Anti-FGFR3 Antibody and Its Use

Abstract

The present invention relates to an anti-FGFR3 (Fibroblast growth factor receptor 3) antibody or an antigen-binding fragment thereof, a nucleic acid encoding the antibody or an antigen-binding fragment thereof, a recombinant expression vector comprising the nucleic acid, a host cell transformed with the recombinant expression vector, a method for producing the antibody or the antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or the antigen-binding fragment thereof, an immune cell engaging bispecific or multispecific antibody comprising an scFv of the antibody and a second binding domain comprising one or more scFvs of an antibody that binds to an immune cell activation antigen, an antibody-drug conjugate (ADC) in which the antibody or the antigen-binding fragment thereof is bound to a drug, a chimeric antigen receptor (CAR) comprising an scFv of the anti-FGFR3 antibody as an antigen-binding site in the extracellular domain, an immune cell into which the chimeric antigen receptor has been introduced, a combination therapeutic composition comprising the immune cell, a combination therapeutic composition comprising the antibody or the antigen-binding fragment thereof, a cancer treatment composition, a cancer treatment method, and a vesicle comprising the antibody or the antigen-binding fragment thereof.

Description

[Technical field]

[0001] The present invention relates to an anti-FGFR3 (Fibroblast growth factor receptor 3) antibody or an antigen-binding fragment thereof, a nucleic acid encoding the antibody or an antigen-binding fragment thereof, a recombinant expression vector comprising the nucleic acid, a host cell transformed with the recombinant expression vector, a method for producing the antibody or the antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or the antigen-binding fragment thereof, an immune cell engaging bispecific or multispecific antibody comprising an scFv of the antibody and a second binding domain comprising one or more scFvs of an antibody that binds to an immune cell activation antigen, an antibody-drug conjugate (ADC) in which the antibody or the antigen-binding fragment thereof is bound to a drug, a chimeric antigen receptor (CAR) comprising an scFv of the anti-FGFR3 antibody as an antigen-binding site in an extracellular domain, an immune cell into which the chimeric antigen receptor has been introduced, a combination therapeutic composition comprising the immune cell, a combination therapeutic composition comprising the antibody or the antigen-binding fragment thereof, a composition for treating cancer, inflammation or an immune disease, a method for treating cancer, inflammation or an immune disease, and a vesicle comprising the antibody or the antigen-binding fragment thereof. [Background technology]

[0002] FGFs (fibroblast growth factors) and their receptor tyrosine kinases (FGFRs) play important roles in embryonic development, homeostasis in various tissues, wound healing processes and metabolic functions. In humans, there are highly homologous FGFRs (FGFR1-4) and 22 FGFs (FGF1-14 and FGF16-23). ​​FGFRs contain an extracellular domain containing three immunodomains D1, D2 and D3, a single transmembrane domain and a split cytoplasmic kinase moiety.

[0003] Dysregulation of signaling by FGFR1-4 is associated with the pathogenesis leading to various types of cancer. Genomic FGFR mutations, including gene amplification, chromosomal translocations and activating mutations, induce aberrant activation of the FGF pathway and promote tumor transformation of normal cells. In particular, amplification of FGFR3 is known to be associated with the induction of bladder cancer. In particular, missense mutations in FGFRs have been observed in multiple types of cancer, and S249C in FGFR3 may enhance FGF-driven signaling and tumor cell growth, indicating a hotspot for somatic mutations.

[0004] The development of cancer drugs has focused on targeting the FGF / FGFR pathway. Meanwhile, fusion proteins caused by chromosomal rearrangements as cancer-inducing mutations have been found in glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell carcinoma, gallbladder cancer, and cervical cancer in recent years. These fusion proteins are generated through the fusion of the FGFR3 gene and the TACC3 (transforming acidic coiled-coil containing protein 3) gene by tandem duplication on chromosome 4. FGFR can be continuously activated by the dimerizing domain of the partner protein, which allows the FGFR monomer in the fusion protein to be close enough to be activated. In FGFR3-TACC3, the coiled-coil domain of TACC3 allows FGFR3 to be autophosphorylated and activated even in the absence of ligand binding.

[0005] TACC3 belongs to the TACC family, and rearrangements at chromosome 4p16, which corresponds to the region containing TACC3 and FGFR3, are frequently observed in multiple myeloma, leading to increased expression of FGFR3 or TACC3 (KN Nelson et al., Oncotarget. 2018 Sep 28; 9(76): 34306-34319).

[0006] Based on the findings above, clinical trials are currently underway for antibodies targeting FGFR3 or fusion genes with the FGFR3 gene.

[0007] B701 (Vofatamab) is a human immunoglobulin G1 monoclonal antibody against FGFR3 that is undergoing clinical trials to determine whether it exhibits antitumor activity and whether it can be used in combination with docetaxel. When administered, the anti-FGFR3 monoclonal antibody B-701 specifically binds to and inhibits both wild-type and mutant forms of FGFR3, inhibiting FGFR3 phosphorylation and thereby suppressing FGFR3 activation and FGFR3-mediated signaling pathways. This inhibits cell growth and induces cell death in FGFR3-expressing tumors.

[0008] LY3076226 is an antibody-drug conjugate (ADC) that combines an anti-FGFR3 antibody with DM4 (microtubule inhibitor), and is undergoing clinical trials to see if it can be used in advanced or metastatic cancers. A phase I clinical trial is underway to see if LY3076226 is effective in advanced or metastatic cancers, including multiple myeloma and lymphoma, with FGFR3 overexpression or mutation, and locally advanced, unresectable or metastatic urinary tract cancer (T Ibrahim et al., Bladder Cancer, vol. 5, no. 2, pp. 87-102, 2019).

[0009] Since such conventional anti-FGFR3 antibodies show low affinity, target resolution and efficacy, the present inventors have endeavored to develop a novel anti-FGFR3 antibody having improved affinity, target resolution and efficacy compared to conventional anti-FGFR3 antibodies. As a result, the inventors have confirmed the characteristics of the antibody, for example, affinity confirmation, FGFR3 binding pattern analysis, FGFR3 resolution confirmation, cancer cell proliferation inhibition results and efficacy test results, and in particular, have selected an antibody having differentiated characteristics through a strategy of selecting an antibody having a more sensitive reactivity to cells derived from brain tumor patients overexpressing FGFR3-TACC3 than known anti-FGFR3 antibodies, and have confirmed that the selected antibody can be used for the intended cancer treatment, thereby completing the present invention. Summary of the Invention

[0010] An object of the present invention is to provide a novel antibody or an antigen-binding fragment thereof against FGFR3 (fibroblast growth factor receptor 3).

[0011] In particular, an object of the present invention is to select an antibody having a more sensitive reactivity to cells derived from brain tumor patients overexpressing FGFR3-TACC3 than known anti-FGFR3 antibodies.

[0012] It is another object of the present invention to provide a nucleic acid encoding said antibody or antigen-binding fragment thereof.

[0013] Another object of the present invention is to provide a recombinant expression vector comprising said nucleic acid or a host cell transformed with said recombinant expression vector.

[0014] Another object of the present invention is to provide a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to FGFR3.

[0015] Another object of the present invention is to provide a bi- or multispecific antibody comprising said antibody or antigen-binding fragment thereof.

[0016] Another object of the present invention is to provide an immune cell engaging bispecific or multispecific antibody comprising said antibody or an antigen-binding fragment thereof.

[0017] Another object of the present invention is to provide an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof is bound to a drug.

[0018] Another object of the present invention is to provide a chimeric antigen receptor (CAR) comprising the scFv of the anti-FGFR3 antibody as an antigen-binding site in the extracellular domain, an immune cell into which the chimeric antigen receptor has been introduced, and a composition for combined therapy comprising the immune cell.

[0019] Another object of the present invention is to provide a combination therapeutic composition comprising said antibody or antigen-binding fragment thereof or said immune cell engaging bispecific or multispecific antibody.

[0020] Another object of the present invention is to provide an antibody or antigen-binding fragment thereof, a bi- or multispecific antibody comprising said antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising said antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising said antibody or antigen-binding fragment thereof, and a composition for cancer treatment or a method for cancer treatment comprising said chimeric antigen receptor.

[0021] Another object of the present invention is to provide a vesicle comprising said antibody or antigen-binding fragment thereof.

[0022] To achieve the above-mentioned object, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to FGFR3 (fibroblast growth factor receptor 3), including: A heavy chain CDR1 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:18; A heavy chain CDR2 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 19 to SEQ ID NO: 41; A heavy chain CDR3 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 42 to SEQ ID NO: 66, SEQ ID NO: 186 to SEQ ID NO: 198; A light chain CDR1 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 67 to SEQ ID NO: 88; a light chain CDR2 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 89 to SEQ ID NO: 109; and A light chain CDR3 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:110 to SEQ ID NO:132, SEQ ID NO:199 to SEQ ID NO:202.

[0023] The invention provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0024] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0025] The present invention also provides a host cell transformed with the recombinant expression vector.

[0026] The present invention also provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to FGFR3, comprising the steps of culturing a host cell to produce the antibody, and isolating and purifying the produced antibody.

[0027] The present invention also provides a bi- or multispecific antibody comprising said antibody or antigen-binding fragment thereof.

[0028] The invention also provides immune cell engage bispecific or multispecific antibodies comprising an scFv composed of an scFv of said antibody and a second binding domain comprising one or more scFvs of an antibody that binds to an activating antigen of an immune cell.

[0029] The present invention also provides an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof is bound to a drug.

[0030] The present invention also provides a chimeric antigen receptor (CAR) comprising an extracellular domain containing an antigen-binding site, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding site of the extracellular domain is an scFv of the antibody.

[0031] The present invention also provides an immune cell comprising said chimeric antigen receptor (CAR).

[0032] The present invention also provides a composition for combined therapy comprising the immune cells and a drug other than an anti-FGFR3 antibody.

[0033] The present invention also provides a combination therapeutic composition comprising the antibody or antigen-binding fragment thereof and one or more selected from the group consisting of: (i) immune cells, (ii) an immune cell comprising an antigen receptor (CAR) that comprises an scFv fragment against an antibody other than an anti-FGFR3 antibody as an extracellular domain; and (iii) Immune checkpoint inhibitor.

[0034] The present invention also provides a combination therapeutic composition comprising said bispecific or multispecific antibody and one or more selected from the group consisting of: (i) immune cells, (ii) an immune cell comprising an antigen receptor (CAR) that comprises an scFv fragment against an antibody other than an anti-FGFR3 antibody as an extracellular domain; and (iii) Immune checkpoint inhibitor.

[0035] The present invention also provides a composition for cancer treatment comprising the antibody or antigen-binding fragment thereof, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell into which the chimeric antigen receptor has been introduced.

[0036] The present invention also provides a composition for cancer therapy comprising the antibody or antigen-binding fragment thereof, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell into which the chimeric antigen receptor has been introduced, for treating a cancer in which the FGFR3 and TACC3 fusion gene has been confirmed to be amplified, mutated, overexpressed, or constitutively activated.

[0037] The present invention also provides a method for treating cancer, comprising the steps of: determining whether a fusion gene of FGFR3 and TACC3 is amplified, mutated, overexpressed, or constitutively activated in a patient-derived sample; and, when a fusion gene of FGFR3 and TACC3 is amplified, mutated, overexpressed, or constitutively activated, administering the antibody or antigen-binding fragment thereof, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell into which the chimeric antigen receptor has been introduced.

[0038] The present invention also provides a vesicle on the exterior of which, or within which, the antibody or antigen-binding fragment thereof is expressed. [Brief description of the drawings]

[0039] FIG. 1 shows a schematic diagram of the phage display-based biopanning used in the present invention. FIG. 1a shows a schematic diagram of cell panning using patient-derived cells overexpressing FGFR3. FIG. 1b shows a schematic diagram of solid-phase biopanning using FGFR3 recombinant protein. FIG. 1c shows a schematic diagram of solution-phase biopanning using FGFR3 recombinant protein. FIG. 1d shows a schematic diagram of magnetic-based semi-automated panning with FGFR3 recombinant protein.

[0040] FIG. 2a shows the biopanning results for antibody screening according to the present invention. FIG. 2b shows the results of ELISA screening for antibody screening according to the present invention. FIG. 2c shows the results of magnetic bead-based panning for antibody screening according to the present invention.

[0041] FIG. 3a shows a schematic of the conversion of scFv to IgG format. Figure 3b shows the results of Expi293-based antibody production and purity and physical property analysis.

[0042] Figures 4a to 4d show the results of affinity ELISA of FGFR3 antibodies (IgG). Figure 4a shows the specific binding ability of the antibodies to the recombinant protein human FGFR3-IIIc, Figure 4b shows the specific binding ability of the antibodies to the recombinant protein human FGFR3-IIIb, Figure 4c shows the specific binding ability of the antibodies to the recombinant protein mouse FGFR3, and Figure 4d shows the specific binding ability of the antibodies to the recombinant protein cynomolgus monkey FGFR3.

[0043] FIG. 5 shows the results of KD value evaluation of FGFR3 antibody (IgG) using Biacore 3000.

[0044] Figure 6 shows the results of FGFR3 expression confirmation and FGFR3-TACC3 fusion analysis of brain tumor patient-derived cells. Figure 6a shows the results of mRNA expression level (RPKM) analysis, protein expression level analysis (western blotting), and cell surface expression analysis (FACS analysis) of patient-derived cells, and Figure 6b shows the confirmation of FGFR3-TACC3 fusion through RT-PCR and fusion-specific primers.

[0045] Figure 7a shows the results of cell surface binding analysis of FGFR3 antibody clones to cells derived from brain tumor patients overexpressing FGFR3 (-TACC3). Figure 7b shows the results of concentration-specific binding pattern analysis of F309 antibody to cells derived from brain tumor patients overexpressing FGFR3 (-TACC3). Figure 7c shows the results of binding strength analysis of F309 and FS306 antibodies to cell lines with no FGFR3 expression.

[0046] FIG. 8 shows the results of FACS-based patient-derived cell surface FGFR3 (-TACC3) resolution analysis and screening of FGFR3 antibodies.

[0047] Figure 9a shows the results of FGFR3 specific binding analysis of F309 and FS306 antibodies and confirmation of their non-competitive ligand properties. Figure 9b shows the results of identification of the unique epitope domains of F309 and FS306 antibodies, Domain I. Figure 9c shows the results of recombinant domain design for epitope mapping of domains.

[0048] Figure 10 shows the results of target resolution analysis of F309 and FS306 antibodies against FGFR3-TACC3 overexpressing brain tumor patient-derived cells and FGFR3 overexpressing cell lines. Figure 10a shows the results of cell surface FGFR3-TACC3 and FGFR3 resolution analysis and FACS analysis of F309 and FS306 antibodies. Figure 10b shows the results of total FGFR3-TACC3 and FGFR3 resolution analysis and Western blotting of F309 and FS306 antibodies.

[0049] Figure 11 shows the results of cellular internalization analysis of F309 and FS306 antibodies. Figure 11a shows the results of cellular internalization analysis and ICC analysis in cells derived from a brain tumor patient with overexpressing FGFR3-TACC3. Figure 11b shows the results of confirming the lysosomal degradation mechanism of F309 and FS306 antibodies through lysosome inhibitor treatment.

[0050] FIG. 12 shows the results of an analysis of FGFR3 sub-signaling inhibition by the F309 and FS306 antibodies.

[0051] Figure 13a shows the results of ligand-dependent cell growth inhibition assay (FGF1, FGF9), and Figure 13b shows the results of ligand-independent cell growth inhibition assay (FGFR3-TACC3 overexpressing brain tumor patient-derived cells, FGFR3 low expression cell line, or brain tumor patient-derived cells).

[0052] Figure 14a shows the results of short-term PK analysis of F309 and FS306. Figure 14b shows the results of antibody efficacy evaluation in mice (PDX) transplanted with cells derived from a brain tumor patient overexpressing FGFR3-TACC3. Figure 14c shows the results of efficacy evaluation of the F309 antibody in a PDX model (cells derived from a brain tumor patient overexpressing FGFR3-TACC3).

[0053] Figure 15a shows a schematic diagram of the 3D and 2D structures and strategic affinity improvement of the F309 antibody. Figure 15b shows the results of affinity comparison of the F309 affinity-improved antibodies by affinity ELISA. Figure 15c shows the results of SPR analysis of the F309 affinity-improved antibodies. Figure 15c shows the results of ligand-independent cell growth inhibition analysis (cells derived from FGFR3-TACC3-overexpressing brain tumor patients) of the F309 improved antibody clones.

[0054] Figure 16a shows a schematic diagram of the production of antibody-drug conjugates (ADCs) for the F309 antibody to confirm their applicability. Figure 16b shows the results of cytotoxicity analysis of F309-vc MMAE against FGFR3-TACC3-overexpressing brain tumor patient-derived cells and FGFR3-overexpressing cell lines. Figure 16c shows the results of cytotoxicity analysis 1 according to the FGFR3 expression level of F309-vc MMAE, and Figure 16d shows the results of cytotoxicity analysis 2 according to the FGFR3 expression level of F309-vc MMAE.

[0055] FIG. 17 shows the Fv structure and epitope domain mapping of affinity matured antibodies and the results of antigen-specific binding analysis.

[0056] FIG. 18 shows the results of an analysis of cell surface antigen expression in cells and cell lines derived from FGFR3-TACC3 brain tumor patients.

[0057] FIG. 19 shows the results of an analysis of the cellular internalization efficiency of affinity matured antibodies.

[0058] FIG. 20 shows the results of an analysis of the FGFR3(-TACC3) target resolution of affinity matured antibodies.

[0059] FIG. 21 shows the results of mouse PK analysis of affinity matured antibodies.

[0060] FIG. 22 shows the results of monkey PK analysis of affinity matured antibodies.

[0061] FIG. 23 shows a schematic diagram of ADC production containing affinity matured antibody (Topoisomerase I inhibitor binding).

[0062] FIG. 24 shows a schematic diagram of ADC production containing an affinity matured antibody (Topoisomerase I inhibitor binding).

[0063] FIG. 25 shows the results of an analysis of the physical properties and binding capacity of ADCs comprising affinity matured antibodies.

[0064] Figure 26 shows the results of an analysis of the physical properties of ADCs containing affinity matured antibodies.

[0065] Figure 27 shows the results of in vitro efficacy evaluation of ADCs comprising affinity matured antibodies.

[0066] Figure 28 shows the results of in vitro efficacy evaluation of ADCs comprising affinity matured antibodies.

[0067] Figure 29 shows the results of in vivo efficacy evaluation of ADCs comprising affinity matured antibodies.

[0068] Figure 30 shows the results of in vivo efficacy evaluation of ADCs comprising affinity matured antibodies.

[0069] Figure 31 shows the results of in vivo efficacy evaluation of ADCs comprising affinity matured antibodies.

[0070] Figure 32 shows the results of ADC efficacy evaluation of ADCs comprising affinity matured antibodies in a FGFR3 (TACC3 fusion) brain tumor stereotactic animal model.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is one that is well known and commonly used in the art.

[0072] Anti-FGFR3 antibody In one aspect, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to FGFR3 (Fibroblast growth factor receptor 3), comprising a heavy chain CDR1 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:18, a heavy chain CDR2 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:19 to SEQ ID NO:41, a heavy chain CDR3 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:42 to SEQ ID NO:66, and SEQ ID NO:186 to 198, a light chain CDR1 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:67 to SEQ ID NO:88, a light chain CDR2 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:89 to SEQ ID NO:109, and a light chain CDR3 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:110 to SEQ ID NO:132, and SEQ ID NO:199 to SEQ ID NO:202.

[0073] The term "antibody" as used herein refers to an anti-FGFR3 antibody that specifically binds to FGFR3. The scope of the present invention includes not only complete antibody forms that specifically bind to FGFR3, but also antigen-binding fragments of said antibody molecules. In some cases, the antibody according to the present invention can target FGFR3-TACC3. In particular, the antibody according to the present invention has been selected as an antibody that has better efficacy against FGFR3-TACC3-overexpressing cancer cells than known anti-FGFR3 antibodies.

[0074] Intact antibodies have a structure that contains two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by disulfide bonds.

[0075] As used herein, the term "heavy chain" refers to both a full-length heavy chain and fragments thereof that includes a variable region domain VH and three constant region domains CH1, CH2 and CH3 that contain an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen, and the term "light chain" refers to both a full-length light chain and fragments thereof that includes a variable region domain VL and a constant region domain CL that contain an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen.

[0076] The whole antibody includes IgA, IgD, IgE, IgM and IgG subtypes, and IgG includes IgG1, IgG2, IgG3 and IgG4. The constant region of the heavy chain has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, with gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2) subclasses. The constant region of the light chain has kappa (κ) and lambda (λ) types.

[0077] Antigen-binding fragments of antibodies or antibody fragments refer to fragments that retain antigen-binding function, and include Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure that has light and heavy chain variable regions, a light chain constant region, and the first constant region of the heavy chain (CH1), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 is generated by disulfide bond formation between the cysteine ​​residues in the hinge region of Fab'.

[0078] Fv is the smallest antibody fragment that has only the heavy and light chain variable regions. In a two-chain Fv, the heavy and light chain variable regions are linked by a non-covalent bond, while in a single-chain Fv (scFv), the heavy and light chain variable regions are generally linked by a covalent bond via a peptide linker or directly at the C-terminus, and can form a dimer-like structure like a two-chain Fv. Such antibody fragments can be produced by using proteolytic enzymes (for example, Fab is obtained by restrictive cleavage of an intact antibody with papain, and F(ab')2 is obtained by cleavage with pepsin) or by using genetic recombination technology.

[0079] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. This region is a dimer of one heavy-chain variable domain and one light-chain variable domain.

[0080] The "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments usually contain a pair of Fab' fragments covalently linked by cysteines in the hinge region located at the C-terminus of the Fab' fragments.

[0081] "Single-chain Fv (scFv)" antibody fragments are structures composed of a single polypeptide chain containing the VH and VL domains of an antibody. A polypeptide linker can further be included between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0082] In one embodiment, the antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFv, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of said antibodies.

[0083] The heavy chain constant region can be selected from any of the isotypes: gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). For example, the constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region can be of the kappa or lambda type.

[0084] The monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies that populate the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigen site. In contrast to conventional (polyclonal) antibodies, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0085] "Epitope" refers to a determinant on a protein to which an antibody can specifically bind. Epitopes are usually composed of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0086] The antibody or antigen-binding fragment thereof according to the present invention can bind to any one selected from the group consisting of D1, D2 and D3, which are three immune domains present in the extracellular region of FGFR3, for example, the D1, D2 or D3 domain. In some cases, the antibody or antigen-binding fragment thereof according to the present invention can specifically bind to domain 1 (SEQ ID NO: 184) of FGFR3 of SEQ ID NO: 183, and may not compete for binding with an FGF ligand.

[0087] The "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (acceptor antibodies) in which residues from a hypervariable region of an acceptor are replaced by residues from a hypervariable region of a non-human species (donor antibody), e.g., mouse, murine, rabbit, or non-human primate, that has the desired specificity, affinity, and capacity.

[0088] The "human antibody" refers to a molecule derived from human immunoglobulin, in which the entire amino acid sequence constituting the antibody, including the complementarity determining region and structural region, is composed of human immunoglobulin.

[0089] Included are "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remaining chain(s) are identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

[0090] The "variable region" of an antibody as used in the present invention refers to the light and heavy chain portions of an antibody molecule that contain the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0091] "Complementary determining region (CDR)" refers to the amino acid residues of an antibody variable domain which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.

[0092] The anti-FGFR3 antibody or antigen-binding fragment thereof according to the present invention is selected as an antibody having superior efficacy against FGFR3-TACC3 overexpressing cancer cells compared to known anti-FGFR3 antibodies, and may include, for example, the following: a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 19, and a heavy chain CDR3 of SEQ ID NO: 42; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 67, a light chain CDR2 of SEQ ID NO: 89, and a light chain CDR3 of SEQ ID NO: 110; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 20, and a heavy chain CDR3 of SEQ ID NO: 43; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 68, a light chain CDR2 of SEQ ID NO: 90, and a light chain CDR3 of SEQ ID NO: 111; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 3, a heavy chain CDR2 of SEQ ID NO: 21, and a heavy chain CDR3 of SEQ ID NO: 44; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 69, a light chain CDR2 of SEQ ID NO: 91, and a light chain CDR3 of SEQ ID NO: 112; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 22, and a heavy chain CDR3 of SEQ ID NO: 45; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 70, a light chain CDR2 of SEQ ID NO: 92, and a light chain CDR3 of SEQ ID NO: 113; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:46; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:186; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:187; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:188; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:189; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:190; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:191; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:192; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:193; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:194; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:195; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:196; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:197; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:198; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:46; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:199; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:46; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:200; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:46; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:201; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:46; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:202; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 24, and a heavy chain CDR3 of SEQ ID NO: 47; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 72, a light chain CDR2 of SEQ ID NO: 94, and a light chain CDR3 of SEQ ID NO: 115; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 6, a heavy chain CDR2 of SEQ ID NO: 25, and a heavy chain CDR3 of SEQ ID NO: 48; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 73, a light chain CDR2 of SEQ ID NO: 95, and a light chain CDR3 of SEQ ID NO: 112; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 26, and a heavy chain CDR3 of SEQ ID NO: 49; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 74, a light chain CDR2 of SEQ ID NO: 96, and a light chain CDR3 of SEQ ID NO: 116; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 8, a heavy chain CDR2 of SEQ ID NO: 27, and a heavy chain CDR3 of SEQ ID NO: 50; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 75, a light chain CDR2 of SEQ ID NO: 97, and a light chain CDR3 of SEQ ID NO: 117; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 9, a heavy chain CDR2 of SEQ ID NO: 28, and a heavy chain CDR3 of SEQ ID NO: 51; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 76, a light chain CDR2 of SEQ ID NO: 98, and a light chain CDR3 of SEQ ID NO: 118; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 29, and a heavy chain CDR3 of SEQ ID NO: 52; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 77, a light chain CDR2 of SEQ ID NO: 97, and a light chain CDR3 of SEQ ID NO: 119; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 30, and a heavy chain CDR3 of SEQ ID NO: 53; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 78, a light chain CDR2 of SEQ ID NO: 99, and a light chain CDR3 of SEQ ID NO: 120; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 54; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 79, a light chain CDR2 of SEQ ID NO: 100, and a light chain CDR3 of SEQ ID NO: 121; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 12, a heavy chain CDR2 of SEQ ID NO: 32, and a heavy chain CDR3 of SEQ ID NO: 55; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 80, a light chain CDR2 of SEQ ID NO: 101, and a light chain CDR3 of SEQ ID NO: 122; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 3, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 56; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 81, a light chain CDR2 of SEQ ID NO: 102, and a light chain CDR3 of SEQ ID NO: 123; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 34, and a heavy chain CDR3 of SEQ ID NO: 57; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 82, a light chain CDR2 of SEQ ID NO: 103, and a light chain CDR3 of SEQ ID NO: 124; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 14, a heavy chain CDR2 of SEQ ID NO: 35, and a heavy chain CDR3 of SEQ ID NO: 58; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 83, a light chain CDR2 of SEQ ID NO: 104, and a light chain CDR3 of SEQ ID NO: 125; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 36, and a heavy chain CDR3 of SEQ ID NO: 59; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 84, a light chain CDR2 of SEQ ID NO: 105, and a light chain CDR3 of SEQ ID NO: 126; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 6, a heavy chain CDR2 of SEQ ID NO: 23, and a heavy chain CDR3 of SEQ ID NO: 60; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 69, a light chain CDR2 of SEQ ID NO: 91, and a light chain CDR3 of SEQ ID NO: 127; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 15, a heavy chain CDR2 of SEQ ID NO: 37, and a heavy chain CDR3 of SEQ ID NO: 61; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 85, a light chain CDR2 of SEQ ID NO: 89, and a light chain CDR3 of SEQ ID NO: 128; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 16, a heavy chain CDR2 of SEQ ID NO: 38, and a heavy chain CDR3 of SEQ ID NO: 62; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 86, a light chain CDR2 of SEQ ID NO: 106, and a light chain CDR3 of SEQ ID NO: 129; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 17, a heavy chain CDR2 of SEQ ID NO: 39, and a heavy chain CDR3 of SEQ ID NO: 63; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 87, a light chain CDR2 of SEQ ID NO: 107, and a light chain CDR3 of SEQ ID NO: 130; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 19, and a heavy chain CDR3 of SEQ ID NO: 64; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 88, a light chain CDR2 of SEQ ID NO: 108, and a light chain CDR3 of SEQ ID NO: 131; a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 18, a heavy chain CDR2 of SEQ ID NO: 40 and a heavy chain CDR3 of SEQ ID NO: 65, a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 85, a light chain CDR2 of SEQ ID NO: 89 and a light chain CDR3 of SEQ ID NO: 128, or A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 16, a heavy chain CDR2 of SEQ ID NO: 41, and a heavy chain CDR3 of SEQ ID NO: 66; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 72, a light chain CDR2 of SEQ ID NO: 109, and a light chain CDR3 of SEQ ID NO: 132.

[0093] "Framework regions (FR)" are those variable domain residues other than the CDR residues. Each variable domain typically has four FRs: FR1, FR2, FR3 and FR4.

[0094] The binding affinity of the anti-FGFR3 antibody to FGFR3 is 10 -5 M to 10 -12 For example, the binding affinity of an anti-FGFR3 antibody to FGFR3 is in the range of 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10-6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10M -7 or 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M or 10 -5 M to 10 -6 It's M.

[0095] The antibody or antigen-binding fragment thereof that binds to FGFR3 may comprise a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 133 to SEQ ID NO: 157, and SEQ ID NO: 203 to SEQ ID NO: 215. The antibody or antigen-binding fragment thereof that binds to FGFR3 may comprise a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 158 to SEQ ID NO: 182, and SEQ ID NO: 216 to SEQ ID NO: 219.

[0096] Specific embodiments according to the present invention may include the following: A heavy chain variable region of SEQ ID NO: 133 and a light chain variable region of SEQ ID NO: 158; A heavy chain variable region of SEQ ID NO: 134 and a light chain variable region of SEQ ID NO: 159; A heavy chain variable region of SEQ ID NO: 135 and a light chain variable region of SEQ ID NO: 160; A heavy chain variable region of SEQ ID NO: 136 and a light chain variable region of SEQ ID NO: 161; A heavy chain variable region of SEQ ID NO: 137 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 203 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 204 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 205 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 206 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 207 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 208 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 209 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 210 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 211 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 212 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 213 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 214 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 215 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 137 and a light chain variable region of SEQ ID NO: 216; A heavy chain variable region of SEQ ID NO: 137 and a light chain variable region of SEQ ID NO: 217; A heavy chain variable region of SEQ ID NO: 137 and a light chain variable region of SEQ ID NO: 218; A heavy chain variable region of SEQ ID NO: 137 and a light chain variable region of SEQ ID NO: 219; A heavy chain variable region of SEQ ID NO: 138 and a light chain variable region of SEQ ID NO: 163; A heavy chain variable region of SEQ ID NO: 139 and a light chain variable region of SEQ ID NO: 164; A heavy chain variable region of SEQ ID NO: 140 and a light chain variable region of SEQ ID NO: 165; A heavy chain variable region of SEQ ID NO: 141 and a light chain variable region of SEQ ID NO: 166; A heavy chain variable region of SEQ ID NO: 142 and a light chain variable region of SEQ ID NO: 167; A heavy chain variable region of SEQ ID NO: 143 and a light chain variable region of SEQ ID NO: 168; A heavy chain variable region of SEQ ID NO: 144 and a light chain variable region of SEQ ID NO: 169; A heavy chain variable region of SEQ ID NO: 145 and a light chain variable region of SEQ ID NO: 170; A heavy chain variable region of SEQ ID NO: 146 and a light chain variable region of SEQ ID NO: 171; A heavy chain variable region of SEQ ID NO: 147 and a light chain variable region of SEQ ID NO: 172; A heavy chain variable region of SEQ ID NO: 148 and a light chain variable region of SEQ ID NO: 173; A heavy chain variable region of SEQ ID NO: 149 and a light chain variable region of SEQ ID NO: 174; A heavy chain variable region of SEQ ID NO: 150 and a light chain variable region of SEQ ID NO: 175; A heavy chain variable region of SEQ ID NO: 151 and a light chain variable region of SEQ ID NO: 176; A heavy chain variable region of SEQ ID NO: 152 and a light chain variable region of SEQ ID NO: 177; A heavy chain variable region of SEQ ID NO: 153 and a light chain variable region of SEQ ID NO: 178; A heavy chain variable region of SEQ ID NO: 154 and a light chain variable region of SEQ ID NO: 179; A heavy chain variable region of SEQ ID NO: 155 and a light chain variable region of SEQ ID NO: 180; a heavy chain variable region of SEQ ID NO: 156 and a light chain variable region of SEQ ID NO: 181; or The heavy chain variable region of SEQ ID NO:157 and the light chain variable region of SEQ ID NO:182.

[0097] scFv An scFv is a structure composed of a single polypeptide chain containing the VH and VL domains of an antibody, and is an antibody fragment, and may further contain a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0098] In one embodiment, in a single chain Fv (scFv) comprising the VH and VL domains of an antibody, the VH and VL domains can be connected via a linker: A heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 133 to SEQ ID NO: 157, SEQ ID NO: 203 to SEQ ID NO: 215 can be connected via a linker to a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 158 to SEQ ID NO: 182, SEQ ID NO: 216 to SEQ ID NO: 219.

[0099] Specific embodiments according to the present invention may include the following: A heavy chain variable region of SEQ ID NO: 133 and a light chain variable region of SEQ ID NO: 158; A heavy chain variable region of SEQ ID NO: 134 and a light chain variable region of SEQ ID NO: 159; A heavy chain variable region of SEQ ID NO: 135 and a light chain variable region of SEQ ID NO: 160; A heavy chain variable region of SEQ ID NO: 136 and a light chain variable region of SEQ ID NO: 161; A heavy chain variable region of SEQ ID NO: 137 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 203 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 204 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 205 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 206 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 207 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 208 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 209 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 210 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 211 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 212 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 213 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 214 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 215 and a light chain variable region of SEQ ID NO: 162; A heavy chain variable region of SEQ ID NO: 137 and a light chain variable region of SEQ ID NO: 216; A heavy chain variable region of SEQ ID NO: 137 and a light chain variable region of SEQ ID NO: 217; A heavy chain variable region of SEQ ID NO: 137 and a light chain variable region of SEQ ID NO: 218; A heavy chain variable region of SEQ ID NO: 137 and a light chain variable region of SEQ ID NO: 219; A heavy chain variable region of SEQ ID NO: 138 and a light chain variable region of SEQ ID NO: 163; A heavy chain variable region of SEQ ID NO: 139 and a light chain variable region of SEQ ID NO: 164; A heavy chain variable region of SEQ ID NO: 140 and a light chain variable region of SEQ ID NO: 165; A heavy chain variable region of SEQ ID NO: 141 and a light chain variable region of SEQ ID NO: 166; A heavy chain variable region of SEQ ID NO: 142 and a light chain variable region of SEQ ID NO: 167; A heavy chain variable region of SEQ ID NO: 143 and a light chain variable region of SEQ ID NO: 168; A heavy chain variable region of SEQ ID NO: 144 and a light chain variable region of SEQ ID NO: 169; A heavy chain variable region of SEQ ID NO: 145 and a light chain variable region of SEQ ID NO: 170; A heavy chain variable region of SEQ ID NO: 146 and a light chain variable region of SEQ ID NO: 171; A heavy chain variable region of SEQ ID NO: 147 and a light chain variable region of SEQ ID NO: 172; A heavy chain variable region of SEQ ID NO: 148 and a light chain variable region of SEQ ID NO: 173; A heavy chain variable region of SEQ ID NO: 149 and a light chain variable region of SEQ ID NO: 174; A heavy chain variable region of SEQ ID NO: 150 and a light chain variable region of SEQ ID NO: 175; A heavy chain variable region of SEQ ID NO: 151 and a light chain variable region of SEQ ID NO: 176; A heavy chain variable region of SEQ ID NO: 152 and a light chain variable region of SEQ ID NO: 177; A heavy chain variable region of SEQ ID NO: 153 and a light chain variable region of SEQ ID NO: 178; A heavy chain variable region of SEQ ID NO: 154 and a light chain variable region of SEQ ID NO: 179; A heavy chain variable region of SEQ ID NO: 155 and a light chain variable region of SEQ ID NO: 180; a heavy chain variable region of SEQ ID NO: 156 and a light chain variable region of SEQ ID NO: 181; or The heavy chain variable region of SEQ ID NO:157 and the light chain variable region of SEQ ID NO:182.

[0100] The linker may be a peptide linker and may have a length of about 10-25 aa, and may include, but is not limited to, hydrophilic amino acids such as glycine and / or serine.

[0101] Specifically, the linker is, for example, (GS) n , (GGS) n , (GSGGS) n or (G n S) m (n and m are each 1 to 10), but the linker can be, for example, (G n S) m (n and m are each 1 to 10). Specifically, the linker may include GGGGS, for example GGGGSGGGGSGGGGSGGGGS of SEQ ID NO: 185, repeated three times.

[0102] "Phage display" is a technique in which mutant polypeptides are displayed on the surface of phages, e.g., filamentous phage particles, as fusion proteins with at least a portion of a coat protein. The utility of phage display is that it allows rapid and efficient sorting of large libraries of randomized protein mutants for sequences that bind with high affinity to a target antigen. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides to find those with specific binding properties.

[0103] Phage display technology has provided a powerful tool for generating and selecting novel proteins that bind to specific ligands (such as antigens). Using phage display technology, large libraries of protein variants can be generated and sequences that bind with high affinity to the target antigen can be rapidly sorted. A nucleic acid encoding a variant polypeptide is fused to a nucleic acid sequence encoding a viral coat protein, such as the gene III protein or the gene VIII protein. A monovalent phage display system has been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused to a nucleic acid sequence encoding a portion of the gene III protein. In the monovalent phage display system, the gene fusion is expressed at low levels and the wild-type gene III protein is also expressed, maintaining particle infectivity.

[0104] Demonstration of peptide expression on the surface of filamentous phages and expression of functional antibody fragments in the periplasm of E. coli is key to developing antibody phage display libraries. Libraries of antibodies or antigen-binding polypeptides have been produced in a number of ways, including by modifying single genes by inserting random DNA sequences or by cloning related gene series. Libraries can be screened for expression of antibodies or antigen-binding proteins with desired characteristics.

[0105] Phage display technology has several advantages over conventional hybridoma and recombinant methods for producing antibodies with desired characteristics. These techniques can generate large antibody libraries with diverse sequences in a short time without the use of animals. Hybridoma production and humanized antibody production can require several months of production time. Also, since no immunization is required, phage antibody libraries can generate antibodies against antigens with low toxicity or antigenicity. Phage antibody libraries can also be used to generate and validate novel therapeutic antibodies.

[0106] Techniques are available for generating human antibodies from immunized, non-immunized humans, germline sequence, or naive B cell Ig repertoires using phage display libraries. Various lymphoid tissues can be used to produce naive or non-immunized antigen-binding libraries.

[0107] A technique that can identify and isolate high affinity antibodies from a phage display library is important in isolating novel antibodies for therapeutic use. Isolating high affinity antibodies from a library depends on the size of the library, the efficiency of production in bacterial cells, and the diversity of the library. The size of the library is reduced by improper folding of the antibody or antigen binding protein and inefficient production due to the presence of stop codons. Expression in bacterial cells can be suppressed if the antibody or antigen binding domain does not fold properly. Expression can be improved by alternating residues on the surface of the variable / invariant interface or selected CDR residues. The sequence of the scaffold region is one element to provide proper folding when generating an antibody phage library from bacterial cells.

[0108] In high affinity antibody isolation, it is important to generate a diverse library of antibodies or antigen binding proteins. It is known that the CDR3 region often participates in antigen binding. The CDR3 region on the heavy chain is quite diverse in terms of size, sequence and structural conformation, which can be exploited to produce a diverse library.

[0109] Diversity can also be generated by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position, which may generate a greater diversity of variant antibody sequences and increase the chances of identifying novel antibodies.

[0110] The antibody or antibody fragment of the present invention may include not only the sequence of the anti-FGFR3 antibody of the present invention described herein, but also its biological equivalents, so long as it can specifically recognize FGFR3. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such changes include, for example, deletion, insertion, and / or substitution of residues in the amino acid sequence of the antibody. Such amino acid mutations are made based on the relative similarity of the amino acid side chain substitutes, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substitutes shows that arginine, lysine, and histidine are all positively charged residues, alanine, glycine, and serine have similar sizes, and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, it can be said that arginine, lysine and histidine, alanine, glycine, serine, and phenylalanine, tryptophan, and tyrosine are biologically functional equivalents.

[0111] Considering the above-mentioned mutations having biologically equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding it is also understood to include a sequence showing substantial identity with the sequence described in the SEQ ID NO. The above-mentioned substantial identity means a sequence showing at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when the above-mentioned sequence of the present invention is aligned to any other sequence so as to correspond to the maximum extent and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) can be accessed at NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST can be accessed at www.ncbi.nlm.nih.gov / BLAST / . Methods for sequence homology comparison using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0112] Based on this, the antibody or antigen-binding fragment of the present invention can have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology compared to the described sequence or the entirety described in the specification. Such homology can be determined by sequence comparison and / or alignment by methods known in the art. For example, the percent sequence homology of the nucleic acid or protein of the present invention can be determined using sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, and visual inspection.

[0113] In another aspect, the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof can be recombinantly produced by isolating a nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention.

[0114] The term "nucleic acid" refers to a nucleic acid that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and the basic building block of nucleic acid, nucleotides, include not only natural nucleotides but also analogues in which the sugar or base moiety is modified. The sequences of the nucleic acids encoding the heavy and light chain variable regions of the present invention can be modified. The modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0115] The DNA encoding said antibody can be readily isolated or synthesized using standard molecular biology techniques (e.g., by using oligonucleotide probes capable of specifically binding to DNA encoding the antibody and the heavy and light chains), and the nucleic acid isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for further expression. Based on this, another aspect of the present invention relates to a recombinant expression vector comprising said nucleic acid.

[0116] The term "vector" as used herein refers to a means for expressing a gene of interest in a host cell, and includes plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, and other viral vectors. Components of a vector typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, and a transcription termination sequence. The nucleic acid encoding the antibody is operably linked to the promoter and transcription termination sequence.

[0117] "Operably linked" refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of binding sites for a transcription factor) and another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0118] When a prokaryotic cell is used as the host, it generally contains a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for the initiation of translation, and a transcription / translation termination sequence. Furthermore, for example, when a eukaryotic cell is used as the host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, which generally have a polyadenylation sequence as a transcription termination sequence.

[0119] In some cases, the vector can be fused to other sequences to facilitate purification of the antibody expressed therefrom, such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

[0120] The vectors contain antibiotic resistance genes commonly used in the art as selection markers, such as genes for resistance to ampicillin, gentamicin, cabenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0121] In another aspect, the present invention relates to a host cell transformed with the recombinant expression vector. The host cell used to produce the antibody of the present invention can be, but is not limited to, a prokaryote, yeast or higher eukaryote cell.

[0122] Prokaryotic host cells such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilus, and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be utilized.

[0123] Of greatest interest, however, are animal cells, examples of useful host cell lines may include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0124] From another aspect, the present invention relates to a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to FGFR3, the method comprising the steps of culturing the host cell to produce an antibody, and isolating and purifying the produced antibody.

[0125] The host cells can be cultured in various media. Any commercially available media can be used as the culture medium without limitation. Any other necessary supplements known to those skilled in the art can also be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc., are already used with the host cells selected for expression and will be apparent to those skilled in the art.

[0126] The antibody or antigen-binding fragment thereof can be recovered by, for example, removing impurities by centrifugation or ultrafiltration, and the resultant can be purified using, for example, affinity chromatography, etc. Further purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., can be used.

[0127] Bispecific or multispecific antibodies From another aspect, the present invention relates to a bi- or multispecific antibody comprising the above-mentioned antibody or an antigen-binding fragment thereof.

[0128] A bispecific antibody refers to an antibody having binding or antagonistic capabilities to one or more targets, and refers to a form in which antibodies having binding or antagonistic capabilities to two different targets are bound, or an antibody in which an antibody having binding capabilities to one target is bound to a substance having antagonistic capabilities to another target.

[0129] A multispecific antibody refers to an antibody that has binding specificities for at least three or more different antigens. Multispecific antibodies can include trispecific or more specific antibodies, for example, trispecific antibodies, tetraspecific antibodies, or antibodies that target more than two targets.

[0130] Antibodies belonging to bispecific or multispecific antibodies can be divided into scFv-based antibodies, Fab-based antibodies, IgG-based antibodies, etc. In the case of bispecific or multispecific antibodies, two or more signals can be simultaneously suppressed or amplified, which is more effective than suppressing / amplifying one signal, and compared to treating each signal with its own signal inhibitor, lower doses can be administered and two or more signals can be suppressed / amplified in the same time and space.

[0131] Methods for producing bispecific or multispecific antibodies are widely known. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two or more immunoglobulin heavy / light chain pairs under conditions where the two or more heavy chains have different specificities.

[0132] In the case of bispecific or multispecific antibodies based on scFv, a diabody can be produced by combining the VL and VH of different scFvs to produce a hybrid scFv in a heterodimeric form, different scFvs can be linked to each other to produce tendem ScFvs, CH1 and CL of Fab can be expressed at the end of each scFv to produce a heterodimeric miniantibody, and some amino acids in the CH3 domain, which is the homodimeric domain of Fc, can be replaced to change it to a "knob into hole" type heterodimeric structure and these changed CH3 domains can be expressed at the end of each different scFv to produce a heterodimeric scFv type minibody.

[0133] In the case of Fab-based bispecific or multispecific antibodies, individual Fab's against a specific antigen can be combined with each other using disulfide bonds or intermediates to produce a heterodimeric Fab, and a specific Fab can be produced with two antigen binding valencies by expressing scFvs against different antigens at the heavy or light chain ends, or a homodimeric Fab can be produced with four antigen binding valencies by placing a hinge region between the Fab and scFv. In addition, a dual-targeting bibody with three antigen binding valencies can be produced by fusing scFvs against different antigens to the light and heavy chain ends of the Fab, a triple-targeting bibody with three antigen binding valencies can be produced by fusing different scFvs to the light and heavy chain ends of the Fab, or by chemically conjugating three different Fabs.

[0134] In the case of IgG-based bispecific or multispecific antibodies, Trion Pharma has produced hybrid hybridomas, also known as quadromas, by crossbreeding mouse and red hybridomas to produce bispecific antibodies. Bispecific antibodies can also be produced in the so-called "holes and knob" form, in which the light chain portion is shared, but some amino acids in the CH3 homodimeric domain of Fc are modified for different heavy chains to produce a heterodimeric form. In addition to heterodimeric bispecific antibodies, two different scFvs can be fused and expressed in the constant domain instead of the variable domains of the light and heavy chains of IgG, to produce a homodimeric form (scFv)4-IgG. ImClone also reported the creation of a bispecific antibody based on IMC-1C11, a chimeric monoclonal antibody against human VEGFR-2, by fusing only a single variable domain against mouse platelet-derived growth factor receptor-α to the amino terminus of the light chain of this antibody. In addition, it is possible to create an antibody with multiple antigen binding valencies against CD20 through the so-called "dock and lock (DNL)" method using the dimerization and docking domain (DDD) of protein kinase A (PKA) R subunit and the anchoring domain of PKA.

[0135] A wide variety of recombinant antibody formats have been developed, including bispecific or multispecific antibodies that are bivalent or more, trivalent or more or quadrivalent or more, such as those described in International Patent Application Publication Nos. WO2001 / 077342, WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, WO2010 / 145793 and WO2011 / 117330. A more than divalent, more than trivalent or more than tetravalent antibody indicates that two or more binding domains, three or more binding domains or four or more binding domains are present in the antibody molecule, respectively.

[0136] In a specific embodiment, the bi- or multispecific antibody according to the present invention may comprise said anti-FGFR3 antibody or antigen-binding fragment, specifically an IgG complete antibody or in the form of a fragment thereof, e.g., a single chain Fv, a VH domain and / or a VL domain, Fab or (Fab)2.

[0137] In addition, an antibody that binds to a target different from the antibody that targets FGFR3, for example, an antibody that targets one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO, specifically, an IgG complete antibody or a fragment form thereof, for example, a single chain Fv, V H Domain and / or V L It may comprise a domain, a Fab or (Fab)2 form.

[0138] Bi- or multispecific antibodies according to the invention make it possible to ensure additional binding specificities induced or mediated by other targets besides FGFR3.

[0139] For example, a bispecific antibody according to the invention may simultaneously target FGFR3 and one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

[0140] For example, a multispecific antibody according to the invention may simultaneously target two or more of the following: FGFR3 and PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), TIGIT, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, MARCO.

[0141] Immune cell engager bispecific or multispecific antibodies In another aspect, the present invention relates to an immune cell engager bispecific or multispecific antibody comprising an scFv composed of an scFv of an antibody and a second binding domain comprising one or more scFvs of an antibody that binds to an immune cell activating antigen.

[0142] The immune cell-engaging bispecific or multispecific antibody temporarily induces a cytolytic synapse between cytotoxic T cells and cancer target cells, resulting in the release of toxins.

[0143] In one embodiment, the immune cells may be one or more selected from the group consisting of T cells, NK cells, cytokine induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor-infiltrating lymphocytes (TIL), and dendritic cells.

[0144] In one embodiment, the immune cell activation antigen may be selected from, for example, the following, and an antibody binding thereto may play a role in immune cell engagement: T cell activation antigens include CD3, TCRα, TCRβ, TCRγ, TCRξ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. NK cell activation antigens include NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b), CRTAM, CD27, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160. B cell activation antigens include OX40, CD40, or CD70, The macrophage activation antigens are CD2 agonists, CD40, CD70, TCR (Toll-like receptor) agonists, CD47, STING or OX40L, or The dendritic cell activating antigens are CD2 agonists, OX40, OX40L, 41BB agonists, TCR agonists, CD47 agonists, or STING agonists.

[0145] Immune cell engagers are specifically described in US Patent Application Publication No. 2017 / 0368169, which is incorporated herein by reference.

[0146] Specifically, said immune cell engaging bispecific or multispecific antibodies comprise tandem scFvs and are capable of binding to the following antigens and to a surface antigen on cancer cells: The surface antigen on cancer cells is FGFR3, which is targeted by the antibody according to the invention. CD3, TCRα, TCRβ, TCRγ, TCRξ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226, NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160, OX40, CD40 or CD70, CD2 agonist, CD40, CD70, TCR (Toll-like receptor) agonist, CD47, STING or OX40L, or CD2 agonist, OX40, OX40L, 41BB agonist, TCR agonist, CD47 agonist, or STING agonist.

[0147] The immune cell engaging bispecific or multispecific antibodies can include, for example, structures in the form of VL(FGFR3)-VH(FGFR3)-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(FGFR3)-VL(FGFR3)-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(CD3 or CD16A)-VL(CD3 or CD16A)-VH(FGFR3)-VL(FGFR3) or VH(CD3 or CD16A)-VL(CD3 or CD16A)-VL(FGFR3)-VH(FGFR3).

[0148] The scFv comprises, for example, a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 133 to SEQ ID NO: 157, and SEQ ID NO: 203 to SEQ ID NO: 215, and a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 158 to SEQ ID NO: 182, and SEQ ID NO: 216 to SEQ ID NO: 219, and the heavy chain variable region and the light chain variable region can be linked by a linker.

[0149] The linker may be a peptide linker and may have a length of about 10-25 aa, and may contain hydrophilic amino acids such as, for example, glycine and / or serine.

[0150] The linker can include, for example, (GS), (GGS), (GGSGGS), or (GnS) (n and m are each 1 to 10). n S) m (n, m each can be 1 to 10). Specifically, the linker can include GGGGS, for example GGGGSGGGGSGGGGSGGGGS of SEQ ID NO: 185 repeated three times.

[0151] Examples of the immune cell-engaging bispecific or multispecific antibodies include blinatumomab (Amgen), which binds to CD3 and CD19, solitomab (Amgen), which binds to CD3 and EpCAM, MEDI 565 (MedImmune, Amgen), which binds to CD3 and CEA, and BAY2010112 (Bayer, Amgen), which binds to CD3 and PSMA. Exemplary DARTs include MGD006 (Macrogenics), which binds to CD3 and CD123, and MGD007 (Macrogenics), which binds to CD3 and gpA33. Exemplary TandAbs can include AFM11 (Affimed Therapeutics), which binds to CD3 and CD19, and AFM13 (Affimed Therapeutics), which binds to CD30 and CD16A.

[0152] Antibody-Drug Conjugates (ADCs) From another aspect, the present invention relates to an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof is bound to a drug.

[0153] In antibody-drug conjugates, the anti-cancer drug must be stably bound to the antibody until it is delivered to the target cancer cell. Once the drug is delivered to the target, it must be released from the antibody and induce the death of the target cell. For this purpose, the drug must be stably bound to the antibody and at the same time, must have sufficient cytotoxicity to induce the death of the target cell when released from the target cell.

[0154] In one embodiment, the antibody can be linked to a drug via a linker, which is a linking moiety between the anti-FGFR3 antibody and the drug, and allows the antibody to be cleavable under intracellular conditions, i.e., to release the drug in the intracellular environment, and ensures that the antibody is stable in the systemic circulation, reflecting the long half-life of the antibody, and that the linker-drug bond does not affect the stability and pharmacokinetics of the antibody.

[0155] The linker may include, for example, a cleavable linker or a non-cleavable linker, such as a peptide linker that can be cleaved by an intracellular peptidase or protease enzyme, for example, a lysosomal or endosomal protease, and a non-cleavable linker, such as a thioether linker, that may release a drug after the antibody is non-selectively degraded by intracellular hydrolysis.

[0156] In one embodiment, the cleavable linker can comprise a peptide linker. The peptide linker has a length of at least two or more amino acids. For example, it can comprise a dipeptide of Val-Cit, Val-Ala, or Val-Cit, or Phe-Leu or Gly-Phe-Leu-Gly. Examples of linkers are specifically described in International Patent Application Publication No. WO2004 / 010957, which can be incorporated herein by reference.

[0157] The antibody-drug conjugate is then internalized in the cancer cell via the endosomal-lysosomal pathway after the antibody domain of the ADC binds to the antigen of the target cancer cell to form an ADC-antigen complex, where the intracellular release of the cytotoxic drug is regulated by the internal environment of the endosome or lysosome.

[0158] In one embodiment, the cleavable linker can be pH sensitive and sensitive to hydrolysis at a certain pH value.Generally, pH sensitive linker indicates that it can be hydrolyzed under acidic conditions.For example, the acid-labile linker that can be hydrolyzed in lysosomes can be, for example, hydrazone, semicabazone, thiosemicabazone, cis-aconitic amide, orthoester, acetal, ketal, etc.

[0159] In other embodiments, the linker may be cleaved under reducing conditions, such as disulfide linkers. Various disulfide bonds can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene). These disulfide linkers may be degraded by disulfide exchange with the thiols of intracellular glutathione.

[0160] The drug and / or drug linker may be randomly conjugated through lysines of the antibody or through cysteines exposed when disulfide bond chains are reduced. In some cases, the linker drug may be attached through cysteines present in a genetically engineered tag, such as a peptide or protein. The genetically engineered tag, such as a peptide or protein, may include an amino acid motif that is recognized by isoprenoid transferase, for example. The peptide or protein may have a deletion at the carboxy terminus of the peptide or protein or a covalent addition of a spacer unit at the carboxy (C) terminus of the peptide or protein.

[0161] The peptide or protein may be linked to the amino acid motif by direct covalent binding or by covalent binding to a spacer unit, which is composed of 1 to 20 amino acids, of which glycine units are preferred.

[0162] The isoprenoid transferase may be, for example, farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase), where FTase and GGTase I can recognize the CAAX motif in the above-mentioned Chemical Formula 1, GGTase II can recognize the XXCC, XCCC or CXX motif, and GGTase II can recognize the XXCC, XCXC or CXX motif (wherein C is cysteine, A is an aliphatic amino acid, and X is an amino acid that determines the substrate specificity of the isoprenoid transferase).

[0163] In another embodiment, the linker may include a β-glucuronide linker that is recognized and hydrolyzed by β-glucuronidase, which is abundantly present in lysosomes or overexpressed in some tumor cells. Unlike peptide linkers, it has a high hydrophilicity and has the advantage of increasing the solubility of the antibody-drug conjugate when conjugated with a drug having a high hydrophobic property.

[0164] In this regard, the β-glucuronide linkers disclosed in International Patent Application Publication No. WO2015 / 182984 can be used, for example β-glucuronide linkers comprising a self-immolative group, the disclosure of which is incorporated by reference.

[0165] In some cases, the linker is, for example, a non-cleavable linker, such that the drug is released in a single step of antibody hydrolysis within the cell, for example to generate an amino acid linker-drug conjugate. This type of linker can be a thioether group or a maleimidocaproyl group, and can remain stable in the blood.

[0166] According to one embodiment of the invention, a linker drug may be attached via a cysteine ​​that is exposed upon reduction of the disulfide bonded chains of the antibody by introducing an antibody end-linking peptide having a random bond or the sequence GGGGGGCVIM.

[0167] The drug (including D in formula (1)) can be bound to the antibody as a drug exhibiting a pharmacological effect, and specifically may be a chemotherapeutic agent, a toxin, a microRNA (miRNA), an siRNA, an shRNA, or a radioisotope. The chemotherapeutic agent can be, for example, a cytotoxic agent or an immune inhibitor. Specifically, it can include a chemotherapeutic agent that can function as a microtubulin inhibitor, a mitotic inhibitor, a topoisomerase inhibitor, or a DNA intercalator. It can also include a drug for treating cancer, inflammation, or immune disease. For example, it can include an immunomodulatory compound, an anticancer drug, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof.

[0168] Examples of the anticancer drugs include maytansinoids, oristatins (including MMAE and MMAF), aminophterin, actinomycin, bleomycin, tallysomycin, camptothecin, N8-acetylspermidine, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazide, esparamycin, etoposide, 6-makaptopurine, dolostatin, trichothecenes, calicheamicin, taxol, taxanes, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocamicin, L -L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustardmustard, cytoxan, etoposide, 5-fluorouracil, CNU (bischloroethylnitrosourea), irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase inase, vinorelbine, chlorambucil, melphalan, carmustine, lomustine, busulfan, treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, crisnatol, mitomycin C C), trimetrexate, mycophenolic acid, thiazopurine, ribavirin, EICAR (5-ethynyl-1-beta-dribofuranosylimidazole-4-carboxamide), hydroxyurea, deferoxamine, flixuridine, doxifluridine, raltitrexed, cytarabine (ara C), cytosine arabinoside (cytosine arabinoside), cyclosporine (cyclosporine ...arabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor factor), gemcitabine, velcade, rebamid, thalamid, lovastatin, 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2 B2), peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil and thapsigargin, nucleases and toxins derived from bacteria, animals and plants, but are not limited thereto.

[0169] The anti-inflammatory agent includes, but is not limited to, steroids that bind to glucocorticoid receptors to reduce inflammation and swelling, nonsteroidal anti-inflammatory agents (NSAIDs) that relieve pain by acting on cyclooxygenase (COX) that synthesizes prostaglandins that cause inflammation, and immunoselective anti-inflammatory derivatives (ImSAIDs) that change the activation and migration of inflammatory cells.

[0170] The immune disease treatment may include, but is not limited to, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, mycophenolate, azathioprine, or methotrexate.

[0171] Specifically, the immune inhibitor may be, but is not limited to, the calcineurin inhibitor tacrolimus, the gene synthesis inhibitor mycophenolate mofetil, or the anti-inflammatory steroid prednisone.

[0172] In some cases, the drug can include one or more nucleophilic groups selected from the group consisting of amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups that can react to form covalent bonds with electrophilic groups on the linker and linker reagent.

[0173] In a specific embodiment of the present invention, an ADC was prepared by linking the antibody or antigen-binding fragment thereof according to the present invention to a drug, such as oristatin (MMAE), via an MC-vc-PAB linker. Such an ADC was confirmed to exhibit the desired cytotoxicity.

[0174] Chimeric antigen receptors (CARs) From another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising an extracellular domain containing an antigen-binding site, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding site of the extracellular domain is an scFv of the antibody.

[0175] Chimeric antigen receptors (CARs) are synthetic constructs designed to induce immune responses against target antigens and cells expressing the antigens. CARs contain an extracellular domain, a transmembrane domain, and an intracellular signaling domain. Genes encoding receptors that recognize cancer cell surface antigens that are specifically expressed on the surface of cancer cells can be introduced into immune cells to kill the cancer cells. Immune cells that contain receptors that bind to antigens specifically expressed on cancer cells can be used to target only the cancer cells and generate immune responses. The CAR contains the scFv of the anti-FGFR3 antibody according to the present invention as an antigen recognition site in the extracellular domain.

[0176] The first generation CAR contains an extracellular domain containing an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain, and only CD3ζ is used as the signaling domain, but it has a problem that the therapeutic effect on cancer is weak and the duration is short. Such a first generation CAR is specifically described in U.S. Patent No. 6,319,494, which is incorporated by reference into the present invention.

[0177] Second-generation CARs were produced by combining costimulatory domains (CD28 or CD137 / 4-1BB) with CD3ζ to improve reactivity to immune cells, and the number of immune cells containing CARs remaining in the body was significantly increased compared to the first-generation CARs. The second-generation CARs utilized one costimulatory domain, whereas the third-generation CARs utilized two or more costimulatory domains. To achieve expansion and persistence of immune cells containing CARs in vivo, the costimulatory domain can be combined with 4-1BB, CD28, or OX40, etc. The second-generation CARs are specifically described in U.S. Patent Nos. 7,741,465, 7,446,190, or 9,212,229, and the third-generation CARs are specifically described in U.S. Patent No. 8,822,647, which are incorporated herein by reference.

[0178] The fourth generation CARs contain additional genes encoding cytokines such as IL-12 or IL-15 to allow further expression of cytokine CAR-based immune proteins, and the fifth generation CARs further contain interleukin receptor chains, e.g., IL-2Rβ, for immune cell enhancement. The fourth generation CARs are specifically described in U.S. Patent No. 10,316,102, and the fifth generation CARs are specifically described in U.S. Patent No. 10,336,810, both of which are incorporated herein by reference.

[0179] In one embodiment, the antigen-binding site of the extracellular domain is an scFv of an antibody. In the scFv comprising the VH and VL domains of an antibody, the VH and VL domains can be connected via a linker. A heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 133 to SEQ ID NO: 157, SEQ ID NO: 203 to SEQ ID NO: 215 can be linked via a linker to a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 158 to SEQ ID NO: 182, SEQ ID NO: 216 to SEQ ID NO: 219.

[0180] The linker may be a peptide linker and may have a length of about 10 to 25 aa, and may contain hydrophilic amino acids such as, for example, glycine and / or serine.

[0181] The linker can include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (n, m are each 1 to 10), and the linker can be, for example, (GnS)m (n, m are each 1 to 10). Specifically, the linker can include GGGGS, for example, GGGGSGGGGSGGGGSGGGGS of SEQ ID NO: 185 repeated three times.

[0182] The transmembrane domain can be derived from a natural or synthetic source. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can include the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS. If the transmembrane domain is synthetic, it can include hydrophobic residues such as leucine and valine, or phenylalanine, tryptophan and valine-containing peptides at each end. A short oligo- or polypeptide linker, 2 to 10 amino acids in length, can form a bond between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine peptide can be used as the linker.

[0183] The signaling domain can induce activation of the normal effector function of the immune cell in which the CAR is located. For example, it can induce cytolytic activation or helper activation through the secretion of cytokines. The signaling domain can include a truncated fragment of the intracellular signaling domain sufficient to transduce an effector function signal.

[0184] The signaling domains may include the cytoplasm of the T cell receptor (TCR) and co-receptors that act in concert to initiate signaling following antigen receptor engagement.

[0185] It is also known that signals generated by the TCR alone are insufficient for full activation of T cells, and that costimulatory signals are required. Thus, T cell activation may involve initiating antigen-dependent primary activation via the TCR, and acting in an antigen-dependent manner to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may include signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs that include primary cytoplasmic signaling sequences include TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0186] In some cases, the cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. For example, it may include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. The cytoplasmic signaling sequence in the cytoplasmic signaling portion of the CAR can be linked via a peptide linker that includes 2 to 10 amino acids, such as glycine and serine.

[0187] From another aspect, the present invention relates to an immune cell into which the chimeric antigen receptor (CAR) has been introduced.

[0188] The immune cells can induce immunity to induce a desired cancer therapeutic effect, and can be selected from the group consisting of, for example, T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor-infiltrating lymphocytes (TIL), and dendritic cells, but are not limited thereto.

[0189] In some cases, a chimeric antigen receptor (CAR) containing an scFv against an antibody other than the anti-FGFR3 antibody as an antigen-binding site in the extracellular domain may be further introduced.

[0190] The antibody other than the anti-FGFR3 antibody may be, for example, an antibody or an antigen-binding fragment thereof that targets one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

[0191] Vesicles and exosomes From another aspect, the present invention relates to endoplasmic reticulum on the exterior of which an antibody or an antigen-binding fragment thereof is expressed or present inside thereof.

[0192] The endoplasmic reticulum comprises a vesicle capable of passing through the cell membrane and directly importing a target protein into the cell, a cell membrane and the antibody or antigen-binding fragment thereof according to the present invention.

[0193] The vesicle according to the present invention can be in the form of containing cell membrane and the target preparation to be delivered.Examples of such vesicles are specifically described in US Patent Publication No. 2018 / 0177725, and can be incorporated by reference into the present invention.

[0194] The endoplasmic reticulum may have an average diameter of, for example, 40 to 500 nm, specifically, 100 to 300 nm, 80 to 200 nm, or 100 nm. The endoplasmic reticulum may be, for example, about 100, 200, 300, 400, or 500 nm.

[0195] The endoplasmic reticulum may not contain other intracellular organelles such as mitochondria, lysosomes or Golgi apparatus, apart from the cell membrane and the antibody or antigen-binding fragment thereof according to the invention.

[0196] The endoplasmic reticulum may be divided into an inside and an outside, including a membrane, and the antibody or antigen-binding fragment thereof may be present in the membrane and expressed on the outside, expressed inside, or separated from the membrane and present inside the endoplasmic reticulum.

[0197] The membrane of the endoplasmic reticulum may vary depending on the target to which the antibody or its antigen-binding fragment is to be applied, and may be, for example, but is not limited to, a cancer cell membrane for delivery to a cancer cell expressing FGFR3 to which the antibody or its antigen-binding fragment according to the present invention binds.

[0198] The cancer cells can include, for example, cancer cells that cause Hodgkin's lymphoma, non-Hodgkin's lymphoma (e.g., B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, epithelial cell lymphoma, marginal zone B-cell lymphoma, bucket lymphoma, lymphoplasmacytic lymphoma, myeloid leukemia), acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, multiple myeloma, or acute lymphocytic leukemia.

[0199] Also included are solid cancers, such as ovarian cancer, rectal cancer, gastric cancer, testicular cancer, anal cancer, uterine cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, esophageal cancer, melanoma, Kaposi's sarcoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or umbilical cord malignant melanoma, uterine cancer, brain stem glioma, pituitary adenocarcinoma, epidermoid carcinoma, cervical squamous cell carcinoma, fallopian tube cancer, endometrial cancer, vaginal cancer, soft tissue sarcoma, urethral cancer, vulvar cancer, penile cancer, bladder cancer, kidney cancer or ureter cancer, renal pelvis cancer, spinal tumors, central nervous system (CNS) neoplasms, primary CNS lymphomas, tumor angiogenesis, and cancer cells causing metastatic lesions of the cancer.

[0200] Additionally, it may include cancer cells causing, for example, glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell carcinoma, gallbladder cancer, or cervical cancer.

[0201] The endoplasmic reticulum can be, for example, an exosome. The exosome refers to a small vesicle with a membrane structure of 50 to 200 nm in size that is secreted outside the cell carrying proteins, DNA, RNA, and the like for intercellular signaling.

[0202] Exosomes contain various intracellular proteins, DNA, RNA, etc. Substances secreted from the cell in exosomes can flow back into other cells by fusion with the cell membrane or endocytosis, and can serve as a communication tool between cells. In addition, the substances secreted from the cell in exosomes can be analyzed and used to diagnose certain diseases.

[0203] According to the present invention, the antibody or antigen-binding fragment thereof is expressed on the outside of the exosome or present inside the exosome. The exosome has a membrane structure, is divided into an inside and an outside, and is present in the exosome membrane, and the antibody or antigen-binding fragment thereof can be present and expressed on the outside, expressed inside, or separated from the membrane and present inside the exosome.

[0204] The exosomes are artificial exosomes derived from immune cells / stem cells having various targeting properties, which are loaded with specific drugs and targeted to specific diseased tissues in the human body for delivery, thereby significantly reducing side effects on other tissues and maximizing therapeutic efficacy even with a small amount of drug. For example, this is specifically described in International Patent Publication No. WO2011 / 002239, which is incorporated herein by reference.

[0205] When the antibody or antigen-binding fragment thereof is present in the exosome membrane and expressed externally or internally, the antibody or antigen-binding fragment thereof can be expressed in a form fused to an exosome-specific marker, and the antibody or antigen-binding fragment thereof can be captured inside the exosome.When the exosome membrane is fused to the cell membrane of a cell expressing FGFR3, the antibody or antigen-binding fragment thereof can be delivered to the cell.

[0206] The antibody or its antigen-binding fragment may be displayed on the surface of the exosome. For example, PTGFRN (Prostaglandin F2 Receptor Negative Regularator) protein may be expressed on the surface of the exosome, and other target proteins may be fused to the PTGFRN protein. Such exosomes are specifically described in International Patent Publication Nos. WO2014 / 076137 and WO2018 / 09119, and are incorporated herein by reference.

[0207] In the case of exosomes containing the antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof can be temporarily bound to the marker protein using a photospecific binding protein to produce the exosomes. The target protein can be captured inside the exosomes in a state where it is not bound to the membrane. When the exosomes are delivered into the cells by internalization, the exosomes are degraded and the antibody or antigen-binding fragment thereof can be delivered to the cells.

[0208] The process for enabling the antibody or its antigen-binding fragment to be present inside is specifically described in International Patent Publication Nos. WO2016 / 178532 and WO2018 / 062973, and is incorporated by reference into the present invention.

[0209] For example, photospecific binding proteins CIBN and CRY2 can be used, and specifically, genes encoding these fusion proteins can be introduced into exosome-producing cells so that CIBN is expressed in a fused form with CD9, one of the marker proteins, and CRY2 is expressed in a fused form with the antibody or an antigen-binding fragment thereof. The CIBN-CD9 fusion protein expressed in the exosome-producing cells is contained in exosomes by CD9, and when the cells are irradiated with blue LED light, the CRY2 domain of the target protein-CRY2 fusion protein expressed in the exosome-producing cells binds to the CIBN domain fused to CD9, forming a reversibly induced fusion protein in the form of target protein-CRY2-CIBN-CD9, and such a fusion protein can be contained inside exosomes by CD9. After exosomes containing the target protein are produced in this way, if the blue LED light is no longer irradiated, the CIBN-CRY2 bond is released and the target protein can be contained inside the exosome without being bound to the exosome cell membrane.

[0210] therapeutic composition From another aspect, the present invention relates to a composition for treating cancer, inflammation or an immune disease, comprising the antibody or antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor.

[0211] The present invention may be, for example, a pharmaceutical composition for preventing or treating cancer, comprising: (a) a pharma- ceutical effective amount of an antibody or antigen-binding fragment thereof against FGFR3 according to the present invention, a bi- or multi-specific antibody comprising said antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising said antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising said antibody or antigen-binding fragment thereof, or an immune cell comprising a chimeric antigen receptor comprising said antibody or antigen-binding fragment thereof, and (b) a pharma-ceutical acceptable carrier. The present invention may also be a method for preventing or treating cancer, comprising the step of administering to a cancer patient an antibody or antigen-binding fragment thereof against FGFR3 according to the present invention, a bi- or multi-specific antibody comprising said antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising said antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising said antibody or antigen-binding fragment thereof, or an immune cell comprising said chimeric antigen receptor.

[0212] "Prevention" refers to any action of suppressing the growth of cancer or slowing its progression by administering the composition according to the present invention, and "treatment" refers to suppressing the progression of cancer, reducing tumor size, or eliminating cancer.

[0213] The cancer includes, for example, Hodgkin's lymphoma, non-Hodgkin's lymphoma (e.g., B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, ectodermal cell lymphoma, marginal zone B-cell lymphoma, bucket lymphoma, lymphoplasmacytic lymphoma, myeloma), acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, multiple myeloma, or acute lymphocytic leukemia.

[0214] The cancers include, for example, ovarian cancer, rectal cancer, gastric cancer, testicular cancer, anal cancer, uterine cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, esophageal cancer, melanoma, Kaposi's sarcoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or guiding malignant melanoma, uterine cancer, brain stem glioma, pituitary adenocarcinoma, epidermoid carcinoma, cervical squamous cell carcinoma, fallopian tube cancer, endometrial cancer, vaginal cancer, soft tissue sarcoma, urethral cancer, vulvar cancer, penile cancer, bladder cancer, kidney or ureter cancer, renal pelvis cancer, spinal tumor, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, metastatic lesions of the cancers, or combinations of the cancers.

[0215] The cancer may be, for example, glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell carcinoma, gallbladder cancer, or cervical cancer. In particular, in the case of glioblastoma, the antibody or its antigen-binding fragment must pass through the blood-brain barrier, which is a barrier formed by tight junctions in the brain capillary endothelial cell membrane that exists between the brain and spinal cord and the surrounding circulatory system. It can be used in conjunction with a carrier to pass through the BBB. To deliver drugs through the BBB, there are methods such as bradykinin or HIFU (high density focus ultrasound) to destroy the osmotic pressure of the BBB. In addition, it includes the use of delivery systems such as glucose and amino acid carriers, insulin or transferrin receptor-mediated transcytosis that are endogenous to cells, or blocking efflux transporters of glycoproteins.

[0216] The inflammation may be, for example, but is not limited to, chronic obstructive pulmonary disease, chronic gastrointestinal disease, conjunctivitis, otitis, allergic rhinitis, gingivitis, glossitis, bronchitis, gastroesophageal reflux disease (GERD), esophagitis, gastritis, enteritis, peptic ulcer, inflammatory bowel disease (IBD), Crohn's disease, irritable bowel syndrome (IBS), enteritis or allergy, urethritis, cystitis, vaginitis, proctoproctitis, eosinophilic gastroenteritis or rheumatoid arthritis.

[0217] The immune disease may be, for example, acquired immune deficiency, autoimmune disease, generalized erythematous cyst, scleroderma, Sjogren's syndrome, polymyositis and dermatomyositis, polymyalgia rheumatica, temporal arteritis, polyarteritis nodosa, or Behcet's syndrome.

[0218] The pharma- ceutically acceptable carrier contained in the composition of the present invention is one that is commonly used in formulation, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, etc. The composition of the present invention may further include, in addition to the above-mentioned components, lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents, preservatives, etc.

[0219] The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc.

[0220] For oral administration, since proteins or peptides are digested, oral compositions must be formulated to coat or protect the active agent from degradation in the stomach. Furthermore, pharmaceutical compositions can be administered by any device that allows the active agent to be transferred to the target cells.

[0221] The appropriate dosage of the composition according to the present invention varies depending on factors such as the formulation method, administration method, age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient, and usually, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present invention is 0.0001 to 100 mg / kg. In this specification, the term "pharmaceutical effective amount" means an amount sufficient to prevent or treat cancer or autoimmune disease.

[0222] The pharmaceutical composition of the present invention can be prepared in a unit dose form or in a multi-capacity container by formulating it using pharma- ceutically acceptable carriers and / or excipients in a manner that can be easily carried out by a person having ordinary skill in the art to which the invention pertains. In this case, the formulation can be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, acidulant, suppository, powder, granule, tablet or capsule, and can further contain a dispersant or stabilizer.

[0223] Combination treatment The present invention relates to a combination therapeutic composition comprising immune cells and a drug other than an anti-FGFR3 antibody.

[0224] In one embodiment, the agent other than an anti-FGFR3 antibody may include a chemotherapeutic agent or an antibody other than an anti-FGFR3 antibody.

[0225] The drugs include maytansinoids, orlistatins (including MMAE, MMAF), aminophterin, actinomycin, bleomycin, tallysomycin, camptothecin, N8-acetylspermidine, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazide, espiramicin, etoposide, 6-mercaptopurine, dolostatin, trichothecenes, calicheamicins, taxol, taxanes, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocamicin, L-asparaginase (L-asparagine), and the like. nase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustardmustard, cytoxan, etoposide, 5-fluorouracil, CNU (bischloroethylnitrosourea), irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase inase, vinorelbine, chlorambucil, melphalan, carmustine, lomustine, busulfan, treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, crisnatol, mitomycin C C), trimetrexate, mycophenolic acid, thiazopurine, ribavirin, EICAR (5-ethynyl-1-beta-dribofuranosylimidazole-4-carboxamide), hydroxyurea, deferoxamine, flixuridine, doxifluridine, raltitrexed, cytarabine (ara C), cytosine arabinoside (cytosine arabinoside), cyclosporine (cyclosporine ...arabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor factor, gemcitabine, velcade, revamid, thalamid, lovastatin, 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2, peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, and thapsigargin, nucleases, and toxins derived from bacteria, animals, or plants.

[0226] In one embodiment, the antibody other than the anti-FGFR3 antibody may be, for example, an antibody or an antigen-binding fragment thereof that targets one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

[0227] The present invention relates to combination therapeutic compositions comprising an antibody or antigen-binding fragment thereof and one or more selected from the group consisting of: (i) immune cells, (ii) an immune cell comprising an antigen receptor (CAR) that comprises an scFv against an antibody other than an anti-FGFR3 antibody as an extracellular domain; and (iii) Immune checkpoint inhibitor.

[0228] The present invention also relates to a combination therapeutic composition comprising said immune cell engaging bispecific or multispecific antibody and one or more selected from the group consisting of: (i) immune cells, (ii) an immune cell comprising an antigen receptor (CAR) that comprises an scFv fragment against an antibody other than an anti-FGFR3 antibody as an extracellular domain; and (iii) Immune checkpoint inhibitor.

[0229] The immune cells are capable of immunotherapy, e.g., inducing immunity to induce a desired cancer treatment effect, and may be selected from the group consisting of, but not limited to, T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor-infiltrating lymphocytes (TIL), and dendritic cells.

[0230] The antibody other than the anti-FGFR3 antibody may be an antibody that targets a target other than FGFR3, and may be, for example, an antibody or an antigen-binding fragment thereof that binds to LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, or MARCO, but is not limited to these.

[0231] The present invention relates to combination therapeutic compositions comprising an antibody or antigen-binding fragment thereof and one or more selected from the group consisting of: (i) immune cells, (ii) an immune cell comprising an antigen receptor (CAR) that comprises an scFv against an antibody other than an anti-FGFR3 antibody as an extracellular domain; and (iii) Immune checkpoint inhibitor.

[0232] The present invention also relates to a combination therapeutic composition comprising said immune cell engaging bispecific or multispecific antibody and one or more selected from the group consisting of: (i) immune cells, (ii) an immune cell comprising an antigen receptor (CAR) that comprises an scFv fragment against an antibody other than an anti-FGFR3 antibody as an extracellular domain; and (iii) Immune checkpoint inhibitor.

[0233] The immune cells are capable of immunotherapy, e.g., inducing immunity to induce a desired cancer treatment effect, and may be selected from the group consisting of, but not limited to, T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor-infiltrating lymphocytes (TIL), and dendritic cells.

[0234] The antibody other than the anti-FGFR3 antibody may be an antibody that targets a target other than FGFR3, for example, an antibody or an antigen-binding fragment thereof that binds to LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, or MARCO, but is not limited to these.

[0235] The immune checkpoint inhibitor refers to a preparation capable of blocking T cell inhibitory signals at the site where antigen presenting cells (APCs) and immune cells, such as T cells, meet, thereby inducing T cell activation. The immune checkpoint inhibitor may be, but is not limited to, a drug targeting, for example, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, or CD200R.

[0236] The first and second components of the co-administration target can be administered simultaneously. The first and second components of the co-administration target can also be administered separately at a certain time interval. The second component of the co-administration target can be administered separately before or after the administration of the first component.

[0237] Companion diagnostics (CDx) Companion diagnostics is a type of molecular diagnostic technology for predicting a patient's responsiveness to a specific drug treatment, and can provide criteria for selecting target patients for drug application, and can provide criteria for treating patients with appropriate drugs by taking into account the specificity and sensitivity of matching diagnostic, prognostic and predictive biomarkers.

[0238] Anti-cancer drugs are highly effective in patients who respond, but the percentage of patients who respond is low, so it is important to select patients who will respond. Companion diagnostics can be used to select patients who will respond to anti-cancer drugs, thereby improving the efficiency of patient treatment.

[0239] From another aspect, the present invention relates to a composition for cancer therapy comprising the antibody or an antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, a chimeric antigen receptor, or an immune cell comprising the chimeric antigen receptor, for treating a cancer in which a fusion gene of FGFR3 and TACC3 has been confirmed to be amplified, mutated, overexpressed, or constitutively activated.

[0240] The present invention also relates to a method for treating cancer, comprising the step of administering the antibody or antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate, a chimeric antigen receptor, or an immune cell comprising the chimeric antigen receptor, when it is confirmed that the fusion gene of FGFR3 and TACC3 is amplified, mutated, overexpressed, or constitively activated in a patient-derived sample.

[0241] The sample refers to a biological specimen obtained from a patient's tissue or body fluid. The source of the tissue sample can be solid tissue, such as from frozen and / or preserved organs, tissue samples, biopsies or aspirates, blood or any blood components (e.g., serum, plasma), bone marrow or any bone marrow components, body fluids such as urine, cerebrospinal fluid, whole blood, plasma and serum. The sample can include a non-cellular fraction (e.g., urine, plasma, serum or other non-cellular body fluids). The sample can include body fluids, such as blood (e.g., whole blood). Specifically, the sample can be a whole blood sample, a whole bone marrow sample, a whole peripheral blood sample, or a whole tumor sample obtained from a patient. The "whole" sample is a sample that is substantially free of components (e.g., cells) that have been removed or separated from the sample. The sample, such as a blood sample, can be diluted (e.g., by a physiological compatibility buffer or medium) before use. In other embodiments, the "whole" sample, such as a whole tissue sample or a whole tumor sample, can substantially maintain the microenvironment from the tissue of origin, e.g., the structure of the tumor or immune microenvironment. Specifically, a sample, such as a tumor sample, may be processed (eg, crushed, compressed, mixed, ground, etc.) into smaller pieces and diluted (eg, with a physiologically compatible buffer or medium).

[0242] The expression of the gene can be confirmed by polymerase chain reaction (PCR) in the process of confirming the presence or absence of the gene and the degree of its expression, and in some cases, it can be confirmed using transcription-polymerase chain reaction (RT-PCR), competitive reverse transcription-polymerase reaction (Competitive RT-PCR), real-time reverse transcription-polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, or DNA chip.

[0243] Polymerase chain reaction (PCR) can be carried out through gene amplification reaction using primers. Multiplex PCR, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chain reaction (IPCR), vectorette PCR and TAIL-PCR (thermal asymmetric interlaced PCR) can be carried out.

[0244] It is also possible to detect the presence or absence of protein expression according to the genetic code. This is the process of confirming the presence or absence of the protein expressed from the gene and the level of its expression. The amount of protein can be confirmed using antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), aptamers, etc. that specifically bind to the protein.

[0245] Analytical methods for this purpose include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), and methods using protein chips. EXAMPLES

[0246] The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are provided solely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0247] Example 1: Selection of phage antibodies specific to FGFR3, cell panning (phage display technology) Our research group performed a cell panning method to select antibodies with cell internalization function using a synthetic scFv phage library {(1)Bai, Xuelian & Kim, Jihye & Kang, Seungmin & Kim, Wankyu & Shim, Hyunbo. (2015).A Novel Human scFv Library with Non-Combinatorial Synthetic CDR Diversity. PloS one.10. e0141045. 10.1371 / journal.pone.0141045., (2) Yang, Hye & Kang, Kyung & TCW, Julia & Shim, Hyunbo. (2009).Construction of a large synthetic human scFv library with six diverse CDRs and high functional diversity. Molecules and Cells.27. 225-35. 10.1007 / s10059-009-0028-9.}. FGFR3 overexpressing cells (682T) and FGFR3 low expressing cells (626T) were verified through RPKM analysis and protein expression confirmation by western blotting to select the corresponding cells. The previously collected phage library stock was treated with FGFR3 low expressing cells and reacted at 4 degrees for 1 hour, and then the supernatant was collected through a centrifuge and treated with previously prepared FGFR3 overexpressing cells and reacted at 4 degrees for 1 hour. Then, nonspecific phages were removed through culture medium, and the cells were cultured at 37 degrees for 30 minutes to recover the antibodies internalized in the cells. The recovery method was to remove nonspecific cell surface-bound phages by washing with a low pH buffer, then lyse the cells with a lysis buffer, recover the antibodies with the function of internalization through FGFR3 specific binding inside the cells, and infect E. coli. The infected E. coli was amplified again and the same cell panning method was carried out twice more for a total of three cell panning rounds, after which ELISA screening was carried out to select FGFR3-specific antibodies. The experimental diagram is shown in Figure 1a.

[0248] Example 2: Selection of phage antibodies specific to FGFR3, antigen immobilization panning (phage display technology) Using the same phage library, we performed immobilized antigen panning, which is commonly used. FGFR3-IIIc / Fc antigen and negative / Fc antigen were coated on immune-tubes at 3ug / ml each (overnight, 4 times), and the previously collected phage library stock was first reacted with pre-blocked negative / Fc antigen at room temperature for 1 hour. The supernatant was then collected and reacted with pre-blocked FGFR3-IIIc / Fc tube at room temperature for 1 hour. After washing with PBST to remove non-specific phages, phages specific to FGFR3-lllc / Fc antigen were collected through low pH buffer. After infection with E.coli, the same panning method was carried out three times after the amplification process, and bio-panning was carried out a total of four times, and ELISA screening was carried out to select FGFR3-specific antibodies. The experimental diagram is shown in Figure 1b.

[0249] Example 3: Phage antibody selection specific to FGFR3, magnetic bead panning, semi-automated panning (phage display technology) Semi-automated bio-panning was performed using the same phage library. This method is magnetic beads-based panning, in which phages are treated with target antigens that have been biotinylated using the conventional manual method, and after a binding process, antigen-specific phages are washed and collected using a column or magnetic chamber (Figure 1c). The inventors introduced this series of processes into a semi-automated system. Phage library stock, biotinylated FGFR3-IIIc / Fc antigen, streptavidin-M280 dynabeads, 0.1% PBST wash buffer, elution buffer, and other reagents were dispensed into Kingfisher Flex 24 deep wells of a Kingfisher Flex (Thermo Scientific) device, and the pre-prepared software protocol (BindIt TM Bio-panning was performed using the ELISA software (Figure 1d). Bio-panning was repeated 3 to 4 times to amplify and recover phages that specifically bind to the FGFR3 antigen. Using these, ELISA screening was performed to select FGFR3-specific antibodies.

[0250] Example 4: Affinity ELISA-based antibody screening The output of each bio-panning method was E. coli host cells (TG1), which were smeared on Agar LB / Ampicillin medium as a single clone, and then optionally inoculated into SB / ampicillin culture medium and cultured (3 hours, 37 degrees). Then, for induction of scFv-pIII protein, each well was treated with IPTG at a final concentration of 1 mM and cultured at 30 degrees overnight. The next day, the supernatant was removed from the 96-well culture plate using a centrifuge, and only E. coli was collected and suspended in 1x TES solution (20% w / v sucrose, 50 mM Tris, 1 mM EDTA, pH 8.0) at 4 degrees for more than 1 hour, and then further treated with 0.2x TES solution and suspended for more than 2 hours to extract the scFv-pIII protein present in the E. coli periplasmic region. The FGFR3 recombinant protein and negative recombinant protein were coated on a 96-well plate at 1ug / ml overnight at 4°C one day in advance, and the scFv-pIII extracted from the coated plate was treated on each plate and reacted at room temperature for 1 hour. After the reaction, the plate was washed with PBST and treated with anti-HA antibody conjugated with HRP for 1 hour, and after washing again, a color reaction (TMB substrate) was induced and the OD value was measured (450nm). The OD ratio of the FGFR3 recombinant protein / negative recombinant protein was calculated to select FGFR3-specific scFv-pIII clones. The selection criteria were a conversion calculation (positive / negative) based on the relative ratio, and antibody sequence analysis was performed on clones with a value of 2 or more.

[0251] In the case of bio-panning using patient-derived cells, 564 single clones were obtained from the completed output phage and screened using ELISA, of which 10 positive clones were confirmed (Figure 2a). In the case of antigen-fixed bio-panning, ELISA screening was performed on 658 single clones in two separate experimental runs (a total of 1,316 clones), of which 328 positive clones were confirmed (Figure 2b). After magnetic-based semi-automated bio-panning was completed, 376 clones were screened using ELISA, of which 26 positive clones were confirmed (Figure 2c).

[0252] Example 5: Antibody sequence analysis The antibody sequences for clones with an OD ratio (FGFR3 antigen / negative antigen) of 2 or more were analyzed based on the affinity ELISA screening analysis. E. coli carrying the phagemid vector of the selected clone was cultured in LB / ampicillin medium in a 14 ml round tube, and plasmid DNA was extracted by DNA prep (mini-prep). The extracted DNA was analyzed using a primer that matches the scFv sequence of the phagemid vector.

[0253] As a result of sequence analysis by each method, two antibody-specific sequences were analyzed by cell panning, 26 antibody-specific sequences by antigen immobilization panning, and eight antibody-specific sequences by semi-automated panning. Based on this, ELISA was repeatedly performed on each corresponding clone in the form of scFv, and finally, 25 clones showing FGFR3 specific binding ability were identified, and the sequences for the heavy and light chains were analyzed.

[0254] [Table 1]

[0255] [Table 2] TIFF0007630646000003.tif89170

[0256] [Table 3]

[0257] [Table 4] TIFF0007630646000006.tif90170

[0258] Example 6: Conversion of scFv antibody fragments to IgG and antibody production Based on the FGFR3 antibody sequence confirmed based on sequence analysis, we cloned it into the heavy chain production vector and light chain production vector owned by this research group to construct an IgG production vector. Based on the constructed production vector, FGFR3 antibody was produced using the mammalian protein expression system (Expi293F) and the affinity chromatography column MabSelect SuRe was used with the AKTA prime plus device. TM The high-purity antibody was collected and purified by the above procedure. The completed sample was analyzed for its high-purity sample characteristics based on SDS-PAGE and size-exclusion chromatography HPLC (SEC-HPLC) analysis (Figures 3a and 3b).

[0259] Example 7: Evaluation of antigen-binding ability of FGFR3 antibody: ELISA Affinity ELISA was performed to confirm the binding ability of the produced antibody (IgG) to the FGFR3 antigen. After coating a 96-well plate with 1-2 ug / ml of FGFR3 antigen (4 times, overnight), blocking was performed with 3% skim milk (PBS) (room temperature, 1 hour). Then, serially diluted antibody samples were treated in duplicate and triplicate and reacted at room temperature for 1 hour. After washing with PBST 2-3 times, anti-human Fab antibody bound to HRP was diluted in skim milk and treated (room temperature, 1 hour), washed with PBST again, and a color reaction (TMB substrate) was performed. Then, the OD was measured at 450 nm to confirm the value.

[0260] The same method was used to confirm the identity of human FGFR3 IIIb (1264-FR-050, R&D), IIIc (766-FR-050, R&D), mouse FGFR3 (9089-FR, R&D), and monkey FGFR3 (90313-C02H, sino biological) antigens.

[0261] The binding ability of each antibody was confirmed at each concentration (Figures 4a to 4d), and the OD values ​​were converted to relative values ​​and graphed. KD values ​​were calculated based on EC50 (Tables 5a to 5d).

[0262] [Table 5a]

[0263] [Table 5b]

[0264] [Table 5c]

[0265] [Table 5d]

[0266] Example 8: Evaluation of antigen-binding ability of FGFR3 antibody; SPR SPR analysis was performed to evaluate the binding ability of the produced antibodies (IgG) to FGFR3 subtypes and subfamilies. SRP-based affinity measurements of FGFR3 antibody candidates to FGFR3 antigens were performed using a Biacore-T200 instrument. The same antigens described in the ELISA binding capacity were directly immobilized on a CM5 biosensor chip by amine coupling, achieving approximately 300 RU. Serial dilutions were injected in sequence (25 times) at approximately 20 nM concentration for 180 seconds of binding time at a flow rate of 30 ug / min, followed by a separation step at the same flow rate for 180 seconds. Between each injection, 10 mM glycine-HCl was injected as a buffer to regenerate the CM5 biosensor chip. The binding and dissociation rates were calculated using a one-to-one Ranmul binding model. The equilibrium dissociation constant (KD) was calculated as the ratio of Koff / Kon. (Figure 5, Table 6)

[0267] [Table 6]

[0268] Example 9: Confirmation of FGFR3 protein expression level; Western blotting, FACS analysis In order to conduct cell surface binding and other assays for the FGFR3 antibody discovered in the present invention, western blotting and FACS analysis were performed to select FGFR3 overexpressing cells. Patient-derived cells and cancer cell lines were selected based on mRNA expression level (RPKM), and a certain number of cells were obtained through subculture. In the case of Western blotting, lysates were prepared and examined, and in the case of FACS analysis, cell surface FGFR3 expression was directly confirmed. First, in the case of Western blotting, each cell was cultured in cComplete TMAfter lysis with Lysis-M (Roche), Bradford protein quantification was performed, and 20 μg of the lysate was subjected to Western blotting. Total FGFR3 (sc-13121, Santa Cruz) and β-actin were confirmed, and the relative FGFR3 protein expression levels between cell lines were compared. For FACS, each subcultured cell was dispensed at 3.0-5.0E+05 cells / tube and reacted with FGFR3-PE direct antibody (FAB766P, R&D) for 1 hour, four times, washed 2-3 times, and then analyzed by BD FACSAria. TM The analysis was performed using a 3-D microscope (BD Biosciences).

[0269] Western blot results confirmed that 682T and 526T patient-derived cells were FGFR3 overexpressing cells. On the other hand, 626T and 559T were confirmed to have almost no FGFR3 expression. The same phenomenon was verified by FACS results. In particular, 682T cells appear to have a very high level of FGFR3 expression on the cell surface (Figure 6a).

[0270] Example 10: FGFR3-TACC3 fusion verification; RT-PCR FGFR3 appears in the form of overexpression, amplification, mutation, and fusion in various carcinomas. Among them, patients with FGFR3-TACC3 fusion have been reported to have poor prognosis in bladder cancer and brain tumors. Therefore, therapeutic drugs for this have the potential to overcome unmet medical needs. Therefore, in the present invention, patient-derived cells were grouped based on FGFR3-TACC3 fusion analysis and confirmed using RT-PCR technique. Patient-derived cells GBM15-682T, GBM14-526T, BT16-1154T, GBM14-606T, and NS10-783T were propagated through subculture, and a certain amount was collected and RNA was extracted using RNeasy Mini Kit (Qiagen). Since the extracted RNA is unstable, RT-PCR (Superscript IV Reverse transcriptase) was performed to synthesize it into cDNA. To analyze the FGFR3-TACC3 fusion site, we used the UCSC genome browser to confirm the fusion sites that had been studied, and created primers to recognize the FGFR3-TACC3-specific region (FW: 5'-TGATCATGCGGGGAGTGCTG-3', RV: 5'-CTCCTCACACCTGACACCTGCTCCTC-3'). PCR experiments were performed on cDNA using the primers created to confirm whether the FGFR3-TACC3 fusion-specific region was amplified. At the same time, GAPDH was also confirmed as a control. As a result, GBM15-682T, GBM14-526T, and BT16-1154T were confirmed as FGFR3-TACC3 fusions (expected PCR size, 400-500 bp). Among them, GBM15-682T and GBM14-526T are expected to be the same fusion, and BT16-1154T is expected to be a different fusion (Figure 6b). We analyzed the properties and anti-cancer effects of additional FGFR3 antibody clones based on cells derived from patients with FGFR3-TACC3 fusion brain tumors.

[0271] Example 11. Evaluation of cell binding ability of FGFR3 antibody; FACS analysis. FACS analysis was performed to confirm the binding ability of the discovery antibody to the patient-derived cells overexpressing FGFR3-TACC3 confirmed in the present invention. Each cell was dispensed at 3.0-5.0E+05 cells / tube, washed (with PBS), and then treated with 100-200 nM discovery antibody at 4°C. The same washing method was then used to remove unattached antibody samples, and the antibody attached to the cell surface was detected by treatment with anti-human alexa-488 conjugate (Catalog # A-11013, Invitrogen) antibody using BD FACSAria. TM The antibodies were detected using a Fluorescence Integrator III (BD Biosciences) to confirm their cell surface binding ability. Each of the discovered antibodies showed cell binding ability with different affinities (Figure 7a). Under the same conditions, the cell surface binding ability of the F309 clone was analyzed according to concentration, and it was confirmed that the cell surface binding ability was proportional to the concentration, verifying that the antibodies had FGFR3-specific binding ability (Figure 7b). It was further confirmed that the antibodies did not bind to cell lines that do not express FGFR3 (Figure 7c).

[0272] Example 12: Evaluation of target resolution of FGFR3 antibodies; FACS The FGFR3 targeting ability of the candidate antibodies discovered in the present invention was screened by FACS. Representatively, FGFR3 overexpressing cells, GBM15-682T patient-derived cells, were subcultured and dispensed at 3.0E+05 cells / tube, and treated with 40ug / ml of each candidate antibody (2 hours, 37 degrees). After washing 2-3 times, FGFR3-PE (FAB766P, R&D) was treated and reacted for 1 hour 4 times. After washing 2-3 times in the same manner, the antibodies were analyzed by BD FACSAria. TM The results were analyzed using a 3D chromatograph III (BD Biosciences). This allows the comparison of the overall FGFR3 expression level on the cell surface after antibody treatment. The results were compared to the IgG control and analyzed based on the Geomean value. The antibodies were treated for different times (4 hours, 2 hours, 1 hour, 30 minutes, and 15 minutes) in the same manner to confirm the ability of the antibodies to degrade cell surface FGFR3 over time. The same procedure was performed with the KMS11 cell line.

[0273] A relative comparison was made between the preferentially discovered antibodies at the same concentration conditions, and based on this, it was confirmed that the F309 and FS306 antibodies effectively reduced FGFR3 on the cell surface (Figure 8). In addition, in the case of the F309 and FS306 clones, the overall FGFR3 expression on the cell surface decreased over time, further confirming that the clones have FGFR3 decomposition ability. It is also believed that F309 and FS306 have better decomposition ability than other antibodies, B701 and LY3076226 (D11) antibodies (Figure 10a).

[0274] Example 13: FGFR3 epitope domain mapping Since FGFR3 is composed of three domains, in order to analyze the FGFR3 binding domain of the discovery antibody, each domain (FGFR3 Ig domain I, domain II, and domain III) was Fc-tagged and produced and purified through a mammalian cell protein expression system (Figure 9c). The binding ability of the antibody to the purified domain was analyzed in the same manner as the ELISA described above, by coating with 1ug / ml, treating the antibody, and inducing a TMB color reaction to measure. It was confirmed that the F309 and FS306 clones specifically bind to FGFR3 domain 1 (Figure 9b). The comparison group antibodies B701 (Vofatamab, Rainier Therapeutics) and D11 (LY3076226, Eli Lilly) were confirmed to bind between domains 2 and 3 as reported in the literature. However, the epitope of the comparison group antibody is contained in domain 2 in the present invention. It has also been reported that FGFR3 and ligands such as FGF1 and FGF9 bind to Ig domain II and domain III of FGFR3. Based on this, we performed a competition ELISA assay. As a result, the B701 and D11 antibodies showed binding to FGFR3 competitively with FGF ligands as expected, while the F309 antibody was observed to bind to FGFR3 independently of FGF ligands. This can be interpreted as evidence supporting the binding to FGFR3 Ig domain I mentioned above (Figure 9a).

[0275] Example 14: Evaluation of target resolution of FGFR3 antibody; Western blotting The FGFR3 target resolution of the candidate antibodies discovered in the present invention was verified through Western blotting. GBM15-682T patient-derived cells were used as representative cells, as in the FACS method. The cells were dispensed into a 6-well plate at 2.0E+06 cells / well, treated with each antibody at a concentration of 30ug / ml, and reacted at 37 degrees for 3 hours. Lysates were then prepared and 20ug of each sample was calculated using the Bradford quantification method, and western blotting was performed. Total FGFR3 was observed with the sc-13121 antibody. The experiment was also performed with the KMS11 cell line at the same concentration and reaction conditions. As with the FACS results, it was confirmed that the total FGFR3 resolution of the F309 and FS306 clones was excellent (Figure 10b).

[0276] Example 15: Antibody cellular internalization; Immunocytochemistry (ICC) The mechanism of antibody internalization was further verified through ICC. GBM15-682T was used as FGFR3 overexpressing cells, and U87MG was used as low expressing cells. GBM15-682T patient-derived cells did not adhere to ICC slides, so they were pre-coated with PLO reagent and cultured on the slides. U87MG cells were adherent cells and not treated with PLO. First, for the internalization condition, each antibody was treated with 30ug / ml of cells attached to ICC slides and incubated at 37 degrees for 2 hours. For the inactivation condition, the same concentration was used for the internalization inactivation at 4 degrees for 2 hours. After that, the cells were washed and fixed with 2% PFA (10 minutes, 4 degrees). The same washing method was repeated, and the cells were permeabilized by reacting with 0.1% Triton-X at room temperature for 3 minutes, and washed again. After that, blocking was performed with 1% BSA for 30 minutes at room temperature, and the primary antibody was treated overnight at 4 degrees. The primary antibodies used were LAMP-1 antibody, a lysosomal marker, and anti-human IgG. The next day, the samples were washed and incubated with the secondary antibody at 4°C for 1 hour in the dark. The secondary antibodies used were fluorescent antibodies that probe the primary antibody (Alexa 488; Anti-human LAMP1, Alexa 594; Anti-human IgG). After washing and staining with DAPI, each sample was analyzed by confocal microscopy. The analysis results confirmed that F309 and FS306 clones were attached to the surface of GBM15-682T cells, which are FGFR3 overexpressing cells, when treated at 4°C, and that the antibody was observed in the same way as the lysosomal location (LAMP-1) because the cellular internalization proceeded at 37°C. Based on this, it is expected that the clones bind to cell surface FGFR3, then are internalized in the cell and degraded in the lysosome (Figure 11a).

[0277] Example 16: Target degradation mechanism of FGFR3 antibody; antibody mechanism analysis by lysosomal inhibition In order to clarify the FGFR3 target degradation mechanism of the antibody discovered in the present invention, verification was performed using a lysosomal inhibitor. When an antibody binds to a specific antigen on the cell surface, it becomes receptor (antigen) mediated cellular internalization (receptor mediated endocytosis), and a mechanism has been reported in which the receptor and antibody are degraded together in the lysosome. Therefore, the degradation in lysosomes of antibodies that have been confirmed to have FGFR3 decomposition ability among FGFR3 antibodies was verified, and the total FGFR3 level for samples treated with and without lysosomal inhibitors was confirmed through western blotting. GBM15-682T was dispensed into a 6-well plate at 1.5E+06 cells / well, and treated with Concanamycin, known as a lysosomal inhibitor, at a concentration of 100 nM for 2 hours at 37 degrees. DMSO was treated as a control group. After 2 hours, the antibodies were treated at 30 ug / ml for 3 hours at 37 degrees. The total FGFR3 level was then confirmed by proceeding in the same manner as the western blotting method. Under the lysosomal inhibitor concanamycin treatment, total FGFR3 did not decrease even when F309 and FS306 clones were treated, whereas total FGFR3 was significantly decreased without treatment. This suggests that F309 and FS306 clones attach to cell surface FGFR3, are internalized within the cells, and are degraded together with FGFR3 in the lysosomes (Fig. 11b).

[0278] Example 17: Signaling pathway inhibition assay; Western blotting FGFR3 sub-signaling inhibition analysis was analyzed by western blotting. The same procedure as the Western blotting method was followed, with GBM15-682T patient-derived cells dispensed into a 6-well plate and treated with 30ug / ml of each antibody (37°C, 3 hours). Then, to activate FGFR3-related sub-signaling, FGF1 and FGF9 representative ligands were treated at 50ng / ml (treated with heparin sulfate 50ug / ml) and reacted at 37°C for 30 minutes. Lysates were then prepared and western blotting was performed on 20ug of each sample. Phosphorylation and activation of the FGFR3 sub-signaling pathway were verified using the following antibodies. p-FGFR3 (ab155960, Abcam), p-ERK (4370s, CST), and p-AKT (9271s, CST). F309 and FS306 clones were confirmed to inhibit FGFR3 sub-signaling pathways p-FGFR3, p-ERK, and p-AKT (Figure 12).

[0279] Example 18: Cancer cell growth inhibition assay; ligand-dependent environment In order to confirm the ability of the antibody discovered in the present invention to inhibit cancer cell growth in a ligand-dependent environment, GBM15-682T was dispensed into a 96-well black-flat bottom plate at 10,000 cells / well under serum-free conditions and starvated for one day. The next day, the ligands FGF1 (232-FA, R&D) and FGF9 (273-F9, R&D) were treated to a final concentration of 50 ng / ml, and the antibody was also diluted stepwise and treated at different concentrations. The cells were then cultured in a 37°C CO2 incubator for 7 days. Cell growth was measured using the CellTiter-Glo(R) Luminescent Cell Viability Assay, and cell growth in the antibody-treated group was compared to that in the control group. It was confirmed that when the cells were treated with the ligand, the cancer growth was accelerated by about 1.5 to 2 times, and when the clones were treated with different concentrations, cell growth was suppressed by 20 to 30% compared to the IgG control (control group) (Figure 13a).

[0280] Example 19: Cancer cell growth inhibition assay; ligand-independent environment Based on the cell growth assay described in this invention, the ability of antibodies to inhibit cancer cell growth in a ligand-independent environment was evaluated. GBM15-682T patient-derived cells were dispensed into a 96-well black-flat bottom plate at 10,000 cells / well, and the antibodies were serially diluted and treated at different concentrations. The cells were then cultured in a 37°C CO2 incubator for 7 days. Cell growth was measured using the CellTiter-Glo(R) Luminescent Cell Viability Assay, and cell growth in the antibody-treated group was compared to that in the control group. When the antibodies were treated in a ligand-free environment, it was confirmed that the F309 and FS306 clones inhibited cell growth by 30-40% compared to the IgG control (control group). No ability to inhibit cancer cell growth was observed in U87MG, a cell line with low FGFR3 expression, and patient-derived cells (626T). This is considered to be a characteristic of growth inhibition due to FGFR3 expression (Figure 13b).

[0281] Example 20: In vivo short-term PK analysis and efficacy evaluation in xenograft models Short-term PK was performed to confirm whether the discovered antibodies had an initial clearance when administered in vivo. Balb / c mice were administered 10 mg / kg of the antibody, and individual blood samples were obtained immediately after administration, and at intervals of 1 hr, 2 hr, 4 hr, 1 day, and 2 days. Plasma was obtained using a centrifuge to remove blood cells from the blood, and the antibody titer in the plasma was quantified using ELISA. As a result, it was confirmed that, like the control antibody, there was no initial clearance pattern for both the F309 and FS306 antibodies, and that there was a normal initial PK for the antibody (Figure 14a).

[0282] To evaluate the in vivo efficacy of the discovered antibody, the research group created a xenograft model. GBM15-682T (1.0E+06 cells) was injected subcutaneously into the flank of Bab / c nude mice to form tumors, which grew to an average size of 100-150 mm. 3 Once tumors were formed, mice were regrouped into groups of 5 mice each. Each group received intraperitoneal injections of antibody and vehicle three times a week or twice a week at different concentrations. Tumor volumes were measured using calipers to obtain V=0.5a × b 2 (a: tumor long axis length, b: tumor short axis length). The mice were also weighed on the same day as the tumor measurements. It was confirmed that F309 and FS306 clones suppressed cancer growth by approximately 50% and 40%, respectively, when administered at 10mg / kg (three times a week) (Figure 14a). In the case of F309, it is considered to suppress cancer growth with excellent anticancer effect when administered twice a week at different concentrations (30mpk, 10mpk, 3mpk) (Figure 14b).

[0283] Example 21: F309 antibody affinity maturation engineering Affinity maturation was carried out to further improve F309's excellent FGFR3 resolution and cancer growth inhibition ability. A phagemid vector displaying F309 scFv fragments (heavy chain variable region; VH, light chain variable region; VL) on the surface of M13 bacteriophage was constructed and used as a library template. For affinity maturation, CDRs H1, H2, H3 and CDRs L1, L2, L3 were selected based on the antibody's Kabat numbering, and an F309 mutant library was constructed using primers containing NNK degeneration codons for each CDR (Figure 15a). The biopanning (antigen immobilization panning) was performed 3 to 4 times for each library, and antibodies with higher affinity than the parent F309 antibody were selected by ELISA-based affinity screening. The clones obtained were re-validated by performing a reproduction experiment, and finally affinity matured mutants of F309 were obtained through antibody sequence analysis. The obtained F309 mutant is judged to have a significantly increased specific binding ability to FGFR3 recombinant protein compared to the parent antibody (Figure 15b). In addition, additional mutants SSM023, SSM024, SSM026, SSM034, SSM036, SSM234, and SSM236 were designed and produced by combining amino acid residues for the SSM001 to SSM010 mutants. ELISA was performed to verify the FGFR3 antigen-specific binding ability analysis of these antibodies, and the results confirmed the further increased FGFR3 antigen affinity (Figures 15b and 15c). Analysis of the biological activity of the mutant antibodies was performed on GBM15-682T, and the results confirmed increased cancer growth inhibition compared to the F309 parent antibody (Figure 15d).

[0284] [Table 7]

[0285] [Table 8]

[0286] [Table 9]

[0287] [Table 10]

[0288] [Table 11]

[0289] [Table 12]

[0290] [Table 13]

[0291] Example 22: Verification of applicability of F309 antibody ADC In the case of antibody-drug conjugates, the antibody specifically binds to an antigen on the cell surface, enters the cell through receptor-mediated cellular internalization, is degraded by lysosomes and proteases inside the cell, and kills the cell with the attached toxic substance. This is a promoter form that can maximize the therapeutic effect, and is widely used as a promoter form for next-generation antibody therapeutics. In the present invention, we attempted to confirm the applicability of clones with excellent internalization and target resolution for FGFR3 and FGFR3-TACC3 fusion cells as antibody-drug conjugates. Since FGFR3 target resolution and cellular internalization were demonstrated for the F309 and FS306 clones as described in the present invention, we determined that they could be applied as antibody-drug conjugate promoter forms, and carried out this study. Among them, the production of antibody-drug conjugates for the F309 clone was carried out using PerKit. TMUsing the Antibody MMAE Conjugation Kit (CellMosaic), this form of the antibody is a first-generation antibody-drug conjugate technology (a method developed by Seattle Genetics: Sun et al. 2005, Bioconjugate Chem.16, 1282-1290) that arbitrarily conjugates -vc MMAE to the interchain disulfide of the antibody (Figure 16a). Various patient-derived cells and cancer cell lines were selected based on FACS expression criteria and relatively grouped into High, Mild, and Low, and cytotoxicity experiments of F309 were performed on them. The evaluation method was measured using the CellTiter-Glo(R) Luminescent Cell Viability Assay as described in this invention. As a result, it was confirmed that the cytotoxicity of F309-vc MMAE was superior in proportion to the amount of FGFR3 expressed on the cell surface (Figures 16b-16c). To further secure competitiveness, the parent antibody LY3076226, which is currently in clinical development as an FGFR3-ADC, was used in the same kit to compare the effects. F309-vc MMAE showed superior cytotoxicity to LY3076266-vc MMAE in GBM15-682T, which is an FGFR3-TACC3 fusion. The growth inhibitory effect of ADC cells according to each FGFR3 expression level was analyzed by IC50 and MAX inhibition and is shown in the table in Figure 16d.

[0292] Example 23: Epitope domain mapping and antigen affinity analysis of affinity matured antibodies The epitope domain mapping previously performed was also performed on the affinity matured antibody F309#SSM236 (hereinafter referred to as AMB302#1.2). In the case of the parent antibody, it was confirmed that it bound to domain D1, and even when the same domains (Domain I, Domain II, Domain III) were used for analysis, it was confirmed that the affinity matured antibody specifically bound to domain D1. In the case of this antibody, it was reconfirmed that it has specific binding ability to human / cyno FGFR3 (Figure 17). The KD values ​​for each antigen are as shown in Table 14 below.

[0293] [Table 14]

[0294] Example 24: Cellular internalization and target resolution assessment of affinity matured antibodies We verified that the affinity-matured F309 mutant efficiently degrades the FGFR3 target on the cell surface. Using the same method as in the previous invention, we first confirmed the FGFR3 cell surface expression level and evaluated its internalization in the cells through FACS, and then performed target resolution through Western blotting. It was confirmed that the affinity-matured mutant had considerable internalization efficiency and target resolution in the cells (Figures 18 to 20).

[0295] Example 25: Mouse / Monkey PK Analysis of Affinity Matured Antibodies To verify the in vivo PK profile of affinity matured antibodies (AMB302#1.2, SSM236), analysis was performed on individual mice / monkeys. BALB / c mice (8 weeks, ~20g, n=3, female) were administered monoclonal antibodies at 10mg / kg, 1mg / kg, and 0.1mg / kg, and blood was collected from the orbit of three live animals at intervals of 10min, 1h, 3h, 5h, 8h, 24h, and on days 2, 3, 7, 10, 14, 17, 21, and 28 immediately after administration. Plasma was collected through a centrifuge to remove blood cells, and the antibody titer in the plasma was quantified using ELISA. As a result, it was confirmed that the AMB302#1.2 antibody has a stable half-life (t 1 / 2) characteristic and CL of more than 10 days in vivo in mice. In addition, no biologically specific clinical side effects or changes in body weight were observed at any administered concentration (Figure 21).

[0296] In one monkey (2-3 years, ~3kg, female, naive), a single dose of 50mg / kg was administered after administration of 25mg / kg, and blood samples were taken at each time point (10 minutes, 1 and 6 hours, 1, 2, 3, 7, 10, 14, 21, and 28 days). The collected blood was centrifuged to remove blood cells, and the plasma was collected, and the antibodies in the plasma were quantified by quantification ELISA. As a result, a stable half-life (more than 10 days) and CL tendency were confirmed, similar to the results in mice. In addition, no specific clinical side effects or changes in body weight were observed at any administered concentration (Figure 22).

[0297] Example 26: Production of affinity matured antibody ADCs In the present invention, ADCs were produced for the mutants obtained through affinity maturation engineering (Figure 23). The purified antibody sample was treated with tris(2-carboxyethyl) phosphine hydrochloride (TCEP) at an equivalent ratio of 8.0 or more and completely reduced at 37°C for 2 hours. After reduction, the buffer was exchanged with PBS pH 7.4, and the linker payload (Topoisomerase I inhibitor, equivalent ratio 10.0) was dissolved in DMA for the sample and conjugated with the reduced antibody at 37°C for 2 hours. After conjugation, the buffer was exchanged through a centrifugal column (Amicon Ultra-15 Centrifugal Filter Units).

[0298] Using the same method, the antibody was partially reduced to prepare an ADC conjugated with a linker payload (Duocarmycin derivatives, linker-AMB401) (Figure 24).

[0299] Example 27: Analysis of affinity matured antibody ADCs (TOPOI) The antigen affinity of the synthesized ADC sample (TOPOI-bound ADC) was confirmed by ELISA, which was performed in the same manner as the affinity ELISA performed in the present invention, and confirmed to have the same antigen binding ability as the parent antibody. In addition, the high purity physical properties of the produced ADC were confirmed by SEC-HPLC analysis.

[0300] DAR analysis was performed via LC-MS, and mass spectrometry was performed on the linker payloads attached to the heavy and light chains of the produced ADCs. When converted, it was confirmed that the DAR was approximately 6 or more (Figure 25).

[0301] Example 28: Analysis of affinity matured antibody ADCs (Duocarmycin, Linker-AMB401) The synthesized ADC sample (duocarmycin, linker-AMB401; ADC) was analyzed for purity through SEC-HPLC, and DAR was analyzed through HIC-HPLC and PLRP analysis. It was confirmed that the substance had uniform physical properties with a purity of over 98% and a DAR of about 4 (Figure 26: 20 mM Histidine-acetate pH 6.0).

[0302] Example 29: Efficacy Assessment of Affinity Matured Mutant ADCs in Patient-Derived Organoid Ex Vivo Models (ADC-TOPOI) The efficacy of the manufactured ADC was evaluated for organoids derived from patients with overexpression of FGFR3 (TACC3 fusion). Cancer cells were isolated from cancer tissue derived from brain tumor patients to create single spheroid organoid models. Two types of patient-derived cells with FGFR3-TACC3 fusion (AMB-BT-0050T, AMB-BT-0112T) and one with no FGFR3-TACC3 fusion (AMB-BT-0013T) were incubated overnight to form single spheroids, which were then treated with ADC and incubated for one week before the results were confirmed. There was no difference in sensitivity between the ADC and control ADC in the negative control, but it was analyzed that the ADC specifically inhibited spheroid growth and induced cell death in proportion to the amount of FGFR3 expression in two types of patient-derived cells with FGFR3-TACC3 fusion compared to the control ADC. In particular, the affinity matured mutant antibody-ADC was confirmed to induce significantly improved cytotoxicity and killing in FGFR3 (TACC3 fusion) overexpressing cells compared to the parent antibody-ADC, showing an IC50 of 0.3 nM or less against FGFR3 (TACC3 fusion) overexpressing cells and an IC50 of 90 nM or more against negative cells (Figure 27).

[0303] Example 30: Efficacy Evaluation of Affinity Matured Mutant ADCs in Patient-Derived Organism Ex Vivo Models (ADC-Duocarmycin, linker-AMB401) The ex vivo efficacy of AMB302#1.2-Duocarmycin ADC was evaluated using the same method as in Example 27. It showed a low IC50 of about 0.3 nM against FGFR3 (TACC3) overexpressing brain tumor patient-derived cells, and a high IC50 of 100 nM or more against negative cells. This indicates that the ADC induces cell death in a manner that is fairly proportional to FGFR3 expression (Figure 28).

[0304] Example 31: Efficacy evaluation of ADC in FGFR3 (TACC3 fusion) brain tumor stereotactic animal model (ADC-TOPOI) Increased survival rate in brain tumor stereotactic animal models (brain tumor, GBM) by ADC treatment was evaluated (Figure 29). FGFR3 overexpressing cells, AMB-BT-0050T (682T) patient-derived cells, were cultured and prepared by mixing 2X10^5 cells with 5 ul of medium. The prepared cell line was injected 1.7 mm to the left and 0.5 mm above the bregma of immunodeficient mice at a depth of 3.2 mm to create a brain tumor mouse model. The mice were divided into 4 groups: Control, Control-TOPOI, and AMB302#1.2-TOPOI (single administration, repeated administration), and evaluation was performed with 7 mice per group. The candidate drug was administered a single dose by intravenous injection on the 7th day after model creation. Intermediate sampling was performed on the 15th day for biomarker analysis, etc., with 2 mice per group. The mice were weighed twice a week, and if there was a weight loss of 20% or more, they were euthanized and the brains were removed. In the case of AMB302#1.2-TOPOI, it was confirmed that even a single administration increased the survival time of mice by 70% or more.

[0305] In the same model, even when AMB302#1.1-TOPO1 was administered twice (1 week apart, 10mg / kg), the survival time of the mice was significantly increased to over 100%. In addition, when the tumor size in the mouse brain was compared through mid-term sampling, no tumors were found in the ADC-treated individuals compared to the control group. It was also confirmed that the affinity-matured ADC induced tumor suppression and increased survival rate that were reliably improved compared to the parent antibody-based ADC (Figure 30).

[0306] Example 32: Efficacy evaluation of ADC in FGFR3 (TACC3 fusion) subcutaneous implantation model (ADC-TOPOI) Bab / c nude mice were injected subcutaneously (sc) with RT112 cells (1.0E+06 cells) into the flank of the mouse to form tumors. When tumors of an average size of 100-150 mm3 were formed, the mice were regrouped into groups of 5 mice each. ADC and vehicle were intravenously injected twice (1 week apart, 5 mp / kg) into each group. Tumor volume was calculated using calipers with the formula V=0.5a x b2 (a: tumor major axis length, b: tumor minor axis length). Mice were weighed on the same day as tumor measurement. It was confirmed that ADC-TOPOI completely inhibited the growth of the tumors, and demonstrated excellent anticancer effects (Figure 31).

[0307] Example 33: ADC efficacy evaluation in FGFR3 (TACC3 fusion) brain tumor stereotactic animal model (ADC-Duocarmycin, linker-AMB401) Using the same model as above, AMB302#1.1-AMB401 was administered either once (10 mg / kg) or repeatedly (10 mg / kg, Q4D) to evaluate efficacy. It was confirmed that the survival rate was significantly increased in the ADC administration group, and in particular, the survival rate increased by more than 30% in the group that received repeated administration (Figure 32). [Industrial Applicability]

[0308] The anti-FGFR3 antibody or antigen-binding fragment thereof according to the present invention exhibits superior binding ability to FGFR3 compared to conventional anti-FGFR3 antibodies, and can be usefully used in the prevention or treatment of target tumors or cancers.

[0309] In particular, by screening cells derived from brain tumor patients who overexpress FGFR3-TACC3, the researchers selected an antibody that has superior efficacy against FGFR3-TACC3 overexpressing cancer cells compared to known anti-FGFR3 antibodies.

[0310] Although the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to FGFR3 (Fibroblast growth factor receptor 3), including: A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 20, and a heavy chain CDR3 of SEQ ID NO: 43; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 68, a light chain CDR2 of SEQ ID NO: 90, and a light chain CDR3 of SEQ ID NO: 111; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:46; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:187; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:188; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:189; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:191; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:192; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:193; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:195; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:197; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:198; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:114; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:46; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:201; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:5, a heavy chain CDR2 of SEQ ID NO:23, and a heavy chain CDR3 of SEQ ID NO:46; a light chain variable region comprising a light chain CDR1 of SEQ ID NO:71, a light chain CDR2 of SEQ ID NO:93, and a light chain CDR3 of SEQ ID NO:202; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 24, and a heavy chain CDR3 of SEQ ID NO: 47; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 72, a light chain CDR2 of SEQ ID NO: 94, and a light chain CDR3 of SEQ ID NO: 115; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 6, a heavy chain CDR2 of SEQ ID NO: 25, and a heavy chain CDR3 of SEQ ID NO: 48; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 73, a light chain CDR2 of SEQ ID NO: 95, and a light chain CDR3 of SEQ ID NO: 112; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 9, a heavy chain CDR2 of SEQ ID NO: 28, and a heavy chain CDR3 of SEQ ID NO: 51; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 76, a light chain CDR2 of SEQ ID NO: 98, and a light chain CDR3 of SEQ ID NO: 118; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 29, and a heavy chain CDR3 of SEQ ID NO: 52; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 77, a light chain CDR2 of SEQ ID NO: 97, and a light chain CDR3 of SEQ ID NO: 119; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 30, and a heavy chain CDR3 of SEQ ID NO: 53; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 78, a light chain CDR2 of SEQ ID NO: 99, and a light chain CDR3 of SEQ ID NO: 120; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 54; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 79, a light chain CDR2 of SEQ ID NO: 100, and a light chain CDR3 of SEQ ID NO: 121; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 12, a heavy chain CDR2 of SEQ ID NO: 32, and a heavy chain CDR3 of SEQ ID NO: 55; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 80, a light chain CDR2 of SEQ ID NO: 101, and a light chain CDR3 of SEQ ID NO: 122; A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 3, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 56; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 81, a light chain CDR2 of SEQ ID NO: 102, and a light chain CDR3 of SEQ ID NO: 123; a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 34 and a heavy chain CDR3 of SEQ ID NO: 57, a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 82, a light chain CDR2 of SEQ ID NO: 103 and a light chain CDR3 of SEQ ID NO: 124, or A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 14, a heavy chain CDR2 of SEQ ID NO: 35, and a heavy chain CDR3 of SEQ ID NO: 58; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 83, a light chain CDR2 of SEQ ID NO: 104, and a light chain CDR3 of SEQ ID NO:

125.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO:134, SEQ ID NO:137 to SEQ ID NO:139, SEQ ID NO:142 to SEQ ID NO:149, SEQ ID NO:204 to SEQ ID NO:206, SEQ ID NO:208 to SEQ ID NO:210, SEQ ID NO:212, and SEQ ID NO:214 to SEQ ID NO:

215.

3. The antibody or antigen-binding fragment thereof of claim 1, comprising a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 162 to SEQ ID NO: 164, SEQ ID NO: 167 to SEQ ID NO: 174, and SEQ ID NO: 218 to SEQ ID NO:

219.

4. The antibody or antigen-binding fragment thereof of claim 1 , comprising a single-chain Fvs (scFv), a single-chain antibody, Fab, F(ab') or a disulfide-linked Fvs (sdFv).

5. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that the scFv comprises a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 134, SEQ ID NO: 137 to SEQ ID NO: 139, SEQ ID NO: 142 to SEQ ID NO: 149, SEQ ID NO: 204 to SEQ ID NO: 206, SEQ ID NO: 208 to SEQ ID NO: 210, SEQ ID NO: 212, and SEQ ID NO: 214 to SEQ ID NO: 215, and a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 162 to SEQ ID NO: 164, SEQ ID NO: 167 to SEQ ID NO: 174, and SEQ ID NO: 218 to SEQ ID NO: 219, linked via a linker.

6. The linker is n S) m (n, m are each 1 to 10),

7. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A recombinant expression vector comprising the nucleic acid of claim 7.

9. A host cell transformed with the recombinant expression vector of claim 8.

10. 10. The host cell of claim 9, which is COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

11. A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to FGFR3, comprising the steps of culturing the host cell described in claim 9 to produce an antibody, and isolating and purifying the produced antibody.

12. A bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

13. 13. The bi- or multispecific antibody of claim 12, comprising one or more partners selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), TIGIT, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, MARCO.

14. An immune cell engager bispecific or multispecific antibody comprising an scFv of an antibody according to any one of claims 1 to 6 and a second binding domain comprising one or more scFvs of an antibody that binds to an immune cell activation antigen.

15. The bispecific or multispecific antibody according to claim 14, characterized in that the scFv comprises a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 134, SEQ ID NO: 137 to SEQ ID NO: 139, SEQ ID NO: 142 to SEQ ID NO: 149, SEQ ID NO: 204 to SEQ ID NO: 206, SEQ ID NO: 208 to SEQ ID NO: 210, SEQ ID NO: 212, and SEQ ID NO: 214 to SEQ ID NO: 215, and a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 162 to SEQ ID NO: 164, SEQ ID NO: 167 to SEQ ID NO: 174, and SEQ ID NO: 218 to SEQ ID NO: 219, linked via a linker.

16. The linker is n S) m (n, m each being 1 to 10).

17. An antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof described in any one of claims 1 to 6 is bound to a drug.

18. The drugs include: (1) maytansinoids, orlistatins (including MMAE and MMAF), aminophterin, actinomycin, bleomycin, tallysomycin, camptothecin, N8-acetylspermidine, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazide, espiramicin, etoposide, 6-mercaptopurine, dolostatin, trichothecenes, calicheamicins, taxol, taxanes, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocamicin, L-asparaginase (L-aspara ginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustardmustard, cytoxan, etoposide, 5-fluorouracil, CNU (bischloroethylnitrosourea), irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase inase, vinorelbine, chlorambucil, melphalan, carmustine, lomustine, busulfan, treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, crisnatol, mitomycin C C), trimetrexate, mycophenolic acid, thiazopurine, ribavirin, EICAR (5-ethynyl-1-beta-dribofuranosylimidazole-4-carboxamide), hydroxyurea, deferoxamine, flixuridine, doxifluridine, raltitrexed, cytarabine (ara C), cytosine arabinoside (cytosine arabinoside), cyclosporine (cyclosporine ...arabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor one or more anticancer drugs selected from the group consisting of: 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2, peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, and thapsigargin, nucleases, and toxins derived from bacteria, animals, or plants; (2) Anti-inflammatory drugs, including steroids, nonsteroidal anti-inflammatory drugs (NSAIDs), or immunoselective anti-inflammatory derivatives (ImSAIDs); or (3) The antibody-drug conjugate according to claim 17, which is a therapeutic drug for immune diseases comprising azathioprine, chlorambucil, cyclophosphamide, cyclosporine, mycophenolate, azathioprine, or methotrexate.

19. The antibody-drug conjugate of claim 17 , wherein the antibody or antigen-binding fragment thereof is conjugated to the drug via a linker.

20. The antibody-drug conjugate of claim 19 , wherein the linker is a cleavable linker or a non-cleavable linker.

21. The antibody-drug conjugate of claim 20, wherein the cleavable linker is an acid-labile linker, a disulfide linker, a peptide linker, or a beta-glucuronide linker, and the non-cleavable linker comprises a thioether group or a maleimidocaproyl group.

22. The antibody-drug conjugate of claim 19, wherein the linker is bound to a cysteine ​​residue that is exposed upon reduction of disulfide bonds of the antibody or a cysteine ​​residue present in a tag bound to the antibody.

23. A chimeric antigen receptor (CAR) comprising an extracellular domain containing an antigen-binding site, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding site of the extracellular domain is an scFv of an antibody according to any one of claims 1 to 6.

24. The composition of claim 23, wherein the scFv comprises a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 134, SEQ ID NO: 137 to SEQ ID NO: 139, SEQ ID NO: 142 to SEQ ID NO: 149, SEQ ID NO: 204 to SEQ ID NO: 206, SEQ ID NO: 208 to SEQ ID NO: 210, SEQ ID NO: 212, and SEQ ID NO: 214 to SEQ ID NO: 215, and a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 162 to SEQ ID NO: 164, SEQ ID NO: 167 to SEQ ID NO: 174, and SEQ ID NO: 218 to SEQ ID NO: 219, linked via a linker.

25. The linker is n S) m The composition of claim 24, comprising: (n, m each being 1 to 10).

26. An immune cell into which the chimeric antigen receptor (CAR) of claim 23 has been introduced.

27. ​​The immune cell described in claim 26, which is one or more selected from the group consisting of T cells, NK cells, cytokine induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor tissue-infiltrating T cells (TIL), and dendritic cells.

28. The immune cell described in claim 26, further comprising a chimeric antigen receptor (CAR) that includes an scFv against an antibody other than an anti-FGFR3 antibody as an antigen-binding site in the extracellular domain.

29. The immune cell of claim 28, wherein the antibody other than an anti-FGFR3 antibody is an antibody targeting one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

30. A combination therapeutic composition comprising the immune cells of claim 26 and a drug other than an anti-FGFR3 antibody.

31. The composition of claim 30, wherein the drug other than an anti-FGFR3 antibody is an antibody targeting one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

32. A combination therapeutic composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and one or more selected from the group consisting of: (i) immune cells, (ii) an immune cell comprising an antigen receptor (CAR) that comprises an scFv against an antibody other than an anti-FGFR3 antibody as an extracellular domain; and (iii) Immune checkpoint inhibitor.

33. The composition according to claim 32, wherein the immune cell is one or more selected from the group consisting of T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor-infiltrating lymphocytes (TIL), and dendritic cells.

34. The composition of claim 32, wherein the antibody other than an anti-FGFR3 antibody of claim 32 is an antibody targeting one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

35. The composition of claim 32, wherein the immune checkpoint inhibitor is a drug targeting one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

36. A combination therapeutic composition comprising a bispecific or multispecific antibody according to claim 12 and one or more selected from the group consisting of: (i) immune cells, (ii) an immune cell comprising an antigen receptor (CAR) that comprises an scFv fragment against an antibody other than an anti-FGFR3 antibody as an extracellular domain; and (iii) Immune checkpoint inhibitor.

37. The composition of claim 36, wherein the antibody other than an anti-FGFR3 antibody is an antibody targeting one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

38. The composition of claim 36, wherein the immune checkpoint inhibitor is a drug targeting one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

39. A composition for treating cancer, inflammation or an immune disease, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, a bispecific or multispecific antibody comprising said antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising said antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising said antibody or antigen-binding fragment thereof, or an immune cell comprising said chimeric antigen receptor.

40. A composition for treating cancer, inflammation or an immune disease, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof.

41. A composition for treating cancer, inflammation or immune disease, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, a bispecific or multispecific antibody comprising said antibody or antigen-binding fragment thereof, an antibody-drug conjugate, a chimeric antigen receptor, or an immune cell comprising said chimeric antigen receptor, for treating a cancer in which it has been confirmed that the fusion gene of FGFR3 and TACC3 is amplified, mutated, overexpressed, or constitutively activated.

42. A vesicle on the exterior of which or the interior of which is present an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

43. The endoplasmic reticulum described in claim 42, which is an exosome.

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