KR100528721B1 - A mouse monoclonal antibody specific for the human asialoglycoprotein receptor, the hybridoma cell line secreting this antibody and the generation and verification method thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody, a fusion cell line secreting the same, and a method for preparing the human desialal group-glycoprotein receptor, and more particularly, a human desialal group-glycoprotein receptor (ASGPR) that is a hepatocyte specific expression protein. 1) An antibody that specifically reacts to a H1 subunit, and relates to a fusion cell line which produces a monoclonal antibody using a peptide antigen selected from the extracellular membrane region of ASGPR, and to produce the same.

Description

Mouse monoclonal antibody specific for the human asialoglycoprotein receptor, the hybridoma cell line secreting this antibody and the generation and verification method

The present invention relates to a monoclonal antibody, a fusion cell line secreting the same, and a method for preparing the human desialal group-glycoprotein receptor, and more particularly, a human desialal group-glycoprotein receptor (ASGPR) that is a hepatocyte specific expression protein. 1) An antibody that specifically reacts to a H1 subunit, and relates to a fusion cell line which produces a monoclonal antibody using a peptide antigen selected from the extracellular membrane region of ASGPR, and to produce the same.

ASGPR functions as an endocytic receptor that captures and transports desylylated glycoproteins to lysosomes as the protein ages, and is primarily used in liver tissue. Distribution [Stockert, RJ. (1995) The asialoglycoprotein receptor; relationships between structure, function, and expression. Physiol. Rev. 75: 591-609. The protein is a hetero-oligomeric complex consisting of H1 and H2 subunits [Yik JH, Saxena A, Weigel PH. (2002) The minor subunit splice variants, H2b and H2c, of the human asialoglycoprotein receptor are present with the major subunit H1 in different hetero-oligomeric receptor complexes. J. Biol. Chem. 277 (25): 23076-23083], in the case of H2 monomers are known to be in the form of splice variants such as H2a, H2b, and H2c. It was confirmed that H2b and H2c isoforms do not coexist in the same ASGPR conjugate, and the types of recycling receptors that mediate the foreign body absorption process are different according to the difference of ASGPR conjugate due to the difference of these monomer combinations. This is known. Endocytosis by ASGPR proceeds with a coated pit pathway mediated by c-src tyrosine kinase [Parker A, Fallon RJ. (2001) c-src tyrosine kinase is associated with the asialoglycoprotein receptor in human hepatoma cells. Mol Cell Biol Res Commun. 4 (6): 331-336.

ASGPR is a cell receptor function against human hepatitis B virus (HBV) and Marburg virus in addition to its original function in hepatocytes [Treichel U., Meyer zum Buschenfelde KH., Stockert RJ. , Poralla T., Gerken G. (1994) The asialoglycoprotein receptor mediates hepatic binding and uptake of natural hepatitis B virus particles derived from viraemic carriers. J. Gen. Virol. 75 (Pt 11), 3021-3029; Becker S., Spiess M., Klenk HD. (1995) The asialoglycoprotein receptor is a potential liver-specific receptor for Marburg virus. J. Gen. Virol. 76 (Pt 2) 393-399], the role of erythroagglutination [Stockert RJ., Morell AG., Scheinberg IH. (1974) Mammalian hepatic lectin. Science 186 (4161) 365-366]. In addition, hepatitis A-specific IgA mediated the hepatocellular invasion process of hepatitis A virus by ASGPR [Dotzauer A., Gebhardt U., Bieback K., Gottke U., Kracke A., Mages J., Lemon SM., Vallbracht A. (2000) Hepatitis A virus-specific immunoglobulin A mediates infection of hepatocytes with hepatitis A virus via the asialoglycoprotein receptor. J Virol. 74 (23): 10950-10957] and mediated hepatitis C virus (HCV) hepatocyte specific infection [Saunier B., Triyatni M., Ulianich L., Maruvada P., Yen P., Kohn LD. (2003) Role of the asialoglycoprotein receptor in binding and entry of hepatitis C virus structural proteins in cultured human hepatocytes. J Virol. 77 (1): 546-559.

In addition, based on the fact that the expression of ASGPR increases when hepatic cell death progresses in studies on the association between liver disease and apoptosis, serum-soluble ASGPR can be used as a marker for apoptosis of liver tissue. And can be used to diagnose liver disease [Kakegawa T, Ise H, Sugihara N, Nikaido T, Negishi N, Akaike T, Tanaka E. (2002) Soluble asialoglycoprotein receptors reflect the apoptosis of hepatocytes. Cell Transplant 11 (5): 407-415. There is also a report on the association with liver cancer, and it has been confirmed that ASGPR is expressed and maintained in the majority of differentiated liver cancer cells. Based on these facts, a method of administering ASGPR-mediated hepatocellular carcinoma cells with a DNA synthesis inhibitor on a galactosyl group carrier has been suggested as a method of cancer cell control [Trere D, Fiume L, De Giorgi LB, Di]. Stefano G, Migaldi M, Derenzini M. (1999) The asialoglycoprotein receptor in human hepatocellular carcinomas: its expression on proliferating cells. Br J Cancer. 1999 Oct; 81 (3): 404-408.

Anti-ASGPR antibodies have been found among several autoimmune antibodies found in autoimmune chronic active hepatitis (AIH) and primary biliary cirrhosis (PBC) [Yoshioka M, Mizuno M, Morisue Y, Shimada M, Hirai M, Nasu J, Okada H, Sakaguchi K, Yamamoto K, Tsuji T. (2002) Anti-asialoglycoprotein receptor autoantibodies, detected by a capture-immunoassay, are associated with autoimmune liver diseases. Acta Med Okayama. 56 (2): 99-105].

In summary, verification of ASGPR expression can be used as a marker for hepatocyte differentiation and proliferative apoptosis, and hepatocyte specific expression may be useful for tissue-specific drug delivery or gene therapy. . Therefore, securing specific antibodies to ASGPR will be an essential factor in developing the above techniques. Until now, when using these techniques, in most cases, a carrier having a galactosyl group has been used as a specific reactant for ASGPR. On the other hand, several ASGPR specific antibodies have been reported, but most of them are polyclonal antibodies and the only foreign monoclonal antibodies reported [Kohgo Y, Kato J, Nakaya R, Mogi Y, Yago H, Sakai Y, Matsushita] H, Niitsu Y. (1993) Production and characterization of specific asialoglycoprotein receptor antibodies. Hybridoma. 12 (5): 591-598] were antibodies prepared using ASGPR purified from hepatocytes as antigens, but were not reactive with ASGPR in rats or rabbits, but had cross-reactivity with ASGPR in mice.

Accordingly, the present inventors have various immunological methods that can verify the deshial group-glycoprotein receptor (ASGPR), namely Western blotting, ELISA, flow cytometry, and immunohistochemistry. To obtain a monoclonal antibody that can be used both, etc., for this purpose, while the equivalent of the carbohydrate-recognition domain (CRD) that plays a functional role in the extracellular membrane region of human ASGPR, Antigen sites with the largest difference were selected and prepared as synthetic peptides and injected with the peptide antigens to prepare monoclonal antibodies against them.

The present invention relates to an anti-ASGPR monoclonal antibody that can be used for the detection and neutralization of a human asialoglycoprotein receptor (ASGPR), a hepatocyte specific expression protein. It features.

The present invention also features an anti-ASGPR antibody secreting fusion cell line using a peptide antigen selected from the extracellular domain of ASGPR, and a method for preparing the same.

In addition, the present invention is characterized by the expression vectors of the maltose binding protein fusion expressing the extracellular membrane region of ASGPR, and the anti-ASGPR antibody using ASGPR-recombinant proteins c2 and c3 produced from the E. coli transformants of the expression vector. Include identification methods.

The present invention also includes a diagnostic reagent for detecting and testing expression of ASGPR H1 in vivo using the anti-ASGPR monoclonal antibody produced from the above.

Referring to the present invention in more detail as follows.

The present invention is an antibody that specifically reacts with a human ASGPR H1 subunit, which is a hepatocyte specific expression protein, to produce a monoclonal antibody using a peptide antigen selected from an extracellular membrane region of ASGPR, and to produce a fusion cell line that produces the same. It relates to a manufacturing method thereof.

The characteristics of the 291 amino acid sequences of the ASGPR H1 subunit were examined to select sites suitable for specific antibody production in human ASGPR sequences. Most receptor protein sequences can be divided into extracellular domains, membrane insertion domains, and intracellular domains. ASGPR H1 is a type II membrane protein, The carboxyl terminus of the protein is directed out of the cell membrane. In order to select a site to be used as an antigen for immunization of the mouse, the following items were considered. First, in order to increase the utilization of the antibody to the receptor, we tried to select high antigenic sites from the extracellular membrane. In addition, since the experimental animal to produce an antibody is a mouse, the site | region in which the sequence crossover with the ASGPR of a mouse is minimum among the human ASGPR sequences was selected. Finally, the sequence overlapping with the carbohydrate recognition domain corresponding to the ligand binding site of ASGPR is selected among these candidate sites, and functional experiments such as future substrate binding neutralization using monoclonal antibodies are performed. It was also intended to be available. As a result, a 30 mer peptide sequence (SEQ ID NO: 3: CRLEDAHLVV VTSWEEQKFV QHHIGPVNTW) of 182 to 211 sites in the human ASGPR sequence was determined as the antigenic site, and the epitope was named "PJ-3" [Fig. One]. For its immunization, this amino acid sequence was subjected to a solid phase method using Fmoc chemistry [Merrifield RB. (1969) Solid-phase peptide synthesis. Adv Enzymol Relat Areas Mol Biol. 1969; 32: 221-296. Ovalbumin was used as a carrier protein for immunization of the synthesized peptides, and synthetic peptides were linked to amino and sulfonyl groups using sulfo-MBS (m-Maleimido benzoyl-N-hydroxy-sulfosuccinimide ester). Bound to the carrier protein.

To prepare a monoclonal antibody-producing fusion cell line, mice (BALB / c) were immunized with a peptide antigen and co-antigen mixed emulsion three times at two week intervals [FIG. 2]. Splenocytes were extracted and subjected to cell fusion with mouse myeloma cells. After cell fusion, fused medium was added to select fused cells. After HAT medium selective colonies were formed, antibody-positive positive clones were selected by ELISA using synthetic peptide as antigen for cell culture medium of HAT medium selective colonies. Limited dilution was performed to separate the monoclones from the positive clones selected in the above procedure. Through this process, the monoclonal antibody finally confirmed anti-ASGPR specificity was named Assa-1, and it was confirmed that it has a kappa light chain as an IgG ₁ type. The mouse B-cell fusion cell line secreting it was deposited with the Korea Biotechnology Research Institute Gene Bank on Dec. 27, 1999 (Accession No .: KCTC 0717BP).

In order to confirm the reactivity of Assa-1, amino acids 62 to 291 corresponding to the extracellular membrane of ASGPR were prepared in a fused form with maltose binding protein. To this end, the corresponding region of ASGPR H1 cDNA was cloned into the pMAL vector, and the sequence analysis of the plasmid gene of several clones obtained through this process showed that c2 clone and one base sequence having the sequence of normal human ASGPR H1 monomer were obtained. This mutated c3 clone could be identified. The c3 clone is a 611 nucleotide sequence A of the ASGPR transcript is replaced with G (CAC-> CGC), resulting in the 204 amino acid sequence is substituted for histidine to arginine (Fig. 4a). This region overlaps the CRD of the ASGPR and the antigen peptide written as immunogen, corresponding to the antigen recognition region of Assa-1. Therefore, in the present invention, in order to determine whether the variation of the amino acid sequence according to the difference between the C2 and C3 clone correlated with the reactivity of Assa-1, a recombinant protein expressed from the two genes was prepared by the following method and the reactivity to these Western blotting (Western Blotting) and confirmed by EILSA (Fig. 4b). As a result, both ASGPR clones c2 and c3 fused to maltose binding protein by genetic recombination are all monoclonal antibodies specific for maltose binding protein HAM-19 [Park JH, Choi EA, Cho EW, Hahm KS, Kim KL. (1998) Maltose binding protein (MBP) fusion proteins with low or no affinity to amylose resins can be single-step purified using a novel anti-MBP monoclonal antibody. Mol Cells. 1998 8 (6): 709-716, Assa-1, an anti-ASGPR specific antibody prepared in the present invention, specifically recognized the c2 clone protein, but did not recognize the c3 clone, an amino acid sequence variant. I could not. It did not react at all with the maltose binding protein-beta-galactosidase alpha peptide used as a negative control. These results suggest that Assa-1 is an antibody specific for the 182-211 region of ASGPR, and has one sequence variation (Histidine sequenced to Arginine) of the antigen recognition region, such as c3 clone protein. It was also found that it is a monoclonal antibody having a very high specificity.

In addition, ELISA was performed to confirm the antigen specificity of the monoclonal antibody Assa-1. As a result, Assa-1 showed an antibody-antigen concentration-dependent response pattern only for the ASGPR c2 clone, and showed no responsiveness to the c3 protein and the maltose-binding protein-beta-galactosidase alpha peptide, which are sequence variants of ASGPR. . The minimum antigen concentration showing the reactivity of Assa-1 was ng level, and even when diluted Assa-1 monoclonal antibody was confirmed that the antigen can be recognized up to ng level [Fig. 5].

Since the monoclonal antibody Assa-1 has an extracellular membrane site of the receptor as an antigen recognition site, it was expected that the monoclonal antibody Assa-1 could be used even when using flow cytometry. To verify this, cell staining using Assa-1 was performed as follows. As a result, as shown in Figure 6 it can be seen that the cell staining by the flow cytometry, similar to the degree of expression of the ASGPR H1 monomer was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR).

Thus, these results confirmed that Assa-1 monoclonal antibody can be effectively used for cell staining for cell assays as well as for ELISA and Western blotting.

Assa-1 monoclonal antibody proposed in the present invention has a high antibody titer, and can be used for verification of ASGPR expression in the human body by applying to ELISA, Western blotting, and oily cytometry.

Therefore, Assa-1 may be applied to research in various ways when studying the physiological functions of ASGPR. First, since ASGPR is used as a receptor for several glycoproteins and as an infection medium for some viruses, neutralizing the ligand binding response to ASGPR using Assa-1 inhibits metabolic processes or virus infection caused by ASGPR function. Effect may be induced. Second, when ASGPR shows hepatocyte-specific expression, it is possible to construct a gene delivery system or a drug delivery system using ASGPR as a carrier, and in general, considering that the in vivo stability of antibody protein is superior to other proteins, It is expected to serve as a carrier suitable for bioapplication than any other carrier. Third, based on the fact that the expression of ASGPR increases when hepatic cell death progresses in the study of the association between liver disease and apoptosis, serum soluble ASGPR can be used as a marker of apoptosis process of liver tissue. In addition, it is possible to construct a liver disease diagnosis kit using Assa-1 having high antibody titer because it has been proved that it can be used for diagnosis of liver disease.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Selection, Synthesis and Preparation of Immunogen Antigen Peptides Derived from Human Thasial Group-Glycoprotein Receptors

The 30mer peptide sequence (SEQ ID NO: 3) of the (182-211) region of the human ASGPR sequence was determined to be the most suitable antigenic site, and this was given a unique name of "PJ-3" epitope, which is referred to as epitope below. As the antibody was prepared using the immunogenicity and specificity of FIG. 1 (FIG. 1A), the solid resin phase method using Fmoc chemistry [Merrifield RB. (1969) Solid-phase peptide synthesis. Adv Enzymol Relat Areas Mol Biol. 1969; 32: 221-296. For peptide synthesis, Fmoc-Wang amino acid resin (Novabiochem, San Diego, CA) was used as a support. For amino acid addition reaction, dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBt, N-hydroybenzotriazole) were used. ) Was used. The side chains of the respective amino acids used a form with a stable protecting group for piperidine (Asp (OtBu), Asn (Trt), Lys (Boc), Trp (Boc), Arg ( Pmc), Ser (tBu), Tyr (tBu)). In the final step of the synthesis, TFA solvent (88% TFA, 5% phenol, 5% H ₂ O, 5% thioanisole, 2.5% 1,2 ethanedithiol and 2% triisopropyl silane) was treated for 2 hours to give a solid resin phase. Peptide synthesized at and removed the residue protecting groups of each amino acid at the same time.

In order to elute the peptide from the synthetic solution containing the peptide after the reaction, a peptide precipitate was obtained using ethyl ether, which was dried using a vacuum dryer. Peptides were purified using preparative reverse phase-high performance liquid chromatography (Waters 15μ Deltapak C18 column (300A, 19 × 300 mm)) and lyophilized. Some of the purified peptides were analyzed again using analytical reverse phase high performance liquid chromatography (Ultrasphere, 5u Beckman, San Raman, CA), and found to have a purity of 99% or more.

For immunization of the synthesized peptide, Ovalbumin was used as a carrier protein, and the synthetic peptides were bound to the carrier protein by an amino group-sulfonyl group linkage method using sulfo-MBS. Dissolve 2 mg of ovalbumin in 200 μl of 50 mM sodium phosphate buffer (pH 8.0), mix 100 μl of MBS solution (10 mg / ml), and react at room temperature for 1 hour. 100 mM sodium phosphate buffer (pH 7.0) was separated on a -50 column using column buffer. Activated ovalbumin (MBS-OVA) was mixed with the peptide and further reacted at room temperature for 4 hours. After the reaction, the reaction mixture was separated by PBS buffer using Sephadex G 50 gel filtration chromatography.

Example 2: Preparation of Single Antibody Producing Clones

In order to prepare a monoclonal antibody-producing fusion cell line, an immunization operation was performed as shown in FIG. 2 using 8-week experimental female mice (BALB / c). First, 50 μl of the peptide conjugate prepared in Example 1 was emulsified in 150 μl of Complete Freund's adjuvant (Sigma), followed by intraperitoneal injection. After the first injection, two more immunizations were performed at 10 day intervals. At the second and third injections, the same amount of peptide conjugate was intraperitoneally injected into an incomplete Freund's adjuvant. Finally, 3 days before the fusion cell line, 20 μg of synthetic peptide was dissolved in PBS and injected into the tail vein.

For the production of fusion cell lines, splenocytes were extracted from the immunized mice and mixed so that the number of cells of mouse myeloma cells P3X63-Ag8.653 (ATCC, USA) was 5: 1. Subsequently, the supernatant was removed by centrifuging the mixed solution, and 1 ml of PEG-1500 [Boehringer Mannheim] was added using a plastic Pasteur pipette and mixed well for 1 minute, and 15 ml of RPMI-1640 culture solution [Gibco Corporation] without serum was added. The mixture was slowly added over 10 minutes and mixed well, followed by addition of RPMI-1640 medium containing the same volume of 10% fetal bovine serum, which was then dispensed into 100-ul plates in 96-well plates at 37 ° C and 5% Incubate for one day under atmospheric conditions of CO ₂ . After 24 hours, 100 μl of 2X HAT medium [Gibco BRL Co., Ltd.] was added to each well and cultured to select the formation of fusion cells. After HAT medium selective colonies were formed, antibody-positive positive clones were selected by ELISA using synthetic peptides as antigens for cell culture medium of HAT medium selective colonies.

Specifically, the human ASGPR-derived peptide used as an immunization antigen was mixed in a surface treatment buffer (0.1 M Na-carbonate buffer, pH 9.5), and each 1 μg of each well of a 96-well ELISA plate (Maxisorp, Nunc) After the addition, the reaction was carried out at 4 ° C for 16 hours. HBV preS2 (120-145) peptide was used as a negative control. After the reaction, each well was washed with TBS (Tris-buffered saline) buffer, and then a blocking buffer (blocking buffer, 3% casein / TBS) was added and reacted at 37 ° C. for 2 hours. Then, the solution was removed and 100 μl of the culture medium of the fusion cell lines was added to each well, followed by an additional 2 hours of reaction at 37 ° C. to remove excess antibody that was not reacted, and horseradish peroxidase (HRP) was added. Bound rabbit-derived anti mouse IgG antibody [Sigma] and 4-chloro-1-naphtol [Sigma] and H ₂ O ₂ The presence of the antibody was confirmed. As a result, hybridoma clones that react with human ASGPR-derived peptides but do not react with HBV preS2 (120-145) peptides, which are used as negative controls of similar size, are positive clones that secrete anti-human ASGPR recognition antibodies. Screened.

Limited dilution was performed to separate the monoclones from the positive clones selected in the above procedure. To this end, first designate one positive clone hybridoma (hybridoma), take all the cell lines on 96-well, and suspend them. Accurately measure the number of cells so that 0.3 cells per well of each 96-well plate can be entered. The cells were aliquoted, followed by three cells per well, followed by one 96-well plate in the order of 30, 300 cells. Here, the clones grown on the plate in which the smallest cell number was dispensed were repopulated and expanded, and the above process was repeated again. Finally, clones grown from the most diluted cell concentration plates were considered monoclonal.

The monoclonal antibody finally tested for anti-ASGPR specificity after the above process was named Assa-1, and the mouse B-cell fusion cell line secreting it was deposited with the Korea Biotechnology Research Institute Gene Bank on December 27, 1999. (Accession number: KCTC 0717BP).

Example 3 Determination of Antibody Type of Monoclonal Antibody Assa-1

To determine the immunoglobulin antibody type of the monoclonal antibody Assa-1, a mouse immunoglobulin type determination kit (Pharmingen) was used. The kit is a kit based on a monoclonal antibody, in which a monoclonal antibody specific for each type of immunoglobulin is first coated on a well of an ELISA plate, and then a sample is added, followed by a normal ELISA to react in a specifically reacted well. Will be searched with anti-mouse immunoglobulin antibody. As a result, as shown in FIG. 3, it was confirmed that the monoclonal antibody Assa-1 had a kappa light chain as an IgG ₁ type.

Example 4 Verification of Antigen Specificity of Assa-1

<1> Preparation of maltose binding protein fusion comprising extracellular membrane region of human deciall-glycoprotein receptor protein

Sequences 2-40 of the human ASGPR H1 subunit consisting of a total of 291 amino acids are the cytoplasmic domain, sequences 41-61 are the transmembrane domain, and sequences 62-291. Is expected to form extracellular domains. In order to prepare a recombinant protein for confirming the reactivity of Assa-1, the protein sequence of the extracellular membrane 62-291 was amplified by PCR and cloned into pMAL-ASGPR (C2) based on the predicted sequence information. pMAL-ASGPR (C2) is a plasmid used to express cDNA by recombination in Escherichia coli and is a vector that is easily purified because its external protein is expressed in a fused form with maltose binding protein. Such a fusion protein has the characteristics of high solubility of maltose binding protein and has the advantage of being able to be purified in one step using amylose resin based on affinity for amylose. have. Specifically, cloning was carried out as follows.

CDNA clones of human ASGPR H1 have been described in Lee DG, Lee SG, Kim KL, Hahm KS. cDNA (1997) Sequence for asialoglycoprotein receptor from human fetal liver. J. Biochem. Mol. Biol. 30, 299-301] in order to amplify only the extracellular membrane sequence, primer 1 (SEQ ID NO: 4) and amino acid sequence of amino acids 62-67 and amino acids 286-291 Primer 2 (SEQ ID NO: 5) corresponding to the sequence was as follows.

       Primer 1: 5'-GGAATTCCAAAACTCCCAGCTGCA-3 '

       Primer 2: 5'-GCGTGTCGACTTAAAGGAGAGGTGGCTCC-3 '

Primer 1 has EcoRI restriction enzyme sequence (GAATTC), Primer 2 has SalI restriction enzyme sequence (GTCGAC). For polymerase reaction (PCR), 10 μl of 10 × PCR buffer solution, 2 μl of dNTP mixture solution (mixture of 10 mM each of ATP, GTP, CTP, and TTP), and 2 and 10 μM of primers 1 and 2, respectively. Prepare a reaction mixture of 1 μl, 2 μl of ASGPR cDNA, 81 μl of distilled water, and 1 μl of Taq DNA polymerase (Qiagen), denature at 96 ° C. for 40 seconds, anneal at 55 ° C. for 1 minute, and 72 The polymerase reaction was carried out under the condition of elongation at 2 ° C. for 2 minutes. After the polymerase reaction, the reaction was electrophoresed on an agarose gel, and the gel fragment containing the PCR product was cut out. To extract gene fragments from the gel fragment, a gel extraction kit (Qiagen) was used. 4] was used. The obtained gene fragment and pMAL-c2X were digested with EcoR I and Sal I. The restriction enzyme reaction composition was 10 μg / 20 μl of DNA, 5 μl of 10X reaction buffer solution, 1 μl of EcoRI, 1 μl of SalI, 23 μl of distilled water, and reacted at 37 ° C. for 4 hours. Restriction enzyme cleaved vector and ASGPR gene fragment were separated on agarose gel and purified by gel extraction method.Then, they were mixed to a molar ratio of 1: 3 and linked using T4 DNA ligase (Promega). A ligation reaction was carried out. The ligation reaction was carried out at 16 ° C. for 16 hours, and after the reaction, the CaCl ₂ method was used for DH5α strain, which is widely used for protein expression for recombinant protein expression [Preparation and transformation of competent E. coli using calcium chloride, (2001) Molecular Cloning : Laboratory Mannual, edited by Sambrook & Russell, p116-1.118, Cold Spring Harbor Laboratory Press) and plated on LB plates containing ampicillin. E. coli, including pMAL-ASGPR, which expresses ampicillin-resistant protein expression genes during transformation, formed colonies.

Colonies obtained by the above process were cultured and plasmids were extracted from them using a DNA miniprep kit (Qiagen), and M13 primers (5'-CGCCAGGGTTTTCCCAGTCACGAC-3 ': SEQ ID NO: 8) were used. The sequencing of them was conducted by the Korea Research Institute of Bioscience and Biotechnology. Two clones obtained from the sequencing results were identified, and the sequence of the ASGPR site fused to maltose binding protein was identified among the sequences of these plasmids. As a result, the C2 clone had a sequence of normal human ASGPR H1 monomer, and the C3 clone was confirmed to have one nucleotide sequence mutated. The C3 clone is a 611 nucleotide sequence A of the ASGPR transcript is substituted with G (CAC-> CGC), as a result, the 204th amino acid sequence was replaced by histidine to arginine (Fig. 4a). In the present invention, the recombinant protein expressed from the two genes were prepared by the following method in order to confirm whether the variation of the amino acid sequence correlated with the reactivity of Assa-1 and confirmed their reactivity by Western blotting and EILSA [ 4b].

The plasmids, pMAL-ASGPR (C2) and pMAL-ASGPR (C3), respectively, were transformed into E. coli DH5α strains to induce protein expression. E. coli DH5α clones transformed with pMAL-ASGPR (C2) and pMal-ASGPR (C3) were incubated for 2 hours, followed by incubation for 2 hours with IPTG (isopropyl β-thiogalactopyronoside) added at a final concentration of 1 mM for optimal growth. Expression of the fusion protein was induced. Each E. coli transformant induced expression using IPTG was recovered by centrifugation at 6000 rpm for 10 minutes, and again, this was a column buffer solution (20 mM Tris-HCl; pH 7.4, 200 mM) of 1/20 volume of the culture medium. NaCl, 1 mM EDTA) and then ultrasonically triturated. After centrifugation of the lysate, the supernatant was passed through Amylose resin (New England Biolabs) to separate only maltose binding protein-ASGPR fusion protein dissolved in the cytoplasm. Proteins that were not specifically bound to the resin were removed by washing with an excess of column buffer, and the desired protein was isolated by passing a column buffer solution containing 20 mM Maltose (Sigma). Expression and purity of each protein were confirmed by performing 10% SDS-PAGE [Fig. 4b].

<2> Validation of antigen specificity of Assa-1 using western blotting

To verify the antigenic specificity of Assa-1, C2 and C3 clonal proteins fused with maltose binding protein (MBP) and ASGPR extracellular membrane and alpha peptide of beta-galactosidase The fused form of protein [New England Biolabs] was electrophoresed on DMF SDS-PAGE and blotted by transfer to polyvinylidene fluoride membrane [Millipore] in tris-methanol buffer. In order to remove the nonspecific reaction, the PVDF membrane was blocked with 5% skim milk powder at room temperature for 2 hours, and then reacted for 2 hours by diluting Assa-1, an anti-ASGPR specific antibody, to an appropriate amount. After washing with TBS buffer containing 0.05% of Tween-20, the anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) was diluted 1: 2000 times and reacted for 2 hours. The reagent was washed again with the same TBS buffer solution as above, followed by luminescence with ECL reagent [Pharmacia]. As a result, both ASGPR clones c2 and c3 fused to maltose binding protein by genetic recombination reacted with HAM-19, a monoclonal antibody specific for maltose binding protein. Assa-1, an anti-ASGPR specific antibody prepared in the present invention, specifically recognized the C2 clone protein, but did not recognize the C3 clone, an amino acid sequence variant. It did not react at all with the MBP-beta-galactosidase alpha peptide used as a negative control. As a result, Assa-1 is an antibody specific for amino acids 182 to 211 corresponding to the sugar recognition site (CRD) of ASGPR, and the sequence variation (Histidine) of the antigen recognition site, such as C3 clone protein, is known as Arginine) is a monoclonal antibody with a very high specificity of whether the antigen is sensitive to one another.

<3> Antigen Specificity of Assa-1 Using Enzyme-linked Immunoassay

In order to confirm the antigen specificity of the monoclonal antibody Assa-1, enzyme-mediated immunoassay (ELISA) was performed. To this end, the purified ASGPR-maltose binding protein fusion proteins C2 and C3 protein and maltose binding protein fusion-beta-galactosidase alpha peptides prepared in <1> were treated with a surface treatment buffer (0.1M Na-carbonate, pH 9.5). ) Was added to each well of a 96-well plate and reacted at 4 ° C. for one day. After coating the antigen, each well was washed with TBS (Tris-buffered saline) buffer, and then added with blocking buffer (5% skim milk powder) and left at 37 ° C. for 2 hours. Thereafter, the solution was removed, and 100 µl of Assa-1 monoclonal antibody dilution was added to each well, followed by further reaction at 37 ° C for 2 hours. Afterwards, unreacted excess antibody was removed, and 100 μl of rabbit-derived anti mouse mouse antibody [Sigma] conjugated with horseradish peroxidase (HRP) as a secondary antibody was added to each well. The reaction was carried out for a time. Afterwards, the excess unreacted antibody was removed again, followed by adding wasabi peroxidase substrate ο-phenylenediamine [Sigma] and hydrogen peroxide (H ₂ O ₂ ) in that order. After the reaction, 100 µl of 2.5MH ₂ SO ₄ was added to stop the color reaction, and the absorbance at 492 nm wavelength was measured. The ELISA was performed in two ways, the first of which fixed the amount of antigen attached to the 96-well plate at 1 µg / ml and the serial amount of the Assa-1 monoclonal antibody from 10 µg / ml to 1/3 in succession. Reactivity was measured for the concentration [FIG. 5A], and the second was 0.5 µg / ml of the amount of Assa-1 monoclonal antibody which was serially diluted from 5 µg / ml to 1/3 of the antigen to be attached and reacted after blocking. The reactivity was examined by fixing with [Fig. 5b]. As a result, Assa-1 showed an antibody-antigen concentration-dependent response pattern only for the ASGPR C2 clone, and was not responsive to the c3 protein and maltose-binding protein fusion-beta-galactosidase alpha peptides, which are sequence variants of ASGPR. Did. It was confirmed that the minimum antigen concentration showing the reactivity of Assa-1 was ng level, and even when diluted Assa-1 monoclonal antibody was able to recognize the antigen up to ng level.

Example 5 Validation of ASGPR for Membrane Expression by Monoclonal Antibody Assa-1

Since monoclonal antibody Assa-1 has an extracellular membrane site of the receptor as an antigen recognition site, it was expected that the monoclonal antibody Assa-1 could be used even when using a flow cytometry. In order to verify this, cell staining using Assa-1 was performed as follows. First, the cells to be subjected to the flow cytometry are suspended in PBS containing cell suspension buffer (0.1% bovine serum albumin (BSA) and 0.01% sodium azide) and 4 × 10 ⁵ cells / 250 μl per tube. After the reaction, Assa-1 monoclonal antibody was added and reacted for 30 minutes at 4 ° C. After the reaction, the cell suspension buffer solution was filled up to 3 ml and centrifuged to obtain only the cells, the supernatant was removed, and the cell suspension buffer solution was again 3 ml filled. After washing the cells well, the cell washing process was repeated twice, followed by centrifugation. The reaction was carried out for 30 minutes at 4 ° C. After the reaction, the cell washing process was performed three times, and the washed cells were suspended in 500 μl of the cell suspension buffer solution, followed by FACSscan cytometer [Becton Dickinson]. The fluorescence staining of the cells was measured using FCS express V2, and the results were analyzed using FCS express V2. As a result, the expression of ASGPR H1 monomer was confirmed by the RT-PCR as shown in Fig. 6. It was confirmed that the cell staining was also confirmed.

Thus, these results confirmed that Assa-1 monoclonal antibody can be effectively used for cell staining for cell assays as well as for ELISA and Western blotting.

Example 6 Diagnosis Using Monoclonal Antibody Assa-1 for Quantification of Soluble ASGPR in Serum

Serum soluble ASGPR can be used as a marker for apoptosis of liver tissues based on the fact that the expression of ASGPR increases when hepatocyte death progresses in studies of liver disease and apoptosis. Since it can be used to diagnose liver disease, it is possible to construct a diagnostic kit for liver disease using Assa-1 having high antibody titer. The diagnostic method for confirming the serum concentration of ASGPR is composed of a competitive ELISA method using Assa-1 monoclonal antibody, the detailed method is as follows.

C2 and C3 proteins, the purified ASGPR-maltose binding protein fusion proteins prepared in Example 1, were mixed in surface treatment buffer (0.1 M Na-carbonate, pH 9.5) for each well of a 96-well plate. After 1 μg addition, the solution is reacted at 4 ° C. for 1 day. After coating the antigen, each well was washed with Tris-buffered saline (TBS) buffer, and then added with a blocking buffer (5% skim milk powder) and left at 37 ° C. for 2 hours. In addition, the samples to check the concentration of water-soluble ASGPR are prepared by diluting to various concentrations and mixed 1: 1 with Assa-1 antibody diluent (200 ng / ㎖). The blocking buffer filled in the 96-well plate is removed, and 100 µl of the mixture of the sample and the Assa-1 antibody is added to each well, and the reaction is further performed at 37 ° C for 2 hours. Thereafter, the solution containing the unreacted antibody is removed, and each well of the plate is washed with TBS solution containing 0.01% of Tween-20. As a secondary antibody, 100 μl of rabbit-derived anti mouse mouse antibody (Sigma) diluted with horseradish peroxidase (HRP) bound (1: 20,000-fold dilution) was added to each well and reacted at 37 ° C. for an additional 2 hours. . Afterwards, the excess unreacted antibody was removed again, followed by adding wasabi peroxidase substrate ο-phenylenediamine [Sigma] and hydrogen peroxide (H ₂ O ₂ ) in that order. After the reaction, the color reaction was stopped by adding 100 µl of 2.5MH ₂ SO ₄ , and the absorbance at 492 nm was measured. At this time, the OD value read in the competitive ELISA on the plate coated with C3 protein is used as a negative control group, and the OD value read from the C2 plate is corrected and the concentration of ASGPR protein in the sample is calculated.

Example 7: ASGPR expressing cell specific drug delivery system using monoclonal antibody Assa-1

Given that ASGPR exhibits specific expression of hepatocytes, especially early in differentiation, it is possible to construct a gene delivery system or a drug delivery system using ASGPR as a carrier, and in general, the in vivo stability of the antibody protein is higher than that of other proteins. Given its superiority, it is expected to serve as a suitable carrier for bioapplications than any other carrier. The basic composition of the drug carrier is a protein sample or drug having a drug effect by covalent bonding using an amine group-kiloxyl group reaction or an amine group-sulfonyl group reaction to a site that does not damage the antigen-binding site of Assa-1 monoclonal antibody. By immobilizing liposomes, etc., which are contained and administered to the living body, the selection of the drug may be selected by what effect is desired for hepatocytes, and in general, may be applied to biopolymers such as proteins and gene fragments. In addition, cloning of the gene encoding the Assa-1 antibody protein from the Assa-1 monoclonal antibody secreting cell line was performed, and only the gene for the antibody binding site (Fv) of Assa-1 was manipulated separately, thereby reducing the drug effect only at the antibody binding site. It can be prepared in a form expressed in the form of a protein sample and a fusion protein to be expressed, and can be used as a hepatocyte specific drug. In this case, it is generally applied to other antibodies. As a method for specifically treating TNFα (Tumor necrosis factor α) to blood vessels of cancer tissue, the antibody against B-FN showing cancer tissue blood vessel specific expression It can be seen that there is a recent study that reports that the protein of the TNFα fused to the antibody binding site (Fv) of the protein is expressed in animal cells, purified and administered in vivo. L, Balza E, Carnemolla B, Sassi F, Castellani P, Berndt A, Kosmehl H, Biro A, Siri A, Orecchia P, Grassi J, Neri D, Zardi L. (2003) Selective targeted delivery of TNF {alpha} to tumor blood vessels. Blood. 2003 Aug 21 [Epub ahead of print].

As described above, the Assa-1 monoclonal antibody of the present invention is a specific antibody against the anti-ASGPR H1 monomers, particularly amino acids 181 to 211 which are part of the carbohydrate recognition domain, and include a kappa chain. It is an IgG ₁ antibody including a light chain, and has a specificity enough to show a difference in reactivity that is very sensitive to a single amino acid sequence of an antigen-response site. In addition, the monoclonal antibody of the present invention is prepared with a high titer and can be usefully used as a diagnostic reagent for ELISA or Western blotting to detect and test the expression of ASGPR, and can also be used for oily cell staining. It can be used for neutralizing or treating various liver-related diseases.

FIG. 1A shows a comparison of the sequences of the human Asialoglycoprotein Receptor H1 subunit and the mouse desial-glycoprotein receptor H1 unit, and the antigen selected and determined by the present inventors from the extracellular region sequence. Epitope peptide sequence,

FIG. 1 b shows a process of chemically synthesizing the peptide to prepare a conjugate with a carrier protein and using this as an immunogen to prepare a monoclonal antibody.

Figure 2 schematically shows the process of producing mouse immunized and monoclonal antibody-producing fusion cell lines using the antigen described in Figure 1.

Figure 3 shows the enzyme-mediated immunoassay (ELISA) results for determining the type of immunoglobulin of the monoclonal antibody produced in the hybridoma, Assa-1, prepared in FIG.

Figure 4a is a preparation of the vector (pMAL-ASGPR (ex)) expressed in the form of the cDNA corresponding to the extracellular membrane region of the human desialal group-glycoprotein receptor H1 unit in the form of fusion to maltose-binding protein (MBP) It is about. A vector prepared to express the human desialal-glycoprotein receptor H1 monomer cDNA as it is is named C2, while the other one modified the Assa-1 antigen site by amino acid single point-mutation. An additional human dealial group-glycoprotein receptor H1 monomer cDNA expression vector was prepared and named C3. 4A compares the amino acid sequences of the two recombinant protein expression clones C2 and C3.

Figure 4b is purified by using the two vectors (maltose-binding protein and C2, c3 clone protein) purified by protein staining (Coomassie staining) and confirmed after anti-maltose binding protein monoclonal antibody HAM-19 and Immunostaining with Assa-1, an anti-human desial group-glycoprotein receptor monoclonal antibody, provided in the present invention [M: molecular weight marker, 1: beta galactosidase alpha peptide fusion maltose binding protein, 2: human desial group-glycoprotein receptor extracellular membrane fusion maltose binding protein (C2 clone), 3: human desial group-glycoprotein receptor extracellular membrane site modified fusion maltose binding protein (C3 clone)].

FIG. 5A is a graph showing the antigen specificity of the anti-human decial group-glycoprotein receptor monoclonal antibody Assa-1 using ELISA, and is a result of confirming the difference in the degree of antibody-antigen response according to the Assa-1 antibody concentration.

FIG. 5B is a graph showing the antigen specificity of the anti-human decial group-glycoprotein receptor monoclonal antibody Assa-1 using ELISA, showing the difference in the degree of antibody-antigen response according to the antigen concentration change [□: betagal Lactosidase alpha peptide fusion maltose-binding protein, ●: human decial group-glycoprotein receptor extracellular membrane fusion maltose-binding protein (C2 clone), ◆: human desitial-glycoprotein receptor extracellular membrane site modified fusion maltose binding protein (C3) Clone)].

Figure 6a is a result of confirming the expression of the human desial group-glycoprotein receptor (ASGPR) by measuring the responsiveness to the cell surface of various human cell lines using Assa-1 monoclonal antibody in oily cell assay,

Figure 6b is a result confirmed by the reverse transcriptional polymerase chain reaction (ASGPR) expression in the cell lines analyzed in Figure 6a.

<110> Korea Research Institute of Bioscience and Biotechnology <120> A mouse monoclonal antibody specific for the human asialoglycoprotein receptor, the hybridoma cell line secreting this antibody and the generation and verification method <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 284 <212> PRT <213> Mus musculus <400> 1 Met Thr Lys Asp Tyr Gln Asp Phe Gln His Leu Asp Asn Asp Asn Asp 11 15 20 25 His His Gln Leu Arg Arg Gly Pro Pro Pro Thr Pro Arg Leu Leu Gln 30 35 40 Arg Leu Cys Ser Gly Ser Arg Leu Leu Leu Leu Ser Ser Ser Leu Ser 45 50 55 Ile Leu Leu Leu Val Val Val Cys Val Ile Thr Ser Gln Asn Ser Gln 60 65 70 Leu Arg Glu Asp Leu Leu Ala Leu Arg Gln Asn Phe Ser Asn Leu Thr 75 80 85 90 Val Ser Thr Glu Asp Gln Val Lys Ala Leu Ser Thr Gln Gly Ser Ser 95 100 105 Val Gly Arg Lys Met Lys Leu Val Glu Ser Lys Leu Glu Lys Gln Gln 110 115 120 Lys Asp Leu Thr Glu Asp His Ser Ser Leu Leu Leu His Val Lys Gln 125 130 135 Leu Val Ser Asp Val Arg Ser Leu Ser Cys Gln Met Ala Ala Phe Arg 140 145 150 Gly Asn Gly Ser Glu Arg Thr Cys Cys Pro Ile Asn Trp Val Glu Tyr 155 160 165 170 Glu Gly Ser Cys Tyr Trp Phe Ser Ser Ser Val Arg Pro Trp Thr Glu 175 180 185 Ala Asp Lys Tyr Cys Gln Leu Glu Asn Ala His Leu Val Val Val Thr 190 195 200 Ser Arg Asp Glu Gln Asn Phe Leu Gln Arg His Met Gly Pro Leu Asn 205 210 215 Thr Trp Ile Gly Leu Thr Asp Gln Asn Gly Pro Trp Lys Trp Val Asp 220 225 230 Gly Thr Asp Tyr Glu Thr Gly Phe Gln Asn Trp Arg Pro Glu Gln Pro 235 240 245 250 Asp Asn Trp Tyr Gly His Gly Leu Gly Gly Gly Glu Asp Cys Ala His 255 260 265 Phe Thr Thr Asp Gly Arg Trp Asn Asp Asp Val Cys Arg Arg Pro Tyr 270 275 280 Arg Trp Val Cys Glu Thr Lys Leu Asp Lys Ala Asn 285 290 <210> 2 <211> 291 <212> PRT <213> Homo sapiens <400> 2 Met Thr Lys Glu Tyr Gln Asp Leu Gln His Leu Asp Asn Glu Glu Ser 12 16 21 26 Asp His His Gln Leu Arg Lys Gly Pro Pro Pro Gln Pro Leu Leu 31 36 41 Gln Arg Leu Cys Ser Gly Pro Arg Leu Leu Leu Leu Ser Leu Gly Leu 46 51 56 Ser Leu Leu Leu Leu Val Val Val Cys Val Ile Gly Ser Gln Asn Ser 61 66 71 Gln Leu Gln Glu Glu Leu Arg Gly Leu Arg Glu Thr Phe Ser Asn Phe 76 81 86 91 Thr Ala Ser Thr Glu Ala Gln Val Lys Gly Leu Ser Thr Gln Gly Gly 96 101 106 Asn Val Gly Arg Lys Met Lys Ser Leu Glu Ser Gln Leu Glu Lys Gln 111 116 121 Gln Lys Asp Leu Ser Glu Asp His Ser Ser Leu Leu Leu His Val Lys 126 131 136 Gln Phe Val Ser Asp Leu Arg Ser Leu Ser Cys Gln Met Ala Ala Leu 141 146 151 Gln Gly Asn Gly Ser Glu Arg Thr Cys Cys Pro Val Asn Trp Val Glu 156 161 166 171 His Glu Arg Ser Cys Tyr Trp Phe Ser Arg Ser Gly Lys Ala Trp Ala 176 181 186 Asp Ala Asp Asn Tyr Cys Arg Leu Glu Asp Ala His Leu Val Val Val 191 196 201 Thr Ser Trp Glu Glu Gln Lys Phe Val Gln His His Ile Gly Pro Val 206 211 216 Asn Thr Trp Met Gly Leu His Asp Gln Asn Gly Pro Trp Lys Trp Val 221 226 231 Asp Gly Thr Asp Tyr Glu Thr Gly Phe Lys Asn Trp Arg Pro Glu Gln 236 241 246 251 Pro Asp Asp Trp Tyr Gly His Gly Leu Gly Gly Gly Glu Asp Cys Ala 256 261 266 His Phe Thr Asp Asp Gly Arg Trp Asn Asp Asp Val Cys Gln Arg Pro 271 276 281 Tyr Arg Trp Val Cys Glu Thr Glu Leu Asp Lys Ala Ser Gln Glu Pro 286 291 296 Pro Leu Leu 301 <210> 3 <211> 30 <212> PRT <213> Homo sapiens <400> 3 Cys Arg Leu Glu Asp Ala His Leu Val Val Val Thr Ser Trp Glu Glu 1 5 10 15 Gln Lys Phe Val Gln His His Ile Gly Pro Val Asn Thr Trp 20 25 30 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer 1 <400> 4 ggaattccaa aactcccagc tgca 24 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer 2 <400> 5 gcgtgtcgac ttaaaggaga ggtggctcc 29 <210> 6 <211> 32 <212> PRT <213> Homo sapiens <400> 6 Ala His Leu Val Val Val Thr Ser Trp Glu Glu Gln Lys Phe Val Gln 1 5 10 15 His His Ile Gly Pro Val Asn Thr Trp Met Gly Leu His Asp Gln Asn 20 25 30 <210> 7 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> MBP-ASGPR (c3) <400> 7 Ala His Leu Val Val Val Thr Ser Trp Glu Glu Gln Lys Phe Val Gln 1 5 10 15 His Arg Ile Gly Pro Val Asn Thr Trp Met Gly Leu His Asp Gln Asn 20 25 30 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> M13 primer <400> 8 cgccagggtt ttcccagtca cgac 24

Claims (9)

A mouse monoclonal antibody against an antigen of 30 mer size (182 to 211) in the human ASGPR sequence produced by the mouse B-cell fusion cell line Assa-1 [KCTC 0717BP]. The method of claim 1, wherein the subtype of an antibody (subclass type) is a monoclonal antibody, characterized in that IgG _1. delete delete delete Mouse B-cell fusion cell line Assa-1 producing the monoclonal antibody of claim 1 [KCTC 0717BP]. (1) preparing a conjugate of an antigen site of 30 mer size (182 to 211) in the human ASGPR sequence represented by SEQ ID NO: 3 with an ovalbumin which is a carrier protein; (2) immunizing the conjugate by injection into the abdominal cavity of the mouse; (3) extracting splenocytes from the immunized mice and fusing them with mouse myeloma cells; And (4) selecting fusion cell lines producing monoclonal antibodies using ELISA Method for producing a mouse B-cell fusion cell line [KCTC 0717BP] producing a monoclonal antibody of claim 1 comprising a. Method for measuring the expression level of the desial group glycoprotein receptor in serum by performing a competitive ELISA method using the monoclonal antibody of claim 1. A drug carrier specific for ASGPR expression using the monoclonal antibody of claim 1.