METHOD FOR POSITIONING, IN CYTOPLASM, ANTIBODY HAVING COMPLETE IMMUNOGLOBULIN FORM BY PENETRATING ANTIBODY THROUGH CELL MEMBRANE, AND USE FOR SAME

Abstract

The present invention relates to a method of localizing an intact immunoglobulin-format antibody in cytosol by permeating membrane of cells. The present invention also relates to a light-chain variable region (VL) that induces an intact immunoglobulin-format antibody to penetrate the membrane of living cells and be localized in the cytosol, and to an antibody comprising the same. The present invention also relates to a biologically active molecule fused to the antibody and selected from the group consisting of peptides, proteins, small-molecule drugs, nanoparticles and liposomes. The present invention also relates to a composition for prevention, treatment or diagnosis of cancer, comprising: the antibody; or a biological active molecule fused to the antibody and selected from the group consisting of peptides, proteins, small-molecule drugs, nanoparticles and liposomes. The present invention also relates to a polynucleotide that encodes the light-chain variable region and the antibody. The present invention also relates to a method for producing an antibody which penetrates cells and is localized in the cytosol.

According to the method of the present invention, which allows an intact immunoglobulin-format antibody to actively penetrate living cells and be localized in the cytosol, the antibody can penetrate living cells and be localized in the cytosol, without having to use a special external protein delivery system. Moreover, the use of the cytosol-penetrating light-chain variable region according to the present invention and the intact immunoglobulin-format antibody comprising the same can penetrate cells and remain in the cytosol, without affecting the high specificity and affinity of a human antibody heavy-chain variable region (VH) for antigens, and thus can be localized in the cytosol which is currently classified as a target in disease treatment based on small-molecule drugs, and at the same time, can exhibit high effects on the treatment and diagnosis of tumor and disease-related factors that show structurally complex interactions through a wide and flat surface between protein and protein. In addition, these can selectively inhibit KRas mutants, which are major drug resistance-associated factors in the use of various conventional tumor therapeutic agents, and at the same time, can be used in combination with conventional therapeutic agents to thereby exhibit effective anticancer activity.

Claims

1.-44. (canceled)

45. A method of localizing an intact immunoglobulin-type antibody in the cytosol of a cell comprising: contacting the cell with an intact immunoglobulin-type antibody whereby said antibody penetrates the membrane of the cell and localizes in the cell's cytosol, wherein the antibody comprises a light-chain variable region (VL) that penetrates the cell membrane comprising: a CDR1 comprising an amino acid sequence having at least 90% homology with an amino acid sequence as set forth in SEQ ID No: 4; and a CDR3 comprising an amino acid sequence having at least 90% homology with an amino acid sequence selected from the group consisting of SEQ ID Nos: 6 and 12.

46. The method of claim 45, wherein the light-chain variable region (VL) penetrates the cell membrane by undergoing endocytosis and subsequently escaping an endosome.

47. The method of claim 45 wherein the 2nd and 4th amino acids starting from the N-terminus of the light-chain variable region are respectively substituted with leucine (L) and methionine (M).

48. The method of claim 45, wherein the 9th, 10th, 13th, 15th, 17th, 19th, 21st, 22nd, 42nd, 45th, 58th, 60th, 79th and 85th amino acids starting from the N-terminus of the light-chain variable region (VL) are respectively substituted with serine (S), serine (S), alanine (A), valine (V), aspartic acid (D), valine (V), isoleucine (I), threonine (T), lysine (K), lysine (K), valine (V), serine (S), glutamine (Q) and threonine (T), (wherein the positions of the amino acids are numbered according to the Kabat numbering system).

49. The method of claim 45, wherein the 89th and 91st amino acids starting from the N-terminus of the light-chain variable region (VL) are respectively substituted with glutamine (Q) and tyrosine (Y) (wherein the positions of the amino acids are numbered according to the Kabat numbering system).

50. The method of claim 45, wherein the light-chain variable region (VL) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 3.

51. The method of claim 45, wherein the antibody binds specifically to a GTP-bound activated RAS (“RAS-GTP”) in the cytosol of the cell via a heavy chain variable region (VH) comprising: a CDR1 comprising an amino acid sequence having at least 90% homology with an amino acid sequence as set forth in SEQ ID No: 14; a CDR2 comprising an amino acid sequence having at least 90% homology with an amino acid sequence as set forth in SEQ ID No: 15; and a CDR3 comprising an amino acid sequence having at least 90% homology with an amino acid sequence as set forth in SEQ ID No: 16.

52. The method of claim 51, wherein the heavy chain variable region (VH) comprises an amino acid sequence as set forth in SEQ ID No: 13.

53. A light-chain variable region (VL) that induces an intact immunoglobulin-type antibody to penetrate the membrane of the cell and localize in the cell's cytosol comprising: a CDR1 comprising an amino acid sequence having at least 90% homology with an amino acid sequence as set forth in SEQ ID No: 4; and a CDR3 comprising an amino acid sequence having at least 90% homology with an amino acid sequence selected from the group consisting of SEQ ID Nos: 6 and 12.

54. The light-chain variable region (VL) of claim 53, wherein the light-chain variable region penetrates the cell membrane by undergoing endocytosis and subsequently escaping an endosome.

55. The light-chain variable region (VL) of claim 53, wherein the 2nd and 4th amino acids starting from the N-terminus of the light-chain variable region are respectively substituted with leucine (L) and methionine (M).

56. The light-chain variable region (VL) of claim 53, wherein the 9th, 10th, 13th, 15th, 17th, 19th, 21st, 22nd, 42nd, 45th, 58th, 60th, 79th and 85th amino acids starting from the N-terminus of the light-chain variable region (VL) are respectively substituted with serine (S), serine (S), alanine (A), valine (V), aspartic acid (D), valine (V), isoleucine (I), threonine (T), lysine (K), lysine (K), valine (V), serine (S), glutamine (Q) and threonine (T), (wherein the positions of the amino acids are numbered according to the Kabat numbering system).

57. The light-chain variable region (VL) of claim 53, wherein the 89th and 91st amino acids starting from the N-terminus of the light-chain variable region (VL) are respectively substituted with glutamine (Q) and tyrosine (Y) (wherein the positions of the amino acids are numbered according to the Kabat numbering system).

58. The light-chain variable region (VL) of claim 53, wherein the light-chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 3.

59. An antibody comprising the light-chain variable region (VL) according to claim 53.

60. The antibody of claim 59, wherein the antibody binds specifically to a GTP-bound activated RAS (RAS-GTP) in the cytosol of a cell, wherein the antibody comprises a heavy chain variable region (VH) that binds specifically to RAS-GTP comprising: a CDR1 comprising an amino acid sequence having at least 90% homology with an amino acid sequence as set forth in SEQ ID No: 14; a CDR2 comprising an amino acid sequence having at least 90% homology with an amino acid sequence as set forth in SEQ ID No: 15; and a CDR3 comprising an amino acid sequence having at least 90% homology with an amino acid sequence as set forth in SEQ ID No: 16.

61. The antibody of claim 60, wherein the heavy chain variable region (VH) comprises an amino acid sequence as set forth in SEQ ID No: 13.

62. A method of preventing or treating a disease in a subject comprising: administering a therapeutically active amount of an intact immunoglobulin-type antibody to the subject whereby said antibody penetrates the membrane of the cell and localizes in the cell's cytosol, wherein the antibody comprises a light-chain variable region (VL) that penetrates the cell membrane comprising: a CDR1 comprising an amino acid sequence having at least 90% homology with an amino acid sequence as set forth in SEQ ID No: 4; and a CDR3 comprising an amino acid sequence having at least 90% homology with an amino acid sequence selected from the group consisting of SEQ ID Nos: 6 and 12, wherein the antibody binds specifically to an antigen in the cytosol which is a target in disease treatment.

63. The method of claim 62, wherein the antibody is fused to a biologically active molecule selected from the group consisting of peptides, proteins, small-molecule drugs, nanoparticles and liposomes.

64. The method of claim 62, wherein the disease comprises a cancer.

65. The method of claim 62, wherein the disease comprises an angiogenesis-related disease.

66. A method of diagnosing a disease comprising: contacting a cell with an intact immunoglobulin-type antibody whereby said antibody penetrates the membrane of the cell and localizes in the cell's cytosol, wherein the antibody comprises a light-chain variable region (VL) that penetrates the cell membrane comprising: a CDR1 comprising an amino acid sequence having at least 90% homology with an amino acid sequence as set forth in SEQ ID No: 4; and a CDR3 comprising an amino acid sequence having at least 90% homology with an amino acid sequence selected from the group consisting of SEQ ID Nos: 6 and 12, wherein the antibody binds specifically to an antigen in the cytosol which is characteristic of the disease and the antibody is bound to a substance for imaging, and detecting a signal from the substance for imaging, whereby the presence or relative strength of the detected signal indicates the presence or characteristics of a disease.

67. The method of claim 65, wherein diagnosing a disease comprises demonstrating the onset or progress of a cancer.

68. The method of claim 65, wherein the substance for imaging is a fluorescent substance.

69. A polynucleotide that encodes the light chain variable region of claim 53.

70. A polynucleotide that encodes the antibody of claim 59.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0136] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0137] FIG. 1 is a schematic view showing the concept of an intact immunoglobulin-format antibody, named “cytotransmab”, which penetrates a cell and localizes in the cytosol.

[0138] FIG. 2A shows the results of analysis of a sequence including a clone used in a process of obtaining the improved, cytosol-penetrating humanized light-chain variable single domain hT3 VL, which binds stably to a humanized antibody heavy-chain variable region, from the mouse light-chain variable region m3D8 VL.

[0139] FIG. 2B compares model structures using the WAM modeling of m3D8 VL, the humanized light-chain variable single domain hT0 VL and its mutants (hT2 VL and hT3 VL) by a superimposing method.

[0140] FIG. 3A shows the results of confocal microscopy observation of the cytosol-penetrating ability of light-chain variable single domains.

[0141] FIG. 3B shows the results of confocal microscopy observation performed to verify the cytosol-penetrating mechanisms of light-chain variable single domains.

[0142] FIG. 4A shows the results of analyzing the amino acid sequence of hT3 VL together with the amino acid sequences of light-chain variable regions (VLs) of conventional human antibody Adalimumab (Humira) and humanized antibody Bevacizumab (Avastin) in order to confirm whether or not hT3 VL can be applied to a variety of human antibody heavy-chain variable regions.

[0143] FIG. 4B shows the results of analyzing interface residues between variable regions in order to construct stable cytotransmab that optimally interacts with a human antibody heavy-chain variable region.

[0144] FIG. 5 is a schematic view showing a method of substituting a light-chain variable region having no cell-penetrating ability with a humanized light-chain variable region having cytosol-penetrating ability in order to construct cytotransmab.

[0145] FIG. 6A shows the results of analyzing cytotransmab by reductive or non-reductive SDS-PAGE after purification.

[0146] FIG. 6B shows the results of an experiment performed using a size exclusion chromatography column (Superdex™200 10/300 GC) (GE Healthcare) by HPLC (high performance liquid chromatography) (The Agilent 1200 Series LC systems and Modules) (Agilent) in order to confirm that cytotransmab is present as a monomer in a natural environment.

[0147] FIG. 6C shows the results of ELISA (enzyme linked immunosorbent assay) performed to measure the affinities of the heavy-chain variable regions of cytotransmab (TMab4, HuT4 or AvaT4) and IgG antibodies (Bevacizumab (Avastin) and Adalimumab (Humira)) for target molecules.

[0148] FIG. 6D shows the results of an agarose gel nucleic acid hydrolysis experiment performed to examine the hydrolysis of nucleic acids in cytotransmab obtained by substitution with a cell-penetrating human light-chain variable region (hT4) grafted with the CDR of an autoimmune mouse antibody.

[0149] FIG. 7A shows the results of observing 1-2 cells in various cell lines by confocal microscopy in order to verify the cytosol-penetrating ability of cytotransmabs having a light-chain variable region substituted with the cytosol-penetrating light-chain region hT4 VL.

[0150] FIG. 7B shows the results of examining cytosol-penetrating ability for several cells, performed at a reduced magnification in order to examine cell-penetrating efficiency in the cytosol-penetrating ability examination experiment by confocal microscopy observation as shown in FIG. 7A.

[0151] FIG. 8A shows the results of observing the degree of cell penetration of TMab4 as a function of the concentration of TMab4 by confocal microscopy.

[0152] FIG. 8B shows the results of observing the degree of cell penetration of TMab4 as a function of time after TMab4 treatment by confocal microscopy.

[0153] FIG. 9A is a graph showing the results obtained by treating HeLa and PANC-1 cell lines with cytotransmab and evaluating the inhibition of growth of the cells in vitro.

[0154] FIG. 9B is an image showing the results obtained by treating HeLa and PANC-1 cell lines with cytotransmab and evaluating the inhibition of growth of the cells in vitro.

[0155] FIG. 10 shows the results of observing the transport and stability of intracellularly introduced TMab4 by pulse-chase and confocal microscopy.

[0156] FIG. 11A shows the results of confocal microscopy observation performed using calcein to indirectly confirm the cytosolic localization of cytotransmab TMab4 or HuT4.

[0157] FIG. 11B is a bar graph showing the results of quantifying the calcein fluorescence of the confocal microscope images shown in FIG. 11A.

[0158] FIG. 12A is a schematic view showing a process in which GFP fluorescence by complementary association of split-GFP is observed when cytotransmab localizes in the cytosol.

[0159] FIG. 12B shows the results of Western blot analysis performed to examine the expression level of streptavidin-GFP1-10 in a constructed stable cell line.

[0160] FIG. 12C shows the results of confocal microscopy observation of the GFP fluorescence of GFP11-SBP2-fused cytotransmab by complementary association of split GFP.

[0161] FIG. 13 is a schematic view showing a process of constructing anti-Ras•GTP iMab by replacing the heavy-chain variable region (VH) of an intact IgG-format Cytotransmab having only cytosol-penetrating ability with a heavy-chain variable region (VH) that binds specifically to GTP-bound KRas.

[0162] FIG. 14 shows the application of an IgG-format cytotransmab having only cytosol-penetrating ability, and is a schematic view showing a strategy of inducing cytotoxicity specific for Ras mutant cells by use of a monoclonal antibody (anti-Ras. GTP iMab: internalizing & interfering monoclonal antibody) which has a heavy-chain variable region (VH) replaced with a heavy-chain variable region (VH) binding specifically to GTP-bound KRas and which penetrates cells and binds specifically to GTP-bound Ras in the cells.

[0163] FIG. 15 is a schematic view showing a library screening strategy for obtaining a humanized antibody heavy-chain variable single domain having a high affinity only for GTP-bound KRas G12D protein.

[0164] FIG. 16 shows the results of FACS analysis of binding under a condition of GTP-bound KRas G12D alone and a condition competitive with GTP-bound KRas G12D in each step of the above-described process for obtaining a high affinity for GTP-bound KRas G12D.

[0165] FIG. 17 shows the results of analyzing anti-Ras•GTP iMab RT4 by 12% SDS-PAGE under reductive or non-reductive conditions after purification.

[0166] FIG. 18 shows the results of ELISA performed to measure affinity for GTP-bound and GDP-bound wild-type KRas and GTP-bound and GDP-bound KRas mutants (KRas G12D, KRas G12V, and KRas G13D).

[0167] FIG. 19 shows the results of analyzing the affinity of anti-Ras•GTP iMab RT4 for GTP-bound KRAS G12D by use of SPR (BIACORE 2000) (GE healthcare).

[0168] FIG. 20 shows the results of confocal microscopy observation performed to examine the cytosol-penetrating ability of anti-Ras•GTP iMab RT4.

[0169] FIG. 21 shows the results obtained by treating NIH3T3, NIH3T3 KRas G12V and NIH3T3 HRas G12V cell lines with anti-Ras•GTP iMab RT4 and evaluating the inhibition of growth of the cells in vitro.

[0170] FIG. 22 shows the results of evaluating the inhibition of growth of non-adherent cells in an NIH3T3 HRas G12V cell line.

[0171] FIG. 23 shows the results of confocal microscopy observation of whether anti-Ras•GTP iMab RT4 is superimposed with activated HRas G12V mutants in cells.

[0172] FIG. 24 shows the results of confocal microscopy observation of whether anti-Ras•GTP iMab RT4 is superimposed with GTP-bound KRas G12V mutants in cells.

[0173] FIG. 25 shows the results obtained by treating HCT116 and PANC-1 cell lines with RGD-TMab4 and RGD-RT4 and evaluating the inhibition of growth of the cells in vitro.

[0174] FIG. 26A shows the results of analyzing the tumor growth inhibitory effect of RGD-fused anti-Ras•GTP iMab RT4 in mice xenografted with HCT116 cells.

[0175] FIG. 26B is a graph showing the results of measuring the body weight of mice in order to examine the non-specific side effects of RGD-fused anti-Ras•GTP iMab RT4.

BEST MODE FOR CARRYING OUT THE INVENTION

[0176] Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention.

Example 1: Rationale for Development of Cytosol-Penetrating Humanized Light-Chain Variable (VL) Single Domain

[0177] FIG. 1 is a schematic view showing the concept of an intact immunoglobulin antibody, named “cytotransmab”, which penetrates a cell and localizes in the cytosol. To realize this antibody and understand the cytosol-penetrating ability of humanized antibody light-chain variable regions, reference was made to conventional studies on the correlations between the cytosol-penetrating ability of the mouse light-chain variable single domain m3D8 VL and CDRs corresponding to light-chain variable region fragments (Lee et al., 2013).

[0178] FIG. 2A shows the results of analysis of a sequence including a clone used in a process of obtaining the improved, cytosol-penetrating humanized light-chain variable single domain hT3 VL, which binds stably to a humanized antibody heavy-chain variable region, from the mouse light-chain variable region m3D8 VL.

[0179] Specifically, based on a comparison of cytosol-penetrating ability between the mouse light-chain variable single domain m3D8 VL and hT0 VL obtained by humanizing the single domain m3D8 VL by use of CDR-grafting technology, it was confirmed that the cytosol-penetrating ability was lost even though the CDR1 sequence of the light-variable variable region (VL) was conserved.

[0180] Thus, in order to improve the structure of CDR1 to have a structure similar to that of m3D8 VL to thereby restore the cytosol-penetrating ability of the humanized antibody light-chain variable single domain, CDR regions (Vernier zones) in the FR (framework) were comparatively analyzed. As a result, it was found that residues 2 and 4 differ from those of mouse m3D8 VL having cytosol-penetrating ability. Particularly, because residues 2 and 4 act as an upper core that greatly influence the CDR1 structure (Vernizer zone), hT2 VL having a CDR1 structure similar to that of m3D8 VL was developed by reverse mutations of hT0 VL (see FIG. 2A).

[0181] Next, in order to construct stable cytotransmab and to create a pair between VH3 and Vκ1 subgroups (that are highly prevalent in stable antibodies) to thereby develop a light-chain variable region that complementarily stably binds to a variety of human antibody heavy-chain variable regions and retains its ability to penetrate into the cytosol, the FR (framework) of hT2 VL and the light-variable region FR (framework) of the humanized therapeutic monoclonal antibody Trastuzumab (Herceptin), which has VH3 and Vκ1 subgroups and is very stable, were comparatively analyzed. As a result, it was shown that 14 residues in the FR (framework) of hT2 VL differ from those in the light chain-variable region FR (framework) of Trastuzumab. These 14 residues were mutated with the sequence of the light chain-variable region FR (framework) of Trastuzumab, thereby developing hT3 VL (see FIG. 2A).

[0182] FIG. 2B compares model structures using the WAM modeling of m3D8 VL, the humanized light-chain variable single domain hT0 VL and its mutants (hT2 VL and hT3 VL) by a superimposing method. It was found that, through reverse mutations at residues 2 and 4 as described above, the structural difference of the CDR1 region from that of m3D8 VL was reduced.

Example 2: Expression and Purification of Humanized Light-Chain Variable (VL) Single Domain Having Cytosol-Penetrating Ability

[0183] To compare the actual cytosol-penetrating abilities of hT2 VL and hT3 VL designed in the above Example, humanized light-chain variable (VL) single domains were purified.

[0184] Specifically, the cytosol-penetrating light-chain variable single domain containing a Pho A signal peptide at the N-terminus and a protein A tag at the C-terminus was cloned into a pIg20 vector by NheI/BamHI restriction enzymes, and then the vector was transformed into E. coli BL21(DE3)plysE for protein expression by electroporation. The E. coli was cultured in LBA medium containing 100 ug/ml of ampicillin at 180 rpm and 37° C. until the absorbance at 600 nm reached 0.6-0.8. Then, the culture was treated with 0.5 mM of IPTG (isopropyl β-D-1-thiogalactopyronoside, and then incubated at 23° for 20 hours to express the protein. After expression, the culture was centrifuged by a high-speed centrifuge at 8,000 rpm for 30 minutes, and the supernatant was collected, and then reacted with IgG-Sepharose resin (GE Healthcare). The resin was washed with 50 ml of TBS (Tris-HCl, 137 mM NaCl, 2.7 mM KCl, pH 7.4), and then washed with 5 ml of 5 mM NH.sub.4Ac (pH 5.0) buffer. Next, the protein was eluted from the resin by use of 0.1 M HAc (pH 3.0) buffer, and the buffer was replaced with TBS (pH 7.4) by dialysis. Then, the concentration of the protein was measured by a BCA (bicinchoninic acid (Pierce)) assay, and the purity of the protein was analyzed by SDS-PAGE.

Example 3: Verification of Cytosol-Penetrating Ability and Cell Penetration Mechanism of Cytosol-Penetrating Humanized Light-Chain Variable (VL) Single Domain

[0185] FIG. 3A shows the results of confocal microscopy observation of the cytosol-penetrating ability of light-chain variable single domains.

[0186] Specifically, in order to verify the cytosol-penetrating abilities of m3D8 VL, hT0 VL, hT2 VL and hT3 VL, a cover slip was added to 24-well plates, and 5×10.sup.4 HeLa cells per well were added to 0.5 ml of 10% FBS (Fetal bovine Serum)-containing medium and cultured for 12 hours under the conditions of 5% CO.sub.2 and 37° C. When the cells were stabilized, each well was treated with 10 μM of m3D8 VL, hT0 VL, hT2 VL or hT3 VL in 0.5 ml of fresh medium, and incubated for 6 hours under the conditions of 37° C. and 5% CO.sub.2. Next, the medium was removed, and each well was washed with PBS, and then treated with a weakly acidic solution (200 mM glycine, 150 mM NaCl, pH 2.5) to remove proteins from the cell surface. Next, each well was washed with PBS, and the cells were fixed in 4% paraformaldehyde at 25° C. for 10 minutes. After washing with PBS, each well was incubated with PBS buffer containing 0.1% saponin, 0.1% sodium azide and 1% BSA at 25° C. for 10 minutes to form pores in the cell membranes. After washing with PBS, each well was incubated with PBS buffer c containing 2% BSA at 25° C. for 1 hour to eliminate nonspecific binding. Then, each well was treated with rabbit-IgG (Sigma) that recognizes the protein A tag of the light-chain variable single domain, and each well was incubated at 25° C. for 2 hours, washed three times with PBS, and then treated with red fluorescence (TRITC)-labeled anti-rabbit antibody (Sigma), followed by incubation at 25° C. for 1 hour. Finally, the nucleus was blue-stained with Hoechst33342 and observed with a confocal microscope. As a result, it was shown that m3D8 VL, hT2 VL and hT3 VL, except for hT0 VL, had cell-penetrating ability.

[0187] FIG. 3B shows the results of confocal microscopy observation performed to verify the cytosol-penetrating mechanisms of light-chain variable single domains.

[0188] Specifically, when HeLa cells were prepared as shown in FIG. 3A and stabilized, a dilution of 10 μM of m3D8 VL, hT2 VL or hT3 VL and 10 ug/ml of Alexa Fluor 488-transferrin (TF, green fluorescence), FITC-cholera toxin B (Ctx-B, green fluorescence) or Oregon green-dextran (Dextran, green fluorescence) in 0.5 ml of fresh medium was added to each well and incubated for 2 hours under the conditions of 37° C. and 5% CO.sub.2. Next, the light-chain variable single domains were stained as shown in FIG. 3A. As shown in FIG. 3B, all the light-chain variable single domains were superimposed with cholera toxin-B, indicating that these domains penetrate the cytosol by caveolae.

Example 4: Development of Cytosol-Penetrating Humanized Light-Chain Variable (VL) Single Domain that Easily Interacts with Human Antibody Heavy-Chain Variable Domain

[0189] FIG. 4A shows the results of analyzing the amino acid sequence of hT3 VL together with the amino acid sequences of light-chain variable domains (VLs) of conventional human antibody Adalimumab (Humira) and humanized antibody Bevacizumab (Avastin) in order to confirm whether or not hT3 VL can be applied to a variety of human antibody heavy-chain variable domains.

[0190] Specifically, VH-VL interface residues that are involved in the interaction between heavy-chain and light-chain variable domains were analyzed. As a result, it was found that lysine (K) at position 89 and serine (S) at position 91 of the CDR3 of the VL domain are consistent with glutamine (Q) at position 89 and tyrosine (Y) in human antibodies.

[0191] To construct a strategy for improving the residues, the effects of VH-VL interface residues on the CDRs of the heavy-chain variable domain and the light-chain variable region were analyzed in more detail.

[0192] FIG. 4B shows the results of analyzing interface residues between variable regions in order to construct stable cytotransmab that optimally interacts with a human antibody heavy-chain variable region.

[0193] Specifically, based on information about the positions of interface residues between human antibody variable regions, the frequency of binding to specific interface residues located in opposite variable regions, and the abundance of interface residues in human antibodies, which were reported in the literature, hT3 VL and the interface residues between the heavy chain and light chain variable regions of Bevacizumab (Avastin) and Adalimumab (Humira), which are antibodies approved by the FDA, were analyzed (Vargas-Madrazo and Paz-Garcia, 2003). The results of the analysis indicated that, in the mouse CDRs of hT3 VL, residues 89 and 91 in CDR3 that is involved in association between variable regions are highly abundant in human antibodies and can influence the CDR3 structure of the heavy-chain variable region (VH). The two residues were mutated with amino acids that are highly abundant in human antibodies, thereby hT4 VL that can optimally bind to human antibody heavy-chain variable regions.

[0194] Tables 1 and 2 below show the sequences of the designed human antibody light-chain variable regions having cytosol-penetrating ability. Table 1 shows the full-length sequences of the human antibody light-chain variable regions, numbered according to the Kabat numbering system, and Table 2 shows the CDR sequences of the antibody sequences shown in Table 1.

TABLE-US-00005 TABLE 1 Full-length seqeunces of cytosol-penetrating human antibody light-chain variable Names of light chain variable regions Sequences SEQ ID NOS: hT2 VL 1        10        20      abcdef  30       40         50 SEQ ID NO: 1 DLVMTQSPATLSLSPGERATLSCKSSQSLFNSRTRKNYLAWYQQKPGQAPRLLIYW          60        70        80        90        100 ASTRESGIPGRFSGSGSGTDFTLTISSLEPEDFAVYYCKQSYYHMYTFGQGTKVEIKR hT3 VL 1        10        20      abcdef  30       40         50 SEQ ID NO: 2 DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW          60        70        80        90        100 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYYHMYTFGQGTKVEIKR hT4 VL 1        10        20      abcdef  30       40         50 SEQ ID NO: 3 DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW          60        70        80        90        100 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYYHMYTFGQGTKVEIKR

TABLE-US-00006 TABLE 2 CDR sequences of cytosol-penetrating human antibody light-chain variable regions. Names of light chain  variables CDR1 SEQ CDR2 SEQ regions Sequence ID Sequence ID Kabat No. 24 25 26 27 27a 27b 27c 27d 27e 27f 28 29 30 31 32 33 34 NO: 50 51 52 53 54 55 56 NO: hT2 VL K S S Q S L F N S R T R K N Y L A  4 W A S T R E S  5 hT3 VL K S S Q S L F N S R T R K N Y L A  7 W A S T R E S  8 hT4 VL K S S Q S L F N S R T R K N Y L A 10 W A S T R E S 11 Names of light chain  variables CDR3 SEQ regions Sequence ID Kabat No. 89  90  91 92 93 94 95 96 97 NO: hT2 VL K Q S Y Y H W Y T  6 hT3 VL K Q S Y Y H W Y T  9 hT4 VL Q Q Y Y Y H W Y T 12

Example 5: Development of Cytotransmab by Substitution with Cytosol-Penetrating Humanized Light-Chain Region (VL), and Expression and Purification of Cytotransmab

[0195] FIG. 5 is a schematic view showing a method of substituting a light-chain variable region having no cell-penetrating ability with a humanized light-chain variable region having cytosol-penetrating ability in order to construct cytotransmab.

[0196] Specifically, in order to construct a heavy-chain expression vector for producing an intact IgG-format monoclonal antibody, a DNA encoding a heavy chain comprising an antibody heavy-chain variable region (Bevacizumab VH, Adalimumab VH, or humanized hT0 VH) and a heavy-chain constant region (CH1-hinge-CH2-CH3), which has a secretion signal peptide-encoding DNA fused to the 5′ end, was cloned into a pcDNA3.4 vector (Invitrogen) by NotI/HindIII. Furthermore, in order to construct a vector that expresses a light chain, a DNA encoding either a cytosol-penetrating light-chain variable region (hT4 VL) or the light-chain variable region (Bevacizumab VL, or Adalimumab VL) and light-chain constant region (CL) of a model antibody, which a secretion signal peptide-encoding DNA fused to the 5′ end, was cloned into a pcDNA3.4 vector (Invitrogen) by use of NotI/HindIII.

[0197] The light-chain and heavy-chain expression vectors were transiently transfected, and the proteins were expressed and purified, followed by comparison of the yield of the proteins. In a shaking flask, HEK293-F cells (Invitrogen) suspension-growing in serum-free FreeStyle 293 expression medium (Invitrogen) were transfected with a mixture of plasmid and polyethylenimine (PEI) (Polyscience). After 200 mL transfection in a shaking flask (Corning), HEK293-F cells were seeded into 100 ml of medium at a density of 2.0×10.sup.6 cells/ml, and cultured at 150 rpm and in 8% CO.sub.2. To produce each monoclonal antibody, a suitable heavy-chain and light-chain plasmid were diluted in 10 ml of FreeStyle 293 expression medium (Invitrogen) (125 μg heavy chain, 125 μg light chain, a total of 250 μg (2.5 μg/ml)), and the dilution was mixed with 10 ml of medium containing 750 μg (7.5 μg/ml) of PEI, and the mixture was incubated at room temperature for 10 minutes. The incubate medium mixture was added to 100 ml of the seeded cell culture which was then cultured at 150 rpm in 8% CO.sub.2 for 4 hours, after which 100 ml of FreeStyle 293 expression was added to the cell culture, followed by culture for 6 days. In accordance with the standard protocol, the protein was purified from the collected cell culture supernatant. The antibody was applied to a Protein A Sepharose column (GE Healthcare), and washed with PBS (pH 7.4). The antibody was eluted using 0.1 M glycine buffer (pH 3.0), and then immediately neutralized with 1M Tris buffer. The eluted antibody fraction was concentrated while the buffer was replaced with PBS (pH 7.4) by dialysis. The purified protein was quantified by measuring the absorbance at 280 nm and the absorption coefficient.

[0198] Table 3 below shows the yields of purified cytotransmabs and proteins produced per liter of culture volume. Three measurements were statistically processed, and ± indicates standard deviation values. With respect to the yields of the obtained proteins, cytotransmabs, including hT4 VL improved to facilitate its interaction with a human heavy-chain variable region (VH), did not greatly differ from the wild-type monoclonal antibodies.

TABLE-US-00007 TABLE 3 Comparison of the purification yields of Cytotransmabs with those of wild-type IgG-format monoclonal antibodies (Adalimumab, and Bevacizumab) IgG purification yield (mg/1-liter of transfected IgG clone VH VL cells) TMab2 h3D8 VH hT2 VL 8.0 ± 0.7 TMab3 h3D8 VH hT3 VL 8.2 ± 0.5 TMab4 h3D8 VH hT4 VL 10.8 ± 1.0  Adalimumab Adalimumab VH Adalimumab VL 11.6 ± 0.3  HuT2 Adalimumab VH hT2 VL 2.1 ± 0.6 HuT3 Adalimumab VH hT3 VL 3.5 ± 0.8 HuT4 Adalimumab VH hT4 VL 10.9 ± 0.8  Bevacizumab Bevacizumab VH Bevacizumab VL 8.8 ± 0.4 AvaT4 Bevacizumab VH hT4 VL 8.0 ± 1.1

[0199] These results indicate that the humanized light-chain variable region (hT4 VL) obtained by additionally modifying interface residues can optimally interact with a humanized antibody heavy-chain variable region, and thus can be stably expressed and purified.

[0200] FIG. 6A shows the results of analyzing cytotransmab by reductive or non-reductive SDS-PAGE after purification.

[0201] Specifically, in a non-reductive condition, a molecular weight of about 150 kDa appeared, and in a reductive condition, the heavy chain showed a molecular weight of about 50 kDa, and the light-chain showed a molecular weight of about 25 kDa. This suggests that the purified cytotransmab and monoclonal antibodies are present as monomers in a solution state, and do not form a dimer or an oligomer by a non-natural disulfide bond.

[0202] FIG. 6B shows the results of an experiment performed using a size exclusion chromatography column (Superdex™200 10/300 GC) (GE Healthcare) by HPLC (high performance liquid chromatography) (The Agilent 1200 Series LC systems and Modules) (Agilent) in order to confirm that cytotransmab is present as a monomer in a natural environment.

[0203] Specifically, high-salt elution buffer (12 mM phosphate, pH 7.4, 500 mM NaCl, 2.7 mM KCl) (SIGMA) was used at a flow rate of 0.5 ml/min in order to eliminate the nonspecific binding to resin caused by electrical attraction due to basic residues. The proteins used as protein size markers were dehydrogenase (150 kDa), albumin (66 kDa), and carbonic anhydrase (29 kDa). A single extreme point was measured in all the monoclonal antibodies and cytotransmab, indicating that these antibodies are present as monomers.

Example 6: Analysis of Affinity of Heavy-Chain Variable Region of Cytotransmab and Analysis of DNA Hydrolysis Ability of Light-Chain Variable Region (VL)

[0204] FIG. 6C shows the results of ELISA (enzyme linked immunosorbent assay) performed to measure the affinities of the heavy-chain variable regions of cytotransmab (TMab4, HuT4 or AvaT4) and monoclonal antibodies (Bevacizumab (Avastin) and Adalimumab (Humira)) for target molecules.

[0205] Specifically, a target molecule (VEGF-A, or TNF-α) was incubated in a 96-well EIA/RIA plate (COSTAR Corning) at 37° C. for 1 hour, and then washed three times with 0.1% PBST PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 12 mM phosphate, 2.7 mM KCl) (SIGMA) for 10 minutes. After incubation with 5% PBSS PBSS (5% Skim milk, pH 7.4, 137 mM NaCl, 12 mM phosphate, 2.7 mM KCl) (SIGMA) for 1 hour, the target molecule was washed three times with 0.1% PBST for 10 minutes. Next, each of cytotransmab and monoclonal antibodies (TMab4, Bevacizumab, Adalimumab, AvaT4, and HuT4) was bound to the target molecule, followed by washing three times with 0.1% PBST for 10 minutes. As a marker antigen, goat alkaline phosphatase-conjugated anti-human mAb (SIGMA) was used. Each of the resulting material was reacted with pNPP (p-nitrophenyl palmitate) (SIGMA), and the absorbance at 405 nm was measured.

[0206] As shown in FIG. 6C, AvaT4 and HuT4 lost their affinity for the target molecule. In the case of Adalimumab and TNF-α, it was shown that the antigen recognition site was involved in all the CDRs located in the heavy chain and the light chain (Shi et al., 2013). In the case of Bevacizumab, it was found that the CDR3 of the heavy-chain variable region (VH) plays an important role in binding to antigen, but the analysis results shown in FIG. 8B indicated that Bevacizumab has the VH7 subgroup. In addition, it was found that residue 96 of the light-chain variable region of Bevacizumab, which greatly influences the heavy-chain variable region (VH) CDR3, did greatly differ from that of hT4 VL (Charlotte et al., 2007).

[0207] FIG. 6D shows the results of an agarose gel nucleic acid hydrolysis experiment performed to examine the hydrolysis of nucleic acids in cytotransmab obtained by replacement with a cell-penetrating human light-chain variable region (hT4) grafted with the CDR of an autoimmune mouse antibody.

[0208] Specifically, in a total mixture volume of 10 μl, a purified pUC19 substrate (2.2 nM) and either m3D8 scFv protein (0.5 μM and 0.1 μM) known to have the ability to hydrolyze nucleic acids, or each of cytotransmab and monoclonal antibodies (TMab4, AvaT4, HuT4 (0.1 μM)), were incubated in TBS reaction buffer (50 mM Tris-HCl, 50 mM NaCl, pH 7.4) (SIGMA). Herein, the TBS buffer contained 2 mM MgCl.sub.2, and another buffer contained 50 mM EDTA (SIGMA) and was used as a control. The prepared samples were incubated at 37° C. After 1 hour, the samples were observed.

[0209] As shown in FIG. 6D, the results of the observation indicated that TMab4, AvaT4 and HuT4 had no nucleic acid-hydrolyzing ability at 0.1 μM. This suggests that when these antibodies penetrate the cytosol and remain in the cytosol, they no cause nonspecific cytotoxicity.

Example 7: Verification of Cytosol-Penetrating Abilities of Cytotransmab

[0210] FIG. 7A shows the results of observing 1-2 cells in various cell lines by confocal microscopy in order to verify the cytosol-penetrating abilities of cytotransmabs having a light-chain variable region replaced with the cytosol-penetrating light-chain region hT4 VL.

[0211] Specifically, in a 24-well plate, 5×10.sup.4HeLa, PANC-1, HT29 or MCF-7 cells per well were added to 0.5 ml of 10% FBS-containing medium, and cultured for 12 hours under the conditions of 5% CO.sub.2 and 37° C. When the cells were stabilized, each well was incubated with a dilution of each of 1 μM of TMab4, Adalimumab (Humira), Bevacizumab (Avastin), HuT4 or AvaT4 in 0.5 ml of fresh medium for 6 hours under the conditions of 37° C. and 5% CO.sub.2. Next, the medium was removed, and each well was washed with PBS, and then treated with a weakly acidic solution (200 mM glycine, 150 mM NaCl (pH 2.5)) to remove proteins from the cell surface. After washing with PBS, the cells were fixed in 4% paraformaldehyde at 25° C. for 10 minutes. Next, each well was washed with PBS, and incubated with PBS buffer containing 0.1% saponin, 0.1% sodium azide and 1% BSA at 25° C. for 10 minutes to pores in the cell membranes. Next, each well was washed with PBS, and then incubated with PBS buffer containing 2% BSA at 25° C. for 1 hour in order to eliminate nonspecific binding. Thereafter, each well was incubated with FITC (green fluorescence)-labeled antibody (Sigma), which specifically recognizes human Fc, at 25° C. for 1.5 hours, and the nucleus was blue-stained with Hoechst33342, and observed with a confocal microscope. Unlike IgG-format monoclonal antibodies (Adalimumab and Bevacizumab) which target extracellularly secreted proteins, TMab4, HuT4 and AvaT4 showed green fluorescence in the cells.

[0212] FIG. 7B shows the results of examining cytosol-penetrating ability for several cells, performed at a reduced magnification in order to examine cell-penetrating efficiency in the cytosol-penetrating ability examination experiment by confocal microscopy observation as shown in FIG. 7A.

[0213] It was shown that the cytotransmab introduced with the cytosol-penetrating humanized light-chain variable region penetrated the cytosol of all the cells and localized in the cytosol.

[0214] FIG. 8A shows the results of observing the degree of cell penetration of TMab4 as a function of the concentration of TMab4 by confocal microscopy. HeLa cells were treated with 10 nM, 50 nM, 100 nM, 500 nM, 1 μM and 2 μM of TMab4, and cultured at 37° C. for 6 hours. In the same manner as described above, the cells were observed with a confocal microscope. When TMab4 was incubated for 6 hours, green fluorescence was observed in the cells, starting from a concentration of 100 nM. As the concentration increased from 100 nM, green fluorescence in the cells increased.

[0215] FIG. 8B shows the results of observing the degree of cell penetration of TMab4 as a function of time after TMab4 treatment by confocal microscopy. HeLa cells were treated with 1 μM of TMab4, and then cultured at 37° C. for 10 min, 30 min, 1 hour, 2 hours, 6 hours, 12 hours 24 hours and 48 hours. The cultured cells were stained in the same manner as described in the above Example, and were observed with a confocal microscope.

[0216] Starting from 30 minutes, TMab4 showed weak green fluorescence in the cells. The green fluorescence gradually increased, and was the strongest at 6 hours. Thereafter, the fluorescence gradually decreased, and became very weak at 48 hours.

Example 8: Evaluation of Cytotoxicity of Cytotransmabs

[0217] In order to examine whether or not the cytotransmabs confirmed to have cytosol-penetrating ability in Example 7 would have cytotoxicity in vitro, HeLa or PANC-1 cells were treated with each of TMab4, HuT4, Adalimumab, AvaT4 and Bevacizumab, and the inhibition of growth of the cells was examined by an MTT assay (Sigma).

[0218] Specifically, in a 96-well plate, 1×10.sup.4 HeLa or PANC-1 cells per well were cultured in 0.1 ml of 10% FBS-containing medium for 12 hours under the conditions of 37° C. and 5% CO.sub.2. Then, each well was treated with 1 μM of each of TMab4, HuT4, Adalimumab, AvaT4 and Bevacizumab for 20 hours or 44 hours, and then 20 μl of MTT solution (1 mg/ml PBS) was added to each well, followed by incubation for 4 hours. The formed formazan was dissolved in 200 μl of DMSO (dimethyl sulfoxide), and the absorbance at 595 nm was measured to determine cell viability.

[0219] FIG. 9A is a graph showing the results obtained by treating HeLa and PANC-1 cell lines with cytotransmab and evaluating the inhibition of growth of the cells in vitro. FIG. 9B is an image showing the results obtained by treating HeLa and PANC-1 cell lines with cytotransmab and evaluating the degree of inhibition of the cells in vitro. As shown in FIGS. 9A and 9B, all the antibodies showed no cytotoxicity. As shown in Example 6 above, cytotransmabs had no nucleic acid-hydrolyzing ability, unlike m3D8 scFv, and thus had no cytotoxicity.

Example 9: Verification of Intracellular Transport and Degradation Mechanisms of Cytotransmab

[0220] FIG. 10 shows the results of observing the transport and stability of intracellularly introduced TMab4 by pulse-chase and confocal microscopy.

[0221] Specifically, HeLa cells were prepared in the same manner as described above. The prepared cells were treated with 3 μM of TMab4 at 37° C. for 30 minutes, and then washed quickly three times with PBS, and cultured in medium at 37° C. for 2 hours, 6 hours and 18 hours. The cells were washed with PBS and a weakly acidic solution in the same manner as described in the above Example, and then subjected to cell fixation, cell perforation and blocking processes. TMab4 was stained with green fluorescence (FITC) or red fluorescence (TRITC)-labeled antibody that specifically recognizes human Fc. Furthermore, the cells were incubated with anti-EEA1 antibody against the early endosome marker EEA1 (Early Endosome Antigen1), anti-caveolin-1 antibody against the caveosome marker caveolin-1, anti-calnexin antibody against the endoplasmic reticulum marker calnexin, or anti-58K Golgi antibody (Santa Cruz) against the Golgi marker 58K Golgi protein, at 4° C. for 12 hours, and incubated with red fluorescence (TRITC)-labeled secondary antibody at 25° C. for 1 hour. At 30 minutes before cell fixation, the cells being cultured were treated directly with 1 μM of LysoTracker® Red DND-99 or 10 μg/ml of Alexa Fluor 488-transferrin. After the staining process, the cells were analyzed with a confocal microscope. As a result, TMab4 was more stable in the cells than transferrin, and penetrated into the cytosol by clathrin and localized in the early endosome up to 2 hours, after which it was not transported into the lysosome and not superimposed with any organelle.

[0222] FIG. 11A shows the results of observing the cytosolic localization of cytotransmab TMab4 or HuT4 by confocal microscopy.

[0223] Specifically, HeLa cells were prepared in the same manner as described above. The prepared cells were incubated with 5 μM of PBS, TMab4, Adalimumab or HuT4 in serum-free medium at 37° C. for 4 hours. After 4 hours, each well containing PBS or the antibody was treated with 50 μM of calcein and incubated at 37° C. for 2 hours. After washing with PBS, the cells were fixed in the same manner as described above and were observed with a confocal microscope. As a result, it was shown that TMab4 and HuT4 showed the green fluorescence of calcein which escaped from the endosome into the cytosol. However, Adalimumab showed no green fluorescence in the cytosol.

[0224] FIG. 11B is a bar graph showing the results of quantifying the calcein fluorescence of the confocal microscope images shown in FIG. 11A.

[0225] Specifically, using Image J software (National Institutes of Health, USA), 15 cells were selected in each condition, and then the obtained mean values of fluorescence are graphically shown.

Example 10: Examination of Cytosolic Retention of Cytotransmab by Recombination of GFP Fragments

[0226] FIG. 12A is a schematic view showing a process in which GFP fluorescence by complementary association of split-GFP is observed when cytotransmab localizes in the cytosol.

[0227] Specifically, to directly confirm that cytotransmab localizes in the cytosol, a split-GFP system was used. If the green fluorescence protein GFP is split into two fragments (GFP 1-10 and GFP 11), the fluorescence property will be removed, and if the distance between the two fragments becomes closer so that they bind to each other, the florescence property can be restored (Cabantous et al., 2005). Based on such characteristics, the GFP 1-10 fragment is expressed in the cytosol, and the GFP 11 fragment is fused to the C-terminus of the heavy chain of Cytotransmab. Thus, the observation of GFP fluorescence indicates that Cytotransmab localizes in the cytosol.

[0228] In addition, in order to assist in the complementary association of split GFP, streptavidin-SBP2 (streptavidin binding peptide 2) with a higher affinity was used (Barrette-Ng et al., 2013). SBP2 with a smaller size was fused to the C-terminus of the GFP 11 fragment via three GGGGS linkers by a genetic engineering method. Furthermore, streptavidin was fused to the N-terminus of the GFP 1-10 fragment via three GGGGS linkers by a genetic engineering method. To realize this system, a stable transgenic cell line expressing streptavidin-GFP1-10 was developed.

[0229] Specifically, a DNA encoding Streptavidin-GFP1-10 was cloned into the Lenti virus vector pLJM1 (Addgene) by SalI/EcoRI. In a cell culture dish, 3×10.sup.6 HEK293T cells were added to 1 ml of 10% FBS-containing medium and cultured for 12 hours under the conditions of 5% CO.sub.2 and 37° C. 40 μl of Lipofectamine 2000 (Invitrogen, USA) was added to 600 μl of Opti-MEM media (Gibco), and the constructed Lenti virus vector and a virus packaging vector (pMDL, pRSV, or pVSV-G (Addgene)) were carefully added thereto and incubated at room temperature for 20 minutes, and then added to the dish. In addition, 9 ml of antibiotic-free DMEM medium was added to the cells which were then cultured for 6 hours under the conditions of 37° C. and 5% CO.sub.2, after which the medium was replaced with 10 ml of 10% FBS-containing DMEM medium, followed by culture for 72 hours. After 60 hours, 1×10.sup.5 HeLa cells were added to 1 ml of 10% FBS-containing medium and cultured for 12 hours under the conditions of 37° C. and 5% CO.sub.2. The medium transiently transfected with the Lenti virus vector was completely filtered, and the viral particles in the medium were added to the prepared cell culture dish containing HeLa cells. To measure antibiotic resistance, puromycin resistance gene was used as a selection marker.

[0230] FIG. 12B shows the results of Western blot analysis performed to analyze the expression level of streptavidin-GFP1-10 in a constructed stable cell line.

[0231] Specifically, in a 6-well plate, 1×10.sup.5 HeLa cells per well were added to 1 ml of 10% FBS-containing medium and cultured for 12 hours under the conditions of 37° C. and 5% CO.sub.2. After culture, lysis buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% SDS, 1 mM EDTA, Inhibitor cocktail(sigma)) was added to the cells to obtain a cell lysate. The cell lysate was quantified using a BCA protein assay kit (Pierce). After SDS-PAGE, the gel was transferred to a PVDF membrane and incubated with antibodies (Santa Cruz) that recognize streptavidin and β-actin, respectively, at 25° C. for 2 hours, after which it was incubated with HRP-conjugated secondary antibody (Santa Cruz) at 25° C. for 1 hour, followed by detection. Analysis was performed using ImageQuant LAS4000 mini (GE Healthcare).

Example 11: Expression and Purification of GFP11-SBP2-Fused Cytotransmab

[0232] For expression of GFP11-SBP2-fused cytotransmab in animal cells, GFP11-SBP2 was fused to the C-terminus of the heavy chain via three GGGGS linkers. Next, an animal expression vector encoding the cytosol-penetrating light-chain and the cytosol-penetrating light-chain with improved endosomal escape and an animal expression vector expressing the GFP11-SBP2-fused heavy-chain were transiently co-transfected into HEK293F protein expression cells. Next, purification of the GFP11-SBP2-fused cytosol-penetrating monoclonal antibody was performed in the same manner as described in Example 5.

Example 12: Examination of GFP Fluorescence of GFP11-SBP2-Fused Cytotransmab by Cytosolic Localization

[0233] FIG. 12C shows the results of confocal microscopy observation of the GFP fluorescence of GFP11-SBP2-fused cytotransmab by complementary association of split GFP.

[0234] Specifically, HeLa cells were prepared in the same manner as described in Example 7. When the cells were stabilized, these cells were cultured with 0.2, 0.4, 0.6, 0.8 and 1 μM of PBS or TMab4-GFP11-SBP2 at 37° C. for 6 hours. According to the same method as described in Example 7, the cells were washed with PBS and a weakly acidic solution, and then fixed. Furthermore, the nucleus was blue-stained with Hoechst33342 and observed with a confocal microscope. It was observed that TMab4 showed GFP fluorescence at 0.8 μM and 1 μM.

[0235] The above results clearly indicate that cytotransmab TMab4 penetrates cells and localizes in the cytosol.

Example 13: Selection of Heavy-Chain Variable Region (VH), which Binds Specifically to GTP-Bound KRas, by High-Diversity Human VH Library

[0236] FIG. 13 is a schematic view showing a process of constructing anti-Ras•GTP iMab by replacing the heavy-chain variable region (VH) of an intact IgG-format Cytotransmab having only cytosol-penetrating ability with a heavy-chain variable region (VH) that binds specifically to GTP-bound KRas.

[0237] FIG. 14 shows the application of an IgG-format cytotransmab having only cytosol-penetrating ability, and is a schematic view showing a strategy of inducing cytotoxicity specific for Ras mutant cells by use of a monoclonal antibody (anti-Ras. GTP iMab: internalizing & interfering monoclonal antibody) which has a heavy-chain variable region (VH) replaced with a heavy-chain variable region (VH) binding specifically to GTP-bound KRas and which penetrates cells and binds specifically to GTP-bound Ras in the cells.

[0238] In order to select a stable humanized heavy-chain variable single domain (VH) which is to be introduced into the anti-Ras•GTP iMab and which binds specifically to GTP-bound KRas, a yeast expression VH library constructed through a previous study was used (Baek and Kim, 2014).

[0239] Specifically, the FR (framework) of the library used was the V gene IGHV3-23*04, J.sub.H4 which is most commonly used in conventional antibodies, and the CDR3 in the library had 9 residues. The construction of the library and a yeast surface display method are described in detailed in a previously reported paper (Baek and Kim, 2014).

Example 14: Preparation of GTP-Bound KRas G12D Protein

[0240] Expression in E. coli and purification, performed to prepare GTP-bound KRas G12D antigen for library screening and affinity analysis, are described in detail in a previously reported paper (Tanaka T et al., 2007).

[0241] Specifically, a DNA encoding residues 1 to 188, which comprises the CAAX motif of each of wild-type KRas and mutant KRas G12D, KRas G12V and KRas G13D (listed in the order of higher to lower mutation frequency), was cloned into the E. coli expression vector pGEX-3X by use of the restriction enzymes BamHI/EcoRI. Herein, the expression vector was designed to have a T7 promoter-GST-KRas. All KRas mutations were induced using an overlap PCR technique, and the expression vector was constructed using the above-described method. The pGEX-3X-KRas vector was transformed into E. coli by electroporation, and selected in a selection medium. The selected E. coli was cultured in LB medium in the presence of 100 μg/ml of an ampicillin antibiotic at 37° C. until the absorbance at 600 nm reached 0.6. Then, 0.1 mM IPTG was added thereto for protein expression, and then the E. coli cells were further cultured at 30° C. for 5 hours. Thereafter, the E. coli cells were collected by centrifugation, and then disrupted by sonication (SONICS). The disrupted E. coli cells were removed by centrifugation, and the remaining supernatant was collected and purified using glutathione resin (Clontech) that specifically purifies GST-tagged protein. The glutathione resin was washed with 50 ml of washing buffer (140 mM NaCl, 2.7 mM KCl, 10 mM NaH.sub.2PO.sub.4, 1.8 mM KH.sub.2PO.sub.4, 1 mM EDTA, 2 mM MgCl.sub.2 pH 7.4) (SIGMA), and then protein was eluted with elution buffer (50 mM Tris-HCl pH8.0, 10 mM reduced glutathione, 1 mM DTT, 2 mM MgCl.sub.2) (SIGMA). The eluted protein was dialyzed to replace the buffer with storage buffer (50 mM Tris-HCl pH8.0, 1 mM DTT, 2 mM MgCl.sub.2) (SIGMA). The purified protein was quantified by measuring the absorbance at a wavelength of 280 nm and the absorption coefficient. SDS-PAGE analysis indicated that the protein had a purity of about 98% or higher.

[0242] Next, in order to bind a GTPλS (Millipore) or GDP (Millipore) substrate to KRas protein, KRas and a substrate at a molecular ratio of 1:20 were reacted in a reaction buffer (50 mM Tris-HCl pH8.0, 1 mM DTT, 5 mM MgCl.sub.2, 15 mM EDTA) (SIGMA) at 30° C. for 30 minutes, and 60 mM MgCl.sub.2 was added thereto to stop the reaction, and then stored at −80° C.

Example 15: Selection of Heavy-Chain Variable Region (VH) Specific for GTP-Bound KRas G12D

[0243] FIG. 15 is a schematic view showing a library screening strategy for obtaining a humanized antibody heavy-chain variable single domain having a high affinity only for GTP-bound KRas G12D protein.

[0244] Specifically, GTP-bound KRas G12D purified in Example 14 was biotinylated (EZ-LINK™ Sulfo-NHS-LC-Biotinylation kit (Pierce Inc., USA)), and then reacted with a heavy-chain variable region library displayed on the yeast cell surface at room temperature for 1 hour. The heavy-chain variable region library on the yeast cell surface, which reacted with the biotinylated GTP-bound KRas G12D, was reacted with Streptavidin (Microbead™ (Miltenyi Biotec) at 4° C. for 20 minutes, and then yeast displaying a heavy-chain variable region having a high affinity for the GTP-KRAS G12D was enriched using MACS (magnetic activated cell sorting). The selected library-displaying yeast was cultured in a selection medium and cultured in SG-CAA+URA (20 g/L Galactose, 6.7 g/L Yeast nitrogen base without amino acids, 5.4 g/L Na2HPO.sub.4, 8.6 g/L NaH.sub.2PO.sub.4, 5 g/L casamino acids, 0.2 mg/L Uracil) (SIGMA) medium to induce protein expression. Next, the yeast was incubated with a yeast displaying the library competitively with GTP-bound KRas G12D alone or non-biotinylated GTP-bound KRas G12D antigen at a concentration 10-fold higher than GTP-bound KRas G12D, at room temperature for 1 hour, after which it was reacted with PE-conjugated Streptavidin (Streptavidin-R-phycoerythrin conjugate (SA-PE) (Invitrogen), and enriched by FACS (fluorescence activated cell sorting) (FACS Caliber) (BD biosciences). After selection of screening conditions by FACS analysis, antigen was bound to the yeast displaying the enriched library under the same conditions as described, and then the yeast was enriched using a FACS aria II sorter. The humanized heavy-chain region library enriched by the first MACS and first FACS screening was mated with a yeast secreting the cytosol-penetrating light-chain variable single domain (hT4 VL), and displayed on the yeast surface in the form of Fab, and then subjected to second FACS and third FACS screening.

[0245] Specifically, in order to construct a yeast which is to be mated with the heavy-chain variable domain (VH) library and which secretes the cytosol-penetrating light-chain variable domain (VL), a DNA encoding the cytosol-penetrating hT4 VL was cloned into the light-chain variable domain yeast secretion vector pYDS-K by the restriction enzymes NheI and BsiWI, thereby obtaining pYDS-K-hT4 VL. The obtained pYDS-K-hT4 VL was transformed into the mating α-type yeast mating strain YVH10 by electroporation, and mated with a yeast cultured in the selection medium SD-CAA+Trp (20 g/L Glucose, 6.7 g/L Yeast nitrogen base without amino acids, 5.4 g/L Na2HPO.sub.4, 8.6 g/L NaH.sub.2PO.sub.4, 5 g/L casamino acids, 0.4 mg/L tryptophan) (SIGMA).

[0246] Specifically, in the case of yeast mating, there are 1×10.sup.7 yeast cells when the absorbance at 600 nm is 1. Among the cultured yeast cells, 1.5×10.sup.7 yeast cells expressing the selected heavy-chain variable domain library and 1.5×10.sup.7 yeast cells containing hT4 VL were added to GTP-bound KRas G12D, and washed three times with YPD YPD (20 g/L Dextrose, 20 g/L peptone, 10 g/L yeast extract, 14.7 g/L sodium citrate, 4.29 g/L citric acid, pH 4.5) (SIGMA). Then, the yeast cells were re-suspended in 100 μl of YPD, and dropped onto an YPD plate so as not to spread, after which these yeast cells were dried and cultured at 30° C. for 6 hours. Next, the dried yeast-coated portion was washed three times with YPD medium, and then incubated in the selection medium SD-CAA at 30° C. for 24 hours to a final yeast concentration of 1×10.sup.6 cells or less, and only mated yeast cells were selected. The selected yeast cells were incubated in SG-CAA medium to induce expression of a humanized antibody Fab fragment, and enriched by second and third FACS such that the yeast cells would be 100-fold competitive with GDP-bound KRas G12D at a GTP-bound KRas G12D concentration of 100 nM.

[0247] FIG. 16 shows the results of FACS analysis of binding under a condition of GTP-bound KRas G12D alone and a condition competitive with GTP-bound KRas G12D in each step of the above-described screening process for obtaining a high affinity for GTP-bound KRas G12D. Accordingly, it was found that it is possible to select a library that can bind specifically to GTP-bound KRas G12D in a manner dependent on the heavy-chain variable domain (VH).

[0248] Through the high-throughput screening as described above, an RT4 clone was finally selected from the library having a high affinity and specificity for GTP-bound KRas G12D protein by individual clone analysis.

[0249] Tables 4 and 5 below show the sequence information and SEQ ID NO of the heavy-chain variable domain RT4 that binds to activated RAS. Table 4 shows the full-length sequence of RT4, numbered according to the Kabat numbering system, and Table 5 shows the CDR sequence of the antibody sequence shown in Table 4.

TABLE-US-00008 TABLE 4 Full-length sequence of heavy-chain variable domain RT4 that binds to activated RAS Names of heavy chain variable regions Sequences SEQ ID NOS: RT4          10        20       30        40         50 A 13 EVQLVESGGGLVQPGGSLRLSCAASGTFSSYAMSWVRQAPGKGLEWVSTISRSGHSTY  60        70        80 abc       90         a100       110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRFGSIVFDYWGQGTLVTVSS

TABLE-US-00009 TABLE 5 CDR sequence of heavy-chain variable domain RT4 that binds to activated RAS Names of light chain variables CDR1 SEQ CDR2 regions Sequence ID Sequence Kabat No. 32 32 33 34 35 NO: 50 51 52 52a 53 54 55 56 57 58 59 RT4 S Y A M S 14 T I S R S G H S T Y Y Names of light chain  variables CDR2 SEQ CDR3 SEQ regions Sequence ID Sequence ID Kabat No. 60 61 62 63 64 65 NO: 95 96 97 98 99 100 100a 101 102  NO: RT4 A D S V K G 15 R F G S I V F D Y 16

Example 16: Expression and Purification of Anti-Ras•GTP iMab, and Analysis of Affinity for KRas Mutations

[0250] In order to express, in animal cells, anti-Ras•GTP iMab that can penetrate cells and specifically target GTP-bound Ras in the cells as a result of replacing the heavy-chain variable region (VH) of cell-penetrating and cytosol-localizing cytotransmab with RT4 VH selected in Example 13, as described in Example 5 above, a DNA, which has a secretion peptide-encoding DNA fused to the 5′ end and comprises an RT4 heavy-chain variable region that binds specifically to GTP-bound KRas and a heavy-chain constant region (CH1-hinge-CH2-CH3), was cloned into a pcDNA3.4 vector (Invitrogen) by NotI/HindIII. Next, an animal expression vector encoding the cytosol-penetrating light-chain, and the constructed animal expression vector encoding a heavy chain comprising a heavy-chain variable region that binds specifically to GTP-bound KRas, were transiently co-transfected into protein-expressing HEK293F cells. Next, purification of anti-Ras•GTP iMab was performed in the same manner as described in Example 5.

[0251] FIG. 17 shows the results of analyzing anti-Ras•GTP iMab RT4 by 12% SDS-PAGE under reductive or non-reductive conditions after purification.

[0252] Specifically, in a non-reductive condition, a molecular weight of about 150 kDa appeared, and in a reductive condition, a heavy-chain molecular weight of about 50 kDa and a light-chain molecular weight of about 25 kDa appeared. This indicates that the expressed and purified anti-Ras•GTP iMab is present as a monomer in a solution state free of a non-covalent bond, and does not form a dimer or an oligomer by a non-natural disulfide bond.

[0253] FIG. 18 shows the results of ELISA performed to measure affinity for GTP-bound and GDP-bound wild-type KRas and GTP-bound and GDP-bound KRas mutants (KRas G12D, KRas G12V, and KRas G13D).

[0254] Specifically, each of GTP-bound KRas mutants and GDP-bound KRas mutants, which are target molecules, was incubated in a 96-well EIA/RIA plate (COSTAR Corning) at 37° C. for 1 hour, and then the plate was washed three times with 0.1% TBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 12 mM Tris, 2.7 mM KCl, 5 mM MgCl.sub.2) (SIGMA) for 10 minutes. Next, each well of the plate was incubated with 4% TBSB (4% BSA, pH7.4, 137 mM NaCl, 12 mM Tris, 2.7 mM KCl, 10 mM MgCl.sub.2) (SIGMA) for 1 hour, and then washed three times with 0.1% TBST for 10 minutes. Thereafter, each well was incubated with anti-Ras•GTP iMab RT4 (and cytotransmab TMab4 having cytosol-penetrating ability only without Ras-binding ability) diluted in 4% TBSB at various concentrations, after which each well was washed three times with 0.1% PBST for 10 minutes. As a marker antibody, goat alkaline phosphatase-conjugated anti-human mAb (SIGMA) was used. Each well was treated with pNPP (p-nitrophenyl palmitate) (SIGMA), and the absorbance at 405 nm was measured.

[0255] In order to further quantitatively analyze the affinity of anti-Ras•GTP iMab RT4 for GTP-bound KRas G12D, SPR (Surface plasmon resonance) was performed using a Biacore 2000 instrument (GE healthcare).

[0256] Specifically, anti-Ras•GTP iMab RT4 was diluted in 10 mM Na-acetate buffer (pH 4.0), and immobilized on a CMS sensor chip (GE Healthcare) at a concentration of about 1100 response units (RU). For analysis, Tris buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl2, 0.005% Tween 20) was flushed at a flow rate of 30 μl/min, and GTP-bound KRas G12D was used at a concentration ranging from 1000 nM to 62.5 nM. After analysis of association and dissociation, regeneration of the CMS chip was performed by flushing a buffer (10 mM NaOH, 1M NaCl, pH10.0) at a flow rate of 30 μl/min for 1.5 minutes. Each of sensorgrams obtained at 3 min of association and 3 min of dissociation was normalized and subtracted from a blank cell, thereby determining affinity.

[0257] FIG. 19 shows the results of analyzing the affinity of anti-Ras•GTP iMab RT4 for GTP-bound KRAS G12D by use of SPR (BIACORE 2000) (GE Healthcare).

Example 17: Examination of Cytosol-Penetrating Ability of Anti-Ras•GTP iMab RT4

[0258] FIG. 20 shows the results of confocal microscopy observation performed to examine the cytosol-penetrating ability of anti-Ras•GTP iMab RT4.

[0259] In cells lines (PANC-1, and HCT116) having mutant KRas and cell lines (HT29, HeLa) having wild-type KRas, the cell-penetrating ability of anti-Ras•GTP iMab RT4 was analyzed.

[0260] Specifically, each cell line was added to a 24-well plate at a density of 5×10.sup.4 cells per well and cultured in 0.5 ml of 10% FBS-containing medium for 12 hours under the conditions of 5% CO.sub.2 and 37° C. When the cells were stabilized, each of TMab4 and RT4, diluted in 0.5 ml of fresh medium at a concentration of 1 μM, was added to each well, followed by incubation for 6 hours under the conditions of 37° C. and 5% CO.sub.2. A subsequent procedure was performed in the same manner as that of the staining procedure described in Example 7. It was observed that anti-Ras•GTP iMab showed fluorescence in the cells, indicating that cytotransmab did not lose its cytosol-penetrating ability, even after it was substituted with the heavy-chain variable region that binds specifically to GTP-bound KRas.

Example 18: Evaluation of Cytotoxicity of Anti-Ras•GTP iMab RT4

[0261] (1) Evaluation of the Effect of Anti-Ras•GTP iMab on Inhibition of Growth of Adherent Cells

[0262] FIG. 21 shows the results obtained by treating NIH3T3, NIH3T3 KRas G12V and NIH3T3 HRas G12V cell lines with anti-Ras•GTP iMab RT4 and evaluating the inhibition of growth of the cells in vitro.

[0263] Specifically, in order to examine whether anti-Ras•GTP iMab has cytotoxicity specific for KRas mutant-dependent cells in vitro, wild-type KRas NIH3T3 mouse fibroblast cells, NIH3T3 KRas G12V cells having artificially overexpressed Ras mutant, NIH3T3 HRas G12V mutant cells, and KRas G13D mutant human pancreatic cells (PANC-1), were treated with 1 μM of each of TMab4 and RT4, and the inhibition of growth of adherent cells was evaluated.

[0264] Specifically, each type of NIH3T3 and PANC-1 cells was added to a 24-well plate at a density of 2×10.sup.3 cells per well and cultured in 0.5 ml of 10% FBS-containing medium for 12 hours under the conditions of 37° C. and 5% CO.sub.2. Next, the cells were treated twice with 1 μM of TMab4 or RT4 for 72 hours each time and observed for a total of 144 hours, and then the number of viable cells was counted, thereby determining the degree of growth of the cells.

[0265] As shown in FIG. 21, the cells treated with TMab4 showed no cytotoxicity, whereas RT4 inhibited the growth of the KRas mutant cell lines (NIH3T3 KRas G12V, and NIH3T3 HRas G12V), and the NIH3T3 cells showed no cytotoxicity. In addition, the growth of the KRas G13D mutant PANC-1 cells was inhibited. Thus, TMab4 had no cytotoxicity, whereas RT4 inhibited cell growth.

[0266] (2) Evaluation of the Effect of Anti-Ras•GTP iMab RT4 on Inhibition of Growth of Non-Adherent Cells

[0267] FIG. 22 shows the results of evaluating the inhibition of growth of non-adherent cells in an NIH3T3 HRas G12V cell line.

[0268] Specifically, in order to examine whether anti-Ras•GTP iMab inhibits the growth of non-adherent cells in KRas mutant cells, NIH3T3 HRas G12V mutant cells were analyzed by a colony formation assay. Specifically, a mixture of 0.5 ml of 2×DMEM medium and 0.5 ml of 1% agrose solution was plated on a 12-well plate and hardened to form 0.5% gel. Then, 0.4 ml of 2×DMEM medium, 0.5 ml of 0.7% agarose, and 0.05 ml of 1×10.sup.3 NIH3T3 HRas G12V cells were mixed with 0.05 ml (20 μM) of PBS, TMab4, RT4 or Lonafarnib (20 μM), and the mixture was plated on the 0.5% agarose gel and hardened. Thereafter, the 0.35% agarose gel was treated with a dispersion of 1 μM of PBS, TMab4, RT4 or Lonafarnib in 0.5 ml of 1×DMEM at 3-day intervals for a total of 21 days. On day 21, the cells were stained with NBT (nitro-blue tetrazolium) solution, and then the number of colonies was counted.

[0269] Similarly to the results of the above-described experiment on the inhibition of growth of adherent cells, RT4 inhibited colony formation, whereas TMab4 did not inhibit colony formation.

[0270] The above results indicate that anti-Ras•GTP iMab RT4 bind specifically to Ras mutants in the cytosol and inhibits the growth of adherent and non-adherent cells.

Example 19: Examination of Whether Anti-Ras•GTP iMab RT4 Binds Specifically to GTP-Bound KRas in Cells

[0271] FIG. 23 shows the results of whether anti-Ras•GTP iMab RT4 is superimposed with activated HRas G12V mutants in cells. FIG. 24 shows the results of confocal microscopy observation of whether anti-Ras•GTP iMab RT4 is superimposed with GTP-bound KRas G12V mutants in cells.

[0272] Specifically, 24-well plates were coated with fibronectin (Sigma), and then a dilution of 0.5 ml of NIH3T3 cells expressing mCherry (red fluorescence) HRas G12V or mCherry (red fluorescence) KRas G12V was added to the plate at a density of 2×10.sup.4 cells per well, and cultured for 12 hours under the conditions of 37° C. and 5% CO.sub.2. Next, the cells were treated with 2 μM of each of TMab4 and RT4 and cultured at 37° C. for 12 hours. Thereafter, the cells were stained under the same conditions as those described in Example 7, and were observed with a confocal microscope.

[0273] As shown in FIGS. 23 and 24, green fluorescent RT4 was superimposed with the cellular inner membrane in which red-fluorescent activated Ras was located, whereas TMab was not superimposed.

[0274] The above experimental results indicate that anti-Ras•GTP iMab RT4 bind specifically to GTP-bound Ras in the cells.

Example 20: Evaluation of Cytotoxicity of RGD-Fused Anti-Ras•GTP iMab RT4

[0275] For in vivo experiments, it is required to impart tumor tissue specificity. Conventional cytotransmabs bind to HSPG on the cell surface, and have no specificity for any other tumor tissue, and for this reason, cannot specifically inhibit the growth of tumors in in vivo experiments. To overcome this problem, an RGD4C peptide (CDCRGDCFC; SEQ ID NO: 17) having specificity for integrin αvβ3 which is overexpressed in angiogenetic cells and various tumors was fused to the N-terminus of the light chain via one GGGGS linker by a genetic engineering method. The RGD4C peptide is characterized in that it has affinity higher than conventional RGD peptides and can be fused using a genetic engineering method, and the specific structure thereof can be maintained even when it is fused to the N-terminus (Koivunen E et al., 1995).

[0276] FIG. 25 shows the results obtained by treating HCT116 and PANC-1 cell lines with RGD-TMab4 and RGD-RT4 and evaluating the inhibition of growth of the cells in vitro.

[0277] In order to examine whether RGD-TMab4 and RGD-RT4 themselves have cytotoxicity in vitro, human colorectal cancer HCT116 cells having a KRas G13D mutant, and human pancreatic cancer PANC-1 cells having a KRas G12D mutant, were treated with each of RGD-TMab4 and RGD-RT4, and the inhibition of growth of the cells was evaluated.

[0278] Specifically, each type of HCT116 and PANC-1 cells was added to a 24-well plate at a density of 5×10.sup.3 cells per well, and cultured in 0.5 ml of 10% FBS-containing medium for 12 hours under the conditions of 37° C. and 5% CO.sub.2. Next, the cells were treated twice with 1 μM of each of RGD-TMab4 and RGD-RT4 for 72 hours each time, and observed for a total of 144 hours, and then the number of the cells was counted, thereby determining the degree of growth of the cells.

[0279] As shown in FIG. 25, RGD-TMab4 inhibited the growth of HCT116 cells by about 20% and inhibited the growth of PANC-1 cells by about 15%, and RGD-RT4 inhibited the growth of HCT116 and PANC-1 cells by about 40% and about 50%, respectively. According to previous studies, the RGD4C peptide has an affinity for integrin αvβ5, which is about 3 times lower than that for integrin αvβ3. However, integrin αvβ3 is overexpressed mainly in angiogenetic cells, and integrin αvβ5 is expressed in various tumor cells. Thus, the RGD4C peptide has the ability to bind αvβ5 of HCT116 and PANC-1 cells to thereby inhibit cell adhesion (Cao L et al., 2008).

[0280] Thus, RGD4C peptide-fused TMab4 does not appear to have cytotoxicity. In addition, a comparison between RGD-TMab4 and RGD-RT4 indirectly confirmed that TMab4 can inhibit Ras-specific cell growth even when the RGD is fused thereto.

Example 21: Examination of the Effect of RGD-Fused Anti-Ras•GTP iMab on Inhibition of Tumor Growth

[0281] FIG. 26A shows the results of analyzing the tumor growth inhibitory effect of RGD-fused anti-Ras•GTP iMab RT4 in mice xenografted with HCT116 cells. FIG. 26B is a graph showing the results of measuring the body weight of mice in order to examine the non-specific side effects of RGD-fused anti-Ras•GTP iMab RT4.

[0282] Specifically, in order to examine the tumor growth inhibitory effect of RGD-RT4 in vivo based on the in vitro experiment results of Example 20, KRas G13D mutant human colorectal HCT116 cells were injected subcutaneously into Balb/c nude mice at a density of 5×10.sup.6 cells per mice. After about 6 days when the tumor volume reached about 50 mm.sup.3, the mice were injected intravenously with 20 mg/kg of each of PBS, RGD-TMab4 and RGD-RT4. The injection was performed a total of 9 times at 2-day intervals, and the tumor volume was measured using a caliper for 18 days.

[0283] As shown in FIG. 26A, unlike the control PBS, RGD-TMab4 and RGD-RT4 inhibited the growth of cancer cells, and RGD-RT4 more effectively inhibited tumor growth compared to RGD-TMab4. In addition, as shown in FIG. 26B, there was no change in the body weight of the test group treated with RGD-RT4, indicating that RGD-RT4 has no other toxicities.