3, 2009 Sheet 68 of 128 US 2009/0297520 Al. PA-PiX) ...-â¢â¢. 0 OS -i. CU: 0 07-. 'â¢..J c. A « r ...... mined by high-throughput 384-well planar patch clamp elec- ...... GVI INVKCKI SAQCL[Cit]PCKDAGMRFGACMNGKCACTPK- 2 515 amide ...... cisco), pp. 79-86 (1983). [0462] Compounds of the present invention can be changed.
Illllllllllllllllllllllllllllllllllllllllllllllllllllllllln US 20090297520A1
(19) United States (12) Patent Application Publication Sullivan et al. (54)
METHODS OF USING CONJUGATED TOXIN PEPTIDE THERAPEUTIC AGENTS
(76)
Inventors:
(22)
(io) Pub. No.: US 2009/0297520 Al (43) Pub. Date: Dec. 3, 2009 Filed:
Oct. 25, 2007 Related U.S. Application Data
John K. Sullivan, Newbury Park, CA (US); Joseph G. McGivern, Oak Park, CA (US); Leslie P. Miranda, Thousand Oaks, CA (US); Hung Q. Nguyen, Thousand Oaks, CA (US); Kenneth W. Walker, Newbury Park, CA (US); Shaw-Fen Sylvia Hu, Thousand Oaks, CA (US); Colin V. Gegg, JR., Newbury Park, CA (US); Taruna Arora Khare, Thousand Oaks, CA (US); Beverly S. Adler, Newbury Park, CA (US); Francis H. Martin, Newbury Park, CA (US)
Correspondence Address: AMGEN INC. MAIL STOP 28-2-C, ONE AMGEN CENTER DRIVE THOUSAND OAKS, CA 91320-1799 (US) (21)
Appl.No.:
11/978,111
(60)
Provisional application No. 60/854,674, filed on Oct. 25, 2006, provisional application No. 60/995,370, filed on Sep. 25, 2007. Publication Classification
(51)
Int. CI. ^62^^9/^95 A61K 38/16 (52) U.S. CI (57)
424/134.1; 514/12 ABSTRACT
Disclosed is a composition of matter comprising an OSK1 peptide analog, and in some embodiments, a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprises the composition and a pharmaceutically acceptable carrier. Also disclosed are DNAs encoding the inventive composition of matter, an expression vector comprising the DNA, and host cells comprising the expression vector. Methods of treating an autoimmune disorder and of preventing or mitigating a relapse of a symptom of multiple sclerosis are also disclosed.
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Patent Application Publication
Dec. 3, 2009 Sheet 14 of 128
US 2009/0297520 Al
FIG. 11A Ndel I CATATGGACAAAACTCACACATGTCCACCTTGTCCAGCTCCGGAACTCCTGGGGGGACCG 5131 + + + + + + 5190 GTATACCTGTTTTGAGTGTGTACAGGTGGAACAGGTCGAGGCCTTGAGGACCCCCCTGGC MDKTHTCPPCPAPELLGGP
5191
TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG + + + + + + 52 50 AGTCAGAAGGAGAAGGGGGGTTTTGGGTTCCTGTGGGAGTACTAGAGGGCCTGGGGACTC SVFLFPPKPKDTLMISRTPE
5251
GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC + + + • + + + 5310 CAGTGTACGCACCACCACCTGCACTCGGTGCTTCTGGGACTCCAGTTCAAGTTGACCATG VTCVVVDVSHEDPEVKFNWY
5311
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC + + + + + + 537 0 CACCTGCCGCACCTCCACGTATTACGGTTCTGTTTCGGCGCCCTCCTCGTCATGTTGTCG VDGVEVHNAKTKPREEQYNS
5371
acgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggag + + + + + + 5430 tgcatggcacaccagtcgcaggagtggcaggacgtggtcctgaccgacttaccgttcctc TYRVVSVLTVLHQDWLNGKE
5 4 31
tacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaa + + + + + + 5490 atgttcacgttccagaggttgtttcgggagggtcgggggtagctcttttggtagaggttt YKCKVSNKALPAPIEKTISK
54 91
gccaaagggcagcccCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTG + + + + + + 5550 cggtttcccgtcgggGCTCTTGGTGTCCACATGTGGGACGGGGGTAGGGCCCTACTCGAC AKGQPREPQVYTLPPSRDEL
5 551
ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC + + + + + + 5610 TGGTTCTTGGTCCAGTCGGACTGGACGGACCAGTTTCCGAAGATAGGGTCGCTGTAGCGG TKNQVSLTCLVKGFYPSDIA
5 611
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG + + + + + + 567 0 CACCTCACCCTCTCGTTACCCGTCGGCCTCTTGTTGATGTTCTGGTGCGGAGGGCACGAC VEWESNGQPENNYKTTPPVL
Patent Application Publication
Dec. 3, 2009 Sheet 15 of 128
US 2009/0297520 Al
FIG. 11B 5 671
GACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG + + + + + + 5730 CTGAGGCTGCCGAGGAAGAAGGAGATGTCGTTCGAGTGGCACCTGTTCTCGTCCACCGTC DSDGSFFLYSKLTVDKSRWQ
57 31
CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG + + + + + + 5790 GTCCCCTTGCAGAAGAGTACGAGGCACTACGTACTCCGAGACGTGTTGGTGATGTGCGTC QGNVFSCSVMHEALHNHYTQ
BsmBI I AAGAGCCTCTCCCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTTGAGACGCTGCAGGACGT 57 91 + + + + + + 5850 TTCTCGGAGAGGGACAGAGGCCCATTTCCACCTCCACCACCAACTCTGCGACGTCCTGCA KSLSLSPGKGGGGG 5851
TGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGT + + + + + + 5910 ACTAGCCGTGCATTCTCCAAGGTTGAAAGTGGTATTACTTTATTCTAGTGATGGCCCGCA
5911
ATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCCAAAATGGAGAAAAAAATCAC + + + + + + 5 970 TAAAAAACTCAATAGCTCTAAAAGTCCTCGATTCCTTCGGTTTTACCTCTTTTTTTAGTG M
5971
E
K
K
I
T
-
TGGATATACCACCgttgatatatcccaatggcatcgtaaagaacatttTGaggcatTtca + + + + + + 6030 ACCTATATGGTGGcaactatatagggttaccgtagcatttcttgtaaaACtccgtaAagt GYTTVDISQWHRKEHFEAFQ-
6031
gtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaa + + + + + + 6090 cagtcaacgagttacatggatattggtctggcaagtcgacctataatgccggaaaaattt SVAQCTYNQTVQLDITAFLK-
60 91
gaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcct + + + + + + 6150 ctggcatttctttttattcgtgttcaaaataggccggaaataagtgtaagaacgggcgga TVKKNKHKFYPAFIHILARL-
6151
gatgaatgctcatccggaattccgtatggcaATGAAAGACGGTGAGCTGGTGATATGGGA + + + + + + 6210 ctacttacgagtaggccttaaggcataccgtTACTTTCTGCCACTCGACCACTATACCCT MNAHPEFRMAMKDGELVIWD-
Patent Application Publication
Dec. 3, 2009 Sheet 16 of 128
US 2009/0297520 Al
FIG. 11C 6211
TAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTATG + + + + + + 6270 ATCACAAGTGGGAACAATGTGGCAAAAGGTACTCGTTTGACTTTGCAAAAGTAGCGATAC SVHPCYTVFHEQTETFSSLW-
6271
GAGTGAATACCACGACGATTCCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTG + + + + + + 6330 CTCACTTATGGTGCTGCTAAGGGCCGTCAAAGATGTGTATATAAGCGTTCTACACCGCAC SEYHDDSRQFLHIYSQDVAC-
6331
TTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTTTC + + + + + + 6390 AATGCCACTTTTGGACCGGATAAAGGGATTTCCCAAATAACTCTTATACAAAAAGCAAAG YGENLAYFPKGFIENMFFVS-
63 91
AGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTT + + + + + + 6450 TCGGTTAGGGACCCACTCAAAGTGGTCAAAACTAAATTTGCACCGGTTATACCTGTTGAA ANPWVSFTSFDLNVANMDNF-
64 51
CTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCC + + + + + + 6510 GAAGCGGGGGCAAAAGTGGTACCCGTTTATAATATGCGTTCCGCTGTTCCACGACTACGG FAPVFTMGKYYTQGDKVLMP-
6511
GCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAA + + + + + + 657 0 CGACCGCTAAGTCCAAGTAGTACGGCAAACACTACCGAAGGTACAGCCGTCTTACGAATT LAIQVHHAVCDGFHVGRMLN-
6571
TGAATTACAACAGTACTGCGATGAGTGGCGGGGCGGGGCGTAATTTTTTTAAGGCAGTTA + + + + + + 6630 ACTTAATGTTGTCATGACGCTACTCACCGCCCCGCCCCGCATTAAAAAAATTCCGTCAAT ELQQYCDEWRGGA* BamHI BsmBI |
I
I
TTGGTGGCGCGCCCGTCTCTTAAGGATCCG 6631 + + + 6660 AACCACCGCGCGGGCAGAGAATTCCTAGGC
Patent Application Publication
Dec. 3, 2009 Sheet 17 of 128
US 2009/0297520 Al
FIG. 11D pAMG21ampR-Fc-Pep 7000 BamHI
7027
BsmBI
6000 BsmBI
2000 Duplex encoding the peptide replaces the CmR cassette fusing the peptide in frame to the C-terminus of Fc
4000
3000
Patent Application Publication
Dec. 3, 2009 Sheet 18 of 128
US 2009/0297520 Al
FIG. 12A BsmBI Ndel
I CATATGCGAGACGGGGCGGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTAT 5131 + + + + + + 5190 GTATACGCTCTGCCCCGCCACAACTGTTAATTAGTAGCCGTATCATATAGCCGTATCATA M
5191
AATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCA + + + + + + 5250 TTATGCTGTTCCACTCCTTGATTTGGTACCGGTTCAACTGGTCACGGCAAGGCCACGAGT MAKLTSAVPVLT-
52 51
CCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGG 1+ + + + + 5310 GGCGCGCGCTGCAGCGGCCTCGCCAGCTCAAGACCTGGCTGGCCGAGCCCAAGAGGGCCC ARDVAGAVEFWTDRLGFSRD-
5311
ACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCG + + + + + + 5370 TGAAGCACCTCCTGCTGAAGCGGCCACACCAGGCCCTGCTGCACTGGGACAAGTAGTCGC FVEDDFAGVVRDDVTLFI
5371
SA-
CGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGG + + + + + + 5430 GCCAGGTCCTGGTCCACCACGGCCTGTTGTGGGACCGGACCCACACCCACGCGCCGGACC VQDQVVPDNTLAWVWVRGLD-
54 31
ACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGC + + + + + + 54 90 TGCTCGACATGCGGCTCACCAGCCTCCAGCACAGGTGCTTGAAGGCCCTGCGGAGGCCCG ELYAEWSEVVSTNFRDASGP-
5 4 91
CGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGG + + + + + + 5550 GCCGGTACTGGCTCTAGCCGCTCGTCGGCACCCCCGCCCTCAAGCGGGACGCGCTGGGCC AMTEIGEQPWGREFALRDPA-
5551
CCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTCCGACGGCGGCGTC + + + + + + sglO GGCCGTTGACGCACGTGAAGCACCGGCTCCTCGTCCTGACTGTGCAGGCTGCCGCCGCAG GNCVHFVAEEQD
Patent Application Publication
Dec. 3, 2009 Sheet 19 of 128
US 2009/0297520 Al
FIG. 12B BsmBI I TCAGGTGGTGGTGGTGGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTC 5 611 + + + + + + 5670 AGTCCACCACCACCACCACTGTTTTGAGTGTGTACGGGTGGCACGGGTCGTGGACTTGAG GGGGGDKTHTCPPCPAPEL 5 671
CTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC + + + + + + 5730 GACCCCCCTGGCAGTCAGAAGGAGAAGGGGGGTTTTGGGTTCCTGTGGGAGTACTAGAGG LGGPSVFLFPPKPKDTLMIS
57 31
CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG + + + + + + 57 90 GCCTGGGGACTCCAGTGTACGCACCACCACCTGCACTCGGTGCTTCTGGGACTCCAGTTC RTPEVTCVVVDVSHEDPEVK
57 91
TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG + + + + + + 5850 AAGTTGACCATGCACCTGCCGCACCTCCACGTATTACGGTTCTGTTTCGGCGCCCTCCTC FNWYVDGVEVHNAKTKPREE
5851
CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG + + + + + + 5910 GTCATGTTGTCGTGCATGGCACACCAGTCGCAGGAGTGGCAGGACGTGGTCCTGACCGAC QYNSTYRVVSVLTVLHQDWL
5911
AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAA + + + + + + 5970 TTACCGTTCCTCATGTTCACGTTCCAGAGGTTGTTTCGGGAGGGTCGGGGGTAGCTCTTT NGKEYKCKVSNKALPAPIEK
5971
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC + + + + + + 6030 TGGTAGAGGTTTCGGTTTCCCGTCGGGGCTCTTGGTGTCCACATGTGGGACGGGGGTAGG TISKAKGQPREPQVYTLPPS
60 31
CGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCC + + + + + + 6090 GCCCTACTCGACTGGTTCTTGGTCCAGTCGGACTGGACGGACCAGTTTCCGAAGATAGGG
Patent Application Publication
Dec. 3, 2009 Sheet 20 of 128
US 2009/0297520 Al
FIG. 12C RDELTKNQVSLTCLVKGFYP
60 91
AGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG + + + + + + 6150 TCGCTGTAGCGGCACCTCACCCTCTCGTTACCCGTCGGCCTCTTGTTGATGTTCTGGTGC SDIAVEWESNGQPENNYKTT
6151
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAG + + + + + + 6210 GGAGGGCACGACCTGAGGCTGCCGAGGAAGAAGGAGATGTCGTTCGAGTGGCACCTGTTC PPVLDSDGSFFLYSKLTVDK
6211
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC + + + + + + 6270 TCGTCCACCGTCGTCCCCTTGCAGAAGAGTACGAGGCACTACGTACTCCGAGACGTGTTG SRWQQGNVFSCSVMHEALHN BaraHI
I CACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAATGGATCC 6271 + + + + 6319 GTGATGTGCGTCTTCTCGGAGAGGGACAGAGGCCCATTTATTACCTAGG HYTQKSLSLSPGK*
Patent Application Publication
Dec. 3, 2009 Sheet 21 of 128
US 2009/0297520 Al
FIG. 12D pAMG21ampR-Pep-Fc
BamHI
6687
1000 BsmBI Duplex encoding the peptide replaces the ZeoR cassette fusing the peptide in frame to the N-terminus of Fc Ndel/ BsmBI 5000
2000
3000
Patent Application Publication
Dec. 3, 2009 Sheet 22 of 128
US 2009/0297520 Al
FIG. 12E BsmBl Ndel 5131
CATATGGGTCGAGACGGGGCGGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAG + + + + + + 5190 GTATACCCAGCTCTGCCCCGCCACAACTGTTAATTAGTAGCCGTATCATATAGCCGTATC M
5191
TATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGC + + + + + + 5250 ATATTATGCTGTTCCACTCCTTGATTTGGTACCGGTTCAACTGGTCACGGCAAGGCCACG *
52 51
G
YDKVRN*TMAKLTSAVPVL-
TCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCC + + + + + + 5310 AGTGGCGCGCGCTGCAGCGGCCTCGCCAGCTCAAGACCTGGCTGGCCGAGCCCAAGAGGG TARDVAGAVEFWTDRLGFSR-
5311
GGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCA + + + + + + 537 0 CCCTGAAGCACCTCCTGCTGAAGCGGCCACACCAGGCCCTGCTGCACTGGGACAAGTAGT DFVEDDFAGVVRDDVTLFIS-
5371
GCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCC + + + + + + 5430 CGCGCCAGGTCCTGGTCCACCACGGCCTGTTGTGGGACCGGACCCACACCCACGCGCCGG AVQDQVVPDNTLAWVWVRGL-
5431
TGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCG + + + + + + 54 90 ACCTGCTCGACATGCGGCTCACCAGCCTCCAGCACAGGTGCTTGAAGGCCCTGCGGAGGC DELYAEWSEVVSTNFRDASG-
5491
GGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACC + + + + + + 5550 CCGGCCGGTACTGGCTCTAGCCGCTCGTCGGCACCCCCGCCCTCAAGCGGGACGCGCTGG PAMTEIGEQPWGREFALRDP-
5551
CGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTCCGACGGCGGC + + + + + + 5610 GCCGGCCGTTGACGCACGTGAAGCACCGGCTCCTCGTCCTGACTGTGCAGGCTGCCGCCG AGNCVHFVAEEQD*
Patent Application Publication
Dec. 3, 2009 Sheet 23 of 128
US 2009/0297520 Al
FIG. 12F BsmBI
5 611
GTCTCAGGTGGTGGTGGTGGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA + + + + + + 5670 CAGAGTCCACCACCACCACCACTGTTTTGAGTGTGTACGGGTGGCACGGGTCGTGGACTT GGGGGDKTHTCPPCPAPE
5 671
CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC + + + + + + 5730 GAGGACCCCCCTGGCAGTCAGAAGGAGAAGGGGGGTTTTGGGTTCCTGTGGGAGTACTAG LLGGPSVFLFPPKPKDTLMI
5731
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC + + + + + + 5790 AGGGCCTGGGGACTCCAGTGTACGCACCACCACCTGCACTCGGTGCTTCTGGGACTCCAG SRTPEVTCVVVDVSHEDPEV
5791
AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG + + + + + + 5850 TTCAAGTTGACCATGCACCTGCCGCACCTCCACGTATTACGGTTCTGTTTCGGCGCCCTC KFNWYVDGVEVHNAKTKPRE
5851
GAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG + + + + + + 5910 CTCGTCATGTTGTCGTGCATGGCACACCAGTCGCAGGAGTGGCAGGACGTGGTCCTGACC EQYNSTYRVVSVLTVLHQDW
5911
CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG + + + + + + 5970 GACTTACCGTTCCTCATGTTCACGTTCCAGAGGTTGTTTCGGGAGGGTCGGGGGTAGCTC LNGKEYKCKVSNKALPAPIE
5971
AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA + + + + + + TTTTGGTAGAGGTTTCGGTTTCCCGTCGGGGCTCTTGGTGTCCACATGTGGGACGGGGGT
6030
KTISKAKGQPREPQVYTLPP 6031
TCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT + + + + + + gogo AGGGCCCTACTCGACTGGTTCTTGGTCCAGTCGGACTGGACGGACCAGTTTCCGAAGATA SRDELTKNQVSLTCLVKGFY
Patent Application Publication
Dec. 3, 2009 Sheet 24 of 128
US 2009/0297520 Al
FIG. 12G 6091
CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC + + + + + + 6150 GGGTCGCTGTAGCGGCACCTCACCCTCTCGTTACCCGTCGGCCTCTTGTTGATGTTCTGG PSDIAVEWESNGQPENNYKT
6151
ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC + + .-- + + + + 6210 TGCGGAGGGCACGACCTGAGGCTGCCGAGGAAGAAGGAGATGTCGTTCGAGTGGCACCTG TPPVLDSDGSFFLYSKLTVD
6211
AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC + + + + + + 6270 TTCTCGTCCACCGTCGTCCCCTTGCAGAAGAGTACGAGGCACTACGTACTCCGAGACGTG KSRWQQGNVFSCSVMHEALH BamHI
6271
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAATGGATCC + + + + + -- 6322 TTGGTGATGTGCGTCTTCTCGGAGAGGGACAGAGGCCCATTTATTACCTAGG NHYTQKSLSLSPGK*
Patent Application Publication
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*'*'*?•*% *yv -c •; • y 4 5' y T'C•* r >* vcrfryyv^.-j-yv-rfT T*«'*t*•?*
Patent Application Publication
Dec. 3, 2009 Sheet 47 of 128
•^KWWiw.-..
rip*
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US 2009/0297520 Al
Patent Application Publication
Dec. 3, 2009 Sheet 48 of 128
US 2009/0297520 Al
FIG. 27E 0.06 r. 0.05^ —i—•—i—-^
39005 56992 Mass (m/z)
73009
90004
Patent Application Publication
Dec. 3, 2009 Sheet 52 of 128
FIG, 28A
••"K-X-XVIMW.
f^
^ ^\ r\
V.^ ^
^w... \.^ ^..rf
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\
US 2009/0297520 Al
Patent Application Publication
Dec. 3, 2009 Sheet 53 of 128
US 2009/0297520 Al
FIG. 28C PA-Peak
310
320
330
Wavelength (nm)
FIG. 28D
u c TO •TO
c=
-O
< >
"%'-W
0.0
-3-2-10 1 2 Fc-L10-ShK[l-35](log,ng/ml) -A- Fc-L10-5hK -B- Fc-L10-5hK+1% Rat Serum
4
3
-JK- Fc control --X-- Fc control+io/o Rat Serum
FIG. 34B
3.0 n 2.5E 2.0o % 1.5 d 1.00.50.0 E
E
Z3
CO
oo oo r^ r^ co co m m
aauepiinqvaAiiepy
s
o m o mom CO CN
CN
in ro
CN
in
Patent Application Publication
Dec. 3, 2009 Sheet 76 of 128
US 2009/0297520 Al
FIG. 42A atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccgac taccttacctcgacccagaaagagaagaaggacagtcattgctgaccacaggtgaggctg MEWSWVFLFFLSVTTGVHSD aaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttc ttttgagtgtgtacgggtggcacgggtcgtggacttgaggacccccctggcagtcagaag KTHTCPPCPAPELLGGPSVF ctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc gagaaggggggttttgggttcctgtgggagtactagagggcctggggactccagtgtacg LFPPKPKDTLMISRTPEVTC gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggc caccaccacctgcactcggtgcttctgggactccagttcaagttgaccatgcacctgccg VVVDVSHEDPEVKFNWYVDG 24i
gtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt + + + + + + 300 cacctccacgtattacggttctgtttcggcgccctcctcgtcatgttgtcgtgcatggca VEVHNAKTKPREEQYNSTYR gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc caccagtcgcaggagtggcaggacgtggtcctgaccgacttaccgttcctcatgttcacg VVSVLTVLHQDWLNGKEYKC
3 61
421
4 81
541
aaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaaggg + + + + + + 42 0 ttccagaggttgtttcgggagggtcgggggtagctcttttggtagaggtttcggtttccc KVSNKALPAPIEKTISKAKG cagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaac + + + + + + 48o gtcggggctcttggtgtccacatgtgggacgggggtagggccctactcgactggttcttg QPREPQVYTLPPSRDELTKN caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgg + + + + + + 54 0 gtccagtcggactggacggaccagtttccgaagatagggtcgctgtagcggcacctcacc QVSLTCLVKGFYPSDIAVEW gagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgac + + + + + + ctctcgttacccgtcggcctcttgttgatgttctggtgcggagggcacgacctgaggctg ESNGQPENNYKTTPPVLDSD
6o0
Patent Application Publication
Dec. 3, 2009 Sheet 77 of 128
US 2009/0297520 Al
FIG. 42B 601
6 61
721
781
341
ggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac + + + + + + 660 ccgaggaagaaggagatgtcgttcgagtggcacctgttctcgtccaccgtcgtccccttg GSFFLYSKLTVDKSRWQQGN gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctc + + + + + + 720 cagaagagtacgaggcactacgtactccgagacgtgttggtgatgtgcgtcttctcggag VFSCSVMHEALHNHYTQKSL tccctgtctccgggtaaaggaggaggaggatccggaggaggaggaagcggcgtgatcatc + + + + + + 780 agggacagaggcccatttcctcctcctcctaggcctcctcctccttcgccgcactagtag SLSPGKGGGGSGGGGSGVII aacgtgaagtgcaagatcagccgccagtgcctggagccctgcaagaaggccggcatgcgc + + + + + + 84 0 ttgcacttcacgttctagtcggcggtcacggacctcgggacgttcttccggccgtacgcg NVKCKISRQCLEPCKKAGMR ttcggcaagtgcatgaacggcaagtgccactgcacccccaagtagtaa +
+
+
+
888
aagccgttcacgtacttgccgttcacggtgacgtgggggttcatcatt FGKCMNGKCHCTPK* *
Patent Application Publication
Dec. 3, 2009 Sheet 78 of 128
US 2009/0297520 Al
FIG. 43A atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccgac taccttacctcgacccagaaagagaagaaggacagtcattgctgaccacaggtgaggctg MEWSWVFLFFLSVTTGVHSD aaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttc ttttgagtgtgtacgggtggcacgggtcgtggacttgaggacccccctggcagtcagaag KTHTCPPCPAPELLGGPSVF ctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc gagaaggggggttttgggttcctgtgggagtactagagggcctggggactccagtgtacg LFPPKPKDTLMISRTPEVTC
181
gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggc + + + + + + 240 caccaccacctgcactcggtgcttctgggactccagttcaagttgaccatgcacctgccg VVVDVSHEDPEVKFNWYVDG gtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt cacctccacgtattacggttctgtttcggcgccctcctcgtcatgttgtcgtgcatggca VEVHNAKTKPREEQYNSTYR gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc caccagtcgcaggagtggcaggacgtggtcctgaccgacttaccgttcctcatgttcacg VVSVLTVLHQDWLNGKEYKC
3 61
aaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaaggg + + + + + + 420 ttccagaggttgtttcgggagggtcgggggtagctcttttggtagaggtttcggtttccc KVSNKALPAPIEKTISKAKG cagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaac gtcggggctcttggtgtccacatgtgggacgggggtagggccctactcgactggttcttg QPREPQVYTLPPSRDELTKN
4 81
541
caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgg + + + + + + 540 gtccagtcggactggacggaccagtttccgaagatagggtcgctgtagcggcacctcacc QVSLTCLVKGFYPSDIAVEW gagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgac + + + + + + 600 ctctcgttacccgtcggcctcttgttgatgttctggtgcggagggcacgacctgaggctg ESNGQPENNYKTTPPVLDSD
Patent Application Publication
Dec. 3, 2009 Sheet 79 of 128
US 2009/0297520 Al
FIG. 43B ggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac ccgaggaagaaggagatgtcgttcgagtggcacctgttctcgtccaccgtcgtccccttg GSFFLYSKLTVDKSRWQQGN 6 61
gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctc + + + + + + 720 cagaagagtacgaggcactacgtactccgagacgtgttggtgatgtgcgtcttctcggag VFSCSVMHEALHNHYTQKSL tccctgtctccgggtaaaggaggaggaggatccggaggaggaggaagcggcgtgatcatc agggacagaggcccatttcctcctcctcctaggcctcctcctccttcgccgcactagtag SLSPGKGGGGSGGGGSGVII aacgtgagctgcaagatcagccgccagtgcctggagccctgcaagaaggccggcatgcgc ttgcactcgacgttctagtcggcggtcacggacctcgggacgttcttccggccgtacgcg NVSCKISRQCLEPCKKAGMR ttcggcaagtgcatgaacggcaagtgccactgcacccccaagtagtaa aagccgttcacgtacttgccgttcacggtgacgtgggggttcatcatt FGKCMNGKCHCTPK* *
Patent Application Publication
Dec. 3, 2009 Sheet 80 of 128
US 2009/0297520 Al
FIG. 44A atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccgac taccttacctcgacccagaaagagaagaaggacagtcattgctgaccacaggtgaggctg MEWSWVFLFFLSVTTGVHSD aaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttc 6]_
+
+
+
+
+
+
3^20
ttttgagtgtgtacgggtggcacgggtcgtggacttgaggacccccctggcagtcagaag KTHTCPPCPAPELLGGPSVF
121
181
24i
ctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc + + + + + + 18 0 gagaaggggggttttgggttcctgtgggagtactagagggcctggggactccagtgtacg LFPPKPKDTLMISRTPEVTC gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggc + + + + + + 24 0 caccaccacctgcactcggtgcttctgggactccagttcaagttgaccatgcacctgccg VVVDVSHEDPEVKFNWYVDG gtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt + + + + + + 300 cacctccacgtattacggttctgtttcggcgccctcctcgtcatgttgtcgtgcatggca VEVHNAKTKPREEQYNSTYR
301
3 61
4 21
gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc + + + + + + 360 caccagtcgcaggagtggcaggacgtggtcctgaccgacttaccgttcctcatgttcacg VVSVLTVLHQDWLNGKEYKC aaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaaggg + + + + + + 420 ttccagaggttgtttcgggagggtcgggggtagctcttttggtagaggtttcggtttccc KVSNKALPAPIEKTISKAKG cagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaac + + + + + + 480 gtcggggctcttggtgtccacatgtgggacgggggtagggccctactcgactggttcttg QPREPQVYTLPPSRDELTKN
4 81
541
caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgg + + + + + + 540 gtccagtcggactggacggaccagtttccgaagatagggtcgctgtagcggcacctcacc QVSLTCLVKGFYPSDIAVEW gagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgac +
+
+
+
+
+
ctctcgttacccgtcggcctcttgttgatgttctggtgcggagggcacgacctgaggctg ESNGQPENNYKTTPPVLDSD
60o
Patent Application Publication
Dec. 3, 2009 Sheet 81 of 128
US 2009/0297520 Al
FIG. 44B ggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac ccgaggaagaaggagatgtcgttcgagtggcacctgttctcgtccaccgtcgtccccttg GSFFLYSKLTVDKSRWQQGN gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctc cagaagagtacgaggcactacgtactccgagacgtgttggtgatgtgcgtcttctcggag VFSCSVMHEALHNHYTQKSL tccctgtctccgggtaaaggaggaggaggatccggaggaggaggaagcggcgtgatcatc agggacagaggcccatttcctcctcctcctaggcctcctcctccttcgccgcactagtag SLSPGKGGGGSGGGGSGVII aacgtgaagtgcaagatcagccgccagtgcctgaagccctgcaaggacgccggcatgcgc ttgcacttcacgttctagtcggcggtcacggacttcgggacgttcctgcggccgtacgcg NVKCKISRQCLKPCKDAGMR ttcggcaagtgcatgaacggcaagtgccactgcacccccaagtagtaa aagccgttcacgtacttgccgttcacggtgacgtgggggttcatcatt FGKCMNGKCHCTPK**
Patent Application Publication
Dec. 3, 2009 Sheet 82 of 128
US 2009/0297520 Al
FIG. 45A atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccgac taccttacctcgacccagaaagagaagaaggacagtcattgctgaccacaggtgaggctg MEWSWVFLFFLSVTTGVHSD aaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttc ttttgagtgtgtacgggtggcacgggtcgtggacttgaggacccccctggcagtcagaag KTHTCPPCPAPELLGGPSVF ctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc gagaaggggggttttgggttcctgtgggagtactagagggcctggggactccagtgtacg LFPPKPKDTLMISRTPEVTC gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggc caccaccacctgcactcggtgcttctgggactccagttcaagttgaccatgcacctgccg VVVDVSHEDPEVKFNWYVDG gtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt cacctccacgtattacggttctgtttcggcgccctcctcgtcatgttgtcgtgcatggca VEVHNAKTKPREEQYNSTYR gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc caccagtcgcaggagtggcaggacgtggtcctgaccgacttaccgttcctcatgttcacg VVSVLTVLHQDWLNGKEYKC aaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaaggg ttccagaggttgtttcgggagggtcgggggtagctcttttggtagaggtttcggtttccc KVSNKALPAPIEKTISKAKG
4 21
4 81
541
cagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaac + + + + + + 4 80 gtcggggctcttggtgtccacatgtgggacgggggtagggccctactcgactggttcttg QPREPQVYTLPPSRDELTKN caggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgg + + + + + + 54 0 gtccagtcggactggacggaccagtttccgaagatagggtcgctgtagcggcacctcacc QVSLTCLVKGFYPSDIAVEW gagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgac + + + + + + 600 ctctcgttacccgtcggcctcttgttgatgttctggtgcggagggcacgacctgaggctg ESNGQPENNYKTTPPVLDSD
Patent Application Publication
Dec. 3, 2009 Sheet 83 of 128
US 2009/0297520 Al
FIG. 45B ggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac ccgaggaagaaggagatgtcgttcgagtggcacctgttctcgtccaccgtcgtccccttg GSFFLYSKLTVDKSRWQQGN gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctc cagaagagtacgaggcactacgtactccgagacgtgttggtgatgtgcgtcttctcggag VFSCSVMHEALHNHYTQKSL tccctgtctccgggtaaaggaggaggaggatccggaggaggaggaagcggcgtgatcatc agggacagaggcccatttcctcctcctcctaggcctcctcctccttcgccgcactagtag SLSPGKGGGGSGGGGSGVII aacgtgagctgcaagatcagccgccagtgcctgaagccctgcaaggacgccggcatgcgc ttgcactcgacgttctagtcggcggtcacggacttcgggacgttcctgcggccgtacgcg NVSCKISRQCLKPCKDAGMR ttcggcaagtgcatgaacggcaagtgccactgcacccccaagtagtaa aagccgttcacgtacttgccgttcacggtgacgtgggggttcatcatt FGKCMNGKCHCTPK* *
Patent Application Publication
Dec. 3, 2009 Sheet 84 of 128
.1, HO kDa
• b ; & >—••
'$QQW>
:
S: ?
•••
S\
US 2009/0297520 Al
Patent Application Publication
Dec. 3, 2009 Sheet 85 of 128
US 2009/0297520 Al
\.
v-.y.-.-.;.-.-.-.;.-.-.-.;.-.-.-.;.-.-.-.v-'-'-'.;-'-'-'.;-'-'-V-'-'- • -ssv. ^. ^v. j. -•. »^A j. A v A v A
250
2G0
270
280
290
300
I'M 'A^'&i?• ^O 1 :"'•
::::!^^:-;-x
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310 >"*^"J :
320
330
Patent Application Publication
Dec. 3, 2009 Sheet 86 of 128
US 2009/0297520 Al
FIG. 49C
0.40
0.40
irT"r~rT~r
0.35
4-4-
0.30 0.25
-t--h-
< 0.20 0.15 0.10 0.05
I
4|--h-44--f-f0.35 I I I I I 0.30 I I I I I 0.25 H--h-|--i-i--h _1 1_LJ_1_L. 0.20 < I I I I I r_r_1_T_r. 0.15 1 4 H-4-44-4--^ ro.io I I I I I :0.05 I I I -y H 1 h :0.00
I
4-4-
0.00
'Mll|llll|llll|llll|llll|llllllllt|llll|llll|lllllllll|llll|llll|llll|llll|lllllllll|lllllllll|lll
0
2
4
6
8 10 12 14 16 18 20 Minutes
FIG. 49D 63875.08
100-1 90-
(M+H)+
r 7037.3
8070en
»
stigj
\ 20- Donor 1459 (10 uMTG) 0i i -6 -5 -4 -3 -2-10 1 Concentration (log, uM) •
i
AShK[l-35] (IC50=0.043nM)
f
• Cyclosporin A (IC50=38.5nM)
Patent Application Publication
Dec. 3, 2009 Sheet 106 of 128 US 2009/0297520 Al
FIG. 66A CD4+T Cells FC=26.4X
u
FIG. 66B
1
1
Dec. 3, 2009 Sheet 123 of 128 US 2009/0297520 Al
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*
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Patent Application Publication
Dec. 3, 2009 Sheet 124 of 128 US 2009/0297520 Al
IMt
Patent Application Publication
Dec. 3, 2009
Sheet 125 of 128 US 2009/0297520 Al
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'v u
05
Patent Application Publication
Cyclosporin A
FIG. 90A
Dec. 3, 2009
iL-2-Piate i
IC50 (uM) =0.236445
o o
Sheet 126 of 128 US 2009/0297520 Al
IFN-g-Plate 1 1001-
+
IC50 (uM) =0.291694
U O a.
0.001 Cone. (uM)
ShK-Ala22
10
IC50 (uM) =0.000205
0.001
1 Cone. (uM)
IFN-g--Plate 4
IC50 (uM)
f
1201- + =0.000262 o o
FIG. 90B
QL
1x10
0.01
1x10 0.01 Cone. (uM)
Cone. (uM)
OSK1-Ala29
+
IL-2--Plate 4
IC50 (UM) =0.000006
90 H-
IFN-g--Plate 4
IC50 (uM) = 0.000007
85
75-
FIG. 90C
u o
o o
60-
CL
45301x10"° 0.01 Cone. (uM)
OSK1-Ala12
IL-2--Plate 4
FIG. 90D
o o CL
75 60
: \
45 3U
0.01
Cone. (uM)
IFN-g--Plate 4
\ IC50 (uM) -\ =0.000004 : \
90
1x10
:
-.
IC50 (uM) =0.000004 o o CL
,
\.
+
iF+f,
1x10^ Cone. (uM)
0.01
1x10' Cone. (uM)
0.01
Patent Application Publication
Dec. 3, 2009
Sheet 127 of 128 US 2009/0297520 Al
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