Methods of using bispecific antigen-binding constructs targeting her2

Abstract

Described herein methods of using antigen-binding constructs to treat HER2+ tumors in a subject such as breast, lung, or head and neck tumors. In some aspects, the tumor volume in the subject after receiving at least seven doses of the antigen binding construct is less than the tumor volume of a control subject receiving an equivalent amount of trastuzumab. In some aspects, the survival of the subject receiving the antigen binding construct is increased as compared to a control subject receiving an equivalent amount of a non-specific control antibody or as compared to a control subject not receiving treatment.

We claim: 
     
         1 . A method of treating a subject having a tumor; inhibiting, reducing or blocking HER2 signaling; or killing or inhibiting the growth of a HER2-expressing tumor cell, the method comprising administering an effective amount of an antigen binding construct comprising:
 a first antigen-binding polypeptide construct which monovalently and specifically binds a HER2 (human epidermal growth factor receptor 2) ECD2 (extracellular domain 2) antigen on a HER2-expressing cell;   a second antigen-binding polypeptide construct which monovalently and specifically binds a HER2 ECD4 (extracellular domain 4) antigen on a HER2-expressing cell;   first and second linker polypeptides, wherein the first linker polypeptide is operably linked to the first antigen-binding polypeptide construct, and the second linker polypeptide is operably linked to the second antigen-binding polypeptide construct;   wherein the linker polypeptides are capable of forming a covalent linkage with each other,   wherein one or both of the first or the second antigen binding polypeptide construct is an scFv,   wherein the dissociation constant (K D ) of the antigen binding construct to murine HER2 extracellular domain as measured by surface plasmon resonance (SPR) is equal to or less than the dissociation constant of a monospecific anti-HER2 ECD4 antibody (v506; SEQ ID NO:1 and SEQ ID NO:317) to murine HER2 extracellular domain as measured by surface plasmon resonance (SPR), and   wherein tumor growth is decreased as compared to a control receiving an equivalent amount of a non-specific control antibody, as compared to a control receiving an equivalent amount of Herceptin/trastuzumab, or as compared to a control not receiving treatment.   
     
     
         2 . The method of  claim 1  wherein the antigen binding construct comprises the full length sequences set forth in SEQ ID NOs 97, 295, and 69 (v10000), and optionally wherein the dissociation constant (K D ) of the construct to murine HER2 extracellular domain as measured by surface plasmon resonance (SPR) is approximately 0.6 nM. 
     
     
         3 . A method of treating a subject having a tumor; inhibiting, reducing or blocking HER2 signaling; or killing or inhibiting the growth of a HER2-expressing tumor cell, the method comprising administering an effective amount of an antigen binding construct comprising:
 a first antigen-binding polypeptide construct which monovalently and specifically binds a HER2 (human epidermal growth factor receptor 2) ECD2 (extracellular domain 2) antigen on a HER2-expressing cell, wherein the first antigen-binding polypeptide construct comprises a first variable light-chain (VL1) domain and a first variable heavy-chain (VH1) domain, wherein the first antigen-binding polypeptide construct comprises VH1 and VL1 CDR sequences that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VH1 and VL1 CDR sequences of v7091 (SEQ ID NOs 223, 225, 227, 37, 39, and 41), and wherein the VL1 domain comprises 1, 2, 3, 4, or 5 amino acid substitutions and/or the VH1 domain comprises 1, 2, 3, 4, or 5 amino acid substitutions;   a second antigen-binding polypeptide construct which monovalently and specifically binds a HER2 ECD4 (extracellular domain 4) antigen on a HER2-expressing cell;   first and second linker polypeptides, wherein the first linker polypeptide is operably linked to the first antigen-binding polypeptide construct, and the second linker polypeptide is operably linked to the second antigen-binding polypeptide construct;   wherein one or both of the first or the second antigen binding polypeptide construct is an scFv,   wherein the linker polypeptides are capable of forming a covalent linkage with each other, and   wherein tumor growth is decreased as compared to a control receiving an equivalent amount of a non-specific control antibody, as compared to a control receiving an equivalent amount of Herceptin/trastuzumab, or as compared to a control not receiving treatment.   
     
     
         4 . The method of any of the above claims, wherein the binding affinity of the antigen binding construct to murine HER2 extracellular domain as measured by surface plasmon resonance (SPR) is greater than the binding affinity of v7091 (SEQ ID NOs 33, 219, and 295) to murine HER2 extracellular domain as measured by surface plasmon resonance (SPR), optionally wherein the antigen binding construct and v7091 bind the same epitope, optionally wherein the antigen binding construct binds the same epitope as pertuzamab, optionally wherein the antigen binding construct has a greater Bmax than v7091, and optionally wherein the antigen binding construct is internalized to a greater extent upon cell surface binding relative to v7091. 
     
     
         5 . The method of any of the above claims, wherein the binding affinity of the antigen binding construct to murine HER2 extracellular domain as measured by surface plasmon resonance (SPR) is equal to or greater than the binding affinity of a monospecific anti-HER2 ECD4 antibody (v506; SEQ ID NO:1 and SEQ ID NO:317) to murine HER2 extracellular domain as measured by surface plasmon resonance (SPR). 
     
     
         6 . The method of any of the above claims, wherein the first antigen-binding polypeptide construct comprises the VH1 and VL1 CDR sequences of v7091 (SEQ ID NOs 223, 225, 227, 37, 39, and 41), wherein the VL1 domain comprises 1, 2, 3, 4, or 5 amino acid substitutions and/or the VH1 domain comprises 1, 2, 3, 4, or 5 amino acid substitutions, optionally wherein the first antigen-binding polypeptide construct comprises a substitution at Y96 in the VL1 domain (SEQ ID NO:35), optionally wherein the first antigen-binding polypeptide construct comprises a Y96A substitution in the VL1 domain (SEQ ID NO:35), optionally wherein the first antigen-binding polypeptide construct comprises substitutions at T30, A49, and/or L69 in the VH1 domain (SEQ ID NO:221), optionally wherein the first antigen-binding polypeptide construct comprises T30A, A49G, and/or L69F substitution(s) in the VH1 domain (SEQ ID NO:221), and optionally wherein the first antigen-binding polypeptide construct comprises T30A, A49G, and L69F substitution(s) in the VH1 domain (SEQ ID NO:221). 
     
     
         7 . The method of any of the above claims, wherein the second antigen-binding polypeptide construct comprises VH2 and VL2 CDR sequences that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VH2 and VL2 CDR sequences of v10000 (SEQ ID NOs 299, 301, 303, 307, 309, and 311), optionally wherein the second antigen-binding polypeptide construct comprises the VH2 and VL2 CDR sequences of v10000 (SEQ ID NOs 299, 301, 303, 307, 309, and 311). 
     
     
         8 . The method of any of the above claims, wherein the antigen binding construct comprises the variable domain sequences set forth in SEQ ID NOs 71 and/or 99, the variable domain sequences set forth in SEQ ID NOs 297 and/or 305, or the variable domain sequences set forth in SEQ ID NOs 71, 99, 297, and 305. 
     
     
         9 . The method of any of the above claims, wherein the antigen binding construct comprises the full length sequence set forth in SEQ ID NO 97, the full length sequence set forth in SEQ ID NO 295, the full length sequence set forth in SEQ ID NO 69, or the full length sequences set forth in SEQ ID NOs 97, 295, and 69 (v10000). 
     
     
         10 . The method of any of the above claims, wherein the first and second linker polypeptide each comprise an immunoglobulin hinge region polypeptide selected from an IgG1, IgG2 or IgG4 hinge region. 
     
     
         11 . The method of any of the above claims, wherein the first and second linker polypeptides are operably linked to a scaffold, optionally an Fc. 
     
     
         12 . The method of any of the above claims, wherein the first and second linker polypeptides are operably linked to a dimeric Fc comprising first and second Fc polypeptides each comprising a CH3 sequence, wherein the first Fc polypeptide is operably linked to the first linker polypeptide and the second Fc polypeptide is operably linked to the second linker polypeptide. 
     
     
         13 . The method of any of the above claims, wherein (i) the first antigen binding polypeptide construct is an scFv and the second antigen binding polypeptide construct is a Fab; or (ii) the first antigen binding polypeptide construct is a Fab and the second antigen binding polypeptide construct is an scFv; or (iii) both the first antigen binding polypeptide construct and the second antigen binding polypeptide construct are scFvs. 
     
     
         14 . The method of any of the above claims, wherein
 i. the first antigen-binding polypeptide construct is a Fab and comprises
 a. a first heavy chain variable polypeptide VH1 comprising the VH of the pertuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NOs 221, 149, 221, 259, and 99, respectively), and 
 b. a first variable light chain polypeptide VL1 comprising the VL of the pertuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NOs 35, 35, and 71 for v5019, v7091, and v10000, respectively); and 
 the second antigen-binding polypeptide construct is an scFv and comprises 
 (a) a second variable heavy chain polypeptide VH2 comprising the VH of the trastuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NOs 171, 205, 297, 171, and 297, respectively), and 
 (b) a second variable light chain polypeptide VL2 comprising the VL of the trastzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NO:35 for v5020); or 
   ii. the first antigen-binding polypeptide construct is an scFv and comprises
 (a) a first variable heavy chain polypeptide VH1 comprising the VH of the pertuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NOs 221, 149, 221, 259, and 99, respectively), and 
 (b) a first variable light chain polypeptide VL1 comprising the VL of the pertuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NOs 35, 35, and 71 for v5019, v7091, and v10000, respectively), and 
 the second antigen-binding polypeptide construct is an Fab and comprises 
 (a) a second heavy chain variable polypeptide VH2 comprising the VH of the trastuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NOs 171, 205, 297, 171, and 297, respectively), and 
 (b) a second variable light chain polypeptide VL2 comprising the VL of the trastuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NO:35 for v5020); or 
   iii. the first antigen-binding polypeptide construct is an scFv and comprises
 (a) a first heavy chain variable polypeptide VH1 comprising the VH of the pertuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NOs 221, 149, 221, 259, and 99, respectively), and 
 (b) a first variable light chain polypeptide VL1 comprising the VL of the pertuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NOs 35, 35, and 71 for v5019, v7091, and v10000, respectively), and 
 the second antigen-binding polypeptide construct is an scFv and comprises 
 (a) a second heavy chain variable polypeptide VH2 comprising the VH of the trastuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NOs 171, 205, 297, 171, and 297, respectively), and 
 (b) a second variable light chain polypeptide VL2 comprising the VL of the trastuzumab arm of v5019, v 5020 v7091, v6717 or v10000 (SEQ ID NO:35 for v5020). 
   
     
     
         15 . The method of any of the above claims, wherein the first antigen-binding polypeptide construct is selected from:
 i. a polypeptide construct comprising three VH CDR sequences comprising the amino acid sequences SEQ ID NO: 335, SEQ ID NO:336 and SEQ ID NO:337, or SEQ ID NO:335, SEQ ID NO:336, and SEQ ID NO:348;   ii. a polypeptide construct comprising three VH CDR sequences comprising amino acid sequences that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the three VH CDR sequences of SEQ ID NO: 335, SEQ ID NO:336 and SEQ ID NO:337, or SEQ ID NO:335, SEQ ID NO:336, and SEQ ID NO:348;   iii. a polypeptide construct comprising three VL CDR sequences comprising the amino acid sequences of the three VL CDR sequences of SEQ ID NO: 338, SEQ ID NO:339 and SEQ ID NO:340, or SEQ ID NO:338, SEQ ID NO:347, and SEQ ID NO:340;   iv. a polypeptide construct comprising three VL CDR sequences that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequences of the three VL CDR sequences are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 338, SEQ ID NO:339 and SEQ ID NO:340, or SEQ ID NO:338, SEQ ID NO:347, and SEQ ID NO:340;   v. a polypeptide construct comprising six CDR sequences comprising the amino acid sequences of the six CDR sequences of SEQ ID NO: 335, SEQ ID NO:336, SEQ ID NO:337, SEQ ID NO: 338, SEQ ID NO:339 and SEQ ID NO:340; or SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:348, SEQ ID NO:338, SEQ ID NO:347, and SEQ ID NO:340; or   vi. a polypeptide construct comprising six CDR sequences comprising the amino acid sequences that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the six CDR sequences of SEQ ID NO: 335, SEQ ID NO:336, SEQ ID NO:337, SEQ ID NO: 338, SEQ ID NO:339 and SEQ ID NO:340; or SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:348, SEQ ID NO:338, SEQ ID NO:347, and SEQ ID NO:340; and   the second antigen-binding polypeptide is selected from   vii. a polypeptide construct comprising three VH CDR sequences comprising the amino acid sequences of the three VH CDR sequences of SEQ ID NO: 341, SEQ ID NO:342 and SEQ ID NO:343;   viii. a polypeptide construct comprising three VH CDR sequences comprising amino acids sequences that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the three VH CDR sequences of SEQ ID NO: 341, SEQ ID NO:342 and SEQ ID NO:343;   ix. a polypeptide construct comprising three VL CDR sequences comprising the amino acid sequences of the three VL CDR sequences of SEQ ID NO: 344, SEQ ID NO:345 and SEQ ID NO:346;   x. a polypeptide construct comprising three VL CDR sequences that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequences of the three VL CDR sequences of SEQ ID NO: 344, SEQ ID NO:345 and SEQ ID NO:346;   xi. a polypeptide construct comprising six CDR sequences comprising the amino acid sequences of the six CDR sequences of SEQ ID NO: 341, SEQ ID NO:342, SEQ ID NO:343, SEQ ID NO: 344, SEQ ID NO:345 and SEQ ID NO:346; or   xii. a polypeptide construct comprising six CDR sequences comprising the amino acid sequences that are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the six CDR sequences of SEQ ID NO: 341, SEQ ID NO:342, SEQ ID NO:343, SEQ ID NO: 344, SEQ ID NO:345 and SEQ ID NO:346.   
     
     
         16 . The method of any of the above claims wherein the first antigen binding polypeptide construct: (i) blocks by 50% or greater the binding of pertuzumab to ECD2, and/or (ii) the second antigen binding polypeptide blocks by 50% or greater the binding of trastuzumab to ECD4. 
     
     
         17 . The method according to any preceding claim wherein the first antigen binding polypeptide construct comprises one of the v5019, v10000, v7091, v5020 or v6717 antigen binding polypeptide constructs specific for HER2 ECD2, and the second antigen binding polypeptide construct comprises one of the v5019, v10000, v7091, v5020 or v6717 antigen-binding polypeptide constructs specific for HER2 ECD4. 
     
     
         18 . The method according to any preceding claim, wherein the first antigen-binding polypeptide construct comprises an amino acid sequence at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the v5019, v10000, v7091, v5020 or v6717 antigen-binding polypeptide construct specific for HER2 ECD2 and the second antigen-binding polypeptide construct comprises an amino acid sequence at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the v5019, v10000, v7091, v5020 or v6717 antigen-binding polypeptide construct specific for HER2 ECD4. 
     
     
         19 . The method according to any preceding claim, selected from v5019, v10000, v7091, v5020 and v6717. 
     
     
         20 . The method according to any preceding claim, wherein the first antigen binding polypeptide construct is an Fab and the second antigen binding polypeptide construct is an scFv, and wherein the antigen binding construct
 (i) induces increased receptor internalization in HER2 3+ cells and/or   (ii) displays higher potency in an ADCC (antibody directed cellular cytotoxicity) assay against HER2 1+ cells, and/or   (iii) comprises one or more of the characteristics described in one or more of the Examples, Tables, and Figures,   as compared to a reference biparatopic antigen binding construct having two Fabs.   
     
     
         21 . The method according to according to any preceding claim, wherein the first and second antigen binding polypeptide constructs are scFvs, and wherein the antigen binding construct induces increased receptor internalization in HER2 1+, 2+ and 3+ cells as compared to a reference antigen binding construct having two Fabs. 
     
     
         22 . The method according to any preceding claim, wherein the antigen-binding construct comprises an Fc, optionally wherein the Fc is a heterodimeric Fc. 
     
     
         23 . The method according to any preceding claim, wherein the antigen-binding construct comprises a heterodimeric Fc, wherein the dimerized CH3 sequences have a melting temperature (Tm) of about 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 77.5, 78, 79, 80, 81, 82, 83, 84, or 85° C. or higher. 
     
     
         24 . The method according to any preceding claim, wherein the antigen-binding construct comprises a heterodimeric Fc formed with a purity greater than about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% when expressed. 
     
     
         25 . The method according to any preceding claim, wherein the antigen-binding construct comprises a heterodimeric Fc formed with a purity greater than about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% when expressed via a single cell. 
     
     
         26 . The method according to any preceding claim, wherein the antigen-binding construct comprises a heterodimeric Fc comprising one or more modifications in at least one of the CH3 sequences. 
     
     
         27 . The method according to any preceding claim, wherein the antigen-binding construct comprises a heterodimeric Fc comprising one or more modifications in at least one of the CH3 sequences that promote the formation of a heterodimer with stability comparable to a wild-type homodimeric Fc. 
     
     
         28 . The method according to any preceding claim, wherein the antigen-binding construct comprises:
 i. a heterodimeric IgG1 Fc having the modifications L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366L_K392M_T394W in the second polypeptide;   ii. a heterodimeric IgG1 Fc having the modifications L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366L_K392L_T394W in the second Fc polypeptide;   iii. a heterodimeric IgG1 Fc having the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392L_T394W in the second Fc polypeptide;   iv. a heterodimeric IgG1 Fc having the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392M_T394W in the second Fc polypeptide;   v. a heterodimeric IgG1 Fc having the modifications T350V_L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide,   vi. a heterodimeric IgG1 Fc having the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366I_N390R_K392M_T394W in the second Fc polypeptide; or   vii. a heterodimeric IgG1 Fc having the modifications L351Y_S400E_F405A_Y405V in the first Fc polypeptide, and the modifications T350V_T366L_K392L_T394W in the second Fc polypeptide;   according to EU numbering compared to a wild-type homodimeric Fc.   
     
     
         29 . The method according to any preceding claim, wherein the antigen-binding construct comprises a heterodimeric Fc comprising at least one CH2 domain. 
     
     
         30 . The method according to  claim 29 , wherein the CH2 domain(s) of the heterodimeric Fc comprises one or more modifications. 
     
     
         31 . The method according to any preceding claim, wherein the antigen-binding construct comprises a heterodimeric Fc comprising one or more modifications to promote selective binding of Fc-gamma receptors. 
     
     
         32 . The method according to any preceding claim, wherein the antigen-binding construct comprises at least one modification, and wherein the modification is afucosylation. 
     
     
         33 . The method according to any preceding claim, wherein the antigen-binding construct is conjugated to a drug. 
     
     
         34 . The method construct according to  claim 33 , wherein the drug is maytansine (DM1). 
     
     
         35 . The method according to  claim 34 , wherein the construct is conjugated to DM1 through an SMCC linker. 
     
     
         36 . The method according to any preceding claim, wherein the antigen-binding construct is formulated in a pharmaceutical composition with a pharmaceutical carrier. 
     
     
         37 . The method  claim 36 , wherein the pharmaceutical carrier comprises a buffer, an antioxidant, a low molecular weight molecule, a drug, a protein, an amino acid, a carbohydrate, a lipid, a chelating agent, a stabilizer, or an excipient. 
     
     
         38 . The method according to any preceding claim, wherein the result of the treatment is shrinking the tumor, inhibiting growth of the tumor, increasing time to progression of the tumor, prolonging disease-free survival of the subject, decreasing metastases, increasing the progression-free survival of the subject, or increasing overall survival of the subject. 
     
     
         39 . The method according to any preceding claim, wherein the tumor comprises cells that express an average of 10,000 or more copies of HER2 per tumor cell, optionally wherein the tumor is HER2 gene-amplified. 
     
     
         40 . The method according to any preceding claim, wherein the tumor is HER2 1+, HER2 2+ or HER2 3+ as determined by immunohistochemistry (IHC). 
     
     
         41 . The method according to any preceding claim, wherein the tumor expresses HER2 at a level of 2+ or lower as determined by IHC. 
     
     
         42 . The method according to any preceding claim, wherein the HER2+ tumor is a breast cancer that expresses HER2 at a 2+ level or lower, as determined by immunohistochemistry (IHC). 
     
     
         43 . The method according to any preceding claim, wherein the tumor is a lung tumor, optionally wherein the tumor is a non-squamous non-small cell lung tumor that is HER2-low, non-HER2 gene amplified. 
     
     
         44 . The method of  claim 43 , wherein the tumor is HER3+. 
     
     
         45 . The method of  claim 43  or  44 , wherein the tumor is EGFR low. 
     
     
         46 . The method of  claim 43 ,  44 , or  45 , wherein the tumor is moderately sensitive to Cisplatin at the MTD. 
     
     
         47 . The method according to any preceding claim, wherein the tumor is a head and neck tumor, optionally wherein the tumor is a squamous cell tumor of the head and neck that is HER2 low, non-HER2 gene amplified. 
     
     
         48 . The method of  claim 47 , wherein the tumor is HER3+ low. 
     
     
         49 . The method of  claim 47  or  48 , wherein the tumor is EGFR+. 
     
     
         50 . The method of  claim 47 ,  48 , or  49 , wherein the tumor is highly sensitive to Cisplatin at the MTD. 
     
     
         51 . The method according to any preceding claim, wherein the tumor is a breast tumor, optionally wherein the tumor is a ER+/PR− breast cancer with a luminal B molecular classification. 
     
     
         52 . The method according to any preceding claim, wherein the tumor is a pancreatic tumor, optionally wherein the pancreatic tumor is HER2 negative as determined by IHC. 
     
     
         53 . The method according to any preceding claim, wherein the tumor is a gastric tumor, optionally wherein the gastric tumor is HER2 3+. 
     
     
         54 . The method according to any preceding claim, wherein the subject has not previously been treated with an anti-HER2 antibody. 
     
     
         55 . The method according to any preceding claim, wherein the tumor is resistant or refractory to pertuzumab, trastuzumab and/or TDM1. 
     
     
         56 . The method according to any preceding claim, wherein the subject has previously been treated with pertuzumab, trastuzumab and/or TDM1. 
     
     
         57 . The method of any one of  claims 1 - 41 , wherein the tumor is (i) a HER2 3+ estrogen receptor negative (ER−), progesterone receptor negative (PR−), trastuzumab resistant, chemotherapy resistant invasive ductal breast cancer, (ii) a HER2 3+ ER−, PR−, trastuzumab resistant inflammatory breast cancer, (iii) a HER2 3+, ER−, PR−, invasive ductal carcinoma or (iv) a HER2 2+ HER2 gene amplified trastuzumab and pertuzumab resistant breast cancer. 
     
     
         58 . The method any one of  claims 1 - 41  wherein the tumor cell is a HER2 1+ or 2+ human pancreatic carcinoma cell, a HER2 3+ human lung carcinoma cell, a HER2 2+ human Caucasian bronchioaveolar carcinoma cell, a human pharyngeal carcinoma cell, a HER2 2+ human tongue squamous cell carcinoma cell, a HER2 2+ squamous cell carcinoma cell of the pharynx, a HER2 1+ or 2+ human colorectal carcinoma cell, a HER2 3+ human gastric carcinoma cell, a HER2 1+ human breast ductal ER+ (estrogen receptor-positive) carcinoma cell, a HER2 2+/3+ human ER+, HER2-amplified breast carcinoma cell, a HER2 0+/1+ human triple negative breast carcinoma cell, a HER2 2+ human endometrioid carcinoma cell, a HER2 1+ lung-metastatic malignant melanoma cell, a HER2 1+ human cervix carcinoma cell, Her2 1+ human renal cell carcinoma cell, or a HER2 1+ human ovary carcinoma cell. 
     
     
         59 . The method of any one of  claims 1 - 41  wherein the tumor cell is a HER2 1+ or 2+ or 3+ human pancreatic carcinoma cell, a HER2 2+ metastatic pancreatic carcinoma cell, a HER2 0+/1+, +3+ human lung carcinoma cell, a HER2 2+ human Caucasian bronchioaveolar carcinoma cell, a HER2 0+ anaplastic lung carcinoma, a human non-small cell lung carcinoma cell, a human pharyngeal carcinoma cell, a HER2 2+ human tongue squamous cell carcinoma cell, a HER2 2+ squamous cell carcinoma cell of the pharynx, a HER2 1+ or 2+ human colorectal carcinoma cell, a HER2 0+, 1+ or 3+ human gastric carcinoma cell, a HER2 1+ human breast ductal ER+ (estrogen receptor-positive) carcinoma cell, a HER2 2+/3+ human ER+, HER2-amplified breast carcinoma cell, a HER2 0+/1+ human triple negative breast carcinoma cell, a HER2 0+ human breast ductal carcinoma (Basal B, Mesenchymal-like triple negative) cell, a HER2 2+ER+ breast carcinoma, a HER2 0+ human metastatic breast carcinoma cell (ER−, HER2− amplified, luminal A, TN), a human uterus mesodermal tumor (mixed grade III) cell, a 2+ human endometrioid carcinoma cell, a HER2 1+ human skin epidermoid carcinoma cell, a HER2 1+ lung-metastatic malignant melanoma cell, a HER2 1+ malignant melanoma cell, a human cervix epidermoid carcinoma vcell, a HER2 1+ human urinary bladder carcinoma cell, a HER2 1+ human cervix carcinoma cell, Her2 1+ human renal cell carcinoma cell, or a HER2 1+, 2+ or 3+ human ovary carcinoma cell, and wherein the antigen-binding construct is conjugated to maytansine (DM1). 
     
     
         60 . The method of any one of  claims 1 - 41  wherein the tumor cell is selected from a HER2 2/3+, gene amplified ovarian cancer cell, a HER2 0+/1+ triple negative breast cancer cell; an ER+, HER2 1+ breast cancer cell; a trastuzumab resistant HER2 2+ breast cancer cell; an ER+, HER2+ breast cancer cell; or a HER2 3+ breast cancer cell. 
     
     
         61 . The method according to any preceding claim, wherein the construct is selected from v5019, v10000, v7091, v5020 or v6717. 
     
     
         62 . The method according to any preceding claim, wherein administering is done by injection or infusion, optionally wherein the administering is intravenous. 
     
     
         63 . The method according to any preceding claim, further comprising administering to the subject an additional agent, optionally a chemotherapeutic agent. 
     
     
         64 . The method of  claim 63 , wherein the additional agent is one or more of bleomycin, carboplatin, cisplatin, nab-paclitaxel, docetaxel, doxorubicin, erlotinib, fluorouracil, gemcitabine, methotrexate, pemetrexed, topotecan, vinorelbine, capecitabine, navelbine, or paclitaxel. 
     
     
         65 . The method of  claim 63 , wherein
 i. the tumor is non-small cell lung cancer, and the additional agent is one or more of cisplatin, carboplatin, paclitaxel, albumin-bound paclitaxel, nab-paclitaxel, docetaxel, gemcitabine, vinorelbine, irinotecan, etoposide, vinblastine, capecitabine, navelbine or pemetrexed; or   ii. the tumor is head and neck cancer, and the additional agent is one or more of paclitaxel, carboplatin, doxorubicin or cisplatin; or   iii. the tumor is a estrogen and/or progesterone positive breast cancer, and the additional agent is one or more of doxorubicin, epirubicin, paclitaxel, nab-paclitaxel, docetaxel, fluorouracil, cyclophosphamide, carboplatin, letrozole, mifepristone, capecitabine, gemcitabine, vinorelbine or tamoxifen; or   iv. the tumor is a pancreatic tumor and the additional agent is nab-paclitaxel, capecitabine, gemcitabine, navelbine or paclitaxel.   
     
     
         66 . The method according to any preceding claim, wherein the subject is a human. 
     
     
         67 . The method according to any preceding claim, wherein the method comprises inhibiting, reducing or blocking HER2 signaling. 
     
     
         68 . The method according to any preceding claim, wherein the method comprises killing or inhibiting the growth of a HER2-expressing tumor cell. 
     
     
         69 . The method according to any preceding claim, wherein the subject is administered at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses. 
     
     
         70 . The method according to any preceding claim, wherein the amount of at least one of the plurality of doses is at least 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg. 
     
     
         71 . The method according to any preceding claim, wherein the amount of each of the plurality of doses is at least 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg. 
     
     
         72 . The method according to any preceding claim, wherein each dose is administered at least daily, weekly, or monthly. 
     
     
         73 . The method according to any preceding claim, wherein each dose is administered at least every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. 
     
     
         74 . The method according to any preceding claim, wherein treatment continues for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks; or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 months. 
     
     
         75 . The method according to any preceding claim, wherein the mean tumor volume in the subject after receiving at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses is less than the mean tumor volume of a control subject receiving an equivalent amount of trastuzumab. 
     
     
         76 . The method according to any preceding claim, wherein overall survival of the subject is significantly increased as compared to a control subject receiving an equivalent amount of a non-specific control antibody or as compared to a control subject not receiving treatment; or wherein the growth of tumor is significantly decreased as compared to a control subject receiving an equivalent amount of a non-specific control antibody, as compared to a control subject receiving an equivalent amount of Herceptin, or as compared to a control subject not receiving treatment. 
     
     
         77 . The method of  claim 76 , wherein the significance is measured by a log rank test. 
     
     
         78 . The method of  claim 76 , wherein the p value is less than 0.5, 0.01, or 0.001. 
     
     
         79 . The method according to any preceding claim, wherein overall survival of the subject is more significantly increased as compared to a control subject receiving an equivalent amount of trastuzumab. 
     
     
         80 . The method of  claim 79 , wherein the antigen-binding construct p value is less than 0.001 and wherein the trastuzumab p value is greater than 0.001. 
     
     
         81 . The method according to any preceding claim, wherein the p value of the significance of the increase relative to the control subject receiving an equivalent amount of a non-specific control antibody is less than the p value of an increase in survival of a second control receiving an equivalent amount of trastuzumab as compared to the control subject receiving an equivalent amount of a non-specific control antibody. 
     
     
         82 . The method of  claim 81 , wherein the antigen-binding construct p value is less than 0.001 and wherein the trastuzumab p value is greater than 0.001. 
     
     
         83 . The method according to any preceding claim, wherein overall survival of the subject after receiving a combination of the antigen-binding construct and an additional agent is significantly increased as compared to a control subject receiving an equivalent amount of trastuzumab alone. 
     
     
         84 . The method according to any preceding claim, wherein overall survival of the subject is significantly increased as compared to a control subject receiving a lesser amount of trastuzumab.