KR101671069B1 - Antibody that reconizes the beta-1 integrin and use of the antibody - Google Patents

Abstract

The present invention relates to a novel monoclonal antibody having high specificity and affinity for beta 1 integrin protein associated with tumor suppression and progression, and its use for the treatment of non-small cell lung cancer. The antibody of the present invention can be used for the treatment of non-small cell lung cancer by inducing apoptosis on non-small cell lung cancer. When the antibody and cisplatin are coadministered, the therapeutic effect on non-small cell lung cancer is maximized, It can be useful for treatment.

Description

Antibodies that specifically recognize beta 1 integrin and uses thereof < RTI ID = 0.0 >

The present invention relates to antibodies that specifically recognize beta 1 integrin and uses thereof.

Primary lung cancer, a malignant tumor originating from the lung, is divided into small cell lung cancer (NSCLC) and non-small cell lung cancer (NSCLC) according to its tissue type. Non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer and is the leading cause of cancer death in the United States, a cure rate of less than 15%, and an average survival rate of 8 to 10 months. Surgical operations are not possible after the initial stage, and only the chemotherapy should be relied on, but NSCLC itself is a very heterogeneous cancer type, so its response to chemotherapy is very low.

Therefore, doublet therapy of cytotoxic drugs is the mainstay of non-small cell lung cancer (NSCLC) treatment. Such anticancer agents include carboplatin, paclitaxel, docetaxel, etoposide, cisplatin, doxorubicin, and the treatment with paclitaxel and carboplatin is preferred . As a target therapeutic agent, Epidermal Growth Factor Receptor tyrosine kinase inhibitor (EGFR TKI), which is a drug that inhibits epidermal growth factor (EGF), is administered to Tarceva, Irresa, etc. (NSCLC) in the treatment of non-small cell lung cancer (NSCLC).

However, combination therapy with cytotoxic drugs has serious limitations and is difficult to apply in patients with low physical function status such as elderly patients. In addition, anticancer efficacy is poor and recurrence rates are high in patients with very early or already advanced non-small cell lung cancer (NSCLC). Therefore, it is necessary to reorganize the treatment of non-small cell lung cancer (NSCLC) centering on an effective anti-cancer drug which has no toxicity and excellent anti-cancer efficacy.

Korean Patent Publication No. 2014-0044362

Accordingly, an object of the present invention is to provide an antibody that specifically binds to a beta-1 integrin comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 2 .

It is an object of the present invention to provide an antibody that specifically binds to a beta-1 integrin comprising the nucleotide sequence of SEQ ID NO: 3 encoding the heavy chain variable region and the nucleotide sequence of SEQ ID NO: 4 encoding the light chain variable region DNA.

It is an object of the present invention to provide a hybridoma cell line (KCTC12693BP) which produces said antibody.

It is also an object of the present invention to provide a pharmaceutical composition for the treatment of non-small cell lung cancer comprising the antibody as an active ingredient.

It is a further object of the present invention to provide a method for treating non-small cell lung cancer, which comprises administering the antibody of claim 1 and cisplatin in combination to a cancer-induced individual other than human.

In order to accomplish the above object, the present invention provides a method for producing a beta-1 integrin comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 1 and a light chain variable region having an amino acid sequence of SEQ ID NO: 2 ≪ / RTI >

The present invention provides an antibody DNA that specifically binds to a beta-1 integrin comprising the nucleotide sequence of SEQ ID NO: 3 encoding the heavy chain variable region and the nucleotide sequence of SEQ ID NO: 4 encoding the light chain variable region to provide.

The present invention provides a hybridoma cell line (KCTC12693BP) that produces said antibody.

The present invention provides a pharmaceutical composition for the treatment of non-small cell lung cancer comprising the antibody as an active ingredient.

In one embodiment of the present invention, cisplatin may be added to the composition.

In addition, the present invention provides a method for treating non-small cell lung cancer, which comprises administering the antibody of claim 1 and cisplatin in combination to a cancer-induced individual other than human.

The present invention relates to a novel monoclonal antibody having high specificity and affinity for beta 1 integrin protein associated with tumor suppression and progression, and its use for the treatment of non-small cell lung cancer. The antibody of the present invention can be used for the treatment of non-small cell lung cancer by inducing apoptosis on non-small cell lung cancer. When the antibody and cisplatin are coadministered, the therapeutic effect on non-small cell lung cancer is maximized, It can be useful for treatment.

FIG. 1 shows the result of immunoprecipitation and immunoblotting for the antigen analysis of P5 monoclonal antibody in a leukemia cell line TF-1 cell lysate.
FIG. 2 shows the result of analysis of the protein sequence of the immunoprecipitated P5 antigen.
FIG. 3 shows the results of comparing the expression of beta 1 integrin with time after treatment with cisplatin in non-small cell lung cancer cell line A549.
FIG. 4 shows the result of measuring cell growth inhibition after P5 monoclonal antibody and cisplatin treatment.
FIG. 5 shows the results of measurement of apoptosis after P5 monoclonal antibody and cisplatin treatment.

"Beta 1 integrin" is a protein known to be involved in tumor suppression and progression. The present invention is characterized by providing a novel antibody capable of specifically recognizing the beta 1 integrin.

Accordingly, the present invention provides an antibody that specifically binds to a beta-1 integrin comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 2 .

The fragment of the antibody that specifically binds to the beta 1 integrin of the present invention is not a whole antibody but a part of the antibody and has the ability to bind to the beta 1 integrin and specifically binds to the beta 1 integrin of the present invention Lt; RTI ID = 0.0 > 1 < / RTI > integrin.

The present invention also includes functional variants of the antibody. Functional variants may optionally be monoclonal antibodies comprising amino acid sequences containing substitutions, insertions, deletions, or combinations thereof of one or more amino acids compared to the amino acid sequence of the parental monoclonal antibody. Moreover, functional variants may comprise truncated forms of the amino acid sequence at one or both of the amino terminus or the carboxy terminus. Functional variants of the present invention may have the same, different, higher or lower binding affinity compared to the parental monoclonal antibody of the invention, but still bind to the beta 1 integrin.

Functional variants within the scope of the present invention also include antibodies that specifically bind to the beta-1 integrin of the present invention at least about 50% to 99%, preferably at least about 60% to 99% , More preferably at least about 80% to 99%, more preferably about 90% to 99%, especially at least about 95% to 99%, and especially at least about 97% to 99% amino acid sequence homology. Gap or Bestfit known to those skilled in the art of computer algorithms can be used to optimally align the amino acid sequences to be compared and to define similar or identical amino acid residues. Functional variants can be obtained by altering the parent monoclonal antibody or a portion thereof by known general molecular biology methods including PCR, oligonucleotide-based mutagenesis and partial mutagenesis, or by organic synthesis methods, no.

The present invention also provides an isolated nucleic acid molecule encoding an antibody that specifically binds to said beta-1 integrin.

The nucleic acid molecule of the present invention includes all of the nucleic acid molecules translated into a polynucleotide sequence as known to those skilled in the art by the amino acid sequence of the antibody provided herein. Therefore, various polynucleotide sequences by ORF (open reading frame) can be produced, and these are all included in the nucleic acid molecule of the present invention.

Further, the present invention provides an expression vector into which the above-mentioned isolated nucleic acid molecule is inserted. As the expression vector, a MarEx vector, which is an expression vector peculiar to Celltrion, and a widely used pCDNA vector, F, R1, RP1, Col, pBR322, ToL, Ti vector; Cosmids; Lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7 and the like; Plant virus, but it is not limited thereto. Any expression vector known to the person skilled in the art as an expression vector can be used in the present invention. In selecting an expression vector, a target host It depends on the nature of the cell. May be performed by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection, or electroporation methods, but not limited thereto, by those skilled in the art An expression vector and a suitable introduction method for the host cell can be selected and used.

Preferably, the vector contains one or more selectable markers, but is not limited thereto, and a vector containing no selectable marker can be used to select the product according to whether the product is produced or not. Selection of the selection marker is selected by the desired host cell, and the present invention is not limited thereto since it uses a method already known to those skilled in the art.

In order to facilitate purification, the nucleic acid molecule of the present invention can be fused by inserting a tag sequence into an expression vector. Such tags include, but are not limited to, hexa-histidine tags, hemagglutinin tags, myc tags, or flag tags, all of which are known to those skilled in the art and that facilitate purification.

The present invention also provides a hybridoma cell line that produces an antibody that specifically binds to beta-1 integrin.

In the present invention, the cell line can include, but is not limited to, cells of mammalian, plant, insect, fungal or cellular origin. The mammalian cells may be selected from the group consisting of CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells and HEK293T cells Although it is preferable to use one as a host cell, it is not limited thereto, and cells usable as a mammalian host cell known to a person skilled in the art are all available.

The present invention also provides a pharmaceutical composition for the treatment of non-small cell lung cancer, comprising an antibody specifically binding to beta-1 integrin of the present invention as an active ingredient.

The composition of the present invention may include pharmaceutically acceptable excipients in addition to an antibody specifically binding to beta 1 integrin, and may include other therapeutic agents that are effective for treating non-small cell lung cancer.

Such therapeutic agents include, but are not limited to, agents for the treatment of non-small cell lung cancer. Such agents may include antibodies, small molecules, organic or inorganic compounds, enzymes, polynucleotide sequences, anti-viral peptides, and the like.

The compositions for the prophylaxis and treatment of the present invention may be sterile and stable under the conditions of manufacture and storage and may be in powder form for reconstitution into suitable pharmaceutically acceptable excipients at the time of delivery or prior to delivery. In the case of bactericidal powders for the preparation of bactericidal injection solutions, the preferred method of preparation is vacuum drying and lyophilization to produce additional desired components from powders of active ingredients and their presterilized-filtered solutions. The compositions of the present invention may be in a solution form and suitable pharmaceutically acceptable excipients may be added and / or mixed prior to or during delivery to provide a unit dose scanning form. Preferably, the pharmaceutically acceptable excipients used in the present invention are suitable for high drug concentrations, maintain adequate flowability, and can delay absorption if desired.

Optimal pathway selection for administration of the compositions for the prevention and treatment of the present invention depends on several factors, including the physico-chemical properties of the active molecules in the composition, the urgency of the clinical situation, and the relationship of the plasma concentration of the active molecule to the desired therapeutic effect Lt; / RTI > For example, the antibodies of the present invention may be formulated with carriers for their rapid release, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used in the present invention. The antibodies of the present invention can also be coated or co-administered with a substance or compound that inhibits the inactivation of the antibody. For example, an antibody of the invention may be administered with a suitable carrier-liposome or diluent.

The method of administering the composition for prevention and treatment of the present invention can be divided into oral and parenteral routes, and the preferred route of administration is intravenous, but not limited thereto.

The oral form can be in the form of tablets, troches, medicinal drops, aqueous or oily suspensions, powders or dispersible granules, emulsions, rigid capsules, soft gelatine capsules, syrups or elixirs, tablets, dragees, Can be formulated as a slurry agent. These formulations include, but are not limited to, pharmaceutical excipients containing inert diluents, granulating or disintegrating agents, binders, polishes, preservatives, colorants, flavoring or sweetening agents, vegetable or mineral oils, wetting agents and thickening agents.

The parenteral form may be in the form of an aqueous or non-aqueous isotonic sterile non-toxic injection or infusion solution or suspension. Solutions or suspensions may be formulated with pharmaceutically acceptable additives such as agents, oils, fatty acids, topical anesthetics, preservatives, buffers, viscosity or solubility enhancement such as 1,3-butanediol, Ringer's solution, Hanks solution, isotonic sodium chloride solution, Soluble antioxidants, oil-soluble antioxidants, and metal chelating agents.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

Hybridoma  Cell manufacturing method

Peripheral blood mononuclear cells (PBMCs) were isolated and washed three times using Ficoll-Hypaque (Pamacia, Sweden: d = 1.077). Then, 10 ⁷ cells in Balb / c mice were injected into the abdominal cavity three times at intervals of 4 weeks-2 weeks-2 weeks to induce an immune response thereto. On the third day after the last addition, the spleen was removed to prepare a cell suspension .

Mouse splenocytes were harvested and splenocytes were obtained. To the DMEM culture medium, 2 × 10 ⁷ mouse spleen cells and mouse myeloma cell line SP2 / O-Ag14 (ATCC, Virginia, USA) were injected into the DMEM culture medium using polyethyleneglycol Lt; / RTI &gt; Cells were fused in 8 wells of 96 well plates and incubated at 37 ° C in 5% CO ₂ , HAT medium (hypoxanthine, aminopterin and thymidine containing media).

When colony formation was observed, the reactivity of human peripheral blood mononuclear cells was confirmed by flow cytometry and the fifth clone among the clones showing reactivity with human peripheral blood mononuclear cells was named P5, dilution was performed to establish a monoclonal hybridoma.

< Example  2>

P5 Monoclonal  Antigen analysis of antibodies

<2-1> P5 Monoclonal  Immunoprecipitation for antigen analysis of antibodies Immunoblotting  Analysis experiment

The present inventors conducted immunoprecipitation and immunoblotting experiments for antigen analysis of P5 monoclonal antibody in the leukemia cell line TF-1 cell lysate as follows.

First, A leukemia cell lines were prepared for TF-1 cells (1 × 10 ^8), twice washed the cells with PBS, a biotin solution _{(20mM NaHCO 3 100μL, 150mM NaCl} , biotin 100μg) into a 1mL from 4 ℃ 30 Min using a rotor. Then, 10 μL of FBS was added and the mixture was further rotated at 4 ° C. for 10 minutes. After washing with PBS twice, 1% NP-40 was added, and the supernatant was centrifuged at 4 ° C overnight using a rotor.

Protein G beads were prepared by washing twice with PBS. After 5 μg of P5 monoclonal antibody and 20 μL of beads were added and the mixture was rotated at 4 ° C. overnight using a rotor, the prepared biotin-conjugated TF-1 cells 500 μL of the lysate was added, and the mixture was rotated at 4 ° C. overnight using a rotor. The antibody-antigen conjugated beads were incubated with NaCl buffer 1, 2, 3 (NaCl buffer 1; 1% NP40, 1 M NaCl, NaCl buffer 2; 0.1% NP40, 0.5 M NaCl, NaCl buffer 3; 0.1% And washed in order.

40 μl of PBS and 10 μl of reducing buffer (5 ×) were added to the bead conjugated with the antibody-antigen in a micro test tube and boiled for 10 minutes. The prepared sample was loaded onto a 10% polyacrylamide gel with a protein marker (Mid-range Pre-stained Marker) and electrophoresed at 10 mA for 3 hours. After electrophoresis, the polyacrylamide gel was placed on a nitrocellulose membrane (WHATMAN) and transfected at 17V for 40 min. The transfected membranes were blocked with 5% skim milk for 1 hour and reacted with 1 μg / mL of HRP-conjugated secondary antibody (streptavidin-HRP) for 30 minutes. After washing with 0.05% PBST and visualization using an ECL kit (Amersham GE Healthcare), a band of about 140 kDa was identified as shown in Figure 1A.

In addition, a commercial anti-beta 1 integrin monoclonal antibody was purchased (Santa Cruz TS2 / 16) and protein G beads were prepared by washing twice with PBS. 5 μg of commercial anti-beta 1 integrin antibody and 20 μl of beads were added and the mixture was rotated at 4 ° C. overnight using a rotor. Then, 500 μl of the prepared TF-1 cell lysate was added, and the mixture was rotated at 4 ° C. overnight using a rotor . The antibody-antigen conjugated beads were incubated with NaCl buffer 1, 2, 3 (NaCl buffer 1; 1% NP40, 1 M NaCl, NaCl buffer 2; 0.1% NP40, 0.5 M NaCl, NaCl buffer 3; 0.1% And washed in order.

40 μl of PBS and 10 μl of reducing buffer (5 ×) were added to the bead conjugated with the antibody-antigen in a micro test tube and boiled for 10 minutes. The prepared sample was loaded onto a 10% polyacrylamide gel with a protein marker (El-Pis Biotech, Mid-range Pre-stained Marker) and electrophoresed at 10 mA for 3 hours. After electrophoresis, the polyacrylamide gel was placed on a nitrocellulose membrane (WHATMAN) and transfected at 17V for 40 min. The transfected membrane was blocked with 5% skim milk for 1 hour and reacted with 1E1 monoclonal antibody at 1 μg / mL for 1 hour. After washing with 0.05% PBST, HRP-conjugated secondary antibody (anti-mouse immunoglobulin (Dako)) was reacted at 1 μg / mL for 30 minutes. After washing with 0.05% PBST and visualization using an ECL kit (Amersham GE Healthcare), a band of 140 kDa was identified as shown in FIG. 1B.

<2-2> LC  Mass analysis

In addition, the present inventors washed the leukemia cell line TF-1 cells twice with PBS and then incubated with 1% NP-40 at 4 ° C for 2 hours using a rotor, and centrifuged Respectively. Protein G beads were prepared by washing twice with PBS. After 5 μg of P5 monoclonal antibody and 20 μL of beads were added, the mixture was rotated overnight at 4 ° C. using a rotor, and then 500 μL of the prepared TF-1 cell lysate was added , And rotated using a rotor at 4 ° C overnight.

The antibody-antigen conjugated beads were incubated with NaCl buffer 1, 2, 3 (NaCl buffer 1; 1% NP40, 1 M NaCl, NaCl buffer 2; 0.1% NP40, 0.5 M NaCl, NaCl buffer 3; 0.1% And washed in order. 40 μl of PBS and 10 μl of reducing buffer (5 ×) were added to the bead conjugated with the antibody-antigen in a micro test tube and boiled for 10 minutes. The prepared sample was loaded onto a 10% polyacrylamide gel with a protein marker (El-Pis Biotech, Mid-range Pre-stained Marker) and electrophoresed at 10 mA for 3 hours. After electrophoresis, the polyacrylamide gel was stained with EZ-Silver Staining Kit (BioWorld). A band of about 140 kDa, which was seen as a band for the P5 monoclonal antibody in the dyed gel, was sliced and commissioned by LC Mass Spectrometry (Proteomtech). As shown in FIG. 2, a band of about 140 kDa It was confirmed that it is integrin.

Therefore, it can be seen from the above experimental result that the antibody of the present invention is an antibody recognizing beta 1 integrin.

< Example  3>

Cisplatin  Beta 1 after processing Integrin  Confirmation of increased expression

2 × 10 ⁵ non-small cell carcinoma cell lines A549 were dispensed into a 6-well plate and cultured for 24 hours, 24 hours, and 72 hours at a concentration of 1 μg / mL in cisplatin at 37 ° C in 5% CO ₂ Lt; / RTI &gt; Cells were detached at predetermined times with 0.05% trypsin / 0.02% EDTA to perform flow cytometry. Negative controls without cisplatin treatment were prepared for each test.

The cisplatin-treated non-small cell carcinoma cell line A549 was removed and placed in a 5 mL assay tube. After washing with cold PBS, 1 μg of P5 monoclonal antibody was added and reacted at 4 ° C for 30 minutes. After completion of the reaction, the cells were washed with cold PBS to remove residual P5 monoclonal antibody and reacted with FITC-conjugated secondary antibody (anti-mouse immunoglobulin (Daino)) at 1 μg / mL for 20 minutes at 4 ° C. The cells were washed with cold PBS, fixed with 1% paraformaldehyde, and analyzed using a flow cytometer (BD).

As a result, as shown in Fig. 3, it was confirmed that beta 1 integrin expression increased with time after cisplatin treatment.

< Example  4>

P5 Monoclonal  Cytotoxicity test of antibody

Experiment I and incubated for a day with 10% bovine serum in an RPMI medium supplemented with non-small cell lung cancer cell line 96-well plate overnight at 4 × 10 ³ per well for gae by the frequency divider and, 37 ℃ 5% CO ₂ condition to be 100 μL A549. On the next day, the culture medium was removed and treated with P5 monoclonal antibody in RPMI medium at a concentration of 1 μg / mL for 10 μg / mL, either alone or in combination, and then incubated for 12 hours 24 hours and 48 hours at 37 ° C in 5% CO ₂ .

After completion of the reaction, 10 μL of EZ-Cytox (Daeil Lab), a cytotoxicity test reagent, was added and reacted at 37 ° C and 5% CO ₂ for 2 hours. Absorbance was measured at 480 nm.

As a result, as shown in Fig. 4A, the highest cytotoxic ability was observed at 48 hours after the combination therapy of cisplatin with the P5 monoclonal antibody.

The present inventors also conducted experiments in the same manner as above to examine whether the cytotoxic ability of P5 monoclonal antibody and cisplatin were different. However, cisplatin was fixed at 1 μg / mL in RPMI medium and P5 monoclonal antibody The absorbance was measured with a combination of cisplatin at 1, 5, 10, and 20 μg / mL, or P5 monoclonal antibody at 1 μg / mL, and cisplatin at 1, 5, and 10 μg / mL. As a result, as shown in FIG. 4B and FIG. 4C, it was found that the cytotoxic ability of P5 monoclonal antibody or cisplatin concentration-dependent cytotoxicity was increased 48 hours after the combination therapy of cisplatin with P5 monoclonal antibody.

< Example  5>

P5 Monoclonal  Antibody Apoptosis  analysis

The present inventors conducted experiments as follows to determine whether the P5 monoclonal antibody of the present invention can induce apoptosis in non-small cell lung cancer cell lines.

First, 2 mL of the non-small-cell lung cancer cell line A549 was dispensed per 1 × 10 ⁵ cells per well in RPMI medium containing 10% rabbit serum in a 6-well plate on the day before the experiment, and the cells were cultured overnight at 37 ° C. in 5% CO ₂ .

The next day, the culture broth was removed, treated with P5 monoclonal antibody to 10 μg / mL in RPMI medium, either alone or in combination with cisplatin at 1 μg / mL, followed by reaction at 37 ° C and 5% CO ₂ for 48 hours. Negative controls were filled with fresh RPMI. At the end of the reaction, flow cytometry was performed with FITC labeled Annexin V and PI.

As a result, it was confirmed that the P5 monoclonal antibody of the present invention had apoptotic ability similar to that of cisplatin known as a therapeutic agent for non-small cell lung cancer. When compared with the case of P5 monoclonal antibody and cisplatin alone, (See Fig. 5).

As a result, the P5 monoclonal antibody of the present invention was found to be effective for the treatment of non-small cell lung cancer, and when the P5 monoclonal antibody of the present invention and cisplatin were coadministered, the therapeutic effect on non-small cell lung cancer was maximized It is characterized by the fact that it has identified.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Korea Biotechnology Research Institute KCTC12693BP 20141015

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Antibody that reconizes the beta-1 integrin and use of the          antibody <130> PN1409-293 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 118 <212> PRT <213> P5 Heavy chain variable region <400> 1 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn Tyr              20 25 30 Trp Ile Glu Trp Ile Val Gln Arg Pro Gly His Gly Leu Glu Trp Ile          35 40 45 Gly Glu Ile Leu Pro Gly Ser Val Asn Thr Asn Tyr Asn Ala Lys Phe      50 55 60 Lys Asp Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Ser  65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                  85 90 95 Ala Leu Ala Thr Pro Tyr Tyr Ala Leu Asp Ser Trp Gly Gln Gly Thr             100 105 110 Ser Val Thr Val Ser Ser         115 <210> 2 <211> 112 <212> PRT <213> P5 Light chain variable region <400> 2 Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Ser Val Thr Pro Gly   1 5 10 15 Glu Ser Val Ser Ile Ser Cys Arg Ser Thr Glu Ser Leu Leu His Ser              20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser          35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Arg Ala Ser Gly Val Pro      50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile  65 70 75 80 Arg Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His                  85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys             100 105 110 <210> 3 <211> 356 <212> DNA <213> P5 Heavy chain variable region <400> 3 caggtgcaac tgcagcagtc tggggcagag ctgatgaagc ctggcgcctc agtgaaaatt 60 tcctgcaaag ctactggcta taccttcagt aactactgga tagagtggat cgtgcagaga 120 ccagggcacg gcctcgagtg gatcggagaa attctgcccg gttccgttaa taccaactat 180 aatgcgaagt tcaaagacaa ggcaacattt actgccgata caagttcaaa cacggcctcc 240 atgcaactga gctcccttac atctgaagat tctgccgtct attactgtgc actcgctacc 300 ccctactacg ctttggactc gtggggccag ggaaccagcg tgaccgtatc cagcgg 356 <210> 4 <211> 336 <212> DNA <213> P5 Light chain variable region <400> 4 gatattgtga tgactcaggc tgcaccctct gtttctgtca ctcctggaga gtcagtatcc 60 atctcctgca ggtctactga gagtctcctg catagtaatg gcaacactta cttgtattgg 120 ttcctgcaga ggccgggcca gtctcctcaa ctcctgatat atcggatgtc caaccgtgcc 180 tcaggagtcc cagacaggtt cagtggcagt gggtcaggaa ctgctttcac actgaaaatc 240 agaagagtgg aggctgagga tgtgggagtt tattactgta tgcaacatct agaatatcct 300 ttcacgttcg gtgctgggac caagctggag ctgaaa 336

Claims (7)

1. A monoclonal antibody that specifically binds to a beta-1 integrin, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 2. (Beta-1 integrin) comprising a nucleotide sequence of SEQ ID NO: 3 encoding the heavy chain variable region of claim 1 and a nucleotide sequence of SEQ ID NO: 4 encoding the light chain variable region of claim 1 Monoclonal antibody DNA. A hybridoma cell line (KCTC12693BP) producing the antibody of claim 1. A pharmaceutical composition for the treatment of non-small cell lung cancer comprising the antibody of claim 1 as an active ingredient. 5. The method of claim 4,
A pharmaceutical composition for the treatment of non-small cell lung cancer, which comprises adding cisplatin to the composition.

delete delete

Images