KR20110098593A - Epitope of cd66c specific to lung adenocarcinoma and antibody recognizing the same - Google Patents

Abstract

The present invention relates to a lung adenocarcinoma-specific epitope of CD66c and an antibody that recognizes the same, an antibody that identifies and recognizes an epitope specific to lung adenocarcinoma in CD66c, a cell line producing the same, and the diagnosis or treatment of lung adenocarcinoma using the same It is about use. The CD66c epitope is specific for lung adenocarcinoma cells, and an antibody or antibody fragment thereof that recognizes the CD66c epitope may be usefully used as a composition for diagnosing, treating or preventing lung adenocarcinoma cells.

Description

CD66c specific adenocarcinoma specific epitope and antibody recognizing the same {Epitope of CD66c specific to lung adenocarcinoma and antibody recognizing the same}

The present invention relates to a lung adenocarcinoma-specific epitope of CD66c and an antibody that recognizes the same, an antibody that identifies and recognizes an epitope specific to lung adenocarcinoma in CD66c, a cell line producing the same, and the diagnosis or treatment of lung adenocarcinoma using the same It is about use.

Lung cancer is one of the most frequently occurring cancers and is known as a life-threatening cancer worldwide. Around 1.2 million people worldwide develop lung cancer, which accounts for 25% of all cancer deaths. In Korea, the incidence and mortality rate of lung cancer is gradually increasing. According to the Korea Central Cancer Registry released in 2008, an average of 132,941 cancers occurred in Korea between 2003 and 2005. Combined, the annual average was 16,123 cases, ranking 2nd with 12.1% of all cancer cases.

Lung cancer is the 5-year survival rates when early detection in the early stage of IA known as about 80% (Mulshine, JL and Sullivan , DC Clinical practice Lung cancer screening N. Engl J. Med 2005; 352:..... 2714 Therefore, it is important to find markers that can be detected early in the detection of specific biomarker markers for new lung cancer. Lung cancer, in particular, is a heterogeneous tumor, which differs in response and prognosis for various forms of treatment. Medically, it is divided into two groups, small cell lung cancer and non-small cell lung cancers (NSCLC) .Non-small cell cancers are divided into lung adenocarcinoma and squamous cell carcinoma. It is divided into squamous cell carcinoma and large cell carcinoma.

CD66c, also known as Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) or non-specific cross-reacting glycoprotein antigen (NCA) -90, is known as CD66c in the blood of patients with lung cancer, pancreatic cancer, breast cancer, rectal cancer, liver cancer, etc. It is known that the amount is high. The CD66c is an important protein associated with cell adhesion and is known to be involved in adhesion to endothelial cells activated by cytokines in the case of neutrophils.

In addition, in the case of normal cells, if cell adhesion is not achieved, apoptosis occurs. This action is called anikis (anoikis). However, tumor cells are resistant to these anoikis, thereby promoting cancer development and cancer metastasis. It has been reported that CD66c inhibits anoikis, and that malignant phenotype of cancer cells is changed by regulating the expression of CD66c. In addition, the results of silencing CD66c gene using small interfering RNA to inhibit the expression of protein have been reported to increase the anokis and inhibit the cancer metastasis in vivo. After all, inhibiting the function of CD66c can play an important role in inhibiting cancer metastasis.

In order to develop an antibody against CD66c expressed in lung adenocarcinoma cells, the present inventors have developed the present invention by developing an antibody against CD66c specific for lung adenocarcinoma and revealing an epitope thereof by immunizing a lung adenocarcinoma cell line with a recombinant antigen of CD66c. I did it.

Accordingly, an object of the present invention is to provide a polypeptide that is an epitope of CD66c (Cluster of Differentiation 66c) represented by the amino acid sequence of SEQ ID NO.

It is also an object of the present invention to provide an antibody or antibody fragment that recognizes the polypeptide as an epitope.

It is also an object of the present invention to provide a cell line for producing the antibody or antibody fragment.

It is another object of the present invention to provide a composition for diagnosing lung adenocarcinoma containing the lung cancer specific antibody or antibody fragment as an active ingredient.

It is another object of the present invention to provide a lung adenocarcinoma diagnostic kit containing the lung adenocarcinoma-specific antibody or antibody fragment as an active ingredient.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating lung cancer, comprising the lung cancer specific antibody or antibody fragment as an active ingredient.

In order to solve the above problems, the present invention provides a polypeptide which is an epitope of CD66c (Cluster of Differentiation 66c) represented by the amino acid sequence of SEQ ID NO.

The present invention also provides an antibody or antibody fragment that recognizes the polypeptide as an epitope.

The present invention also provides a cell line for producing the antibody or antibody fragment.

In another aspect, the present invention provides a composition for diagnosing lung adenocarcinoma containing the lung cancer specific antibody or antibody fragment as an active ingredient.

The present invention also provides a lung adenocarcinoma diagnostic kit containing the lung cancer specific antibody or antibody fragment as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating lung adenocarcinoma containing the lung cancer specific antibody or antibody fragment as an active ingredient.

Hereinafter, the present invention will be described in more detail.

In the present invention, CD66c (Cluster of Differentiation 66c) is a protein also known as CEACAM 6 (carcinoembryonic antigen-related cell adhesion molecule 6) or NCA (non-specific cross-reacting glycoprotein antigen) -90, cell adhesion and It is known to be an important protein involved and may be preferably represented by the amino acid sequence of SEQ ID NO: 1 (Genebank Protein No. AAH05008).

In the present invention, the epitope is an epitope that is specifically recognized by an antibody, but is not limited thereto. Preferably, the epitope may be a linear epitope.

In the present invention, an antibody refers to a polypeptide, or a fragment thereof, comprising a framework region from an immunoglobulin gene, wherein the antibody specifically binds to and recognizes an antigen. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as a number of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. In particular, the antibody in the present invention includes both chimeric and humanized antibodies.

In the present invention, chimeric antibodies are antibodies in which variable region sequences are derived from one species and constant region sequences are derived from another species, eg, variable region sequences are derived from mouse antibodies and constant region sequences are derived from human antibodies. Mean antibody.

In the present invention, the humanized antibody refers to an antibody that retains the activity of a non-human antibody while having low immunity in humans. This can be prepared, for example, by maintaining non-human CDR regions and replacing the rest of the antibody with human counterparts. For example, reference is made to Morrison et al, Proc. Natl. Acad. ScL USA, 81: 6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44: 65-92 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988); Padlan, Molec. Immun., 28: 489-498 (1991); Padlan, Molec. Immun., 31 (3): 169-217 (1994).

In the present invention, the antibody fragment may include, but is not limited to, if the CD66c epitope is selectively recognized, including the antibody light chain variable region (V _L ) and the antibody heavy chain variable region (V _H ), but Fab, Fab ′, F (ab ′). ), ScFv, dsFv and CDR may be selected from the group consisting of. In particular, the scFv is an antibody fragment made of a single chain by linking the heavy chain variable region (V _H ) and light chain variable region (V _L ) with a linker polypeptide.

Monoclonal antibodies in the present invention are terms known in the art and are highly specific antibodies directed against a single antigenic site. Unlike polyclonal antibodies, which typically include different antibodies directed against different determinants (epitopes), monoclonal antibodies are directed against a single determinant on an antigen. Monoclonal antibodies of the invention can be prepared using conventional cloning and cell fusion techniques. For example, an immunogen of interest (antigen) can be administered to wild type or breeding mice (eg BALB / c) to produce natural or human monoclonal antibodies. Such antigens can be administered alone, in admixture with an adjuvant, can be expressed from a vector and can elicit an immune response as a DNA or fusion protein. Fusion proteins include carrier proteins coupled with peptides intended for an immune response, such as β-galactosidase, glutathione S-transferase, keyhole limpet hemocyanin (KLH), and bovine serum albumin, Carrier proteins are not limited thereto. In this case the peptide acts as a hapten for the carrier protein.

Briefly describing the monoclonal antibody production method is as follows. After boosting the animal, the spleen is removed and the spleen cells are extracted by methods known in the art [Kohler and Milstein, Nature 256: 495-497 (1975); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988)]. The hybrid cells obtained are cloned by conventional methods, for example by limiting dilution, and the resulting clones are cultured to produce the desired monoclonal antibody. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and because they are synthesized by hybridoma culture, they also have another advantage that they are not contaminated by other immunoglobulins. Have

Hybridoma cells in the present invention are known in the art and refer to cells formed by fusion of antibody producing cells with immortal cells, such as myeloma cells. The hybrid cell can continuously supply the antibody.

In the present invention, the polyclonal antibody is a conventional antigen containing an epitope of the present invention, preferably a part of CD66c of SEQ ID NO: 2 as an antigen, injected into an animal and collected from the animal to obtain a serum containing the antibody. Can be produced by the method. Such polyclonal antibodies can be purified by any method known in the art and can be made from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs and the like.

The detection label used in the present invention means a label for detection of the reaction product of the antigen-antibody, specific examples include radioisotope labels, enzymes, chemoluminescent compounds, fluorescein, picobiliprotein, Fluorescent materials such as rare earth chelates, rhodamines, enzyme cofactors, biotin, and the like, but are not necessarily limited thereto.

The polypeptide found to be an epitope of CD66c in the present invention is characterized by being represented by the amino acid sequence of SEQ ID NO: 7.

The epitope of CD66c is not limited thereto, but may preferably be specific for lung adenocarcinoma.

In one embodiment of the invention, a monoclonal antibody against CD66c specific for lung adenocarcinoma is first developed (see <Example 2>) by immunizing a lung adenocarcinoma cell line with a recombinant antigen of CD66c (see <Example 1>), and its This is the first time that an epitope is represented by the amino acid sequence of SEQ ID NO. 7 (see <Example 3>). In particular, the epitope of the CD66c of the present invention is an epitope specifically recognized by the monoclonal antibody (8F5) of the present invention, unlike the 9A6 (Santa cruz biotechnology) or AP11 (Dinona) antibody known as the antibody of the conventional CD66c. See Example 3)

Therefore, the epitope of CD66c represented by the amino acid sequence of SEQ ID NO: 7 of the present invention can be used as an active ingredient of a composition for inducing antibody formation specific to lung adenocarcinoma.

The epitope of the CD66c can be isolated from a biological sample, chemically synthesized or produced by genetic engineering techniques, and the method can be easily carried out by known techniques.

Meanwhile, the antibody or antibody fragment of the present invention is characterized by recognizing the epitope of the present invention.

The antibody of the present invention is not limited thereto, but may preferably be a monoclonal antibody or a polyclonal antibody, and the monoclonal antibody may be more preferably produced by hybridoma cells (Accession Number: KCLRF-BP-00230). have.

In particular, the antibody of the present invention is not limited thereto but preferably belongs to IgG1, and may be classified as including a kappa light chain (see <Example 2-2>).

The antibody of the present invention may be, but is not limited to, an antibody specific for lung adenocarcinoma.

In one embodiment of the present invention, in order to produce an antibody specific for lung adenocarcinoma, a part of CD66c represented by SEQ ID NO: 2 and a recombinant antigen of Fc of human immunoglobulin G are immunized to a lung adenocarcinoma cell line, and By screening for specific antibodies, the antibodies of the present invention were developed (see <Example 2>).

Meanwhile, the cell line of the present invention is characterized by producing the antibody or antibody fragment.

The cell line may be, but is not limited to, a hybridoma cell with preferably Accession Number KCLRF-BP-00230. The hybridoma cells are cells producing the monoclonal antibody, and a method for producing the same is known in the art, and preferably, may be prepared by fusion of antibody-producing cells with immortal cells, for example, myeloma cells. Although not limited thereto, it may be preferably prepared through cell fusion of cells injected with the CD66c antigen (preferably CHO-K1 cells) and mouse spleens immunized with lung cancer cell lines.

The hybridoma cells were deposited with the accession number KCLRF-BP-00230 on February 22, 2010 as '8F5' to the Korean Cell Line Research Foundation (KCLRF).

Meanwhile, the composition for diagnosing lung adenocarcinoma of the present invention is characterized in that it contains the lung cancer specific antibody or antibody fragment as an active ingredient.

The lung adenocarcinoma-specific antibody or antibody fragment specifically recognizes an epitope of CD66c (Cluster of Differentiation 66c) represented by the amino acid sequence of SEQ ID NO: 7, thereby effectively detecting the CD66c antigen, in particular the CD66c antigen This expressed lung adenocarcinoma can be effectively detected. (See <Example 4>.)

When the lung adenocarcinoma specific antibody or antibody fragment is used in the diagnostic composition, the antibody or antibody fragment is selected from the group consisting of enzymes, radioisotopes, fluorescent materials, chromogens, and staining materials. Can be labeled. Fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (allophycocyanin, APC) or biotin may be used as the fluorescent material, but is not limited thereto. It is not.

The diagnostic composition may include a carrier used for immunological analysis in addition to the lung adenocarcinoma-specific antibody or antibody fragment, a detection label that can generate a detectable signal, a dissolving agent, and a cleaning agent. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Such carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers (eg PBS) or insoluble carriers known in the art such as polystyrene, polyethylene, polypropylene, polyesters, polyacrylonitrile , Fluorine resins, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated metals on lactex, other papers, glass, metals, agarose and combinations thereof.

Meanwhile, the diagnostic kit of the present invention may effectively diagnose lung cancer by including the lung cancer specific antibody or antibody fragment as an active ingredient.

Kit systems for use in the present invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatography and radiation split immunoassay devices, flow-through devices, and the like. Preferably, a diagnostic kit in the form of a strip or a device using immunochromatography can be used. Diagnosis using immunochromatography involves the reaction of an antigen in the serum of a sample with a tracer antibody bound to colloidal gold particles, followed by a nitrocellulose membrane by capillary action. While moving through the micropores of the microorganisms, a color band is formed by combining with a capture antibody fixed to the inner surface of the micropores, thereby making it possible to visually discriminate the positive and the negative.

The kit may include, but is not limited to, enzyme-linked immunoassay (ELISA) or lateral flow immunographic assay. As described above, when using sandwich immunoassay and lateral flow immunographic assay, antibody pairs are prepared using lung cancer specific antibodies or antibody fragments of the present invention, and lung cancer is prepared using the same. It can be diagnosed effectively.

The kit of the present invention is not limited thereto, as long as it is typically used in the kit. For example, if ELSIA is used, a solid-phase support, a lung cancer-specific antibody or antibody fragment of the present invention, an enzyme-labeled antibody solution for reaction with an antigen And an enzyme-linked immunoassay (ELSIA) reaction solution containing a coloring solution that exhibits an enzyme reaction. More specifically, the enzyme-labeled antibody solution may be goat anti-mouse Ig-HRP, the coloring solution may be tetramethylbenzidine (TMB), and the reaction blocking solution may be HCl or H ₂ SO ₄ .

Meanwhile, the pharmaceutical composition for preventing or treating lung adenocarcinoma of the present invention is characterized by containing the lung cancer specific antibody or antibody fragment as an active ingredient.

Lung adenocarcinoma-specific antibodies or antibody fragments of the present invention are very excellent in suppressing tumors, particularly lung adenocarcinoma (see <Experiment 5).

Therefore, the pharmaceutical composition of the present invention can be effectively used for preventing or treating lung adenocarcinoma by containing the lung adenocarcinoma specific antibody or antibody fragment as an active ingredient.

The content of the antibody or antibody fragment as an active ingredient in the pharmaceutical composition of the present invention can be appropriately controlled according to the use form and purpose, patient condition, type and severity of symptoms, preferably 0.1 to 50% by weight, more preferably Preferably it may be 1 to 20% by weight, but is not limited thereto.

The pharmaceutical compositions of the present invention can be administered to a mammal, including humans, by various routes. The mode of administration can be any of the routinely used methods and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection. The pharmaceutical compositions of the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, transdermals, suppositories, and sterile injectable solutions in accordance with conventional methods. Can be formulated and used.

The pharmaceutical composition of the present invention may further contain an adjuvant such as a pharmaceutically suitable and physiologically acceptable carrier, excipient and diluent in addition to the extract. Carriers, excipients and diluents which may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants can be used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, and the like in the original, solid and long-eye extracts. It may be prepared by mixing sucrose or sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, transdermal agents and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition of the present invention may be administered alone to a person, but generally may be administered in admixture with a pharmaceutical carrier selected in view of the mode of administration and standard phamaceutical practice. For example, the pharmaceutical compositions of the present invention may be in the form of tablets containing starch or lactose, or in the form of capsules containing alone or excipients, or elixirs or suspending agents containing chemicals that flavor or color. Oral, oral or sublingual. Such liquid preparations may be suspending agents (e.g., semisynthetic glycerides such as methylcellulose, witepsol or mixtures of apricot kernel oil with PEG-6 esters or PEG-8 with caprylic / capric Pharmaceutically acceptable additives such as glyceride mixtures such as mixtures of glycerides).

The dosage of the pharmaceutical composition of the present invention may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and may be administered once or several times a day at regular intervals. For example, the daily dosage may be 1 to 20 mg / kg, preferably 5 to 10 mg / kg, based on the active ingredient content. The above dosages are illustrative of the average case and may be high or low depending on individual differences. If the daily dose of the pharmaceutical composition of the present invention is less than the dose, a significant effect is not obtained, and if it exceeds the above, it is uneconomical and there is a concern that undesirable side effects may occur if it is out of the range of the amount used. Since it may arise, it is preferable to set it as said range.

The CD66c epitope of the present invention is specific for lung adenocarcinoma cells, and antibodies or fragments thereof which recognize the same may be usefully used as a composition for diagnosing, treating or preventing lung adenocarcinoma cells.

1 is a diagram showing the result of setting up a CD66c recombination site.
2 is a diagram showing a schematic diagram of pSegTag-CD66c-HuIgFc cloning.
3 shows the results of confirming pSegTagB-CD66c-HuIgFc cloning.
Figure 4 shows the results confirmed by ELISA whether the CD66c-HuIgFc recombinant antigen is expressed in the transformed CHO-K1 cell line.
5 is a result of western blot confirming the purified CD66c-HuIgFc recombinant antigen.
6 shows the results of confirming CD66c activity of recombinant CD66c-HuIgFc.
7 is a schematic diagram of the development of a monoclonal antibody against lung adenocarcinoma-specific CD66c.
8 is a result of confirming the titer of the A549 cell immune rat serum to A549 cell.
Figure 9 shows the result of confirming the titer of CD66c-HuIgFcd in the serum of A549 cells.
10 is a result of comparing western blotting results of 8F5 and AP11.
11 shows the result of analyzing the binding of 8F5 monoclonal antibody in peripheral blood.
12 ELISA analysis of CD66c-HuIgFc.
13 shows the result of confirming whether 8F5 staining in lung cancer tissue.
14 shows the results obtained by performing the MOLDI-TOF epitope of the monoclonal antibody of the present invention.
15 is a schematic diagram showing a method for verifying epitopes of monoclonal antibodies of the present invention.
Figure 16 shows the results of verifying the epitope of the monoclonal antibody of the present invention.
17 is a schematic diagram comparing the epitope of the present invention found through MOLDI-TOF of the present invention and the result of verifying the same.
Figure 18 shows the result of detecting the lung adenocarcinoma containing the CD66c antigen using the monoclonal antibody of the present invention.
19 shows the anticancer activity of the monoclonal antibody of the present invention.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1>

CD66c  And Human immunoglobulin Fc region sign C _H 2 - C _H 3 end Combined  Recombinant antigen ( CD66c - HuIgFc Manufacturing

<1-1> gene Cloning

Obtaining the CD66c-HuIgFc Recombinant Antigen First, a primer for the CD66c gene was prepared by constructing 34 to 310aa region (SEQ ID NO: 2) from which the hydrophobic portion was removed from the entire CD66c cDNA (SEQ ID NO: 1) (Fig. 1). Primers were prepared for the Fc (Human immunoglobulin Fc region C _H 2 -C _H 3; HuIgFc) portion of immunoglobulin G, specifically, the portion having basicity represented by SEQ ID NO: 11 (Table 1). A schematic diagram of the overall cloning process is shown in FIG. 2.

Gene Primer SEG ID Sequence CD66c Hnd66c 5 ' SEG ID No: 3 AAG CTT AAG CTC ACT ATT GAA TCC ACG 66cEcRV 3 ' SEG ID No: 4 GAT ATC AGT GAC TGT GGT CCT ATT GA Human Ig Fc
(CH2-CH3) EcrVFc 5 ' SEG ID No: 5 GAT ATC GAC GTC GAG TCC AAA TCT TGT FcXhO1 3 ' SEG ID No: 6 CTC GAG TTT ACC CGG AGA CAG GGA GA

The CD66c gene was obtained by extracting RNA from a human lung adenocarcinoma cell line A549 (ATCC CCL-185) using a QIAGEN RNEasy Mini spin kit and performing RT-PCR using the primers described in Table 1 above. Specifically, the RT-PCR was a Novagen first cDNA synthesis kit, and more specifically, RNA 1ug and Oligo d (T) primer 1ul (10pmol / ul) was added to the reaction for 10 minutes at 70 ℃ and then cooled and 5X buffer, 100mM DTT cDNA was synthesized by adding reverse transcriptase and reacting at 37 ° C. for 1 hour. Thus synthesized cDNA using a Bioneer PCR premix as a primer of Table 1 PCR amplified by 30 cycles for 1 minute at 94 ℃, 1 minute at 60 ℃, 2 minutes at 72 ℃ to secure the CD66c gene.

As a result of the experiment, about 850 bp of DNA for the CD66c gene encoding the hydrophobic region of the entire CD66c cDNA was identified (FIG. 3a) and cloned into a pGEM T (Promega, USA) vector and the restriction enzyme EcoR Confirmed by I and confirmed by sequencing the nucleotide sequence of SEQ ID NO: 8.

In addition, the Human Ig Fc region (HuIgFc) was obtained by extracting RNA from human B cells (ATCC CTL-1834) with a QIAGEN RNEasy Mini spin kit and performing the RT-PCR using the primers described in Table 1 above.

PGEM T-CD66c gene cloned with the CD66c gene was cut with restriction enzymes (Promega, USA) Hind III and EcoR V to prepare an insert, and HuIgFc was cut with EcoR V and Xho I to prepare two inserts. A pSegTagB (invitrogen, USA) vector having an Igk leader sequence as an expression vector was cut with restriction enzymes Hind III and Xho I and cloned by inserting two inserts simultaneously.

The final pSegTagB-CD66c-HuIgFc recombinant gene was treated with the restriction enzyme Bgl II to confirm that the DNA fragment of 1300 bp appeared to confirm that the CD66c-HuIgFc recombinant gene was cloned (FIG. 3b).

<1-2> Transformation

PSecTag-CD66c-HuIgFc DNA prepared in Example 1-1 was transformed into CHO-K1 cells (ATCC CRL-9618) to develop a cell line expressing a recombinant antigen. Detailed experimental method is as follows.

First, the day before transformation, CHO-K1 cells were inoculated in 6-well plates at a concentration of 1 × 10 ⁶ cells / ml, and DMEM medium containing 10% Fetal bovine serum (Gibco, USA) (Dulbecco's modified Eagle's medium, Gibco, USA) 3ml was added and incubated for 18 hours at 37 ℃, 5% CO ₂ conditions. PSecTag-CD66c-HuIgFc DNA prepared in Example 1-1 was transformed into CHO-K1 cells using an Effectene transfection reagent kit (QIAGEN, Hilden, Germany).

Three days after the transformation, the supernatant was taken and subjected to ELISA assay using CD66c and HuIg to confirm the amount of CD66c-HuIgFc expressed and selected using 150 ug / ml Zeocin (Gibco, USA) to make a stable cell line. The process was carried out. After the screening process, limiting dilution was performed to establish a final cell line to obtain a single colony.

<1-3> Purification of Recombinant Antigens

In the case of the CD66c-HuIgFC recombinant antigen, the supernatant of the cell line expressing the same can be easily purified by applying the protein G-affinity chromatography. Detailed experimental method is as follows.

The supernatant of the transformed cell line selected in <Example 1-2> was collected, and the protein G-agarose (Thermo Fisher Scientific Inc. USA) was added thereto to bind overnight at 4 ° C, and then packed on a column. ) And washed with 15 ml of wash buffer (20 mM Phosphate buffer pH 7.2). Thereafter, 5 ml of dilution buffer (Elution Buffer, 0.1M Glycine pH 2.9) was added to the column to receive 1 ml of fractions, and 100ul of neutralizing buffer (1M TrisCl pH 8.0) was neutralized and purified. CD66c-HuIgFc recombinant protein was obtained.

The obtained recombinant protein was measured for absorbance at 280 nm, eluted, and dialyzed with Phosphate buffered saline (PBS) solution, followed by concentration using a BCA Protein Assay Kit (Thermo Fisher Scientific Inc. USA).

As described above, 2ug, 5ug, and 10ug of purified antigen were loaded onto 10% Tris-glycine gel by protein G-affinity chromatography, transferred to nitrocellulose membrane, and western blot was detected by human Ig-HRP. The protein of molecular weight 105kDa was confirmed. (FIG. 5)

<1-4> Of recombinant antigens in transgenic cell lines Expression  Confirm

In order to confirm the expression of the CD66c-HuIgFc recombinant antigen in the CHO-K1 cell line transformed in <Example 1-2>, Goat anti-human Fc fragment and Goat anti, which are antibodies to AP11 and HuIgFc, to CD66c Three sandwich ELISAs using human Ig were used (see Table 2, mAb: monoclonal antibody).

method # 1 method # 2 method # 3 coating anti-CD66c mAb (AP11) anti-Human Ig Fc anti-Human Ig detecting anti-Human Ig Fc-HRP Mouse anti-CD66c mAb Goat anti-mouse IgM-HRP

Specifically, 100ng / well of AP11 mAb (DINONA, Korea) and 400ng / well of Goat anti-human Fc fragment, Goat anti-human Ig on 96-well microtitration plates (Maxisorp; Nunc, Roskilde, Denmark) Each antibody was diluted in PBS buffer and then coated by incubating at 37 ° C. for one hour. Then, 1 × blocking buffer (blocking buffer, Sigma) was added to 200ul per well and incubated at 37 ° C. for one hour for blocking. 100 μl of the culture supernatant of the transformed CHO-K1 cell line in the blocked well was incubated at 37 ° C. for one hour to induce binding to the coated antibody and not to bind the antibody. Washed with water three times. Hydroperoxidase (HRP) conjugated goat anti-human Fc fragment specific Ab (Jackson Immuno Research Laboratories, West Grove, PA) was added to the wells in which the AP11 mAb and the supernatant were combined, followed by reaction at 37 ° C. for 30 minutes, and anti-human After injection of mouse anti-CD66c mAb into a well combined with Ig Fc and anti-human Ig, the supernatant was reacted for 1 hour at 37 ° C, washed three times with PBS, and then HRP conjugated goat anti mouse IgM (Jackson Immuno Research Laboratories). , West Grove, PA) was added and reacted at 37 ° C. for 30 minutes. After binding HRP as described above, the wells were washed with PBS to remove unbound HRP conjugate, and 50 μl of TMB (3, 3 ', 5, 5'-tetramethylbenzidene) substrate solution was added per well for 10 minutes at room temperature. The reaction was then stopped with 2N sulfuric acid. The degree of reaction was measured by absorbance at 450nm using an ELISA reader and the results are shown in FIG. At this time, the cell supernatant obtained by transforming only the pSegTag vector and another antigen PD1 (Dinona Co., Ltd.) combined with HuIgFc were used.

As shown in FIG. 4, it can be seen that the CD66c-HuIgFc recombinant antigen is expressed in the CHO-K1 cell line transformed in Example 1-2.

<1-5> Functional check of recombinant antigens

In order to confirm whether the recombinant antigen (CD66c-HuIgFc) purified in Example 1-3 functions as a CD66c antigen, the recombinant antigen prevents the binding of the anti-CD66c antibody AP11 to the A549 cell surface. It was confirmed by flow cytometry and the results are shown in FIG. 6.

Specifically, the recombinant antigens 0ug, 10ug, 50ug, 100ug and AP11 antibody were combined for 30 minutes at 4 ° C, and then reacted with A549 cells for 30 minutes at 4 ° C, followed by centrifugation at 1500 rpm for 3 minutes and washing with 3ml of PBS. To identify the bound antibody, 200-fold diluted goat anti-Mouse IgM FITC (Dinona), a secondary antibody, reacted at 4 ° C. for 15 minutes, washed with PBS 3ml in the same manner as above, and measured by flow cytometry. .

As shown in FIG. 6, when the CD66c-HuIgFc was not added (FIG. 6B), the recombinant antigen (CD66c-HuIgFc) 10ug, 50ug, 100ug and anti-CD66c mAb were first bound, When bound to A549 cells, it can be seen that the blocking phenomenon occurs in proportion to the dose (Fig. 6C, d, e).

< Example  2>

Lung adenocarcinoma  Specific Monoclonal  Development of Antibodies

<2-1> Hybridoma  Cells and Monoclonal  Preparation of Antibodies

To develop monoclonal antibodies against specific CD66c expressed directly in lung adenocarcinoma, immunizing the lung adenocarcinoma A549 cell line without directly immunizing the recombinant CD66c-HuIgFc and then injecting the recombinant CD66c-HuIgFc in the boosting step against CD66c Antibodies were amplified, and antibodies against CD66c expressed in lung adenocarcinoma were selected by selecting antibodies positive for both CD66c-HuIgFc and A549 cells during hybridoma screening (see FIG. 7).

Lung cancer-specific to the 6-week-old Balb / c female cut mice maridang lung cancer cell line A549 (ATCC CCL-185) to develop a monoclonal antibody mouse abdominal cavity by the number of 1x10 ^7; a (IP intraperitoneal cavity) for three weeks Serum was isolated by injecting three times at intervals and taking a blood sample from the vein. 100ng / well of the CD66c-HuIgFc purified in <Example 1> was added to the separated serum and reacted at 37 ° C. for 1 hour, and then coated with 200ul of blocking buffer (sigma) per well for 1 hour at 37 ° C. Reaction was blocked. The serum was diluted in the coated plate and reacted at 37 ° C. for 1 hour, and the unbound antibody was washed with PBS. Finally, to confirm the antibody bound to the secondary antibody goat anti-Mouse Ig-HRP (Jackson) diluted 2000 times and put in 100ul per well and reacted at 37 ℃ and washed with PBS and TMB (3,3 ', 5,5'-Tetramethylbenzidine) was added at 50ul per well and reacted at room temperature for 10 minutes. After stopping the reaction with 2N H ₂ SO ₄ (sigma), the titer was confirmed by measuring the absorbance at 450nm and diluted in A549 cells. Serum was added and reacted at 4 ° C. for 30 minutes. Then, 3 ml of PBS was added and centrifuged at 1500 rpm for 3 minutes to wash the unbound antibody, and to confirm the bound antibody, goat anti-Mouse Ig-FITC (Dinona) was used. After diluting 200 times, the reaction was performed at 4 ° C. for 15 minutes, washed with PBS 3ml in the same manner as above, and measured by flow cytometry to confirm the titer on CD66c. The results are described in FIGS. 8 and 9. Specifically, three days before cell fusion, 50 ug anti-CD40 agonist mAb was added to increase the immune response and 100 ug CD66c-HuIgFc was injected to induce amplification of the antibody against CD66c.

As shown in FIG. 8 and FIG. 9, the titers of A549 cells were confirmed to be high as a result of flow cytometry (FIG. 8). Serum immunized with A549 cells even though the antigens against CD66c were not directly immunized. It was confirmed that the positive for CD66c-HuIgFc was shown in (Fig. 9).

The spleens of the immunized rats were excised to obtain single cell suspension, washed twice with RPMI (GIBCO), mixed with 0.4% trypan blue (sigma) at 1: 1 (v / v), and then microscopically. Cell counts were counted by tryphan blue staining, which measures unstained cells. SP2 / 0 (ATCC CRL-1581) or X63 moue myeloma cell line (ATCC CRL-1580) was used as cell fusion partner cells and the number of cells after washing was counted in the same manner as the splenocytes.

The myeloma cells and splenocytes were mixed at a ratio of 1: 5 and the supernatant was removed after centrifugation. 1 ml of 50% PEG (polyethylene glycol) 1500, previously preheated to 37 ° C., was slowly added over 1 minute. It was allowed to stand for about 1 minute and then diluted gradually by adding RPMI medium slowly. After centrifugation, the mixture was suspended in RPMI (20% FBS, hypoxanthine-aminopterin-thymidine) containing 1xHAT, and was incubated in a 37-well 5% CO ₂ incubator after dispensing at 100ul / well in a 96-well plate. After the fusion, the HAT feeding was performed for a certain period of time, and when the wells that formed the colonies were observed, the culture supernatant was added 100ul of hybridoma culture supernatant to A549 cells, which are lung adenocarcinoma cell lines, and reacted at 4 ° C. for 30 minutes. And centrifuged at 1500 rpm for 3 minutes to wash the unbound antibody, and 200-fold dilution of the secondary antibody goat anti-Mouse Ig-FITC (Dinona) was carried out for 15 minutes at 4 ° C. After washing with PBS 3ml in the same manner as above, and measured by flow cytometry, the purified CD66c-HuIgFc using CD66c-HuIgFc was added to 100ng / well and reacted for 1 hour at 37 ℃ coating and the blocking buffer (sigma) 200ul per well was blocked by reacting for 1 hour at 37 ℃. 100 μl per well of the hybridoma culture supernatant was added to the coated plate and reacted at 37 ° C. for 1 hour, and the unbound antibody was washed with PBS. Finally, to confirm the antibody bound to the secondary antibody goat anti-Mouse Ig-HRP (Jackson) diluted 2000 times and put in 100ul per well, reacted at 37 ℃ and washed with PBS and TMB (3,3 ' 50 μl of 5,5'-Tetramethylbenzidine per well was reacted at room temperature for 10 minutes, followed by ₂ N H ₂ SO ₄ (sigma), followed by enzyme-linked immunosorbent assay (ELISA) by measuring absorbance at 450 nm. It was.

In both methods, 8F5 monoclonal antibodies positive were selected and finally, hybridoma cells expressing a single colony of 8F5 mAb were obtained through limiting dilution.

<2-2> Monoclonal  Antibody Isotype ( isotype ) decision

In order to determine the isotype of the 8F5 monoclonal antibody prepared in <Example 2-1>, it was analyzed by mouse immunoglobulin isotyping ELISA kit (BD Biosciences, USA). Specifically, isotyping was performed using rabbit anti-murine isotype specific antisera (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, Kappa, Lamda) and peroxidase-labeled goat anti-rabbit IgG. The color reaction was induced using ortho-phenylenediamine (OPD) and hydrogen peroxide substrate and the absorbance at 450 nm was measured to confirm the value.

As a result, 8F5 monoclonal antibody was identified as mouse IgG1 / kappa light chain. (Result not shown)

<2-3> Monoclonal  Antibody CD66c  Antigenic Cum

Western blotting of A549 cell lysate and recombinant antigen (CD66c-HuIgFc) was performed using 8F5 monoclonal antibody and anti-CD66c AP11 antibody of the present invention.

Specifically, the lung adenocarcinoma cell line A549 of 1x10 ⁷ cells with 100ul of lysis buffer (1% Nonidet P-40; NP-40 in 50mM Tris-HCl, pH 7.4, 50mM EDTA, and 1mM phenyl-methyl-sulfonyl-fluoride; PMSF) After suspension, the cells were lysed at room temperature for 15 minutes, centrifuged to remove cell residues, and the supernatant was collected to prepare a lysate. The prepared A549 lysate and the recombinant antigen (CD66c-HuIgFc) obtained in Example 1 were boiled for 3 minutes and then loaded under 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. . After electrophoretic transfer to nitrocellulose membrane, blocking with 5% skim milk (sigma) solution and binding to 8F5 and AP11 monoclonal antibodies, respectively, combined with 8F5 monoclonal antibody were washed three times with PBS and then peroxidase-conjugated goat anti -mouse IgG (Sigma, Saint Louis, USA) was bound and AP11 monoclonal antibody reacted with peroxidase-conjugated goat anti-mouse IgM (Sigma, Saint Louis, USA). After washing the nitrocellulose membrane with PBS, the band was confirmed with an enhanced chemiluminescence detection system (ECL, Amersham, Sweden) and the results are shown in FIG. 10.

As shown in FIG. 10, a band of approximately 75 kDa was confirmed in both non-reduced and reduced conditions, and recombinant CD66c-HuIgFc showed a band of about 210 kDa in both non-reduced conditions and 105 kDa in reduced conditions. You can check. The difference in non-reduced and reduced size of recombinant CD66c-HuIgFc was due to the dimer formation in HuIgFc region.

As a result, it can be seen that the Western blotting results of AP11 and 8F5, which are antibodies to the conventional anti-CD66c, are consistent. Therefore, it can be seen that the CD66c antigen can be detected using the monoclonal antibody of the present invention.

The CD66c antigen is known to be expressed in granulocytes of blood. In order to confirm the binding of 8F5 to peripheral blood, 1ug of 8F5 antibody was added to PBMC (peripheral blood mononuclear cells) and reacted at 4 ° C for 30 minutes, and then 3 ml of PBS was added and centrifuged at 1500 rpm for 3 minutes. After washing the non-antibodies and checking the bound antibody, 200-fold dilution of goat anti-Mouse Ig-FITC (Dinona), which is a secondary antibody, was reacted at 4 ° C for 15 minutes, and then washed with PBS 3ml in the same manner as above. Measured by flow cytometry and the results are shown in FIG.

As shown in FIG. 11, it was confirmed that the CD66c antigen was expressed in almost all granulocytes by 98% by using 8F5 monoclonal antibody, and was hardly expressed in lymphocytes and monocytes. Able to know.

In addition, a monoclonal antibody against anti-CD66c was used to confirm binding to 8F5 and CD66c-HuIgFc using a well-known 9A6 clone (SantaCrutz, USA) in addition to AP11. After the reaction was coated and put blocking buffer (sigma) 200ul per well was blocked by reacting for 1 hour at 37 ℃. 100 μl of each of the 9A6, 8F5, and AP11 antibodies at a concentration of 1 ug / ml was added to the coated plate and reacted at 37 ° C. for 1 hour, and the unbound antibody was washed with PBS. Finally, to confirm the antibody bound to the secondary antibody goat anti-Mouse Ig-HRP (Jackson) diluted 2000 times and put in 100ul per well and reacted at 37 ℃ and washed with PBS and TMB (3,3 ' 50 μl of 5,5'-Tetramethylbenzidine per well was reacted at room temperature for 10 minutes, and then the reaction was stopped with 2N H ₂ SO ₄ (sigma) and confirmed by ELISA, a method for measuring absorbance at 450 nm. It described in 12.

As shown in FIG. 12, all three antibodies show high values for CD66c-HuIgFc, but it can be seen that the three clones have different activities (FIG. 12).

<2-4> 8F5 Monoclonal  Antibody Lung adenocarcinoma  Cell line CD66c  Expression Analysis of Antigens

Flow cytometry confirmed the binding of 8F5 monoclonal antibodies to various cancer cell lines obtained through Korean Cell Line Bank (KCLB) and Seoul National University (SNU).

Specifically, cancer cell lines were obtained through Korean Cell Line Bank (KCLB) and Seoul National University (SNU), and 10% heat inactivated fetal bovine serum (L-132, SW-900, DU145, LNCap, MDA-MB231, MCF-7) FBS; GIBCO, invitrogen) added Dubco's MEM (GIBCO, invitrogen) medium, A549, NCI-H460, NCI-H417, DLD-1, HCT116, HT-29, SW-480, SW-620, PC-3 Was prepared using 5% CO ₂ at 37 ° C using RPMI 1640 (GIBCO, invitrogen) medium supplemented with 10% heat inactivated FBS _. The culture was carried out using a state incubator.

Put the 8F5 monoclonal antibody of the present invention in the cultured cancer cell line and incubated for 30 minutes at 4 ℃, washed with PBS, and incubated for 15 minutes at 4 ℃ put FITC-conjugated goat anti-mouse Ig G (DiNona Inc, Korea) It was. After washing with PBS again and analyzed by FACScaliber (Becton Dickinson, USA) the results are shown in Table 3.

As shown in Table 3 above, the 8F5 monoclonal antibody of the present invention binds highly in lung adenocarcinoma cell line A549, while four cells except L-132, a normal lung cell line, and NCI-H417, HT-29, a small cell carcinoma cell line. The colon cancer cell line, three prostate cell lines, and two breast cancer cell lines did not bind to each other. The colon cancer cell line HT-29 was found to be weakly bound.

<2-5> 8F5 Monoclonal  Lung Cancer Tissues Using Antibodies CD66c  Expression Analysis of Antigens

Immunity to 8F5 with frozen thymus, lymph node, tonsil, spleen, kidney, urethra and skin frozen tissue obtained from Chungbuk National University Hospital Histochemical staining was performed, and the lung cancer tissues secured in the clinic were fixed with 10% formalin (sigma) and embedded in paraffin.

Specifically, tissue staining was performed by adding 8F5 monoclonal antibody to blocking buffer (4% skim milk, 0.1% Tween-20, PBS) at 4 ° C. overnight, followed by biotinylated goat anti-mouse IgG and HRP conjugated streptavidin (Dako, Denmark) was added and allowed to bind at room temperature for 20 minutes. Color reaction was performed using 3,3'-diaminobenzidine (Sigma, Saint Louis. USA) and the results are shown in Table 4 and FIG.

As shown in Table 4, 8F5 monoclonal antibody staining for normal frozen tissue obtained through Chungbuk National University Hospital can be seen that all tissues are negative.

However, brown positivity was detected in lung adenocarcinoma tissues (FIGS. 13A, B), squamous cell carcinoma (squamous cell carcinoma, FIG. 13C) and small cell carcinoma (small cell carcinoma, FIG. 13D), and negative in tissues. It can be seen that the antibody can be used to detect lung adenocarcinoma tissue.

< Example  3>

Of the present invention Monoclonal  Antibody Epitope ( epitope ) decision

<3-1> of the present invention Monoclonal  Antibody Epitope ( epitope ) decision

In order to determine the epitope of the 8F5 antibody of the present invention prepared in <Example 2> monoclonal antibody, protein peptide analysis using the MOLI-TOF method was used.

First, 2-5 ug of the 8F5 antibody and 20ul of Protein A resin (amersham phamarcia) were added and bound at 4 ° C for at least 12 hours. Then, 500ul of A549 lysate was added and rotated at 4 ° C for at least 4 hours. After washing with PBS, only proteins bound to the antibody remained in protein A resin. This was loaded onto an SDS-PAGE gel to separate proteins and trypsinization followed by MOLDI-TOF (Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Shevchenko A, Wilm M, Vorm O, Mann M. Anal Chem Peptides were analyzed by 1996 Mar 1; 68 (5): 850-8.). The analyzed screen was captured and described in FIG. 14.

As shown in FIG. 14, the epitope of the monoclonal antibody of the present invention can be seen that the amino acid sequence of SEQ ID NO: 7 (RNDAGSYECEIQNPASANR).

<3-2> of the present invention Monoclonal  Antibody Epitope ( epitope ) Verification

It was confirmed that the antigen for the 8F5 antibody determined by the proteome analysis of <Example 3-1> was CD66c, and as shown in FIG. 15 to verify the epitope, the <Example 3-1> Recombinant antigens with or without the determined epitope region were constructed to perform an immune response against the 8F5 antibody.

<3-2-1> CD66c mutant  Recombinant gene manufacturing

Using the pSec-Tag-CD66cfull-hFc recombinant gene, a CD66c mutant recombinant gene was generated using the restriction enzyme BamHI present in the epitope region of the 8F5 antibody. Specifically, the CD66c mutant recombinant gene was cleaved by inserting the entire nucleotide sequence of the CD66c antigen represented by SEQ ID NO: 8 and the gene to which hFc is bound in pSec-Tag, and cleaving it using restriction enzyme BamHI present in the epitope region of the 8F5 antibody. Secured.

More specifically, the CD66c mutant (BI / BHI) not containing the epitope of the 8F5 antibody was cut by BamHI, and then produced by a method of removing 252bp between 535 and 787bp of the CD66c gene of SEQ ID NO: 8 and relinking the remaining fragments. It was. The sequence of the prepared CD66c mutant (BI / BHI) is represented by the nucleotide sequence of SEQ ID NO.

The CD66c mutant (HPK / HdIII / PstI / klenow), which contains an epitope of the 8F5 antibody but lacks the N- and N-domains of the N-terminal, was cut with Hind III and PstI, and then removed a 479bp fragment from the N-terminal. Klenow was applied to the remaining gene fragments to fill the sticky ends by HindIII and PstI, and thus, the blunt ends were connected to each other. The sequence of the prepared CD66c mutant (HPK / HdIII / PstI / klenow) is represented by the nucleotide sequence of SEQ ID NO: 10.

<3-2-2> CD66c mutant  Expression of Recombinant Genes

The pSec-Tag vector inserted with the CD66cfull-hFc, CD66c mutant (BI / BHI) -hFc, and CD66c mutant (HPK / HdIII / PstI / klenow) -hFc was inserted into CHO-K1 cells using Effectene (Qiagen). By transformation.

More specifically, the genes and the Effectene complexes were plated one day before, sprinkled with CHO cells, which were ground with fresh medium, and cultured for 48 hours. Two days after the transfection, the culture supernatant was harvested and the expression was confirmed using a sandwich ELISA detecting human Fc (hFc). (Result not shown)

<3-2-3> of the present invention Monoclonal  Antibody Epitope ( epitope ) Verification

In order to verify the epitope of the CD66c of the monoclonal antibody of the present invention, 100ng of known anti-CD66c antibodies, 9A6 (Santa cruz) and AP11 (Dinona), were reacted for one hour at 37 ° C and coated as a capture antibody. Then, 200 μl of 1 × blocking solution (sigma) was added per well and blocked by reaction at 37 ° C. for 1 hour. The prepared supernatant of CD66c full-hFc, CD66c mutant (BI / BHI) -hFc and CD66c mutant (HPK / HdIII / PstI / klenow) -hFc prepared in Example 3-2-2 was prepared in the prepared plate. After adding 100ul per well, the reaction was performed at 37 ° C for one hour and washed with PBS to remove the non-binding antibody. After diluting anti-human Ig-HRP (Jackson) and reacting for 1 hour, rinsed with PBS and rinsed with PBS and reacted for 10 minutes by adding 50ul of TMB solution per well, and stopping the reaction with 50ul of sulfuric acid. It was. In order to confirm the presence of the CD66c mutant-hFc protein in the culture supernatant, Capture & detect Sandwich ELISA was performed using an anti-human Ig antibody as a control and the experimental results are shown in Table 5 and FIG. 16.

Capture antibody 8F5 9A6 AP11 a-huIg Upper layer
Culture CD66c full-hFc 2.873 2.838 2.913 3.123 CD66c mutant (HPK / HdIII / PstI / klenow) -hFc 2.804 0.049 0.097 3.113 CD66c mutant (BI / BHI) -hFc 0.019 1.924 2.662 3.149 negative 0.017 0.027 0.247 3.161

As described in Table 5 and FIG. 16, the 8F5 antibody of the present invention is a CD66c full-hFc or CD66c mutant (HPK / HdIII / PstI / klenow)-containing an epitope determined in Example 3-1. It can be seen that there is activity for binding to hFc, but little to CD66c mutant (BI / BHI) -hFc from which the epitope has been removed. However, it can be seen that the antibodies of the existing CD66c 9A6, AP11 mainly binds to the CD66c mutant (BI / BHI) -hFc from which the epitope is removed. Therefore, it can be seen that the 8F5 antibody of the present invention is represented by the amino acid sequence of SEQ ID NO: 7, which is a different epitope from the conventional 9A6 and AP11 antibodies.

As shown in FIG. 17, it can be seen that the epitope region of the 8F5 antibody revealed through MOLDI-TOF is exactly the same as the epitope region.

< Example  4>

Of the present invention Monoclonal  Antibody Lung cancer  detection

CD66c antigen was detected by immunoassay with the monoclonal antibody of the present invention.

Specifically, AP11, which is an antibody against the conventional CD66c, was put in 100ng per well and reacted at 37 ° C. for one hour to coat as a capture antibody, and 200 × of 1 × blocking solution (sigma) was added per well for 1 hour at 37 ° C. for blocking. Into the plate prepared as described above, lysate of pulmonary adenocarcinoma cell line A549, small cell lung cancer cell line H417, gastric cancer cell line KatoIII (ATCC HB-103), and blood cancer cell line K562 (ATCC CCL-243) was put in 100 ugs per well, at 37 ° C. The reaction was removed for 1 hour, washed with PBS, and the antibody was not bound. After diluting the HRP-conjugated antibody into the 8F5 antibody and reacting for 1 hour, washing with PBS and rinsing with PBS for 50 minutes per well for 10 minutes, stopping the reaction by adding 50ul of sulfuric acid and checking the value at 450nm. It was. At this time, a value (detection Ag) derived from CD66c full-hFc protein as a standard was quantitatively calculated, and the results are shown in FIG. 18.

As shown in FIG. 18, it can be seen that lung cancer can be effectively detected through the 8F5 antibody of the present invention and thus can be effectively used as a lung cancer-specific biomarker.

< Example  5>

Of the present invention Monoclonal  Anticancer Activity of Antibodies

Mice were 6 weeks old (18 g) male Nude-mouse (Central Experimental Animal). The lung adenocarcinoma cell line A549 was suspended with 1xtrypsin-EDTA (GIBCO), washed twice with Hanks balanced salt solution (HBSS) and 1x10. Tumor lines were obtained by injecting ⁷ cells into one nude mouse in the back of the mouse forelimb in a volume of 100ul. Tumor sections of rapidly growing stage were made with sufficient blood supply before the central necrosis of the tumor occurred.They were placed on the skin to confirm the location of the tumor and observed growth at least twice a week. Mice that reached 100 mm ³ were used in this experiment.

To obtain statistical significance, mice with a volume of 100 mm ³ were selected and 10 mice were assigned to each group, and monoclonal antibodies of the present invention were injected into mouse tail vein diluted in 100 ug / 100 ul of 1 × PBS per subject and injected. Intervals were administered seven times at 0, 3, 7, 14, 21, 28, and 35 days.

Tumor volume was measured by calipers twice a week on the basis of the monoclonal antibody administration date of the present invention, and the length and shortening of the tumor were measured for 4 to 5 weeks on a weekly basis to determine the effect of the test substance. The result is shown in FIG.

As shown in FIG. 19, the 8F5 antibody of the present invention is an antigen-free antibody expressed in A549 cells, and has an tumor suppression effect of about 30% or more compared to an isotype control antibody (Dinona), an antibody having an isotype such as 8F5. It can be seen.

Korea Cell Line Research Foundation KCLRFBP00230 20100222

<110> DiNonA Inc. <120> Epitope of CD66c specific to lung adenocarcinoma and antibody recognizing the same <130> DPP20103373KR <150> KR2010-0018078 <151> 2010-02-26 <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 344 <212> PRT <213> CD66c <400> 1 Met Gly Pro Pro Ser Ala Pro Pro Cys Arg Leu His Val Pro Trp Lys   1 5 10 15 Glu Val Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr              20 25 30 Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly          35 40 45 Lys Glu Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Arg Ile Gly      50 55 60 Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val  65 70 75 80 Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser                  85 90 95 Gly Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val             100 105 110 Thr Gln Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp         115 120 125 Leu Val Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu     130 135 140 Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys 145 150 155 160 Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Val Gln Asn Thr Thr Tyr                 165 170 175 Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Leu Gln             180 185 190 Leu Ser Asn Gly Asn Met Thr Leu Thr Leu Leu Ser Val Lys Arg Asn         195 200 205 Asp Ala Gly Ser Tyr Glu Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn     210 215 220 Arg Ser Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp Val Pro 225 230 235 240 Thr Ile Ser Pro Ser Lys Ala Asn Tyr Arg Pro Gly Glu Asn Leu Asn                 245 250 255 Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe             260 265 270 Ile Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn         275 280 285 Ile Thr Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala His Asn Ser     290 295 300 Ala Thr Gly Leu Asn Arg Thr Thr Val Thr Met Ile Thr Val Ser Gly 305 310 315 320 Ser Ala Pro Val Leu Ser Ala Val Ala Thr Val Gly Ile Thr Ile Gly                 325 330 335 Val Leu Ala Arg Val Ala Leu Ile             340 <210> 2 <211> 276 <212> PRT <213> Recombinant CD66c <400> 2 Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys Glu   1 5 10 15 Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Arg Ile Gly Tyr Ser              20 25 30 Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val Gly Tyr          35 40 45 Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly Arg      50 55 60 Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln  65 70 75 80 Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Val                  85 90 95 Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu Pro Lys             100 105 110 Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys Asp Ala         115 120 125 Val Ala Phe Thr Cys Glu Pro Glu Val Gln Asn Thr Thr Tyr Leu Trp     130 135 140 Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser 145 150 155 160 Asn Gly Asn Met Thr Leu Thr Leu Leu Ser Val Lys Arg Asn Asp Ala                 165 170 175 Gly Ser Tyr Glu Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn Arg Ser             180 185 190 Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp Val Pro Thr Ile         195 200 205 Ser Pro Ser Lys Ala Asn Tyr Arg Pro Gly Glu Asn Leu Asn Leu Ser     210 215 220 Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe Ile Asn 225 230 235 240 Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr                 245 250 255 Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala His Asn Ser Ala Thr             260 265 270 Gly leu asn arg         275 <210> 3 <211> 27 <212> DNA <213> CD66c gene forward primer <400> 3 aagcttaagc tcactattga atccacg 27 <210> 4 <211> 26 <212> DNA <213> CD66c gene reverse primer <400> 4 gatatcagtg actgtggtcc tattga 26 <210> 5 <211> 27 <212> DNA <213> HuIgFc forward primer <400> 5 gatatcgacg tcgagtccaa atcttgt 27 <210> 6 <211> 26 <212> DNA <213> HuIgFc reverse primer <400> 6 ctcgagttta cccggagaca gggaga 26 <210> 7 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> epitope of 8F5 monoclonal antibody <400> 7 Arg Asn Asp Ala Gly Ser Tyr Glu Cys Glu Ile Gln Asn Pro Ala Ser   1 5 10 15 Ala Asn Arg             <210> 8 <211> 831 <212> DNA <213> Artificial Sequence <220> <223> CD66c cDNA-hydrophobic region <400> 8 actattgaat ccacgccgtt caatgtcgca gaggggaagg aggttcttct actcgcccac 60 aacctgcccc agaatcgtat tggttacagc tggtacaaag gcgaaagagt ggatggcaac 120 agtctaattg taggatatgt aataggaact caacaagcta ccccagggcc cgcatacagt 180 ggtcgagaga caatataccc caatgcatcc ctgctgatcc agaacgtcac ccagaatgac 240 acaggattct ataccctaca agtcataaag tcagatcttg tgaatgaaga agcaaccgga 300 cagttccatg tatacccgga gctgcccaag ccctccatct ccagcaacaa ctccaacccc 360 gtggaggaca aggatgctgt ggccttcacc tgtgaacctg aggttcagaa cacaacctac 420 ctgtggtggg taaatggtca gagcctcccg gtcagtccca ggctgcagct gtccaatggc 480 aacatgaccc tcactctact cagcgtcaaa aggaacgatg caggatccta tgaatgtgaa 540 atacagaacc cagcgagtgc caaccgcagt gacccagtca ccctgaatgt cctctatggc 600 ccagatggcc ccaccatttc cccctcaaag gccaattacc gtccagggga aaatctgaac 660 ctctcctgcc acgcagcctc taacccacct gcacagtact cttggtttat caatgggacg 720 ttccagcaat ccacacaaga gctctttatc cccaacatca ctgtgaataa tagcggatcc 780 tatatgtgcc aagcccataa ctcagccact ggcctcaata ggaccacagt c 831 <210> 9 <211> 600 <212> DNA <213> Artificial Sequence <220> <223> CD66c mutant (BI / BHI) <400> 9 aagcttaagc tcactattga atccacgccg ttcaatgtcg cagaggggaa ggaggttctt 60 ctactcgccc acaacctgcc ccagaatcgt attggttaca gctggtacaa aggcgaaaga 120 gtggatggca acagtctaat tgtaggatat gtaataggaa ctcaacaagc taccccaggg 180 cccgcataca gtggtcgaga gacaatatac cccaatgcat ccctgctgat ccagaacgtc 240 acccagaatg acacaggatt ctatacccta caagtcataa agtcagatct tgtgaatgaa 300 gaagcaaccg gacagttcca tgtatacccg gagctgccca agccctccat ctccagcaac 360 aactccaacc ccgtggagga caaggatgct gtggccttca cctgtgaacc tgaggttcag 420 aacacaacct acctgtggtg ggtaaatggt cagagcctcc cggtcagtcc caggctgcag 480 ctgtccaatg gcaacatgac cctcactcta ctcagcgtca aaaggaacga tgcaggatcc 540 tatatgtgcc aagcccataa ctcagccact ggcctcaata ggaccacagt cacggatatc 600                                                                          600 <210> 10 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> CD66c mutant (HPK / HdIII / PstI / klenow) <400> 10 aagctgctgt ccaatggcaa catgaccctc actctactca gcgtcaaaag gaacgatgca 60 ggatcctatg aatgtgaaat acagaaccca gcgagtgcca accgcagtga cccagtcacc 120 ctgaatgtcc tctatggccc agatgtcccc accatttccc cctcaaaggc caattaccgt 180 ccaggggaaa atctgaacct ctcctgccac gcagcctcca acccacctgc acagtactct 240 tggtttatca atgggacgtt ccagcaatcc acacaagagc tctttatccc caacatcact 300 gtgaataata gcggatccta tatgtgccaa gcccataact cagccactgg cctcaatagg 360 accacagtca cggatatc 378 <210> 11 <211> 705 <212> DNA <213> Human immunoglobulin Fc region CH2-CH3 <400> 11 gacgtcgagt ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 60 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180 aagttcagct ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 240 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420 tcccgagagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540 acgcctcccg tgctggactc cgacggctcc ttcttcctct atagcaagct caccgtggac 600 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 660 aaccactaca cgcagaagag cctctccctg tctccgggta aatga 705

Claims (10)

A polypeptide which is an epitope of CD66c (Cluster of Differentiation 66c) represented by the amino acid sequence of SEQ ID NO. The polypeptide of claim 1, wherein the CD66c epitope is specific for lung adenocarcinoma. An antibody or antibody fragment that recognizes the polypeptide of claim 1 as an epitope. The antibody or antibody fragment of claim 3, wherein the antibody is a monoclonal antibody or polyclonal antibody. The antibody or antibody fragment of claim 4, wherein the monoclonal antibody is produced by hybridoma cells (Accession No .: KCLRF-BP-00230). A cell line producing the antibody or antibody fragment of any one of claims 3 to 5. The cell line of claim 6, wherein said cell line is a hybridoma cell (Accession Number: KCLRF-BP-00230). A lung adenocarcinoma diagnostic composition comprising the lung adenocarcinoma-specific antibody or antibody fragment of any one of claims 3 to 5 as an active ingredient. A lung adenocarcinoma diagnostic kit comprising the lung adenocarcinoma-specific antibody or antibody fragment of any one of claims 3 to 5 as an active ingredient. A pharmaceutical composition for preventing or treating lung adenocarcinoma, comprising the lung adenocarcinoma-specific antibody or antibody fragment of any one of claims 3 to 5 as an active ingredient.

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