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(12) United States Patent (10) Patent No.: US 9,499,622 B2 Cheong Et Al

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(12) United States Patent (10) Patent No.: US 9,499,622 B2 Cheong Et Al USOO9499622B2 (12) United States Patent (10) Patent No.: US 9,499,622 B2 Cheong et al. (45) Date of Patent: Nov. 22, 2016 (54) ANTI-EGFR/ANTI-HER2 BISPECIFIC 5,821,337 A 10, 1998 Carter et al. ANTIBODIES WITH ANT-EGFR DARPNS 6,165,464 A 12/2000 Hudziak et al. 7,417,130 B2 8/2008 Stumpp et al. 8,071,730 B2 12/2011 Goetsch et al. (71) Applicant: Samsung Electronics Co., Ltd., 8, 110,653 B2 2/2012 Stumpp et al. Suwon-si, Gyeonggi-do (KR) 8,329,873 B2 12/2012 Adams et al. 8,524,244 B2 9/2013 Camphausen et al. (72) Inventors: Kwang Ho Cheong, Seoul (KR): 9,234,028 B2 1/2016 Camphausen et al. Seung Hyun Lee, Suwon-si (KR): 9,359.440 B2 * 6/2016 Cheong ................ CO7K 14,705 Powei Lin, Hwaseong-si (KR); Saet 2009/0010840 A1 1/2009 Adams et al. Byoul Lee, Seoul (KR); Jae Woong 2010/0178298 A1 7/2010 Lindhofer 2012fOO2O966 A1* 1/2012 Barbas, III ........... CO7K 14,515 Hwang, Seoul (KR) 424,134.1 2012/0156191 A1 6/2012 Goetsch et al. (73) Assignee: SAMSUNGELECTRONICS CO., 2012,0277143 A1 1 1/2012 Jacobs et al. LTD., Suwon-Si (KR) 2013, OO17200 A1 1/2013 Scheer et al. 2015.OO30596 A1* 1/2015 Cheong ................ CO7K 14,705 (*) Notice: Subject to any disclaimer, the term of this 424,134.1 patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS EP 1332.209 B2 11/2009 (21) Appl. No.: 14/625,296 EP 21496.04 A1 2, 2010 JP 2011-5 17314 A 6, 2011 (22) Filed: Feb. 18, 2015 KR 2009-0088878 A 8, 2009 KR 2012-01.23299. A 11 2012 (65) Prior Publication Data WO WO 2012/069655 A2 5, 2012 US 2015/O232573 A1 Aug. 20, 2015 OTHER PUBLICATIONS (30) Foreign Application Priority Data HogenEsch et al. J. Controlled Release 2012; 164:183-186.* Tang et al. Cancer Letters 2016; 370:85-90.* Feb. 18, 2014 (KR) ........................ 10-2014-0018649 Kataja et al., Ann Oncol 2009; 20(sup 4): ivl 0-14.* Nelson et al., Ann. Intern Med. 2009; 151:727-737.* (51) Int. Cl. Balmana et al. Ann Oncol 2009; 20(supp 4):iv19-20.* C07K 6/30 (2006.01) Boersma et al., J. Biol. Chem., 2011:286: 41273-85.* Steiner et al. J. Mol. Biol. 2008; 382:1211-27 and Supplemental A 6LX 39/395 (2006.01) Figures.* C07K 6/28 (2006.01) C07K 6/32 (2006.01) * cited by examiner (52) U.S. Cl. CPC ........... C07K 16/2863 (2013.01); C07K 16/32 Primary Examiner — Jessica H Roark (2013.01); C07K 23.17/31 (2013.01); C07K (74) Attorney, Agent, or Firm — Leydig, Voit & Mayer, 2317/73 (2013.01); C07K 2317/77 (2013.01); Ltd. C07K 2317/92 (2013.01) (58) Field of Classification Search (57) ABSTRACT CPC .............. C07K 16/00–16/468; C07K 23.17/31 An anti-EGFR/anti-HER2 bispecific antibody including an See application file for complete search history. anti-EGFR DARPin and an anti-HER2 antibody, a pharma ceutical composition including the bispecific antibody, a (56) References Cited method of preparing the bispecific antibody, and a method of U.S. PATENT DOCUMENTS reducing a side effect and/or enhancing efficacy of an anti-HER2 antibody using an anti-EGFR DARPin. 5,677,171 A 10, 1997 Hudziak et al. 5,772,997 A 6, 1998 Hudziak et al. 19 Claims, 5 Drawing Sheets U.S. Patent Nov. 22, 2016 Sheet 1 of 5 US 9,499,622 B2 FG, Ai-HER Antibody ti-ER DARPS U.S. Patent Nov. 22, 2016 Sheet 2 of 5 US 9,499 622 B2 •ooogoogz00:ºzpozz00:0z00:2100:91ooptoozi00:0109:8900:øz00:0 53?nä??ae |-ovoro U.S. Patent Nov. 22, 2016 Sheet 3 of 5 US 9,499,622 B2 FG. 3 3. ic: s . - s w 2 : S as .. * s ::s S 8.8 s s 8s 8S as | w Antibody: gai U.S. Patent Nov. 22, 2016 Sheet 4 of 5 US 9,499,622 B2 w 120 100 control Erbitux hierceptin H2E-O1 U.S. Patent Nov. 22, 2016 Sheet S of 5 US 9,499,622 B2 FG. 6 US 9,499,622 B2 1. 2 ANT-EGFR/ANTI- HER2 BISPECIFIC Another embodiment provides a pharmaceutical compo ANTIBODES WITH ANT-EGFR DARPNS sition including the bispecific antibody. Another embodiment provides a method of preventing CROSS-REFERENCE TO RELATED and/or treating a cancer including administering the bispe APPLICATIONS cific antibody to a subject in need of preventing and/or treating the cancer. This application claims the benefit of Korean Patent Another embodiment provides a method of reducing a Application No. 10-2014-0018649 filed on Feb. 18, 2014, in side effect (such as agonism) and/or enhancing an efficacy of the Korean Intellectual Property Office, the entire disclosure an anti-HER2 antibody, including binding a DARPinto the of which is hereby incorporated by reference. 10 antibody. INCORPORATION-BY-REFERENCE OF BRIEF DESCRIPTION OF THE DRAWINGS MATERIAL ELECTRONICALLY SUBMITTED FIG. 1 is a schematic view showing processes of prepar Incorporated by reference in its entirety herein is a com 15 ing an anti-EGFR/anti-HER2 bispecific antibody. puter-readable nucleotide?amino acid sequence listing Sub FIG. 2 is a graph showing properties of an anti-EGFR/ mitted herewith and identified as follows: One 18,994 bytes anti-HER2 bispecific antibody. ASCII (Text) file named “719349 ST25-revised.TXT,” cre FIG. 3 is a graph showing the degree of proliferation ated Feb. 18, 2015 Apr. 29, 2016. inhibition of MKN45 cells by an anti-EGFR/anti-HER2 bispecific antibody. BACKGROUND OF THE INVENTION FIG. 4 is a graph showing the degree of proliferation inhibition of SNU638 cells by an anti-EGFR/anti-HER2 1. Field bispecific antibody. Provided is an anti-EGFR/anti-HER2 bispecific antibody 25 FIG. 5 is a graph showing the degree of proliferation including an anti-EGFR DARPin and an anti-HER2 anti inhibition of N87 cells by an anti-EGFR/anti-HER2 bispe body, a pharmaceutical composition including the bispecific cific antibody. antibody, a method of preparing the bispecific antibody, and FIG. 6 is a set of fluorescence images showing internal a method of reducing a side effect and/or enhancing an ization of EGFR and HER2 by an anti-EGFR/anti-HER2 efficacy of an anti-HER2 antibody using an anti-EGFR 30 bispecific antibody. DARPin. 2. Description of the Related Art DETAILED DESCRIPTION OF THE In living cells, various proteins interact with each other INVENTION and are participants in various disease-causing mechanisms. If at least two of such proteins are simultaneously inhibited, 35 Bispecific antibodies have been developed in various a greater effect of treating a disease and a greater possibility kinds and forms and are expected as a new drug antibody of overcoming a resistance against an inhibitor against each having excellent therapeutic effects compared to pre-exist protein can be obtained, compared with the case of inhib ing monoclonal antibodies, due to its dual (multi-) binding iting a single protein. For these reasons, various antibodies activity to at least two different antigens. Herein, a bispecific capable of inhibiting at least two proteins have been devel 40 antibody obtained by binding a DARPinto an IgG form oped. antibody is disclosed. Although many bispecific antibodies have been devel DARPin (designed ankyrin repeat protein) refers to an oped, most of the bispecific antibodies cannot be commer antibody mimetic protein having high specificity and high cialized as antibody medicaments, because their therapeutic binding affinity to a target protein, which is prepared via effects are not clinically verified or various side effects are 45 genetic engineering. DARPin is originated from natural observed. In addition, the developed bispecific antibodies ankyrin protein, and has a structure where at least 2 or at have defects in stability and large scale production, which is least 3 ankyrin repeat motifs, for example, 3, 4, 5, 6, 8 or 10 an obstacle in commercialization. The early developed ankyrin repeat motifs are repeated. For example, the bispecific antibodies having IgG form have difficulties in DARPins including 3, 4 or 5 ankyrin repeat motifs may have isolation and purification, since light chains and heavy 50 a molecular weight of about 10 kDa, about 14 kDa, and chains are randomly combined during producing processes, about 18 kDa, respectively. DARPin includes a core part leading to problems in large scale production. In addition, in which acts structural function and a target binding part the case of bispecific antibodies having other form than IgG, outside of the core which binds to a target. The core part the stability as a medicine in respect of protein folding, includes conserved amino acid sequence and the target pharmacokinetics, and the like has not been Verified. 55 binding part, which is positioned outside of the core part, Therefore, there is a need for developing a bispecific includes different amino acid sequence depending on the antibody having increased stability and improved properties target. as a medicine. DARPin has target specificity, which is similar to an antibody, and thus, a new form of a bispecific antibody can BRIEF SUMMARY OF THE INVENTION 60 be made by attaching at least one DARPinto an antibody having various forms such as an IgG (e.g., IgG1, IgG2, IgG3 One embodiment provides a bispecific antibody against or IgG4) form, a schv-Fc form, and the like. EGFR and HER2, including an anti-EGFR DARPin and an The “EGFR (epidermal growth factor receptor) is a anti-HER2 antibody.
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