Bispecific antibodies against her2 and cd3

Abstract

Bispecific antibodies which comprise one antigen-binding region binding to an epitope of human epidermal growth factor receptor 2 (HER2) and one antigen-binding region binding to human CD3, and related antibody-based compositions and molecules, are disclosed. Pharmaceutical compositions comprising the antibodies and methods for preparing and using the antibodies are also disclosed.

1 . A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, which second antigen-binding region binds an epitope on human CD3 and the first antigen-binding region binds an epitope on human epidermal growth factor receptor 2 (HER2) and blocks the binding to HER2, optionally soluble HER2, of a reference antibody 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:1 and a variable light (VL) region comprising the sequence of SEQ ID NO:5,   c) an antibody comprising a VH region comprising the sequence of SEQ ID NO:165 and a VL region comprising the sequence of SEQ ID NO:169, 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 the first antigen-binding region blocks the binding to soluble HER2 of an antibody of (a). 
     
     
         3 . The bispecific antibody of  claim 1 , wherein the first antigen-binding region blocks the binding to soluble HER2 of an antibody of (b), (c), or (d). 
     
     
         4 - 5 . (canceled) 
     
     
         6 . The bispecific antibody of  claim 1 , wherein the first antigen-binding region comprises VH CDR1, CDR2, and CDR3 sequences selected from the group consisting of
 a) SEQ ID NOs: 2, 3 and 4, respectively;   b) SEQ ID NOS: 9, 10 and 11, respectively;   c) SEQ ID NOs:16, 17 and 18, respectively;   d) SEQ ID NOs: 23, 24 and 25, respectively;   e) SEQ ID NOs:30, 163, and 31, respectively;   f) SEQ ID NOs: 36, 37 and 38, respectively:   g) SEQ ID NOs: 43, 44 and 45, respectively;   h) SEQ ID NOs:50, 51 and 52, respectively;   i) SEQ ID NOs: 57, 58 and 59, respectively;   j) SEQ ID NOs:64, 65 and 66, respectively;   k) SEQ ID NOs: 71, 72 and 73, respectively;   l) SEQ ID NOs: 166, 167 and 168, respectively;   m) SEQ OD NOs: 173, 174, and 175, respectively;   n) SEQ ID NOs: 180, 181, and 182, respectively;   o) SEQ ID NOs:187, 188, and 189, respectively;   p) SEQ ID NOs:194, 195, and 196, respectively; and   q) SEQ ID NOs:201, 202, and 203, respectively.   
     
     
         7 . A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, which second antigen-binding region binds an epitope on human CD3 and the first antigen-binding region binds an epitope on HER2, wherein the first antigen-binding region comprises a VH region and a VL region selected from the group consisting of
 a) 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;   b) 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;   c) 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;   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;   e) 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;   f) 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;   g) 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;   h) 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;   i) 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;   j) 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;   k) 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;   l) 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;   m) 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;   n) 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;   o) 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;   p) 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;   q) 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.   
     
     
         8 . The bispecific antibody of claim  5 , wherein the first antigen-binding region comprises a VH region and a VL region selected from the group consisting of
 a) 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;   b) 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;   c) 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, and   d) 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.   
     
     
         9 . A bispecific antibody of  claim 1  comprising a first antigen-binding region and a second antigen-binding region, which second antigen-binding region binds an epitope on human CD3 and the first antigen-binding region binds HER2 and comprises
 a) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 2, 3 and 4, respectively, and, optionally a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:6, DAS, and SEQ ID NO:7, respectively; 
 b) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:64, 65 and 66, respectively; and, optionally, a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:68, DAS, and SEQ ID NO:69, respectively; 
 c) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:166, 167 and 168, respectively; and, optionally, a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:170, GAS, and SEQ ID NO:171, respectively; or 
 d) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:23, 24 and 25, respectively; and, optionally, a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:27, AAS, and SEQ ID NO:28, respectively. 
 
     
     
         10 - 12 . (canceled) 
     
     
         13 . A bispecific antibody of  claim 1  comprising a first antigen-binding region and a second antigen-binding region, which second antigen-binding region binds an epitope on human CD3 and the first antigen-binding region binds HER2 and comprises:
 a) a VH region comprising the sequence of SEQ ID NO:1 and, optionally a VL region comprising SEQ ID NOs:5; 
 b) a VH region comprising the sequence of SEQ ID NO:63 and, optionally a VL region comprising SEQ ID NOs:67; 
 c) a VH region comprising the sequence of SEQ ID NO:165, and, optionally, a VL region comprising the sequence of SEQ ID NO:169; or 
 d) a VH region comprising SEQ ID NO:22, and, optionally, a VL region comprising the sequence of SEQ ID NO:26. 
 
     
     
         14 - 16 . (canceled) 
     
     
         17 . A bispecific antibody of  claim 1 , wherein the second antigen-binding region blocks the binding to CD3 of a reference antibody selected from the group consisting of
 a) an antibody comprising a VH region comprising the sequence of SEQ ID NO:240 and a VL region comprising the sequence of SEQ ID NO:241;   b) an antibody comprising a VH region comprising the sequence of SEQ ID NO:234 and a VL region comprising the sequence of SEQ ID NO:235;   c) an antibody comprising a VH region comprising the sequence of SEQ ID NO:238 and VL region comprising the sequence of SEQ ID NO:239; and   d) an antibody comprising a VH region comprising the sequence of SEQ ID NO:236 and a VL region comprising the sequence of SEQ ID NO:237.   
     
     
         18 . A bispecific antibody of  claim 1 , wherein the second antigen-binding region blocks the binding to CD3 of a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:240 and a VL region comprising the sequence of SEQ ID NO:241. 
     
     
         19 . A bispecific antibody of  claim 1 , wherein the second antigen-binding region comprises a VH region selected from the group consisting of:
 a) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:242, 243 and 244, respectively;   b) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:234;   c) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:238; and   d) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:236.   
     
     
         20 . A bispecific antibody of  claim 1 , wherein the second antigen-binding region comprises a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:242, 243 and 244, respectively. 
     
     
         21 . A bispecific antibody of  claim 1  comprising a second antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO:244 and a second antigen-binding region comprising the VH CDR3 sequence of SEQ ID NO:4. 
     
     
         22 . The bispecific antibody of  claim 21 , wherein the second antigen-binding region comprises the VL CDR3 sequence of SEQ ID NO:246 and the second antigen-binding region comprises the VL CDR3 sequence of SEQ ID NO:7. 
     
     
         23 . The bispecific antibody of  claim 1 , wherein the second antigen-binding region comprises the VH CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:242, 243 and 244, respectively; and, optionally, the VL CDR1, CDR2 and CDR3 sequences of 245, DTS and 246, respectively. 
     
     
         24 . The bispecific antibody of  claim 1 , wherein the second antigen-binding region comprises a VH region comprising the sequence of SEQ ID NO: 240 and, optionally, a VL region comprising the sequence of SEQ ID NO:241. 
     
     
         25 . The bispecific antibody of  claim 1 , wherein said bispecific antibody further comprises a first Fc region and a second Fc region. 
     
     
         26 . The bispecific antibody of  claim 1 , 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. 
     
     
         27 . The bispecific antibody of  claim 1 , 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. 
     
     
         28 . The bispecific antibody of  claim 26 , wherein the isotypes of the first and second Fc-regions are independently selected from IgG1, IgG2, IgG3, and IgG4. 
     
     
         29 . The bispecific antibody of  claim 28 , wherein the isotypes of the first and second Fc-regions are independently selected from IgG1 and IgG4. 
     
     
         30 . The bispecific antibody of  claim 29 , wherein one of the first and second Fc-regions is of an IgG1 isotype and one is of an IgG4 isotype; or wherein the isotypes of the first and second Fc regions are IgG1. 
     
     
         31 . (canceled) 
     
     
         32 . The bispecific antibody of  claim 1 , which is effector-function deficient. 
     
     
         33 . The bispecific antibody of  claim 26 , 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 407, 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. 
     
     
         34 . The bispecific antibody of  claim 26 , 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 amino 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;   c) 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 position 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;   j) 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 Tip 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;   q) 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 Tip 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, Gln, 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; or   t) the first Fc-region has a Tyr at position 407 and an Arg 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.   
     
     
         35 - 53 . (canceled) 
     
     
         54 . The bispecific antibody of  claim 26 , 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, or   (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.   
     
     
         55 . The bispecific antibody of  claim 26 , wherein the first Fc-region has an Arg, Ala, His or Gly at position 409, and the second homodimeric protein 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,   (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.   
     
     
         56 . The bispecific antibody of  claim 26 , 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.   
     
     
         57 . The bispecific antibody of  claim 26 , wherein said first and second CH3 regions, except for the specified mutations, comprise the sequence of SEQ ID NO:256. 
     
     
         58 . The bispecific antibody of  claim 26 , wherein neither said first nor said second Fc-region comprises a Cys-Pro-Ser-Cys sequence in the hinge region. 
     
     
         59 . The bispecific antibody of  claim 26 , wherein both of said first and said second Fc-region comprise a Cys-Pro-Pro-Cys sequence in the hinge region. 
     
     
         60 . (canceled) 
     
     
         61 . The bispecific antibody of  claim 26 , wherein said first and second Fc region, except for the specified mutations, comprise a sequence independently selected from the group consisting of SEQ ID NOs: 247, 248, 249, 250, 251, 252, 253, 254, and 255. 
     
     
         62 . 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. 
     
     
         63 . The bispecific antibody of  claim 1 , wherein the first and second antigen-binding regions are from heavy-chain antibodies. 
     
     
         64 . The bispecific antibody of  claim 1 , wherein the first and second antigen-binding regions comprise a first and second light chain. 
     
     
         65 . The bispecific antibody of  claim 64 , wherein said first and second light chains are different. 
     
     
         66 . The bispecific antibody of  claim 26 , wherein the first and/or the second Fc-region comprises a mutation removing the acceptor site for Asn-linked glycosylation. 
     
     
         67 . The bispecific antibody of  claim 1 , which is conjugated to one or more other moieties, or contains one or more acceptor group for the same. 
     
     
         68 . The bispecific antibody of  claim 67 , 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. 
     
     
         69 . An in vitro method for generating a bispecific antibody, said method comprising the steps of:
 a) providing a first antibody binding to an epitope on HER2 and comprising a first Fc region, said Fc region comprising a first CH3 region,   b) providing a second antibody binding to an epitope on human CD3 and comprising a second Fc region, said Fc region comprising a second CH3 region,   c) incubating said first antibody together with said second 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.   
     
     
         70 . The method of  claim 69 , wherein the first antibody 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:1 and a VL region comprising the sequence of SEQ ID NO:5,   b) a VH region comprising the sequence of SEQ ID NO:63 and a VL region comprising the sequence of SEQ ID NO:67,   c) a VH region comprising the sequence of SEQ ID NO:165 and a VL region comprising the sequence of SEQ ID NO:169, and   d) a VH region comprising the sequence of SEQ ID NO:22 and a VL region comprising the sequence of SEQ ID NO:26.   
     
     
         71 . The method of  claim 69 , 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 407, 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. 
     
     
         72 . A bispecific antibody obtainable by the method of  claim 69 . 
     
     
         73 . A recombinant eukaryotic or prokaryotic host cell which produces a bispecific antibody as defined in  claim 1 . 
     
     
         74 . A pharmaceutical composition comprising a bispecific antibody as defined in  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         75 . (canceled) 
     
     
         76 . A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the bispecific antibody of  claim 1 . 
     
     
         77 . The method of  claim 76 , 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. 
     
     
         78 . The method of  claim 76 , further comprising administering one or more additional therapeutic agents. 
     
     
         79 . (canceled) 
     
     
         80 . A method for inhibiting growth and/or proliferation of one or more tumor cells expressing HER2, comprising administering to a subject in need thereof the bispecific antibody according to  claim 1 . 
     
     
         81 . A method for treating cancer, comprising
 a) selecting a subject suffering from a cancer comprising tumor cells co-expressing HER2, and   b) administering to the subject the bispecific antibody according to  claim 1 .   
     
     
         82 . The method of  claim 81 , 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. 
     
     
         83 . A method for producing a bispecific antibody of  claim 68 , said method comprising the steps of
 a) culturing a host cell of  claim 73 , and   b) purifying the bispecific antibody from the culture media.