KR100388942B1 - A Single Chain Variable Fragment of an Antibody for Protein Expressed on Cell Surface of Human Cortical Thymocyte and Leukemic Cell - Google Patents

Abstract

The present invention relates to a recombinant antibody that specifically reacts with cell surface proteins expressed in human cortical thymic cells, and is expressed only in tumor cells of cortical thymic cells, leukemia, and T-lymphoblastic lymphoma. Segmented antibodies comprising an antigen recognition site that selectively recognizes and selectively binds JL-1 antigen, a protein having a molecular weight of about 120,000 Daltons (120 kDa) revealed through staining and flow cytometry, and a method of manufacturing the same and leukemia It is about the method used for the diagnosis and treatment of the.

Description

A Single Chain Variable Fragment of an Antibody for Protein Expressed on Cell Surface of Human Cortical Thymocyte and Leukemic Cells

The present invention relates to antibodies and their clinical applications that specifically react to surface proteins of human cortical thymic cells and leukemia tumor cells. More specifically, the present invention provides a protein having a molecular weight of about 120,000 Daltons (120 kDa) expressed on the surface of cortical thymic cells and tumor cells of leukemia and T-lymphocytic lymphoma (this protein is Korean Patent Application Publication No. 94-26067). Segmented antibodies comprising antigen-recognition sites (CDRs) that specifically respond to JL-1 antigens) and methods of making the same.

Recently developed antibodies against the JL-1 antigen (the antibody is designated as JL-1 antibody in Korean Patent No. 97-55593), which react with human thymic cells by injecting human thymic cells as antigens into white mice After producing and isolating the antibody, it was prepared by screening the antibody that reacts with the cortical thymocytes through immunohistochemical staining with the separated antibody, deposited hybridoma No. No. KCLRF-BP-00010 It is a monoclonal antibody produced by.

Existing single antibodies developed in the process of treating leukemia and tumors and to investigate their carcinogenic mechanisms have not undergone humanization of antibody-producing genes or fragmentation (single-chain variable region antibodies). A rabbit-derived antibody, which is recognized as a heterologous protein in the human body, which may induce hypersensitivity reactions or immunorejection reactions, and due to its large molecular weight, access to the site of action slows access to the site of action, There was a problem that was difficult to use as a diagnostic agent.

Therefore, in recent years, a single antibody derived from a mouse has a low immune rejection response in the human body such as a chimeric antibody, a humanized antibody, a single chain variable fragment (scFv), or a single chain variable region antibody, and has a high efficiency in antibody treatment. Attempts to administer to humans after transformation into recombinant antibodies are actively underway.

An object of the present invention is to provide a fragment antibody comprising an antigen recognition site of an antibody that recognizes and selectively binds a JL-1 antigen (named JL-1 antibody by Korean Patent No. 97-55593) and a linker linking them. It provides a method of manufacture and a method of using the same for the diagnosis and treatment of leukemia.

The JL-1 fragment antibody consists of a part of an antibody including an antigen recognition site that recognizes a protein expressed in human cortical thymic cells, in particular, can recognize the antigen specifically, the variable region including the antigen recognition site of the antibody It consists of a single-chain variable region in which the heavy and light chains corresponding to the linker are linked by a linker.

JL-1 antibody is a protein that can reveal the differentiation stage of cells, and specifically responds to proteins expressed only in cortical thymic cells and tumor cells of leukemia and T-lymphoblastic lymphoma. Tumor cells derived from thymic cells and hematopoietic organs can be diagnosed and treated effectively and easily.

1 is a genetic map of plasmid (pTFvSN) for producing the fragment antibody of the present invention.

Figure 2 is an electrophoresis picture of the fragment antibody of the present invention prepared using the plasmid shown in Figure 1.

Figure 3 is an enzyme immunoassay (EIA) results performed in Example 4.

Figure 4 is a genetic map of plasmid (pTFvCSN) to which the cysteine tag (Cys-tag) is attached to the fragment antibody of the present invention.

Fig. 5 is a genetic map of plasmid (pTFvASN) for attaching and expressing angiogenin sequence to JL-1 segmental antibody (scFv) of the present invention.

FIG. 6 shows electrophoresis (SDS-PAGE) of JL-1 fragment antibody (JL-1 scFv-Cys) and angiogenin-expressed antibody (JL-1 scFv-Ang) including the cysteine tag of FIGS. 4 and 5 (Left) and western blot (right).

7 shows enzyme immunoassay results of the JL-1 fragment antibody.

The fragment antibody of the present invention is a protein having a molecular weight of about 30,000 Daltons, and the base sequence having information of a diprotein is composed of 732 bases. This is a form in which the heavy chain (V _H ) and light chain (V _L ) variable region proteins, each containing three antigen recognition region sequences capable of recognizing the JL-1 antigen, are linked in a single chain by a linker. .

The manufacturing method of the fragment antibody in this invention is as follows. MRNA was purified from a hybridoma cell line secreting a single antibody against the JL-1 antigen (deposited under Accession No. KCARF-BP-00010H to the Korea Cell Line Research Foundation) (Quick Prep mRNA Purification Kit). : Purified by Pharmacia, Sweden, and amplified by reverse transcriptase-polymer (RT-PCR: Reverse Transcriptase-Polymerase Chain React-ion) of the immunoglobulin heavy chain and light chain PCR was performed again with the DNA linker having the information of two variable region base chains Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser Connect it. The DNA thus prepared is inserted into a pCANTAB5E vector (product of Swedish Pharmacia) to infect E. coli for phage screening. Phage recovery using M13K07 supplemental phage yields phages in which a single chain variable region protein is conjugated to the gIII protein of pCANTAB5E. These phages were plated on purified JL-1 antigen (Molt-4 cells or thymic cells) onto an enzyme immunoassay plate to repeat the binding and washing of antigen and phage expressing antibodies. How to select only the antibody with good binding ability) to screen for the antibody and good binding ability. Candidate clones are obtained through the insertion of the selected antibody gene in E. coli, and the clones showing affinity to the JL-1 antigen are selected by Western blot and enzyme immunoassay, and the nucleotide sequence is confirmed by sequencing. The DNA obtained from these phages is then used as a soluble single chain variable region protein in several possible bacterial expression systems to obtain the desired fragment antibody. The protein of the present invention has the amino acid sequence of SEQ ID NO: 1 and the DNA of the present invention has the nucleotide sequence of SEQ ID NO: 2.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are provided only to explain the present invention more easily, and the scope of the present invention is not limited to these examples.

Example 1 Extraction of mRNA from Hybridoma Cells Secreting Antibody to JL-1 Antigen

1.5 ml of extract buffer (buffer solution containing guanidine thiocyanate and N-lauryl sarcosine) was added to 1 × 10 ⁷ centrifuged hybridoma cells, and the whole DNA was digested by inhalation with a syringe. . 3 ml of elution buffer solution (10 mM Tris. PH 7.4,1 mM EDTA) was added and again aspirated several times with a syringe. The cell homogenate was centrifuged to separate only the supernatant and stored.

Purified mRNA was obtained from the supernatant using an mRNA purification kit (Sweden Pharmacia), and then the absorbance at 260 nm and 280 nm was measured to confirm the purity and purified amount of mRNA.

Example 2 Synthesis of cDNA, Amplification of Heavy and Light Chain Variable Regions and Preparation of Single Chain Variable Regions

CDNA was obtained from mRNA using a first strand cDNA synthesis kit (manufactured by Swedish Pharmacia), and amplified heavy and light chain cDNA by PCR. Then, each reaction solution amplified by PCR of heavy and light chain cDNA was extracted with phenol / chloroform and quantified by 1% agar gel electrophoresis. Thereafter, gel extraction of each cDNA was performed to recover the amplified DNA. After using the heavy chain and light chain variable region cDNA and linker PCR primers (linker PCR) to perform the linker (linker PCR) by PCR and the resulting single chain variable region (V _H + linker + V _L ) Once again amplified using PCR. At this time, the Sfi I restriction enzyme site was added to the 5 ′ region of the posterior primer and the Not I restriction enzyme site was added to the 5 ′ region of the translocation primer to facilitate cloning into the pCANTAB5E vector (Sweden Pharmacia).

Example 3: Fabrication of a phage display library in which a single chain variable region is bonded

250ng of the single-chain variable region cDNAA 200k and 250CVCA vector obtained from the above procedure were cut into Sfi I and Not I, and the cut vector and single-chain variable region base chain were subjected to 1% agar gel electrophoresis, followed by gel extraction for each DNA band. Purification by After binding the cDNA to the cut vector and the single chain variable region (Maniatis et al. 1989. Molecular cloning: A Laboratory manual. CSH Lab., NY, USA), the DNA was recovered and infected with the host cell Escherichia coli XL1-Blue. Here, 3 ml of SOC medium (2% bacto-tryptone, 0.5% bacto-yeast extract, 0.05% NaCl, 20 mM glucose) was added and incubated at 37 ° C. for 1 hour, followed by 20 μg / ml carbenicillin, tetra Incubated for 1 hour in SB medium (3% bacto-tryptone, 2% bacto-east extract, 1% Mops 94% distilled water, pH 7.0) to which 10 μg / ml of tracycline was added and helper phage M13K07 Was added by 10 ¹² plaques and incubated for 2 hours. Afterwards, 70 μg / ml of kanamycin was added and cultured at 37 ° C. for 15 hours to recover phage. The culture solution was centrifuged at 4000 rpm for 15 hours, and then polyethylene glycol 8000 was added to the supernatant to a final concentration of 4% (w / v), sodium chloride was added to a concentration of 3% (w / v), and allowed to stand on ice for 30 minutes. After centrifugation at 4 ° C. for 20 minutes, the precipitated phage is dissolved in 2 ml of PBS (phosphate buffered saline: 50 mM phosphoric acid, pH 7.2, 150 mM, sodium chloride) and centrifuged for 3 minutes in a microcentrifuge to remove residues from bacteria. Was removed.

A package the phagemid (packaged phagemid) purified as above in SB medium to 10 ^-3, 10 ^-5, and diluted by a factor of 10 ^-8 take 1μl, just manufactured E. coli XL1- Blue of 50μl (at OD 660nm = Mixed with 1). This was incubated for 15 minutes in SB medium containing 10 μg / ml of tetracycline and then applied to Luria Bertani / carbenicillin (Luria Bertani / carbenicillin) plate medium. After culturing for 15 hours, the number of phages was determined by counting colonies. Microtiter plates were coated with 25 μl of JL-1 antigen solution (10 μg / ml in 100 mM bicarbonate buffer, pH 8.6, such as thymic cells or Molt-4 cells) for 12 hours per well. Thereafter, each well was blocked with 200 μl of 1% BSA / PBS solution. 50 μl of phage display library (typically 10 ¹¹ plaques) was added to each well and incubated at 37 ° C. for 2 hours. The plate was first washed once with distilled water and then at least 10 times over 1 hour with a solution comprising 50 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 0.5% Tween 20. The plate was washed once again with distilled water, and the attached phage was added to 50 μl of elution buffer (0.1 M hydrochloric acid, adjusted to pH 2.2 with solid glycine and added 0.1% BSA) and incubated for 10 minutes at room temperature. The solution from each well was then mixed well several times with a pipette and collected in one tube. Thereafter, 3 μl of 2M tris-hydrochloric acid solution was added to each 50 μl and neutralized. The recovered phage was added to 2 ml of immediately prepared E. coli XL1-blue (OD at 600 nm = 1), and allowed to stand at room temperature for 15 minutes for infection, followed by carbenicillin (20 µg / ml) and tetracycline (10 µg / ml). 10 ml of containing SB medium was added. After another one hour of incubation at 300rpm, 37 ° C, 100 ml of carbenicillin (50 μg / ml) and tetracycline (10 μg / ml) containing SB medium was added and incubated for 1 hour. Supplemental phage M13K07 (10 ¹² plaques) was added thereto and incubated for 2 hours. Kanamycin (70 μg / ml) was then added and incubated at 37 ° C. for 12 hours. Since phage preparation and panning was the same as the above process, the phage culture rate was repeated until it is no longer high. Phage culture rate was calculated by dividing by the number of phage added to the number of culture phage.

The phage selected in the last panning was infected with E. coli XL1-Blue in the same manner, and incubated at 37 rpm for 1 hour by adding 10 ml of SB medium. 100 μl of this was taken and diluted to 1/10, 1/100, and 1/1000, and 100 μl of each was plated on LB / carbenicillin plate medium, and then incubated at 37 ° C. for 12 hours. Each well of the sterilized microtiter plate was loaded with SB medium containing 100 μl of carbenicillin (50 μg / ml) and tetracycline (10 μg / ml), and then phage on the LB / carbenicillin plate medium in each well. The colonies were transferred and incubated for 12 hours with slow shaking. Meanwhile, the auxiliary phage M13K07 phage (1.05 × 10 ¹⁰ plaques) was diluted in an SB medium containing 21 ml of carbenicillin (50 μg / ml) and tetracycline (10 μg / ml), and then immediately sterilized microtiter. Put 200 μl into each well of the plate. As described above, 20 μl of the culture medium containing the phage in which the antibody was cloned is transferred to each well of the plate containing the auxiliary phage. Incubate at 37 ° C. for 2 hours with slow shaking, then centrifuge the plate. All supernatants were aspirated off and 200 μl of SB medium containing kanamycin was added to each well. After incubation at 37 ° C. for 2 hours with slow shaking the plates were centrifuged. Some of the supernatant containing the packaged phage was well stored, and enzyme immunoassay was performed using only 50 μl of the supernatant.

Example 4 Phage Selection Using Enzyme Immunoassay

100 μl of 1 mg / ml poly-L-lysine-hydrobromide (TypeI-B, molecular weight 400,000, dissolved in PBS) per microwell platter was added and removed after 30 minutes. Thereafter, malt-4 cells (ATCC CRL-1582), a cell line expressing JL-1 antigen, were washed twice with DMEM solution, and then adjusted to 5 × 10 ⁶ / ml. 100 μl of this cell solution was added to each well and centrifuged. The solution was removed after standing at room temperature for 15 minutes. Thereafter, the washing process was repeated three times to add and remove 350 μl of PBS buffer solution per well. Each well was then blocked with 200 μl of 1% BSA / PBS solution. 50 μl of the phage supernatant obtained in Example 3 was added to each well and maintained at 37 ° C. for 2 hours. The plate was first washed once with distilled water and then with a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM sodium chloride and 0.5% Tween 20. The plate was washed once with distilled water, and 50 μl of each of the anti-M13 phage antibodies (Horseradish peroxidase conjugated sheep anti-M13 phage antibody (made in Sweden Pharmacia) diluted in 1 / 1,000 in 1% BSA / PBS was added to each well and 37 ° C. It was kept for 2 hours at. The plate was first washed once with distilled water and then with a solution containing 50 mM tris-hydrochloric acid (pH 7.5), 150 mM sodium chloride and 0.5% Tween 20. The plate was washed once again with distilled water and a color substrate solution containing 50 μl of O-phenylenediamine was added. After sufficient color development, 50 µl of 0.5N sulfuric acid solution was added to stop the color reaction. The developed solution was transferred to an enzyme immunoassay plate, and then absorbance was measured at a wavelength of 495 nm using an enzyme-linked immunoassay analyzer.

Competence (reaction) enzyme immunoassay (competition EIA) is almost the same method as above, but the phageclone and anti-JL-1 antibody to see the tendency to bind to the antigen, was performed as follows. When the phage supernatant was added to the enzyme immunoassay well, the anti-JL-1 antibody was added to a final concentration of 10 μg / ml per clone and the wells to which the anti-JL-1 antibody was not added. It was done. Among them, Western blot and sequencing were performed by selecting three clones with a large difference in absorbance compared to the other clones and a large difference in absorbance between the wells with and without JL-1. This absorbance analysis data is shown in FIG.

Example 5: Determination of the base sequence of the heavy chain and light chain variable region

In this example, three phage clones identified as positive on enzyme immunoassay and Western blot were selected to obtain DNA in phagemid state, followed by sequencing. The sequencing primer was used as the base sequence of the pCANTAB5E vector and the base sequence of the linker. The obtained nucleotide sequence was analyzed using the DNAsis program to determine the V _H and V _L sequences of the JL-1 antibodies shown in SEQ ID NO: 1 and SEQ ID NO: 2. In the case of V _H belonging to the subgroup: IIA, family: V, it was confirmed that the same base sequence has not been reported so far in the blast search (Blast sear_ch). For V _L , a subgroup; V, family; It was confirmed that the same base sequence belonging to XIII and also in blast search has not been reported to date.

Example 6; Preparation of Segmented Antibody Expression Vectors

In order to mass express the JL-1 fragment antibody, the variable region antibody gene cloned in the pCANTAB5E vector was transferred to the pTFvSN plasmid vector and replicated. This pTFvSN (Fig. 1, Accession No. KCTC 0791BP) can be obtained by binding the variable region antibody gene to the pRSET B vector (commercially available from Invitrogen, Inc.). The pCANTAB5E vector and pTFvSN vector, which were cloned with the JL-1 single chain gene, were digested with Sfi I and Not I restriction enzymes, and then subjected to 1% agar gel electrophoresis. After extracting each DNA from the gel to recover the variable region antibody gene and vector, and transfer the plasmid obtained by binding to the E. coli BL21 (DE3) strain to produce the fragment antibody of the present invention (Accession No. KCTC 0790BP) Was prepared.

In the same way, the JL-1 fragment antibody gene was cloned into the pTFvCSN vector (FIG. 4) to facilitate the technetium-99m labeling. scFv-Cys) synthetic vector was made. In addition, the JL-1 fragment antibody gene was cloned into the pTFvASN vector (FIG. 5), and a JL-1 fragment antibody (JL-1 scFv-Ang) in which angiogenin, a human-derived toxin, was attached to the C-terminus in the form of a fusion protein. Expression vectors were made. JL-1 scFv-Ang can be viewed as a kind of immunotoxin.

Example 7 Expression of Recombinant JL-1 Segmented Antibody

The recombinant JL-1 antibody plasmid in Example 6 was transferred to E. coli BL21 (DE3) (Takara, Japan) suitable as antibody overexpressing strain. This was fermented in a fermentor containing Luria Bertani medium (IPTG 0.3mM, 30 ° C) for 10 hours at 200 rpm, and in the process, the antibody was synthesized in the form of an inclusion body in the strain. It became.

Example 8: Purification of Recombinant JL-1 Segmented Antibody

The conditions for obtaining the purified solution single chain variable region protein from the inclusion body (inclusion body) are as follows. First, the strains induced with antibody expression were suspended in crushing buffer (50 mM Tris-HCl, 1.0 mM EDTA, 100 mM potassium chloride, 0.1 mM PMSF, pH 8.0), and then sufficiently disrupted. The E. coli lysate was centrifuged and the supernatant was removed. This washing process was repeated six times to obtain an inclusion body pellet.

Dissociation buffer (6M guanidine hydrochloride, 50 mM Tris-hydrochloric acid, 10 mM calcium chloride, 50 mM potassium chloride, pH 8.0) was added to complete dissolution and centrifugation to obtain supernatant. After measuring the absorbance at 280 nm, the dissociation complete layer solution was added so that the absorbance became 25 or less. Slowly add refolding buffer (50 mM Tris-HCl, 10 mM calcium chloride, 50 mM potassium chloride, pH 8.0) to 10 times the original volume. The addition time was 2 hours or more, left to stand for 20 hours or more, and dialyzed into the refolding buffer.

Ammonium sulfate was added to the dialysis solution to 40% saturation concentration and centrifuged to obtain a supernatant. Ammonium sulfate was added to the supernatant to 80% saturated concentration and centrifuged to obtain a precipitate. The precipitate was suspended in 100 ml of PBS and the protein amount was quantified. In addition, the purity was confirmed through sodium dodecyl sulfate polyacrylamide gel electrophoresis. Figure 2 shows the electrophoresis picture of sodium dodecyl sulfate polyacrylamide gel confirming JL-1 scFv expressed using the pTFvSN vector. Cell lysate and purified samples showed that the JL-1 scFv band was absent in BL21 (DE3) Escherichia coli. Sodium dodecyl sulfate polyacrylamide of JL-1 (JL-1 scFv-Cys) containing cysteine tag and angiogenin-expressed antibody (JL-1 scFv-Ang) using the plasmid shown in FIGS. 4 and 5 Figure 6 shows a Western blot photograph (right) performed using an gel electrophoresis photograph (left) and an antibody that recognizes an express tag contained in the JL-1 antibody. In both photographs, it was confirmed that the JL-1 scFv band, which was not visible in BL21 (DE3) cell lysate, was formed at the position of the desired molecular weight.

Example 9: Analysis of affinity of JL-1 fragment antibody against JL-1 antigen

The JL-1 fragment antibody prepared in Example 8 was examined to show affinity for the JL-1 antigen. 100 μl of 1 mg / ml poly-al-lysine hydrobromide per microtiter plate was added and removed after 30 minutes. Thereafter, malt-4 cells, a cell line expressing the JL-1 antigen on the cell membrane surface, were washed twice with DMEM solution, and then adjusted to a concentration of 1 × 10 ⁷ / ml. 100 μl of this cell solution was added to each well and centrifuged. The solution was removed after standing at room temperature for 15 minutes. Thereafter, the washing process was repeated three times, adding and removing 350 μl of buffer solution (PBS) for each well. 100 μl of 2% glutaaldehyde solution was added thereto for about 30 minutes to fix the cells. Each well was then blocked with 200 μl of 1% BSA / PBS solution. Thereafter, the JL-1 fragment antibody obtained in Example 10 was mixed in a 1: 1 ratio with 6% BSA-containing buffer solution (PBS) by concentration, and 50 μl was added to each well, and maintained at 37 ° C. for 2 hours. The plate was first washed once with distilled water and then with a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM sodium chloride and 0.5% Tween 20. After the treatment, the alpha-express antibody that can recognize the express tag of the JL-1 fragment antibody attached to the JL-1 antigen was mixed with 3% BSA-containing buffer (PBS) in a ratio of 1: 5,000 and added to each well. After incubation at room temperature for 2 hours, the cells were washed as described above. To generate an enzyme reaction against the antibody, 50 μl of horseradish peroxidase (Pharmacia, Sweden) diluted in 1/500 in 3% BSA / PBS in each well was added to each well. Kept for hours. The plate was washed once with distilled water and with a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM sodium chloride and 0.5% Tween 20. 1-STEP ABTS substrate solution (product of Pierce Co., Ltd.) was added to develop a color reaction, and after sufficient color development, the developed solution was transferred to a new enzyme immunoassay plate and absorbance was measured at 405 nm with an enzyme-linked immunoassay absorbance analyzer. . JL-1 fragment antibody concentration, mean absorbance, and standard deviation measured through the above procedure are shown in FIG. 7.

Example 10 Preparation of Immunotoxins Using JL-1 Segmented Antibody

In this embodiment, JL-1 immunotoxin was prepared by combining toxic proteins such as gelonin, angiogenin, lysine and the like for the clinical application of leukemia treatment of JL-1 fragment antibody. . Thus prepared JL-1 immunotoxin can obtain a therapeutic effect by selectively binding and removing leukemia cells. The binding of the JL-1 fragment antibody and the toxic protein was performed using the SPDP method provided by Pierce, USA.

SPDP (US Pierce, N-succinimidy3- (2-pridyldithio) propiionate) 3.Omg / ml aqueous solution or sulfo-LC-SPDP (US Pierce, Sulfosuccinimidyl 6- [3- (2-pyridyldithio) propionamido] hexanote) 5.2 mg / ml aqueous solution was prepared. It acts to activate the antibody to react easily with the amine groups in the antibody protein to form double sulfur bonds. The JL-1 fragment antibody was dissolved in physiological saline at pH 7.4 at a concentration of 10 mg / ml, and then mixed with the previously prepared SPDP solution or sulfo-LC-SPDP solution at a volume ratio of 10: 1 (SPDP solution: antibody solution) at room temperature for 30 minutes. The reaction was carried out and purified by gel filtration chromatography (using a Sephadex G-25 column manufactured by Pharmacia Sweden).

Triethanolamine-EDTA buffer (Triethanolamine-EDTA buffer.TEA-HCl 50mM, EDPA 10mM, pH 8.0) removes dissolved gases by nitrogen injection at 0.33mM concentration and 20mg / ml 2-iminothiolane (iminothiolane) ) A thiol group was introduced into the toxic protein by reacting with an aqueous solution (traut's reagent, manufactured by Pierce, USA) at a volume ratio of 100: 7 (toxic protein: 2-Iminothiolane) for 1 hour, and purified by gel filtration chromatography.

After reacting the above toxic protein and the activated antibody at a molar ratio of 5: 1 for 20 hours in a refrigerated state, 2mM iodoacetamide was added and the reaction was stopped at room temperature for 1 hour to stop the reaction. The prepared immunotoxin was confirmed by the production of sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then purified by gel filtration chromatography and ion exchange chromatography to obtain JL-1 immunotoxin.

Example 11: Radioisotope Iodine-125 Labeling with JL-1 Segmented Antibody

Radioactive isotopes were labeled on JL-1 fragment antibodies for the application of JL-1 fragment antibodies for radiographic imaging and radiotherapy. The labeling method was carried out as described in the literature (Radio- isotope in Biology; A Practical Approch, Slater, IRL Press) and in the instructions for use of the relevant radioisotope kit (Amersham Corporation MDP kit).

Iodine-125 was labeled using Chloramine-T method (Radioisotope in Biology; A Practical Approach, R. J. Slater, IRL Press) such as Greenwood. First, 50 μl of a JL-1 fragment antibody solution at a concentration of 2 mg / ml and 500 μl of 0.5 M sodium phosphate solution at pH 7.5 were mixed, and then 100 μCi of iodine-125 was added thereto. 3 μl of a solution of Chloramine-T (1 mg / ml) dissolved in 0.05 M sodium phosphate solution at pH 7.5 was gently mixed, reacted at room temperature for 2 minutes, and dissolved in sodium metabidium at 0.05 M sodium phosphate solution at pH 7.5. The reaction was stopped by adding 7 μl of a solution of sodium metabisulphite dissolved at a concentration of 1 mg / ml. Since the Sephadex G-25 PD-10 column (Sweden Pharmacia Co., Ltd.) was purified by radiolabeled antibody can be obtained.

Example 12 Radioisotope Technium-99m Labeling on JL-1 Segmented Antibodies

Technicium-99m label was performed as follows. To 2 mg of antibody, 12 μl 0.3 M EDTA solution with pH 7.0 and 12 μl 1.0 M sodium bicarbonate solution were added and mixed well. 15 μl of 1.5 M beta-mercaptoethanol was added thereto, followed by 30 The reaction was reduced for a minute to reduce the antibody. The antibody thus reduced was purified using Sephadex G-25 PD-10 column (manufactured by Swedish Pharmacia), filled with nitrogen gas, and stored and used at -70 ° C. 5 ml of physiological saline was added to the reagent for the radioisotope labeling kit manufactured by Amersham, USA, and 150 μl of the aliquot was added to 200 μg of the reduced and stored antibody, followed by 200mCi / ml of technium-99m pertechnate

technetate) was added and reacted at room temperature for 30 minutes to label technicium-99m on the fragment antibody of the present invention.

Example 13: Radioactive Isotope Rhenium-188 Labels on JL-1 Segmented Antibodies

The rhenium-188 label to the fragment antibody of the present invention was prepared by the use of a tin: tartrate molar ratio of 1: 9, and a reduced antibody that prepared and stored the amount corresponding to 422 μg of tin ions in the above. Rhenium-188 was added thereto at a concentration of 20 mCi / ml and reacted for 1 hour at room temperature to prepare a fragment antibody of JL-1 labeled with rhenium-188.

The antibody labeling efficiency of the radioisotope prepared in Examples 11, 12, and 13 was calculated by measuring the radioactivity of each portion of the chromatography separated and developed by thin layer chromatography (TLC) with an 85% methanol solution. Labeled antibodies can be purified by gel filtration chromatography.

Since the fragment antibody of the present invention has a molecular weight of about 30,000 Daltons, which is about 1/4 of the molecular weight of the JL-1 antibody disclosed in Korean Patent Application No. 97-55593, it is much easier to access the site of action. There is no constant region, which greatly reduces the body's immune rejection reaction, and isotopes, toxins, genes, etc. can be attached to the C-terminus of the JL-1 fragment antibody for diagnosis and treatment. Application is significantly easier than conventional JL-1 antibodies.

The present invention is intended to develop and industrially apply chimeric antibodies, humanized antibodies, single chain variable region antibodies comprising the V _H and V _L regions and antigen recognition site sequences, which are the most important recognition sites of antibodies that recognize the JL-1 antigen. Can be. On the other hand, the identification of such an antigen recognition region has an effect that can also contribute to the development of antibodies with higher antigen affinity through modification of some amino acids of this region.

Claims (7)

JL-1 fragment antibody (Accession No. KCTC 0790BP) represented by the amino acid sequence set forth in SEQ ID NO: 1. The JL-1 fragment antibody according to claim 1, wherein the radioisotope is bound to a terminal. 3. The JL-1 fragment antibody according to claim 2, wherein the radioisotope is a radioisotope selected from the group consisting of iodine (125), technium (99m) and rhenium (188). The JL-1 fragment antibody according to claim 1, wherein the toxic protein is terminally bound. Plasmid (pTFvSN) represented by the nucleotide sequence set forth in SEQ ID NO: 2 (Accession No. KCTC 0791BP). Purifying mRNA in a cell line (KCARF-BP-00010H), Amplifying the variable region of the heavy chain and the light chain by reverse transcriptional PSA method by preparing cDNA from the purified mRNA, Connecting the variable region of the heavy chain and the light chain variable region with a DNA linker Method for producing an anti-JL-1 recombinant segmental antibody comprising a. Inserting randomly synthesized recombinant DNA into a phage expression vector for selection, Infection with the host strain to prepare a phage display library, Binding the JL-1 antigen to the phage display library prepared above to obtain DNA of high binding phage, Analyzing the nucleotide sequence of the phage thus obtained Method of confirming the gene sequence in the JL-1 antibody comprising a.