KR101994977B1 - An anti-c-Myc monoclonal antibody and use of the same - Google Patents

Abstract

The present invention relates to a monoclonal antibody, or a functional fragment thereof, which specifically recognizes a c-Myc protein or a part of the protein, wherein the monoclonal antibody or a functional fragment thereof is any one selected from the group consisting of the following polypeptide sequences Or a functional fragment thereof: 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 heavy chain comprising SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR3 region of SEQ ID NO: 6, wherein the antibody of the present invention decreases Myc-activity in various cancers Can act as an antagonist, and thus can be employed as a therapeutic antibody candidate for cancer.

Description

c-Myc-specific monoclonal antibody and its use {An anti-c-Myc monoclonal antibody and use of the same}

The present invention relates to monoclonal antibodies specific for c-Myc and uses thereof.

Cancer is a complex, multi-step process linked to a change in the expression level of the cell-oncogenes gene, including the one-oncogenic gene c-myc (Denhardt, DT, 1999; Pendergast GC, 1999). c-myc regulates cell growth, differentiation, and apoptosis, and its abnormal expression is associated with many cancers including lung cancer, colon cancer, breast cancer, bladder cancer, leukemia, and lung cancer (Dang et al., 1999).

Reports involving uncontrolled expression or abnormal expression of the basic-spiral-loop-spiral nucleus c-myc in multiple cancers lead to pre-clinical and clinical studies evaluating c-myc inhibitory effects using a number of approaches .

Myc gene is a regulator gene that affects the coding of transcription factors. It is also a cancer gene that encodes a DNA binding protein, and there are two types, v-Myc and c-Myc. v-Myc (v-myc avian myelocytomatosis viral oncogene homolog) is a cancer gene in birds that causes bone marrow cell tumor in algae.

On the other hand, c-Myc has a structure of a basic region-helical loop-helical-leucine zipper region (bHLH-LZ) and amplifies the expression of genes in various cancer cells and is located in the region of human chromosome 24 . The protein expressed by c-Myc plays a crucial role in the cycle, death, and transformation of the cell, which is a phosphoprotein. The gene promotes cell proliferation and transformation. In other words, acetylation of histone causes loosening of contact with DNA, thereby relieving the transcriptional repression state of the gene, thereby allowing Oct3 / 4 and Sox2 to bind to specific gene positions and induce transcription.

When mutations occur in c-Myc and become overactive, the cells divide and form tumors. These c-Myc-dependent cancer cells die when c-Myc is removed. Therefore, researchers have been trying to develop anticancer drugs by focusing on these vulnerabilities of cancer cells, but they fail to develop antibodies that specifically or effectively bind to Myc protein because c-Myc does not have an effective binding site for the antibody to be.

[Prior Patent Literature]

Korean Patent Publication No. 1020160133368

The present invention has been made in view of the above needs, and an object of the present invention is to provide a monoclonal antibody specific for c-Myc.

Another object of the present invention is to provide cancer-related uses of said antibody.

In order to achieve the above object, the present invention provides a monoclonal antibody, or a functional fragment thereof, which specifically recognizes a c-Myc protein or a part of the protein, wherein the monoclonal antibody or the functional fragment thereof is composed of the following polypeptide sequence Or a functional fragment thereof: 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 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.

In one embodiment of the invention, the functional fragment is preferably a single side chain antibody (scFv)

In another embodiment of the present invention, the functional fragment is preferably an antigen-binding fragment (Fab), and the functional fragment is preferably a light or heavy chain comprising the CDR region of the present invention, But it is not limited thereto.

In another embodiment of the present invention, the monoclonal antibody is preferably a heavy chain comprising the polypeptide sequence of SEQ ID NO: 7 and a light chain comprising the polypeptide sequence of SEQ ID NO: 8,

In another embodiment of the present invention, a portion of the c-Myc protein is preferably a peptide sequence of SEQ ID NO: 9, but is not limited thereto.

The present invention also provides a composition for treating cancer comprising an anti-c-Myc monoclonal antibody of the present invention as an active ingredient.

The present invention also provides a cancer diagnostic composition comprising a conjugate obtained by conjugating a marker substance to a monoclonal antibody against c-Myc of the present invention.

In one embodiment of the present invention, the labeling substance is preferably selected from the group consisting of enzymes, luciferase, magnetic particles, fluorescent substances and radioactive isotopes, but is not limited thereto.

The present invention also relates to a method for preparing a monoclonal antibody comprising contacting a sample with the monoclonal antibody of the present invention; And 2) detecting the reaction between the monoclonal antibody and the sample.

The present invention also relates to the above monoclonal antibody of the present invention; And 2) a container.

The present invention also relates to the cancer diagnostic composition of the present invention; And 2) a container.

The diagnostic composition according to the present invention may include a monoclonal antibody of the present invention and a reagent used for immunological analysis. Reagents used in immunological assays include suitable carriers for use in all known quantitative assay methods based on antigen-antibody binding, labels capable of generating detectable signals, solubilizers, and detergents. Examples of the quantitative analysis method include, but are not limited to, immunoblotting, immunoprecipitation, enzyme immunoassay, protein chip, rafepress and microarray methods.

Suitable carriers herein include, but are not limited to, soluble carriers such as any one of the physiologically acceptable buffers known in the art (e.g., PBS) or insoluble carriers such as polystyrene, polyethylene, polypropylene, Polyester, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, glass, metal, agarose, and combinations thereof.

Enzymes, fluorescent substances, luminescent substances and radioactive substances can be used as markers capable of generating detectable signals. Examples of the enzyme include peroxidase, alkaline phosphatase,? -D-galactosidase, glucose oxidase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, And the fluorescent substance may be a fluorescent substance such as fluorescein isothiocyanate, phycobilin protein, rhodamine, phycoerythrin, phycocyanin and orthophthalic acid, Orthophthalic aldehyde and the like can be used. Isoluminol, lucigenin and the like can be used as the luminescent material, and 131I, 14C, 3H and the like can be used as the radioactive material. However, in addition to those exemplified above, any of them can be used as long as they can be used for immunological assays.

Specifically, according to the present invention, a method for diagnosing cancer development in a sample by a sandwich enzyme immunoassay includes the following steps:

(1) immobilizing a first antibody specific to human c-Myc protein to a solid body,

(2) reacting the solid solution with a sample solution to be tested so that c-Myc in the sample binds to the antibody,

(3) washing and removing unreacted material using a washing liquid,

(4) adding a second antibody specific to c-Myc protein bound to the chromogenic enzyme to the solid to react with the complex of c-Myc and the first antibody bound in step (2)

(5) washing and removing unreacted materials using a washing liquid,

(6) adding an enzyme substrate solution to the washed solid to cause a color reaction, and

(7) Measuring the degree of color development of the reaction, and measuring the concentration of c-Myc in the sample.

In addition, the kit of the present invention for measuring c-Myc by the above sandwich type enzyme immunoassay comprises an anti-c-Myc first antibody immobilized on a solid body, an anti-c-Myc second antibody bound to an enzyme, Sample diluent solution, enzyme substrate solution, wash solution and c-Myc standard solution.

It is preferable to use horseradish peroxidase as an enzyme for quantitatively measuring the concentration of c-Myc by the chromogenic reaction. In this case, as the substrate solution of the enzyme, ortho-phenylene Diamine (OPD) solution is used.

Alternatively, the antibody of the present invention may be directly contacted with a fluorescent substance (for example, Alexa488) for quantitative analysis.

In addition, a well of a microplate is generally used as a solid body.

In addition to the enzyme immunoassay described above, the immunoassay using the anti-c-Myc monoclonal antibody according to the present invention may also be used to measure the presence or absence of c-Myc in a sample using immunochromatography.

The immunochromatographic method is a method of immunologically reacting an anti-c-Myc antibody bound to colored microparticles with a sample, and performing a continuous movement of the colored microparticle-antibody-c-Myc complex by capillary action, By allowing the complex to bind to a separate anti-c-Myc antibody band previously immobilized, the presence or absence of c-Myc in the sample is detected depending on whether the band position is colored.

The method described above is characterized in that the sample member, the immunoreactive member in which the anti-c-Myc first antibody bound to the colored fine particles are temporarily immobilized, the anti-c-Myc second antibody band (judgment line) Line), and an immunochromatographic kit comprising an absorbing member.

Each of the members is arranged in series in order to partially overlap the sample solution by continuous capillary action, and the control line is spaced apart from the determination line at a predetermined distance.

In the immune response member, the anti-c-Myc first antibody bound to the colored microparticles is temporarily fixed on the membrane to form a complex by an immune reaction with c-Myc in the sample solution absorbed by the sample member, And is continuously moved onto the result display member by the capillary phenomenon.

In addition, a total of two antibody bands composed of the anti-c-Myc second antibody band and the control antibody band are fixed on the surface at regular intervals in the result display member.

Wherein the anti-c-Myc second antibody band is a resultant line indicative of the presence of c-Myc in the sample, wherein the colored microparticle-antibody-c-Myc complex formed in the immunoreactive member passes through this band point -c-Myc antibody to specifically bind to the second antibody to form a complex, thereby causing coloration at the band position.

On the other hand, the control antibody band, usually located at a position of about 0.5 to 2 cm from the bottom of the second antibody band in the sample movement direction, contains a general antibody such as goat anti-mouse IgG, By immunologically reacting with the sample components, it confirms whether this test is normally performed.

Thereafter, the remaining sample components are continuously moved by the capillary phenomenon and absorbed by the absorption member.

The anti-c-Myc first antibody and the second antibody used in the immunochromatographic kit of the present invention are the same or different monoclonal antibodies specifically binding to c-Myc produced from the clones according to the present invention, It is more preferable to use it.

The present invention also provides a pharmaceutical composition comprising the above antibody or single-chain antibody (scFv) fragment of the present invention as an active ingredient.

The present invention also provides a composition for preventing or treating cancer, which comprises the antibody of the present invention or a single side chain antibody (scFv) fragment thereof as an active ingredient.

The present invention also provides an anticancer composition comprising the antibody of the present invention or a single side chain antibody (scFv) fragment thereof as an active ingredient.

The pharmacology of the present invention may further comprise pharmaceutically acceptable excipients, carriers, diluents, and the like.

Carriers which can be used in the present invention include macromolecules that are slowly metabolized such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers and inert viral particles. Salts of inorganic acids such as, for example, hydrochloride, hydrobromide, phosphate and sulfate; Pharmaceutically acceptable salts such as salts of organic acids such as acetate, propionate, malonate and benzoate; Liquids such as water, saline, glycerol and ethanol, and auxiliary materials such as wetting agents, emulsifying agents or pH buffering substances may be used.

Pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, 1991.

In addition, the composition may be formulated into a unit dosage form suitable for intra-body administration of a patient according to a conventional method in a pharmaceutical field, preferably a formulation useful for administration of a protein drug, Or intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, vaginal, or rectal routes But are not limited to, the parenteral route of administration.

Formulations suitable for this purpose include various oral dosage forms such as tablets, pills, dragees, powders, capsules, syrups, solutions, gels, suspensions, emulsions and microemulsions; And injectable preparations such as injectable ampoules, injecting agents and spray agents such as hypospores are preferred. In the case of injection or infusion formulations, it may take the form of a suspension, solution or emulsion, and may contain formulatory agents such as suspending, preserving, stabilizing and / or dispersing agents. In addition, the antibody molecules may be formulated in a dried form that can be used by appropriate sterile liquid roll reshaping prior to use.

Since the composition of the present invention contains an antibody molecule that is easily decomposed in the gastrointestinal tract as an active ingredient, when the composition is to be administered by a route using the gastrointestinal tract, the antibody is protected from degradation. After the antibody is released, And the like.

The present invention also provides a method of preventing or treating cancer, comprising administering an antibody of the invention to an animal, preferably a mammal, more preferably a human, in a variety of ways as described above.

As an active ingredient of the composition or pharmaceutical preparation of the present invention, the antibody may be administered to a mammal, including a human, in an amount of 0.001 to 50 mg / kg body weight, preferably 0.1 to 20 mg / kg body weight per day, Lt; / RTI > However, the actual dosage of the active ingredient will depend on a variety of factors such as the disease to be prevented or treated, the severity of the disease, the route of administration, the patient's body weight, age and sex, drug combination, response sensitivity and tolerance / Should be understood to be determined, and thus the dose is not intended to limit the scope of the invention in any way.

The functional antibody fragments of the present invention include light chains, heavy chains, variable regions, peptides containing Fab, Fab ', F (ab') 2, scFv, Diabody, Tribody, dsFv and CDR.

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

The Fab of the present invention can be obtained by treating the antibody of the present invention with protease papain. Alternatively, a Fab can be produced by inserting a DNA encoding the Fab of the antibody into an expression vector for prokaryotic or eukaryotic expression, and introducing the vector into a prokaryote or eukaryote.

F (ab ') 2 is slightly larger than that bound to the Fab via the SS bonds in the hinge region, among the fragments obtained by treating IgG with protease pepsin (truncated to amino acid residue 234 of the H chain) Is an antibody fragment having an antigen-binding activity of about 100,000 in molecular weight.

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

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) having 12 or more residues, and is an antibody fragment having an antigen binding activity.

The scFv of the present invention is obtained by obtaining a cDNA encoding VH and VL of the antibody of the present invention, constructing a DNA encoding the scFv, inserting the DNA into an expression vector for prokaryotic or eukaryotic expression, Prokaryotic or eukaryotic organisms.

A diabody is an antibody fragment in which the scFv having the same or different antigen binding specificity forms a dimer, and is an antibody fragment having a bivalent antigen binding activity to the same antigen or a bifunctional antigen binding activity to a different antigen.

The diabody of the present invention can be obtained by, for example, obtaining a cDNA encoding VH and VL of the antibody of the present invention, constructing a DNA encoding scFv having 3 to 15 residues of a polypeptide linker and expressing the DNA in a prokaryotic expression Vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism to express the diabody.

When linker P length is 3-10, tribody is formed and can be included as tribody.

dsFv means that a polypeptide in which one amino acid residue of VH and VL is substituted with a cysteine residue is bound via S-S bond between the cysteine residues. The amino acid residue substituted with the cysteine residue can be selected based on the stereostructure prediction of the antibody according to the method described by Reiter et al. (Protein Engineering, 7, 697 (1994)).

Hereinafter, the present invention will be described.

Epitope  Select and 2E2F12 Monoclonal  Production of antibodies

B-cell epitope peptides Myc362 and Myc387 were selected in consideration of protein-protein interaction and antigenicity of c-Myc and Max (Fig. IA). BALB / c mice were immunized with a complex of each epitope peptide and Lipoplex (O). ELISA analysis indicated that the Myc362 immunized group derived serum had a significant production of antibody compared to that of Myc387 (FIG. 1B and C). Next, Myc362-immunized mouse spleen cells were extracted and fused with SP2 / 0 cells for hybridoma cell production. Additional selection of Myc362 clones with HAT (Fig. 2A) and HT medium resulted in the knockout of 2E2A8, 2E2F12, 2E2G12, and 2E2H11 (Fig. 2B). To investigate the efficiency of these clones, HEK293 cells were transfected into empty vectors or c-Myc and Max expression vectors. The cell lysate was immunoprecipitated with the culture supernatant of each clone and immunoblotted with rabbit anti-c-Myc antibody (Fig. 2C). All antibody clones immunoprecipitated recombinant c-Myc. We chose 2E2F12 clones for further study because of the more efficient antibody production from multiple pools and greater stability of the clones.

2E2F12 Monoclonal  Purification and Characterization of Antibodies

2E2F12 hybridoma cells were injected into pristine-primed mice to produce ascites and to confirm the robust generation of anti-c-Myc antibody (FIG. 3A) in order to develop a clear monoclonal antibody bearing anti-c-Myc antibody . Next, purification was performed using protein A agarose column chromatography. Additional confirmation was performed by running the purified antibody on SDS-PAGE and staining with Coomassie brilliant blue (Fig. 3B). For determination of IgG isotype, monoclonal antibodies were assayed by ELISA. The results indicated that the 2E2F12 mAb was IgG2a (Figure 3C). The binding affinity of the 2E2F12 antibody to the Myc362 peptide was estimated to be Kd of ~ 37 nM (Figure 3D). Thereby, the present inventors successfully produced and characterized the 2E2F12 monoclonal antibody.

anti- Myc362 Peptides - specific Monoclonal  The variable domain of the antibody Cloning

CDNA sequences encoding the heavy and light chain variable domains (V _H and V _L ) were cloned from hybridoma cells (2E2F12) producing anti-Myc362 peptide-specific monoclonal antibodies using conventional heavy and light chain primers . The sequences were confirmed by DNA sequencing as shown in Fig. Sequences were analyzed for known sequences and homology by the protein BLAST program (http://www.ncbi.nlm.nih.gov). CDNAs encoding the variable domains of the 2E2F12 heavy and light chains showed about 80-95% and 93-98% homology with the reported immunoglobulin variable heavy and light chain domains, respectively.

Western With blut 2E2F12 Monoclonal  Immunoprecipitation analysis with antibodies

To analyze the specificity of the 2E2F12 mAb, Western blot analysis was performed using 2E2F12 monoclonal antibodies using conventional rabbit and mouse anti-c-Myc antibodies. The data showed that anti-c-Myc antibodies typically detected c-Myc protein overexpressed in HEK293 cells, while antibodies of the present invention could not detect c-Myc (data not shown). Immunoprecipitation was performed using the rabbit anti-c-Myc antibody and the 2E2F12 monoclonal antibody (Fig. 5A) in order to investigate whether the antibody of the present invention can identify c-Myc in its native form. This experiment suggests that the 2E2F12 monoclonal antibody can adequately immunoprecipitate the c-Myc protein and that the 2E2F12 monoclonal antibody can effectively identify the c-Myc protein in its native state. Next, we analyzed the immunoprecipitated complexes using anti-Max antibodies and found that the 2E2F12 monoclonal antibody can detect and coimmunoprecipitate c-Myc-Max complexes endogenously expressed in Jurkat and LoVo cells (Fig. 5B).

2E2F12 Monoclonal  The expression of the promoter-c- Myc -Max Dimer  Inhibition of interaction

c-Myc forms a heterodimer with Max before binding to a specific DNA sequence and is involved in several normal cellular processes (Nair SK and Burley SK: Cell 2003; 112 (2): 193-205; Blackwood EM and Eisenman RN: Science 1991; 251 (4998): 1211-1217). In addition, c-Myc is involved in the important aspects of cancer cell survival and metabolism (Hsieh AL, Walton ZE, Altman BJ, Stine ZE and Dang CV: Semin Cell Dev Biol 2015; 43: 11-21). Thus, an ELISA-based DNA-protein interaction assay was performed to determine if 2E2F12 antibody could inhibit c-Myc binding to DNA. The results show reduced DNA binding in the presence of the 2E2F12 mAb compared to the untreated isotype IgG-treated sample or the untreated group (FIG. 6). Thus, these results suggest that anti-c-Myc antibody significantly diminishes the binding of heterodimers to DNA or the dimerization of c-Myc and Max in dimerization or all.

As can be seen from the present invention, the inventors have successfully developed a monoclonal antibody against c-Myc that can be developed as an antagonist that reduces Myc-activity in various cancers, It can be adopted as a candidate substance.

Figure 1 shows the production of monoclonal antibodies against the B cell epitope of c-Myc. (A) Model showing the location of Myc and Max interactions and Myc epitopes. (B and C) mice were immunized with B-cell epitope peptides of Myc362 and Myc387 in Lipoplex (O) and sera were analyzed by ELISA for the detection of anti-c-Myc antibodies.
Figure 2 shows screening of a hybridoma clone producing an anti-Myc362 peptide-specific mAb. (A) Three BALB / c mice were intraperitoneally immunized with Myc362 peptide co-encapsulated and MB-ODN 4531 (O) in DOPE: CHEMS complex four times at 10 day intervals. ELISA results from initial screening (HAT medium) of cell fusion experiments using myc362 peptide immunized mouse spleen cells. (B) Hybridoma clones were selected for the production of monoclonal antibodies and subcloned by limiting dilution in HT medium. (C) HEK293 cells were transfected into empty vector (E) or c-Myc and Max expression vector (M). Cell lysate was immunoprecipitated using cell culture supernatants of Myc362 clones, 2E2A8, 2E2F12, 2E2G12, and 2E2H11 and immunoblotted with rabbit anti-c-Myc antibody. The cells were immunoblotted with mouse anti-c-Myc antibody and anti-beta-Actin to indicate the amount of protein starting the whole cell lysate.
Figure 3 shows the purification and characterization of the 2E2F12 mAb. (A) A plurality of titration curves were examined by ELISA. 2E2F12 clones were injected into pristane-primed mice for the production of multiple. (B) The 2E2F12 mAb was purified from ascites using protein-A agarose column chromatography, analyzed by SDS-PAGE, and stained with Coomassie Blue. R, reduction conditions. NR, non-reducing conditions. (C) Purified 2E2F12 monoclonal antibodies were subjected to isotype IgG ELISA for determination of epitope specific IgG1, IgG2a, IgG2b, and IgG3. (D) The binding affinity of 2E2F12 mAb to Myc362 was evaluated by ELISA.
Figure 4 shows the cDNA sequence of the variable domains of the heavy and light chains separated from the hybridoma cell clone 2E2F12. (A) the sequence of the heavy chain variable domain. (B) the sequence of the light chain variable domain. The predicted amino acid sequence is listed at the bottom of the cDNA sequence.
Figure 5 shows the characteristics of 2E2F12 mAb by Western blot and immunoprecipitation assay. (A) HEK293 cells were transfected into empty vector (E) or c-Myc and Max expression vector (M). Cell lysate was immunoprecipitated with rabbit anti-c-Myc antibody or 2E2F12 monoclonal antibody and Western blot was performed usually with rabbit and mouse anti-c-Myc antibody. (B) Immunoprecipitated samples obtained using 2E2F12 monoclonal antibody and mouse anti-c-Myc antibody were analyzed by Western blotting with rabbit anti-c-Myc antibody and goat anti-Max antibody. J, Jurkat cell lysate. L, LoVo cell lysate.
Figure 6 shows the inhibition of the promoter-c-Myc-Max dimmer interaction by the 2E2F12 monoclonal antibody. Carbonate buffer containing Avidin was coated on 96-well immunoplates and biotinylated oligonucleleotide encoding the E box sequence was cultured on the plate. Whole extracts of c-Myc and Max co-transfected cells were cultured with 2E2F12 monoclonal antibody or isotype control antibody and then cell lysate was cultured on plate. The plates were incubated with rabbit anti-c-Myc antibody and incubated with peroxidase-conjugated goat anti-rabbit IgG. The levels of promoter-c-Myc-Max dimer interactions were determined by the phenomenon of peroxidase activity.

Hereinafter, the present invention will be described in detail with reference to non-limiting examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: cell  culture

HEK293 (human embryonic kidney cell) was purchased from ATCC (American Type Culture Collection, Manassas, Va., USA), and human colorectal cancer cell line LoVo was obtained from Korean cell line bank (Seoul, Korea). Jurkat, an immortalized human T-lymphocyte cell line, was obtained from Prof. Geun-Wook Lee of Hallym University. HEK293 cells were maintained in DMEM (Dulbecco's modified Eagle's medium, Life Technologies, Grand Island, NY, USA). LoVo and Jurkat cells were maintained in RPMI1640 (Life Technologies) medium. All media were supplemented with 10% fetal bovine serum (FBS, Life Technologies), 25 mM HEPES, 100 U / ml penicillin and 100 ug / ml streptomycin. All cell lines were treated with 95% air and 5% CO ₂ at 37 ° C. Lt; / RTI >

Example  2: Recombinant c- Myc  And Max protein expression

c-Myc and Max were expressed as myc-histidine (His) -tagged and FLAG-tagged proteins, respectively. In summary, total mRNA was obtained for c-Myc and Max from Huh-7 and PANC1 cells, respectively. c-Myc cDNA was amplified by PCR using the following primer set: sense 5'-GGA TCC ATG GAT TTT TTT CGG GTA GTG G (SEQ ID NO: 10) -3 'and anti-sense 5'-CTC GAG CGC ACA AGA GTT CCG TAG C-3 '(SEQ ID NO: 11). Max cDNA was amplified using the following primer sets: sense 5'-GAA CAT ATG GCC GAC GAA AAG CCC A-3 '(SEQ ID NO: 12) and anti-sense 5'- GAA CTC GAG GTA GAC ACC TCC CGT CTG C - 3 ' (SEQ ID NO: 13). The amplified cDNA fragments were ligated to the expression vector pcDNA3.1-Myc-His (Thermo Fisher Scientific, Waltham, MA, USA) for c-Myc and the expression vector pDream 2.1-FLAG (GeneScript, Piscataway, Lt; / RTI > The c-Myc and Max expression plasmids were co-transfected into HEK293 cell lines with Fugene 6 transfection reagent (Promega, Madison, USA). After transfection, the cell lysate was centrifuged and loaded onto SDS-PAGE for Western blot analysis.

Example  3: B cells Epiphone Of peptide  Produce

In order to select B cell epitope peptides for c-Myc specific antibody production, we selected peptide sequences for the peptides Myc362 and Myc387, respectively, as follows: ³⁶² SSDTEENVKRRTHNVLERQRRNELK ³⁸⁶ (SEQ ID NO: 9) and ³⁸⁷ RSFFALRDQIPELENNEKAPKVVIL ⁴¹¹ (SEQ ID NO: 14). Each peptide was synthesized using an automated peptide synthesizer (Peptron III-R24, Peptron, Daejeon, Korea). The peptides were purified by reverse phase HPLC (Prominence HPLC, Shimadzu Corp., Tokyo, Japan) and purified to a purity of 90% or more.

Example  4: Mouse and Immunization

Four-week-old female BALB / c (H-2 ^b ) mice were purchased from Nara-Biotec (Seoul, Korea). This animal test was approved by the Committee for Animal Experiments at Hallym University (Hallym2015-56). The liposome complex (Lipoplex (O)) of CpG-DNA and B-cell epitope peptide co-encapsulated in DOPE: CHEMS was prepared by the previously described method (Kim D, Kwon S, Ahn CS, Lee Y, HY and Kwon HJ: BMB Rep 2011; 44 (11): 758-763; Kim D, Kwon HJ and Lee Y: BMB Rep 2011; 44 (9): 607-612). The mouse was intraperitoneally injected with 50 μg of peptide epitope mixed with 50 μg of CpG-DNA encapsulated in liposomes, such as Kim D, Lee Y and Kwon HJ: Methods Mol Biol 2015; 1348: 127-135. All mice were injected three times at 10-day intervals.

Example  5: antigen-specific Ig ELISA Assay

Mouse serum was collected by orbital bleeding and assayed for antigen-specific antibody production. 5 ug / ml of the peptide was coated on 96-well immunoplates (Thermo Fisher Scientific) and blocked with 0.05% Tween-20 in PBS containing 1% BSA. Antibody production levels were measured as in Park BK, Choi SH, Kim YE, Park S, Lee Y, Lee KW and Kwon HJ: Monoclon Antib Immunodiagn Immunotherapy 2015; 34 (2): 101-109.

Example  6: anti-c- Myc Monoclonal  Preparation of antibodies

Hybridoma cells were selected to produce anti-Myc362 peptide-specific monoclonal antibody (2E2F12) according to standard hybridoma technology (Kwon S, Choi KC, Kim YE, Ha YW, Kim D, Park BK, Wu G, Kim DS, Lee Y and Kwon HJ: Cancer Res 2014; 74 (14): 3844-3856; Yokoyama WM, Christensen M, Santos G D and Miller D: Curr Protoc Immunol 2006; Myc362 peptide (50 ug) and MB-ODN 4531 (O) (50 ug) encapsulated in DOPE: CHEMS complex were intraperitoneally injected into BALB / c mice three times at intervals of 10 days. Immunized mouse derived spleens were used for fusion according to standard hybridoma technology. Hybridoma clones in HAT medium and HT medium were selected as standard limiting dilution protocols to obtain clonal cell populations. To obtain ascites, the selected hybridomas clone was injected intraperitoneally into BALB / c mice. Anti-Myc362 peptide-specific mAb was purified from peritoneal fluid by protein-A column chromatography. To determine the isotype of the anti-Myc362 peptide-specific mAb, an isotyping kit (Southern Biotechnology Associates Inc, Birmingham, USA) was used.

Example  7: Measurement of binding affinity

To confirm the binding affinity of the 2E2F12 mAb, 5 ug / ml Myc362 peptide was coated on a 96-well immunoplate and treated with PBST containing 1% BSA. Antibodies were started at 1 nM, added to the top row of each plate, serially diluted 1: 5 in PBST and then placed in the column. The plates were incubated at room temperature for 2 hours, washed with PBST, and incubated with anti-IgG antibody conjugated with horseradish peroxidase. The plates were developed with TMB (tetramethylbenzidine) peroxidase substrate (KPL, Gaithersburg, MD, USA) and the reaction was stopped with TMB-stop solution (KPL, Gaithersburg, MD, USA) (Molecular Devices, Sunnyvale, Calif., USA) at 405 nm.

Example  8: anti- relaxin -2 Monoclonal  Variable of antibody Heavy chain  And Light chain  Of the domain Cloning

Hybridoma cells (2E2F12) producing anti-Myc362 peptide-specific mAb were cultured and isotyped using a mouse monoclonal antibody isotyping kit (Dipstick format, Bibco BRL or Roche, Mannheim, Germany). Total RNAs were extracted from hybridoma cells (2E2F12) using RNeasy Mini Kit (Qiagen) to prepare cDNAs. The cDNAs produced to clone the sequences for the variable heavy and light chain domains (V _H and V _L ) of the anti-Myc362 peptide-specific mAb were amplified with the following primer sets using Vent polymerase (NEB). 3 ') (SEQ ID NO: 15) and 5'MH2 (5'-CTT CCG GAA TTC SAR GTN MAG CTG SAG SAG TCG GG-5' -GGA AGA TCT CTT GAC CAG GCA TCC TAG AGT CA- 3 ') (SEQ ID NO: 16) was used. 3 ') (SEQ ID NO: 17) and 5' Mk (5'-GG GAG CTC GAY ATT GTG MTS ACM CAR WCT MCA (5'-GGT GGA TGC GGA TAC AGT TGG TGC AGC ATC- -3 ') (SEQ ID NO: 18) was used. Standard PCR reactions were performed for 25 cycles. The PCR products were ligated to the pGEM-T easy vector (Promega). Cloned mouse Ig inserts were analyzed by DNA sequencing.

Example  9: Western Blotting  And immunoprecipitation

To identify the c-Myc-Max complex, disruption fluids were prepared from c-Myc and Max-overexpressing cells. The cell lysates were incubated with 2E2F12, rabbit anti-c-Myc antibody (Cell Signaling Technology, Danvers, MA, USA) or mouse anti-c-Myc antibody (Santa Cruz Biotechnology, Dallas, After incubation, protein A beads were pulled down. The samples were loaded onto SDS-PAGE and detected with 2E2F12, rabbit anti-c-Myc antibody, mouse anti-c-Myc antibody or goat anti-Max antibody (Novus Biologicals, Littleton, CO, USA). Jurkat and LoVo cell lysates were used for comparison of over-expressing cells.

Example 10: ELISA  Based DNA-protein interaction measurement Assay

DNA-protein interaction assay based on ELISA was performed using the method described in Sohn WJ, Kim D, Lee KW, Kim MS, Kwon S, Lee Y, Kim DS and Kwon HJ: Mol Immunol 2007; 44 (13): 3273-3282 . Briefly, sense: 5-GCG CAA CGA GCA CGT GGC CTG GGG CGC CAA GTG CGC-3 (SEQ ID NO: 19), antisense: 5-GCG CAC TTG, which is oligoglucuronide for binding of c- GCG CCC CAG GCC ACG TGC TCG TTG CGC-3 (SEQ ID NO: 20) was hybridized with heat shock (95 ° C for 5 minutes) in the form of a double strand (sense strand tagged with biotin) and cooled at room temperature. Carboate buffer (10 ug / ml) containing Avidin was coated on 96-well immunoplates (Thermo Fisher scientific) and incubated overnight at 4 캜. Plates were washed 3 times with PBST and blocked with PBS containing 1% BSA for 1 hour at room temperature. 50 μl of PBS containing 10 pmol of biotinylated oligonucleotide was incubated at 37 ° C for 1 hour. c-Myc and Max co-transfected cells were disrupted and a total of 20 ul of extract (2 ug / ml) was incubated with 2E2F12 monoclonal antibody or isotype control antibody for 1 hour at 4 ° C. All samples were incubated with buffer A (2 mM HEPES, pH 7.5, 100 mM KCL, 8% glycerol, 5 mM DTT, 0.2% BSA and 0.016% salmon sperm DNA) for 1 hour at room temperature and then washed three times with PBST . The plates were incubated with rabbit anti-c-Myc antibody (1: 1000) in buffer B (10 mM phosphate buffer, pH 7.4, 20 mM NaCl and 1% skim milk) for 1 hour at room temperature and washed with PBST. Chlorine anti-rabbit IgG (1: 5000) with peroxidase in buffer B was added to the wells and incubated at room temperature for 1 hour before development.

<110> INDUSTRY ACADEMIC COOPERATION FOUNDATION HALLYM UNIVERSITY An anti-c-Myc monoclonal antibody and use of the same <130> P17-0046HS <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 1 Gly Tyr Thr Phe Thr Ser Tyr Trp Met His   1 5 10 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 2 Ile Asn Pro Ser Ser Gly Gly Thr Asn Tyr Asn Glu Asn Phe Lys Arg   1 5 10 15 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 3 Thr Ile His Arg Ser Tyr   1 5 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 4 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn   1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 5 Leu Val Ser Lys Leu Asp Ser   1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 6 Trp Gln Gly Thr His Phe Pro Tyr Thr   1 5 <210> 7 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 7 Leu Pro Glu Phe Glu Val Glu Leu Gln Glu Ser Gly Thr Glu Leu Val   1 5 10 15 Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr              20 25 30 Phe Thr Ser Tyr Trp Met His Trp Val Lys Leu Arg Pro Gly Gln Gly          35 40 45 Phe Glu Trp Ile Gly Glu Ile Asn Pro Ser Ser Gly Gly Thr Asn Tyr      50 55 60 Asn Glu Asn Phe Lys Arg Lys Ala Thr Leu Thr Val Asp Arg Ser Ser  65 70 75 80 Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala                  85 90 95 Val Tyr Tyr Cys Thr Ile His Arg Ser Tyr Trp Gly Gln Gly Thr Thr             100 105 110 Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Ser Ser Tyr Pro Leu         115 120 125 Ala Pro Arg Ser Ser     130 <210> 8 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> kappa chain <400> 8 Glu Leu Asp Ile Val Met Thr Gln Ser Pro Leu Thr Leu Ser Val Thr   1 5 10 15 Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu              20 25 30 Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly          35 40 45 Gln Ser Pro Lys Arg Leu Ile Phe Leu Val Ser Lys Leu Asp Ser Gly      50 55 60 Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu  65 70 75 80 Lys Ile Thr Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp                  85 90 95 Gln Gly Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu             100 105 110 Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ala Cys Thr         115 120 125 <210> 9 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Myc362 <400> 9 Ser Ser Asp Thr Glu Glu Asn Val Lys Arg Arg Thr His Asn Val Leu   1 5 10 15 Glu Arg Gln Arg Arg Asn Glu Leu Lys              20 25 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ggatccatgg atttttttcg ggtagtgg 28 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctcgagcgca caagagttcc gtagc 25 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gaacatatgg ccgacgaaaa gccca 25 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gaactcgagg tagacacctc ccgtctgc 28 <210> 14 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 14 Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu Glu Asn Asn   1 5 10 15 Glu Lys Ala Pro Lys Val Val Ile Leu              20 25 <210> 15 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ggaagatctc ttgaccaggc atcctagagt ca 32 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 cttccggaat tcsargtnma gctgsagsag tcwgg 35 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ggtgcatgcg gatacagttg gtgcagcatc 30 <210> 18 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gggagctcga yattgtgmts acmcarwctm ca 32 <210> 19 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gcgcaacgag cacgtggcct ggggcgccaa gtgcgc 36 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gcgcacttgg cgccccaggc cacgtgctcg ttgcgc 36

Claims (13)

In a monoclonal antibody, or a functional fragment thereof, which specifically recognizes a c-Myc protein or a part of the protein,
Wherein the monoclonal antibody, or a functional fragment thereof, comprises all of the following heavy and light chain CDR sequences, or a functional fragment thereof:
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, a CDR1 region represented by SEQ ID NO: 4, a CDR2 region represented by SEQ ID NO: 5, A monoclonal antibody or a functional fragment thereof comprising a light chain comprising a CDR3 region represented by SEQ ID NO: 6. 2. The monoclonal antibody of claim 1, wherein the functional fragment is a single side chain antibody (scFv), or a functional fragment thereof. 2. The monoclonal antibody of claim 1, wherein the functional fragment is an antigen binding segment (Fab), or a functional fragment thereof. 2. The monoclonal antibody or the functional fragment thereof according to claim 1, wherein the functional fragment is a light chain or heavy chain comprising the CDR region of claim 1. The monoclonal antibody or the functional fragment thereof according to claim 1, wherein the functional fragment is a variable domain comprising the CDR region of claim 1. The monoclonal antibody according to claim 1, wherein said monoclonal antibody is a light chain comprising a heavy chain comprising the polypeptide sequence shown in SEQ ID NO: 7 and a polypeptide sequence represented by SEQ ID NO: 8. The monoclonal antibody according to claim 1, wherein a part of the c-Myc protein is a peptide sequence represented by SEQ ID NO: 9. delete A composition for diagnosing cancer comprising a conjugate obtained by conjugating a marker substance to a monoclonal antibody against c-Myc of claim 1. The cancer diagnostic composition according to claim 9, wherein the labeling substance is any one selected from the group consisting of enzymes, luciferase, magnetic particles, fluorescent substances and radioactive isotopes. 1) contacting the sample with the monoclonal antibody of any one of claims 1 to 7; And
2) detecting the reaction of the sample with the monoclonal antibody
A method for providing information on whether or not a cancer has occurred in a subject. 1) a monoclonal antibody according to any one of claims 1 to 7; And
2) container
Wherein the cancer diagnostic kit comprises: (1) The cancer diagnostic composition of (9); And
2) container
Wherein the cancer diagnostic kit comprises:

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