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
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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. member of the receptor tyrosine kinases (RTKs) of HER Another embodiment provides a method of preparing a 65 family. Over-expression, gene amplification, mutation, or bispecific antibody against EGFR and HER2, including rearrangement of EGFR are frequently observed in several linking an anti-HER2 antibody and an anti-EGFR DARPin. human malignant tumors and are related to poor prognosis US 9,499,622 B2 3 4 of cancer treatment and bad clinical outcomes. For Such growth factor binds thereto, leading to activation of various reasons, the EGFR becomes an important target in antican signal transduction pathways, thereby inducing apoptosis, cer therapy. survival, or proliferation of cells. Therefore, in an embodiment, provided is a fusion protein The HER2 protein may be originated from a mammal, for comprising or consisting essentially of an anti-EGFR DAR example, HER2 originated from primates, such as human Pin (or an EGFR binding DARPin) which specifically binds HER2, monkey HER2, and the like, or HER2 originated to EGFR and an anti-HER2 antibody. The fusion protein from rodents, such as mouse HER2, rat HER2, and the like. may be used as a bispecific antibody specifically recognizing For example, the HER2 protein may be human HER2 (e.g., and/or binding to EGFR and HER2. Therefore, another encoded by the nucleotide sequence (mRNA) of GenBank embodiment provides an anti-EGFR/anti-HER2 bispecific 10 antibody comprising or consisting essentially of an anti Accession Number NM 004448), mouse HER2 (e.g., EGFR DARPin and an anti-HER2 antibody. The anti-HER2 encoded by the nucleotide sequence (mRNA) of GenBank antibody may be in an IgG form, a schv-Fc form, or a Accession Number NM_001003817), or rat HER2 (e.g., combination thereof. As used herein, the term “IgG form’ encoded by the nucleotide sequence (mRNA) of GenBank may refer to a protein complex composed of four peptide 15 Accession Number NM 017003). chains, i.e., two identical heavy chains and two identical The antibody having an IgG form may be in a form of light chains, arranged in a Y-shape. As used herein, the term IgG1, IgG2, IgG3 or IgG4 Subtype of a mammal, for “schv-Fc' may refer to an antibody fragment comprising example, IgG1 or IgG2 subtype. The antibody having an IgG ScFv (single-chain variable fragment; a fusion protein of the form includes two heavy chains and two light chains, and the variable regions of the heavy (generally at N-terminus) and heavy chain and the light chain are linked to each other via light chains (generally at C-terminus) of immunoglobulins, disulfide bond, forming two heavy chain-light chain struc connected with each other directly (through a covalent bond tures. The formed two heavy chain-light chain structures are Such as a peptide bond) or via a peptide linker) and Fc region linked to each other at Fc region of the heavy chain via (fragment crystallizable region) which is linked to the C-ter disulfide bond. minus of the scFv directly (through a covalent bond such as 25 a peptide bond) or via a peptide linker. The antibody having a scFv-Fc form may be in a mono The anti-EGFR DARPin may be any DARPin having meric form comprising a Sclv-Fc fragment comprising an DARPin's own unique structure and specifically binding to antigen-binding region specifically recognizing and/or bind EGFR. For example, the anti-EGFR DARPin may include at ing to HER2 or in a dimeric form comprising two scFv-Fc least one selected from the group consisting of the following 30 fragments comprising antigen-binding regions specifically four anti-EGFR DARPins: recognizing and/or binding to HER2, where the two schv-Fc fragments are linked to each other at Fc region. The Fc region may be derived from subtype IgG1, IgG2, IgG3 or anti-EGFR DARPin- O1 (SEQ ID NO: 1) : IgG4 of a mammal, for example, IgG1 or IgG2. diligkkilleaaragoddevrilmangadvnaddtwdwtp.lhlaaygghlei 35 IgG1, IgG2, IgG3, or IgG4 may originate from a mam vevillking advnaydyigwtp.lhlaadghlei vevillking advnasdyig mal. Such as a primate including human, a monkey, and the dtplhlaahnghileivevillkhgadvnaqdkfgktafdisidingnedlae like, or a rodent including a mouse, a rat, and the like, and for example, may be human IgG1, IgG2, IgG3, or IgG4 ild 40 Subtype. anti-EGFR DARPin- 67 (SEQ ID NO: 2) : The anti-HER2 antibody may be (1) an antibody or (2) an diligkkilleaaragoddevrilmangadvnatolindgntplhlsa Wighlei IgG type antibody or a scv-Fc type antibody comprising an antigen-binding region of the antibody (1), wherein the vevillkhgadvnaddllgmtp.lhlaadtghleivevillkygadvnardtr antibody (1) may be selected from the group consisting of 45 gktplhlaardghleivevillkhdadvnaqdkfgktafidisidingnedla i) trastuzumab comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and eild a light chain variable region comprising the amino acid anti-EGFR DARPin- 68 (SEQ ID NO : 3) : sequence of SEQ ID NO: 8, diligkkilleaaragoddevrilmangadvnafdywgmtp.lhlaadinghlei 50 ii) pertuZumab comprising a heavy chain comprising the vevillkhgadvnas dnfgiftpilhlaafyghleivevillkhgadvnafdmw amino acid sequence of SEQ ID NO: 9 and a light chain gntplhlaaqnghileivevillkingadvnaqdkfgktafidisidingnedla comprising the amino acid sequence of SEQ ID NO: 10, iii) trastuzumab emtansine (T-DM1), and eild 55 iv) an anti-HER2 antibody comprising a heavy chain anti-EGFR DARPin- 69 (SEQ ID NO : 4) : variable region comprising the amino acid sequence of SEQ diligkkilleaaragoddevrilmangadvnaddnagirtplhlaanfghlei ID NO: 5 and a light chain variable region comprising the vevillking advnakghhentiplhlaawaghleivevillkygadvnaddde amino acid sequence of SEQ ID NO: 6. gytplhlaadigdleivevillkygadvnawdmygrtplhlaasaghleiv 60
U Sample Antigen Flow Cell R (RU) K (nM) k (1/Ms) k (1/s) Chi Value T(K) T(K) H2E-O1 EGFR-FC fia-ii1 78.89 0.03 1.0 x 10 <2.8 x 10 1.58 95 6.2 x 102 1.2 130522 Her2-FC #2-#1 80.45 <0.01 6.9 x 10 <7.5 x 10 1.56 95 9.8 x 102 1.4
As shown in Table 1, the bispecific antibody H2E-01 Trastuzumab (HERCEPTIN antibody, Roche), CETUX prepared in Example 1 exhibits very high affinity to EGFR 65 IMAB antibody (Erbitux, #ET509081213, Merck), and anti and HER2 as KD=0.03 nM and <0.01 nM, respectively, as HER2/anti-EGFR DARPin bispecific antibody H2E-01 pre measured by Biacore. pared in Example 1 were treated alone or in combination at US 9,499,622 B2 15 16 the amount of 1 ug/ml per each well (when treated in context. The use of the term “at least one’ followed by a list combination, each treated amount is 1 ug/ml), and incubated of one or more items (for example, “at least one of A and B) at 37° C. for 2 hours. The incubated cells were treated with is to be construed to mean one item selected from the listed 4% (v/v) formaldehyde for 15 minutes, to be immobilized on items (A or B) or any combination of two or more of the 5 listed items (A and B), unless otherwise indicated herein or plate, and then, washed three times with PBS. Thereafter, the clearly contradicted by context. The terms “comprising.” resulted cells were treated with blocking buffer (0.5% (v/v) “having,”99 “including,& and “containing are to be construed triton X-100 and 5% (v/v) donkey serum) for 1 hour at room as open-ended terms (i.e., meaning “including, but not temperature, and then, treated with primary antibodies limited to.”) unless otherwise noted. Recitation of ranges of respectively against HER2 and EGFR (primary antibody for values herein are merely intended to serve as a shorthand HER2: # 280003Z, Invitrogen, primary antibody for EGFR: 10 method of referring individually to each separate value #5616, Cell signaling) at the amount of 100 ul (1:100 falling within the range, unless otherwise indicated herein, diluted) at 4° C. for 15 hours. The resultant was washed and each separate value is incorporated into the specification three times with PBS, treated with a secondary antibody as if it were individually recited herein. All methods (#A21433, Invitrogen) at the amount of 100 ul (1:2000 described herein can be performed in any suitable order diluted) at room temperature for 1 hour, and washed again 15 unless otherwise indicated herein or otherwise clearly con three times with PBS, to prepare a plate with mounting tradicted by context. The use of any and all examples, or medium (HH-1200, Vector). The cells in the prepared plate exemplary language (e.g., “such as”) provided herein, is were observed by a confocal microscope (Zeiss, LSM710). intended merely to better illuminate the invention and does The obtained results are shown in FIG. 6. As shown in not pose a limitation on the scope of the invention unless FIG. 6, when Herceptin and Erbitux are treated in combi otherwise claimed. No language in the specification should nation, EGFR and HER2 still remain on cell membrane, be construed as indicating any non-claimed element as whereas when H2E-01 is treated, both of HER2 and EGFR essential to the practice of the invention. move into a cell. Preferred embodiments of this invention are described In conclusion, the anti-HER2/anti-EGFR DARPin bispe herein, including the best mode known to the inventors for cific antibody with an anti-EGFR DARPin inhibits EGFR 25 carrying out the invention. Variations of those preferred and HER2 is believed to act by different mechanism from embodiments may become apparent to those of ordinary that of pre-existing anti-EGFR or anti-HER2 antibody. skill in the art upon reading the foregoing description. The All references, including publications, patent applica inventors expect skilled artisans to employ such variations tions, and patents, cited herein are hereby incorporated by as appropriate, and the inventors intend for the invention to reference to the same extent as if each reference were 30 be practiced otherwise than as specifically described herein. individually and specifically indicated to be incorporated by Accordingly, this invention includes all modifications and reference and were set forth in its entirety herein. equivalents of the Subject matter recited in the claims The use of the terms “a” and “an and “the' and “at least appended hereto as permitted by applicable law. Moreover, one' and similar referents in the context of describing the any combination of the above-described elements in all invention (especially in the context of the following claims) 35 possible variations thereof is encompassed by the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or otherwise clearly con unless otherwise indicated herein or clearly contradicted by tradicted by context.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS : 17
<21 Os SEQ ID NO 1 &211s LENGTH: 153 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic anti-EGFR DARPin-O1 <4 OOs SEQUENCE: 1 Asp Lieu. Gly Lys Llys Lieu. Lieu. Glu Ala Ala Arg Ala Gly Glin Asp Asp 1. 5 10 15 Glu Val Arg Ile Lieu Met Ala Asn Gly Ala Asp Val Asn Ala Asp Asp 2O 25 3O Thir Trp Gly Trp Thr Pro Leu. His Lieu Ala Ala Tyr Glin Gly His Lieu. 35 4 O 45
Glu Ile Val Glu Val Lieu. Lieu Lys Asn Gly Ala Asp Wall Asn Ala Tyr SO 55 6 O Asp Tyr Ile Gly Trp Thr Pro Lieu. His Lieu Ala Ala Asp Gly His Lieu. 65 70 7s 8O
Glu Ile Val Glu Val Lieu. Lieu Lys Asn Gly Ala Asp Wall Asn Ala Ser 85 9 O 95 Asp Tyr Ile Gly Asp Thr Pro Lieu. His Lieu Ala Ala His Asn Gly. His US 9,499,622 B2 17 - Continued
1OO 105 11 O Lieu. Glu Ile Val Glu Val Lieu. Lieu Lys His Gly Ala Asp Val Asn Ala 115 12 O 125 Glin Asp Llys Phe Gly Llys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 13 O 135 14 O Asn Glu Asp Lieu Ala Glu Ile Lieu. Glin 145 150
<210s, SEQ ID NO 2 &211s LENGTH: 154 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Anti-EGFR DARPin-67 <4 OOs, SEQUENCE: 2 Asp Lieu. Gly Lys Llys Lieu. Lieu. Glu Ala Ala Arg Ala Gly Glin Asp Asp 1. 5 1O 15 Glu Val Arg Ile Lieu Met Ala Asn Gly Ala Asp Val Asn Ala Thr Asp 2O 25 3 O Asn Asp Gly Asn Thr Pro Lieu. His Lieu. Ser Ala Trp Ile Gly. His Lieu 35 4 O 45 Glu Ile Val Glu Val Lieu Lleu Lys His Gly Ala Asp Wall Asn Ala Asp SO 55 60 Asp Lieu. Lieu. Gly Met Thr Pro Lieu. His Lieu Ala Ala Asp Thr Gly His 65 70 7s 8O Lieu. Glu Ile Val Glu Val Lieu Lieu Lys Tyr Gly Ala Asp Val Asn Ala 85 90 95 Arg Asp Thir Arg Gly Llys Thr Pro Lieu. His Lieu Ala Ala Arg Asp Gly 1OO 105 11 O His Lieu. Glu Ile Val Glu Val Lieu. Lieu Lys His Asp Ala Asp Wall Asn 115 12 O 125 Ala Glin Asp Llys Phe Gly Llys Thr Ala Phe Asp Ile Ser Ile Asp Asn 13 O 135 14 O Gly Asn. Glu Asp Lieu Ala Glu Ile Lieu. Glin 145 150
<210s, SEQ ID NO 3 &211s LENGTH: 154 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Anti-EGFR DARPin- 68 <4 OOs, SEQUENCE: 3 Asp Lieu. Gly Lys Llys Lieu. Lieu. Glu Ala Ala Arg Ala Gly Glin Asp Asp 1. 5 1O 15 Glu Val Arg Ile Lieu Met Ala Asn Gly Ala Asp Val Asn Ala Phe Asp 2O 25 3 O Tyr Trp Gly Met Thr Pro Lieu. His Lieu Ala Ala Asp Asn Gly His Lieu. 35 4 O 45 Glu Ile Val Glu Val Lieu Lleu Lys His Gly Ala Asp Wall Asn Ala Ser SO 55 60 Asp Asin Phe Gly Phe Thr Pro Leu. His Leu Ala Ala Phe Tyr Gly His 65 70 7s 8O Lieu. Glu Ile Val Glu Val Lieu. Lieu Lys His Gly Ala Asp Val Asn Ala 85 90 95 US 9,499,622 B2 19 20 - Continued
Phe Asp Met Trp Gly Asn Thir Pro Lieu. His Lieu Ala Ala Glin Asn Gly 1OO 105 11 O
His Luell Glu Ile Wall Glu Wall Luell Lieu Lys Asn Gly Ala Asp Wall Asn 115 12 O 125
Ala Glin Asp Llys Phe Gly Lys Thir Ala Phe Asp Ile Ser Ile Asp Asn 13 O 135 14 O
Gly Asn Glu Asp Lieu. Ala Glu Ile Luell Glin 145 150
SEQ ID NO 4 LENGTH: 1.87 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Anti-EGFR DARPin-69
<4 OOs, SEQUENCE: 4
Asp Lieu. Gly Llys Llys Lell Lell Glu Ala Ala Arg Ala Gly Glin Asp Asp 1. 5 15
Glu Wall Arg Ile Lieu. Met Ala Asn Gly Ala Asp Wall Asn Ala Asp Asp 2O 25 3 O
Asn Ala Gly Arg Thr Pro Lell His Luell Ala Ala Asn Phe Gly His Lieu. 35 4 O 45
Glu Ile Wall Glu Wall Lell Lell Asn Gly Ala Asp Wall Asn Ala Lys SO 55 60
Gly His His Cys Asn Thir Pro Lel His Luell Ala Ala Trp Ala Gly His 65 70
Lell Glu Ile Wall Glu Wall Lell Lel Tyr Gly Ala Asp Wall Asn Ala 85 90 95
Asp Asp Asp Glu Gly Thir Lel His Luell Ala Ala Asp Ile Gly 1OO 105 11 O
Asp Luell Glu Ile Wall Glu Wall Lel Lel Tyr Gly Ala Asp Wall Asn 115 12 O 125
Ala Trp Asp Met Tyr Gly Arg Thir Luell His Lell Ala Ala Ser Ala 13 O 135 14 O
Gly His Luell Glu Ile Wall Glu Wall Lel Luell Lys Gly Ala Asp Wall 145 150 155 160
Asn Ala Glin Asp Llys Phe Gly Thir Ala Phe Asp Ile Ser Ile Asp 1.65 17s
Asn Gly Asn Glu Asp Lell Ala Glu Ile Luell Glin 18O 185
SEO ID NO 5 LENGTH: 120 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic heavy chain variable region of anti-HER2 antibody
<4 OOs, SEQUENCE: 5
Glu Wall Glin Lieu Wall Glu Ser Gly Gly Gly Lieu. Wall Glin Pro Gly Gly 1. 5 15
Ser Luell Arg Luell Ser Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 2O 25 3 O
Tyr Ile His Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Val 35 4 O 45
Ala Arg Ile Tyr Pro Thir Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Wall US 9,499,622 B2 21 - Continued
SO 55 60 Lys Gly Arg Phe Thir Ile Ser Ala Asp Thir Ser Lys Asn. Thir Ala Tyr 65 70 7s 8O Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Glin 1OO 105 11 O Gly Thr Lieu Val Thr Val Ser Ser 115 12 O
<210s, SEQ ID NO 6 &211s LENGTH: 108 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic light chain variable region of anti-HER2 antibody
<4 OOs, SEQUENCE: 6 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1. 5 1O 15 Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Asp Wall Asn. Thir Ala 2O 25 3 O Val Ala Trp Tyr Glin Gln Llys Pro Gly Lys Ala Pro Llys Lieu. Lieu. Ile 35 4 O 45 Tyr Ser Ala Ser Phe Lieu. Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly SO 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Ser Leu Gln Pro 65 70 7s 8O Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Glin Gly Thr Lys Val Glu Ile Lys Arg 1OO 105
<210s, SEQ ID NO 7 &211s LENGTH: 220 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Heavy chain variable region of Trastuzumab
<4 OO > SEQUENCE: 7 Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Asn. Ile Lys Asp Thr 2O 25 3 O Tyr Ile His Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val 35 4 O 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val SO 55 60
Lys Gly Arg Phe Thir Ile Ser Ala Asp Thir Ser Lys Asn. Thir Ala Tyr 65 70 7s 8O
Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Glin 1OO 105 11 O
Gly Thr Lieu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val US 9,499,622 B2 23 24 - Continued
115 12 O 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 13 O 135 14 O Lieu. Gly Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala Val 1.65 17O 17s Lieu. Glin Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val Pro 18O 185 19 O Ser Ser Ser Leu Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His Lys 195 2OO 2O5 Pro Ser Asn. Thir Lys Val Asp Llys Llys Val Glu Pro 21 O 215 22O
<210s, SEQ ID NO 8 &211s LENGTH: 214 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Light chain variable region of Trastuzumab
<4 OOs, SEQUENCE: 8 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1. 5 1O 15 Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Asp Wall Asn. Thir Ala 2O 25 3 O Val Ala Trp Tyr Glin Gln Llys Pro Gly Lys Ala Pro Llys Lieu. Lieu. Ile 35 4 O 45 Tyr Ser Ala Ser Phe Lieu. Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly SO 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Ser Leu Gln Pro 65 70 7s 8O Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Glin Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 1OO 105 11 O Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 12 O 125 Thir Ala Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe Tyr Pro Arg Glu Ala 13 O 135 14 O Llys Val Glin Trp Llys Val Asp Asn Ala Lieu. Glin Ser Gly Asn. Ser Glin 145 150 155 160 Glu Ser Val Thr Glu Glin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 1.65 17O 17s Ser Thr Lieu. Thir Lieu. Ser Lys Ala Asp Tyr Glu Lys His Llys Val Tyr 18O 185 19 O Ala Cys Glu Val Thr His Glin Gly Lieu Ser Ser Pro Val Thr Lys Ser 195 2OO 2O5
Phe Asn Arg Gly Glu. Cys 21 O
<210s, SEQ ID NO 9 &211s LENGTH: 448 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 9,499,622 B2 25 26 - Continued <223> OTHER INFORMATION: Synthetic Heavy chain of Pertuzumab <4 OOs, SEQUENCE: 9 Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 2O 25 3 O Thir Met Asp Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val 35 4 O 45 Ala Asp Val Asn Pro Asn. Ser Gly Gly Ser Ile Tyr Asn. Glin Arg Phe SO 55 60 Lys Gly Arg Phe Thr Lieu. Ser Val Asp Arg Ser Lys Asn. Thir Lieu. Tyr 65 70 7s 8O Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Lieu. Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Glin Gly 1OO 105 11 O Thr Lieu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 12 O 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 13 O 135 14 O Gly Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala Val Lieu. 1.65 17O 17s Gln Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 18O 185 19 O Ser Ser Leu Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His Llys Pro 195 2OO 2O5 Ser Asn. Thir Lys Val Asp Llys Llys Val Glu Pro Llys Ser Cys Asp Llys 21 O 215 22O Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly Gly Pro 225 23 O 235 24 O Ser Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 26 O 265 27 O Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 27s 28O 285 Ala Lys Thir Lys Pro Arg Glu Glu Glin Tyr Asn. Ser Thr Tyr Arg Val 29 O 295 3 OO Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu 3. OS 310 315 32O Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu Lys 3.25 330 335
Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr 34 O 345 35. O
Lieu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser Lieu. Thr 355 360 365
Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 37 O 375 38O
Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. 385 390 395 4 OO US 9,499,622 B2 27 28 - Continued Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Lys Lieu. Thr Val Asp Llys 4 OS 41O 415 Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 42O 425 43 O Ala Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 44 O 445
<210s, SEQ ID NO 10 &211s LENGTH: 214 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Light chain of Pertuzumab <4 OOs, SEQUENCE: 10 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1. 5 1O 15 Asp Arg Val Thir Ile Thr Cys Lys Ala Ser Glin Asp Wal Ser Ile Gly 2O 25 3 O Val Ala Trp Tyr Glin Gln Llys Pro Gly Lys Ala Pro Llys Lieu. Lieu. Ile 35 4 O 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly SO 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Ser Leu Gln Pro 65 70 7s 8O
Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin Tyr Tyr Ile Tyr Pro Tyr 85 90 95
Thr Phe Gly Glin Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 1OO 105 11 O
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 12 O 125
Thir Ala Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe Tyr Pro Arg Glu Ala 13 O 135 14 O
Llys Val Glin Trp Llys Val Asp Asn Ala Lieu. Glin Ser Gly Asn. Ser Glin 145 150 155 160
Glu Ser Val Thr Glu Glin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 1.65 17O 17s Ser Thr Lieu. Thir Lieu. Ser Lys Ala Asp Tyr Glu Lys His Llys Val Tyr 18O 185 19 O
Ala Cys Glu Val Thr His Glin Gly Lieu Ser Ser Pro Val Thr Lys Ser 195 2OO 2O5
Phe Asn Arg Gly Glu. Cys 21 O
<210s, SEQ ID NO 11 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic modified hinge region (U7-HC6)
<4 OOs, SEQUENCE: 11 Glu Pro Ser Cys Asp Llys His Cys Cys Pro Pro Cys Pro 1. 5 1O
<210s, SEQ ID NO 12 &211s LENGTH: 13 US 9,499,622 B2 29 30 - Continued
212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic modified hinge region (U6-HC7) <4 OOs, SEQUENCE: 12 Glu Pro Llys Ser Cys Asp Cys His Cys Pro Pro Cys Pro 1. 5 1O
<210s, SEQ ID NO 13 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic modified hinge region (U3 -HC9)
<4 OOs, SEQUENCE: 13 Glu Arg Lys Cys Cys Val Glu. Cys Pro Pro Cys Pro 1. 5 1O
<210s, SEQ ID NO 14 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic modified hinge region (U6-HC8)
<4 OOs, SEQUENCE: 14 Glu Pro Arg Asp Cys Gly Cys Llys Pro Cys Pro Pro Cys Pro 1. 5 1O
<210s, SEQ ID NO 15 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic modified hinge region (U8-HC5
<4 OOs, SEQUENCE: 15 Glu Lys Cys Asp Llys Thr His Thr Cys Pro Pro Cys Pro 1. 5 1O
<210s, SEQ ID NO 16 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic human hinge region
<4 OOs, SEQUENCE: 16 Glu Pro Llys Ser Cys Asp Llys Thr His Thr Cys Pro Pro Cys Pro 1. 5 1O 15
<210s, SEQ ID NO 17 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic
<4 OOs, SEQUENCE: 17 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1. 5 1O US 9,499,622 B2 31 32 What is claimed is: (2) a schv-Fc antibody comprising an antigen-binding 1. An anti-EGFR/anti-HER2 bispecific antibody compris fragment of trastuzumab, pertuzumab, trastuzumab 19. emtansine, or an anti-HER2 IgG antibody comprising a an anti-EGFR DARPin, and heavy chain variable region comprising the amino acid an anti-HER2 antibody, wherein the anti-HER2 antibody is an IgG antibody or an scFv-Fc antibody, or a sequence of SEQID NO: 5 and a light chain variable combination thereof. region comprising the amino acid sequence of SEQID 2. The bispecific antibody of claim 1, wherein the anti NO: 6. EGFR DARPin comprises at least one of SEQ ID NO: 1, 9. A method of enhancing the efficacy of an anti-HER2 SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, and antibody, comprising binding an anti-EGFR DARPinto an wherein at least one EGFR DARPin is, optionally, repeated 10 anti-HER2 antibody, wherein the anti-HER2 antibody is an 2 to 10 times. IgG antibody, an scFV-Fc antibody, or a combination 3. The bispecific antibody of claim 1, wherein the anti thereof. HER2 antibody is 10. The method of claim 9, wherein the anti-EGFR (1) trastuzumab, pertuzumab, trastuzumab emtansine, or DARPin comprises at least one of SEQID NO: 1, SEQ ID an anti-HER2 IgG antibody comprising a heavy chain 15 NO: 2, SEQ ID NO:3, and SEQID NO: 4, and wherein at variable region comprising SEQID NO: 5 and a light least one DARPin is, optionally, repeated 2 to 10 times. chain variable region comprising SEQ ID NO: 6; or 11. The method of claim 9, wherein the anti-HER2 (2) an scFV-Fc antibody comprising an antigen-binding antibody is fragment of trastuzumab, pertuzumab, trastuzumab (1) trastuzumab, pertuzumab, trastuzumab emtansine, or emtansine, or an anti-HER2 IgG antibody comprising a an anti-HER2 IgG antibody comprising a heavy chain heavy chain variable region comprising SEQID NO: 5 variable region comprising SEQ ID NO: 5 and a light and a light chain variable region comprising SEQ ID chain variable region comprising SEQ ID NO: 6; or NO: 6. (2) a schv-Fc antibody comprising an antigen-binding 4. A pharmaceutical composition comprising the anti fragment of trastuzumab, pertuzumab, trastuzumab 25 emtansine, or an anti-HER2 IgG antibody comprising a EGFR/anti-HER2 bispecific antibody of claim 1. heavy chain variable region comprising SEQID NO: 5 5. A method of treating a cancer in a subject, comprising and a light chain variable region comprising SEQ ID administering the anti-EGFR/anti-HER2 bispecificantibody NO: 6. of claim 1 to the subject, wherein the cancer is characterized 12. A nucleic acid encoding the bispecific antibody of by the expression of EGFR and HER2. 30 6. A method of preparing an anti-EGFR/anti-HER2 bispe claim 1, optionally in a vector. cific antibody of claim 1, comprising linking 13. A cell comprising the nucleic acid of claim 12. an anti-EGFR DARPin, and 14. A method of preparing a bispecific antibody of claim an anti-HER2 antibody, wherein the anti-HER2 antibody 1 comprising expressing a nucleic acid encoding the bispe is an IgG antibody, a scFv-Fc antibody, or a combina cific antibody in a cell. 35 15. The method of claim 5, wherein the subject is a cancer tion thereof. patient having resistance against an EGFR antagonist. 7. The method of claim 6, wherein the anti-EGFR DAR 16. The method of claim 5, wherein the subject is a cancer Pin comprises at least one of SEQ ID NO: 1, SEQ ID NO: patient having resistance against an anti-HER2 antibody. 2, SEQID NO:3, or SEQID NO: 4, and wherein at least one 17. The method of claim 5, wherein the subject is a cancer EGFR DARPin is, optionally, repeated 2 to 10 times. 40 8. The method of claim 6, wherein the anti-HER2 anti patient having resistance against an EGFR antagonist and an body is anti-HER2 antibody. (1) trastuzumab, pertuzumab, trastuzumab emtansine, or 18. The antibody of claim 1, wherein treatment of a an anti-HER2 IgG antibody comprising a heavy chain gastric cancer cell with the antibody causes internalization Variable region comprising the amino acid sequence of of HER2 and EGFR. SEQ ID NO: 5 and a light chain variable region 19. The method of claim 5, wherein the antibody causes comprising the amino acid sequence of SEQID NO: 6: internalization of HER2 and EGFR. Or ck ck ck ck *k