ANTI-BETA 1 INTEGRIN HUMANIZED ANTIBODY, AND PHARMACEUTICAL COMPOSITION FOR TREATING CANCER, COMPRISING SAME

Abstract

The present invention relates to a monoclonal antibody or fragment thereof that recognizes and binds specifically to beta 1 integrin as an antigen. The present invention also relates to a pharmaceutical composition for preventing or treating cancer including the monoclonal antibody or fragment thereof. The monoclonal antibody of the present invention is useful in preventing or treating cancer due to its ability to inhibit the proliferation and angiogenesis of cancer cells and effectively induce apoptosis

Claims

1. A monoclonal antibody or fragment thereof that recognizes and binds specifically to beta 1 integrin as an antigen wherein the monoclonal antibody or fragment thereof comprises replaced amino acids at the following positions according to the Kabat EU numbering system: a) A9S, 120V, T25S, S30T, K66R, S75T, N76D, and Q81E in a heavy chain variable region (VL) having the sequence set forth in SEQ ID NO: 1; and b) V11L, F36Y, and R39K in a light chain variable region (VL) having the sequence set forth in SEQ ID NO: 2.

2. The monoclonal antibody or fragment thereof according to claim 1, wherein the monoclonal antibody or fragment thereof comprises a heavy chain variable region having the sequence set forth in SEQ ID NO: 3 and a light chain variable region having the sequence set forth in SEQ ID NO: 4.

3. The monoclonal antibody or fragment thereof according to claim 1, wherein the monoclonal antibody or fragment thereof is a single-chain variable fragment (scFv).

4. A multispecific antibody or antibody-drug conjugate (ADC) comprising the monoclonal antibody or fragment thereof according to claim 1.

5. A nucleic acid molecule encoding the monoclonal antibody or fragment thereof according to claim 1.

6. A vector comprising the nucleic acid molecule according to claim 5.

7. A host cell comprising the vector according to claim 6.

8. A method for preventing or treating cancer comprising administering a pharmaceutical composition comprising the monoclonal antibody or fragment thereof according to claim 1, a nucleic acid molecule encoding the monoclonal antibody or fragment thereof, or a vector comprising the nucleic acid molecule.

9. The method according to claim 8, wherein the cancer is resistant to cytotoxic chemotherapy.

10. The method according to claim 8, wherein the cancer is lung cancer, breast cancer or colon cancer.

11. A method for quantifying beta 1 integrin in a sample, comprising treating the sample with the monoclonal antibody or fragment thereof according to claim 1.

12. A method for providing information for the diagnosis of a disease caused by overexpression of beta 1 integrin, comprising (a) separating a sample from a subject, (b) treating the sample with the monoclonal antibody or fragment thereof according to claim 1, and (c) determining whether the expression level of beta 1 integrin in the sample from the subject is higher than that of beta 1 integrin in a normal sample.

13. The method according to claim 12, wherein the disease caused by overexpression of beta 1 integrin is a cancer.

14. A kit for quantifying beta 1 integrin comprising the monoclonal antibody or fragment thereof according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG. 1 shows the amino acid sequences of a heavy chain variable region and a light chain variable region of a monoclonal antibody (GP5) according to the present invention.

[0067] FIG. 2 confirms the purity (FIG. 2A) and homogeneity (FIG. 2B) of a monoclonal antibody (GP5) according to the present invention.

[0068] FIG. 3 shows the affinity of a monoclonal antibody (GP5) according to the present invention for recombinant human beta 1 integrin (FIG. 3A), the affinity y of the monoclonal antibody (GP5) for recombinant mouse beta 1 integrin (FIG. 3B), and the specificity of the monoclonal antibody (GP5) for beta 1 integrin (FIG. 3C).

[0069] FIG. 4 confirms the expressions of beta 1 integrin on the surfaces of non-small cell lung cancer cell line A549, breast cancer cell line MDA-MB-231, and colorectal cancer cell line HCT116.

[0070] FIG. 5 shows the apoptotic activity of a monoclonal antibody (GP5) according to the present invention (FIG. 5A), the inhibitory activity of the monoclonal antibody GP5 for cell growth (FIG. 5B), and signaling pathways inhibited by the monoclonal antibody GP5 (FIG. 5C).

[0071] FIG. 6 confirms the induction of internalization of beta 1 integrin on the surface of cancer cells by a monoclonal antibody (GP5) of the present invention (FIG. 6A: 120 min; FIG. 6B: time-dependent results in cell line A549).

[0072] FIG. 7 shows an improvement in the sensitivity to gefitinib in PC9GR, a gefitinib-resistant non-small cell lung cancer cell line, to a level in parental PC9 cells when a monoclonal antibody (GP5) of the present invention was used in combination with gefitinib (FIG. 7A confirms the expressions of beta 1 integrin on the surfaces of PC9 and PC9GR; FIG. 7B confirms the degrees of apoptosis in PC9 and PC9GR induced by a combination of gefitinib and the monoclonal antibody GP5).

[0073] FIG. 8 shows the anticancer activity of an antibody (GP5) according to the present invention in a mouse model xenografted with a non-small cell lung cancer cell line (FIG. 8A: comparison of tumor volumes; FIG. 8B: comparison of tumor sizes).

[0074] FIG. 9 shows the inhibitory activity of an antibody (GP5) according to the present invention for the proliferation of tumor cells (FIG. 9A), the inhibitory activity of the antibody (GP5) for intratumoral angiogenesis (FIG. 9B), and the ability of the antibody (GP5) to induce tumor apoptosis (FIG. 9C).

MODE FOR CARRYING OUT THE INVENTION

[0075] The present invention will be more specifically explained with reference to the following examples. It will be evident to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

EXAMPLES

<Example 1> Development of Humanized Antibody with Higher Activity for Cancer Apoptosis than P5

[0076] The present inventors performed the following experiment to develop a humanized antibody with higher activity for cancer apoptosis than P5 (Kim M Y et al. J Biomed Res, 2016, 30(3): 217-24).

[0077] For the modification of P5, mutations were introduced into 4 FRs (HFR1, HFR2, HFR3, and HFR4) of the heavy chain variable region and 4 FRs (LFR1, LFR2, LFR3, and LFR4) of the light chain variable region by the following procedure:

[0078] 1) Specifically, amino acids in HFR1, HFR2, HFR3, and HFR4 were replaced with different amino acids from those in the original antibody using the sequences of IGHV7-4-1*03, IGHV4-30-4*06, IGHV1-69-2*01, and IGHJ6*01, respectively, taking into consideration the similarity or dissimilarity in physicochemical properties of amino acids. The replaced amino acids were 120V, T25S, and S30T in IGHV7-4-1*03, R40H and H43K in IGHV4-30-4*06, K66R, A67V, F691, S75T, N76D, S79Y, Q81E, T83R, and S87T in IGHV1-69-2*01, and S108T in IGHJ6*01. After the modified heavy chain variable region was again aligned with IGHV1-2*02, mutations expected to have the best performances were selected.

[0079] 2) Amino acids in LFR1, LFR2, LFR3, and LFR4 were replaced using IGKV2-18*01, IGKV2-18*01, IGKV2-28*01, and IGKJ2*01, respectively, as follows. The replaced amino acids were A8P, V11L, T14N, S18P, and V19A in IGKV2-18*01, R39K in IGKV2-18*01, nothing in IGKV2-28*01, and L1061 in IGKJ2*01. After the modified light chain variable region was again aligned with IGKV2D-29*02, mutations expected to have the best performances were selected.

[0080] 3) The positions of the selected mutations were as follows:

[0081] {circle around (1)} Heavy chain variable region: A9S, 120V, T25S, 530T, K66R, S75T, N76D, Q81E.

[0082] {circle around (2)} Light chain variable region: V11L, F36Y, R39K.

[0083] (The amino acid residues in the antibody domains are numbered according to the Kabat EU numbering system commonly used in the art (Kabat et al., “Sequences of Proteins of Immunological Interest” 5th Ed., U.S. Department of Health and Human Services, NIH

[0084] Publication No. 91-3242, 1991).

[0085] The antibody was constructed by combining the humanized heavy chain variable region with a human IgG1 heavy chain constant region (CH1, CH2, CH3) and combining the humanized light chain variable region with a human light chain constant region (Ckappa).

[0086] Finally, the humanized antibody was called “GP5” and its amino acid sequence is shown in Table 1. Information on the replaced amino acids can be found in FIG. 1.

TABLE-US-00001 TABLE 1 Amino acid sequences of P5 and GP5 heavy and light chain variable regions Heavy chain variable region P5 QVQLQQSGAELMKPGASVKISCKATGYTFSNYWIEWIVQRPGHGLE WIGEILPGSVNTNYNAKFKDKATFTADTSSNTASMQLSSLTSEDSA VYYCALATPYYALDSWGQGTSVTVSS (SEQ ID NO: 1) GP5 QVQLQQSGSELMKPGASVKVSCKASGYTFTNYWIEWIVQRPGHGLE WIGEILPGSVNTNYNAKFKDRATFTADTSTDTASMELSSLTSEDSA VYYCALATPYYALDSWGQGTSVTVSS (SEQ ID NO: 3) Light chain variable region P5 DIVMTQAAPSVSVTPGESVSISCRSTESLLHSNGNTYLYWFLQRPG QSPQLLIYRMSNRASGVPDRFSGSGSGTAFTLKIRRVEAEDVGVYY CMQHLEYPFTFGAGTKLELK (SEQ ID NO: 2) GP5 DIVMTQAAPSLSVTPGESVSISCRSTESLLHSNGNTYLYWYLQKPG QSPQLLIYRMSNRASGVPDRFSGSGSGTAFTLKIRRVEAEDVGVYY CMQHLEYPFTFGAGTKLELK (SEQ ID NO: 4)

[0087] In the above table, the positions of the replaced amino acids are highlighted in bold (A9S, 120V, T25S, S30T, K66R, S75T, N76D, Q81E, V11L, F36Y, and R39K according to the Kabat EU numbering system).

<Example 2> Conversion of GP5 Clone into Full Antibody and Expression/Purification

[0088] The DNA of the variable region of GP5 developed in Example 1 was synthesized in the form of scFv (Cosmogenetech, Korea) and converted into a full antibody (IgG) by PCR. First, fragments of the heavy and light chain variable and constant regions were obtained from a scFv-containing pUC vector (Cosmogenetech, Korea) by PCR using combinations of the VH, CH, VL, and CK primers shown in Table 2. The heavy and light chains of GP5 with the variable and constant regions of the antibody were obtained by PCR using combinations of the HC and LC primers shown in Table 2. The heavy chain was treated with EcoRl and NotI (New England Biolab, UK) and ligated into a pCMV vector (Thermo Fisher SCIENTIFIC, USA) for animal cell expression treated with the same restriction enzymes. The light chain was treated with XbaI (New England Biolab, UK) and ligated into a pCMV vector with the same restriction enzyme. The ligated plasmids were transformed to DH5α competent Escherichia coli cells (New England Biolab, UK) by the application of a thermal shock, and colonies were obtained and mass cultured to obtain plasmids.

TABLE-US-00002 TABLE 2 List of primers used for cloning of GP5 full antibody Primer Sequence SEQ ID NO: V.sub.H Forward1 CAG AAT TCA CTC TAA CCA TGG AAT GGA GCT GGG 5 TCT TTC TCT TCT TCC TGT CAG TAA CTA CAG V.sub.H Forward2 CTT CCT GTC AGT AAC TAC AGG TGT CCA CTC CCA 6 GGT GCA ACT GCA GCA GTC V.sub.H Reverse1 CCA GCG TGA CCG TAT CCA GCG CCT CCA CCA AGG 7 GCC CCA V.sub.H Reverse2 CCA GCG TGA CCG TAT CCA GCG CCT CCA CCA AGG 8 GCC CCA C.sub.H Forward1 GGG CCC TTG GTG GAG GCG CTG GAT ACG GTC ACG 9 CTG G C.sub.H Reverse1 GCA TTG TCT GAG TAG GTG TC 10 HC Forward1 CAG AAT TCA CTC TAA CCA TGG AAT GGA GCT GGG 11 TCT TTC TCT TCT TCC TGT CAG TAA CTA CAG HC Reverse1 GCA TTG TCT GAG TAG GTG TC 12 V.sub.L Forward1 AAG CTT CGG CAC GAG CAG ACC AGC ATG GGC ATC 13 AAG ATG GAG ACA CAT TCT CAG GTC TTT GTA TAC AT V.sub.L Forward2 TCT CAG GTC TTT GTA TAC ATG TTG CTG TGG TTG 14 TCT GGT GTT GAA GGA GAT ATT GTG ATG ACT CAG GC V.sub.L Reverse1 GGA CCA AGC TGG AGC TGA AAC GTA CGG T 15 V.sub.L Reverse2 GGA CCA AGC TGG AGC TGA AAC GTA CGG T 16 c.sub.k Forward1 TGG GGC CCT TGG TGG AGG CGC TGG ATA CGG TCA 17 CGC TGG c.sub.k Reverse1 CAT TTT GTC TGA CTA GGT GTC C 18 LC Forward1 AAG CTT CGG CAC GAG CAG ACC AGC ATG GGC ATC 19 AAG ATG GAG ACA CAT TCT CAG GTC TTT GTA TAC AT LC Reverse1 CAT TTT GTC TGA CTA GGT GTC C 20

[0089] Each of the plasmids of the heavy and light chains of the full antibody was transfected into HEK293F cells (Invitrogen, USA) using polyethylenimine (PEI) (Polysciences, USA) and 150 mM NaCl, followed by culture in Freestyle 293 expression medium (Invitrogen, USA) at 37° C. temperature, 8% CO.sub.2, and 55% humidity for 7 days. The expressed cell culture was centrifuged at 4,000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm filter. The filtrate was allowed to bind to 1 ml of Protein A resin (GenScript, China) at 4° C. The bound resin was washed with 10 cv (column volume) of PBS solution, eluted with 100 mM glycine-HCl (pH 2.7), and neutralized with 1 M Tris-HCl (pH 9.0). After buffer change with PBS at pH 7.2-7.4, the sizes and purities of the light and heavy chains of the purified antibody were determined by SDS-PAGE. The results are shown in FIG. 2A. The molecular weights of the light and heavy chains of the purified monoclonal antibody GP5 were found to agree with theoretical calculations. The purity of the monoclonal antibody GP5 was found to be high. In addition, the homogeneity of the purified antibody was found to be 95%, as determined by size exclusion chromatography (SEC) (GE Healthcare, USA). The results are shown in FIG. 2B.

<Example 3> Analysis of Affinity of the Monoclonal Antibody GP5 for Beta 1 Integrin

[0090] The affinity of the monoclonal antibody GP5 produced in Example 2 for beta 1 integrin was determined by direct ELISA. Since the monoclonal antibody GP5 is a humanized antibody and P5 is a mouse antibody, each antibody was labeled with HRP using a peroxidase labeling kit-NH.sub.2 (Dojindo, Japan) for direct affinity comparison. The direct ELISA was performed by the following procedure. First, each of recombinant human beta 1 integrin (Sino biological, China) and recombinant mouse beta 1 integrin (MyBioSource, USA) was diluted to 1 μg/ml in 50 μl of PBS, plated in a 96-well immune plate (Corning, USA), and stored at 4° C. overnight for its adsorption. After incubation with a buffer containing 3% bovine serum albumin (Millipore, USA) at 37° C. for 1 h, the wells were treated with the HRP-labeled antibody sequentially diluted to concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, 100, 300, and 1000 nM (50 μl/well). The well plate was incubated at 37° C. for 2 h to allow the antibody to bind to the antigen and washed 3 times with a buffer containing 0.5% Tween 20 (Amresco, USA). 50 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) (Life technologies, USA) was plated in each well and allowed to develop color for 30 min. Absorbance was measured at 450 nm using a spectrophotometer (Biotek, USA). The results are shown in FIGS. 3a and 3b.

[0091] The affinities of the inventive monoclonal antibody GP5 for recombinant human beta 1 integrin and recombinant mouse beta 1 integrin were comparable to those of P5 (FIGS. 3a and 3b).

[0092] The affinities of the monoclonal antibody GP5 for various integrins were measured by indirect ELISA to determine the specificity of the monoclonal antibody GP5 for beta 1 integrin. Each of recombinant human αVβ1 integrin (R&D Systems, USA), αVβ3 integrin (R&D Systems, USA), αVβ5 integrin (R&D Systems, USA), αVβ6 integrin (R&D Systems, USA), αVβ8 integrin (R&D Systems, USA), α5β1 integrin (R&D Systems, USA), and α2bβ3 integrin (R&D Systems, USA) was diluted to 1 μg/ml in 50 μl of PBS, plated in a 96-well immune plate (Corning, USA), and stored at 4° C. overnight for its adsorption. After incubation with a buffer containing 3% bovine serum albumin (Millipore, USA) at 37° C. for 1 h, the wells were treated with the monoclonal antibody GP5 sequentially diluted to concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, 100, 300, and 1000 nM (50 μl/well). The well plate was incubated at 37° C. for 2 h to allow the antibody to bind to the antigen and washed 3 times with a buffer containing 0.5% Tween 20 (Amresco, USA). The wells were treated with HRP-labeled anti-human Fc IgG secondary antibody diluted 1:3000 with PBS (50 μl/well). The well plate was incubated at 37° C. for 1 h and washed 3 times with a buffer containing 0.5% Tween 20 (Amresco, USA). 50 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) (Life technologies, USA) was plated in each well and allowed to develop color for 30 min. Absorbance was measured at 450 nm using a spectrophotometer (Biotek, USA). The results are shown in FIG. 3C.

[0093] The inventive monoclonal antibody GP5 was found to specifically bind to only integrins whose β chain is beta 1 regardless of their a chain (FIG. 3C).

[0094] As demonstrated above, the modified monoclonal antibody (GP5) did not show a decrease in affinity, which is common during antibody humanization, and was specific for beta 1 integrin. Therefore, the modified monoclonal antibody (GP5) is expected to be useful in treating various cancers, including non-small cell lung cancer, like the parent antibody (P5).

<Example 4> Confirmation of Expression of Beta 1 Integrin in Various Cancer Cell Lines, Including Non-Small Cell Lung Cancer Cell Line

[0095] The present inventors conducted an experiment to confirm the expression of beta 1 integrin in various cancer cell lines, including a non-small cell lung cancer cell line.

[0096] Specifically, non-small cell lung cancer cell line A549, breast cancer cell line MDA-MB-231, and colorectal cancer cell line HCT116 were suspended at a density of 5×10.sup.5 cells/sample in PBS with or without the monoclonal antibody GP5 at a concentration of 10 μg/ml and cultured at 4° C. for 1 h. The culture was centrifuged at 3,500 rpm for 5 min, washed with 200 μl of PBS, and centrifuged again at 3,000 rpm for 5 min. Cells were treated with goat anti-human IgG antibody, Alexa Fluor 488 (ThermoFisher Scientific, USA) diluted 1:200 with PBS, followed by culture at 4° C. in the dark for 30 min. The fluorescently stained cells were washed with PBS, suspended in 500 μl of PBS, and analyzed by flow cytometry using an Attune NxT flow cytometer (ThermoFisher Scientific, USA). The results are shown in FIG. 4.

[0097] The flow cytometry analysis results revealed that beta 1 integrin was overexpressed on the cell surfaces of the non-small cell lung cancer cell line A549, the breast cancer cell line MDA-MB-231, and the colorectal cancer cell line HCT116 (FIG. 4).

<Example 5> Confirmation of Apoptotic Activity and Cell Growth Inhibitory Effect of Monoclonal Antibody GP5 in Cancer Cell Lines and Analysis of Anticancer Effect Mechanism

[0098] The present inventors conducted an experiment to determine whether P5 and the inventive monoclonal antibody GP5 can induce apoptosis in various cancer cell lines, including a beta 1 integrin-expressing non-small cell lung cancer cell line.

[0099] Specifically, on the day before the experiment, each of non-small cell lung cancer cell line A549, breast cancer cell line MDA-MB-231, and colorectal cancer cell line HCT116 was plated on RPMI medium (WELGENE, Korea) supplemented with 10% bovine serum (GIBCO, USA) in a 24-well plate at a density of 5×10.sup.4 cells/well in 1 ml medium and cultured at 37° C. and 5% CO.sub.2 overnight.

[0100] On the next day, the supernatant was discarded and the RPMI medium (WELGENE, Korea) was treated with each of P5 and the monoclonal antibody GP5 until a concentration of 10 or 20 μg/ml was reached, followed by incubation at 37° C. and 5% CO.sub.2 for 48 h. Fresh RPMI medium (WELGENE, Korea) was used as a negative control. After completion of the incubation, cells were washed with PBS, detached with 0.05% Trypsin-EDTA (Gibco, USA), placed in an EP tube, and washed again with PBS. Thereafter, cells were centrifuged at 3,500 rpm for 5 min. The cell pellets were collected and analyzed with an Attune NxT flow cytometer (ThermoFisher Scientific, USA) using an FITC Annexin V apoptosis detection kit with 7-AAD (BioLegend, USA). The results are shown in FIG. 5.

[0101] The inventive monoclonal antibody GP5 was found to have higher apoptotic activity than P5 and showed a concentration-dependent apoptotic effect in the non-small cell lung cancer cell line A549 (FIG. 5A).

[0102] The present inventors also conducted an experiment to determine whether the inventive monoclonal antibody GP5 can inhibit cell growth in various cancer cell lines, including a beta 1 integrin-expressing non-small cell lung cancer cell line.

[0103] Specifically, on the day before the experiment, each of non-small cell lung cancer cell line A549, breast cancer cell line MDA-MB-231, and colorectal cancer cell line HCT116 was plated on RPMI medium (WELGENE, Korea) supplemented with 10% bovine serum (GIBCO, USA) in a 12-well plate at a density of 1×10.sup.5 cells/well in 1 ml medium and cultured at 37° C. and 5% CO.sub.2 overnight.

[0104] On the next day, the supernatant was discarded and the RPMI medium (WELGENE, Korea) was treated with the monoclonal antibody GP5 until a concentration of 10, 20 or 50 μg/ml was reached, followed by incubation at 37° C. and 5% CO.sub.2 for 48 h. Fresh RPMI medium (WELGENE, Korea) was used as a negative control. After completion of the incubation, cells were washed with PBS, treated with 200 μl of 4% paraformaldehyde (Biosesang, Korea) per well, and incubated at 4° C. for 10 min for their immobilization. The immobilized cells were washed with PBS and treated with 300 μl of 0.5% crystal violet (Sigma, USA) per well, followed by incubation in an orbital shaker for 30 min. After that, cells were washed with triple-distilled water until purple color did not appear in the washing solution. After drying, the dried plate was treated with 300 μl of 1% sodium dodecyl sulfate (Amresco, USA) per well to lyse the cells. Absorbance was measured at 570 nm using a spectrophotometer (Biotek, USA). The results are shown in FIG. 5B.

[0105] The inventive monoclonal antibody GP5 was found to have good inhibitory activity for cell growth and a concentration-dependent inhibitory effect on cell growth in the non-small cell lung cancer cell line A549, the breast cancer cell line MDA-MB-231, and the colorectal cancer cell line HCT116 (FIG. 5B).

[0106] The present inventors also conducted an experiment to investigate the anticancer mechanism of the monoclonal antibody GP5.

[0107] Beta 1 integrin is known to activate the Akt and ERK pathways involved in the survival and growth of cancer cells (Blandin A F, Renner G, Lehmann M, et al. β1 integrin as therapeutic targets to disrupt hallmarks of cancer. Front Pharmacol, 2015; 6:279.). Thus, the inhibitory activity of the monoclonal antibody GP5 on signaling pathways induced by beta 1 integrin was analyzed by immunoblotting. First, after A549 cell pellets were treated or untreated with the monoclonal antibody GP5 (20 μg/ml) for 48 h, Western blotting was performed according to the procedure described in the literature (Lee M S, Lee J C, Choi C Y et al. Production and characterization of monoclonal antibody to botulinum neurotoxin type B light chain by phage display. Hybridoma (Larchmt), 2008; 27(1): 18-24). At this time, AKT, pAKT, ERK, pERK (1:1000 dilution; Cell Signaling Technology, USA) and β-actin (1:3000 dilution; Santa Cruz Biotechnology) antibodies were used as primary antibodies, and HRP-labeled anti-rabbit IgG (1:5000 dilution; Abcam, UK) or HRP-labeled anti-mouse IgG (1:5000 dilution; Abcam, UK) was used as a secondary antibody. The blots were visualized using an enhanced chemiluminescence system (ThermoFisher Scientific, USA) according to the manufacturer's guidelines. The results are shown in FIG. 5C. As shown in FIG. 5C, the expressions of pAKT and pERK were significantly reduced in A549 cells treated with the monoclonal antibody GP5.

[0108] In conclusion, the inventive monoclonal antibody GP5 has apoptotic and cell growth inhibitory activities due to its ability to inhibit the AKT and ERK pathways involved in the survival and growth of cancer cells activated by beta 1 integrin.

[0109] The above results reveal that the inventive monoclonal antibody GP5 has a therapeutic effect on various cancers, including non-small cell lung cancer. In addition, the superior apoptotic activity of the inventive monoclonal antibody GP5 compared to P5 demonstrates efficient modification of GP5.

<Example 6> Analysis of Internalization of Beta 1 Integrin on the Surface of Cancer Cells by the Monoclonal Antibody GP5

[0110] The present inventors conducted an experiment to evaluate the effects of P5 and the inventive monoclonal antibody GP5 on the induction of internalization of beta 1 integrin on the surface of various cancer cell lines, including a non-small cell lung cancer cell line. Specifically, 5×10.sup.5 cells of each of non-small cell lung cancer cell line A549, breast cancer cell line MDA-MB-231, and colorectal cancer cell line HCT116 detached from T75 flasks (SPL, Korea) by treatment with 0.05% Trypsin-EDTA (Gibco, USA) (5×10.sup.5 cells) were placed in an EP tube, centrifuged at 3500 rpm for 5 min, and washed with PBS. Thereafter, the obtained cell pellets were treated with 100 μl of P5 or the monoclonal antibody GP5 diluted to 10 μg/ml in PBS. After incubation at 4° C. for 1 h, the non-small cell lung cancer cell line A549 continued to incubate at 37° C. for 0, 40, 60, 80, 90, 120, and 150 min and each of the breast cancer cell line MDA-MB-231 and the colorectal cancer cell line HCT116 continued to incubate at 37° C. for 120 min. After completion of the incubation, cells were washed with PBS and treated with 100 μl of FITC-labeled anti-mouse antibody (Sigma, USA) diluted 1:100 with PBS in the P5-treated EP tube or 100 μl of FITC-labeled anti-human antibody (Life technologies, USA) diluted 1:200 with PBS in the GP5-treated EP tube. Cells were incubated at 4° C. in the dark for 30 min, washed with PBS, and analyzed with an Attune NxT flow cytometer (ThermoFisher Scientific, USA). The results are shown in FIG. 6. Specifically, FIG. 6A shows the results of incubation of the non-small cell lung cancer cell line A549, the breast cancer cell line MDA-MB-231, and the colorectal cancer cell line HCT116 at 37° C. for 120 min and FIG. 6B shows the results of incubation of the non-small cell lung cancer cell line A549 with time after incubation at 37° C. Referring to FIG. 6, the proportions of surface beta 1 integrin on the A549, MDA-MB-231 and HCT116 cells treated with the monoclonal antibody GP5 were significantly reduced compared to those of surface beta 1 integrin on the A549, MDA-MB-231 and HCT116 cells treated with P5.

[0111] These results indicate that the binding of the monoclonal antibody GP5 to beta 1 integrin induces internalization of beta 1 integrin and suggest that the monoclonal antibody GP5 can bind to and be internalized in beta 1 integrin overexpressing cells as well as non-small cell lung cancer cells. This internalization effect is attributed to the modification of the inventive antibody and explains the superior anticancer activity of the monoclonal antibody GP5 compared to P5.

<Example 7> Analysis of Apoptosis in Gefitinib-Resistant Cell Lines by the Monoclonal Antibody GP5

[0112] Beta 1 integrin is known to cause resistance to cytotoxic chemotherapy in a variety of cancers (Park C C et al. Cancer Res, 2006, 66(3): 1526-35). Thus, the present inventors conducted an experiment to determine the degrees of apoptosis induction in a non-small cell lung cancer cell line resistant to gefitinib used in cytotoxic chemotherapy when the monoclonal antibody GP5 was used alone or in combination with gefitinib.

[0113] First, the expressions of beta 1 integrin in gefitinib-resistant non-small cell lung cancer cell line PC9GR and parental non-small cell lung cancer cell line PC9 were confirmed in the same manner as in Example 4.

[0114] The flow cytometry analysis revealed that the peak corresponding to the binding of the monoclonal antibody GP5 to beta 1 integrin in the gefitinib-resistant non-small cell lung cancer cell line PC9GR more shifted to the right than that in the parental non-small cell lung cancer cell line PC9, demonstrating that beta 1 integrin was more expressed in PC9GR than in the parental PC9 (FIG. 7A).

[0115] The abilities of the monoclonal antibody GP5 or a combination thereof with gefitinib to induce apoptosis in the cell lines PC9 and PC9GR were investigated. Specifically, on the day before the experiment, each of cell lines PC9 and PC9GR was plated on RPMI medium (WELGENE, Korea) supplemented with 10% bovine serum (GIBCO, USA) in a 12-well plate at a density of 1×10.sup.5 cells/well in 1 ml medium and cultured at 37° C. and 5% CO.sub.2 overnight.

[0116] On the next day, the supernatant was discarded and the RPMI medium (WELGENE, Korea) was treated with gefitinib (Sigma, USA) and/or the monoclonal antibody GP5 until a concentration of 2 (gefitinib) or 10 μg/ml (GP5) was reached, followed by incubation at 37° C. and 5% CO.sub.2 for 24 h. Fresh RPMI medium (WELGENE, Korea) was used as a negative control. After completion of the incubation, cells were washed with PBS, detached with 0.05% Trypsin-EDTA (Gibco, USA), placed in an EP tube, and washed again with PBS. Thereafter, cells were centrifuged at 3,500 rpm for 5 min. The cell pellets were collected and analyzed with an Attune NxT flow cytometer (ThermoFisher Scientific, USA) using an FITC Annexin V apoptosis detection kit with 7-AAD (BioLegend, USA). The results are shown in FIG. 7B. As shown in FIG. 7B, gefitinib induced high apoptosis (>50%) in the parental PC9 cells but lower apoptosis (˜30%) in PC9GR. The combination of the monoclonal antibody GP5 and gefitinib induced high apoptosis (˜50%) in PC9GR.

[0117] The sensitivity to gefitinib was found to be lower in the gefitinib-resistant cell line PC9GR than in the parental PC9. The lowered sensitivity to gefitinib in PC9GR was restored to the level in the cell line PC9 when the combination of gefitinib and the monoclonal antibody GP5 was used (FIG. 7B).

[0118] Based on these results, it was concluded that the inventive monoclonal antibody GP5 can suppress resistance to anticancer drugs due to its ability to block beta 1 integrin, which is a cause of resistance to anticancer drugs.

<Example 8> Analysis of Anticancer Activity of the Monoclonal Antibody GP5 in Human A549 Non-Small Cell Lung Cancer Xenograft Model

[0119] The present inventors conducted an experiment to determine whether P5 and the inventive monoclonal antibody GP5 exhibit anticancer activities in nude mice xenografted with a non-small cell lung cancer cell line.

[0120] Specifically, non-small cell lung cancer cell line A549 was inoculated subcutaneously into the flanks of female Balb/c nude mice (SLC, Japan) at 5×10.sup.6 cells/mouse. Mice were weighed twice a week and the tumor volume was calculated by using the formula: V=width×width×length/2. When the tumor volume reached ˜80 mm.sup.3 7 days after inoculation, mice were divided randomly, 6 animals per group. PBS (negative control), P5 or the monoclonal antibody GP5 at a dose of 1 mg/kg or cisplatin (Sigma, USA) at a dose of 2.5 mg/kg was administered intraperitoneally to mice twice a week for 5 weeks. For a combined treatment group, 1 mg/kg of P5 or the monoclonal antibody GP5 and 2.5 mg/kg of cisplatin (Sigma, USA) were administered to mice twice a week for 5 weeks. Thereafter, the tumor size and weight were measured twice a week for 3 weeks without antibody and cisplatin administration. The tumor volume was calculated for each drug administration. The results are shown in FIG. 8A (see the arrows (↓): time points of administration, *: P<0.05 compared to the negative control using the Student's t-test, ***: P<0.001 compared to the negative control using the Student's t-test). Mice were sacrificed and cancer tissues were excised for the subsequent example 9. FIG. 8B shows images of the excised cancer tissues.

[0121] As shown in FIGS. 8a and 8b, the inventive monoclonal antibody GP5 was found to have superior anticancer activity compared to P5 when administered alone and showed higher anticancer activity than cisplatin, which is known as a therapeutic drug for non-small cell lung cancer. In addition, combined administration of the monoclonal antibody GP5 and cisplatin produced superior anticancer activity compared to single administration of the monoclonal antibody GP5. The tumor volume did not increase even after the drug administration was stopped. That is, both single and combined administration of the monoclonal antibody GP5 led to an increase in anticancer efficacy compared to the single administration of P5 or cisplatin.

<Example 9> Histopathological Studies Based on Immunohistochemistry

[0122] Immunohistochemical staining was performed in the LOGONE Bio-Convergence Research Foundation (Korea) and histopathological analysis was performed in SG Medical Inc (Korea). At the end of the experiment (Day 60), all mice were sacrificed for tissue processing, immunohistochemical staining, and histological analysis. The experimental animals were subjected to laparotomy under deep anesthesia and blood was collected from the heart. Thereafter, tissues were excised, fixed in 4% formaldehyde solution, embedded in paraffin, and sectioned to a thickness of 4 μm at the largest tumor area. The paraffin was removed, followed by rehydration. For immunoperoxidase labeling, intracellular peroxidase was inhibited by exposure to 0.3% H.sub.2O.sub.2 for 15 min. Then, the sections were placed in an antigen retrieval solution (TE pH 9.0) (Sigma, USA) for antigen retrieval, heated in a pressure cooker (Bio SB, USA) for 30 min, and exposed to a blocking solution for 20 min to exclude non-specific immune responses.

[0123] Immunohistochemical staining of human Ki67 was performed using a primary rabbit antibody to human Ki67 (Abcam, UK) to evaluate the degree of tumor cell proliferation. The primary antibody was diluted and incubated on the tissue section treated with the blocking solution at room temperature for 1 h to form an antigen-antibody complex. The antigen-antibody complex was conjugated with an HRP-labeled secondary antibody (EnVision+ System-HRP labeled polymer anti-rabbit (Dako, USA)) and allowed to develop color using 3,3′-diaminobenzidine (DAB) as a substrate with a liquid DAB+ substrate chromogen system (Dako, USA). Hematoxylin (Sigma, USA) staining was used as a counterstaining for the DAB staining. Images were observed using an optical microscope (ix71, Olympus, Japan). The proportions of the Ki67 stained sites were calculated using ImageJ software (NIH, USA). The results are shown in FIG. 9A (*: P<0.05 compared to the negative control using the Student's t-test, ***: P<0.001 compared to the negative control using the Student's t-test).

[0124] Immunohistochemical staining of mouse CD31 was performed using a primary rabbit antibody to CD31 (Abcam, UK) to evaluate changes in tumor blood vessels. The immunohistochemical staining was performed in the same manner as described above for Ki67 staining. Images were observed using an optical microscope (ix71, Olympus, Japan). The proportions of the CD31 stained sites were calculated using ImageJ software (NIH, USA). The results are shown in FIG. 9B (*: P<0.05 compared to the negative control using the Student's t-test, ***: P<0.001 compared to the negative control using the Student's t-test).

[0125] Terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL) staining was performed using an ApopTag peroxidase in situ apoptosis detection kit (Chemicon, USA) to evaluate the degree of tumor apoptosis. Color development was performed with a liquid DAB+ substrate chromogen system (Dako, USA). Hematoxylin staining was performed as a counterstaining for the DAB staining. Images were observed using an optical microscope (ix71, Olympus, Japan). The proportions of the apoptotic sites were calculated using ImageJ software (NIH, USA). The results are shown in FIG. 9C (*: P<0.05 compared to the negative control using the Student's t-test).

[0126] As a result of the immunohistochemical staining, the expressions of Ki67 and CD31 were the highest in the negative control (FIGS. 9a and 9b) and almost no TUNEL-stained cells were observed (FIG. 9C), indicating active proliferation and angiogenesis of cancer cells in the negative control. The expressions of Ki67 and CD31 were lower in the group administered with the monoclonal antibody GP5 alone than in the group administered with the monoclonal antibody P5 (FIGS. 9a and 9b). A larger number of TUNEL-stained cells were observed in the group administered with the monoclonal antibody GP5 alone than in the group administered with the monoclonal antibody P5 (FIG. 9C). These results indicate that the monoclonal antibody GP5 is effective in inhibiting cancer cell proliferation and angiogenesis and inducing apoptosis compared to P5. This effect was more pronounced when the monoclonal antibody GP5 and cisplatin were co-administered than when administered alone (FIGS. 9a, 9b, and 9c). These results indicate that the monoclonal antibody GP5 possesses anticancer activity due to its ability to inhibit cancer cell proliferation and angiogenesis and induce apoptosis and maximizes this activity when combined with cisplatin.

[0127] Although the particulars of the present invention have been described in detail, it will be obvious to those skilled in the art that such particulars are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.