US 20060253913A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0253913 A1 Huang et al. (43) Pub. Date: Nov. 9, 2006 (54) PRODUCTION OF HSA-LINKED (60) Provisional application No. 60/344.295, filed on Dec. BUTYRYLCHOLINESTERASES IN 21, 2001. TRANSGENIC MAMMALS Publication Classification (76) (51) Int. Cl. Inventors: Yue-Jin Huang, Montreal (CA); Costas AOIK 67/027 (2006.01) N. Karatzas, Beaconsfield (CA); C7H 2L/04 (2006.01) Anthoula Lazaris, Beaconsfield (CA) CI2P 2/06 (2006.01) CI2N 9/18 (2006.01) Correspondence Address: CI2N 5/06 (2006.01) Alan J. Grant, Esq. (52) U.S. Cl. ............................. 800/14: 800/18: 435/69.1; c/o Carella, Byrne, Bain, Gilfillan, Cecchi, 435/197; 435/354; 435/455; Stewart & Olstein 536/23.2 5 Becker Farm Road Roseland, NJ 07068 (US) (57) ABSTRACT Appl. No.: 11/401,390 The present invention provides methods for the large-scale (21) production of recombinant butyrylcholinesterase fused to (22) Filed: Apr. 10, 2006 human serum albumin in cell culture, and in the milk and/or urine of transgenic mammals. The recombinant butyrylcho Related U.S. Application Data linesterase-albumin fusion protein of this invention can be used to treat and/or prevent organophosphate pesticide poi (63) Continuation-in-part of application No. 10/326,892, Soning, nerve gas poisoning, cocaine intoxication, and Suc filed on Dec. 20, 2002. cinylcholine-induced apnea. Patent Application Publication Nov. 9, 2006 Sheet 1 of 21 US 2006/0253913 A1 ATGCATAGCAAAGTCACAATCATATGCATCAGATTTCTCTTTTGGTTTCTTTTGCTCTGC M H S K W T I I C I R F L. F W F L L L C 5 ATGCTTATTGGGAAGTCACATACTGAAGATGACATCATAATTGCAACAAAGAATGGAAAA 21 M L G. K. S. H. T E D D A T K N G K 121 GTCAGAGGGATGAACTTGACAGTTTTTGGTGGCACGGTAACAGCCTTTCTTGGAATTCCC 4. W R G M N L T W F. G G T W T A F L G P 181 TATGCACAGCCACCTCTTGGTAGACTTCGATTCAAAAAGCCACAGTCTCTGACCAAGTGG 6. Y A Q. P P L G R L R F K K P Q S L T K W 241 TCTGATATTTCGAATGCCACAAAATATGCAAATTCTTGCTGTCAGAACATAGATCAAAGT 8. S D I W N A T K Y. A N S C C C N I D O S 3O TTTCCAGGCTTCCATGGATCAGAGATGTGGAACCCAAACACTGACCTCAGTGAAGACTGT O F P G F H G S E M W N P N T D L S E D C 36 TTATATCTAAATGTATGGATTCCAGCACCTAAACCAAAAAATGCCACTGTATTGATATGG 12 L. Y. L. N. W. W. I P A P K P K N A T W L I W 421 ATTTAGGTGGTGGTTTTCAAACTGGAACATCATCTTTACATGTTTATGATGGCAAGTTT 141 I Y G G G F C T G T S S L H V Y D G K . F 48 CTGGCTCGGGTTGAAAGAGTTATTGTAGTGTCAATGAACTATAGGGTGGGTGCCCTAGGA 16 L. A R V E R v I V V S M N Y R v G A L G 541 TTCTTAGCTTGCCAGGAAATCCTGAGGCTCCAGGGAACATGGGTTTATTTGACAACAG 8. F L. A. L. P G N P E A P G. N. M. G. L. F D O O. 6 Ol TTGGCTCTTCAGTGGGTTCAAAAAAATATAGCAGCCTTTGGTGGAAATCCTAAAAGTGTA 2O L. A. L. O. W. W. O. K. N. A A F G G N P K S W 661 ACTCTCTTTGGAGAAAGTGCAGGAGCAGCTTCAGTTAGCCTGCATTTGCTTTCTCCTGGA 221 T L. F. G E S A G A A S W S H L E S P G 72 AGCCATTCATTGTTCACCAGAGCCATTCTGCAAAGTGGTTCCTTTAATGCTCCTTGGGCG 24. S H S L. F T R A I L Q S G S F N A P W A 78. GTAACATCTCTTTATGAAGCTAGGAACAGAACGTTGAACTTAGCTAAATTGACTGGTTGC 26 W T S Y E A R N R T L N A K L T G C 84 TCTAGAGAGAATGAGACTGAAATAACAAGTGTCTAGAAATAAAGATCCCCAAGAAATT 28 S R E N E T E I I K C L R N K D P O E I FIGURE 1A Patent Application Publication Nov. 9, 2006 Sheet 2 of 21 US 2006/0253913 A1 90 CTTCTGAATGAAGCATTTGTTGTCCCCTATGGGACTCCTTTGTCAGTAAACTTTGGTCCG 30 L. L N E A F W V P Y G T P L S. V N F G P 96. ACCGTGGATGGTGATTTTCTCACTGACATGCCAGACATATTACTTGAACTTGGACAATTT 321 T W D G D F L T D M P D . L. L. E L G O F 1021 AAAAAAACCCAGATTTTGGTGGGTGTTAATAAAGATGAAGGGACAGCTTTTTTAGTCTAT 34 K K T Q I L W G V N K D E G T A F L V Y O8. GGTGCTCCTGGCTTCAGCAAAGATAACAATAGTATCATAACTAGAAAAGAATTTCAGGAA 361 G A P G F S K D N N S I I T R K E F Q E ll 4l GGTTTAAAAATATTTTTTCCAGGAGTGAGTGAGTTTGGAAAGGAATCCATCCTTTTTCAT 381 G. L. K. I. F. F. P G W S E F. G K E S F. H. 20 TACACAGACTGGGTAGATGATCAGAGACCTGAAAACTACCGTGAGGCCTTGGGTGATGTT 4 Ol Y T D W W D D C R P E N Y R E A L G D V 126l GTTGGGGATTATAATTTCATATGCCCTGCCTTGGAGTTCACCAAGAAGTTCTCAGAATGG 421 V G D Y N F C P A L E F T K K F. S. E. W. 1321 GGAAATAATGCCTTTTTCTACTATTTTGAACACCGATCCTCCAAACTTCCGTGGCCAGAA 441 G N N A F F Y Y F E H R S S K L P W P E 138 TGGATGGGAGTGATGCATGGCTATGAAATTGAATTTGTCTTTGGTTTACCTCEGGAAAGA 4 61 W M G W M H G Y E E. F. W. F. G P L E R 1441 AGAGATAATTACACAAAAGCCGAGGAAATTTTGAGTAGATCCATAGTGAAACGGTGGGCA 481 R D N Y T K A E E I L S R S I v K R W A SO1 AATTTTGCAAAATATGGGAATCCAAATGAGACTCAGAACAATAGCACAAGCTGGCCTGTC 50 N F A K Y G N P N E T O N N S T S W P V 1561. TTCAAAAGCACTGAACAAAAATATCTAACCTTGAATACAGAGTCAACAAGAATAATGACG 52. F K S T E O K. Y. L T L N T E S T R I. M T. 62 AAACTACGTGCTCAACAATGTCGATTCTGGACATCATTTTTTCCAAAAGTCTTGGAAATG 541 K L R A Q O C R F W T S F F P K V L E M l68l ACAGGAAATATTGATGAAGCAGAATGGGAGTGGAAAGCAGGATTCCATCGCTGGAACAAT 56. T. G. N. D E A E W E W K. A. G. F. H. R. W. N. N. 1741 TACATGATGGACTGGAAAAATCAATTTAACGATTACACTAGCAAGAAAGAAAGTTGTGTG 581 Y M. M. D. W. K. N. Q F N D Y T S K K E S C W 80. GGTCTCTAA 601 G k FIGURE 1B Patent Application Publication Nov. 9, 2006 Sheet 4 of 21 US 2006/0253913 A1 1 2 3 4 5 6 Dimer BChE-hSA a Tetramer BChE - Monomer BChE-hSA FIGURE 3 Patent Application Publication Nov. 9, 2006 Sheet 5 of 21 US 2006/0253913 A1 naturt protein 214 ATG (lo) TAA (1938) XXXXXXXXXXXX Noti (16908) & XhoI + 3-cascin SS Xh PCR 5' 8-casein HHR 1. Xhol digestion XhoI digestion 2. Cloning into pGEM-T 1. Ligation V 2. Transformation XhoI digestion Not insulator p3CNN/BChE 17399. a. XhoI p (4647) (12404) 6-casein SS 1 BChE XhoI (10675) 5' 3-casein FIGURE 4 Patent Application Publication Nov. 9, 2006 Sheet 6 of 21 US 2006/0253913 A1 BChE encoding sequence 5' UTR (47nt) ATG STOP Goat 3-casein genomic locus 1000bp FIGURES Patent Application Publication Nov. 9, 2006 Sheet 7 of 21 US 2006/0253913 A1 Dimer rBChE < Endogenous BChE dimer Monomer rBChE FIGURE 6 Patent Application Publication Nov. 9, 2006 Sheet 8 of 21 US 2006/0253913 A1 1- Tetramer BChE -- Dimer BChE -- Monomer BChE FIGURE 7 Patent Application Publication Nov. 9, 2006 Sheet 9 of 21 US 2006/0253913 A1 1.2 Monomer BChE FIGURE 8 Patent Application Publication Nov. 9, 2006 Sheet 10 of 21 US 2006/0253913 A1 Noti (14920) Not (2228) WAP Coding insulator pWAP/BChE 15583bp BamHt (466) Pnie (10706) Signal Sequence FIGURE 9 Patent Application Publication Nov. 9, 2006 Sheet 11 of 21 US 2006/0253913 A1 seve T 8H - V r O , & & r O A : Patent Application Publication Nov. 9, 2006 Sheet 12 of 21 US 2006/0253913 A1 Not I Fsel Sgfl insulator pUM/SS13 HBChE PCR product (4647) -Digest with Fsel & Sgfl -Ligate UM promoter Not I Insulator FIGURE 11 Patent Application Publication Nov. 9, 2006 Sheet 13 of 21 US 2006/0253913 A1 Not UM 3' Insulator Asc Fsel pUM/BChEmod Asc Sgf Mouse UPll PCR product UPI Promoter BCE UM promoter Digested with Asci-Fsel & ligation Sgfl Notl Insulator III pUPII/BChE1nt Mouse UPI PCR product 4kb ASCI 3' genomic sequence Digested with Sgfl-Notl & ligation UPI promoter Not I UP 3 Insulator Ascl Sgfi W8 BChE Fse UPII promoter UPII SS FIGURE 12 Patent Application Publication Nov. 9, 2006 Sheet 16 of 21 US 2006/0253913 A1 Figure 13C 21 Oil CAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATG 701 Q H K D D N P N L. P R L V R P E V D V M 2161 TGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCC 72 C T A F H D N E E T F. L. K. K. Y L. Y. E . A 2221 AGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCT 741 R. R. H P Y F. Y. A P E L L. F. F. A K R Y K. A 2281 GCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGAT 761. A F T E C C Q A A D K. A. A. C. L. L. P K L D 234 GAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTC 781 E L R D E G K A S S A K Q R L. K. C. A. S. L. 24 O1 CAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTT 801 Q K F. G E R A F K. A. W. A. V. A R L S Q R F 2461 CCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACG 821 P. K. A. E. F. A E V S K L W T D L T K W H T 252 GAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTAT 841 E. C. C. H. G D L L E C A D D R A D L. A. K. Y 2581 ATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTG 861 I C E N Q D S I S S K L K E C C E K P L. 2641 TTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCT 881 E. K. S. H. C. I. A E V E N D E M P A D L. P 27 OL TCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAG 901 S L. A. A D F W E S K D V C. K. N. Y. A E A K. 276 GATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTC 921 D V F L G M F. L. Y. E. Y. A R R H P D Y S V 2821 GTGCTGCTGCTGAGACTTGCCAAGACAATGAAACCACTCTAGAGAAGTGCTGTGCCGCT 941 W L L L R I, A K T Y.