Monoclonal antibodies against aminoacyl-tRNA synthetases and uses thereof

Abstract

The present application relates to the field of monoclonal antibody, in particular to the monoclonal antibody against aminoacyl-tRNA synthetase. The present application provides the monoclonal antibody against aminoacyl-tRNA synthetase, the amino acid sequence of the three CDR regions of the heavy chain has the amino acid sequence as shown in SEQ ID NO:1, 2 and 3 respectively; and the amino acid sequence of the three CDR regions of the light chain has the amino acid sequence as shown in SEQ ID NO:4, 5 and 6 respectively. The present application utilizes phage display technology to obtain the aminoacyl-tRNA synthetase monoclonal antibody. The monoclonal antibody has high specificity, high affinity, high detection sensitivity, no matrix effect, solves the interchangeability and consistency of quality control products. Through identification, the monoclonal antibody is used for repeated detection, and has good stability and high accuracy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of monoclonal antibodies, and in particular to monoclonal antibodies against aminoacyl-tRNA synthetases. BACKGROUND

[0002] Autoimmunity is the reaction of the immune system to self-antigens, producing autoantibodies and autoreactive T cells. In normal physiological conditions, autoimmunity can timely eliminate aged and dead self-cells, maintaining the stability of the internal environment. In certain pathological conditions, autoreactive T cells and autoantibodies, which directly or indirectly damage self-tissues, can be produced, causing pathological changes and dysfunction of the corresponding organs and tissues, which are called autoimmune diseases. Common autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, multiple dermatomyositis, mixed connective tissue disease, hyperthyroidism, juvenile diabetes, idiopathic thrombocytopenia, autoimmune hemolytic anemia, ulcerative colitis, and many skin diseases, chronic liver disease, etc.

[0003] Anti-Jo-1 antibody is commonly found in polymyositis (PM), and the positive detection rate can reach 40% to 50%; in patients with PM and DM (dermatomyositis) overlap, the positive detection rate of anti-Jo-1 antibody is 25%; in patients with DM alone, the detection rate of anti-Jo-1 antibody is less than 10%; in other autoimmune diseases, anti-Jo-1 antibody is negative, so anti-Jo-1 antibody has specificity for diagnosing PM. In addition, in patients with PM and scleroderma overlap, the positive rate of anti-Jo-1 antibody can be as high as 85%; in patients with progressive systemic sclerosis (PSS) and PM overlap, the positive rate of anti-Jo-1 antibody is about 25%, and in patients with PM with pulmonary interstitial fibrosis, the positive rate of anti-Jo-1 antibody can reach 60%. Jo-1 antibody is one of histidyl-tRNA synthetase antibodies. This cytoplasmic enzyme catalyzes the esterification of histidine and tRNA, and exists in the cytoplasm in the form of homodimer, with a subunit molecular weight of about 50 kD. The antigenic determinant site of Jo-1 is at the N-terminus, which is a region of about 32 amino acids with a helical structure. Jo-1 antibody is rarely found in non-myositis patients. There is evidence that Jo-1 antibody is related to MHC class II genes, and the antibody titer is related to the degree of disease activity, suggesting that the body's response to Jo-1 is related to the pathogenesis of myositis in some way.

[0004] Quality control products are samples with known concentration content, which are used to identify whether the instrument measurement result is accurate. The JO-1 quality control product will undergo a period of storage and transportation before use, and even if it is used according to the reagent storage conditions and expiration date, there is still a great risk of instability, which will cause trouble to the use of the matching reagent. Therefore, providing an anti-Jo-1 antibody with strong stability and high reactivity can solve the above problems. SUMMARY

[0005] Therefore, the present application provides a monoclonal antibody against aminoacyl-tRNA synthetase. The present application uses phage display technology to obtain the aminoacyl-tRNA synthetase monoclonal antibody described in the present application. The monoclonal antibody has high specificity, high affinity, high detection sensitivity, and no matrix effect, solving the interchangeability and consistency problems of quality control products. After identification, the monoclonal antibody is used for repeated detection, and has good stability and high accuracy.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides a monoclonal antibody against aminoacyl-tRNA synthetase, (1) the amino acid sequences of the three CDR regions of the heavy chain have the amino acid sequences shown in SEQ ID NO: 1, 2 and 3, respectively; and

[0008] (2) the amino acid sequences of the three CDR regions of the light chain have the amino acid sequences shown in SEQ ID NO: 4, 5 and 6, respectively; or

[0009] (3) the amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence of (1) or (2), and the amino acid sequence which is functionally the same or similar to the amino acid sequence shown in (1) or (2); or

[0010] (4) an amino acid sequence having at least 80% homology with the sequence of (1), (2) or (3).

[0011] In some embodiments of the present application, the sequence of SEQ ID NO: 1 is GFTFSSY W.

[0012] In some embodiments of the present application, the sequence of SEQ ID NO: 2 is IKQDGSE K.

[0013] In some embodiments of the present application, the sequence of SEQ ID NO: 3 is ARDLGSG WPHEY.

[0014] In some embodiments of the present application, the sequence of SEQ ID NO: 4 is QSISSY.

[0015] In some embodiments of the present application, the sequence of SEQ ID NO: 5 is AAS.

[0016] In some embodiments of the present application, the sequence of SEQ ID NO: 6 is QXSYSTP YT; wherein: X can be Q or H.

[0017] In some embodiments of the present application, the monoclonal antibody, (5), has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 and / or SEQ ID NO: 8; and

[0018] (6), has a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 9 and / or SEQ ID NO: 10; or

[0019] (7), the amino acid sequence of (5) or (6) is obtained by substitution, deletion or addition of one or more amino acids, and an amino acid sequence functionally identical or similar to the amino acid sequence shown in (5) or (6); or

[0020] (8), an amino acid sequence having at least 80% homology with the sequence of (5), (6) or (7).

[0021] In some embodiments of the present application, the sequence of SEQ ID NO: 7 is EVQLLES GGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGS EKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGSGWP HEYWGQGTLVTVSS.

[0022] In some embodiments of the present application, the sequence of SEQ ID NO: 8 is QVQLVQS GGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGS EKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGSGWP HEYWGQGTLVTVSS.

[0023] In some embodiments of the present application, the sequence of SEQ ID NO: 9 is DIVMTQS PSSLSASIGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVDIK.

[0024] In some embodiments of the present application, the sequence of SEQ ID NO: 10 is: NIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSYSTPYTFGQGTKVDIK.

[0025] In some embodiments of the present application, the number of the monoclonal antibodies is 2, 3, 4 or 5.

[0026] The present application also provides nucleic acid molecules encoding the monoclonal antibodies.

[0027] The present application also provides expression vectors comprising the nucleic acid molecules.

[0028] The present application also provides hosts transformed and / or transfected with the expression vectors.

[0029] The present application also provides uses of the monoclonal antibodies, the nucleic acid molecules, the expression vectors and / or the hosts in any of the following:

[0030] (I) preparing products for detecting aminoacyl-tRNA synthetases; and / or

[0031] (II) preparing products for detecting Jo-1 antibody content; and / or

[0032] (III) preparing products for detecting diseases related to eosinophil airway inflammation; and / or

[0033] (IV) preparing products for detecting autoimmune diseases.

[0034] The present application also provides medicaments comprising the monoclonal antibodies, the nucleic acid molecules, the expression vectors and / or the hosts and pharmaceutically acceptable adjuvants.

[0035] The present application also provides kits comprising the monoclonal antibodies, the nucleic acid molecules, the expression vectors and / or the hosts and acceptable adjuvants.

[0036] The present application utilizes phage display technology to obtain the aminoacyl-tRNA synthetase monoclonal antibodies. The monoclonal antibodies have high specificity, high affinity and high detection sensitivity, and have no matrix effect, thus solving the problems of interchangeability and consistency of quality control products. It has been identified that the monoclonal antibodies have good stability and high accuracy when used for repeated detection. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or the prior art description will be briefly introduced.

[0038] Figure 1 Figure 2 shows a recombinant antibody purification SDS-PAGE electrophoresis map. DETAILED DESCRIPTION

[0039] The present application discloses a monoclonal antibody against aminoacyl-tRNA synthetase.

[0040] It should be understood that the expression "one or more of something" includes each of the objects recited after the expression and various combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in conjunction with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0041] The terms "comprising", "having", or "including", or their grammatical variants thereof, when used in this specification, including the claims, are generally understood to be taken as specifying the presence of one or more stated elements or steps, but not excluding the presence of one or more additional elements or steps. The use of "including" and "comprising" and variations thereof herein is meant to be broad and encompass the various options discussed herein or in any appended claims.

[0042] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0043] The use of any and all examples, or exemplary language herein, for example, only the better illustrate the present application, and unless otherwise claimed, does not pose a limitation on the scope of the present application. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present application.

[0044] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are merely intended to convey general information as to the scope of the application. Consistent with the application as claimed, the numerical ranges and parameters should be construed in light of the number of requests for the particular values in the respective examples. Therefore, unless otherwise stated, all ranges, numbers, values and percentages used herein are to be understood as "about" any stated value, unless otherwise explicitly stated. Herein, "about" generally means within ±10%, ±5%, ±1%, or ±0.5% of a stated value or range.

[0045] The present application provides a monoclonal antibody against aminoacyl-tRNA synthetase and its application. The antibody of the present application is mainly used for detecting the content of aminoacyl-tRNA synthetase by western blot.

[0046] To achieve the above object, the present application provides the following technical solutions.

[0047] The present application provides a monoclonal antibody against aminoacyl-tRNA synthetase,

[0048] (1), the CDR1, CDR2 and CDR3 of its heavy chain have the amino acid sequences shown as SEQ ID No: 1, 2 and 3 in order; and

[0049] (2), the CDR1, CDR2 and CDR3 of its light chain have the amino acid sequences shown as SEQ ID No: 4, 5 and 6 in order; or

[0050] (3), a sequence in which one or more amino acids are substituted, deleted, added and / or replaced on the basis of the amino acid sequences shown in (1) or (2); or

[0051] (4), a sequence having more than 80% homology with the amino acid sequences shown in any one of (1) to (3).

[0052] In some embodiments of the present application, the monoclonal antibody,

[0053] (5), the heavy chain variable region thereof has the amino acid sequence shown as SEQ ID No: 7 or as SEQ ID No: 8; and

[0054] (6), the light chain variable region thereof has the amino acid sequence shown as SEQ ID No: 9 or as SEQ ID No: 10; or

[0055] (7), a sequence in which one or more amino acids are substituted, deleted, added and / or replaced on the basis of the amino acid sequences shown in (5) or (6); or

[0056] (8), a sequence having more than 80% homology with the amino acid sequences shown in any one of (5) to (7).

[0057] The light chain of monoclonal antibody J233 is:

[0058] NIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSYSTPYTFGQG TKVDIK (shown as SEQ ID NO: 11);

[0059] Light chain QSISSY (as shown in SEQ ID NO: 12) AAS (as shown in SEQ ID NO: 13) QHSYSTPYT (as shown in SEQ ID NO: 14) ;

[0060] Heavy chain: QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPG KGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDT AVYYCARDLGSGWPHEYWGQGTLVTVSS (as shown in SEQ ID NO: 15) ;

[0061] Heavy chain GFTFSSYW (as shown in SEQ ID NO: 16) IKQDGSEK (as shown in SEQ ID NO: 17) ARDLGSGWPHEY (as shown in SEQ ID NO: 18).

[0062] The application also provides application of the monoclonal antibody in preparation of a reagent or kit for detecting aminoacyl-tRNA synthetase.

[0063] The application also provides application of the monoclonal antibody in preparation of a reagent and / or kit for diagnosing Jo-1 antibody content.

[0064] The application also provides application of the monoclonal antibody in preparation of a reagent and / or kit for detecting a disease related to eosinophil airway inflammation.

[0065] The autoimmune diseases include diseases such as polymyositis (PM) and dermatomyositis (DM).

[0066] On the basis of the above research, the application also provides a reagent comprising the monoclonal antibody.

[0067] In the embodiments 1-5 of the application, the raw materials and reagents used can be purchased from the market.

[0068] The application will be further described below in combination with examples:

[0069] Example 1: Screening of human serum positive samples

[0070] Preparation of Anti-Jo-1 IgG detection kit, detection of 20 human serum samples, 20 μL sample + 380 μL diluent dilution, 10 μL, add 20 μL magnetic beads, 90 μL sample dilution, incubate for 15 min, then wash with magnet, add 100 μL enzyme conjugate, incubate for 17 min, wash with magnet, add substrate to detect signal value, the test results are shown in Table 1, one positive sample 18 is detected, the lymphocytes of the person are separated, the total RNA of the lymphocytes is extracted by Trizol method, and the cDNA is synthesized by reverse transcription.

[0071] Table 1 Positive sample screening

[0072]

[0073] Example 2 scFv gene splicing and phage screening construction

[0074] 1. scFv gene splicing:

[0075] PCR amplifies the light chain variable region and the heavy chain variable region of the antibody, and the PCR reaction program is shown in Table 2: The PCR product is recovered by 1% agarose gel. The amplified light chain variable region and the heavy chain variable region are spliced into scFv using overlap-PCR method, and the product is stored at-20℃ after recovery.

[0076] Table 2 PCR amplification program

[0077]

[0078]

[0079] 2. Construction and screening of phage single-chain antibody library:

[0080] The phagemid vector pcomb3XSS and the purified ScFv fragment are subjected to enzyme digestion with SfiI to construct a recombinant plasmid, the recombinant plasmid is electroporated into TG1 competent cells to construct an immune single-chain antibody library, and a primary phage single-chain antibody library is prepared; the primary phage single-chain antibody library is subjected to enrichment screening for 3 rounds to obtain a specific phage single-chain antibody library with high affinity and strong specificity; 558 monoclonal antibodies are picked, and a monoclonal phage supernatant is prepared, and positive clones are identified by Phage-ELISA to obtain positive sequences. The titers of 10 phage supernatants screened by library construction and washing all meet the requirements, as shown in Table 3.

[0081] Table 3 Phage supernatant reactivity

[0082] Number Luminous value Y201 50,446,292 Y216 57,744,461 Y218 51,714,830 Y226 58,224,202 Y232 55,083,209 Y233 48,395,436 Y249 48,627,015 Y253 53,386,107 Y266 55,618,318 Y273 62,397,770 Y274 69,184,845 Y280 50,257,598

[0083] Example 3 Recombinant monoclonal antibody expression and antibody purification

[0084] 1. Construction of transient cell strain

[0085] The antibody variable region gene was obtained by PCR amplification of the antibody heavy chain variable region and light chain variable region, respectively. The heavy chain antibody gene and the light chain antibody gene were homologously recombined with the RGFc-PCMV3 and RCL-PCMV3 vectors digested by KasI / BamHI, respectively, to obtain an expression vector of the human whole antibody sequence. After recombination, the whole antibody plasmid was transformed into competent DH5a cells, and positive clones were selected for sequencing and plasmid extraction. The extracted plasmid was transfected into HEK293 cells. After 48 h of transfection, ELISA was used to detect the expression of the antibody, and then the medium was supplemented. After 7 days of transfection, the cell supernatant was collected to obtain the target protein supernatant.

[0086] 2. Antibody SPA purification and SDS-PAGE identification column equilibration

[0087] The flow rate was set to 6.4 mL / min, the equilibration buffer 0.02 mol / L PBS pH 7.4 was used to flush the column, the flow rate was set to 3.8 mL / min, and the sample was loaded for purification. 10 μL of sample from the flow tube was added to 200 μL of G250 dye solution to detect blue color. When the flow tube was placed in a 250 mL conical flask, the flow was collected, and the column was equilibrated again: the flow rate was set to 6.4 mL / min, and the equilibration buffer 0.02 mol / L PBS pH 7.4 was used to flush the column again until no protein flowed out. Dissociation: 10 4 mL centrifuge tubes were placed on the dissociation tube rack, 200 μL of 1 mol / L Tris (pH 8.5) was added to each tube, the constant flow pump was set to a flow rate of 6.4 mL / min, and the target protein was dissociated with dissociation buffer 0.2 mol / L Gly + 0.15 mol / L NaCl pH 2.7. When 10 μL of sample from the flow tube was added to 200 μL of G250 dye solution and blue color was detected, manual collection was started. Each tube collected 4 mL, and when 10 μL of sample from the flow tube was added to 200 μL of G250 dye solution and no color was detected, the collection was stopped. The collected protein was dialyzed into 10 mM PBS PH 7.2 buffer, and the concentration was adjusted to 2 mg / mL. Then, SDS-PAGE detection was performed, as shown in Figure 1 The purified antibody had high purity.

[0088] Example 4: Screening of high-reactivity recombinant human J0-1 antibody using Antu self-produced kit

[0089] Prepare Anti-Jo-1 IgG detection kit, detect recombinant antibody reactivity, 20 μL sample + 380 μL diluent dilution, take 10 μL, add 20 μL magnetic beads, 90 μL sample dilution, incubate for 15 min, then wash with magnet, add 100 μL enzyme conjugate, incubate for 17 min, then wash with magnet, add substrate to detect signal value, test results are shown in Table 4, 4 strains of purified antibodies are diluted 500 times, and are all higher than 200 AU / mL, which can further evaluate the universality of the antibodies.

[0090] Table 4 Recombinant Jo-1 antibody reactivity

[0091]

[0092] Example 5 Jo-1 antibody stability detection

[0093] Use the Anti-Jo-1 IgG magnetic microparticle luminescence kit on the market to detect 4 strains of recombinant antibodies, and detect the stability of the antibodies. The experimental results are as follows: the stability of 266 is the strongest.

[0094] Table 5

[0095]

[0096] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A monoclonal antibody against histyl-tRNA synthetase, characterized in that, (1) The amino acid sequences of its heavy chain CDR1, CDR2 and CDR3 are as shown in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively; and (2) The amino acid sequences of its light chain CDR1, CDR2 and CDR3 are shown in SEQ ID NO:12, AAS and SEQ ID NO:14, respectively.

2. The monoclonal antibody as described in claim 1, characterized in that, (5) Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO:8; and (6) Its light chain variable region has an amino acid sequence as shown in SEQ ID NO:10; or Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences described in (7), (5), or (6), and which have the same or similar function as the amino acid sequences shown in (5) or (6); or (8) An amino acid sequence that is at least 80% homologous to the sequence described in (5), (6) or (7).

3. The monoclonal antibody as described in claim 2, characterized in that, The "multiple" refers to 2, 3, 4, or 5.

4. A nucleic acid molecule encoding the monoclonal antibody as described in any one of claims 1 to 3.

5. An expression vector, characterized in that, Includes the nucleic acid molecules as described in claim 4.

6. The host, characterized in that, Transformation and / or transfection of the expression vector as described in claim 5.

7. The use of the monoclonal antibody as described in any one of claims 1 to 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, and / or the host as described in claim 6 in any one of the following: ( ), and prepare products for detecting histyl-tRNA synthetase; and / or ( ), and prepare products for detecting Jo-1 antibody content; and / or ( ), and prepare products for the detection of polymyositis.

8. A kit comprising a monoclonal antibody as described in any one of claims 1 to 3, a nucleic acid molecule as described in claim 4, an expression vector as described in claim 5, and / or a host as described in claim 6, as well as acceptable adjuvants.

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