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(12) United States Patent (10) Patent No.: US 9,708.635 B2 Murphy Et Al

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(12) United States Patent (10) Patent No.: US 9,708.635 B2 Murphy Et Al USOO9708635B2 (12) United States Patent (10) Patent No.: US 9,708.635 B2 Murphy et al. (45) Date of Patent: *Jul.18, 2017 (54) METHODS OF MAKING ANUCLEC ACID 15/907 (2013.01); A0IK 2207/15 (2013.01); ENCOOING A HUMAN VARIABLE REGION A0IK 2217/05 (2013.01); A0IK 2227/105 (2013.01); A0IK 2267/01 (2013.01); C07K (71) Applicant: Regeneron Pharmaceuticals, Inc., 2317/10 (2013.01); C07K 2317/21 (2013.01); Tarrytown, NY (US) C07K 2317/24 (2013.01); C07K 2317/51 (72) Inventors: Andrew J. Murphy, (2013.01); C07K 2317/515 (2013.01); C07K Croton-On-Hudson, NY (US); George 2317/56 (2013.01); C12N 2800/204 (2013.01) D. Yancopoulos, Yorktown Heights, NY (58) Field of Classification Search (US); Margaret Karow, Santa Rosa CPC ............ A01K 67/0278: A01K 67/0275: A01 K Valley, CA (US); Lynn Macdonald, 2227/105: A01K 2267/01: A01K 2207/15; White Plains, NY (US): Sean Stevens, A01K 2227/10; C07K 16/00; C07K San Diego, CA (US); Aris N. 16/462; C12N 15/85; C12N 15/907; Economides, Tarrytown, NY (US); C12N 15/902; C12N 2800/30; A61 K David M. Valenzuela, Yorktown 49/OO Heights, NY (US) See application file for complete search history. (73) Assignee: Regeneron Pharmaceuticals, Inc., Tarrytown, NY (US) (56) References Cited (*) Notice: Subject to any disclaimer, the term of this U.S. PATENT DOCUMENTS patent is extended or adjusted under 35 5,202,238 A 4/1993 Fell, Jr. et al. U.S.C. 154(b) by 0 days. 5,436,149 A 7/1995 Barnes This patent is Subject to a terminal dis 5,545,807 A * 8/1996 Surani et al. ..................... 800/6 claimer. 5,614,396 A 3/1997 Bradley et al. 5,625,126 A * 4/1997 Lonberg et al. ................ 800, 18 5,770.429 A 6/1998 Lonberg et al. (21) Appl. No.: 13/719,819 5,789,215 A 8, 1998 Berns et al. (22) Filed: Dec. 19, 2012 (Continued) FOREIGN PATENT DOCUMENTS (65) Prior Publication Data EP 438 474 A1 7, 1991 US 2013/0210137 A1 Aug. 15, 2013 EP 1204 740 B1 5, 2002 Related U.S. Application Data (Continued) (60) Continuation of application No. 13/154,976, filed on OTHER PUBLICATIONS Jun. 7, 2011, which is a continuation of application No. 1 1/595,427, filed on Nov. 9, 2006, which is a Clark et al (Nature Reviews: Genetics. 2003, 825-833.* continuation of application No. 10/624,044, filed on (Continued) Jul. 21, 2003, now abandoned, which is a division of application No. 09/784,859, filed on Feb. 16, 2001, now Pat. No. 6,596,541, which is a Primary Examiner — Anoop Singh continuation-in-part of application No. 09/732,234, (74) Attorney, Agent, or Firm — Brendan T. Jones; Neil filed on Dec. 7, 2000, now Pat. No. 6,856,251. Miyamoto; Foley Hoag LLP (60) Provisional application No. 60/244,665, filed on Oct. 31, 2000. (57) ABSTRACT (51) Int. C. A method for engineering and utilizing large DNA vectors to AIK 67/027 (2006.01) target, via homologous recombination, and modify, in any C07K 6/00 (2006.01) desirable fashion, endogenous genes and chromosomal loci C07K I6/46 (2006.01) in eukaryotic cells. These large DNA targeting vectors for CI2N 15/90 (2006.01) eukaryotic cells, termed LTVECs, are derived from frag CI2P 2L/00 (2006.01) ments of cloned genomic DNA larger than those typically CI2N 15/67 (2006.01) used by other approaches intended to perform homologous CI2N 15/85 (2006.01) targeting in eukaryotic cells. Also provided is a rapid and C07K 6/28 (2006.01) convenient method of detecting eukaryotic cells in which the (52) U.S. C. LTVEC has correctly targeted and modified the desired CPC .......... CI2P21/00 (2013.01); A0IK 67/0275 endogenous gene(s) or chromosomal locus (loci) as well as (2013.01); A0IK 67/0278 (2013.01); C07K the use of these cells to generate organisms bearing the 16/00 (2013.01); C07K 16/28 (2013.01); C07K genetic modification. I6/462 (2013.01): CI2N 15/67 (2013.01); CI2N 15/85 (2013.01); C12N 15/8509 (2013.01); C12N 15/902 (2013.01); CI2N 10 Claims, 7 Drawing Sheets US 9,708.635 B2 Page 2 (56) References Cited WO WO-2014/071397 A2 5, 2014 WO WO-2015,088643 A1 6, 2015 U.S. PATENT DOCUMENTS WO WO-2016,081923 A2 5, 2016 5,877,397 A 3, 1999 Lonberg et al. 5,928,914 A 7, 1999 E. et al. OTHER PUBLICATIONS 3.252 A ge. Shepati et al. Niemann et al (Rev. Sci, Tech. Off. Int. Spiz. 2005, (24), 285-298.* 6,069,010 A 5, 2000 Choi Ristevski et al Molecular Biotechnology, 2005, 153-163.* 6,075,181 A 6/2000 Kucherlapati et al. Gama Sosa et al (Brain Struct Funct, 2010, 214:91-109.* 6,096,878 A 8/2000 Honjo et al. 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