KR20150063859A - Antibody that recognizes the ICAM4 on erythroleukemia and use of the antibody - Google Patents

Abstract

The present invention relates an antibody specifically coupled to intercellular adhesion molecule 4 (ICAM4) and use of the antibody, and more specifically to: a monoclonal antibody specifically coupled to ICAM4; a diagnostic composition of erythroleukemia containing the antibody; a diagnostic kit; a method for detecting erythroleukemia markers using the antibody; a method for providing information on diagnosis of erythroleukemia using the antibody; a composition for treating erythroleukemia containing the antibody; and a hybridoma cell for producing the antibody. A monoclonal antibody according to the present invention not only demonstrates superior specificity and affinity to ICAM4 expressed in erythroleukemic cells to be employed usefully in diagnosis of erythroleukemia, but also gives rise to antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular Phagocytosis (ADCP) to be employed usefully in treatment of erythroleukemia.

Description

The present invention relates to antibodies that recognize ICAM4 and to the use thereof for the production of ICAM4 in erythroleukemic cells,

The present invention relates to antibodies that specifically bind to ICAM4 (intercellular adhesion molecule 4) and uses thereof, and more particularly to monoclonal antibodies that specifically bind to ICAM4; A composition for diagnosing an autoimmune leukemia comprising the same; Diagnostic kit; A method for detecting an autoimmune leukemia marker using the same; A method of providing information for the diagnosis of leukemia using the same; A composition for treating an autoimmune leukemia comprising the same; And a hybridoma cell producing the same.

ICAM-4 is known as CD242 and is known as the Intercellular Adhesion Molecule (ICAM) protein family and the LW blood group antigen (Daniels, Geoff: Human Blood Groups (2nd ed.). Malden Mass.: Blackwell Blackwell, ISBN 978-0-3232-05646-0, 2002.], [Klein, Harvery G .; Mollison, PL; Asnstee, David J .: Mollison's Blood Transfusion in Clinical Medicine (11th ed. -0-632-06454-0, 2005).

ICAM4 is a 40-42 kDa glycoprotein with sialic acid residues expressed on human erythrocyte membranes. It was first known as LW blood type antigen by Landsteiner and Weiner in 1940, but recently ICAM4 has been known to be similar to ICAM It was named. ICAM4 is also strongly associated with Rh blood type, which is reported to be significantly higher in Rh + blood type RBC than in Rh-type blood (Cartron, J.-P.: Defining the Rh blood group antigens. Biochemistry and molecular genetics. 8: 199-212, 1994).

In detail, the ICAM4 structure consists of two domains, both of which have CD11a / CD18 (LFA-1) and CD11b / CD18 (Mac-1) integrin binding sites. Recently, studies on the interaction between ICAM-4 and various integrins using amino acid deletion mutants of each domain have been carried out.

ICAM4 has a selective elimination function of aging red blood cells, and ICAM-4 of sickle cell red blood cells has been reported to be phosphorylated differently from normal red blood cells (Ihanus, E .; Uotila, LM; Toivanen, A.; ; Gahmberg, CG: Red-cell ICAM-4 is a ligand for the monocyte / macrophage integrin CD11c / CD18: characterization of the binding sites on ICAM-4. Blood 109; 802810, 2007] [Anne Toivanen, Eveliina Ihanus, Minna Mattila; Hans U. Lutz; Carl G. Gahmberg: Importance of molecular studies on major blood groups Intercellular adhesion molecule-4, a blood group antigen involved in multiple cellular interactions. Biochimica et Biophysica Acta 1780; 456466, 2008).

On the other hand, erythroid leukemia is a rare form of acute myelogenous leukemia with prominent proliferation (> 50%) of erythroid system, accounting for 5% of total acute myelogenous leukemia. Currently, the World Health Organization (WHO) has developed two erythroleukemia, erythroblast and proerythroblast pure erythroid leukemia in the presence of immature erythrocytes and myelosuppressive cells (pure erythroid leukemia) subtype). Most patients are diagnosed at age 50 or older and are reported to have a slightly higher incidence in men. It is known that the prognosis is worse among acute myelogenous leukemia, with a long - term survival rate of less than 10%, accompanied by many chromosomal abnormalities of leukemia cells. There is no specific drug that can be used as a standard treatment for current leukemia, and new drug development is required.

Thus, the present inventors have found that ICAM4 (= CD242) is not expressed in T cells and B cell leukemia cells but is expressed in red blood cells. On the basis of this fact, ICAM4 (= CD242) (ICAM-4) (= CD242) and exhibited high specificity and affinity for ICAM4-expressing leukemia cells and antibody-dependent cytotoxicity of ICAM4-expressing leukemia cells Antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP), thereby completing the present invention.

Korean Patent Publication No. 10-1999-0022011 Korean Patent Publication No. 10-2003-0076448

It is an object of the present invention to provide a monoclonal antibody capable of specifically recognizing and binding to an ICAM4 antigenic determinant site.

It is still another object of the present invention to provide a composition for the diagnosis of red leukemia comprising the monoclonal antibody.

Another object of the present invention is to provide an anti-leukemia diagnostic kit comprising the monoclonal antibody

It is still another object of the present invention to provide a method for effectively detecting an enemy leukemia marker using the monoclonal antibody.

It is still another object of the present invention to provide a method for effectively diagnosing red leukemia using the monoclonal antibody.

It is still another object of the present invention to provide a composition for treating canine leukemia comprising the monoclonal antibody.

It is still another object of the present invention to provide a hybridoma cell producing said monoclonal antibody.

In order to achieve the above object, the present invention provides a monoclonal antibody that specifically binds ICAM4 (intercellular adhesion molecule 4).

In one embodiment of the invention, the monoclonal antibody comprises a heavy chain CDR H1 as defined in SEQ ID NO: 1; CDR H2 as defined in SEQ ID NO: 2; And a heavy chain variable region comprising the heavy chain CDR H3 defined by SEQ ID NO: 3 and a light chain CDR L1 defined by SEQ ID NO: 4; A light chain CDR L2 defined by SEQ ID NO: 5; And a light chain variable region comprising the light chain CDR L3 defined by SEQ ID NO: 6.

The present invention also provides a composition for the diagnosis of red leukemia comprising the monoclonal antibody.

The present invention also provides an anti-leukemia diagnostic kit comprising the monoclonal antibody.

The present invention also provides a method for detecting an anti-leukemia marker by contacting said monoclonal antibody with a biological sample to detect antigen-antibody complex formation.

In one embodiment of the present invention, the antigen-antibody complex formation can be detected by RIA, ELISA, immunofluorescence, hue binding or chemiluminescent substance binding method.

The present invention also relates to a method for producing a biological sample, comprising: obtaining a biological sample from the individual; Contacting the monoclonal antibody to a biological sample; Detecting ICAM4 bound to a monoclonal antibody; And determining that the level of expression of the detected ICAM4 protein is higher than that of a normal control individual and that the individual is at risk of developing an autoimmune leukemia.

In one embodiment of the present invention, the biological sample may be selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid and urine.

In one embodiment of the present invention, the monoclonal antibody may induce antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) of ICAM4-expressing leukemia cells have.

The present invention also provides a hybridoma cell producing said monoclonal antibody.

In one embodiment of the present invention, the cell may be the accession number KCLRF-BP-00303.

The monoclonal antibody of the present invention exhibits a high degree of specificity and affinity for ICAM4 expressed in red leukemia cells, and thus can be used not only for the diagnosis of autoimmune leukemia, a type of myeloid leukemia, but also for the diagnosis of ICAM4- Induced cell cycle cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP), which may be useful in the treatment of leukemia.

1 is a result of immunoblotting a red blood cell lysate with the anti-ICAM4 monoclonal antibody 1E1 of the present invention.
2 is a result of immunoprecipitation of the cell lysate of the leukemia cell line K562 with the anti-ICAM4 monoclonal antibody 1E1 of the present invention.
FIG. 3 is a result of LC mass spectrometry of Immunoprecipitation of an anti-ICAM4 monoclonal antibody 1E1 of the present invention to a leukemia cell line K562 cell lysate.
FIG. 4 shows the result of immunoprecipitation with an anti-ICAM4 monoclonal antibody 1E1 of the present invention after immune precipitation with an anti-leukemia cell line K562 cell lysate and a commercial anti-ICAM4 polyclonal antibody.
FIG. 5 is a table showing the reactivity of anti-ICAM4 monoclonal antibody 1E1 in various cell lines.
FIG. 6 is a flow cytometry graph showing the reactivity of anti-ICAM4 monoclonal antibody 1E1 against the leukemia cell line K562 (black line represents negative control and red line represents 1E1 monoclonal antibody).
Figure 7 is a graph showing the interalization characteristics of anti-ICAM4 monoclonal antibody 1E1.
Figure 8 is a graph showing the antibody-dependent cytotoxicity (ADCC) effect of anti-ICAM4 monoclonal antibody 1E1.
FIG. 9 is a graph showing the antibody-dependent phagocytic action (ADCP) effect of anti-ICAM4 monoclonal antibody 1E1.
Figure 10 shows the variable region sequence of anti-ICAM4 monoclonal antibody 1E1.
FIG. 11 shows the results of confirming the variable region sequence of the anti-ICAM4 monoclonal antibody 1E1 through the hibaviation assay after preparing the 1E1 chimeric antibody of the present invention.

The present invention provides an anti-ICAM4 antibody or fragment thereof that selectively recognizes ICAM4 (intercellular adhesion molecule 4) and can specifically bind to its antigenic determinant site.

The term " antibody " as used in the present invention includes immunoglobulin molecules immunologically reactive with specific antigens, and includes both polyclonal antibodies and monoclonal antibodies. The term also includes forms produced by genetic engineering such as chimeric antibodies (e. G., Humanized murine antibodies) and heterologous binding antibodies (e. G., Bispecific antibodies).

As used herein, the term "chimeric" antibody refers to the most preferred antibody derived using recombinant deoxyribonucleic acid techniques and refers to a human (immunologically "related" species, including chimpanzee) Includes all. Thus, the constant region of the chimeric antibody is most preferably consistent with the constant region of the native human antibody; The variable region of the chimeric antibody is most preferably derived from a non-human source and retains the desired antigen specificity for the target antigen (ICAM4). A non-human source can be any vertebrate source that can be used to generate antibodies against the ICAM4 antigen. Such non-human sources include, but are not limited to, rodents (such as rabbits, rats, mice, etc., such as those disclosed in U.S. Patent No. 4,816,567, incorporated herein by reference) and non-human primates such as Old World monkeys, apes, For example, U.S. Patent Nos. 5,750,105 and 5,756,096, incorporated herein by reference).

The term " antibody fragment " as used herein refers to a specific part of an antibody that is capable of binding to a target antigen, in this case ICAM4 or a specific part thereof. For example, antibody fragments include F (ab ') 2, Fab, Fab' and Fv fragments. These can be produced conventionally using recombinant DNA technology or using conventional methods of degrading antibody proteins with papain or pepsin (CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley and Sons Coligan et al., Eds. (1991-1992)).

As used herein, the term " monoclonal antibody " refers to a highly specific antibody directed against a single antigenic site as is known in the art. Typically, unlike polyclonal antibodies that contain different antibodies directed against different epitopes (antigenic determinants), monoclonal antibodies are directed against a single determinant on the antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays that utilize antigen-antibody binding and also have other advantages that are not contaminated by other immunoglobulins because they are synthesized by hybridoma cultures.

Typically, immunoglobulins have a heavy chain and a light chain, and each heavy and light chain comprises a constant region and a variable region (the region also known as a "domain"). The variable regions of the light and heavy chains include three variable regions and four "framework regions" called "complementarity determining regions" (hereinafter referred to as "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 " variable " as used herein is used to refer to the fact that the sequence of a particular region between antibodies is significantly different and is used to indicate the binding specificity of each particular antibody to a particular antigen. The variability of the antibody is centered on the CDR rather than being uniformly distributed throughout the variable domains of the antibody. The heavy and light chains of the monoclonal antibody each have three CDRs, and these regions form an antigen-antibody complex by recognizing ICAM4. These CDRs have a characteristic sequence for each monoclonal antibody and one or both of these six CDRs can interact to allow a single monoclonal antibody to recognize a particular epitope.

The anti-ICAM4 antibody of the present invention is a monoclonal antibody that specifically binds to an antigenic determinant region of ICAM4, and may include variable regions of light and heavy chains.

More specifically, the heavy chain variable region of the monoclonal antibody comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3, or at least 80% homology with this sequence in the CDR region, 90% or more, and most preferably 95% or more; And / or wherein the light chain variable region comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 4 to 6, or at least 80%, preferably 90% or more, And most preferably 95% or more.

In one embodiment of the invention, the monoclonal antibody comprises a heavy chain CDR H1 as defined in SEQ ID NO: 1; CDR H2 as defined in SEQ ID NO: 2; And a heavy chain variable region comprising the heavy chain CDR H3 defined by SEQ ID NO: 3 and a light chain CDR L1 defined by SEQ ID NO: 4; A light chain CDR L2 defined by SEQ ID NO: 5; And a light chain variable region comprising the light chain CDR L3 defined by SEQ ID NO: 6.

The present inventors have found that ICAM4 (= CD242) is not expressed in T cells and B cell leukemia cells but is expressed in red blood cells. In the following experiment, the anti-ICAM4 monoclonal antibody according to the present invention is actually expressed in cells In order to examine whether the antibody specifically binds to ICAM4 expressing the antibody, the leukemic cell line K562 was analyzed by immunoblotting, immunoprecipitation and LC mass spectrometry. As a result, the monoclonal antibody according to the present invention exhibited ICAM4 (See Figs. 1 to 4).

Monoclonal antibodies specific for ICAM4 can be made by a fusion method well known in the art (Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519). For example, a monoclonal antibody of the invention can be prepared by immunizing an animal with ICAM4 as an immunogen, fusing splenocytes from an immunized animal with myeloma cells to produce a hybridoma, and selectively expressing a monoclonal antibody recognizing ICAM4 Producing hybridomas, cultivating selected hybridomas, and separating the antibodies from the hybridoma culture. The hybridoma cell line can be cultured in vitro or in vivo to separate the antibody.

The monoclonal antibody produced by the hybridoma may be used without purification. However, in order to obtain the best results, the monoclonal antibody produced by the hybridoma may be purified to a high purity (for example, 95% or more) according to a method well known in the technical field of the present invention . Such a purification technique can be separated from a culture medium or a plurality of solutions by using a purification method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography and the like.

As an example of the production of a monoclonal antibody using a hybridoma, a hybridoma is inserted into the abdominal cavity of a mouse, and the hybridoma is separated and purified. The separation and purification of the monoclonal antibody can be carried out using affinity chromatography such as ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column or protein A column.

Hybridoma cells producing the monoclonal antibody according to the present invention were named '1E1-2B2' and deposited at the Korean Cell Line Research Foundation on Oct. 10, 2013, and deposited with Deposit No. KCLRF-BP-00303 dated October 25, .

The present invention also provides a diagnostic composition, preferably an autoimmune leukemia diagnostic composition, for a disease or an ICAM4 mediated disease associated with the expression or degree of expression of ICAM4 comprising said monoclonal antibody.

The present invention also relates to a method for detecting an infectious leukemia marker by contacting said monoclonal antibody with a biological sample to detect antigen-antibody complex formation. That is, the ICAM4 protein can be detected by reacting the monoclonal antibody of the present invention with a biological sample and detecting antigen-antibody complex formation.

The present invention also relates to a method for producing a biological sample, comprising: obtaining a biological sample from the individual; Contacting the biological sample with the monoclonal antibody of the present invention; Detecting ICAM4 bound to a monoclonal antibody; And determining that the level of expression of the detected ICAM4 protein is higher than that of a normal control individual, thereby determining that the individual is at risk of developing red blood cell leukemia.

As used herein, the term "biological sample" refers to a tissue, cell, whole blood, serum, plasma, tissue autopsy sample (brain, skin, lymph node, spinal cord, etc.), urine, cell culture supernatant, ruptured eukaryotic cell, But is not limited thereto. These biological samples can be reacted with the antibody of the present invention without manipulation or manipulation to confirm the presence of ICAM4 protein and the presence or absence of leukemia.

As used herein, the term " antigen-antibody complex " refers to an ICAM4 protein antigen in a sample and a combination of the monoclonal antibody according to the present invention recognizing it. The formation of such an antigen-antibody complex can be performed by a colormetric method, An arbitrary one selected from the group consisting of an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual assessment, and a scintillation counting method. . ≪ / RTI > However, various applications and applications are possible without being limited thereto.

In the present invention, various markers can be used to detect an antigen-antibody complex. Specific examples include, but are not necessarily limited to, enzymes, minerals, ligands, emitters, microparticles, and radioactive isotopes.

Examples of the enzyme used as the detection label include acetylcholine esterase, alkaline phosphatase,? -D-galactosidase, horseradish peroxidase,? -Lactamase and the like, , Eu < 3 + >, Eu < 3 + >, chelate or cryptate, and ligands include biotin derivatives and the like. Examples of the emitters include acridinium ester, isoluminol derivatives, Colored latex and the like, and the radioactive isotope may include 57Co, 3H, 125I, 125I-Bonton Hunter reagent and the like, but is not particularly limited thereto.

Preferably, the antigen-antibody complexes can be detected using enzyme immunoassay (ELISA). Enzyme Immunosorbent Assay (ELISA) includes direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of an antibody recognizing an antigen attached to a solid support , A direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antigen attached to the solid support, and the other antibody recognizing the antigen in the complex of the antibody and the antigen attached to the solid support, And indirect sandwich ELISA using labeled secondary antibodies recognizing the antibody. The monoclonal antibody can have a detection label, and when it does not have a detection label, it can capture another monoclonal antibody and can be identified by treating another antibody having the detection label.

The present invention also provides an anti-leukemia diagnostic kit comprising the monoclonal antibody.

The monoclonal antibodies used in the anti-leukemia diagnostic kit of the present invention can also use fragments of monoclonal antibodies as long as the antibodies can selectively recognize ICAM4. Such antibody fragments may include F (ab ') 2, Fab, Fab', Fv fragments, and the like.

The anti-leukemia diagnostic kit of the present invention may include monoclonal antibodies selectively recognizing ICAM4 or fragments thereof and tools / reagents used in immunological analysis.

Tools / reagents used in immunological assays include suitable carriers, labeling agents capable of producing detectable signals, solubilizers, detergents, and the like. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring the enzyme activity and a reaction terminator.

Suitable carriers include, but are not limited to, soluble carriers, e. G., Physiologically acceptable buffers such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

The assay system for use in the detection methods and diagnostic kits of the present invention may be applied to a variety of assay systems including, but not limited to, ELISA plates, dip-stick devices, immunochromatographic test strips and split- through devices and the like.

The present invention also provides a composition for treating a disease or ICAM4 mediated disease associated with the expression or degree of expression of ICAM4 comprising said monoclonal antibody and a pharmaceutically acceptable carrier. The disease is preferably leukemia.

The composition of the present invention may be administered in single or multiple doses in a pharmaceutically effective amount. The therapeutic composition may be administered in the form of a solution, a powder, an aerosol, a capsule, an intravaginal tablet or a capsule or a suppository. Routes of administration include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, endothelial, oral, topical, intranasal, intrarectal, and the like. However, upon oral administration, the peptide will be digested and the oral composition should be formulated to coat the active agent or protect it from degradation from above. The pharmaceutical composition may also be administered by any device capable of transferring the active agent to the target cell.

When the composition is formulated, the anti-ICAM4 monoclonal antibody is considered to retain the desired biological activity at a given point in time. If the desired biological activity at that time is within about 30%, and preferably within about 20%, of the desired biological activity occurring within that time, the pharmaceutical composition may be used as determined in a suitable assay for the desired biological activity . Assays for measuring the desired biological activity of an anti-ICAM4 monoclonal antibody can be performed as described in the Examples herein. Schultze et al. (1998) Proc. Natl. Acad. ScL USA 92: 8200-8204; Denton et al. (1998) Pediatr. Transplant. 2: 6-15; Evans et al. (200O) < / RTI > J Immunol. 164: 688-697; Noelle (1998) Agents Actions Suppl. 49: 17-22; Lederman et al. (1996) Curr. Opin. Hematol. 3: 77-86; Coligan et al. (1991) Current Protocols in Immunology 13:12; Kwekkeboom et al. (1993) Immunology 79: 439-444; And U.S. Pat. Nos. 5,674,492 and 5,847,082; Which is incorporated herein by reference.

The anti-ICAM4 monoclonal antibodies of the invention can be formulated into liquid pharmaceutical formulations. Anti-ICAM4 monoclonal antibodies can be prepared using any known method and include the methods disclosed herein. In one embodiment of the invention, the anti-ICAM4 monoclonal antibody was recombinantly produced in the hybridoma cell line KCLRF-BP-00303.

If anti-ICAM4 monoclonal antibodies are stored prior to their formulation, they can be frozen, for example, ≤-20 degrees C, and then thawed at room temperature for further formulation. Liquid pharmaceutical formulations may comprise a therapeutically effective amount of the monoclonal antibody. The amount of these antibodies present in the formulation takes into account the route of administration and the desired dosage volume.

The composition of the present invention is administered in a pharmaceutically effective amount. By " pharmaceutically effective amount " is meant an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prophylaxis, and the effective dosage level will vary depending on factors such as the severity of the disease, The composition of the present invention can be used in combination with the composition of the present invention, including drugs used in combination with the composition of the present invention, and other medical fields including, for example, body weight, health, sex, sensitivity of the patient to the drug, May be determined according to well known factors. In addition, the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

In this manner, the liquid pharmaceutical composition comprises an anti-ICAM4 monoclonal antibody at a concentration of from about 0.1 mg / ml to about 50.0 mg / ml, from about 0.5 mg / ml to about 40.0 mg / ml, from about 1.0 mg / ml to about 30.0 mg / ml, about 5.0 mg / ml to about 25.0 mg / ml, about 5.0 mg / ml to about 20.0 mg / ml, or about 15.0 mg / ml to about 25.0 mg / ml. In one embodiment of the invention, the liquid pharmaceutical composition comprises about 0.1 mg / ml to about 5.0 mg / ml, about 5.0 mg / ml to about 10.0 mg / ml, about 10.0 mg / ml to about 15.0 mg / About 15.0 mg / ml to about 20.0 mg / ml, about 20.0 mg / ml to about 25.0 mg / ml, about 25.0 mg / ml to about 30.0 mg / ml, about 30.0 mg / / ICAM4 monoclonal antibody at a concentration of about 40.0 mg / ml, about 40.0 mg / ml to about 45.0 mg / ml, or about 45.0 mg / ml to about 50.0 mg / ml. In another embodiment of the invention, the liquid pharmaceutical composition comprises about 15.0 mg / ml, about 16.0 mg / ml, about 17.0 mg / ml, about 18.0 mg / ml, about 19.0 mg / ml, about 20.0 mg / ICAM4 monoclonal antibody at a concentration of about 21.0 mg / ml, about 22.0 mg / ml, about 23.0 mg / ml, about 24.0 mg / ml, or about 25.0 mg / ml. The liquid pharmaceutical composition comprises an anti-ICAM4 monoclonal antibody and an anti-ICAM4 monoclonal antibody at about pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and other such values may include a buffer to maintain the pH of the formulation at a pH in the range of about pH 5.0 to about pH 7.0, which is in the range of about pH 5.0 to about pH 7.0. The buffer may be added to the formulation in order to maintain the pH of the formulation at about pH 5.0 to about pH 6.5, about pH 5.0 to about pH 6.0, about pH 5.0 to about pH 5.5, about pH 5.5 to about 7.0, about pH 5.5 to about pH 6.5, To about pH 6.0.

Any suitable buffer that maintains the pH of a liquid anti-ICAM4 monoclonal antibody formulation ranging from about pH 5.0 to about pH 7.0, to the extent that it retains the physiochemical stability and desired biological activity of the antibody as described above, can be used for formulation have.

Suitable buffers include, but are not limited to, conventional acids and salts thereof, wherein the counterion may be, for example, sodium, potassium, ammonium, calcium, or magnesium. Examples of conventional acids and salts thereof that can be used as buffers in pharmaceutical liquid formulations include succinic or succinic acid salts, citric acid or citric acid salts, acetic acid or acetic acid salts, tartaric acid or tartaric acid salts, phosphoric acid or phosphate salts, gluconic acid or gluconic acid salts , Glutamic acid or glutamate, aspartic acid or aspartate, maleic acid or maleate, and malic or malate buffers. The buffer concentration in the formulation can be from about 1 mM to about 50 mM and is about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 45 mM, 50 mM, or about 1 mM to about 50 mM. In one embodiment of the invention, the buffer concentration in the formulation is about 5 mM to about 15 mM, and about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 14 mM, 15 mM, or from about 5 mM to about 15 mM.

When a proximal isotonic liquid pharmaceutical formulation is desired, the liquid pharmaceutical formulation comprising the anti-ICAM4 monoclonal antibody and the buffer may additionally comprise an amount of isotonic agent sufficient to impart proximity to the formulation. By "proximal isotonicity" is meant that the aqueous formulation is about 240 mmol / kg to about 360 mmol / kg, preferably about 240 to about 340 mmol / kg, more preferably about 250 to about 330 mmol / Quot; means having an osmolarity of about 260 to about 320 mmol / kg, even more preferably about 270 to about 310 mmol / kg. Methods for determining the isotonicity of a solution are known to those skilled in the art. See, for example, Setnikar et al. (1959) J. Am. Pharm. Assoc. 48: 628.

Those skilled in the art are familiar with various pharmaceutically acceptable solutes useful in providing isotonicity within pharmaceutical compositions. The isotonic agent may be any reagent capable of controlling the osmotic pressure of the liquid pharmaceutical formulation of the present invention at levels equivalent to body fluids. It is preferred to use physiologically acceptable isotonic agents. Thus, a liquid pharmaceutical formulation comprising a therapeutically effective amount of an anti-ICAM4 monoclonal antibody and a buffer can comprise an ingredient that can be used to provide isotonicity, for example, sodium chloride; Amino acids such as alanine, valine, and glycine; Sugars and sugar alcohols (polyols) including but not limited to glucose, dextrose, fructose, sucrose, maltose, mannitol, trehalose, glycerol, sorbitol, and xylitol; Acetic acid, other organic acids or salts thereof, and relatively small amounts of citric acid salts or phosphates. Those skilled in the art are aware of additional agents suitable for providing the rigidity of liquid formulations.

Further, in one preferred embodiment of the present invention, the liquid pharmaceutical formulation comprising the anti-ICAM4 monoclonal antibody and the buffer may further comprise sodium chloride as an isotonic agent. The sodium chloride concentration in the formulation will depend on the contribution of the other ingredients to the rigidity. For example, the concentration of sodium chloride may range from about 50 niM to about 300 mM, from about 50 mM to about 250 mM, from about 50 mM to about 200 mM, from about 50 mM to about 175 mM, from about 50 mM to about 150 mM, from about 100 mM to about 200 mM, from about 100 mM to about 150 mM, from about 125 mM to about 175 mM, from about 125 mM to about 175 mM, from about 75 mM to about 175 mM, from about 75 mM to about 150 mM, from about 100 mM to about 175 mM, About 150 mM, about 130 mM to about 170 mM, about 130 mM to about 160 mM, about 135 mM to about 155 mM, about 140 mM to about 155 mM, or about 145 mM to about 155 mM.

In the treatment of the liquid pharmaceutical formulations of the present invention, freeze thaw or mechanical shear-induced proteolysis can be suppressed by incorporating the surfactant into the formulation to lower the surface tension at the solution-air interface. Thus, in one embodiment of the invention, the liquid pharmaceutical formulation comprises a therapeutically effective amount of an anti-ICAM4 monoclonal antibody, a buffer, and additionally a surfactant. In another embodiment of the invention, the liquid pharmaceutical formulation comprises an anti-ICAM4 monoclonal antibody, a buffer, an isotonic agent, and may further comprise a surfactant.

Typical surfactants used are nonionic surfactants, such as polyoxyethylene sorbitol esters such as polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20); Polyoxypropylene-polyoxyethylene esters such as Pluronic F68; Polyoxyethylene alcohols such as Brij 35; Simethicone; Polyethylene glycol such as PEG 400; Lysophosphatidylcholine; And polyoxyethylene-p-t-octylphenol, such as Triton X-100. The stabilization of traditional medicaments by surfactants or emulsifiers is described, for example, by Levine et al. (1991) J Parenteral Sci. Technol. 45 (3): 160-165, which is incorporated herein by reference.

When a surfactant is included, it will generally contain from about 0.001% to about 1.0% (w / v), from about 0.001% to about 0.5%, from about 0.001% to about 0.4%, from about 0.001% to about 0.3% About 0.005% to about 0.2%, about 0.01% to about 0.2%, about 0.01% to about 0.2%, about 0.03% to about 0.5%, about 0.03% to about 0.3% %, From about 0.05% to about 0.5%, or from about 0.05% to about 0.2%.

When the composition of the present invention is administered in a pharmaceutically effective amount, the monoclonal antibody of the present invention having a strong affinity for the ICAM4 protein specifically binds to the ICAM4 protein expressed in the acute leukemia cells to inhibit antibody-dependent cytotoxicity (ADCC) and antibody-dependent phagocytic action (ADCP). On the other hand, the monoclonal antibody can bind to toxic substances to form a fusion protein in order to enhance the therapeutic effect of the anti-leukemia. The toxin protein may be ricin, saporin, gelonin, momordin, diphtheria toxin, or pseudomonas toxin, but the type of the toxin is not particularly limited.

The present invention also provides the use of a composition comprising an anti-ICAM4 monoclonal antibody as an active ingredient for the manufacture of a medicament for the treatment of enemas leukemia. The composition of the present invention comprising the active ingredient as described above can be used for the preparation of a medicament for the treatment of red leukemia.

The present invention also relates to a method for the treatment of red leukemia comprising the step of administering said composition (composition for treating an anti-leukemia comprising an anti-ICAM4 monoclonal antibody and a pharmaceutically acceptable carrier) in an individual.

The term "animal" as used herein refers to a mammal such as a human, horse, dog, cat, pig, goat, rabbit, hamster, monkey, guinea pigs, rat, mouse, lizard, And any animal (e. G., Human), including birds.

As used herein, the term "treatment" refers to any action that alleviates or alleviates an adverse leukemia symptom by administration of a composition comprising an anti-ICAM4 monoclonal antibody according to the present invention and a pharmaceutically acceptable carrier.

The anti-ICAM4 monoclonal antibodies of the invention may be used with other antibodies, biologically active agents or materials for various purposes. For example, the anti-ICAM4 monoclonal antibodies of the invention may be used in combination with known anti-leukemia inhibiting / therapeutic agents.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.

< Example  1>

One E1 Monoclonal  For antibody production target  Site design

The present inventors have screened new surface proteins of human red blood cells in order to prepare new antibodies capable of easily and accurately diagnosing the infantile leukemia. As a result, it was found that ICAM4 was overexpressed on the surface of the cells that had induced leukemia. Therefore, the gene and protein sequence of ICAM4 was confirmed in NCBI BLAST to prepare antibody against ICAM4, A monoclonal antibody capable of binding to a site specific to ICAM-4 and having high affinity was prepared through the same experiment as the following examples, and was named '1E1' in the present invention.

< Example  2>

Hybridoma  Cell manufacturing and 1 E1 Monoclonal  Antibody production

<2-1> Hybridoma  Cell Manufacturing

Blood from healthy adults was separated and washed three times with Ficoll-Hypaque (Pamacia, Sweden: d = 1.077), and then 10 ⁷ cells were injected into the abdominal cavity three times at 2-week intervals in Balb / c mice And the immune response was induced. At 3 days after the last injection, the spleen was removed to prepare a cell suspension. Spleen by harvesting the mouse spleen was obtained for cells (splenocyte), polyethyene glycol with DMEM to mouse spleen cells 10 ⁸ and a mouse myeloma cell line SP2 / O-Ag14 (ATCC, Virginia, USA) culture medium using fused to 2 × 10 ⁷ gae . Cells were fused in 8 96-well plates and incubated at 37 ° C in 5% CO2, HAT medium (hypoxanthine, aminopterin and thymidine containing media). When colonization was observed, reactivity was confirmed by flow cytometry on human RBCs. Among the clones showing reactivity with human RBC, the clone in the first row of the first plate was named '1E1 clone' and was established as a monoclonal hybridoma by performing limiting dilution 23 times. On the other hand, the hybridoma cells were deposited with the Korean Cell Line Research Foundation on Oct. 10, 2013 and received Deposit No. KCLRF-BP-00303 dated October 25,

<2-2> Hybridoma  1 of the present invention using cells E1 Monoclonal  Antibody production

Hybridoma cultures were obtained by culturing in a cell-bag (Xcellerex, Malborough, MA, USA) for 2 weeks and filtered with a 0.22 μm filter. Prepared samples were transferred to a Histrap Protein G 5 mL column (GE Healthcare, Little Chalfont, UK), and 20 mM sodium phosphate (pH 7.2) was added to 10 column volumes (50 mL) pH 3.0) and eluted with 5 column volumes. The purified antibody was dialyzed with phosphate buffered saline (pH 7.4) and used as a sample in the following experiment.

< Example  3>

Monoclonal  Antigen analysis for antibodies

<3-1> Immunoblotting ( Immunoblotting )

3-1-1. Manufacture of erythrocyte lysine

Human blood was collected and added with ficoll solution (blood: ficoll = 1: 2). After centrifugation at 2000 rpm for 20 minutes, 1% NP-40 was added to the obtained red blood cells. ), And centrifuged to obtain only the supernatant.

3-1-2. Perform Western Blotting

40 μL of the red blood cell lysate was added to 2 micro test tubes, and 10 μL of Reducing buffer (5 ×) and 10 μL of Non-Reducing buffer (5 ×) were added and boiled for 10 minutes. Two samples were loaded onto a 10% polyacrylamide gel with a protein marker (Mid-range Pre-stained Marker) and electrophoresed at 10 mA for 3 hours. After the periodic electrophoresis, place the polyacrylamide gel on a nitrocellulose membrane (WHATMAN) and transpose at 17V for 40 minutes. The transfected membrane was blocked with 5% skim milk for 1 hour and reacted with 1E1 monoclonal antibody at 1 μg / mL for 1 hour. After washing with 0.05% PBST, HRP-conjugated secondary antibody (anti-mouse immunoglobulin (Dako)) was reacted at 1 μg / mL for 30 minutes. After washing with 0.05% PBST, the cells were visualized using an ECL kit (Amersham GE Healthcare).

As a result, as shown in Fig. 1, a band of about 40 kDa was confirmed.

<3-2> Immunoprecipitation ( Immunoprecipitation )

3-2-1 Production of biotin-binding cell lysate of the leukemia cell line K562

The leukemia cell line K562 cells were prepared (1 × 10 ⁸ ). After washing the cells twice with PBS, 1 mL of biotin solution (20 μM NaHCO _3, 100 μL, 150 mM NaCl, 100 μg biotin) was added, and the mixture was rotated at 4 ° C. for 30 minutes using a rotor. And rotated for 10 minutes. After washing with PBS twice, 1% NP-40 was added, and the supernatant was centrifuged at 4 ° C overnight using a rotor.

3-2-2 Perform immunoprecipitation

Protein G beads were prepared by washing twice with PBS. 5 μg of 1E1 monoclonal antibody and 20 μl of beads were added, and the mixture was rotated at 4 ° C. overnight using a rotor. Thereafter, 500 μl of the prepared biotin-conjugated K562 cell lysate was added, and the mixture was rotated at 4 ° C. overnight by using a rotor. The antibody-antigen conjugated beads were incubated with NaCl buffer 1, 2, 3 (NaCl buffer 1; 1% NP40, 1 M NaCl, NaCl buffer 2; 0.1% NP40, 0.5 M NaCl, NaCl buffer 3; 0.1% And washed in order.

3-2-3 Performing Western Blotting

40 μl of PBS and 10 μl of reducing buffer (5 ×) were added to the bead conjugated with the antibody-antigen in a micro test tube and boiled for 10 minutes. The prepared sample was loaded onto a 10% polyacrylamide gel with a protein marker (Mid-range Pre-stained Marker) and electrophoresed at 10 mA for 3 hours. After the periodic electrophoresis, the polyacrylamide gel was placed on a nitrocellulose membrane (WHATMAN) and transfected at 17V for 40 min. The transfected membranes were blocked with 5% skim milk for 1 hour and reacted with 1 μg / mL of HRP-conjugated secondary antibody (streptavidin-HRP) for 30 minutes. After washing with 0.05% PBST, the cells were visualized using an ECL kit (Amersham GE Healthcare).

As a result, as shown in Fig. 2, a band of about 40 kDa was confirmed.

<3-3> LC  Mass analysis

3-3-1 Production of cell lysate of leukemia cell line K562

The leukemia cell line K562 cells were prepared (1 × 10 ⁸ ). Cells were washed twice with PBS, and 1% NP-40 was added and incubated at 4 ° C for 2 hours with a rotor, followed by centrifugation of the supernatant.

3-3-2 Perform immunoprecipitation

Protein G beads were prepared by washing twice with PBS. 5 μg of 1E1 monoclonal antibody and 20 μl of beads were added, and the mixture was rotated at 4 ° C. overnight using a rotor. Thereafter, 500 μl of the prepared K562 cell lysate was added, and the mixture was rotated at 4 ° C. overnight by using a rotor. The antibody-antigen conjugated beads were incubated with NaCl buffer 1, 2, 3 (NaCl buffer 1; 1% NP40, 1 M NaCl, NaCl buffer 2; 0.1% NP40, 0.5 M NaCl, NaCl buffer 3; 0.1% And washed in order.

3-3-3 Perform Silver staining

40 μl of PBS and 10 μl of reducing buffer (5 ×) were added to the bead conjugated with the antibody-antigen in a micro test tube and boiled for 10 minutes. The prepared sample was loaded onto a 10% polyacrylamide gel with a protein marker (Mid-range Pre-stained Marker) and electrophoresed at 10 mA for 3 hours. After the end of the period, the polyacrylamide gel was stained with EZ-Silver Staining Kit (Bio-World). A band of about 40 kDa, which is seen as a band for the 1E1 monoclonal antibody, was sliced from the dyed gel and submitted to LC Mass Spectrometry (Proteomtech).

As a result, as shown in Fig. 3, it was confirmed that the band of about 40 kDa was CD242.

<3-4> ICAM4 &Lt; / RTI &gt;

3-4-1 Preparation of Commercial Antibody

A commercial anti-CD242 polyclonal antibody was purchased (Abnova H00003386-B01P).

3-4-2 Perform immunoprecipitation

Protein G beads were prepared by washing twice with PBS. 5 μg of the commercial anti-CD242 polyclonal antibody and 20 μL of beads are added and the mixture is rotated at 4 ° C. overnight using a rotor. Then, 500 μL of the prepared K562 cell lysate is added, and the mixture is rotated at 4 ° C. overnight by using a rotor. The antibody-antigen conjugated beads were incubated with NaCl buffer 1, 2, 3 (NaCl buffer 1; 1% NP40, 1 M NaCl, NaCl buffer 2; 0.1% NP40, 0.5 M NaCl, NaCl buffer 3; 0.1% And washed in order.

3-4-3 Performing Western Blotting

40 μl of PBS and 10 μl of reducing buffer (5 ×) were added to the bead conjugated with the antibody-antigen in a micro test tube and boiled for 10 minutes. The prepared sample was loaded onto a 10% polyacrylamide gel with a protein marker (Mid-range Pre-stained Marker) and electrophoresed at 10 mA for 3 hours. At the end of the periodic electrophoresis, the polyacrylamide gel was placed on a nitrocellulose membrane (WHATMAN) and transfected at 17V for 40 min. The transfected membrane was blocked with 5% skim milk for 1 hour and reacted with 1E1 monoclonal antibody at 1 μg / mL for 1 hour. HRP-conjugated secondary antibody (anti-mouse immunoglobulin (Dako)) was reacted at a concentration of 1 μg / mL for 30 minutes. After washing with 0.05% PBST, the cells were visualized using an ECL kit (Amersham GE Healthcare).

As a result, a band of 40 kDa was confirmed as shown in Fig.

As a result of the above-mentioned experiment, it was proved that the antibody of the present invention is an antibody recognizing ICAM4 (= CD242).

< Example  4>

In many tumor cell lines Monoclonal  Reactivity of antibodies

<4-1> In various tumor cell lines, 1 E1 Monoclonal  Investigation of antibody reactivity

In this experiment, reactivity of 1E1 mAb to various tumor cell lines was examined by flow cytometry.

Several cell lines were prepared at 2x10 &lt; ^{5 &} gt ;. 1 [mu] g monoclonal antibody was reacted at 4 [deg.] C for 30 minutes, washed with cold PBS, and reacted with FITC-conjugated secondary antibody (anti-mouse immunoglobulin (Dynona)) for 20 minutes at 4 [deg.] C. Washed with cold PBS and flow cytometry was performed using FACScalibur. The staining intensity was measured in logarithm of the fluorescence intensity and expressed in units of 10 watt. In the control group, only secondary antibody except 1E1 monoclonal antibody was used after staining. This method was used to determine a positive sample with a positive rate of 10% or more.

As a result, a prominent positive reaction was confirmed in red blood cells, as shown in Figures 5 and 6. FIG. 5 is a table summarizing the reactivity of 1E1 monoclonal antibody in various tumor cell lines. FIG. 6 shows a case of the K562 strain of a hematopoietic cell line as a representative example of the result in FIG. The black line is the negative control and the red line is the 1E1 monoclonal antibody.

< Example  5>

Monoclonal  Characterization of antibodies

<5-1> Internalization internalization )

1E1 monoclonal antibody was investigated by flow cytometry.

In detail, red leukemic cell line K562 was prepared in ⁵ × 10 ⁵ micro test tubes. One μg of 1E1 monoclonal antibody was incubated at 37 ° C for 0, 30, 60, and 90 minutes, and fixed with 1% paraformaldehyde. Washed with cold PBS, and the secondary antibody conjugated with PE (anti-mouse immunoglobulin (Dynonona)) was reacted at 4 ° C for 20 minutes. Washed with cold PBS and flow cytometry was performed using FACScalibur. The staining intensity was measured in logarithm of the fluorescence intensity and expressed in units of 10 watt. The positive rate was measured by this method and plotted on a time - by - time basis.

As a result, as shown in Fig. 7, it was confirmed that the 1E1 monoclonal antibody was internalized in an acute leukemia cell line.

< Example  6>

Monoclonal  Antibody Red leukemia  Therapeutic effect

<6-1> Antibody-dependent cytotoxicity test ADCC )

6-1-1 Preparation of effector cell

Human blood was collected and added with ficoll solution (blood: ficoll = 1: 2) and centrifuged at 2000 rpm for 20 minutes to obtain PBMC (Peripheral Blood Mononuclear Cell). The obtained PBMCs were stored at 37 ° C in RPMI supplemented with 5% FBS.

6-1-2 Antibody-dependent cytotoxicity test

Five microtest tubes (micro test tubes) were used to prepare target cell leukemia cell line K562 (5 × 10 ⁴ cells). 1E1 monoclonal antibody was added to each tube at 0 μg / mL, 0.5 μg / mL, 5 μg / mL, and 50 μg / mL, and reacted at 4 ° C. for 30 minutes. 1E1 monoclonal antibody-bound target cells (K562) were dispensed into a 96-well plate at a rate of 1 × 10 ⁴ per well, and the prepared effector cells were placed at 5 × 10 ⁵ per well (50 times the target cells) And cultured for 3 hours at 37 ° C. As a positive control, a lysis buffer was added, followed by incubation at 37 ° C for 45 minutes, followed by centrifugation at 2500 rpm for 4 minutes to obtain only supernatant. LDH (lactate dehydrogenase) dissolved from the target cells was measured and the degree of cell lysis was calculated (Promega assay kit).

As a result, as shown in Fig. 8, it was confirmed that the 1E1 monoclonal antibody of the present invention exhibited antibody-dependent cytotoxicity.

<6-2> Test for antibody-dependent phagocytosis ADCP )

6-2-1 Preparation of effector cell

Cells in the abdominal cavity of the rats were collected by injecting 10 mL of sterilized PBS into the abdominal cavity of the rats and drawing them with a syringe again. After washing twice with PBS, the cells were cultured in RPMI supplemented with 10% FBS overnight at 37 ° C. On the next day, the floating cells were removed and only the macrophages of the differentiated rats were prepared.

6-2-2 Antibody-dependent phagocytosis assay

1 × 10 ⁵ of target cell leukemia cell line K562 was prepared in three micro test tubes. CFSE was added to each tube and incubated at 37 ° C for 15 minutes. Washed with PBS, and further incubated at 37 DEG C for 15 minutes. 1E1 monoclonal antibody and a positive control (an antibody known to cause macrophage) B6H12 were added and reacted at 4 ° C for 30 minutes. The reacted antibody-binding target cells were transferred to a 24-well plate, and 2 x 10 ⁵ (two times the target cells) of the effector mouse macrophages were added thereto, followed by incubation at 37 ° C for 2 hours or more. After incubation, the cells were washed with cold PBS and PE-conjugated anti-CD11b antibody was reacted at 4 ° C for 20 minutes. After washing with cold PBS, flow cytometry was performed using FACScalibur.

As a result, as shown in Fig. 9, it was confirmed that the 1E1 monoclonal antibody of the present invention exhibited antibody-dependent phagocytosis.

< Example  7>

Chimeric  Preparation of antibodies

<7-1> Antibody Cloning

RNA was extracted from hybrid cells producing 1E1 monoclonal antibodies (QIAGEN, RNeasy Plus Mini Kit). The variable region (Fv) DNA of the 1E1 mAb was obtained using Mouse Ig-Primer Set (Novagen), which is shown in Fig. The 1E1 monoclonal antibody variable region DNA was transferred to the expression vector pOptiVec and pcDNA3.3 vector (Dainna Inc.) which were designed to express a constant region of human antibody (Fc) together and introduced into 293T cells.

<7-2> Kai Meric  Antibody identification

Flow cytometry was used to determine if the 1E1 chimeric antibody was properly produced. 2 × 10 ⁵ of the leukemia cell line K562 was prepared in a micro test tube. 10 [mu] g of 1E1 monoclonal antibody was reacted at 4 DEG C for 30 minutes. After washing with cold PBS, 100 μL of the culture obtained by introducing 1E1 chimeric antibody vector into 293T was reacted at 4 ° C for 20 minutes. Washed with cold PBS, and reacted with a secondary antibody (anti-human immunoglobulin (Dynona)) conjugated with FITC for 20 minutes at 4 ° C. Washed with cold PBS and flow cytometry was performed using FACScalibur. The staining intensity was measured in logarithm of the fluorescence intensity and expressed in units of 10 watt.

As a result, as shown in Fig. 11, it was confirmed that the 1E1 chimeric antibody prepared in the above experiment had the same variable region as the 1E1 monoclonal antibody.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

1E1: Anti-ICAM4 monoclonal antibody of the present invention
CDR: complementarity determining region
ADCC: antibody dependent cellular cytotoxicity
ADCP: antibody dependent Cellular Phagocytosis

Korea Cell Line Research Foundation KCLRFBP00303 20131011

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> The antibody that recognizes the ICAM4 on erythroleukemia and use          of the antibody <130> PN1309-327 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> anti-ICAM4 antibody CDR H1 polypeptide sequence <400> 1 Gly Phe Ser Leu Thr Asp Tyr Gly   1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> anti-ICAM4 antibody CDR H2 polypeptide sequence <400> 2 Met Trp Gly Gly Gly Arg Thr   1 5 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> anti-ICAM4 antibody CDR H3 polypeptide sequence <400> 3 Val Lys Thr Gly Gln Asp Tyr Ala Val Asp Tyr   1 5 10 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> anti-ICAM4 antibody CDR L1 polypeptide sequence <400> 4 Lys Ser Ile Ser Lys Tyr   1 5 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> anti-ICAM4 antibody CDR L2 polypeptide sequence <400> 5 Ser Gly Ser   One <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> anti-ICAM4 antibody CDR L3 polypeptide sequence <400> 6 Gln Gln His Asn Val Tyr Pro Trp Thr   1 5

Claims (12)

A monoclonal antibody that specifically binds to ICAM4 (intercellular adhesion molecule 4). The method according to claim 1,
Said monoclonal antibody comprises a heavy chain CDR H1 consisting of the amino acid sequence of SEQ ID NO: 1; A CDR H2 consisting of the amino acid sequence of SEQ ID NO: 2; And a heavy chain variable region comprising a heavy chain CDR H3 consisting of the amino acid sequence of SEQ ID NO: 3 and a light chain CDR L1 consisting of the amino acid sequence of SEQ ID NO: 4; A light chain CDR L2 consisting of the amino acid sequence of SEQ ID NO: 5; And a light chain variable region comprising a light chain CDR L3 consisting of the amino acid sequence of SEQ ID NO: 6. A composition for diagnosing an autoimmune leukemia comprising the monoclonal antibody of any one of claims 1 and 2. 4. An anti-leukemia diagnostic kit comprising the monoclonal antibody of claim 1 or 2. 12. A method for detecting an anti-leukemia marker by contacting a monoclonal antibody of claim 1 or 2 with a biological sample to detect antigen-antibody complex formation. 6. The method of claim 5,
Wherein said antigen-antibody complex formation is detected by RIA, ELISA, immunofluorescence, hue binding, or chemiluminescent substance binding method. 1) obtaining a biological sample from the individual;
2) contacting the sample with the monoclonal antibody of claim 1 or 2;
3) detecting ICAM4 bound to the monoclonal antibody of claim 1; And
4) determining that the level of expression of ICAM4 protein detected in step 3) is higher than that of the normal control individual, and that the individual is at increased risk of developing leukemia. 8. The method of claim 7,
Wherein the biological sample is selected from the group consisting of tissue, cells, whole blood, feces, serum, plasma, saliva, cerebrospinal fluid and urine. 10. A composition for treating an erythroleukemia comprising the monoclonal antibody of claim 1 or 2 and a pharmaceutically acceptable carrier. 10. The method of claim 9,
Wherein said monoclonal antibody causes antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) of ICAM4-expressing leukemia cells. A hybridoma cell producing the monoclonal antibody of any of claims 1 or 2. 12. The method of claim 11,
Wherein said cell is the accession number KCLRF-BP-00303.

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