HER2 antibody combination

Abstract

The present invention is directed to HER2 antibodies directed against an epitope at amino acids 342-652 of human HER2 for use in the treatment of HER2-associated disorders in combination with a second HER2 inhibitor. More specifically, the present invention relates to methods and uses of MAB270 or antibodies with the same CDRs as MAB270 in combination with trastuzumab or pertuzumab in HER2-positive cancers.

Description

[Technical field]

[0001] The present invention is directed to HER2 antibodies directed to an epitope of amino acids 342-652 of human HER2 for use in the treatment of HER2-associated disorders in combination with one or more other agents. More specifically, the present invention relates to methods and uses of MAB270 or antibodies with the same CDRs as MAB270 in combination with trastuzumab or pertuzumab in HER2-positive cancers. [Background technology]

[0002] The receptor tyrosine-protein kinase erbB-2, also known as the proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (human), is a protein encoded by the ERBB2 gene in humans and is often referred to as HER2 (for human epidermal growth factor receptor 2) or HER2 / neu.

[0003] HER2 is a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. Known as a proto-oncogene, HER2 is located on the long arm of human chromosome 17 (17ql2). Amplification or overexpression of this oncogene has been shown to play an important role in the development and progression of certain aggressive types of cancer.

[0004] Thus, in recent years it has become an important biomarker and therapeutic target for many cancer types, including breast cancer.

[0005] The ErbB family consists of four plasma membrane-bound receptor tyrosine kinases. One of them is EGFR, and the other members are epidermal growth factor receptor, HER3 (neuregulin-binding; lacks kinase domain) and HER4. All four contain an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain that can interact with many signaling molecules and exhibit both ligand-dependent and ligand-independent activity. HER2 can heterodimerize with any of the other three receptors and is thought to be the preferred dimerization partner of the other ErbB receptors.

[0006] Dimerization results in autophosphorylation of tyrosine residues in the cytoplasmic domain of the receptor, initiating various signaling pathways, including the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K / Akt), phospholipase Cy / protein kinase C (PKC), and signal transducer and activator of transcription (STAT) pathways. Thus, signaling through ErbB family receptors promotes cell proliferation, differentiation, and survival and must be tightly regulated to prevent uncontrolled cell growth.

[0007] Amplification or overexpression of the HER2 gene occurs in approximately 15-30% of breast cancers. It is strongly associated with increased disease recurrence and poor prognosis. Overexpression is also known to occur in ovarian, gastric, bladder, lung, head and neck cancers, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. For example, HER2 is overexpressed in approximately 7-34% of gastric cancer patients and approximately 30% of salivary duct carcinomas.

[0008] Various structural changes have been identified that lead to ligand-independent firing of the receptor even in the absence of receptor overexpression. For example, a valine to glutamic acid substitution in the transmembrane domain can lead to the protein constitutively dimerizing in the absence of ligand. HER2 mutations have also been found in non-small cell lung cancer (NSCLC).

[0009] Given its central role in tumor development, different strategies targeting HER2 are used in the clinic: 1.) antibodies directed against the extracellular domain of the receptor, and 2.) small molecule inhibitors acting on the intracellular kinase domain.

[0010] Trastuzumab, a humanized monoclonal antibody against the extracellular juxtamembrane domain IV of HER2, causes a decrease in HER2 signaling and suppression of cell growth by several proposed mechanisms: (I) a decrease in ligand-independent signaling, (II) an increase in the destruction of HER2 after endocytosis, (III) immune activation, and (IV) inhibition of extracellular domain shedding. Trastuzumab has been proposed to induce antibody-dependent cellular cytotoxicity (ADCC) because its IgG1 Fc heavy chain domain can bind and activate Fc receptors on immune effector cells. Consistent with this hypothesis, the antitumor activity of trastuzumab was reduced in mice lacking FcγR, whereas enhancing natural killer cell responses enhanced antitumor activity.

[0011] Following the approval of trastuzumab, lapatinib, an oral small molecule tyrosine kinase inhibitor, became available for patients with HER2-positive metastatic breast cancer whose disease had progressed on prior treatment with trastuzumab, a taxane, and an anthracycline. In contrast to trastuzumab, lapatinib binds to the intracellular adenosine triphosphate-binding domains of HER1 and HER2. Thus, clinical synergy has been demonstrated with the combination of trastuzumab and lapatinib in patients whose disease had progressed on trastuzumab.

[0012] Pertuzumab is a recombinant humanized monoclonal antibody that targets HER2. Unlike trastuzumab, which binds to HER2 at the juxtamembrane domain IV, pertuzumab binds to HER2 at the extracellular dimerization subdomain II, which is important for homo- and heterodimerization. Thus, pertuzumab blocks the HER2 receptor from dimerizing with Her2 or other HER family members, including EGFR, HER1, HER3, and HER4.

[0013] Despite the overall encouraging activity in HER2-positive breast cancer, only half of patients had a tumor response and 50% of patients had disease progression within 1 year, indicating the existence of de novo and acquired resistance to HER2-targeted therapy. Trastuzumab-resistant tumors generally display continued HER2 amplification, high HER2 protein levels, and dependence on HER2 signaling.

[0014] Many mechanisms of resistance to anti-HER2 therapy have been proposed. For example, primary resistance is mainly the failure to respond positively to therapy and may be due to redundancy, inactive target receptors (such as truncated HER2 receptors that do not have the extracellular trastuzumab-binding domain), alternative dimerization patterns within the HER receptor family, or incomplete inhibition of the HER2 signaling pathway. Acquired resistance has also been reported, which is caused by the ability to reactivate pathway signals at or downstream of the receptor layer, such as activation of HER, intrinsic changes in HER2, or loss of negative regulatory mechanisms of downstream pathways. Other reported mechanisms are upregulation of other tyrosine kinase receptors or crosstalk between estrogen receptor and HER2 pathway.

[0015] There is therefore a need for the development of new, more effective antibodies and treatment methods that can be used in follow-up therapy when the results of the gold standard therapy with trastuzumab (and chemotherapy) are not satisfactory or as an alternative therapy in combination with existing antibodies. Summary of the Invention

[0016] In the present invention, it has surprisingly been found that a combination of an anti-HER2 antibody specific for the epitope of amino acids 342-652 of human HER2 or a functional fragment or functional derivative thereof, together with a further HER2 inhibitor, is particularly useful for therapeutic and diagnostic applications. [Brief description of the drawings]

[0017] [Figure 1] Apoptosis induction was measured as Annexin V staining after incubation of KPL-4 cells with antibodies trastuzumab or MAB270. Annexin V positive apoptotic cells are shown compared to the positive control camptothecin (positive control is set at 100%). Data are presented as mean ± SD (n=3). [Diagram 2] Tumor volumes (cm3) of individual mice 33 (A) and 62 (B) days after tumor cell injection (treatment started on day 15). KPL-4 xenografted mice were treated with vehicle (circles), co-administration of trastuzumab and pertuzumab (squares), MAB270 monotherapy (triangles), or co-administration of MAB270 and trastuzumab (inverted triangles). Mean ± SD is shown. *, P<0.05 (Mann-Whitney test). [Diagram 3] Individual data points of tumor volume (cm3) before (day 15) and at the end of the experiment (day 78) for Trastuzumab / Pertuzumab co-administration and / Trastuzumab co-administration are shown. Mice showing tumor regression are highlighted. [Figure 4] Kaplan-Meier survival curves of tumor-bearing mice treated as indicated. Mice were sacrificed when tumor volume reached 1.5 cm3, became moribund, or lost weight. ***, P<0.0001 for MAB270 / trastuzumab vs. control, *, P=0.0224 for MAB270 vs. control (log-rank test). [Diagram 5]Epitope mapping of Pertuzumab, Trastuzumab, and B100. B100 anti-HER2 was analyzed in a binding ELISA to different domains of the ECD of HER2. B100 binds to a different domain than trastuzumab and pertuzumab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The epitope recognized by the antibody of the present invention is preferably located in domain III of human HER2. Preferably, the specific anti-HER2 antibody is directed against an epitope at amino acids 342 to 510 of human HER2. Particularly preferred is a humanized or human antibody.

[0019] Specific antibodies for use according to the present invention are characterized by six complementarity determining regions, as described herein below: - a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence which differs therefrom by one or two amino acids; - a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence which differs therefrom by one or two amino acids; - a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence which differs therefrom by one or two amino acids, - a light chain complementarity determining region 1 (CDRL1) having the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence which differs therefrom by one or two amino acids, - a light chain complementarity determining region 2 (CDRL2) having the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence which differs therefrom by one or two amino acids, - a light chain complementarity determining region 3 (CDRL3) having the amino acid sequence as set forth in SEQ ID NO: 6 or 7, or an amino acid sequence which differs therefrom by one or two amino acids.

[0020] Preferably, an antibody for use according to the invention comprises a heavy chain comprising a combination of CDRH1, CDRH2, and CDRH3 as shown in Table 1.

[0021] [Table 1]

[0022] It is further preferred according to the invention that the antibody comprises a light chain comprising a combination of CDRL1, CDRL2, and CDRL3 as shown in Table 2. It is understood that each row of this table represents one particular combination of CDRL1, CDRL2, and CDRL3.

[0023] [Table 2]

[0024] Most preferably, an antibody for use in accordance with the present invention comprises a heavy chain comprising a combination of CDRH1 set forth in SEQ ID NO:1, CDRH2 set forth in SEQ ID NO:2, and CDRH3 set forth in SEQ ID NO:3, and a light chain comprising a combination of CDRL1 set forth in SEQ ID NO:4, CDRL2 set forth in SEQ ID NO:5, and CDRL3 set forth in SEQ ID NO:6 or 7.

[0025] In the present invention, it was found that antibodies having the above-defined complementarity determining regions exhibited exceptionally strong apoptosis induction in the partially HER2-resistant breast cancer cell line KPL-4, in contrast to trastuzumab, and the induction of apoptosis was even stronger than that of the positive control camptothecin.

[0026] Furthermore, in a KPL-4 in vivo breast cancer model, treatment with an antibody having the CDRs defined above in combination with trastuzumab significantly inhibited tumor growth and was superior to the combination of trastuzumab and pertuzumab.

[0027] Furthermore, the antibodies with the above-defined CDRs specifically bind to HER2 within a different epitope than the antibodies Trastuzumab and Pertuzumab, and exhibit a different and unique mechanism of action. In in vitro assays, the antibodies with the above-defined CDRs can induce apoptosis and strongly induce the FcR-mediated signaling pathway, which results in the activation of antibody-dependent cellular cytotoxicity (ADCC). Trastuzumab and Pertuzumab can also induce ADCC, but the combination of both agents does not increase ADCC efficiency (Scheuer et al., 2009).

[0028] Thus, the present invention comprises an antibody having a CDR as defined above or a functional fragment or derivative thereof, combined with one or more second HER2 inhibitor(s), either in one single formulation or in two formulations, for treating HER2 positive disease in a patient comprising co-administering, simultaneously or sequentially, an antibody having a CDR as defined above or a functional fragment or derivative thereof with at least one further HER2 inhibitor to said patient who does not respond to monotherapy with a HER2 inhibitor or to sequential monotherapy with at least two HER2 inhibitors.

[0029] The present invention further comprises an antibody with a CDR as defined above or a functional fragment or derivative thereof combined with trastuzumab and / or pertuzumab, either in one single formulation or in two formulations, for treating HER2 positive cancer or metastasis of HER2 positive cancer in a patient comprising co-administering, simultaneously or sequentially, an antibody with a CDR as defined above or a functional fragment or derivative thereof with trastuzumab or pertuzumab to a patient who does not respond to monotherapy with trastuzumab or pertuzumab or to sequential monotherapy with trastuzumab and pertuzumab.

[0030] The present invention further comprises an antibody having a CDR as defined above or a functional fragment or derivative thereof in combination with trastuzumab and / or pertuzumab for treating HER2 positive cancer or metastasis of HER2 positive cancer in a patient who does not respond to first line monotherapy with trastuzumab, comprising co-administering, simultaneously or sequentially, an antibody having a CDR as defined above or a functional fragment or derivative thereof with trastuzumab or pertuzumab to said patient.

[0031] The present invention further comprises an antibody having a CDR as defined above or a functional fragment or derivative thereof in combination with trastuzumab and / or pertuzumab for treating HER2 positive cancer or metastasis of HER2 positive cancer in a patient who does not respond to first line monotherapy with pertuzumab, comprising co-administering, simultaneously or sequentially, an antibody having a CDR as defined above or a functional fragment or derivative thereof with trastuzumab or pertuzumab to said patient.

[0032] The present invention further comprises an antibody having a CDR as defined above or a functional fragment or derivative thereof in combination with trastuzumab and / or pertuzumab for treating HER2 positive cancer or metastasis of HER2 positive cancer in a patient who does not respond to monotherapy with trastuzumab or to monotherapy with pertuzumab, comprising co-administering, simultaneously or sequentially, an antibody having a CDR as defined above or a functional fragment or derivative thereof with trastuzumab or pertuzumab to said patient.

[0033] In the present invention, it has surprisingly been found that a combination of an antibody having the above defined CDRs or a functional fragment or derivative thereof with one or more agent(s) capable of blocking HER2 activity is superior to the combination of trastuzumab and pertuzumab.

[0034] Thus, the present invention relates to an antibody having a CDR as defined above, or a functional fragment or derivative thereof, in combination with a second agent that reduces the activity of HER2, for use in the prevention, alleviation and / or treatment of diseases, in particular diseases associated with overexpression, amplification and / or hyperactivity of HER2.

[0035] The antibodies of the present invention may be of various immunoglobulin (Ig) types, such as IgA, IgD, IgE, IgG or IgM-type, preferably of IgG or IgM type, including but not limited to IgG1, IgG2, IgG3, IgG4, IgM1 and IgM2 types. In a preferred embodiment, the antibody is of IgG1 type.

[0036] As mentioned above, the complementarity determining regions (CDRs) of an antibody may be adjacent to the framework regions. An antibody heavy or light chain containing three CDRs, for example, contains four framework regions.

[0037] Most preferably, an antibody of the invention comprises a heavy chain comprising four framework regions, wherein the combinations of FR-H1, FR-H2, FR-H3, and FR-H4 are selected from those shown in Table 3. It is understood that each row of this table represents one specific combination of FR-H1, FR-H2, FR-H3, and FR-H4.

[0038] [Table 3]

[0039] Most preferably, an antibody of the invention comprises a light chain comprising four framework regions, wherein the combinations of FR-L1, FR-L2, FR-L3, and FR-L4 are selected from those shown in Table 4. It is understood that each row of this table represents one specific combination of FR-L1, FR-L2, FR-L3, and FR-L4.

[0040] [Table 4]

[0041] Furthermore, the antibody of the present invention is a) a heavy chain variable region (VH) comprising framework regions FR-H1, FR-H2, FR-H3, and FR-H4, the FR-H1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 16; the FR-H2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 25; the FR-H3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 to 34; The FR-H4 region comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 43; b) a light chain variable region (VL) comprising the framework regions FR-L1, FR-L2, FR-L3, and FR-L4, the FR-L1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44 to 52; the FR-L2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 61; the FR-L3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 62 to 70; The FR-L4 region comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 to 79. may include:

[0042] Furthermore, the present invention also encompasses antibodies that recognize the same epitope in human HER2 as the specific antibodies characterized by the above-mentioned heavy and / or light chain CDRs.Functional fragments and functional derivatives of these antibodies are also within the scope of the present invention.

[0043] To determine the epitope in HER2 recognized by an antibody, a chemically prepared array of short peptides derived from the protein sequence derived from the amino acid sequence of the extracellular domain of human HER2 can be used to locate and identify the antibody epitope (Reinicke W., Methods Mol. Biol. 2004, 248: 443-63). Further methods for mapping the epitope in the HER2 extracellular domain bound by the antibody of the present invention include Snaps / SELDI (Wang et al., Int. J. Cancer, 2001, June 15; 92 (6): 871-6), or a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) may be performed.

[0044] The antibodies of the present invention are preferably humanized or human antibodies.

[0045] In a preferred embodiment of the present invention, the human antibody comprises a heavy chain variable region (VH) shown in any one of SEQ ID NOs: 80 to 88, or a sequence which differs therefrom by one or two amino acids.

[0046] Furthermore, the human antibody of the present invention preferably comprises a light chain variable region (VL) shown in any one of SEQ ID NOs: 89 to 97, or a sequence which differs therefrom by one or two amino acids.

[0047] Particularly preferred is a human antibody comprising a heavy chain variable region shown in any one of SEQ ID NOs: 80 to 88 and a light chain variable region shown in any one of SEQ ID NOs: 89 to 97. In particular, it is preferable to use any one of the antibodies disclosed below.

[0048] [Table 5]

[0049] [Table 6]

[0050] Particularly preferred is a human antibody (MAB270) comprising a heavy chain comprising CDRH1 shown in SEQ ID NO: 1, CDRH2 shown in SEQ ID NO: 2, and CDRH3 shown in SEQ ID NO: 3, and a light chain comprising CDRL1 shown in SEQ ID NO: 4, CDRL2 shown in SEQ ID NO: 5, and CDRL3 shown in SEQ ID NO: 7. Also preferred are human antibodies in which one or more amino acids in the CDRs differ by one or two, or antibodies that recognize the same epitope in human HER2.

[0051] In a particularly preferred embodiment, the human antibody comprises a heavy chain variable region sequence according to SEQ ID NO: 88 and a light chain variable region sequence according to SEQ ID NO: 97. Also preferred are human antibodies in which the sequences of the heavy and / or light chain variable regions differ by one or two amino acids from those shown in SEQ ID NOs: 88 and 97.

[0052] The monoclonal antibody according to the invention may be a rabbit antibody. In a preferred embodiment, the antibody according to the invention is a rabbit / human chimeric antibody. In a further preferred form, the antibody is a humanized antibody.

[0053] The present invention also encompasses antibodies, or fragments or derivatives thereof, that specifically bind to HER2, or polypeptides containing at least a portion of the antibodies sufficient to confer HER2-binding specificity, which antibodies bind to human Fc receptors and induce FcR-mediated signaling pathways.

[0054] Preferably, the antibodies according to the invention show increased induction of FcR-mediated signalling pathways compared to trastuzumab or pertuzumab.

[0055] In KPL-4 cells, the antibodies of the present invention preferably show a stimulation of apoptosis 100% higher than untreated cells, more preferably 110% higher than untreated cells, and most preferably 120% higher than untreated cells. This reflects a much higher potency than the HER2 antibody trastuzumab, which shows a comparable increase in apoptosis, 69% of untreated cells (Figure 1).

[0056] This increased activity of the antibody according to the invention compared to trastuzumab and pertuzumab, which are used in cancer therapy, clearly demonstrates the superiority and great potential of the antibody for use in the treatment of HER2-mediated diseases.

[0057] The term "rabbit" according to the present invention refers to an animal that is a member of the taxonomic order Lagomorpha, which includes hares and rabbits, and the family Ochotonidae (pikas), preferably the genus Oryctolagus. The term "antibody" encompasses the various forms of antibody structures, including but not limited to whole antibodies and antibody fragments, so long as they exhibit the properties according to the present invention.

[0058] The term "rabbit monoclonal antibody" according to the present invention refers to a monoclonal antibody produced by immunizing a rabbit and isolated from the antigen-producing cells of said rabbit, as well as to such antibodies which have been further modified, preferably humanized antibodies, chimeric antibodies, fragments thereof, or further genetically engineered and recombinantly produced antibodies, as long as the characteristic properties according to the present invention are retained. Preferably, the antibodies are derived from the B cells of said rabbit or rabbit hybridoma cells.

[0059] The term "antibody-producing cells" according to the present invention means rabbit B cells, preferably B cells or rabbit hybridoma cells, that produce antibodies.

[0060] A "native antibody" is usually a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, but the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0061] "Variable region (or domain)" (variable region of the light chain (VL), variable region of the heavy chain (VH) as used herein means each of the pair of light and heavy chain regions that are directly involved in binding the antibody to the antigen. The variable light and variable heavy chain regions have the same general structure, and each region contains four framework (FR) regions whose sequences are widely conserved and which are connected by three complementarity determining regions, CDRs.

[0062] The term "antigen-binding portion of an antibody" refers to amino acid residues that are involved in antigen binding of the antibody. The antigen-binding portion of an antibody preferably comprises amino acid residues from the "complementarity determining regions" or "CDRs". CDR sequences are defined according to Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of, or insertion into, the FRs or CDRs of the variable region. For example, a heavy chain variable region may contain one amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 30 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues for a given antibody can be determined by aligning the antibody sequence with the "standard" Kabat numbering sequence in the homology region.

[0063] The "constant domain (constant part)" is not directly involved in binding of the antibody to an antigen, but also exerts, for example, effector functions. The heavy chain constant region corresponding to human IgG1 is called the γ1 chain. The heavy chain constant region corresponding to human IgG3 is called the γ3 chain. The human constant γ heavy chain is described in detail by Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991) and Brueggemann, M., et al., J. Exp. Med. 166 (1987) 1351-1361; Love, TW, et al., Methods Enzymol. 178 (1989) 515-527. The constant domain of IgG1 or IgG3 type is glycosylated at Asn297. By "Asn297" according to the present invention is meant the amino acid asparagine located at about position 297 of the Fc region; depending on the slight sequence diversity of antibodies, Asn297 may also be located a few amino acids upstream or downstream (usually +3 amino acids or less).

[0064] The term "antibody effector function(s)" or "effector function" as used herein refers to a function mediated by the Fc effector domain(s) of IgG (e.g., the Fc region of an immunoglobulin). Such a function may be exerted, for example, by the Fc effector domain(s) binding to an Fc receptor on an immune cell with phagocytic or lytic activity, or by the Fc effector domain(s) binding to a component of the complement system. Exemplary effector functions are ADCC, ADCP, and CDC. An "antibody fragment" refers to a molecule other than an intact antibody that includes a portion of an intact antibody that binds to an antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0065] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcR (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibody bound to a target cell and subsequently cause lysis of the target cell. NK cells, the primary cells for mediating ADCC, express only FcyRIII, whereas monocytes express FcyRI, FcyRII, and FCYRIII. Expression of FCRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, and Kinet, Annu. Rev. Immunol 9 (1991) 457-492. The terms "antibody-dependent cellular phagocytosis" and "ADCP" refer to the process of internalization, in whole or in part, of antibody-coated cells by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of immunoglobulin.

[0066] "Clq" is a polypeptide that contains a binding site for the Fc region of immunoglobulins. Clq, together with two serine proteases Clr and Cls, forms the complex Cl, which is the first component of the complement-dependent cytotoxicity (CDC) pathway. Fluman Clq can be purchased commercially, for example, from Quidel (San Diego, California).

[0067] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA5, and IgA2. The heavy chain constant domains that correspond to the various classes of immunoglobulins are called α, 6, s, y, and p, respectively.

[0068] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0069] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions.

[0070] Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0071] A "variant Fc region" comprises an amino acid sequence which differs from that of a "native" or "wild-type" sequence Fc region by virtue of at least one "amino acid modification", as defined herein.

[0072] The term "Fc variant" as used herein refers to a polypeptide that contains modifications in the Fc domain. All positions discussed in this invention are numbered according to the EU index. The EU index or EU numbering scheme, such as the EU index or Kabat, refers to the numbering of EU antibodies (Edelman, et al., Proc Natl Acad Sei USA 63 (1969) 78-85, incorporated herein by reference in its entirety). Modifications may be additions, deletions, or substitutions. Substitutions may include naturally occurring and non-naturally occurring amino acids. Variants may also include non-naturally occurring amino acids.

[0073] The term "Fc region-containing polypeptide" refers to a polypeptide such as an antibody or immunoadhesin (see definition below) that comprises an Fc region.

[0074] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. FcRs (gamma receptors) that bind IgG antibodies include receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcyRII receptors include FcyRIIA ("activating receptor") and FcyRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review in Daeron, M., Annu. Rev. Immunol. 15 (1997) 203-234). FcRs are reviewed in Ravetch, and Kinet, Annu. Rev. Immunol 9 (1991) 457-492; Capel, et al., Immunomethods 4 (1994) 25-34; and de Flaas, et al., J. Lab. Clin. Med. 126 (1995) 330-41. Other FcRs, including those that may be identified in the future, are also encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer, et al., J. Immunol. 117 (1976) 587 and Kim, et al., J. Immunol. 24 (1994) 249). "IgG Fc ligand" as used herein refers to any biologically derived molecule, preferably a polypeptide, that binds to the Fc region of an IgG antibody to form an Fc / Fc ligand complex. Fc ligands include, but are not limited to, FcyR, FcyR, FcyRn, Clq, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcyR.Fc ligands also include Fc receptor homologs (FcRH), a family of Fc receptors that are homologous to FcyR (Davis, et al., Immunological Reviews 190 (2002) 123-136, incorporated herein by reference in its entirety). Fc ligands may also include undiscovered molecules that bind to Fc. In particular, Fc ligands for IgG are FcRn and Fc gamma receptors. "Fc ligand" as used herein means any biologically derived molecule, preferably a polypeptide, that binds to the Fc region of an antibody to form an Fc / Fc ligand complex. "Fc gamma receptor", "FcyR", or "FcgammaR" as used herein means any member of the family of proteins that bind to the Fc region of an IgG antibody and are encoded by the FcyR gene. In humans, this family includes, but is not limited to, FcyRI (CD64), which includes isoforms FcyRIA, FcyRIB, and FcyRIC; FcyRII (CD32), which includes isoforms FcyRIIA (including allotypes H131 and R131), FcyRIIB (including FcyRIIB-1 and FcyRIIB-2), and FcyRIIc; and FcyRIII (CD16), which includes isoforms FcyRIIIA (including allotypes VI58 and F158) and FcyRI11b (including allotypes FcyRIIB-NAI and FcyRIIB-NA2) (Jefferis, et al., Immunol Lett 82 (2002) 57-65, which is incorporated herein by reference in its entirety), as well as any undiscovered human FcyR or isoform or allotype of FcyR. FcyRs may be from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcyRs include, but are not limited to, FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), and FcyRIII-2 (CD16-2), as well as any undiscovered mouse FcyR or FcyR isoforms or allotypes.

[0075] "FcRn" or "neonatal Fc receptor" as used herein refers to a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn may be from any organism, including but not limited to human, mouse, rat, rabbit, and monkey. As known in the art, a functional FcRn protein comprises two polypeptides, often referred to as heavy and light chains. The light chain is β-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, FcRn or FcRn protein refers to the complex of FcRn heavy chain and β-2-microglobulin.

[0076] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its target antigen in a competitive assay. The possibility of overlapping epitopes of two antibodies that bind to the same target antigen can be detected using a competitive test system. For this purpose, for example, an enzyme immunoassay is used to test the extent to which a new antibody competes with a known antibody for binding to an immobilized target antigen. For this purpose, a suitably immobilized target antigen is incubated with a labeled form of the known antibody and an excess of the target antibody. By detecting the bound label, the extent to which the target antibody can displace the known antibody from the binding site (=epitope) can be easily ascertained. If, with reference to the known antibody, more than 10%, preferably more than 20%, is displaced at the same concentration or at a higher concentration, preferably 10-fold excess, of the target antibody, there is an overlap of epitopes. This means that the target antibody binds to the same epitope as the known antibody.

[0077] Immunoassays are well known to those skilled in the art. Methods for carrying out such assays as well as practical applications and procedures are summarized in relevant textbooks. Examples of relevant textbooks are Tijssen, R, Preparation of enzyme-antibody or other enzyme-macromolecule conjugates in Practice and theory of enzyme immunoassays, Burdon, RH and v. Knippenberg, PH (eds.), Elsevier, Amsterdam (1990) pp. 221-278; and various volumes of Methods in Enzymology, Colowick, SP and Caplan, NO (eds.), Academic Press, dealing with immunological detection methods, in particular volumes 70, 73, 74, 84, 92 and 121.

[0078] The term "epitope" as used herein means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. Depending on the size of the antigen to which the epitope belongs, more than one epitope may be available per antigen, so that there may be more than one antibody binding site (= epitope) per antigen.

[0079] By "immunoconjugate" is meant an antibody conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin, another antibody, or a radioisotope.

[0080] An "antibody fragment" comprises a portion of a full-length antibody, preferably the variable region thereof, or at least the antigen-binding site thereof. Examples of antibody fragments include diabodies, Fab fragments, and single-chain antibody molecules. scFv antibodies are described, for example, in Fluston, JS, Methods in Enzymol. 203 (1991) 46-88.

[0081] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.

[0082] The term "humanized antibody" or "humanized form of an antibody" refers to an antibody in which both the heavy and light chains have been humanized as a result of genetic engineering of the antibody. A humanized chain is typically a chain in which the amino acid sequence of the V region has been altered so that, when analyzed as a whole, it has closer homology to the human germline sequence than to the germline sequence of the original species. The assessment of humanization is based on the resulting amino acid sequence, not the methodology per se.

[0083] The term "specifically binds to a target or anti-target antibody" as used herein refers to the binding of an antibody to the respective antigen (target) or antigen-expressing cells as measured by ELISA, which preferably involves coating the respective antigen to a solid support, adding the antibody under conditions allowing the formation of an immune complex with the respective antigen or protein, and detecting the immune complex by measuring the optical density value (OD) using a secondary antibody that binds to the antibody of the invention and peroxidase-mediated color development.

[0084] The term "antigen" according to the present invention refers to an antigen used for immunization or a protein that includes said antigen as part of its protein sequence. For example, a fragment of the extracellular domain of the protein (e.g., the first 20 amino acids) may be used for immunization, whereas the extracellular domain of the protein or the full-length protein may be used for detection / assay etc.

[0085] As used herein, the terms "specifically bind" or "specifically recognized" mean that an antibody exhibits a recognized affinity for an antigen and preferably does not exhibit significant cross-reactivity.

[0086] An "appreciable" binding affinity is at least 10 -7 M, specifically at least 10 -8 M, more specifically at least 10 -9 M, or even more specifically at least 10 -10 It involves binding with an affinity of M.

[0087] An antibody that "does not exhibit significant cross-reactivity" is one that does not appreciably bind to other undesired proteins. Specific binding can be determined according to any art-recognized means for determining such binding, for example, by competitive binding assays such as ELISA. All protein terms as used herein refer to human proteins. When proteins of other species are meant, this is clearly stated.

[0088] The term "cancer" as used herein includes, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer, and the like. cancer), colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory forms of any of the above cancers, or a combination of one or more of the above cancers. Preferably, such cancer is breast cancer, colon cancer, lung cancer, or pancreatic cancer.

[0089] In the context of the present invention, additional other cytotoxic, chemotherapeutic or anti-cancer agents, or compounds which enhance the effect of such agents, may be used in the combination treatment of HER2-positive cancer or metastasis of HER2-positive cancer with an antibody having a CDR as defined above.

[0090] Such agents include, for example, alkylating agents or agents having alkylating properties, such as cyclophosphamide (CTX; for example, Cytoxan®).

[0091] In the context of the present invention, anti-hormonal agents may be used in combination treatment of HER2-positive cancer or metastasis of HER2-positive cancer with an antibody having the CDR defined above. As used herein, the term "anti-hormonal agent" includes natural or synthetic organic or peptidic compounds that act to regulate or inhibit hormone action on tumors.

[0092] In the context of the present invention, additional anti-proliferative agents may be used in the combination treatment of HER2-positive cancer or metastasis of HER2-positive cancer with an antibody having a CDR as defined above.

[0093] In the context of the present invention, an effective amount of ionizing radiation may be administered and / or a radiopharmaceutical may be used in the combination treatment of HER2-positive cancer or metastasis of HER2-positive cancer with an antibody having a CDR as defined above.

[0094] The present invention further relates to a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a therapeutically effective amount of an antibody having a CDR as defined above, in combination with a second agent according to the invention, which can be administered to a patient in a method of treating a HER-2 mediated disease according to the invention.

[0095] The invention also encompasses administering the pharmaceutical composition to a patient. As is well known in the medical art, the dosage for any one patient depends on many factors, including the patient's size, body surface and area, age, the specific compound being administered, sex, time and route of administration, general health, and other drugs being administered concomitantly. Depending on the type and severity of the condition being treated, about 1 pg / kg to 15 mg / kg of active ingredient may be administered to a patient in need thereof, for example, by one or more divided doses or continuous infusion. A typical daily dose may range from about 1 μg / kg to about 100 mg / kg, depending on the factors mentioned above. When administered repeatedly over several days or longer, treatment is sustained until a desired suppression of the disease or symptoms occurs, depending on the condition being treated. The composition may be administered by any suitable route, for example, parenterally, subcutaneously, intranasally, intravascularly, intravenously, intraarterially, or intrathecally, by injection or infusion. Progress can be monitored by periodic evaluation. The compositions of the invention may be administered locally or systemically. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases.

[0096] The present invention also relates to a kit comprising an antibody having a CDR as defined above, a second agent, and a package insert instructing a user to co-administer the antibody having a CDR as defined above and the second agent to a patient suffering from a HER2-positive cancer that does not respond to monotherapy with the second agent.

[0097] The following examples and figures, including the experiments performed and results obtained, are provided for illustrative purposes only and should not be construed as limiting the present teachings. EXAMPLES

[0098] Example 1: Induction of apoptosis in KPL-4 cell line To analyze the induction of apoptosis, an in vitro Annexin V staining assay was then performed. KPL-4 cells were seeded in 2 mL of medium (4x104 cells / well) and incubated for 72 h before treatment, followed by incubation with 5 μg / mL of antibody or assay medium as control at 37°C for 72 h. 2 mM camptothecin was used as a positive control (incubated for 24 h). Cells and supernatants were collected, centrifuged and washed with PBS. Cells were then resuspended in pre-cooled binding buffer (0,1 M HEPES, 1.4 M NaCl, 25 mM CaCl2), mixed with Annexin (Annexin V FITC conjugate from Immunotools) and incubated for 20 min on ice / dark. DRAQ7 was used for staining dead cells (incubated for 7 min on ice / dark). Annexin staining was determined by flow cytometry analysis. The tested MAB270 shows exceptionally strong apoptosis induction in contrast to trastuzumab: the antibody is able to induce apoptosis in a greater number of KPL-4 cells compared to the positive control camptothecin, whereas trastuzumab shows a lower activity (Figure 1).

[0099] Example 2: Antitumor effects in KPL-4 breast cancer xenograft model To evaluate the antitumor efficacy of MAB270 alone and in combination with trastuzumab, compared to trastuzumab in combination with pertuzumab, a breast cancer xenograft model was applied. 6-8 week-old female SCID beige mice (Charles River Laboratories) were inoculated with 3 × 10 6KPL-4 tumor cells were injected into the intramammary fat pad. Tumor cell injection corresponds to day 0 of the experiment. Tumor-bearing mice were placed approximately 100 cm 3 Tumors were stratified according to tumor size (n=8 per group) and treatment was initiated on day 15. All tested antibodies were provided in the same buffer (20 mM histidine, 140 mM NaCl, pH 6.0). The vehicle group received reference buffer intraperitoneally (ip) once a week. MAB270 was administered ip at a loading dose of 30 mg / kg, followed by a maintenance dose of 15 mg / kg once a week. The combination of MAB270 and trastuzumab or the combination of trastuzumab and pertuzumab was administered at the same dose regimen and schedule as monotherapy. Animals were weighed and tumor volumes were measured twice weekly throughout the experiment. Tumors were 0.01 to 0.05 cm in volume and 0.05 to 0.05 cm in volume. 3 Mice were sacrificed when they reached 0.1 mg / kg, became moribund, or lost weight.

[0100] On day 33, tumors in control animals had a mean tumor volume of 1.0 cm 3 The tumor growth rate reached 100% and individual mice had to be sacrificed after day 33. Treatment with MAB270 alone at 15 mg / kg once weekly for three cycles (initial cycle at 30 mg / kg) significantly reduced tumor growth compared to the vehicle group (Figure 2) and significantly increased survival time before mice had to be sacrificed due to tumor volume (Figure 4). Treatment with the combination therapy significantly inhibited tumor growth compared to the control (Figure 2). The combination of MAB270 and trastuzumab (15 mg / kg once weekly for eight cycles; initial at 30 mg / kg) was superior to the control and to MAB270 monotherapy (Figure 2) and increased survival time before mice had to be sacrificed due to tumor volume. Furthermore, the combination therapy showed significantly improved efficacy compared to the combination of trastuzumab and pertuzumab (Figure 3). In the group treated with MAB270 + trastuzumab, individual mice showed tumor regression, whereas in the group treated with trastuzumab + pertuzumab, individual mice had to be sacrificed due to tumor volume (Fig. 4 ).

[0101] Example 3: Epitope Mapping The binding of anti-HER2 monoclonal antibodies trastuzumab, pertuzumab, and B100 to the full-length extracellular domain of HER2 or to different subdomains of HER2 was tested in a biochemical ELISA. Recombinant Her2 domains (full-length ECD purchased from Biozol, ECD domains produced by Mab Discovery) were coated onto 384-well Nunc™ MaxiSorp™ plates at optimized concentrations (0.25-1 mg / mL) in PBS for 1 h at room temperature. After washing three times with washing buffer (PBS, 0.1% Tween), the plates were blocked with PBS, 2% BSA, 0.05% Tween for 1 h at room temperature. The plates were washed again three times with washing buffer and incubated with antibodies at a concentration of 10 μg / mL in PBS, 0.5% BSA, 0.05% Tween for 1 h at room temperature. After washing three times with wash buffer, the wells were incubated with 12.5 μL of goat anti-human peroxidase-conjugated species-specific F(ab)2 fragment (AbD Serotec) diluted 1:5000 in ELISA buffer for 1 h at room temperature. The wells were washed six times with wash buffer and 15 μL / well of TMB substrate solution (Invitrogen) was added. After 10 min at room temperature, 15 μL of stop solution (1 M HCl) was added per well and absorbance was measured at wavelengths of 450 nm and 620 nm using a Tecan M1000 microplate reader.

[0102] All antibodies show binding to the full-length extracellular domain (ECD) of HER2. For trastuzumab, which is reported to bind to subdomain IV, binding could only be observed to fusion proteins containing subdomains III and IV. Trastuzumab does not bind to subdomain I or subdomain III. Pertuzumab does not bind to any of the HER2 domains I, III, or IV tested. Pertuzumab is reported to bind to domain II. From the results shown in FIG. 5, it can be considered that the B100 antibody binds to subdomain III. Binding could be observed to the full-length ECD, domain III, and domain III-IV fusion proteins, but no binding was observed to subdomain I. ELISA binding assays indicate that B100 binds to a novel epitope of HER2 that is distinct from the subdomains of pertuzumab and trastuzumab.

Claims

1. 1. A combination of a monoclonal anti-HER2 antibody, or a functional fragment or derivative thereof, with a second HER2 inhibitor for use in the prevention or treatment of a disease associated with overexpression, amplification and / or overactivity of HER2, comprising: The anti-HER2 antibody or a functional fragment or derivative thereof, heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO:1; a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO:2; heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO:3; a light chain complementarity determining region 1 (CDRL1) having the amino acid sequence set forth in SEQ ID NO:4; a light chain complementarity determining region 2 (CDRL2) having the amino acid sequence set forth in SEQ ID NO:5; and Light chain complementarity determining region 3 (CDRL3) having the amino acid sequence set forth in SEQ ID NO: 6 or 7 Including, the second HER2 inhibitor is trastuzumab or pertuzumab; A combination.

2. The monoclonal anti-HER2 antibody is a) a heavy chain variable region (VH) comprising framework regions FR-H1, FR-H2, FR-H3, and FR-H4, the FR-H1 region comprises the amino acid sequence shown in SEQ ID NO:8, the FR-H2 region comprises the amino acid sequence shown in SEQ ID NO:17, the FR-H3 region comprises the amino acid sequence shown in SEQ ID NO:26, and the FR-H4 region comprises the amino acid sequence shown in SEQ ID NO:35; or the FR-H1 region comprises the amino acid sequence set forth in SEQ ID NO:9, the FR-H2 region comprises the amino acid sequence set forth in SEQ ID NO:18, the FR-H3 region comprises the amino acid sequence set forth in SEQ ID NO:27, and the FR-H4 region comprises the amino acid sequence set forth in SEQ ID NO:36; or the FR-H1 region comprises the amino acid sequence set forth in SEQ ID NO: 10, the FR-H2 region comprises the amino acid sequence set forth in SEQ ID NO: 19, the FR-H3 region comprises the amino acid sequence set forth in SEQ ID NO: 28, and the FR-H4 region comprises the amino acid sequence set forth in SEQ ID NO: 37; or the FR-H1 region comprises the amino acid sequence set forth in SEQ ID NO: 11, the FR-H2 region comprises the amino acid sequence set forth in SEQ ID NO: 20, the FR-H3 region comprises the amino acid sequence set forth in SEQ ID NO: 29, and the FR-H4 region comprises the amino acid sequence set forth in SEQ ID NO: 38; or the FR-H1 region comprises the amino acid sequence set forth in SEQ ID NO: 12, the FR-H2 region comprises the amino acid sequence set forth in SEQ ID NO: 21, the FR-H3 region comprises the amino acid sequence set forth in SEQ ID NO: 30, and the FR-H4 region comprises the amino acid sequence set forth in SEQ ID NO: 39; or the FR-H1 region comprises the amino acid sequence set forth in SEQ ID NO: 13, the FR-H2 region comprises the amino acid sequence set forth in SEQ ID NO: 22, the FR-H3 region comprises the amino acid sequence set forth in SEQ ID NO: 31, and the FR-H4 region comprises the amino acid sequence set forth in SEQ ID NO: 40; or the FR-H1 region comprises the amino acid sequence set forth in SEQ ID NO: 14, the FR-H2 region comprises the amino acid sequence set forth in SEQ ID NO: 23, the FR-H3 region comprises the amino acid sequence set forth in SEQ ID NO: 32, and the FR-H4 region comprises the amino acid sequence set forth in SEQ ID NO: 41; or the FR-H1 region comprises the amino acid sequence set forth in SEQ ID NO: 15, the FR-H2 region comprises the amino acid sequence set forth in SEQ ID NO: 24, the FR-H3 region comprises the amino acid sequence set forth in SEQ ID NO: 33, and the FR-H4 region comprises the amino acid sequence set forth in SEQ ID NO: 42; or a heavy chain variable region (VH) in which the FR-H1 region comprises the amino acid sequence shown in SEQ ID NO: 16, the FR-H2 region comprises the amino acid sequence shown in SEQ ID NO: 25, the FR-H3 region comprises the amino acid sequence shown in SEQ ID NO: 34, and the FR-H4 region comprises the amino acid sequence shown in SEQ ID NO: 43; b) a light chain variable region (VL) comprising framework regions FR-L1, FR-L2, FR-L3, and FR-L4, the FR-L1 region comprises the amino acid sequence set forth in SEQ ID NO:44, the FR-L2 region comprises the amino acid sequence set forth in SEQ ID NO:53, the FR-L3 region comprises the amino acid sequence set forth in SEQ ID NO:62, and the FR-L4 region comprises the amino acid sequence set forth in SEQ ID NO:71; or the FR-L1 region comprises the amino acid sequence set forth in SEQ ID NO:45, the FR-L2 region comprises the amino acid sequence set forth in SEQ ID NO:54, the FR-L3 region comprises the amino acid sequence set forth in SEQ ID NO:63, and the FR-L4 region comprises the amino acid sequence set forth in SEQ ID NO:72; or the FR-L1 region comprises the amino acid sequence set forth in SEQ ID NO:46, the FR-L2 region comprises the amino acid sequence set forth in SEQ ID NO:55, the FR-L3 region comprises the amino acid sequence set forth in SEQ ID NO:64, and the FR-L4 region comprises the amino acid sequence set forth in SEQ ID NO:73; or the FR-L1 region comprises the amino acid sequence set forth in SEQ ID NO:47, the FR-L2 region comprises the amino acid sequence set forth in SEQ ID NO:56, the FR-L3 region comprises the amino acid sequence set forth in SEQ ID NO:65, and the FR-L4 region comprises the amino acid sequence set forth in SEQ ID NO:74; or the FR-L1 region comprises the amino acid sequence set forth in SEQ ID NO:48, the FR-L2 region comprises the amino acid sequence set forth in SEQ ID NO:57, the FR-L3 region comprises the amino acid sequence set forth in SEQ ID NO:66, and the FR-L4 region comprises the amino acid sequence set forth in SEQ ID NO:75; or the FR-L1 region comprises the amino acid sequence set forth in SEQ ID NO:49, the FR-L2 region comprises the amino acid sequence set forth in SEQ ID NO:58, the FR-L3 region comprises the amino acid sequence set forth in SEQ ID NO:67, and the FR-L4 region comprises the amino acid sequence set forth in SEQ ID NO:76; or the FR-L1 region comprises the amino acid sequence set forth in SEQ ID NO:50, the FR-L2 region comprises the amino acid sequence set forth in SEQ ID NO:59, the FR-L3 region comprises the amino acid sequence set forth in SEQ ID NO:68, and the FR-L4 region comprises the amino acid sequence set forth in SEQ ID NO:77; or the FR-L1 region comprises the amino acid sequence set forth in SEQ ID NO:51, the FR-L2 region comprises the amino acid sequence set forth in SEQ ID NO:60, the FR-L3 region comprises the amino acid sequence set forth in SEQ ID NO:69, and the FR-L4 region comprises the amino acid sequence set forth in SEQ ID NO:78; or a light chain variable region (VL), wherein the FR-L1 region comprises the amino acid sequence shown in SEQ ID NO:52, the FR-L2 region comprises the amino acid sequence shown in SEQ ID NO:61, the FR-L3 region comprises the amino acid sequence shown in SEQ ID NO:70, and the FR-L4 region comprises the amino acid sequence shown in SEQ ID NO:79; 2. The combination for use according to claim 1 , comprising:

3. The combination for use according to claim 1 or 2, wherein the monoclonal anti-HER2 antibody comprises a heavy chain variable region (VH) as set forth in any one of SEQ ID NOs: 80 to 88, and / or a light chain variable region (VL) as set forth in any one of SEQ ID NOs: 89 to 97.

4. The combination for use according to any one of claims 1 to 3, wherein said monoclonal anti-HER2 antibody is a humanized or human antibody.

5. 5. The combination for use according to any one of claims 1 to 4, in combination with one or more further cytotoxic, chemotherapeutic or anti-cancer agents, such as alkylating agents, anti-hormonal agents, anti-proliferative agents, radiopharmaceuticals or ionising radiation.

6. 6. The combination for use according to any one of claims 1 to 5, for administration to a patient who does not respond to monotherapy with said monoclonal anti-HER2 antibody or to monotherapy with said second HER2 inhibitor.

7. The combination for use according to any one of claims 1 to 6, wherein the disease associated with overexpression, amplification and / or hyperactivity of HER2 is HER2-positive cancer or metastasis of HER2-positive cancer.

8. The cancer is selected from the group consisting of lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer, and the like.

8. The combination for use according to claim 7, which is selected from the group consisting of cancer of the following cancers: colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, refractory forms of any of the above cancers, or a combination of one or more of the above cancers.

9. A pharmaceutical composition or kit comprising a combination as defined in any one of claims 1 to 5, either in a single formulation or in two separate formulations.

Images