Patent Description:
Receptor tyrosine-protein kinase erbB-<NUM>, also known as CD340 (cluster of differentiation <NUM>), proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (human) is a protein that in humans is encoded by the ERBB2 gene, which is also frequently called HER2 (from human epidermal growth factor receptor <NUM>) or HER2/neu.

HER2 is a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. HER2, a known proto-oncogene, is located at the long arm of human chromosome <NUM> (17q12). Amplification or overexpression of this oncogene has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. In recent years, the protein has become an important biomarker and target of therapy for approximately <NUM>% of breast cancer patients.

The ErbB family consists of four plasma membrane-bound receptor tyrosine kinases. One of which is erbB-<NUM>, and the other members being epidermal growth factor receptor, erbB-<NUM> (neuregulin-binding; lacks kinase domain), and erbB-<NUM>. All four contain an extracellular ligand binding domain, a transmembrane domain, and an intracellular domain that can interact with a multitude of signaling molecules and exhibit both ligand-dependent and ligand-independent activity. HER2 can heterodimerise with any of the other three receptors and is considered to be the preferred dimerisation partner of the other ErbB receptors.

Dimerisation results in the autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptors and initiates a variety of signaling pathways. These include the mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide <NUM>-kinase (Pl3K/Akt) pathway, phospholipase C γ-, protein kinase C (PKC)-, and the Signal transducer and activator of transcription (STAT) pathways. Therefore, signaling through the ErbB family of receptors promotes cell proliferation and opposes apoptosis, and consequently must be tightly regulated to prevent uncontrolled cell growth from occurring.

Amplification or over-expression of the ERBB2 gene occurs in approximately <NUM>-<NUM>% of breast cancers. It is strongly associated with increased disease recurrence and a poor prognosis. Over-expression is also known to occur in ovarian, stomach, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. For example, HER-<NUM> is overexpressed in approximately <NUM>-<NUM>% of patients with gastric cancer and in <NUM>% of salivary duct carcinomas.

Diverse structural alterations have been identified that cause ligand-independent firing of this receptor, doing so in the absence of receptor over-expression.

HER2 is found in a variety of tumors and some of these tumors carry point mutations in the sequence specifying the transmembrane domain of HER2. Substitution of a valine for a glutamic acid in the transmembrane domain can result in the constitutive dimerization of this protein in the absence of a ligand. HER2 mutations have also been found in non-small-cell lung cancers (NSCLC) and can direct treatment.

HER2 is the target of the monoclonal antibody trastuzumab (marketed as Herceptin). Trastuzumab is effective only in cancers where HER2 is over-expressed. One year of trastuzumab therapy is recommended for all patients with HER2-positive breast cancer who are also receiving chemotherapy. An important downstream effect of trastuzumab binding to HER2 is an increase in p27, a protein that halts cell proliferation. Trastuzumab, a humanized IgG1 antibody, is disclosed in European patent application <CIT>. <NPL> describe that Trastuzumab mediates antibody-dependent cell-mediated cytotoxicity via binding to Fc gamma RIIA on leucocytes. <NPL> describe in vivo efficacy of afucosylated Trastuzumab in the treatment of HER2-amplified breast cancer.

Another monoclonal antibody, pertuzumab, which inhibits dimerization of HER2 and HER3 receptors, was approved by the FDA for use in combination with trastuzumab in June <NUM>.

Additionally, NeuVax (Galena Biopharma) is a peptide-based immunotherapy that directs "killer" T cells to target and destroy cancer cells that express HER2. It has entered phase <NUM> clinical trials.

HER2 testing is performed in breast cancer patients to assess prognosis and to determine suitability for trastuzumab therapy. It is important that trastuzumab is restricted to HER2-positive individuals as it is expensive and has been associated with cardiac toxicity. For HER2-negative tumors, the risks of trastuzumab clearly outweigh the benefits.

Thus, there is a need for the development of novel, more effective antibodies that can be used in follow-up therapies when results of the gold-standard therapy with trastuzumab (and chemotherapy) are not satisfying or as an alternative in combination with existing antibodies.

Aim of the study underlying the present invention was to generate a large quantity of high-affinity antibodies against HER2 in order to find new molecules with a novel mechanism of action in comparison to existing antibodies and therapies, such as trastuzumab and pertuzumab.

The present invention relates to a monoclonal antibody that specifically binds to HER2, or a fragment or derivative thereof or a polypeptide that contains at least a portion of said antibody that is sufficient to confer specific HER2 binding to the polypeptide, wherein said antibody binds to the human Fc receptor and induces FcR mediated signaling pathways. The antibody of the invention is defined in claims <NUM>-<NUM>. Other antibodies are described for comparative purposes only.

The antibody according to the invention binds to a different epitope than trastuzumab.

The present document also describes a method of treating an HER-<NUM> mediated disease in a patient, comprising administering to a patient a pharmaceutically effective amount of the antibody according to the invention. It is understood that any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the invention for use in a method for treatment.

The present invention further relates to a pharmaceutical composition defined in claim <NUM>, comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the antibody according to the invention. Said pharmaceutical composition can be administered to a patient in a method of treating cancer. Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.

The term "rabbit" according to the invention means an animal of the members of the taxonomic order Lagomorpha, which includes the families (hares and rabbits) and Ochotonidae (pikas), preferably of genus Oryctolagus.

The term "antibody" encompasses the various forms of antibody structures including, but not being limited to, whole antibodies and antibody fragments as long as it shows the properties according to the invention.

The term "rabbit monoclonal antibody "according to the invention means a monoclonal antibody produced by immunizing a rabbit and isolated from an antigen producing cell of said rabbit as well as such an antibody which is further modified, preferably a humanized antibody, a chimeric antibody, a fragment thereof, or a further genetically engineered and recombinant produced antibody as long as the characteristic properties according to the invention are retained. Preferably the antibody is from a B cell or a rabbit hybridoma cell of said rabbit.

The term "antibody producing cell" according to the invention means a rabbit B cell which produce antibodies, preferably a B cell or rabbit hybridoma cell.

"Native antibodies" are usually heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

"Percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-<NUM>, ALIGN or Megalign (DNASTAR) software.

The terms "Fc receptor" or "FcR" as used here refers to a human receptor that binds to the Fc region of an antibody. FcRs bind IgG antibodies and include receptors of the FcyRI, FcyRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRll receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review <NPL>)). FcRIIIA (CD16a) mediaties ADCC. FcRs are reviewed in <NPL>); <NPL>); and <NPL>). These and all other FcRs are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (<NPL>) and <NPL>)) and mediates slower catabolism, thus longer half-life.

The "constant domains (constant parts)" are not involved directly in binding of an antibody to an antigen, but exhibit e.g. also effector functions. The heavy chain constant region that corresponds to human IgG1 is called γ1 chain. The heavy chain constant region that correspond to human IgG3 is called y3 chain. Human constant γ heavy chains are described in detail by<NPL>), and by <NPL>; <NPL>. Constant domains of IgG1 or IgG3 type are glycosylated at Asn297. "Asn <NUM>" according to the invention means amino acid asparagine located at about position <NUM> in the Fc region; based on minor sequence variations of antibodies, Asn297 can also be located some amino acids (usually not more than +<NUM> amino acids) upstream or downstream.

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

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by <NUM>% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by <NUM>% or more. An exemplary competition assay is provided herein.

"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell- mediated reaction in which nonspecific cytotoxic cells that express FcRs (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FCYRIII. FCR expression on hematopoietic cells is summarized in Table <NUM> on page <NUM> of <NPL>. The term "Antibody-dependent cellular phagocytosis" and "ADCP" refer to a process by which antibody-coated cells are internalized, either in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils and dendritic cells) that bind to an immunoglobulin Fc region.

C1q" is a polypeptide that includes a binding site for the Fc region of an immunoglobulin. C1q together with two serine proteases, C1r and C1s, forms the complex C1, the first component of the complement dependent cytotoxicity (CDC) pathway. Human C1q can be purchased commercially from, e.g. Quidel, San Diego, California.

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 may be further divided into subclasses (isotypes), e.g., IgG<NUM>, IgG<NUM>, IgG<NUM>, IgG<NUM>, IgA<NUM>, and IgA<NUM>. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, δ, ε, γ, and µ, respectively.

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.

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

Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in <NPL>).

A "variant Fc region" comprises an amino acid sequence which differs from that of a "native" or "wildtype" sequence Fc region by virtue of at least one "amino acid modification" as herein defined.

The term "Fc-variant" as used herein refers to a polypeptide comprising a modification in the Fc domain. For all positions discussed in the present invention, numbering is according to the EU index. The EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody (<NPL>. ) The modification can be an addition, deletion, or substitution. Substitutions can include naturally occurring amino acids and non-naturally occurring amino acids. Variants may comprise non-natural amino acids.

The term "Fc region-containing polypeptide" refers to a polypeptide, such as an antibody or immunoadhesin (see definitions below), which comprises an Fc region.

The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of an antibody. A FcR which binds an IgG antibody (a gamma receptor) includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see review in <NPL>). FcRs are reviewed in <NPL>; <NPL>; and <NPL>. Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (<NPL> and <NPL>).

By "IgG Fc ligand" as used herein is meant a molecule, preferably a polypeptide, from any organism 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 FcyRs, FcyRs, FcyRs, FcRn, Clq, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcyR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that are homologous to the FcyRs (<NPL>). Fc ligands may include undiscovered molecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gamma receptors. By "Fc ligand" as used herein is meant a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc/Fc ligand complex.

By "Fc gamma receptor", "FcyR" or "FcgammaR" as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and is encoded by an FcyR gene. In humans this family includes but is not limited to FcyRI (CD64), including isoforms FcyRIA, FcyRIB, and FcyRIC; FcyRII (CD32), including isoforms FcyRIIA (including allotypes H131 and R131), FcyRIIB (including FcyRIIB-I and FcyRIIB-<NUM>), and FcyRllc; and FcyRIII (CD <NUM>), including isoforms FcyRIIIA (including allotypes VI <NUM> and F158) and FcyRlllb (including allotypes FcyRIIB-NAI and FcyRIIB-NA2) (<NPL>), as well as any undiscovered human FcyRs or FcyR isoforms or allotypes. An FcyR 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 (CD <NUM>), and FCYRIII-<NUM> (CD <NUM>-<NUM>), as well as any undiscovered mouse FcyRs or FcyR isoforms or allotypes.

By "FcRn" or "neonatal Fc Receptor" as used herein is meant a protein that binds the IgG antibody Fc region and is encoded at least in part by an FcRn gene. The FcRn may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, the functional FcRn protein comprises two polypeptides, often referred to as the heavy chain and light chain. The light chain is beta-<NUM>-microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise noted herein, FcRn or an FcRn protein refers to the complex of FcRn heavy chain with beta-<NUM>-microglobulin.

An "immunoconjugate" means 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 radioactive isotope.

"Antibody fragments" comprise a portion of a full-length antibody, preferably the variable regions 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, e.g., described in <NPL>.

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 "humanized antibody" or "humanized version of an antibody" refers to antibodies for which both heavy and light chains are humanized as a result of antibody engineering. A humanized chain is typically a chain in which the V-region amino acid sequence has been changed so that, analyzed as a whole, is closer in homology to a human germline sequence than to the germline sequence of the species of origin. Humanization assessment is based on the resulting amino acid sequence and not on the methodology per se.

The terms "specifically binding, against target, or anti-target antibody ", as used herein, refer to binding of the antibody to the respective antigen (target) or antigen-expressing cell, measured by ELISA, wherein said ELISA preferably comprises coating the respective antigen to a solid support, adding said antibody under conditions to allow the formation of an immune complex with the respective antigen or protein, detecting said immune complex by measuring the Optical Density values (OD) using a secondary antibody binding to an antibody according to the invention and using a peroxidase-mediated color development.

The term "antigen" according to the invention refers to the antigen used for immunization or a protein comprising said antigen as part of its protein sequence. For example, for immunization a fragment of the extracellular domain of a protein (e.g. the first <NUM> amino acids) can be used and for detection/assay and the like the extracellular domain of the protein or the full length protein can be used.

The term "specifically binding" or "specifically recognized" herein means that an antibody exhibits appreciable affinity for an antigen and, preferably, does not exhibit significant cross-reactivity.

"Appreciable" binding affinity includes binding with an affinity of at least <NUM>-<NUM>M, specifically at least <NUM>-<NUM>M, more specifically at least <NUM>-<NUM>M, or even yet more specifically at least <NUM>-<NUM>M.

An antibody that "does not exhibit significant cross-reactivity" is one that will not appreciably bind to an undesirable other protein. Specific binding can be determined according to any art-recognized means for determining such binding, e.g. by competitive binding assays such as ELISA.

All protein terms as used herein refers to the human proteins. If a protein from another species is meant, this is explicitly mentioned.

The "variable region (or domain) of an antibody according to the invention" (variable region of a light chain (VL), variable region of a heavy chain (VH)) as used herein denotes each of the pair of light and heavy chain regions which are involved directly in binding the antibody to the antigen. The variable light and heavy chain regions have the same general structure and each region comprises four framework (FR) regions whose sequences are widely conserved, connected by three complementary determining regions, CDRs.

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 preferably amino acid residues from the "complementary determining regions" or "CDRs". The CDR sequences are defined according to <NPL>). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable region. For example, a heavy chain variable region may include a single amino acid insert (residue 52a according to Kabat) after residue <NUM> of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue <NUM>. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

The term "cancer" as used herein may be, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric 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 cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. Preferably such cancer is a breast cancer, colon cancer, lung cancer, or pancreatic cancer.

The present invention originates from making use of the technologies established by the inventors, which allow for the production of a large amount of diverse molecules with different properties.

The inventors produced more than <NUM> supernatants of B-cells and tested them, out of which they identified more than <NUM> antibodies, which bind to HER-receptors mono- or oligospecific in ELISA assays.

The RNA and amino acid sequences of <NUM> molecules were determined, and respective antibodies were cloned, expressed and purified, generating about <NUM> recombinant chimeric antibodies. This genetic modification and purification of the antibodies allows for specific biochemical testing of defined amounts of antibody, thus appropriate quantitative evaluation and for consequently for in vivo use as unwanted immune defense reactions are minimized.

The recombinant monoclonal antibodies were tested and compared in vitro and in vivo. Surprisingly, several molecules were found that show at least a <NUM>-fold increase of FcyRllla mediated activity in JIMT-<NUM> cells, i.e. a higher activation of the NFAT pathway than trastuzumab /Herceptin. Moreover, some of the antibodies were found to bind to different epitopes than the gold-standard antibodies, trastuzumab (TZ) and pertuzumab (PZ), for breast cancer therapy.

Therefore, the present invention relates to a monoclonal humanized antibody that specifically binds to HER2, or a fragment or derivative thereof or a polypeptide that contains at least a portion of said antibody that is sufficient to confer specific HER2 binding to the polypeptide, comprising:.

Other antibodies are described for comparative purposes only.

In one embodiment, the antibody according to the invention is a monoclonal IgG antibody. Preferably, the antibody according to the invention is a monoclonal IgG1 antibody.

Preferably, the antibody comprises a heavy chain variable (VH) region that is at least <NUM>%, or <NUM>% identical to SEQ ID NO: <NUM>, and most preferred the VH region comprises SEQ ID NO: <NUM>.

In certain embodiments, a VH sequence having at least <NUM>%, or <NUM>% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, whereby the antibody retains the ability to bind specifically according to the invention to the respective antigen. In certain embodiments, a total of <NUM> to <NUM> amino acids have been substituted, inserted and/or deleted in each of said VH sequences. Substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

Most preferred, the VH sequence is SEQ ID NO:<NUM>.

Preferably, the antibody comprises a light chain variable (VL) region that is at least <NUM>%, or <NUM>% identical, and most preferred the VL region comprises SEQ ID NO: <NUM>.

In some embodiments, a VL sequence having at least <NUM>%, or <NUM>%o identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, whereby the antibody retains the ability to bind specifically to the respective antigen. In certain embodiments, a total of <NUM> to <NUM> amino acids have been substituted, inserted and/or deleted in said VL sequences. The substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

Most preferred, the VL sequence is SEQ ID NO:<NUM>.

Further preferred is that the VL sequences according to the invention comprise a mutation at position <NUM>. Preferably, the VL sequences comprising a sequence of SEQ ID NO: <NUM>, comprise a mutation at position <NUM>. Preferably the mutation is a Cysteine to Serine mutation. However, it can also be a different amino acid substitution. The VL sequences may of course also comprise further mutations as detailed above.

The antibody of the invention comprises the VH region and VL region comprising the respective CDR1, CDR2 and CDR3 regions and the respective FR1, FR2, FR3, and FR4 regions of the antibody designated as B100 (corresponding to MABD B100).

A monoclonal antibody according to the invention is a humanized antibody.

The present invention also encompasses an antibody that specifically binds to HER2, or a fragment or derivative thereof or a polypeptide that contains at least a portion of said antibody that is sufficient to confer HER2 binding specificity, wherein said antibody binds to the human Fc receptor and induces FcR mediated signaling pathways, as defined in claim <NUM>.

Preferably, the antibodies according to the invention show an increased induction of FcR mediated signaling pathway, when compared to commercially available antibodies.

Surprisingly, the inventors identified several molecules that show at least <NUM> - fold increase (Fol: Fold of induction) of FcyRllla mediated activity (cf. <FIG>, Example <NUM>), i.e. at least a <NUM>-fold higher activation of the NFAT pathway than trastuzumab. Trastuzumab exhibits a maximum Fold of induction (Fol) of <NUM> (cf.

More specifically, the antibody according to the invention may increase the Fc receptor signaling activity in an FcyRllla assay by at least <NUM>-fold, preferably at least <NUM>-fold, more preferably at least <NUM>-fold, most preferably <NUM>-fold or more (cf. Example <NUM>, <FIG>) c)).

It is preferred that an antibody according to the invention increases the Fc receptor signaling activity in an FcyRllla assay by <NUM> -fold, preferably by <NUM> -fold, more preferably by <NUM> -fold and most preferably by <NUM> -fold.

The antibodies according to the invention are also more potent as commercially available antibodies.

In SBKR-<NUM> cells, the antibodies according to the invention, show a stimulation of FcR signaling that is preferably more than <NUM>-fold, more preferably more than <NUM>-fold, <NUM>-fold and more preferably more than <NUM>-fold at an EC50 of <NUM> ng/ml. A preferred antibody may increase FcR signaling by <NUM>-fold at an EC50 of <NUM> ng/ml (cf. This reflects a much higher signaling potency as Trastuzumab. Trastuzumab exhibits a comparable increase of signaling at an EC50 of <NUM> ng/ml.

This increased activity in comparison to conventional antibodies used in cancer therapy clearly shows its superiority and outstanding potential for the use in the treatment of HER2-mediated diseases.

In order to find novel, more effective monoclonal antibodies than those that are commercially available and conventionally used in cancer therapy, the inventors selected the most promising candidate antibodies and tested them in competition assays (Example <NUM>).

Surprisingly, several molecules were found to bind to different epitopes as the gold-standard antibodies for breast cancer therapy, trastuzumab (TZ) and pertuzumab (PZ), i.e. exhibiting a different and unique mode of action. In combination with their increased activity in NFAT pathway stimulation assays (Example <NUM>, <FIG>), the differential binding characteristic makes them ideal novel reagents for the use in treating HER2-mediated diseases.

Therefore, the antibody according to the invention binds to a different epitope than trastuzumab. Preferably, the antibody according to the invention also bind to a different epitope than pertuzumab.

This means that the antibody according to the invention does not compete with trastuzumab in an epitope competition assay (<FIG>).

Also preferred is an antibody according that does not compete with pertuzumab in an eptitope competition assay. With the antibodies designated as B106 and B115 being an exception, it is preferred that the antibodies do not compete with pertuzumab in an epitope competition assay (<FIG>).

The antibodies according to the invention have the advantage to be very potent when it comes to binding to their target. They exhibit a strong binding capacity to their antigen, HER <NUM>, but not to other receptors. The binding properties of the antibodies were studied in biochemical enzyme-linked immunosorbent assays (ELISA- cf. Example <NUM> and <NUM>), and are exemplified in <FIG> and <FIG>.

Preferred antibodies according to the invention, show a half maximal effective concentration (EC50) of less than <NUM> ng/ml, preferably of more than <NUM> ng/ml in experiments as described in Example <NUM>). A preferred antibody, MABD B100, shows an EC50 of <NUM>,<NUM> ng/ml which is comparable to the EC50 of Trastuzumab and Pertuzumab (cf.

Strikingly, the antibodies according to the invention also show a very strong binding to their antigen in experiments in which HER <NUM> is expressed in the SK-BR-<NUM> cell line (cf. Example <NUM>). Preferably, the antibodies exhibit an EC50 of less than <NUM> ng/ml, preferably less than <NUM> ng/ml. A preferred antibody according to the invention, MABD B100, shows an EC50 of <NUM> ng/ml which is comparable to the EC50 of Trastuzumab and Pertuzumab.

The antibodies according to the invention are also very specific in their binding properties. The show a strong binding to HER <NUM>, but not to the homologous receptors HER1, HER3 or HER <NUM>. This is examplied in <FIG>. Strikingly, the inventors found that this is independent of the concentrations used. All antibodies tested show specific binding to HER2 within the concentration range of <NUM> ng/mL to <NUM> ng/mL. Even at a concentration of more than <NUM>-fold the EC50 of HER2 ELISA, no signal of binding to HER1, HER3 and HER4 was detected.

The inventors also found that the antibodies according to the invention do not only bind to human HER2, but may also be capable of binding to HER2 orthologues. It is preferred that antibodies according to the invention show strong binding to human and cynomolgus HER2 receptors. They may show partial binding to rat HER2 receptor. As it is shown in <FIG>), the binding of various antibody according to the invention, to human and cynomolgus HER2 receptors was at a comparable strenght, with similar EC50 values and the antibodies tested also showed a partial reactivity for rat HER2 (EC50 >100ng/mL), but no reactivity to murine HER2.

Surprisingly, and in contrast to commercially available antibodies and antibodies of prior art, the inventors found that the antibodies of this invention are capable of inducing apoptosis with an efficacy comparable to a cytotoxic drug like camptothecin. This is an outstanding activity that will be additive to the activity of other HER2 antibodies with different modes of action. There is no risk for additional toxicities. In contrast to Trastuzumab and Pertuzumab, the antibodies of the invention are capable of inducing apoptosis in at least <NUM>%, preferably more than <NUM>%, more than <NUM>%, <NUM>%, <NUM>% and most preferred more than <NUM>% of cells in SK-BR-<NUM> cell line experiments compared to the positive control camptothecin. In one embodiment of the invention, the antibody shows an induction of apoptosis in <NUM>% of cells, in contrast to only <NUM>% for Trastuzumab and <NUM>% for Pertuzumab (<FIG>).

As said before, a monoclonal antibody according to the invention is a humanized antibody.

The humanized antibodies according to the invention maintain the favorable properties of their chimeric versions. For example, they remain their strong binding capacity and potency. Preferred humanized antibodies according to the invention show an EC50 of less than <NUM> ng/ml. In other embodiments, the antibody exhibits an EC <NUM> of less than <NUM> ng/ml, less than <NUM> ng/ml, less than 7ng/ml or less than <NUM> ng/ml, most preferred of <NUM> ng/ml. <FIG> shows examples of some of the preferred antibodies of the invention and their potency.

Also, the humanized versions of the antibodies according to the invention show strong binding capacity in SK-BR-<NUM> experiments (cf. e.g. <FIG>, Example <NUM>). Preferred humanized antibodies according to the invention show an EC50 of less than <NUM> ng/ml. In other embodiments, the antibody exhibits an EC <NUM> of less than <NUM> ng/ml, less than <NUM> ng/ml, less than <NUM> ng/ml or less than <NUM> ng/ml. or most preferred, less than <NUM> ng/ml. <FIG> shows example of some of the preferred antibodies of the invention and their potency.

The humanized antibodies according to the invention are also capable of inducing strong Fcy-receptor signaling. Preferably, the Fcy signaling is comparable to the signaling of the chimeric versions of the antibodies or more potent. It is also preferred that the induction of Fcy signaling is stronger as for commercially available antibodies. In certain embodiments of the invention, the humanized antibodies have an EC <NUM> of less than <NUM> ng/ml, preferably less than <NUM> ng/ml, less than <NUM> nt/ml, less than <NUM> ng/ml and most preferably less than <NUM> ng/ml.

As noted before, in contrast to commercially available antibodies and antibodies of prior art, the antibodies of this invention are capable of inducing apoptosis. This holds also true for the humanized version of the antibodies. The humanized antibodies of the invention show an induction of apoptosis of at least <NUM>%, preferably more than <NUM>%, more than <NUM>%, <NUM>%, <NUM>% and most preferred more than <NUM>% (<FIG>, Example <NUM>).

Strikingly and in contrast to the commercially available antibodies the inventors surprisingly found that the antibodies of the invention are capable of greatly reducing tumor burden in mice. In a HTM-SK-BR-<NUM> tumor model, the antibodies according to the invention are capable of reducing the size of a tumor and the amount of cancer cells dramatically, in contrast to Trastuzumab and Pertuzumab.

Preferred antibodies of the invention can reduce the tumor cell number by more than <NUM>%, preferably more that <NUM>% and most preferred of more than <NUM>%, when compared to Trastuzumab.

The inventors found a strong anti-tumor and anti-metastatic activity of the antibodies according to the invention also when analyzing histological sections and tumor cell number of different tissues via flow cytometry. In contrast to commercially available antibodies such as Trastuzumab and Pertuzumab and other antibodies of prior art, the antibodies of the invention strongly reduce tumor cell numbers. For example, tumor cell number is reduced in lung, liver and brain tissue after treatment with said antibodies (cf.

Furthermore, the antibodies of the invention can efficiently inhibit metastasis of tumor cells, in contrast to commercially available antibodies and to other antibodies of prior art. For example, the antibodies can inhibit the dissemination of tumor cells into the bone marrow in contrast to Trastuzumab and Pertuzumab (<FIG>).

Due to the specific and favorable properties, the antibodies according to the invention are particularly suited in the treatment of a disease in which the dysregulation of their target antigen is the underlying reason. Due to these specific properties, they are much better suited than commercially available antibodies and other antibodies of prior art.

Therefore, the antibodies according to the invention are especially useful for the treatment of diseases where the dysregulation of the HER2 is the underlying reason.

Therefore, the invention also encompasses an antibody according to the invention for the use in the treatment of cancer.

Thus, the present document also describes a method of treating an HER-<NUM> mediated disease in a patient, comprising administering to a patient a pharmaceutically effective amount of the antibody according to the invention. The method of treatment is not within the scope of the invention.

Moreover, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the antibody according to the invention, as defined in claim <NUM>.

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. The step of administration is not within the scope of the present invention. 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.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. 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.

One aspect of the invention is a pharmaceutical composition according to the invention for use in the treatment of cancer, as defined in this application.

Another aspect of the disclosure which is not claimed is a method of treating an HER-<NUM> mediated disease in a patient, comprising administering to a patient the pharmaceutical composition according to the invention. Such HER-<NUM> mediated diseases may include cancer.

The term "cancer" as used herein may be, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric 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 cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. Preferably such cancer is a breast cancer, colon cancer, lung cancer, or pancreatic cancer. Most preferably the cancer is breast cancer.

The following examples are used in conjunction with the figures and tables to illustrate the invention. Examples about other antibodies than defined in claims <NUM>-<NUM> are provided for comparative purposes only.

The FcyRllla signaling assay, which is commercially available e.g. from Promega, allows for early detection of FcyRllla receptor signaling, thus for effective antibody candidate selection.

Target cells (Jurkat), exhibiting the respective antigen (HER2), are incubated with sample antibodies and JIMT-<NUM> cells. The Jurkat cell line stably expresses the FcyRllla receptor, V158, possessing a high affinity to human IgG Fc fragment and contains a NFAT-RE-Promotor site, allowing for the induction of luciferase expression. Upon binding of the antibody to the target (HER2 expressing) cells, Fc mediated binding of the antibody to the FcyRllla receptor, activates the NFAT pathway, thus the expression of luciferase. Addition of the luciferase substrate (Luciferin), activates a pathway leading to the photoluminescence, which can be measured, and correlated qualitatively and semi-quantitatively to the binding of the antibody to the receptor, i.e. to its immunological activity. Signal strength of Luciferase correlates with the number of receptors activated.

Candidate antibodies were selected in respect to their Fold induction (Fol) of FcyRllla activation for the subsequent generation of chimeric antibodies. The resulting recombinant antibodies were then tested in follow-up experiments.

<FIG>: Sequences (amino acids in one letter code).

Complete sequences of Variable Regions (VR):.

The VL sequences may comprise a Cysteine to Serine mutation at position <NUM>. Complementary Determining Regions (CDR):.

<FIG>: Antibody selection through FcγRIIIa signaling assay
Details of the assay procedure are disclosed in example <NUM>. Candidate antibodies were selected in respect to their Fold of Induction (Fol) of FcyRllla signaling.

<FIG>: Epitope competition assay
None of the selected antibodies compete with trastuzumab for its epitope, while Herceptin (positive control) reduces the POD signal by over <NUM>% (+++). As positive control, the presence of pertuzumab in the pre-incubation mix reduces the POD signal over <NUM>% (+++) in a concentration dependent manner. B106 shows epitope competition with pertuzumab, while B115 shows partial competition for the pertuzumab epitope. No epitope competition is observed with any other of the selected candidate antibodies. <FIG>: HER2 biochemical ELISA.

<FIG>: Receptors binding in ELISA experiments
Antibody binding to different receptors was tested in biochemical ELISA experiments. The experiments were carried out according to the protocol as detailed in Example <NUM> (Protocol <NUM>).

<FIG>: Apoptosis induction on SK-BR-<NUM> cell line
<FIG> shows the results of experiments that were carried out according to the protocol of Example <NUM>. Apoptosis induction was measured as Annexin staining after in-vitro incubation. Camptothecin was used as positive control (set as <NUM>%). MABD B100 shows a uniquely strong apoptosis induction (<NUM>%), in contrast to Trastuzumab (<NUM>%) and Pertuzumab (<NUM>%). <FIG>: HER2 biochemical ELISA of humanized variants.

<FIG>: Binding to SK-BR-<NUM> cell line of humanized Mab variants.

<FIG>: Fcy-receptor signaling of humanized Mab variants
Experiments were carried out according to the protocol as described in Example <NUM>. The four humanized leads maintain in vitro properties of the chimeric version. <FIG>: Apoptosis induction of humanized Mab variants
Experiments were carried out according to the Protocol as described in Example <NUM>. Camptothecin (second row) was used as positive control (set as <NUM>%). The preferred humanized antibodies show a strikingly strong apoptosis induction (<NUM>% to <NUM>%). <FIG>: In vivo experiments
In vivo experiments were carried out in well-established human transplant mouse models (HTM) ( Wege et al. <NUM>/<NUM>/<NUM>). Specifically, the HTM model with the SK-BR-<NUM> tumor cell line was employed. This model is resistant to trastuzumab treatment and shows strong dissemination and metastasis. The experiments comprised <NUM> weeks of establishment of immune system, tumor and metastases and <NUM> weeks of treatment with <NUM>/kg/week i. antibody or placebo. <FIG>: In vivo Profile of MABD B100: Tumor burden after treatment.

<FIG>: In vivo Profile of MABD B100: Anti-Tumor and Anti-Metastatic Activity
Experiments were carried out in the HTM-SK-BR-<NUM> model and results were evaluated qualitatively (Y/N): Metastases by IHC (a) and by flow cytometry (b).

Claim 1:
Monoclonal humanized antibody that specifically binds to HER2, or a fragment or derivative thereof or a polypeptide that contains at least a portion of said antibody that is sufficient to confer HER2 binding specificity, wherein the antibody, antibody fragment or antibody derivative comprises:
a) a heavy chain variable (VH) region that is at least <NUM> % identical to the VH region of SEQ ID NO: <NUM> and comprises a CDR-H1 region of SEQ ID NO: <NUM>, a CDR-H2 region of SEQ ID NO: <NUM> and a CDR-H3 region of SEQ ID NO: <NUM>, and
b) a light chain variable (VL) region that is at least <NUM>% identical to the VL region of SEQ ID NO: <NUM> and comprises a CDR-L1 region of SEQ ID NO: <NUM>, a CDR-L2 region of SEQ ID NO: <NUM> and a CDR-L3 region of SEQ ID NO: <NUM>.