BISPECIFIC ANTIBODIES AGAINST HER2

• US 20140170148A1
• Filed: 04/20/2012
• Published: 06/19/2014
• Est. Priority Date: 04/20/2011
• Status: Abandoned Application

• Current Assignee: Genmab A/S
• Original Assignee: Genmab A/S
• Inventors: De Goeij, Bart, Van Berkel, Patrick, Labrijn, Aran Frank, Neijssen, Joost J., Meesters, Joyce I., Parren, Paul, Schuurman, Janine, Strumane, Kristin
• Application Number:
  US14/112,848
• Publication Number:
  US 20140170148A1
• Time in Patent Office: Days
• US Class Current: 424/136.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 39/395 Antibodies agglutinins A61K38/36 ; as drug carriers A61K47/50; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• CPC Class Codes: A61K 47/6805 the drug being a vinca alkaloid
• CPC Class Codes: A61K 47/6809 Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
• CPC Class Codes: A61K 47/6813 the drug being a peptidic cytokine, e.g. an interleukin or interferon
• CPC Class Codes: A61K 47/6825 Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin
• CPC Class Codes: A61K 47/6827 Ricin A
• CPC Class Codes: A61K 47/6829 Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
• CPC Class Codes: A61K 47/6855 the tumour determinant being from breast cancer cell
• CPC Class Codes: A61K 47/6879 the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/30 characterized by aspects of specificity or valency
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/33 Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/732 : Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/77 : Internalization into the cell
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

Bispecific antibodies which comprise antigen-binding regions binding to two different epitopes of human epidermal growth factor receptor 2 (HER2), and related antibody-based compositions and molecules, are disclosed. Pharmaceutical compositions comprising the antibodies and methods of preparing and using the antibodies are also disclosed.

Claims

1. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, which first and second antigen-binding regions bind different epitopes on human epidermal growth factor receptor 2 (HER2), and wherein each of the first and second antigen-binding region block the binding to HER2, optionally soluble HER2, of a reference antibody independently selected from the group consisting of:

a) an antibody comprising a VH region comprising the sequence of SEQ ID NO;

63 and a VL region comprising the sequence of SEQ ID NO;

67,

b) an antibody comprising a variable heavy (VH) region comprising the sequence of SEQ ID NO;

165 and a variable light (VL) region comprising the sequence of SEQ ID NO;

169,

c) an antibody comprising a VH region comprising the sequence of SEQ ID NO;

1 and a VL region comprising the sequence of SEQ ID NO;

5, and

d) an antibody comprising a VH region comprising the sequence of SEQ ID NO;

22 and a VL region comprising the sequence of SEQ ID NO;

26.

2. The bispecific antibody of claim 1, wherein at least one of the first and second antigen-binding regions block the binding to soluble HER2 of an antibody of a), b), c), or d).

6. The bispecific antibody of claim 1, wherein

(i) the first antigen-binding region blocks the binding to soluble HER2 of an antibody of (a) and the second antigen-binding region blocks the binding to soluble HER2 of an antibody of (b), or vice versa;

(ii) the first antigen-binding region blocks the binding to soluble HER2 of an antibody of (a) and the second antigen-binding region blocks the binding to soluble HER2 of an antibody of (c), or vice versa;

(iii) the first antigen-binding region blocks the binding to soluble HER2 of an antibody of (a) and the second antigen-binding region blocks the binding to soluble HER2 of an antibody of (d), or vice versa;

(iv) the first antigen-binding region blocks the binding to soluble HER2 of an antibody of (b) and the second antigen-binding region blocks the binding to soluble HER2 of an antibody of (c), or vice versa;

(v) the first antigen-binding region blocks the binding to soluble HER2 of an antibody of (b) and the second antigen-binding region blocks the binding to soluble HER2 of an antibody of (d), or vice versa;

(vi) the first antigen-binding region blocks the binding to soluble HER2 of an antibody of (c) and the second antigen-binding region blocks the binding to soluble HER2 of an antibody of (d), or vice versa.

7. The bispecific antibody of claim 1, wherein the first and second antigen-binding regions each comprises VH CDR1, CDR2, and CDR3 sequences independently selected from the group consisting of

a) SEQ ID NOs:

64, 65 and 66, respectively;

b) SEQ ID NOS;

43, 44 and 45, respectively;

c) SEQ ID NOs;

50, 51 and 52, respectively;

d) SEQ ID NOs;

57, 58 and 59, respectively;

e) SEQ ID NOs;

71, 72 and 73, respectively;

f) SEQ ID NOs;

166, 167 and 168, respectively;

g) SEQ ID NOS;

173, 174, and 175, respectively;

h) SEQ ID NOS;

180, 181, and 182, respectively;

i) SEQ ID NOS;

187, 188, and 189, respectively;

j) SEQ ID NOS;

194, 195, and 196, respectively;

k) SEQ ID NOS;

201, 202, and 203, respectively;

1) SEQ ID NOs;

2, 3 and 4, respectively;

m) SEQ ID NOS;

9, 10 and 11, respectively;

n) SEQ ID NOs;

16, 17 and 18, respectively;

o) SEQ ID NOS;

23, 24 and 25, respectively;

p) SEQ ID NOs;

30, 163, and 31, respectively;

q) SEQ ID NOs;

36, 37 and 38, respectively;

r) the VH CDR1, CDR2 and CDR3 sequences of trastuzumab; and

s) the VH CDR1, CDR2 and CDR3 sequences of pertuzumab,

with the proviso that when the first antigen-binding region is from trastuzumab, the second antigen-binding region is not from pertuzumab, and vice versa.

8. The bispecific antibody of claim 7, wherein the first and second antigen-binding regions each comprise a VH region and a VL region independently selected from the group consisting of

a) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:

64, 65 and 66, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

68, DAS, and SEQ ID NO;

69, respectively;

b) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

43, 44 and 45, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

47, AAS, and SEQ ID NO;

48, respectively;

c) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

50, 51 and 52, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

54, AAS, and SEQ ID NO;

55, respectively;

d) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

57, 58 and 59, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

60, AAS, and SEQ ID NO;

61, respectively;

e) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

71, 72 and 73, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

75, DAS, and SEQ ID NO;

76, respectively;

f) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

166, 167 and 168, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

170, GAS and SEQ ID NO;

171, respectively;

g) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

173, 174 and 175, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

177, DAS, and SEQ ID NO;

178, respectively;

h) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

180, 181 and 182, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

184, GAS, and SEQ ID NO;

185, respectively;

i) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

187, 188 and 189, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

191, GAS, and SEQ ID NO;

192, respectively;

j) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

194, 195 and 196, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

198, GAS, and SEQ ID NO;

199, respectively;

k) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

201, 202 and 203, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

205, GAS, and SEQ ID NO;

206, respectively;

l) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

2, 3 and 4, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

6, DAS, and SEQ ID NO;

7, respectively;

m) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

9, 10 and 11, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

13, AAS, and SEQ ID NO;

14, respectively;

n) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

16, 17 and 18, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

20, VAS, and SEQ ID NO;

21, respectively;

o) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

23, 24 and 25, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

27, AAS, and SEQ ID NO;

28, respectively;

p) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

30, 163 and 31, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

33, AAS, and SEQ ID NO;

34, respectively;

q) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

36, 37 and 38, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

40, DAS, and SEQ ID NO;

41, respectively;

t) a VH region comprising the VH CDR1, CDR2 and CDR3 sequences of trastuzumab and a VL region comprising the VL CDR1, CDR2 and CDR3 sequences of trastuzumab; and

u) a VH region comprising the VH CDR1, CDR2 and CDR3 sequences of pertuzumab and a VL region comprising the VL CDR1, CDR2 and CDR3 sequences of pertuzumab;

with the proviso that when the first antigen-binding region is from trastuzumab, the second antigen-binding region is not from pertuzumab, and vice versa.

9. The bispecific antibody of claim 8, wherein the first and the second antigen-binding regions each comprise a VH region and a VL region independently selected from the group consisting of

a) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:

64, 65 and 66, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

68, DAS, and SEQ ID NO;

69, respectively;

b) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

166, 167 and 168, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

170, GAS, and SEQ ID NO;

171, respectively;

c) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

2, 3 and 4, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

6, DAS, and SEQ ID NO;

7, respectively; and

d) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

23, 24 and 25, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

27, AAS, and SEQ ID NO;

28, respectively.

10. A bispecific antibody according to claim 1, wherein said bispecific antibody is selected from a bispecific antibody comprising

(i) a first antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO:

66 and a second antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO;

168, or vice versa;

(ii) a first antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO;

66 and a second antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO;

4, or vice versa;

(iii) a first antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO;

168 and a second antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO;

4, or vice versa;

(iv) a first antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO;

25 and a second antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO;

168, or vice versa;

(v) a first antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO;

25 and a second antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO;

66, or vice versa;

or

(iv) a first antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO;

25 and a second antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO;

4, or vice versa.

11. The bispecific antibody of claim 1, wherein

(i) the first antigen-binding region further comprises the VL CDR3 sequence of SEQ ID NO:

69, and the second antigen-binding region further comprises the VL CDR3 sequence of SEQ ID NO;

171;

(ii) the first antigen-binding region further comprises the VL, CDR3 sequence of SEQ ID NO;

69 and the second antigen-binding region further comprises the VL CDR3 sequence of SEQ ID NO;

7;

(iii) the first antigen-binding region further comprises the VL CDR3 sequence of SEQ ID NO;

171, and the second antigen-binding region further comprises the VL CDR3 sequence of SEQ ID NO;

7;

(iv) the first antigen-binding region further comprises the VL CDR3 sequence of SEQ ID NO;

28, and the second antigen-binding region further comprises the VL CDR3 sequence of SEQ ID NO;

17;

(v) the first antigen-binding region further comprises the VL CDR3 sequence of SEQ ID NO;

28, and the second antigen-binding region further comprises the VL CDR3 sequence of SEQ ID NO;

69;

or

(vi) the first antigen-binding region further comprises the VL CDR3 sequence of SEQ ID NO;

28, and the second antigen-binding region further comprises the VL CDR3 sequence of SEQ ID NO;

7.

12. The bispecific antibody of claim 1, wherein

(i) the first antigen-binding region further comprises the VH CDR1 sequence of SEQ ID NO:

64 and the VH CDR2 sequence of SEQ ID NO;

65, and the second antigen-binding region further comprises the VH CDR1 sequence of SEQ ID NO;

166 and a VH CDR2 sequence of SEQ ID NO;

167;

(ii) the first antigen-binding region further comprises the VH CDR1 sequence of SEQ. ID NO;

64 and the VH CDR2 sequence of SEQ ID NO;

65, and the second antigen-binding region further comprises the VH CDR1 sequence of SEQ ID NO;

2 and a VII CDR2 sequence of SEQ ID NO;

3;

(iii) the first antigen-binding region further comprises the VH CDR1 sequence of SEQ ID NO;

166 and the VH CDR2 sequence of SEQ ID NO;

167, and the second antigen-binding-region further comprises the VH CDR1 sequence of SEQ ID NO;

2 and a VH CDR2 sequence of SEQ ID NO;

3;

(iv) the first antigen-binding region further comprises the VH CDR1 sequence of SEQ ID NO;

23 and the VH CDR2 sequence of SEQ ID NO;

24, and the second antigen-binding region further comprises the VH CDR1 sequence of SEQ ID NO;

166 and a VH CDR2 sequence of SEQ ID NO;

167;

(v) the first antigen-binding region further comprises the VII CDR1 sequence of SEQ ID NO;

23 and the CDR2 sequence of SEQ ID NO;

24, and the second antigen-binding region further comprises the VH CDR1 sequence of SEQ ID NO;

64 and a VH CDR2 sequence of SEQ ID NO;

65;

or

(vi) the first antigen-binding region further comprises the VH CDR1 sequence of SEQ ID NO;

23 and the VH CDR2 sequence of SEQ ID NO;

24, and the second antigen-binding region further comprises the VH CDR1 sequence of SEQ ID NO;

2 and a VH CDR2 sequence of SEQ ID NO;

3.

13. A bispecific antibody according to claim 1, wherein the bispecific antibody is selected from a bispecific antibody comprising

(i) a first antigen-binding region comprising a VH region comprising SEQ ID NO:

63 and a VL region comprising SEQ ID NO;

67, and a second antigen-binding region comprising a VH region comprising SEQ ID NO;

165 and a VL region comprising SEQ ID NO;

169, or vice versa;

(ii) a first antigen-binding region comprising a VH region comprising SEQ ID NO;

63 and a VL region comprising SEQ ID NO;

67, and a second antigen-binding region comprising a VH region comprising SEQ ID NO;

1 and a VL region comprising SEQ ID NO;

5, or vice versa;

(iii) a first antigen-binding region comprising a VH region comprising, SEQ ID NO;

165 and a VL region comprising SEQ ID NO;

169, and a second antigen-binding region comprising a region comprising SEQ ID NO;

1 and a VL region comprising SEQ ID NO;

5, or vice versa;

(iv) a first antigen-binding region comprising a VII region comprising SEQ ID NO;

22 and a VL region comprising SEQ ID NO;

26, and a second antigen-binding region comprising a VH region comprising SEQ ID NO;

165 and a VL region comprising SEQ ID NO;

169, or vice versa;

(v) a first antigen-binding region comprising a VH region comprising SEQ NO;

22 and a VL region comprising SEQ ID NO;

26, and a second antigen-binding region comprising a VH region comprising SEQ ID NO;

63 and a VL region comprising SEQ ID NO;

67, or vice versa;

or

(vi) a first antigen-binding region comprising a VH region comprising SEQ ID NO;

22 and a VL region comprising SEQ ID NO;

26, and a second antigen-binding region comprising a VII region comprising SEQ ID NO;

1 and a VL region comprising SEQ ID NO;

5, or vice versa.

75. The bispecific antibody of claim 1, wherein the first and second antigen-binding regions comprise human antibody VH sequences and, optionally, human antibody VL sequences.

76. The bispecific antibody of claim 1, wherein the first and second antigen-binding regions are from heavy-chain antibodies.

77. The bispecific antibody of claim 1, wherein the first and second antigen-binding regions comprise a first and second light chain.

78. The bispecific antibody of claim 77, wherein said first and second light chains are different.

80. The bispecific antibody of claim 1, which is conjugated to one or more other moieties, such as a drug, radioisotope, cytokine or cytotoxic moiety, or contains one or more acceptor group for the same.

81. The bispecific antibody of claim 80, wherein at least one cytotoxic moiety is selected from the group consisting of taxol;

cytochalasin B;

gramicidin D;

ethidium bromide;

emetine;

mitomycin;

etoposide;

tenoposide;

vincristine;

vinblastine;

colchicin;

doxorubicin;

daunorubicin;

dihydroxy anthracin dione;

a tubulin-inhibitor such as maytansine or an analog or derivative thereof;

mitoxantrone;

mithramycin;

actinomycin D;

1-dehydrotestosterone;

a glucocorticoid;

procaine;

tetracaine;

lidocaine;

propranolol;

puromycin;

calicheamicin or an analog or derivative thereof an antimetabolite such as methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabin, 5 fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine;

an alkylating agent such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, carboplatin, duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), or an analog or derivative thereof;

an antibiotic such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC));

an antimitotic agent such as monomethyl auristatin E or F or an analog or derivative thereof;

diphtheria toxin and related molecules such as diphtheria A chain and active fragments thereof and hybrid molecules, ricin toxin such as ricin A or a deglycosylated ricin A chain toxin, cholera toxin, a Shiga-like toxin such as SLT I, SLT II, SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins such as PAPI, PAPII, and PAP S, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins;

ribonuclease (RNase);

DNase I, Staphylococcal enterotoxin A;

pokeweed antiviral protein;

diphtherin toxin; and

Pseudomonas endotoxin.

82. The bispecific antibody of claim 80, which is conjugated to at least one cytotoxic moiety selected from the group consisting of maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), monomethyl auristatin E, monomethyl auristatin F or an analog, derivative, or prodrug of any thereof.

83. The bispecific antibody of claim 80, which is conjugated to a cytokine selected from the group consisting of IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα

, IFNβ

, IFNγ

, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα

.

84. The bispecific antibody of claim 80, which is conjugated to a radioisotope.

89. A recombinant eukaryotic or prokaryotic host cell which produces a bispecific antibody of claim 1.

90. A pharmaceutical composition comprising a bispecific antibody of claim 1 and a pharmaceutically acceptable carrier.

92. A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the bispecific antibody of claim 1.

93. The method of claim 92, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer, squamous cell carcinoma of the head and neck, cervical cancer, pancreatic cancer, testis cancer, malignant melanoma and soft-tissue cancer.

94. The method of claim 92, wherein the bispecific antibody is for the treatment of cancer in combination with one or more further therapeutic agent, such as a chemotherapeutic agent.

96. A method for inhibiting growth and/or proliferation of one or more tumor cells expressing HER2 comprising administering to a subject the of claim 1.

97. A method for treating cancer, comprising

a) selecting a subject suffering from a cancer comprising tumor cells expressing HER2, and

b) administering to the subject the bispecific antibody of claim 1.

98. The method of claim 97, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, endometrial/cervical cancer, lung cancer, malignant melanoma, ovarian cancer, pancreatic cancer, prostate cancer, testis cancer, a soft-tissue tumor such as synovial sarcoma, and bladder cancer.

99. A method for producing a bispecific antibody of claim 1, said method comprising the steps of

a) culturing a host cell which produces the antibody, and

b) purifying the bispecific antibody from the culture media.

3-5. -5. (canceled)

14-33. -33. (canceled)

34. A bispecific antibody comprising a first antigen-binding region which binds an epitope in HER2Domain II and a second antigen-binding region which binds an epitope in HER2 Domain III or IV.

35. The bispecific antibody of claim 34, wherein the second antigen-binding region binds an epitope in HER2Domain III.

36. The bispecific antibody of claim 34, wherein the second antigen-binding region binds an epitope in HER2Domain IV.

37. The bispecific antibody of claim 34, wherein the first antigen-binding region blocks the binding to soluble HER2 of a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:

63 and a VL region comprising the sequence of SEQ ID NO;

67.

38. The bispecific antibody of claim 34, wherein the first and/or second antigen-binding region comprises a VH region and, optionally, a VL region, of a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, which first and second antigen-binding regions bind different epitopes on human epidermal growth factor receptor 2 (HER2), and wherein each of the first and second antigen-binding region block the binding to HER2 of a reference antibody independently selected from the group consisting of:

(a) an antibody comprising a VH region comprising the sequence of SEQ NO;

63 and a VL region comprising the sequence of SEQ ID NO;

67,

(b) an antibody comprising a variable heavy (VH) region comprising the sequence of SEQ ID NO;

165 and a variable light (VL) region comprising the sequence of SEQ ID NO;

169, and

(c) an antibody comprising a VH region comprising the sequence of SEQ ID NO;

1 and a VL region comprising the sequence of SEQ ID NO;

5 and

(d) an antibody comprising a VH region comprising the sequence of SEQ NO;

22 and a VL region comprising the sequence of SEQ ID NO;

26.

39. The bispecific antibody of claim 34, wherein said bispecific antibody further comprises a first Fc region and a second Fc region.

40. The bispecific antibody of claim 34, comprising a first Fab-arm comprising the first antigen-binding region and a first Fc-region, and a second Fab-arm comprising the second antigen-binding region and a second Fc-region.

41. The bispecific antibody of claim 34, comprising a first Fab-arm comprising the second antigen-binding region and a first Fc-region, and a second Fab-arm comprising the first antigen-binding region and a second Fc-region.

42. The bispecific antibody of claim 34, wherein the isotypes of the first and second Fab-arms are independently selected from IgG1, IgG2, IgG3, and IgG4.

43. The bispecific antibody of claim 42, wherein the isotypes of the first and second Fc-regions are independently selected from IgG1 and IgG4, wherein one of the first and second Fc-regions is of an IgG1 isotype and one is of an IgG4 isotype or the isotypes of the first and second Fc-region are of IgG1 isotype.

46. The bispecific antibody of claim 39, wherein the first Fc-region has an amino acid substitution at a position selected from the group consisting of 409, 366, 368, 370, 399, 405 and 409, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of 405, 366, 368, 370, 399, 407, and 409, and wherein said first Fc-region and said second Fc-region are not substituted in the same positions.

47. The bispecific antibody of claim 39, wherein

a) the first Fc-region has an amino acid other than Lys, Leu or Met at position 409 and the second Fc-region has an amino acid substitution at a position selected from the group consisting of 405, 366, 368, 370, 399 and 407,

b) the first Fc-region has an ammo acid other than Lys, Leu or Met at position 409 and the second Fc region has an amino acid other than Phe at position 405;

the first Fc-region has an amino acid other than Lys, Leu or Met at position 409 and the second Fc-region has an amino acid other than Phe, Arg or Gly at position 405;

d) the first Fc-region comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409 and said second Fc-region comprises an amino acid other than Phe at position 405 and a Lys at position 409;

e) the first Fc-region comprises a Phe at on 405 and an amino acid other than Lys,Leu or Met at position 409 and the second Fc-region comprises an amino acid other than Phe, Arg or Gly at position 405 and a Lys at position 409;

f) the first Fc-region comprises a Phe at position 405 and an amino acid other than Lys Leu or Met at position 409 and the second Fc-region comprises a Leu at position 405 and a Lys at position 409;

g) the first Fc-region comprises a Phe at position 405 and an Arg at position 409 and said second Fc-region comprises an amino acid other than Phe, Arg or Gly at position 405 and a Lys at position 409;

h) the first Fc-region comprises Phe at position 405 and an Arg at position 409 and the second Fc-region comprises a Leu at position 405 and a Lys at position 409;

i) the first Fc-region comprises an amino acid other than Lys, Leu or Met at position 409 and the second Fc-region comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405;

the first Fc-region comprises an Arg at position 409 and the second Fc-region comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405;

k) the first Fc-region comprises a Lys at position 370, a Phe at position 405 and an Arg at position 409 and the second Fc-region comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405;

l) the first Fc-region has an amino acid other than Lys, Leu or Met at position 409 and the second Fc-region has an amino acid other, than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407;

m) the first Fc-region has an amino acid other than Lys, Leu or Met at position 409 and the second Fc-region has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407;

n) the first Fc-region has an amino acid other than Lys, Leu or Met at position 409 and the second Fc-region has a Gly, Leu, Met, Asn or Trp at position 407;

o) the first Fc-region has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and the second Fc-region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys at position 409;

p) the first Fc-region has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and the second Fc-region has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at position 409;

the first Fc-region has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and the second Fc-region has a Gly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409;

r) the first Fc-region has a Tyr at position 407 and an Arg at position 409 and the second Fc-region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Glu, Arg, Ser or Thr at position 407 and a Lys at position 409;

s) the first Fc-region has a Tyr at position 407 and an Arg at position 409 and the second Fc-region has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at position 409; and

t) the first Fc-region has a Tyr at position 407 and an Arg at position 409 and the second Fc-region has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at position 409.

67. The bispecific antibody of claim 39, wherein the first Fc-region has an amino acid other than Lys, Leu or Met at position 409, and the second Fc-region has

(i) an amino acid other than Phe, Leu and Met at position 368,

(ii) a Trp at position 370,

(iii) an amino acid other than Asp, Cys, Pro, Glu or Gln at position 399, or

(iv) an amino acid other than Lys, Arg, Ser, Thr, or Trp at position 366.

68. The bispecific antibody of claim 39, wherein the first Fc-region has an Arg, Ala, His or Gly at position 409, and the second Fc-region has

(i) a Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or

(ii) a Trp at position 370, or

(iii) an Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position 399, or

(iv) an Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu, Met, or Tyr at position 366.

69. The bispecific antibody of claim 39, wherein the first Fc-region has an Arg at position 409, and the second Fc-region has

(i) an Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or

(ii) a Trp at position 370, or

(iii) a Phe, His, Lys, Arg or Tyr at position 399, or

(iv) an Ala, Asp, Glu, His, Asn, Val, Gln at position 366.

70. The bispecific antibody of claim 39, wherein said first and second Fc regions, except for the specified mutations, comprise the sequence of SEQ ID NO:

236.

71. The bispecific antibody of claim 39, wherein neither said first nor said second Fc-region comprises a Cys-Pro-Ser-Cys sequence in the hinge region.

72. The bispecific antibody of claim 39, wherein both of said first and said second Fc-region comprise a Cys-Pro-Pro-Cys sequence in the hinge region.

74. The bispecific antibody of claim 40, wherein said first and second Fab arm, except for the specified mutations, comprise a sequence selected from the group consisting of SEQ ID NOS:

234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244 and 245.

79. The bispecific antibody of claim 37, wherein the first and/or the second Fc-region comprise a mutation removing the acceptor site for Asn-linked glycosylation.

87. The method of claim 39, wherein the first Fc-region has an amino acid substitution at a position selected from the group consisting of 409, 366, 368, 370, 399, 405 and 409, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of 405, 366, 368, 370, 399, 407, and 409, and wherein said first Fc-region and said second Fc-region are not substituted in the same positions.

44-45. -45. (canceled)

48-66. -66. (canceled)

73. (canceled)

85. An in vitro method for generating a bispecific antibody, said method comprising the steps of:

a) providing a first HER2 antibody comprising a first Fc region, said Fc region comprising a first CH3 region,

b) providing a second HER2 antibody comprising a second Fc region, said Fc region comprising a second CH3 region,

c) incubating said first HER2 antibody together with said second HER2 antibody under reducing conditions, and

d) obtaining said bispecific antibody,

wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions.

86. The method of claim 85, wherein at least one of the first and second antibodies blocks the binding to soluble human epidermal growth factor receptor 2 (HER2) of an antibody comprising:

a) a VH region comprising the sequence of SEQ ID NO;

165 and a VL region comprising the sequence of SEQ ID NO;

169,

b) a VH region comprising the sequence of SEQ ID NO;

22 and a VL region comprising the sequence of SEQ ID NO;

26,

c) a VH region comprising the sequence of SEQ ID NO;

63 and a VL region comprising the sequence of SEQ ID NO;

67, and

d) a VH region comprising the sequence of SEQ ID NO;

1 and a VL region comprising the sequence of SEQ ID NO;

5.

88. A bispecific antibody obtainable by the method of claim 86.

91. (canceled)

95. (canceled)