TW201706308A - Human anti-FGFR4 antibody - Google Patents

Abstract

The present invention relates to novel antibodies against the FGF receptor 4 (FGFR4) and to the medical use thereof, in particular for the diagnosis prevention or treatment of diseases associated with FGFR expression, overexpression or hyperactivity.

Description

Human anti-FGFR4 antibody

The present invention relates to novel antibodies against FGF receptor 4 (FGFR4) and their medical use, particularly for the diagnosis, prevention or treatment of diseases associated with FGFR expression, overexpression or hyperactivity.

Fibroblast growth factor (FGF) is a family of growth factors with different biological activities, and its members are involved in angiogenesis, wound healing, embryonic development and various endocrine signaling pathways. The human FGF family contains 18 members, which are structurally related signaling molecules. They exert their biological activities by interacting with their homologous receptor (FGFR), a family of tyrosine kinase receptors. The mammalian FGFR family has four members: FGFR1-4, each consisting of three extracellular immunoglobulin-type domains (D1-D3), a single transmembrane domain, and an intracellular cleavage tyrosine kinase domain. composition. FGF interacts primarily with the D2 and D3 domains. Each FGFR binds to a specific subset of FGF. Most FGFs can bind to several different FGFR subtypes, while other FGFs can specifically activate an isoform of a receptor or receptor.

Receptor-ligand interactions result in receptor dimerization and autophosphorylation, complexing with membrane-associated proteins and cytoplasmic accessory proteins and initiating several signaling cascades. The FGFR-FGF signaling system plays an important role in development and tissue repair by regulating cellular functions and processes such as growth, differentiation, migration, morphogenesis, and angiogenesis.

FGFR4 signaling can be activated by several FGFs that also activate other members of the FGFR family ( Ornitz et al., 1996, J. Biol. Chem., 1996, 271: 15292-7 ), while the FGF19 line is specific for FGFR4. ( Xie et al., 1999, Cytokine, 11(10): 720-35 ). Activation of the FGFR4 receptor results in several types of cellular signaling, including the initiation of a signaling pathway mediated by the phosphorylation cascade following stimulation of FGFR4 by FGF. When the ligand binds to the extracellular domain of FGFR4, receptor dimerization and subsequent phosphorylation of tyrosine kinase residues leads to activation of the signaling pathway by inducing binding of the signaling molecule to the receptor ( Vainikka et al) .1992, EMBO, 11(12): 4273-4280, and 1994, J. Biol. Chem. 269: 18320-18326 ). For example, FGFR4 is linked to PLCγ1, and MAP kinase activation and DNA synthesis are increased when FGF stimulation has been observed. Further interaction with members of other human FGF growth factor receptor families may amplify the signaling potential of FGFR4 and this method is not only used for signal diversification but also for amplifying signals ( McKeehan WL and Kan M., 1994, Mol. Reprod. Dev .39:69-82 ). The 85 kDa serine kinase has been found to negatively regulate tyrosine phosphorylation of FGFR4, but its exact function has not been elucidated ( Vainikka et al., 1996, J. Biol. Chem. 271: 1270-1273 ). The association of FGFR4 with NCAM has been shown to mediate integrin-dependent adhesion ( Callallaro et al., 2001, Nat. Cell Biol. 3: 650-657 ), which may have a decisive role in tumor metastasis.

FGFR4 has been reported to have several cellular roles. The receptor is involved in controlling the differentiation processes of various cells in the tube and in vivo, such as skeletal muscle differentiation and regeneration, mesenchymal differentiation or osteogenesis, or alveolar formation during postnatal liver development. In addition, FGFR4 is described as controlling bile acid and cholesterol homeostasis and is thought to be involved in controlling obesity. In addition, FGFR4 controls the balance between bile production and cholesterol production in glass tubes and in vivo. FGFR4 is also involved in certain tumor phenomena, such as the development of hepatocellular carcinoma or colon cancer, or the proliferation of breast fibroadenoma cells or breast cancer epithelial cells, such as breast cancer or colorectal cancer cells. FGFR4 has also been described as being overexpressed in certain pancreatic cancer cell lines and is associated with malignant astrocytoma.

FGFR4 is involved in a variety of diseases and thus makes this receptor an important target for diagnostic and therapeutic applications. In this context, an effective strategy is to use antibodies against FGFR4. In particular, antibodies that interfere with FGFR4-mediated signaling are desirable. Examples of anti-FGFR4 antibodies are described, for example, in the international application WO 03/063893, WO 2012/138975, WO 2013/0183319, and US application US 2011/0150903. Bumbaca et al. (mAbs 3:4, 1-11; 2011) describe a humanized anti-FGFR4 antibody that binds FGF receptor 4 with high specificity. This antibody is referred to herein as a comparative example and is referred to as GT-13. However, there is still a need for new anti-FGFR4 antibodies.

A fundamental problem of the present invention is the provision of novel anti-FGFR4 antibodies and methods of using them to diagnose, prevent, and/or treat diseases associated with FGFR4 expression, overexpression, and/or overactivity.

In particular, there is a need for new human antibodies against FGFR4. A problem that often occurs with recombinantly produced antibodies is that the protein sequence of an antibody produced in a non-human immune system is partially different from that of a naturally occurring homologous antibody in the human body. Therefore, it may be immunogenic when administered to a human patient. Thus, it has been found that monoclonal antibodies designed to be administered to humans are preferably modified to increase their similarity to naturally occurring antibody variants of the human body. Particularly advantageous are those antibodies that are fully human, ie, they do not contain any part of a non-human source. Accordingly, it is an object of the present invention to provide human anti-FGFR4 antibodies which are advantageous for human use in diagnostic and therapeutic applications. Preferably, the desired antibody is to prevent FGFR4 from signaling. The invention described herein satisfies this need and provides additional benefits.

A first aspect of the invention is directed to an amino acid between 119 and 248 of human FGFR4 (preferably between 152 and 240 amino acid (Ig-like domain 2), more preferably an amine) A human antibody at an epitope of between 230 and 240, or a functional fragment or functional derivative of the antibody.

The antibody is suitable for use in medicine, especially human medicine, and more particularly, for the diagnosis, prevention and/or treatment of diseases associated with FGFR4 expression, overexpression and/or overactivity.

The invention also provides conjugated conjugates comprising an antibody as described herein. In particular, antibody drug conjugates are the subject of the present invention.

Another aspect of the invention is a fusion protein, wherein the anti-system of the invention Connect IL-2 or its functional fragment.

Another aspect of the invention is a nucleic acid molecule encoding the antibody, which is operably linked to an expression control sequence.

Another aspect of the invention is a host, particularly a recombinant cell comprising the nucleic acid molecule. This cell can be used to prepare the antibody.

Still another aspect of the invention is a pharmaceutical composition comprising the antibody, the nucleic acid molecule or host, and optionally a pharmaceutically acceptable carrier.

Yet another aspect of the invention is a method for diagnosing, preventing, and/or treating a disease associated with FGFR4 expression, overexpression, and/or overactivity.

The present invention relates to human antibodies against FGFR4 or functional fragments or functional derivatives thereof. The term "antibody" specifically refers to a molecule comprising at least one immunoglobulin heavy chain and at least one immunoglobulin light chain. Each heavy and light chain can comprise a variable domain and a constant domain. The antigen binding site can be formed from variable domains of the heavy and light chains. The variable region (also referred to as a variable domain) comprises a complementarity determining region (CDR), such as the CDR1, CDR2 and CDR3 regions and a framework region (FR) adjacent to the CDR. The term " complementarity determining region " is readily understood by those skilled in the art (see, for example, Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSHL press, Cold Spring Harbor, NY, 1988 ) and refers to antibodies. Within the variable domain, an extension of the amino acid that is primarily in contact with the antigen and determines the specificity of the antibody. This area is also known as the hypervariable area.

The term "human antibody" encompasses whole human antibodies or humanized antibodies, with whole human antibodies being preferred. Human antibodies can be prepared from genetically engineered animals, such as animals comprising a heterologous immune system or from an antibody display library according to known techniques. Human antibodies are generally described in van Dijk and van de Winkel (Curr. Opin. Pharmacol. 5: 368-74 (2001)) and Lonberg (Curr. Opin. Immunol. 20: 450-459 (2008) . The immunogen is administered to a transgenic animal that has been modified to produce an intact human antibody or an intact antibody having a human variable region in response to an antigen challenge. Such animals typically contain all or a portion of a human immunoglobulin locus, These human immunoglobulin loci can be substituted for endogenous immunoglobulin loci, either extrachromosomally or randomly integrated into the chromosome of an animal. In these transgenic mice, the endogenous immunoglobulin locus is roughly The above has been deactivated. A review of methods for obtaining human antibodies from transgenic animals is described, for example, in Lonberg, Nat. Biotech. 23: 1117-1125 (2005) . Human variable from intact antibodies produced by such animals The regions can be further modified, for example, by combining with different human constant regions.

Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques And Applications, pp . 51-63 ). Human antibodies produced by human B cell hybridoma technology are also described in Li et al, Proc. Natl. Acad. Sci., USA 103: 3557-3562 (2006) .

Human antibodies can also be produced by phage display methods (see, for example, US 6,248,516, US 5,403,484, US 5,969,108, US 5,885,793, US 6,696,248, US 5,849,500). Techniques for selecting human antibodies from antibody libraries are known in the art. (See, for example, Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p. 189-203; and Siriwardena, D. et al., Ophthalmology (2002) 109 (3) , p.427-431). For example, a phage display method can be used which involves displaying a human antibody variable region in the form of a single-chain antibody (scFv) on the surface of a phage and selecting an antigen-binding phage (Nature (1991), 352, (6336), p. 624-628, Journal of Molecular Biology (1992), 227, (2), p. 381-388, and Nature Biotechnology (2005), 23, (9), p. 1105-1116). Similarly, another phage display method can be used which involves displaying a human antibody Fab (antigen-binding fragment) on the surface of a phage and selecting an antigen-binding phage (WO 97/08320 and WO 01/05950). A gene encoding a phage selected based on antigen binding can be analyzed to determine a DNA sequence encoding a variable region of a human antibody that binds to the antigen. When the DNA sequence of the scFv or Fab that binds to the antigen is clear, the CDR sequence is extracted therefrom and a expression vector having the sequence can be prepared and introduced into a suitable host, followed by expression of the gene to obtain a human antibody (WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388, Annu. Rev. Immunol (1994) 12, p. 433-455 and Nature Biotechnology (2005) ) 23 (9), p. 1105-1116).

Human antibodies can also be generated by isolating a variable domain sequence of an Fv selection strain selected from a human-derived phage display library. These variable domain sequences can then be combined with the desired human constant domain. For antibodies The library of techniques for selecting human antibodies is known in the art.

Humanized antibodies can be prepared by humanizing monoclonal antibodies according to known techniques. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Humanized antibodies and methods of making same can be examined , for example, in Alamagro and Fransson, Front. Biosci. 13: 1619-1633 (2008) .

The invention also encompasses fragments of human antibodies, such as portions of the above antibodies, which comprise at least one antigen binding site. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments, bifunctional antibodies or single chain antibody molecules and other fragments as long as they exhibit the desired ability to bind human FGFR4. For review of certain antibody fragments, see Hudson et al., Nat. Met. 9: 129-134 (2003) .

A bifunctional antibody is an antibody fragment that has two antigen binding sites, possibly bivalent or bispecific. See, for example, Hudson et al, (2003). A single-chain antibody is an antibody fragment comprising all or part of a heavy chain variable domain, or all or part of a light chain variable domain. Antibody fragments can be made by a variety of techniques including, but not limited to, proteolytic cleavage of intact antibodies and production by recombinant hosts (e.g., E. coli or phage) as described herein.

In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites.

In certain embodiments, one of the binding specificities is for FGFR4 and the other binding specificity is for any other antigen.

In certain embodiments, the bispecific antibody binds to FGFR4 Two different epitopes. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express FGFR4. Bispecific antibodies can be prepared in the form of full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinantly displaying two immunoglobulin heavy chain-light chain pairs and "knob in hole" genetic engineering with different specificities. Multispecific antibodies can also be made by the following methods: genetically engineered electrostatic steering effects for the production of antibody Fc-heterodimer molecules; cross-linking of two or more antibodies or fragments; use of leucine zippers to make bispecific Sex antibodies; preparation of trispecific antibodies using the "bifunctional antibody" technique used to make bispecific antibodies and the use of single chain Fvs and instructions. Also included herein are genetically engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies."

In certain embodiments, amino acid sequence variants of the antibodies provided herein can be considered as long as they exhibit the desired ability to bind to human FGFR4. For example, it may be desirable to increase the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications in the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion of residues and/or insertion residues and/or substitution residues from the amino acid sequence of the antibody. Deletions, insertions, and substitutions of any combination can be made to obtain the final construct, except that the final construct possesses the desired characteristics, such as binding to an antigen.

The term "bind or binding" of an antibody means an antigen at least temporarily associated with the target (ie, a fragment of human FGFR4 comprising an epitope) Interaction or association.

In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of μ1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (eg, 10 ^-8 M or less). , for example, from 10 ^-8 M to 10 ^-13 M, for example 10 ^-9 M to 10 ¹³ M).

In one embodiment, the Kd value is measured by radiolabeled antigen binding assay (radioimmunoassay, RIA), which is performed in the form of the Fab of the desired antibody and its antigen.

According to another embodiment, the Kd system is measured using surface plasmon resonance analysis and immobilized antigen. According to a preferred embodiment of the invention, the antibody is a human monoclonal antibody against an epitope of human FGFR4 as described herein.

The inventors of the present application have found that antibodies directed against epitopes between amino acids 119 to 284 of human FGFR4, or functional fragments or functional derivatives thereof, are particularly useful in therapeutic and diagnostic applications. Particularly preferred are antibodies directed against an epitope between 152 and 240 of human FGFR4, and more preferably between 230 and 240 of the amino acid of human FGFR4. According to a particularly preferred embodiment, the anti-system of the invention is directed to an epitope comprising the amino acid sequence RYNY, consisting essentially of the amino acid sequence RYNY or consisting of the amino acid sequence RYNY.

Preferably, the epitope determined by the human antibody of the invention is located in the Ig-like domain 2 of human FGFR4.

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

In certain embodiments of the invention, the antibody may comprise a specific heavy chain complementarity determining region, CDRH1, CDRH2 and/or CDRH3, as described below.

In one embodiment, the human antibody comprises an amino acid sequence having the sequence shown in any one of SEQ ID NOS: 1 to 6 or an amine derived from the sequence but having 1 or 2 amino acids. The heavy chain complement of the base acid sequence determines the region 1 (CDRH1).

In a further embodiment, the antibody comprises an amino acid sequence having the sequence shown in any one of SEQ ID NOs: 7 to 12 or an amine derived from the sequence but having 1 or 2 amino acids. The heavy chain complement of the base acid sequence complements the region 2 (CDRH2).

In still further embodiments, the antibody comprises or consists of an amino acid sequence as shown in any of SEQ ID NOs: 13 to 20 but differs from one or two amino acids. The heavy chain complement of the amino acid sequence determines the region 3 (CDRH3).

An antibody according to the invention may also comprise a specific light chain complementarity determining region, CDRL1, CDRL2 and/or CDRL3.

Thus, in one embodiment, the antibody comprises or has an amino acid sequence as shown in any of the sequences of SEQ ID NOs: 21 to 23 and 68 but has one or two amino acid phases The light chain complementarity determining region 1 (CDRL1) of the heteroamino acid sequence.

In a further embodiment, the antibody comprises or is derived from an amino acid sequence as set forth in any of SEQ ID NOs: 24-27 The light chain complementarity determining region 2 (CDRL2) of an amino acid sequence of the same sequence but having 1 or 2 amino acids.

In still further embodiments, the antibody comprises or consists of an amino acid sequence as shown in any of SEQ ID NOs: 28 to 35 but differs from one or two amino acids. The light chain complementarity of the amino acid sequence determines region 3 (CDRL3).

Preferably, an antibody of the invention may comprise a particular CDR combination (ie, a combination of CDRH1, CDRH2, and CDRH3) within one heavy chain.

Thus, in a preferred embodiment, the antibody comprises a heavy chain comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, wherein the CDRH1 is selected from or derived from the sequences set forth in SEQ ID NOs: 1 to 6. There are 1 or 2 amino acid-amino acid sequences, and the CDRH2 is selected from the sequences shown in SEQ ID NOS: 7 to 12 or amines having the same sequence but having 1 or 2 amino acids. The base acid sequence, and the CDRH3 is selected from the sequences shown in SEQ ID NOS: 13 to 20 or amino acid sequences derived from the sequences but having 1 or 2 amino acids.

Most preferably, the antibody of the invention comprises a heavy chain comprising three CDRs, wherein the combination of CDRH1, CDRH2 and CDRH3 is selected from those shown in Table 1. It will be understood that each row in Table 1 represents a specific combination of CDRH1, CDRH2 and CDRH3.

More preferably, the antibody comprises a particular combination of CDRs (i.e., a particular combination of CDRL1, CDRL2, and CDRL3) within a light chain, in accordance with the invention.

Thus, in a preferred embodiment, the antibody comprises a light chain comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein the CDRL1 has an amino group as shown in any one of SEQ ID NOs: 21 to 23 and 68 An acid sequence or an amino acid sequence derived from the sequence but having 1 or 2 amino acid dissimilarities, and the CDRL2 has an amino acid sequence as shown in any of SEQ ID NOs: 24 to 27 or from such a sequence but having 1 or 2 amino acid-amino acid sequences, and the CDRL3 has an amino acid sequence as shown in any of SEQ ID NOs: 28 to 35 or from the sequences but has 1 or Two amino acid different amino acid sequences.

Most preferably, the antibody of the invention comprises a light chain comprising three CDRs, wherein the combination of CDRL1, CDRL2 and CDRL3 is selected from those shown in Table 2. It should be understood that each row in Table 2 represents a particular combination of CDRL1, CDRL2, and CDRL3.

As described above, the complementarity determining region (CDR) of the antibody can abut the framework region. The heavy or light chain of an antibody containing three CDRs contains, for example, four framework regions.

Furthermore, the invention also encompasses those antibodies which recognize an epitope on the same human FGFR4 as a specific antibody characterized by the above-described heavy and/or light chain CDRs. Functional fragments and functional derivatives of those antibodies are also within the scope of the invention. To determine the epitope on FGFR4 recognized by the antibody, a short peptide array derived from a chemically prepared protein sequence derived from the amino acid sequence of the extracellular domain of human FGFR4 can be used to locate and identify the antigen of the antibody. Decision site (Reinicke W., Methods Mol. Biol. 2004, 248: 443-63). Another method of indicating the location of an epitope in the extracellular domain of FGFR4 to which an antibody of the invention binds comprises Snaps/SELDI (Wang et al., Int. J. Cancer , 2001, June 15; 92(6): 871 -6) Alternatively, routine cross-blocking analysis as described in Antibodies, A Laboratory Manual , Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) can be performed.

According to a particularly preferred embodiment, the human antibody of the invention comprises a heavy chain comprising at least one CDR selected from the group consisting of: (a) CDRH1 as set forth in SEQ ID NOs: 1 to 6, or from Iso-sequence but with 1 or 2 amino acid-differentiated CDRH1 sequences, (b) CDRH2 as shown in SEQ ID NOs: 7 to 12, or from such sequences but having 1 or 2 amino acids different a CDRH2 sequence, and (c) a CDRH3 as set forth in SEQ ID NOS: 13 to 20, or a CDRH3 sequence derived from the sequence but having 1 or 2 amino acids, and/or comprising at least one selected from The light chain of the CDRs of the group consisting of (d) is the CDRL1 shown in SEQ ID NO: 21 to 23 or 68, or the sequence of the CDRL1 from the sequence but having 1 or 2 amino acids, (e) a CDRL2 as set forth in SEQ ID NOs: 24 to 27, or a sequence of CDRL2 derived from such sequences but having 1 or 2 amino acids, and (f) as in SEQ ID NOs: 28 to 35 The indicated CDRL3, or a sequence of CDRL3 derived from such sequences but having 1 or 2 amino acids.

In a preferred embodiment of the invention, the human antibody comprises a sequence as shown in any one of SEQ ID NOs: 52 to 59 or a sequence different from the sequence of one or two amino acids. Chain variable region (VH). Furthermore, preferably, the human antibody of the present invention comprises a light chain variable region as shown in the sequence of any one of SEQ ID NOS: 60 to 67 or a sequence different from one or two amino acids of the sequences. (VL). Particularly good human antibodies are included as SEQ ID NO: an antibody of a heavy chain variable region shown in any one of 52 to 59 and a light chain variable region as shown in any one of SEQ ID NOS: 60 to 67.

Particularly preferred is a human antibody (U4-1) comprising a CDRH1 as set forth in SEQ ID NO: 1, a CDRH2 as set forth in SEQ ID NO: 7, and a CDRH3 as set forth in SEQ ID NO: The heavy chain further comprises a light chain comprising a CDRL1 as set forth in SEQ ID NO: 21, a CDRL2 as set forth in SEQ ID NO: 24, and a CDRL3 as set forth in SEQ ID NO: 28. The invention also encompasses human antibodies in which one or more CDRs have one or two amino acids differing or an antibody that recognizes the same epitope on human FGFR4. In a particularly preferred embodiment, the human antibody comprises a heavy chain variable region according to SEQ ID NO: 52 and a light chain variable region according to SEQ ID NO: 60. The invention also encompasses human antibodies in which the sequence of the heavy and/or light chain variable region differs from the sequence set forth in SEQ ID NOS: 52 and 60 by one or two amino acids.

Particularly preferred is a human antibody (U4-2) comprising a CDRH2 as set forth in SEQ ID NO: 2, a CDRH2 as set forth in SEQ ID NO: 7, and a CDRH3 as set forth in SEQ ID NO: The heavy chain further comprises a light chain comprising a CDRL1 as set forth in SEQ ID NO: 22, a CDRL2 as set forth in SEQ ID NO: 24, and a CDRL3 as set forth in SEQ ID NO:29. The invention also encompasses human antibodies in which one or more CDRs have one or two amino acids differing or an antibody that recognizes the same epitope on human FGFR4. In a particularly preferred embodiment, the human antibody comprises a heavy chain variable region according to SEQ ID NO: 53 and Light chain variable region of SEQ ID NO:61. The present invention also encompasses human antibodies in which the sequence of the variable region of the heavy and/or light chain differs from the sequence shown in SEQ ID NOS: 53 and 61 by one or two amino acids.

Particularly preferred is a human antibody (U4-3) comprising a CDRH2 as set forth in SEQ ID NO: 3, a CDRH2 as set forth in SEQ ID NO: 8, and a CDRH3 as set forth in SEQ ID NO: 15. The heavy chain further comprises a light chain comprising a CDRL1 as set forth in SEQ ID NO: 23, a CDRL2 as set forth in SEQ ID NO: 25, and a CDRL3 as set forth in SEQ ID NO:30. The invention also encompasses human antibodies in which one or more CDRs have one or two amino acids differing or an antibody that recognizes the same epitope on human FGFR4. In a particularly preferred embodiment, the human antibody comprises a heavy chain variable region according to SEQ ID NO: 54 and a light chain variable region according to SEQ ID NO: 62. The invention also encompasses human antibodies in which the sequence of the variable region of the heavy and/or light chain differs from the sequence shown in SEQ ID NOS: 54 and 62 by one or two amino acids.

Particularly preferred is a human antibody (U4-4) comprising a CDRH2 as set forth in SEQ ID NO: 4, a CDRH2 as set forth in SEQ ID NO: 9, and a CDRH3 as set forth in SEQ ID NO: The heavy chain further comprises a light chain comprising a CDRL1 as set forth in SEQ ID NO: 22, a CDRL2 as set forth in SEQ ID NO: 24, and a CDRL3 as set forth in SEQ ID NO:31. The invention also encompasses human antibodies in which one or more CDRs have one or two amino acids differing or an antibody that recognizes the same epitope on human FGFR4. In a particularly preferred embodiment, the human antibody comprises a heavy chain variable region according to SEQ ID NO: 55 and Light chain variable region of SEQ ID NO:63. The invention also encompasses human antibodies in which the sequence of the variable region of the heavy and/or light chain differs from the sequence shown in SEQ ID NOS: 55 and 63 by one or two amino acids.

Particularly preferred is a human antibody (U4-5) comprising a CDRH1 as set forth in SEQ ID NO: 5, a CDRH2 as set forth in SEQ ID NO: 10, and a CDRH3 as set forth in SEQ ID NO: The heavy chain further comprises a light chain comprising a CDRL1 as set forth in SEQ ID NO: 22, a CDRL2 as set forth in SEQ ID NO: 24, and a CDRL3 as set forth in SEQ ID NO:32. The invention also encompasses human antibodies in which one or more CDRs have one or two amino acids differing or an antibody that recognizes the same epitope on human FGFR4. In a particularly preferred embodiment, the human antibody comprises a heavy chain variable region according to SEQ ID NO: 56 and a light chain variable region according to SEQ ID NO: 64. The invention also encompasses human antibodies in which the sequence of the variable regions of the heavy and/or light chains differs from the sequences set forth in SEQ ID NOS: 56 and 64 by one or two amino acids.

Particularly preferred is a human antibody (U4-6) comprising a CDRH1 as set forth in SEQ ID NO: 6, a CDRH2 as set forth in SEQ ID NO: 12, and a CDRH3 as set forth in SEQ ID NO: 19. The heavy chain comprises a light chain comprising a CDRL1 as set forth in SEQ ID NO: 22, a CDRL2 as set forth in SEQ ID NO: 26, and a CDRL3 as set forth in SEQ ID NO:34. The invention also encompasses human antibodies in which one or more CDRs have one or two amino acids differing or an antibody that recognizes the same epitope on human FGFR4. In a particularly preferred embodiment, the human antibody comprises a heavy chain variable region according to SEQ ID NO: 58 and Light chain variable region of SEQ ID NO:66. The invention also encompasses human antibodies wherein the sequence of the variable regions of the heavy and/or light chains differs from the sequences set forth in SEQ ID NOS: 58 and 66 by one or two amino acids.

Particularly preferred is a human antibody (U4-7) comprising a CDRH2 as set forth in SEQ ID NO: 3, a CDRH2 as set forth in SEQ ID NO: 7, and a CDRH3 as set forth in SEQ ID NO: The heavy chain further comprises a light chain comprising a CDRL1 as set forth in SEQ ID NO: 68, a CDRL2 as set forth in SEQ ID NO: 27, and a CDRL3 as set forth in SEQ ID NO:35. The invention also encompasses human antibodies in which one or more CDRs have one or two amino acids differing or an antibody that recognizes the same epitope on human FGFR4. In a particularly preferred embodiment, the human antibody comprises a heavy chain variable region according to SEQ ID NO: 59 and a light chain variable region according to SEQ ID NO: 67. The invention also encompasses human antibodies in which the sequence of the variable regions of the heavy and/or light chains differs from the sequences set forth in SEQ ID NOS: 59 and 67 by one or two amino acids.

Particularly preferred is a human antibody (U4-8) comprising a CDRH2 as set forth in SEQ ID NO: 4, a CDRH2 as set forth in SEQ ID NO: 11, and a CDRH3 as set forth in SEQ ID NO: 18. The heavy chain further comprises a light chain comprising a CDRL1 as set forth in SEQ ID NO: 22, a CDRL2 as set forth in SEQ ID NO: 24, and a CDRL3 as set forth in SEQ ID NO:33. The invention also encompasses human antibodies in which one or more CDRs have one or two amino acids differing or an antibody that recognizes the same epitope on human FGFR4. In a particularly preferred embodiment, the human antibody comprises a heavy chain variable region according to SEQ ID NO: 57 and Light chain variable region of SEQ ID NO:65. The present invention also encompasses human antibodies in which the sequence of the variable region of the heavy and/or light chain differs from the sequence shown in SEQ ID NOS: 57 and 65 by one or two amino acids.

Also preferred is a human antibody (U4-9) comprising CDRH1 as set forth in SEQ ID NO: 3, CDRH2 as set forth in SEQ ID NO: 8 and as set forth in SEQ ID NO: The heavy chain of CDRH3 is shown and comprises a light chain comprising a CDRL1 as set forth in SEQ ID NO:68, a CDRL2 as set forth in SEQ ID NO:27, and a CDRL3 as set forth in SEQ ID NO:35. The invention also encompasses human antibodies in which one or more CDRs have one or two amino acids differing or an antibody that recognizes the same epitope on human FGFR4. In a particularly preferred embodiment, the human antibody comprises a heavy chain variable region according to SEQ ID NO: 54 and a light chain variable region according to SEQ ID NO: 67. Preferably, antibody U4-9 comprises a heavy chain specific to antibody U4-3 and a light chain specific to antibody U4-7. In the experiments provided in this application, antibody U4-9 demonstrated higher affinity for FGFR4 than U4-3.

As described above, the antibody of the present invention activates other antitumor agents and/or tumors in terms of its binding specificity and biological activity, particularly its ability to recognize human anti-FGFR4 epitopes to reduce cell growth and cell migration. Cells exhibit advantageous properties in terms of their ability to be sensitized to therapeutic treatment. The antibodies U4-1 to U4-9 of the present invention have endogenous antitumor activity. It specifically binds to FGFR4 and preferably shows no cross-reactivity with other FGF receptors FGFR1-3b, c.

This antibody is capable of inhibiting ligand binding to human FGFR4. Antibody pair ligand The effect of binding to FGFR4 can be determined by incubating cells expressing this receptor (e.g., MDA-MB453 breast cancer cells) with radiolabeled ligand in the absence (control) or in the presence of an anti-FGFR4 antibody. Antibodies that reduce the binding affinity of the ligand to the FGFR4 receptor or antibodies that block ligand binding to FGFR4 can be identified. The FGFR4 ligands known include FGF1, FGF2, FGF4, FGF6, FGF8, FGF9, FGF17, FGF18, FGF19 and FGF20. The cell line endogenously expressing FGF19 is Huh-7. The antibodies of the invention are capable of at least partially inhibiting the binding of these ligands to human FGFR4. The particularly preferred antibody of the present invention is capable of blocking the binding of one or more of the above ligands by at least 60%, preferably at least 70%, more preferably at least 80% or 87%. Particularly preferred antibodies of the invention are those which are capable of blocking the binding of FGF19 to human FGFR4 by at least 80%, more preferably at least 85% or at least 90%.

Blocking ligand binding to human FGFR4 results in inhibition of FGFR4 signaling. To select antibodies that reduce ligand-induced FGFR4 phosphorylation, cells are pre-incubated with buffer (control) or antibodies and treated with ligand or control buffer. The cells can then be lysed and the crude lysate product centrifuged to remove insoluble material. The supernatant was incubated with antibody-specific pure FGFR4 and protein A-agarose to achieve effective precipitation. After washing, the immunoprecipitates can be separated by SDS-PAGE. Then, the Western blotting of the gel was detected with an anti-phosphotyrosine antibody. After visualization, the spot ink was peeled off and probed again with anti-FGFR4 antibody. Reflective scanning densitometry of the gel can be performed to quantify the effect of the antibody in question on the formation of complexes induced by HRG. Choose those relative to the control group (untreated cells) An antibody that reduces FGFR4 phosphorylation.

Intravitreal experiments can be performed to determine the ability of the antibodies of the invention to inhibit ligand-stimulated cell proliferation. The appropriate number of desired cells are incubated with antibodies diluted in a suitable medium. The ligand was directly added to the antibody solution to stimulate the cells, which were then allowed to grow for 72 hours. Adding ^{Alamar Blue TM (Thermo Fisher Scientific,} Waltham, MA; USA) and at 37 ℃, incubated in the dark. The absorbance at 590 nm was measured every 30 minutes.

In order to select antibodies that reduce FGFR4-mediated cell migration, transmigration experiments can be performed. Serum starved cells are incubated with antibodies. An appropriate number of cells can be placed in the upper chamber of a coated transwell disk (BD Biosciences, Inc., San Jose, Calif.) having 8 [mu]m wells. In the case of using a stimulation medium alone or containing a chemotactic agent, it is used in the bottom chamber. The cells are moved and subsequently stained. The stained nuclei were counted; the percentage inhibition was expressed relative to the degree of inhibition of the control antibody.

We have found that the antibodies of the invention have enhanced anti-tumor activity compared to known anti-FGFR4 antibodies. The anti-tumor efficacy of this therapeutic antibody can be assessed in human xenograft tumor studies. In these studies, human tumor lines were grown in immunodeficient mice in the form of xenografts, and therapeutic efficacy was measured by the degree of tumor growth inhibition (TGI). To determine if the FGFR4 antibody of the invention interferes with tumor growth in human cancer cells in nude mice, cells are implanted in nude mice. The tumor line grows subcutaneously on the back of the animal or on the side of the animal. Treatment can begin immediately or when the tumor reaches a certain average body The time begins to accumulate. Prior to the first treatment, mice were randomized and statistically tested to ensure uniformity (mean, median, and standard deviation) of the initial tumor volume between treatment groups. At the beginning of the treatment, a loading dose of 25 mg/kg was administered first, followed by a weekly injection of 25 mg/kg via the intraperitoneal route.

The antibodies of the invention are suitable for use in medicine, especially for human medical care. Antibodies can be used to prevent and/or treat diseases associated with FGFR4 expression, overexpression, and/or overactivity. Examples of diseases which can be prevented and/or treated using the antibodies of the present invention are described in detail below.

Furthermore, the antibodies of the invention may be used as diagnostic agents, for example, for the diagnosis of diseases associated with FGFR4 expression, overexpression and/or overactivity. Examples of diseases that can be diagnosed using the antibodies of the invention are described below.

An antibody of the invention can be coupled to a heterologous group, such as an effector group. Such antibody conjugates are particularly suitable for therapeutic applications. The term " effector group " can refer to a cytotoxic group, such as a radioisotope or radionuclide, a toxin, a therapeutic group, or another effector group known in the art. A conjugate conjugate in which the antibody is coupled to a therapeutic group, the so-called antibody-drug conjugate (ADC), is particularly preferred. Alternatively, an antibody of the invention can be coupled to a labeling group. Such antibody conjugates are particularly suitable for use in diagnostic applications. As the term is used herein refers to a detectable marker of "labeling group", for example radiolabelled amino acids by any other type of marker, or biotin moiety, a fluorescent label, an enzyme, or known in the present art. Attachment of a radioisotope to an antibody or antibody fragment of the invention can, for example, provide a benefit to tumor treatment. Unlike chemotherapy and other forms of cancer treatment, radioimmunotherapy or the administration of radioisotope-antibody compositions can target cancer cells with minimal damage to surrounding normal healthy tissue.

Another embodiment of the invention is a fusion protein wherein the antibody as defined above is fused to interleukin-2 (IL-2). Interleukin 2 is a 15 kDa cytokine produced by T helper cells that stimulates cytotoxic T lymphocytes and NK cells. IL-2 has been clinically used to treat melanoma and renal cell carcinoma to stimulate the immune system of cancer patients. Achieving long-term high doses of IL-2 in tumors can induce long-lasting anti-tumor responses leading to rejection of tumors that may be fatal. It has been demonstrated in the present invention that IL-2 is incorporated into a fusion protein comprising an antibody of the present invention, while maintaining its activity. Thus, a fusion protein in which the antibody of the present invention is fused to IL-2 is suitable as a delivery system which retains antigen binding specificity and possesses the full activity of IL-2.

The invention also relates to nucleic acid molecules encoding antibodies as described above. The term " nucleic acid molecule " encompasses DNA, such as single or double stranded DNA or RNA. The DNA can be of genomic, cDNA or synthetic origin or a combination thereof. A nucleic acid molecule of the invention can be operably linked to a performance control sequence, i.e., linked to a sequence necessary to effect the performance of the encoding nucleic acid sequence. Such expression control sequences can include promoters, enhancers, ribosome binding sites, and/or transcription termination sequences. Specific examples of suitable performance control sequences are known in the art.

According to a preferred embodiment, the invention is directed to an isolated nucleic acid molecule selected from the group consisting of

(a) a nucleic acid sequence encoding an antibody, fragment or derivative thereof as defined above,

(b) A nucleic acid sequence as set forth in any one of SEQ ID NOS: 36 to 43 and SEQ ID NOS: 44 to 51.

(c) a nucleic acid sequence complementary to any of (a) or (b), and

(d) A nucleic acid sequence capable of hybridizing to (a), (b) or (c) under stringent conditions.

According to a particularly preferred embodiment of the invention, the nucleic acid molecule comprises a sequence encoding a heavy chain variable region of an antibody and a sequence encoding a light chain variable region of the antibody. In an alternate embodiment, a combination of two nucleic acid molecules is provided, wherein one nucleic acid molecule encodes the light chain of the antibody and the other nucleic acid molecule encodes the heavy chain of the antibody. Preferably, the nucleic acid sequence encoding the heavy chain variable region is selected from the sequences set forth in any one of SEQ ID NOs: 36 to 43. Preferably, the nucleic acid sequence encoding the variable region of the light chain of the antibody is selected from the sequences set forth in any one of SEQ ID NOs: 44 to 51. Particularly preferred are combinations comprising at least one of the sequences set forth in SEQ ID NOS: 36 to 43 and at least one of the sequences set forth in SEQ ID NOS: 44 to 51. The nucleic acid sequence may be present within an isolated nucleic acid molecule or may be within a combination of two separate nucleic acid molecules.

The term " hybridization under stringent conditions " means that two nucleic acid fragments are, for example, in, for example, Sambrook et al., "Expression of cloned Genes in E. coli" in Molecular Cloning: A Laboratory Manual (1989), Cold Spring Harbor Laboratory Press, Hybridization to each other under standardized hybridization conditions as described in New York, USA . These conditions are, for example, hybridization in 6.0 x SSC (Saline Sodium Citrate) at about 45 ° C, followed by a washing step at 2.0 ° SSC at 50 ° C, preferably at 65 It is washed with 2.0 x SSC at ° C or at 0.2 ° SSC at 50 ° C, preferably at 0.2 ° SSC at 65 ° C.

The nucleic acid molecule of the invention may be on a vector which may additionally contain a replication origin and/or selectable marker gene. Examples of vectors are, for example, plasmids, cosmids, phages, viruses, and the like. Accordingly, a further embodiment of the invention is a vector comprising a nucleic acid sequence as disclosed herein. Preferably, the vector is an expression vector. The vector can be, for example, a phage, plasmid, virus or retroviral vector. Retroviral vectors can be replication competent or replication defective. In the latter case, viral propagation usually occurs only in complementary hosts/cells.

The nucleic acid molecules of the invention may be linked to a vector containing a selectable marker for propagation in a host. In general, the plasmid vector is in a precipitate, such as a calcium phosphate precipitate or a ruthenium chloride precipitate or in a complex complexed with a charged lipid or in a carbon cluster such as fullerene. Introduced. If the vector is a virus, it can be packaged in a glass tube using a suitable packaging cell line prior to administration to the host cell.

Preferably, the vector of the invention is an expression vector, wherein the nucleic acid molecule is operably linked to one or more control sequences which permit transcription and alternative expression in prokaryotic and/or eukaryotic host cells. The expression of the nucleic acid molecule comprises transcription of the nucleic acid molecule, preferably into a translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. It typically comprises a regulatory sequence that ensures initiation of transcription and, optionally, a poly A signal that ensures transcription termination and transcript stabilization. Additional regulatory elements can include transcriptional and translational enhancers. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, the lac, trp or tac promoter in E. coli, while examples of regulatory elements that allow expression in eukaryotic host cells are AOXI or GAL in yeast. 1 promoter or CMV (cytomegalovirus)-, SV40 (simian virus 40)-, RSV promoter (Lloyd's sarcoma virus), CMV-enhancer, SV40 enhancer or globin in mammalian and other animal cells ( Globin) intron. In addition to the elements responsible for transcription initiation, such regulatory elements may also comprise a transcription termination signal, such as the SV40 poly A site or the tk-poly A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art, such as the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 or pSPORTI (Thermo Fisher Scientific). Preferably, the vector is a performance vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses, such as retroviruses, vaccinia virus, adeno-associated virus, herpes virus or bovine papilloma virus, can be used to deliver a polynucleotide or vector of the invention into a target cell population. Persons familiar with methods well known in the art of the present can be used to construct recombinant viral vectors; see, e.g. Sambrook, Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001,3 rd edition), NY and Ausubel, Current Protocols in Molecular Biology , Techniques described in Green Publishing Associates and Wiley Interscience, NY (1994) . Alternatively, a nucleic acid molecule of the invention can be reconstituted into a liposome for delivery to a target cell. Furthermore, the invention relates to a host comprising a nucleic acid molecule or vector as described above. The nucleic acid molecule or vector can be introduced into the host by transformation, transfection or transduction according to any method known in the art.

The host can be a prokaryotic or eukaryotic cell or a non-human transgenic animal. The polynucleotide or vector of the invention present in the host can be integrated into the genome of the host or it can be kept extrachromosomally. In this regard, it is also to be understood that the nucleic acid molecules of the invention can be used in " gene targeting " and/or " gene replacement " for restoring mutant genes or mutant genes created by homologous recombination; see, for example, Mouellic , Proc. Natl. Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, Oxford University Press .

The host can be any prokaryotic or eukaryotic cell, such as a bacterium, insect, fungus, plant, animal, mammal or preferably human cell. Preferred fungal cells are, for example, fungal cells of the genus Saccharomyces, especially fungal cells of the Saccharomyces cerevisiae species. The term "prokaryotic" is intended to include all bacteria which can be transformed or transfected with a polynucleotide for the expression of a variant polypeptide of the invention. Prokaryotic hosts may include Gram-negative and Gram-positive bacteria such as, for example, E. coli , Salmonella typhimurium , Serratia marcescens , and Bacillus subtilis ( Bacillus). Subtilis ). Polynucleotides encoding mutant forms of the variant polypeptides of the invention can be transformed or transfected into a host using any of the techniques generally known to those of ordinary skill in the art. Methods for preparing fused operably linked genes and expressing them in bacterial or animal cells are well known in the art ( Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001, 3rd Edition )). The genetic constructs and methods described therein can be used to express variant antibodies, antibody fragments or derivatives thereof of the invention, for example, in a prokaryotic host. In general, a promoter sequence containing a nucleic acid molecule that facilitates the insertion is used. The expression vector for efficient transcription is associated with a host. The expression vector typically contains an origin of replication, a promoter and a terminator, and a specific gene that provides for phenotypic selection of the transformed cell. The transformed prokaryotic host can be according to techniques known in the art. Growing and culturing in a fermenter for optimal cell growth. The antibodies, antibody fragments or derivatives thereof of the invention can then be isolated from growth media, cell lysates or cell membrane fractions. The antibody, antibody fragment or derivative thereof of the present invention can be isolated and purified by any conventional means, , For example, preparative chromatographic separations and immunological separation, such as those involving the use of monoclonal or polyclonal antibodies in.

According to one embodiment of the invention, the host is a human, bacterial, animal, fungal, amphibious or plant cell. Preferred animal cells include, but are not limited to, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), mouse embryonic fibroblasts (NIH-3T3), and many other cell lines, including Human cells.

In another preferred embodiment, the animal cell is an insect cell. Preferred insect cells include, but are not limited to, cells derived from SF9 cell lines.

Preferably, the cell is a mammalian cell, such as a hamster, rabbit or human cell. Most preferably, the cell is a human cell. The human cells include, but are not limited to, human embryonic kidney cells (HEK293, 293T, 293 freestyle). Furthermore, the human cell strain includes, but is not limited to, HeLa cells, human hepatocyte cancer cells (eg, HEPG2, Huh-7), A549 cells. According to another embodiment, the host of the invention is a non-human transgenic animal. The invention provides transgenic non-human animals comprising one or more nucleic acid molecules of the invention, which are useful in the manufacture of antibodies of the invention. The antibody can be produced and recovered from the milk, blood or urine of tissue or body fluids such as goats, cows, horses, pigs, rats, mice, rabbits, hamsters or other mammals. No. 5, 756, 690; As described above, a non-human transgenic animal comprising a human immunoglobulin locus can be prepared by immunization with FGFR4 or a portion thereof.

The antibody of the present invention can be produced by a method in which the anti-system is obtained from a host as described above. Accordingly, a further embodiment of the present invention is a method for producing an antibody comprising culturing a host of the present invention under conditions permitting synthesis of the antibody and recovering the antibody from the culture.

The transformed host can be grown in a fermenter and cultured to achieve optimal cell growth according to techniques known in the art. Once expressed, the whole antibodies, dimers, individual light and heavy chains or other immunoglobulin forms of the invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, coagulation. Glue electrophoresis, etc.; see Scopes, "Protein Purification", Springer-Verlag, NY (1982) . The antibody of the invention or its corresponding immunoglobulin chain can then be isolated from the growth medium, cell lysate or cell membrane fraction. Isolation and purification of antibodies or immunoglobulin chains of the invention, for example, by microorganisms, can be carried out by any conventional means, such as, for example, preparative chromatography and immunological separation, such as those involving the use of, for example, antibodies of the invention Individual or multiple antibody antibodies in the constant region.

It will be apparent to those skilled in the art that the antibodies of the invention can be further coupled to other moieties such as drug targeting and imaging applications. Such coupling may be performed chemically after the antibody or antigen is present on the adhesion side or the coupling product may be genetically engineered into the DNA level of the antibody or antigen of the invention. The DNA is then displayed in a suitable host system and the protein of the expression is collected and, if necessary, reconstituted.

According to one embodiment, the recombinant cells as described above are cultured under conditions permitting expression of the nucleic acid molecule encoding the antibody. The antibody can be collected from the cultured cells or culture supernatant. Preferably, the anti-system is prepared from a mammal, especially from a human cell.

Still another aspect of the invention pertains to pharmaceutical compositions comprising an antibody, conjugate conjugate, fusion protein, nucleic acid molecule, vector or host, as described above, optionally together with a pharmaceutically acceptable carrier.

The term "carrier" includes those agents which are non-toxic to the cells or mammals that are exposed to them at the dosages and concentrations employed, such as diluents, stabilizers, adjuvants or other types of excipients. Examples of pharmaceutically acceptable carriers are well known in the art and include phosphate buffered physiological saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Typically, the pharmaceutically acceptable carrier is an aqueous pH buffer solution that can be used to deliver a drug, particularly for delivery of antibody molecules. The pharmaceutical composition can be formulated by well known methods, i.e., by mixing the active agent with a carrier and, optionally, other agents which are often included in the preparation. For example, the composition can be formulated in the form of a lyophilized preparation, an aqueous solution, a dispersion, or a solid preparation.

Administration of a suitable composition can be effected by various methods, for example by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The compositions of the invention may also be administered directly to the site of the target, such as by a biolistic delivery to a site of an external or internal target, such as the brain. The dosage regimen will be determined by the attending physician and clinical factors.

The invention also encompasses administration of a pharmaceutical composition to an individual in need of the pharmaceutical composition, particularly a human patient suffering from a condition associated with FGFR4 expression, overexpression, and/or overactivity. As is well known in the art of pharmacy, the dosage for any patient depends on a number of factors, including the size, body surface and area of the patient, age, particular compound to be administered, sex, time and route of administration, general health. Conditions and other drugs that are to be administered at the same time. Depending on the type and severity of the condition to be treated, the active ingredient may be administered, for example, by one or more separate administrations or by continuous infusion to a subject in need thereof from about 1 [mu]g/kg to 15 mg/kg. Typical daily doses may range from about 1 [mu]g/kg to about 100 mg/kg, depending on the above factors. In order to repeat the administration over a period of days or longer, depending on the condition to be treated, the treatment continues until the desired disease or condition is inhibited. The composition can be administered by any suitable route, for example by parenteral, subcutaneous, intranasal, intravascular, intravenous, intraarterial or intrathecal injection or infusion.

Progress can be monitored by periodic assessments. The compositions of the invention may be administered via local or systemic routes. Formulations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are polyethylene, propylene glycol, polyethylene glycol, value oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carrier includes water, alcohol/water soluble Liquid, emulsion or suspension, including physiological saline and buffered media. The gastrointestinal external carrier includes sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like.

The active agents according to the invention may be administered with other active agents. The additional active agent can be administered alone or as part of a pharmaceutical composition of the invention.

According to a preferred embodiment of the present invention, the pharmaceutical composition of the present invention may contain other reagents depending on the intended use of the pharmaceutical composition. Particularly preferably, the pharmaceutical composition comprises other active agents such as, for example, additional anti-tumor agents, small molecule inhibitors, anti-tumor agents or chemotherapeutic agents. The invention also relates to a pharmaceutical composition comprising a combination of an antibody of the invention and at least one other anti-tumor agent. These combinations are effective, for example, to inhibit abnormal cell growth.

Many anti-tumor agents are currently known in the art. In one embodiment, the anti-tumor agent is selected from the group of therapeutic proteins, including, but not limited to, antibodies or immunomodulating proteins.

In another embodiment, the anti-tumor agent is selected from the group consisting of a small molecule inhibitor or a chemotherapeutic agent consisting of a mitotic inhibitor, a kinase inhibitor, an alkylating agent, an antimetabolite, and an insertion. Antibodies, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, histone deacetylase inhibitors, anti-survival agents, biological response modifiers, anti-hormones such as anti-androgens and anti-angiogenesis Agent.

Of course, the above additional active agents can be administered not only together with the antibodies of the present invention in a shared pharmaceutical composition, but they can also be administered separately.

In yet another embodiment, the invention is directed to a diagnostic method comprising determining the amount and/or localization of FGFR4 in a patient tissue or patient sample. In the present embodiment, it is particularly preferred to use an antibody carrying a labeling group as described above. Comparing the results of the resulting patient tissue or patient sample to reference data may allow diagnosis of a disease associated with FGFR4 performance, overexpression, and/or overactivity. The diagnostic method of the invention can be used to detect the performance, overexpression or hyperactivity of unwanted human FGFR4 in different cells, tissues or other suitable samples. Thus, the onset or disease state of the disease associated with FGFR4 performance, overexpression, and/or overactivity can be assessed. The diagnostic method of the present invention may also include the step of establishing a treatment regimen for the patient based on the results obtained.

In another embodiment, the invention relates to a method of assessing the presence of a cell expressing FGFR4 comprising contacting an antibody of the invention with a cell or tissue suspected of having FGFR4 on its surface. A suitable method for detecting the expression of FGFR4 in a sample may be enzyme-linked immunosorbent assay (ELISA) or immunohistochemistry (IHC).

ELISA assays can be performed in a microtiter plate format in which, for example, the anti-FGFR4 antibody is adsorbed in the wells of a microtiter plate. The wells are rinsed with a blocking agent, such as milk protein or albumin, to prevent non-specific adsorption of the analyte. Next, the wells are treated with the test sample. After rinsing off the test sample or standard, the wells are treated with a secondary anti-FGFR4 antibody labeled (eg, bound to biotin). After the excess secondary antibody is washed and removed, the mark is detected For example, horseradish peroxidase (HRP) conjugated with avidin is detected and a suitable chromogenic receptor is detected. The concentration of the FGFR4 antigen in the test sample was determined by comparison to a standard curve developed from a standard sample.

In the case of IHC, a paraffin-embedded tissue can be used, wherein the tissue is, for example, dewaxed in xylene, then dehydrated (for example using ethanol) and rinsed in distilled water. The antigenic epitope determined by formalin fixation and paraffin embedding can be exposed by masking, enzymatic decomposition or saponin removal of the epitope. To remove the mask of the epitope, the paraffin can be sliced in a steamer, water bath or microwave oven and heated in an epitope-repairing solution (eg 2N HCL solution, pH 1.0) for 20 to 40 minutes. In the case of enzymatic breakdown, tissue sections can be incubated for 1030 minutes at 37 ° C in different enzyme solutions (such as proteinase K, trypsin, pronase, pepsin, etc.).

After rinsing off the epitope-recovering solution or excess enzyme, the tissue sections were treated with blocking buffer to prevent non-specific interactions. Add a suitable concentration of primary anti-FGFR4 antibody. Excess primary antibody was rinsed off and the sections were incubated for 10 minutes in a peroxidase blocking solution at room temperature. After another washing step, the tissue sections are incubated with labeled secondary antibodies (eg, labeled with a group that can serve as an anchor for the enzyme). Thus, an example thereof is a biotinylated secondary antibody that is recognized by horseradish peroxidase coupled to streptavidin. Detection of the antibody/enzyme complex can be achieved by incubation with a suitable chromogenic host.

In another embodiment, the invention relates to blocking FGFR4 function A method comprising contacting an antibody of the present invention with a cell or tissue suspected of having FGFR4 on its surface under conditions in which the antibody is capable of blocking FGFR4 function. This contact can be carried out in a glass tube or in vivo.

Furthermore, the invention relates to kits for diagnosing or treating diseases associated with FGFR4 expression, overexpression and/or overactivity. The kit of the invention comprises at least one antibody, nucleic acid molecule and/or vector as described above. Additionally, the kit may further comprise at least one other active agent or other component. Preferably, the diagnostic kit of the invention comprises a labeled antibody as described above. Additionally, the diagnostic kit can include reference data regarding the amount and/or location of FGFR4 in the same type of tissue or sample. Reference data may be obtained from one or more healthy individuals and/or from one or more individuals with known disease states. Comparing the reference data may allow for the diagnosis of diseases associated with FGFR4 performance, overexpression, and/or overactivity. A treatment plan for the patient can be established based on the results obtained.

According to the present invention, diseases associated with FGFR4 expression, overexpression and/or hyperactivity are, for example, hyperproliferative diseases such as cancer. Cancers that can be diagnosed, prevented, and/or treated in accordance with the present invention may be selected from the group consisting of hepatocellular carcinoma, breast cancer, gastric cancer, colon cancer, rhabdomyosarcoma, prostate cancer, ovarian cancer. , soft tissue sarcoma, melanoma, head and neck squamous cell carcinoma and lung adenocarcinoma and other cancers and tumor metastasis that express or overexpress FGFR4.

According to another embodiment, the disease associated with FGFR4 expression, overexpression and/or overactivity is a metabolic disease, in particular metabolic syndrome or obesity.

According to still another embodiment, the disease associated with FGFR4 expression, overexpression, and/or hyperactivity is ventricular hypertrophy, cardiac hypertrophy, or chronic kidney disease.

The invention will now be illustrated in more detail by the following figures and examples.

Figure 1 shows the determination of antibodies U4-3, U4-5 and U4-6 binding to Huh-7 using a phycoerythrin-labeled anti-human IgG antibody.

Figure 2 shows the results of measurement of the anti-FGFR4 antibody U4-3 and U4-9 binding to the extracellular domain of FGFR4.

Figure 3 shows the results of the interaction analysis of the antigen with the reversibly captured antibody U4-3.

Figure 4 shows the species specificity of the antibody U4-3 of the present invention and the antibody GT-13.

Figure 5 is a graph showing the binding of human antibody U4-3 to different FGF receptors. It can be seen from the figure that the antibody U4-3 recognizes human FGFR4 with high specificity and does not cross-react with other members of the FGFR family.

Figure 6 is a bar graph showing the binding of antibodies to different extracellular domains of FGFR4.

Figure 7 is a graph showing the determination of FGFR4 binding epitopes for antibodies U4-3 and GT-13. As can be seen from the figure, the emphasized tetrapeptide sequence RYNY can be recognized by U4-3 but cannot be recognized by GT-13.

Figure 8 is a bar graph showing the binding of the assay antibody U4-3 to FGFR4. The results were obtained by alanine scanning mutagenesis of the FGFR4 extracellular domain. As can be seen from the figure, the binding of FGFR4 is highly specific and mutagenesis in the D2 domain results in a significant decrease in binding.

Figure 9 is a graph showing the binding of the anti-FGFR4 antibody U4-3 to the ligand. The hIgG shown is a comparative example.

Figure 10 (A) shows the use of different U4-3 antibody concentrations to determine concentration-dependent inhibition of b-FGF mediated phosphorylation of ERK.

Fig. 10(B) is a histogram showing the quantized data from Fig. 10(A).

Figure 11 shows inhibition of endogenous FGFR4 phosphorylation in Huh-7 cells by anti-FGFR4 antibody U4-3. A: immunoprecipitation (IP); B: total cell lysate; C: 11A quantification.

Figure 12 shows the NIH 3T3 sphere growth assay using antibodies U4-3 and U4-9.

Figure 13 is a bar graph showing inhibition of soft agar colony growth in Huh-7 cells in the presence of anti-FGFR4 antibody U4-3 or control IgG antibody.

Fig. 14 is a bar graph showing the results of cell growth analysis of ZR-75-1 cells cultured in the presence of antibody U4-3 and control IgG.

Figure 15 is a bar graph showing the results of MG-63 spheroid growth analysis using different concentrations of U4-3 and control IgG.

Figure 16 shows the anti-FGFR4 antibody in a mouse tumor model. A graph of U4-3 or GT-13 mediated growth inhibition results. A: dose-response; B: different antibodies, C: compared to GT-13.

Figure 17 Inhibition of in vivo growth using antibody U4-3 in Huh-1 (A) and Hep3B (B) tumor models.

Figure 18 Antibody U4-3 was used to inhibit in vivo growth in the SNU-761 human liver xenograft model.

Figure 19 In vivo efficacy in a gastric xenograft tumor model derived from a patient.

Figure 20 In vivo efficacy in human colon xenograft tumor model SW620.

Example 1: Generation of FGFR4-specific antibodies Isolation of Fab antibody fragments

The Fab antibody fragment recognizing human FGFR4 was isolated using the n-CoDeR Fab library-encoded text (Biolnvent, Lund, Sweden). Targeting human FGFR4-Fc (R & D system, Minneapolis, Minnesota; USA), human FGFR4-His/Myc, or rat myogenic cell line L6 (ATCC, Manassas, stably expressing human FGFR4) Virginia; USA) Screening as a differential panning method. Other families of human FGFR-Fc (R & D system), human Fc (Jackson Immuno Research, Newmarket; UK) and L6 cells were used in the negative selection. In liquid panning, human FGFR4-Fc and humans were EZ-Link NHS-Chromogenic-Biotin (Thermo Fisher Scientific) FGFR4-His/Myc biotinylation. Dynabeads M-280 Streptavidin (Thermo Fisher Scientific) was added to recover FGFR4 binding phage. Human solid FGFR4-Fc and human FGFR4-His/Myc were directly immobilized on polystyrene spheres in solid panning. The phage were incubated with the beads, and the L6 cells and unbound fractions of FGFR4 were removed by washing. The bound phage was released and amplified in E. coli by incubation with trypsin. After five different pannings, E. coli (TOP10F') was transformed with a Fab-expressing plasmid, and finally individual Fab selection strains were expressed.

Example 2: Identification of unique FGFR4 binding Fab

The binding of the Fab from the third round of panning described above was tested as follows: 1 Pimol FGFR4-Fc or FGFR4-His/Myc coating was applied to each well of the ELISA plate and incubated overnight at 4 °C. . After washing and blocking, the medium expressing the Fab of E. coli TOP10F' was added to the wells and incubated for 1 hour. Finally, the bound Fab was detected by incubation with a peroxidase-conjugated anti-His antibody (R&D system) or a peroxidase-conjugated anti-human Fab (Jackson Immuno Research). Signals were detected by the addition of Super Signal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Standard sequencing techniques were used to analyze FGFR4-binding Fab sequences to identify unique selection strains and to analyze the binding of purified unique Fabs to L6 or 293T cells expressing human FGFR4 (transient or stable expression) by flow cytometry. . It was confirmed that the FGFR4-binding Fab strain was transformed into the IgG format by using standard recombinant techniques.

Example 3: Testing the signal neutralization activity of FGFR4 binding Fab via Elk1 luciferase reporter gene assay

The functionality of Fab-binding FGFR4 was tested via Elk1 luciferase reporter gene analysis, and the Elk1 luciferase reporter gene analysis was established as follows: HEK293 cells stably expressing integrin αv and integrin β3 were seeded in The 96-well plates were transiently transfected with Lipofectamine 2000 according to the manufacturer's protocol using the following groups: pcDNA-DEST40-hFGFR4, pcDNA-DEST40, pFA2-Elk1 (Agilent Technologies, Santa Clara) , California; USA), pFR-Luc2CP, GAL4 containing 5×pFR-Luc at the selection site of pGAL4.12[luc2CP] and pGL4.74[hRluc/TK] (Promega, Madison, Wisconsin, USA) Binding components (Agilent Technologies). On the next day, the cells were incubated with Fab (diluted to a concentration of 10, 1, 0.1 μg/ml in DMEM 2% FBS) for 1 hour at 37 ° C, and then with ligand 10 ng / ml (recombinant human FGF-17) (rhFGF17), R&D system) were incubated for 6 hours. Finally, luciferase activity was determined by the addition of Dual Glo Luciferase Assay System (Promega). The ratio of specific (Firefly luciferase) activity to signal normalization (renilla luciferase) was calculated and the values of the different Fabs are shown as a percentage of the control values (Table 3). All Fabs tested showed significant inhibitory activity, with a signal reduction of more than 50% at 1 [mu]g/ml and a decrease of more than 35% at 10 [mu]g/ml.

Example 4: KD of anti-FGFR4 antibody in Huh-7 cells was determined by flow cytometry.

Huh-7 cells (obtained from the JCRB cell bank) were cultured in DMEM containing 10% FBS under standard conditions at 37 ° C and 5% CO ₂ . Cells were harvested in 5 mM EDTA (in PBS) and 200,000 cells per sample were incubated with the indicated anti-FGFR4 antibody (1:3 serial dilution; initial concentration 30 [mu]g/ml) for 30 minutes at 4 °C. After incubation, the cells were washed and the bound antibodies were assayed using a phycoerythrin-labeled anti-human IgG antibody (Jackson Immuno Research). Huh-7-conjugated antibodies were measured using a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, Calif.; USA) and analyzed using Accuri C6 software. Subsequently, the equilibrium dissociation constant (K _D ) was determined using Prism (GraphPad Software, La Jolla, California; USA). The calculated K _D value U3 Pharma antibodies is very similar (between 0.2 to 1.6nM). FIG 1 shows a first representative experiment and measured K _D values for the remaining antibody independently. As a result, it was found that the affinity of GT-13 was low when compared with all tested U3 Pharma antibodies (Table 4).

Example 5: Determination of the antibody U4-3 and U4-9 against the extracellular domain (ECD) of FGFR4 by ELISA _D .

Mouse anti-His tag antibodies were obtained from Bio-Rad (Hercules, CA; USA) and plated at a concentration of 2 μg/ml on a MaxiSorp® flat-bottom 96-well ELISA plate (Sigma, St. Louis, Missouri; USA). After coating, the extracellular domain of human FGFR4 at a concentration of 0.01 μg/ml (labeled by myc- and His) was added. After removal of unbound protein, the indicated anti-FGFR4 antibody was added (1:4 serial dilution, initial concentration 10 μg/ml), and finally, Fluostar Omega from BMG Labtech (Ortenberg; Germany) On a fluorescent disc analyzer, alkaline phosphatase-conjugated goat anti-human IgG F (ab') fragment was used and binding was detected with an AttoPhos® AP Fluorescence System (Promega). Representative results are shown in Figure 2. Next, the dissociation constants of U4-3, -5, and -9 were calculated using Graph Pad Prism (Table 5). All antibodies tested showed excellent KD, and the observed U4-3 values were similar to those observed previously. By.

Example 6: Determination of K of U4-3 using Biacore analysis _D .

A goat anti-human IgG capture S-CM5 sensor wafer was prepared using standard amine coupling (the reference flow cell also contained the capture antibody). The carboxymethylated dextran (CMD) surface was activated by injecting a mixture of 200 mM EDC and 50 mM NHS for 7 minutes (10 μl/min). Subsequently, 30 μg/ml of anti-human IgG antibody diluted in 10 mM sodium acetate pH 5.0 was injected for 7 minutes (10 μl/min). The remaining unreacted ester was quenched with 1 M ethanolamine pH 8.5 (7 min, 10 μl/ml). The surface density obtained from anti-human IgG antibodies was 17892-18376 RU.

BiacoreTM T100 instrument (GE Healthcare Bio-Sciences (Piscataway, New Jersey; USA)) was used for antigen and HBS assay buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 50 μM EDTA, 0.05% Tween 20) at 25 °C. Interaction analysis of reversibly captured antibodies. A specific antibody of 37-38 RU was captured on the flow cell. Subsequently, 5 series of triplicate dilutions (3.7, 11.1, 33.3, 100 and 300 nM) of the antigen were separately injected in a single cycle mode for 2 minutes (50 μl/min). Dissociation was recorded for 10 minutes at the same flow rate. A flow cell containing anti-human IgG capture antibody was used as a reference. By repeated injection A final percentage of 5% 1,4-dioxane 10 mM glycine pH 1.7 was used to complete complete regeneration (removal of specific antibodies from the capture surface along with the bound antigen).

Software version 2.0.3 was evaluated using Biacore T100 and data processing (eg, blank run subtraction) and evaluation were performed by a full fit using a bivalent analyte binding model. The equilibrium dissociation constant KD1 is calculated by dividing kd1 by ka1. FIG. 3 shows a representative measurement of antibodies U4-3; K _D value of the antibody and U4-9 (average value of 3 measurements) are reported in Table 6.

Example 7: Species specificity was determined by flow cytometry.

Hek293 cells (Cell Lines Service, Eppelheim; Germany) were obtained from CLS and cultured in DMEM containing 10% FBS under standard conditions at 37 ° C and 5% CO ₂ . On day 0, at 4 × 10 ⁵ cells / 10cm dish of cells plated at a density. After 24 hours, the cells were transfected with a plasmid encoding FGFR4 from rat, mouse, human or cynomolgus monkey. The cells were allowed to express the protein for 48 hours, and then the cells were isolated from the culture dish with 5 mM EDTA in PBS, followed by incubation with the indicated concentrations of anti-FGFR4 antibody U4-3 or GT-13. Binding was determined using an anti-human IgG antibody (Jackson Immuno Research Europe) conjugated to phycoerythrin. Data were acquired with a BD Accuri C6 flow cytometer (BD Biosciences) and analyzed using Accuri C6 software. Antibody titration experiments (starting concentration of 10 μg/ml; 1:3 dilution) allowed calculation of the dissociation constant of the antibody (see table below). This antibody showed a similar affinity for all of the tested species at very low concentrations [1μg / ml] and K _D value of between species exhibit protein with human and cynomolgus best combination equivalent to each other. However, when compared to GT-13, U4-3 showed a better binding between all species tested.

Example 8: Binding to other FGFRs.

According to the instructions (extracellular domain, Fc chimera), the recombinant human FGFR (or BSA as a control) obtained from the R & D system was coated with a Nunc MaxiSorp® flat-bottom 96-well ELISA plate from Thermo Fisher Scientific, and finally The concentration was 100 μg/well and was processed according to the manufacturer's instructions. After incubation with the anti-FGFR4 antibody U4-3, the AttoPhos® AP Fluorescent Substrate System purchased from Promega was used to homogenize the goat anti-human IgG F with alkaline phosphatase. The binding was detected (ab') and detected by Fluostar Omega Fluorescence Analyzer from BMG Labtech. The results are shown in Figure 5.

Antibody U4-3 recognizes human FGFR4 with high specificity and, importantly, does not cross-react with other members of the FGFR family.

Example 9: Assays Antibodies bind to different extracellular domains of FGFR4.

Hek293 cells were obtained from CLS and cultured in DMEM containing 10% FBS under standard conditions at 37 ° C, 5% CO ₂ . On day 0, cells were seeded and cells were transfected 24 hours later with a plasmid encoding FGFR4 containing the indicated domain. The cells were allowed to express the protein for 48 hours, and then the cells were isolated from the culture dish with 5 mM EDTA in PBS, followed by incubation with the designated anti-FGFR4 antibody. Binding was determined using an anti-human IgG antibody (Jackson Immuno Research Europe) conjugated to phycoerythrin. Data were acquired with a BD Accuri C6 flow cytometer (BD Biosciences) and analyzed using Accuri C6 software. The binding of U4-3 to FGFR4 occurs mostly in the Ig-like domain 2 of the receptor and no binding is observed at Ig-like domains 1 (amino acids 22 to 180) and 3 (amino acids 249 to 349). The results are shown in Figure 6.

Example 10: Determination of the epitope that binds to FGFR4.

The peptides were synthesized according to the amino acid sequence of the target protein (domain 2 of the human FGFR4 extracellular domain) using standard Fmoc chemistry and the trifluoroacid of the scavenger was deprotected. The restriction peptide was synthesized on a chemical scaffold using the Chemically Linked Peptides on Scaffolds (CLIPS) technique to reconstitute the opioid epitope. By using a 0.5 mM CLIPS template solution (such as in ammonium bicarbonate (20 mM, pH 7.9) / acetonitrile (1:1 (v/v)) on a polypropylene PEPSCAN card (455 peptide format/card) - Bis(bromomethyl)benzene) is reacted to couple the side chains of multiple cysteine acids in the peptide to the CLIPS template. The card was gently shaken in the solution for 30 to 60 minutes while completely covering the solution. Finally, the card was extensively washed with excess H ₂ O and sonicated for 30 minutes at 70 ° C in interrupt buffer containing 1% SDS/0.1% β-mercaptoethanol in PBS (pH 7.2). It was then reprocessed by ultrasonication for 45 minutes in H ₂ O. The binding of the antibody to each peptide was tested in a PEPSCAN-based ELISA. A 455-well credit card format polypropylene card containing covalently linked peptides and a primary antibody solution (eg, 1 μg/ml diluted in blocking solution, eg 4% horse serum in PBS/1% Tween, 5% egg albumin (w/v) was incubated together. After washing (3 x 10 min), the 1:1000 dilution of the peptide and antibody peroxidase conjugate was incubated for 1 hour at 25 °C (or streptavidin-HRP). After washing (3 x 10 minutes), peroxidase was added with 2,2'-azino-di-3-ethylbenzenethiazoline sulfonate (ABTS) and 2 μl of 3% H ₂ O ₂ . After one hour, color development was measured and quantified with an electrical coupling element (CCD)-camera and image processing system. Figure 7 shows the difference in peptide recognition of the tested antibodies; in particular, the emphasized tetrapeptide sequence RYNY can be recognized by U4-3 and GT-13 is not recognized.

Example 11: Alanine scanning mutagenesis of the FGFR4 extracellular domain.

Hek293 cells were obtained from CLS and cultured in DMEM containing 10% FBS under standard conditions at 37 ° C and 5% CO ₂ . On day 0 to 2 × 10 ⁵ cells / 60mm dish of cells plated at a density. After 24 hours, cells were transfected with 4.8 μg of plasmid encoding the extracellular domain 2 (D2) of FGFR4 (Table 7). The expressed protein carries a flag tag and differs in the amino acid sequence as described below. The cells were allowed to express the protein for 60 hours (with medium change between them), and finally the cells were separated from the culture dish with 5 mM EDTA in PBS. Cells were incubated with the indicated concentrations of anti-FGFR4 antibody U4-3 and binding was determined using anti-human IgG antibodies (Jackson Immuno Research Europe) conjugated to phycoerythrin. Data were acquired with a BD Accuri C6 flow cytometer (BD Biosciences) and analyzed using Accuri C6 software. The fluorescence intensity is normalized to the performance of the flag tag. The results are shown in Figure 8.

The binding of FGFR4 is highly specific and single amino acid mutagenesis in the D2 structural region results in a significant decrease in binding.

Example 12: Inhibition of ligand binding by an anti-FGFR4 antibody.

Recombinant human FGFR4 (extracellular domain, Fc chimera) obtained from the R&D system was coated with a Nunc Maxi Sorp® flat-bottom 96-well ELISA plate from Thermo Fisher Scientific according to the manufacturer's instructions at a final concentration of 4 μg/well. And deal with it. Each well was incubated with 1.2 μg/ml of recombinant FGF-19 from the R & D system, followed by the indicated concentrations FGFR4 antibody U4-3 was processed. After incubation and washing, the bound FGF-19 was detected with a biotinylated anti-FGF19 antibody (R & D system) at 0.2 μg/ml, and then incubated with alkaline phosphatase conjugated streptavidin. The enzyme catalytic reaction was triggered by incubation with the AttoPhos® AP Fluorescence System (Promega) and detected by a Fluostar Omega Fluorescence Analyzer from BMG Labtech. The data shows that U4-3 is able to compete with FGF-19 for binding to receptors. The results are shown in Figure 9.

Example 13: Inhibition of ERK phosphorylation induced by b-FGF by U4-3.

L6 cells (ATCC) stably expressing full-length human FGFR4 were plated in 12-well culture dishes (1 × 10 ⁵ cells) using DMEM containing 10% FBS under standard tissue culture conditions (37 ° C, 5% CO ₂ ). /hole). The next day, the medium was replaced with growth medium containing the indicated concentrations of anti-FGFR4 or control antibody. After 1 hour, b-FGF (R & D system) at a concentration of 20 ng/ml was added to the designated wells. After 10 minutes of incubation, the wells were quickly washed with cold PBS and lysed in buffers containing phosphatase (Merck, Darmstadt, Germany) and protease (Roche, Basel; Switzerland) inhibitors. Finally, the samples were subjected to gel electrophoresis and immunoblotted (on a nitrocellulose membrane) using anti-ERK and phospho-ERK antibodies from Cell Signaling (Danvers, Inc.; Massachusetts). Bands were detected and quantified using an Odyssey detector from Li-Cor (Lincoln, Nebraska; USA) along with appropriate fluorescently labeled secondary antibodies. The bands were quantified by Odyssey software and plotted as the ratio between pERK and total ERK intensity. Figure 10 (A) shows a representative immunodot ink and 10 (B) shows subsequent quantified data. U4-3 was shown to inhibit b-FGF-mediated ERK phosphorylation in a concentration-dependent manner.

Example 14: Inhibition of endogenous FGFR4 phosphorylation by incubation with U4-3 antibodies.

Huh-7 cells (obtained from the JCRB cell bank) cultured in DMEM containing 10% FBS under standard conditions at 37 ° C and 5% CO ₂ were seeded in 60 mm culture dishes (2 × 10 per culture dish) ⁵ cells). On the next day, the medium was removed and replaced with growth medium containing anti-FGFR antibody U4-3 (0.1, 1, 3 and 10 μg/ml) or control IgG as indicated. After 6 days of treatment, the cells were washed twice with PBS and the cells were lysed in IP lysis buffer (containing a phosphatase inhibitor from Merck and a protease inhibitor from Roche). Total FGFR4 was immunoprecipitated using anti-FGFR4 antibody (mouse) and rProtein A Sepharose Fast Flow matrix from GE Healthcare Bio-Sciences. The substrate is washed and finally, the sample is separated by electrophoresis. The protein is transferred from the gel to the nitrocellulose membrane and spotted with an antibody that recognizes the phosphorylated tyrosine residue (detection of pFGFR4) or with an antibody that recognizes total FGFR4 (Santa Cruz Dallas, Texas; USA). To ensure equal loading of the samples. Bands were detected using appropriate secondary antibodies and an Odyssey detector from Li-Cor (Lincoln, Nebraska; USA). A representative scan of immunoprecipitation is shown in Figure 11(A), while the immuno spot ink of whole cell lysate is shown in Figure 11(B). After scanning the bands, the ratio between FGFR4 and pFGFR4 was determined by Odyssey software and normalized to IgG control values, see Figure 11 (C). The anti-FGFR4 antibody U4-3 showed strong inhibition of FGFR4 phosphorylation at all concentrations tested.

Example 15: NIH3T3 Sphere Growth Analysis

NIH3T3 cells stably expressing human FGFR4 and human FGF19 were cultured in RPMI1640 containing 10% FBS, 500 μg/ml G418, and 150 μg/ml hygromycin. On the first day of the experiment, 2000 cells were seeded in each well of a 96-well PrimeSurface disk (Sumitomo Bakelite, Tokyo; Japan) containing the specified concentration of U4-3 or U4-9 antibody. After incubation for 5 days in a standard tissue culture incubator (37 ° C and 5% CO ₂ ), CellTiter-Glo® luminescence cell viability was performed using a VICTOR analyzer from Perkin Elmer (Waltham, MA; USA) Cell growth was measured by CellTiter-Glo® Luminescent Cell Viability Assay (Promega). The values were normalized to untreated samples, and both antibodies were effective in reducing cell growth by more than 70%. The IC50 values were calculated and determined to be 24.7 nM and 10.6 nM (U4-9), respectively. The results are shown in Figure 12.

Example 16: Inhibition of soft agar colony growth of Huh-7 liver cancer cells

Huh-7 cells (JCRB cell bank) were cultured in DMEM containing 10% FBS under standard conditions at 37 ° C and 5% CO ₂ . The cells were harvested with PBS/0.05% trypsin and resuspended in the upper agar medium (Iscove's modified Dulbecco's medium (IMDM), 0.4 agar, 20% FBS) and carefully placed on the solidified agar medium (0.75%). Agar, 20% FBS in IMDM). The 96-well plate contains 2,000 cells per well. These wells were incubated in the presence of anti-FGFR4 antibody U4-3 (maximum concentration 30 [mu]g/ml) or control IgG antibody; colony formation was observed over time (about 3 weeks). Viable cells were stained using MTT reagent from Sigma (St. Louis, Missouri; USA) and colonies were quantified using a colony counting system from Oxford Optronix (Abingdon; UK) and normalized to the untreated control. The tested anti-FGFR4 antibody U4-3 showed inhibition of Huh-7 cell growth in a concentration dependent manner when compared to the control antibody. The results are shown in Figure 13.

Example 17: Growth analysis of ZR-75-1 breast cancer cells.

ZR-75-1 cells (CLS) were cultured in RPMI 1640 supplemented with 10% FBS, 10 μg/ml insulin, and 2 mM glutamine. On the day of the experiment, 2000 cells were seeded in each well of a 6-well plate containing the indicated amount of antibody. The cells were cultured for a total of 16 days (37 ° C and 5% CO ₂ ) and the medium and antibody were changed twice a week. Finally, the medium was removed and stained with 0.5% crystal violet solution (Carl Roth, Karlsruhe, Germany). The visible colonies were counted by the Plant Counting System (Oxford Optronix) and normalized to wells that did not receive any treatment (NT). The results are shown in Figure 14. U4-3 was effective in inhibiting about 50% of visible colony formation when compared to the control treatment.

Example 18: Analysis of MG-63 osteosarcoma sphere growth.

1000 MG-63 cells (ATCC) cultured in MEM (10% FBS) were inoculated into each well of a 96-well PrimeSurface disk (Sumitomo Bakelite) containing the indicated amount of antibody. After 5 days of incubation in a standard tissue culture incubator (37 ° C and 5% CO ₂ ), cell growth was measured by CellTiter-Glo® Luminescent Cell Viability Assay (Promega) using a VICTOR analyzer from Perkin Elmer. The results are shown in Figure 15. Antibody U4-3 reduced cell growth by approximately 25%; this result was observed at a concentration of 0.4 μg/ml.

Example 19: Inhibition of Huh-7 tumor growth in vivo.

Growth inhibition mediated by anti-FGFR4 antibodies in a mouse tumor model. Female NU/NU nude mice were obtained from the Charles River Laboratory (Boston, Massachusetts; USA). Six weeks after birth, via subcutaneous injection 5x10 ⁶ th Huh-7 cells (in Matrigel (of Matrigel) in, BD Biosciences Company) animals. Fourteen days after the injection, animals of similar tumor volume were pooled and treated with anti-FGFR4 antibody (q3dx3) via intraperitoneal injection. On days 15, 18, 21 and 24 after tumor inoculation, the tumor volume (in mm ³ ) was measured using the formula [volume = 0.52 × (width) ² × (length)]. Figure 16 (A) shows that the tumor growth inhibition line when treated with Huh-7 was dose-dependent, and maximum growth inhibition (about 68%) was observed at a dose of 25 mpk. At a dose of 10 mpk, U4-3 and U4-9 showed comparable tumor growth inhibition (Fig. 16(B)). When U4-3 and GT-13 were directly compared using the same dosing regimen, U4-3 showed superior efficacy and statistically significantly better inhibition of tumor growth (Fig. 16(C)).

Example 20: In vivo growth inhibition in Huh-1 and Hep3B tumor models . Example A: Growth inhibition mediated by anti-FGFR4 antibodies in the Huh-1 tumor model.

Via the subcutaneous route of female Balb / c nude mice (5-6 weeks old) th injection of 5 × 10 ⁶ Huh-1 cells. After the tumor size reached 150 mm ³ , the anti-FGFR4 antibody [25 mpk] was administered by intraperitoneal injection to treat the animals twice a week for 3 weeks. The tumor volume (measured in mm ³ ) was measured by a caliper using the formula (L x W ² )/2 as indicated. The results are shown in Figure 17(A).

Example B: Growth inhibition mediated by anti-FGFR4 antibodies in a Hep3B tumor model.

Animals were treated as above, but Hep3B cells were used and implanted using Matrigel (BD Biosciences). Tumor volume was determined at the indicated times as described above. The results are shown in Figure 17(B).

Tumor growth was inhibited by U4-3, and in the case of Huh-1 cells, tumor growth was inhibited by more than 50%.

Example 21: Inhibition of SNU-761 tumor growth in vivo.

Growth inhibition mediated by anti-FGFR4 antibodies in another mouse liver tumor model.

Female BALB/c nude mice were obtained from Shanghai Lingchang Biotechnology Co., Ltd. (LC, Shanghai, China). Seven weeks after birth, via the subcutaneous route in 0.1ml PBS injected animals in the 1 × 10 ⁷ th SNU-761 tumor cells (1: 1 in Matrigel, BD Biosciences Corporation, San Jose, CA; USA). Treatment was started when the average tumor size reached 119 m ³ . Anti-FGFR4 antibody was administered by intraperitoneal injection twice a week for 5 weeks. Since tumor volume can affect the effectiveness of any given treatment, mice were assigned to each group using a randomized block design based on the tumor volume of the mice. This ensures that all groups have comparable baselines. The randomized block design was used to assign experimental animals, ensuring that each animal had the same probability of being assigned to any given treatment group, thus minimizing systematic errors. Using a caliper, the tumor volume was measured twice a week in two dimensions, and the formula: V = 0.5 a × b ² (where a and b are the long and short diameters of the tumor, respectively), and the volume is expressed in mm ³ .

The tumor volume expressed in mm ^{3 was} measured from day 95 to day 130, and the measurement interval was about 3 to 4 days. Figure 18 shows the inhibition of tumor growth in human liver SNU-761 xenograft cancer cells when treated with anti-FGFR4 antibody U4-3. In the subcutaneous SNU-761 human liver xenograft model, U4-3 showed significant antitumor activity at a dose level of 25 mg/kg. Tumor growth inhibition was calculated as %TGI=1-(Ti-T0)/(Vi-VO))*100; Ti is the geometric mean tumor volume of the treatment group on the measurement day; T0 is the geometric mean tumor volume of the treatment group at D1 Vi is the geometric mean tumor volume of the control group on the measurement day; V0 is the geometric mean tumor volume of the control group at D1.

Example 22: In vivo efficacy in a gastric xenograft tumor model derived from a patient.

In the patient-derived gastric xenograft tumor model GA0080 FGFR4 antibody mediated growth inhibition.

Female BALB/c nude mice (eight to nine weeks old) were obtained from Shanghai Lingchang Biotechnology Co., Ltd. (LC, Shanghai, China). This efficacy study was performed by selecting the HuPrime® gastric cancer model GA0080 from a female patient. The pathology of this model is moderate to poorly differentiated adenocarcinoma.

Tumor fragments from breeding mice inoculated with selected primary human gastric tissue were harvested and used for inoculation into BALB/c nude mice. Tumors were developed on the right side of each mouse (2 to 4 mm in diameter) via the subcutaneous route. Treatment was initiated when the average tumor size reached approximately 153 mm ³ . Mice were randomly divided into two experimental groups according to their tumor size. From day 1 to day 22, FGFR4 antibody U4-3 was administered to tumor-bearing mice on a time schedule of 1, 4, 8, 11, 15, 18, and 22. The secondary tumor size was measured as described above.

Figure 19 shows the inhibition of tumor growth in the HuPrime® gastric cancer model GA0080 when treated with the anti-FGFR4 antibody U4-3. U4-3 demonstrated an anti-tumor activity of approximately 25% in a gastric xenograft model.

Example 23: In vivo efficacy in a human colon cancer xenograft tumor model.

Growth inhibition mediated by anti-FGFR4 antibodies in a human colon cancer (SW620) xenograft tumor model.

Female NMRI nude mice (five to six weeks old) were obtained from Charles River (Sulzfeld, Germany). Twenty female NMRI nude mice were inoculated 5×10 ⁶ SW620 tumor cells (ATTC No. CCL-227) in 100 μl PBS on the left side via the subcutaneous route. On the 12th day, when the average tumor size reached about 150 to 200 mm ³ , the 20 tumor-bearing mice were randomly divided into 2 groups of 10 animals each according to the tumor size. From day 1 to day 38, tumor-bearing mouse secondary anti-FGFR4 antibody U4-3 was administered weekly via the intraperitoneal route according to the schedules of 13, 17, 20, 24, 27, 31, 34 and 38. The secondary tumor size was measured weekly as described above. Figure 20 shows the inhibition of tumor growth when treated with anti-FGFR4 antibody U4-3. U4-3 demonstrated significant inhibition of primary tumor growth.

<110> U3 Pharma GmbH

<120> Human anti-FGFR4 antibody

<130> 56549P WO

<150> EP 14180555

<151> 2014-08-11

<160> 68

<170> PatentIn version 3.5

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<220>

<223> U4-1-VL CDRL3

<400> 28

<210> 29

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> U4-2-VL CDRL3

<400> 29

<210> 30

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> U4-3-VL CDRL3

<400> 30

<210> 31

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> U4-4-VL CDRL3

<400> 31

<210> 32

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> U4-5-VL CDRL3

<400> 32

<210> 33

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> U4-8-VL CDRL3

<400> 33

<210> 34

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> U4-6-VL CDRL3

<400> 34

<210> 35

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> U4-7-VL and U4-9-VL CDRL3

<400> 35

<210> 36

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> U4-1-VH

<400> 36

<210> 37

<211> 399

<212> DNA

<213> Artificial sequence

<220>

<223> U4-2-VH

<400> 37

<210> 38

<211> 399

<212> DNA

<213> Artificial sequence

<220>

<223> U4-3-VH and U4-9-VH

<400> 38

<210> 39

<211> 402

<212> DNA

<213> Artificial sequence

<220>

<223> U4-4-VH

<400> 39

<210> 40

<211> 408

<212> DNA

<213> Artificial sequence

<220>

<223> U4-5-VH

<400> 40

<210> 41

<211> 399

<212> DNA

<213> Artificial sequence

<220>

<223> U4-8-VH

<400> 41

<210> 42

<211> 393

<212> DNA

<213> Artificial sequence

<220>

<223> U4-6-VH

<400> 42

<210> 43

<211> 399

<212> DNA

<213> Artificial sequence

<220>

<223> U4-7-VH

<400> 43

<210> 44

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> U4-1-VL

<400> 44

<210> 45

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> U4-2-VL

<400> 45

<210> 46

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> U4-3-VL

<400> 46

<210> 47

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> U4-4-VL

<400> 47

<210> 48

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> U4-5-VL

<400> 48

<210> 49

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> U4-8-VL

<400> 49

<210> 50

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> U4-6-VL

<400> 50

<210> 51

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> U4-7-VL and U4-9-VL

<400> 51

<210> 52

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> U4-1-VH

<400> 52

<210> 53

<211> 133

<212> PRT

<213> Artificial sequence

<220>

<223> U4-2-VH

<400> 53

<210> 54

<211> 133

<212> PRT

<213> Artificial sequence

<220>

<223> U4-3-VH and U4-9-VH

<400> 54

<210> 55

<211> 134

<212> PRT

<213> Artificial sequence

<220>

<223> U4-4-VH

<400> 55

<210> 56

<211> 136

<212> PRT

<213> Artificial sequence

<220>

<223> U4-5-VH

<400> 56

<210> 57

<211> 133

<212> PRT

<213> Artificial sequence

<220>

<223> U4-8-VH

<400> 57

<210> 58

<211> 131

<212> PRT

<213> Artificial sequence

<220>

<223> U4-6-VH

<400> 58

<210> 59

<211> 133

<212> PRT

<213> Artificial sequence

<220>

<223> U4-7-VH

<400> 59

<210> 60

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> U4-1-VL

<400> 60

<210> 61

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> U4-2-VL

<400> 61

<210> 62

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> U4-3-VL

<400> 62

<210> 63

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> U4-4-VL

<400> 63

<210> 64

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> U4-5-VL

<400> 64

<210> 65

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> U4-8-VL

<400> 65

<210> 66

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> U4-6-VL

<400> 66

<210> 67

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> U4-7-VL and U4-9-VL

<400> 67

<210> 68

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> U4-7 and U4-9 CDRL1

<400> 68

Claims (36)

A human antibody directed against an epitope of between 119 and 248 amino acids of human FGFR4, preferably between 152 and 240 amino acids, more preferably between 230 and 240 amino acids. Or a functional fragment or functional derivative of the antibody. An antibody according to claim 1 which binds to the Ig-like domain 2 of the FGFR4, in particular to an epitope comprising the amino acid sequence RYNY. An antibody according to claim 1 or 2, which comprises (i) a heavy chain comprising: having the sequence shown in any one of SEQ ID Nos: 1 to 6 or having 1 or 2 with the sequences Heavy chain complementarity determining region 1 (CDRH1) of an amino acid-incompatible sequence, having the sequence shown in any one of SEQ ID Nos: 7 to 12 or having 1 or 2 amino acids with the sequences The heavy chain complementarity determining region 2 (CDRH2) of the different sequence and/or having the sequence shown in any of the sequences of SEQ ID Nos: 13 to 20 or having one or two amino acids different from the sequences a heavy chain complementarity determining region 3 (CDRH3), and/or (ii) a light chain comprising: a sequence having the sequence shown in any one of SEQ ID Nos: 21 to 23 and 68 or Light chain complementarity determining region 1 (CDRL1) having 1 or 2 amino acid-incompatible sequences, having the sequence shown in any of SEQ ID Nos: 24-27, or having 1 or 2 with the sequences Light chain complementarity determining region 2 (CDRL2) of an amino acid-incompatible sequence, and/or having the sequence shown in any of SEQ ID Nos: 28 to 35 or Light chain complementarity determining region 3 (CDRL3) having one or two amino acid-incompatible sequences, or a monoclonal antibody recognizing the same epitope on human FGFR4. An antibody according to claim 3, which comprises the CDRH1 having the sequence shown in any one of SEQ ID Nos: 1 to 6, having the sequence shown in any one of SEQ ID Nos: 7 to 12. CDRH2 of the sequence, CDRH3 having the sequence shown in any one of SEQ ID Nos: 13 to 20, and CDRL1 having the sequence shown in any one of SEQ ID Nos: 21 to 23 and 68 have The CDRL2 of the sequence shown in any one of SEQ ID Nos: 24 to 27 and the CDRL3 having the sequence shown in any one of SEQ ID Nos: 28 to 35. An antibody according to any one of the preceding claims, comprising a heavy chain comprising: (a) CDRH1 as set forth in SEQ ID NO: 1, or 1 or 2 amines from SEQ ID NO: 1. a CDRH1 sequence differing from the base acid, (b) a CDRH2 as shown in SEQ ID NO: 7, or a CDRH2 sequence having one or two amino acids different from SEQ ID NO: 7, and (c) SEQ. CDRH3 shown in ID NO: 13, or a CDRH3 sequence having SEQ ID NO: 13 having 1 or 2 amino acids, and/or a light chain comprising (d) SEQ ID NO: 21 The CDRL1 shown in the above, or the sequence of the CDRL1 from SEQ ID NO: 21 having 1 or 2 amino acids, (e) the CDRL2 shown in SEQ ID NO: 24, or from SEQ ID NO: 24 has 1 or 2 amino acid-differentiated CDRL2 sequences, and (f) has a CDRL3 as shown in SEQ ID NO: 28, or 1 or 2 amino acid phases from SEQ ID NO: 28. A different CDRL3 sequence, or a monoclonal antibody that recognizes the same epitope on human FGFR4. The antibody of any one of claims 1 to 5, which comprises a heavy chain comprising: (a) CDRH1 as shown in SEQ ID NO: 2, or 1 from SEQ ID NO: Or a CDRH1 sequence differing between two amino acids, (b) a CDRH2 as set forth in SEQ ID NO: 7, or a CDRH2 sequence having one or two amino acids different from SEQ ID NO: 7, and c) CDRH3 as shown in SEQ ID NO: 14, or a CDRH3 sequence having SEQ ID NO: 14 having 1 or 2 amino acids, and/or a light chain comprising: (d) The CDRL1 shown in SEQ ID NO: 22, or the CDRL1 sequence having SEQ ID NO: 22 having 1 or 2 amino acids different, (e) the CDRL2 as shown in SEQ ID NO: 24, or from SEQ ID NO: 24 has 1 or 2 amino acid-differentiated CDRL2 sequences, and (f) CDRL3 as shown in SEQ ID NO: 29, or 1 or 2 amino acid phases from SEQ ID NO: 29. A different CDRL3 sequence, or a monoclonal antibody that recognizes the same epitope on human FGFR4. The antibody of any one of claims 1 to 5, which comprises a heavy chain comprising: (a) CDRH1 as shown in SEQ ID NO: 3, or CDRH1 sequence differing from SEQ ID NO: 3 with 1 or 2 amino acids, (b) CDRH2 as shown in SEQ ID NO: Or a CDRH2 sequence differing from SEQ ID NO: 8 with 1 or 2 amino acids, and (c) CDRH3 as shown in SEQ ID NO: 15, or 1 or 2 from SEQ ID NO: 15. An amino acid-differentiated CDRH3 sequence, and/or a light chain comprising: (d) a CDRL1 as set forth in SEQ ID NO: 23, or one or two amino acids from SEQ ID NO: a different sequence of CDRL1, (e) a CDRL2 as set forth in SEQ ID NO: 25, or a sequence of 1 or 2 amino acid differs from SEQ ID NO: 25, and (f) as SEQ ID NO The CDRL3 shown in 30, or the CDRL3 sequence from SEQ ID NO: 30 having 1 or 2 amino acids, or a monoclonal antibody recognizing the same epitope on human FGFR4. The antibody of any one of claims 1 to 5, which comprises a heavy chain comprising: (a) CDRH1 as shown in SEQ ID NO: 4, or 1 from SEQ ID NO: Or a CDRH1 sequence differing between two amino acids, (b) a CDRH2 as shown in SEQ ID NO: 9, or a CDRH2 sequence having one or two amino acids different from SEQ ID NO: 9, and c) a CDRH3 as shown in SEQ ID NO: 16, or a CDRH3 sequence having SEQ ID NO: 16 having 1 or 2 amino acid dissimilarities, and/or a light chain comprising: (d) a CDRL1 as shown in SEQ ID NO: 22, or a CDRL1 sequence having SEQ ID NO: 22 having 1 or 2 amino acids different, (e) a CDRL2 as set forth in SEQ ID NO: Or a CDRL2 sequence having 1 or 2 amino acids different from SEQ ID NO: 24, and (f) a CDRL3 as shown in SEQ ID NO: 31, or 1 or 2 from SEQ ID NO: 31 Amino acid-differentiated CDRL3 sequences, or monoclonal antibodies that recognize the same epitope on human FGFR4. The antibody of any one of claims 1 to 5, which comprises a heavy chain comprising: (a) CDRH1 as shown in SEQ ID NO: 5, or 1 from SEQ ID NO: Or a CDRH1 sequence differing between two amino acids, (b) a CDRH2 as set forth in SEQ ID NO: 10, or a CDRH2 sequence having one or two amino acids different from SEQ ID NO: 10, and c) CDRH3 as shown in SEQ ID NO: 17, or a CDRH3 sequence having SEQ ID NO: 17 having 1 or 2 amino acids, and/or a light chain comprising: (d) The CDRL1 shown in SEQ ID NO: 22, or the CDRL1 sequence having SEQ ID NO: 22 having 1 or 2 amino acids different, (e) the CDRL2 as shown in SEQ ID NO: 24, or from SEQ ID NO: 24 has 1 or 2 amino acid-differentiated CDRL2 sequences, and (f) has a CDRL3 as shown in SEQ ID NO: 32, or 1 or 2 amino acid phases from SEQ ID NO: 32 a different CDRL3 sequence, or a single resistance that recognizes the same epitope on human FGFR4 body. The antibody of any one of claims 1 to 5, which comprises a heavy chain comprising: (a) CDRH1 as shown in SEQ ID NO: 4, or 1 from SEQ ID NO: Or a CDRH1 sequence differing between two amino acids, (b) a CDRH2 as set forth in SEQ ID NO: 11, or a CDRH2 sequence having one or two amino acids different from SEQ ID NO: 11, and c) a CDRH3 as shown in SEQ ID NO: 18, or a CDRH3 sequence having SEQ ID NO: 18 having 1 or 2 amino acids, and/or a light chain comprising: (d) The CDRL1 shown in SEQ ID NO: 22, or the CDRL1 sequence having SEQ ID NO: 22 having 1 or 2 amino acids different, (e) the CDRL2 as shown in SEQ ID NO: 24, or from SEQ ID NO: 24 has 1 or 2 amino acid-differentiated CDRL2 sequences, and (f) has a CDRL3 as shown in SEQ ID NO: 33, or 1 or 2 amino acid phases from SEQ ID NO: 33 A different CDRL3 sequence, or a monoclonal antibody that recognizes the same epitope on human FGFR4. The antibody of any one of claims 1 to 5, which comprises a heavy chain comprising: (a) CDRH1 as shown in SEQ ID NO: 6, or 1 from SEQ ID NO: Or a CDRH1 sequence of two amino acid dissimilarities, (b) a CDRH2 as set forth in SEQ ID NO: 12, or a CDRH2 sequence having one or two amino acids different from SEQ ID NO: 12, and (c) CDRH3 as shown in SEQ ID NO: 19, or a CDRH3 sequence having SEQ ID NO: 19 having 1 or 2 amino acids, and/or a light chain comprising: (d) CDRL1 as shown in SEQ ID NO: 22, or a CDRL1 sequence differing from SEQ ID NO: 22 with 1 or 2 amino acids, (e) CDRL2 as shown in SEQ ID NO: 26, or from SEQ ID NO: 26 has 1 or 2 amino acid-differentiated CDRL2 sequences, and (f) is a CDRL3 as set forth in SEQ ID NO: 34, or 1 or 2 amino acids from SEQ ID NO: 34 A distinct CDRL3 sequence, or a monoclonal antibody that recognizes the same epitope on human FGFR4. The antibody of any one of claims 1 to 5, which comprises a heavy chain comprising: (a) CDRH1 as shown in SEQ ID NO: 3, or 1 from SEQ ID NO: Or a CDRH1 sequence differing between two amino acids, (b) a CDRH2 as set forth in SEQ ID NO: 7, or a CDRH2 sequence having one or two amino acids different from SEQ ID NO: 7, and c) a CDRH3 as shown in SEQ ID NO: 20, or a CDRH3 sequence having SEQ ID NO: 20 having 1 or 2 amino acids, and/or a light chain comprising: (d) The CDRL1 shown in SEQ ID NO: 68, or the CDRL1 sequence differing from SEQ ID NO: 68 by 1 or 2 amino acids, (e) the CDRL2 as shown in SEQ ID NO: 27, or from SEQ ID NO: 27 has 1 or 2 amino acid-related CDRL2 sequences, and (f) a sequence of CDRL3 as shown in SEQ ID NO: 35, or a sequence of 1 or 2 amino acid different from SEQ ID NO: 35, or a single epitope that recognizes the same epitope on human FGFR4 Strain antibody. The antibody of any one of claims 1 to 5, which comprises a heavy chain comprising: (a) CDRH1 as shown in SEQ ID NO: 3, or 1 from SEQ ID NO: Or a CDRH1 sequence of two amino acid dissimilarities, (b) a CDRH2 as set forth in SEQ ID NO: 8, or a CDRH2 sequence having one or two amino acids different from SEQ ID NO: 8, and c) a CDRH3 as shown in SEQ ID NO: 15, or a CDRH3 sequence having SEQ ID NO: 15 having 1 or 2 amino acids, and/or a light chain comprising: (d) The CDRL1 shown in SEQ ID NO: 68, or the CDRL1 sequence differing from SEQ ID NO: 68 by 1 or 2 amino acids, (e) the CDRL2 as shown in SEQ ID NO: 27, or from SEQ ID NO: 27 has 1 or 2 amino acid-differentiated CDRL2 sequences, and (f) has a CDRL3 as shown in SEQ ID NO: 35, or 1 or 2 amino acid phases from SEQ ID NO: 35 A different CDRL3 sequence, or a monoclonal antibody that recognizes the same epitope on human FGFR4. The antibody of any one of claims 1 to 13 which comprises a heavy chain variable region as shown in any one of SEQ ID Nos: 52 to 59 and/or as SEQ ID Nos: 60 to 67 Light as shown in any of the sequences Chain variable region. An antibody according to any one of the preceding claims, which is a Fab fragment, a Fab' fragment, an F(ab') fragment, an Fv fragment, a diabody or a single chain antibody molecule. An antibody according to any one of the preceding claims, which is an antibody of the IgG1 type, IgG2 type, IgG3 type or IgG4 type. The antibody of any one of the preceding claims, wherein the antibody is an inhibitor of FGFR4 signaling, particularly wherein the antibody is an inhibitor of a ligand that binds to FGFR4. The antibody of claim 17, wherein the antibody inhibits binding of FGF1, FGF2, FGF4, FGF6, FGF8, FGF9, FGF17, FGF18, FGF19, FGF20, FGF21 and/or FGF23 to FGFR4. An antibody according to any one of the preceding claims, wherein the antibody does not have cross-reactivity to FGFR1-3b, FGFR1-3c. A conjugate, comprising an antibody according to any one of the preceding claims, wherein the anti-system is coupled to a labeling group and/or an effector group, preferably a therapeutic group. A fusion protein comprising an antibody fused to IL-2 according to any one of claims 1 to 19. An isolated nucleic acid molecule selected from the group consisting of: (a) an antibody, antibody fragment or derivative thereof according to any one of claims 1 to 19, or as claimed Item 20 a conjugated conjugate or a nucleic acid sequence of the fusion protein of claim 21, (b) a nucleic acid sequence as set forth in any one of SEQ ID NOS: 36 to 43 and SEQ ID NO: 44 to 51, c) a nucleic acid sequence complementary to any of (a) or (b); and (d) a nucleic acid sequence capable of hybridizing to (a), (b) or (c) under stringent conditions. A vector comprising the nucleic acid sequence of claim 22, which is preferably an expression vector, wherein the nucleic acid sequence is operably linked to a control sequence. A host comprising a nucleic acid as in claim 22 or a carrier as in claim 23 of the patent application. The host of claim 24, which is a human, a bacterium, an animal, a fungus, an amphibian or a plant cell. A host of claim 25, which is a non-human transgenic animal. A method of producing an antibody according to any one of claims 1 to 19, a conjugate of claim 20, or a fusion protein according to claim 21 of the patent application, which comprises, for example, a patent application The step of obtaining the antibody, antibody conjugate conjugate or fusion protein by the host of any one of items 24 to 26. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 19, a conjugate conjugate as claimed in claim 20, or a fusion protein according to claim 21 of the patent application, such as an application Nucleic acid molecule of claim 22, as set forth in item 23 of the patent application The host of any one of claims 24 to 26, or the antibody produced by the method of claim 27 of the patent application. A pharmaceutical composition according to claim 28, which comprises another active agent. An antibody according to any one of claims 1 to 19, a conjugate according to claim 20, a fusion protein as claimed in claim 21, a nucleic acid molecule as claimed in claim 22 The carrier of claim 23, the host of any one of claims 24 to 26, and the pharmaceutical composition according to any one of claims 28 to 29, for diagnosis , preventing and/or treating diseases associated with FGFR4 performance, overexpression, and/or overactivity. An antibody, conjugate conjugate, fusion protein, nucleic acid molecule, vector, host or pharmaceutical composition for use according to claim 30, wherein the disease is a hyperproliferative disease. An antibody, a conjugate conjugate, a fusion protein, a nucleic acid molecule, a vector, a host or a pharmaceutical composition for use according to claim 31, wherein the hyperproliferative disease is cancer, preferably selected from the group consisting of Group of cancer: hepatocellular carcinoma, breast cancer, gastric cancer, colon cancer, rhabdomyosarcoma, prostate cancer, ovarian cancer, soft tissue sarcoma, melanoma, head and neck squamous cell carcinoma and lung adenocarcinoma and other manifestations Or excessive expression of FGFR4 in cancer and tumor metastasis. An antibody according to any one of claims 1 to 19, a conjugate according to claim 20, a fusion protein as claimed in claim 21, a nucleic acid molecule as claimed in claim 22 , The carrier of claim 23, the host of any one of claims 24 to 26, or the pharmaceutical composition according to any one of claims 28 to 29, for diagnosis, Prevent and/or treat metabolic diseases, especially metabolic syndrome or obesity. An antibody according to any one of claims 1 to 19, a conjugate according to claim 20, a fusion protein as claimed in claim 21, a nucleic acid molecule as claimed in claim 22 The carrier of claim 23, the host of any one of claims 24 to 26, or the pharmaceutical composition according to any one of claims 28 to 29, for diagnosis , prevention and / or treatment of left ventricular hypertrophy, cardiac hypertrophy or chronic kidney disease. A method for diagnosing a condition associated with performance, over-expression, and/or hyperactivity of FGFR4, comprising administering a sample to an antibody according to any one of claims 1 to 19, as claimed in claim 20 The conjugate is conjugated or the fusion protein as in claim 21 of the patent application is contacted and the presence of FGFR4 is detected. A kit comprising the antibody of any one of claims 1 to 19, the conjugate of claim 20, the fusion protein of claim 21, as claimed in the patent application. The nucleic acid molecule of item 22 or the carrier of claim 23, and another anti-tumor agent.