Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 1 of 61
`
`Exhibit 5
`
`

`

`USOO7622443B2
`
`(12) United States Patent
`Anderson et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7.622,443 B2
`*Nov. 24, 2009
`
`(54) METHOD FOR INHIBITING
`PRO-ANGIOGENIC ACTIVITIES OF
`ENDOTHELIAL CELLS SELECTIVELY AT A
`SITE OF NEOANGOGENESIS IN A
`MAMMAL BY ADMINISTRATION OF THE
`EXTRACELLULAR DOMAIN OFD1-1
`POLYPEPTIDES
`
`(75) Inventors: David J. Anderson, Altadena, CA (US);
`Hai U. Wang, Folsom, CA (US);
`Donghun Shin, Pasadena, CA (US)
`
`(73) Assignee: California Institute of Technology,
`Pasadena, CA (US)
`
`(*) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 55 days.
`This patent is Subject to a terminal dis
`claimer.
`
`(21) Appl. No.: 11/437,755
`
`(22) Filed:
`
`May 18, 2006
`
`(65)
`
`Prior Publication Data
`US 2007/OO82OOO A1
`Apr. 12, 2007
`
`Related U.S. Application Data
`(63) Continuation-in-part of application No. 10/424,986,
`filed on Apr. 28, 2003, now Pat. No. 7,538,088.
`(60) Provisional application No. 60/375,904, filed on Apr.
`26, 2002, provisional application No. 60/682.542,
`filed on May 18, 2005.
`
`(51) Int. Cl.
`(2006.01)
`A6 IK 38/17
`(2006.01)
`C07K I4/47
`(2006.01)
`C07K I4/55
`(52) U.S. Cl. .......................... 514/12:530/300; 530/324
`
`Human D1-1 Polypeptides
`
`(58) Field of Classification Search ....................... None
`See application file for complete search history.
`References Cited
`
`(56)
`
`U.S. PATENT DOCUMENTS
`
`2004/O120955 A1
`
`6/2004 Anderson et al. ........ 424,146.1
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`WO
`WO
`WO
`WO
`WO
`
`9, 1993
`O56.1172 A1
`O682113 A2 11/1995
`WO-OOf 55.173 A1
`9, 2000
`WO-0061623 A1 10, 2000
`WO-01 57.190 A2
`8, 2001
`WO-O 1/77 289 A2 10, 2001
`WO-O2/O79492 A2 10, 2002
`
`OTHER PUBLICATIONS
`
`Delisser et al., “Platelet Endothelial Cell Adhesion Molecule
`(CD31).” Current Topics In Microbiology and Immunology 184:37
`45(1993).
`Marra et al., Accession No. AA267694 (Mar. 21, 1997).
`(Continued)
`Primary Examiner Bridget E Bunner
`Assistant Examiner Zachary C Howard
`(74) Attorney, Agent, or Firm McCarter & English, LLP
`
`(57)
`
`ABSTRACT
`
`The disclosure provides, among other things, novelangiogen
`esis-related nucleic acids, polypeptides and methods of use.
`
`5 Claims, 17 Drawing Sheets
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 2 of 61
`
`A. Full-length sequence (SEQD NO:10)
`
`MGTAGAMOLCww.LGFLLFRGHNSQPEMTOTSSSQGGLGGLSLTTEPWSS 50
`NPGYIPSSEANRPSHLSSTGPGASWPSSGRDGGTSRDTFTWPPNSTM 100
`SLSMRECATILESRTSETWLTWAAFGVSFIWWWWWIITWGWWSLRFK 150
`CRKSKESEDPQKPGssGLSESCSTANGEKDSITLESMKNINMNNGKQSLS 200
`AEKW 2.05
`
`B. Extracellular portion (SEQID NO:11)
`OPTMTQTSssogg.GGLSLTTEPVSSNPGYIPSSEANRPSHLSSTGTPGA
`GwpsSCRDGGTSRDTFTwPPNSTTMSLSMREDATILPSPTSETVLT
`
`C. Conserved portion (SEQID NO:12)
`
`CSTANGEKDSITLISMKNNMNNGKQSSAEKWL
`
`

`

`US 7,622.443 B2
`Page 2
`
`OTHER PUBLICATIONS
`Osborn et al., “Direct Expression Cloning of Vascular Cell Adhesion
`Molecule1. A Cytokine-Induced Endothelial Protein That Binds to
`Lymphocytes.” Cell 59:1203-1211(1989).
`Genbank ref. No. XM 148854.
`Genbank ref No. XP 148854.
`Bork and Bairoch, 1996, Go hunting in sequence databases but watch
`out for the traps, Trends in Genet. 12(10):425427.
`Bork, 2000, Powers and Pitfalls in Sequence Analysis: The 70%
`Hurdle, Genome Res. 10:398-400.
`Brenner, 1999, Errors in genome annotation, Trends in Genet.
`15:132-133.
`Doerks, 1998, Protein annotation: detective work for function pre
`diction, Trends in Genet. 14(6):248-250.
`Ngo et al., 1995. The Protein Folding Problem and Tertiary Structure
`Prediction, Chapter 14: Computational Complexity Protein Structure
`Prediction, and the Levinthal Paradox, pp. 492-495.
`Skolnick and Fetrow, 2000. From genes to protein structure and
`function: novel applications of computational approached in the
`genomic era, Trends in Genet. 18:34-39.
`Smith and Zhang, 1997. The challenges of genome sequence anno
`tation of “The devil is in the details.' Nature Biotech. 15:1222-1223.
`
`Staton et al., 2004. Current methods for assaying angiogenesis in
`vitro and in vivo, Int. J. Exp. Path. 85:233-248.
`Wells, 1990. Additivity of Mutational Effects in Proteins, Biochem.
`29(37):8509-8517.
`Antikainen et al., “Altering protein specificity: techniques and
`applicaitons.” Bioorganic & Medicinal Chemistry, 13:2701-2716
`(2005).
`Armstrong, et al., “ECSM2. An Endothelial Specific Filamin A Bind
`ing Protein That Mediates Chemotaxis.” Arterioscler Thromb Vasc
`Biol. 28:1-7 (2008).
`Auerbach, et al. "Angiogenisis Assays: A Critical Overview.” Clini
`cal Chemistry, 49(1):32-40 (2003).
`Ferrer-Costa, et al., “Characterization of Compensated Mutations in
`Terms of Structural and Physico-Chemical Properties,” J. Mol. Biol.
`365:249-256 (2007).
`Ponce et al., “The Chick Chorioallantoic Membrane as an In Vivo
`Angiogenesis Model.” Current Protocols in Cell Biology, Supple
`ment 18:19.5.1-19.5.6 (2003).
`Genbank ref. No. XM 148854, Date: May 17, 2002.
`Genbank refNo. XP 148854, Date: May 17, 2002.
`* cited by examiner
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 3 of 61
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 1 of 17
`
`US 7.622,443 B2
`
`D1-1 Alignment : E
`
`Sicna P
`
`f
`
`Ser-OGly
`
`ne
`In
`human
`bow line
`porcine
`
`Inu ine
`human
`bow line
`porcine
`
`nure
`human
`bo Wilne
`porcine
`
`Inuille
`human
`bo wine
`porcine
`
`s ss is MRLsAILLILL Qeyssor--TTTT's EILQEssovsws
`- - - - - - - - as a s - a
`- - - - - - - - - - - - EEE TSSG GIGGLSLTTEP
`-GCLPVADQADMGSWEETLRAILL ELQGAPs------ OSS - - - - e s - is
`QPR
`l PrvRASIPRP TomasvestocealerilLogg-- - - - - - ---
`- - - - - - - - ITIPS
`
`--r
`O
`41 - - QPWTPassTDKQSLs. PDLMSFQP- - -KETL g--PgrGTPE - - - - - - - - - - - - - -
`49 EEEEEEEEE
`-
`- -
`-
`-
`- -
`-
`-
`-
`-
`-
`-
`-
`- TGTPG- - - - - - - - - - - - - -
`s
`EEEEEEEEEES TASETsgs.gift ssTVADHLPss PGP Tssos
`43 P- - gEspsessssels- - - - - - - - - - - - - - - - - - - - - - SSTSS - - - - - - - - - - - - - -
`for
`is
`90 - - - - - - - - - Rssssssssss:
`PSPETTSLTERMTIILTELPTPTSESLT
`- - - - - - - - AG FSSGRESSRT
`PTTSs.
`lO3 QEHT3GLSADWPSs
`DSSDTSHNWTST's PressRT
`55 - - - - - - - - - - - - sssssssTTTPhingtsspTy
`
`Livs vs. ReageSED pressessics race KD
`AWEST -
`AISL -IIL www.gll RFECREsk RSEDP RPGSSGLSR.Sc.gTAGRED
`AAEGWESFIAIL
`TIVL’s vis, RFECREREs BDP PasscLsses TAGE
`WAAF GWIS FIWIL
`IILWSWWSLREECRENKESBDPQKPGSSGL S RSC STANGERD
`
`l90 SILISM-IVFSGSSABI
`Inur in a
`lel sITLISMIKN-INNGEssary
`human
`bowie 220 SMERSEPs
`porcine l. 69 SITLISM-INMES- - - - - - - - - -
`
`m
`
`m
`
`
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 4 of 61
`
`Figure l
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 2 of 17
`
`US 7.622,443 B2
`
`
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 5 of 61
`
`Figure 2
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 3 of 17
`
`US 7.622,443 B2
`
`
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 6 of 61
`
`Figure 3
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 4 of 17
`
`US 7.622,443 B2
`
`
`
`Control
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 7 of 61
`
`Figure 4
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 5 Of 17
`
`US 7.622,443 B2
`
`Figure 5: Murine D1-1 Nucleic Acids
`
`A. cDNA sequence (SEQID NO:1)
`
`GAGCCTGCTACACACCCAGCTGATCTGGGGACCAGCGGAGCCATGAGGCTGGG
`TTCAGCAATTCTCGGTTTACTCCTGCTCCAAGGCTACAGCTCTCAACCTACGA
`CAACTCAGACCTCGCAGGAAATTCTACAGAAGTCTTCTCAGGTCTCCTTGGTA
`TCCAATCAGCCTGTGACACCAAGGTCAAGCACCATGGATAAACAGTCCCTTTC
`CTTGCCTGACTTGATGTCCTTCCAGCCACAGAAGCACACACTGGGACCTGGCA
`CAGGAACCCCAGAAAGGAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGGAGA
`GGAGAAGCATCTCTGGATGCTACTCCCAGTCCAGAAACCACCAGCCTTCAGAC
`AAAAAAGATGACCATCCTGCTGACCATCCTGCCTACCCCCACATCAGAGTCAG
`TGCTAACTGTGGCTGCCTTTGGTGTCATCAGCTTCATTGTCATCCTGGTGGTT
`
`GTGACCTGGAGTCGCCATGGGTCCACGTGTGCGGCTGTCCCCTGGCCATGAGG
`AAGGAGAGGAGACGAGATTGGGGGAGGCAGCGGACCACACATAAATTATTTGA
`TGTCATGCCTGCTCCCAGTTCTAAAGGACATGAGATTCCTCTAGATCCAGAAG
`AACCTACCACACAAGAGACTCCTTCCCACTTGGAAGCCATGCTAGACACTTGG
`CCTGCTCCCCCTCCTCCTGCTGCTCAGAAACTCAGGAACGAGGAGTCAATAGA
`GCAAGACTTAAGGAAATAATGAGGTAGATTGTCCATTCTACTAGAATTAAAAT
`TATTTTCTGGCCTGG
`
`B. Sequence encoding extracellular portion (SEQID NO:2)
`
`CAACCTACGACAACTCAGACCTCGCAGGAAATTCTACAGAAGTCTTCTCAGGT
`CTCCTTGGTATCCAATCAGCCTGTGACACCAAGGTCAAGCACCATGGATAAAC
`AGTCCCTTTCCTTGCCTGACTTGATGTCCTTCCAGCCACAGAAGCACACACTG
`GGACCTGGCACAGGAACCCCAGAAAGGAGCAGCAGCAGCAGCAGCAGCAGCAG
`CAGCAGGAGAGGAGAAGCATCTCTGGATGCTACTCCCAGTCCAGAAACCACCA
`GCCTTCAGACAAAAAAGATGACCATCCTGCTGACCATCCTGCCTACCCCCACA
`TCAGAGTCAGTGCTAACT
`
`C. Sequence encoding conserved portion (SEQ ID NO:3)
`
`GTGGCTGCCTTTGGTGTCATCAGCTTCATTGTCATCCTGGTGGTTGTAGTGAT
`CATCCTGGTCAGTGTGGTCAGTCTAAGATTTAAGTGTCGGAAGAACAAGGAGT
`CTGAAGATCCACAGAAACCAGGGAGTTCAGGACTGTCTGAAAGCTGCTCAACA
`GCCAATGGAGAGAAAGACAGCATCACACTCATCTCCATGAGGAACATCAACGT
`GAACAACAGCAAAGGCAGCATGTCAGCAGAGAAGATTCTT
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 8 of 61
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 6 of 17
`
`US 7.622,443 B2
`
`Figure 6: Murine Dl-l Polypeptides
`
`A. Full-length sequence (SEQID NO:4)
`
`MRLGSAILGLLILLOGYSSOPTTTOTSOEILOKSSOWSLVSNOPWTPRSST 50
`MDKOSLSLPDLMSFOPOKHTLGPGTGTPERSSSSSSSSSSRRGEASLDAT 100
`PSPETTSLOTKKMTILLTILPTPTSESVLTVAAFGVISFIVILVVVVIIL 150
`VSVVSLRFKCRKNKESEDPQKPGSSGLSESCSTANGEKDSITLISMRNIN 200
`WNNSKGSMSAEKIL 214
`
`B. Extracellular portion (SEQID NO:5)
`
`SQPTTTOTSQEILQKSSQVSLVSNQPWTPRSSTMDKQSLSLPDLMSFQPQK
`HTLGPGTGTPERSSSSSSSSSSRRGEASLDATPSPETTSLQTKKMTILLTI
`LPTPTSESVLT
`
`C. Conserved portion (SEQID NO:6)
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 9 of 61
`
`VAAFGVISFIVILVVVVIIILVSVVSLRFKCRKNKESEDPQKPGSSGLSESC
`STANGEKDSITLSMRNINVNNSKGSMSAEKIL
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 7 of 17
`
`US 7.622,443 B2
`
`Figure 7: Human Dl-1 Nucleic Acids
`
`A. cDNA sequence (SEQ ID NO: 7)
`
`GAGCCTCCACTGAGCTGCTGCCTGCCCGCCACATACCCAGCTGACATGGGCAC
`CGCAGGAGCCATGCAGCTGTGCTGGGTGATCCTGGGCTTCCTCCTGTTCCGAG
`GCCACAACTCCCAGCCCACAATGACCCAGACCTCTAGCTCTCAGGGAGGCCTT
`GGCGGTCTAAGTCTGACCACAGAGCCAGTTTCTTCCAACCCAGGATACATCCC
`TTCCTCAGAGGCTAACAGGCCAAGCCATCTGTCCAGCACTGGTACCCCAGGCG
`CAGGTGTCCCCAGCAGTGGAAGAGACGGAGGCACAAGCAGAGACACATTTCAA
`ACTGTTCCCCCCAATTCAACCACCATGAGCCTGAGCATGAGGGAAGATGCGAC
`CATCCTGCCCAGCCCCACGTCAGAGACTGTGCTCACTGTGGCTGCATTTGGTG
`
`TANAGGACACTAGCATTGCCCCAGATCTGGGGAGCAGCTACCAACAGGGGGAG
`ACTCTTTTCCTGTATGGACAGCTGCTGTGGAAAATACTGGCCTGGCTTCTCCC
`CACTCCTCAGAGC
`
`B. Sequence encoding the extracellular portion (SEQID NO:8)
`
`CAGCCCACAATGACCCAGACCTCTAGCTCTCAGGGAGGCCTTGGCGGTCTAAG
`TCTGACCACAGAGCCAGTTTCTTCCAACCCAGGATACATCCCTTCCTCAGAGG
`CTAACAGGCCAAGCCATCTGTCCAGCACTGGTACCCCAGGCGCAGGTGTCCCC
`AGCAGTGGAAGAGACGGAGGCACAAGCAGAGACACATTTCAAACTGTTCCCCC
`CAATTCAACCACCATGAGCCTGAGCATGAGGGAAGATGCGACCATCCTGCCCA
`GCCCCACGTCAGAGACTGTGCTCACT
`
`C. Sequence encoding the conserved portion (SEQID NO:9)
`
`GTGGCTGCATTTGGTGTTATCAGCTTCATTGTCATCCTGGTGGTTGTGGTGAT
`CATCCTAGTTGGTGTGGTCAGCCTGAGGTTCAAGTGTCGGAAGAGCAAGGAGT
`CTGAAGATCCCCAGAAACCTGGGAGTTCAGGGCTGTCTGAAAGCTGCTCCACA
`GCCAATGGAGAGAAAGACAGCATCACCCTTATCTCCATGAAGAACATCAACAT
`GAATAATGGCAAACAAAGTCTCTCAGCAGAGAAGGTTCTTTANAAGCAACTTT
`GGGTCCCCATGAGTCCAAGGATGATGCAGCTGCCCTGTGACTACAAGGAGGAA
`GAGATGGAATTAGTAGAGGCAATGAACCACATGTAAATTATTTTATTGTTTCA
`TGTCTGCTTC
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 10 of 61
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 8 of 17
`
`US 7.622,443 B2
`
`Figure 8: Human D1-1 Polypeptides
`
`A. Full-length sequence (SEQID NO:10)
`
`MGTAGAMOLCWWILGFLLFRGHNSOPTMTQTSSSQGGLGGLSLTTEPVSS 50
`NPGYIPSSEANRPSHLSSTGTPGAGVPSSGRDGGTSRDTFQTVPPNSTTM loo
`SLSMREDATILPSPTSETVLTVAAFGWISFIVILVVV VIIILVGVVSLRFK 150
`CRKSKESEDPQKPGSSGLSESCSTANGEKDSITLISMKNINMNNGKQSLS 2 OO
`AEKVL 2 O5
`
`B. Extracellular portion (SEQID NO:11)
`
`QPTMTOTSSSQGGLGGLSLTTEPVSSNPGYIPSSEANRPSHLSSTGTPGA
`GVPSSGRDGGTSRDTFQTVPPNSTTMSLSMREDATILPSPTSETVLT
`
`C. Conserved portion (SEQID NO:12)
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 11 of 61
`
`WAAFGVISFIVILVVVVIILVGVVSLRFKCRKSKESEDPQKPGSSGLSES
`CSTANGEKDSITLISMKNINMNNGKQSLSAEKVL
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 9 of 17
`
`US 7.622,443 B2
`
`Figure 9: Bovine D1-1 Nucleic Acids
`
`A. cDNA sequence (SEQ ID NO:13)
`
`ACAGAGGCTGCCTGCCGGTTGCAGACCAAGCTGACATGGGGAGTGTCAGAGAAA
`CGCAGCTGCGCTGGGCCATCCTGGGCTTCCTCCTGCTCCAAGGAGCCTTCAGCA
`GTCAAAGTTCAACCACACAGCCAGCTTCCCCTGAAACAAGTCCTTCCACAGAGG
`CCAACAGCTTAAGCCCTCTGTCCGGCACCTGGACCACAGCAGCATCAGAGACGC
`CCTCACAGTTCTCCACGGAAGCCATGACTCTGAGTTCAAGCACCGTGGCTGATC
`ACTTGCCGTCCTCTCCGGGACCGACTTGGTCCCAGTCACAGAAACACACGTCAG
`GACTCAGCGCTGATGTTCCGAGCAGTGGCAGGAGCAGCGACAGCATGAGTGGAG
`ACACCTCTCACAATGTTACTTCCACATCACCCAACATGAGTTTTAGGACGACAG
`CAGACTCCACTGTCCCACCCAGCCCCACGTCAGAGACGGTGCTCACTGTGGCTG
`
`B. Sequence encoding extracellular portion (SEQID NO:14)
`
`CAAAGTTCAACCACACAGCCAGCTTCCCCTGAAACAAGTCCTTCCACAGAGGCC
`AACAGCTTAAGCCCTCTGTCCGGCACCTGGACCACAGCAGCATCAGAGACGCCC
`TCACAGTTCTCCACGGAAGCCATGACTCTGAGTTCAAGCACCGTGGCTGATCAC
`TTGCCGTCCTCTCCGGGACCGACTTGGTCCCAGTCACAGAAACACACGTCAGGA
`CTCAGCGCTGATGTTCCGAGCAGTGGCAGGAGCAGCGACAGCATGAGTGGAGAC
`ACCTCTCACAATGTTACTTCCACATCACCCAACATGAGTTTTAGGACGACAGCA
`GACTCCACTGTCCCACCCAGCCCCACGTCAGAGACGGTGCTCACT
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 12 of 61
`
`C. Sequence encoding conserved portion (SEQ ID NO:15)
`
`GTGGCTGCATTTGGTGTTATCAGCTTCATTGCCATCCTAGTGGTTGTGGTGATT
`GTCCTGGTCAGTGTGGTCAGTCTAAGGTTTAAGTGTCGGAAGAACAAGGAGTCT
`GAAGATCCCCAGAAACCTGGGAGTTCAGGGCTCTCTGAAAGCGGTTCCACAGCC
`AATGGAGAGAAAGAGAGCATCACTCTTATCTCGATGAAGAACATCAACATGAAT
`AACAGCAAAGGATGCCCCTCA
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 10 of 17
`
`US 7.622,443 B2
`
`Figure 10: Bovine D1-1 Polypeptides
`
`A. Full-length sequence (SEQ ID NO:16)
`
`RGCLPVADQADMGSVRETOLRWAILGFLLLQGAFSSQSSTTOPASPETSP
`STEANSLSPLSGTWTTAASETPSOFSTEAMTLSSSTVADHLPSSPGPTWS
`OSOKHTSGLSADVPSSGRSSDSMSGDTSHNWTSTSPNMSFRTTADSTVPP
`SPTSETVLTVAAFGVISFIAILVVVVTIVLVSVVSLRFKCRKNKESEDPQ
`KPGSSGLSESGSTANGEKESITLISMKNINMNNSKGCPS
`
`B. Extracellular portion (SEQID NO:17)
`
`SOSSTTOPASPETSPSTEANSLSPLSGTWTTAASETPSQFSTEAMTLSSS
`TVADHLPSSPGPTWSOSOKHTSGLSADVPSSGRSSDSMSGDTSHNWTSTS
`
`PNMSFRTTADSTVPPSPTSETVLT
`
`C. Conserved portion (SEQED NO:18)
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 13 of 61
`
`VAAFGVISFIAILVVVVTIVLVSVVSLRFKCRKNKESEDPQKPGSSGLSES
`GSTANGEKESTLISMKNIINMNNSKGCPS
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 11 of 17
`
`US 7.622,443 B2
`
`Figure 1: Porcine D1-1 Nucleic Acids
`
`A. cDNA sequence (SEQ ID NO:19)
`
`CCCACGCGTCCGCGCCAGCCTGCCCCGTCCCACTGACATGGGGAGCGTCGGAGAAACGCA
`GCTGTGCTGGGCCATCCTGGGCTTCCTCCTGCTCCAAGGCCACGGCTCCCAGCTCACAAT
`ACCTAGCCCTCAGGGAGAGAGTCCTTCCGCAGAGTCCAACAGCTCAAGCCCTCTATCCAG
`CAGCACCAGCAGCAGCAGCAACAGCAGCAGCAGCACCAGCACCACAGACACCCCTCACAA
`TGGTACGTCCACGTCACCCACCGTGAGTCTGAGAACCAGAGAAGACCCGACCGTCCTGCC
`CAGCCCCACGTCAGAGACGGTGCTCACAGTGGCCGCCTTTGGTGTCATCAGCTTCATTGT
`
`B. Sequence encoding extracellular portion (SEQ ID NO:20)
`
`CAGCTCACAATACCTAGCCCTCAGGGAGAGAGTCCTTCCGCAGAGTCCAACAGCTCAAGC
`CCTCTATCCAGCAGCACCAGCAGCAGCAGCAACAGCAGCAGCAGCACCAGCACCACAGAC
`ACCCCTCACAATGGTACGTCCACGTCACCCACCGTGAGTCTGAGAACCAGAGAAGACCCG
`ACCGTCCTGCCCAGCCCCACGTCAGAGACGGTGCTCACA
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 14 of 61
`
`C. Sequence encoding conserved portion (SEQID NO:21)
`
`GTGGCCGCCTTTGGTGTCATCAGCTTCATTGTCATCCTGCTGGTTGTGGTGATCATCCTG
`GTCAGCGTGGTCAGTCTAAGGTTTAAGTGTCGGAGGAACAAGGAATCTGAAGATCCCCAG
`AAACCTGGGAGTTCGGGGCTCTCTGAAAGCTGCTCCACAGCCAATGGAGAGAAAGACAGC
`
`ATCACCCTCATCTCCATGAAAAATATCAACATGAATAACAGC
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 12 of 17
`
`US 7.622,443 B2
`
`Figure 12: Porcine D1-1 Polypeptide Sequences
`
`A. Sequence (SEQID NO:22)
`
`PRVRASLPRPTDMGSVGETOLCWAILGFL.LLOGHGSQLTIPSPQGESPSA
`ESNSSSPLSSSTSSSSNSSSSTSTTDTPHNGTSTSPTVSLRTREDPTVLP
`SPTSETVLTVAAFGVISFIVILLVVVIILVSVVSLRFKCRRNKESEDPOK
`PGSSGLSESCSTANGEKDSITLISMKNINMNNS
`
`B. Extracellular portion (SEQID NO:23)
`
`QLTIPSPOGESPSAESNSSSPLSSSTSSSSNSSSSTSTTDTPHNGTSTSPT
`VSLRTREDPTVLPSPTSETVLT
`
`C. Conserved portion (SEQ ID NO:24)
`
`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 15 of 61
`
`VAAFGVISFIVILLVVVIIILVSVVSLRFKCRRNKESEDPQKPGSSGLSESC
`STANGEKDSITLISMKNNMNNS
`
`

`

`U.S. Patent
`
`Nov. 24, 2009
`
`Sheet 13 of 17
`
`US 7.622,443 B2
`
`
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`Case 6:20-cv-00947-ADA Document 1-5 Filed 10/13/20 Page 16 of 61
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`U.S. Patent
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`Nov. 24, 2009
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`U.S. Patent
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`Nov. 24, 2009
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`Sheet 15 Of 17
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`US 7.622,443 B2
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`Figure 15
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`U.S. Patent
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`Nov. 24, 2009
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`Sheet 16 of 17
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`US 7.622,443 B2
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`U.S. Patent
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`Nov. 24, 2009
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`Sheet 17 Of 17
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`US 7.622,443 B2
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`US 7,622,443 B2
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`1.
`METHOD FOR INHIBITING
`PRO-ANGIOGENIC ACTIVITIES OF
`ENDOTHELIAL CELLS SELECTIVELY AT A
`SITE OF NEOANGOGENESIS INA
`MAMMAL BY ADMINISTRATION OF THE
`EXTRACELLULAR DOMAN OFD1-1
`POLYPEPTIDES
`
`RELATED APPLICATIONS
`
`This application is a Continuation-in-part of U.S. patent
`application Ser. No. 10/424,986, filed Apr. 28, 2003, which
`claims the benefit of U.S. Provisional Application No.
`60/375,904, filed Apr. 26, 2002. This application also claims
`the benefit of the filing date of U.S. Provisional Application
`No. 60/682,542, filed May 18, 2005. All of the aforemen
`tioned patent applications are incorporated by reference
`herein in their entirety.
`
`STATEMENT OF FEDERALLY SPONSORED
`RESEARCH
`
`This invention was made with Government funding under
`Grant No. 5 RO1 HL66221 awarded by the National Institutes
`of Health. The United States Government has certain rights in
`this invention.
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`BACKGROUND
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`What is needed therefore are compositions and methods
`which can modulate angiogenesis according to specific situ
`ations. These include methods and compositions which can
`inhibit the unwanted growth of blood vessels, especially the
`newly formed blood vessels that support tumor growth. Fur
`thermore, it is also desirable to have methods and composi
`tions which can promote angiogenesis to treat inadequate
`angiogenesis in certain diseases and processes.
`
`SUMMARY
`
`In certain aspects, the present invention features a family of
`proteins, D1-1 proteins, that are novel Type I transmembrane
`proteins involved in angiogenesis, and nucleic acids encoding
`D1-1 proteins. In certain embodiments of the present inven
`tion provides D1-1 proteins and nucleic acids from a variety
`of different species, as well as variants, including fragments
`and fusion proteins. In certain embodiments, the invention
`provides truncated extracellular D1-1 polypeptides, option
`ally engineered to be fusion proteins comprising a heterolo
`gous polypeptide. Such as an Fc domain, and in certain
`embodiments. Such polypeptides may be used as inhibitors of
`angiogenesis. In certain aspects, the invention provides meth
`ods, compositions and new model systems for manipulating
`angiogenesis and diseases involving angiogenic disorders. In
`certain aspects, the invention provides screening methods for
`identifying therapeutic agents useful for the prevention or the
`treatment of angiogenesis-related disorders. In certain
`aspects, the invention provides methods for selectively
`detecting neovascularization in a tissue by detecting the
`expression of D1-1 nucleic acid or protein.
`In part, the disclosure relates to the finding that D1-1 is
`selectively expressed in endothelial cells at sites of neoangio
`genesis. Accordingly, in one aspect, the invention provides
`methods for inhibiting pro-angiogenic activities of endothe
`lial cells selectively at a site of neoangiogenesis in a mammal.
`Pro-angiogenic activities of endothelial cells include prolif
`eration, migration and secretion of pro-angiogenic factors. A
`method may comprise administering a pharmaceutical prepa
`ration comprising an agent selected from the group consisting
`of: (a) an antibody that binds specifically to the extracellular
`domain of D1-1 and inhibits D1-1 signaling; (b) a polypep
`tide comprising a truncated extra cellular D1-1; (c) an anti
`body that binds specifically to the extracellular domain of
`D1-1 that is conjugated with a second therapeutic agent; and
`(d) a nucleic acid construct that decreases expression of D1-1.
`An antibody of for use may bind specifically to an extracel
`lular portion of a D1-1 polypeptide selected from the group
`consisting of SEQID Nos. 5, 11, 17 and 23. The polypeptide
`comprising a truncated extracellular D1-1 may comprise an
`amino acid sequence selected from the group consisting of
`(i) an amino acid sequence that is at least 60% identical to an
`amino acid sequence of one or more of SEQID Nos. 5, 11, 17
`and 23; (ii) an amino acid sequence encoded by a nucleic acid
`sequence that is at least 60% identical to a nucleic acid
`sequence of one or more of: SEQID Nos. 2, 8, 14 and 20; and
`(iii) at least 30 consecutive amino acids from an amino acid
`sequence of one or more of: SEQID Nos. 5, 11, 17 and 23.
`The truncated extracellular D1-1 polypeptide may be a fusion
`protein further comprising a heterologous sequence. The het
`erologous polypeptide may be selected from the group con
`sisting of a portion of an immunoglobulin, a multimerization
`domain, a stabilizing domain, a targeting domain and a puri
`fication domain. The heterologous polypeptide may be an Fc
`portion of an immunoglobulin (including variants of Fc por
`tions, such as those engineered to decrease FcR binding or
`complement activation). The second therapeutic agent con
`
`Angiogenesis, the process of forming new blood vessels, is
`critical in many normal and abnormal physiological states.
`Under normal physiological conditions, humans or animals
`undergo angiogenesis in specific and restricted situations. For
`example, angiogenesis is normally observed in wound heal
`ing, fetal and embryonic development and formation of the
`corpus luteum, endometrium and placenta.
`Undesirable or inappropriately regulated angiogenesis
`occurs in a many disorders, in which abnormal endothelial
`growth may cause or participate in the pathological process.
`For example, abnormal endothelial cell proliferation has been
`postulated to contribute to atherosclerosis. Angiogenesis also
`participates in the growth of many tumors. Deregulated
`angiogenesis has been implicated in pathological processes
`Such as rheumatoid arthritis, retinopathies, hemangiomas,
`and psoriasis. The diverse pathological disease states in
`which unregulated angiogenesis is present have been catego
`rized as angiogenesis-dependent orangiogenesis-associated
`diseases.
`Inadequate angiogenesis is implicated in several diseases
`and biological processes. Inadequate vascularization of the
`uterine endometrium and associated infertility, wound repair,
`healing of cuts and incisions, are some of the examples of
`problems caused by or associated with inadequate angiogen
`esis.
`Both controlled and uncontrolled angiogenesis are thought
`to proceed in a similar manner. Endothelial cells and peri
`cytes, Surrounded by a basement membrane, form capillary
`blood vessels. Angiogenesis begins with the erosion of the
`basement membrane by enzymes released by endothelial
`cells and leukocytes. The endothelial cells, which line the
`lumen of blood vessels, then protrude through the basement
`membrane. Angiogenic stimulants induce the endothelial
`cells to migrate through the eroded basement membrane. The
`migrating cells form a “sprout' protruding from the parent
`blood vessel, where the endothelial cells undergo mitosis and
`proliferate. Endothelial sprouts merge with each other to form
`capillary loops, creating the new blood vessel.
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`jugated to an anti-D1-1 antibody may be any agent that inhib
`its a pro-angiogenic activity of endothelial cells, including a
`cytotoxic agent, a cytostatic agent, an anti-angiogenic agent
`and a sensitizing agent. A nucleic acid construct may com
`prise a nucleic acid that hybridizes with a sequence of at least
`18 consecutive nucleotides of a nucleic acid sequence of one
`or more of SEQID Nos. 1, 2, 13, 14, 19 or 20. The nucleic acid
`construct may be selected from the group consisting of an
`siRNA probe, an antisense nucleic acid and a ribozyme.
`In certain aspects, the invention provides methods for
`selectively identifying sites of neoangiogenesis in a mammal.
`A method may comprise detecting expression of a D1-1
`polypeptide, a D1-1 nucleic acid or a marker gene operably
`linked to a D1-1 promoter. The site of neoangiogenesis may
`be, for example, a tumor or an injured tissue. Expression of a
`D1-1 polypeptide may be done by detecting an extracellular
`portion of the D1-1 polypeptide in an extracellular fluid, such
`as blood, urine or lymph. Detection of a polypeptide may
`employ an antibody, often an antibody labeled with a label
`that is detectable ex vivo or in vivo (e.g., an MRI-detectable
`label). Detection of nucleic acids and proteins may be per
`formed on a sample obtained from the mammal or in vivo.
`An aspect of the invention provides D1-1 polypeptides and
`fragments thereof. An aspect of the invention encompasses
`truncated extracellular D1-1 polypeptides and derivatives
`thereof, especially angiogenesis-inhibiting peptide frag
`ments. The truncated extracellular D1-1 fragments can be
`provided as a fusion protein which includes a second
`polypeptide portion, e.g. the second polypeptide having an
`amino acid sequence unrelated (heterologous) to D1-1. For
`example, the heterologous polypeptide portion may include a
`multimerization domain (e.g. a dimerization, trimerization or
`tetramerization domain), a stabilizing domain (e.g. a domain
`that stabilizes or aids in the solubility of the fusion protein), a
`targeting domain (e.g. a domain that targets the fusion protein
`to a particular cell or tissue type) and a purification domain
`(e.g. a domain that facilitates purification of the fusion pro
`tein). For example, the second polypeptide portion may be the
`Fc portion of an immunoglobulin, or glutathione-S-trans
`ferase, or an enzymatic activity Such as alkaline phosphatase,
`or an epitope tag. The invention also includes D1-1 antibody,
`D1-1 ligands, D1-1 agonists or antagonists and other D1-1
`associated proteins.
`An aspect of the invention provides nucleic acid sequences
`that code for D1-1 amino acid sequences, and complementary
`45
`sequences thereof In certain embodiments, nucleic acids of
`the invention encode angiogenesis-inhibiting D1-1 polypep
`tides. In certain embodiments, the invention also includes
`antibodies directed against D1-1 or fragments thereof. In
`specific embodiments, the invention includes antibodies
`directed against an extracellular region, or an intracellular
`region of the D1-1 protein.
`Another aspect of the invention provides for a non-human
`transgenic animal comprising a mutation in the D1-1 gene. In
`a specific embodiment, the mutation is a deletion. The muta
`tion may affect one allele or bothalleles. In a specific embodi
`ment, the animal is a rodent and preferably amouse. In certain
`embodiments, the transgenic animals according to the inven
`tion provide model systems for studying angiogenesis asso
`ciated diseases and for screening and/or testing agents useful
`for treating and/or preventing these diseases. Organs from the
`transgenic animals (such as retina, aortas, etc.) are useful for
`screening and/or testing such agents. Cells from the trans
`genic animals are also useful for Screening and/or testing Such
`agents.
`It is yet another aspect of the present invention to provide
`methods and compositions for treating angiogenesis-associ
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`ated diseases, processes and other pathologies in which D1-1
`is involved. An important example of an angiogenesis-asso
`ciated disease is cancer.
`In certain embodiments, the invention provides methods
`and compositions for treating diseases and processes medi
`ated by undesired and uncontrolled angiogenesis by admin
`istering to a human or animal a composition comprising a
`substantially purified truncated extracellular D1-1 polypep
`tides in a dosage Sufficient to inhibit angiogenesis. In certain
`aspects, the methods and compositions may be used for treat
`ing or repressing the growth of tumors, and particularly pre
`vascularized tumors. The method also relates to the co-ad
`ministration of the truncated extracellular D1-1 polypeptides
`of the present invention and another anti-angiogenesis com
`pound, for example, angiostatin or endostatin. In certain
`embodiments, the invention provides a new form of birth
`control, wherein an effective amount of truncated extracellu
`lar D1-1 is administrated to a female subject such that uterine
`endometrial vascularization is inhibited and embryo implan
`tation cannot occur, or be sustained.
`In certain aspects, the invention also features methods and
`compositions that may promote endothelialization and vas
`cularization. Antagonists of the truncated extracellular D1-1
`polypeptides with angiogenesis-inhibitory activity may act in
`Such an angiogenesis-stimulating fashion. Accordingly, Such
`agents will be useful in situations of inadequate vasculariza
`tion of the uterine endometrium and associated infertility,
`wound repair, healing of cuts and incisions, treatment of
`vascular problems in diabetics, especially retinal and periph
`eral vessels, promotion of vascularization in transplanted tis
`Sue including muscle and skin, promotion of vascularization
`of cardiac muscle especially following transplantation of a
`heart or heart tissue and after bypass Surgery, promotion of
`vascularization of Solid and relatively avascular tumors for
`enhanced cytotoxin delivery, and enhancement of blood flow
`to the nervous system, including but not limited to the cere
`bral cortex and spinal cord.
`In certain aspects, the present invention also includes diag
`nostic methods and kits for detecting D1-1 polypeptide in
`biological fluids. In certain aspects, the present invention also
`includes antibodies specific for D1-1 and antibodies that
`inhibit the binding of antibodies specific for D1-1. These
`antibodies can be polyclonal antibodies or monoclonal anti
`bodies. The antibodies specific for the D1-1 can be used in
`diagnostic kits to detect the presence and quantity of D1-1
`which may be diagnostic or prognostic for the occurrence or
`recurrence of cancer or other diseases mediated by angiogen
`esis. Antibodies specific for D1-1 may also be administered to
`a human or animal to passively immunize the human or
`animal against D1-1.
`Another aspect of the invention includes diagnostic meth
`ods and kits for detecting the presence and quantity of anti
`bodies that bind D1-1 in body fluids. The diagnostic method
`and kit can be in any configuration well known to those of
`ordinary skill in the art.
`Angiogenesis-inhibiting agents can be given prophylacti
`cally to individuals known to be at high risk for developing
`new or re-current tumors or other disorders or conditions
`associated with unwanted angiogenesis. Accordingly, an
`aspect of the invention encompasses methods for prophylac
`tic prevention of unwanted angiogenesis in a Subject, com
`prising administrating to the Subject an effective amount of a
`truncated extracellular D1-1 polypeptide.
`In certain aspects, the invention further includes methods
`for modulating angiogenesis in Vivo, using the D1-1 protein
`or variants thereof, or nucleic acids corresponding to the D1-1
`proteins and variants, or antibodies that bind specifically to
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`5
`the proteins of the present invention. Without limitations,
`these methods may, for example, comprise inhibiting expres
`sion of an endogenous D1-1 nucleic acid by contacting a cell
`expressing D1-1 with a siRNA, an antisense nucleic acid or a
`ribozyme. These methods may further comprise increasing
`D1-1 expression by causing a cell to express a heterologous
`D1-1 nucleic acid, and administrating to a subject at least o
`portion of D1-1 protein or variants thereof.
`Another aspect of the invention provides methods for
`assessing the ability of an agent to bind to D1