WO2014069681A1 - Cancer-targeting antibody and composition for preventing or treating cancer comprising same - Google Patents

Abstract

The present invention relates to a monoclonal antibody for inducing apoptosis by binding to a cancer cell and a medical use thereof, and the antibody according to the present invention exhibits activity for cancer cell line-specific binding and thus can be used in cancer cell-targeted diagnosis or as a drug conjugate to effectively diagnose or treat cancer. Also, the antibody according to the present invention exhibits the characteristic of inducing apoptosis by binding to cancer cells, particularly lymphoma cells and ovarian cancer cells, and thus the antibody according to the present invention can be used alone as an anticancer drug.

Description

Cancer target antibody and composition for preventing or treating cancer comprising the same

The present invention relates to monoclonal antibodies that bind to cancer cells and induce apoptosis and their medical use.

Cancer is one of the most deadly threats to human health. In the United States, nearly 1,300,000 new patients get cancer each year, causing about one in four deaths following heart disease. It is also predicted that cancer can surpass cardiovascular disease as the first cause of death within five years. Solid cancer accounts for most of these deaths. Although significant progress has been made in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved only about 10% over the past 20 years. Cancer or malignant tumors metastasize and grow rapidly in an uncontrolled manner, making it very difficult to detect and treat in a timely manner. In addition, cancer can occur from almost any tissue in the body through one or several normal cells in the tissue through malignant transformation, and differ from one type of cancer with a particular tissue origin.

Current methods for treating cancer are relatively non-selective. Surgery to remove diseased tissue; Radiation treatment reduces solid tumors; Chemotherapy kills rapidly dividing cells. In particular, chemotherapy produces a variety of side effects, which in some cases are so severe that they limit the dose that can be administered and eventually rule out the use of potentially effective drugs. In addition, cancer often develops resistance to chemotherapy drugs. Therefore, specific and more effective cancer treatments are urgent.

Tumor cells differ in antigenicity from normal cells due to the presence of "tumor antigens". They may be specific to tumor cells, or are recognized as "tumor-associated antigens" (TAAs) by being expressed differently or in excess. Tumor cells not only differ in protein primary structure, i.e. from amino acid sequences, from normal cell changes, but also in secondary and tertiary due to changes in post-translational modifications, such as changes in glycosylation and phosphorylation. The structure may also differ from normal cells, which continually change the antigenicity of the protein and also specify it as a tumor-associated antigen. One typical example is protein mucin from the mammary gland, which itself is not a change present in tumor cells, but autoantibodies to it are found in breast cancer patients (sometimes in ovarian cancer). The reason is presumably because the protein is highly glycosylated in normal cells and is not exposed at all due to the dense and thick coating of carbohydrate chains. Poor glycosylation in tumor cells results in exposure of protein fragments that act as antigenic determinants to the immune system.

TAA is currently thought to be a molecule that can be associated with certain tumors, such as lymphomas, carcinomas, sarcomas, or melanoma, and can elicit cellular and / or humoral immune responses against tumors and, rarely, against tumors. It also defends the host. Such TAAs are currently classified into three classes, which are highly specific for certain tumors that are present in only one or only a few individuals and are not found in normal cells, for example tumor-specific transplant antigens (class 1). ); Present in a large number of related tumors of different patients (second class); And present in normal cells and malignant cells and expressed in large quantities in malignant cells (third class). Since the second class of TAA is present in many tumors and rarely observed in normal subjects, it is considered to have the greatest potential for clinically useful analyses.

Therefore, the use of antibodies targeting the tumor cell specific antigens as a specific and more effective cancer treatment method, and the development of cancer therapeutic agents that induce apoptosis of cancer cells through the antibody is currently of interest. In connection with the above technology, Korean Laid-Open Patent Publication 10-2010-0045903 discloses an antibody targeting anti DR5, which is an apoptosis receptor for cancer cells.

Therefore, the present invention is to provide a novel antibody targeting a tumor cell specific antigen and a method for treating cancer inducing apoptosis of cancer cells through the antibody.

In order to solve the above problems, the present invention binds to cancer cells as a monoclonal antibody inducing apoptosis,

A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3;

Provided is an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.

In one embodiment of the invention, the antibody is a single-chain variable fragment (scFv, single-chain variable fragment) antibody, and may have an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 21. .

The present invention also provides a gene encoding the single chain variable region antibody.

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

The present invention also provides a cancer cell targeting diagnostic or therapeutic conjugate comprising the antibody, wherein one or more diagnostic or anticancer agents are bound to the antibody.

The antibody of the present invention has an activity of specifically binding to a cancer cell line, and can be used as a cancer cell targeting diagnosis or therapeutic conjugate to effectively diagnose or treat cancer. In addition, the antibody of the present invention shows a property of inducing apoptosis by binding to cancer cells, particularly lymphoma cells and ovarian cancer cells, through which the antibody of the present invention can be used as an anticancer agent.

1 is a flow cytometry result for the IM9 cell line of Example 1.

Figure 2 shows the nucleotide and amino acid sequences of the scFv clone ITTab1-1 and CDRs and linker regions thereof according to the present invention.

Figure 3 shows the nucleotide and amino acid sequences of the scFv clone ITTab1-2 and its CDR and linker regions according to the present invention.

Figure 4 shows the nucleotide and amino acid sequence of the scFv clone ITTab1-3 and its CDR and linker regions according to the present invention.

Figure 5 shows the nucleotide and amino acid sequences of the scFv clone ITTab1-4 and its CDR and linker regions according to the present invention.

6 is a photograph of confocal microscopy results of examining the binding of the antibody of the present invention to NK lymphoma cells.

Figure 7 is a flow cytometry test results of examining apoptosis of NK lymphoma cells by the antibody treatment of the present invention.

8 is a flow cytometry test result of examining the binding of the antibody of the present invention to ovarian cancer cells.

9 is a flow cytometry test result of examining apoptosis of ovarian cancer cells by the antibody treatment of the present invention.

The present invention is a monoclonal antibody that binds to cancer cells and induces apoptosis,

A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3; Provided is an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.

The inventors of the present invention, while studying a novel antibody targeting a tumor cell specific antigen, constructed a hybridoma cell that specifically binds to B cell tumor cells (IM9) using PBMC stimulated with ConA, The monoclonal antibody prepared by this specifically binds to cancer cells, in particular NK lymphoma cells and ovarian cancer cells, and confirmed that it also induces apoptosis, thereby completing the present invention.

In one embodiment of the invention, the light chain variable region may consist of the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15 or SEQ ID NO: 19.

In another embodiment of the present invention, the heavy chain variable region may be composed of the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16 or SEQ ID NO: 20.

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. Antibodies in the form of single-chain variable fragments (scFv) are preferred. 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.

In another embodiment of the invention, the antibody of the invention is a single chain variable region antibody, which may have an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 21.

In addition, the antibodies of the present 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, and 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.

The present invention also provides a gene encoding each of the single-chain variable region antibodies having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 21.

Gene 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.

More specifically, the gene encoding the single-chain variable region antibody having the amino acid sequence of SEQ ID NO: 9 may be composed of the nucleotide sequence of SEQ ID NO: 10.

In addition, the gene encoding the single-chain variable region antibody having the amino acid sequence of SEQ ID NO: 13 may be composed of the nucleotide sequence of SEQ ID NO: 14.

In addition, the gene encoding the single-chain variable region antibody having the amino acid sequence of SEQ ID NO: 17 may be composed of the nucleotide sequence of SEQ ID NO: 18.

In addition, the gene encoding the single-chain variable region antibody having the amino acid sequence of SEQ ID NO: 21 may be composed of the nucleotide sequence of SEQ ID NO: 22.

In one embodiment of the present invention, the host cell is transformed with the gene and the expression vector including the same, and cultured under appropriate conditions to express and isolate the antibody, thereby producing an antibody molecule.

The antibody of the present invention exhibits a property of specifically binding to cancer cells, and in particular, binds to lymphoma cells and ovarian cancer cells and induces cell death, and thus can be used for the prevention or treatment of cancer.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer containing the antibody as an active ingredient, the use of the antibody for the manufacture of an anticancer agent, and administering to the subject a therapeutically effective amount of the antibody to a subject. To provide.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating cancer containing the antibody as an active ingredient is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, It may further comprise one or more additives selected from the group consisting of lubricants, lubricants, flavors, antioxidants, buffers, bacteriostatics, diluents, dispersants, surfactants, binders and lubricants.

Specifically, the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules. And the like, and such solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the composition. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used. Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used as the base material of the suppository.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating cancer containing the antibody as an active ingredient is a granule, powder, coated tablet, tablet, pill, capsule, suppository, gel, syrup, juice It can be formulated as a suspension, emulsion, drop or liquid.

According to one embodiment of the invention the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, nasal, inhaled, topical, rectal, oral, intraocular or intradermal Via the route can be administered to the subject in a conventional manner.

The preferred dosage of the antibody may vary depending on the condition and weight of the subject, the type and extent of the disease, the form of the drug, the route of administration, and the duration and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg. Administration may be administered once a day or divided into several times, thereby not limiting the scope of the invention.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

The cancer may be any one selected from liver cancer, lung cancer, colon cancer, ovarian cancer, breast cancer, prostate cancer, leukemia, lymphoma and pancreatic cancer.

The present invention also provides a cancer cell targeting diagnostic or therapeutic conjugate comprising the antibody and binding one or more diagnostic or anticancer agents to the antibody.

The diagnostic agent may be any one or two or more selected from the group consisting of radionuclides, paramagnetic metal ions, superparamagnetic nanoparticles, chromophores, chromophores, luminescent materials, and fluorescent materials, and the contrast agent may be CT contrast agent, MRI. It may be any one selected from the group consisting of a contrast agent, an optical contrast agent, and an ultrasound contrast agent.

The anticancer agent may be used without limitation as long as it is conventionally used for the treatment of cancer, for example cisplatin, 5-fluorouracil, adriamycin, methotrexate, vinblastine, busulfan, chlorambucil, cyclophosphamide, Melphalan, nitrogen mustard, nitrosourea, taxol, paclitaxel, docetaxel, 6-mercaptopurine, 6-thioguanine, bleomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitomycin-C And hydroxyurea can be used any one or two or more selected from the group consisting of. The linkage between the anticancer agent and the antibody of the present invention can be carried out through methods known in the art, such as covalent bonds, crosslinking and the like.

In addition, the anticancer agent may be a therapeutic radionuclide, for example, ³² P, ⁴⁷ Sc, ⁶⁴ Ga, ⁶⁴ Cu, ⁶⁶ Cu, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ⁹¹ Y, ¹⁰³ Pd, ^{103 m} Rh, ¹⁰⁵ Rh, ¹¹¹ Ag, ¹¹¹ In, ^117m Sn, ¹²⁵ I, ¹³¹ I, ¹⁴⁵ Sm, ¹⁴⁹ Pm, ¹⁵³ Sm, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷⁶ Lu, ¹⁷⁷ Lu, ¹⁸² Ta, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁸⁸ W , ¹⁹² Ir, ^{195 m} Pt, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹² Bi, ²¹³ Bi, ²²³ Ra, ²²⁵ Ac, ²³⁰ U, ²⁵² Cf, or a combination thereof.

The amount of the antibody of the present invention included in the cancer cell targeting therapeutic conjugate of the present invention may vary depending on the type and amount of the anticancer agent to be bound. Preferably, the amount of the anticancer agent administered for treatment may be an amount sufficient to deliver the target cancer disease site. However, since the drug is determined not only by the route of administration and the number of treatments, but also by various factors such as the age, weight, health condition, sex, severity of the disease, diet, and excretion rate, the effective dose to the patient is determined. In view of this, one of ordinary skill in the art will be able to determine an appropriate effective amount depending on the use of the antibody for treating cancer diseases by combining the antibody with an anticancer agent.

The cancer cell targeting therapeutic conjugate of the present invention may include a suitable buffer solution for maintaining / preserving the stability of the antibody. The cancer cell targeting therapeutic conjugate of the present invention is not particularly limited to its formulation, route of administration, and method of administration so long as the desired effect is achieved in the present invention. For example, the conjugates of the present invention can be administered parenterally, such as subcutaneous injection, intramuscular injection, intravenous injection, urethral injection, intrabronchial inhalation, intrauterine dural or cerebrovascular injection, and the like.

In addition, the cancer cell targeting therapeutic conjugates of the present invention include appropriate carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bactericides, diluents, It may further comprise one or more additives selected from the group consisting of dispersants, surfactants, binders and lubricants.

In addition, the cancer cell targeting therapeutic conjugate of the present invention may be formulated into granules, powders, coated tablets, tablets, pills, capsules, suppositories, gels, syrups, juices, suspensions, emulsions, drops or solutions according to conventional methods. Can be used. For example, injectables can be prepared in the form of unit dose ampoules or multiple dose inclusions.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Preparation Example 1 Preparation of ITTab1 Antibody as a scFv (single chain Fv)

1. Hybridoma Cell Line Construction

Mice were 5-6 week old female Balb / c mice purchased from Orient Bio. The purchased mouse was purified for 1 week by supplying sufficient solid feed and water in an indoor environment with a temperature of 20-25 ° C., a humidity of 60%, and an illuminance of 200-300 Lux .

PBMC 1X10 ⁷ isolated from Ficoll-gradient method from healthy volunteers was stimulated with Con A (concanavalin A) 10 ug / ml and immunized with mouse. After 2 months, spleens of mice were isolated and 1X10 ⁸ splenocytes were isolated. This was obtained and fused with 10 ⁷ SP 2 / 0-Ag14 mouse myeloma cells using polyethylene glycol (PEG 4000; Sigma, St Louis, MO) method. The method is described in detail in Kohler G, Milstein C. Nature 256 (1975) 495-497. FACS staining was performed using the obtained culture supernatant of 100 hybridoma cell lines to obtain hybridoma cells reactive to B cell tumor cells (IM9, Raju, Ramos).

2. Fabrication and activity validation of scFv (single chain Fv) ITTab1 antibody from hybridoma cell line

ITT-ab1 (Inje Tumor Targeting-antibody1) mouse antibody of the present invention, the library (library) production step (A); It was prepared, characterized in that it comprises an antibody function confirmation step (B).

In the library production step (A), scFv (single-chain Fv) forms from hybridoma cells (hereinafter, hybridoma F6 cells) obtained in the method 1 of production of antibodies that bind to B cell tumor cells (IM9). To prepare a non-immune mouse antibody fragment library of

Separation and purification step (a1) for purifying RNA; A polymerase chain reaction amplification step (a2) for amplifying the first strand cDNA by a polymerase chain reaction of a mouse antibody gene; A cell fabrication step (a3) of fabricating a competent cell; It is composed of a library completion step (a4) for completing a single chain Fv (scFv) library based on the production step. The following describes the process for each step.

1) Separation and purification step (a1)

Separation and purification step (a1) is to purify RNA, cultivate hybridoma F6 cells, add TRIZOL 2ml to F6 cell 1x10 ⁷ using TRIZOL reagent for 5 minutes at room temperature, and then 200ul of chloroform per 1ml TRIZOL. I vortex for about 15 seconds. And centrifuged for 15 minutes at 13000rpm at 4 ℃ using a centrifuge. Then, the supernatant was carefully transferred to a microcentrifuge tube so as not to pick the middle layer, and isopropyl alcohol was mixed in the same ratio and reacted at room temperature for 10 minutes. After removing the supernatant by centrifuging at 12000 xg for 10 minutes at 4 ℃, washed with 1ml of 75% ethanol once more centrifuged at 12000 xg for 15 minutes at 4 ℃ and remove the supernatant 30 DEL treated distilled water 30ul RNA was recovered. RNA was electrophoresed on 0.8% agarose gel to confirm RNA purity, which was quantified using a spectrophotometer and used for experiments.

2) polymerase chain reaction amplification step (a2)

Polymerase chain reaction amplification step (a2) is a step of preparing the first strand cDNA and amplifying the polymerase chain reaction of the mouse antibody gene, and the purified RNA 2 ug in the purification step (a1) and 0.5 ug oligo (dT) and After mixing and adding distilled water to a final volume of 10 ul and reacted for 5 minutes at 75 ℃. Thereafter, 15 ul of cDNA reaction solution (5 ul of 5 x M-MLV reaction solution, 100 units of M-MLV reverse transcriptase, 10 mM dNTP 1.25 ul, and RNase Inhibitor 4 units) were added and vortex mixed with an RNA sample. The reaction was carried out in a chain polymerization apparatus at 37 ° C. for 1 hour and at 95 ° C. for 5 minutes to amplify the polymerase chain reaction with an mGAPDH primer.

The antibody gene was amplified by polymerase chain reaction with a mouse antibody specific primer. Amplify the variable regions of the light and heavy chains, bind them with overlapping polymerase chain reaction using linker-specific primers, and clone them into the Sfi I restriction site of the pComb3X-TT expression vector. Was produced. The primer used at this time was determined by a manual (Barbas et al., Phage Display; A Laboratory Manual, Cold Spring Harbor Laboratory, (2001), New York), and was synthesized by making a request to Bioneer (Korea).

Polymerase chain reaction conditions were pre-denaturated at 94 ° C. for 5 minutes, followed by 25 cycles of reaction at 94 ° C. 30 sec, 57 ° C. 15 sec, and 72 ° C. 30 sec. The presence or absence of amplification was confirmed by 1% agarose gel electrophoresis, purified using Qiagen gel extraction kit, and the amplified light chain variable region and heavy chain variable region were subjected to the following overlapping polymerase chain reaction.

The polymerase chain reaction product was purified by DNA from agarose with Qiagen gel extraction kit, restriction enzyme digestion with Sfi I, and then gel extraction. Sfi I was added to a mixture of the cut antibody gene 30 ng and Sfi I cleavage pComb3X-TT 30 ng after 10X buffer and a ligase (ligase, Gibco-BRL, USA ) 10 unit and the reaction at room temperature for 2 hours after TOP10F Transformed competent cells.

Mouse IgG specific primers used in the development of the synthetic human unicellular antibody of the present invention are described in detail in the Phage Display manual (Barbas et al., Phage Display; A Laboratory Manual, Cold Spring Harbor Laboratory, (2001), New York). It is.

3) Cell manufacturing step (a3)

Cell production step (a3) is a step of producing a competent cell (Electrocompetent cell), incubation of E. coli ER2537 in a minimal plate (minimal plate) to induce the expression of the F 'cilia after the production of water-soluble cells (competent cell) Used. ER2537 was inoculated in 2 ml of SB medium (10 g MOPS, 30 g, bacto-trypton, 20 g yeast extract in 1 L) and incubated at 37 ° C. for 18 hours, followed by OD ₆₀₀ to 3.54. 1 ml of the pre-culture was incubated in 100 ml of LB medium (10 g NaCl, 10 g, bacto-trypton, 5 g yeast extract in 1 L) so that the OD ₆₀₀ was 0.4. Thereafter, centrifuge was performed for 10 minutes at 4,000 rpm at 4 ℃. The cells were suspended with 20 ml of 50 mM CaCl ₂ and then reacted for 30 minutes on ice, and then centrifuged at 4,000 rpm for 10 minutes at 4 ° C. The supernatant was removed and 3 ml of (10% glycerol + 50 mM CaCl ₂ ) was added to the cells, followed by aliquoting of 50ul.

4) Library Completion Step (a4)

The library completion step (a4) is a step of completing the scFv (single chain Fv) library based on the preparation step, and 100 μg / mL of ampicillin after infecting the recovered phage antibody with Escherichia coli ER2537. Plates were added to the added LB agar plates and incubated at 37 ° C for 18 hours. The colonies formed were inoculated into 5 mL of SB (SB / amp) liquid medium to which 100 μg / mL of ampicillin was added and shaken at 37 ° C. at 250 rpm for 4 hours. For phage formation, 10 ¹⁰ pfu of VCSM13 was added and allowed to stand at 37 ° C for 30 minutes for infection, followed by incubation at 250 rpm at 37 ° C for 1 hour 30 minutes, and kanamycin was added to 70 μg / mL for 18 hours at 37 ° C. Incubated. Thereafter, the culture solution was centrifuged at 2,500 rpm for 10 minutes to recover the supernatant containing phage.

Subsequently, the antibody function checking step (B) confirms the function of the antibodies completed in the library completion step (a4), and treats phage antibodies that recognize IM-9 cells with α-M13 and α-mIgG-PE. After FACS staining was confirmed whether the binding to IM9 B cell tumor cells.

Example 1 Examination of the binding capacity of ITTab1 B cell tumor cells (IM9)

In order to determine whether the ITTab1 obtained by phage display has the same characteristics as the antibody derived from the secured hybridoma, FACS staining was confirmed for binding to IM9 cell line, B cell tumor cells. IM-9 cells (10 ⁶ ) were treated with 1 ug of ITTab1 subclonal antibodies (ITTab 1-1 to ITTab 1-4), respectively, and washed with PBS after 30 min binding. The washed cells were combined with FITC-conjugated goat anti-mouse IgG, (Sigma-Aldrich) for 30 minutes, washed with PBS three times, and observed with FACS Calibur (BD Pharmingen). The results are shown in FIG.

Referring to FIG. 1, it was observed that all of the subclonal antibodies of ITTab1 bind to the surface of IM-9 cells. These results confirm that the target antigen for this antibody is present in IM-9 cells, and that the antibody obtained from previously obtained hybridoma cells and the ITTab1 antibody of the present invention obtained by phage display recognize the same antigen. Could.

Example 2 Sequence Identification and Total Nucleic Acid and Amino Acid Sequence of the Antibody CDR Region of ITTab1

Sequencing was performed to identify sequences of CDR regions of ITTab1-1 to ITTab1-4, which are subclones of ITTab1 obtained by phage display, and the characteristics were observed using the obtained information. Complimentarily-determining region (CDR) in the variable region of ITTab1-1 to ITTab1-4 of scFv (short chain Fv) type was identified.The underlined portion is a linker region, which is divided into a light chain and a heavy chain, and the heavy chain. From the sites marked in boldface at the top of the chain, CDR1, CDR2 and CDR3 of the heavy chain were determined. The sites shown in bold at the front of the light chain bordering the linker were determined by CDR1, CDR2 and CDR3 of the light chain.

1) ITTab1-1

Figure 2 shows the base and amino acid sequence of ITTab1-1 and its CDR and linker regions.

Referring to Figure 2, ITTab1-1 is a light chain CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), CDR3 (SEQ ID NO: 3), heavy chain CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 1) No. 5), CDR3 (SEQ ID NO: 6).

 In addition, the light chain amino acid sequence of ITTab1-1 is shown in SEQ ID NO: 7, and the heavy chain amino acid sequence of SEQ ID NO: 8,

The entire amino acid sequence of ITTab1-1 is shown in SEQ ID NO: 9, and the entire nucleotide sequence is shown in SEQ ID NO: 10.

2) ITTab1-2

3 shows the base and amino acid sequence of ITTab1-2 and its CDR and linker regions.

Referring to Figure 3, ITTab1-2 is a light chain CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), CDR3 (SEQ ID NO: 3), heavy chain CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 1) No. 5), CDR3 (SEQ ID NO: 6).

 In addition, the light chain amino acid sequence of ITTab1-2 is shown in SEQ ID NO: 11, and the heavy chain amino acid sequence of SEQ ID NO: 12,

The entire amino acid sequence of ITTab1-2 is shown in SEQ ID NO: 13, and the entire nucleotide sequence is shown in SEQ ID NO: 14.

3) ITTab1-3

4 shows the base and amino acid sequence of ITTab1-3 and its CDR and linker regions.

Referring to Figure 4, ITTab1-3 is a light chain CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), CDR3 (SEQ ID NO: 3), heavy chain CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 1) No. 5), CDR3 (SEQ ID NO: 6).

 In addition, the light chain amino acid sequence of ITTab1-3 is shown in SEQ ID NO: 15, and the heavy chain amino acid sequence of SEQ ID NO: 16,

The entire amino acid sequence of ITTab1-3 is shown in SEQ ID NO: 17, and the entire nucleotide sequence is shown in SEQ ID NO: 18.

4) ITTab1-4

5 shows the base and amino acid sequences of ITTab1-4 and their CDRs and linker regions.

Referring to Figure 5, ITTab1-4 is in the order of bold font CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), CDR3 (SEQ ID NO: 3), CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 4) of the heavy chain No. 5), CDR3 (SEQ ID NO: 6).

 In addition, the light chain amino acid sequence of ITTab1-4 is shown in SEQ ID NO: 19, and the heavy chain amino acid sequence of SEQ ID NO: 20,

The entire amino acid sequence of ITTab1-4 is shown in SEQ ID NO: 21, and the entire nucleotide sequence is shown in SEQ ID NO: 22.

Example 3 Identification of Antigen Expression of ITTab1 on NK Lymphoma Cells

In order to confirm the reactivity with the antigen of ITTab1 and the expression pattern for tumor cells, the presence of the antigen of ITTab1 on NK lymphoma cells was analyzed by immunofluorescence and confocal microscopy.

After NK lymphoma cells were incubated with 1 ug of ITTab1 for 30 minutes, the cells were recovered and washed with PBS. The washed cell lines were incubated with FITC-conjugated goat anti-mouse IgG (Sigma-Aldrich) for 30 minutes and washed three times with PBS. The washed cells were placed on a microscope slide under the cover-slip using fluorescent mounting medium (DakoCytomation, Glostrup, Denmark). Fluorescent stained cells were observed with 400 × original magnification using a Confocal Laser-Scanning microscope (Carl ZEISS, 510 META, Jena, Germany) and analyzed by confocal Microscopy Software Release 3.0 (Carl ZEISS, 510 META).

The results are shown in FIG. Referring to FIG. 6, it can be seen from the confocal microscopy results that the cell surface and the cytoplasm are stained with green fluorescence by ITTab1. Through this, the target antigen for the ITTab1 antibody is present in NK lymphoma cells. there was.

Example 4 Examining the Effect of ITTab1 on Apoptosis of NK Lymphoma Cells

To clarify the function of ITTab1 antigen expressed on NK lymphoma cells on NK lymphoma cells, the effect of ITTab1 on tumor cells after treatment with tumor cells was analyzed by an annexin-V assay.

NK lymphoma cells (10 ⁶ cells) were incubated with each concentration of ITTab1 for 1 hour, and then the cells were recovered and washed twice with PBS. The washed cell lines were suspended with 100 ul of Annexin-V binding buffer (10 mM EPES / NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl ₂ ), followed by 2ul of FITC-conjugate Annexin-V (BD Pharmingen) and 7- ADD was processed. Treated NK lymphoma cells were incubated for 15 minutes at room temperature in the dark, followed by the addition of 400 ul of Annexin-V binding buffer and analyzed using FACS Calibur (BD Pharmingen).

The results are shown in FIG. Referring to FIG. 7, it was observed that apoptosis was induced in a concentration-dependent manner in which the ITTab1 antibody was increased, thereby suggesting that ITTab1 of the present invention can be developed as an apoptosis-inducing antibody in NK lymphoma cells.

Example 5 Identification of Antigen Expression of ITTab1 on Ovarian Cancer Cells

In order to confirm the reactivity with the antigen of ITTab1 and the expression pattern for tumor cells, the presence of ITTab1 antigen in ovarian cancer cells was stained with an antibody and observed with FACS calibur (BD Pharmingen).

Ovarian cancer cells were incubated with 1 ug of ITTab1 for 30 minutes and then cells were recovered and washed with PBS. The washed cell lines were incubated with FITC-conjugated goat anti-mouse IgG (Sigma-Aldrich) for 30 minutes and washed three times with PBS. Washed cells were analyzed using FACS calibur (BD Pharmingen).

The results are shown in FIG. Referring to FIG. 8, in the flow cytometry results, it was confirmed that the antigen of ITTab1 was expressed by ITTab1 on both the cell surface and the cytoplasm, and the target antigen for the antibody of the present invention was present in ovarian cancer cells. Could know.

Example 6 Examination of the Effect of ITTab1 on Apoptosis of Ovarian Cancer Cells

To investigate the function of the ITTab1 antigen expressed in ovarian cancer cells on ovarian cancer cells, the effect of ITTab1 on tumor cells after treatment with tumor cells was analyzed by cell cycle analysis by propidium iodide (PI) treatment.

Ovarian cancer cells (10 ⁶ cells) were incubated with 5ug of ITTab1 for 1 hour and then recovered and washed twice with PBS. The washed cell lines were treated with propidium iodine (PI) and the cells were analyzed using FACS caliber (BD Pharmingen).

The results are shown in FIG. Treatment of ovarian cancer cells with ITTab1 showed that PI apoptosis cells in the subG1 phase increased with increasing antibody concentrations compared to those in isotype control (MOPC) cells. Through this, it can be seen that ITTab1 of the present invention can be used as an apoptosis-inducing antibody in ovarian cancer cells.

Having described the specific part of the present invention in detail, it will be apparent to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (17)

1. A monoclonal antibody that binds to cancer cells and induces apoptosis,

   A light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence of SEQ ID NO: 1, a light chain CDR2 consisting of an amino acid sequence of SEQ ID NO: 2, and a light chain CDR3 consisting of an amino acid sequence of SEQ ID NO: 3;

   An antibody comprising a heavy chain variable region comprising a heavy chain CDR1 consisting of an amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 consisting of an amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 consisting of an amino acid sequence of SEQ ID NO: 6.
2. The method of claim 1,

   The light chain variable region is

   The antibody having the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO: 19.
3. The method of claim 1,

   The heavy chain variable region is

   The antibody having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 20.
4. The method of claim 1,

   The antibody is a single-chain variable fragment (scFv) antibody,

   The antibody having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, or SEQ ID NO: 21.
5. A gene encoding a single-chain variable fragment (scFv) antibody having the amino acid sequence of SEQ ID NO.
6. The method of claim 5,

   The gene is one consisting of the nucleotide sequence of SEQ ID NO: 10.
7. A gene encoding a single-chain variable fragment (scFv) antibody having the amino acid sequence of SEQ ID NO: 13.
8. The method of claim 7, wherein

   The gene is one consisting of the nucleotide sequence of SEQ ID NO: 14.
9. A gene encoding a single-chain variable fragment (scFv) antibody having the amino acid sequence of SEQ ID NO: 17.
10. The method of claim 9,

    The gene is one consisting of the nucleotide sequence of SEQ ID NO: 18.
11. A gene encoding a single-chain variable fragment (scFv) antibody having the amino acid sequence of SEQ ID NO: 21.
12. The method of claim 11,

    The gene is one consisting of the nucleotide sequence of SEQ ID NO: 22.
13. A pharmaceutical composition for preventing or treating cancer containing the antibody according to any one of claims 1 to 4 as an active ingredient.
14. The method of claim 13,

    The cancer is any one selected from liver cancer, lung cancer, colon cancer, ovarian cancer, breast cancer, prostate cancer, leukemia, lymphoma and pancreatic cancer pharmaceutical composition for cancer prevention or treatment.
15. A cancer cell targeting diagnostic or therapeutic conjugate comprising an antibody according to any one of claims 1 to 4, wherein one or more diagnostic or anticancer agents are bound to the antibody.
16. The method of claim 15,

    The diagnostic agent is any one or two or more selected from the group consisting of radionuclides, paramagnetic metal ions, superparamagnetic nanoparticles, chromophores, chromophores, luminescent materials and fluorescent materials, cancer cell targeting diagnostic or therapeutic conjugate.
17. The method of claim 15,

    The anticancer agent is cisplatin, 5-fluorouracil, adriamycin, methotrexate, vinblastine, busulfan, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, nitrosourea, taxol, paclitaxel, docetaxel, Cancer cell which is any one or more selected from the group consisting of 6-mercaptopurine, 6-thioguanine, bleomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitomycin-C and hydroxyurea Targeted diagnostic or therapeutic conjugates.

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