KR102259298B1 - An antibody specific for protein in cancer stem cell membrane and a use thereof - Google Patents

Abstract

The present invention is a monoclonal antibody specific for cell membrane proteins of cancer stem cells selected from the group consisting of monoclonal antibodies of a), b) and c), or functional fragments thereof: a) CDR1 region represented by SEQ ID NO: 1, SEQ ID NO: A heavy chain comprising a CDR2 region shown in 2 and a CDR3 region shown in SEQ ID NO: 3 and a light chain comprising a CDR1 region shown in SEQ ID NO: 4, a CDR2 region shown in SEQ ID NO: 5, and a CDR3 region shown in SEQ ID NO: 6 A monoclonal antibody comprising; b) a heavy chain comprising a CDR1 region shown in SEQ ID NO: 7, a CDR2 region shown in SEQ ID NO: 8, and a CDR3 region shown in SEQ ID NO: 9, and a CDR1 region shown in SEQ ID NO: 10, and a CDR2 region shown in SEQ ID NO: 11 And a monoclonal antibody comprising a light chain comprising a CDR3 region set forth in SEQ ID NO: 12; And c) a heavy chain comprising a CDR1 region represented by SEQ ID NO: 13, a CDR2 region represented by SEQ ID NO: 14, and a CDR3 region represented by SEQ ID NO: 15, and a CDR1 region represented by SEQ ID NO: 16, and a CDR2 represented by SEQ ID NO: 17. It relates to a monoclonal antibody comprising a light chain comprising a region and a CDR3 region set forth in SEQ ID NO: 18.

Description

Antibody specific to cancer stem cell membrane protein and its application {AN ANTIBODY SPECIFIC FOR PROTEIN IN CANCER STEM CELL MEMBRANE AND A USE THEREOF}

The present invention relates to antibodies specific for cancer stem cell membrane proteins and their applications, and more particularly, to cancer-related cancer obtained by a method of simultaneously detecting an antibody binding to cancer stem cells and a druggable target protein for cancer stem cells. It is about antigens, novel antibody-based substances that bind to antigens, and diagnosis and treatment of various cancers and diseases that express antigens.

Currently, the biggest problem in anticancer treatment is that the cancer recurs after cancer treatment or continuous treatment is impossible due to resistance to anticancer drugs due to continuous administration of anticancer drugs. In particular, the most vulnerable and problem is that targets for developing cancer treatments and improving treatments that can overcome these problems are extremely limited. Global pharmaceutical companies are investing in immune response-based treatments to solve the problem of drug resistance and recurrence, but the number of targets being used in the clinical stage is less than 60, so the development of a combination treatment method is intensively focused on existing targets. And there is a limit to overcoming the current problems.

Therefore, there is a desperate need for novel anticancer treatments, anticancer resistance of current cancer treatments, and innovative novel or novel targets that can overcome the recurrence of cancer and treatments for them.

However, a problem in the development of targeted antibody therapeutics is that most of the proteins overexpressed in cancer cells, that is, recombinant proteins for pre-selected targets, and therapeutic antibodies for them are being developed. This may differ from the structure of the target antigen recognized in vivo and the tumor microenvironment (TME), and as a reflection of this, sometimes lower anticancer effects than preclinical tests are reported in clinical trials.

In addition, existing anticancer drugs are therapeutics for proteins/targets overexpressed in diseased cancer cells, and because they cannot target and kill cancer stem cells, which are the core of cancer cells, cancer stem cells contribute a lot to recurrence of cancer and anticancer drug resistance and recurrence. It is recognized as doing. Accordingly, there is a need to discover specific cell membrane proteins of cancer stem cells, discover new target therapeutic targets that specifically apply them, and develop new anticancer drugs applied.

[Prior patent literature]

Republic of Korea Patent Publication 10-2017-0104619

The present invention was conceived by the above necessity, and an object of the present invention is to provide a novel anticancer agent target antigen and an antibody reflecting the phenotypic structural characteristics of the target antigen.

In order to achieve the above object, the present invention

A monoclonal antibody specific for a cell membrane protein of cancer stem cells and/or cancer cells selected from the group consisting of monoclonal antibodies of a), b) and c) below, or a functional fragment thereof:

a) a heavy chain comprising a CDR1 region represented by SEQ ID NO: 1, a CDR2 region represented by SEQ ID NO: 2, and a CDR3 region represented by SEQ ID NO: 3, and

A monoclonal antibody comprising a light chain comprising a CDR1 region represented by SEQ ID NO: 4, a CDR2 region represented by SEQ ID NO: 5, and a CDR3 region represented by SEQ ID NO: 6;

b) a heavy chain comprising a CDR1 region represented by SEQ ID NO: 7, a CDR2 region represented by SEQ ID NO: 8, and a CDR3 region represented by SEQ ID NO: 9, and

A monoclonal antibody comprising a light chain comprising a CDR1 region represented by SEQ ID NO: 10, a CDR2 region represented by SEQ ID NO: 11, and a CDR3 region represented by SEQ ID NO: 12; And

c) a heavy chain comprising a CDR1 region represented by SEQ ID NO: 13, a CDR2 region represented by SEQ ID NO: 14, and a CDR3 region represented by SEQ ID NO: 15, and

It provides a monoclonal antibody comprising a light chain comprising the CDR1 region of SEQ ID NO: 16, the CDR2 region of SEQ ID NO: 17, and the CDR3 region of SEQ ID NO: 18.

In one embodiment of the present invention, the cancer stem cell and/or the cell membrane protein of the cancer cell is preferably CD 44 or CD71, but is not limited thereto.

Functional antibodies of the present invention include IgG, IgA, IgD, IgE, IgM isotypes and subclasses.

Functional antibody fragments of the present invention include light chain, heavy chain, variable region, Fab, Fab', F(ab')2, single variable segment (scFv), Diabody, Tribody, double chain variable segment ( dsFv), scFv-Fc (Maxibody), single chain IgG (scIgG), a peptide containing a multiplex or CDR (Complementarity Determining Region) is preferred, but is not limited thereto.

Fab is a fragment obtained by treating IgG with the protease papain (cut to the 224th amino acid residue of the H chain), about half of the N-terminal side of the H chain and the entire L chain are bound by a disulfide bond (SS bond). It is an antibody fragment having an antigen-binding activity of about 50,000 molecular weight.

The Fab of the present invention can be obtained by treating the antibody of the present invention with the protease papain. Alternatively, the DNA encoding the Fab of the antibody is inserted into a prokaryotic expression vector or an expression vector for eukaryotes, and the vector is introduced into a prokaryote or eukaryote to express it to prepare a Fab.

F(ab')2 is a fragment obtained by treating IgG with the protease pepsin (cut to the 234th amino acid residue of the H chain), which is slightly larger than that of the Fab bound through the SS bond of the hinge region. It is an antibody fragment having an antigen-binding activity of about 100,000 molecular weight.

F(ab')2 of the present invention can be obtained by treating the antibody of the present invention with the protease pepsin. Alternatively, the following Fab' can be constructed by thioether binding or S-S binding. Fab' is an antibody fragment having an antigen-binding activity of about 50,000 molecular weight by cleaving the S-S bond of the hinge region of F(ab')2.

Single chain IgG (scIgG) is a single chain Fab (scFab) is a polypeptide linked to Fc, and is an antibody composed of one polypeptide having antigen-binding activity. Here, the single chain Fab is a single chain Fab (scFab) that connects one VH-CH1 and VL-CL using a suitable peptide linker (P) of 30 or more, and is VH-CH1-P-VL-CL to VL-CL-P- It is an antibody fragment having antigen-binding activity as a VH-CH1 polypeptide.

scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using a suitable peptide linker (P) of 12 or more residues, and is an antibody fragment having antigen-binding activity.

The scFv of the present invention obtains cDNA encoding VH and VL of the antibody of the present invention, constructs a DNA encoding scFv, and inserts the DNA into an expression vector for prokaryote or an expression vector for eukaryote to obtain the expression vector. It can be produced by expression by introduction into prokaryotes or eukaryotes.

Diabody is an antibody fragment in which scFvs having the same or different antigen-binding specificities form a dimer, and is an antibody fragment having a bivalent antigen-binding activity for the same antigen or a bispecific antigen-binding activity for different antigens.

Diabody of the present invention, for example, obtains cDNA encoding VH and VL of the antibody of the present invention, constructs a DNA encoding scFv having a polypeptide linker of 3 to 15 residues, and expresses the DNA for prokaryote By inserting into a vector or expression vector for eukaryotes and introducing the expression vector into a prokaryote or eukaryote, it can be prepared by expressing a Diabody.

In addition, when the linker P length is 3-10, a tribody is formed and may be included as a tribody.

dsFv refers to a polypeptide obtained by substituting a cysteine residue for one of the amino acid residues of VH and VL through an S-S bond between the cysteine residues. Amino acid residues substituted with cysteine residues can be selected based on prediction of the conformational structure of an antibody according to the method described by Reiter et al. (Reference: Protein Engineering, 7, 697 (1994)).

In one embodiment of the present invention, the a) monoclonal antibody preferably comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 19 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 20,

The b) monoclonal antibody preferably comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 21 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 22,

The c) monoclonal antibody preferably includes a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 23 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 24, but is not limited thereto.

In addition, the present invention provides a composition for diagnosis of cancer comprising the monoclonal antibody of the present invention or a functional fragment thereof as an active ingredient.

In one embodiment of the present invention, the cancer is breast cancer, lung cancer, ovarian cancer, kidney cancer, pancreatic cancer, colon cancer, colorectal cancer, prostate cancer, renal cell carcinoma, clear cell carcinoma, small cell carcinoma, squamous cell carcinoma, gastric cancer, lymphoma , It is preferably a cancer selected from the group consisting of leukemia and multiple myeloma, but is not limited thereto.

In addition, the present invention provides a composition for treating cancer comprising the monoclonal antibody of the present invention or a functional fragment thereof as an active ingredient.

The composition of the present invention may contain other drugs or other immune adjuvants to provide additional immune stimulating effects. Types of adjuvants are well known in the art (Vaccine Design-The Subunit and Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, MF, and Newman, MJ, Plenum Press, New York and London, ISBN. 0-306-44867-X). Preferably, the adjuvant included in the composition of the present invention comprises an aluminum salt or a calcium salt (eg, hydroxide or phosphate).

Specific examples of preferred adjuvants are as follows: aluminum salt or calcium salt (hydroxide or phosphate), oil-in-water emulsion (WO 95/17210, EP 0 399 843) or fine particles such as liposomes. Sex carrier (WO 96/33739), an immunologically adjuvant activity saponin fraction derived from the South American tree Quillaja Saponaria Molina (e.g., Quil A), 3 De-O- Silylated monophosphoryl lipid A, muramyl dipeptide, 3DMPL (3-O-deacylated monophosphoryl lipid A).

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenteral, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. have.

A suitable dosage of the pharmaceutical composition of the present invention is formulated in various ways depending on factors such as the formulation method, the mode of administration, the patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity. Can be. Meanwhile, the oral dosage of the pharmaceutical composition of the present invention is preferably 0.001-10,000 mg/kg (body weight) per day.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Alternatively, it may be prepared by enclosing it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

In addition, the present invention provides a conjugate or CAR-T in which a specific substance is bound to the monoclonal antibody of the present invention or a functional fragment thereof.

In one embodiment of the present invention, the conjugate is preferably an antibody-fluorescent material conjugate, antibody-toxin, antibody-drug conjugate, or antibody-antibody conjugate, but is not limited thereto.

Hereinafter, the present invention will be described.

The discovery of druggable targets for cancer stem cell membranes that reflect the phenotypic structural characteristics of cancer stem cell membrane proteins and development of antibodies thereto can be a method of effectively enhancing the clinical efficacy of cancer treatment drugs, as well as new diagnosis, treatment, and treatment improvement.

Accordingly, the present invention simultaneously develops an innovative druggable novel target or a new target that can solve the difficulties of the current cancer treatment and treatment, and simultaneously develops an anticancer treatment that reflects the phenotypic structural characteristics of the target. The furnace is to apply disease cell lines, especially cancer stem cell lines.

By simultaneously identifying an antibody that recognizes a target protein related to cancer stem cells or a target protein with phenotypic structural characteristics overexpressed in cancer stem cells and a target protein, it helps to cure cancer by contributing to anti-cancer therapy, anti-cancer resistance control and recurrence inhibition, and early cancer diagnosis. As it was predicted to be possible, the study was conducted with the strategy shown in FIG. 1.

The method for simultaneous antibody/antigen identification of the present invention is referred to as PhAbomics Technology (platform).

Although cancer stem cells were used as examples of the present invention, cells and stem cells for other specific disease cell lines (e.g., arthritis, multiple sclerosis, lung cancer, pancreatic cancer, etc.) are also used as the technique of the present invention. Diagnosis and therapeutics can be developed by securing specific antigens and antibody-based substances for cell lines.

Antibodies discovered simultaneously with the technology of the present invention can be applied to detection of target proteins, diagnosis of cancer, and development of various anticancer therapeutics (e.g., cancer stem cells and breast cancer, triple negative breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, etc.). .

1 is a schematic diagram of the simultaneous identification of a phenotypic cancer antigen and antibody pair,
Figure 2 is a verification of the antibody pool specifically binding to the cancer stem cell membrane. (A) Binding histogram for cancer stem cells (CSC) and PBMC of each round of antibody pools. Control is a historgram in which a mouse anti-M13 phage antibody and an anti-mouse-fluorescent-labeled antibody are bound to the corresponding cells. (B) Relative binding level of antibody pool by round
3 is a selection antibody specific for a cancer stem cell line,
Figure 4 (A) analysis of the scFv PCR product (~770bp) by agarose electrophoresis. (B) analyzing purified scFv-Fc by reducing (R) and non-reducing (NR) SDS-PAGE,
5 is a specific binding force of the scFv-Fc antibody to the cancer stem cell line. (A) flow cytometry and (B) immunofluorescence staining for 1G11 antibody that specifically binds to the surface of cancer stem cells,
6 is a process for identifying the target antigen of the selection antibody,
7 is (A) LC-MS/MS analysis of selected cancer stem cell 3C7 antibody and (B) antigen protein search result of 3C7 antibody. Antigen verification of 3C7 target antigen using a cell line knocked out of the antigen gene and a commercial antibody,
Figure 8 is (A) LC-MS/MS results of the selected antibody 1E12. (B) The binding to the antigens of the selection antibodies 1E12 and 2E1 against the target antigen was verified by ELISA,
9 is a breast cancer binding specificity of the 3C7 antibody,
Figure 10 is the binding specificity of 1E12 and 2E1 antibodies to breast cancer cell lines,
11 shows the binding specificity of the 1E12 antibody to gastric cancer, pancreatic cancer, and ovarian cancer cell lines,
12 is a correlation analysis of the expression level of the target protein. (A) Relative expression levels of target antigens per cell known from the Proteomics database. (B) the level of relative expression of the target antigen per cell, as determined by flow cytometry,
Figure 13 is the in vitro cell proliferation inhibitory effect of the 1E12 antibody and the 3C7 antibody (A) verification of the cytostatic efficacy of the 1E12 and 3C7 antibodies against cancer stem cells and parent cancer cells. (B) Evaluation of changes in efficacy according to the presence or absence of a ligand of the target antigen of the 1E12 antibody in a cancer stem cell line. (C) Verification of the cell proliferation inhibitory efficacy of 1E12 antibody against 4 subtypes of breast cancer cell lines and 1 type of gastric cancer cell line,
Fig. 14 is an evaluation of efficacy of 1E12 and 2E1 antibodies labeled with ADC antibody secondary antibodies. (A) When only the ADC secondary antibody was treated with MDA-MB-453 (CSC) and MDA-MB-231 (TNBC), it was confirmed that there was no cytotoxic effect on cells. (B) MDA-MB-453 (CSC) or (C) MDA-MB-231 (TNBC) was treated with 1E12 antibody and Fab anti-Fc-MMAF (30 nM) at the same time to evaluate the cell proliferation control efficacy of 1E12 antibody. (D) Evaluation of the cell proliferation control efficacy of 2E1 antibody when treated with 2E1 antibody and IgG anti-Fc-MMAF (6.7 nM) to MDA-MB-453 (CSC) simultaneously, and
15 is a diagram showing the amino acid sequences of a) 3C7 antibody, b) 1E12 antibody, and c) 2E1 antibody. CDR regions are indicated in underlined blue.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are described with the intention of illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

All cell lines used in the present invention were cultured at 37°C and 5% CO2 using a culture medium containing 10% FBS and 1% penicillin/streptomycin according to the supplier's instructions unless otherwise specified. Breast cancer stem cell lines and breast cancer cell lines contain DMEM (Gibco) containing 10% FBS, 1% penicillin/streptomycin, 10% FBS for CHO-DG44 cells, 1% penicillin/streptomycin, and HT supplement (sodium hypoxanthine, thymidine mixture). DMEM/F12 HEPES (Gibco) was incubated at 37°C and 5% CO2 incubator, respectively. The HEK293F cell line was cultured using freestyle 293F (GIBCO) as a protein expression medium under conditions of 37°C and 8% CO2.

Example 1: Cancer Identification and verification of antibodies and antigens for stem cells

1) Discovery and verification of antibodies that bind to cancer stem cell lines

To discover an antibody that recognizes the phenotypic structure/microenvironment of the target protein of cancer stem cell membranes, the existing well-known (George Smith, Science 228, 1315-1317 (1985); McCafferty et al., Nature) 348, 552-554 (1990); Azzazy and Highsmith, Clin Biochem 35, 425-445 (2002); Smith and Petrenko, Phage Display, Chem Rev 97, 391-410 (1997); Daugherty, Curr Opin Struct Biol 17, 474-480 (2007)) biopanning was performed on cancer stem cell lines by applying a phage display technique.

In order to confirm whether an antibody binding to a cancer stem cell line was discovered, a pool of phage antibodies was expressed for each round, and the binding force to the cancer stem cells and PBMC cells was determined by flow cytometry as follows.

After expressing each of the phage antibody pools from round 1 to round 4 secured by biopanning, ^{incubation with 1x10 6} cells for 1 hour at 4 °C to bind and buffer (1% BSA, 0.03% NaN3, pH 7.4 in PBS). Washed 3 times. Mouse anti-M13 phage antibody (Thermo scientific) and Alexa Flour® 647 goat anti-mouse antibody (Jackson Immunoresearch) were respectively added in turn, and the phage antibody specifically bound to the cells was fluorescently labeled.

Using FACS calibur (BD Bioscience) and software CellQuest Pro, fluorescence detection and data analysis were performed on the antibody.

As a result of the above example, it was confirmed by flow cytometry that the binding force to the cancer stem cell line increased as the biopanning round was repeated, but the binding force to PBMC did not increase. Therefore, it was confirmed that antibodies that specifically bind to cancer stem cells and an antibody pool were secured through biopanning (FIG. 2).

2) Selection of antibodies that bind to cancer stem cell lines

In order to select monoclonal antibodies that specifically bind to cancer stem cell lines, single colonies were isolated from the antibody pool, and the binding strength of each monoclonal antibody to cancer stem cell lines was analyzed in the same manner as in the flow cytometry method described above.

Among the analyzed monoclonal antibodies, monoclonal antibodies that specifically bind to cancer cell lines without binding to normal cells were selected, and the analysis results of representative antibodies are shown in FIG. 3.

3) Antibody expression and purification against cancer stem cells

In order to produce the selected antibody in the form of scFv-Fc antibody that functions similar to whole IgG in phage-scFv, the scFv of the antibody was amplified by PCR method and sub-cloned with scFv-Fc antibody in an animal cell expression vector. . After cloning was completed, transiently transfected into HEK293F, expressed for 6 days, secured the supernatant, verified the expression of the antibody by western blot, and purified and concentrated each antibody by affinity chromatography using Protein A resin. .

In order to sub-cloning into the parent vector based on animal cell expression, the amplified scFv fragment of each antibody was confirmed at a size of approximately 770 bp (example Fig. 4A), and the purified antibody through affinity chromatography was reduced or SDS-reduced under non-reducing conditions, respectively. As a result of PAGE analysis, the antibody band was confirmed at a size similar to that of the predicted antibody size based on the antibody sequence (Fig. 4B, reduced form: ~50kDa, non-reduced form: ~100kDa).

Therefore, the scFv-Fc antibody was obtained and verified from the phage antibody.

4) Characterization of cancer stem cell antibodies (FACS and IF)

The binding force of the scFv-Fc antibody to the cancer stem cell line was carried out in the same manner by the flow cytometry method described above. In addition, the localization of the antibody on the surface of cancer stem cells was observed by immunofluorescence staining by the following method.

10 μg/mL scFv-Fc antibody was added to each of cancer stem cells, cancer cells, and CHO-DG44 cultured with about 70% confluence in a culture dish, and binding was performed on ice for 1 hour. After washing twice with PBS to remove unbound antibody, 4% paraformaldehyde was added and the Fix process was performed at room temperature for 20 minutes. After the fix was finished, it was washed twice with PBS and then immediately labeled scFv-Fc with Alexa594 anti-human IgG (Jackson Immunoresearch). After two washings, 300nM DAPI was added to stain the nucleus. After all reactions were completed, the cells were washed three times, and then the cells were observed with a fluorescence microscope (Olympus 1x71) and the image was observed.

As a result of flow cytometric analysis of the scFv-Fc type antibody, it was confirmed that all the verified antibodies strongly bind to the cancer stem cell line in the same way as the phage antibody, while having relatively weak binding power in the cancer cell line, and not completely binding in the normal cell (CHO). (Figure 5A).

In addition, the antibody selected by immunofluorescence staining strongly binds to the surface of cancer stem cells, and fluorescence is observed only in a small part of cancer cell lines, and is not fluorescently labeled in normal cells. It is shown in Fig. 5B as an example of an antibody that binds only with specificity on the surface of cancer stem cells.

Therefore, the difference in antibody binding ability to cancer stem cells and cancer cells identified through flow cytometry was re-verified through immunofluorescence staining, and it was verified that the selected cancer stem cell membrane antibodies have higher specificity for cancer stem cells compared to cancer cells. .

5) Identification and verification of cancer stem cell membrane target antigen of cancer stem cell antibody ( In situ IP and LC-MS/MS)

In order to identify the target antigen of the selective antibody that specifically binds to cancer stem cells, the cancer cell membrane antigen recognized by the cancer stem cell antibody was pulled down by the following method by the in-situ immunoprecipitation technique (FIG. 5).

After culturing cancer stem cells and cancer cells on a culture plate, 40 μg of IgG or scFv-Fc antibody fragment was added to each cell line, followed by antibody-binding reaction at 4°C for 1.5 hours. Antibody not bound to the cell membrane antigen was removed by washing 3 times with PBS, and then cancer stem cells and cancer cell lysate were obtained using lysis buffer (25mM HEPES, 150mM NaCl, 1% NP-40, 5mM MgCl2, protease inhibitor). Cell lysates were spin down at 4500 rpm for 5 minutes to separate the supernatant. The separated supernatant was pulled down with protein A resin, and then the cell surface protein-antibody conjugate purified using 50mM glycine was isolated from proein A resin. The obtained cell surface protein-antibody conjugate sample was analyzed by western blot and SDS-PAGE, in-gel digestion, and then LC-MS/MS peptide analysis to identify specific cancer stem cell membrane proteins recognized by cancer stem cell antibodies ( Fig. 6).

The ELISA analysis method for validation of the identified cancer stem cell target protein was performed as follows.

After coating 100 nM of recombinant protein on a 96-well ELISA plate overnight with 50 mM NaHCO3, pH9 buffer, washing with PBS buffer, blocking with PBS/3% milk and washing with PBS again, the selected antibody and huIgG antibody were respectively added. It was put at 1 μg/mL and reacted at room temperature for 1.5 hours. After washing with PBS, the anti-huIgG-HRP antibody was diluted 1/5000, put into each plate well, and reacted at room temperature for 1 hour. After washing with PBS six times, TMB substrate was added and reacted, and the HRP reaction was stopped with 0.25M sulphuric acid to detect a signal at 450nm.

The selected cancer stem cell antibody and pull-down cancer stem cell membrane target antigen were analyzed by LC-MS/MS. The mass/charge ratio analysis of the detected peptide and the peptide sequence were identified using a protein search tool such as PD 2.2, PEAKS, and Sorcerer to identify the cancer stem cell membrane target antigen recognized by the antibody.

[Example 1] In the case of the selected 3C7 antibody, the cancer stem cell membrane target antigen was identified by LC-MS/MS analysis, and CD44-specific commercial antibody and 3C7 antibody were CD44-expressing cell lines (CD44+), and CD44 gene knockout. By comparative analysis of binding to the cell line (CD44-), it was verified that the cancer stem cell membrane target protein recognized by the 3C7 antibody was CD44 (FIG. 7).

[Example 2] The selected 1E1 antibody was subjected to an antigen identification experiment, and the peptide obtained by LC-MS/MS was analyzed to identify CD71 as an antigen candidate group (FIG. 8A).

As a result of analysis by ELISA to see if the selection antibody binds to the identified target antigen CD71, it was observed that the 1E12 antibody and the 2E1 antibody specifically had strong binding power to the CD71 protein (Pro1). It did not bind to BSA or CTLA-4 and other cell membrane proteins (Pro2 to Pro6) used as a negative control (FIG. 8B).

Cancer stem cell membrane proteins to which cancer stem cell antibodies specifically bind were known to be closely related to tumors such as tumor growth, cancer metastasis, invadopodia formation, and lymphocyte activation (Table 1).

Specific CD44 has been applied as a cancer stem cell biomarker and has been reported to promote multi-drug resistance (Reference: Ravindranath et al., CD44 promotes multi-drug resistance by protecting P-glycoprotein from FBXO21-mediated ubiquitination, Oncotarget 22, 26308-26321 (2015)).

Therefore, by simultaneously discovering overexpressed or specifically expressed proteins and antibodies against cancer stem cells and other specific disease cells by the present technology method, discovery and diagnosis and treatment of targets for the development of biomarkers and therapeutic agents are carried out. It was proved that it was possible to find a suitable antibody.

Table 1 shows the target exclusive spectrum count of cancer stem cell-specific antibodies and cancer-related functions of the reported target antigens.

Example 2: excavated Cancer stem cells Antigen and Application method for antibodies example

The following method demonstrates that the protein discovered by the method of the present invention can be applied as a target for the development of biomarkers and therapeutic agents for breast cancer and other cancer diseases, and at the same time, the antibody discovered by the method of the present invention can be applied as a diagnosis and therapeutic agent. I did.

1) Binding of selection antibodies against breast cancer, gastric cancer, pancreatic cancer, and ovarian cancer cell lines

Selective antibodies 3C7, 1E12, 2E1 antibodies were analyzed by flow cytometry to determine whether they bind to various cell lines such as breast cancer subtype, gastric cancer, pancreatic cancer, ovarian cancer, etc., and proceeded in the same manner as the analysis of cancer stem cell binding ability of the scFv-Fc antibody described above. . Breast cancer subtype cells include triple negative breast cancer (TNBC; MDA-MB-468, MDA-MB-231, BT-20, MDA-MB-453) and ZR-75-1, BT-474, MCF-7, T47D, SKBR-3 (Table 2) was used, and as gastric cancer, MKN45, NCI-87, pancreatic cancer PANC-1, and ovarian cancer SKOV-3 cell lines (FIGS. 9 to 12) were used.

Table 2 shows subtypes of breast cancer cell lines

[A] Cancer stem cell line It was re-verified that the 3C7 antibody that recognizes CD44, known as a biomarker, as a target antigen has the highest binding ability specifically for cancer stem cell lines (FIG. 9). In addition, among breast cancer cells, it was observed that it binds only to BT-20 very weakly, and not all other breast cancer cell lines.

Therefore, it was re-verified that the method of the present invention can discover and select antigens and antibodies specific to cancer stem cell lines, and it was verified that the method of the present invention can simultaneously discover new therapeutic target antigens and antibodies thereto, thereby enabling diagnosis and treatment development.

In addition, interestingly, as it is observed that the 3C7 antibody weakly binds to NCI-87 due to this study, it is recognized that it is possible to develop a gastric cancer treatment agent targeting CD4.

CD44 background

The CD44 antigen targeted by the 3C7 antibody is a protein expressed on the cell membrane and is present on the extracellular matrix (ECM), hyaluronic acid (HA), osteopontin (OPN), chondroitin, collagen, fibronectin, and serglycin/sulfated proteoglycan. Among them, HA is reported as the most specific ligand for CD44 activation (Reference: Chen Chen, Shujie Zhao, Anand Karnad and James W. Freeman Journal of Hematology & Oncology 2018; 11 :64).

The combination of CD44 and HA causes structural changes to facilitate the binding of the intracellular cytoplasmic tail of CD44 to the adapter molecule, and through these structural changes, cell interactions and signal transduction pathways such as Ras, MAPK, PI3K, Rho and NFκB are activated. And contributes to the development of tumor diseases. In particular, the above signaling pathway is known to be involved in the activation of cell cycle and proliferation, cytoskeleton reconstruction and cell migration, cell survival, and signaling pathways such as inflammatory response, and the expression level of D44 is EMT, an important factor in cancer metastasis. Has been reported to be associated with, and this may potentially increase the tendency of tumor invasiveness and induction of angiogenesis through ECM (References: Cho SH, Park YS, Kim HJ, Kim CH, Lim SW, Huh JW, Lee JH, Kim HR. Int J Oncol. 2012;41(1):211-8).

The binding of CD44 and HA in breast cancer cells has been reported to contribute to the increased expression of the multidrug resistance gene P-glycoprotein and the anti-apoptotic gene Bcl, and as a result promote the proliferation and survival of cancer cells (References: Bourguignon LY, Xia. W, Wong G. J Biol Chem. 2009;284(5):2657-71).

CD44 is also overexpressed in cancer patients who do not respond to chemotherapy and cancer patients with low survival rates (References: Gao Y, Feng Y, Shen JK, Lin M, Choy E, Cote GM, Harmon DC, Mankin HJ, Hornicek FJ, Duan Z. Sci Rep. 2015;5:11365).

This can be expected to be able to establish new treatment guidelines for cancer patients and measure patient prognosis based on the expression level of CD44.

Overall, it can be seen that CD44 is heavily involved in the process of cancer development, and among them, there is a high association with drug resistance. Therefore, if the antibody composition developed in the present invention recognizes the surface expression of CD44 on the surface of cancer cells and at the same time appears to contribute to the inhibition of cancer cell growth and metastasis, a very useful diagnosis and treatment process will be realized.

[B] It was confirmed that the 1E12 antibody and the 2E1 antibody specifically recognizing CD71 showed specific binding properties to the parent cell line MDA-MB-453 of breast cancer stem cells (FIG. 10A).

In addition, when FACS analysis of the CD71 expression rate for various breast cancer cell lines for 1E12 antibody binding, it was confirmed that it binds more strongly to all other breast cancer cell lines, including triple negative breast cancer, than the control antibody. This is because CD71 is expressed in various breast cancer cell lines. It was verified that it is also suitable as a target (FIG. 10).

In addition, it was confirmed that the 1E12 antibody specifically binds to gastric cancer, pancreatic cancer, and ovarian cancer.

Therefore, it was confirmed that the antibody targeting CD71 strongly binds not only to cancer stem cell lines, but also to various carcinoma cells and controls it to be used in the treatment of breast cancer and other cancer diseases (FIG. 11, Table 3).

Table 3 shows the specific binding of cancer stem cell target antibodies to various carcinoma cell lines

2) Antigen verification through flow cytometry and correlation analysis of target protein expression

The expression level of the target protein protein X1B1 was compared and analyzed by flow cytometry for 4 breast cancer cell lines and ovarian cancer cell lines in which the expression level of the cell membrane target protein X1B1 discovered in the present invention was reported.

It was confirmed that the reported target protein protein X1B1 expression ratio in ProteomicsDB (https://www.ProteomicsDB.org ) and the protein expression ratio obtained by the experiment were consistently correlated with each other (Fig. 12).

Based on these results, the binding and specificity of the target protein and antibody obtained through LC-MS/MS analysis could be re-verified.

3) Cell growth inhibition efficacy evaluation of the discovered antibody (In-vitro cell based assay)

Dispense 3x10 ³ cells into each well of a 96-well plate, _{incubate at 37°C, 5% CO 2} for 24 hours, replace with new growth media, and add serially diluted 1E12 antibody or control antibody to each well. Then, the cells were incubated at 37°C and 5% CO ₂ for about 4-6 days, and then treated with AlamarBlue (Invitrogen), and fluorescence values were measured in Ex530 and Em590 every 2, 4 and 6 hours (PerkinElmer Victor x4). . EC50 values were analyzed with Prism4 (GraphPad).

[example]: The 3C7 antibody discovered in the present invention has the ability to inhibit cell growth by more than 30% (p=0.0032, t-test) compared to a control cell line (parent cancer cell) that does not express CD44 in a cancer stem cell line (CSC) in which CD44 expression is reported. Was confirmed (Fig. 13A).

The growth rate of cells not treated with antibodies to each cell line of CD44+ or CD44- was calculated as 100%.

[Example]: When the 1E12 antibody discovered in the present invention was treated with a cancer stem cell line (CSC), it was confirmed that cell growth was inhibited by up to 32%. A similar cell growth inhibitory effect was shown not only in cancer stem cell lines but also in parent cancer cells (FIG. 13 ).

There was no efficacy in CSC and cancer cells to which the control antibody human IgG (hIgG) was added. As a result of analyzing the EC50 value for each cell, it was found to be 53.28 nM in the cancer stem cell line and 18.33 nM in the parent cancer cell line (Fig. 13A).

CD71, which is targeted by the 1E12 antibody, is a receptor expressed on the cell membrane, and has the property of contributing to cell growth by binding to iron-conjugated transferrin (transferrin) ligand. Therefore, it was confirmed that the 1E12 antibody binds to CD71 even in the presence of the transferrin ligand to control the binding of the ligand to the CD71, and thereby inhibit cell growth. It was observed that even when the ligand was added, the effect of inhibiting the proliferation of cancer stem cells was maintained (FIG. 13B).

In addition, it was confirmed that the 1E1 antibody inhibited the activation of cancer cell line proliferation mediated through the transferrin ligand, as a higher concentration of 1E12 antibody was required in the presence of the transferrin ligand than in the absence of the ligand for cancer stem cell death.

In addition, it was observed that the 1E12 antibody strongly binds to various cancer and breast cancer cell line subtypes, and thus cell proliferation inhibitory efficacy was observed even when cell proliferation inhibition analysis of 4 subtypes of breast cancer cell lines and 1 type of gastric cancer cell line was performed (Fig. 13C). ). Therefore, it is possible to develop various cancer treatments by targeting the CD71 protein discovered in the present invention, and the 1E12 antibody developed in the present invention was verified for the applicability of triple negative breast cancer and other anticancer drugs. Table 4 summarizes the efficacy evaluation results for each cell line of the 1E12 antibody.

Table 4 shows the efficacy of the 1E12 antibody for each cell line.

3) Cell growth inhibition efficacy evaluation of Antibody-Drug Conjugate (In-vitro cell based assay)

In addition to the naked antibody (whole IgG, fragment antibody, etc.) as the therapeutic plateform for the target discovered in the present invention, various therapeutic agents such as Antibody-drug conjugate (ADC) can be developed, so the anti-CD71 antibody developed in the present invention, 1E12 Antibody and 2E1 antibody were tested for inhibition of proliferation of cancer cell lines in "ADC" form.

For the efficacy evaluation of the target antibody labeled with the ADC secondary antibody, the cells were treated with the antibody under the same conditions as for the cell growth inhibitory efficacy evaluation of the antibody, and additionally treated with 30 nM Fab aHFc-MMAF or 6.6 nM IgG aHFc-MMAF. After day incubation, AlamarBlue (Invitrogen) was treated, and fluorescence values were measured in Ex530 and Em590 every 2, 4 and 6 hours (Victor x4, PerkinElmer), and the EC50 values were analyzed with (Prism4, GraphPad).

It was proved in the 1E12 and 2E1 antibodies that the anti-CD71 antibody (1E12 or 2E1) and the MMAF-conjugated secondary antibody were treated with the cancer stem cell line compared to the existing naked antibody. , And D).

In addition, it was verified that the 1E12 antibody targeting CD71 also inhibits cell proliferation in the triple negative breast cancer (TNBC) cell line MDA-MB-231 (FIG. 14C).

On the other hand, at the concentration of the MMAF secondary antibody used in this experiment, it was observed that there was no inhibition of cell proliferation when only the secondary antibody bound with MMAF was treated.

Therefore, it was confirmed that the antibodies simultaneously discovered by the technology of the present invention can be applied for development as various anticancer therapeutic agents for target proteins (e.g., cancer stem cells and breast cancer, triple negative breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, etc.)

<110> KNU-Industry Cooperation Foundation <120> ANTIBODY SPECIFIC FOR PROTEIN IN CANCER STEM CELL MEMBRANE AND A USE THEREOF <130> P18-0052HS <160> 24 <170> KopatentIn 2.0 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VH <400> 1 Gly Asp Tyr Gly Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VH <400> 2 Gly Ile Ser Trp Asn Ser Asp Thr Ile Gly Leu Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VH <400> 3 Pro Ala Phe Ile Ser Gly Trp Tyr Pro Gly Phe Phe Asp Ile 1 5 10 <210> 4 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VL <400> 4 Ser Gly Thr Thr Arg Asp Val Gly Arg Tyr Asp Tyr Val Ser 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VL <400> 5 Asp Val Ser Lys Arg Pro Ser 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VL <400> 6 Cys Ser Tyr Ala Gly Asn Ser Thr Tyr Val 1 5 10 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VH <400> 7 Asp Tyr Tyr Met His 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VH <400> 8 Gly Ile Ser Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Asn Phe Gln 1 5 10 15 Gly <210> 9 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VH <400> 9 Asp Gly Ala Gly Pro Glu Tyr Tyr Tyr Tyr Gly Leu Asp Val 1 5 10 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VL <400> 10 Thr Gly Thr Ser Ser Asp Ile Gly Gly Tyr Asn Tyr Val Ser 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VL <400> 11 Glu Val Thr Lys Arg Pro Ser 1 5 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VL <400> 12 Ser Ser Tyr Ser Asp Ser Asp Asn Leu Leu 1 5 10 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VH <400> 13 Ser Tyr Ala Ile Ser 1 5 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VH <400> 14 Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VH <400> 15 Glu Gln Asp Gly Gly Tyr Phe Asp Tyr 1 5 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 OF VL <400> 16 Gly Gly Asp Asn Ile Gly Arg Glu Ser Val His 1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 OF VL <400> 17 Tyr Asp Ser Asp Arg Pro Ser 1 5 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 OF VL <400> 18 Gln Val Trp Asp Ser Ser Ser Asp His Val Val 1 5 10 <210> 19 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 19 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Asp Thr Ile Gly Leu Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Ala Phe Ile Ser Gly Trp Tyr Pro Gly Phe Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 20 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 20 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Ser Gly Thr Thr Arg Asp Val Gly Arg Tyr 20 25 30 Asp Tyr Val Ser Trp Tyr Arg Gln Tyr Pro Gly Glu Ala Pro Gln Leu 35 40 45 Leu Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Phe Asp Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Asn 85 90 95 Ser Thr Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 21 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 21 Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ser Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Asn Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Asn Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ala Gly Pro Glu Tyr Tyr Tyr Tyr Gly Leu Asp Val 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 22 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 22 Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Ile Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln His His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Val Ile Ser Glu Val Thr Lys Arg Pro Ser Gly Val Pro Gly Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ser Asp Ser 85 90 95 Asp Asn Leu Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 23 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Glu Gln Asp Gly Gly Tyr Phe Asp Tyr Trp Ser Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 24 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 24 Gln Leu Val Leu Thr Gln Ser Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asp Asn Ile Gly Arg Glu Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Ala Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Tyr Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Trp Val Asp Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr His Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105

Claims (12)

A heavy chain comprising a CDR1 region represented by SEQ ID NO: 1, a CDR2 region represented by SEQ ID NO: 2, and a CDR3 region represented by SEQ ID NO: 3, and
A monoclonal antibody specific for cancer stem cells and/or cell membrane proteins of cancer cells comprising a light chain comprising the CDR1 region of SEQ ID NO: 4, the CDR2 region of SEQ ID NO: 5, and the CDR3 region of SEQ ID NO: 6, or Its functional fragment. The monoclonal antibody or functional fragment thereof according to claim 1, wherein the cancer stem cell and/or the cell membrane protein of the cancer cell is CD 44. The method of claim 1, wherein the functional fragment is Fab, Fab', F(ab')2, single variable segment (scFv), Diabody, Tribody, double chain variable segment (dsFv), single A monoclonal antibody, or a functional fragment thereof, characterized in that it is a variable segment-C fragment (scFv-Fc), or a single chain IgG (scIgG). The monoclonal antibody of claim 1, wherein the monoclonal antibody comprises a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 19 and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 20, or a functional fragment thereof. delete delete A composition for diagnosis of cancer comprising the monoclonal antibody of any one of claims 1 to 4, or a functional fragment thereof as an active ingredient. The method of claim 7, wherein the cancer is breast cancer, lung cancer, ovarian cancer, kidney cancer, pancreatic cancer, colon cancer, rectal colon cancer, prostate cancer, renal cell carcinoma, clear cell carcinoma, small cell carcinoma, squamous cell carcinoma, gastric cancer, lymphoma, leukemia, and Cancer diagnosis composition, characterized in that the cancer selected from the group consisting of multiple myeloma. A composition for treating cancer comprising the monoclonal antibody of any one of claims 1 to 4, or a functional fragment thereof as an active ingredient. The method of claim 9, wherein the cancer is breast cancer, lung cancer, ovarian cancer, kidney cancer, pancreatic cancer, colon cancer, rectal colon cancer, prostate cancer, renal cell carcinoma, clear cell carcinoma, small cell carcinoma, squamous cell carcinoma, gastric cancer, lymphoma, leukemia, and Cancer treatment composition, characterized in that the cancer selected from the group consisting of multiple myeloma. A conjugate in which a specific substance and/or a genetically recombinant substance is bound to the monoclonal antibody of any one of claims 1 to 4, or a functional fragment thereof. The conjugate according to claim 11, wherein the conjugate is an antibody-fluorescent substance conjugate, antibody-toxin, antibody-drug conjugate, or antibody-antibody conjugate.

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