USOO9464286B2 (12) United States Patent (10) Patent No.: US 9,464,286 B2 Zhu et al. (45) Date of Patent: Oct. 11, 2016 (54) HIGH THROUGHPUT GENERATION AND 5,767,260 A 6/1998 Whitlow et al. AFFINITY MATURATION OF HUMANIZED 5,780,279 A 7/1998 Matthews et al. 5,798,208 A 8, 1998 Crea ANTIBODY 5,811,238 A 9, 1998 Stemmer et al. 5,814,476 A 9, 1998 Kauffman et al. (75) Inventors: Li Zhu, Palo Alto, CA (US); 5,817483. A 10, 1998 Kauffman et al. Shuanghong Wei, San Jose, CA (US); 5,821,047 A 10, 1998 Garrard et al. Shaobing B. Hua, Cupertino, CA (US) 5,824,514 A 10, 1998 Kauffman et al. 5,830,663 A 11/1998 Embleton et al. 5,837,242 A 11/1998 Holliger et al. (73) Assignee: Adimab, LLC, Lebanon, NH (US) 5,837,500 A 11/1998 Ladner et al. 5,840,479 A 11/1998 Little et al. (*) Notice: Subject to any disclaimer, the term of this 5,846,765 A 12/1998 Matthews et al. patent is extended or adjusted under 35 5,858,657 A 1/1999 Winter et al. 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CI2N 15/1041 (2013.01); CI2N 15/1062 MacCallum et al. J. Mol. Biol., 262, 732-745, 1996.* (2013.01); C12N 15/1086 (2013.01) Casset et al. Biochemical and Biophysical Research Communica (58) Field of Classification Search tions, 307: 198-205, 2003.* None (Continued) See application file for complete search history. (56) References Cited Primary Examiner — Laura B Goddard Assistant Examiner — Meera Natarajan U.S. PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Brenda Herschbach 4,230,685 A 10/1980 Senyei et al. Jarrell; Robert N. Sahr; Choate Hall & Stewart LLP 4,452,773. A 6/1984 Molday 4,661,454. A 4, 1987 Botstein et al. (57) ABSTRACT 4,770,183 A 9, 1988 Groman et al. Compositions, methods, and kits are provided for efficiently 4,816,567 A 3/1989 Cabilly et al. 4.946,778 A 8, 1990 Ladner et al. generating and screening humanized antibody with high 4.959,317 A 9, 1990 Sauer affinity against a specific antigen. The library of humanized 5,118,605 A 6, 1992 Urdea antibody is generated by mutagenizing a chimeric antibody 5,223,409 A 6, 1993 Ladner et al. template that combines human antibody framework and 5,283,173 A 2f1994 Fields et al. 5,380,833. A 1, 1995 Urdea antigen binding sites of a non-human antibody. Alterna 5,506,121 A 4, 1996 Skerra et al. tively, the library of humanized antibody is generated by 5,525.490 A 6, 1996 Erickson et al. grafting essential antigen-recognition segment(s) such as 5.530,101 A * 6/1996 Queen et al. CDRs of the non-human antibody into the corresponding 5,565,332 A 10/1996 Hoogenboom et al. position(s) of each member of a human antibody library. 5,571,698 A 11/1996 Ladner et al. 5,618,920 A 4/1997 Robinson et al. This library of humanized antibody is then screened for high 5,658,727 A 8, 1997 Barbas et al. affinity binding toward a specific antigen in vivo in organism 5,688,666 A 11/1997 Bass et al. Such as yeast or in vitro using techniques such as ribosome 5,695,941 A 12/1997 Brent et al. display or mRNA display. The overall process can be 5,712, 120 A 1/1998 Rodriguez et al. efficiently performed in a high throughput and automated 5,723,323 A 3, 1998 Kauffman et al. 5,723,584 A 3, 1998 Schatz manner, thus mimicking the natural process of antibody 5,733,743 A 3, 1998 Johnson et al. affinity maturation. 5,739,281 A 4, 1998 Thogersen et al. 5,750,373 A 5, 1998 Garrard et al. 18 Claims, 13 Drawing Sheets US 9,464,286 B2 Page 2 (56) References Cited 6,696,248 B1 2/2004 Knappik et al. 6,696.251 B1 2/2004 Wittrup et al. 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