KR100341827B1 - Antibodies against amino-rna and method of preparation for the same - Google Patents

Abstract

The present invention provides an antibody against amino-RNA using an in vitro immunoassay and a method for producing the same, wherein the antibody against RNA that cannot be produced by using bioimmunity with normal RNA is relatively stable amino-RNA in vitro immunity. In addition, the obtained antibody is specifically and strongly binds to specific autoantibodies of autoimmune diseases to prevent autoimmune diseases, and thus is useful as a therapeutic agent for autoimmune diseases. It can induce the production of can be developed as a vaccine against autoimmune diseases.

Description

ANTIBODIES AGAINST AMINO-RNA AND METHOD OF PREPARATION FOR THE SAME

The present invention relates to an antibody against RNA and a method for producing the same, and more particularly, to an antibody against amino-RNA and a method for producing the same using an in vitro immunoassay.

In autoimmune diseases, autoantibodies refer to antibodies made against autologous proteins, such as their own molecules, such as DNA, RNA or many receptors. Although many studies have been conducted on antibodies that bind DNA or proteins, antibodies to RNA are rarely found. However, autoantibodies which are conjugated to several RNA molecules are known. However, this is not directly confirmed, but derived from random RNA libraries derived from RNA sequences that bind well to autoantibodies, and then obtained by indirect evidence that the base sequence of the selected RNA is similar to the site within the U1 RNA. lost. This means that autoantibodies can be cross reactive with natural RNA antigen determining sites. The discovery of RNA libraries that specifically bind to antibodies, particularly autoantibodies, in random RNA libraries, and if the selected RNA sequences have a structure similar to natural RNA, then antibodies capable of recognizing RNA may exist and are likely to exist. Can be said to be quite high.

However, few attempts have been made to directly prepare antibodies to specific RNA or DNA molecules. The first reason is that since these RNAs or DNAs are their own molecules and not foreign substances, antibodies to these automolecules are unlikely to be produced by normal immune systems. The second reason is that even if a specific DNA or RNA is produced and immunized, it is difficult to cause these molecules to be easily degraded by DNA or RNA degrading enzymes in physiological conditions to generate an immune response.

In most cases, the autoimmune disease, which has a half-life of about 2 weeks to generate an immune response, generates autoantibodies that are conjugated to acetylcholine receptors in humans and causes various diseases, called Mysthenia gravis (hereinafter, MG). There is). As a method of their treatment, RNA was chosen as a potent conjugate for antibodies that bind to such receptors. The selected RNA molecule is capable of strong binding to autoantibodies, so that the RNA administered through in vivo experiments binds to the autoantibodies, thereby releasing acetylcholine receptors so that signaling through the receptors is normally performed. The treatment of autoimmune diseases using RNA with high adhesion to certain autoantibodies is now on track.

However, since RNA is easily degraded by RNA degrading enzymes, there is a fundamental problem as a therapeutic agent. Fortunately, the preparation of RNA substituted at the 2 ′ position of the RNA with an amino group instead of a hydroxyl group increases the in vivo stability and at least a half-life of at least 3 days. Experimental results have shown that these amino-RNAs that withstand RNA degrading enzymes strongly bind to autoantibodies obtained from patients with MG disease (Seong-Wook Lee and Bruce A. Sullenger, Nature Biotechnol. 15: 41, 1997 and J. Exp. Med. 184: 315-32, 1998). The amino RNA thus produced is therefore particularly useful as a therapeutic agent.

The present invention seeks to provide antibodies against amino-RNAs and methods of making them using in vitro immunity. The production method of the present invention, in view of the characteristics that RNA is easily degraded under normal conditions and is recognized as an autologous material, and thus the production of antibodies is not easy in a normal immune system, amino-RNA has a relatively long half-life and thus an antibody can be prepared. In addition, unlike the method of producing a monoclonal antibody using a mouse, and because it is made in vitro, there is an advantage that the RNA degrading enzymes can not function in vivo and immune system is not impaired.

In addition, using the in vitro immunoassay of the present invention, the antibody against the amino-RNA and the nucleotide sequence encoding the same are specific and strongly bound to specific autoantibodies of autoimmune diseases to prevent autoimmune diseases, which is useful as a therapeutic agent for autoimmune diseases. In addition, the amino-RNA can be administered in vivo to induce the production of antibodies against it, and thus can be developed as a vaccine against autoimmune diseases.

Figure 1 shows the nucleotide sequence secondary structure of the amino-RNA for the MS autoantibody.

The present invention relates to an antibody against RNA and a method for producing the same, and more particularly, to an antibody against amino-RNA and a method for producing the same using an in vitro immunoassay.

The present invention is a method for producing a monoclonal antibody using a hybridoma, using an in vitro immunoassay to incubate immunized splenocytes in a medium to which amino-RNA obtained from autoantibodies of autoimmune diseases is added. Provided is a method for preparing an antibody against amino-RNA, and in one embodiment of the invention, V _H and SEQ ID NO: 3 that specifically bind to the amino-RNA acetylcholine esterase receptor and are encoded by SEQ ID NO: 1 a method of producing antibodies using the vitro immunization of the antibody consisting of V _L coding for.

Specifically, the present invention relates to a method for preparing an antibody selected for amino-RNA against an autoantibody of MG disease. In the process of transcription from template DNA to RNA, amino nucleotides were prepared by substituting an amine group for UTP and CTP in each nucleotide (Seong-Wook Lee et al., Supra), and 6-week-old BALB / c without amino RNA immunization. In vitro immunization technique (Suga, H., Tanimoto, et al.), Which extracts spleen cells of female mice to obtain splenocytes and incubates splenocytes for 3 days in medium containing amino RNA obtained from MG autoantibodies. N., Sinskey, AJ and Masamune, SJ Am. Chem. Soc. 116: 11197, 1994; Stahl, M., Goldie, B., Bourne, S. and Thomas, NRJ Am. Chem. Soc. 117: 5164; Yu, JH et al., J. Chem. Soc., Chem. Commun. P1957, 1997). The immunized splenocytes were fused with SP2 / o-Ag14 myeloma cells using PEG1500, and then cultured in about 200 wells to obtain 30 or more hybridoma cells. Identification of hybridoma clones producing antibodies that bind amino RNA was selected by ELISA method. Amino RNA was added to each well of the ELISA plate and bound. Then, the antigen-binding portion was blocked using Tween 20, and hybridoma culture solution was added and allowed to react at room temperature. After reacting with goat anti-mouse IgG conjugated with Horse-radish peroxidase (Gibco BRL), OPD was added to show the degree of antigen antibody binding of each well by using enzymatic activity. The degree of uptake was measured to examine the degree of reaction, and hybridoma clones producing antibodies that bind amino RNA were selected. Primary hybridoma clones were obtained. In order to generate monoclonal antibodies using five clones whose priority is binding, among the hybridoma clones, the dilution is sufficiently divided into three cell culture wells, and the spleen feeder cell medium is included. The cells were cultured until the cells grew. Cultures in well-grown wells are picked up by ELISA analysis to obtain a single hybridoma clone, cloned and sequenced.

Whether to prepare antibodies against amino-RNAs that withstand RNA degrading enzymes. Obviously, under normal physiological conditions, however, such amino-RNAs will not be sustained continuously, so the present invention attempted an immune response in vitro for only 3 days via in vitro immunization. Because it is expected that no damage. The production of an antibody against RNA thus proves that an antibody to any desired RNA can be made. Therefore, any amino acid sequence can produce an antibody against amino-RNA, and the antibody can be produced in vivo. Prove that it can be made. Therefore, such amino-RNA can be used as an antigen, thus opening the possibility of using it as a vaccine preparation. This possibility supports the importance of amino-RNAs and the fact that the selection of specific RNAs from random RNA libraries using amino-RNAs can be of great importance.

To date, the production of antibodies to automolecules, including RNA or DNA, has been produced only very limitedly. In particular, even in the normal immune system, these attempts have been rarely performed. The antibody against DNA was not immunized with DNA, but after fusion of a B cell library of the MRL / lpr system, an autoimmune disease mouse in which many DNA antibodies are found, to the myeloma cell cells, The most binding antibody was obtained by ELISA.

The evolutionary maturation process occurs mainly in the normal immune system, in which the antibody mutates the original gene to increase its binding to the antigen, mutating the amino acids at the antigen binding site of the antibody to create enhanced antigen-antibody binding. Immune action. In the present invention, although a normal immune system was used, in vitro immunization was attempted for a non-living system for 3 days which is much shorter than the normal immunization period of 2-4 weeks. The discovery of a complete antibody in the IgG form, but not in the IgM form, which is strongly conjugated to the amino-RNA, is the result of a breakthrough.

The present invention seeks to provide an antibody against RNA using in vitro immunoassay and a nucleotide sequence encoding the same.

Antibodies were prepared using the RNAs set forth in SEQ ID NO: 5 using the method for preparing amino-RNAs for the autoantibodies of the MG disease and for producing antibodies to these amino RNAs. The sequence of the obtained antibody peptide is shown in SEQ ID NO: 1 to 2.

The nucleotide sequence of the light chain of the antibody MGN11a4d selected for the amino-RNA against the autoantibody of MG disease was compared with that of the existing antibody (BLSAT investigation). The light chain is very similar to the antibody against P24 of the HIV protein surrounding the RNA. It has high similarity and high similarity with DNA specific antibody found in MRL / lpr . The discovery of this similarity strongly suggests that at least the antibody obtained in the present invention can recognize RNA or DNA. Therefore, according to the results of the present study, at least amino-RNA can be produced using any sequence of the antibody, and the antibody similarity to the amino-RNA can be produced in vivo. Reconfirmed in the quest.

Therefore, the present invention has confirmed the possibility of using amino-RNA as a vaccine as an antigen capable of inducing antibody with a kind of regulated RNA. If the amino-RNA can induce the antibody, it is possible to prepare an amino-RNA having a specific base sequence of a retrovirus such as HIV to prepare an antibody against it, which is another good way to combat the virus, namely a vaccine. The importance of amino-RNA as a therapy can be emphasized. In addition, it is possible to select amino-RNAs that bind strongly to biomaterials from random libraries and to make antibodies against them. Therefore, such antibodies are an important tool for biological research because the result is an antibody that can replace biomaterials. Can be used.

Example

Example 1: In Vitro Immunization Against Amino-RNA

Spleen cells from 6-week-old non-immunized BALB / c female mice were harvested to obtain splenocytes, followed by 'Isolation of a nuclease-resistant decoy RNA that can protect human acetylcholine' (Lee, SW, etc.). Receptors from myasthenic antibodies' Nat. Biotechnol. 1997 Jan; 15 (1): 41-5) were added directly to splenocyte cultures without linkage to carriers and incubated for 3 days to immunize. Splenocytes immunized in this manner were fused with SP2 / o-Ag14 myeloma cells using PEG1500, and then cultured in about 200 wells to obtain 50 or more hybridoma cells. At least 50 hybridoma cells were obtained from each fusion by selective culture in a medium to which HAT (hyperanthin, aminopterin, thymidine) was added.

Among the antibodies produced by these hybridomas, ELISA method was used to select antibodies that bind amino-RNA. Specifically, an appropriate amount of each amino-RNA was reacted with a 96-well ELISA plate for 2 hours or more at room temperature, and then bound, and then blocked with a portion where the antigen was not bound using 0.05% Tween20, and hybridoma culture solution. Was added and reacted at room temperature. React with alkaline phosphatase-bound goat anti-mouse IgG, add p-nitrophenylphosphate (hereinafter referred to as pNPP) to show the degree of antigen antibody binding of each well using enzymatic activity, and then absorbance at 405 nm. The hybridoma clones producing antibodies that bind to each amino-RNA were selected by measuring the reaction rate. Limit dilution was used to purely isolate hybridoma clones that produced only a single antibody specific for amino-RNA using some of the primary hybridoma clones. This is achieved by diluting the primary hybridoma clones cultured in a medium containing an average of one cell per three cell wells using a medium, and then dividing the cells in a medium containing a Spleen feeder cell culture supernatant. Incubate until growth. Cultures were taken from well-grown wells and analyzed by ELISA to obtain a single hybridoma clone that produced antibodies that bound amino-RNA. Five replicate ELISA methods were used to select the six most clones. The clones were named MGN8c8g, MGN11a4d, MGN11f6f, MGN11e10e, MGN11a4b, and MGN8c9f, respectively.

Example 2: Preparation of cDNA of Antibody Genes

MRNA was extracted from hybridoma cells with high adhesion to amino-RNA using a quick prep micro mRNA purification kit (Pharmacia). Specifically, the cells to be extracted are cultured by replenishing the medium the day before, and then collected by centrifugation of 1 × 10 ⁷ cells. After washing once with 1 X salt buffer (hereinafter referred to as PBS), 0.4 ml of extraction buffer (guanidinium thiocyanate, N-lauryl sarcosine) was added and mixed thoroughly. Centrifuge it, mix it slowly with oligo (dT), and then centrifuge again to discard the supernatant, and add high concentration buffer solution (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 3 M NaCl) The oligo (dT) was washed twice using separation. The oligo (dT) washed with high concentration buffer was washed in the same manner with low concentration buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 M NaCl), followed by a microspum column. Moved to. This was centrifuged with elution buffer (10 mM Tris-HCl (pH 7.5), 1. mM EDTA) to extract only mRNA and precipitated with 70% ethanol. The isolated mRNA was made into cDNA using a single cDNA strand synthesis kit (cDNA Synthesis Kit; Pharmacia).

The antibody gene consists of a light chain and a heavy chain, of which only the genes of each variable region (Vk, Vh) are selected from the light primer mix (Pharmacia) and the heavy primer mix (Pharmacia). Amplified by PCR (polymerase chain reaction) (Perkin-Elmer). These primer mixtures are mixtures of different kinds of primers, and are designed so that all antibodies can be amplified regardless of various kinds of antibody change sites. Reaction conditions of gene amplification (PCR) were first denatured at 95 ° C. for 5 minutes, and then amplified DNA by repeating 30 times of 1 minute at 94 ° C., 2 minutes at 55 ° C., and 2 minutes at 72 ° C., and finally 72 The reaction was terminated after extending the reaction at 10 DEG C for 10 minutes. After the completion of the reaction, electrophoresis was performed on 1% agarose gel to confirm the presence of 0.3 kb sized DNA. Cut the bands corresponding to these DNA fragments, place them in a dialysis bag (Sigma), and electrophoresis for 30 seconds to extract DNA. It was. The extracted DNA was extracted twice with phenol / chloroform solution, precipitated with ethanol, purified and recovered.

Example 3: Cloning of Antibody Genes

The DNA corresponding to 300 bp obtained in Example 2 was cloned using the pGEM ^R -T Easy vector system (Promega). DNA products obtained by amplification by PCR were cloned using the pGEM ^R- T Easy vector system using the property of generating an extra adenosine at the 3 'end. Since the T-vector has one extra thymidine at the 3 'end, it has advantages in ligation with the PCR DNA (insert) product as well as multiple cloning sites at the beta-galactosidase coding site. The cloning site) and the T7 and SP6 promoters are listed so that the presence of a 300bp fragment can be easily identified by color conversion. Moreover, the vector has a pUC / M13 5 'primer binding site and a 3' primer binding site, which can be used for sequencing. 1 μl of pGEM ^R- T easy vector (50 ng / μl), 1 μl of T4 DNA ligation 10 × buffer, 16.5 ng of DNA obtained by PCR, 1 μl of T4 DNA ligation (3 units / μl), and water Was added to a final volume of 10 μl. Optimal ligation efficiency was achieved when the molar ratio between the insert DNA and the vector was 3: 1. Thus, 16.5 nanograms of PCR products with 300 bp were used in light of 50 ng of pGEM ^R- T Easy Vector having a size of about 3 kb. The ligation reaction was reacted at 4 ° C. for one day.

Competent cells used for transformation were prepared as follows. Colonies of E. Coli DH5-alpha cells were inoculated in 2 ml of LB medium, incubated and transferred to a flask containing 100 ml LB media incubated for 3-4 hours in a stirrer at 37 ° C until the UV absorbance was 0.4 at 400 nm. It was then transferred to a 50 ml tube and left on ice for 10 minutes. The solution was centrifuged at 4 ° C. and 4000 RPM for 10 minutes, then the supernatant was completely removed and the pellet was dissolved in 100 ml of 0.1 M CaCl ₂ , pre-cooled, placed on ice for 15 minutes and then centrifuged at 4 ° C. 4000 RPM for 10 minutes. Separated. The supernatant of this solution was discarded and the pellet was dispensed with 5 ml of 0.1 M CaCl ₂ in an eppendorf tube kept on ice and stored at -70 ° C. 2 μl of the ligated mixture was added to 100 μl of the prepared competent cells, mixed, and then transformed by standing on ice for 30 minutes. It was left for 2 minutes at 42 ℃ for complete transformation experiments and transferred to ice again and left for 2 minutes. In order to improve the state of the cells, 1 ml of LB medium was added and incubated for 1 hour in a 37 ° C stirrer. Incubated 4 μl of isopropylthio-β-D-galactoside (200 mg / ml) and x-gal (5-bromo-4-chloro-3-indoly-β-D-galactoside, 20 mg / ml dimethylformamide) Smear evenly in an LB medium containing 40 μL of ampicillin and incubated at 37 ° C. for one day. Using ampicillin as the marker, a single white population was selected and shaken in LB medium containing ampicillin to select clones. Only white colonies growing apart from relatively different colonies were taken and incubated for 3-4 hours in tubes containing 3 ml of LB medium, and then transferred to 15 ml tubes for one day in a 37 ° C. incubator. After confirming that the culture was cloudy, only 200 µl of the culture was taken, transferred to an Eppendorf tube containing 0.8 ml of LB medium (including 40% glycerol), and stored at -70 ° C.

Example 4 DNA Preparation by Mini-prep Method

In addition to the culture solution of Example 3, 2.8 ml of the culture solution was used for convenient plasmid DNA preparation. In this experiment, plasmid DNA was obtained by alkaline lysis. The composition of Solution I, II, III necessary for this experiment is as follows. (Solution I, 50 mM glucose, 25 mM Tris-HCl, pH 8.0), 10 mM EDTA, pH 8.0; Solution II, 0.2 N NaOH, 1% SDS; Solution III, 60 ml of 5M KOAc, 11.5 ml of acetic acid). First, 1.4 ml of the culture solution was transferred to two eppendorf tubes and centrifuged at 8000 RPM for 2 minutes to discard the supernatant, leaving only the pellet, and then stored at 4 ° C. 200 μl of Solution II was added to the solution, the tube was shaken well, left for 5 minutes on ice, and finally, 150 μl of Solution III was added, mixed well, and left for 5 more minutes on ice. After centrifugation of this solution at 8000 RPM for 5 minutes, only the supernatant containing plasmid RNA was taken and transferred to each new Eppendorf tube. Once again centrifuged for 5 min at 8000 RPM, the cell debris was allowed to settle under the tube, the supernatant was taken and transferred back to a new Endofdorf tube, followed by DNAase-free RNase A (DNase-free RNase A; 1 mg). / ml) 25 μl was added and incubated at 37 ° C. for 3 hours, and phenol was used to remove RNA remaining in the supernatant. Ethanol was concentrated and each plasmid was dropped into pellets, and each was dissolved in 30 μl of water, followed by 1 μl of 9 μL of water and 2 μL of 6X DNA dye (0.25% bromophenol blue, 30% glycerol, 0.5 x TBE). The mixture was then electrophoresed in a 0.8% agarose gel and the concentration of each band on the gel was observed to determine the extraction and relative concentration of the plasmid. 2 μL of each plasmid DNA was taken, cut with restriction enzyme EcoR1, and electrophoresed on 1.2% agarose gel to confirm the presence of 300 base pairs of nucleotides.

Example 5: Identification of base sequence

ABI PRISM ^TM Dye Terminator cycle sequencing Ready Reaction Kit (part # 402080, Perkin Elmer) was used as a template using plasmid DNA obtained as a mini-prep as a primer and using M13 broad forward oligonucleotide as a primer. DNA was prepared by using a Perkin-Elmer Model 2400 thermal cycler PCR to obtain an amount of DNA to confirm the base sequence. The sequence was confirmed using an ABI 373 automated DNA sequencer (applied Biosystems, USA). The PCR reaction was denatured at 98 ° C. for 5 minutes and then repeated 25 times for 10 seconds at 98 ° C., 5 seconds at 50 ° C., and 4 minutes at 60 ° C., and then maintained at 4 ° C. after the reaction. The PCR reaction solution was prepared by adding 8.0 μl of Terminator Ready Reaction Mix, 200-500 nanograms of plasmid DNA, 4 picomoles of M13 forward oligonucleotide, and water to adjust the total reaction solution to 20 μl. Terminator ReadyReaction Mix contains A-dye terminator, C-dye terminator, G-dye terminator, T-dye terminator, dITP, dATP, dCTP, dTTP, Tris-HCl (pH 9.0), MgCl ₂ , thermal stability fatigue Phosphatase, and AmpliTaq DNA Polymerase, FS.

The present invention seeks to provide antibodies against amino-RNAs and methods of making them using in vitro immunity. The production method of the present invention, in view of the characteristics that RNA is easily degraded under normal conditions and is recognized as an autologous material, and thus the production of antibodies is not easy in a normal immune system, amino-RNA has a relatively long half-life and thus an antibody can be prepared. In addition, unlike the method for producing a monoclonal antibody using a mouse, because it is made in vitro, there is an advantage that in vivo RNAase can not work and immune system is not impaired.

In addition, using the in vitro immunoassay of the present invention, the antibody against the amino-RNA and the nucleotide sequence encoding the same are specific and strongly bound to specific autoantibodies of autoimmune diseases to prevent autoimmune diseases, which is useful as a therapeutic agent for autoimmune diseases. In addition, the amino-RNA can be administered in vivo to induce the production of antibodies against it, and thus can be developed as a vaccine against autoimmune diseases.

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Claims (6)

In the method for producing monoclonal antibody using hybridoma, in vitro, in vitro, amino-RNA using in vitro immunoassay in which splenocytes are cultured and immunized in a medium to which amino-RNA obtained from autoantibodies of autoimmune diseases is added. Method for producing an antibody against. The antibody of claim 1, wherein the amino-RNA specifically binds to an acetylcholine esterase receptor and the antibody to the amino-RNA is encoded by V _H and SEQ ID NO: 2, which encodes SEQ ID NO: 1. A method for producing an antibody against amino-RNA using in vitro immunity, characterized in that the antibody consists of V _L. An antibody against an amino-RNA selected for an autoantibody specific for an acetylcholine esterase receptor. The antibody of claim 3, wherein the antibody consists of V _H encoded by SEQ ID NO: 1 and V _L encoded by SEQ ID NO: 2. A DNA molecule encoding the V _H of the antibody of claim 3 and having the sequence shown in SEQ ID NO: 3. A DNA molecule encoding the V _L of the antibody of claim 3 and having the sequence shown in SEQ ID NO: 4.