WO2018124851A1 - Pharmaceutical composition for preventing or treating cancer, containing antibody specifically binding to l1cam protein and pyrimidine analog and/or platin-based anticancer drug - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, containing: an antibody specifically binding to an L1CAM protein; and a pyrimidine analogue, a platin-based anticancer drug, or a combination thereof. Compared to when administrating each ingredient individually, when co-administering an antibody specifically binding to an L1CAM protein with gemcitabine, which is a type of pyrimidine analogue, and/or cisplatin, which is a type of platin-based anticancer drug, synergistic effects are exhibited on the inhibition of bile duct cancer growth, and thus the pharmaceutical composition, of the present invention, containing the antibody and the drug can be usable in the treatment of biliary tract cancer.

Description

Antibodies that specifically bind to L1CAM protein; And pyrimidine analogues and / or platinum-based anticancer agents

The present invention provides an antibody that specifically binds to L1CAM protein; And pyrimidine analogues and / or platinum-based anticancer agents.

Biliary tract cancer is a malignant tumor of the ductal epithelium of the bile tract, which is anatomically anatomical of intrahepatic cholangiocarcinoma (ICC) or extrahepatic cholangiocarcinoma (ECC). (Khan SA, et al., Cholangiocarcinoma. Lancet. 2005; 366 (9493): 1303-14.). Biliary cancer is difficult to diagnose with recent clinical symptoms and has a poor prognosis because it is resistant to conventional chemotherapy and radiation therapy. The overall 5-year survival rate for patients with biliary tract cancer is 5 to 10% and includes curable surgery. In all cases, the reported 5-year survival rate is 25-30%. Thus, there is a need for new and effective treatment regimens that improve the survival of biliary tract cancer patients.

As a chemotherapy drug for biliary tract cancer, gemcitabine, a pyrimidine analog that inhibits DNA synthesis, has been extensively studied. In stage 2 clinical trials, it has been known that monotherapy of gemcitabine has shown efficacy in patients with advanced or metastatic biliary tract cancer (Okusaka T, et al, Cancer Chemother Pharmacol. 2006; 57 (5): 647-53.). In addition, a drug for biliary cancer comprising gemcitabine, 5-fluorouracil, and amino acids has been developed (Korean Patent Publication No. 2014-0079831), and the combination therapy of gemcitabine and cisplatin is used for patients with locally advanced or metastatic biliary tract cancer. Although survival has been reported to be significantly higher than gemcitabine monotherapy in, the response to chemotherapy in patients with biliary tract cancer is limited and still has a low 5-year survival rate (Rizvi S, et al., Semin Liver Dis. 2014; 34 (4): 456-64.

In this regard, the use of drugs targeting epidermal growth factor receptor (EGFR), which regulates bile epithelial cell growth and is overexpressed in biliary tract cancer, is increasing. For example, additional administration of cetuximab, an anti-EGFR antibody, to gemcitabine and oxaliplatin has recently been attempted, but efficacy in patients with advanced biliary tract cancer has not improved in phase II trials. In addition, combination therapy of gemcitabine and oxaliplatin with EGFR tyrosine kinase inhibitor erlotinib has been tried, but the recently released Phase III clinical trials did not show an improved effect compared to chemotherapy. . Thus, there is a need for new targeted therapies along with chemotherapy that improves patient survival.

On the other hand, L1 cell adhesion molecule (L1CAM) is a 200-220 kDa transmembrane glycoprotein, including six immunoglobulin-like domains, five fibronectin-type III domains, transmembrane stretch and It consists of a short cellular tail. L1CAM is a neuronal adhesion molecule of the central nervous system that plays an important role in the movement of cerebellar cells and the growth of neurites in many cancers, including melanoma, neuroblastoma, ovarian cancer, colon cancer, cholangiocarcinoma and gallbladder cancer. Has been reported to play an important role in the growth and metastasis of cancer cells, and that L1CAM overexpression is associated with poor prognosis of cancer (Raveh et al., Cancer Letters 282: 137-145, 2009).

The present inventors have diligently studied to develop a method for effectively treating cancer, specifically, biliary tract cancer. As a result, when the present invention is administered with a combination of Ab417, an antibody targeting L1CAM, and gemcitabine and / or cisplatin, the biliary tract cancer, It confirmed that the synergistic effect was shown with respect to the growth inhibition of, and completed this invention.

One object of the invention is an antibody that specifically binds to an L1 cell adhesion molecule (L1CAM) protein; And it provides a pharmaceutical composition for the prevention or treatment of cancer, including an anticancer agent, specifically pyrimidine analog, a platinum-based anticancer agent or a combination thereof.

Another object of the present invention is to provide a kit comprising the composition.

Another object of the present invention is the antibody; And administering a pyrimidine analog, a platinum anticancer agent, or a combination thereof to a subject in need thereof.

In the case of co-administration of an antibody and gemcitabine and / or cisplatin that specifically bind to L1CAM protein, it shows a remarkable effect on the growth inhibition of biliary tract cancer as compared to the case of administration alone, thus including the antibody and the drug. The pharmaceutical composition of the present invention can be usefully used for the treatment of biliary tract cancer.

1 shows the anticancer effect of Ab417 in Choi-CK xenograft model. Ab417 (10 mg / kg), as a comparison group anti-L1CAM antibody Hz2.2 (10 mg / kg) or isotype control (hFc, 3.3 mg / kg) in nude mice with Choi-CK cells ( n = 8 ) Intravenously three times a week. A is the tumor volume and B is the tumor weight. Each point represents 'mean ± standard deviation', and p <0.05 (*) and p <0.01 (**) by Dunnett's t-test show significant differences compared to homologous controls. it means.

2 shows the anticancer effect of Ab417 in Choi-CK xenograft model. Ab417 (10 mg / kg) or isotype control (hFc, 3.3 mg / kg) was injected intravenously twice a week in nude mice with Choi-CK cells ( n = 8). A is tumor volume, B is tumor weight and C is body weight. Each point p by "mean ± standard deviation" a represents, Dunnett t- test (Dunnett's t-test) p <0.05 (*) and p <0.01 (**), and Steel test (Steel's test) according to the < 0.05 ( ^# ) means significant difference compared to the homologous control. D represents the area stained with anti-human Ki-67 antibody in the tumor section. The Ki-67 index is expressed as a percentage of the positive staining site relative to the total area.

3 shows the appearance of Ab417 bound to Choi-CK expressed L1CAM, and the effect of reducing the membrane-L1CAM level of Ab417. A shows confocal microscopy analysis of Ab417 internalization. SCK-L1 cells were pretreated with Ab417 (10 μg / mL) for the indicated time, Ab417 bound to membrane L1CAM was anti-human IgG (H + L) Cross Adsorbed DyLight 594 (red), and internalized Ab417 was anti-human Detection was done using IgG (Fc-specific) -FITC (green). B represents Choi-CK cells treated with Ab417 (10 μg / mL) for the indicated time. Membrane L1CAM was detected by Western blot analysis using A10-A3, and pan-cadherin was used as loading control. Cells not treated with Ab417 were used as a control (C). C is a 400x magnified confocal microscopy image stained with L1CAM for Choi-CK tumors treated with PBS (top) or Ab417 (bottom). It is expressed as a percentage of the positive staining site relative to the total area. D represents the region stained with an antibody specific for the N-terminal region of human L1CAM in the tumor section. It is expressed as a percentage of the positive staining area relative to the total area.

4 shows the anticancer effects of gemcitabine or cisplatin in Choi-CK heterogeneous model. Intraperitoneal injections of the indicated doses of the drug twice a week for three or four weeks. A is the tumor volume, B is the tumor weight. Each point represents 'mean ± standard deviation', p <0.05 (*) and p <0.01 (**) by Dunnett test, and p <0.05 ( ^# ) and p <0.01 ( ^## ) by Steel test Means a significant difference compared to the control (saline).

Figure 5 shows the anticancer effect of the combination of Ab417 and gemcitabine, or cisplatin in Choi-CK xenograft model. A and C represent tumor volume, B and D represent tumor weight. Each point represents 'mean ± standard deviation', p <0.05 (*) and p <0.01 (**) by Dunnett test, and p <0.05 ( ^# ) and p <0.01 ( ^## ) by Steel test Means a significant difference compared to the control (saline).

FIG. 6 shows the degree of weight change of Ab417 and gemcitabine, or cisplatin combined administration in Choi-CK xenograft model. A relates to administration of gemcitabine or cisplatin alone, B to administration of Ab417 or gemcitabine alone, and combination of Ab417 and gemcitabine, C to administration of Ab417 or cisplatin alone, and the combination of Ab417 and cisplatin. Each point represents a mean ± standard deviation.

7 shows membrane L1CAM levels suppressed by Ab417 in TFK-1 cells.

8 shows the anticancer effect of Ab417 in the TFK-1 xenograft model. A is the tumor volume and B is the tumor weight. Each point represents 'mean ± standard deviation', and p <0.01 (**) by Dunnett's t-test indicates a significant difference compared to the homologous control (hFc).

9 shows the anticancer effect of gemcitabine or cisplatin in TFK-1 xenograft model. A is the tumor volume and B is the tumor weight. Each point represents 'mean ± standard deviation', meaning that p <0.05 (*) and p <0.01 (**) by Dunnett's test indicate a significant difference compared to the saline.

10 shows the anticancer effect of the combination of Ab417 and gemcitabine, or cisplatin in TFK-1 xenograft model. A is the tumor volume and B is the weight. Each point represents 'mean ± standard deviation', meaning that p <0.05 (*) and p <0.01 (**) by Dunnett's test indicate a significant difference compared to the saline.

In order to achieve the above object, one embodiment of the present invention comprises an antibody that specifically binds to L1 cell adhesion molecule (L1CAM) protein; And it provides a pharmaceutical composition for the prevention or treatment of cancer, including an anticancer agent, specifically pyrimidine analog (pyrimidine analog), a platinum-based anticancer agent or a combination thereof.

In the present invention, when a combination of an antibody binding to L1CAM protein and a pyrimidine analog, gemcitabine or cisplatin, a platinum anticancer agent, the treatment efficiency of biliary tract cancer is significantly increased as compared with the administration of the antibody and drug alone. Confirmed.

As used herein, the term "L1 CAM" refers to one of the intrinsic membrane glycoproteins belonging to immunoglobulin superfamily cell adhesion molecules (CAMs). The L1CAM is expressed in a variety of cancers, including melanoma, neuroblastoma, ovarian cancer, colorectal cancer, cholangiocarcinoma and gallbladder cancer, and plays an important role in the growth and metastasis of cancer cells. Relevance has been reported (Raveh et al., Cancer Letters 282: 137-145, 2009).

Since the antibody of the present invention binds to both human and mouse L1CAM proteins with high affinity and exhibits significant cancer growth inhibitory effect, it can be usefully used in the field of prevention or treatment of cancer in which L1CAM is overexpressed.

As used herein, the term “antibody” refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, including polyclonal antibodies, monoclonal antibodies, It includes both whole antibodies and antibody fragments. The term also includes chimeric antibodies (eg, humanized murine antibodies) and bivalent or bispecific molecules (eg, bispecific antibodies), diabodies, triabodies, and tetrabodies. The term further includes artificial antibodies based on protein scaffolds and single chain antibodies, caps, analogs of antibody constant regions that possess binding functions for FcRn. The total antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds. The total antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype, including IgG1, IgG2, IgG3 and IgG4. The antibody fragment refers to a fragment having an antigen binding function, and includes Fd, Fab, Fab ', F (ab') ₂ and Fv. Fd means the heavy chain portion included in the Fab fragment. The Fab has one antigen binding site in a structure having a variable region of the light and heavy chains, a constant region of the light chain, and a first constant region of the heavy chain (CH1 domain). Fab 'differs from Fab in that it has a hinge region comprising at least one cysteine residue at the C terminus of the heavy chain CH1 domain. F (ab ') ₂ antibodies are produced when the cysteine residues of the hinge region of Fab' form disulfide bonds. Fv (variable fragment) means a minimum antibody fragment having only the heavy chain variable region and light chain variable region. Double disulfide Fv (dsFv) is a disulfide bond is linked to the heavy chain variable region and the light chain variable region, and short chain Fv (scFv) is generally covalently linked to the variable region of the heavy chain and the light chain through a peptide linker. Such antibody fragments can be obtained using proteolytic enzymes (e.g., the entire antibody can be restricted to papain and Fab can be obtained, and pepsin can yield F (ab ') ₂ fragment). Can be produced through genetic recombination techniques.

In the present invention, the 'immunoglobulin' has a heavy chain and a light chain, each heavy and light chain comprises a constant region and a variable region (the region is also known as a domain). The variable regions of the light and heavy chains comprise three multivariable regions and four framework regions called complementarity-determining regions (hereinafter referred to as "CDRs"). The CDRs mainly serve to bind epitopes of antigens. The CDRs of each chain are typically called CDR1, CDR2, CDR3, starting sequentially from the N-terminus and also identified by the chain in which the particular CDR is located.

As used herein, the term "an antibody that specifically binds to an L1 cell adhesion molecule (L1CAM) protein" refers to an antibody that binds to an L1CAM protein and inhibits the activity of the L1CAM protein. For the purposes of the present invention, the antibody that specifically binds to L1CAM is an antibody that binds to the Ig5 region of L1CAM, but is not limited thereto.

Specifically, the antibody specifically binding to the L1CAM protein may be an antibody or a functional variant thereof comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 5.

As used herein, the term "functional variant of an antibody comprising a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5" refers to a parent antibody comprising a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5 Compared with the antibody having a difference in at least one amino acid of the heavy chain variable region and / or light chain variable region, it may be an antibody having improved physical properties compared to the parent antibody. In addition, the functional variant may retain biological activity equivalent to that of the parent antibody.

In particular, the antibody is an antibody having a difference in 1 to 10 amino acids of the heavy chain variable region and / or the light chain variable region as compared to the parent antibody comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 5, Specifically, at least one amino acid, in particular 1 to 10, more specifically 1 in the heavy chain variable region and / or light chain variable region of the parent antibody comprising a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: To 6 amino acids may be substituted.

Such antibodies may be monoclonal antibodies, and may be in the form of full length antibodies or antibody fragments thereof.

More specifically, such antibodies include heavy chain CDR1 as set forth in SEQ ID NO: 2; Heavy chain CDR2 set forth in SEQ ID NO: 3 or 10; And a heavy chain variable region comprising a heavy chain CDR3 as set out in SEQ ID NO: 4 and a light chain CDR1 as set out in SEQ ID NO: 6 or 12; Light chain CDR2 set forth in SEQ ID NO: 7; And a light chain variable region comprising the light chain CDR3 set forth in SEQ ID NO: 8, but is not limited thereto. In addition, the antibody comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 1, 9 and 13; And a light chain variable region selected from the group consisting of SEQ ID NOs: 5, 11, 14, and 15, but is not limited thereto.

The antibody specifically binding to L1CAM may be an antibody disclosed in Korean Patent Application Laid-Open No. 10-2014-0066101 A, WO2014-077648 A1, or US 2015-0344571 Al. The entire specification of the patent is incorporated herein by reference in its entirety.

Specifically, the antibody specifically binding to L1CAM may be as follows, but is not particularly limited thereto:

(1) a heavy chain CDR1 set forth in SEQ ID NO: 2; Heavy chain CDR2 set forth in SEQ ID NO: 3; And a heavy chain variable region comprising a heavy chain CDR3 as set out in SEQ ID NO: 4 and a light chain CDR1 as set out in SEQ ID NO: 6; Light chain CDR2 set forth in SEQ ID NO: 7; And a light chain variable region comprising the light chain CDR3 described in SEQ ID NO: 8, and more specifically, an antibody comprising a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5; Or an antibody comprising a heavy chain variable region of SEQ ID NO: 13 and a light chain variable region of SEQ ID NO: 15. An antibody comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 5 is named Ab417 in the present invention, and the antibody comprising the heavy chain variable region of SEQ ID NO: 13 and the light chain variable region of SEQ ID NO: 15 is provided herein. Is designated Ab417-H5L1.

(2) a heavy chain CDR1 set forth in SEQ ID NO: 2; A heavy chain CDR2 set forth in SEQ ID NO: 10; And a heavy chain variable region comprising a heavy chain CDR3 as set out in SEQ ID NO: 4 and a light chain CDR1 as set out in SEQ ID NO: 12; Light chain CDR2 set forth in SEQ ID NO: 7; And it may include a light chain variable region comprising a light chain CDR3 described in SEQ ID NO: 8, More specifically, the antibody comprising a heavy chain variable region of SEQ ID NO: 9 and a light chain variable region of SEQ ID NO: 11; Or an antibody comprising a heavy chain variable region of SEQ ID NO: 9 and a light chain variable region of SEQ ID NO: 14. The antibody comprising the heavy chain variable region of SEQ ID NO: 9 and the light chain variable region of SEQ ID NO: 11 is named Ab417-H6L2 in the present invention, and the antibody comprising the heavy chain variable region of SEQ ID NO: 9 and the light chain variable region of SEQ ID NO: 14 It is named Ab417-H6L6 in the present invention.

Ab417-H5L1, -H6L2, and -H6L6 described above are all properties of the applicant compared to Ab417 in the prior patents of the applicant, that is, Korean Patent Application Publication No. 10-2014-0066101A, WO2014-077648 A1, or US 2015-0344571 Al. It was developed as an improved antibody and can show an effect equivalent to Ab417.

The sequences used in the present invention are to be construed to include sequences that exhibit substantial identity with the sequences listed in the Sequence Listing, in view of variations that are biologically equivalent. The term 'substantial identity' means that if the alignment of the sequence of the present invention with any other sequence is maximally matched, the aligned sequence is analyzed using algorithms commonly used in the art. By 60% homology, more specifically 70% homology, even more specifically 80% homology, most specifically 90% homology.

Therefore, an amino acid sequence having high homology with the sequence represented by SEQ ID NOS: 1 to 15, for example, an amino acid having a high homology of 70% or more, specifically 80% or more, more specifically 90% or more. Sequences should also be construed as falling within the scope of the present invention.

As used herein, the term "pyrimidine analog" refers to a nucleoside analog compound that mimics the structure of metabolic pyrimidine. The pyrimidine analogs can be used as intracellular chemotherapeutic agents, intracellular anticancer agents, anti-viral agents or nucleic acid components for DNA viruses, and the like.

For the purposes of the present invention, the pyrimidine analogue may be used as an anticancer agent, and may be, for example, gemcitabine, but is not limited thereto.

As used herein, the term "platinum-based anticancer drugs" refers to anti-cancer drugs used as drugs that damage DNA as a heavy metal compound containing platinum. It is mainly used to treat various kinds of cancers such as breast cancer, bladder cancer, gastric cancer and cervical cancer, and combines with two adjacent guanines on DNA strands to form an interstrand crosslink to inhibit DNA synthesis. Known. Specifically, the platinum-based anticancer agent is cisplatin (cisplatin), oxaliplatin (oxaliplatin), tetraplatin (tetraplatin), heptaplatin (heptaplatin), paraplatin (carplatin) (carboplatin) or nanoplatin ( nanoplatin), more specifically cisplatin, but is not limited thereto.

In the present invention, the type of cancer is not particularly limited, but may be a cancer in which L1CAM is overexpressed. Specifically, melanoma, neuroblastoma, ovarian cancer, colorectal cancer, biliary tract cancer (biliary duct cancer or gallbladder cancer), but is not limited thereto.

As used herein, the term "biliary tract cancer" refers to "biliary duct cancer" and "gallbladder cancer" and refers to a malignant tumor occurring in epithelial cells surrounding the inside of the gallbladder. Bile duct cancer is a cancer that occurs in the bile duct that is the passage of bile discharged from the liver, and gallbladder cancer refers to a cancer that occurs in the gallbladder, which is a place where the bile is first stored. Biliary cancers can be divided into intrahepatic biliary and extrahepatic biliary tract cancers, depending on the location of the tumor, which may be divided into peripheral cholangiocarcinoma and hepatic cholangiocarcinoma; Extrahepatic biliary tract cancer may be classified into upper (proximal), middle, and lower (distal) biliary tract cancers according to the occurrence site.

Biliary cancers are difficult to diagnose early because they do not show any characteristic symptoms until they progress significantly. In addition, various anatomical structures in the biliary tract and vasculature, and it is difficult to determine the exact extent of tumor invasion before or even during surgery, resulting in curative resection (to remove all areas where cancer exists or is likely to exist). Sometimes it is impossible. Chemotherapy is used to prevent the growth of cancer cells that may remain after surgery or metastases due to cancer metastasis. Although many advances have been made in the treatment of biliary tract cancer, the effects have not yet been proven.

As used herein, the term "prevention" refers to the antibody; And all actions of inhibiting or delaying cancer by administration of a composition comprising a pyrimidine analog, a platinum anticancer agent, or a combination thereof.

As used herein, the term “treatment” refers to the antibody; And any action in which the symptoms of cancer improve or benefit altered by administration of a composition comprising a pyrimidine analog, a platinum anticancer agent, or a combination thereof.

In one embodiment of the present invention, the administration of Ab417 or gemcitabine alone showed 35.3% or 44.4% cancer growth inhibition, respectively, against Choi-CK cells, which are ICC cells, but 88.8% It was confirmed that the inhibition rate of (Fig. 5). In addition, when Ab417 or gemcitabine alone was administered to TFK-1 cells, which are ECC cells, cancer growth inhibition was 10.7% or 21.7%, respectively, but when they were used in combination, they showed an inhibition rate of 52.2%. (FIG. 10).

The pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition, and the carrier may comprise a non-naturally occuring carrier. have.

The term 'pharmaceutically acceptable' means to exhibit properties that are not toxic to cells or humans exposed to the composition.

Specifically, the pharmaceutical composition may be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. However, if the formulation is used in the art for the prevention or treatment of cancer, it is not limited thereto.

Examples of carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone (PCL; polycaprolactone), polylac Polylactic acid (PLA), poly-L-lactic acid (PLLA), Mineral oil and the like.

When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may comprise at least one excipient such as starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The antibody included in the pharmaceutical composition of the present invention; And the content of pyrimidine analogues, platinum anticancer agents or combinations thereof may be included in an amount of 0.0001 to 50% by weight, more specifically 0.01 to 20% by weight, based on the total weight of the final composition, but is not particularly limited thereto.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount.

The term `` pharmaceutically effective amount '' means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and an effective dose level refers to the severity of the disease, the activity of the drug, the patient Age, body weight, health, sex, sensitivity of the patient to the drug, time of administration of the composition of the invention used, route of administration and rate of release, duration of treatment, drugs comprising the drug in combination or concurrently with the composition of the invention used and It may be determined according to factors well known in other medical fields. The pharmaceutical composition of the present invention may be administered alone or in combination with known biliary tract cancer therapeutics. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects.

The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered at about 0.1 ng / kg to about 100 mg / kg, specifically about 1 ng / kg to about 10 mg / kg, per adult Frequency of administration is not particularly limited, but may be administered once a day or several times in divided doses. The dosage does not limit the scope of the invention in any aspect.

Another embodiment is the antibody; And administering to the individual a pharmaceutically effective amount of a pyrimidine analog, a platinum based anticancer agent, or a combination thereof.

As used herein, the term "individual" may include, without limitation, mammals, farmed fish, and the like, including rats, livestock, humans, and the like, which may or may not develop cancer.

In the method for preventing or treating cancer of the present invention, the route of administration and mode of administration for administering the composition is not particularly limited and may be in accordance with any route of administration and mode of administration so long as the composition can reach the desired site of interest. Can be.

Specifically, the composition may be administered through various routes of oral or parenteral, non-limiting examples of the route of administration, eye, mouth, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal And intranasal administration or inhalation.

In addition, the composition may be administered by any device in which the active substance may migrate to the target cell.

In addition, the antibody; And pyrimidine analogs, platinum anticancer agents or combinations thereof may be administered in combination.

As used herein, the term "combination" should be understood to denote simultaneous, separate or sequential administration. If the administration is sequential or separate, the interval of administration of the secondary components should be such that the beneficial effect of the combination is not lost.

Each of the agents may be administered simultaneously, sequentially, or in reverse order, and may be administered simultaneously in any combination of appropriate effective amounts, but is not limited to any particular method or order of administration.

The method of the present invention may comprise administering the active ingredient in a pharmaceutically effective amount. Suitable total daily doses may be determined by the treating physician within the scope of good medical judgment and may be administered once or in divided doses. However, for the purposes of the present invention, the specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and extent of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health of the patient, It is desirable to apply differently depending on various factors and similar factors well known in the medical field, including sex and diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used with or co-specific with the specific composition.

Another aspect provides a pharmaceutical kit comprising the composition.

In the present invention, the pharmaceutical kit may be in tablet, capsule or injection form, but is not limited thereto.

In addition, the kit may comprise a pharmaceutically effective dosage of the antibody; And pyrimidine analogs, platinum-based anticancer agents, or combinations thereof.

The kit can also include instructions for the administration of the components.

Hereinafter, the present invention will be described in more detail in the following Examples. However, these examples are only for aiding the understanding of the present invention, and the present invention is not limited thereto.

Experimental Example 1. Cell line and culture method used

The human intrahepatic cholangiocarcinoma (ICC) cell lines Choi-CK and SCK-L1 were cultured in DMEM (Welgene, Republic of Korea) supplemented with 10% FBS (Atlas Biologics, USA). Choi-CK was provided by Dr. Dae-Gon Kim of Chonbuk National University. TFK-1, an extrahepatic cholangiocarcinoma (ECC) cell line, was purchased from German Collection of Microorganisms and Cell Cultures, Human and Animal Cell Lines, Braunschweig, Germany, and cultured in RPMI (Thermo Fisher Scientific, USA). . CHO-DG44 cell line # 9-20 expressing Ab417 antibody (Cho S, et al., MAbs. 2016; 8 (2): 414-25.) Was 5% (v / v) dialyzed FBS (Thermo Fisher Scientific) ) Was supplemented with MEM-α (Welgene) supplemented and replaced with medium without serum (SFM4CHO, GE Lifesciences, USA) for the production of antibodies. All cell lines were cultured in a wet incubator at 37 ° C. with 5% CO ₂ .

Experimental Example 2. Production method of Ab417 antibody

Ab417 antibody (hereinafter referred to as 'Ab417') was produced in CHO-DG44 cell line (# 9-20). The culture supernatant was centrifuged and filtered using a bottle top filter (0.22 μm PES, Sartorius) and affinity chromatography was performed on Protein A-Agarose (GenScript, USA) for purification. Antibodies bound to protein A were eluted with 0.1 M sodium citrate containing 150 mM sodium chloride, pH 3.6. The eluted antibody was replaced with 25 mM sodium citrate buffer (pH 6.4) containing 150 mM sodium chloride and 0.007% Tween-20 (pH 6.4) and stored. Protein concentration was measured with a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific).

Experimental Example 3. In vivo ( In vivo Anticancer Effect Analysis

Nude mice (BALB / c Slc- nu , 5 weeks old) were purchased from SLC, Inc., Japan and stored in a specific pathogen-free environment for 7 days according to the guidelines of the Animal Control Committee of Biotoxtech (Korea).

In order to confirm the anticancer effect on Ab417, the drug (gemcitabine; gemcitabine or cisplatin), or Ab417, and a combination of the above drugs, prior approval of the committee was obtained (B13698, B13933, or B13934, respectively).

Specifically, Choi-CK cells (1 × 10 ⁶ ) were inoculated subcutaneously in the right flank of each mouse. Choi-CK tumor tissue (3 × 3 × 3 mm ³ ) was inoculated subcutaneously in the back of new nude mice. When the tumor volume reached about 100 mm ³ , mice were divided into arbitrary groups and Ab417 or drug (gemcitabine or cisplatin) was administered twice a week (n = 8 per group) intravenously (iv) or intraperitoneally (ip) Injection. Gemcitabine (Gemcitabine hydrochloride, Sigma Aldrich) and cisplatin (cis-Diammineplatinum (II) dichloride, Sigma Aldrich) were dissolved in an appropriate concentration using sodium chloride (Choongwae, Republic of Korea). Additional animals not used were euthanized using CO ₂ gas and fatal bleeding.

Tumor growth was measured in length and width with a caliper and the tumor volume was calculated using the following formula: TV (mm ³ ) = L (mm) x W ² (mm ² ) x 1/2. In this case, L means length and W means width. Animal weights were also measured. Animals were anesthetized using isoflurane and the tumor tissue was taken out and weighed. Tumor growth inhibition rate (IR) was calculated using the following formula: IR (%) = (1-T / C) x 100, where T is the mean of the tumor for the test substance group Weight, C means volume or weight for the negative control group.

In order to elucidate the mechanism of anticancer activity, nude mice (BALB / c-nude, 6 weeks old) were purchased from NARA Biotech (Republic of Korea) and followed for 7 days according to the instructions of Kangwon National University IACUC (KW-160429-1). Stored in an environment free of specific pathogens.

Choi-CK cells (1 × 10 ⁶ ) were inoculated subcutaneously in the right flank of nude mice ( n = 3). When the tumor volume reached about 110 mm ³ , mice were divided into any two groups and injected intraperitoneally with antibody or PBS three times a week. Ten days after injection, tumors were removed and immunofluorescence stained for immunohistochemistry for detection of Ki-67 and detection of L1CAM. After the experiment, the animals were euthanized using CO ₂ gas and fatal bleeding.

On the other hand, for the heterogeneous model of TFK-1, NOD / SCID mice (5 weeks old) were purchased from Animal Resources Center (ARC, Australia) and specified according to the guidelines of the Animal Ethics Committee of Asan Medical Center (Korea). Stored in a pathogen free environment. TFK-1 cells (5 × 10 ⁶ ) were implanted into the right hind limb of each mouse. Mice were divided into arbitrary groups ( n = 8 per group) and injected Ab417 or drug twice a week.

Experimental Example 4 Immunohistochemical Analysis

After the mice were anesthetized and tissues were obtained, the tissues obtained for paraffin sections were fixed with formalin. Paraffin-embedding tissue sections were de-paraffinized with xylene and 95%, 90%, 70% and 50% ethanol and washed with PBS. The de-paraffinized slides were heated in 0.1 mol / L citric acid buffer (pH 6.0) for 30 minutes and incubated for 15 minutes in 0.3% (v / v) hydrogen peroxide contained in methanol. Sections were blocked in normal horse serum at room temperature and blocked, and immunostaining was performed overnight at 4 ° C. with primary antibodies against Ki-67 (C-term) (1: 200, Acris, Germany). Target proteins were visualized using ABC and DAB kits (Vector Laboratories, USA) and cross-stained with hematoxylin.

For immunofluorescence staining, immunostaining was performed overnight at 4 ° C. with anti-NCAM-L1 (N-14) mAb (1:50, Santa Cruz Biotechnology, USA), and donkey anti-goat Incubated with Alexa 488-conjugated secondary antibody (1: 200, Thermo Fisher Scientific) for 1 hour at room temperature. Cell nuclei were labeled with DAPI (5 μΜ, Sigma Aldrich). Images were taken using a Zeiss confocal microscope and the degree of staining was quantitatively assessed using Image J software (version 1.45).

Experimental Example 5. Antibody internalization assay

Internalization of Ab417 was measured using a confocal laser scanning microscope. Medium containing Ab417 (10 μg / mL) was added to L1CAM-expressing SCK-L1 cells loaded on slide glass. After incubation at 37 ° C. for 0.5 or 4 hours, the cells were placed on ice, rinsed with 4 ° C. PBS and fixed with 4% formaldehyde (Sigma Aldrich).

As a zero-time control, Ab417 was added to formaldehyde fixed cells. Cell surface antibodies were detected with a rabbit anti-human IgG (H + L) Cross Adsorbed DyLight 594 polyclonal antibody (1: 100, v / v, Thermo Fisher Scientific). Cell surface secondary antibodies were fixed for 5 minutes using formaldehyde. After washing, cells were blocked and permeabilized for 30 minutes at room temperature with 10% goth serum and 0.1% Triton X-100 contained in PBS. To label internalized L1CAM, cells were treated with anti-human IgG (Fc-specific) -FITC (1: 500, v / v) in PBS with 5% goth serum and 0.02% Triton X-100 at room temperature. Incubate for minutes. Slides were washed three times with PBS and mounted using SlowFade® Antifade kit (Thermo Fisher Scientific). Fluorescence signals were detected with Olympus LX70 FV300 05-LGP-193 (OLYMPUS, Japan).

Experimental Example 6. Analysis of cell membrane L1CAM levels in Ab417-treated cells

Choi-CK or TFK-1 cells were incubated for a time designated as Ab417 (10 μg / mL) and membrane protein fractions were obtained with the MEM-PER ^™ Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific). The obtained protein sample was quantified via a BCA protein quantitative assay and SDS-PAGE was performed. Proteins were transferred to nitrocellulose membranes and then blocked with Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% skim milk (BD Biosciences). L1CAM was detected using mouse mAb and goth anti-mouse IgG-HRP conjugates (1: 8,000, v / v, Thermo Fisher Scientific) bound to human L1CAM. Pan-cadherin to Pan-cadherin Rabbit mAb (1: 2,500, v / v, Cell Signaling Technology, USA) and anti-rabbit IgG-HRP conjugate (1: 5,000, v / v, Cell Signaling Technology's loading control. The immunoreactive bands were then visualized using SuperSignal ^™ West Femto Maximum Sensitive Substrate (Thermo Fisher Scientific).

Experimental Example 7. Statistical Analysis

Results are expressed as mean ± standard deviation, and the statistical comparison between groups

Dunnett t-test (Dunnett's t-test) or Steel test (Steel's test) followed by one-way analysis. The value of p <0.05 was judged to be statistically significant.

Example 1 Confirmation of Anticancer Activity in ICC Xenograft Mouse Model

Example 1-1. Anticancer activity of Ab417

In our previous study, we measured the anticancer activity of Ab417 when intravenous Ab417 antibody (10 mg / kg) was injected intravenously into nude mice ( n = 8) with Choi-CK xenografts as ICC cells. As a result, as shown in A and B of Figure 1, compared with recombinant human Fc (hFc) as a homologous control, it was confirmed that the cancer growth inhibitory activity of 68.6% based on the average of the tumor weight. In addition, Ab417 did not affect the weight, it was confirmed that does not cause other side effects (Cho S, et al. MAbs. 2016; 8 (2): 414-25.).

Therefore, in the present invention, in order to confirm whether Ab417 has a dose-dependent anticancer effect, according to Experimental Example 3, Ab417 (10 mg / kg) or hFc (3.3 mg / kg) in the same model ( n = 8) Intravenously twice a week.

As a result, as can be seen from A to C of Figure 2, it was confirmed that Ab417 inhibits cancer growth without affecting the weight. After 22 days of infusion, the average tumor volume and weight of the Ab417-treated group were 278.3 mm ³ ( p <0.05) and 0.17 g ( p <0.05), respectively, and the control group was 574.5 mm ³ and 0.26 g, the control group (hFc). Compared with the Ab417 treated group, the cancer growth inhibition rate was 35.6%.

The results show that Ab417 inhibits tumor growth in a dose-dependent manner in the Choi-CK xenograft model.

In addition, to confirm whether tumor growth inhibition by Ab417 is a result of inhibition of cancer cell proliferation, according to Experimental Examples 3 and 4, immunostaining was performed with Ki-67, a marker of cell proliferation.

As a result, as shown in D of FIG. 2, Ab417 treated tumors showed a lower Ki-67 index than the control.

The results indicate that Ab417 inhibits tumor growth by inhibiting proliferation of cancer cells in vivo .

Example 1-2. Analysis of the effect of Ab417 on membrane L1CAM

Down regulation of L1CAM expression is known to inhibit proliferation and migration of ICC cells in vitro and to inhibit tumor growth in vivo (Min JK, et al., Clin Cancer Res. 2010; 16 (14): 3571-80 .). Thus, in order to confirm the mechanism of tumor cell proliferation inhibitory activity by Ab417, according to Experimental Example 5, an antibody internalization assay was performed using SCK-L1 cells.

As a result, as shown in FIG. 3A, it was confirmed that the internalization of Ab417 into the cytoplasm was detected 4 hours after the antibody was added to the cells.

Then, to confirm that membrane L1CAM levels were reduced by internalization of Ab417, according to Experiment 6, Choi-CK cells were incubated with antibodies for each designated time (1, 2, 4 or 6 hours), Membrane protein fractions of treated cells were analyzed by Western blot using murine anti-L1CAM mAb (A10-A3) that binds to the Ig1 domain of human L1CAM.

As a result, as shown in B of Figure 3, Ab417 was confirmed to down-regulate the membrane L1CAM level in a concentration-dependent manner. In addition, as shown in FIGS. 3C and 3D, through immunofluorescence staining of Choi-CK tumors performed with mAbs specific for the N-terminal region of human L1CAM, Ab417-treated tumors were treated with hFc-treated tumors. It was confirmed that the L1CAM level is significantly reduced compared to.

The results showed that Ab417 internalized to inhibit the proliferation of cancer cells by reducing membrane L1CAM levels.

Example 1-3. Inhibition of cancer cell proliferation by gemcitabine or cisplatin

Since gemcitabine or cisplatin is considered to be the standard treatment for advanced biliary tract cancer, the effect of this drug on the in vivo proliferation of Choi-CK cells was confirmed.

As a result, gemcitabine or cisplatin inhibited the proliferation of Choi-CK cells in a dose-dependent manner, and the IC ₅₀ value of gemcitabine or cisplatin was 1.5 μg / mL or 3.0 μg / mL, respectively.

In addition, flow cytometry was performed to confirm the mechanism of tumor cell proliferation inhibitory activity by the drug.

As a result, it was confirmed that when treated with gemcitabine or cisplatin with an IC ₅₀ value, the cell cycle was stopped in the G2 / M state. In addition, when treated with cisplatin than gemcitabine, it was confirmed that the cell cycle was further increased.

Through the above results, it was found that gemcitabine or cisplatin inhibits the proliferation of cells by stopping the cycle of cancer cells.

Example 1-4. Anticancer Effects of Gemcitabine or Cisplatin

For the combined administration of Ab417, and gemcitabine or cisplatin, we identified the anticancer effect of gemcitabine or cisplatin in the Choi-CK tumor xenograft model. Specifically, according to Experimental Example 3, nude mice having Choi-CK xenografts with gemcitabine (10, 25, or 50 mg / kg), cisplatin (0.5 or 1.5 mg / kg) or saline as a control Intraperitoneal injection twice a week at ( n = 8). Twenty two days after the injection, the cancerous tissue was removed and weighed.

As a result, as shown in A and B of Figure 4, 10 or 25 mg / kg gemcitabine showed a growth inhibition rate of 55.4% or 80.7%, respectively, it was confirmed that inhibits cancer growth in a dose-dependent method It was. On the other hand, 0.5 or 1.5 mg / kg of cisplatin showed a cancer growth inhibition rate of 39.8% or 36.7%, respectively, it was confirmed that the same anti-cancer effect even at different concentrations.

Example 1-5. Confirmation of anticancer effect of Ab417 and gemcitabine or cisplatin combination

When Ab417 and drug were administered in combination, it was confirmed whether the tumor growth inhibitory effect was superior to that of antibody or drug alone.

Specifically, Ab417, and the maximum dose of gemcitabine or cisplatin were injected into the Choi-CK xenograft model, according to Experiment 3, Ab417 (10 mg / kg) alone; Gemcitabine (10 mg / kg) alone; Or cisplatin (0.5 mg / kg) alone, or Ab417 (10 mg / kg) and gemcitabine (10 mg / kg); Ab417 (10 mg / kg) and cisplatin (0.5 mg / kg); Or Ab417 (10 mg / kg) and saline as a control were injected into mice with Choi-CK xenografts twice a week for 3 weeks.

As a result, as shown in Figures A and B of Figure 5, when combined with Ab417 and gemcitabine cancer growth was completely inhibited, based on the tumor weight, it was confirmed that the cancer growth inhibition rate of 88.8% compared to the control group It was. On the other hand, Ab417 or gemcitabine was confirmed to exhibit cancer growth inhibition of 35.3% or 44.4%, respectively.

In addition, as shown in C and D of Figure 5, when combined with Ab417 and cisplatin, it was confirmed that the cancer growth inhibition rate of 79.2% ( p <0.01) compared to the control based on the tumor weight. Ab417 or cisplatin, on the other hand, exhibited cancer growth inhibition of 39.7% or 51.2%, respectively.

In addition, as shown in Figures A to C of 6, it was confirmed that the combination or alone administration does not affect the weight.

From the above results, it was found that the combined administration of Ab417, and cisplatin or gemcitabine showed a significantly superior synergistic effect compared to the administration of antibody or drug alone.

Example 2. Confirmation of anticancer activity in ECC xenograft mouse model

Example 2-1. Analysis of the effect of Ab417 on membrane L1CAM

Being an ECC cell by internalization of Ab417 We tried to determine if the membrane L1CAM levels of TFK-1 cells decreased.

Specifically, according to Experiment 6, TFK-1 cells were incubated with the antibody for a designated time (1, 2, 4, 6 hours), and the membrane protein fraction of the treated cells was bound to the Ig1 domain of human L1CAM. Analysis was by Western blot using murine anti-L1CAM mAb (A10-A3).

As a result, it can be seen that Ab417 downregulates membrane L1CAM levels in a concentration dependent manner.

The results indicate that Ab417 inhibits the proliferation of cancer cells by internalizing even ECC cells to reduce membrane L1CAM levels.

Example 2-2. Anticancer activity of Ab417

In order to confirm the anticancer effect of Ab417 in the ECC model, according to Experimental Example 3 above, Ab417 (10 mg / kg), hFc (3.3 mg / kg) or NOD / SCID mice having TFK-1 xenografts with saline as a control group ( n = 8) twice a week intravenously for 4 weeks.

As a result, as can be seen in Figures A and B of Figure 8, Ab417 compared with the control group, it was confirmed that the anti-cancer activity of 65.4% based on the weight of the tumor.

The results indicate that Ab417 exhibits excellent tumor growth inhibitory activity even in ECC cells.

Example 2-3. Identify anticancer effects of gemcitabine or cisplatin

In order to confirm the anticancer effect of Ab417, and gemcitabine or cisplatin combined administration, first, the anticancer effect of gemcitabine or cisplatin alone was administered in the ECC tumor model. According to Experimental Example 3 above, gemcitabine (5 or 10 mg / kg), cisplatin (0.5 or 2 mg / kg) or saline as a control (saline) NOD / SCID mice with TFK-1 xenograft ( n = 8 ) Intraperitoneally twice a week. Twenty two days after the injection, the cancerous tissue was removed and weighed.

As a result, as can be seen in Figures A and B of 5, Gemcitabine of 5 or 10 mg / kg was confirmed to show cancer growth inhibition rate of 43.2% or 77.0%, respectively, based on tumor weight, dose-dependent method It was confirmed that inhibiting cancer growth.

On the other hand, 0.5 or 2.0 mg / kg of cisplatin showed 54.1% or 63.5% cancer growth inhibition, respectively, based on tumor weight, but only 8.3% or 20.4% inhibition of cancer growth based on tumor volume. It confirmed that it was shown.

Example 2-4. Confirmation of anticancer effect of Ab417 and gemcitabine or cisplatin combination

When Ab417 and drug were administered in combination, it was confirmed whether the tumor growth inhibitory effect was superior to that of antibody or drug alone.

Specifically, since 10 mg / kg of Ab417 showed an excellent cancer growth inhibitory effect in the TFK-1 model, a lower dose of 5 mg / kg of Ab417 was used in the present invention.

According to Experiment 3, Ab417 (5 mg / kg) alone; Gemcitabine (5 mg / kg) alone; Or cisplatin (0.5 mg / kg) alone, or Ab417 (5 mg / kg) and gemcitabine (5 mg / kg); Ab417 (5 mg / kg) and cisplatin (0.5 mg / kg); Or Ab417 (5 mg / kg) and saline as a control was injected into NOD / SCID mice ( n = 8) with TFK-1 xenografts twice a week for 3 weeks.

As a result, as shown in Figures A and B of Figure 10, in combination with Ab417 and gemcitabine cancer growth was completely inhibited, based on the tumor volume, it was confirmed that the cancer growth inhibition rate of 52.2% compared to the control group It was. On the other hand, when Ab417 or gemcitabine alone was administered, it was confirmed that the cancer growth inhibition rate of 10.7% or 21.7%, respectively.

From the above results, it was found that the combined administration of Ab417 and gemcitabine showed a synergistic effect, which was significantly better than the antibody or drug alone administration.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (11)

1. (a) an antibody that specifically binds to an L1 cell adhesion molecule (L1CAM) protein; And

   (b) A pharmaceutical composition for the prevention or treatment of biliary tract cancer, comprising a pyrimidine analog, a platinum anticancer agent or a combination thereof.
2. The pharmaceutical composition of claim 1, wherein the antibody that specifically binds to an L1 cell adhesion molecule (L1CAM) protein is an antibody comprising a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5, or a functional variant thereof. Composition.
3. The method of claim 2, wherein the functional variant is substituted with one or more amino acids in the heavy chain variable region, light chain variable region, or both of the antibody comprising a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5 The improved physical properties compared to the antibody comprising a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5.
4. The antibody of claim 1, wherein the antibody comprises a heavy chain CDR1 as set forth in SEQ ID NO: 2; Heavy chain CDR2 set forth in SEQ ID NO: 3 or 10; And a heavy chain variable region comprising a heavy chain CDR3 as set out in SEQ ID NO: 4 and a light chain CDR1 as set out in SEQ ID NO: 6 or 12; Light chain CDR2 set forth in SEQ ID NO: 7; And a light chain variable region comprising the light chain CDR3 set forth in SEQ ID NO: 8.
5. The method according to claim 4, wherein the antibody is selected from the group consisting of SEQ ID NO: 1, 9 and 13, heavy chain variable region; And

   A pharmaceutical composition comprising a light chain variable region selected from the group consisting of SEQ ID NOs: 5, 11, 14 and 15.
6. The pharmaceutical composition of claim 1, wherein the pyrimidine analog is gemcitabine.
7. The method of claim 1, wherein the platinum-based anticancer agent is cisplatin (cisplatin), oxaliplatin (oxaliplatin), tetraplatin (heptraplatin), heptalatin (paratapin), carboplatin (carboplatin) or nano Platinum (nanoplatin), pharmaceutical composition.
8. The pharmaceutical composition of claim 1, wherein the biliary tract cancer is intrahepatic biliary tract cancer or extrahepatic biliary tract cancer.
9. (a) an antibody that specifically binds to an L1 cell adhesion molecule (L1CAM) protein; And

   (b) A pharmaceutical kit for preventing or treating biliary tract cancer, comprising a pyrimidine analog, a platinum anticancer agent or a combination thereof.
10. (a) an antibody that specifically binds to an L1 cell adhesion molecule (L1CAM) protein; And

    (b) administering a pyrimidine analog, a platinum anticancer agent, or a combination thereof to a subject in need thereof.
11. The method of claim 10, wherein the antibody of (a) and the pyrimidine analog of (b), the platinum-based anticancer agent or a combination thereof are administered simultaneously, separately or sequentially. .

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