KR20180040842A - Radiolabeled anti-CD55 antibody for the diagnosis and therapy of cancer and use thereof - Google Patents

Abstract

The present invention relates to an anti-CD55 antibody having a radioactive isotope labeled to a monoclonal antibody having high affinity to a decay-accelerating factor (CD55 or DAF) which is a receptor suppressing immune mechanisms, and an antigen-binding fragment thereof; a method for preparing the same; a cancer diagnostic kit comprising the anti-CD55 antibody labeled with a radioactive isotope; a method for providing information necessary for the diagnosis of cancer; a pharmaceutical composition for preventing or treating cancer, comprising the anti-CD55 antibody labeled with a radioactive isotope; and a functional health food composition for preventing or alleviating cancer, comprising the anti-CD55 antibody labeled with a radioactive isotope. According to the present invention, the anti-CD55 antibody having a radioactive isotope labeled to a monoclonal antibody having high affinity to a decay-accelerating factor (CD55 or DAF) and an antigen-binding fragment thereof have excellent binding affinity to CD55, and thus bind to CD55 with high specificity; and have extremely high sensitivity and specificity, and thus find useful applications in screening patients with various cancers including lung cancer. Further, compared with monoclonal antibodies, the antibody bound to a radioactive isotope creates stronger effects in killing cancer cells and suppressing metastases of cancer cells, and thus may show superior treatment effects in the treatment of various cancers including lung cancer.

Description

[0002] Radiolabeled anti-CD55 antibody for the diagnosis and treatment of cancer and use thereof,

The present invention relates to a CD55 target antibody and a antigen-binding fragment thereof, wherein the radioisotope is labeled with a monoclonal antibody having a high binding affinity for a decay-accelerating factor (CD55 or DAF) which is an immunosuppressive receptor, A cancer diagnostic kit including an isotope-labeled CD55 target antibody and an antigen-binding fragment thereof, and information necessary for diagnosis of cancer, a method for preventing or treating cancer including the above-mentioned radioactive isotope-labeled CD55 target antibody and antigen binding fragments thereof A pharmaceutical composition thereof, and a health functional food composition for preventing or ameliorating cancer comprising the radioactive isotope-labeled CD55 target antibody and an antigen-binding fragment thereof.

Conventional anticancer drugs have a therapeutic mechanism that attacks rapidly dividing cancer cells. However, such a therapeutic mechanism causes normal, rapidly dividing cells such as intestinal epithelium to be attacked, resulting in serious side effects such as diarrhea and vomiting. To overcome these side effects, targeted chemotherapy technology is being developed. Target chemotherapy is an effective chemotherapy that can selectively reduce the side effects of normal cells because it selectively attacks cancer cells. In particular, the target chemotherapy using a radioisotope exhibits an excellent therapeutic effect by binding a radioisotope to an antibody or a peptide targeting the cancer.

Lung cancer is one of the easily metastatic cancers, and it causes malignant pleural effusion especially in the thoracic cavity. Malignant pleural effusion may cause lung cancer to spread to the chest wall, and the survival period is usually about four months. Therefore, effective chemotherapy is needed to treat fatal metastatic lung cancer such as malignant pleural effusion.

Decay-accelerating factor (CD55 or DAF) is a receptor that inhibits complement immunity. This receptor is expressed in many cases such as colon cancer, lung cancer, stomach cancer, breast cancer, ovarian cancer, leukemia, head and neck cancer, and the complement immune system in the body inhibits the destruction of cancer cells, thereby promoting the proliferation of cancer cells. Therefore, suppression of this receptor is expected to help the cancer cells to destroy the complement system in the body.

We have developed an antibody that has a high binding capacity for decay-accelerating factor (CD55 or DAF) and can induce a therapeutic effect. An antibody conjugated with a radioactive isotope may induce a higher cancer cell killing effect than a monoclonal antibody. Therefore, there is a need for a therapeutic technique that can induce a higher therapeutic effect by binding a radioisotope to a decay-accelerating factor (CD55 or DAF) target antibody.

An object of the present invention is to provide a radioactive isotope-labeled CD55 target antibody or antigen-binding fragment thereof capable of exerting an improved cancer cytotoxic effect as compared to a single antibody.

Another object of the present invention is to provide a cancer diagnostic kit comprising the above-mentioned radioactive isotope-labeled CD55 target antibody or an antigen-binding fragment thereof.

Yet another object of the present invention is to provide a method for providing information necessary for cancer diagnosis using the radioactive isotope-labeled CD55 target antibody or antigen-binding fragment thereof.

Yet another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises the above-mentioned radioactive isotope-labeled CD55 target antibody or antigen-binding fragment thereof as an active ingredient.

Yet another object of the present invention is to provide a health functional food composition for preventing or ameliorating cancer, which comprises the above-mentioned radioactive isotope-labeled CD55 target antibody or antigen-binding fragment thereof as an active ingredient.

In order to solve the above-mentioned problems, the present invention can provide a radioactive isotope-labeled CD55 target antibody or an antigen-binding fragment thereof, which comprises the following general formula 1:

[Formula 1]

Radioisotope-R1-R2

Wherein R1 is a chelator and R2 is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD55.

Another embodiment of the present invention can provide a cancer diagnostic kit comprising a radioactive isotope labeled CD55 target antibody or an antigen-binding fragment thereof.

Another embodiment of the present invention is a method for detecting a cancer, comprising the steps of: (a) contacting a radioactive isotope labeled CD55 target antibody or antigen-binding fragment thereof of claim 1 with a biological sample isolated from a subject suspected of having cancer; (b) measuring the level of expression of a radioactive isotope labeled CD55 target antibody or antigen-binding fragment thereof CD55 protein in said biological sample through antigen-antibody complex formation; And (c) comparing the expression level of the CD55 protein measured in the step (b) with that of the control, and when the expression level of the protein is higher than that of the control, determining that the patient is a cancer patient; A method for providing information necessary for cancer diagnosis, including cancer diagnosis, can be provided.

The present invention can also provide a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a CD55 target antibody or an antigen-binding fragment thereof labeled with a radioactive isotope.

The present invention can also provide a health functional food composition for preventing or ameliorating cancer, which comprises a radioactive isotope-labeled CD55 target antibody or an antigen-binding fragment thereof as an active ingredient.

The present invention relates to a CD55 target antibody and an antigen-binding fragment thereof, which are radioisotope labeled with a monoclonal antibody having a high binding affinity for a decay-accelerating factor (CD55 or DAF), and exhibit excellent binding affinity to CD55, And it is very useful for screening lung cancer and various cancer patients because of its high sensitivity and specificity. Furthermore, the antibody conjugated with the radioactive isotope compared to the monoclonal antibody has a higher effect of inhibiting cancer cell killing and cancer cell metastasis, and thus has a higher therapeutic effect in various cancer treatments including lung cancer.

1 is a diagram showing the expression of decay-accelerating factor (CD55 or DAF) in lung cancer.
2 is a diagram showing the expression of a decay-accelerating factor (CD55 or DAF) in a lung cancer cell line.
FIG. 3 is a graph showing the binding strength of a decay-accelerating factor (CD55 or DAF) target antibody (4-1H) to a lung cancer cell line.
Figure 4 is a plot showing radiochemical purity (> 95%) and radiochemical stability of Lu-177-DTPA-anti CD55 Ab.
Figure 5 is a diagram showing the immunoreactivity of Lu-177-DTPA-anti CD55 Ab to lung cancer cells.
FIG. 6 is a graph showing the binding force of Lu-177-DTPA-anti CD55 Ab and lung cancer cells using the same.
FIG. 7 is a graph showing the binding strength between Lu-177-DTPA-anti CD55 Ab and various kinds of lung cancer cells.
Fig. 8 shows the internalization and exogenous kinetics of Lu-177-DTPA-anti CD55 Ab and lung cancer cells.
FIG. 9 is an illustration showing the results of anatomy and expression of CD55 in an animal model of lung cancer metastasis in a thoracic cavity. FIG.
FIG. 10 is a diagram showing the in vivo distribution of Lu-177-DTPA-anti CD55 Ab in an animal model of lung cancer metastasis in the thorax.
Fig. 11 is a chart showing the residual amounts and the amounts of Lu-177-DTPA-anti CD55 Ab in an animal model of lung cancer metastasis.
FIG. 12 is a graph showing the tumor uptake rate of Lu-177-DTPA-anti CD55 Ab according to the fasting time before administration of the drug.
FIG. 13 shows SPECT / CT images of Lu-177-DTPA-anti CD55 Ab in an animal model of lung cancer metastasis.
14 is a chart showing the therapeutic effect of Lu-177-DTPA-anti CD55 Ab in lung cancer cells.
Fig. 15 is a chart showing the evaluation of in vivo lung cancer (malignant pleural effusion) treatment effect of Lu-177-DTPA-anti CD55 Ab.
16 is a diagram showing high expression of CD55 in squamous cell carcinoma.
17 is a graph showing the inhibitory effect of Lu-177-DTPA-anti CD55 Ab on in vitro lung cancer metastasis.
Fig. 18 is a chart showing the therapeutic effect of Lu-177-DTPA-anti CD55 Ab and cisplatin for in vitro lung cancer combination.
Fig. 19 shows the therapeutic effect of Lu-177-DTPA-anti CD55 Ab and cisplatin for in vivo lung cancer.

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

The present inventors found that CD55 is expressed at low levels in normal tissues and normal cells, but is highly expressed in cancer tissues and cancer cells, and that CD55 is important for cancer diagnosis and treatment. In particular, it was confirmed that CD55 is expressed low in normal tissues and normal cells, but highly expressed in lung cancer tissues and lung cancer cells, and it was found that CD55 is important for diagnosis and treatment of lung cancer (FIGS. 1 and 2).

Accordingly, the present inventors have developed a radioactive isotope-labeled CD55-labeled antibody useful for diagnosis and treatment of various cancers including lung cancer by binding specifically to CD55 through further studies. The monoclonal antibody of the present invention has excellent binding affinity to CD55, binds specifically to CD55, and has very high affinity. Therefore, it can be useful for diagnosis and treatment of various cancers including lung cancer. Furthermore, the antibody conjugated with the radioactive isotope compared to the monoclonal antibody has a higher effect of inhibiting cancer cell killing and cancer cell metastasis, and thus has a higher therapeutic effect in various cancer treatments including lung cancer.

The present invention provides a CD55 target antibody or antigen-binding fragment thereof labeled with a radioisotope.

As used herein, the term "antibody" means a protein molecule that acts as a receptor that specifically recognizes an antigen, including immunoglobulin molecules immunologically reactive with specific antigens, and includes polyclonal antibodies, monoclonal antibodies, Whole antibody and antibody fragments are all included. The term also includes chimeric antibodies (e. G., Humanized murine antibodies) and bivalent or bispecific molecules (e. G., Bispecific antibodies), diabodies, triabodies, and tetrabodies. The whole antibody is a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype and includes IgG1, IgG2, IgG3 and IgG4. The antibody fragment refers to a fragment having an antigen-binding function, and includes Fab, Fab ', F (ab') 2, Fv, and the like. The Fab has one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1 domain) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Fv (variable fragment) refers to the smallest antibody fragment that has only a heavy chain variable region and a light chain variable region. The double-stranded Fv (dsFv) is linked by a disulfide bond to a light chain variable region and a light chain variable region. A single-chain Fv (scFv) is generally linked to a variable region of a heavy chain and a variable region of a light chain via a peptide linker by a covalent bond. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, an F (ab ') 2 fragment can be obtained by cutting a whole antibody into papain and obtaining a Fab and digesting with pepsin) Can be produced through recombinant DNA technology.

The radioactive isotope labeled CD55 target antibody or antigen binding fragment thereof may be a radioactive isotope labeled CD55 target antibody or antigen binding fragment thereof,

[Formula 1]

Radioisotope-R1-R2

Wherein R1 is a chelator and R2 is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD55.

 The monoclonal antibody or antigen-binding fragment thereof specifically binding to the CD55 of the present invention comprises a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence of SEQ ID NO: A light chain variable region comprising a light chain CDR3 and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5 and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6 can do. The sequence listing is shown in Table 1 below.

The light and heavy CDR amino acid sequences of 4H-1 LCDR1 LCDR2 LCDR3 SGGGGSYG
SEQ ID NO: 1) WNDKRPS
(SEQ ID NO: 2) GGWDSSTYAI
(SEQ ID NO: 3) HCDR1 HCDR2 HCDR3 DRGMA
(SEQ ID NO: 4) GISSSGRYTYYAPAVKG
(SEQ ID NO: 5) NAGAGGWHAAYIDA
(SEQ ID NO: 6)

Typically, immunoglobulins have a heavy chain and a light chain, and each heavy and light chain comprises a constant region and a variable region, which region is also known as a domain. The variable region of the light chain and the heavy chain comprises three variable regions and four framework regions called complementarity-determining regions (CDRs). The CDRs mainly serve to bind to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, CDR3 starting from the N-terminus and sequentially identified by the chain in which the particular CDR is located.

The term "monoclonal antibody specifically binding to CD55" in the present invention means an antibody capable of binding to CD55 and resulting in inhibition of the biological activity of CD55, and can be used in combination with "anti- have. Monoclonal antibodies that specifically bind to CD55 include, without limitation, monoclonal antibodies that specifically inhibit CD55 to CD55. The form of the monoclonal antibody may include both whole antibodies and antibody fragments as described above.

The monoclonal antibody or antigen-binding fragment thereof that specifically binds to the CD55 of the present invention may comprise a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 and an amino acid sequence of SEQ ID NO:

The light and heavy chain variable region amino acid sequences of 4H-1 Light chain variable region ALTQPSSVSANLGGTVKITCSGGGGSYGWFQQKSPGSAPVTVIYWNDKRPSNIPSRFSGSKSGSTATLTITGVQAEDEAVYYCGGWDSSTYAIFGAGTTLTVL (SEQ ID NO: 7) Heavy chain variable region AVTLDESGGGLQTPGGALSLVCKASGFSFSDRGMAWVRQAPGKGLEWVAGISSSGRYTYYAPAVKGRATISRDNGQSTVRMQLNNLRAEDTGTYYCAKNAGAGGWHAAYIDAWGHGTEVIVSS (SEQ ID NO: 8)

Naturally occurring antibody structural units usually include tetramers. Each of the conjugates typically consists of two pairs of identical polypeptide chains, each pair having one full length light chain (usually a molecular weight of about 15 kDa) and one full length heavy chain (usually a molecular weight of about 50 to 70 kDa) . The amino-terminal portion of each of the light and heavy chains typically comprises a variable region of about 100 to 110 or more amino acids that are responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that is involved in effector function. Human light chains are typically classified as kappa (kappa) and lambda (lambda) light chains. The heavy chain is usually classified as mu, delta, gamma, alpha or epsilon and defines the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. Within the full-length light and heavy chains, variable and constant regions are typically joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. The variable region of each light chain / heavy chain pair usually forms an antigen-binding site. According to a preferred embodiment of the present invention, the heavy chain of the monoclonal antibody of the present invention may be an IgG1, IgG2a, IgG2b, IgG3, IgA or IgM isotype and the light chain may be carfamed or lambda chain, Light chain and IgGl heavy chain.

In the monoclonal antibody or antigen-binding fragment thereof of the present invention, "an antigen-binding fragment" means a fragment which has an antigen-binding function and, Fab, F (ab ') , F (ab') 2 and Fv, or Single chain antibody molecule molecules, and the like. Among the antibody-binding fragments, Fab has one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain. F (ab ') differs from Fab in that it has a hinge region containing at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. F (ab ') ₂ is produced by disulfide bonding of cysteine residues in the hinge region of Fab'. Fv refers to the smallest antibody fragment that has only the heavy chain variable region and the light chain variable region. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme. For example, when the whole antibody is cleaved with papain, a Fab can be obtained. When cut with pepsin, an F (ab ') ₂ fragment can be obtained, Can be produced through recombinant DNA technology.

A nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD55 of the present invention comprises a nucleotide sequence encoding a heavy chain of SEQ ID NO: 7 and a nucleotide sequence comprising a nucleotide sequence encoding a light chain of SEQ ID NO: Lt; / RTI >

The term "nucleic acid molecule" in the present invention encompasses both DNA (gDNA and cDNA) and RNA molecules. The nucleotide as a basic constituent unit in the nucleic acid molecule includes not only natural nucleotides but also analogs and an analogue. The sequence of the nucleic acid molecule encoding the heavy and light chain variable regions of the invention can be modified. Such modifications include addition, deletion, or non-conservative substitution or conservative substitution of nucleotides.

The nucleic acid molecule of the present invention is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence described above. The above-mentioned substantial identity is determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. % Homology, at least 90% homology in one particular example, and at least 95% homology in another specific example.

The chelator of the present invention may be a chelator capable of binding with a trivalent metal.

The term "chelator" in the present invention refers to a molecule that forms a complex with a radioactive isotope, wherein the complex is stable under physiological conditions. More specifically, the chelator may be a molecule that is synthesized with a radioisotope and is physiologically stable and has at least one reactive functional group for bonding with the spacer. The chelator may also comprise a single amino acid between the metal chelator and the spacer to couple with the spacer.

The chelator may be any one selected from the group consisting of a DTPA chelator, a NOTA chelator, a DOTA chelator, a DFO chelator NODAGA chelator, an N2S2 chelator, a TETA chelator, and a HYNIC chelator. Species, and most preferably DTPA-type.

The chelator of the present invention may be a chelator capable of binding with a trivalent metal. The chelator may be selected from the group consisting of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA (1,4,7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DFO (3- [6,17-dihydroxy-7,10,18,21-tetraoxo-27- [N-acetylhydroxylamino) -6,11,17 , 22-tetraazaheptaeicosane] thiourea), diethylenetriaminepentaacetic acid (DTPA), diaminedithiol N2S2, p-SCN-Bn-NOTA 2- (4'- isothiocyanatobenzyl) -1,4,7- triazacyclononane- triacetic acid), NODAGA (1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid), p-SCN-Bn-DOTA (2- (4'-isothiocyanatobenzyl) Tetraacacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), p-SCN-Bn-DTPA (2- (4-isothiocyanatobenzyl) -diethylenetriaminepentaacetic acid), p-SCN-Bn-DFO (1- (4-Isothiocyanatophenyl) -3- [6,17-dihydroxy-7,10,18,21-tetraoxo-27- [Nacetylhydroxylamino) 6,11,17,22-tetraazaheptaeicosane] thiourea), p-SCN-Bn-CHX-A'-DTPA ([(R) -2-Amino-3- (4-isothiocyanato phenyl) propyl] -trans- (S, S) -cyclohexane-1,2-diamine-pentaacetic acid and HYNIC (hyrazinonicotinic acid). Preferably, the metal chelator for the trivalent metal may be a DTPA-based chelator and analog thereof. Most preferably, the DTPA-based chelator can be p-SCN-Bn-CHX-A " -DTPA (macrocyclics).

The molar ratio of the monoclonal antibody or antigen-binding fragment thereof specifically binding to the CD55 to the chelator may be from 5: 1 to 500: 1, preferably from 25: 1 to 250: 1, Lt; / RTI > can be 50: 1.

The isotope is selected from the group consisting of ¹⁷⁷ Lu, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ¹¹¹ In, ¹⁰⁵ Rh, ¹⁷⁶ Yb, ¹⁶⁹ Gd, ¹⁷⁷ Lu, ¹¹¹ Ag, ¹²⁵ I, ¹²⁹ I and ¹⁶⁶ Ho Or more, and most preferably ¹⁷⁷ Lu.

The ratio of the radioactive isotope of the present invention to the above-mentioned radioisotope for R1-R2 may be 1 to 100 mCi, preferably 1 to 50 mCi, and most preferably 20 mCi Lt; / RTI >

In another embodiment of the present invention, a cancer diagnostic kit comprising a radioactive isotope labeled CD55 target antibody or an antigen-binding fragment thereof can be provided. The cancer may be selected from the group consisting of melanoma, lymphoma, lung, thyroid, breast, large intestine, prostate, head and neck, stomach, liver, bladder, kidney, cervix, pancreas, leukemia, bone marrow, ovary, uterus, sarcoma, And most preferably may be lung cancer.

As used herein, the term "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm whether or not the cancer has been invented. In particular, CD55 protein is a cancer diagnostic marker, and it is highly expressed in lung cancer, and moreover, in non-small cell lung cancer and metastatic lung cancer cell, compared with normal tissue.

Antibodies to CD55, included in the kits of the invention, can be immobilized, preferably using a variety of methods, as described in the literature, to suitable carriers or supports, examples of suitable carriers or supports include PBS, polystyrene, polyethylene, poly But are not limited to, polyacrylonitrile, polyacrylonitrile, fluorocarbon resin, agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethylcellulose, polyacrylamide, polysterine, , Plastic films, plastic tubes, polyamine-methyl vinyl ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylons, metals, glass, glass beads or magnetic particles. Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes. The support may have any possible shape, for example spherical (bead), cylindrical (test tube or well inner surface), planar (sheet, test strip).

A label capable of producing a detectable signal enables the formation of an antigen-antibody complex to be qualitatively or quantitatively measurable, and examples thereof include an enzyme, a fluorescent substance, a ligand, a luminescent material, a microparticle, a redox molecule, Isotopes and the like can be used. Examples of the enzymes include? -Glucuronidase,? -D-glucosidase, urease, peroxidase (such as hosadradia peroxidase), alkaline phosphatase, acetylcholinesterase, glycosidoxidase, Dehydrogenase, glucose-6-phosphate dehydrogenase, invertase and the like can be used. Examples of the fluorescent substance include fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, fluorine synthase, and the like. Examples of the ligand include biotin derivatives, and examples of the luminescent material include acridinium ester, luciferin, and luciferase. Examples of the fine particles include colloidal gold and colored latex. Examples of redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone and the like. Radioactive isotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I and ¹⁸⁶ Re. However, in addition to those exemplified above, any of them can be used as long as they can be used for immunological assays.

In another example of the present invention, a method for providing information necessary for diagnosis to cancer of the present invention can be performed in the following steps.

(a) contacting the radioactive isotope labeled CD55 target antibody or antigen-binding fragment thereof of claim 1 with a biological sample isolated from a subject suspected of having cancer;

(b) measuring the level of expression of a radioactive isotope labeled CD55 target antibody or antigen-binding fragment thereof CD55 protein in said biological sample through antigen-antibody complex formation; And

(c) comparing the expression level of the CD55 protein measured in the step (b) with the control group, and determining that the expression level of the protein is higher than that of the control group as a cancer patient. The cancer may be selected from the group consisting of melanoma, lymphoma, lung, thyroid, breast, large intestine, prostate, head and neck, stomach, liver, bladder, kidney, cervix, pancreas, leukemia, bone marrow, ovary, uterus, sarcoma, , And most preferably, it may be lung cancer.

In the present invention, the term "isolated biological sample" refers to a tissue sample (cancer tissue), cancer cells, plasma, serum, blood, saliva, synovial fluid, urine, sputum, Lymphocytes, cerebrospinal fluid, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatant, or ruptured eukaryotic cells and includes samples derived from metastatic lesions as well as primary lesions of cancer. These biological samples may be manipulated or manipulated to react with the monoclonal antibody labeled with the radionuclide element of the present invention to confirm the expression level of the CD55 protein.

The term "individual" in the present invention includes mammals, birds, and the like, including cows, pigs, sheep, chickens, dogs,

In another embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a CD55 target antibody or an antigen-binding fragment thereof labeled with a radioisotope may be provided. The cancer may be selected from the group consisting of melanoma, lymphoma, lung, thyroid, breast, large intestine, prostate, head and neck, stomach, liver, bladder, kidney, cervix, pancreas, leukemia, bone marrow, ovary, uterus, sarcoma, , And most preferably, it may be lung cancer.

The pharmaceutical composition of the present invention may be an antibody having an effect of inhibiting apoptosis induction or metastatic growth of cancer cells.

In the present invention, the term "prevention" means any action that inhibits or delays the onset of cancer by the administration of the composition. "Treatment" refers to any action that improves or alters the symptoms of cancer by administration of the composition. it means.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier that is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added and formulated into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The pharmaceutical composition may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories It can have one formulation.

The radioactive isotope-labeled CD55 target antibody or antigen-binding fragment thereof may be used together with other anticancer agents.

The other anticancer agent may be any one or more selected from the group consisting of Avastin, Erbitux, Opdivo, cisplatin, Herceptin, Mabthera, and Rituxan. And most preferably cisplatin.

A health functional food composition for preventing or ameliorating cancer comprising a radioactive isotope-labeled CD55 target antibody or an antigen-binding fragment thereof as an active ingredient. The cancer may be selected from the group consisting of melanoma, lymphoma, lung, thyroid, breast, large intestine, prostate, head and neck, stomach, liver, bladder, kidney, cervix, pancreas, leukemia, bone marrow, ovary, uterus, sarcoma, , And most preferably, it may be lung cancer.

Yet another embodiment of the present invention can provide a method for producing a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD55 described above.

The step of generating a conjugate by binding a chelator with a monoclonal antibody or an antigen-binding fragment thereof specifically binding to the CD55, the step of contacting the CD55 with the monoclonal antibody or its antigen-binding fragment and the chelator Can be from 5: 1 to 500: 1, preferably from 25: 1 to 250: 1, and most preferably 50: 1. In addition, in the step of binding the CD55-binding monoclonal antibody or its antigen-binding fragment to the chelator to produce a conjugate, the pH may be from pH 5.0 to pH 11.5 using sodium bicarbonate buffer, pH 6.5 to pH 9.0, and most preferably pH 8.0 to pH 8.5. Further, the temperature at which the metal chelator and the antibody are added and reacted may be 27 캜 to 47 캜, preferably 32 캜 to 42 캜, and most preferably 37 캜. The time for the metal chelator to react with the antibody can be from 1 hour to 5 hours, and most preferably 3 hours. The conjugate may be DTPA-anti CD55 Ab.

In the step of binding the conjugate with the radioactive isotope, the ratio of the conjugate to the radioactive isotope may be 20 mCi per 1 mg of the conjugate. In the step of binding the conjugate to the radioisotope, the pH may be from pH 2.0 to pH 9.0 using 0.1 M ammonium acetate buffer, preferably from pH 3.5 to pH 7.5, and most preferably, pH 5.0 to pH 6.0. Metal chelator-Decay-accelerating factor (CD55 or DAF) The temperature at which the target antibody and the radioactive isotope are reacted may be room temperature. The time for reacting with the metal chelator-decay-accelerating factor (CD55 or DAF) target antibody and the radioisotope may be from 1 minute to 60 minutes, preferably from 1 minute to 30 minutes, and most preferably from 10 minutes .

The radioactive isotope labeled CD55 target antibody and the antigen-binding fragment thereof can be taken after the fasting for 1 hour to 100 hours, preferably 12 to 60 hours, most preferably 24 hours to 48 hours .

Example 1 Expression of CD55 in Lung Cancer Tissue and Lung Cancer Cells

40 lung cancer patients and 10 general patient tissues were immunochemically stained for quantification. Immunochemical staining analyzes were performed using the services provided by the SuperBioChips laboratory. Anti-CD55 polyclonal anti-body (AP14798A; Abgent) diluted 1: 200 was used for immunochemical staining analysis. Tissue array slices from various lung cancer patients were immunostained and photographed by digital equipment. Blind and quantification of immunochemical staining was performed by a pathologist. As shown in Fig. 1A, CD55 was expressed in various lung cancer tissues, but it was found to be hardly expressed in normal lung tissues. Furthermore, the types of lung cancer are classified into non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC) and metastasis. 1A, expression of CD55 is particularly highly expressed in non-small cell lung cancer (76.47%).

 Lung cancer tissues from various lung cancer patients were immunochemically stained by the same method. 1b shows, in order: i) normal lungs; Ii) squamous cell carcinoma; Iii) from lung cancer to lymph node metastasis; Iv) Mucinous epidermoid carcinoma; V) pulmonary squamous cells; Vi) abolished cancer; Ⅶ) Hodg cell cancer; And ⅷ) lung bronchial cancer. As shown in FIG. 1B, unlike the normal lung, high expression of CD55 was confirmed in various types of non-small cell lung cancer.

Immunoblotting was performed to examine the expression of CD55 in lung cancer cells. The lung cancer cell line used was H460 (CD55-positive), bronchogenic carcinoma H358, and small-cell lung carcinoma H69 (CD55-negative) from pleural fluid. In addition, WI-38 was used as normal lung cells. (ATCC CCL-75) cells were maintained in RPMI-1640 medium supplemented with 10% FBS and maintained in RPMI-1640 medium supplemented with 10% FBS. And maintained in DMEM containing FBS. In the Western blot, CD55 anti-body was obtained from Abcam and tubulin was obtained from Santa Cruz Biotechnology. As a result, the expression of CD55 was found to be very high in H460, which is a non-small cell lung cancer cell line, and somewhat higher, in H358 (FIG. 2).

Example 2. Expression and purification of decay-accelerating factor (CD55 or DAF) target antibody (4-1H)

Vectors of the variable and constant regions of the heavy and light chains from pComb3x containing scFv of 4-1H were obtained by PCR using the primer combinations in Table 3 below.

The variable and constant regions of the antibodies were obtained by PCR using the HC and LC primer combinations in Table 3, respectively, and the pcDNA3.3TM-TOPO.TTA cloning kit and the pOPTIVECTM-TOPO.TTA cloning kit (Invitrogen, USA ) Into a mammalian cell expression plasmid. 1 μl of each vector (pcDNA3.3TM-TOPO® vector and pOPTIVECTM-TOPO® vector) and a slice were added to a buffer solution containing 200 mM NaCl and 10 mM MgCl2 in a total volume of 6 μl, and reacted at room temperature for 5 minutes. DH 5α competent cells were transformed with heat shock to obtain colonies, followed by mass culture to obtain plasmids.

The plasmids prepared above were used to transfect HEK293F cells (Invitrogen, USA) and incubated for 7 days. The antibodies obtained were purified using protein A column (GE, USA). The culture was loaded on the column, and the antibody (IgG) in the culture broth was bound to Protein A and eluted with 20 mM sodium citrate buffer (pH 3.0).

primer order SEQ ID NO: V _H Forward GCT AGC CGC CAC CAT GGG CTG GTC CTG CAT CAT CCT GTT CCT GGT GGC CAC CGC CAC CGG CGC CGT GAC GTT GGA CGA GTC CGG G 9 Reverse GGG CCC TTG GTG GAG GCG GAG GAG ACG ATG ACT TCG GTC CC 10 C _H Forward GCC TCC ACC AAG GGC CCC TC 11 Reverse CGG GAT CCC TTG CCG GCC GT 12 HC Forward GCT AGC CGC CAC CAT GGG 13 Reverse GGA TCC CTT GCC GGC CGT CGC ACT CAC TT 14 V _L Forward AAG CTT GCC GCC ACC ATG GGC TGG TCC TGC ATC ATC CTG TTC CTG GTG GCC ACC GCC ACC GCC CTCC ACT CAG CCG TCC TCG GTG 15 Reverse GAG GGG GCG GCC ACG GTC CGT AGG ACG GTC AGG GTT GTC CCG GC 16 C _k Forward CGG ACC GTG GCC GCC CCC TC 17 Reverse GCT CTA GAC TAG CAC TCG C 18 LC
Forward AAG CTT GCC GCC ACC ATG 19 Reverse TCT AGA CTA GCA CTC GCC 20

Example 3 Evaluation of Binding Capacity of Decay-Accelerating Factor (CD55 or DAF) Target Antibody (4-1H) to Lung Cancer Cell Lines

 The binding capacity of decay-accelerating factor (CD55 or DAF) target antibody (4-1H) and lung cancer cell line was determined by flow cytometry (FACS). The lung cancer cell line used was H460 of large cell lung cancer and H69 of small cell lung cancer from pleural fluid. The H460 cells (ATCC HTB-177) and H69 (ATCC HTB-119) cells were maintained in RPMI-1640 medium supplemented with 10% FBS. Cells were stained with Alexa Fluor 647 (A-20186; Molecular Probes) -conjugated anti-CD55 antibody or isotype human control IgG (I4506; Sigma; blue histograms) II. As a result, the H460 cell line expressing high levels of decay-accelerating factor (CD55 or DAF) showed a very high level of binding force (FIG. 3).

Example 4. Production of radioactive isotope labeled CD55 antibody

was prepared in a molar ratio of [50: 1] of [p-SCN-Bn-CHX-A "DTPA: CD55 target antibody (4-1H)] in bicarbonate buffer at pH 8.0- Lt; 0 > C for 3 hours. The reaction product was purified using Amicon Ultracel-50K (millipore) to prepare DTPA-anti CD55 Ab. DTPA-anti-CD55 Ab was prepared by reacting 10 mCi of radioactive isotope Lu-177 per 0.1 mg ammonium acetate buffer at pH 5.0-pH 6.0 at room temperature for 10 minutes. The reaction results were evaluated by TLC-SG using 10 Mm sodium citrate buffer as a developing solvent, and a radioactive compound having a radiochemical purity of 95% or more was prepared and used. The prepared radioactive compound also maintained a radiochemical purity of 90% or more for 7 days in physiological saline or 37 ° C serum (FIG. 4).

Example 5. Evaluation of immunoreactivity of H460 of lung cancer cells with Lu-177-DTPA-anti CD55 Ab using Lindmo assay.

The immunoreactivity of Lu-177-DTPA-anti CD55 Ab to H460 was determined by the Lindmo assay. 0.074 MBq of Lu-177-DTPA-anti CD55 Ab was added to the H460 cells (ATCC HTB-177) at various concentrations (0 to 6.0 × 10 ⁶ cells) and mixed well using a rotation mixer at room temperature. After 1 hour, the cells were washed three times with RPMI-1640 medium supplemented with 10% FBS and the remaining counts per minute were analyzed using a Wallac 1470 automated gamma counter (PerkinElmer Life Science). When the immunoreactivity was evaluated using the Lindmo assay, it was confirmed that the immunoreactivity was maintained at 78.9 ± 5.4% (FIG. 5).

Example 6. Evaluation of Binding Capacity for Lu-177-DTPA-anti CD55 Ab and H460 in Lung Cancer Cells

Binding capacity of Lu-177-DTPA-anti CD55 Ab and H460 of lung cancer cells was determined by saturation binding assay. (0 - 25 nmol / L) of Lu-177-DTPA-anti CD55 Ab was added to 3 × 10 ⁶ of the H460 cells (ATCC HTB-177) and mixed well using a rotation mixer at room temperature. After 1 hour, the cells were washed three times with RPMI-1640 medium supplemented with 10% FBS and the remaining counts per minute were analyzed using a Wallac 1470 automated gamma counter (PerkinElmer Life Science). The Kd value was 7.149 ± 5.144 nmol / L and the Bmax value was 30 ± 7.218 fmol / mg when the binding force of Lu-177-DTPA-anti CD55 Ab to H460 lung cancer cells was evaluated using the saturation binding assay (Fig. 6. This shows the specific binding activity of Lu-177-DTPA-anti CD55 Ab with lung cancer cells.

Example 7. Evaluation of the binding force between Lu-177-DTPA-anti CD55 Ab and various kinds of lung cancer cells

The binding of Lu-177-DTPA-anti CD55 Ab to various types of lung cancer cells was performed by binding assay. The lung cancer cell line used was H460 (CD55 ^high ), bronchogenic carcinoma H3589 (CD55 ^normal ) and H69 (CD55 ^low ) of large cell lung cancer from pleural fluid. In addition, WI-38 (CD55 ^low ) was used as a normal lung cancer cell. The cells were maintained in RPMI-1640 medium supplemented with H460 cells (ATCC HTB-177), H358 (ATCC CRL5807), H69 (ATCC HTB-119) and 10% FBS, And maintained in DMEM containing FBS. After loading 0.074 MBq of Lu-177-DTPA-anti CD55 Ab into 1 × 10 ⁶ of the above H460, H358, H69 and WI-38 cells, a sample with 50 fold unlabeled anti CD55 Ab Mix well using a mixer. After 1 hour, the cells were washed 3 times with RPMI-1640 medium supplemented with 10% FBS, and then the Wallac 1470 automated gamma counter (PerkinElmer Life Science). Was used to analyze the remaining counts per minute in the sample. binding activity of Lu-177-DTPA-anti CD55 Ab to various types of lung cancer cells was assessed by binding assay, and it was confirmed to bind to H460 cells at the highest level (FIG. 7).

Example 8. Internalization of Lu-177-DTPA-anti CD55 Ab and Lung Cancer Cells and Evaluation of Exogenous Kinetics

 The internalization kinetics of Lu-177-DTPA-anti CD55 Ab and lung cancer cells were performed in the following manner. H460 was used for the lung cancer cell line from large-cell lung cancer, and the H460 cells (ATCC HTB-177) were cultured in a 6-well plate in RPMI-1640 medium supplemented with 10% FBS. Lu-177-DTPA-anti CD55 Ab of 25,000 CPM (counts per minute) was incubated in the wells and incubated at 0, 15, 30, 60, 90, Ab were assayed for counts per minute using a Wallac 1470 automated gamma counter (PerkinElmer Life Science). The results are calculated as percentage (%) of internalized radioactive / total cells. As a result, the rate of internalization of Lu-177-DTPA-anti CD55 Ab and lung cancer cells was about 33.5% of the total cell-related activity in 30 minutes (Fig. 8a)

The ex vivo kinetics of Lu-177-DTPA-anti CD55 Ab and lung cancer cells were performed in the following manner. Exogenous products were prepared by adding 25,000 CPM (counts per minute) of Lu-177-DTPA-anti CD55 Ab in RPMI-1640 medium supplemented with 10% FBS in H360 cells (ATCC HTB-177) For a period of time. The radioactivity on the cell surface was removed with pH 2.5 acetic acid / saline and the exogenous radioactivity was measured using a Wallac 1470 automated gamma counter (PerkinElmer Life Science) at 0, 15, 30, 60, 90, The minutes per minute was analyzed. The above results are calculated as percentage (%) of exogenous radioactive / total cells. As a result, the excretion rate of Lu-177-DTPA-anti CD55 Ab and lung cancer cells was 74.6% after 2 hours. This shows that the internalized Lu-177-DTPA-anti CD55 Ab slowly released (Figure 8b)

Example 9. Anatomical results and evaluation of expression of CD55 in an animal model of lung cancer metastasis in thoracic cavity

To induce a malignant pleural fluid (MPE) mouse model, 1 × 10 ⁷ H460 cells were suspended in 200 μl of phosphate-buffered saline and injected into the chest cavity of male balb / c nude mice and cultured for 10 days. Metastatic cancer developed 10 days later. As shown in FIGS. 9a and 9b, the pleural metastasized tumor infiltrated the lateral lung, chest wall or bone, and immunohistochemistry showed positive expression of CD55 as in 9c.

Example 10. In vivo distribution of Lu-177-DTPA-anti CD55 Ab in an animal model of lung cancer metastasis in thoracic cavity

FIG. 10A is a table showing the in vivo distribution of Lu-177-DTPA-anti CD55 Ab in a model of lung cancer metastasis in a thoracic cavity, and FIG. 10B is a graph summarized in a graph. To induce a malignant pleural fluid (MPE) mouse model, 1 × 10 ⁷ H460 cells were suspended in 200 μl of phosphate-buffered saline and injected into the thoracic cavity of male balb / c nude mice and cultured for 10 days. Animal models of metastatic lung cancer were prepared. Lu-177-DTPA-anti CD55 Ab was administered to the chest cavity of the prepared animal model. The in vivo distribution was measured at 1, 6, 24, 72, 120, and 168 hours after injection. At each time point, rats were sacrificed and the relevant organs, tissues and blood removed, quantified, and the remaining minute counts of the samples were analyzed using a Wallac 1470 automated gamma counter (PerkinElmer Life Science). The results of evaluating the body distribution are shown in Fig. 10B. The rate of tumor uptake was 15.92 ± 8.75% ID / g at 1 hour after administration and 8.70 ± 0.20% ID / g at 7 days, confirming the target efficiency of very high lung cancer (chest wall and metastatic lung cancer). As shown in Fig. 11, the total residual radioactivity in the mice gradually decreased.

Example 11. Tumor uptake rate of Lu-177-DTPA-anti CD55 Ab according to fasting time before drug administration

To induce a malignant pleural fluid (MPE) mouse model, 1 × 10 ⁷ H460 cells were suspended in 200 μl of phosphate-buffered saline and injected into the thoracic cavity of male balb / c nude mice and cultured for 10 days. Animal models of metastatic lung cancer were prepared. The animals were fasted at 0 hr, 12 hr and 24 hr, and then Lu-177-DTPA-anti CD55 Ab was administered to the thoracic cavity. The tumor uptake rate of Lu-177-DTPA-anti CD55 Ab was then measured. Interestingly, the uptake of Lu-177-DTPA-anti CD55 Ab was stimulated up to 10.47 fold in Kia (Fig. 12). These results suggest that fasting may enhance the tumor lethal effects of Lu-177-DTPA-anti CD55 Ab, considering that the efficacy of radiopharmaceuticals is proportional to absorption.

Example 12. SPECT / CT image of Lu-177-DTPA-anti CD55 Ab in a model of lung cancer metastasis

To induce a malignant pleural fluid (MPE) mouse model, 1 × 10 ⁷ H460 cells were suspended in 200 μl of phosphate-buffered saline and injected into the thoracic cavity of male balb / c nude mice and cultured for 10 days. Animal models of metastatic lung cancer were prepared. The animal model chest cavity was treated with Lu-177-DTPA-anti CD55 Ab. The animals were injected with 177Lu-anti-CD55 antibody (7.4 MBq) directly into the thoracic cavity and photographed with a NanoSPECT / CT system for 3 hours and 24 hours. Images were obtained with PMOD v3.6 software and Bioscan in vivo Scope software. As shown in Fig. 13, it was possible to effectively diagnose the lung cancer portion transferred into the chest wall and the abortion.

Example 13. Therapeutic effect of Lu-177-DTPA-anti CD55 Ab on lung cancer cells

The therapeutic effect of Lu-177-DTPA-anti CD55 Ab on lung cancer cells was evaluated by MTT assay. The H460 cells (ATCC HTB-177) and H358 (ATCC CRL5807) were maintained in RPMI-1640 medium supplemented with 10% FBS. H460 and H358 3 × 10 ^Four cancer cells were plated in 96-well plates and incubated with various concentrations of Lu-177-DTPA-anti CD55 Ab or unlabeled anti-CD55 Ab and human complement system (Sigma, Cat #: S1764) was added and cultured at 37 ° C / 5% CO ₂ for 24 hours. After 24 hours, 10 νl of Cell Counting Kit-8 (CCK-8, DOJINDO, Cat #: CK04-11) solution was added to the plate and reacted at 37 ℃ / 5% CO ₂ incubator for 1 hour in dark condition Next, the cancer cell killing effect was observed by measuring absorbance at 450 nm in a microplate reader. When the therapeutic effect of Lu-177-DTPA-anti CD55 Ab was evaluated in cells using MTT assay, H460 cancer cells were killed with a small amount of radioactive (0.07 MBq). In addition, the CD55 specific neutralizing antibody (IgG) without radioactivity did not affect the survival rate of H358, whereas the anti-CD55 Ab inhibited the survival of H358 cells (Fig. 14).

Example 14 Evaluation of in vivo lung cancer (malignant pleural effusion) treatment of Lu-177-DTPA-anti CD55 Ab

15A is a graph showing the survival rate of in vivo lung cancer (malignant pleural effusion) of Lu-177-DTPA-anti CD55 Ab. Fig. 15B shows the weight change of in vivo lung cancer (malignant pleural effusion) of Lu-177-DTPA-anti CD55 Ab. H460 lung cancer cells were injected into the thoracic cavity of 6-week-old nude mice to prepare a malignant pleural fluid animal model. 200 μCi Lu-177-DTPA-anti CD55 Ab was intraperitoneally injected into the thoracic cavity to treat malignant pleural effusion Respectively. As a result, the treatment effect of Lu-177-DTPA-anti CD55 Ab was higher than that of monoclonal antibody, and the median survival time was extended 2.1 times as compared with the control group. In addition, the weight was also increased when compared with the control group.

Example 15. Evaluation of Expression of CD55 in Squamous Cell Cancer.

16 is a view showing CD55 in squamous cell carcinoma through immunochemical staining. Immunochemical staining analyzes were performed using the services provided by the SuperBioChips laboratory. Anti-CD55 polyclonal anti-body (AP14798A; Abgent) diluted 1: 200 was used for immunochemical staining analysis. Tissue array slices from squamous cell carcinoma patients were immunostained and photographed by digital equipment. As a result, high expression of CD55 was confirmed in the front part of squamous cell carcinoma (Fig. 16). Therefore, it was confirmed that the infiltration and migration of Lu-177-DTPA-anti CD55 Ab were dose-dependently treated with lung cancer cells, as shown in FIG.

Example 16. Therapeutic effect of Lu-177-DTPA-anti CD55 Ab and cisplatin for lung cancer in vitro

FIG. 18 is a chart showing the therapeutic effect of Lu-177-DTPA-anti CD55 Ab and cisplatin for lung cancer in vitro. The therapeutic effect of Lu-177-DTPA-anti CD55 Ab and cisplatin in lung cancer cells was evaluated by MTT assay. The H460 cells (ATCC HTB-177) and H358 (ATCC CRL5807) were maintained in RPMI-1640 medium supplemented with 10% FBS. H460 and H358 3 × 10 ⁴ cancer cells were dispensed into 96-well plates, and 0.37 MBq of Lu-177-DTPA-anti CD55 Ab or 3 νM cisplatin and human complement system (Sigma, Cat #: S1764) at 37 ℃ / 5% CO ₂ and incubated for 24 hours. After 24 hours, 10 νl of Cell Counting Kit-8 (CCK-8, DOJINDO, Cat #: CK04-11) solution was added to the plate and reacted at 37 ℃ / 5% CO ₂ incubator for 1 hour in dark condition Next, the cancer cell killing effect was observed by measuring absorbance at 450 nm in a microplate reader. The efficacy of Lu-177-DTPA-anti CD55 Ab in lung cancer was similar or higher than that of cisplatin when MTT assay was used to evaluate the therapeutic effects of Lu-177-DTPA-anti CD55 Ab and cisplastin in lung cancer. The therapeutic effect was greatly improved when the combination was administered.

Example 17. Therapeutic effect of Lu-177-DTPA-anti CD55 Ab and cisplatin for lung cancer in vivo

Figure 19 is a graph showing the survival rate of 15Lu-177-DTPA-anti CD55 Ab and cisplatin in vivo lung cancer. H460 lung cancer cells were injected into the thoracic cavity of 6-week-old nude mice and a malignant pleural fluid animal model was prepared. 200 μCi Lu-177-DTPA-anti CD55 Ab and cisplatin were intraperitoneally injected into the thoracic cavity to treat metastatic malignant pleural effusion The effects were evaluated. As a result, the survival rate of Lu-177-DTPA-anti CD55 Ab and cisplatin was increased by 32%.

<110> Korea Atomic Research Institute <120> Radiolabeled anti-CD55 antibody for the diagnosis and therapy of          cancer and use thereof <130> 1061295 <160> 20 <170> KoPatentin 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of a light chain variable region of 4-1H <400> 1 Ser Gly Gly Gly Gly Ser Tyr Gly   1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of a light chain variable region of 4-1H <400> 2 Trp Asn Asp Lys Arg Pro Ser   1 5 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of a light chain variable region of 4-1H <400> 3 Gly Gly Trp Asp Ser Ser Thr Tyr Ala Ile   1 5 10 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of a heavy chain variable region of 4-1H <400> 4 Asp Arg Gly Met Ala   1 5 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of a heavy chain variable region of 4-1H <400> 5 Gly Ile Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly     <210> 6 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of a heavy chain variable region of 4-1H <400> 6 Asn Aly Gly Aly Gly Gly Trp His Ala Ala Tyr Ile Asp Ala   1 5 10 <210> 7 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> a light chain variable region of 4-1H <400> 7 Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Leu Gly Gly Thr Val   1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Gly Ser Tyr Gly Trp Phe Gln Gln              20 25 30 Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Trp Asn Asp Lys          35 40 45 Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser      50 55 60 Thr Ala Thr I Thr Ile Thr Gly Val Gln Ala Glu Asp Glu Ala Val  65 70 75 80 Tyr Tyr Cys Gly Gly Trp Asp Ser Ser Thr Tyr Ala Ile Phe Gly Ala                  85 90 95 Gly Thr Thr Leu Thr Val Leu             100 <210> 8 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> a heavy chain variable region of 4-1H <400> 8 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly   1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Ser Phe Ser Asp Arg              20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Gly Ile Ser Ser Ser Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val      50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg  65 70 75 80 Met Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys                  85 90 95 Ala Lys Asn Ala Gly Ala Gly Gly Trp His Ala Ala Tyr Ile Asp Ala             100 105 110 Trp Gly His Gly Thr Glu Val Ile Val Ser Ser         115 120 <210> 9 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Foward primer for amplification for a heavy chain variable          region <400> 9 gctagccgcc accatgggct ggtcctgcat catcctgttc ctggtggcca ccgccaccgg 60 cgccgtgacg ttggacgagt ccggg 85 <210> 10 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for the amplification for a heavy chain variable          region <400> 10 gggcccttgg tggaggcgga ggagacgatg acttcggtcc c 41 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for the amplification for a heavy chain constant          region <400> 11 gcctccacca agggcccctc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for the amplification for a heavy chain constant          region <400> 12 cgggatccct tgccggccgt 20 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Foward primer for amplification for a heavy chain variable          region and constant region <400> 13 gctagccgcc accatggg 18 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for the amplification for a heavy chain variable          region and constant region <400> 14 ggatcccttg ccggccgtcg cactcactt 29 <210> 15 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplification for a light chain variable          region <400> 15 aagcttgccg ccaccatggg ctggtcctgc atcatcctgt tcctggtggc caccgccacc 60 ggcgccctga ctcagccgtc ctcggtg 87 <210> 16 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for the amplification for a light chain variable          region <400> 16 gagggggcgg ccacggtccg taggacggtc agggttgtcc cggc 44 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplification for a constant region of          kappa light chain <400> 17 cggaccgtgg ccgccccctc 20 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for the amplification for a constant region of          kappa light chain <400> 18 gctctagact agcactcgc 19 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplification for a light chain variable          region and constant region of kappa <400> 19 aagcttgccg ccaccatg 18 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for the amplification for a light chain variable          region and constant region of kappa <400> 20 tctagactag cactcgcc 18

Claims (20)

A radioactive isotope labeled CD55 target antibody or antigen-binding fragment thereof,
[Formula 1]
Radioisotope-R1-R2
In this formula,
R1 is a chelator;
R2 is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD55. 2. The antibody of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof specifically binding to CD55 comprises light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, A heavy chain variable region comprising a light chain CDR3 comprising the sequence of SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5 and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6 Region, wherein the CD55 target antibody or antigen-binding fragment thereof is a radioactive isotope labeled antibody. 7. The antibody of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD55 comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: A radioactive isotope labeled CD55 target antibody or antigen binding fragment thereof. The radioactive isotope labeled CD55 target antibody or antigen binding fragment thereof according to claim 1, wherein the chelator is a chelator capable of binding to a trivalent metal. The method of claim 1, wherein the chelator is selected from the group consisting of a DTPA chelator, a NOTA chelator, a DOTA chelator, a DFO chelator, a NODAGA chelator, an N2S2 chelator, a TETA chelator, and a HYNIC chelator A radioactive isotope-labeled CD55 target antibody or an antigen-binding fragment thereof. The method of claim 1, wherein the chelator is selected from the group consisting of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane- tetraacetic acid), DFO (3- [6,17-dihydroxy-7,10,18,21-tetraoxo-27- [N-acetylhydroxylamino) -6,11,17,22- tetraazaheptaeicosane] thiourea), DTPA (diethylenetriaminepentaacetic acid ), N2S2 (diaminedithiol), p-SCN-Bn-NOTA (2- (4'- isothiocyanatobenzyl) -1,4,7- triazacyclononane- 1,4,7- triacetic acid), NODAGA (1,4,7- triazacyclononane, 1-glutaric acid-4,7-acetic acid), p-SCN-Bn-DOTA (2- (4'-isothiocyanatobenzyl) -1,4,7,10-tetraazacyclododecane- tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), p-SCN-Bn-DTPA (2- (4-isothiocyanatobenzyl) -diethylenetriaminepentaacetic acid) -Bn-DFO (1- (4-Isothiocyanatophenyl) -3- [6,17-dihydroxy-7,10,18,21-tetraoxo-27- [Nacetylhydroxylamino] -6,11,17,22- tetraazaheptaeicosane] thiourea) (p-SCN-Bn-CHX-A'-DTPA ([(R) -2-Amino-3- (4-isothiocyanatophenyl) propyl] -trans- (S, S) -cyclohexane- pentaacetic a cid) and HYNIC (hyrazinonicotinic acid), wherein the radioactive isotope-labeled CD55 target antibody or antigen-binding fragment thereof is at least one selected from the group consisting of: The method of claim 1, wherein the monoclonal antibody or the antigen-binding fragment thereof specifically binding to the CD55 is in a molar ratio of the chelator to the CD55 of from 5: 1 to 500: 1, the radioactive isotope labeled CD55 target antibody, snippet. The method of claim 1, wherein the isotope is selected from the group consisting of ¹⁷⁷ Lu, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ¹¹¹ In, ¹⁰⁵ Rh, ¹⁷⁶ Yb, ¹⁶⁹ Gd, ¹¹¹ Ag, ¹²⁵ I, ¹²⁹ I and ¹⁶⁶ Ho A CD55 target antibody or an antigen-binding fragment thereof, which is labeled with a radioactive isotope. The radioactive isotope-labeled CD55 target antibody and the antigen-binding fragment thereof according to claim 1, wherein the ratio of the radioisotope to the R1-R2 is 1 to 100 mCi per 1 mg of the R1-R2. 10. A cancer diagnostic kit comprising a CD55 target antibody or antigen-binding fragment thereof labeled with a radioisotope of any one of claims 1 to 9. The method of claim 10, wherein the cancer is selected from the group consisting of melanoma, lymphoma, lung, thyroid, breast, large intestine, prostate, head and neck, liver, bladder, kidney, cervix, pancreas, leukemia, bone marrow, ovary, uterus, And endometrial cell carcinoma. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; (a) contacting a radioactive isotope labeled CD55 target antibody or an antigen-binding fragment thereof of any one of claims 1 to 9 with a biological sample isolated from a subject suspected of having cancer;
(b) measuring the level of expression of a radioactive isotope labeled CD55 target antibody or antigen-binding fragment thereof CD55 protein in said biological sample through antigen-antibody complex formation; And
(c) comparing the expression level of the CD55 protein measured in the step (b) with the control group, and determining that the expression level of the protein is higher than that of the control group as a cancer patient;
The method comprising the steps of: The method of claim 12, wherein the cancer is selected from the group consisting of melanoma, lymphoma, lung, thyroid, breast, large intestine, prostate, head and neck, liver, bladder, kidney, cervix, pancreas, leukemia, bone marrow, ovary, uterus, And endometrial cell carcinomas. The method according to any one of claims 1 to 3, A pharmaceutical composition for preventing or treating cancer, which comprises the CD55 target antibody or antigen-binding fragment thereof of any one of claims 1 to 9 as an active ingredient. The method of claim 14, wherein the cancer is selected from the group consisting of melanoma, lymphoma, lung, thyroid, breast, large intestine, prostate, head and neck, liver, bladder, kidney, cervix, pancreas, leukemia, bone marrow, ovary, uterus, And endometrial carcinoma. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; 15. The pharmaceutical composition for preventing or treating cancer according to claim 14, wherein the pharmaceutical composition has an effect of inhibiting apoptosis induction or metastatic growth of cancer cells. The pharmaceutical composition for preventing or treating cancer according to claim 14, wherein the pharmaceutical composition is used together with other anticancer agents. 18. The method of claim 17, wherein the other anti-cancer agent is selected from the group consisting of Avastin, Erbitux, Opdivo, cisplatin, Herceptin, Mabthera, and Rituxan. Or a pharmaceutically acceptable salt thereof. A health functional food composition for preventing or ameliorating cancer, which comprises the CD55 target antibody or antigen-binding fragment thereof of any one of claims 1 to 9 as an active ingredient. The method of claim 19, wherein the cancer is selected from the group consisting of melanoma, lymphoma, lung, thyroid, breast, large intestine, prostate, head and neck, stomach, bladder, kidney, cervix, pancreas, leukemia, bone marrow, ovary, uterus, And endometrial cell carcinoma, wherein the composition is at least one selected from the group consisting of:

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