JP6655673B2 - Non-competitive and allosteric anti-human HER3 antibodies against neuregulin and uses thereof - Google Patents

Description

Field of the Invention:
The present invention relates to anti-human HER3 antibodies that are non-competitive for neuregulin (NRG) and their use in diagnostics and therapeutics.

BACKGROUND OF THE INVENTION The human epidermal growth factor receptor ErbB / HER family of receptor tyrosine kinases (RTKs) comprises four members: EGFR (ErbB1 / HER1), HER2 (c-Neu, HER2), HER3 (HER3). ) And HER4 (HER4). The HER receptor has an intracellular C-terminal portion containing an extracellular glycosylation domain consisting of four structural domains, indicated by 1-4, followed by a transmembrane domain, and a kinase domain for coupling to signal transduction pathways. Including. With the exception of HER3, the intracellular region contains tyrosine kinase activity. Signaling is mediated through ligand-induced receptor dimerization, followed by phosphorylation, thereby activating intracytoplasmic signaling pathways. HER2 has no specific ligand. Because it is in a natural "active" conformation. Other HER receptors exist as inactive monomers and the molecule is folded to prevent dimerization. Ligands binding to domains 1 and 3 induce large conformational changes, ultimately exposing the dimerization loop of domain 2 of the receptor. Exposure of this dimerization loop allows for receptor dimerization.

  The HER3 receptor, first described in 1990, is the only HER family member receptor that lacks intrinsic kinase activity and downstream signaling is achieved through heterodimerization. Therefore, the HER3 receptor as a monomer is called "non-self" and cannot form a homodimer. Binding of the ligand neuregulin (NRG) to the HER3 receptor triggers heterodimerization of HER3 with other HER family receptors, preferentially HER2. Within the heterodimer, the HER3 kinase domain acts as an allosteric activator of its HER family pair.

  HER3 is involved in tumorigenesis of various cancers including breast cancer and ovarian cancer (Lee-Hoeflich ST, Cancer Res. 2008; McIntyre E, Breast Cancer Res Treat. 2010; Tanner B, J Clin Oncol. 2006). . HER3 expression correlates with tumor progression in malignant melanoma and metastasis and reduced patient survival, and is associated with reduced survival in ovarian cancer. Importantly, in breast cancer, tumors with low HER2 expression that are not eligible for treatment with Herceptin are often "programmed" to strongly express HER3 (Smith et al. Br. J. Cancer 2004), HER2 ++ tumors that became resistant to Herceptin after prolonged treatment are "reprogrammed" to strongly express HER3 (Narayan, Cancer Res. 2009). Resistance to cetuximab in lung cancer (Wheeler, Oncogene 2008) and colorectal cancer (Lu Cancer Res 2007) was also associated with HER3 overexpression, as well as dysregulation of EGFR internalization / degradation. In recent years, overexpression of HER3 has been significantly associated with worse metastasis-free survival of colorectal cancer (Ho-Pun-Cheung, Int J Cancer 2010). Thus, overexpression of HER3 and compensatory signaling through activation of the PI3K / AKT pathway not only led to the development of resistance to treatment with HER targeted therapies (antibodies and TKIs) (Wheeeler 2008, Lu 2007 , Narayan, 2009, Sergina, 2007), IGFR targeted therapy (Desbois-Mouthon, Clin Cancer Res 2009) and the development of resistance to treatment with chemotherapeutic agents (Kruser, Exp Cell Res 2010).

  All of these findings suggest that HER3 targeting agents, particularly antibodies, may help to further understand the role of HER3 signaling in cancer and may be used as particularly effective immunotherapeutics.

  Currently, no therapeutic anti-HER3 antibodies are commercially available, but the scientific literature strongly emphasizes the benefits of targeting HER3 in tumor therapy. Two human antibodies are currently available at Merrimack Pharmaceuticals, Inc./Sanofiventis (MM-121 antibody; PCT Publication No. 2008/100624) and U3 Pharma AG / Daiichi Sankyo / Amgen (U3- 1287 or AMG-888; PCT Publication No. 2007/077028). The MM-121 antibody has been implicated in phase I clinical trials for non-small cell lung cancer and in phase I / II studies for ER + PR + HER2-breast cancer. The U3-1287 antibody is in phase I of non-small cell lung cancer with erlotinib. The EGFR / HER3 bispecific antibody MEHD7945A (Genentech; PCT Publication No. 2010/108217) is still under research and development. The HER2 / HER3 bispecific antibody MM-111 (Merimac Pharmaceuticals; PCT Publication No. WO 2005/117977, WO 2006/091209) alone in HER2-amplified breast cancer, Or in combination with trastuzumab or lapatinib in phase I / II clinical trials.

  Since all of the above antibodies block the heregulin binding site of the HER3 receptor, treatment of ligand-dependent tumors with these antibodies is reduced. Targeting HER3 with an antibody that is not directed to the heregulin binding site of HER3 allows one to bypass resistance to targeted therapy or chemotherapy in HER2 amplified and resistant breast cancer, To enable the application of targeted therapies for HER2 low breast cancers that are not currently eligible for treatment, or to treat ternary negative breast cancers that express HER3 and for which targeted therapies are not yet available Would be possible.

SUMMARY OF THE INVENTION:
The present invention relates to anti-human HER3 antibodies that are non-competitive against neuregulin (NRG) and their use in diagnostics and therapeutics.

DETAILED DESCRIPTION OF THE INVENTION:
We have characterized a mouse anti-human HER3 antibody designated 9F7-F11 that has unique specificity for HER3 positive cells in the presence of the ligand neuregulin. In particular, the affinity of 9F7-F11 / HER3 is not inhibited in the presence of neuregulin (the 9F7-F11 antibody is non-competitive for neuregulin), and furthermore, the affinity of 9F7-F11 / HER3 We have shown that sex is allosterically enhanced when neuregulin is present in the environment of HER3 positive cells (9F7-F11 is an allosteric anti-HER3 antibody). The 9F7-F11 antibody, which is non-competitive to NRG, inhibits the MAPK, AKT and p53 pathways, blocks the G1 phase of the cell cycle, inhibits cell proliferation, and restores apoptosis of tumor cells. This unique 9F7-F11 antibody reduces tumor growth of NRG-dependent pancreatic cancer and HER2-amplified or triple-negative breast cancer, and was used alone or in combination with the trastuzumab / pertuzumab HER2 antibody. It is more effective to combine with the HER2 specific antibody pertuzumab than the HER2 antibody.

  Therapeutic 9F7-F11 antibodies have a broader range than antibodies that compete for ligands or that are non-competitive for ligands that do not have allosteric effects, targeting a more restricted mechanism of HER3 activation. Will have clinical utility in tumors. Due to its allosteric effect, the 9F7-F11 antibody, which is non-competitive for NRG, can be used to generate other antibodies if neuregulin is secreted by the tumor (autocrine) or by the microenvironment (paracrine). Would be more effective against ligand-dependent tumors. Due to its allosteric effect, the 9F7-F11 antibody would be more effective if resistance mediated by upregulation of neuregulin (ie, resistance to cetuximab in colorectal cancer) occurred. Due to its allosteric effect, the binding of 9F7-F11 can be improved if the receptor is heterodimerized after activation by a ligand. In sum, the non-competitive and allosteric anti-human HER3 antibody 9F7-F11 for NRG can be used to treat conditions where existing therapeutic antibodies are clinically ineffective.

In conclusion, the antibodies of the present invention provide the following advantages over the anti-HER3 antibodies described in the prior art:
-They are allosteric antibodies,
They are non-competitive for neuregulin,
-They have a broader range of effects (for both ligand-independent and ligand-dependent cancers)
They are more effective against autocrine or paracrine ligand dependent tumors (due to their allosteric effect),
They are more effective when resistance (eg, resistance to antibodies or TKIs, or chemotherapy, or antihormones) mediated by up-regulation of HER3 ligand occurs
-They can be used to treat conditions in which existing therapeutic antibodies are clinically ineffective, for example, triple negative breast cancer, pancreatic cancer, other niches such as renal cell carcinoma.

Definition:
The term “neuregulin” has its general meaning in the art and is often used as a synonym for the term “heregulin”. The heregulin family comprises α, β and γ heregulin (Holmes et al., Science, 256: 1205-1210 (1992); US Patent No. 5,641,869; and Schaefer et al. Oncogene 15: 1385-1394 ( 1997)); neu differentiation factor (NDF), glial cell growth factor (GGF); acetylcholine receptor-inducing activity (ARIA); and sensory and motor neuron-derived factor (SMDF). For a review, see Groenen et al. Growth Factors 11: 235-257 (1994); Lemke, G. Molec. & Cell. Neurosci. 7: 247-262 (1996) and Lee et al. Pharm. Rev. 47: 51-. 85 (1995); Falls and D. (2003). "Neuregulins: functions, forms, and signaling strategies." See Experimental Cell Research 284 (1): 14-30.

  The term "HER3" refers to the human HER3 receptor as described in Plowman et al., Proc. Natl. Acad. Sci. USA, 87: 4905-4909 (1990); Kani et al., Biochemistry 44: See also 15842-857 (2005), Cho and Leahy, Science 297: 1330-1333 (2002). HER3 is also known as "HER3".

  The term "anti-human HER3 antibody" refers to an antibody directed against human HER3.

  According to the present invention, “antibody” or “immunoglobulin” have the same meaning and are used equivalently in the present invention. As used herein, the term "antibody" refers to an immunoglobulin molecule and an immunologically active portion of the immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds to an antigen. . Thus, the term antibody encompasses not only intact antibody molecules, but also antibody fragments and variants of antibodies and antibody fragments, including derivatives. In a natural antibody, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (l) and kappa (k). There are five major classes of heavy chains (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinctly different sequence domains. The light chain contains two domains, a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine binding recognition and specificity for the antigen. The constant region domains of the light (CL) and heavy (CH) chains are important in biology, including antibody chain association, secretion, transplacental migration, complement binding, and binding to the Fc receptor (FcR). To provide specific characteristics. The Fv fragment is the N-terminal part of the Fab fragment of immunoglobulin and consists of one light chain and one heavy chain variable portion. Antibody specificity resides in the structural complementarity between the antibody's binding site and the antigenic determinant. The binding site of an antibody is made up primarily of residues from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) affect the overall domain structure and thus the binding site. Complementarity determining region, or CDR, refers to an amino acid sequence that defines both the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The immunoglobulin light and heavy chains each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, the antigen binding site comprises six CDRs, including the CDR set from each V region of the heavy and light chains. Framework region (FR) refers to the amino acid sequence inserted between CDRs.

  The term "chimeric antibody" refers to an antibody that comprises the VH and VL domains of an antibody derived from the 9F7-F11 antibody, and the CH and CL domains of a human antibody.

  According to the present invention, the term "humanized antibody" refers to an antibody that has the framework and constant regions of the variable region of a human antibody, but retains the CDRs of the 9F7-F11 antibody.

  The term "Fab" has a molecular weight of about 50,000 and an antigen-binding activity, and among the fragments obtained by treating IgG with the protease papain, about half and completely about the N-terminal side of the heavy chain. 1 shows antibody fragments in which different light chains are linked to each other through disulfide bonds.

  The term "F (ab ') 2" has a molecular weight of about 100,000 and an antigen-binding activity, and among the fragments obtained by treating IgG with the protease pepsin, disulfide bonds in the hinge region are identified. Refers to an antibody fragment slightly larger than the Fab bound via the

  The term "Fab '" refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, obtained by breaking a disulfide bond in the hinge region of F (ab') 2.

  Single-chain Fv ("scFv") polypeptides are covalently linked VH: VL heterologous proteins that are normally expressed from a gene fusion comprising a VH-encoding gene and a VL-encoding gene joined by a peptide-encoding linker. It is a dimer. "DsFv" is a VH: VL heterodimer stabilized by disulfide bonds. Bivalent and multivalent antibody fragments can be formed spontaneously by association of monovalent scFvs, or can be made by linking monovalent scFvs with peptide linkers, such as bivalent sc (Fv) 2. .

  The term "diabody" refers to a small antibody fragment having two antigen-binding sites, wherein the fragment comprises a heavy chain variable domain (VL) connected to a light chain variable domain (VL) within the same polypeptide chain (VH-VL). Domain (VH). By using a linker that is too short to pair between two domains on the same chain, the domains are paired with complementary domains on another chain, creating two antigen-binding sites.

  When referring to an antibody or nucleotide sequence according to the present invention by "purified" and "isolated", the molecules shown are substantially similar to other biological macromolecules of the same type. It means that it exists in the absence. As used herein, the term "purified" refers to a homologous biological macromolecule that is preferably at least 75%, more preferably at least 85%, even more preferably at least 95%, most preferably Means at least 98% by weight. An “isolated” nucleic acid molecule that encodes a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide; It may include some additional bases or moieties that do not deleteriously affect the characteristics.

Antibodies of the invention:
The present invention provides anti-HER3 antibodies or fragments thereof that are non-competitive and allosteric for isolated neuregulin (NRG). In particular, we have generated hybridomas that produce a mouse anti-HER3 antibody (9F7-F11). We cloned and characterized the variable domains of the light and heavy chains of the 9F7-F11 monoclonal antibody, and thus determined the complementarity determining region (CDR) domains of the antibody as described in Table 1. .

  Thus, the present invention provides a heavy chain (variable domain comprising a sequence selected from the group consisting of SEQ ID NO: 2 for H-CDR1, SEQ ID NO: 3 for H-CDR2, and SEQ ID NO: 4 for H-CDR3. A monoclonal antibody having specificity for HER3.

  The present invention also provides a light chain wherein the variable domain has at least a sequence selected from the group consisting of SEQ ID NO: 6 for L-CDR1, SEQ ID NO: 7 for L-CDR2, and SEQ ID NO: 8 for L-CDR3. A monoclonal antibody having specificity for HER3 (including one CDR).

  The monoclonal antibody of the present invention comprises a heavy chain (variable domain having a sequence selected from the group consisting of SEQ ID NO: 2 for H-CDR1, SEQ ID NO: 3 for H-CDR2, and SEQ ID NO: 4 for H-CDR3). And the light chain (comprising at least one CDR) having a variable domain selected from the group consisting of SEQ ID NO: 6 for L-CDR1, SEQ ID NO: 7 for L-CDR2, and SEQ ID NO: 8 for L-CDR3. Including at least one CDR having a sequence).

  In particular, the present invention provides a heavy chain variable region comprising SEQ ID NO: 2 in the H-CDR1 region, SEQ ID NO: 3 in the H-CDR2 region, and SEQ ID NO: 4 in the H-CDR3 region; and SEQ ID NO: 6 in the L-CDR1 region. And a light chain variable region comprising SEQ ID NO: 8 in the L-CDR2 region and SEQ ID NO: 8 in the L-CDR3 region.

  In one particular embodiment, the heavy chain variable region of the antibody has the amino acid sequence shown as SEQ ID NO: 1 and / or the light chain variable region has the amino acid sequence shown as SEQ ID NO: 5.

  In another embodiment, the monoclonal antibody of the present invention is a chimeric antibody, preferably a chimeric mouse / human antibody. In particular, the mouse / human chimeric antibody may comprise the variable domains of the 9F7-F11 antibody as defined above.

  In another embodiment, the monoclonal antibodies of the invention are humanized antibodies. In particular, in the humanized antibody, the variable domains comprise a human acceptor framework region, and, if present, a human constant domain, and a non-human donor CDR, eg, a mouse CDR as defined above.

  The present invention further provides anti-HER3 antibodies directed against HER3, including, but not limited to, Fv, Fab, F (ab ') 2, Fab', dsFv, scFv, sc (Fv) 2 and diabodies. Provide HER3 fragment.

  In another aspect, the invention has a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. , Polypeptides.

Methods of Making Antibodies of the Invention Anti-human HER3 antibodies of the invention include, but are not limited to, any chemical, biological, genetic, or enzymatic technology alone or in combination. , Can be made by any technique known in the art.

  Once the amino acid sequence of the desired sequence is known, one skilled in the art can easily produce the antibody by standard polypeptide production techniques. For example, they can be synthesized using well-known solid phase methods, preferably using a commercially available peptide synthesizer (eg, an apparatus manufactured by Applied Biosystems, Foster City, Calif.) According to the manufacturer's instructions. Can be done. Alternatively, the antibodies of the present invention can be synthesized by recombinant DNA techniques well known in the art. For example, an antibody can be prepared by incorporating a DNA sequence encoding the antibody into an expression vector and introducing the vector into a suitable eukaryotic or prokaryotic host that will express the desired antibody, followed by expression as a DNA expression product. And can be subsequently isolated using well-known techniques.

  Therefore, a further object of the present invention relates to a nucleic acid sequence encoding an antibody according to the present invention.

  In one particular embodiment, the invention relates to a nucleic acid sequence encoding the VH domain of an antibody obtainable from hybridoma 9F7-F11, or the VL domain of an antibody obtainable from hybridoma 9F7-F11.

  In one particular embodiment, the invention relates to a nucleic acid sequence comprising the sequence of SEQ ID NO: 9.

  In one particular embodiment, the invention relates to a nucleic acid sequence comprising the sequence of SEQ ID NO: 10.

  Typically, the nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

  The terms "vector," "cloning vector," and "expression vector" refer to a vehicle, by which a DNA or RNA sequence (eg, a foreign gene) can be introduced into a host cell, thereby transforming the host. Can promote the expression (eg, transcription and translation) of the introduced sequence.

  Thus, a further object of the invention relates to a vector comprising the nucleic acid of the invention.

  Such vectors may include regulatory sequences, such as promoters, enhancers, terminators, etc., to cause or direct expression of the antibody upon administration to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), the Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987). ), An immunoglobulin heavy chain promoter (Mason JO et al. 1985) and an enhancer (Gillies SD et al. 1983).

  Any expression vector for animal cells can be used as long as the gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSG1βd2-4- (Miyaji H et al. 1990). Other examples of plasmids include replication plasmids that include an origin of replication, or integration plasmids, such as pUC, pcDNA, pBR, and the like. Other examples of viral vectors include adenovirus vectors, retrovirus vectors, herpes virus vectors, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, for example, by transfection of packaging cells, or by transient transfection using a helper plasmid or virus. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv + cells, 293 cells, and the like. Detailed protocols for producing such replication defective recombinant viruses are described, for example, in WO 95/14785, WO 96/22378, US Pat. No. 5,882,877, US Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056, and WO 94/19478.

  A further object of the invention relates to a host cell transfected, infected or transformed by a nucleic acid and / or a vector according to the invention.

  The term "transformation" refers to the introduction of a DNA or RNA sequence of a "foreign" (ie, exogenous or extracellular) gene into a host cell, whereby the host cell expresses the introduced gene or sequence. To produce the desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA has been “transformed”.

  The nucleic acids of the invention can be used to produce antibodies of the invention in a suitable expression system. The term "expression system" means a vector compatible with host cells under conditions suitable for the expression of, for example, a protein encoded by the foreign DNA carried by the vector and introduced into the host cell.

  General expression systems include E. coli. E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (eg, bacteria) and eukaryotic cells (eg, yeast cells, mammalian cells, insect cells, plant cells, etc.). As a specific example, E.I. E. coli, Kleberomyces or Saccharomyces yeast, mammalian cell lines (eg, Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (eg, lymphoblasts, fibroblasts, (Produced from embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), dihydrofolate reductase gene (hereinafter referred to as "DHFR gene"). Deficient CHO cells (Urlaub G et al; 1980), rat YB2 / 3HL. P2. G11.16Ag. 20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells").

  The present invention also relates to a method of producing a recombinant host cell expressing an antibody according to the present invention, said method comprising: (i) transforming a recombinant nucleic acid or vector as described above in vitro or ex vivo, Introducing a host cell, (ii) culturing the obtained recombinant host cell in vitro or ex vivo, and (iii) optionally selecting a cell that expresses and / or secretes the antibody. Such a recombinant host cell can be used for producing the antibody of the present invention.

In another specific embodiment, the method comprises:
(I) culturing hybridoma 9F7-F11 under conditions suitable for allowing expression of the 16D3-C1 antibody; and (ii) collecting the expressed antibody.

  The antibodies of the present invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

  In one particular embodiment, the human chimeric antibodies of the invention obtain nucleic acid sequences encoding VL and VH domains, as described previously, and replace them with the genes encoding human antibody CH and human antibody CL. Can be produced by constructing a human chimeric antibody expression vector by inserting it into an expression vector for animal cells having the following, and expressing the coding sequence by introducing the expression vector into animal cells.

  As the CH domain of the human chimeric antibody, it can be any region belonging to human immunoglobulin, but those of IgG class are suitable, and any one of subclasses belonging to IgG class, for example, IgG1, IgG2, IgG3 and IgG4 Can be used. The CL of the human chimeric antibody can be any region belonging to Ig, and a κ class or λ class can be used.

  Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques well known in the art (Morrison SL. Et al. (1984) and patent documents US Pat. No. 5,202,238). And U.S. Patent No. 5,204,244).

  The humanized antibodies of the present invention obtain nucleic acid sequences encoding CDR domains as described previously, and combine them with (i) the same heavy chain constant region as a human antibody and (ii) the same light chain as a human antibody. It can be produced by constructing a humanized antibody expression vector by inserting it into an expression vector for animal cells having a gene encoding a chain constant region, and expressing the gene by introducing the expression vector into animal cells.

  Humanized antibody expression vectors are of the type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain are present on separate vectors, or of the type in which both genes are present on the same vector (tandem type). Either one may be used. From the viewpoint of ease of construction of a humanized antibody expression vector, ease of introduction into animal cells, and balance between the expression levels of the H chain and L chain of the antibody in animal cells, expression of a tandem humanized antibody is preferred. Vectors are preferred (Shitara K et al. 1994). Examples of tandem humanized antibody expression vectors include pKANTEX93 (WO 97/10354), pEE18, and the like.

  Methods for making humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, eg, Riechmann L. et al. 1988; Neuberger MS. Et al. 1985). . Antibodies can be used, for example, by CDR grafting (European Patent 239,400; PCT Publication No. WO 91/09967; US Patent No. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. Et al. (1994)), and humanization using various techniques known in the art, including the chain shuffling method (US Pat. No. 5,565,332). Can be done. General recombinant DNA techniques for the preparation of such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).

  The Fab of the present invention can be obtained by treating an antibody that specifically reacts with human HER3 with protease papain. Further, Fab is prepared by inserting a DNA encoding the Fab of an antibody into a vector for a prokaryotic or eukaryotic cell expression system, introducing the vector into a prokaryotic or eukaryotic cell (as appropriate), and It can also be produced by expression.

  The F (ab ') 2 of the present invention can be obtained by treating an antibody that specifically reacts with human HER3 with the protease pepsin. Further, F (ab ') 2 can also be produced by binding the following Fab' via a thioether bond or a disulfide bond.

  The Fab 'of the present invention can be obtained by treating F (ab') 2 which specifically reacts with human HER3 with a reducing agent dithiothreitol. Fab ′ is obtained by inserting a DNA encoding the Fab ′ fragment of an antibody into an expression vector for prokaryotic cells or an expression vector for eukaryotic cells, and introducing the vector into prokaryotic cells or eukaryotic cells (as appropriate). It can also be produced by performing its expression.

  The scFvs of the present invention can be obtained as described previously by obtaining cDNAs encoding VH and VL domains, constructing DNAs encoding the scFvs, and transforming the DNAs into expression vectors for prokaryotic cells or eukaryotic cells. It can be produced by inserting into an expression vector and then introducing the expression vector into prokaryotic or eukaryotic cells (as appropriate) to express the scFv. To make humanized scFv fragments, a well-known technique called CDR grafting can be used, which selects complementarity determining regions (CDRs) from donor scFv fragments and identifies them with a three-dimensional structure. Including grafting into the human scFv fragment framework (eg, WO 98/45322; WO 87/02671; US Pat. No. 5,859,205; US Pat. No. 5,585,089). No .: U.S. Pat. No. 4,816,567; European Patent No. 0173494).

  Modifications of the amino acid sequence of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. If the humanized antibody is produced by simply grafting only the CDRs in the VH and VL of the antibody from the non-human animal to the FRs of the VH and VL of the human antibody, the antigen binding activity will It is known to be reduced compared to the original antibody from which it is derived. Several amino acid residues of VH and VL in the FRs as well as in the CDRs of non-human antibodies are also thought to be directly or indirectly involved in antigen binding activity. Thus, by substituting these amino acid residues with different amino acid residues from the human antibody VH and VL FRs, the binding activity will be reduced. In order to solve the problem, in the human CDR-grafted antibody, the human antibody VH and VL FR amino acid sequences that are directly related to the Efforts must be made to identify amino acid residues that are active or that maintain the three-dimensional structure of the antibody and are directly involved in binding to the antigen. Reduced antigen binding activity could be increased by replacing the identified amino acids with amino acid residues of the original antibody from a non-human animal.

  Modifications and changes can be made in the structures of the antibodies of the present invention, and the DNA sequences encoding them, and still obtain a functional molecule encoding the antibody with the desired characteristics.

  In changing the amino acid sequence, the hydropathic index of the amino acid can be considered. The importance of the hydropathic index of amino acids in conferring interactive biological function on a protein is generally understood in the art. The relative hydrophilicity / hydrophobicity characteristics of amino acids contributes to the secondary structure of the resulting protein, which, in turn, interacts with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Are defined to define the interaction of Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1). Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6) Histidine (-3.2); Glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); Asparagine (-3.5); Lysine (-3.9) And arginine (-4.5).

  A further object of the present invention also encompasses function-conservative variants of the antibodies of the present invention.

  A “function conservative variant” is one that changes a given amino acid residue in a protein or enzyme without altering the overall conformation and function of the polypeptide, including: Substitution with an amino acid having similar properties (eg, polarity, hydrogen bonding ability, acidic, basic, hydrophobic, aromatic, etc.). Amino acids other than those indicated as being conserved may be different in the proteins, and thus the similarity of the protein or amino acid sequence between any two proteins having similar functions may be different, for example, According to the alignment scheme, for example, as determined by the Cluster method or the like, it can be 70% -99%, and the similarity is based on the MEGALIGHT algorithm. "Function-conservative variants" also refer to at least 60%, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, even more preferably the percentage of amino acid identity as determined by the BLAST or FASTA algorithm. Also includes polypeptides having at least 95% and having the same or substantially similar properties or functions as the native or parent protein to which it is compared.

  The two amino acid sequences are more than 80%, preferably more than 85%, preferably more than 90%, identical or more than about 90%, preferably more than 95% similar over the full length of the shorter sequence "Substantially homologous" or "substantially similar" when they are (functionally identical). Preferably, similar or homologous sequences are obtained using, for example, a GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pile-up program, or a sequence comparison algorithm such as BLAST, FASTA. Identified by the alignment.

  For example, certain amino acids in the protein structure may be replaced with other amino acids without any appreciable loss of activity. Since the ability and nature of a protein to interact define the biological functional activity of the protein, it is possible to make certain amino acid substitutions within the protein sequence and, of course, within its DNA coding sequence. A protein with similar properties is obtained. Accordingly, it is contemplated that various changes may be made to the antibody sequence of the present invention, or the corresponding DNA sequence encoding the antibody, without an appreciable decrease in its biological activity.

  Certain amino acids can be substituted with other amino acids having similar hydrophobicity indices or scores and still obtain a protein having similar biological activity, i.e., a protein that is still biologically functionally equivalent Is known in the art.

  Thus, as outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. . Exemplary substitutions that take into account various of the above features are well known to those of skill in the art and include arginine and lysine; glutamic and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Is mentioned.

Accordingly, the present invention also provides an antibody comprising a heavy chain,
Variable domains are
H-CDR1, having at least 90% or 95% identity to the sequence shown as SEQ ID NO: 2,
An H-CDR2 having at least 90% or 95% identity to the sequence shown as SEQ ID NO: 3,
An H-CDR3 having at least 90% or 95% identity to the sequence shown as SEQ ID NO: 4,
An L-CDR1 having at least 90% or 95% identity to the sequence shown as SEQ ID NO: 6,
An L-CDR2 having at least 90% or 95% identity to the sequence shown as SEQ ID NO: 7,
An L-CDR3 having at least 90% or 95% identity to the sequence shown as SEQ ID NO: 8
Including
-Heavy chains (variable domains include SEQ ID NO: 2 for H-CDR1, SEQ ID NO: 3 for H-CDR2, and SEQ ID NO: 4 for H-CDR3) and light chains (variable domain is L-CDR1 Has substantially the same affinity as an antibody comprising SEQ ID NO: 6, SEQ ID NO: 7 for L-CDR2, and SEQ ID NO: 8 for L-CDR3, more preferably substantially the same as the mouse anti-HER3 antibody 9F7-F11. Specifically binds to HER3 with the same affinity.

  The antibodies can be assayed for specific binding by any method known in the art. Many different competitive binding assay formats (s) can be used for epitope binning. Immunoassays that can be used include Western blots, radioimmunoassays, ELISAs, "sandwich" immunoassays, immunoprecipitation assays, precipitin assays, gel diffusion sediment assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and complement binding Examples include, but are not limited to, competitive assay systems using techniques such as assays. Such assays are routine and well known in the art (see, eg, Ausubel et al., Eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & sons, Inc., New York. reference). For example, Biacore® (GE Healthcare, Piscataway, NJ) is one of a variety of surface plasmon resonance assay formats conventionally used for epitope binning a panel of monoclonal antibodies. In addition, conventional cross-blocking assays, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988, can be performed.

  Engineered antibodies of the present invention include, for example, antibodies in which framework residues in the VH and / or VL have been modified to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation are likely to contain framework residues that differ from the germline sequence from which the antibody was derived. Such residues can be identified by comparing the framework sequence of the antibody to the germline sequence from which the antibody was derived. "Backmutating" a somatic mutation to a sequence on the germline, eg, by site mutagenesis or PCR-mediated mutagenesis, to return the sequence of the framework region to its germline arrangement. Can be. Such "backmutated" antibodies are also intended to be encompassed by the present invention. Another type of framework modification is to mutate one or more residues in the framework regions, or even one or more CDR regions, to remove T cell epitopes, thereby resulting in a possible antibody And reducing immunogenicity. This approach is also referred to as "deimmunization" and is described in further detail in US Patent Publication No. 20030153043 by Carr et al.

  In addition to or in lieu of modifications made within the framework or CDR regions, the antibodies of the present invention typically comprise one or more functional properties of the antibody, such as serum half-life, complementation with complement. The modifications can be engineered to include alterations in the Fc region, such as to alter binding, binding to Fc receptors, and / or antigen-dependent cellular damage. Furthermore, the antibodies of the invention can be chemically modified (eg, one or more chemical moieties can be attached to the antibody) to re-modify one or more functional properties of the antibody, or Alternatively, it can be modified to alter its glycosylation. Each of these embodiments is described in further detail below. The numbering of the residues in the Fc region is that of the Kabat EU index.

  In one embodiment, the hinge region of CH1 has been modified such that the number of cysteine residues in the hinge region is altered, eg, increased or decreased. This approach is further described in US Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CH1 has been modified, for example, to promote association of the light and heavy chains or to increase or decrease the stability of the antibody.

  In another embodiment, the Fc hinge region of the antibody has been mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are such that the binding of the antibody to SpA is impaired, as compared to the binding of the native Fc-hinge domain to staphylococcal protein A (SpA). It has been introduced into the interface region of the CH2-CH3 domain of the Fc-hinge fragment. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

  In another embodiment, the antibody has been modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in US Pat. No. 6,277,375 to Ward et al. Alternatively, the antibodies may be modified in the CH1 or CL region to increase biological half-life, as described in US Pat. Nos. 5,869,046 and 6,121,022 to Presta et al. Can contain an epitope that binds to a salvage receptor taken from two loops of the CH2 domain of the IgG Fc region.

  In yet another embodiment, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue, such that the antibody has an altered affinity for the effector ligand, but retains the antigen-binding ability of the parent antibody. I have. An effector ligand, the affinity of which is altered, is, for example, the Cl component of the Fc receptor or complement. This approach is described in further detail in US Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

  In another embodiment, one or more amino acids selected from the amino acid residues can be replaced with a different amino acid residue, whereby the antibody binds to the modified C1q and / or has a reduced or Has disappeared complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 by ldusogie et al.

  In another embodiment, one or more amino acid residues are altered, thereby altering the ability of the antibody to bind to complement. This approach is further described in PCT Publication WO 94/29351 by Bodmer et al.

  In yet another embodiment, the Fc region comprises one or more Fc regions to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fc receptors. It has been modified by modifying amino acids. This approach is further described in PCT publication WO 00/42072 by Presta. Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped, and variants with improved binding have been described (Shields, RL et al., 2001 J. Biol. Chen. 276: 6591-6604, WO 2010106180).

  In yet another embodiment, the glycosylation of the antibody has been modified. For example, a deglycosylated antibody can be made (ie, the antibody lacks glycosylation). Glycosylation can be modified, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that eliminate a glycosylation site in the framework of one or more variable regions, thereby eliminating glycosylation at that site. Such deglycosylation can increase the affinity of the antibody for the antigen. Such an approach is described in further detail in US Pat. Nos. 5,714,350 and 6,350,861 by Co et al.

  Additionally or alternatively, such as hypofucosylated or non-fucosylated antibodies with reduced or no fucosyl residues, or antibodies with increased bisecting GlcNac structure, Antibodies with altered types of glycosylation can be made. Such altered glycosylation patterns have been demonstrated to enhance the ADCC ability of antibodies. Such a sugar chain modification can be achieved, for example, by expressing the antibody in a host cell having an altered glycosylation mechanism. Cells having an altered glycosylation mechanism have been described in the art and are used as host cells to express the recombinant antibodies of the invention, thereby producing an antibody having an altered glycosylation. be able to. For example, European Patent No. 1,176,195 by Hang et al. Describes a cell line in which the FUT8 gene encoding fucosyltransferase has been functionally disrupted, and thus the antibody expressed in such a cell line. Shows low fucosylation or lacks fucosyl residues. Thus, in one embodiment, the antibodies of the invention are recombinantly expressed in a cell line that exhibits a hypofucosylated or non-fucosylated pattern, eg, a mammalian cell line deficient in expression of the FUT8 gene encoding fucosyltransferase. Can be produced. PCT Publication WO 03/035835 by Presta describes Lecl3 cells, a mutant CHO cell line that has a reduced ability to attach fucose to sugar chains linked to Asn (297), also here. It also results in hypofucosylation of antibodies expressed in the host cell (see also Shields, RL et al., 2002 J. Biol. Chem. 277: 26733-26740). PCT Publication No. WO 99/54342 by Umana et al. Expresses a glycosyltransferase that modifies glycoproteins (eg, β (1,4) -N-acetylglucosaminyltransferase III (GnTIII)). Thus, an engineered cell line is described, and the antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structure, resulting in increased ADCC activity of the antibody (Umana et al. ., 1999 Nat. Biotech. 17: 176-180). Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies having a modified mammalian glycosylation pattern lacking fucosyl residues (http: // www. eurekainc.com/a&boutus/companyoverview.html). Alternatively, the antibodies of the invention can be produced in yeast or filamentous fungi that can be engineered for a mammalian-like glycosylation pattern and produce antibodies that lack fucose as the glycosylation pattern ( See, for example, EP 1 297 172 B1).

  Another modification of the antibodies herein contemplated by the present invention is PEGylation. Antibodies can be PEGylated, for example, to increase the biological (eg, serum) half-life of the antibody. In order to PEGylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), eg, PEG, under conditions where one or more PEG groups become attached to the antibody or antibody fragment. React with ester or aldehyde derivatives. PEGylation can be performed by an acylation or alkylation reaction with a reactive PEG molecule (or a similarly reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to any form of PEG used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol -Intended to include maleimides. In certain embodiments, the antibody to be PEGylated is a deglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present invention. See, for example, European Patent 0154316 by Nishimura et al. And European Patent 0401384 by Ishikawa et al.

  Another modification of an antibody contemplated by the present invention is a conjugate or protein fusion of at least the antigen binding region of the antibody of the present invention and a serum protein, such as human serum albumin or a fragment thereof, resulting in a resulting molecule. The half-life of is extended. Such an approach is described, for example, in Ballance et al. EP 0 322 094.

  Another possibility is the fusion of at least the antigen-binding region of the antibody according to the invention with a protein capable of binding to serum proteins, for example human serum albumin, which increases the half-life of the resulting molecule. . Such an approach is described in Nygren et al., EP 0486525.

Immune complex:
Antibodies of the invention can be conjugated to a detectable label to form an anti-HER3 immune complex. Suitable detectable labels include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, enzyme labels, bioluminescent labels, or colloidal gold. Methods for making and detecting such detectably labeled immune complexes are well known to those of skill in the art and are described in more detail below.

The detectable label can be a radioisotope detected by autoradiography. Isotopes that are particularly useful for the purposes of the present invention are ³ H, ¹²⁵ I, ¹³¹ I, ³⁵ S and ¹⁴ C.

  The anti-HER3 immune complex can also be labeled using a fluorescent compound. The presence of the fluorescently labeled antibody is determined by exposing the immune complex to light of the appropriate wavelength and detecting the resulting fluorescence. Fluorescently labeled compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

  Alternatively, the anti-HER3 immunoconjugate can be detectably labeled by coupling the antibody to a chemiluminescent compound. The presence of the chemiluminescent-tagged immune complex is determined by detecting the presence of luminescence that occurs during the course of the chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalic esters.

  Similarly, bioluminescent compounds can be used to label anti-HER3 immunoconjugates of the invention. Bioluminescence is a type of chemiluminescence found in biological systems in which catalytic proteins increase the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Bioluminescent compounds useful for labeling include luciferin, luciferase and aequorin.

  Alternatively, the anti-HER3 immune complex can be detectably labeled by linking an anti-human HER3 monoclonal antibody to the enzyme. When the anti-HER3 antibody-enzyme conjugate is incubated in the presence of a suitable substrate, the enzyme moiety reacts with the substrate to produce a chemical moiety, which can be, for example, a spectrophotometric, fluorometric, Or by a visual method. Examples of enzymes that can be used to detectably label multispecific immune complexes include β-galactosidase, glucose oxidase, peroxidase, and alkaline phosphatase.

  Those skilled in the art will also know other suitable labels that can be used in accordance with the present invention. Binding of the marker moiety to the anti-human HER3 monoclonal antibody can be accomplished using standard techniques known in the art. Typical methods in this regard are Kennedy et al., Clin. Chim. Acta 70: 1, 1976; Schurs et al., Clin. Chim. Acta 81: 1, 1977; Shih et al., Int'l J. Cancer 46: 1101, 1990; Stein et al., Cancer Res. 50: 1330, 1990; and Coligan, supra.

  Furthermore, the convenience and versatility of immunochemical detection can be enhanced by using anti-human HER3 monoclonal antibodies conjugated to avidin, streptavidin, and biotin (see, eg, Wilchek et al. (eds.), “Avidin-Biotin Technology,” Methods In Enzymology (Vol. 184) (Academic Press 1990); Bayer et al., “Immunochemical Applications of Avidin-Biotin Technology,” in Methods In Molecular Biology (Vol. 10 ) 149-162 (see Manson, ed., The Humana Press, Inc. 1992).

  Methods for performing immunoassays are well established (eg, Cook and Self, “Monoclonal Antibodies in Diagnostic Immunoassays,” in Monoclonal Antibodies: Production, Engineering, and Clinical Application 180-208 (Ritter and Ladyman, eds. , Cambridge University Press 1995); Perry, “The Role of Monoclonal Antibodies in the Advancement of Immunoassay Technology,” in Monoclonal Antibodies: Principles and Applications 107-120 (Birch and Lennox, eds., Wiley-Liss, Inc. 1995); Diamandis, Immunoassay (Academic Press, Inc. 1996)).

  In another aspect, the invention provides an anti-human HER3 monoclonal antibody-drug conjugate. As used herein, "anti-human HER3 monoclonal antibody-drug conjugate" refers to an anti-human HER3 monoclonal antibody according to the invention conjugated to a therapeutic agent. Such anti-human HER3 monoclonal antibody-drug conjugates are typically administered alone, but also in combination with other therapeutic agents, when administered to a subject, for example, a subject having a HER3-expressing cancer. Has a clinically beneficial effect on HER3-expressing cells.

  In an exemplary embodiment, an anti-human HER3 monoclonal antibody is conjugated to a cytotoxic agent, so that the resulting antibody-drug conjugate is capable of expressing HER3 when taken up or internalized by cells. It exerts a cytotoxic or cytostatic effect on cells (eg, HER3-expressing cancer cells). Particularly suitable moieties for conjugation to an antibody are chemotherapeutic agents, prodrug converting enzymes, radioisotopes or compounds, or toxins. For example, conjugating an anti-human HER3 monoclonal antibody to a cytotoxic agent such as a chemotherapeutic agent or toxin (eg, a cytostatic or cell-killing agent such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin). Can be.

  Useful classes of cytotoxic agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (eg, platinum complexes such as cisplatin, mononuclear platinum, dinuclear platinum, And trinuclear platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapeutic sensitizers, duocarmycin, etoposide, pyrimidine fluoride, ionophore, lexitropsin, nitrosoureas, Platinol, pre-forming compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids and the like.

  Examples of individual cytotoxic agents include androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065 ( Li et al., Cancer Res. 42: 999-1004, 1982), chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinonomycin). , Daunorubicin, decarbazine, docetaxel, doxorubicin, estrogen, 5-fluorodeoxyuridine, etoposide phosphate (VP-16), 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin , Ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, plicamycin, procarbazine, streptozotocin, tenoposide (VM-26) , 6-thioguanine, thiotepa, topotecan, vinblastine, vincristine, and vinorelbine.

  Particularly suitable cytotoxic agents include, for example, dolastatin (eg, auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (eg, enediyne and lexitropsin), duocarmycin, taxanes (eg, paclitaxel and docetaxel) , Puromycin, vinca alkaloid, CC-1065, SN-38 (7-ethyl-10-hydroxy-camptothecin), topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, ethothilone A And B, estramustine, cryptophysin, semadotin, maytansinoids, discodelmolide, eluterobin, and mitoxantrone.

  In certain embodiments, the cytotoxic agent is a conventional chemotherapeutic agent, for example, doxorubicin, paclitaxel, melphalan, vinca alkaloid, methotrexate, mitomycin C, or etoposide. In addition, potent agents such as CC-1065 analogs, calicheamicin, maytansine, analogs of dolastatin 10, rhizoxin, and palitoxin can be linked to anti-HER3 expressing antibodies.

  In a specific variation, the cytotoxic or cytostatic agent is auristatin E (also known in the art as dolastatin-10) or a derivative thereof. Typically, an auristatin E derivative is, for example, an ester formed between auristatin E and a keto acid. For example, auristatin can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP (dimethylvaline-valine-drysoloinine-doraproin-phenylalanine-p-phenylenediamine), MMAF (dovaline-valine-drysoloinine-doraproin-phenylalanine), and MAE ( Monomethylauristatin E). The synthesis and structure of auristatin E and its derivatives are described in U.S. Patent Application Publication No. 20030083263; International Patent Publication Nos. 2002/0888172 and 2004/010957; and U.S. Patent No. 6,884,869. No. 6,323,315; No. 6,239,104; No. 6,034,065; No. 5,780,588; No. 5,665,860; No. 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; No. 410,024; No. 5,138,036; No. 5,076,973; No. 4,986,988; No. 4,978,744; No. 4,879,278; No. 4,816. 4 They are described in, and No. 4,486,414; No. 4.

  In another variation, the cytotoxic agent is a DNA minor groove binder (see, eg, US Pat. No. 6,130,237). For example, in certain embodiments, the minor groove binder is a CBI compound. In another embodiment, the minor groove binder is enediyne (eg, calicheamicin).

  In certain embodiments, the antibody-drug conjugate comprises an anti-tubulin agent. Examples of anti-tubulin agents include, for example, taxanes (eg, Taxol® (paclitaxel), Taxotea® (docetaxel)), T67 (Tularik), vinca alkaloids (eg, vincristine, vinblastine, vindesine, and Vinorelbine), and dolastatin (eg, auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Other anti-tubulin agents include, for example, baccatin derivatives, taxane analogs (eg, epothilones A and B), nocodazole, colchicine and colcemide, estramustine, cryptophysin, semadotin, maytansinoids, combretastatin, discodelmo Ride, and eluterobin. In some embodiments, the cytotoxic agent is another group of anti-tubulin agents, maytansinoids. For example, in a specific embodiment, the maytansinoid is maytansine or DM-1 (see also ImmunoGen, Inc .; Chari et al., Cancer Res. 52: 127-131, 1992).

  In another embodiment, the cytotoxic agent is an antimetabolite. Antimetabolites include, for example, purine antagonists (eg, azathioprine or mycophenolate mofetil), dihydrofolate reductase inhibitors (eg, methotrexate), acyclovir, ganciclovir, zidovudine, vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine, It can be dideoxyuridine, iododeoxyuridine, foscarnet or trifuridine.

  In another embodiment, an anti-human HER3 monoclonal antibody is conjugated to a prodrug converting enzyme. The prodrug converting enzyme may be recombinantly fused to the antibody using known methods, or may be chemically conjugated. Exemplary prodrug converting enzymes are carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase, and carboxypeptidase A.

  Techniques for conjugating therapeutic agents to proteins, particularly antibodies, are well known (eg, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker, Inc., 2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. Eds., 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy, "in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. Eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62: 119-58. See, for example. See also PCT Publication No. WO 89/12624. I want to.)

Diagnostic applications:
A further object of the present invention relates to an anti-human HER3 antibody of the present invention for diagnosing and / or monitoring a cancer disease associated with HER3 expression. Cancer diseases associated with HER3 expression typically include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, Cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colorectal cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, kidney cancer, renal cancer, prostate cancer , Vulvar cancer, thyroid cancer, hepatocellular carcinoma, and various types of head and neck cancer. In one particular embodiment, the cancer diagnosed using the method of the present invention is breast or ovarian cancer. In one particular embodiment, the antibodies of the invention are useful for diagnosing breast and ovarian cancer.

  In one particular embodiment, the antibodies of the invention may be labeled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule, or any other label known in the art as described above. . For example, an antibody of the invention can be labeled with a radioactive molecule by any method known in the art. For example, radioactive molecules include, but are not limited to, radioactive atoms for scintigraphic studies, such as Il23, Il24, In111, Re186, Re188. The antibodies of the present invention may also include spin labels for nuclear magnetic resonance (NMR) imaging (magnetic resonance imaging, also known as mir), such as iodine-123, iodine-131, indium-111, fluorine-. It may be labeled with 19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. After administration of the antibody, the distribution of the antibody in the patient is detected. Methods for detecting the distribution of any particular label are known to those of skill in the art, and any suitable method can be used. Some non-limiting examples include computed tomography (CT), positron tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence, and sonography.

  The antibodies of the invention may be useful for staging cancer diseases associated with HER3 expression (eg, in radiographic imaging). For example, the antibodies of the invention may be useful for staging breast or ovarian cancer. They can be used alone or in combination with other breast or ovarian cancer markers, including but not limited to HER2, CA125, HE4 and mesothelin.

  Typically, the diagnostic method involves the use of a biological sample obtained from the patient. The term "biological sample" as used herein encompasses various sample types obtained from a subject, which can be used in a diagnostic or monitoring assay. Biological samples include, but are not limited to, blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens, or tissue culture fluid or cells obtained therefrom, and progeny Not done. For example, a biological sample comprises cells obtained from a tissue sample collected from an individual suspected of having a cancer disease associated with HER3 expression from the breast or ovary in one particular embodiment. Therefore, biological samples include clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

In one particular embodiment, the invention is a method for diagnosing a cancer disease associated with HER3 expression in a subject by detecting HER3 on cells from the subject using the antibodies of the invention. In particular, the diagnostic method comprises:
(A) A biological sample of a subject having a high possibility of suffering from a cancer disease related to HER3 expression is obtained by combining the antibody according to the present invention with the cells of a biological sample expressing HER3 and the antibody. Contacting under conditions sufficient to form a body;
(B) detecting and / or quantifying the complex, whereby detecting the complex may comprise a step consisting of indicating a cancer disease associated with HER3 expression.

  To monitor cancer disease, the diagnostic methods described in the present invention can be repeated at various time intervals to determine whether the binding of the antibody to the sample has increased or decreased, thereby progressing the cancer disease. Is determined.

Therapeutic uses:
The antibodies, fragments, or immunoconjugates of the invention may be useful for treating any HER3 expressing cancer. The antibodies of the present invention can be used alone or in combination with any suitable agent.

  Examples of cancers expressing HER3 include, but are not limited to, cell carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovary Cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, prostate cancer, vulvar cancer, Thyroid cancer, hepatocellular carcinoma, and various types of head and neck cancers. In one particular embodiment, the cancer treated using the method of the invention is a breast or ovarian cancer.

  Accordingly, an object of the present invention relates to a method for treating a cancer associated with HER3 expression, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody, fragment or immunoconjugate of the present invention.

  In some embodiments, the antibodies of the invention are particularly suitable for treating ligand (ie, NRG) -independent and ligand-dependent cancers.

  In some embodiments, the antibodies of the invention are particularly suitable for treating autocrine or paracrine ligand dependent tumors (due to their allosteric effects).

  In some embodiments, the antibodies of the invention are particularly suitable for treating cancer that is resistant to treatment with an antibody, a tyrosine kinase inhibitor (TKI), a chemotherapeutic agent, or an antihormonal agent.

  In some embodiments, the antibodies of the invention are particularly suitable for treating a cancer selected from the group consisting of ternary negative breast cancer, pancreatic cancer, and renal cell carcinoma.

  In the context of the present invention, the term “treat” or “treatment” as used herein, refers to the disease or condition to which such term applies, or one or more symptoms of such disease or condition. Ameliorate, ameliorate, arrest or prevent its progression.

  According to the present invention, the term "patient" or "patient in need thereof" refers to a human or non-human mammal suffering from or likely to suffer from a cancer involving the expression of human HER3 cancer with the expression of human HER3. Means animal.

  By "therapeutically effective amount" of an antibody of the invention is meant an amount of the antibody sufficient to treat the cancer, at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the antibodies and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will be the disease to be treated and the severity of the disease; the activity of the specific antibody used; the specific composition used, the age, weight, General health, gender and diet; time of administration, route of administration, and excretion rate of the particular antibody used; treatment period; drugs used in combination with or simultaneously with the particular antibody used; It will depend on a variety of factors, including similar factors well known in the medical arts. For example, starting doses of a compound at a lower level than required to achieve a desired therapeutic effect, and gradually increasing the dose until the desired effect is achieved, is within the skill of the art. Well known within.

  In certain embodiments, an anti-human HER3 monoclonal antibody or antibody-drug conjugate is used in combination with a second agent for the treatment of a disease or disorder. When used to treat cancer, the anti-human HER3 monoclonal antibodies or antibody-drug conjugates of the invention can be used in combination with conventional cancer therapies, such as surgery, radiation therapy, chemotherapy, or combinations thereof. . In certain aspects, other therapeutic agents useful for combination cancer therapy with the anti-HER3 antibodies or antibody-drug conjugates of the present invention include anti-angiogenic agents. In some aspects, an antibody or antibody-drug conjugate according to the invention is co-administered with a cytokine (eg, a cytokine that stimulates an immune response to a tumor).

  In some other aspects, other therapeutic agents useful for combination therapy include antagonists of a particular factor involved in tumor growth (eg, EGFR, HER2, or HER4).

  In one particular embodiment, an anti-human HER3 monoclonal antibody or antibody-drug conjugate of the invention is used in combination with an anti-human HER2 monoclonal antibody, such as trastuzumab or pertuzumab.

  In some embodiments, an anti-human HER3 monoclonal antibody or antibody-drug conjugate described herein is used in combination with a tyrosine kinase inhibitor (TKI). BAY43-9006 (Sorafenib, Nexavar®) and SU11248 (Sunitinib, Sutent®) are two such approved TKIs. Other TKIs include imatinib mesylate (Gleevec®, Novartis); gefitinib (Iressa®, AstraZeneca); erlotinib hydrochloride (Tarceva®, Genentech); vandetanib (Zactima) (Registered trademark), AstraZeneca), tipifarnib (Zanestra (registered trademark), Janssen Sirag); dasatinib (Sprycel (registered trademark), Bristol-Myers Squibb); ronafanib (Sarasar (registered trademark), Scheringplough) ); Batalanib succinate (Novatis, Schering AG); lapatinib (Tykerb®, GlaxoSmithKline); nilotinib (Novatis); restaurtinib (Cefalon); pazopanib hydrochloride (glaxosumi) Axitinib (Pfizer); Canertinib dihydrochloride (Pfizer); Peritinib (US National Cancer Institute, Weiss); Tandutinib (Millennium); Bostinib (Weiss); Semaxanib (Sugen, Taiho Pharmaceutical) AZD-2171 (AstraZeneca); VX-680 (Merck, Vertex); EXEL-0999 (Exelixis); ARRY-142886 (Array BioPharma, AstraZeneca); PD-0325901 (Pfizer) AMG-706 (Amgen); BIBF-1120 (Boehringer Ingelheim); SU-6668 (Daiho Pharmaceutical); CP-547632 (OSI); AEE-788 (Novatis); BMS-58664 (Buri) Stall Myers JNK-401 (Celgene); R-788 (Rijel); AZD-1152 HQPA (AstraZeneca); NM-3 (Genzyme Oncology); CP-868596 (Pfizer); BMS- 599626 (Bristol-Myers Squibb); PTC-299 (PTC Therapeutics); ABT-869 (Abbott); EXEL-2880 (Exelixis); AG-024322 (Pfizer); XL-820 (Exelixis) OSI-930 (OSI); XL-184 (Exerixis); KRN-951 (Kirin Brewery); CP-724714 (OSI); E-7080 (Eisai); HKI-272 (Weiss). CHIR-258 (Chiron); ZK-3 4709 (Schering AG); EXEL-7647 (Exerixis); BAY-57-9352 (Bayer); BIBW-2992 (Boehringer Ingelheim); AV-412 (AVEO); YN-968D1 (Advenchen Laboratories) Midostaurin (Novatis); Perifosine (Eterna Zentalis, Celix, National Cancer Institute, USA); AG-024322 (Pfizer); AZD-1152 (AstraZeneca); ON-01910Na (Onconova); And AZD-0530 (AstraZeneca), but are not limited thereto.

Pharmaceutical composition:
For administration, the anti-human HER3 monoclonal antibody or antibody-drug conjugate is formulated as a pharmaceutical composition. Pharmaceutical compositions comprising anti-human HER3 monoclonal antibodies or antibody-drug conjugates can be formulated according to known methods for preparing pharmaceutically useful compositions, wherein the therapeutic molecule is used. Combined with a pharmaceutically acceptable carrier in a mixture. A composition is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by the recipient patient. Sterile phosphate buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known to those skilled in the art. (See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent loss of protein on vial surfaces, and the like.

  The dosage form, route of administration, dosage, and regimen of the pharmaceutical composition will necessarily depend on the condition to be treated, the severity of the affliction, the age, weight, and sex of the patient.

  The pharmaceutical composition of the present invention can be formulated for topical administration, oral administration, parenteral administration, intranasal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intraocular administration.

  Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These are, in particular, isotonic, sterile saline solutions (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts) or, as the case may be, It can be a dried, especially lyophilized composition which allows the reconstitution of the injection solution by the addition of sterile water or saline.

  The dose used for administration may be adapted as a function of various parameters, in particular as a function of the dosage form used, the condition involved, or alternatively the duration of treatment desired.

  To prepare a pharmaceutical composition, an effective amount of the antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

  Dosage forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

  Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylmethylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

  The antibodies of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), for example, inorganic acids such as hydrochloric acid or phosphoric acid, or acetic, oxalic, tartaric acids , And mandelic acid. Salts formed with the free carboxyl groups also include inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide, and isopropylamine, trimethylamine, histidine, procaine. And the like.

  The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Protection of the action of microorganisms can be provided by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

  Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent, as required with various other ingredients enumerated above, followed by sterile filtration. Generally, dispersions incorporate the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. Prepared by In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and lyophilization techniques, whereby powders of the active ingredient and any additional desired ingredients from the previously sterile filtered solution Is obtained.

  The preparation of more concentrated or highly concentrated solutions (assuming use of DMSO as solvent) for direct injection is planned, which results in a very rapid penetration and a high concentration of activity The drug is delivered to a small tumor area.

  Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are readily administered in a variety of dosage forms, eg, in the type of injectable solutions described above, although drug release capsules and the like may be used.

  For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or dextrose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion therapy or injected at the proposed site of injection (eg, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). See). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The administration director will determine the appropriate dose for the individual subject in any event.

  Antibodies of the invention may contain from about 0.0001 to 1.0 mg, or about 0.001 to 0.1 mg, or about 0.1 to 1.0 mg, or even about 10 mg, per dose, within a therapeutic mixture. It can be formulated to include. Multiple doses may be administered.

  In addition to compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable dosage forms include, for example, tablets or other solid dosage forms for oral administration; Release capsules; and any other dosage forms currently used.

  In certain embodiments, the use of liposomes and / or nanoparticles is contemplated for introducing antibodies into host cells. The formation and use of liposomes and / or nanoparticles is known to those skilled in the art.

  Nanocapsules can generally encapsulate compounds in a stable and reproducible manner. Such ultra-fine particles (about 0.1 μm in size) are generally designed using polymers that can be degraded in vivo to avoid side effects due to polymer overloading into cells. Is done. Biodegradable polyalkyl-cyanoacrylic acid nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be easily made.

  Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilamellar vesicles (also called multilamellar vesicles (MLV)). MLVs typically have a diameter between 25 nm and 4 μm. The sonication of MLVs forms small unilamellar vesicles (SUVs) having a diameter in the range of 200-500 ° containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

kit:
Finally, the present invention also provides a kit comprising at least one antibody of the present invention. Kits containing the antibodies of the invention find use in detecting HER3 expression or in therapeutic or diagnostic assays. The kits of the invention may contain the antibody bound to a solid support, such as a tissue culture plate or beads (eg, Sepharose beads). Kits containing antibodies for detecting and quantifying HER3 in vitro, eg, in an ELISA or Western blot, may be provided. Such antibodies useful for detection can be provided with a label, such as a fluorescent or radiolabel.

  The present invention will be further described by the following figures and examples. However, these examples and figures should not be construed as limiting the scope of the invention in any way.

FIG. 1 shows the allosteric interaction of the 9F7-F11 monoclonal antibody with NRG for binding to the HER3 receptor on cells. 9F7-F11 binding to HER3 increases on SKBR3 cells when various concentrations of NRG are added. In contrast, antibody A is unaffected by NRG binding and antibody B is blocked by NRG binding (panel A). Conversely, NRG binding to HER3 is increased on HER2 / HER3 transfected 3T3 fibroblasts when various concentrations of the 9F7-F11 monoclonal antibody are added, while irrelevant IgG is Does not affect the binding of In contrast, free NRG displaces labeled NRG for binding to HER3 (panel B). FIG. 2 quantifies the affinity of 9F7-F11 for the HER3 receptor in the presence or absence of NRG. A 6-fold increased affinity was measured in the presence of NRG (0.47 ± 0.07 nM) compared to the affinity measured in the absence of NRG (2.33 ± 0.30 nM). FIG. 3 shows the inhibition of HER2 / HER3 receptor phosphorylation and the inhibition of downstream PI3K / Akt and ERK signaling by using the anti-HER3 monoclonal antibody 9F7-F11 in BxPC3 pancreatic cancer cells. FIG. 4 shows the effect of 9F7-F11 monoclonal antibody on p53 / MDM2 expression and phosphorylation demonstrated by Western blot (A) and quantified by Image J (B). FIG. 5 shows the effect of the 9F7-F11 monoclonal antibody on the expression of p53-inducible genes p21, Cyclin A2, PERP, Puma, and Bcl-2, as demonstrated by quantitative PCR. Relative mRNA expression was normalized to GAPDH expression. FIG. 6 shows the effect of the 9F7-F11 monoclonal antibody on cell cycle arrest of BxPC3 pancreatic cancer cells. FIG. 7 shows the effect of the 9F7-F11 monoclonal antibody on apoptosis of BxPC3 pancreatic cancer cells (A). Cleavage of caspase-9, which initiates mitochondrial apoptosis, is demonstrated by western blot of cell lysates of BxPC3 cells treated with 9F7-F11 (B). FIG. 8 shows the effect of the 9F7-F11 monoclonal antibody, alone or in combination with cetuximab or trastuzumab, on the growth of BxPC3 pancreatic cancer cells compared to the effect observed with trastuzumab or cetuximab alone. FIG. 9 shows the ADCC effect of 9F7-F11 monoclonal antibody versus trastuzumab on target MDA-MB-453 breast cancer cells. FIG. 10 shows inhibition of tumor progression by 9F7-F11 monoclonal antibody in nude mice xenografted with HER2 amplified / PIK3CA-mut MDA-MB-361 breast cancer cells. FIG. 11 shows the inhibition of tumor progression by a 9F7-F11 monoclonal antibody in nude mice xenografted with three negative PTEN-mut / p53-mut MDA-MB-468 breast cancer cells (A). Kaplan-Meier survival curves were calculated when MDA-MB-468 tumors reached a volume of 2000 mm ³ (B). FIG. 12 shows NRG-dependent, p53-mut-HER2 ^low BxPC3 pancreatic cancer cells xenografted and treated with the 9F7-F11 monoclonal antibody used alone (9F7) or in combination with pertuzumab (P + 9F7). In addition, the tumor progression and survival of nude mice are shown in comparison to pertuzumab alone (P) or vehicle (control) (panel A) and trastuzumab and pertuzumab combination (P + T) (panel B). FIG. 13 shows inhibition of tumor progression by 9F7-F11 monoclonal antibody in nude mice xenografted with shHER3- or shLuc (control) -knockout BxPC3 pancreatic cancer cells (A). HER3 silencing is confirmed by Western blot on xenografts recovered from treated mice (B).

Example 1 Allosteric Effect of 9F7-F11 on HER3 Binding Transfected with HER2 / HER3, which had been previously stimulated with neuregulin β1 (NRG) to promote HER2 / HER3 heterodimer formation The NIH3T3 cell line (about 2 × 10 ⁶ cells) was injected intraperitoneally into one Balb / c mouse. Spleen cells from immunized mice were fused using myeloma PX63Ag8.653 according to the protocol described previously (Salhi et al. Biochem. J. 2004). 1 was of 10 ⁵ per well fused cells were cultured in plates containing HAT medium for the hybridomas selection. Twelve days after fusion, screening of hybridoma supernatants was performed by ELISA using the protein HER3-Fc as antigen. In controls, screening is performed simultaneously using only the distinguishing antigens HER2-Fc and Fc fragments.

Cytometry competition experiments were performed to quantify the ability of NRG to inhibit binding of the antibody to HER3 in a SKBR3 cell-based assay. To this end, 10 ⁵ SKBR3 cells on ice for 1.5 hours preincubated with various concentrations of competing NRG ligands. After washing once with PBS-1% BSA, a concentration of the selected anti-HER3 monoclonal antibody 9F7-F11 that resulted in 50% of maximum binding was added to each well on ice for 1 hour. In some experiments, the NRG ligand and the anti-HER3 antibody 9F7-F11 were co-incubated on ice for 2 hours. The cells were then washed and further incubated with a secondary antibody (Sigma) conjugated to the appropriate FITC at a dilution of 1:60 on ice for 45 minutes, followed by a Quanta device (Beckman Coulter). Was analyzed by cytometry. Competition experiments by FACS demonstrated that the 9F7-F11 antibody did not compete with NRG, suggesting that the antibody did not bind to the NRG binding site (FIG. 1A). The binding of the 9F7-F11 antibody, which is non-competitive for NRG, was even enhanced up to 160% when NRG was added, thus demonstrating the allosteric effect of the 9F7-F11 monoclonal antibody on binding to HER3. In contrast, the binding of the positive control antibody A was not altered by incubation with NRG, and the positive control antibody B showed NRG-dependent binding (FIG. 1A).

Conversely, 10 ⁵ 3T3 fibroblasts transfected with HER3 in a cultured for 2 days, then was starved, then at + 4 ° C. in KREBS buffer with various concentrations of 9F7-F11 monoclonal antibody Incubated for 45 minutes. NRG labeled with d2 cryptate dye was added for an additional 45 minutes. After washing with KREBS, the fluorescence at 620/670 nm was measured using a Pherastar microplate reader. As demonstrated in FIG. 1B, 9F7-F11 binding induced an increase in NRG binding to HER3, whereas irrelevant IgG did not. As a control, different concentrations of free NRG displaced the binding of labeled NRG to the HER3 receptor (FIG. 1B), and therefore allosteric interaction between 9F7-F11 and NRG for binding to HER3. Proven.

Example 2: Addition of NRG induces a 6-fold increase in the affinity of 9F7-F11 for the HER3 receptor
Use Tag-Lite technology developed by CisBio BioAssay Inc., 10 ⁴ HER3 of SNAP tagged HEK cells were labeled with Lumi4- terbium cryptate donor, then, of NRG and various concentrations d2 Co-incubated with acceptor labeled 9F7-F11 monoclonal antibody. After 16 hours of incubation, the fluorescence of Lumi4-terbium and d2 was measured at 620 nm and 665 nm, respectively (60 μs time difference, 400 μs integration time) on a Pherastar FS instrument at 337 nm excitation. As demonstrated in FIG. 2, in the presence of NRG, the dose-dependent increase in 9F7-F11 binding to the HER3 receptor was compared to the lower binding to HER3 measured in the absence of NRG. Was observed. The _Kd value of 9F7-F11 binding to HER3 was calculated to be 0.47 ± 0.07 nM after co-incubation with NRG, while the _Kd was 2.33 ± 0 in the absence of NRG. .30 nm, thus demonstrating that the addition of NRG allosterically induced a 6-fold increase in the affinity of 9F7-F11 for the HER3 receptor.

Example 3: 9F7-F11, an anti-HER3 antibody that is non-competitive to NRG, inhibits HER2 and HER3 phosphorylation, as well as blocks ERK1 / 2 and AKT phosphorylation 500 and 1,000 BxPC3 pancreatic tumor cells were added to each well of a 6-well culture plate at 37 ° C. for 24 hours. After starvating the serum for 16 hours in RPMI complete medium containing 1% FCS and washing further, the cells were incubated with the 9F7-F11 antibody at a concentration of 50 μg / ml, or a negative control antibody at 37 ° C. for 15 minutes or Pre-incubated for 1 hour, then washed, and subsequently stimulated or not stimulated with 100 ng / ml NRG diluent. Thereafter, the cells were washed, peeled off, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.5 mM MgCl ₂ , 1 mM EDTA, 1% triton, 10% glycerol, 0.1 mM phenylmethylsulfonyl chloride, 100 mM fluoride Solubilization was carried out using a buffer containing sodium, 1 mM sodium orthovanadate (Sigma-Aldrich) and one complete protease inhibitor mixture tablet (Roche Diagnostics, Indianapolis, IN). After an incubation time of 30 minutes, the insoluble fraction was removed from the samples by centrifugation and the protein concentration in the cell lysate was determined by the Bradford assay. These protein lysates were mixed directly with Laemmli buffer (1-20 μg total protein depending on target and cell line) and heated at 95 ° C. for 5 minutes. After electrophoresis on 7% SDS-PAGE under reducing conditions, the proteins were transferred to a polyvinylidene difluoride membrane (Millipore), which was then transferred to a TNT buffer (Tris (25 mM pH 7.4, NaCl 150 mM, Tween 0.1%) at 25 ° C. for 1 hour. Primary antibodies directed against the kinase receptor or signaling kinase, and its phosphorylated form, were incubated at 4 ° C. in TNT-5% BSA buffer for 18 hours. After washing five times in TNT buffer, a rabbit, goat or mouse polyclonal antibody conjugated to peroxidase (Sigma-Aldrich) is optionally added to a TNT buffer containing 5% nonfat dry milk at 25 ° C. Added for 1 hour. After washing five times with TNT buffer, blots were visualized using a chemiluminescent substrate (Western lightning Plus-ECL, PerkinElmer).

  Remarkably, the 9F7-F11 antibody blocked ligand-induced phosphorylation on Y1289-HER3 and Y1242-HER2 (FIG. 3). At the same time, the allosteric, non-competitive 9F7-F11 antibody induced inhibition of Akt phosphorylation on Ser473 and of ERK1 / 2 phosphorylation on Thr202 / 204.

Example 4: Anti-HER3 antibody 9F7-F11, which is non-competitive and allosteric for NRG, inhibits MDM2 expression and phosphorylation, along with regulation of the p53 pathway. Similar to BxPC3 described above. Tumor cells were added to each well of a 6-well culture plate at 37 ° C. for 24 hours. After starving the serum for 16 hours in RPMI complete medium containing 1% FCS and washing further, the cells were incubated with the 9F7-F11 antibody at a concentration of 50 μg / ml, or a negative control antibody, at 37 ° C. for 24 hours. Or pre-incubated for 72 hours, followed by washing, followed by stimulation with or without 100 ng / ml NRG diluent. Subsequently, cells were either lysed for SDS-PAGE and Western blot or subjected to RNA extraction, reverse transcription, and quantitative PCR using appropriate primers. As demonstrated in FIG. 4 by Western blot, treatment with 9F7-F11 inhibited MDM2 expression and phosphorylation and increased p53 expression. In this context, treatment with 9F7-F11, as demonstrated by Q-PCR (FIG. 5), results in the expression of p53-inducible genes such as p21 involved in cell cycle and proliferation blockade, and apoptosis. Puma and PERP were adjusted to positively increase. In contrast, expression of the CyclinA2 and Bcl2 genes, which promote proliferation and inhibit apoptosis, respectively, decreased after treatment with the 9F7-F11 antibody, which is non-competitive for NRG (FIG. 5).

Example 5: 9F7-F11, an anti-HER3 antibody that is non-competitive to NRG, blocks the G1 phase of the cell cycle, inhibits proliferation and restores apoptosis 9F7-F11, a HER3-specific antibody against the cell cycle Was evaluated using staining with propidium iodide. Briefly, 300,000 BxPC3 tumor cells per well were cultured in 6-well microtiter plates for 24 hours, after which the serum was starved and taken for an additional 24 hours in RPMI medium without FCS. Synchronization was followed by co-incubation with 100 μg / ml anti-HER3 antibody 9F7-F11 and 100 ng / ml NRG. Permeabilized cells were stained 24 hours later with propidium iodide and then analyzed by cytometry. For proliferation and apoptosis assays, 50,000 BxPC cells / well were seeded (in RPMI-1% FCS) one day before starvation. Then, HER3-specific antibodies 9F7-F11 and NRG were added for 120 hours. Cell proliferation was measured by incorporation of 5-ethynyl-2'-deoxyuridine (EdU) (Invitrogen) conjugated to Alexa Fluor 488 during the last 30 hours of culture. Cell apoptosis was assessed by incubation with fluorescently conjugated Annexin V and 7-aminoactinomycin D (7-AAD; Beckman Coulter). All experiments were performed three times. For caspase-9 analysis, BxPC3 cells were treated and lysed as described above. After SDS-PAGE and Western blot of the cell lysate, activation of caspase-9 through cleavage of the proenzyme was demonstrated using appropriate antibodies. As shown in FIG. 6, treatment of the allosteric 9F7-F11 antibody for 24 hours blocks the G1 phase of the cell cycle, leaving G1 cells untreated cells or cells treated with a control antibody. From 36-38% for BxPC3 cells treated with 9F7-F11. Treatment with 9F7-F11 simultaneously reduced the proportion of BxPC3 cells in S and G2 / m phases (FIG. 6). Treatment of BxPC3 cells with 9F7-F11 monoclonal antibody increased early (18%) and late (12%) apoptosis compared to untreated cells and cells treated with control antibody (FIG. 7A). And simultaneously cleaved pro-caspase-9, which initiates mitochondrial apoptosis (FIG. 7B). Finally, BxPC cell proliferation was inhibited after treatment with the 9F7-F11 antibody for 120 hours. Using the anti-HER2 antibody trastuzumab alone on HER2 ^low BxPC3 cells, no specific effect on cell proliferation was observed, whereas the anti-EGFR antibody cetuximab was more effective than the anti-HER3 antibody 9F7-F11. It was low (FIG. 8). In contrast, the combination of the 9F7-F11 monoclonal antibody and trastuzumab is more effective at inhibiting cell proliferation than the 9F7-F11 / cetuximab combination, indicating that cetuximab is effective against HER2 ^low tumor cells. It suggests that the combination of / 9F7-F11 is additive while the combination of trastuzumab / 9F7-F11 is synergistic. In summary, these results indicate that the NRG non-competitive and allosteric anti-HER3 antibody 9F7-F11 blocks the G1 phase of the cell cycle and restores early and late mitochondrial apoptosis through pro-caspase-9 cleavage. And demonstrated that it inhibited the growth of tumor cells. In this case, the combination of the 9F7-F11 monoclonal antibody and the anti-HER2 antibody may have significant advantages in HER2 ^low tumors.

Example 6: 9F7-F11, an anti-HER3 antibody that is non-competitive and allosteric to NRG, induces antibody-dependent cytotoxicity in breast cancer cells
One day before the ADCC assay, 20,000 MDA-MB-453 tumor target cells obtained from basal-like type 3 negative breast cancer were seeded in a flat-bottom 96-well microplate per well. The MDA-MB-453 cell line expresses about 180,000 HER2 receptors and 21,000 HER3 receptors, but no EGFR expression is observed. After washing with culture medium, 10 μg / ml 9F7-F11 monoclonal antibody was added during 30 minutes, followed by effector cells obtained from peripheral mononuclear cells (PBMC). PBMC were prepared by density gradient centrifugation of healthy donor blood samples obtained from the "Etablissement Francais du Sang". Effector cells / target cells (E / T) were incubated for 24 hours at a 15/1 E / T ratio in a humidified cell incubator. Killing of MDA-MB-453 target cells was performed using lactate dehydrogenase (LDL) from injured cells using a cytotoxicity detection kit (LDH detection kit; Promega G-1780) according to the manufacturer's instructions. Was assessed by measuring the release of Briefly, 50 μl of cell supernatant was carefully transferred to a new flat bottom 96-well microplate and the kit LDH reaction mixture (50 μl / well) was added to each well. After incubation at 37 ° C. for 30 minutes, 50 μl of stop solution (provided in the kit) was added and the absorbance at 490 nm was measured. The following controls were made in each experiment: PBMC only, MDA-MB-453 target cells only (spontaneous release of LDH), PBMC and target cells (antibody-dependent spontaneous release), lysis buffer And target cells (maximum LDH release), antibodies and PBMC, antibodies and target cells. The specific lysis rate for each sample was determined using the following formula: specific lysis rate = (sample value−spontaneous release) / (maximum release−spontaneous release) × 100. As shown in FIG. 9, the 9F7-F11 antibody induced 5-10% specific cytolysis of MDA-MB-453 breast cancer cells using PBMCs from healthy donors 1 and 2. In the same experiment, the positive control trastuzumab induced about 40% lysis due to the fact that MAD-MB-453 expresses 10 times more HER2 receptor than HER3 receptor.

Example 7: Monotherapy of 9F7-F11 in mice xenografted with HER2 amplified MDA-MB-361 breast cancer and three negative MDA-MB-468 breast cancer Athymic 6-8 week old female BALB / c Mice were purchased from Janvier and Charles River Laboratories. HER2-amplified / PIK3CA-mut breast cancer cells MDA-MB361 (10 × 10 ⁶ ) and HER-unamplified / PTEN-mut / p53-mut / ER− / PR− triple-negative breast cancer cells MDA-MB -468 to (3.5 × 10 ⁶ cells) were injected subcutaneously into the right flank of athymic BALB / c nude mice. They both expressed low levels of the HER3 receptor (approximately 10,000 receptors / cell). All in vivo experiments were performed in compliance with the French guidelines for the study of laboratory animals (consent number B34-172-27).

Mice bearing tumors were randomized into different treatment groups when the tumors reached a volume of about 100 mm ³ . Mice were treated by intraperitoneal injection of the HER3 specific 9F7-F11 antibody versus vehicle (PBS). The amount of antibody injected was 300 μg (15 mg / kg) per injection three times a week for 6 consecutive weeks (Q2D-6W). Tumor size was measured twice a week using calipers and volume was calculated by the formula D1 × D2 × D3 / 2. Tumor progression was calculated using the formula [(final volume)-(initial volume)] / (initial volume). The results were also expressed by a Kaplan-Meier survival curve, using the time required for the tumor to reach a final volume determined to be 2,000 mm ³ . The median time difference was defined as the time at which 50% of the mice had the tumor reaching the determined volume.

  As shown in FIG. 10, we compared 55 days after tumor implantation (corresponding to the end date of antibody treatment) compared to the average tumor size measured in mice treated with vehicle. At 40 days), a significant 47 ± 5% decrease in MDA-MB-361 tumor growth in mice treated with 9F7-F11 was observed (p <0.001). At the end of the experiment (97 days), a smaller but significant 21 ± 2% reduction in tumor size was observed in the 9F7-F11 treated group, probably because treatment with 9F7-11 was discontinued after 57 days. .

  As shown in FIG. 11A, the mean tumor volume was significantly lower in mice treated with 9F7-F11 xenografted MDA-MB-468 cells than in the control (vehicle) (9F7-F11). (A 35% volume reduction 105 days after xenotransplantation). Treatment with the 9F7-F11 antibody delayed median survival by 50% for 20 days in animals xenografted with MDA-MB-468 cells (one animal in the 9F7-F11 treated group at the end of the experiment, ie, 190 days later). Of mice were stabilized; p <0.05). In summary, these results indicate that the non-competitive and allosteric anti-HER3 antibody to NRG, 9F7-F11, is a tumor growth in mice xenografted with either HER2 amplified or three negative breast cancer cell lines. Was delayed.

Example 8: Combination therapy of pertuzumab and 9F7-F11 in mice xenografted with NRG-dependent BxPC3 pancreatic cancer We have previously shown that the combination of the therapeutic antibody trastuzumab with other targeted therapies. Demonstrated a synergistic effect on pancreatic cancer with low HER2 expression (Larbouret, 2007, 2010). The present inventors have now evaluated the combination therapy of the allosteric anti-HER3 antibody 9F7-F11 and the anti-HER2 antibody pertuzumab in HER2 ^low pancreatic cancer. Six weeks old female athymic mice were injected subcutaneously with HER2 ^low BxPC-3 pancreatic cells (4.5 × 10 ⁶ ) (NRG-dependent) secreting neuregulin into the right flank. Mice bearing tumors were randomized into different treatment groups when the tumors reached a volume of 100 mm ³ (at least 6 animals / group), followed by 2 or 10 mg / kg pertuzumab, 10 mg / kg Treatment was with 9F7-F11 or a combination of pertuzumab and 9F7-F11 (10 mg / kg of each monoclonal antibody). Antibodies were administered intraperitoneally (ip) twice a week for 4 weeks (Q3D-4W). Tumor volume formula: was calculated by _{D 1 × D 2 × D 3} /2. Tumors were sacrificed when they reached a volume of 1000 mm ³ for survival comparison.

Both antibodies alone slowed tumor growth significantly compared to the untreated group (p <0.001), and no significant difference was observed between the anti-HER3 antibodies 9F7-F11 and pertuzumab (P = 0.6488) (FIG. 12A). The median 50% survival was significantly delayed compared to controls for 17 days in mice treated with 9F7-F11 and 23 days in mice treated with pertuzumab (FIG. 12A). Furthermore, co-treatment with 9F7-F11 and pertuzumab inhibited tumor growth much more than the respective antibody alone (pertuzumab versus 9F7-F11 / pertuzumab; p = 0.004). At the end of 4 weeks of treatment, tumor volume continued to increase in mice treated with 9F7-F11 or pertuzumab alone, but was still very stable in animals receiving the 9F7-F11 / pertuzumab combination ( FIG. 12A). Median survival was longer for animals treated with the combination of the two antibodies (9F7) than for control animals (obtained after 42 days; p = 0.0001) or mice that received a single antibody. -F11 / pertuzumab vs. 9F7-F11 p = 0.0013; 9F7-F11 / pertuzumab vs. pertuzumab p = 0.0355) (FIG. 12A). Finally, the combination of the anti-HER3 antibodies 9F7-F11 and pertuzumab is significantly more effective than the pertuzumab / trastuzumab combination, and the average survival time is greater than for animals treated with pertuzumab / trastuzumab (13 days). Animals treated with 9F7-F11 / pertuzumab (42 days) were longer (FIG. 12B). In summary, these results indicate that the combination therapy using the non-competitive and allosteric NHER3 anti-HER3 antibody 9F7-F11 and the anti-HER2 antibody pertuzumab was more effective than the combination therapy using two HER2 specific antibodies. demonstrated to be more effective against ^low tumors.

Example 9: HER3 knockout abolishes the efficacy of 9F7-F11 in vivo in mice xenografted with NRG-dependent BxPC3 pancreatic cancer
Based on the work of Lee-Hoeflich et al. (2008), two short hairpin oligonucleotides were selected to knock down HER3 mRNA levels as described in Supporting Materials and Methods. The control vector (shCTRL) pSIREN-shLuc was kindly provided by L. Le Cam, which has been described previously (Le Cam et al., 2006). Next, pSIREN-shHER3 and pSIREN-shLuc, which contain the puromycin N-acetyltransferase resistance gene, were transfected into the amphotropic packaging cell line AmphoPack-293 (Clontech). Two days later, the supernatant containing the replication-defective virus particles was collected and used to infect BxPC3 cells. Antibiotic selection (10 μg / ml puromycin) was started two days later. Seven days after selection, cells were subcloned and selected based on lack of endogenous HER3 protein expression. Six-week-old female athymic mice purchased from Harlan (Le Malcourlet, France) have parental shHER3 BxPC-3 cells as described above (3.5 × 10 ⁶ ) or control shLuc BxPC- Three cells (4.5 × 10 ⁶ ) were injected subcutaneously. Mice bearing tumors were randomized into different treatment groups when the tumors reached a volume of 100 mm ³ (at least 6 animals / group) and then weekly with 10 mg / kg 9F7-F11 for 2 weeks. Intraperitoneally (Q3D-4W) for 4 weeks.

  To confirm in vivo the relationship between HER3 expression and 9F7-F11 therapeutic efficacy, mice were xenografted with shHER3 BxPC-3 or shCTRL BxPC-3 cells (FIG. 13A). Consistent with our in vitro data, the anti-HER3 antibody, 9F7-F11, which is non-competitive for NRG, is associated with shCTRL BxPC-3 tumor xenograft growth as compared to untreated controls. Was significantly inhibited (p <0.0001 and p = 0.0015) (FIG. 13A). In contrast, no significant regression of tumor growth was observed in mice xenografted with shHER3 BxPC-3 cancer cells and treated with the 9F7-F11 antibody compared to untreated controls ( (FIG. 13A). At the end of the experiment, HER3 expression was still silenced in shHER3 BxPC-3 tumor xenografts isolated from treated mice (FIG. 13B). These results indicate that in vivo HER3 knockdown abolishes the therapeutic efficacy of 9F7-F11, an anti-HER3 antibody that is non-competitive for NRG.

References:
Throughout this application, various references describe the state of the art in the art to which this invention pertains. The disclosures of these references are incorporated herein by reference to the present disclosure.

Claims (9)

A non-competitive, allosteric anti-human HER3 antibody against neuregulin comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 5
- combination of anti-human HER2 monoclonal antibody. The combination of anti-human HER2 monoclonal antibody is trastuzumab or pertuzumab claim 1 Symbol placement. Pharmaceutical composition comprising a combination of 請 Motomeko 1 Symbol placement. For use definitive in the treatment of cancer, according to claim 3 pharmaceutical composition. Cancer is a cancer comprising NRG, for use according to claim 4, a pharmaceutical composition. Cancer is a NRG-dependent cancers, for use according to claim 4, a pharmaceutical composition. Cancer is NRG Adikutido cancer, for use according to claim 4, a pharmaceutical composition. 3. The pharmaceutical composition according to Me other uses which definitive diagnosis of cancer. Cancer is a cancer comprising NRG, for use according to claim 8, a pharmaceutical composition.