g., NT-3, NT-4, NT-5), growth OH differentiation factors (e.g., GDF-5), glial cell line-derived -o O neurotrophic factor (GDNF), brain derived neurotrophic fac NH l O tor (BDNF), nerve growth factor (NGF), von Willebrand fac OH 1.N. o1-k n-1N-Q. tor (vWF) protease, Factor VII, Factor VIa, Factor VIII, Fac O tor IX, Factor X, Factor XI, B-domain deleted Factor VIII, VWF-Factor VIII fusion protein having full-length Factor In another exemplary embodiment, Q is a member selected VIII, VWF-Factor VIII fusion protein having B-domain from Hand CH. In another exemplary embodiment, wherein deleted Factor VIII, erythropoietin (EPO), granulocyte said glycosyl linking group has the formula: colony stimulating factor (G-CSF), Granulocyte-Macroph age Colony Stimulating Factor (GM-CSF)interferon alpha, interferon beta, interferon gamma, C.-antitrypsin (ATT, or C-1 protease inhibitor), glucocerebrosidase, Tissue-Type Plasminogen Activator (TPA), Interleukin-2 (IL-2), uroki (GlcNAc-Gal)-R'; and (GlcNAc-Gal)-Sia-R's nase, human DNase, insulin, Hepatitis B surface protein (Hb SAg), human growth hormone, TNF Receptor-IgGFc region fusion protein (EnbrelTM), anti-HER2 monoclonal antibody wherein R' is said sialyl linking group; and the index p is an (HerceptinTM), monoclonal antibody to Protein F of Respira integer selected from 1 to 10. In an exemplary embodiment, tory Syncytial Virus (SynagisTM), monoclonal antibody to the glycosyl linking group comprises the formula: TNF-C. (RemicadeTM), monoclonal antibody to glycoprotein

OH HO

HO HOOC O O GlcNAc--Gal--O H NH S N r N-re O ---"f OH O wherein the index b is an integer selected from 0 and 1. In an IIb/IIIa (ReoproTM), monoclonal antibody to CD20 (Rit exemplary embodiment, the index S is 1; and the index fis an uxanTM), anti-thrombin III (ATIII), human Chorionic Gona integer selected from about 200 to about 300. dotropin (hCG), alpha-galactosidase (FabrazymeTM), alpha iduronidase (AldurazymeTM), follicle stimulating hormone, II. D. Modifying Groups beta-glucosidase, anti-TNF-alpha monoclonal antibody, glu 0169. The peptide conjugates of the invention comprise a cagon-like peptide-1 (GLP-1), glucagon-like peptide-2 modifying group. This group can be covalently attached to a (GLP-2), beta-glucosidase, alpha-galactosidase A and fibro peptide through an amino acid or a glycosyl linking group. In blast growth factor. “Modifying groups' can encompass a another exemplary embodiment, when the modifying group variety of structures including targeting moieties, therapeutic includes the moiety: moieties, biomolecules. Additionally, "modifying groups” US 2010/0041872 A1 Feb. 18, 2010 36 include polymeric modifying groups, which are polymers which can alter a property of the peptide Such as its bioavail -continued ability or its half-life in the body. H 0170 In an exemplary embodiment, the polymeric modi H (OCH2CH2)OCH fying group has a structure including a moiety according to the following formulae: CHO(CH2CH2O). H H O H O (OCH2CH2)A hi, "Null H A’(CH2CH2O) H 0173 For the purposes of convenience, the modifying O O groups in the remainder of this section will be largely based on polymeric modifying groups such as water Soluble and r water insoluble polymers. However, one of skill in the art will itsul 3, recognize that other modifying groups, such as targeting moi N and eties, therapeutic moieties and biomolecules, could be used in (OCH2CH2)A place of the polymeric modifying groups. In addition, those of skill will appreciate that the reliance on branched PEG hi, structures set forth above is simply for clarity of illustration, the PEG can be replaced by substantially any polymeric moi A’(CH2CH2O) H ety, including, without limitation those species set forth in the definition of “polymeric moiety’ found herein. II. D. i. Linkers of the Modifying Groups 0.174. The linkers of the modifying group serve to attach the modifying group (ie polymeric modifying groups, target ing moieties, therapeutic moieties and biomolecules) to the Ox peptide. In an exemplary embodiment, the polymeric modi fying group is bound to a glycosyl linking group, generally 0171 In another exemplary embodiment according to the through a heteroatom, e.g., nitrogen, on the core through a formula above, the polymeric modifying group includes a linker, L, as shown below: moiety according to the following formula:

H (R)-L H (OCH2CH2)A.

A’(CH2CH2O) H R" is the polymeric moiety and L is selected from a bond and a linking group. The index w represents an integer selected H La from 1-6, preferably 1-3 and more preferably 1-2. Exemplary H linking groups include Substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl moieties and sialic acid. An exemplary component of the linker is an acyl moiety. In an exemplary embodiment, A' and A are each members 0.175. An exemplary compound according to the invention selected from —OH and —OCH. has a structure according to Formulae I, Ia, II or IIa above, in 0172 Exemplary polymeric modifying groups according which at least one of R,R,R,R,R or Rhas the formula: to this embodiment include the moiety:

H NH-L-RI. H (OCH2CH2)OCH

CHO(CH2CH2O). H (0176) In another example according to this embodiment at O least one of R,R,R,R,R or R has the formula: H H O HN\- HN and in which s is an integer from 0 to 20 and R' is a linear polymeric modifying moiety. US 2010/0041872 A1 Feb. 18, 2010 37

0177. In an exemplary embodiment, the polymeric modi molecule (U.S. Pat. No. 4,412.989), ribonuclease and super fying group-linker construct is a branched structure that oxide dismutase (Veronese at al., App. Biochem. Biotech. 11: includes two or more polymeric chains attached to central 141-45 (1985)). moiety. In this embodiment, the construct has the formula: 0183 Exemplary water-soluble polymers are those in which a substantial proportion of the polymer molecules in a sample of the polymer are of approximately the same molecu lar weight; such polymers are "homodisperse.” (R)-L 0.184 The present invention is further illustrated by refer ence to a poly(ethylene glycol) conjugate. Several reviews and monographs on the functionalization and conjugation of PEG are available. See, for example, Harris, Macronol. in which R" and L are as discussed above and w' is an integer Chem. Phys. C25: 325-373 (1985); Scouten, Methods in from 2 to 6, preferably from 2 to 4 and more preferably from Enzymology 135:30-65 (1987); Wong et al., Enzyme Microb, 2 to 3. Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992); 0.178 When L is a bond it is formed between a reactive Zalipsky, Bioconjugate Chem. 6: 150-165 (1995); and functional group on a precursor of R' and a reactive func Bhadra, et al., Pharmazie, 57:5-29 (2002). Routes for prepar tional group of complementary reactivity on the saccharyl ing reactive PEG molecules and forming conjugates using the core. When L is a non-zero order linker, a precursor of L can reactive molecules are known in the art. For example, U.S. be in place on the glycosyl moiety prior to reaction with the Pat. No. 5,672,662 discloses a water soluble and isolatable R" precursor. Alternatively, the precursors of R' and L can be conjugate of an active ester of a polymer acid selected from incorporated into a preformed cassette that is Subsequently linear or branched poly(alkylene oxides), poly(oxyethylated attached to the glycosyl moiety. As set forth herein, the selec polyols), poly(olefinic alcohols), and poly(acrylomorpho tion and preparation of precursors with appropriate reactive line). functional groups is within the ability of those skilled in the 0185 U.S. Pat. No. 6,376,604 sets forth a method for art. Moreover, coupling the precursors proceeds by chemistry preparing a water-soluble 1-benzotriazolylcarbonate ester of that is well understood in the art. a water-soluble and non-peptidic polymer by reacting a ter 0179. In an exemplary embodiment, L is a linking group minal hydroxyl of the polymer with di(1-benzotriazoyl)car that is formed from an amino acid, or small peptide (e.g., 1-4 bonate in an organic solvent. The active ester is used to form amino acid residues) providing a modified Sugar in which the conjugates with a biologically active agent such as a protein polymeric modifying group is attached through a substituted or peptide. alkyl linker. Exemplary linkers include glycine, lysine, serine 0186 WO 99/45964 describes a conjugate comprising a biologically active agent and an activated water Soluble poly and cysteine. The PEG moiety can be attached to the amine mer comprising a polymer backbone having at least one ter moiety of the linker through an amide or urethane bond. The minus linked to the polymer backbone through a stable link PEG is linked to the sulfur or oxygen atoms of cysteine and age, wherein at least one terminus comprises a branching serine through thioether or ether bonds, respectively. moiety having proximal reactive groups linked to the branch 0180. In an exemplary embodiment, Rincludes the poly ing moiety, in which the biologically active agent is linked to meric modifying group. In another exemplary embodiment, at least one of the proximal reactive groups. Other branched Rincludes both the polymeric modifying group and a linker, poly(ethylene glycols) are described in WO 96/21469, U.S. L., joining the modifying group to the remainder of the mol Pat. No. 5,932,462 describes a conjugate formed with a ecule. As discussed above, L can be a linear or branched branched PEG molecule that includes a branched terminus structure. Similarly, the polymeric modifying group can be that includes reactive functional groups. The free reactive branched or linear. groups are available to react with a biologically active spe II. D. ii. Water-Soluble Polymers cies, such as a protein or peptide, forming conjugates between 0181 Many water-soluble polymers are known to those of the poly(ethylene glycol) and the biologically active species. skill in the art and are useful in practicing the present inven U.S. Pat. No. 5,446,090 describes a bifunctional PEG linker tion. The term water-soluble polymer encompasses species and its use in forming conjugates having a peptide at each of Such as saccharides (e.g., dextran, amylose, hyalouronic acid, the PEG linker termini. poly(sialic acid), heparans, heparins, etc.); poly (amino 0187 Conjugates that include degradable PEG linkages acids), e.g., poly(aspartic acid) and poly(glutamic acid); are described in WO99/34833; and WO 99/14259, as well as nucleic acids; synthetic polymers (e.g., poly(acrylic acid), in U.S. Pat. No. 6,348,558. Such degradable linkages are poly(ethers), e.g., poly(ethylene glycol); peptides, proteins, applicable in the present invention. and the like. The present invention may be practiced with any 0188 The art-recognized methods of polymer activation water-soluble polymer with the sole limitation that the poly set forth above are of use in the context of the present inven mer must include a point at which the remainder of the con tion in the formation of the branched polymers set forth herein jugate can be attached. and also for the conjugation of these branched polymers to 0182 Methods for activation of polymers can also be other species, e.g., Sugars, Sugar nucleotides and the like. found in WO 94/17039, U.S. Pat. No. 5,324,844, WO 0189 An exemplary water-soluble polymer is poly(ethyl 94/18247, WO 94/04.193, U.S. Pat. No. 5,219,564, U.S. Pat. ene glycol), e.g., methoxy-poly(ethylene glycol). The poly No. 5,122,614, WO 90/13540, U.S. Pat. No. 5,281,698, and (ethylene glycol) used in the present invention is not more WO93/15189, and for conjugation between activated restricted to any particular form or molecular weight range. polymers and peptides, e.g. Coagulation Factor V111 (WO For unbranched poly(ethylene glycol) molecules the molecu 94/15625), hemoglobin (WO 94/09027), oxygen carrying lar weight is preferably between 500 and 100,000. A molecu US 2010/0041872 A1 Feb. 18, 2010 lar weight of 2000-60,000 is preferably used and preferably of from about 5,000 to about 40,000. -continued II. D. iii. Branched Water Soluble Polymers O 0190. In another embodiment the polymeric modifying moiety is a branched PEG structure having more than one HO O-(CH2CH2O)CH linear or branched PEG moieties attached. Examples of branched PEGs are described in U.S. Pat. No. 5,932,462; U.S. NHC(O)CCH2CH2(OCH2CH2)AOCHs Pat. No. 5,342,940; U.S. Pat. No. 5,643,575; U.S. Pat. No. O 5,919,455; U.S. Pat. No. 6,113,906; U.S. Pat. No. 5,183,660; WO 02/09766: Kodera Y., Bioconjugate Chemistry 5: 283 HO O-(CH2CH2O)CH: 288 (1994); and Yamasaki et al., Agric. Biol. Chem., 52: NHC(O)CHCHOCH 2125-2127, 1998. O 0191 Representative polymeric modifying moieties include structures that are based on side chain-containing amino acids, e.g., serine, cysteine, lysine, and Small peptides, HO S-(CH2CH2O)CH3; and e.g., lys-lys. In some embodiments, the polymeric modifying NHC(O)OCH moiety is a branched PEG moiety that is based upon an O oligo-peptide. Such as a tri-lysine peptide. Exemplary amino acids Suitable for use include lysine, cysteine, and serine. In Such embodiments, each polymeric subunit attached to the HO S-(CH2CH2O)CH3. peptide structure may be either a linear PEG moiety or a NHC(O)CH branched PEG moiety. For example, the tri-lysine can be mono-, di-, tri-, or tetra-PEG-ylated with linear PEG moi eties, branched PEG moieties, or a combination of linear and Those of skill will appreciate that the free amine in the di branched PEG moieties. Exemplary branched structures lysine structures can also be pegylated through an amide or include the following moieties: urethane bond with a either a linear PEG moiety or a branched PEG moiety. O O (0192 It will be appreciated by one of skill in the art that in addition to the linear PEG structures shown above, the branched polymers exemplified in the previous sections can ------, also be attached to a branching moiety (e.g., cysteine, serine, lysine, and oligomers of lysine) in place of one or more of the iss---f linear PEG structures. In addition, those of skill will appre O ciate that the reliance on PEG structures set forth above is simply for clarity of illustration, the PEG can be replaced by Substantially any polymeric moiety, including, without limi tation those species set forth in the definition of “polymeric moiety’ found herein. (0193 PEG of any molecular weight, e.g. 1 kD, 2 kD, 5kD, 10 kD, 15 kD, 20 kD, 25 kD, 30 kD, 35 kD, 40 kD and 45 kD is of use in the present invention. PEG of a larger molecular weight can also be used in the present invention, including up to about 200kD, such as at least about 180 kD, about 160 kD, about 140 kD, about 120 kD, about 100 kD, about 90 kD, about 80 kD, and about 70 kD. In certain embodiments the molecular weight of PEG is about 80 kD. In other embodi ments, the molecular weight of PEG is at least about 200 kD. at least about 180 kD, at least about 160 kD, or at least about 140 kD. 0194 Each PEG moiety of the branched polymeric modi fying moiety may have a molecular weight as defined above or the total molecular weight of all PEG moieties of the polymeric modifying moiety may be as defined above. For example, in certain embodiments each PEG moiety of the branched polymeric modifying moiety may be about 80kDor the total molecular weight of all PEG moieties of the poly meric modifying moiety may be about 80 kD. Likewise, in certain embodiments each PEG moiety of the branched poly meric modifying moiety may be about 200 kD or the total molecular weight of all PEG moieties of the polymeric modi fying moiety may be about 200 kD. US 2010/0041872 A1 Feb. 18, 2010 39

0.195 Exemplary species according to this embodiment have the formulae:

O NHC(O)OCH2CH2(OCH2CH2)OCH HO use).--Nicoloristol and

O g

use).--NicollectorO

in which the indices e. f and f are independently selected integers from 1 to 2500; and the indices q, q and q" are (IIIa) independently selected integers from 1 to 20. (OCH2CH2)A. 0196. As will be apparent to those of skill, the branched polymers of use in the invention include variations on the CAA themes set forth above. For example the di-lysine-PEG con (CAA),5. A 6 jugate shown above can include three polymeric Subunits, the third bonded to the O-amine shown as unmodified in the structure above. Similarly, the use of a tri-lysine functional Accio-(CAA) a ized with three or four polymeric subunits labeled with the polymeric modifying moiety in a desired manner is within the CA10All Scope of the invention. 0.197 As discussed herein, the PEG of use in the conju l gates of the invention can be linear or branched. An exem 0198 The branched polymer species according to this for plary precursor of use to form the branched PEG containing mula are essentially pure water-soluble polymers. X is a peptide conjugates according to this embodiment of the moiety that includes an ionizable (e.g., OH, COOH, HPO, invention has the formula: HSO, HPO, and salts thereof, etc.) or other reactive func tional group, e.g., infra. C is carbon. X. R'' and R'' are independently selected from non-reactive groups and poly meric moieties (e.g. poly(alkylene oxide), e.g., PEG). Non reactive groups include groups that are considered to be (III) essentially unreactive, neutral and/or stable at physiological R 16 X2 pH, e.g., H, substituted or unsubstituted alkyl, substituted or X-C-X3. unsubstituted heteroalkyl and the like. An exemplary poly meric moiety includes the branched structures set forth in Formula IIIa and its exemplars. One of skill in the art will appreciate that the PEG moiety in these formulae can be replaced with other polymers. Exemplary polymers include those of the poly(alkylene oxide) family. (e.g., H. unsubsti tuted alkyl, unsubstituted heteroalkyl) and polymeric arms Another exemplary precursor of use to form the branched (e.g., PEG). X and X’ are linkage fragments that are prefer PEG containing peptide conjugates according to this embodi ably essentially non-reactive under physiological conditions, ment of the invention has the formula: which may be the same or different. An exemplary linker US 2010/0041872 A1 Feb. 18, 2010 40 includes neither aromatic nor ester moieties. Alternatively, 0203 Exemplary polymeric modifying groups according these linkages can include one or more moiety that is to this embodiment include the moiety: designed to degrade under physiologically relevant condi tions, ;: esters, disulfides, etc. X and X join polymeric arms R and R7 to C. When X is reacted with a reactive functional group of complementary reactivity on a linker, H sugar or linker-sugarcassette, X is converted to a component H (OCH2CH2)OCH3 and oflinkage fragment X. (0199 Exemplary linkage fragments for X, X and X’ are CHO(CH2CH2O). H independently selected and include S, SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH and NHC(O)C, and OC(O)NH, CHS, CHO, CHCHO, CHCHS, (CH)O,

(CH)S or (CH)Y-PEG wherein, Y is S, NH, NHC(O), C(O)NH, NHC(O)O, OC(O)NH, or O and o is an integer from 1 to 50. In an exemplary embodiment, the linkage frag ments X and X’ are different linkage fragments. 0200. In an exemplary embodiment, the precursor (For mula III), or an activated derivative thereof, is reacted with, and thereby bound to a Sugar, an activated Sugar or a Sugar nucleotide through a reaction between X and a group of complementary reactivity on the Sugar moiety, e.g., an amine. Alternatively, X reacts with a reactive functional grip of precursor to linker, L. One or more of R,R,R,R,R or R of Formulae I, Ia, II or IIa can include the branched polymeric CHO(CH2CH2O). modifying moiety, or this moiety bound through L. 0201 In an exemplary embodiment, the polymeric modi fying group has a structure including a moiety according to the following formulae:

(OCH2CH2)A hi, 0204. In an exemplary embodiment, the moiety: A’(CH2CH2O) H X2 O rO ill itsuls N >, and (OCH2CH2)A is the linker arm, L. In this embodiment, an exemplary linker hi, is derived from a natural or unnatural amino acid, amino acid analogue or amino acid mimetic, or a small peptide formed A’(CH2CH2O) H from one or more Such species. For example, certain branched polymers found in the compounds of the invention have the formula: CX (IV) 0202 In another exemplary embodiment according to the X-. formula above, the branched polymer has a structure accord ing to the following formula: 0205 X is a linkage fragment that is formed by the reac H tion of a reactive functional group, e.g., X, on a precursor of H (OCH2CH2)A. the branched polymeric modifying moiety and a reactive functional group on the Sugar moiety, or a precursor to a A’(CH2CH2O) H linker. For example, when X is a carboxylic acid, it can be activated and bound directly to an amine group pendent from H La an amino-saccharide (e.g., Sia, GalNH, GlcNH, ManNH, H etc.), forming a X that is an amide. Additional exemplary reactive functional groups and activated precursors are described hereinbelow. The index c represents an integer In an exemplary embodiment, A' and A are each selected from 1 to 10. The other symbols have the same identity as from —OH and —OCH. those discussed above. US 2010/0041872 A1 Feb. 18, 2010

0206. In another exemplary embodiment, X* is a linking moiety formed with another linker: -continued

WIa (OCH2CH2)A

---- As5. A 6 (CAA), in which X* is a second linkage fragment and is indepen dently selected from those groups set forth for X', and, simi Accio-Hi89 lar to L., L' is a bond, substituted or unsubstituted alkyl or sawCA10All O O% n substituted or unsubstituted heteroalkyl. La 0207 Exemplary species for X and X include S, SC(O) NH, HNC(O)S, SC(O)O, O, NH, NHC(O), C(O)NH and NHC(O)O, and OC(O)NH. R4 R3 (0208. In another exemplary embodiment, X is a peptide bond to R', which is an amino acid, di-peptide (e.g., Lys Vb Lys) or tri-peptide (e.g., Lys-Lys-Lys) in which the alpha (OCH2CH2)A amine moiety(ies) and/or side chain heteroatom(s) are modi fied with a polymeric modifying moiety. CH2 0209. In a further exemplary embodiment, the peptide Accio- (- (R) conjugates of the invention include a moiety, e.g., an R' CH O R2 moiety that has a formula that is selected from: na O ; and R5 R3 V (R) R4 R 16 -X2 O R2 X-C-La O ; and WIb R17 -X R5 R3 (OCH2CH2)A CH R4 Accio-, - (R) % CH2 O O VI NLa (R) R 16 -X2 O O X-C-La R4 R3 R17 -X

R4 R3 WC R16-X2

Wa X--- O R2 OCH2CH2)A R17-X O ; and s R5 R3 5. A 6 Accio-HA(CAA), R4 (CAA) (R6) WIC CAA10 All O R2 Na X-C-Le O O O ; and US 2010/0041872 A1 Feb. 18, 2010 42

L' moieties include substituted or unsubstituted alkyl chains -continued that include one or more OH or NH. Vc 0210. In yet another exemplary embodiment, the inven tion provides peptide conjugates having a moiety, e.g., an R' CAA moiety with formula: (CAA),5. A 6 Accio-Hi VIII (CAA) O CA10All R16-X2

X4 r

WId IX (OCH2CH2)A O CAA R16-X2 % (CAA),5. A 6 L 5 O O X4 Accio-Hi(CAA) r R4 R3 VAlan y L O O The identity of the radicals represented by the various sym bols is the same as that discussed hereinabove. As those of skill will appreciate, the linker arm in Formulae VIII and IX is R4 R3 equally applicable to other modified Sugars set forth herein. In We exemplary embodiment, the species of Formulae VIII and IX (OCH2CH2)A are the R' moieties attached to the glycan structures set forth CH herein. 0211. In yet another exemplary embodiment, the peptide Accio- (- conjugate includes a R' moiety with a formula which is a CH2 member selected from:

OH

OH

OH WIe O R2 (OCH2CH2)A R 16 X2 O CH x | NLa R3 R17-X Accio-Hi R4 CH2V 7. (OCH2CH2)A CAA (CAA),5. A 6 pH

Accio-HA(CAA) in which the identity of the radicals represented by the various CA10All symbols is the same as that discussed hereinabove. L' is a bond or a linker as discussed above for Land L', e.g., substi tuted or unsubstituted alkyl or substituted or unsubstituted La heteroalkyl moiety. In an exemplary embodiment, L' is a moiety of the side chain of sialic acid that is functionalized with the polymeric modifying moiety as shown. Exemplary US 2010/0041872 A1 Feb. 18, 2010

A. A" and A' are members independently selected from H, -continued substituted or unsubstituted alkyl, substituted or unsubsti tuted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, CH OH - NA 12A, OA'? and SiA' A' A'° and A' are members independently selected from substituted or unsub A’(CH2CH2O). H OH O R2 stituted alkyl, substituted or unsubstituted heteroalkyl, sub CH stituted or unsubstituted cycloalkyl, substituted or unsubsti O tuted heterocycloalkyl, substituted or unsubstituted aryl, and La substituted or unsubstituted heteroaryl. R3 0212. The embodiments of the invention set forth above R4 are further exemplified by reference to species in which the polymer is a water-soluble polymer, particularly poly(ethyl ene glycol) ("PEG), e.g., methoxy-poly(ethylene glycol). Those of skill will appreciate that the focus in the sections that in which the identities of the radicals are as discussed above. follow is for clarity of illustration and the various motifs set An exemplary species for La is (CH),C(O)NH(CH),C forth using PEG as an exemplary polymer are equally appli (O)NH , in which the indices hand j are independently cable to species in which a polymer other than PEG is uti selected integers from 0 to 10. A further exemplary species is lized. —C(O)NH-. The indices m and n are integers indepen (0213. In an exemplary embodiment, the R' moiety has a dently selected from 0 to 5000. A", A, A, A, A, A, A, A, formula that is a member selected from the group:

HOOC O CH(OH)CH(OH)CH2OH *4.O OH

HOOC O CH(OH)CH(OH)CH2OH O O “4. NHC(O)(CH2)NH OH

HOOC O CH(OH)CH(OH)CHNH(CH)NH O “4. NHC(O)CH OH

HOOC O

OH US 2010/0041872 A1 Feb. 18, 2010 44

In each of the structures above, the linker fragment - NH kD, 20 kD, 25 kD, 30 kD, 35 kD, 40 kD and 45 kD. PEG of a (CH2) - can be present or absent. larger molecular weight can also be used in the present inven 0214. In other exemplary embodiments, the peptide con tion, including up to about 200 kD, such as at least about 180 jugate includes an R' moiety selected from the group: kD, about 160 kD, about 140 kD, about 120 kD, about 100

HO OH

HOOC O OH O O NH NH ls 1-k N-1NNo1 Q; OH O HO OH

HOOC O OH O | NH"1 ON-n O -N-99:f O HO OH

HOOC O O OH O -(N-9)

NH NH OH O "'N-ri,~)- O HO OH

HOOC O O OH O -(N-0)

NH NH OH O "N--- and O O

HO OH NH lsO 1n-N-1N e O-Q.

HOOC O OH O O

NH NH OH O

O

0215. In each of the formulae above, the indices e and fare kD, about 90 kD, about 80 kD, and about 70 kD. In certain independently selected from the integers from 1 to 2500. In embodiments the molecular weight of PEG is about 80 kD. In further exemplary embodiments, e and fare selected to pro other embodiments, the molecular weight of PEG is at least vide a PEG moiety that is about 1 kD, 2 kD, 5 kD, 10 kD, 15 about 200kD, at least about 180 kD, at least about 160 kD, or US 2010/0041872 A1 Feb. 18, 2010 at least about 140 kD. The symbol Q represents substituted or In each of the figures above, the indices e. f. f and f" represent unsubstituted alkyl (e.g., C-C alkyl, e.g., methyl), Substi integers independently selected from 1 to 2500. The indices q, tuted or unsubstituted heteroalkyl or H. 0216. Other branched polymers have structures based on q' and q" represent integers independently selected from 1 to di-lysine (Lys-Lys) peptides, e.g.: 20. It will be appreciated by one of skill in the art that in addition to the linear PEG structures shown above, the branched polymers exemplified in the previous sections can also be attached to a branching moiety (e.g., lysine, and oligomers of lysine) in place of one or more of the linear PEG Structures. 0217 In another exemplary embodiment, the modifying group:

has a formula that is a member selected from: -r-g-O |-r-soO and tri-lysine peptides (Lys-Lys-LyS), e.g.:

O

-- g O

NH

NH

La g

O

NH

NH US 2010/0041872 A1 Feb. 18, 2010 46

wherein Q is a member selected from H and substituted or -continued unsubstituted C-C alkyl. The indices e. f and fare integers O independently selected from 1 to 2500, and q and q are integers independently selected from 1 to 20. La aO-(CH2CH2O)-Q; and 0219. In another exemplary embodiment, the branched polymer has a structure including a moiety according to the NHC(O)CH2CH2(OCH2CH2)(OQ following formula: O

La aO-(CH2CH2O)-Q

(IIIa) (OCH2CH2)A wherein Q is a member selected from H and substituted or CAA unsubstituted C-C alkyl. The indices e and fare integers independently selected from 1 to 2500, and the index q is an (CAA), integer selected from 0 to 20. 0218 In another exemplary embodiment, the modifying accio(CAA) group: CA10All

La X5-1- Li has a formula that is a member selected from: in which the indices m and n are integers independently selected from 0 to 5000. A.A.A.A.A.A.A.A.A.A.' and A' are members independently selected from H, substi tuted or unsubstituted alkyl, substituted or unsubstituted het eroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubsti tuted aryl, substituted or unsubstituted heteroaryl, - NA'A'. - OA'° and SiA' A' A' and A' are mem bers independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted hetero cycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. 0220. Formula IIIa is a subset of Formula III. The struc tures described by Formula IIIa are also encompassed by Formula III. 0221. In an exemplary embodiment, the polymeric modi fying group has a structure including a moiety according to the following formulae:

O (OCH2CH2)A and CH O

i-C, s

US 2010/0041872 A1 Feb. 18, 2010 48

The indices a, b and d are integers from 0 to 20. The index c is an integer from 1 to 2500. The structures set forth above can be components of R". 0225. In another illustrative embodiment, a primary hydroxyl moiety of the sugar is functionalized with the modi fying group. For example, the 9-hydroxyl of sialic acid can be converted to the corresponding amine and functionalized to provide a compound according to the invention. Formulae according to this embodiment include:

The structures set forth above can be components of R". 0226. Although the present invention is exemplified in the preceding sections by reference to PEG, as those of skill will appreciate, an array of polymeric modifying moieties is of use in the compounds and methods set forth herein. 0227. In selected embodiments, R' or L-R' is a branched PEG, for example, one of the species set forth above. In an exemplary embodiment, the branched PEG structure is based on a cysteine peptide. Illustrative modified Sugars according to this embodiment include: US 2010/0041872 A1 Feb. 18, 2010 49

-continued HOOC O CH(OH)CH(OH)CHOH HO O

NHC(O)(CH2)NH S-(CH2CH2O)CH OH NHC(O)XCH2CH2(OCH2CH2)(OCH3: O

HOOC O CH(OH)CH(OH)CHNH(CH2)NH S-(CH2CH2O)CH HO NHC(O)xCH2CH2(OCH2CH2).OCHs: NHC(O)CH OH O

HOOC O CH(OH)CH(OH)CH-NH(CH2)NHC(O)O(CH2)(OCH2CH2)O(CH2)NH S-(CH2CH2O)CH HO NHC(O)XCH2CH2(OCH2CH3),OCH, NHC(O)CH OH in which X* is a bondor O. In each of the structures above, the Gal to the mannose core and glycopegylated using a sialic alkylamine linker —(CH2)NH- can be present or absent. acid bearing a linear PEG moiety, affording a peptide that The structures set forth above can be components of R/R'. comprises at least one moiety having the formula:

OH HO

HO HOOC O O (Fuc), Man-GlcNAc-Gal-O H GlcNAc-GlcNAc-Man NH ---rNY N-re tr-, -n-och Man OH O

0228. As discussed herein, the polymer-modified sialic in which the index t is an integer from 0 to 1; the index s acids of use in the invention may also be linear structures. represents an integer from 1 to 10; and the index frepresents Thus, the invention provides for conjugates that include a an integer from 1 to 2500. sialic acid moiety derived from a structure Such as: 0231. In one embodiment, the present invention provides a peptide conjugate comprising the following glycosyl linking group: OH HO HO OH HOOC O O HO COOM O OCH O NH O HO g 8 O HO O G-N H in which the indices q and e are as discussed above. OH 0229. Exemplary modified sugars are modified with water-soluble or water-insoluble polymers. Examples of use D is a member selected from -OH and R'-L-HN : G is a ful polymer are further exemplified below. member selected from R'-L-and-C(O)(C-C)alkyl-R'; R' 0230. In another exemplary embodiment, the peptide is is a moiety comprising a member selected from a straight derived from insect cells, remodeled by adding GlcNAc and chain poly(ethylene glycol) residue and branched poly(eth US 2010/0041872 A1 Feb. 18, 2010 50 ylene glycol) residue; and M is a member selected from H, a salt counterion and a single negative charge; L is a linker -continued which is a member selected from a bond, substituted or O unsubstituted alkyl and substituted or unsubstituted het eroalkyl. In an exemplary embodiment, when D is OH, G is a O-(CH2CH2O)CH R'-L-. In another exemplary embodiment, when G is C(O) (C-C)alkyl, D is R'-L-NH-. NHC(O)CCH2CH2(OCH2CH2)AOCH3: 0232. In an exemplary embodiment, L-R' has the formula:

whereine, f, mand n are integers independently selected from 1 to 2500; and q is an integer selected from 0 to 20. R1-HN 'd 0234. In an exemplary embodiment, R' has a structure that is a member selected from: wherein a is an integer selected from 0 to 20. O 0233. In an exemplary embodiment, R' has a structure that g NHC(O)OCH2CH2(OCH2CH2)OCH includes a moiety selected from: NH2 HN H q' NHC(OOCH2CH2(OCH2CH2)(OCH3: O (OCH2CH)OCHs. O NHC(O)CH2CH2(OCH2CH2)OCH CHO(CH2CH2O). H g NH H O HN H q' NHC(O)CH2CH2(OCH2CH2)(OCH3: O O

O O ls 1n-N-1N HN y-ricocciocicloch. and g N O e ‘O-Q; O O

HN y-rocinogion. O O

a S-(CH2CH2O)CH whereine, fand fare integers independently selected from 1 to 2500; and q and q'are integers independently selected from NHC(O)CH2CH2(OCH2CH2)(OCH3: 1 to 20. O 0235) In another exemplary embodiment, R' has a struc ture that is a member selected from: a S-(CH2CH2O)CH US 2010/0041872 A1 Feb. 18, 2010

0236. In another exemplary embodiment, R' has a struc -continued ture that is a member selected from:

O

whereine and fare integers independently selected from 1 to 25OO. 0237. In another exemplary embodiment, the glycosyl linker has the formula:

OH

COOH O HO O-Ga.

G-HN

OH

wherein the variables are as described above. whereine, fand fare integers independently selected from 1 0238. In another exemplary embodiment, the peptide con to 2500; and q and q'are integers independently selected from jugate comprises at least one of said glycosyl linker according 1 to 20. to a formula selected from:

OH

COOH HO O-Gali-GalNAc- M (Fuc), G-HN Man-GlcNAc-GlcNAc AA OH l olo. and

OH Man (Fuc) COOH Man-GlcNAc-GlcNAc AA HO O-Gali-GalNAc-Man olo

G - HIN

OH US 2010/0041872 A1 Feb. 18, 2010 52 wherein D and Gareas described above, AA is an amino acid residue of said peptide conjugate and t is an integer selected from 0 and 1. 0239. In another exemplary embodiment, the peptide con jugate comprises at least one of said glycosyl linker wherein each of said glycosyl linker has a structure which is a member independently selected from the following formulae:

OH D

O COOH HO O-Ga-GlcNAc-Man

G-HN (Fuc), OH opo OH Man-GlcNAc-GlcNAc-AA D olo O COOH HO O-Gal-GlcNAc-Man

G-HN

OH OH D

O COOH HO O-Gal-GlcNAc-Man G- N (Fuc), OH |opo Man-GlcNAc-GlcNAc-AA OH D olo O COOH HO O-Ga-GlcNAc-Man – HN O OH OH D

O COOH HO H3C O-Gal-GlcNAc-Man X- N (Fuc), O OH OH Man-GlcNAc-GlcNAc-AA D

O COOH HO O-Gal-GlcNAc-Man

OH