ANTIBODY SPECIFICALLY BINDING TO WRS PROTEIN, AND USE THEREOF
20220268787 · 2022-08-25
Inventors
- Sumi BAE (Chungcheongbuk-do, KR)
- Sunghwa SON (Chungcheongbuk-do, KR)
- Yunsun KIM (Chungcheongbuk-do, KR)
- Jieun PARK (Chungcheongbuk-do, KR)
- Min Chul PARK (Gyeonggi-do, KR)
- Su Jin KANG (Gyeonggi-do, KR)
Cpc classification
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to an antibody specifically binding to a tryptophanyl-tRNA synthetase (WRS) protein and, more specifically, to: an antibody, or a fragment of the antibody, specifically binding to a polypeptide of an amino acid sequence represented by SEQ ID NO: 2 in a WRS protein; a polynucleotide encoding the antibody and a vector comprising same; a cell transformed using same; and a use thereof.
Claims
1. An antibody or a fragment thereof specifically binding to a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2 in a WRS (tryptophanyl-tRNA synthetase) protein.
2. The antibody or the fragment thereof according to claim 1, wherein the antibody comprises an antibody light-chain variable region (VL) comprising a complementarity-determining region (CDR) L1 comprising an amino acid sequence represented by SEQ ID NO: 3, a complementarity-determining region (CDR) L2 comprising an amino acid sequence represented by SEQ ID NO: 4, and a complementarity-determining region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 5, and an antibody heavy-chain variable region (VH) comprising a complementarity-determining region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: 6, a complementarity-determining region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 7, and a complementarity-determining region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 8.
3. The antibody or the fragment thereof according to claim 1, wherein the antibody or the fragment thereof comprises a light-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 9 and a heavy-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 10.
4. The antibody or the fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody.
5. The antibody or the fragment thereof according to claim 1, wherein the antibody is selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.
6. The antibody or the fragment thereof according to claim 1, wherein the fragment of the antibody is selected from the group consisting of diabody, Fab, Fab′, F(ab)2, F(ab′)2, Fv, and scFv.
7. A polynucleotide encoding the antibody or the fragment thereof according to claim 1.
8. A vector comprising the polynucleotide according to claim 7.
9. A cell transformed with the vector according to claim 8.
10. A method of producing an antibody or a fragment thereof binding to WRS, comprising: producing a polypeptide comprising light-chain and heavy-chain variable regions by culturing the cell according to claim 9 under conditions in which a polynucleotide is expressed; and recovering the polypeptide from the cell or a culture medium in which the cell is cultured.
11. A composition for diagnosing cancer comprising the antibody or the fragment thereof according to claim 1.
12. A composition for diagnosing an infectious disease or infectious complications comprising the antibody or the fragment thereof according to claim 1.
13. The composition according to claim 12, wherein the infectious disease is an infectious inflammatory disease.
14. The composition according to claim 13, wherein the infectious inflammatory disease is sepsis or septic shock.
15. Use of the antibody or the fragment thereof according to claim 1. for manufacture of an agent for diagnosing cancer.
16. A method of diagnosing cancer, comprising: a) obtaining a sample from a subject; b) measuring a WRS protein expression level in the sample using the antibody or the fragment thereof according to claim 1; and c) determining that the subject has cancer when the protein expression level measured in step b) is increased.
17. Use of the antibody or the fragment thereof according to claim 1 for manufacture of an agent for diagnosing an infectious disease or infectious complications.
18. A method of diagnosing an infectious disease or infectious complications, comprising: a) obtaining a sample from a subject; b) measuring a WRS protein expression level in the sample using the antibody or the fragment thereof according to claim 1; and c) determining that the subject has an infectious disease or infectious complications when the protein expression level measured in step b) is increased.
Description
DESCRIPTION OF DRAWINGS
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MODE FOR INVENTION
[0133] A better understanding of the present invention may be obtained through the following examples. These examples are merely set forth to illustrate the present invention, and are not to be construed as limiting the scope of the present invention.
Example 1
Production of Monoclonal Antibody
[0134] (1) Production of Hybridoma Cell
[0135] 1) Animal Immunization and Cell Fusion [0136] Preparation of immunogen: 1.5 to 2 mg of WRS protein (purity >75%, concentration >0.4 mg/ml) [0137] Animal immunization: Antibody production was induced by inoculating the immunogen into Balb/c mice. [0138] Cell fusion: At least 10,000 hybridoma cells were obtained by electro-fusion of mouse B cells and mouse myeloma cells.
[0139] 2) Selection of Hybridoma Cell [0140] Primary selection: Hybridoma cells producing an antigen-binding antibody were selected through indirect ELISA. [0141] Secondary selection: One hybridoma cell line binding to an antigen was selected through Western blotting using the positive clones obtained in the primary selection, and the antibody produced from the selected hybridoma cell line was named 4D10G6. [0142] Isotyping: Five clones having the best results in the selection process were subjected to isotyping.
[0143] 3) Subcloning, cell expansion, freezing storage, and antibody production
[0144] Subcloning, cell expansion, and freezing storage: Clones having good results were subjected to subcloning, cell expansion, and freezing storage. [0145] Antibody production: An antibody was produced in an amount of at least 2 mg from the hybridoma cell line having the best results in the selection process.
[0146] 2. Formation of Ascites
[0147] 1) After adaptation of mice for at least 3 days, a pristane adjuvant was administered in an amount of 100 pl/mouse thereto. The hybridoma cell line was cultured so that it could be injected 5 to 7 days after administration of the pristane adjuvant.
[0148] 2) The cultured hybridoma cell line was collected in a 50 ml tube, washed three times with 10 ml PBS, and centrifuged.
[0149] 3) After centrifugation, the supernatant was removed by suction, after which the number of cells required per 100 pl was calculated, added with 1× PBS, mixed well, and then transferred into a 1.5 ml tube.
[0150] 4) The above solution was placed in a 1 ml syringe, and the air in the syringe was removed by turning the syringe needle upwards.
[0151] 5) 100 μl of the solution was injected intraperitoneally to each Balb/c mouse, after which the mice were placed in a cage, and whether ascites was present was observed.
[0152] 6) From 5 days after injection of the hybridoma cell line into the mice, abdominal bloating was observed every day.
[0153] 7) When abdominal bloating was noted, ascites fluid was collected from the abdominal cavity of the mice using a product with an injection needle of 23 G or less (using a 3 ml or 5 ml syringe).
[0154] 8) The ascites fluid, collected and placed in a tube, was incubated at RT for 10 min to allow red blood cells to aggregate, followed by centrifugation.
[0155] 9) After centrifugation, only the supernatant was placed in a new 1.5 ml tube and stored at −70° C.
[0156] 3. Production of Antibody
[0157] 1) The produced ascites fluid was taken out at −70° C. and thawed at 4° C., and the type of beads to be used was determined by confirming the subtype of the antibody to be purified. The amount of beads used was 0.5 the volume of ascites fluid.
[0158] 2) Well-mixed Protein A beads or G beads were placed in the calculated amount in a 5 ml chromatography column, and bead washing was performed by allowing 5 ml of 1× PBS to flow into the column.
[0159] 3) After completion of washing, the thawed ascites fluid was placed in the column, and the column was capped.
[0160] 4) Rotation binding was performed at 4° C. for 1 hr so that the beads and the antibody were bound to each other.
[0161] 5) After rotation binding, the entire solution was subjected to a flow-through process.
[0162] 6) Column washing was performed using 100 ml of 1× PBS.
[0163] 7) 100 μl of a neutralization buffer was added to a 1.5 ml tube, and 1 ml of an IgG elution buffer was added to the column to enable neutralization immediately after IgG elution. A total of 10 fractions were obtained under the same conditions.
[0164] 8) A portion of each fraction was loaded on a 12% SDS-PAGE gel, and the band was confirmed through gel staining. During staining, fractions were stored at 4° C.
[0165] 9) The fractions having distinct bands were collected, placed in dialysis tubing, and sealed with a clip to prevent leakage. The dialysis tubing and a stirrer bar were placed in a beaker containing 1 L of 1× PBS, and dialysis was performed at 4° C. for 1 hr using a stirrer.
[0166] 10) Dialysis was performed using 1 L of fresh 1× PBS overnight (15 hr) under the same conditions as in 9) above.
[0167] 11) The next day, the solution was collected from the dialysis tubing and immediately quantified using a BCA assay kit.
Example 2
Sequencing
[0168] Total RNA was isolated from the hybridoma cells according to the technical manual of a TRIzol reagent. Total RNA was reverse-transcribed into cDNA using universal primers according to the technical manual for a PrimeScript 1st Strand cDNA Synthesis Kit. The antibody fragments of a heavy-chain variable region (VH) and a light-chain variable region (VL) were amplified through RACD (rapid amplification of cDNA ends). The amplified antibody fragment was cloned separately into a standard cloning vector. Colony PCR was performed to screen clones having inserts of the correct size. At least 5 colonies having inserts of the correct size were sequenced for each fragment. The sequences of the different clones were aligned, and consensus sequences of these clones were provided.
[0169] The light-chain and heavy-chain variable region amino acid sequences of the sequenced monoclonal antibody of the present invention and the sequences of polynucleotide encoding the same are illustrated in
Example 3
Identification of Polypeptide in WRS to Which Monoclonal Antibody Specifically Binds
[0170] In order to identify the polypeptide region recognized by the monoclonal antibody produced in Example 1, the WRS protein (1-471) of SEQ ID NO: 1 consisting of 471 amino acids and the protein fragments (48-471, 1-104, 1-154, and 45-154) were prepared.
[0171] 1) In order to purify the recombinant WRS protein and fragment peptides thereof, competent cells for protein expression were transformed with the plasmid in which the WRS protein and fragment genes thereof were cloned into a pET28a vector.
[0172] 2) The transformed cells were spread on an LB (+Kanamycin) plate, followed by culture at 37° C. for 15 hr.
[0173] 3) The next day, a single colony was inoculated into 3 ml of LB (+Kan), followed by culture at 200 rpm and 37° C. for 3 hr.
[0174] 4) All of the small cultured cells were placed in 500 ml of LB (+Kan), followed by culture at 37° C. and 200 rpm for 4 hr.
[0175] 5) When 0.8<OD value<1 was measured, 250 μl of a 1 M IPTG stock was added thereto (final 0.5 mM IPTG), followed by induction at 18° C. and 200 rpm overnight (15 hr).
[0176] 6) The next day, the induction-treated cells were centrifuged at 4,000 rpm for 10 min.
[0177] 7) The supernatant was removed, and the pellets were suspended in 10 ml of washing buffer 1.
[0178] 8) The cells were lysed using a sonicator. Treatment with 35% AMPL for 2 sec and storage on ice for min were performed. This process was repeated 14 times (a total of 15 sonications).
[0179] 9) Centrifugation was performed at 15,000 rpm and 4° C. for 30 min to separate pellets and the supernatant from each other.
[0180] 10) 200 pl of Ni-NTA beads were placed in poly-prep chromatography columns, and 5 ml of washing buffer 1 was added to reach equilibrium.
[0181] 11) After centrifugation, the supernatant was filtered using a 0.45 μm filter in a 50 ml tube and was allowed to flow into the column containing the beads. This procedure was performed once more.
[0182] 12) Washing was performed using washing buffer 1.
[0183] 13) Washing was performed using washing buffer 2.
[0184] 14) Washing was performed using washing buffer 3.
[0185] 15) Washing was performed using washing buffer 4.
[0186] 16) The washed column was placed on a 1.5 ml tube and an elution buffer was then passed therethrough, and thus an eluate was collected.
[0187] 17) A 5× sample buffer and DW were placed in a 5 ml tube and subjected to a flow-through process, and a washing buffer and the eluate were added thereto and then boiled in a heat block for 5 min.
[0188] 18) A premade 15-well comb and 15% SDS-PAGE gel were assembled in a cassette, the cassette was placed in the tank, and the gel and the tank were filled with a 1× running buffer.
[0189] 19) The protein marker and sample were sequentially loaded.
[0190] 20) During gel loading, dialysis tubing was heated in a DW bath at 100° C. for 10 min. The DW was replaced with fresh DW and the heating process in a DW bath was repeated twice more, followed by cooling using 200 ml of cold 1× PBS.
[0191] 21) After loading, the gel was separated from the cassette, and staining was performed by pouring instant blue until the gel was submerged (
[0192] Western blotting was performed according to a typical method using the WRS protein produced through the above method, fragments thereof, and the monoclonal antibody produced in Example 1 as the primary antibody.
[0193] As a result, as shown in
Example 4
Analysis of Binding Affinity of Antibody
[0194] In order to evaluate binding affinity of the monoclonal antibody produced in Example 1 and two commercial antibodies (Abnova, anti-WRS antibody (Cat# H00007453-M02) and Novus biological, anti-WRS antibody (Cat#NBP2-32186)), indirect ELISA assay was performed on the full-length WRS protein of SEQ ID NO: 1.
[0195] Briefly, the binding affinity of the antibodies was evaluated according to the following method.
[0196] 1) The WRS protein was diluted to 1 μg/ml in PBS, loaded in an amount of 100 μl/well into a 96-well plate, and reacted at room temperature for 1 hr, whereby the wells were coated therewith.
[0197] 2) After completion of coating, washing was performed once with a PBST (0.05% Tween-20) buffer, and 3% BSA and PBST (0.1% tween-20) were dispensed, followed by a blocking reaction at room temperature for 1 hr.
[0198] 3) The biotin-attached antibody was diluted with a blocking buffer according to each concentration and then reacted at room temperature for 1 hr.
[0199] 4) Washing was performed with PBST (0.05% Tween-20).
[0200] 5) Streptavidin-HRP was diluted with a blocking buffer, followed by reaction at room temperature for 1 hr.
[0201] 6) Washing was performed five times with PBST (0.05% Tween-20) to remove all unattached residue.
[0202] 7) 50 μl/well of TMB was added thereto, followed by reaction at room temperature for 5 min, after which the same amount of 2 M H2504 was added to terminate the reaction.
[0203] 8) Absorbance was measured using a spectrophotometer (Sunrise, Tecan) (450 nm).
[0204] 9) The EC.sub.50 values were calculated from the results of 8) above.
[0205] The results thereof are shown in Table 1 below.
TABLE-US-00001 TABLE 1 4D10G6 Abnova Novus EC.sub.50 40.5 1655.6 532.8
[0206] As is apparent from Table 1, it was confirmed that the antibody according to the present invention exhibited very high affinity to the WRS protein compared to the two commercial antibodies.
Example 5
Analysis of Binding Specificity of Antibody
[0207] In order to evaluate the binding specificity of the monoclonal antibody produced in Example 1 and two commercial antibodies (Abnova, anti-WRS antibody (Cat# H00007453-M02) and Novus biological, anti-WRS antibody (Cat#NBP2-32186)), 20 μg of an HCT116 cell lysate was treated with each of a primary antibody and a secondary antibody under the following conditions, and Western blotting was performed according to a typical method. [0208] Primary antibody (room temperature, 1 hr)
[0209] 4D10G6: 1 μg/ml
[0210] Abnova Ab: 1:5,000 dilution
[0211] Novus Ab: 1:10,000 dilution [0212] Secondary antibody (room temperature, 1 hr)
[0213] Anti-mouse HRP (Millipore, AP181P): 1:10,000 dilution : Abnova, 4D10G6
[0214] Anti-rabbit HRP (Millipore, AP187P): 1:10,000 dilution : Novus
[0215] The results thereof are shown in
[0216] As shown in
[0217] Therefore, it was confirmed that the antibody according to the present invention exhibited very high binding specificity compared to the commercial antibodies.
Example 6
Validation of Cross-Reactivity
[0218] In order to evaluate whether the monoclonal antibody produced in Example 1 exhibit cross-reactivity with CRS (cysteinyl-tRNA synthetase), AIMP1 (aminoacyl tRNA synthase complex-interacting multifunctional protein 1), GRS (glycyl tRNA synthetase), and KRS (lysyl tRNA synthetase), which are other ARS (aminoacyl-tRNA synthetase) proteins secreted from the cells, in addition to WRS, indirect ELISA assay was performed according to the following method.
[0219] 1) Antigen coating: 1 μg/ml in PBS, 100 μl/well, 4° C., overnight coating
[0220] 2) Washing: 0.05% PBST (0.05% Tween 20), 200 μl/well, 3 times
[0221] 3) Blocking: 0.5% BSA in 0.05% PBST, 200 μl/well, RT, 1 hr
[0222] 4) Primary antibody binding: 500 ng/ml in 0.05% PBST, 100 μl/well, RT, 1 hr
[0223] 5) Secondary antibody binding: anti-mouse HRP (AP160P) 1:10,000 in 0.05% PBST, 100 μl/well, RT, 1 hr
[0224] 6) TMB detection
[0225] 7) Reaction termination (2 M H.sub.2SO.sub.4)
[0226] 8) Absorbance measurement: 450 nm
[0227] The results thereof are shown in
[0228] As shown in
INDUSTRIAL APPLICABILITY
[0229] The antibody or the fragment thereof according to the present invention specifically binds to WRS and has no cross-reactivity with other proteins included in the same ARS family, making it possible to detect and inhibit WRS, and can thus be effectively used for detecting WRS and diagnosing WRS-related diseases such as cancer, inflammatory diseases, or infectious diseases, thereby exhibiting high industrial applicability.