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WO2017102789A1 - Combination therapy of anti-her3 antibodies and anti-her2 antibodies - Google Patents

Combination therapy of anti-her3 antibodies and anti-her2 antibodies Download PDF

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Publication number
WO2017102789A1
WO2017102789A1 PCT/EP2016/080890 EP2016080890W WO2017102789A1 WO 2017102789 A1 WO2017102789 A1 WO 2017102789A1 EP 2016080890 W EP2016080890 W EP 2016080890W WO 2017102789 A1 WO2017102789 A1 WO 2017102789A1
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Prior art keywords
antibody
her2
her3
human
seq
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French (fr)
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Birgit Bossenmaier
Maurizio CEPPI
Max Hasmann
Martin Weisser
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F Hoffmann La Roche AG
Hoffmann La Roche Inc
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F Hoffmann La Roche AG
Hoffmann La Roche Inc
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
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    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • A61K31/5685—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone having an oxo group in position 17, e.g. androsterone
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Definitions

  • the present invention relates to the combination therapy of anti-HER3 antibodies and anti-HER2 antibodies, in patients suffering from a HER2 low, HER3 positive and hormone receptor (HR) positive breast cancer including dose schedules and additional therapies.
  • HR hormone receptor
  • Human HER3 encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases which also includes HER1 (also known as EGFR), HER2, and HER4 (Kraus, M.H. et al, PNAS 86 (1989) 9193-9197; Plowman, G.D. et al, PNAS 87 (1990) 4905-4909; Kraus, M.H. et al,
  • the transmembrane receptor HER3 consists of an extracellular ligand- binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD) and a C-terminal phosphorylation domain.
  • ECD extracellular ligand- binding domain
  • TKD transmembrane domain
  • HER3 has a heregulin (HRG) binding domain within the extracellular domain but its kinase domain is inactive. Therefore, when activated by binding of its ligand, HER3 can form heterodimers with other HER family members, but cannot transphosphorylate the partner protein.
  • HRG heregulin
  • heterodimerization of HER3 leads to the activation of the receptor-mediated signaling pathway through transphosphorylation of its intracellular domain by the dimerization partner. Dimer formation between HER family members expands the signaling potential of HER3 and is a means not only for signal diversification but also signal amplification.
  • the HER2/HER3 heterodimer induces one of the most important mitogenic signals via the PI3K and AKT pathway among HER family members (Sliwkowski M.X., et al, J. Biol. Chem. 269 (1994) 14661-14665;
  • WO 97/35885 relates to HER3 antibodies.
  • WO 2003/013602 relates to inhibitors of HER activity, including HER antibodies.
  • WO2012022814, WO2012031198, WO2012044612, WO2012052230, WO2012059858 relate to HER3 antibodies.
  • Human HER2 refers to 185-kDa growth factor receptor also referred to as neu and c-erbB-2 (Slamon, et al, Science 235 (1987) 177-182; Swiss-Prot P04626) whose function is related to neoplastic transformation in human breast cancer cells.
  • Overexpression of this protein has been identified in 20-30% of breast cancer patients where it correlates with regionally advanced disease, increased probability of tumor recurrence, and reduced patient survival. As many as 30-40% of patients having gastric, endometrial, salivary gland, non-small cell lung, pancreatic, ovarian, peritoneal, prostate, or colorectal cancers may also exhibit overexpression of this protein.
  • HER2 comprises four domains, Domain I (amino acid residues from about 1-195), Domain II (amino acid residues from about 196-320), Domain III (amino acid residues from about 321 488), and Domain IV (amino acid residues from about 489-632) (residue numbering without signal peptide).
  • Domain I amino acid residues from about 1-195
  • Domain II amino acid residues from about 196-320
  • Domain III amino acid residues from about 321 488
  • Domain IV amino acid residues from about 489-632
  • Trastuzumab (e.g. Herceptin®) is a recombinant humanized anti-HER2 monoclonal antibody used for the treatment of HER2 over-expressed/HER2 gene amplified metastatic breast cancer. Trastuzumab binds specifically to the same epitope of HER2 as the murine anti-HER2 antibody 4D5 described in Hudziak, et al., Mol. Cell. BioL 9 (1989) 1165-1172.
  • Trastuzumab is a recombinant humanized version of the murine anti-HER2 antibody 4D5, referred to as rhuMAb 4D5 or trastuzumab) and has been clinically active in patients with HER2-overexpressing metastatic breast cancers that had received extensive prior anticancer therapy. (Baselga, et al, J. Clin. Oncol. 14 (1996) 737-744). Trastuzumab and its method of preparation are described in US 5,821,337.
  • Pertuzumab is a recombinant humanized version of the murine anti- HER2 antibody 2C4 (referred to as rhuMAb 2C4 or pertuzumab) and it is described together with the respective method of preparation in WO 01/00245 and
  • epitope 2C4 is the region in the extracellular domain of HER2 to which the antibody 2C4 binds.
  • a routine cross-blocking assay such as that described in "Ed. Harlow and David Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory,
  • epitope mapping can be performed to assess whether the antibody binds to the 2C4 epitope of HER2 (e.g. any one or more residues in the region from about residue 22 to about residue 584 of HER2, inclusive).
  • Epitope 2C4 comprises residues from domain II in the extracellular domain of HER2.
  • 2C4 and pertuzumab bind to sub-domain II of the extracellular domain of HER2 near the junction of domains I, II and III. See also Franklin, et al., Cancer Cell 5 (2004) 317-328.
  • the invention relates to an antibody which binds to human HER3 for use in the treatment of breast cancer in combination with an antibody binding to human HER2 and inhibiting dimerization of HER2 , wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive.
  • the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive. embodiment additionally either
  • an anti-hormonal agent is co-administered , wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), like e.g. fulvestrant, or b) a selective estrogen receptor modulator (SERM); and such as
  • SESD selective estrogen receptor degrader
  • SERM selective estrogen receptor modulator
  • aromatase inhibitor is letrozole, anastrozole or exemestane
  • chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative b) cyclophosphamide, c) cisplatin or carboplatin d) mitomycin C, e) methotrexate, f) 5-fluorouracil or capecitabine, g) doxorubicin or its liposomal formulation, or daunorubicin or its liposomal formulation, h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
  • the combination is administered as a first-line treatment.
  • the antibody which binds to human HER3 is characterized in that the heavy chain variable domain comprises a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDRIH region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6 or a CDR1L region of SEQ ID NO:7.
  • the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7.
  • the antibody which binds to human HER3 is characterized in that the heavy chain variable domain VH is SEQ ID NO:8; and the light chain variable domain VL is SEQ ID NO: 10.
  • the antibody which binds to human HER3 described above is further characterized in that the antibody is of IgGl subclass and is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65 % or lower.
  • the antibody binding to human HER2 and inhibiting dimerization of HER2 is pertuzumab.
  • the antibody which binds to human HER3 is administered at a dose of 400-600 mg every 3 weeks and the antibody which binds to human HER2 is administered at a dose of 300-500 mg every 3 weeks.
  • Another aspect of the invention is an antibody binding to human HER2 and inhibiting dimerization of HER2 for use in the treatment of breast cancer in combination with an antibody which binds to human HER3, wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive.
  • the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive.
  • Another aspect of the invention is the use of an antibody which binds to human HER3 for the manufacture of a medicament for the treatment of breast cancer wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive and wherein the treatment is in combination with an antibody binding to human HER2 and inhibiting dimerization of HER2.
  • the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive.
  • Another aspect of the invention is the use of an antibody binding to human HER2 and inhibiting dimerization of HER2 for the manufacture of a medicament for the treatment of breast cancer wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive and wherein the treatment is in combination with an antibody which binds to human HER3.
  • the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive.
  • Another aspect of the invention is a method of treating a patient suffering from of breast cancer wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive, the method comprising the administration of (an effective amount of ) an antibody which binds to human HER3 and the coadministration of (an effective amount of) an antibody binding to human HER2 and inhibiting dimerization of HER2 .
  • the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive.
  • an anti-HER3 antibody described above with an antibody which binds to human HER2 and which inhibits dimerization of HER2 showed strong tumor growth inhibition of HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) breast cancers, even in tumors where the antibody which binds to human HER2 and which inhibits dimerization of HER2, only showed low to medium tumor growth inhibition when administered alone.
  • HR hormone receptor
  • MCF7 cells by anti-HER3 antibodies in different concentrations are produced by anti-HER3 antibodies in different concentrations.
  • Figure 3A,B and C Western blot after immune precipitation (IP)-HER2 and blotting against ER or pER ERa from HEK 293 cells expressing HER2 and ER.
  • 3B shows Immunoprecipitation with ant-ER and blotting against HER2 in the same cells.
  • Figure 4A,B and C Western blot after immune precipitation (IP) f ER or HER3 expressing HEK 293 cells with anti HER3 blotted against ER (4A) and pER (4B).
  • IP immune precipitation
  • Fig.5B after 91 days (with end of treatment on day 61).
  • Fig.5C ex vivo analyse of MAPK at day 91
  • Fig.5D ex vivo analyse of AKT at day 91.
  • Figure 6 In vivo antitumor efficacy of anti-HER3 -antibody
  • HER3-antibody Mab205.10.2 in combination with pertuzumab in only first line (1L) HER2 low, ER+, HER3 + metastatic breast cancer patients. All patients are HER2 low, ER+, HER3 +.
  • SD Stable Disease
  • PR Partial Response
  • CR Complete Response
  • PD Progressive Disease
  • ORR Overall Response Rate, DCR Disease Control rate. The best overall RECIST responses and the line of treatment are indicated.
  • the invention relates to an antibody which binds to human HER3 for use in the treatment of, or the manufacture of a medicament for the treatment of, or a method of treatment of,
  • breast cancer in combination with an antibody which binds to human HER3, wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive.
  • the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive .
  • the invention comprises an antibody which binds to human HER3, characterized in that the heavy chain variable domain comprises a CDR3H region of SEQ ID NO: 1
  • the invention further comprises an antibody which binds to human HER3 according to the invention characterized in that the heavy chain variable domain VH is SEQ ID NO:8; and the light chain variable domain VL is SEQ ID NO:9, or the light chain variable domain VL is SEQ ID NO: 10, or the light chain variable domain VL is SEQ ID NO: 11; or a humanized version thereof for use in the combination therapies described herein.
  • the antibody which binds to human HER3 according to the invention is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDRIH region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO: 6 for use in the combination therapies described herein.
  • the antibody which binds to human HER3 according to the invention is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDRIH region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO: 7 for use in the combination therapies described herein.
  • Such “chimeric” antibodies are also referred to as "class-switched antibodies.”
  • Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See, e.g., Morrison, S.L., et al, Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.
  • humanized antibody or “humanized version of an antibody” refers to antibodies in which the framework or "complementarity determining regions"
  • the framework region can be modified by further mutations.
  • Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies.
  • Preferably such humanized version is chimerized with a human constant region (see e.g. Sequences SEQ ID NO: 12-16).
  • human antibody is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001)
  • Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al, Nature 362 (1993) 255-258; Brueggemann, M.D., et al., Year Immunol.
  • human antibody as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to Clq binding and/or FcR binding, e.g. by "class switching” i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation).
  • class switching i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation).
  • an "antibody which binds to human HER3" as used herein refers to an antibody specifically which binds to the human HER3 antigen with a binding affinity of KD 1.0 x 10 "8 mol/1 or lower (in one embodiment of KD 1.0 x 10 "8 mol/1 - 1.0 x 10 "13 mol/1) at 25°C.
  • binding to human HER2 As used herein, the terms "binding to human HER2”, “specifically binding to human HER2”, “which binds to human HER2”, “which specifically binds to human HER2”, or “ anti-HER2 antibody” are interchangeable and refer to an antibody which specifically binds to the human HER2 antigen with a binding affinity of KD- value of 1.0 x 10 "8 mol/1 or lower at 25°C, in one embodiment of a KD-value of 1.0 xlO "9 mol/1 or lower at 25°C.
  • the binding affinity is determined with a standard binding assay at 25°C, such as surface plasmon resonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden).
  • an "antibody which binds to human HER2" as used herein refers to an antibody specifically which binds to the human HER2 antigen with a binding affinity of KD 1.0 x 10 "8 mol/1 or lower (in one embodiment of KD 1.0 x 10 "8 mol/1 - 1.0 x 10 "13 mol/1) at 25°C.
  • MAPK Mitogen-activated protein kinase
  • PI3K Phosphoinositide 3-kinase
  • an antibody which binding to human HER2 and inhibiting dimerization of HER2 refer to an anti-HER2 antibody which specifically binds to the human HER2 antigen and which inhibits/blocks ligand-dependent HER2 homodimerization and HER2 heterodimerization with HER1, HER3, and HER4, and especially inhibits HER2/HER3 dimerization (see e.g. Perjeta® Prescribing Information. Genentech, Inc. June 2012. Baselga J, et al; N Engl J Med. 2012;366: 109-119; Baselga J, et al Nat Rev Cancer. 2009;9:463-475; Hynes NE, et al Nat Rev Cancer. 2005;5:341-
  • Human HER3 encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases which also includes FIERI (also known as EGFR), HER2, and HER4 (Kraus, M.H. et al, PNAS 86 (1989), 9193-9197; Plowman, G.D. et al, PNAS 87 (1990), 4905-4909;
  • EGFR epidermal growth factor receptor
  • the transmembrane receptor HER3 consists of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD) and a C-terminal phosphorylation domain.
  • ECD extracellular ligand-binding domain
  • TKD transmembrane domain
  • This membrane-bound protein has HER3 a Heregulin (HRG) binding domain within the extracellular domain but not an active kinase domain. It therefore can bind this ligand but not convey the signal into the cell through protein phosphorylation.
  • HRG Heregulin
  • heterodimers with other HER family members which do have kinase activity. Heterodimerization leads to the activation of the receptor-mediated signaling pathway and transphosphorylation of its intracellular domain. Dimer formation between HER family members expands the signaling potential of HER3 and is a means not only for signal diversification but also signal amplification. For example the HER2/HER3 heterodimer induces one of the most important mitogenic signals via the PI3K and AKT pathway among HER family members (Sliwkowski, M.X., et al, J. Biol. Chem.
  • Anti-HER3 antibodies Mab205.10.1, Mab205.10.2, and Mab205.10.3 showed a competitive binding with the ligand Heregulin (HRG) to HER3. Expression of HER3 gene and/or expression of its protein have been reported in numerous cancers, including prostate, bladder, and breast tumors.
  • Alternate transcriptional splice variants encoding different isoforms have been characterized.
  • One isoform lacks the intermembrane region and is secreted outside the cell. This form acts to modulate the activity of the membrane-bound form. Additional splice variants have also been reported, but they have not been thoroughly characterized.
  • the HER receptor will generally comprise an extracellular domain, which may bind an HER ligand; a lipophilic transmembrane domain, a conserved intracellular tyrosine kinase domain, and a carboxyl-terminal signaling domain harboring several tyrosine residues which can be phosphorylated.
  • the extracellular domain of HER2 comprises four domains, Domain I (amino acid residues from about 1-195),
  • Domain II amino acid residues from about 196-320
  • Domain III amino acid residues from about 321 488
  • Domain IV amino acid residues from about 489-632
  • Pertuzumab (e.g. Perjeta®) is a recombinant humanized anti-HER2 monoclonal antibody used for the treatment of HER2 positive cancers. Pertuzumab binds specifically to the 2C4 epitope, a different epitope on the extracellular domain of HER2 than trastuzumab. Pertuzumab is the first in a new class of HER dimerization inhibitors (HDIs).
  • HDIs HER dimerization inhibitors
  • pertuzumab Through its binding to the HER2 extracellular domain, pertuzumab inhibits dimerization of HER2 (especially ligand-activated heterodimerization with other HER family members), thereby inhibiting downstream signaling pathways and cellular processes associated with tumor growth and progression (Franklin, M.C., et al. Cancer Cell 5 (2004) 317-328 and
  • Pertuzumab is a recombinant humanized version of the murine anti-HER2 antibody 2C4 (referred to as rhuMAb 2C4 or pertuzumab) and it is described together with the respective method of preparation in WO 01/00245 and WO 2006/007398.
  • rhuMAb 2C4 refers to an antibody that binds to the 2C4 epitope and preferably comprising the variable light and variable heavy amino acid sequences disclosed in WO 2006/044908, more particularly the humanized 2C4 version 574 disclosed in Fig. 2 of WO 2006/044908.
  • Pertuzumab encompasses all corresponding anti-HER2 antibodies that fulfill the requirements necessary for obtaining a marketing authorization as an identical or biosimilar product in a country or territory selected from the group of countries consisting of the USA, Europe and Japan.
  • the "epitope 2C4" is the region in the extracellular domain of HER2 to which the antibody 2C4 binds.
  • a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed.
  • epitope mapping can be performed to assess whether the antibody binds to the 2C4 epitope of HER2.
  • Epitope 2C4 comprises residues from domain II in the extracellular domain of HER2.
  • 2C4 and Pertuzumab bind to the extracellular domain of HER2 at the junction of domains I, II and III (Franklin et al. Cancer Cell 5:317-328 (2004)).
  • epitope includes any polypeptide determinant capable of specific binding to an antibody.
  • epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by an antibody.
  • variable domain of an antibody according to the invention denotes each of the pair of light and heavy chain domains which are involved directly in binding the antibody to the antigen.
  • the variable light and heavy chain domains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions” (or complementary determining regions, CDRs).
  • the framework regions adopt a ⁇ -sheet conformation and the CDRs may form loops connecting the ⁇ -sheet structure.
  • the CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site.
  • the antibody's heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.
  • the term "antigen-binding portion of an antibody” when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the antigen-binding portion of an antibody comprises amino acid residues from the "complementary determining regions" or "CDRs".
  • the term "antigen-binding portion" of an antibody of the invention contains six complementarity determining regions (CDRs) which contribute in varying degrees to the affinity of the binding site for antigen.
  • CDRH1 denotes the CDR1 region of the heavy chain variable region calculated according to Kabat.
  • CDRL2 and CDRL3 mean the respective regions from the heavy (H) or light(L) chain.
  • the extent of CDR and framework regions (FRs) is determined by comparison to a compiled database of amino acid sequences in which those regions have been defined according to variability among the sequences according to Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health
  • the "Fc part” of an antibody is not involved directly in binding of an antibody to an antigen, but exhibit various effector functions.
  • a “Fc part of an antibody” is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies.
  • antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgGl, IgG2, IgG3, and IgG4, IgAl, and IgA2.
  • the different classes of immunoglobulins are called a, ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the Fc part of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement- dependent cytotoxicity) based on complement activation, Clq binding and Fc receptor binding.
  • the term "complement-dependent cytotoxicity (CDC)” denotes a process initiated by binding of complement factor Clq to the Fc part of most IgG antibody subclasses. Binding of Clq to an antibody is caused by defined protein- protein interactions at the so called binding site. Such binding sites are known in the state of the art and described e.g.
  • binding sites are e.g. L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to EU index of Kabat, E.A., see below).
  • Antibodies of subclass IgGl, IgG2 and IgG3 usually show complement activation and Clq and C3 binding, whereas IgG4 do not activate the complement system and do not bind Clq and C3.
  • the antibody according to the invention comprises a Fc part derived from human origin and preferably all other parts of the human constant regions.
  • Fc part derived from human origin denotes a Fc part which is either a Fc part of a human antibody of the subclass IgGl, IgG2, IgG3 or IgG4, e.g. a Fc part from human IgGl subclass, a mutated Fc part from human IgGl subclass (preferably with a mutation on L234A + L235A), a Fc part from human IgG4 subclass or a mutated Fc part from human IgG4 subclass (preferably with a mutation on S228P).
  • the anti-HER3 according to the invention is of human IgGl subclass. In one embodiment the anti-HER2 according to the invention is of human IgGl subclass. In one embodiment the anti-HER3 and the anti-HER2 antibody according to the invention are both of human IgGl subclass. In one embodiment the anti-HER3 and the anti-HER2 antibody according to the invention are both characterized in that the constant chains are of human origin. Such constant chains are well known in the state of the art and e.g. described by Kabat, E.A., (see e.g. Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218). For example, a useful human heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 13. For example, a useful human light chain constant region comprises an amino acid sequence of a kappa-light chain constant region of SEQ ID NO: 12.
  • amino acid denotes the group of naturally occurring carboxy a-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp,
  • nucleic acid or "nucleic acid molecule”, as used herein, are intended to include DNA molecules and R A molecules.
  • a nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid.
  • DNA for a presequence or secretory leader is operable linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operable linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operable linked to a coding sequence if it is positioned so as to facilitate translation.
  • operable linked means that the DNA sequences being linked are colinear, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the expressions "cell”, “cell line”, and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived there from without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.
  • the anti-HER3 antibody described herein is preferably characterized in that the constant chains are of human origin.
  • Such constant chains are well known in the state of the art and described, e.g., by Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).
  • a useful human light chain constant region comprises an amino acid sequence of a kappa-light chain constant region of SEQ ID NO: 12.
  • useful human heavy chain constant region comprises SEQ ID NO: 13 to 16.
  • an anti-HER3 antibody for the respective combination therapy comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 8 and SEQ ID NO: 10, respectively,; and having one or more of the following properties (determined in assays as described in Example 3 and 2): the anti-HER3 antibody inhibits the HER3 phosphorylation in tumor cells such as MCF7 cells, FaDu cells or Mel-Juso cell (in one embodiment the anti-HER3 antibody shows an inhibition of the HER3 phosphorylation in MCF7 cells of at least 80% (in one embodiment at least 90%) at a concentration of 1.0 ⁇ / ⁇ 1 ; in one embodiment the anti-HER3 antibody shows an inhibition of the HER3 phosphorylation in FaDu cells of at least 80% (in one embodiment at least 90%) at a concentration of 0.1 ⁇ / ⁇ 1 ; in one embodiment the anti-
  • the anti-HER3 antibody inhibits the proliferation of MDA-MB-175 cells with an IC50 value of less than 10 ⁇ g/ml
  • the anti-HER3 antibody binds to HER3 with a KD value of less than 5.0x lO ⁇ 9 M, in one embodiment with a KD value of less than 3. Ox 10 ⁇ 9 M.
  • an anti-HER3 antibody for the respective combination therapy is a bispecific anti-HER3/anti-HERl antibody as described in US 2010/0255010.
  • the bispecific anti-HER3/anti-HERl antibody is characterized comprising by the characteristic amino acid sequences disclosed in US 2010/0255010, i.e. A) (a) HVR-Hl comprising the amino acid sequence of
  • HVR-H2 comprising the amino acid sequence of VGEISAAGGYTD
  • HVR-H3 comprising the amino acid sequence of ARESRVSFEAAMDY
  • HVR-Ll comprising the amino acid sequence of NIATDVA
  • HVR-L2 comprising the amino acid sequence of SASF
  • HVR-L3 comprising the amino acid sequence of SEPEPYT
  • B) (a) a heavy chain variable domain with the amino acid sequence of SEQ ID NO: 30 as disclosed in US2010/0255010; (b) a light chain variable domain with the amino acid sequence of SEQ ID NO: 29 as disclosed in US2010/0255010.
  • ADCC antibody-dependent cellular cytotoxicity
  • IgGl type antibodies the most commonly used therapeutic antibodies, are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain.
  • ADCC antibody dependent cellular cytotoxicity
  • the antibody according to the invention is afucosylated which means the antibody is glycosylated (if it comprises an Fc part of IgGl subclass) with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 80% or lower (Numbering according to Kabat), e.g. between 80% and 1 %. In another embodiment is the amount of fucose within said sugar chain is 65 % or lower, in one embodiment between 5% and 65%, in one embodiment from 0% to 65%, and in one embodiment the amount of fucose within said sugar chain is 0%.
  • Such antibodies are referred to in the following as
  • afucosylated antibodies or “non-fucosylated antibodies”. Such afucosylated antibodies show enhanced ADCC whereas other antibody properties remain substantially unaffected.
  • the amount of N-glycolylneuraminic acid (NGNA) is 1% or less and/or the amount of N-terminal alpha-l,3-galactose is 1% or less within said sugar chain.
  • the sugar chain show preferably the characteristics of N-linked glycans attached to Asn297 of an antibody recombinantly expressed in a CHO cell.
  • Asn297 according to the invention means amino acid asparagine located at about position 297 in the Fc region. Based on minor sequence variations of antibodies, Asn297 can also be located some amino acids (usually not more than +3 amino acids) upstream or downstream of position 297, i.e. between position 294 and 300.
  • the sugar chains show characteristics of N-linked glycans attached to Asn297 of an antibody recombinantly expressed in a CHO cell
  • NGNA glycosylation of human IgGl occurs at Asn297 as core fucosylated biantennary complex oligosaccharide glycosylation terminated with up to two Gal residues.
  • Human constant heavy chain regions of the IgGl subclass are reported in detail by Kabat, E., A., et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), and by Brueggemann, M., et al, J. Exp. Med.
  • the modified oligosaccharides of the full length parent antibody may be hybrid or complex.
  • the bisected, reduced/not- fucosylated oligosaccharides are hybrid.
  • the bisected, reduced/not-fucosylated oligosaccharides are complex.
  • amount of fucose means the amount of said sugar within the sugar chain at Asn297, related to the sum of all glycostructures attached to Asn297 (e.g. complex, hybrid and high mannose structures) measured by
  • the relative amount of fucose is the percentage of fucose-containing structures related to all glycostructures identified in an N-Glycosidase F treated sample (e.g. complex, hybrid and oligo- and high- mannose structures, resp.) by MALDI-TOF.
  • the antibodies according to the invention are preferably produced by recombinant means. Such methods are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable purity.
  • nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells, such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, yeast, or E. coli cells, and the antibody is recovered from the cells (from the supernatant or after cells lysis).
  • the antibodies may be present in whole cells, in a cell lysate, or in a partially purified, or substantially pure form.
  • Transient expression is described by, e.g., Durocher, Y., et al, Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R., et al, Proc. Natl. Acad. Sci. USA 86 (1989) 3833- 3837; Carter, P., et al, Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; Norderhaug, L., et al, J. Immunol. Methods 204 (1997) 77-87.
  • a preferred transient expression system (HEK 293) is described by Schlaeger, E.-J.
  • Monoclonal antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA and RNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures.
  • the hybridoma cells can serve as a source of such DNA and RNA.
  • the DNA may be inserted into expression vectors, which are then transfected into host cells, such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in the host cells.
  • host cells such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein.
  • Antibodies obtainable from said cell lines are preferred embodiments of the invention.
  • Afucosylated antibodies are preferably prepared via glycoengineering as described above.
  • the heavy and light chain variable domains according to the invention are combined with sequences of promoter, translation initiation, constant region, 3' untranslated region, polyadenylation, and transcription termination to form expression vector constructs.
  • the heavy and light chain expression constructs can be combined into a single vector, co-trans fected, serially transfected, or separately transfected into host cells which are then fused to form a single host cell expressing both chains.
  • the antibodies of the combination are administered as a pharmaceutical composition comprising the respective antibody.
  • the present invention provides a composition, e.g. a pharmaceutical composition, containing an antibody according to the present invention, formulated together with a pharmaceutical carrier.
  • pharmaceutical carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
  • a composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
  • the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • breast cancer as used herein including any metastatic or refractory versions.
  • Another aspect of the invention is an anti-HER3 -antibody according to the invention for the treatment of cancer in combination with an antibody which binds to human HER2 and which inhibits dimerization of HER2, wherein the cancer is a HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) breast cancer.
  • Another aspect of the invention is the use of an antibody which binds to human HER3 for the manufacture of a medicament for the treatment of breast cancer in combination with an antibody which binds to human HER2 and which inhibits dimerization of HER2, wherein the cancer is a is HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) cancer.
  • Another aspect of the invention is a method of treatment of a patient suffering from breast cancer by administering an anti-HER3 -antibody antibody according to the invention to said patient in the need of such treatment in combination with an antibody which binds to human HER2 and which inhibits dimerization of HER2, wherein the cancer is a HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) cancer.
  • an anti-HER3 -antibody antibody according to the invention to said patient in the need of such treatment in combination with an antibody which binds to human HER2 and which inhibits dimerization of HER2, wherein the cancer is a HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) cancer.
  • HR hormone receptor
  • the anti-HER3 antibody used in this combination is characterized in comprising as VH an amino acid sequence of SEQ ID NO: 8 and an as VL an amino acid sequence of SEQ ID NO: 10, b) the anti-HER2 antibody used in this combination is pertuzumab, and c) the cancer is HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) breast cancer.
  • HER3 positive refers to a breast cancer or tumorous tissue which comprises cells which are characterized by HER3 protein expression and/or gene amplification. This means that the respective breast cancer samples show a detectable level of the HER3 protein expression and/or gene amplification.
  • the expression level of HER3 may be detected by an immunohistochemical method, whereas said HER3 gene amplification status can be measured with in situ hybridization methods, like fluorescence in situ hybridization techniques (FISH).
  • FISH fluorescence in situ hybridization techniques
  • HER3 can, inter alia, be detected by an immunohistochemical method.
  • immunohistochemical methods are well known in the art (see e.g. analogous methods and test for HER2 expression levels below or e.g. WO 2015/049355).
  • Other methods like qRT-PCR might be used to detect levels of HER3 gene amplification.
  • HER3 positive refers to the HER3 protein expression detected by an immunohistochemical method, which means the respective breast cancer samples show a detectable level of the HER3 protein in an corresponding immunohistochemistry (IHC) assay.
  • IHC immunohistochemistry
  • HER2 low cancer In the context of the combination therapy of an anti-HER3 antibody with an anti- HER2 antibody, wherein the anti-HER2 antibodies inhibits HER2 dimerization, the term "HER2 low” cancer also referred to as “HER2 low cancer” as used herein refers to a cancer or tumorous tissue which comprises cells which express low levels of HER2. Thus, HER2 low cancer cells do not overexpress HER2 (as defined for HER2 -positive cancer) and are not negative for HER2 expression.
  • HER2 low cancer is a cancer with an immunohistochemistry (IHC) score of 2+ and an in situ hybridization (ISH) amplification ratio ⁇ 2.0 (i.e.
  • HER2 low cancer is present if a low (IHC 1+) or moderate (IHC 2+) HER2 (protein) expression level detected e.g.
  • HER2 low cancer is defined as an immunohistochemistry (IHC) score of HER2(2+) and ISH negative or immunohistochemistry (IHC) score of HER2(1+) and ISH negative (IHC l+/ISH-negative or IHC 2+/ISH-negative).
  • the expression level of HER2 may be detected by an immunohistochemical method, whereas said HER2 gene amplification status can be measured with in situ hybridization methods, like fluorescence in situ hybridization techniques (FISH).
  • FISH fluorescence in situ hybridization techniques
  • the expression level of HER2 can, inter alia, be detected by an immunohistochemical method. Such methods are well known in the art and corresponding commercial kits are available. Exemplary kits which may be used in accordance with the present invention are, inter alia, HerceptTestTM produced and distributed by the company Dako or the test called Ventana PathwayTM. The level of HER2 protein expression may be assessed by using the reagents provided with and following the protocol of the HercepTestTM. A skilled person will be aware of further means and methods for determining the expression level of HER2 by immunohistochemical methods; see for example WO 2005/117553. Therefore, the expression level of HER2 can be easily and reproducibly determined by a person skilled in the art without undue burden. However, to ensure accurate and reproducible results, the testing must be performed in a specialized laboratory, which can ensure validation of the testing procedures.
  • the expression level of HER2 can be classified in a low expression level, an intermediate expression level and a high expression level.
  • HER2 low disease is defined by a low or weak expression level of HER2 (e.g. HER2(1+ or 2+) by IHC) and a negative ISH result, for example determined in a sample of a cancer patient. Therefore parallel testing using immunohistochemistry and in situ hybridisation is preferred.
  • HER2(0), HER2(+), HER2(++) and HER2(+++) is as follows:
  • HER2(+), HER2(++) and HER2(+++) used herein are equivalent to the terms HER2(1+), HER2(2+) and HER2(3+).
  • a "low HER2 protein expression level" used in context of this invention corresponds to a 1+ score ("negative assessment” according to the table shown herein above), and a 2+ score "weakly positive". As described herein above in detail, the evaluation of the protein expression level (i.e. the scoring system as shown in the table) is based on results obtained by immunohistochemical methods.
  • the HER2 status is, accordingly, performed by immunohistochemistry with one of two FDA-approved commercial kits available; namely the Dako HerceptestTM and the Ventana PathwayTM. These are semi-quantitative assays which stratify expression levels into 0 ( ⁇ 20,000 receptors per cell, no expression visible by IHC staining), 1+ (-100,000 receptors per cell, partial membrane staining, ⁇ 10% of cells overexpressing HER2), 2+ (-500,000 receptors per cell, light to moderate complete membrane staining, > 10%> of cells overexpressing HER2), and 3+ (-2,000,000 receptors per cell, strong complete membrane staining, > 10% of cells overexpressing HER2).
  • HER2 low disease is defined by a low or weak expression level of HER2 (e.g. HER2(1+ or 2+) by IHC) and a negative ISH result.
  • the sample to be assessed can be (obtained) from a patient with HER2 low cancer as defined above.
  • the sample may be obtained from a tumorous tissue, (a) tumor(s) and, accordingly, is (a) tumor cell(s) or (a) tumor tissue(s) suspected of being HER2 expressing tumour, like a breast tumor.
  • a person skilled in the art is in the position to identify such tumors and/or individuals/patients suffering from corresponding cancer using standard techniques known in the art and methods disclosed herein.
  • said tumor cell or cancer cell may be obtained from any biological source/organism, particularly any biological source/organism, suffering from the above-mentioned cancer.
  • particular useful cells are, preferably, human cells.
  • the tumor/cancer/tumor cell/cancer cell is a solid tumor/ cancer/tumor cell/cancer cell.
  • the cancer/tumor cell may be a breast cancer/tumor cell or said sample comprises a cancer/tumor cell, such as a breast cancer/tumor cell.
  • said tumor/cancer may be a breast tumor/cancer.
  • Hormone receptor (HR) positive or HR+ refers to Estrogen Receptor (ER) positive and/or Progesterone Receptor (PgR)-positive cancer/tumouros tissue, such as Breast Cancer.
  • ER Estrogen Receptor
  • PgR Progesterone Receptor
  • Breast Cancer are considered negative for ER or PgR if it is detected that ⁇ 1% of tumor cell nuclei are immunoreactive in the presence of evidence that the sample can express ER or PgR (positive intrinsic controls can be detected), as determined by IHC.
  • Breast Cancer are considered uninterpretable for ER or PgR if it is detected that no tumor nuclei are immunoreactive and that internal epithelial elements present in the sample or separately submitted from the same sample lack any nuclear staining, as determined by IHC.
  • the breast cancer is estrogen receptor (ER) positive (ER+).
  • Estrogen receptor (ER) positive or ER+ refers to breast cancer which is considered positive for ER if finding of > 1% of tumor cell nuclei are immunoreactive, as determined by Immunohistochemistry
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • composition must be sterile and fluid to the extent that the composition is deliverable by syringe.
  • carrier preferably is an isotonic buffered saline solution.
  • a method of treating when applied to, for example, cancer refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of a cancer.
  • a method of treating does not necessarily mean that the cancer cells or other disorder will, in fact, be eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will, in fact, be alleviated.
  • a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of a patient, is nevertheless deemed an overall beneficial course of action.
  • both antibodies When both antibodies are administered simultaneously the dose is administered on the same day in one administration, e.g. during one continuous infusion.
  • both antibodies When both antibodies are administered sequentially the dose is administered either on the same day in two separate administrations, e.g. two separate continuous infusions, or one of the antibodies is administered on day 1 and the second antibody is administered on day 2 to day 7, preferably on day 2 to 4.
  • the terms "co-administration” or “coadministering" with respect to the maintenance doses of the first antibody and the second antibody mean that the maintenance doses can be either administered simultaneously, e.g. during one continuous infusion, if the treatment cycle is appropriate for both antibodies.
  • the maintenance doses are administered sequentially, either within one or several days, e.g. the maintenance dose of the first antibody is administered every 3 weeks, and the maintenance dose of the second is administered every 2 weeks.
  • other treatment cycles /usually from 1 to 4 weeks, preferably from 2 to 3 weeks, may be used for both antibodies.
  • paclitaxel derivatives like nab-paclitaxel (protein-bound paclitaxel) (e.g. Abraxane®) or docosahexaenoic acid (DHA) -paclitaxel, (e.g. Taxoprexin®)), or docetaxel (e.g. Taxotere®), b) cyclophosphamide (CTX) (e.g. Cytoxan®), c) cisplatin (e.g. Platinol®) or carboplatin (e.g. Paraplatin® and
  • Paraplatin-AQ® d) mitomycin C (e.g. Mutamycin®), e) methotrexate (MTX) (e.g. Trexall®), f) 5-fluorouracil (5-FU) (e.g. Adrucil®) or capecitabine (e.g.
  • ixabepilone e.g. Ixempra ®
  • additional an anti-hormonal agents may be used in combination treatment of the present invention.
  • agents include, for example: a) a selective estrogen receptor degrader (SERD) like e.g. fulvestrant, or b) a selective estrogen receptor modulators (SERM), such as tamoxifen, raloxifene, toremifene or lasofoxifene, or c) an aromatase inhibitor like e.g. letrozole, anastrozole or exemestane).
  • SESD selective estrogen receptor degrader
  • SERM selective estrogen receptor modulators
  • AC-Taxol AC given 3 -weekly for 4 cycles followed by paclitaxel given either 3- weekly for 4 cycles or weekly (at a smaller dose) for 12 weeks
  • TAC Taxotere (docetaxel), Adriamycin (doxorubicin), and cyclophosphamide given 3 -weekly for 4-6 cycles
  • FEC 5-fluorouracil, epirubicin and cyclophosphamide given 3 -weekly for 6 cycles
  • FEC FEC given 3 -weekly for 3 cycles followed by docetaxel given 3 -weekly for
  • TC Taxotere (docetaxel) and cyclophosphamide given 3 -weekly for 4 or 6 cycles
  • Fulvestrant exemestane alone or in combination with everolimus, anastrozole, letrozole, tamoxifene. Recently the palbociclib has been approved in combination with letrozole and fulvestrant.
  • Radioactive atoms for use in the context of this invention can be selected from the group including, but not limited to, radium, cesium- 137, iridium- 192, americium- 241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131, and indium- 111.
  • the combination treatment of the present invention is used without such additional ionizing radiation.
  • a typical course of treatment for a patient undergoing radiation therapy will be a treatment schedule over a 1 to 6 week period, with a total dose of between 10 and 80 Gy administered to the patient in a single daily fraction of about 1.8 to 2.0 Gy, 5 days a week.
  • a preferred embodiment of this invention there is synergy when tumors in human patients are treated with the combination treatment of the invention and radiation.
  • the antibodies are administered to a patient according to known methods, by intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, or intrathecal routes. Intravenous or subcutaneous administration of the antibodies is preferred.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, the growth of tumors, tumor metastases, or other cancer-causing or neoplastic cells in a patient.
  • treatment refers to the act of treating.
  • a method of treating or its equivalent, when applied to, for example, cancer refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of a cancer.
  • first-line treatment refers to the first type of drug therapy given for the treatment of cancer or metastasis. This can be an adjuvant or neoadjuvant or palliative chemotherapy or immunotherapy offered initially following diagnosis and/or surgery or following diagnosis of metastatic disease.
  • the first-line treatment refers to the first cancer treatment using e.g. chemical or biochemical substances, like cytotoxic drugs, targeted therapies with antibodies, or enzyme inhibitors/antagonist/or modulators.
  • the present invention further provides an article of manufacture comprising a container, a composition within the container comprising an anti-HER3 antibody and a package insert instructing the user of the composition to administer said anti-
  • HER3 antibody to a patient suffering from HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) breast cancer in combination with an anti-HER2 antibody which inhibits the dimerization of HER2.
  • HR hormone receptor
  • the article of manufacture containers may further include a pharmaceutically acceptable carrier.
  • the article of manufacture may further include a sterile diluent, which is preferably stored in a separate additional container.
  • an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), (in one embodiment the estrogen receptor inhibitor is fulvestrant), b) a selective estrogen receptor modulator (SERM), (in one embodiment the selective estrogen receptor modulator is tamoxifen, raloxifene, toremifene or lasofoxifene), and c) an aromatase inhibitor; (in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane);
  • SESD selective estrogen receptor degrader
  • SERM selective estrogen receptor modulator
  • an aromatase inhibitor in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane
  • chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
  • CX cyclophosphamide
  • CX cyclophosphamide
  • an anti-hormonal agent is co-administered, and wherein the anti- hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), b) a selective estrogen receptor modulator (SERM); and c) an aromatase inhibitor.
  • SELD selective estrogen receptor degrader
  • SERM selective estrogen receptor modulator
  • a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
  • CTX cyclophosphamide
  • the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel, docetaxel ( in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel).
  • the antibody according to any one of embodiments 1 to 8, wherein the combination is administered as a first-line treatment.
  • an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), (in one embodiment the selective estrogen receptor degrader (SERD) is fulvestrant), b) a selective estrogen receptor modulator (SERM), (in one embodiment the selective estrogen receptor modulator is tamoxifen, raloxifene, toremifene or lasofoxifene), and c) an aromatase inhibitor; (in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane);
  • SEMD selective estrogen receptor degrader
  • SERM selective estrogen receptor modulator
  • an aromatase inhibitor in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane
  • the antibody which binds to human HER3 is characterized in that the heavy chain variable domain VH is SEQ ID NO: 8; and the light chain variable domain VL is SEQ ID NO: 10. 12.
  • the antibody according to any one of embodiments 1 to 12, wherein the antibody binding to human HER2 and inhibiting dimerization of HER2 is pertuzumab.
  • the use according to embodiment 1, wherein the breast cancer is HER2 low HER3, and positive ER (estrogen receptor) positive is .
  • the use according to any one of embodiments 1 to 2 wherein
  • an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), (in one embodiment the selective estrogen receptor degrader (SERD) is fulvestrant), b) a selective estrogen receptor modulator (SERM), (in one
  • a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and
  • breast cancer is HER2 low HER3, and positive ER (estrogen receptor) positive .
  • chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
  • CTX cyclophosphamide
  • CX c) cisplatin or carboplatin
  • mitomycin C e
  • an aromatase inhibitor c) an aromatase inhibitor.
  • the anti-hormonal agent is selected from the group of: a) fulvestrant, b) tamoxifen, raloxifene, toremifene or lasofoxifene; and
  • the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel, docetaxel ( in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel).
  • combination is administered as a first-line treatment.
  • the antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7.
  • the selective estrogen receptor modulator is tamoxifen, raloxifene, toremifene or lasofoxifene), and c) an aromatase inhibitor; (in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane);
  • chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
  • CX cyclophosphamide
  • CX cyclophosphamide
  • an anti-hormonal agent is co-administered, and wherein the anti- hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), b) a selective estrogen receptor modulator (SERM); and
  • anti-hormonal agent is selected from the group of: a) fulvestrant, b) tamoxifen, raloxifene, toremifene or lasofoxifene; and
  • a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
  • CTX cyclophosphamide
  • the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel, docetaxel ( in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel).
  • paclitaxel nab-paclitaxel
  • docetaxel in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel.
  • the antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7.
  • an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), (in one embodiment the selective estrogen receptor degrader (SERD) is fulvestrant), b) a selective estrogen receptor modulator (SERM), (in one embodiment the selective estrogen receptor modulator is tamoxifen, raloxifene, toremifene or lasofoxifene), and c) an aromatase inhibitor; (in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane);
  • SEMD selective estrogen receptor degrader
  • SERM selective estrogen receptor modulator
  • an aromatase inhibitor in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane
  • chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
  • CTX cyclophosphamide
  • CX c) cisplatin or carboplatin
  • mitomycin C e
  • an anti-hormonal agent is co-administered, and wherein the anti- hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), b) a selective estrogen receptor modulator (SERM); and
  • an aromatase inhibitor c) an aromatase inhibitor.
  • the anti-hormonal agent is selected from the group of: a) fulvestrant, b) tamoxifen, raloxifene, toremifene or lasofoxifene; and
  • a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
  • CTX cyclophosphamide
  • the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel, docetaxel ( in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel).
  • paclitaxel nab-paclitaxel
  • docetaxel in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel.
  • the antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7.
  • an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), (in one embodiment the selective estrogen receptor degrader (SERD) is fulvestrant), b) a selective estrogen receptor modulator (SERM), (in one
  • the selective estrogen receptor modulator is tamoxifen, raloxifene, toremifene or lasofoxifene), and c) an aromatase inhibitor; (in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane);
  • chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
  • CX cyclophosphamide
  • CX cyclophosphamide
  • an anti-hormonal agent is co-administered, and wherein the anti- hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), b) a selective estrogen receptor modulator (SERM); and
  • the anti-hormonal agent is selected from the group of: a) fulvestrant, b) tamoxifen, raloxifene, toremifene or lasofoxifene; and
  • a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
  • CTX cyclophosphamide
  • the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel, docetaxel ( in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel).
  • antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7.
  • the heavy chain variable domain VH is SEQ ID NO: 8; and the light chain variable domain VL is SEQ ID NO: 10.
  • antibody which binds to human HER3 is of human IgGl subclass and is characterized in that the antibody is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65 % or lower.
  • antibody binding to human HER2 and inhibiting dimerization of HER2 is pertuzumab.
  • antibody which binds to human HER3 is administered at a dose of 400-600 mg every 3 weeks and the antibody which binds to human HER2 is administered at a dose of 300-500 mg every 3 weeks.
  • SEQ ID NO 12 human kappa light chain constant region
  • SEQ ID NO 13 human heavy chain constant region derived from IgGl
  • SEQ ID NO 14 human heavy chain constant region derived from IgGl mutated on L234A and L235A
  • SEQ ID NO 16 human heavy chain constant region derived from IgG4 mutated on S228P
  • SEQ ID NO 18 human HER2 (including signal peptide)
  • NMRI mice were immunized with hHER3-ECD (in-house) and boosted with hu-HER3-ECD. The immune response was monitored by testing serum samples against the HER1/2/3- ECD-ELISA. Spleen cells from mice with sufficient titers of anti-HER3 immunoglobulin were frozen for later immortalization by fusion with mouse myeloma cell line P3X63 Ag8.653. One fusion was done and hybridoma supematants screened by HER1/2/-ECD- ELISA showing no cross-reactivity, but binding to HER3-ECD and anti-HER3 selective hybridomas were selected. The relevant hybridomas were cloned by single cell FACS sorting.
  • Single cell clones from different hybridomas were cultured in vitro to produce antibody in tissue culture medium for characterization.
  • Antibodies were selected by determining their ability to inhibit HER3 phosphorylation, AKT phosphorylation and tumor cell proliferation of MDA-MB-175 cells (see Examples below). From the obtained antibodies, one was further humanized to give the following antibodies Mab 205.10.1, Mab 205.10.2 and Mab 205.10.3 with their respective VH and VL or CDRs.
  • such antibodies were prepared using constant regions of human origin e.g. SEQ ID NO: 12-13.
  • Binding assays a) Antigen specific ELISA for binding to human HER3 ECD
  • Soluble human HER3 extracellular domain fused to Streptavidin Binding Protein was captured on a streptavidine plate.
  • SBP Streptavidin Binding Protein
  • CM-5 sensor chip For affinity measurements, 30 ⁇ g/ml of anti Fey antibodies (from goat, Jackson Immuno Research) were coupled to the surface of a CM-5 sensor chip by standard amine-coupling and blocking chemistry on a SPR instrument (Biacore T100). After conjugation, anti-HER3 antibodies were injected at 25°C at a flow rate of 5 ⁇ / ⁇ , followed by a dilution series (0 nM to 1000 nM) of human HER3 ECD at 30 ⁇ / ⁇ . As running buffer for the binding experiment PBS/0.1% BSA was used. The chip was then regenerated with a 60s pulse of 10 mM glycine-
  • Mab205.10.1, Mab205.10.2, and Mab205.10.3 all showed binding to the same epitope.
  • the anti-HER3 -antibodies Ul-7, U-53 and Ul-59 described in WO 2007/077028 and Ab#6 described in WO 2008/100624 were investigated in such assay and revealed to bind to different epitopes than antibodies Mab205.10.1. Mab205.10.2, and Mab205.10.3.
  • HER3 antibodies on receptor phosphorylation in MCF7 cells is shown below and in Fig IB.
  • HER ECD in w0 2003/013602 were investigated in Mel- Juso cells. Assays in Mel- Juso cells were performed according to the aforementioned protocol for MCF7 and FaDu cells. Cell numbers and media volumes were adapted to 12-well plates Percent (%) inhibition of anti-HER3 antibodies on receptor phosphorylation in Mel- Juso cells is shown below and in Figure 1C.
  • Assays were performed in MCF7 cells according to the following protocol: Seed MCF7 cells with 30000 cells/well into Poly-D-Lysine coated 96-well plate in RPMI 1640 medium with 10% FCS and incubate for 24h. Remove medium by tapping on a clean paper towel, wash carefully with 200 ⁇ 1 serum-free medium, incubate overnight with ⁇ /well RPMI 1640 with 0.5% FCS. Remove medium as above; add antibodies in ⁇ RPMI 1640 with 0.5% FCS and incubate 1.5h. Add HRG-lb (final concentration 5ng/ml) for 10 min. Remove medium as above.
  • HER3 antibodies Mab205.10.1, Mab205.10.2, and Mab205.10.3 in a cell proliferation assay was assessed.
  • MDA-MB-175 cells VII Human Breast Carcinoma Cells, ATCC catalog no. HTB-25
  • 20,000 cells per well were seeded into sterile 96 well tissue culture plates with DMEM/F12 cell culture medium, containing 10% FCS and incubated at 37°C ⁇ 1°C with 5 % ⁇ 1% C0 2 for one day.
  • the cells are slow growing cells with a doubling time of ca. 1.5 days.
  • Anti-HER3 antibodies were added in dilution series and further incubated for 6 days. Cell viability was then assessed using the alamarBlue® readout.
  • IC50 values were calculated using means of triplicates for each antibody concentration; otherwise, if the % inhibition of cell viability at the highest concentration was below 50%, no IC50 could be calculated and it is indicated that IC 50
  • the target cells KPL4 (ADCC), breast carcinoma, cultivation in RPMI1640 + 2 mM L-alanyl-L-Glutamine + 10 % FCS ) were collected with trypsin/EDTA (Gibco # 25300-054) in exponential growth phase. After a washing step and checking cell number and viability, the aliquot needed was labeled for 30 min at
  • ⁇ calcein-stained target cells were plated in round-bottom 96-well plates, 50 ⁇ 1 diluted, afucosylated antibody (Mab205.10.1, Mab205.10.2, Mab205.10.3, preparation see below) which was added and 50 ⁇ 1 effector cells.
  • the target cells were mixed with Redimune® NF Liquid (ZLB Behring) at a concentration of 10 mg/ml Redimune.
  • the killing of target cells was assessed by measuring LDH (Lactate Dehydrogenase) release from damaged cells using the Cytotoxicity Detection kit (LDH Detection Kit, Roche # 1 644 793) according to the manufacturer's instruction. Briefly, 100 ⁇ supernatant from each well was mixed with 100 ⁇ substrate from the kit in a transparent flat bottom 96 well plate. The Vmax values of the substrate's colour reaction was determined in an ELISA reader at 490 nm for at least 10 min. Percentage of specific antibody-mediated killing was calculated as follows: (A - SR)/(MR - SR)xl00, where A is the mean of Vmax at a specific antibody concentration, SR is the mean of Vmax of the spontaneous release and
  • MR is the mean of Vmax of the maximal release.
  • the afucosylated antibody (Mab205.10.1, Mab205.10.2, Mab205.10.3) were prepared by co-transfection with four plasmids, two for antibody expression, one for a fusion GnTIII polypeptide expression (a GnT-III expression vector), and one for mannosidase II expression (a Golgi mannosidase II expression vector) at a ratio of 4:4: 1 : 1, respectively in HEK293 or CHO cells.
  • the full antibody heavy and light chain DNA sequences were subcloned into mammalian expression vectors (one for the light chain and one for the heavy chain) under the control of the MPSV promoter and upstream of a synthetic polyA site, each vector carrying an EBV OriP sequence.
  • Antibodies were produced by co- transfecting HEK293-EBNA cells or CHO cells with the antibody heavy and light chain expression vectors using a calcium phosphate-transfection approach. Exponentially growing HEK293-EBNA cells were trans fected by the calcium phosphate method.
  • the cells were co-transfected with four plasmids, two for antibody expression, one for a fusion GnTIII polypeptide expression (a GnT-III expression vector), and one for mannosidase II expression (a Golgi mannosidase II expression vector) at a ratio of 4:4: 1 : 1, respectively.
  • Cells were grown as adherent monolayer cultures in T flasks using DMEM culture medium supplemented with 10% FCS, and were transfected when they were between 50 and 80% confluent.
  • a solution of DNA, CaC12 and water was prepared by mixing 188 ⁇ g total plasmid vector DNA (four plasmids, two for antibody expression (one light chain and one heavy chain), one for a fusion GnTIII polypeptide expression (a GnT-III expression vector), and one for mannosidase II expression (a Golgi mannosidase II expression vector) at a ratio of 4:4: 1 :1, respectively), water to a final volume of 938 ⁇ and 938 ⁇ of a 1M CaC12 solution.
  • the cells were incubated at 37°C, 5%> C02 for about 17 to 20 hours, then medium was replaced with 25 ml DMEM, 10%> FCS.
  • the conditioned culture medium was harvested 7 days post-transfection by centrifugation for 15 min at 210 x g, the solution was sterile filtered (0.22 ⁇ filter) and sodium azide in a final concentration of 0.01 %> w/v was added, and kept at 4°C.
  • the secreted afucosylated antibodies were purified and the oligosaccharides attached to the Fc region of the antibodies were analysed e.g. by MALDI/TOF-MS (as described in e.g. WO 2008/077546).
  • MALDI/TOF-MS as described in e.g. WO 2008/077546.
  • oligosaccharides were enzymatically released from the antibodies by PNGaseF digestion, with the antibodies being either immobilized on a PVDF membrane or in solution.
  • the resulting digest solution containing the released oligosaccharides either prepared directly for MALDI/TOF-MS analysis or was further digested with EndoH glycosidase prior to sample preparation for MALDI/TOF-MS analysis.
  • the analyzed amount of fucose within the sugar chain at Asn297 was between 50-20%.
  • HER2/HER3 and ER estrogen receptor crosstalk (cross-signaling)
  • Cells were immunoprecipitated with and anti HER2 antibody (trastuzumab) and blotted against ERa (Cell signaling mAb # 8644) or vice versa, immunoprecipitated with an anti ERa antibody (Cell signaling mAb # 8644) and blotted against HER2 (Calbiochem Ab 3).
  • HER2 and ERa can form a complex and in this complex ERa is phosphorylated. Results are shown in Figure 3A,3B and 3C.
  • ERa expressing cells were generated. Cells were starved overnight in DEMEM 0, 5% FCS. After 48h cells were stimulated with HRG (black bars) for 10 min and lysed by a Triton XI 00 lyses buffer containing Aprotinin ⁇ /mL, PMSF ⁇ /mL and Orthovanadate 2 ⁇ 1/ ⁇ 1. Cells were immunoprecipitated with and anti HER3 antibody (Ab) and blotted against ERa antibody (Cell signaling mAb # 8644) or vice versa, immunoprecipitated with an anti ERa antibody (Cell signaling mAb # 8644) and blotted against HER3 (Ab208 Roche). HER3 and ERa can form a complex in the presence and absence of HRG, but ER is strongly phosphorylated only in the presence of HRG. Results are shown in Figure 4A,4B and 4C.
  • HER3 and HER2 can form a complex with the estrogen receptor.
  • HRG activated HER2/HER3 heterodimers mediate phosphorylation of estrogen receptor.
  • - HRG is an ER regulated gene.
  • ER signaling in breast cancer induces HRG expression which in turn drives mitogenic signaling via HER2/HER3 heterodimers.
  • HER2 can activate estrogen receptor
  • the in vivo antitumor efficacy of the antibody Mab205.10.2 in combination with pertuzumab was detected in a fragment based HER2 low, ER+, HER3 + breast cancer patient-derived xenograft model (PDX) transplanted on nude mice.
  • PDX patient-derived xenograft model
  • Mab205.10.2 and pertuzumab were provided as stock solution from Roche, Penzberg, Germany.
  • Antibody buffer included histidine.
  • Antibody solution was diluted appropriately in buffer from stock prior injections. Tumor fragments
  • HBCx-19 breast cancer tumor fragments were originally obtained from a metastasis of a lobular carcinoma. Tumors of the same passage were transplanted subcutaneously onto 6-24 donor mice. When these tumors reached 1000 to 2000 mm 3 , donor mice were sacrificed and tumors excised, dissected and cut into fragments measuring of approximately 20 mm 3 . Thereafter fragments were grafted subcutaneous.
  • mice Female athymic nude mice, 6- to 9-week-old at the beginning of the experimental phase, were obtained from Harlan Laboratories (France). Animals were maintained in specific pathogen-free animal housing at the Center for Exploration and Experimental Functional Research (CERFE, Evry, France) animal facility. Animals were delivered to the laboratory 7 days before the experiments during which time they were acclimatized to laboratory conditions. Mice were housed inside individually ventilated cages (IVC) under a light-dark cycle (14-hour circadian cycle of artificial light) and controlled room temperature and humidity.
  • IVC individually ventilated cages
  • Antibody Mab205.10.2 was administered as single agent and in combination with pertuzumab at 10 mg/kg i.p. q7d once weekly until D61.
  • Pertuzumab was administered as single agent and in combination once weekly at
  • HBCx-19 breast cancer xenograft bearing mice were treated with antibody Mab205.10.2 as single agent and in combination with pertuzumab from study D26 to D61.
  • treatment with Mab205.10.2 antibody as single agent showed strong significant anti-tumor efficacy with tumors stasis until D61 and a Relative Tumor Volume (RTV) of 100%.
  • RTV Relative Tumor Volume
  • Monotherapy with pertuzumab translated into partial HBCx-19 tumor regression at 61 >.
  • superior efficacy was observed with Mab205.10.2 in combination with pertuzumab resulting in strongest tumor regression (RTV 10%).
  • the in vivo antitumor efficacy of the antibody Mab205.10.2 in combination with pertuzumab and selective estrogen receptor degrader (SERD) fulvestrant was detected in a fragment based HER2 low, ER+, HER3+ breast cancer patient- derived xenograft model (PDX) transplanted on nude mice.
  • SELD selective estrogen receptor degrader
  • Test agents Mab205.10.2 and pertuzumab were provided as stock solution from Roche,
  • Antibody buffer included histidine. Antibody solution was diluted appropriately in buffer from stock prior injections. Fulvestrant (Faslodex, Astra Zeneca) was purchased commercially.
  • Tumor fragments HBCx-19 breast cancer tumor fragments were originally obtained from a metastasis of a lobular carcinoma. Tumors of the same passage were transplanted subcutaneously onto 6-24 donor mice. When these tumors reached 1000 to 2000 mm 3 , donor mice were sacrificed and tumors excised, dissected and cut into fragments measuring of approximately 20 mm 3 . Thereafter fragments were grafted subcutaneous. Animals
  • mice Female athymic nude mice, 6- to 9-week-old at the beginning of the experimental phase, were obtained from Harlan Laboratories (France). Animals were maintained in specific pathogen-free animal housing at the Center for Exploration and Experimental Functional Research (CERFE, Evry, France) animal facility. Animals were delivered to the laboratory 7 days before the experiments during which time they were acclimatized to laboratory conditions. Mice were housed inside individually ventilated cages (IVC) under a light-dark cycle (14-hour circadian cycle of artificial light) and controlled room temperature and humidity.
  • IVC individually ventilated cages
  • Antibody Mab205.10.2 was administered at 3 mg/kg as single agent once weekly (6x) as single agent and in dual combination with pertuzumab (3 mg/kg i.p. q7dx6) or in triple combination with fulvestrant (50 mg/kg, im, q7dx6) until D57.
  • Pertuzumab was administered as single agent and in combination once weekly at 3 mg/kg and fulvestrant given im at 50 mg/kg once weekly. The corresponding vehicle was administered on the same days.
  • HBCx-19 HER2 low, ER+, HER3+ breast cancer xenograft bearing mice were treated with antibody Mab205.10.2 as single agent and in combination with pertuzumab and anti-estrogen fulvestrant from study D22 to D57.
  • the triple combination (Mab205.10.2 + pertuzumab + fulvestrant) was the most efficacious combination with tumor control of HBCx-19 BC xenografts (RTV 118%).
  • the monotherapies and dual combinations were less efficacious and HBCx-19 tumors progressed. Results are shown in Figure 6.
  • Glycoengineered Mab205.10.2 was investigated in combination with pertuzumab and paclitaxel in patients with MBC expressing HER3 and HER2 protein.
  • Anti-HER3 antibody Mab205.10.2 was administered at a dose of 500 mg on a q3w schedule (i.e., on day 1 of 3-weekly cycles).
  • Pertuzumab was administered at a loading dose of 840 mg during cycle 1 followed by 420 mg for the remaining cycles in a q3w schedule.
  • Paclitaxel was be administered at 80 mg/m 2 in a qw schedule.
  • a dose of Mab205.10.2 of 1000 mg q3w diarrhea as side effect could be detected.
  • PD Progressive Disease
  • ORR Overall Response Rate, DCR Disease Control rate. The best overall RECIST responses and the line of treatment are indicated.
  • Figure 8 In vivo antitumor efficacy (reduction of lesions) anti-HER3 -antibody Mab205.10.2 in combination with pertuzumab in only first line (1L) HER2 low,
  • Mab205.10.2 was administered at a dose of 500 mg on a q3w schedule (i.e., on day 1 of 3-weekly cycles).
  • Pertuzumab was administered at a dose of 420 mg in a q3w schedule.
  • Paclitaxel was be administered at 80 mg/m 2 in a qw schedule (data not shown).

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Abstract

The present disclosure relates to the combination therapy of anti-HER3 antibodies with certain anti-HER2 antibodies.

Description

Combination therapy of anti-HER3 antibodies and anti-HER2 antibodies
The present invention relates to the combination therapy of anti-HER3 antibodies and anti-HER2 antibodies, in patients suffering from a HER2 low, HER3 positive and hormone receptor (HR) positive breast cancer including dose schedules and additional therapies. Background of the Invention
Human HER3 (ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor tyrosine-protein kinase erbB-3, SEQ ID NO: 17) encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases which also includes HER1 (also known as EGFR), HER2, and HER4 (Kraus, M.H. et al, PNAS 86 (1989) 9193-9197; Plowman, G.D. et al, PNAS 87 (1990) 4905-4909; Kraus, M.H. et al,
PNAS 90 (1993) 2900-2904). Like the prototypical epidermal growth factor receptor, the transmembrane receptor HER3 consists of an extracellular ligand- binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD) and a C-terminal phosphorylation domain. HER3 has a heregulin (HRG) binding domain within the extracellular domain but its kinase domain is inactive. Therefore, when activated by binding of its ligand, HER3 can form heterodimers with other HER family members, but cannot transphosphorylate the partner protein. However, heterodimerization of HER3 leads to the activation of the receptor-mediated signaling pathway through transphosphorylation of its intracellular domain by the dimerization partner. Dimer formation between HER family members expands the signaling potential of HER3 and is a means not only for signal diversification but also signal amplification. For example the HER2/HER3 heterodimer induces one of the most important mitogenic signals via the PI3K and AKT pathway among HER family members (Sliwkowski M.X., et al, J. Biol. Chem. 269 (1994) 14661-14665;
Alimandi M, et al, Oncogene. 10 (1995) 1813-1821; Hellyer, N.J., J. Biol. Chem. 276 (2001) 42153-4261; Singer, E., J. Biol. Chem. 276 (2001) 44266-44274; Schaefer, K.L., Neoplasia 8 (2006) 613-622).
Expression of this gene and/or expression of its protein have been reported in numerous cancers, including prostate, bladder, and breast tumors. Alternate transcriptional splice variants encoding different isoforms have been characterized. One isoform lacks the intermembrane region and is secreted outside the cell. This form acts to modulate the activity of the membrane-bound form. Additional splice variants have also been reported, but they have not been thoroughly characterized.
WO 97/35885 relates to HER3 antibodies. WO 2003/013602 relates to inhibitors of HER activity, including HER antibodies. WO 2007/077028, WO 2008/100624 , WO2011076683, WO2011044311, WO2011136911, WO2012019024,
WO2012022814, WO2012031198, WO2012044612, WO2012052230, WO2012059858 relate to HER3 antibodies.
Human HER2 refers to 185-kDa growth factor receptor also referred to as neu and c-erbB-2 (Slamon, et al, Science 235 (1987) 177-182; Swiss-Prot P04626) whose function is related to neoplastic transformation in human breast cancer cells.
Overexpression of this protein has been identified in 20-30% of breast cancer patients where it correlates with regionally advanced disease, increased probability of tumor recurrence, and reduced patient survival. As many as 30-40% of patients having gastric, endometrial, salivary gland, non-small cell lung, pancreatic, ovarian, peritoneal, prostate, or colorectal cancers may also exhibit overexpression of this protein.
The HER receptor will generally comprise an extracellular domain, which may bind an HER ligand; a lipophilic transmembrane domain, a conserved intracellular tyrosine kinase domain, and a carboxyl-terminal signaling domain harboring several tyrosine residues which can be phosphorylated. The extracellular domain of
HER2 comprises four domains, Domain I (amino acid residues from about 1-195), Domain II (amino acid residues from about 196-320), Domain III (amino acid residues from about 321 488), and Domain IV (amino acid residues from about 489-632) (residue numbering without signal peptide). See Garrett, et al, Mol. Cell. 11 (2003) 495-505, Cho, et al, Nature 421 (2003) 756-760, Franklin, et al, Cancer
Cell 5 (2004) 317-328, or Plowman, et al, Proc. Natl. Acad. Sci. 90 (1993) 1746- 1750 and WO 2006/007398.
Trastuzumab (e.g. Herceptin®) is a recombinant humanized anti-HER2 monoclonal antibody used for the treatment of HER2 over-expressed/HER2 gene amplified metastatic breast cancer. Trastuzumab binds specifically to the same epitope of HER2 as the murine anti-HER2 antibody 4D5 described in Hudziak, et al., Mol. Cell. BioL 9 (1989) 1165-1172. Trastuzumab is a recombinant humanized version of the murine anti-HER2 antibody 4D5, referred to as rhuMAb 4D5 or trastuzumab) and has been clinically active in patients with HER2-overexpressing metastatic breast cancers that had received extensive prior anticancer therapy. (Baselga, et al, J. Clin. Oncol. 14 (1996) 737-744). Trastuzumab and its method of preparation are described in US 5,821,337.
Pertuzumab (e.g. Perjeta®) is another recombinant humanized anti-HER2 monoclonal antibody used for the treatment of HER2 positive cancers. Pertuzumab binds specifically to sub-domain II, a different epitope on the extracellular domain of HER2 than trastuzumab's binding site on sub-domain IV. Pertuzumab is the first in a new class of HER2 dimerization inhibitors (HDIs). Through its binding to the HER2 extracellular domain, pertuzumab inhibits dimerization of HER2 (with other HER family members), thereby inhibiting downstream signaling pathways and cellular processes associated with tumor growth and progression (Franklin, M.C., et al. Cancer Cell 5 (2004) 317-328 and Friess, T, et al. Clin Cancer Res 11 (2005) 5300-5309). Pertuzumab is a recombinant humanized version of the murine anti- HER2 antibody 2C4 (referred to as rhuMAb 2C4 or pertuzumab) and it is described together with the respective method of preparation in WO 01/00245 and
WO 2006/007398.
The "epitope 2C4" is the region in the extracellular domain of HER2 to which the antibody 2C4 binds. In order to screen for antibodies which bind to the 2C4 epitope, a routine cross-blocking assay such as that described in "Ed. Harlow and David Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory,
(1988)", can be performed. Alternatively, epitope mapping can be performed to assess whether the antibody binds to the 2C4 epitope of HER2 (e.g. any one or more residues in the region from about residue 22 to about residue 584 of HER2, inclusive). Epitope 2C4 comprises residues from domain II in the extracellular domain of HER2. 2C4 and pertuzumab bind to sub-domain II of the extracellular domain of HER2 near the junction of domains I, II and III. See also Franklin, et al., Cancer Cell 5 (2004) 317-328.
Summary of the Invention
The invention relates to an antibody which binds to human HER3 for use in the treatment of breast cancer in combination with an antibody binding to human HER2 and inhibiting dimerization of HER2 , wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive.
In one preferred embodiment the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive. embodiment additionally either
A) an anti-hormonal agent is co-administered , wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), like e.g. fulvestrant, or b) a selective estrogen receptor modulator (SERM); and such as
tamoxifen, raloxifene, toremifene or lasofoxifene, or c) an aromatase inhibitor; (in one preferred embodiment the
aromatase inhibitor is letrozole, anastrozole or exemestane;
or
B) a chemotherapeutic agent is co-administered , wherein the
chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative b) cyclophosphamide, c) cisplatin or carboplatin d) mitomycin C, e) methotrexate, f) 5-fluorouracil or capecitabine, g) doxorubicin or its liposomal formulation, or daunorubicin or its liposomal formulation, h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone. In on embodiment the combination is administered as a first-line treatment.
In one embodiment the antibody which binds to human HER3 is characterized in that the heavy chain variable domain comprises a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDRIH region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6 or a CDR1L region of SEQ ID NO:7.
In one embodiment the antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDRIH region of SEQ ID
NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7.
In one embodiment the antibody which binds to human HER3 is characterized in that the heavy chain variable domain VH is SEQ ID NO:8; and the light chain variable domain VL is SEQ ID NO: 10.
In one embodiment the antibody which binds to human HER3 described above is further characterized in that the antibody is of IgGl subclass and is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65 % or lower.
In one embodiment the antibody binding to human HER2 and inhibiting dimerization of HER2 is pertuzumab.
In one embodiment the antibody which binds to human HER3 is administered at a dose of 400-600 mg every 3 weeks and the antibody which binds to human HER2 is administered at a dose of 300-500 mg every 3 weeks.
Another aspect of the invention is an antibody binding to human HER2 and inhibiting dimerization of HER2 for use in the treatment of breast cancer in combination with an antibody which binds to human HER3, wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive. In one preferred embodiment the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive. Another aspect of the invention is the use of an antibody which binds to human HER3 for the manufacture of a medicament for the treatment of breast cancer wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive and wherein the treatment is in combination with an antibody binding to human HER2 and inhibiting dimerization of HER2. In one preferred embodiment the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive.
Another aspect of the invention is the use of an antibody binding to human HER2 and inhibiting dimerization of HER2 for the manufacture of a medicament for the treatment of breast cancer wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive and wherein the treatment is in combination with an antibody which binds to human HER3. In one preferred embodiment the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive.
Another aspect of the invention is a method of treating a patient suffering from of breast cancer wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive, the method comprising the administration of (an effective amount of ) an antibody which binds to human HER3 and the coadministration of (an effective amount of) an antibody binding to human HER2 and inhibiting dimerization of HER2 . In one preferred embodiment the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive. Surprisingly it was found that the combination therapy an anti-HER3 antibody described above with an antibody which binds to human HER2 and which inhibits dimerization of HER2 showed strong tumor growth inhibition of HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) breast cancers, even in tumors where the antibody which binds to human HER2 and which inhibits dimerization of HER2, only showed low to medium tumor growth inhibition when administered alone.
Description of the Figures
Figure 1A and B: Percent (%) inhibition HER3 receptor phosphorylation in
MCF7 cells by anti-HER3 antibodies in different concentrations.
Figure 1C Percent (%) inhibition of HER receptor phosphorylation in
Mel-Juso cells by anti-HER3 antibodies in different concentrations. Figure 2: Western blot of total cell lysate blotted against ER, pER
(Ser 118) and HER2.
Figure 3A,B and C: Western blot after immune precipitation (IP)-HER2 and blotting against ER or pER ERa from HEK 293 cells expressing HER2 and ER. 3B shows Immunoprecipitation with ant-ER and blotting against HER2 in the same cells. Figure 4A,B and C: Western blot after immune precipitation (IP) f ER or HER3 expressing HEK 293 cells with anti HER3 blotted against ER (4A) and pER (4B). 4C shows IP with anti ER blotted gains HER3.
Figure 5: In vivo antitumor efficacy of anti-HER3 -antibody
Mab205.10.2 in combination with pertuzumab in a fragment based HER2 low, ER+, HER3 + breast cancer patient-derived xenograft model (HBCx-19). Fig. 5A: after 61 days (=end of treatment) Fig.5B: after 91 days (with end of treatment on day 61). Fig.5C: ex vivo analyse of MAPK at day 91 and Fig.5D: ex vivo analyse of AKT at day 91. Figure 6: In vivo antitumor efficacy of anti-HER3 -antibody
Mab205.10.2 in combination with pertuzumab and the selective estrogen receptor degrader (SERD) fulvestrant in a fragment based HER2 low, ER+, HER3 + breast cancer patient-derived xenograft model (HBCx-19).
Figure 7: In vivo antitumor efficacy (reduction of lesions) of anti-
HER3-antibody Mab205.10.2 in combination with pertuzumab in pretreated (second (2L) to fourth line (4L) and first line (1L) HER2 low, ER+, HER3 + metastatic breast cancer patients. All patients are HER2 low, ER+, HER3 + except patients 1071, 1072 and 1375 who are estrogen receptor negative (ER-). Results in terms of RECIST criterias (SD = Stable Disease, PR = Partial
Response, CR = Complete Response, PD = Progressive Disease; ORR =Overall Response Rate, DCR Disease Control rate. The best overall RECIST responses and the line of treatment are indicated.
Figure 8: In vivo antitumor efficacy (reduction of lesions) of anti-
HER3-antibody Mab205.10.2 in combination with pertuzumab in only first line (1L) HER2 low, ER+, HER3 + metastatic breast cancer patients. All patients are HER2 low, ER+, HER3 +. Results in terms of RECIST criterias (SD = Stable Disease, PR = Partial Response, CR = Complete Response, PD = Progressive Disease; ORR =Overall Response Rate, DCR Disease Control rate. The best overall RECIST responses and the line of treatment are indicated.
Detailed Description of the Invention
The invention relates to an antibody which binds to human HER3 for use in the treatment of, or the manufacture of a medicament for the treatment of, or a method of treatment of,
breast cancer in combination with an antibody binding to human HER2 and inhibiting dimerization of HER2, wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive. In one embodiment the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive.
The invention also relates to an antibody binding to human HER2 and inhibiting dimerization of HER2 for use in
the treatment of, or the manufacture of a medicament for the treatment of, or a method of treatment of,
breast cancer in combination with an antibody which binds to human HER3, wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive. In one embodiment the breast cancer is HER2 low, HER3 positive and ER (estrogen receptor) positive .
The invention comprises an antibody which binds to human HER3, characterized in that the heavy chain variable domain comprises a CDR3H region of SEQ ID
NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6 or a CDR1L region of SEQ ID NO: 7 for use in the combination therapies described herein.
The invention further comprises an antibody which binds to human HER3 according to the invention characterized in that the heavy chain variable domain VH is SEQ ID NO:8; and the light chain variable domain VL is SEQ ID NO:9, or the light chain variable domain VL is SEQ ID NO: 10, or the light chain variable domain VL is SEQ ID NO: 11; or a humanized version thereof for use in the combination therapies described herein.
The invention further comprises an antibody which binds to human HER3 according to the invention characterized in that the heavy chain variable domain VH is SEQ ID NO:8; and the light chain variable domain VL is SEQ ID NO:9, or the light chain variable domain VL is SEQ ID NO: 10, or the light chain variable domain VL is SEQ ID NO: 11 for use in the combination therapies described herein.
In one embodiment the antibody which binds to human HER3 according to the invention is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDRIH region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO: 6 for use in the combination therapies described herein. In one embodiment the antibody which binds to human HER3 according to the invention is characterized in that the heavy chain variable domain VH is SEQ ID NO: 8; and the light chain variable domain VL is SEQ ID NO: 9 or the light chain variable domain VL is SEQ ID NO: 11 for use in the combination therapies described herein. In one embodiment the antibody which binds to human HER3 according to the invention is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDRIH region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO: 7 for use in the combination therapies described herein.
In one embodiment the antibody which binds to human HER3 according to the invention is characterized in that the heavy chain variable domain VH is SEQ ID NO: 8; and the light chain variable domain VL is SEQ ID NO: 10 for use in the combination therapies described herein. In one embodiment such anti-HER3 antibody is monoclonal. In one embodiment such antibody is humanized or human. In one embodiment such anti-HER3 antibody is of IgGl or IgG4 subclass. In one embodiment such anti-HER3 antibody is a monoclonal humanized antibody of IgGl subclass. In one embodiment such anti-HER3 antibody is of IgGl subclass and is characterized in that said antibody is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65 % or lower (in one embodiment, the amount of fucose is between 65 % an 5 %).
The invention comprises the humanized anti-HER3 antibodies Mab 205.10.1, Mab 205.10.2 and Mab 205.10.3 with their respective VH and VL or CDRs for use in the combination therapies described herein.
Figure imgf000011_0001
Figure imgf000011_0002
In one embodiment such anti-HER3 antibodies comprise constant regions of human origin e.g. SEQ ID NO: 12-16, preferably of SEQ ID NO: 12-13.
The invention comprises an antibody which binds to human HER2, characterized in comprising the heavy chain variable domain VH (SEQ ID NO: 20) and the light chain variable domain VL (SEQ ID NO:21) of pertuzumab for use in the combination therapies described herein. In one embodiment the antibody which binds to human HER2 for use in the combination therapies described herein is pertuzumab.
The term "antibody" encompasses the various forms of antibody structures including, but not being limited to, whole antibodies and antibody fragments. The antibody according to the invention is preferably a human antibody, humanized antibody, chimeric antibody, or further genetically engineered antibody as long as the characteristic properties according to the invention are retained. "Antibody fragments" comprise a portion of a full length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof. Examples of antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are, e.g., described in Huston, J.S., Methods in Enzymol. 203 (1991) 46-88. In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VR domain, namely being able to assemble together with a VL domain, or of a VL domain binding to the respective antigen being able to assemble together with a VR domain to a functional antigen binding site and thereby providing the properties of an antibody according to the invention.
The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.
The term "chimeric antibody" refers to a monoclonal antibody comprising a variable region, i.e., binding region, from mouse and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a mouse variable region and a human constant region are especially preferred. Such rat/human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding rat immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of "chimeric antibodies" encompassed by the present invention are those in which the class or subclass has been modified or changed from that of the original antibody. Such "chimeric" antibodies are also referred to as "class-switched antibodies." Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See, e.g., Morrison, S.L., et al, Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.
The term "humanized antibody" or "humanized version of an antibody" refers to antibodies in which the framework or "complementarity determining regions"
(CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. In a preferred embodiment, the CDRs of the VH and VL are grafted into the framework region of human antibody to prepare the "humanized antibody." See e.g. Riechmann, L., et al, Nature 332 (1988) 323-327; and Neuberger, M.S., et al, Nature 314 (1985) 268-270. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies. Human heavy and light chain variable framework regions are listed e.g. in Lefranc, M.-P., Current Protocols in Immunology (2000) - Appendix IP A.1P.1-A.1P.37 and are accessible via IMGT, the international ImMunoGeneTics information system® (http://imgt.cines.fr) or via http://vbase.mrc-cpe.cam.ac.uk. Optionally the framework region can be modified by further mutations. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies. Preferably such humanized version is chimerized with a human constant region (see e.g. Sequences SEQ ID NO: 12-16). The term "humanized antibody" as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to Clq binding and/or FcR binding, e.g. by "class switching" i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or
IgGl/IgG4 mutation).
The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001)
368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al, Nature 362 (1993) 255-258; Brueggemann, M.D., et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al, J. Mol. Biol. 222 (1991) 581- 597). The techniques of Cole, A., et al. and Boerner, P., et al. are also available for the preparation of human monoclonal antibodies (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, A.L., p. 77 (1985); and Boerner, P., et al, J. Immunol. 147 (1991) 86-95). As already mentioned for and humanized antibodies according to the invention the term "human antibody" as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to Clq binding and/or FcR binding, e.g. by "class switching" i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation).
As used herein, the terms "which binds to human HER3", "which specifically binds to human HER3", or " anti-HER3 antibody" are interchangeable and refer to an antibody which specifically binds to the human HER3 antigen with a binding affinity of KD-value of 1.0 x 10"8 mol/1 or lower at 25°C, in one embodiment of a KD-value of 1.0 xlO"9 mol/1 or lower at 25°C. The binding affinity is determined with a standard binding assay at 25°C, such as surface plasmon resonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden). A method for determining the KD-value of the binding affinity is described in Example 2b). Thus an "antibody which binds to human HER3" as used herein refers to an antibody specifically which binds to the human HER3 antigen with a binding affinity of KD 1.0 x 10"8 mol/1 or lower (in one embodiment of KD 1.0 x 10"8 mol/1 - 1.0 x 10"13 mol/1) at 25°C.
As used herein, the terms "binding to human HER2", "specifically binding to human HER2", "which binds to human HER2", "which specifically binds to human HER2", or " anti-HER2 antibody" are interchangeable and refer to an antibody which specifically binds to the human HER2 antigen with a binding affinity of KD- value of 1.0 x 10"8 mol/1 or lower at 25°C, in one embodiment of a KD-value of 1.0 xlO"9 mol/1 or lower at 25°C. The binding affinity is determined with a standard binding assay at 25°C, such as surface plasmon resonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden). A method for determining the KD-value of the binding affinity is described in Example 2b). Thus an "antibody which binds to human HER2" as used herein refers to an antibody specifically which binds to the human HER2 antigen with a binding affinity of KD 1.0 x 10"8 mol/1 or lower (in one embodiment of KD 1.0 x 10"8 mol/1 - 1.0 x 10"13 mol/1) at 25°C.
The pairing of HER receptors on the cell surface is referred to as "dimerization". Besides its homodimerization, HER2 dimerizes with the other members of the HER family, including HER1, HER3, and HER4; HER2:HER3 dimerization is believed to produce the strongest mitogenic signaling and activate 2 key pathways that regulate cell survival and growth (Mitogen-activated protein kinase (MAPK) pathway and Phosphoinositide 3-kinase (PI3K) pathway). As used herein, the term "an antibody which binding to human HER2 and inhibiting dimerization of HER2" refer to an anti-HER2 antibody which specifically binds to the human HER2 antigen and which inhibits/blocks ligand-dependent HER2 homodimerization and HER2 heterodimerization with HER1, HER3, and HER4, and especially inhibits HER2/HER3 dimerization (see e.g. Perjeta® Prescribing Information. Genentech, Inc. June 2012. Baselga J, et al; N Engl J Med. 2012;366: 109-119; Baselga J, et al Nat Rev Cancer. 2009;9:463-475; Hynes NE, et al Nat Rev Cancer. 2005;5:341-
354; Yarden Y, et al, Nat Rev Mol Cell Biol. 2001;2: 127-137; Hsieh AC, et al, Br J Cancer. 2007;97:453-457; Soltoff SP, et al, Mol Cell Biol. 1994;14:3550-3558). Examples of such anti-HER2 antibodies which inhibit HER2 dimerization are described e.g. in WO 01/00245 and WO 2006/007398 wherein pertuzumab (referred to as rhuMAb 2C4 or pertuzumab) is described as one example.
Human HER3 (ErbB-3, ERBB3, c-erbB-3,c-erbB3, receptor tyrosine-protein kinase erbB-3, SEQ ID NO: 17 including signal peptide) encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases which also includes FIERI (also known as EGFR), HER2, and HER4 (Kraus, M.H. et al, PNAS 86 (1989), 9193-9197; Plowman, G.D. et al, PNAS 87 (1990), 4905-4909;
Kraus, M.H. et al, PNAS 90 (1993), 2900-2904). Like the prototypical epidermal growth factor receptor, the transmembrane receptor HER3 consists of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD) and a C-terminal phosphorylation domain. This membrane-bound protein has HER3 a Heregulin (HRG) binding domain within the extracellular domain but not an active kinase domain. It therefore can bind this ligand but not convey the signal into the cell through protein phosphorylation. However, it does form heterodimers with other HER family members which do have kinase activity. Heterodimerization leads to the activation of the receptor-mediated signaling pathway and transphosphorylation of its intracellular domain. Dimer formation between HER family members expands the signaling potential of HER3 and is a means not only for signal diversification but also signal amplification. For example the HER2/HER3 heterodimer induces one of the most important mitogenic signals via the PI3K and AKT pathway among HER family members (Sliwkowski, M.X., et al, J. Biol. Chem. 269 (1994) 14661-14665; Alimandi, M., et al, Oncogene 10 (1995) 1813-1821; Hellyer, N.J., J. Biol. Chem. 276 (2001) 42153-421561; Singer, E., J. Biol. Chem. 276 (2001) 44266-44274; Schaefer, K.L., Neoplasia 8 (2006) 613-622). Anti-HER3 antibodies Mab205.10.1, Mab205.10.2, and Mab205.10.3 showed a competitive binding with the ligand Heregulin (HRG) to HER3. Expression of HER3 gene and/or expression of its protein have been reported in numerous cancers, including prostate, bladder, and breast tumors. Alternate transcriptional splice variants encoding different isoforms have been characterized. One isoform lacks the intermembrane region and is secreted outside the cell. This form acts to modulate the activity of the membrane-bound form. Additional splice variants have also been reported, but they have not been thoroughly characterized.
The term "human HER2" according to the invention refers to 185-kDa growth factor receptor also referred to as neu and c-erbB-2 (Slamon, et al., Science 235 (1987) 177-182; Swiss-Prot P04626; SEQ ID NO: 18 including signal peptide) whose function is related to neoplastic transformation in human breast cancer cells.
The HER receptor will generally comprise an extracellular domain, which may bind an HER ligand; a lipophilic transmembrane domain, a conserved intracellular tyrosine kinase domain, and a carboxyl-terminal signaling domain harboring several tyrosine residues which can be phosphorylated. The extracellular domain of HER2 comprises four domains, Domain I (amino acid residues from about 1-195),
Domain II (amino acid residues from about 196-320), Domain III (amino acid residues from about 321 488), and Domain IV (amino acid residues from about 489-632) (residue numbering without signal peptide). See Garrett, et al, Mol. Cell. 11 (2003) 495-505, Cho, et al, Nature 421 (2003) 756-760, Franklin, et al, Cancer Cell 5 (2004) 317-328, or Plowman, et al, Proc. Natl. Acad. Sci. 90 (1993) 1746-
1750 and WO 2006/007398.
Pertuzumab (e.g. Perjeta®) is a recombinant humanized anti-HER2 monoclonal antibody used for the treatment of HER2 positive cancers. Pertuzumab binds specifically to the 2C4 epitope, a different epitope on the extracellular domain of HER2 than trastuzumab. Pertuzumab is the first in a new class of HER dimerization inhibitors (HDIs). Through its binding to the HER2 extracellular domain, pertuzumab inhibits dimerization of HER2 (especially ligand-activated heterodimerization with other HER family members), thereby inhibiting downstream signaling pathways and cellular processes associated with tumor growth and progression (Franklin, M.C., et al. Cancer Cell 5 (2004) 317-328 and
Friess, T, et al. Clin Cancer Res 11 (2005) 5300-5309). Pertuzumab is a recombinant humanized version of the murine anti-HER2 antibody 2C4 (referred to as rhuMAb 2C4 or pertuzumab) and it is described together with the respective method of preparation in WO 01/00245 and WO 2006/007398. Herein, "Pertuzumab" and "rhuMAb 2C4" refer to an antibody that binds to the 2C4 epitope and preferably comprising the variable light and variable heavy amino acid sequences disclosed in WO 2006/044908, more particularly the humanized 2C4 version 574 disclosed in Fig. 2 of WO 2006/044908. The term "Pertuzumab" encompasses all corresponding anti-HER2 antibodies that fulfill the requirements necessary for obtaining a marketing authorization as an identical or biosimilar product in a country or territory selected from the group of countries consisting of the USA, Europe and Japan. The "epitope 2C4" is the region in the extracellular domain of HER2 to which the antibody 2C4 binds. In order to screen for antibodies which bind to the 2C4 epitope, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping can be performed to assess whether the antibody binds to the 2C4 epitope of HER2. Epitope 2C4 comprises residues from domain II in the extracellular domain of HER2. 2C4 and Pertuzumab bind to the extracellular domain of HER2 at the junction of domains I, II and III (Franklin et al. Cancer Cell 5:317-328 (2004)).
The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody.
The "variable domain of an antibody according to the invention" (variable domain of a light chain (VL), variable domain of a heavy chain (VR)) as used herein denotes each of the pair of light and heavy chain domains which are involved directly in binding the antibody to the antigen. The variable light and heavy chain domains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions" (or complementary determining regions, CDRs). The framework regions adopt a β-sheet conformation and the CDRs may form loops connecting the β-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site. The antibody's heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention. The term "antigen-binding portion of an antibody" when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding. The antigen-binding portion of an antibody comprises amino acid residues from the "complementary determining regions" or "CDRs". The term "antigen-binding portion" of an antibody of the invention contains six complementarity determining regions (CDRs) which contribute in varying degrees to the affinity of the binding site for antigen. There are three heavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs (CDRLl, CDRL2 and CDRL3). The term "CDRH1" denotes the CDR1 region of the heavy chain variable region calculated according to Kabat. CDRH2, CDRH3, CDRLl,
CDRL2 and CDRL3 mean the respective regions from the heavy (H) or light(L) chain. The extent of CDR and framework regions (FRs) is determined by comparison to a compiled database of amino acid sequences in which those regions have been defined according to variability among the sequences according to Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health
Service, National Institutes of Health, Bethesda, Md. (1991).
The "Fc part" of an antibody is not involved directly in binding of an antibody to an antigen, but exhibit various effector functions. A "Fc part of an antibody" is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgGl, IgG2, IgG3, and IgG4, IgAl, and IgA2. According to the heavy chain constant regions the different classes of immunoglobulins are called a, δ, ε, γ, and μ, respectively. The Fc part of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement- dependent cytotoxicity) based on complement activation, Clq binding and Fc receptor binding. The term "complement-dependent cytotoxicity (CDC)" denotes a process initiated by binding of complement factor Clq to the Fc part of most IgG antibody subclasses. Binding of Clq to an antibody is caused by defined protein- protein interactions at the so called binding site. Such binding sites are known in the state of the art and described e.g. by Boackle, R.J., et al, Nature 282 (1979) 742-743, Lukas, T.J., et al, J. Immunol. 127 (1981) 2555-2560, Brunhouse, R., and Cebra, J.J., Mol. Immunol. 16 (1979) 907-917, Burton, D.R., et al, Nature 288 (1980) 338-344, Thommesen, J.E., et al, Mol. Immunol. 37 (2000) 995-1004,
Idusogie, E.E., et al, J. Immunol.164 (2000) 4178-4184, Hezareh, M., et al, J. Virology 75 (2001) 12161-12168, Morgan, A., et al, Immunology 86 (1995) 319- 324, EP O 307 434. Such binding sites are e.g. L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to EU index of Kabat, E.A., see below). Antibodies of subclass IgGl, IgG2 and IgG3 usually show complement activation and Clq and C3 binding, whereas IgG4 do not activate the complement system and do not bind Clq and C3.
In one embodiment the antibody according to the invention comprises a Fc part derived from human origin and preferably all other parts of the human constant regions. As used herein the term "Fc part derived from human origin" denotes a Fc part which is either a Fc part of a human antibody of the subclass IgGl, IgG2, IgG3 or IgG4, e.g. a Fc part from human IgGl subclass, a mutated Fc part from human IgGl subclass (preferably with a mutation on L234A + L235A), a Fc part from human IgG4 subclass or a mutated Fc part from human IgG4 subclass (preferably with a mutation on S228P). Preferred are the human heavy chain constant regions from human IgGl subclass.
In one embodiment the anti-HER3 according to the invention is of human IgGl subclass. In one embodiment the anti-HER2 according to the invention is of human IgGl subclass. In one embodiment the anti-HER3 and the anti-HER2 antibody according to the invention are both of human IgGl subclass. In one embodiment the anti-HER3 and the anti-HER2 antibody according to the invention are both characterized in that the constant chains are of human origin. Such constant chains are well known in the state of the art and e.g. described by Kabat, E.A., (see e.g. Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218). For example, a useful human heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 13. For example, a useful human light chain constant region comprises an amino acid sequence of a kappa-light chain constant region of SEQ ID NO: 12.
The term "amino acid" as used within this application denotes the group of naturally occurring carboxy a-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp,
D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V). The terms "nucleic acid" or "nucleic acid molecule", as used herein, are intended to include DNA molecules and R A molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid. For example, DNA for a presequence or secretory leader is operable linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operable linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operable linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operable linked" means that the DNA sequences being linked are colinear, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. As used herein, the expressions "cell", "cell line", and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived there from without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.
The anti-HER3 antibody described herein is preferably characterized in that the constant chains are of human origin. Such constant chains are well known in the state of the art and described, e.g., by Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). For example, a useful human light chain constant region comprises an amino acid sequence of a kappa-light chain constant region of SEQ ID NO: 12. For example, useful human heavy chain constant region comprises SEQ ID NO: 13 to 16.
In another aspect, an anti-HER3 antibody for the respective combination therapy is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 8 and SEQ ID NO: 10, respectively,; and having one or more of the following properties (determined in assays as described in Example 3 and 2): the anti-HER3 antibody inhibits the HER3 phosphorylation in tumor cells such as MCF7 cells, FaDu cells or Mel-Juso cell (in one embodiment the anti-HER3 antibody shows an inhibition of the HER3 phosphorylation in MCF7 cells of at least 80% (in one embodiment at least 90%) at a concentration of 1.0 μ§/ι 1 ; in one embodiment the anti-HER3 antibody shows an inhibition of the HER3 phosphorylation in FaDu cells of at least 80% (in one embodiment at least 90%) at a concentration of 0.1 μ§/ι 1 ; in one embodiment the anti-HER3 antibody shows an inhibition of the HER3 phosphorylation in Mel-Juso cells of at least 60% (in one embodiment at least 70%) at a concentration of 0.1 μ§/ι 1) the anti-HER3 antibody inhibits the AKT phosphorylation in tumor cells such as Mel-Juso cell (in one embodiment the anti-HER3 antibody inhibits the AKT phosphorylation in Mel-Juso cells with an IC50 value of less than 0.50 μ^ιηΐ, in one embodiment with IC50 value of less than 0.35 μ§/ηι1) - the anti-HER3 antibody inhibits the proliferation of tumor cells such as
MDA-MB-175 cells (in one embodiment the anti-HER3 antibody inhibits the proliferation of MDA-MB-175 cells with an IC50 value of less than 10 μg/ml) the anti-HER3 antibody binds to HER3 with a KD value of less than 5.0x lO~9 M, in one embodiment with a KD value of less than 3. Ox 10~9 M.
In another aspect, an anti-HER3 antibody for the respective combination therapy is a bispecific anti-HER3/anti-HERl antibody as described in US 2010/0255010. In one embodiment, the bispecific anti-HER3/anti-HERl antibody is characterized comprising by the characteristic amino acid sequences disclosed in US 2010/0255010, i.e. A) (a) HVR-Hl comprising the amino acid sequence of
LSGDWIH; (b) HVR-H2 comprising the amino acid sequence of VGEISAAGGYTD; and (c) HVR-H3 comprising the amino acid sequence of ARESRVSFEAAMDY; and (d) HVR-Ll comprising the amino acid sequence of NIATDVA; (e) HVR-L2 comprising the amino acid sequence of SASF; and (f) HVR-L3 comprising the amino acid sequence of SEPEPYT, or B) (a) a heavy chain variable domain with the amino acid sequence of SEQ ID NO: 30 as disclosed in US2010/0255010; (b) a light chain variable domain with the amino acid sequence of SEQ ID NO: 29 as disclosed in US2010/0255010. The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to lysis of human target cells by an antibody according to the invention in the presence of effector cells. ADCC is measured preferably by the treatment of a preparation of HER3 expressing cells with an antibody according to the invention in the presence of effector cells such as freshly isolated PBMC or purified effector cells from buffy coats, like monocytes or natural killer (NK) cells or a permanently growing NK cell line.
Cell-mediated effector functions like ADCC of monoclonal antibodies can be enhanced by engineering their oligosaccharide component as described in Umana, P., et al, Nature Biotechnol. 17 (1999) 176-180, and US 6,602,684. IgGl type antibodies, the most commonly used therapeutic antibodies, are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain. The two complex biantennary oligosaccharides attached to Asn297 are buried between the CH2 domains, forming extensive contacts with the polypeptide backbone, and their presence is essential for the antibody to mediate effector functions such as antibody dependent cellular cytotoxicity (ADCC) (Lifely, M.R., et al, Glycobiology 5 (1995) 813-822; Jefferis, R., et al, Immunol. Rev. 163 (1998) 59- 76; Wright, A., and Morrison, S.L., Trends Biotechnol. 15 (1997) 26-32). Umana, P., et al, Nature Biotechnol. 17 (1999) 176-180 and WO 99/54342 showed that overexpression in Chinese hamster ovary (CHO) cells of B(l,4)-N- acetylglucosaminyltransferase III ("GnTIII"), a glycosyltransferase catalyzing the formation of bisected oligosaccharides, significantly increases the in vitro ADCC activity of antibodies. Alterations in the composition of the Asn297 carbohydrate or its elimination affect also binding to FcyR and Clq (Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180; Davies, J., et al, Biotechnol. Bioeng. 74 (2001)
288-294; Mimura, Y., et al, J. Biol. Chem. 276 (2001) 45539-45547; Radaev, S., et al, J. Biol. Chem. 276 (2001) 16478-16483; Shields, R.L., et al, J. Biol. Chem. 276 (2001) 6591-6604; Shields, R.L., et al, J. Biol. Chem. 277 (2002) 26733- 26740; Simmons, L.C., et al, J. Immunol. Methods 263 (2002) 133-147). Methods to enhance cell-mediated effector functions of monoclonal antibodies via glycoengineering are reported e.g. in WO 2005/044859, WO 2004/065540, WO2007/031875, Umana, P., et al, Nature Biotechnol. 17 (1999) 176-180, WO 99/154342, WO 2005/018572, WO 2006/116260, WO 2006/1 14700, WO 2004/065540, WO 2005/011735, WO 2005/027966, WO 1997/028267, US 2006/0134709, US 2005/0054048, US 2005/0152894, WO 2003/035835 and WO 2000/061739 or e.g. in Niwa, R., et al, J. Immunol. Methods 306 (2005) 151- 160; Shinkawa, T., et al, J Biol Chem, 278 (2003) 3466-3473; WO 03/055993 and US 2005/0249722.
In one embodiment of the invention, the antibody according to the invention is afucosylated which means the antibody is glycosylated (if it comprises an Fc part of IgGl subclass) with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 80% or lower (Numbering according to Kabat), e.g. between 80% and 1 %. In another embodiment is the amount of fucose within said sugar chain is 65 % or lower, in one embodiment between 5% and 65%, in one embodiment from 0% to 65%, and in one embodiment the amount of fucose within said sugar chain is 0%. Such antibodies are referred to in the following as
"afucosylated antibodies" or "non-fucosylated antibodies". Such afucosylated antibodies show enhanced ADCC whereas other antibody properties remain substantially unaffected.
In a further embodiment the amount of N-glycolylneuraminic acid (NGNA) is 1% or less and/or the amount of N-terminal alpha-l,3-galactose is 1% or less within said sugar chain. The sugar chain show preferably the characteristics of N-linked glycans attached to Asn297 of an antibody recombinantly expressed in a CHO cell.
"Asn297" according to the invention means amino acid asparagine located at about position 297 in the Fc region. Based on minor sequence variations of antibodies, Asn297 can also be located some amino acids (usually not more than +3 amino acids) upstream or downstream of position 297, i.e. between position 294 and 300.
The term "the sugar chains show characteristics of N-linked glycans attached to Asn297 of an antibody recombinantly expressed in a CHO cell" denotes that the sugar chain at Asn297 of the full length parent antibody according to the invention has the same structure and sugar residue sequence except for the fucose residue as those of the same antibody expressed in unmodified CHO cells, e.g. as those reported in WO 2006/103100.
The term "NGNA" as used within this application denotes the sugar residue N-glycolyl-neuraminic acid. Glycosylation of human IgGl occurs at Asn297 as core fucosylated biantennary complex oligosaccharide glycosylation terminated with up to two Gal residues. Human constant heavy chain regions of the IgGl subclass are reported in detail by Kabat, E., A., et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), and by Brueggemann, M., et al, J. Exp. Med. 166 (1987) 1351-1361; Love, T.W., et al, Methods Enzymol. 178 (1989) 515-527. These structures are designated as GO, Gl (a- 1,6- or a- 1,3-), or G2 glycan residues, depending from the amount of terminal Gal residues (Raju, T.S., Bioprocess Int. 1 (2003) 44-53). CHO type glycosylation of antibody Fc parts is e.g. described by Routier, F.H., Glycoconjugate J. 14 (1997) 201-207. Antibodies which are recombinantly expressed in non-glycomodified CHO host cells usually are fucosylated at Asn297 in an amount of at least 85%. The modified oligosaccharides of the full length parent antibody may be hybrid or complex. Preferably the bisected, reduced/not- fucosylated oligosaccharides are hybrid. In another embodiment, the bisected, reduced/not-fucosylated oligosaccharides are complex.
According to the invention "amount of fucose" means the amount of said sugar within the sugar chain at Asn297, related to the sum of all glycostructures attached to Asn297 (e.g. complex, hybrid and high mannose structures) measured by
MALDI-TOF mass spectrometry (e.g. in LC/MS system) and calculated as average value (see e.g WO 2008/077546). The relative amount of fucose is the percentage of fucose-containing structures related to all glycostructures identified in an N-Glycosidase F treated sample (e.g. complex, hybrid and oligo- and high- mannose structures, resp.) by MALDI-TOF.
The antibodies according to the invention are preferably produced by recombinant means. Such methods are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable purity. For the protein expression nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells, such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, yeast, or E. coli cells, and the antibody is recovered from the cells (from the supernatant or after cells lysis). Recombinant production of antibodies is well-known in the state of the art and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al, Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-161; Werner, R.G., Drug Res. 48 (1998) 870-880. The antibodies may be present in whole cells, in a cell lysate, or in a partially purified, or substantially pure form. Purification is performed in order to eliminate other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including, column chromatography and others well known in the art (see Ausubel, F., et al, ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)). Expression in NSO cells is described by, e.g., Barnes, L.M., et al, Cytotechnology 32 (2000) 109-123; Barnes, L.M., et al,
Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g., Durocher, Y., et al, Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R., et al, Proc. Natl. Acad. Sci. USA 86 (1989) 3833- 3837; Carter, P., et al, Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; Norderhaug, L., et al, J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J. and Christensen, K., in Cytotechnology 30 (1999) 71-83, and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199. Monoclonal antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures. The hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA may be inserted into expression vectors, which are then transfected into host cells, such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in the host cells. Antibodies obtainable from said cell lines are preferred embodiments of the invention. Afucosylated antibodies are preferably prepared via glycoengineering as described above.
The heavy and light chain variable domains according to the invention are combined with sequences of promoter, translation initiation, constant region, 3' untranslated region, polyadenylation, and transcription termination to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-trans fected, serially transfected, or separately transfected into host cells which are then fused to form a single host cell expressing both chains.
In one aspect of the invention the antibodies of the combination are administered as a pharmaceutical composition comprising the respective antibody. In another aspect, the present invention provides a composition, e.g. a pharmaceutical composition, containing an antibody according to the present invention, formulated together with a pharmaceutical carrier.
As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
The term "breast cancer" as used herein including any metastatic or refractory versions.
Another aspect of the invention is an anti-HER3 -antibody according to the invention for the treatment of cancer in combination with an antibody which binds to human HER2 and which inhibits dimerization of HER2, wherein the cancer is a HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) breast cancer. Another aspect of the invention is the use of an antibody which binds to human HER3 for the manufacture of a medicament for the treatment of breast cancer in combination with an antibody which binds to human HER2 and which inhibits dimerization of HER2, wherein the cancer is a is HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) cancer.
Another aspect of the invention is a method of treatment of a patient suffering from breast cancer by administering an anti-HER3 -antibody antibody according to the invention to said patient in the need of such treatment in combination with an antibody which binds to human HER2 and which inhibits dimerization of HER2, wherein the cancer is a HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) cancer.
In one embodiment, a) the anti-HER3 antibody used in this combination is characterized in comprising as VH an amino acid sequence of SEQ ID NO: 8 and an as VL an amino acid sequence of SEQ ID NO: 10, b) the anti-HER2 antibody used in this combination is pertuzumab, and c) the cancer is HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) breast cancer.
In the context of the combination therapy of an anti-HER3 antibody with an anti- HER2 antibody, wherein the anti-HER2 antibodies inhibits HER2 dimerization, the term "HER3 positive", "HER3+" or "HER3 expression" refers to a breast cancer or tumorous tissue which comprises cells which are characterized by HER3 protein expression and/or gene amplification. This means that the respective breast cancer samples show a detectable level of the HER3 protein expression and/or gene amplification. The expression level of HER3 may be detected by an immunohistochemical method, whereas said HER3 gene amplification status can be measured with in situ hybridization methods, like fluorescence in situ hybridization techniques (FISH). Corresponding assays and kits are well known in the art, for protein expression assays as well as for the detection of gene amplifications. The expression level of HER3 can, inter alia, be detected by an immunohistochemical method. Such methods are well known in the art (see e.g. analogous methods and test for HER2 expression levels below or e.g. WO 2015/049355). Other methods like qRT-PCR might be used to detect levels of HER3 gene amplification. In one preferred embodiment the term "HER3 positive", "HER3+" or "HER3 expression" refers to the HER3 protein expression detected by an immunohistochemical method, which means the respective breast cancer samples show a detectable level of the HER3 protein in an corresponding immunohistochemistry (IHC) assay.
In the context of the combination therapy of an anti-HER3 antibody with an anti- HER2 antibody, wherein the anti-HER2 antibodies inhibits HER2 dimerization, the term "HER2 low" cancer also referred to as "HER2 low cancer" as used herein refers to a cancer or tumorous tissue which comprises cells which express low levels of HER2. Thus, HER2 low cancer cells do not overexpress HER2 (as defined for HER2 -positive cancer) and are not negative for HER2 expression. For the purpose of the present invention, "HER2 low cancer" is a cancer with an immunohistochemistry (IHC) score of 2+ and an in situ hybridization (ISH) amplification ratio <2.0 (i.e. is ISH-negative) or an immunohistochemistry (IHC) score of 1+ and an in situ hybridization (ISH) amplification ratio <2.0 (i.e. is ISH- negative) . Accordingly, HER2 low cancer is present if a low (IHC 1+) or moderate (IHC 2+) HER2 (protein) expression level detected e.g. by immunohistochemical methods and no HER2 gene amplification, detected by in-situ-hybridization (ISH negative, like a HER2 gene copy <4 copies of the HER2 gene per tumor cell or ratio of < 2.0 for the number of HER2 gene copies to the number of signals for CEP 17.), is found in samples obtained from the patients such as breast tissue biopsies or breast tissue resections or in tissue derived from metastatic sites. In one embodiment "HER2 low cancer" is defined as an immunohistochemistry (IHC) score of HER2(2+) and ISH negative or immunohistochemistry (IHC) score of HER2(1+) and ISH negative (IHC l+/ISH-negative or IHC 2+/ISH-negative).
The expression level of HER2 may be detected by an immunohistochemical method, whereas said HER2 gene amplification status can be measured with in situ hybridization methods, like fluorescence in situ hybridization techniques (FISH).
Corresponding assays and kits are well known in the art, for protein expression assays as well as for the detection of gene amplifications. Alternatively other methods like qRT-PCR may be used to detect levels of HER2 gene expression.
The expression level of HER2 can, inter alia, be detected by an immunohistochemical method. Such methods are well known in the art and corresponding commercial kits are available. Exemplary kits which may be used in accordance with the present invention are, inter alia, HerceptTest™ produced and distributed by the company Dako or the test called Ventana Pathway™. The level of HER2 protein expression may be assessed by using the reagents provided with and following the protocol of the HercepTest™. A skilled person will be aware of further means and methods for determining the expression level of HER2 by immunohistochemical methods; see for example WO 2005/117553. Therefore, the expression level of HER2 can be easily and reproducibly determined by a person skilled in the art without undue burden. However, to ensure accurate and reproducible results, the testing must be performed in a specialized laboratory, which can ensure validation of the testing procedures.
The expression level of HER2 can be classified in a low expression level, an intermediate expression level and a high expression level. In context of this invention it is preferred that HER2 low disease is defined by a low or weak expression level of HER2 (e.g. HER2(1+ or 2+) by IHC) and a negative ISH result, for example determined in a sample of a cancer patient. Therefore parallel testing using immunohistochemistry and in situ hybridisation is preferred.
The recommended scoring system to evaluate the IHC staining patterns in breast cancer which reflect the expression levels of HER2 designated herein HER2(0), HER2(+), HER2(++) and HER2(+++), is as follows:
The below IHC staining patterns are recommended for determining HER2 status in breast cancer (see Dako HercepTestTM package insert).
Figure imgf000029_0001
The above IHC staining patterns are routinely used in determining HER2 status in breast cancer. The terms HER2(+), HER2(++) and HER2(+++) used herein are equivalent to the terms HER2(1+), HER2(2+) and HER2(3+). A "low HER2 protein expression level" used in context of this invention corresponds to a 1+ score ("negative assessment" according to the table shown herein above), and a 2+ score "weakly positive". As described herein above in detail, the evaluation of the protein expression level (i.e. the scoring system as shown in the table) is based on results obtained by immunohistochemical methods. As a standard or routinely, the HER2 status is, accordingly, performed by immunohistochemistry with one of two FDA-approved commercial kits available; namely the Dako Herceptest™ and the Ventana Pathway™. These are semi-quantitative assays which stratify expression levels into 0 (<20,000 receptors per cell, no expression visible by IHC staining), 1+ (-100,000 receptors per cell, partial membrane staining, < 10% of cells overexpressing HER2), 2+ (-500,000 receptors per cell, light to moderate complete membrane staining, > 10%> of cells overexpressing HER2), and 3+ (-2,000,000 receptors per cell, strong complete membrane staining, > 10% of cells overexpressing HER2).
Alternatively, further methods for the evaluation of the protein expression level of HER2 may be used, e.g. Western Blots, ELISA-based detection systems and so on.
The below IHC staining patterns are recommended for determining HER2 status in gastric cancer (see Dako Herceptest package insert):
Staining Surgical specimen Biopsy specimen - HER2
Intensity staining pattern staining pattern Overexpressio
Score n Assessment
No reactivity or no No reactivity or no
0 membranous reactivity in membranous reactivity in Negative
< 10%) of tumour cells any tumour cell
Faint / barely perceptible Tumour cell cluster (> 5
membranous reactivity cells) with a faint / barely
in > 10% of tumour perceptible membranous
1+ Negative
cells; cells are reactive reactivity irrespective of
only in part of their percentage of tumour cells
membrane stained
Weak to moderate Tumour cell cluster (> 5
complete, basolateral or cells) with a weak to
lateral membranous moderate complete,
2+ reactivity in > 10% of basolateral or lateral Equivocal tumour cells membranous reactivity
irrespective of percentage
of tumour cells stained Staining Surgical specimen Biopsy specimen - HER2
Intensity staining pattern staining pattern Overexpressio
Score n Assessment
Strong complete, Tumour cell cluster (> 5
basolateral or lateral cells) with a strong
membranous reactivity in complete, basolateral or
3+ > 10% of tumour cells lateral membranous Positive
reactivity irrespective of
percentage of tumour cells
stained
HER2 low disease is defined by a low or weak expression level of HER2 (e.g. HER2(1+ or 2+) by IHC) and a negative ISH result.
In contrast HER2 positive disease is defined by a high expression level of HER2 as HER2 (3+) by IHC or HER2 (2+) by IHC and positive ISH result. And HER2 negative disease is defined as HER2 (0) by IHC and a negative ISH result.
In accordance with the above, the sample to be assessed can be (obtained) from a patient with HER2 low cancer as defined above. For example, the sample may be obtained from a tumorous tissue, (a) tumor(s) and, accordingly, is (a) tumor cell(s) or (a) tumor tissue(s) suspected of being HER2 expressing tumour, like a breast tumor. A person skilled in the art is in the position to identify such tumors and/or individuals/patients suffering from corresponding cancer using standard techniques known in the art and methods disclosed herein. Generally, said tumor cell or cancer cell may be obtained from any biological source/organism, particularly any biological source/organism, suffering from the above-mentioned cancer. In context of this invention particular useful cells are, preferably, human cells. These cells can be obtained from e.g. biopsies or from biological samples. The tumor/cancer/tumor cell/cancer cell is a solid tumor/ cancer/tumor cell/cancer cell. In accordance with the above, the cancer/tumor cell may be a breast cancer/tumor cell or said sample comprises a cancer/tumor cell, such as a breast cancer/tumor cell. In line with the above, said tumor/cancer may be a breast tumor/cancer.
In the context of the combination therapy of an anti-HER3 antibody with anti- HER2 antibody, wherein the anti-HER2 antibody inhibits HER2 dimerization, the term "Hormone receptor (HR) positive or HR+" as used herein refers to Estrogen Receptor (ER) positive and/or Progesterone Receptor (PgR)-positive cancer/tumouros tissue, such as Breast Cancer. Breast Cancer are considered positive for ER or PgR if finding of > 1% of tumor cell nuclei are immunoreactive, as determined by Immunohistochemistry (IHC). Breast Cancer are considered negative for ER or PgR if it is detected that < 1% of tumor cell nuclei are immunoreactive in the presence of evidence that the sample can express ER or PgR (positive intrinsic controls can be detected), as determined by IHC. Breast Cancer are considered uninterpretable for ER or PgR if it is detected that no tumor nuclei are immunoreactive and that internal epithelial elements present in the sample or separately submitted from the same sample lack any nuclear staining, as determined by IHC. In one preferred embodiment in the combination therapy of an HER3 antibody with an anti-HER2 antibody, wherein the anti-HER2 antibody inhibits HER2 dimerization, the breast cancer is estrogen receptor (ER) positive (ER+).
The term "Estrogen receptor (ER) positive or ER+" according to the invention refers to breast cancer which is considered positive for ER if finding of > 1% of tumor cell nuclei are immunoreactive, as determined by Immunohistochemistry
(IHC). Breast cancer is considered negative for ER if finding of < 1% of tumor cell nuclei are immunoreactive in the presence of evidence that the sample can express ER (positive intrinsic controls are seen), as determined by IHC. Breast cancer is considered uninterpretable for ER if finding that no tumor nuclei are immunoreactive and that internal epithelial elements present in the sample or separately submitted from the same sample lack any nuclear staining, as determined by IHC.
Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier preferably is an isotonic buffered saline solution.
Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.
The term "treating" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, the growth of tumors, tumor metastases, or other cancer-causing or neoplastic cells in a patient. The term "treatment" as used herein, unless otherwise indicated, refers to the act of treating.
The phrase "a method of treating" or its equivalent, when applied to, for example, cancer refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of a cancer. "A method of treating" cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will, in fact, be eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will, in fact, be alleviated. Often, a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of a patient, is nevertheless deemed an overall beneficial course of action.
It is self-evident that the antibodies are administered to the patient in therapeutically effective amount which is the amount of the subject compound or combination that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "in combination with" refers to the "co-administration" or "co-administering" of the anti-HER3 antibody which is administered additionally to the anti-HER2 antibody in either order. The "co-administration" means that the first antibody is administered additionally to the second antibody either simultaneously or sequentially. The co-administration can be simultaneous or sequential in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. When both antibodies are administered simultaneously the dose is administered on the same day in one administration, e.g. during one continuous infusion. When both antibodies are administered sequentially the dose is administered either on the same day in two separate administrations, e.g. two separate continuous infusions, or one of the antibodies is administered on day 1 and the second antibody is administered on day 2 to day 7, preferably on day 2 to 4. The terms "co-administration" or "coadministering" with respect to the maintenance doses of the first antibody and the second antibody mean that the maintenance doses can be either administered simultaneously, e.g. during one continuous infusion, if the treatment cycle is appropriate for both antibodies. Or the maintenance doses are administered sequentially, either within one or several days, e.g. the maintenance dose of the first antibody is administered every 3 weeks, and the maintenance dose of the second is administered every 2 weeks. Also other treatment cycles /usually from 1 to 4 weeks, preferably from 2 to 3 weeks, may be used for both antibodies.
The amount of antibody co-administration and the timing of administration may depend on the type (species, gender, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated. Usually typical dosages antibodies are used.
For example, the dosages for administration of the anti-HER3 antibody according to the invention can be about 400 to 600 mg of antibody by one or more separate administrations, or by continuous infusion, typically in a 2 to 4 weeks cycle, and especially in a 3 weeks cycle. In a preferred aspect, the anti-HER3 antibody is administered every two to three weeks, at a dose ranged from about 400 to 600 mg, in one preferred embodiment at a dose of 500 mg every 3 weeks administered as continuous infusion until disease progression is detected. It was detected that by reducing the dose of the anti-HER3 antibody according to the invention from 1000 mg every 3 weeks to 500 mg every 3 weeks the side effect of severe diarrhea could be significantly reduced, while maintaining the effectiveness of the antibody combination with the anti-HER2 antibody according to the invention (which was dosed at 420 mg every 3 weeks)
For example, the dosages for administration of the anti-HER2 antibody according to the invention can be about 300 to 500 mg of antibody by one or more separate administrations, or by continuous infusion, typically in a 2 to 4 weeks cycle, and especially in a 3 weeks cycle. In a preferred aspect, the anti-HER2 antibody is administered every two to three weeks, at a dose ranged from about 300 to 500 mg, in on preferred embodiment at a dose of 420 mg every 3 weeks administered as continuous infusion until disease progression is detected.
In the context of this invention, additional chemotherapeutic agents may be used in combination treatment of the present invention. Such agents include, for example: a) a taxane or taxane derivative like paclitaxel (e.g. Taxol®) and
paclitaxel derivatives (like nab-paclitaxel (protein-bound paclitaxel) (e.g. Abraxane®) or docosahexaenoic acid (DHA) -paclitaxel, (e.g. Taxoprexin®)), or docetaxel (e.g. Taxotere®), b) cyclophosphamide (CTX) (e.g. Cytoxan®), c) cisplatin (e.g. Platinol®) or carboplatin (e.g. Paraplatin® and
Paraplatin-AQ®), d) mitomycin C (e.g. Mutamycin®), e) methotrexate (MTX) (e.g. Trexall®), f) 5-fluorouracil (5-FU) (e.g. Adrucil®) or capecitabine (e.g.
Xeloda®), g) doxorubicin (DXR) (e.g. Adriamycin®) or its liposomal formulation (doxorubicin lipo) (e.g. Doxil®), or daunorubicin (e.g. Cerubidine®) or its liposomal formulation (daunorubicin lipo) (e.g. Daunoxome®), h) gemcitabine (e.g. Gemzar®), i) mitoxantrone (e.g. Novantrone ®), j) vinorelbine (e.g. Navelbine®), k) eribulin (e.g. Halaven®), and
1) ixabepilone (e.g. Ixempra ®).
In the context of this invention, additional an anti-hormonal agents may be used in combination treatment of the present invention. Such agents include, for example: a) a selective estrogen receptor degrader (SERD) like e.g. fulvestrant, or b) a selective estrogen receptor modulators (SERM), such as tamoxifen, raloxifene, toremifene or lasofoxifene, or c) an aromatase inhibitor like e.g. letrozole, anastrozole or exemestane).
The use of the chemotherapeutic or anti-hormonal agents described above in chemotherapeutic/ anti-hormonal regimens is generally well characterized in the cancer therapy arts, and their use herein falls under the same considerations for monitoring tolerance and effectiveness and for controlling administration routes and dosages, with some adjustments. For example in breast cancer disease with metastases in organs like the liver or lung, a high tumor burden and/or relapse after anti-hormonal therapy (typically anti-estrogen treatment), or rapidly progressing disease, a chemotherapeutic agent might be co-administered with the HER3/HER2 antibody combination described herein. In breast cancer disease with only metastases in bone ( and not in organs) and/or lower tumor burden anti-hormonal therapy (typically an anti-estrogen treatment), or slowly progressing disease, an anti-hormonal agent might be co-administered with the HER3/HER2 antibody combination described herein. The actual dosages of the agents may vary depending upon the patient's cultured cell response determined by using histoculture methods. Generally, the dosage will be reduced compared to the amount used in the absence of additional other agents. The "co-administration" or "co -administering" means that the chemotherapeutic or anti-hormonal agent is administered additionally to the antibody combination either simultaneously or sequentially. The co-administration can be simultaneous or sequential in either order, wherein preferably there is a time period while all active agents simultaneously exert their biological activities. The following is a list of commonly used chemotherapy for breast cancer:
CMF: cyclophosphamide, methotrexate, and 5-fluorouracil given 4-weekly for 6 cycles FAC (or CAF): 5-fluorouracil, doxorubicin, cyclophosphamide given 3 -weekly for 6 cycles
AC (or CA): Adriamycin (doxorubicin) and cyclophosphamide given 3 -weekly for 4 cycles AC-Taxol: AC given 3 -weekly for 4 cycles followed by paclitaxel given either 3- weekly for 4 cycles or weekly (at a smaller dose) for 12 weeks
TAC: Taxotere (docetaxel), Adriamycin (doxorubicin), and cyclophosphamide given 3 -weekly for 4-6 cycles
FEC: 5-fluorouracil, epirubicin and cyclophosphamide given 3 -weekly for 6 cycles FECD: FEC given 3 -weekly for 3 cycles followed by docetaxel given 3 -weekly for
3 cycles
TC: Taxotere (docetaxel) and cyclophosphamide given 3 -weekly for 4 or 6 cycles
The following is a list of commonly used anti-hormonal therapy for breast cancer:
Fulvestrant, exemestane alone or in combination with everolimus, anastrozole, letrozole, tamoxifene. Recently the palbociclib has been approved in combination with letrozole and fulvestrant.
Typical dosages of an effective agent can be in the ranges recommended by the manufacturer, and where indicated by in vitro responses or responses in animal models, can be reduced by up to about one order of magnitude concentration or amount. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based on the in vitro responsiveness of the primary cultured malignant cells or histocultured tissue sample, or the responses observed in the appropriate animal models. In the context of this invention, an effective amount of ionizing radiation may be carried out and/or a radiopharmaceutical may be used in addition to combination treatment of the present invention. The source of radiation can be either external or internal to the patient being treated. When the source is external to the patient, the therapy is known as external beam radiation therapy (EBRT). When the source of radiation is internal to the patient, the treatment is called brachytherapy (BT). Radioactive atoms for use in the context of this invention can be selected from the group including, but not limited to, radium, cesium- 137, iridium- 192, americium- 241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131, and indium- 111. In one embodiment the combination treatment of the present invention is used without such additional ionizing radiation.
Radiation therapy is a standard treatment for controlling unresectable or inoperable tumors and/or tumor metastases. Improved results have been seen when radiation therapy has been combined with chemotherapy. Radiation therapy is based on the principle that high-dose radiation delivered to a target area will result in the death of reproductive cells in both tumor and normal tissues. The radiation dosage regimen is generally defined in terms of radiation absorbed dose (Gy), time and fractionation, and must be carefully defined by the oncologist. The amount of radiation a patient receives will depend on various considerations, but the two most important are the location of the tumor in relation to other critical structures or organs of the body, and the extent to which the tumor has spread. A typical course of treatment for a patient undergoing radiation therapy will be a treatment schedule over a 1 to 6 week period, with a total dose of between 10 and 80 Gy administered to the patient in a single daily fraction of about 1.8 to 2.0 Gy, 5 days a week. In a preferred embodiment of this invention there is synergy when tumors in human patients are treated with the combination treatment of the invention and radiation.
In other words, the inhibition of tumor growth by means of the agents comprising the combination or single therapy of the invention is enhanced when combined with radiation, optionally with additional chemotherapeutic or anticancer agents. Parameters of adjuvant radiation therapies are, for example, contained in International Patent Publication WO 99/60023.
The antibodies are administered to a patient according to known methods, by intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, or intrathecal routes. Intravenous or subcutaneous administration of the antibodies is preferred.
The term "treating" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, the growth of tumors, tumor metastases, or other cancer-causing or neoplastic cells in a patient. The term "treatment" as used herein, unless otherwise indicated, refers to the act of treating. The phrase "a method of treating" or its equivalent, when applied to, for example, cancer refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of a cancer. "A method of treating" cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will, in fact, be eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will, in fact, be alleviated. Often, a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of a patient, is nevertheless deemed an overall beneficial course of action.
It is self-evident that the antibodies are administered to the patient in therapeutically effective amount which is the amount of the subject compound or combination that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
The term "first-line treatment" as used herein refers to the first type of drug therapy given for the treatment of cancer or metastasis. This can be an adjuvant or neoadjuvant or palliative chemotherapy or immunotherapy offered initially following diagnosis and/or surgery or following diagnosis of metastatic disease. The first-line treatment refers to the first cancer treatment using e.g. chemical or biochemical substances, like cytotoxic drugs, targeted therapies with antibodies, or enzyme inhibitors/antagonist/or modulators.
The present invention further provides an article of manufacture comprising a container, a composition within the container comprising an anti-HER3 antibody and a package insert instructing the user of the composition to administer said anti-
HER3 antibody to a patient suffering from HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) breast cancer in combination with an anti-HER2 antibody which inhibits the dimerization of HER2.
The present invention further provides an article of manufacture comprising a container, a composition within the container comprising an anti-HER3 antibody and a package insert instructing the user of the composition to administer said anti- HER3 antibody to a patient suffering from HER2 low, HER3 positive and HR (hormone receptor) positive (e.g. ER positive) breast cancer in combination with an anti-HER2 antibody which inhibits the dimerization of HER2. The term "package insert" refers to instructions customarily included in commercial packages of therapeutic products, which may include information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
In one embodiment, the article of manufacture containers may further include a pharmaceutically acceptable carrier. The article of manufacture may further include a sterile diluent, which is preferably stored in a separate additional container.
In the following one series of embodiments of the invention is listed:
1. An antibody which binds to human HER3 for use in the treatment of breast cancer in combination with an antibody binding to human HER2 and inhibiting dimerization of HER2 , wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive.
2. The antibody according to embodiment 1, wherein the breast cancer is HER2 low HER3, and positive ER (estrogen receptor) positive .
3. The antibody according to any one of embodiments 1 to 2, wherein
additionally either
A) an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), (in one embodiment the estrogen receptor inhibitor is fulvestrant), b) a selective estrogen receptor modulator (SERM), (in one embodiment the selective estrogen receptor modulator is tamoxifen, raloxifene, toremifene or lasofoxifene), and c) an aromatase inhibitor; (in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane);
or
B) a chemotherapeutic agent is co-administered, wherein the
chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
The antibody according to any one of embodiments 1 to 2, wherein additionally an anti-hormonal agent is co-administered, and wherein the anti- hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), b) a selective estrogen receptor modulator (SERM); and c) an aromatase inhibitor.
The antibody according to embodiment 4, wherein the anti-hormonal agent is selected from the group of: a) fulvestrant, b) tamoxifen, raloxifene, toremifene or lasofoxifene; and c) letrozole, anastrozole or exemestane. The antibody according to embodiment 4, wherein the anti-hormonal agent is fulvestrant. The antibody according to any one of embodiments 1 to 2, wherein additionally a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone. The antibody according embodiment 7, wherein
the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel, docetaxel ( in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel). The antibody according to any one of embodiments 1 to 8, wherein the combination is administered as a first-line treatment. The antibody according to any one of embodiments 1 to 9, wherein the antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7. The antibody according to any one of embodiments 1 to 9, wherein the antibody which binds to human HER3 is characterized in that the heavy chain variable domain VH is SEQ ID NO: 8; and the light chain variable domain VL is SEQ ID NO: 10. The antibody according to any one of embodiments 1 to 11, wherein the antibody which binds to human HER3 is of human IgGl subclass is characterized in that is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65 % or lower. The antibody according to any one of embodiments 1 to 12, wherein the antibody binding to human HER2 and inhibiting dimerization of HER2 is pertuzumab. The antibody according to any one of embodiments 1 to 13, wherein the antibody which binds to human HER3 is administered at a dose of 400-600 mg every 3 weeks and the antibody which binds to human HER2 is administered at a dose of 300-500 mg every 3 weeks. following another series of embodiments of the invention is listed: An antibody binding to human HER2 and inhibiting dimerization of HER2 for use in the treatment of breast cancer in combination with an antibody which binds to human HER3, wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive. The antibody according to embodiment 1, wherein the breast cancer is HER2 low HER3, and positive ER (estrogen receptor) positive . The antibody according to any one of embodiments 1 to 2, wherein additionally either
A) an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), (in one embodiment the selective estrogen receptor degrader (SERD) is fulvestrant), b) a selective estrogen receptor modulator (SERM), (in one embodiment the selective estrogen receptor modulator is tamoxifen, raloxifene, toremifene or lasofoxifene), and c) an aromatase inhibitor; (in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane);
or
B) a chemotherapeutic agent is co-administered, wherein the
chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone. The antibody according to any one of embodiments 1 to 2, wherein additionally an anti-hormonal agent is co-administered, and wherein the anti- hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), b) a selective estrogen receptor modulator (SERM); and c) an aromatase inhibitor. The antibody according to embodiment 4, wherein the anti-hormonal agent is selected from the group of: a) fulvestrant, b) tamoxifen, raloxifene, toremifene or lasofoxifene; and c) letrozole, anastrozole or exemestane. The antibody according to embodiment 4, wherein the anti-hormonal agent is fulvestrant. The antibody according to any one of embodiments 1 to 2, wherein additionally a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
8. The antibody according embodiment 7, wherein
the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel, docetaxel ( in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel).
9. The antibody according to any one of embodiments 1 to 8, wherein the
combination is administered as a first-line treatment. 10. The antibody according to any one of embodiments 1 to 9, wherein the
antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7.
11. The antibody according to any one of embodiments 1 to 9, wherein the
antibody which binds to human HER3 is characterized in that the heavy chain variable domain VH is SEQ ID NO: 8; and the light chain variable domain VL is SEQ ID NO: 10. 12. The antibody according to any one of embodiments 1 to 11, wherein the antibody which binds to human HER3 is of human IgGl subclass and is characterized in that the antibody is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65 % or lower. The antibody according to any one of embodiments 1 to 12, wherein the antibody binding to human HER2 and inhibiting dimerization of HER2 is pertuzumab. The antibody according to any one of embodiments 1 to 13, wherein the antibody which binds to human HER3 is administered at a dose of 400-600 mg every 3 weeks and the antibody which binds to human HER2 is administered at a dose of 300-500 mg every 3 weeks. following another series of embodiments of the invention is listed: An antibody which binds to human HER3 and/ or an antibody binding to human HER2 and inhibiting dimerization of HER2, for use in the manufacture of a medicament for the combination treatment of breast cancer wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive. The use according to embodiment 1, wherein the breast cancer is HER2 low HER3, and positive ER (estrogen receptor) positive . The use according to any one of embodiments 1 to 2, wherein
additionally either
A) an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), (in one embodiment the selective estrogen receptor degrader (SERD) is fulvestrant), b) a selective estrogen receptor modulator (SERM), (in one
embodiment the selective estrogen receptor modulator is tamoxifen, raloxifene, toremifene or lasofoxifene), and c) an aromatase inhibitor; (in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane);
or B) a chemotherapeutic agent is co-administered, wherein the
chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone. The use according to any one of embodiments 1 to 2, wherein
additionally an anti-hormonal agent is co-administered, and wherein the anti- hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), b) a selective estrogen receptor modulator (SERM); and
c) an aromatase inhibitor. The use according to embodiment 4, wherein the anti-hormonal agent is selected from the group of: a) fulvestrant, b) tamoxifen, raloxifene, toremifene or lasofoxifene; and
c) letrozole, anastrozole or exemestane. The use according to embodiment 4, wherein the anti-hormonal agent is fulvestrant. The use according to any one of embodiments 1 to 2, wherein
additionally a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and
1) ixabepilone. The use according embodiment 7, wherein
the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel, docetaxel ( in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel). The use according to any one of embodiments 1 to 8, wherein the
combination is administered as a first-line treatment. The use according to any one of embodiments 1 to 9, wherein the antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7. The use according to any one of embodiments 1 to 9, wherein the antibody which binds to human HER3 is characterized in that the heavy chain variable domain VH is SEQ ID NO: 8; and the light chain variable domain VL is SEQ ID NO: 10. The use according to any one of embodiments 1 to 11, wherein the antibody which binds to human HER3 is of human IgGl subclass and is characterized in that the antibody is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65 % or lower. The use according to any one of embodiments 1 to 12, wherein the antibody binding to human HER2 and inhibiting dimerization of HER2 is pertuzumab. The use according to any one of embodiments 1 to 13, wherein the antibody which binds to human HER3 is administered at a dose of 400-600 mg every 3 weeks and the antibody which binds to human HER2 is administered at a dose of 300-500 mg every 3 weeks. following another series of embodiments of the invention is listed: The use of antibody which binds to human HER3 and/ or an antibody binding to human HER2 and inhibiting dimerization of HER2, for the manufacture of a medicament for the combination treatment of breast cancer wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive.
The use according to embodiment 1 , wherein the breast cancer is HER2 low HER3, and positive ER (estrogen receptor) positive .
The use according to any one of embodiments 1 to 2, wherein
additionally either
A) an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), (in one embodiment the selective estrogen receptor degrader (SERD) is fulvestrant), b) a selective estrogen receptor modulator (SERM), (in one embodiment the selective estrogen receptor modulator is tamoxifen, raloxifene, toremifene or lasofoxifene), and c) an aromatase inhibitor; (in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane);
or
B) a chemotherapeutic agent is co-administered, wherein the
chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone. The use according to any one of embodiments 1 to 2, wherein
additionally an anti-hormonal agent is co-administered, and wherein the anti- hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), b) a selective estrogen receptor modulator (SERM); and
c) an aromatase inhibitor. The use according to embodiment 4, wherein the anti-hormonal agent is selected from the group of: a) fulvestrant, b) tamoxifen, raloxifene, toremifene or lasofoxifene; and
c) letrozole, anastrozole or exemestane. The use according to embodiment 4, wherein the anti-hormonal agent is fulvestrant. The use according to any one of embodiments 1 to 2, wherein
additionally a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and
1) ixabepilone. 8. The use according embodiment 7, wherein
the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel, docetaxel ( in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel). 9. The use according to any one of embodiments 1 to 8, wherein the
combination is administered as a first-line treatment.
10. The use according to any one of embodiments 1 to 9, wherein the antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7.
11. The use according to any one of embodiments 1 to 9, wherein the antibody which binds to human HER3 is characterized in that the heavy chain variable domain VH is SEQ ID NO: 8; and the light chain variable domain VL is SEQ ID NO: 10.
12. The use according to any one of embodiments 1 to 11, wherein the antibody which binds to human HER3 is of human IgGl subclass and is characterized in that the antibody is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65 % or lower. The use according to any one of embodiments 1 to 12, wherein the antibody binding to human HER2 and inhibiting dimerization of HER2 is pertuzumab. The use according to any one of embodiments 1 to 13, wherein the antibody which binds to human HER3 is administered at a dose of 400-600 mg every 3 weeks and the antibody which binds to human HER2 is administered at a dose of 300-500 mg every 3 weeks. following another series of embodiments of the invention is listed: The use of antibody which binds to human HER3 for the manufacture of a medicament for the treatment of breast cancer wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive, and wherein the treatment is in combination with an antibody binding to human HER2 and inhibiting dimerization of HER2. The use according to embodiment 1, wherein the breast cancer is HER2 low HER3, and positive ER (estrogen receptor) positive . The use according to any one of embodiments 1 to 2, wherein
additionally either
A) an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), (in one embodiment the selective estrogen receptor degrader (SERD) is fulvestrant), b) a selective estrogen receptor modulator (SERM), (in one
embodiment the selective estrogen receptor modulator is tamoxifen, raloxifene, toremifene or lasofoxifene), and c) an aromatase inhibitor; (in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane);
or B) a chemotherapeutic agent is co-administered, wherein the
chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
The use according to any one of embodiments 1 to 2, wherein
additionally an anti-hormonal agent is co-administered, and wherein the anti- hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), b) a selective estrogen receptor modulator (SERM); and
c) an aromatase inhibitor.
The use according to embodiment 4, wherein the anti-hormonal agent is selected from the group of: a) fulvestrant, b) tamoxifen, raloxifene, toremifene or lasofoxifene; and
c) letrozole, anastrozole or exemestane.
The use according to embodiment 4, wherein the anti-hormonal agent is fulvestrant.
The use according to any one of embodiments 1 to 2, wherein
additionally a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
The use according embodiment 7, wherein
the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel, docetaxel ( in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel). The use according to any one of embodiments 1 to 8, wherein the
combination is administered as a first-line treatment. The use according to any one of embodiments 1 to 9, wherein the antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7. The use according to any one of embodiments 1 to 9, wherein the antibody which binds to human HER3 is characterized in that the heavy chain variable domain VH is SEQ ID NO: 8; and the light chain variable domain VL is SEQ ID NO: 10. The use according to any one of embodiments 1 to 11, wherein the antibody which binds to human HER3 is of human IgGl subclass and is characterized in that the antibody is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65 % or lower. The use according to any one of embodiments 1 to 12, wherein the antibody binding to human HER2 and inhibiting dimerization of HER2 is pertuzumab. The use according to any one of embodiments 1 to 13, wherein the antibody which binds to human HER3 is administered at a dose of 400-600 mg every 3 weeks and the antibody which binds to human HER2 is administered at a dose of 300-500 mg every 3 weeks. following another series of embodiments of the invention is listed: The use of an antibody binding to human HER2 and inhibiting dimerization of HER2, for the manufacture of a medicament for the treatment of breast cancer wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive and wherein the treatment is in combination with an antibody which binds to human HER3. The use according to embodiment 1, wherein the breast cancer is HER2 low HER3, and positive ER (estrogen receptor) positive . The use according to any one of embodiments 1 to 2, wherein
additionally either
A) an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), (in one embodiment the selective estrogen receptor degrader (SERD) is fulvestrant), b) a selective estrogen receptor modulator (SERM), (in one embodiment the selective estrogen receptor modulator is tamoxifen, raloxifene, toremifene or lasofoxifene), and c) an aromatase inhibitor; (in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane);
or
B) a chemotherapeutic agent is co-administered, wherein the
chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone. The use according to any one of embodiments 1 to 2, wherein
additionally an anti-hormonal agent is co-administered, and wherein the anti- hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), b) a selective estrogen receptor modulator (SERM); and
c) an aromatase inhibitor. The use according to embodiment 4, wherein the anti-hormonal agent is selected from the group of: a) fulvestrant, b) tamoxifen, raloxifene, toremifene or lasofoxifene; and
c) letrozole, anastrozole or exemestane. The use according to embodiment 4, wherein the anti-hormonal agent is fulvestrant. The use according to any one of embodiments 1 to 2, wherein
additionally a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone. The use according embodiment 7, wherein
the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel, docetaxel ( in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel). The use according to any one of embodiments 1 to 8, wherein the
combination is administered as a first-line treatment. The use according to any one of embodiments 1 to 9, wherein the antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7. The use according to any one of embodiments 1 to 9, wherein the antibody which binds to human HER3 is characterized in that the heavy chain variable domain VH is SEQ ID NO: 8; and the light chain variable domain VL is SEQ ID NO: 10. The use according to any one of embodiments 1 to 11, wherein the antibody which binds to human HER3 is of human IgGl subclass and is characterized in that the antibody is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65 % or lower. The use according to any one of embodiments 1 to 12, wherein the antibody binding to human HER2 and inhibiting dimerization of HER2 is pertuzumab. The use according to any one of embodiments 1 to 13, wherein the antibody which binds to human HER3 is administered at a dose of 400-600 mg every 3 weeks and the antibody which binds to human HER2 is administered at a dose of 300-500 mg every 3 weeks. following another series of embodiments of the invention is listed: A method of treating a patient suffering from of breast cancer wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive, the method comprising the administration of (an effective amount of ) an antibody which binds to human HER3 and the co-administration of (an effective amount of) an antibody binding to human HER2 and inhibiting dimerization of HER2 . The method according to embodiment 1, wherein the breast cancer is HER2 low HER3, and positive ER (estrogen receptor) positive . The method according to any one of embodiments 1 to 2, wherein
additionally either
A) an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), (in one embodiment the selective estrogen receptor degrader (SERD) is fulvestrant), b) a selective estrogen receptor modulator (SERM), (in one
embodiment the selective estrogen receptor modulator is tamoxifen, raloxifene, toremifene or lasofoxifene), and c) an aromatase inhibitor; (in embodiment the aromatase inhibitor is letrozole, anastrozole or exemestane);
or B) a chemotherapeutic agent is co-administered, wherein the
chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
The method according to any one of embodiments 1 to 2, wherein additionally an anti-hormonal agent is co-administered, and wherein the anti- hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), b) a selective estrogen receptor modulator (SERM); and
c) an aromatase inhibitor.
The method according to embodiment 4, wherein the anti-hormonal agent is selected from the group of: a) fulvestrant, b) tamoxifen, raloxifene, toremifene or lasofoxifene; and
c) letrozole, anastrozole or exemestane.
The method according to embodiment 4, wherein the anti-hormonal agent is fulvestrant.
The method according to any one of embodiments 1 to 2, wherein
additionally a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative (like paclitaxel and paclitaxel derivatives, or docetaxel), b) cyclophosphamide (CTX), c) cisplatin or carboplatin, d) mitomycin C, e) methotrexate (MTX), f) 5-fluorouracil (5-FU) or capecitabine, g) doxorubicin (DXR) or its liposomal formulation (doxorubicin lipo), or daunorubicin or its liposomal formulation (daunorubicin lipo), h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
The method according embodiment 7, wherein
the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel, docetaxel ( in one embodiment the chemotherapeutic agent is paclitaxel, nab-paclitaxel, in one embodiment the chemotherapeutic agent is paclitaxel). 9. The method according to any one of embodiments 1 to 8, wherein the combination is administered as a first-line treatment.
10. The method according to any one of embodiments 1 to 9, wherein the
antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7.
11. The method according to any one of embodiments 1 to 9, wherein the
antibody which binds to human HER3 is characterized in that the heavy chain variable domain VH is SEQ ID NO: 8; and the light chain variable domain VL is SEQ ID NO: 10.
12. The method according to any one of embodiments 1 to 11, wherein the
antibody which binds to human HER3 is of human IgGl subclass and is characterized in that the antibody is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65 % or lower.
13. The method according to any one of embodiments 1 to 12, wherein the
antibody binding to human HER2 and inhibiting dimerization of HER2 is pertuzumab.
14. The method according to any one of embodiments 1 to 13, wherein the
antibody which binds to human HER3 is administered at a dose of 400-600 mg every 3 weeks and the antibody which binds to human HER2 is administered at a dose of 300-500 mg every 3 weeks.
The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.
Description of the Sequence Listing
SEQ ID NO: 1 heavy chain CDR3H, Mab 205.10
SEQ ID NO: 2 heavy chain CDR2H, Mab 205.10 SEQ ID NO 3 heavy chain CDR1H, Mab 205.10
SEQ ID NO 4 light chain CDR3L, Mab 205.10
SEQ ID NO 5 light chain CDR2L, Mab 205.10
SEQ ID NO 6 light chain CDR1L (variant 1), Mab 205.10
SEQ ID NO 7 light chain CDR1L (variant 2), Mab 205.10
SEQ ID NO 8 heavy chain variable domain VH, Mab 205.10
SEQ ID NO 9 light chain variable domain VL, Mab 205.10.1
SEQ ID NO 10 light chain variable domain VL, Mab 205.10.2
SEQ ID NO 11 light chain variable domain VL, Mab 205.10.3
SEQ ID NO 12 human kappa light chain constant region
SEQ ID NO 13 human heavy chain constant region derived from IgGl
SEQ ID NO 14 human heavy chain constant region derived from IgGl mutated on L234A and L235A
SEQ ID NO 15 human heavy chain constant region derived from IgG4
SEQ ID NO 16 human heavy chain constant region derived from IgG4 mutated on S228P
SEQ ID NO 17 human HER3 (including signal peptide)
SEQ ID NO 18 human HER2 (including signal peptide)
SEQ ID NO 19 human ER (including signal peptide)
SEQ ID NO 20 heavy chain variable domain VH, pertuzumab
SEQ ID NO 21 light chain variable domain VL, pertuzumab
Examples
Example 1
Immunisation
NMRI mice were immunized with hHER3-ECD (in-house) and boosted with hu-HER3-ECD. The immune response was monitored by testing serum samples against the HER1/2/3- ECD-ELISA. Spleen cells from mice with sufficient titers of anti-HER3 immunoglobulin were frozen for later immortalization by fusion with mouse myeloma cell line P3X63 Ag8.653. One fusion was done and hybridoma supematants screened by HER1/2/-ECD- ELISA showing no cross-reactivity, but binding to HER3-ECD and anti-HER3 selective hybridomas were selected. The relevant hybridomas were cloned by single cell FACS sorting. Single cell clones from different hybridomas were cultured in vitro to produce antibody in tissue culture medium for characterization. Antibodies were selected by determining their ability to inhibit HER3 phosphorylation, AKT phosphorylation and tumor cell proliferation of MDA-MB-175 cells (see Examples below). From the obtained antibodies, one was further humanized to give the following antibodies Mab 205.10.1, Mab 205.10.2 and Mab 205.10.3 with their respective VH and VL or CDRs.
Figure imgf000066_0001
Figure imgf000066_0002
In one embodiment such antibodies were prepared using constant regions of human origin e.g. SEQ ID NO: 12-13.
Example 2
Binding assays a) Antigen specific ELISA for binding to human HER3 ECD
Soluble human HER3 extracellular domain fused to Streptavidin Binding Protein (SBP) was captured on a streptavidine plate. To define optimal binding of the antibody to SPB-CDCP1, 384-well polystyrene plates (NUNC, streptavidin-coated) delivered by MicroCoat, Bernried, Germany (ID-No.1734776-001) have been coated with pure and stepwise diluted HEK293 supernatant (in BSA/IMDM buffer: 100 mg/ml BSA Fraction V, Roche 10735078001, dissolved in Iscove's Modified Dulbeccos Medium). Using mouse a calibration curve of chimeric 205 antibodies the optimal dilution factor of the HEK293 supernatant in relation to the streptavidin binding capacity of the microtiter plate was identified. For the standard coating, SBP-HER3 containing HEK293 supernatant was diluted (between 1 : 15 and 1 : 40) and incubated overnight at 2-80C (25 μΐ per well). Intensive washing of the microtiter plate is necessary to remove remaining unbound SBP-HER3. Antibodies according to the invention antibody were tested either undiluted or using a 12-step-dilution. 12.5μ1 per well of each sample was incubated for 90 min at room temperature. After intensive washing using PBS-T (0.1% Tween 20 in PBS) 25 μΐ goat anti-human IgG antibodies coupled with HRP (Jackson ImmunoResearch, Code No: 109-036-098, dilution 1 : 10000) for human antibodies were added and incubated for 1 hour. After intensive washing the binding of the antibodies was detected with ABTS tablets (Roche Diagnostics GmbH, Cat.No.: 1112422). Absorbance at 405 nm/492 nm was measured using a standard photometer. The table shows the relative binding ratios of the different antibodies.
Figure imgf000067_0001
b) Characterization of the binding of anti-HER3 antibodies to a extracellular- domain-(ECD) fragment of human HER3 by Biacore analyses:
For affinity measurements, 30 μg/ml of anti Fey antibodies (from goat, Jackson Immuno Research) were coupled to the surface of a CM-5 sensor chip by standard amine-coupling and blocking chemistry on a SPR instrument (Biacore T100). After conjugation, anti-HER3 antibodies were injected at 25°C at a flow rate of 5 μί/ηιίη, followed by a dilution series (0 nM to 1000 nM) of human HER3 ECD at 30 μΕ/ιηίη. As running buffer for the binding experiment PBS/0.1% BSA was used. The chip was then regenerated with a 60s pulse of 10 mM glycine-
HC1, pH 2.0 solution.
Calculation of thermodynamic parameters (KD, binding constant to HER3) were calculated using a Langmuir 1 : 1 binding model. Binding Affinity
Antibody
KD [M]
Mab 205.10.1 2.0x 10"9
Mab 205.10.2 l . lxlO"9
Mab 205.10.3 2.0xl0"9
In a competitive binding assay (Biacore) Mab205.10.1, Mab205.10.2, and Mab205.10.3 all showed binding to the same epitope. The anti-HER3 -antibodies Ul-7, U-53 and Ul-59 described in WO 2007/077028 and Ab#6 described in WO 2008/100624 were investigated in such assay and revealed to bind to different epitopes than antibodies Mab205.10.1. Mab205.10.2, and Mab205.10.3.
Example 3
a) Inhibition of HER3 phosphorylation in MCF7, FaDu and Mel-Juso cells
Assays were performed in MCF7 and FaDu cells according to the following protocol: Seed cells with 500,000 cells/well into Poly-D-Lysine coated 6-well plate in RPMI1640 medium with 10% FCS. Incubate for 24h. Remove medium by aspirating, incubate overnight with 500μ1Λνε11 RPMI 1640 with 0.5% FCS. Add antibodies in 500 μΐ RPMI 1640 with 0.5% FCS. Incubate for lh. Add HRG-lb (final concentration 500ng/ml) for 10 min. To lyse the cells remove medium and add 80 μΐ ice cold Triton-X-100 cell lysis buffer and incubate for 5 minutes on ice.
After transferring the lysate into 1.5 ml reaction tube and centrifugation at 14000 rpm for 15 min at 4°C, transfer supernatant into fresh reaction tubes. Samples containing equal amounts of protein in SDS loading buffer were separated on SDS PAGE and blotted by using a semi-dry Western Blot to nitrocellulose membranes. Membranes were blocked by IxNET -buffer + 0.25% gelatine for lh hour and pHER3 is detected by the antibody aPhospho-HER3/ErbB3 (Tyrl289)(21D3), Cell Signaling, #479 land HER3 by the antibody aErbB3 (C-17), Santa Cruz, #sc-285 respectively. After washing und detection of the signals by an POD coupled secondary antibody, bands were densometricaly scanned. The anti-HER3 antibodies Mab205.10.1, Mab205.10.2, and Mab205.10.3 and also anti-HER3 antibodies Ul-7, U-53 and Ul-59 described in WO 2007/077028 and Ab#6 described in WO 2008/100624 were investigated. Percent (%) inhibition of anti- HER3 antibodies on receptor phosphorylation in MCF7 cells is shown below and in Fig 1 A. % Inhibition of HER3 phosphorylation in MCF7 cells
Figure imgf000069_0001
In a further experiment the anti-HER3 antibody Mab205.10.2, and also the anti- HER3-antibodies 8B8.2D9 described in WO 97/35885, and 1B4C3 and 2D1D12 described in WO 2003/013602 were investigated. Percent (%) inhibition of anti-
HER3 antibodies on receptor phosphorylation in MCF7 cells is shown below and in Fig IB.
% Inhibition of HER3 phosphorylation in MCF7 cells
Figure imgf000069_0002
Percent (%) inhibition of anti-HER3 antibodies on receptor phosphorylation FaDu cells is shown below. % Inhibition of HER3 phosphorylation in FaDu cells
Figure imgf000070_0001
In a further experiment, the anti-HER3 antibody Mab205.10.2, and also the anti- HER3-antibodies 8B8.2D9 described in WO 97/35885, and 1B4C3 and 2D1D12 described in WO 2003/013602, and 105.5 from (Millipore, Cat.no. 05-47, named <x-
HERECD in w0 2003/013602) were investigated in Mel- Juso cells. Assays in Mel- Juso cells were performed according to the aforementioned protocol for MCF7 and FaDu cells. Cell numbers and media volumes were adapted to 12-well plates Percent (%) inhibition of anti-HER3 antibodies on receptor phosphorylation in Mel- Juso cells is shown below and in Figure 1C.
% Inhibition of HER3 phosphorylation in Mel- Juso cells
Figure imgf000070_0002
b) AKT phosphorylation (ELISA)
Assays were performed in MCF7 cells according to the following protocol: Seed MCF7 cells with 30000 cells/well into Poly-D-Lysine coated 96-well plate in RPMI 1640 medium with 10% FCS and incubate for 24h. Remove medium by tapping on a clean paper towel, wash carefully with 200μ1 serum-free medium, incubate overnight with ΙΟΟμΙ/well RPMI 1640 with 0.5% FCS. Remove medium as above; add antibodies in ΙΟΟμΙ RPMI 1640 with 0.5% FCS and incubate 1.5h. Add HRG-lb (final concentration 5ng/ml) for 10 min. Remove medium as above.
To lyse the cells add ΙΟΟμΙ ice cold cell lysis buffer on ice and resuspended by pipetting ca.5x. Centrifuge plate at 3000rpm for lOmin at 4°C and transfer 80μ1 supernatant (or aliquots) into fresh polypropylene plate and shock-freeze in LN2. Store at -80°C until assay.
AKTl,2(phospho-Ser473) EIA Kit Assay Designs #900-162:Samples (1 : 10 diluted) are added to the plate coated with a mouse monoclonal antibody specific for the N-terminus of AKT. Incubation lh at RT with shaking. Wash 5x, incubation with biotinylated anti-phospho-AKT(Ser473) lh at RT with shaking. Wash 5x, incubation with streptavidin-HRP conjugate 30min at RT with shaking. Wash 5x, incubate with TMB substrate 30min at RT with shaking. Stop and read at 450nm. Mab 205.10.2 showed an IC50 of the AKT phosphorylation inhibition of
0.06 μg/ml.
In an pAKT ELISA in Mel-Juso cell performed as described for MCF7 cells Mab 205.10.2 showed an IC50 of AKT phosphorylation inhibition of 0.28 μg/ml all the other analyses antibodies show an IC50 above (>) 50.
% AKT phosphorylation inhibition in Mel-Juso cells
Figure imgf000071_0001
c) Inhibition of tumor cell proliferation
The anti-tumor efficacy of HER3 antibodies Mab205.10.1, Mab205.10.2, and Mab205.10.3 in a cell proliferation assay, using MDA-MB-175 cells (VII Human Breast Carcinoma Cells, ATCC catalog no. HTB-25), was assessed. 20,000 cells per well were seeded into sterile 96 well tissue culture plates with DMEM/F12 cell culture medium, containing 10% FCS and incubated at 37°C±1°C with 5 % ±1% C02 for one day. The cells are slow growing cells with a doubling time of ca. 1.5 days. Anti-HER3 antibodies were added in dilution series and further incubated for 6 days. Cell viability was then assessed using the alamarBlue® readout. If the cell viability was reduced to more than 50 % of control, IC50 values were calculated using means of triplicates for each antibody concentration; otherwise, if the % inhibition of cell viability at the highest concentration was below 50%, no IC50 could be calculated and it is indicated that IC50 |^g/ml] is above (>) the highest concentration. Also the anti-HER3 -antibodies Ul-59 described in WO 2007/077028 and Ab#6 described in WO 2008/100624 were investigated.
Figure imgf000072_0001
In a further experiment the anti-HER3 antibodies 8B8.2D9 described WO 97/35885, and 1B4C3 described in WO 2003/013602 were investigated.
Figure imgf000072_0002
Example 4
In vitro ADCC in KPL-4 tumor cells by ^ /ml specific Lysis %
The target cells KPL4 (ADCC), breast carcinoma, cultivation in RPMI1640 + 2 mM L-alanyl-L-Glutamine + 10 % FCS ) were collected with trypsin/EDTA (Gibco # 25300-054) in exponential growth phase. After a washing step and checking cell number and viability, the aliquot needed was labeled for 30 min at
37°C in the cell incubator with calcein (Invitrogen #C3100MP; 1 vial was resuspended in 50 μΐ DMSO for 5 Mio cells in 5 ml medium). Afterwards, the cells were washed three times with AIM-V medium, the cell number and viability was checked and the cell number adjusted to 0.3 Mio/ml. Meanwhile, PBMC (Peripheral Blood Mononuclear Cells) as effector cells were prepared by density gradient centrifugation (Histopaque-1077, Sigma # H8889) according to the manufacturer's protocol (washing steps lx at 400g and 2x at 350g 10 min each). The cell number and viability was checked and the cell number adjusted to 15 Mio/ml.
ΙΟΟμΙ calcein-stained target cells were plated in round-bottom 96-well plates, 50μ1 diluted, afucosylated antibody (Mab205.10.1, Mab205.10.2, Mab205.10.3, preparation see below) which was added and 50μ1 effector cells. In some experiments the target cells were mixed with Redimune® NF Liquid (ZLB Behring) at a concentration of 10 mg/ml Redimune.
As controls served the spontaneous lysis, determined by co-culturing target and effector cells without antibody and the maximal lysis, determined by 1 % Triton X-100 lysis of target cells only. The plate was incubated for 4 hours at 37°C in a humidified cell incubator.
The killing of target cells was assessed by measuring LDH (Lactate Dehydrogenase) release from damaged cells using the Cytotoxicity Detection kit (LDH Detection Kit, Roche # 1 644 793) according to the manufacturer's instruction. Briefly, 100 μΐ supernatant from each well was mixed with 100 μΐ substrate from the kit in a transparent flat bottom 96 well plate. The Vmax values of the substrate's colour reaction was determined in an ELISA reader at 490 nm for at least 10 min. Percentage of specific antibody-mediated killing was calculated as follows: (A - SR)/(MR - SR)xl00, where A is the mean of Vmax at a specific antibody concentration, SR is the mean of Vmax of the spontaneous release and
MR is the mean of Vmax of the maximal release.
As additional readout the calcein retention of intact target cells was assessed by lysing the remaining target cells in borate buffer (5 mM sodium borate + 0.1 % Triton) and measuring the calcein fluorescence in a fluorescence plate reader. Mab205.10.1, Mab205.10.2, Mab205.10.3 showed and ADCC [KPL-4] by ^g/ml of specific Lysis of about 40-60%.
The afucosylated antibody (Mab205.10.1, Mab205.10.2, Mab205.10.3) were prepared by co-transfection with four plasmids, two for antibody expression, one for a fusion GnTIII polypeptide expression (a GnT-III expression vector), and one for mannosidase II expression (a Golgi mannosidase II expression vector) at a ratio of 4:4: 1 : 1, respectively in HEK293 or CHO cells.
The full antibody heavy and light chain DNA sequences were subcloned into mammalian expression vectors (one for the light chain and one for the heavy chain) under the control of the MPSV promoter and upstream of a synthetic polyA site, each vector carrying an EBV OriP sequence. Antibodies were produced by co- transfecting HEK293-EBNA cells or CHO cells with the antibody heavy and light chain expression vectors using a calcium phosphate-transfection approach. Exponentially growing HEK293-EBNA cells were trans fected by the calcium phosphate method. For the production of the glycoengineered antibody, the cells were co-transfected with four plasmids, two for antibody expression, one for a fusion GnTIII polypeptide expression (a GnT-III expression vector), and one for mannosidase II expression (a Golgi mannosidase II expression vector) at a ratio of 4:4: 1 : 1, respectively. Cells were grown as adherent monolayer cultures in T flasks using DMEM culture medium supplemented with 10% FCS, and were transfected when they were between 50 and 80% confluent. For the trans fection of a T150 flask, 15 million cells were seeded 24 hours before transfection in 25 ml DMEM culture medium supplemented with FCS (at 10%> V/V final), and cells were placed at 37°C in an incubator with a 5% C02 atmosphere overnight. For every antibody to be produced, a solution of DNA, CaC12 and water was prepared by mixing 188 μg total plasmid vector DNA (four plasmids, two for antibody expression (one light chain and one heavy chain), one for a fusion GnTIII polypeptide expression (a GnT-III expression vector), and one for mannosidase II expression (a Golgi mannosidase II expression vector) at a ratio of 4:4: 1 :1, respectively), water to a final volume of 938 μΐ and 938 μΐ of a 1M CaC12 solution. To this solution, 1876 μΐ of a 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HP04 solution at pH 7.05 were added, mixed immediately for 10 sec and left to stand at room temperature for 20 sec. The suspension was diluted with 46 ml of DMEM supplemented with 2%> FCS, and divided into two T150 flasks in place of the existing medium.
The cells were incubated at 37°C, 5%> C02 for about 17 to 20 hours, then medium was replaced with 25 ml DMEM, 10%> FCS. The conditioned culture medium was harvested 7 days post-transfection by centrifugation for 15 min at 210 x g, the solution was sterile filtered (0.22 μιη filter) and sodium azide in a final concentration of 0.01 %> w/v was added, and kept at 4°C.
The secreted afucosylated antibodies were purified and the oligosaccharides attached to the Fc region of the antibodies were analysed e.g. by MALDI/TOF-MS (as described in e.g. WO 2008/077546). For this analysis oligosaccharides were enzymatically released from the antibodies by PNGaseF digestion, with the antibodies being either immobilized on a PVDF membrane or in solution. The resulting digest solution containing the released oligosaccharides either prepared directly for MALDI/TOF-MS analysis or was further digested with EndoH glycosidase prior to sample preparation for MALDI/TOF-MS analysis. The analyzed amount of fucose within the sugar chain at Asn297 was between 50-20%. Example 5
HER2/HER3 and ER (estrogen receptor) crosstalk (cross-signaling)
HER2 and ER (estrogen receptor) crosstalk
5xl05 HEK 293 cells in DMEM 10% FCS per 6 well were seeded. 2μg of each cDNA was transfected with a standard Fugene protocol. Control vector, HER2 and ERa expressing cells were generated. After 48h cells were lysed by a Triton XI 00 lyses buffer containing Aprotinin ΙΟμΙ/mL, PMSF ΙΟμΙ/mL and Orthovanadate 2μ1/ιη1. 20μg of total cell lysate was separated on a SDS PAGE and blotted against pERa (Cell Signaling mAb #2511, ERa (Cell Signaling mAb #8644) and HER2 (Calbiochem Ab 3) as indicated. All proteins are expressed at comparable levels. Only in cells expressing ERa and HER2 at the same time, ERa is phosphorylated on serine 118. Results are shown in Figure 2.
HER2 and ER complex
5xl05 HEK 293 cells in DMEM 10% FCS per 6 well were seeded. 2μg of each cDNA was transfected with a standard Fugene protocol. Control vector, HER2 and ERa expressing cells were generated. After 48h cells were lysed by a Triton XI 00 lyses buffer containing Aprotinin ΙΟμΙ/mL, PMSF ΙΟμΙ/mL and Orthovanadat 2μ1/ιη1. Cells were immunoprecipitated with and anti HER2 antibody (trastuzumab) and blotted against ERa (Cell signaling mAb # 8644) or vice versa, immunoprecipitated with an anti ERa antibody (Cell signaling mAb # 8644) and blotted against HER2 (Calbiochem Ab 3). HER2 and ERa can form a complex and in this complex ERa is phosphorylated. Results are shown in Figure 3A,3B and 3C.
HER3 and ER (estrogen receptor) crosstalk
5xl05 HEK 293 cells in DMEM 10% FCS per 6 well were seeded. 2μg of each cDNA was transfected with a standard Fugene protocol. Control vector, HER3 and
ERa expressing cells were generated. Cells were starved overnight in DEMEM 0, 5% FCS. After 48h cells were stimulated with HRG (black bars) for 10 min and lysed by a Triton XI 00 lyses buffer containing Aprotinin ΙΟμΙ/mL, PMSF ΙΟμΙ/mL and Orthovanadate 2μ1/ηι1. Cells were immunoprecipitated with and anti HER3 antibody (Ab) and blotted against ERa antibody (Cell signaling mAb # 8644) or vice versa, immunoprecipitated with an anti ERa antibody (Cell signaling mAb # 8644) and blotted against HER3 (Ab208 Roche). HER3 and ERa can form a complex in the presence and absence of HRG, but ER is strongly phosphorylated only in the presence of HRG. Results are shown in Figure 4A,4B and 4C.
In summary: We could demonstrate this ER cross talk for HER2 and HER3, however no crosstalk could be detected for HER1 (data not shown). Several complementary findings support a model of HER2/HER3 and ER cross talk
- HER3 pathway is activated under long lasting tamoxifen therapy.
- HER3 and HER2 can form a complex with the estrogen receptor.
- HRG activated HER2/HER3 heterodimers mediate phosphorylation of estrogen receptor. - HRG is an ER regulated gene.
ER signaling in breast cancer induces HRG expression which in turn drives mitogenic signaling via HER2/HER3 heterodimers. HER2 can activate estrogen receptor
Example 6
In vivo antitumor efficacy of anti-HER3 therapy in combination with pertuzumab
The in vivo antitumor efficacy of the antibody Mab205.10.2 in combination with pertuzumab was detected in a fragment based HER2 low, ER+, HER3 + breast cancer patient-derived xenograft model (PDX) transplanted on nude mice. As example data is shown for the HBCx-19 PDX xenograft model.
Test agents
Mab205.10.2 and pertuzumab were provided as stock solution from Roche, Penzberg, Germany. Antibody buffer included histidine. Antibody solution was diluted appropriately in buffer from stock prior injections. Tumor fragments
HBCx-19 breast cancer tumor fragments were originally obtained from a metastasis of a lobular carcinoma. Tumors of the same passage were transplanted subcutaneously onto 6-24 donor mice. When these tumors reached 1000 to 2000 mm3, donor mice were sacrificed and tumors excised, dissected and cut into fragments measuring of approximately 20 mm3. Thereafter fragments were grafted subcutaneous.
Animals
Female athymic nude mice, 6- to 9-week-old at the beginning of the experimental phase, were obtained from Harlan Laboratories (France). Animals were maintained in specific pathogen-free animal housing at the Center for Exploration and Experimental Functional Research (CERFE, Evry, France) animal facility. Animals were delivered to the laboratory 7 days before the experiments during which time they were acclimatized to laboratory conditions. Mice were housed inside individually ventilated cages (IVC) under a light-dark cycle (14-hour circadian cycle of artificial light) and controlled room temperature and humidity.
Monitoring
Animals were controlled daily for clinical symptoms and detection of adverse effects. For monitoring throughout the experiment body weight of animals was documented.
Treatment of animals
Animal treatment started at randomisation (D26) when median tumor size was about 100- 150mm3. Antibody Mab205.10.2 was administered as single agent and in combination with pertuzumab at 10 mg/kg i.p. q7d once weekly until D61. Pertuzumab was administered as single agent and in combination once weekly at
15 mg/kg with a 2-fold loading dose first. The corresponding vehicle was administered on the same days. Antibody efficacy
HBCx-19 breast cancer xenograft model
HBCx-19 breast cancer xenograft bearing mice were treated with antibody Mab205.10.2 as single agent and in combination with pertuzumab from study D26 to D61. As a result, treatment with Mab205.10.2 antibody as single agent showed strong significant anti-tumor efficacy with tumors stasis until D61 and a Relative Tumor Volume (RTV) of 100%. Monotherapy with pertuzumab translated into partial HBCx-19 tumor regression at 61 >. Notably, superior efficacy was observed with Mab205.10.2 in combination with pertuzumab resulting in strongest tumor regression (RTV 10%).
Antibody treatment discontinued at D61. In the follow-up tumor regrowth of HBCx-19 xenografts was observed in both monotherapy groups with Mab205.10.2 or pertuzumab (RTV 728%) and 131%, respectively), however, in contrast to this combination of Mab205.10.2 with pertuzumab maintained long lasting tumor remission (RTV 10%>). Results are shown in Figure 5.
Example 7
In vivo antitumor efficacy of anti-HER3 therapy in combination with pertuzumab and anti-hormone therapy
The in vivo antitumor efficacy of the antibody Mab205.10.2 in combination with pertuzumab and selective estrogen receptor degrader (SERD) fulvestrant was detected in a fragment based HER2 low, ER+, HER3+ breast cancer patient- derived xenograft model (PDX) transplanted on nude mice. As example data is shown for the HBCx-19 PDX xenograft model.
Test agents Mab205.10.2 and pertuzumab were provided as stock solution from Roche,
Penzberg, Germany. Antibody buffer included histidine. Antibody solution was diluted appropriately in buffer from stock prior injections. Fulvestrant (Faslodex, Astra Zeneca) was purchased commercially.
Tumor fragments HBCx-19 breast cancer tumor fragments were originally obtained from a metastasis of a lobular carcinoma. Tumors of the same passage were transplanted subcutaneously onto 6-24 donor mice. When these tumors reached 1000 to 2000 mm3, donor mice were sacrificed and tumors excised, dissected and cut into fragments measuring of approximately 20 mm3. Thereafter fragments were grafted subcutaneous. Animals
Female athymic nude mice, 6- to 9-week-old at the beginning of the experimental phase, were obtained from Harlan Laboratories (France). Animals were maintained in specific pathogen-free animal housing at the Center for Exploration and Experimental Functional Research (CERFE, Evry, France) animal facility. Animals were delivered to the laboratory 7 days before the experiments during which time they were acclimatized to laboratory conditions. Mice were housed inside individually ventilated cages (IVC) under a light-dark cycle (14-hour circadian cycle of artificial light) and controlled room temperature and humidity.
Monitoring Animals were controlled daily for clinical symptoms and detection of adverse effects. For monitoring throughout the experiment body weight of animals was documented.
Treatment of animals
Animal treatment started at randomisation (D22) when median tumor size was about 100-150mm3. Antibody Mab205.10.2 was administered at 3 mg/kg as single agent once weekly (6x) as single agent and in dual combination with pertuzumab (3 mg/kg i.p. q7dx6) or in triple combination with fulvestrant (50 mg/kg, im, q7dx6) until D57. Pertuzumab was administered as single agent and in combination once weekly at 3 mg/kg and fulvestrant given im at 50 mg/kg once weekly. The corresponding vehicle was administered on the same days.
Antibody efficacy
HBCx-19 breast cancer xenograft model
HBCx-19 HER2 low, ER+, HER3+ breast cancer xenograft bearing mice were treated with antibody Mab205.10.2 as single agent and in combination with pertuzumab and anti-estrogen fulvestrant from study D22 to D57. The triple combination (Mab205.10.2 + pertuzumab + fulvestrant) was the most efficacious combination with tumor control of HBCx-19 BC xenografts (RTV 118%). The monotherapies and dual combinations were less efficacious and HBCx-19 tumors progressed. Results are shown in Figure 6.
Example 8
In vivo antitumor efficacy of anti-HER3 therapy ( glycoengineered
Mab205.10.2) in combination with pertuzumab and chemotherapy (paclitaxel) ( clinical trial data)
Glycoengineered Mab205.10.2 was investigated in combination with pertuzumab and paclitaxel in patients with MBC expressing HER3 and HER2 protein. Anti-HER3 antibody Mab205.10.2 was administered at a dose of 500 mg on a q3w schedule (i.e., on day 1 of 3-weekly cycles). Pertuzumab was administered at a loading dose of 840 mg during cycle 1 followed by 420 mg for the remaining cycles in a q3w schedule. Paclitaxel was be administered at 80 mg/m2 in a qw schedule. In another setting at a dose of Mab205.10.2 of 1000 mg q3w diarrhea as side effect could be detected. By a reduced dose of Mab205.10.2 of 500 mg q3w in comparison with 1000 mg q3w schedule diarrhea e.g. could be significantly reduced. Results in terms of RECIST criterias (SD = Stable Disease, PR = Partial Response, CR = Complete Response, PD = Progressive Disease; ORR =Overall Response Rate, DCR Disease Control rate, are shown in Figures 7 and 8. Figure 7: In vivo antitumor efficacy (reduction of lesions) anti-HER3 -antibody
Mab205.10.2 in combination with pertuzumab in pretreated (second (2L) to fourth line (4L) and first line (1L) HER2 low, ER+, HER3 + metastatic breast cancer patients. All patients are HER2 low, ER+, HER3 + except patients 1071, 1072 and 1375 who are estrogen receptor negative ( ER-). Results in terms of RECIST criterias (SD = Stable Disease, PR = Partial Response, CR = Complete
Response, PD = Progressive Disease; ORR =Overall Response Rate, DCR Disease Control rate. The best overall RECIST responses and the line of treatment are indicated.
Figure 8: In vivo antitumor efficacy (reduction of lesions) anti-HER3 -antibody Mab205.10.2 in combination with pertuzumab in only first line (1L) HER2 low,
ER+, HER3 + metastatic breast cancer patients. All patients are HER2 low, ER+, HER3 + Results in terms of RECIST criterias (SD = Stable Disease, PR = Partial Response, CR = Complete Response, PD = Progressive Disease; ORR =Overall Response Rate, DCR Disease Control rate. The best overall RECIST responses and the line of treatment are indicated.
In another setting Mab205.10.2 was administered at a dose of 500 mg on a q3w schedule (i.e., on day 1 of 3-weekly cycles). Pertuzumab was administered at a dose of 420 mg in a q3w schedule. Paclitaxel was be administered at 80 mg/m2 in a qw schedule (data not shown). By a reduced dose of Mab205.10.2 of 500 mg q3w in comparison with 1000 mg q3w schedule side effects like diarrhea could be significantly reduced.

Claims

Patent Claims
An antibody which binds to human HER3 for use in the treatment of breast cancer in combination with an antibody binding to human HER2 and inhibiting dimerization of HER2, wherein the breast cancer is HER2 low, HER3 positive and HR (hormone receptor) positive.
The antibody according to claim 1, wherein the breast cancer is HER2 low HER3, and positive ER (estrogen receptor) positive .
The antibody according to any one of claims 1 to 2, wherein
additionally either
A) an anti-hormonal agent is co-administered, wherein the anti-hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), or b) a selective estrogen receptor modulators (SERM); and such as tamoxifen, raloxifene, toremifene or lasofoxifene, or c) an aromatase inhibitor;
or
B) a chemotherapeutic agent is co-administered, wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative b) cyclophosphamide, c) cisplatin or carboplatin d) mitomycin C, e) methotrexate, f) 5-fluorouracil or capecitabine, g) doxorubicin or its liposomal formulation, or daunorubicin or its liposomal formulation, - S ll) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and
1) ixabepilone.
The antibody according to any one of claims 1 to 2, wherein
additionally an anti-hormonal agent is co-administered, and wherein the anti- hormonal agent is selected from the group of: a) a selective estrogen receptor degrader (SERD), b) a selective estrogen receptor modulator (SERM), and c) an aromatase inhibitor.
The antibody according to claim 4, wherein the anti-hormonal agent is selected from the group of: a) fulvestrant, b) tamoxifen, raloxifene, toremifene or lasofoxifene, and c) letrozole, anastrozole or exemestane.
The antibody according to claim 4, wherein the anti-hormonal agent is fulvestrant.
The antibody according to any one of claims 1 to 2, wherein
additionally a chemotherapeutic agent is co-administered, and wherein the chemotherapeutic agent is selected from the group of: a) a taxane or taxane derivative b) cyclophosphamide, c) cisplatin or carboplatin d) mitomycin C, e) methotrexate, f) 5-fluorouracil or capecitabine, g) doxorubicin or its liposomal formulation, or daunorubicin or its liposomal formulation, h) gemcitabine, i) mitoxantrone, j) vinorelbine, k) eribulin, and 1) ixabepilone.
8. The antibody according claim 7, wherein
the chemotherapeutic agent is a taxanes or taxane derivative selected from the group of paclitaxel, nab-paclitaxel and docetaxel.
9. The antibody according to any one of claims 1 to 8, wherein the combination is administered as a first-line treatment.
10. The antibody according to any one of claims 1 to 9, wherein the antibody which binds to human HER3 is characterized in comprising as heavy chain variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and the light chain variable domain comprises a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:7.
11. The antibody according to any one of claims 1 to 9, wherein the antibody which binds to human HER3 is characterized in that the heavy chain variable domain VH is SEQ ID NO:8; and the light chain variable domain VL is SEQ ID NO: 10.
12. The antibody according to any one of claims 1 to 11, wherein the antibody which binds to human HER3 is of human IgGl subclass and is characterized in that the antibody is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65 % or lower.
13. The antibody according to any one of claims 1 to 12, wherein the antibody binding to human HER2 and inhibiting dimerization of HER2 is pertuzumab.
14. The antibody according to any one of claims 1 to 13, wherein the antibody which binds to human HER3 is administered at a dose of 400-600 mg every 3 weeks and the antibody which binds to human HER2 is administered at a dose of 300-500 mg every 3 weeks.
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