WO2010047509A9 - Anti-dr5 antibody with improved affinity and stability, and composition for cancer prevention or treatment including same - Google Patents

Abstract

The present invention relates to an anti-DR5 antibody with improved affinity and stability that specifically binds to the death receptor 5 (DR5) to destroy cancer cells effectively and the composition for cancer prevention or treatment including the same. The anti-DR5 antibody of the present invention causes mutageneses on the amino acid residues of VH-CDR2 and VH-CDR3 within the heavy chain variable region and the amino acid residue of VL-CDR3 within the light chain variable region of HW1 which is the known anti-DR5 antibody, and replaces the framework within the heavy chain variable region from a VH6 subtype to a VH3 subtype and replaces the framework within the light chain variable region with the framework part of Vk1 or Vk3 so that affinity and stability are improved over the known anti-DR5 antibody, HW1. In addition, the anti-DR5 antibody of the present invention effectively induces cell death with respect to TRAIL-sensitive cancer cells that express DR5 and TRAIL-resistant cancer cells that express DR5 by autophagy so that it may be usefully employed in preventing or treating cancers caused by DR5 expression.

Description

Anti-DR5 antibody with improved affinity and stability, and a composition for preventing or treating cancer comprising the same

The present invention specifically binds to a death receptor 5 (hereinafter referred to as "DR5"), anti-DR5 antibody having improved affinity and stability for effectively killing various cancer cells, and a composition for preventing or treating cancer comprising the same. It is about.

Cell death activated by TNF-related apoptosis inducing ligand (TRAIL) in the apoptosis pathway via protein p53-independent tumor necrosis factor receptor (TNFR). The apoptosis mechanism through the receptor 5 (DR5) pathway has been regarded as an important target for cancer drug development because it induces apoptosis specifically in cancer cells while having fewer side effects in normal cells (Ashkenazi et al. J. Clin. Invest ., 104 : 155-162, 1999; and Ashkenazi et al . , Nat. Rev. Cancer , 2: 420-430, 2002).

Research into the development of cancer cell-specific therapeutics targeting DR5 includes the use of recombinant TRAIL (eg, amino acid residues 114-281 of TRAIL), ligands of the apoptosis receptors, and specific apoptosis receptors. There is a method for developing an agonist antibody in a mouse or human complete antibody (eg mAb or IgG) (Pollack et al., Clin. Cancer Res., 7: 1362-1369, 2001; Jo et al., Nat. Med., 6: 564-567, 2000; Ichikawa et al., Nat. Med., 7: 954-960, 2001; and Walczak et al., Nat. Med., 5: 157-161, 1999). However, recombinant TRAIL is highly unstable and forms a soluble oligomer, so that apoptosis activity is reduced by 20 to 100 times, and cells such as astrocytes, hepatocytes, and keratinocytes are normal cells. Toxic and immune, causing side effects (Jo et al . Nat. Med. , 6: 564-567, 2000). TRAIL also does not induce apoptosis in more than 50% of malignant tumor cells (Zhang et al., Cancer Gene Ther., 12: 228-237, 2005). In general, cancer cells killed by TRAIL are called TRAIL-sensitive cancer cells, and cancer cells that do not die by TRAIL are called TRAIL-resistant cancer cells.

Antibodies having affinity specific for DR5, which have been developed to induce apoptosis associated with DR5 to date, include TRA-8 (mouse-derived IgG), a humanized antibody developed from a mouse-derived monoclonal antibody (Walczak et al., Nat. Med., 5: 157-161) and AD5-10 (rat IgG) (Guo et al., J. Biol. Chem., 280: 41940-41952, 2005), HGS-ETR2 (human IgG1), a human-derived monoclonal antibody (Georgakis et al., Br. J. Haematol. , 130: 501-510, 2005) and KMTR2 (human IgG4) (Motoki et al. , Clin. Cancer Res. , 11: 3126-3135, 2005).

All of these antibodies induced apoptosis only in TRAIL-sensitive cancer cells and did not induce apoptosis in TRAIL-resistant cancer cells. In addition, Fab or scFv forms with a monoantigen binding site (monovalent) did not induce apoptosis in cancer cells (eg KMTR2), and showed cytotoxicity only with IgG forms with a double antigen binding site (divalent) ( Examples: HGS-ETR2 and AD5-10) Apoptosis could only be induced by using IgG as a cross-linker (Chuntharapai et al., J. Immunol., 166: 4891-4898, 2001; Motoki et al., Clin. Cancer Res., 11: 3126-3135, 2005; and Wajant et al., Oncogene, 20: 4101-4106, 2001).

Therefore, many studies have been conducted on the development of an antibody having affinity specific for DR5 in place of the existing ligand TRAIL and inducing apoptosis (Walczak et al . , Nat. Med. , 5: 157-161). , 1999; and Guo et al. , J. Biol. Chem. , 280: 41940-41952, 2005; and Georgakis et al. , Br. J. Haematol. , 130: 501-510, 2005). As an example of this study, unlike antibodies previously developed by substituting TRAIL for DR5, TRAIL-sensitive cell lines as well as TRAIL-resistant cell lines are effectively toxic to normal cells through apoptosis pathway by autophagy. An antibody that induces death has been developed (Park et al., Cancer Research , 1; 67 (15): 7237-34, 2007; in Korean Patent No. 10-0847010), and the antibody is applied to the prevention and treatment of various cancers. Can be.

Apoptosis of eukaryotic cells is characterized by three mechanisms, namely apoptosis, autophagy and necrosis, depending on morphological and biochemical characteristics (Kromer G et al. (2005), Cell Death Differ. 12 Suppl 2: 1463-1467). Among these, apoptosis is classified as type II programmed cell death and is increased by nutrient starvation, environmental stress, or various compounds, and the process is called endoplasmic. reticulum (ER) or a newly synthesized double-membrane structure is created, which forms autophagosomes while isolating intracellular organelles (such as mitochondria) or cytoplasmic proteins, ultimately lysosomes and Fusion and destruction (Kondo Y et al. (2005). Nat. Rev. Cancer. 5: 726-734). If the child action is above a certain level, it causes apoptosis, and the development of cancer treatments using such apoptosis is attracting attention (Martinet W et al. (2009), Clinical Science. 116: 697-712).

The V fragment of the variable region of the antibody is a fragment that occupies most of the antibody sequence, and there are various subtypes. In the heavy chain variable region (VH), VH3 is the most stable subtype, and VH1a, VH1b, and VH5 are intermediate. Position, and VH2, VH4 and VH6 fall in terms of stability. In the light chain variable region (VL), Vk3 and Vk1 are known to be the most stable forms. Thus, studies on thermodynamic stability and antibody production yield for each subtype have been preceded (Ewert S et al., J. Mol. Biol., 325: 531-553, 2003; Ewert S et al., Biochemistry, 42: 1517- 1528, 2003). Among these studies, the CDR grafting method is remarkable, as it shows the results of the studies that improved the thermodynamic stability of antibodies by grafting CDRs of less stable antibodies to subtypes having relatively higher thermodynamic stability and yield of antibody production (Jung et al. , Protein.Eng . , 10: 959-966, 1997).

In addition, the improvement of the affinity of the therapeutic antibody can be expected to reduce the single dose, it can lead to a comprehensive treatment efficiency, such as cost reduction and increased efficacy, reduced side effects. However, an increase in ligand-receptor binding affinity in agonists is not necessarily associated with biological activity (Jones et al . , Trends. Biotechnol. , 26: 498-505, 2008). As an example of this, when a variant was prepared in which the affinity to the receptor was increased about 400 times compared to wild-type human growth hormone, but there was no improvement in cell growth (Pearce et al., Biochemistry , 38: 81-89, 1999), or developed an epidermal growth factor (EGF) that binds to the epidermal growth factor receptor (EGFR) in comparison to the wild type, but exhibits an antagonistic activity. Visible cases (Coco et al . , Nat. Biotechnol. , 20: 1246-1250, 2002).

Therefore, various methods have been studied to improve the affinity of the antibody. Specifically, 1) a method of first selecting a variant having improved affinity by randomly changing the whole antibody sequence (Boder ET et al., Proc Natl Acad Sci USA , 26; 97 (20): 10701-5, 2000 ), 2) methods of constructing variants by selecting specific amino acids that are expected to affect affinity enhancement based on the structure of the antibody and previous studies (Barderas R et al., Proc Natl Acad Sci USA, 105 (26): 9029-34, 2008), and 3) a method of mutating and selecting only Complementarity Determining Regions (CDRs), which are sites affecting specific binding to antigens (Garcia-Rodriguez C et al., Nat Biotechnol , 25 ( 1): 107-16, 2007), and each method can be used independently or in combination with each other to have a better effect on improving affinity.

The inventors of the present invention, while studying to develop an anti-DR5 antibody with improved affinity and stability than the existing anti-DR5 antibody, and produced a variant of the existing anti-DR5 human antibody, HW1, and the affinity and The stability was improved more than the existing HW1 and confirmed that effectively induce apoptosis by the progeny action on TRAIL-sensitive cancer cells expressing DR5 or TRAIL-resistant cancer cells expressing DR5, and completed the present invention.

The present invention is to provide an anti-DR5 antibody with improved affinity and stability.

In addition, the present invention is to provide a DNA encoding the anti-DR5 antibody.

In addition, the present invention is to provide a cell transformed with the DNA or an expression vector comprising the same.

In addition, the present invention is to provide a composition for preventing or treating cancer containing the anti-DR5 antibody as an active ingredient.

FIG. 1 is a diagram illustrating primers of VH-CDR2, VH-CDR3, and VL-CDR3 for improving affinity of HW1, an anti-DR5 human antibody.

Figure 2 is a diagram showing the manufacturing process of a variant library for improving the affinity of the anti-DR5 human antibody HW1.

3 is a diagram analyzing the process of selecting a high affinity variant using the FACS from the variant library of HW1.

4 is a diagram analyzing the affinity of the finally selected variants from the variants library of HW1 and HW1 again using FACS.

5 is a diagram showing the entire nucleotide and amino acid sequence of the antibody AU11 (where the underlined portions are in turn VH-CDR1, VH-CDR2, VH-CDR3, linker, VL-CDR1, VL-CDR2, VL-CDR3).

Figure 6 is a view showing a result of comparing the amino acid sequence of the AU11 finally improved the affinity and stability of the existing antibody HW1 (* indicates amino acid residues are changed according to affinity improvement in CDR).

7 is a diagram showing the entire nucleotide and amino acid sequence of the antibody AU12 (where the underlined portions are in turn VH-CDR1, VH-CDR2, VH-CDR3, linker, VL-CDR1, VL-CDR2, VL-CDR3).

FIG. 8 is a diagram showing the results of comparative analysis of the amino acid sequences of AU11 and AU12 which finally improved the affinity and stability of the existing antibody HW1.

9 is a diagram showing the results of analyzing the size and purity of purified antibodies (AU11 and AU12) by reducing SDS-PAGE and non-reducing SDS-PAGE.

10 is a diagram showing the results of analyzing purified antibodies (AU11 and AU12) by size exclusion chromatography.

11 is a diagram showing the result of quantifying the affinity of AU11 and AU12 for DR5 using SPR and comparing the affinity with HW1.

12 shows the results of measuring the stability of AU11 and AU12.

Figure 13 is a diagram showing the result of measuring the same antigen binding site between HW1 and AU11 or AU12 using a competitive ELISA.

14 is a diagram showing the results of measuring the cross-reactivity of AU11 and AU12 against the target antigen DR5 antigen and other analogues (DR4, DcR1, DcR2) by ELISA.

FIG. 15 shows the binding affinity between total DR5 and DR5 fragments CRD2 (residues 97-137) and CRD3 (residues 139-180) and HW1, AU11, AU12, TRAIL having binding capacity to them using ELISA. Figure 1 shows the results.

FIG. 16 is a diagram showing the results of comparing and evaluating the degree of apoptosis of HW1, AU11, and AU12 in HCT116, a human colorectal cancer cell line, and U87MG, a human neurotumor cell line, by MTT-assay.

17 shows MTT-assay of apoptosis ability of HW1, AU11 and AU12 in the presence of various inhibitors (Z-VAD, SP600125, SB203580, 3-MA, Chloroguine) in human colon cancer cell line HCT116 and human neurotumor cell line U87MG. It is a figure which shows the result of analysis.

Fig. 18 shows the results of fluorescence microscopy observation of LC3-GFP aggregates by treatment of AU11 and AU12 to U87MG cancer cell lines expressing LC3-GFP.

The present invention provides an anti-DR5 antibody that specifically binds to DR5, which has the amino acid sequence of SEQ ID NO: 7 or 39.

The present invention also provides a DNA encoding the anti-DR5 antibody.

The present invention also provides a cell transformed with the DNA or an expression vector comprising the same.

The present invention also provides a composition for preventing or treating cancer containing the anti-DR5 antibody as an active ingredient.

Hereinafter, the present invention will be described in detail.

Anti-DR5 antibody according to the present invention, the amino acid residues and light chain variable VH-CDR2, VH-CDR3 in the heavy chain variable region (heavy chain variable region) of the anti-DR5 human antibody HW1 (Korean Patent No. 10-0847010) Mutating amino acid residues of VL-CDR3 in the light chain variable region to enhance affinity for DR5, as well as replacing the framework in the heavy chain variable region from the VH6 subtype to the VH3 subtype, and light chain variable It is characterized by improving the stability by substituting the structure in the region with the structural part of Vk1 or Vk3.

In the present invention, "apoptotic receptor-5 (DR5) protein" means a member of the tumor necrosis factor (TNF) receptor family and binds TRAIL and has an intracellular death domain at the C-terminus ( Pan et al., Science, 277: 815-818, 1997). When DR5 binds to TRAIL, it is known to induce apoptosis in TRAIL-sensitive cancer cells and to increase apoptosis when DR5 is overexpressed, but not to induce apoptosis in normal cells. DR5 includes any protein having the above characteristics, but may be, for example, having an amino acid sequence described in US Pat. No. 6,872,568, but is not limited thereto.

In the present invention, an "antibody" may be an entire form of an antibody ("antibody") or a functional fragment thereof. The whole antibody may be in the form of a monomer or a multimer in which two or more whole antibodies are bound. The functional fragment of the antibody is an antibody having the heavy and light chain variable regions of the whole antibody, which means to recognize substantially the same epitope that the whole antibody recognizes. Functional fragments of the antibody include, but are not limited to, single chain variable region fragments (scFv), (scFv) ₂ , Fab, Fab 'and F (ab') ₂ and the like, and scFv is preferred in the present invention. The single chain variable region (scFv) refers to an antibody fragment in which a heavy chain variable region and a light chain variable region are linked through a linker peptide to take the form of a single chain polypeptide.

The antibody can be generated using methods known in the art, such as phage display methods or yeast cell surface expression systems.

The method described in US Pat. Nos. 4,946,778 and 5,258,498 can be used as a method for preparing scFv, and WO 92/22324 is a method for recombinantly generating Fab, Fab 'and F (ab') ₂ fragments. The method described in the above can be used.

Antibodies of the invention may be derived from any animal, including mammals, birds, and the like, including humans. Preferably, the antibody may be a human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken antibody. Wherein the human antibody is an antibody having an amino acid sequence of human immunoglobulin, which includes an antibody isolated from a human immunoglobulin library or an antibody isolated from an animal transfected against one or more human immunoglobulins and not expressing an endogenous immunoglobulin ( US Pat. No. 5,939,598).

Antibodies of the present invention may be conjugated to enzymes, fluorescent materials, radioactive materials and proteins, but are not limited thereto. In addition, methods of conjugating such materials to antibodies are well known in the art.

Anti-DR5 antibody according to the present invention, the heavy chain variable region having complementary determining region CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 1, 2 and 3 and the complementarity determining region CDR1, CDR2 and CDR3 are SEQ ID NO: 4, 5, respectively And a light chain variable region having the amino acid sequence of 6, specifically binding to DR5 only.

In the present invention, "specifically binds" means that the antibody of the present invention binds only to the DR5 antigen and does not substantially bind to DR5 (death receptor 4), death decoy receptor 1 (DcR1) and DcR2, which are DR5-like antigens. it means.

The light chain variable region has an amino acid sequence of SEQ ID NO: 27 or 37, and the heavy chain variable region has an amino acid sequence of SEQ ID NO: 28 or 38.

The anti-DR5 antibodies of the present invention are AU11 or AU12, which are scFv antibodies having an amino acid sequence of SEQ ID NO: 7 or 39, which in turn comprise CDR1 to CDR3 of the heavy chain variable region, linker oligopeptide and CDR1 to CDR3 of the light chain variable region, respectively. . The antibody is preferably a monoclonal antibody.

Anti-Dr5 antibodies of the present invention, AU11 or AU12, bind to DR5 with higher affinity than HW1, and the equilibrium dissociation constant ( K _D ) shows 1.17 × 10 ⁻⁸ M and 9.17 × 10 ⁻⁹ M, respectively. In addition, the anti-Dr5 antibody AU11 or AU12 is relatively higher in stability than HW1, and similarly to HW1, a single molecule scFv form induces apoptosis by progeny.

The present invention provides DNA encoding AU11 or AU12, which is an anti-DR5 antibody. The DNA may be DNA encoding an scFv having an amino acid sequence of SEQ ID NO: 7 or 39, preferably DNA having a nucleotide sequence of SEQ ID NO: 8 or 40.

DNA sequences encoding the antibodies of the invention can be obtained by methods well known in the art. For example, based on DNA sequences or corresponding amino acid sequences encoding portions or all of the heavy and light chains of the antibody, oligonucleotide synthesis techniques well known in the art, for example site-directed mutagenesis And the polymerase chain reaction (PCR) method.

The present invention also provides a cell transformed with the DNA or an expression vector comprising the same.

The DNA or expression vector comprising the same can be delivered to an appropriate host cell using methods known in the art, such as viral transfection or non-viral based techniques. At this time, the introduction of the DNA or expression vector includes, but is not limited to, adenovirus transformation, gene gun, liposome-mediated transformation and retrovirus or lentiviral-mediated transformation, plasmid, adeno-associated virus, and the like. It may be performed by any known technique. In addition, the cells can be transplanted with a suitable carrier material capable of releasing or delivering the gene to the cells for a long time.

The cells transformed with the DNA or the expression vector including the same may be cultured under appropriate conditions to express and isolate the antibody to produce antibody molecules.

The antibody molecule may accumulate in the cytoplasm of the cell, be secreted from the cell, or may be targeted to periplasm or extracellular medium by an appropriate signal sequence, among which is targeted to periplasm or extracellular medium. desirable. It is also desirable to refold the produced antibody molecules and to have functional conformation using methods well known to those skilled in the art.

When only the heavy or light chain polypeptide of the antibody molecule needs to be produced, a single vector comprising a sequence encoding the heavy or light chain polypeptide is transduced into the host cell, and in order to produce an antibody including both the heavy and light chain polypeptides. To introduce into the host cell two vectors, a first vector encoding the light chain polypeptide and a second vector encoding the heavy chain polypeptide, or a single vector comprising both the light chain and heavy chain polypeptide sequences. It may be.

The present invention also provides a composition for preventing or treating cancer containing the anti-DR5 antibody as an active ingredient.

The anti-DR5 antibodies (AU11 and AU12) according to the present invention bind TRDR-sensitive cancer cells or DR5 that specifically bind to the DR5 antigen with a higher affinity than the existing anti-DR5 antibodies (HW1), and have relatively high stability and express DR5. By effectively inducing apoptosis by the progeny effect on the expressing TRAIL-resistant cancer cells, it is possible to expect the effect of reducing the cost and improving efficiency by reducing the single dose. Therefore, the anti-DR5 antibody of the present invention can be usefully used for the prevention or treatment of cancer caused by the expression of DR5.

Cancer caused by the DR5 expression may include both TRAIL-sensitive cancer cells and TRAIL-resistant cancer cells, specifically, hematologic cancer, lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, Uterine cancer, ovarian cancer, rectal cancer, colon cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, Urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, solid tumors of childhood, differentiated lymphoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, primary central nervous system lymphoma, spinal contraction tumor, brain stem glioma or pituitary gland Adenomas and the like, but is not limited thereto.

The composition of the present invention may contain one or more known active ingredients having an anticancer effect together with the anti-DR5 antibody.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as an antioxidant, buffer And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dose of the anti-DR5 antibody is about 0.01 to 50 mg / kg, preferably about 0.1 to 20 mg / kg, and more preferably administered once to several times a day.

The composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for the prevention and treatment of cancer.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1  : Construction of variant library for improving affinity of HW1

1. Design and fabrication of template and primer for constructing variant library of HW1

In order to enhance the affinity of the anti-DR5 human antibody HW1 (Korean Patent No. 10-0847010), CDR (Complementarity Determining Region) sites, which are known to be important for binding to the antigen, among the heavy chain variable regions Mutant library was designed by giving priority to CDR2, and the vector for expressing the variant on the yeast surface was used pCTCON, a yeast surface expression vector, and random nucleotide sequences were assigned to CDR2 of the heavy chain variable region as shown in FIG. . In this case, in order to preserve the ratio of each wild-type amino acid constituting the heavy chain variable CDR2 to a predetermined level or more, the ratio of wild-type bases is 70% instead of the base mixtures of 25% each of the four bases generally used. A base mixture was used (for example, adenine 70% adenine, guanine, cytosine, thymine each 10% instead of the base mixture containing 25% adenine, guanine, cytosine and thymine at the site where the wild type base is adenine (A)). Variants were constructed using the base mixture contained thick). In the same manner as described above, a primer (fragment 1-SEQ ID NO: 9, SEQ ID NO: 12; fragment 2-SEQ ID NO: 11, SEQ ID NO: 10) (Gennotek, Korea) was finally prepared so that the retention rate of each amino acid was higher than a predetermined level. Subsequently, variants with improved affinity were selected from the mutant library of heavy chain variable CDR2, and the primers (heavy chain variable CDR3: fragment) were selected in the heavy chain variable CDR3 and light chain variable CDR3 sites, respectively, using the selected antibodies as templates. 1-SEQ ID NO: 9, SEQ ID NO: 14, fragment 2-SEQ ID NO: 13, SEQ ID NO: 10; light chain variable CDR3: fragment 1-SEQ ID NO: 9, SEQ ID NO: 16, fragment 2: SEQ ID NO: 15, SEQ ID NO: 10) Two libraries have been built to further enhance affinity. Primer sequence numbers and sequences used to construct variants for improving affinity are shown in Table 1 below.

TABLE 1

※ The numbers in SEQ ID NOs: 11, 13, and 15 in the sequence mean the following (1: 70% A + 10% G + 10% C + 10% T, 2: 10% A + 70% G + 10% C + 10% T, 3: 10% A + 10% G + 70% C + 10% T, 4: 10% A + 10% G + 10% C + 70% T, 5: 33% C + 33% G + 33% T).

2. Amplification and binding of each fragment of antibody gene

In order to introduce mutations into the heavy chain variable CDR2 of the anti-DR5 antibody, two fragments were prepared as shown in FIG. 2. The first fragment is 5'-GGT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GCT AGC-3 '(SEQ ID NO: 9) and 5'-CCT TCC CAG CCA CTC AAG GCC-3 as reverse primer. (SEQ ID NO: 12) was used to perform a polymerase chain reaction (PCR). The second fragment is 5'-GGC CTT GAG TGG CTG GGA AGG 135 415 415 325 435 115 425 415 115 215 TAT GCA GTA TCT GTG AAA AGT-3 '(SEQ ID NO: 11) with forward primer and 5'-GAT CTC with reverse primer. GAG CTA TTA CAA GTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC-3 '(SEQ ID NO: 10) was prepared by PCR method. PCR was performed using pfu polymerase (Intron, Korea), and the reaction of 40 seconds at 94 ° C, 30 seconds at 55 ° C, and 40 seconds at 72 ° C was repeated 30 times.

Similarly, fragments for the variants of the heavy chain variable CDR3 and light chain variable CDR3 sites, respectively, were prepared in the same manner as above. The first fragment of heavy chain variable CDR3 was 5'-GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GCT AGC-3 '(SEQ ID NO: 9) as the forward primer, and 5'-ACA GTA ATA GAC GGC as the reverse primer. CGT GTC-3 '(SEQ ID NO: 14) was used, and the second fragment was a 5'-GAC ACG GCC GTC TAT TAC TGT 235 125 215 335 215 235 225 125 225 235 445 215 145 TGG GGC CAA GGG ACC ACG GTC -3 '(SEQ ID NO: 13), 5'-GAT CTC GAG CTA TTA CAA GTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC-3' (SEQ ID NO: 10) as a reverse primer was prepared by the PCR method. The first fragment of the light chain variable CDR3 was 5'-GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GCT AGC-3 '(SEQ ID NO: 9) as the forward primer, 5'-ACA GTA ATA AAC TGC as the reverse primer AAA ATC-3 '(SEQ ID NO: 16) was used, and the second fragment was a 5'-GAT TTT GCA GTT TAT TAC TGT 315 315 325 125 115 425 335 335 325 235 245 TTC GGC CAA GGG ACA CGA CTG-3 '(SEQ ID NO: 15), 5'-GAT CTC GAG CTA TTA CAA GTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC-3' (SEQ ID NO: 10) as a reverse primer was prepared by the PCR method.

Each of the two fragments prepared was electrophoresed on a 1% agarose gel and purified using an agarose gel purification kit (Intron, Korea). The purified two fragments were mixed in the same amount by 10 μM each, and then, as shown in FIG. 2, overlap extension PCR was performed to prepare a mutated whole scFv gene product. The PCR was performed using pfu polymerase (Intron, Korea), and the reaction of 40 seconds at 94 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C was repeated 30 times.

3. Construction of Mutated scFv Antibody Gene Library

Mutant-inserted scFv antibody gene library (10 µg / µl) and scFv yeast surface expression vector pCTCON (1 µg / µl) were mixed, and then, electroporation was performed to the yeast EBY100 strain (Invitrogen, USA). Transformed. The transformed yeast EBY100 strain was selected as SD-CAA (-ura, -trp; 20 g / l glucose, YNB without 6.7 g / l amino acid (BD, USA), 5.4 g / l Na ₂ Library inducing cell surface expression of scFv in SG-CAA medium substituted with glucose to galactose after inoculation with HPO ₄ , 8.56 g / L NaH ₂ PO ₄ H ₂ O and 5 g / L casamino acid) Were screened. Cells containing the library were diluted 10-fold in SD-CAA medium stepwise to determine library size.

As a result, it was confirmed that the scFv antibody library size was each constructed at about 2 × 10 ⁷ .

Example 2  : Screening for Improved Affinity Variants from Constructed HW1 Variant Library

Anti-c-myc mAb (Ig therapy, Korea) and biotin label diluted 1: 100 to select scFv variants showing high affinity to DR5 from the variant library of HW1 constructed in Example 1 above Biotin-labeled DR5 was added to PBS (PBSB, pH 7.4) with 0.2 mg of 1 mg / mL BSA using the kit (EZ-LINK ^™ Sulfo-NHS-LC-Biotinylation kit, Pierce, USA). Then, incubated for 30 minutes at 25 ℃. After washing the cells with chilled PBSB, the streptavidin conjugated with FITC-labeled anti-mouse IgG (diluted at 1:25) and phycoerythrin as a secondary antibody (SA- PE), Molecular probes, USA; diluted 1: 100). After the labeled cells were washed and resuspended in PBSB, fluorescence activated cell sorting (FACS) was performed sequentially to select high affinity variants. Selected variants were incubated in SD-CAA, a selection medium, relabeled in the same manner as described above, and the selection process was repeated four times. At this time, the concentration of biotin-labeled DR5 used in the selection process was gradually lowered from the initial 1 μM, and the concentration of 10 nM was used for the final 4th screening. Nucleotide sequences of the selected variants were forward primer 5'-GTT CCA GAC TAC GCT CTG CAG G-3 '(SEQ ID NO: 17) and reverse primer 5'-GAT TTT GTT ACA TCT ACA CTG TTG-3' (SEQ ID NO: 18) After analysis using the amino acid sequence was determined and compared with the amino acid sequence of the wild type.

The results are shown in FIG.

As shown in FIG. 3, the affinity of the HW1 variant was increased for each selection step.

Example 3  : Affinity binding of selected antibodies

The amino acid sequence of the variants selected from each library was determined and the changed CDRs of the selected wildtypes were respectively combined to finally produce new variants with the most affinity. A variant derived from heavy chain variable CDR3 was used as a template, and 5'-GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GCT AGC-3 '(SEQ ID NO: 9) and 5'- as reverse primer. Fragments were prepared by PCR using ACA GTA ATA AAC TGC AAA ATC-3 ′ (SEQ ID NO: 16). Using a variant derived from the light chain variable CDR3 as a template, 5'-GAT TTT GCA GTT TAT TAC TGT-3 '(SEQ ID NO: 19) as the forward primer, 5'-GAT CTC GAG CTA TTA CAA GTC CTC TTC AGA as the reverse primer A fragment was prepared by PCR using AAT AAG CTT TTG TTC GGA TCC-3 ′ (SEQ ID NO: 10), and the reaction was repeated 30 times at 40 ° C. at 94 ° C., 30 seconds at 55 ° C., and 50 seconds at 72 ° C. Each of the two fragments prepared was electrophoresed on a 1% agarose gel and purified using an agarose gel purification kit (Intron, Korea), and the two purified fragments were mixed in equal amounts by 10 μM each, and then superimposed. Extended PCR was performed to finally produce a variant whole scFv gene product with improved affinity. The PCR was repeated 30 times 40 minutes at 94 ℃, 30 seconds at 55 ℃, 1 minute at 72 ℃. The mutant scFv product thus prepared was introduced into the yeast surface expression vector pCTCON (Colby et al., Methods Enzymol., 388: 348-358, 2004) using the restriction enzyme Nhe I / BamH I (NEB, USA). The plasmid was transformed into yeast EBY100 strain (Invitrogen).

The affinity was analyzed again using FACS in the same manner as described in Example 2 were selected variants and finally improved affinity variants prepared above. As a control, HW1 was compared by labeling under the same conditions.

The results are shown in FIG.

As shown in FIG. 4, the final variant to which each variant was bound showed the highest affinity than the other variants.

Example 4  : Improved antibody stability of affinity variants

1. Preparation of AU11, an antibody with improved affinity and stability

HW1, an anti-DR5 human antibody, is composed of the VH6 subtype for the heavy chain variable region and Vk3 for the light chain variable region, and the VH6 subtype is compared to other subtypes, particularly VH3. It is known that the stability is low. Thus, in order to improve the antibody stability of the variants with improved affinity, the VH6 subtype was substituted with the VH3 subtype. First, in order to prepare the antibody of the VH3-Vk3 form, the whole gene was divided into three fragments and each fragment was prepared. The first fragment is a forward primer 5'-TTC GCT AGC GAG GTG CAG CTG GTG GAG TCT GGG GGA GGC CTG GTA CAG CCT GGA GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA GAC AGT GTC TCT AGC ACC ACT GTT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG GAG TGG GTC-3 '(SEQ ID NO: 20), 5'-ACA GTA ATA CAC AGC CGT GTC CTC GGC TCT CAG GCT GTT CAT TTG CAG ATA CAG GGT GTT CTT GGA ATT GTC TCT GGA GAT GGT GAA CCG GCC CTT CAC GGA GTC CGC ATA GTA ATT ATA CCA CTT CGA CCT ATA ATA GAT CGC TGA GAC CCA CTC CAG CCC CTT CCC-3 '(SEQ ID NO: 21). The second fragment is 5'-GAG GAC ACG GCT GTG TAT TAC TGT-3 '(SEQ ID NO: 22) with forward primer, 5'-TTC CCC TGG AGA CAA AGA CAG GGT GGC TGG AGA CTG GGT CAA TAC AAT ATC CGA with reverse primer Produced using CCC GCC ACC GCC GCT GCC ACC-3 '(SEQ ID NO: 23). The third fragment is 5'-ACC CTG TTG TCT TTG TCT CCA GGG GAA-3 '(SEQ ID NO: 24) with forward primer, 5'-CGT GGA TCC ACG TTT GAT TTC CAC CTT TGT CCC TTG GCC GAA GAC CGC CCG- It was produced using 3 '(SEQ ID NO: 25). In addition, as a linker linking the heavy chain variable region and the light chain variable region, 4 glycine and 1 serine is repeated three times, using a (G ₄ S) ₃ linker consisting of a total of 15 amino acids (SEQ ID NO: 26). Each fragment thus prepared was subjected to overlap extension PCR to finally prepare a variant whole scFv gene product having improved stability. The PCR was repeated 30 times 40 minutes at 94 ℃, 30 seconds at 55 ℃, 1 minute at 72 ℃. Subsequently, valine # 101 and # 102 serine in the CDR3 region of the heavy chain variable region were replaced with aspartic acid and isoleucine to finally prepare AU11, an antibody having improved affinity and stability. Primer sequence numbers and sequences used to improve stability with antibody AU11 are shown in Table 2 below.

Schematic representation of the entire nucleotide and amino acid sequence of antibody AU11 is shown in FIG. 5 (wherein the underlined portions are in turn VH-CDR1, VH-CDR2, VH-CDR3, linker, VL-CDR1, VL-CDR2, A comparison of the amino acid sequence of AU11 with improved affinity and stability with the existing antibody HW1 is shown in FIG. 6 (* is an amino acid residue which is changed according to affinity improvement in CDR). ).

TABLE 2

2. Preparation of AU12, an antibody with improved affinity and stability

In the light chain variable region, Vk3 and Vk1 are known to be the most stable, and many Vk1 subtypes exist in various mouse-derived humanized antibodies commercially approved by the US FDA (Carter et al., Proc Natl Acad). Sci USA , 10; 89: 4285-4289, 1992; Werther et al ., J Immunol , 11; 157: 4986-4995, 1996; Presta et al ., J Immunol 5; 151: 2623-2632, 1993). In addition, vernier zone residues contribute to thermodynamic stability in antigen binding, and are known as sites that modulate the structural entropy changes of antibodies and consequently have a significant effect on antigen binding (Makabe et al. J Biol Chem , 11; 283 (2): 1156-1166, 2008), three-dimensional simulation analysis of the residues of the Vernier region and other antibody structures among the amino acids constituting the Vk1 subtype (http: // antibody) .bath.ac.uk) was selected to further select residues considered to be structurally important. In addition to the VH3-Vk3 antibody AU11, VH3- was added to the serine by replacing isoleucine, the second amino acid of the modified chain, with serine. Antibodies having the Vk1 subtype were produced. With AU11 as template, the first fragment was 5'-TTC GCT AGC GAG GTG CAG CTG GTG GAG TCT GGG-3 '(SEQ ID NO: 29) with forward primer, 5'-TGG AGA CTG GGT CAT CTG AGA GTC with reverse primer -3 '(SEQ ID NO: 30) was used, and the second fragment was a 5'-GAC TCT CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC GTT AGC-3 '(SEQ ID NO: 31), 5'-CTT AGG GGC TTT CCC TGG TTT CTG CTG ATA CCA GGC TAA GTG GCT-3' (SEQ ID NO: 32) was used as the reverse primer, and the third fragment was a forward primer 5'-AAA GCC CCT AAG CTC CTG ATC TAT GGT GCA TCC AGC-3 '(SEQ ID NO: 33), 5'-GAT GGT GAG AGT GAA ATC TGT CCC AGA TCC ACT GCC ACT GAA CCT TGA TGG GAC CCC AGT GGC CCT-3 '(SEQ ID NO: 34) was used, and the fourth fragment was 5'-TTC ACT CTC ACC ATC AGC AGT CTG CAA CCT GAA GAT TTT GCA ACT TAC TAC TGT CAA CAG CGT GCA AAC GAT TTC CCG CGG G CG GTC-3 '(SEQ ID NO: 35) and 5'-CGT GGA TCC ACG TTT GAT TTC CAC-3' (SEQ ID NO: 36) were prepared as reverse primers. Like AU11, as a linker linking the heavy chain variable region and the light chain variable region, (G ₄ S) ₃ linker in which ₄ glycine and 1 serine was repeated three times was used. Each fragment thus prepared was subjected to overlap extension PCR to finally prepare a variant whole scFv gene product having improved stability. In the PCR, AU12 was finally produced by repeating a reaction of 40 seconds at 94 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C for 30 times. Primer sequence numbers and sequences used in the preparation of AU12 are shown in Table 3 below.

A schematic of the entire nucleotide and amino acid sequence of antibody AU12 is shown in FIG. 7 (wherein the underlined portions are in turn VH-CDR1, VH-CDR2, VH-CDR3, linker, VL-CDR1, VL-CDR2, VL-CDR3), and comparing the amino acid sequence of AU11 and AU12 finally improved affinity and stability with the existing antibody HW1 is shown in FIG.

TABLE 3

Example 5  : Expression and Purification of AU11 and AU12 scFv Antibodies

Antibodies with improved affinity and stability, AU11 and AU12, were introduced into the E. coli expression vector pKJ1 in-frame using Nhe I / BamH I. At this time, the E. coli expression vector was designed to have a T7 promoter-pelB targeting sequence-AU11 or AU12 scFv-flag tag-6X his tag.

After transforming the expression vector into Escherichia coli BL21 (DE3), LB [5 g / L yeast extract (BD company, USA), 10 g / L tryptone (BD company, USA) to which 50 μg / ml of antibiotic ampicillin was added ), 10 g / L sodium chloride] medium at 37 ° C. and 200 rpm to incubate the OD ₆₀₀ at 0.8 to 1.0, followed by addition of 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) and 23 ° C., 150 rpm for 16 hours. Incubated under the conditions of. After expression, only the supernatant was taken by centrifugation, and then, the antibody expressed using Talon-resin (Clontech) was purified, and the size and purity of the purified antibody were analyzed by reducing SDS-PAGE and non-reducing SDS-PAGE.

The results are shown in FIG.

As shown in FIG. 9, antibodies AU11 and AU12 have a molecular weight of about 29 kDa. It was confirmed that the purity was 99% or more.

Experimental Example 1  : Morphology Identification in AU11 and AU12 scFv Antibodies

To determine whether purified AU11 and AU12 scFv antibodies exist in monomeric or oligomeric form in solution, size-exclusion HPLC, reducible SDS-PAGE, and non-reducing SDS-PAGE Was performed.

For size exclusion HPLC, the elution buffer is PBS (50 mM phosphate, pH 7.4, 150 mM NaCl), flow rate 0.5 ml / min, column 280 nm using SuperdexTM 200 10/300 GC (GE, USA) and Agilent 1100 HPLC instrument Absorbance was measured at. Reducing SDS-PAGE was performed using 15% acrylamide gel with 1 mM DTT added to the sample buffer and non-reducing SDS-PAGE was performed with 15% acrylamide gel without 1 mM DTT added to the sample buffer, 150 volts. Analyze by loading.

Purified AU11 and AU12 scFv antibodies were analyzed by reducing SDS-PAGE and non-reducing SDS-PAGE and the results of size exclusion HPLC measurement are shown in FIGS. 9 and 10, respectively.

As shown in FIG. 9, the purified AU11 and AU12 scFv antibodies were free of multimolecular forms with non-natural disulfide bonds in reducing SDS-PAGE and non-reducing SDS-PAGE.

In addition, as shown in FIG. 10, when injected with 500 μg / ml of purified AU11 and AU12 scFv antibodies in size exclusion HPLC, the mixture was eluted as pure single molecule as HW1.

Experimental Example 2  : Affinity Measurement of Antibodies AU11 and AU12

In order to confirm the binding affinity of AU11 and AU12 for the DR5 antigen, it was measured using a Biacore 2000 surface Plasmon resonance (SPR) biosensor (GE, USA). About 0.5 mg / ml of antigen and negative control group BSA (bovine serum albumin) were immobilized on a carboxymethylated dextran surface chip (CM5) at about 2,000 response units, and then the antibodies AU11 and AU12 diluted at various concentrations were added. Interaction with antigen was quantified by injection into the chip for 2 minutes at a rate of 30 μl / min. In addition, in order to compare affinity with HW1, HW1 was prepared at a concentration of 50nM, AU11 and AU12 at 25nM, respectively, and injected at the same time. The surface of the chip was regenerated with 1M NaCl / 20mM NaOH, BIA evaluation ver. Using the 3.2 software to obtain the affinity to the movement rate constants (k _on and k _off) and the equilibrium dissociation constant (K _D).

The results of quantification of the affinity of AU11 and AU12 for DR5 using SPR and the comparison of the affinity with HW1 are shown in FIG. 11, and the affinity of AU11 and AU12 for DR5 was determined by the kinematic velocity constant (k _on). And k _off ) and the equilibrium dissociation constant (K _D ) are shown in Table 4.

TABLE 4

As shown in Figure 11 and Table 4, AU11 and AU12 showed affinity about 17-22 times higher than HW1 (K _D : about 200nM). In addition, simultaneous analysis of HW1, AU11, and AU12 at the same concentration showed that AU11 and AU12 bind more strongly in the dissociation interval than HW1.

Experimental Example 3  : Stability Measurement of Antibodies AU11 and AU12

The stability of AU11 and AU12 against HW1 was measured using a fluorescence spectrometer (JASCO, Japan). HW1, AU11 scFv, and AU12 scFv were prepared by expression and purification in E. coli, and the prepared proteins were dissociated into guanidine-HCl (guanidine-HCl) in 0.3M units from 0M to 4.2M, respectively, to give a final concentration of 10 µg / ml. After reacting at room temperature for 24 hours at a concentration, an excitation wave length of 280 nm, an emission wave length of 290 to 400 nm, and a measurement rate of 250 nm / min were repeated three times. The relative maximum wavelength (normalized l _max ) was plotted by normalizing the wavelength at the highest value for each guanidine-HCl concentration. HW1 has a median value at a concentration of about 1.64M guanidine-HCl, and AU1 and AU12 have a median value at a concentration of about 2.53M and 2.45M, respectively. According to Ewert S et al., Biochemistry , 42: 1517-1528, 2003, the relative maximum wavelength increases as the protein is denatured by a protein denaturation sample such as guanidine-HCl.

The results are shown in FIG.

As shown in FIG. 12, it can be seen that AU11 and AU12 have relatively higher stability than HW1.

Experimental Example 4  : Identification of the same epitope between HW1, AU11, and AU12 for DR5

To determine whether AU11 and AU12 had the same antigen-binding site as HW1 for DR5, a competitive ELISA between AU11 or AU12 and HW1 was performed.

Specifically, 50 μl (20 μg / ml) of antigen was coated on a 96-well plate for ELISA, followed by incubation at 37 ° C. for 1 hour. After washing three times with PBS (pH 7.4, with 0.05% Tween 20 and 0.02% sodium azide) and incubated for 1 hour at 37 ℃ in PBS containing 1% BSA. Thereafter, the biotin-labeled HW1 was immobilized at 30 µg / ml, the concentration of AU11 or AU12 was 5 to 300 µg / ml, mixed and added to each well, followed by incubation at 37 ° C for 1 hour, and the plate was After washing, an anti-biotin antibody conjugated with alkaline phosphatase (anti-biotin mAb AP conjugated, Sigma) was added and reacted at 37 ° C. for 1 hour. After washing three times, 50 μl of pNPP (Sigma.) Was added to the substrate, and the absorbance was measured at 405 nm.

The results are shown in FIG.

As shown in FIG. 13, as the concentration of AU11 or AU12 increased, the binding strength of HW1 to the antigen was weakened. Thus, it can be seen that AU11 or AU12 has the same antigen binding site as HW1.

Experimental Example 5  : Cross-Reactivity Analysis of AU11 and AU12 for DR5 and Amusement Park

ELISA was performed to confirm the cross-reactivity of AU11 and AU12 against DR5, the target antigen, and DR4, DcR1, DcR2, the other antigens.

Specifically, antigens, namely DR5, DR4, DcR1 and DcR2, were coated in 50 μl (20 μg / ml) in 96-well plates for ELISA, followed by incubation at 37 ° C. for 1 hour. After washing three times with 300 μl of PBST (pH 7.4, with 0.1% Tween 20), 300 μl of PBST (pH 7.4, with 0.1% Tween 20) containing 5% skim milk was added for 1 hour at room temperature. Each of the antibodies AU11, AU12, and TRAIL, a ligand capable of binding to all receptors, was added to each of the receptors, and 50 µl (3 µg / ml) of each antibody was incubated at 37 ° C for 1 hour. After washing three times with 300 μl of PBST, incubated at 37 ° C. for 1 hour by adding a mouse anti-FLAG-TAG (mouse anti-FLAG tag antibody, sigma) antibody derived from rats, and then washed three times with 200 μl of PBST. Rabbit-derived anti-mouse mAb AP conjugated (pierce) antibody was added and incubated at 37 ° C. for 1 hour. After washing three times with 300 μl of PBST, 50 μl of pNPP (Sigma.) Was added as a substrate, and the absorbance was measured at 405 nm.

The results are shown in FIG.

As shown in Figure 14, AU11 and AU12 specifically bound only to the target antigen DR5, it was confirmed that the positive control TRAIL binds to all DR5, DR4, DcR1 and DcR2. Thus, it can be seen that the anti-DR5 antibody of the present invention is an antibody that specifically binds to the DR5 antigen.

Experimental Example 6  : Determination of binding between TRAIL, HW1, AU11, and AU12 for DR5 and DR5 fragments

DR5 can be subdivided into sub-domains that each have three disulfide bonds, structurally called cysteine-rich domains (CRDs). Among these subdomains, CRD2 (residues 97-137) and CRD3 (residues 139-180) each contain 50s loop and 90s loop regions that are critical for binding to TRAIL (Hymowitz et al . , Mol. Cell. , 4: 563-571, 1999). Therefore, CRD2, CRD3, and DR5 were purified from E. coli BL21 (DE3) to determine whether they bind to TRAIL, HW1, AU11, and AU12 by ELISA. Specifically, 50 μl (20 μg / ml) of antigens, namely CRD2, CRD3, and DR5, were coated on a 96-well plate for ELISA, followed by incubation at 37 ° C. for 1 hour. After washing three times with 300 μl of PBST (pH 7.4 with 0.1% Tween 20), 200 μl of PBST (pH 7.4 with 0.1% Tween 20) containing 5% skim milk was incubated at room temperature for 1 hour, and each antibody HW1 , AU11, AU12, and TRAIL were added to 50 µl (20 µg / ml) and incubated at 37 ° C for 1 hour. After washing three times with 300 μl of PBST, incubated at 37 ° C. for 1 hour by adding an anti-FLAG-TAG (mouse anti-FLAG tag antibody, sigma) antibody derived from a mouse, and then washed three times with 300 μl of PBST. Rabbit-derived anti-mouse mAb AP conjugated (pierce) antibody was added and incubated at 37 ° C. for 1 hour. After washing three times with 300 μl PBST, 50 μl of pNPP (Sigma.) Was added as a substrate, and the absorbance was measured at 405 nm.

The results are shown in FIG.

As shown in FIG. 15, TRAIL showed a stronger binding force to CRD2 than CRD3, but HW1, AU11 and AU12 bound to each antigen with similar binding force for both CRD2 and CRD3. Therefore, it can be seen that the degree of effect of CRD2 and CRD3 on TRAIL or antibodies differs in the binding between TRAIL-DR5 and the binding between HW1, AU11, and AU12-DR5.

Experimental Example 7  : Confirmation of apoptosis induction of antibody AU11 and AU12 in cancer cells

In order to confirm the possibility of inducing apoptosis in various cancer cells of the antibodies AU11 and AU12, the following experiment was performed.

As cancer cells, HCT116, a human colon cancer cell line, and U87MG, a human glioma cancer cell line, were used. The cell lines stored in liquid nitrogen were taken out, dissolved rapidly at 37 ° C., and centrifuged to remove the cryopreservation medium. The cells obtained from the culture medium were cultured with DMEM medium containing 10% FBS, 100unit / ml penicillin and 100µg / ml streptomycin. (Welgene) was used to cultivate the culture flask. After 3 days of stabilization, 1 ml of TE buffer was treated for 3 to 5 minutes, and then the reaction of TE buffer was stopped with 5 ml of DMEM medium containing 10% FBS, followed by centrifugation to recover the cells. The recovered cells were each resuspended in the same culture medium, and the cells per well were dispensed in 96-well plates by 1 × 10 ⁴ (100 μl), incubated for 24 hours, and used for MTT analysis.

The results are shown in FIG.

As shown in FIG. 16, when AU11, AU12, and control HW1 were cultured at the same concentration in HCT116, a human colon cancer cell line, and U87MG, a human neurotumor cell line, at the same concentration, both AU11 and AU12 showed apoptosis effect. , Compared with the apoptosis effect of HW1 confirmed that apoptosis occurs more efficiently.

Experimental Example 8  : Apoptosis Pathway Assay of Antibodies AU11 and AU12

In order to analyze the apoptosis mechanisms of the antibodies AU11 and AU12, the following experiment was performed.

Human colon cancer cell line HCT116 and human neural tumor cell line, U87MG to a 96-well plate at 1 × 10 ⁴ gae added and for 10% FBS DMEM medium is put 100㎕ is added 5% CO _2, at 37 ℃ 2 il Culture was stabilized. Next, 10 μM of Z-VAD (Pan-caspase inhibitor, Santacruz), 10 μM of SP600125 (JNK inhibitor, calbiochem), 10 μM of SB203580 (p38 inhibitor, calbiochem), 10 μM of 3-MA 100 (Autophagic cell death inhibitor, sigma), Chloroguine ( Autophagic cell death inhibitor (sigma) 10μM was added and pretreated for 1 hour. AU11 and AU12 were added to 30µg / ml, respectively, and then cultured at 5% CO ₂ and 37 ° C for 40 hours to measure the degree of cell death through MTT-assay.

The results are shown in FIG.

As shown in Figure 17, AU11 and AU12 significantly reduced apoptosis in the presence of SP600125 (JNK inhibitor), 3-MA and Chloroquine in human colon cancer cell line HCT116 and human neuronal tumor cell line U87MG, Z-VAD There was no change in the degree of apoptosis in the presence of and SB203580 (p38 inhibitor). The results are the same as the scFv antibody HW1. Therefore, it can be seen that AU11 and AU12 having improved affinity and stability induce apoptosis by progeny.

Experimental Example 9  : LC3-GFP aggregation by antibodies AU11 and AU12

A typical feature of apoptosis by apoptosis is the accumulation of intracellular LC3 proteins in or on the vacuole membrane. In order to determine whether these traits were characterized by treatment with AU11 and AU12, U87MG cancer cell lines (TRAIL-resistant, human glioma cell lines) expressing LC3-GFP were added to 4 × 10 ⁴ cells in 24-well plates. 500 μl of DMEM containing 10% FBS was added thereto, followed by stabilization by incubating at 5% CO ₂ for 24 hours at 37 ° C. for 2 days. Then, AU11 and AU12 were added to 30 µg / ml, respectively, and incubated at 5% CO ₂ , 37 ° C for 30 hours, and observed with a fluorescence microscope (Axiovert 200M, Carl Zeiss, Germany).

The results are shown in FIG.

As shown in FIG. 18, it was observed that LC3-GFP aggregated in the child acting vesicle membrane and the inside of U87MG cancer cell lines treated with AU11 and AU12. Thus, it can be seen that AU11 and AU12 induce apoptosis by child action.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation Example 1  : Preparation of powder

0.1 g of anti-DR5 antibody

Lactose 1.5 g

Talc 0.5 g

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2  : Preparation of Tablet

0.1 g of anti-DR5 antibody

Lactose 7.9 g

1.5 g of crystalline cellulose

0.5 g of magnesium stearate

After mixing the above components, a tablet was prepared by a direct tableting method.

Formulation Example 3 : Preparation of Capsule

0.1 g of anti-DR5 antibody

5 g of corn starch

4.9 g of carboxy cellulose

After the powder was prepared by mixing the above components, the powder was filled in a hard capsule according to a conventional method for preparing a capsule to prepare a capsule.

Formulation Example 4  : Preparation of Injection

0.02-0.2 g of anti-DR5 antibody

Appropriate sterile distilled water for injection

pH adjuster

Stabilizer

According to the conventional method for preparing an injection, the amount of the above-mentioned ingredient was prepared per ampoule (2 ml).

Formulation Example 5  : Manufacture of liquid

0.1 g of anti-DR5 antibody

10 g of isomerized sugar

5 g of mannitol

Purified water

Each component was added to and dissolved in purified water according to the conventional method for preparing a liquid, and lemon flavor was added appropriately, followed by mixing the above components. Then, purified water was added thereto to adjust the total volume to 100 ml, and filled into a brown bottle and sterilized to prepare a liquid.

The anti-DR5 antibody according to the present invention has a higher affinity than a conventional anti-DR5 antibody, specifically binds to a DR5 protein, has a relatively high stability, and is a TRAIL-sensitive cancer cell expressing DR5 or a TRAIL-resistant cancer cell expressing DR5. By effectively inducing apoptosis by progeny, it can be usefully used for the prevention or treatment of cancer caused by DR5 expression. In addition, the anti-DR5 antibody according to the present invention can be expected to reduce costs and improve efficiency by reducing the single dose.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION

<120> Anti-death receptor 5 antibody with improved affinity and

         stability, and composition for preventing or treating cancer

         configuring the same

<130> P09-07023

<150> KR10-2008-0104737

<151> 2008-10-24

<160> 40

<170> KopatentIn 1.71

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<223> VH-CDR1

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Gly Asp Ser Val Ser Ser Thr Thr Val Ala

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Ala Arg Glu Gly Ser Gly His Tyr Gly Ala Phe Asp Ile

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Gln Gln Arg Ala Asn Asp Phe Pro Arg Ala Val

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<220>

<223> amino acid sequence of AU11 scFv

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Ser Val Ser Ser Thr

             20 25 30

Thr Val Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

         35 40 45

Trp Val Ser Ala Ile Tyr His Arg Ser Tyr Trp Asp Lys Tyr Tyr Ala

     50 55 60

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

 65 70 75 80

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

                 85 90 95

Tyr Tyr Cys Ala Arg Glu Gly Ser Gly His Tyr Gly Ala Phe Asp Ile

            100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

        115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln

    130 135 140

Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser

145 150 155 160

Cys Arg Ala Ser Gln Ser Val Ser Ser Ser His Leu Ala Trp Tyr Gln

                165 170 175

Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser

            180 185 190

Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

        195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val

    210 215 220

Tyr Tyr Cys Gln Gln Arg Ala Asn Asp Phe Pro Arg Ala Val Phe Gly

225 230 235 240

Gln Gly Thr Lys Val Glu Ile Lys Arg

                245

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<213> Artificial Sequence

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223 nucleotide sequence of AU11 scFv

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gaggtgcagc tggtggagtc tgggggaggc ctggtacagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggaga cagtgtctct agcaccactg ttgccatgag ctgggtccgc 120

caggctccag ggaaggggct ggagtgggtc tcagcgatct atcacaggtc gtattgggac 180

aagtactatg cggactccgt gaagggccgg ttcaccatct ccagagacaa ttccaagaac 240

accctgtatc tgcaaatgaa cagcctgaga gccgaggaca cggctgtgta ttactgtgca 300

agagagggta gtggccacta tggggctttt gatatctggg gccagggaac cctggtcacc 360

gtttcttcag gtggaggcgg ttcaggcgga ggtggcagcg gcggtggcgg gtcggatatt 420

gtattgaccc agtctccagc caccctgtct ttgtctccag gggaaagagc caccctctcc 480

tgcagggcca gtcagagtgt tagcagcagc cacttagcct ggtaccagca gaaacctggc 540

caggctccca ggctcctcat ctatggtgca tccagcaggg ccactggcat cccagacagg 600

ttcagtggca gtgggtctgg gacagacttc actctcacca tcagcagcct agagcctgaa 660

gattttgcag tttattactg tcagcagcgt gcaaacgatt tcccgcgggc ggtcttcggc 720

caagggacaa aggtggaaat caaacgt 747

<210> 9

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer

<400> 9

ggtggtggtg gttctggtgg tggtggttct ggtggtggtg gttctgctag c 51

<210> 10

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer

<400> 10

gatctcgagc tattacaagt cctcttcaga aataagcttt tgttcggatc c 51

<210> 11

<211> 72

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer

<400> 11

ggccttgagt ggctgggaag gnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ntatgcagta 60

tctgtgaaaa gt 72

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer

<400> 12

ccttcccagc cactcaaggc c 21

<210> 13

<211> 81

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer

<400> 13

gacacggccg tctattactg tnnnnnnnnn nnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60

tggggccaag ggaccacggt c 81

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer

<400> 14

acagtaatag acggccgtgt c 21

<210> 15

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer

<400> 15

gattttgcag tttattactg tnnnnnnnnn nnnnnnnnnnnn nnnnnnnnnn nnnnttcggc 60

caagggacac gactg 75

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer

<400> 16

acagtaataa actgcaaaat c 21

<210> 17

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> forward sequencing primer

<400> 17

gttccagact acgctctgca gg 22

<210> 18

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse sequencing primer

<400> 18

gattttgtta catctacact gttg 24

<210> 19

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer

<400> 19

gattttgcag tttattactg t 21

<210> 20

<211> 159

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer for manufacturing AU11

<400> 20

ttcgctagcg aggtgcagct ggtggagtct gggggaggcc tggtacagcc tggagggtcc 60

ctgagactct cctgtgcagc ctctggagac agtgtctcta gcaccactgt tgccatgagc 120

tgggtccgcc aggctccagg gaaggggctg gagtgggtc 159

<210> 21

<211> 168

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer for manufacturing AU11

<400> 21

acagtaatac acagccgtgt cctcggctct caggctgttc atttgcagat acagggtgtt 60

cttggaattg tctctggaga tggtgaaccg gcccttcacg gagtccgcat agtaattata 120

ccacttcgac ctataataga tcgctgagac ccactccagc cccttccc 168

<210> 22

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer for manufacturing AU11

<400> 22

gaggacacgg ctgtgtatta ctgt 24

<210> 23

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer for manufacturing AU11

<400> 23

ttcccctgga gacaaagaca gggtggctgg agactgggtc aatacaatat ccgacccgcc 60

accgccgctg ccacc 75

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer for manufacturing AU11

<400> 24

accctgtctt tgtctccagg ggaa 24

<210> 25

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer for manufacturing AU11

<400> 25

cgtggatcca cgtttgattt ccacctttgt cccttggccg aagaccgccc g 51

<210> 26

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of (G4S) 3 linker

<400> 26

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

  1 5 10 15

<210> 27

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of AU11 VL

<400> 27

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

  1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

             20 25 30

His Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

         35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

     50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ala Asn Asp Phe

                 85 90 95

Pro Arg Ala Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

            100 105 110

<210> 28

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of AU11 VH

<400> 28

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

  1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Ser Val Ser Ser Thr

             20 25 30

Thr Val Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

         35 40 45

Trp Val Ser Ala Ile Tyr His Arg Ser Tyr Trp Asp Lys Tyr Tyr Ala

     50 55 60

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

 65 70 75 80

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

                 85 90 95

Tyr Tyr Cys Ala Arg Glu Gly Ser Gly His Tyr Gly Ala Phe Asp Ile

            100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

        115 120

<210> 29

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer for manufacturing AU12

<400> 29

ttcgctagcg aggtgcagct ggtggagtct ggg 33

<210> 30

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer for manufacturing AU12

<400> 30

tggagactgg gtcatctgag agtc 24

<210> 31

<211> 90

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer for manufacturing AU12

<400> 31

gactctcaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcgttagc 90

<210> 32

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer for manufacturing AU12

<400> 32

cttaggggct ttccctggtt tctgctgata ccaggctaag tggct 45

<210> 33

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer for manufacturing AU12

<400> 33

aaagccccta agctcctgat ctatggtgca tccagc 36

<210> 34

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer for manufacturing AU12

<400> 34

gatggtgaga gtgaaatctg tcccagatcc actgccactg aaccttgatg ggaccccagt 60

ggccct 66

<210> 35

<211> 87

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer for manufacturing AU12

<400> 35

ttcactctca ccatcagcag tctgcaacct gaagattttg caacttacta ctgtcaacag 60

cgtgcaaacg atttcccgcg ggcggtc 87

<210> 36

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer for manufacturing AU12

<400> 36

cgtggatcca cgtttgattt ccac 24

<210> 37

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of AU12 VL

<400> 37

Asp Ser Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

  1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

             20 25 30

His Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

         35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ser Arg Phe Ser

     50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

 65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ala Asn Asp Phe

                 85 90 95

Pro Arg Ala Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

            100 105 110

<210> 38

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of AU12 VH

<400> 38

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

  1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Ser Val Ser Ser Thr

             20 25 30

Thr Val Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

         35 40 45

Trp Val Ser Ala Ile Tyr His Arg Ser Tyr Trp Asp Lys Tyr Tyr Ala

     50 55 60

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

 65 70 75 80

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

                 85 90 95

Tyr Tyr Cys Ala Arg Glu Gly Ser Gly His Tyr Gly Ala Phe Asp Ile

            100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

        115 120

<210> 39

<211> 249

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of AU12 scFv

<400> 39

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

  1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Ser Val Ser Ser Thr

             20 25 30

Thr Val Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

         35 40 45

Trp Val Ser Ala Ile Tyr His Arg Ser Tyr Trp Asp Lys Tyr Tyr Ala

     50 55 60

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

 65 70 75 80

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

                 85 90 95

Tyr Tyr Cys Ala Arg Glu Gly Ser Gly His Tyr Gly Ala Phe Asp Ile

            100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

        115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ser Gln Met Thr Gln

    130 135 140

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

145 150 155 160

Cys Arg Ala Ser Gln Ser Val Ser Ser Ser His Leu Ala Trp Tyr Gln

                165 170 175

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Ser

            180 185 190

Arg Ala Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

        195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

    210 215 220

Tyr Tyr Cys Gln Gln Arg Ala Asn Asp Phe Pro Arg Ala Val Phe Gly

225 230 235 240

Gln Gly Thr Lys Val Glu Ile Lys Arg

                245

<210> 40

<211> 747

<212> DNA

<213> Artificial Sequence

<220>

223 nucleotide sequence of AU12 scFv

<400> 40

gaggtgcagc tggtggagtc tgggggaggc ctggtacagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggaga cagtgtctct agcaccactg ttgccatgag ctgggtccgc 120

caggctccag ggaaggggct ggagtgggtc tcagcgatct atcacaggtc gtattgggac 180

aagtactatg cggactccgt gaagggccgg ttcaccatct ccagagacaa ttccaagaac 240

accctgtatc tgcaaatgaa cagcctgaga gccgaggaca cggctgtgta ttactgtgca 300

agagagggta gtggccacta tggggctttt gatatctggg gccaagggac cctggtcacc 360

gtttcttcag gtggaggcgg ttcaggcgga ggtggcagcg gcggtggcgg gtcggactct 420

cagatgaccc agtctccatc ctccctgtct gcatctgtag gagacagagt caccatcact 480

tgccgggcaa gtcagagcgt tagcagcagc cacttagcct ggtatcagca gaaaccaggg 540

aaagccccta agctcctgat ctatggtgca tccagcaggg ccactggggt cccatcaagg 600

ttcagtggca gtggatctgg gacagatttc actctcacca tcagcagtct gcaacctgaa 660

gattttgcaa cttactactg tcaacagcgt gcaaacgatt tcccgcgggc ggtcttcggc 720

caagggacca aagtggaaat caaacgt 747

Claims (17)

1. An anti-DR5 antibody that specifically binds to DR5, having the amino acid sequence of SEQ ID NO: 7.
2. An anti-DR5 antibody that specifically binds to DR5, having the amino acid sequence of SEQ ID NO.
3. 3. The antibody of claim 1 or 2, wherein the antibody has a heavy chain variable region having complementary determining region CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NOs: 1, 2 and 3, and the complementary determining region CDR1, CDR2 and CDR3, respectively, An anti-DR5 antibody comprising a light chain variable region having the amino acid sequences of Nos. 4, 5, and 6.
4. The anti-DR5 antibody according to claim 3, wherein the light chain variable region has an amino acid sequence of SEQ ID NO: 27 or 37.
5. 4. The anti-DR5 antibody according to claim 3, wherein the heavy chain variable region has an amino acid sequence of SEQ ID NO: 28 or 38.
6. The anti-DR5 antibody according to claim 1 or 2, wherein the antibody induces apoptosis by progeny against TRAIL-sensitive cancer cells expressing DR5 or TRAIL-resistant cancer cells expressing DR5.
7. The anti-DR5 antibody of claim 1 or 2, wherein the antibody is selected from the group consisting of single-chain variable region fragments (scFv), (scFv) ₂ , Fab, Fab ', and F (ab') ₂ . Antibodies.
8. The anti-DR5 antibody according to claim 1 or 2, wherein the antibody is a monoclonal antibody.
9. DNA having a nucleotide sequence of SEQ ID NO: 8 encoding an amino acid sequence of SEQ ID NO: 7 of the antibody of claim 1.
10. DNA having a nucleotide sequence of SEQ ID NO: 40 encoding an amino acid sequence of SEQ ID NO: 39 of the antibody of claim 2.
11. A cell transformed with the DNA of claim 9 or an expression vector comprising the same.
12. Cell transformed with the DNA of claim 10 or an expression vector comprising the same.
13. Complementarity determining region CDRs of the heavy chain variable region of the anti-DR5 antibody, consisting of CDR1, CDR2, CDR3 having the amino acid sequence of SEQ ID NO: 1, 2, 3, respectively.
14. Complementarity determining region CDRs of the light chain variable region of the anti-DR5 antibody, consisting of CDR1, CDR2, CDR3 having the amino acid sequence of SEQ ID NO: 4, 5, 6, respectively.
15. A composition for preventing or treating cancer, comprising the DDR5 antibody of claim 1 as an active ingredient.
16. The method for preventing or treating cancer according to claim 15, wherein the cancer is cancer caused by DR5 expression.
17. The method of claim 16, wherein the cancer caused by the DR5 expression is hematological cancer, lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, rectal cancer, colon cancer, colon cancer, breast cancer , Uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia Cancer prevention characterized in that it is selected from the group consisting of childhood solid tumors, differentiated lymphoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, primary central nervous system lymphoma, spinal contraction tumor, brainstem glioma and pituitary adenoma Or a therapeutic composition.

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