Humanized anti-beta2-GP1 monoclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to the technical field of antibodies, in particular to a humanized anti-beta2-GP1 monoclonal antibody as well as a preparation method and application thereof. The full-length IgG / IgM / IgA type antibody sequence is obtained by constructing a natural full human source library, adopting a phage display technology, screening out a high-affinity antigen binding fragment, and assembling a variable region sequence and an IgG / IgM / IgA constant region. An anti-beta2-GPI antibody is expressed through transient transfection, a protein A / L affinity purification method is adopted for preparation, and binding activity determination is carried out. Results show that the specific beta2-GPI antibody obtained by the method is high in antigen binding activity, high in specificity and good in affinity.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a humanized anti-β2-GP1 monoclonal antibody, a preparation method and an application thereof. Background Art

[0002] Antiphospholipid antibodies (aPLs) are a group of autoantibodies that use phospholipids and / or phospholipid-binding proteins as target antigens. aPLs are mainly found in patients with autoimmune diseases such as antiphospholipid syndrome (APS) and are the most characteristic laboratory indicators of APS. aPLs are also risk factors for thrombosis and pathological pregnancy. At the same time, aPLs can be seen in malignant tumors, infectious diseases, after the use of certain drugs, and even in some healthy people. Among them, lupus anticoagulant (LA), anticardiolipin antibodies (aCL), and anti-β2-glycoprotein I (β2-GPI) antibodies are laboratory indicators in the APS classification standard. They are currently widely used in clinical practice and have become one of the most common autoantibody detection items in clinical laboratories. The standardization of aPLs detection is crucial to its clinical application. Regarding the standardization of the clinical application of LA, aCL, and anti-β2-GPI antibody detection, relevant consensus and guidelines have been published by different international academic organizations.

[0003] aPLs are mainly divided into the following categories according to the characteristics of the target antigen: (1) LA: a group of immunoglobulins that can bind to negatively charged phospholipids and phospholipid protein complexes. They are detected mainly based on the fact that LA can prolong the coagulation test time of different phospholipid-dependent pathways in vitro; (2) Anti-negatively charged phospholipid antibodies: aCL antibodies, anti-phosphatidylserine antibodies, anti-phosphatidic acid antibodies, anti-phosphatidylinositol antibodies, etc.; (3) Anti-neutral phospholipid antibodies: anti-phosphatidylcholine antibodies, etc.; (4) Anti-amphoteric phospholipid antibodies: anti-phosphatidylethanolamine antibodies, etc.; (5) Anti-phospholipid-binding protein antibodies: anti-β2-GPI antibodies, anti-prothrombin antibodies, anti-protein C antibodies, anti-protein S antibodies, anti-annexin A2 antibodies, anti-annexin A5 antibodies, etc. Among them, LA, aCL-IgG antibody, aCL-IgM antibody, anti-β2-GPI-IgG antibody, and anti-β2-GPI-IgM antibody have been used as laboratory indicators in the APS classification standard revised by the International Society on Thrombosis and Hemostasis (ISTH) in 2006.

[0004] The detection of aCL antibodies and anti-β2-GPI antibodies usually includes IgG, IgM, and IgA subtypes. It is recommended to detect aCL-IgG antibodies and aCL-IgM antibodies, anti-β2-GPI-IgG antibodies, and anti-β2-GPI-IgM antibodies. aCL-IgA antibodies and anti-β2-GPI-IgA antibodies have not yet been included in the APS classification criteria. High titers of aCL-IgA antibodies and anti-β2-GPI-IgA antibodies are rarely seen clinically, and their exact clinical significance remains to be further studied. If the IgG and IgM types of aCL antibodies and anti-β2-GPI antibodies are negative, but APS is suspected clinically, it is recommended to test aCL antibodies and anti-β2-GPI-IgA antibodies.

[0005] ELISA is a conventional detection method for aCL and aβ2-GPI antibodies and is currently widely used in clinical practice. The ELISA detection method mainly relies on the calibrators and controls in the kit to compare and analyze the serum samples to be tested and to distinguish positive. At present, these calibrators and controls are made by mixing and preparing a certain concentration gradient with the positive serum of patients and the serum of healthy people. This requires a stable source of serum, and the serum raw materials must be representative. Considering the issue of biosafety, major hospitals implement strict management of serum samples, resulting in a shortage of serum raw materials in the production process of ELISA kits. On the other hand, the serum raw materials collected clinically have serious individual differences, which often lead to fluctuations in the test data of the kit and inconsistent interpretation of the results during use. This is a problem that is difficult to avoid in the application of ELISA kits produced using clinical serum. At the same time, the test results of ELISA kits provided by different manufacturers vary greatly, and the false positive rate is high. The cause of false positives may come from the interference of nonspecific antibodies and the preparation of calibrators and controls used in the kits. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a humanized anti-β2-GP1 monoclonal antibody, a preparation method and an application thereof.

[0007] The present invention provides an anti-β2-GP1 monoclonal antibody.

[0008] The amino acid sequences of the three CDR regions of its heavy chain are shown in SEQ ID NOs: 1, 2 and 3, respectively, and the amino acid sequences of the three CDR regions of its light chain are shown in SEQ ID NOs: 4, 5 and 6, respectively.

[0009] In some embodiments, the anti-β2-GP1 monoclonal antibody provided by the present invention is

[0010] Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7;

[0011] The light chain variable region thereof has the amino acid sequence shown in SEQ ID NO:8.

[0012] In some embodiments, in the anti-β2-GP1 monoclonal antibody provided by the present invention, the constant region of the heavy chain is any one of the IgG, IgA and IgM subtypes, and the constant region of the light chain is κ type.

[0013] Immunizing animals with antigens to obtain polyclonal antiserum is a classic method for preparing antibodies, but the preparation period is long and the amount of antibodies obtained is limited. The present invention uses phage display technology to screen and obtain the anti-β2-GP1 monoclonal antibody of the present invention, which has strong specificity, high sensitivity and good affinity.

[0014] The present invention provides a biomaterial comprising at least one of the following:

[0015] 1) A nucleic acid encoding the anti-β2-GP1 monoclonal antibody;

[0016] 2) an expression vector comprising the nucleic acid;

[0017] 3) transforming or transfecting host cells with the expression vector;

[0018] 4) culturing the host cells to obtain a culture containing the anti-β2-GP1 monoclonal antibody.

[0019] In some embodiments, the present invention further provides an expression module comprising the nucleic acid, wherein the expression module comprises a promoter, a terminator and the nucleic acid of the present invention.

[0020] In some embodiments, the expression module further comprises an expression module formed by combining a single or multiple nucleic acids of the present invention in series, fusion expression or other feasible manners, and the present invention is not limited to this.

[0021] In some embodiments, the present invention also provides a transcription unit, which refers to a DNA sequence starting from a promoter and ending at a terminator. A regulatory segment may also be included on both sides or between the promoter and the terminator, and the regulatory segment may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, a transmembrane signal peptide, and a homologous recombination site operably connected to the nucleic acid sequence, such as an enhancer of a promoter, an ITR sequence, polyA, a MIS signal peptide, etc.

[0022] In some embodiments, the host cells described in the present invention are derived from plants, animals, microorganisms or viruses, which are not limited in the present invention. The present invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells, so that the transformed host cells have the ability to replicate protein-encoding vectors or express desired proteins.

[0023] In some embodiments, the transformation method includes chemical transformation and electroporation; the transfection method includes calcium phosphate coprecipitation, artificial liposome method, viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0024] The invention provides a labeled antibody, comprising a label and the anti-β2-GP1 monoclonal antibody.

[0025] In some embodiments, the markers include chemical markers and biological markers;

[0026] The chemical markers include isotopes and / or chemical drugs;

[0027] The biomarker includes biotin, avidin or enzyme; the enzyme includes horseradish peroxidase or alkaline phosphatase.

[0028] The invention provides a conjugate, comprising a conjugation medium and the anti-β2-GP1 monoclonal antibody.

[0029] In some embodiments, the coupling medium comprises a solid medium or a semi-solid medium.

[0030] In some embodiments, the coupling medium is selected from colloidal gold, polystyrene plates or beads.

[0031] The present invention provides the use of at least one of the following i-iii in the preparation of a product for detecting antiphospholipid syndrome:

[0032] i. the anti-β2-GP1 monoclonal antibody;

[0033] ii. the biological material;

[0034] iii. the labeled antibody;

[0035] iv. the conjugate.

[0036] The present invention provides a product for detecting antiphospholipid syndrome, comprising at least one of the following ① to ④:

[0037] ①, the anti-β2-GP1 monoclonal antibody;

[0038] ②, the biological material;

[0039] ③, the labeled antibody;

[0040] ④. The conjugate.

[0041] The present invention collects whole blood from APS patients, especially patients with positive β2-GPI antibodies, obtains lymphocytes through lymphocyte separation, constructs a natural all-human library, uses phage display technology to screen high-affinity antigen-binding fragments, assembles variable region sequences with IgG / IgM / IgA constant regions, and obtains full-length IgG / IgM / IgA antibody sequences. Anti-β2-GPI antibodies are expressed by transient transfection into HEK 293T cells, and protein L affinity purification is used to prepare and measure binding activity and cell activity. The results show that a specific β2-GPI antibody is obtained by the above method, and the antibody has strong antigen binding activity, strong specificity, high sensitivity, and good affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Shown is the reducing SDS-PAGE image of β2-GPI-IgG, β2-GPI-IgM, and β2-GPI-IgA antibodies, in which the 5 lanes from left to right are Marker, GP144-IgA, GP144-IgG, GP144-IgG, and GP144-IgM. DETAILED DESCRIPTION

[0043] The present invention provides humanized anti-β2-GP1 monoclonal antibodies, preparation methods and applications thereof. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0044] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0045] Example 1 Production of anti-β2-GPI antibodies

[0046] Using phage display technology, specific antibodies against human β2-GPI protein were screened from a natural fully human library. For this purpose, 400 mL of 2×YT / ampicillin medium was inoculated with glycerol bacteria from a natural library of phage-displayed fully human single-chain antibodies to a cell density of OD600 = 0.1, and cultured at 37°C and 200 rpm until the cell density reached OD600 = 0.5. 12pfu of M13KO7 helper phage (purchased from Invitrogen) was infected and cultured for 30 minutes. After adding 50 mg / L kanamycin, the cells were shaken and cultured at 37°C and 200 rpm for 30 minutes, and then the precipitate was separated by centrifugation, resuspended in 400 mL 2×YT / ampicillin / kanamycin medium, and cultured at 37°C and 200 rpm for 16 hours. Finally, the cells were separated and precipitated by centrifugation (20 minutes, 5000×g, 4°C) and discarded. After the supernatant was filtered with a 0.45 μm filter membrane, 1 / 4 volume of 20% (w / v) PEG8000, 2.5M NaCl solution was added and kept in an ice bath for 1 hour to precipitate the phage particles. Then centrifuge and precipitate (20 minutes, 8000×g, 4°C), discard the supernatant, resuspend the phage in 25mL pre-cooled PBS (137mM NaCl, 2.7mMKCl, 8mMNa2HPO4, 2mM KH2PO4), and centrifuge (5 minutes, 20000×g, 4°C). Add 1 / 4 volume of 20% (w / v) PEG8000, 2.5MNaCl solution to the supernatant, and ice bath for 30 minutes to precipitate the phage particles again. Centrifuge and precipitate (30 minutes, 20000×g, 4°C), resuspend the phage precipitate in 2mL pre-cooled PBS again, keep on ice for 30 minutes and centrifuge (30 minutes, 17000×g, 4°C). The supernatant is mixed with PBS solution containing 4% (w / v) BSA in a ratio of 1:1, placed on a rotating mixer, incubated at room temperature for 30 minutes, and then used directly for screening.

[0047] Using the above phage antibody library, four rounds of directional screening were carried out for the human β2-GPI antigen (Antu Bio) coated with magnetic beads. The screening scheme was as follows: the phage antibody library was incubated with the magnetic beads (Antu Bio) coated with the human β2-GPI antigen at room temperature for 2 hours. The magnetic beads were then washed with PBST (containing 0.1% Tween-20) buffer to remove non-specific binding or weak binding phages. Phages with strong binding ability were eluted from the magnetic beads with glycine-hydrochloric acid (pH 2.2), neutralized with Tris neutralizing solution (pH 9.1), used to infect Escherichia coli ER2738 in the middle of logarithmic growth, and used for the next round of screening. In the four rounds of screening, the amount of magnetic beads used was 50 μL, 20 μL, 10 μL and 10 μL, respectively, and the number of PBST washings was 10 times, 10 times, 15 times and 20 times, respectively.

[0048] 94×2 clones were randomly selected from the clones obtained in the fourth round of screening, and their binding ability to human β2-GPI antigen was tested by single phage ELISA. For this purpose, 300 μL of 2×YT / ampicillin medium (containing 2% glucose) was inoculated into a 96-well deep-well culture plate for each single colony and cultured at 37°C and 250 rpm for 16 hours. 20 μL of culture was inoculated into 500 μL of 2×YT / ampicillin medium (containing 0.1% glucose) and cultured at 37°C and 250 rpm for 1.5 hours. Prepare the helper phage solution, take 75 μL of M13KO7 (titer of 3×10 12 pfu / mL) was mixed into 15mL 2×YT medium, and 50μL / well was added to the culture plate. Cultured at 37°C and 150rpm for 30 minutes, then 50μL / well of the prepared kanamycin solution was added (180μL of 50mg / mL kanamycin was added to 15mL 2×YT medium), and cultured at 37°C and 250rpm for 16 hours. Finally, the cells were centrifuged (30 minutes, 5000×g, 4°C), and the supernatant was transferred to a new 96-well deep-well culture plate.

[0049] To conduct single phage ELISA, the above phage supernatant was detected using an anti-β2-GPI antibody detection kit (Antu Biotechnology, magnetic microparticle chemiluminescence method). The enzyme conjugate component in the kit was replaced with an HRP-labeled anti-M13 antibody (naked antibody purchased from Sino-Bio) diluted 1 / 5k with PBST, and the test was performed using a fully automatic chemiluminescence analyzer. The clone number and signal value detection results are shown in Table 1 below, among which the signal values ​​of numbers GP58, GP108 and GP144 are relatively strong, and GP144 has the strongest reactivity. The antibody sequence information of GP58, GP108 and GP144 is shown in Table 2

[0050] Table 1 Detection results of β2-GPI phage monoclonal supernatant

[0051]

[0052]

[0053]

[0054] Table 2 Preferred antibody sequence information

[0055]

[0056]

[0057] Example 2. Expression of fully human anti-β2-GPI antibodies

[0058] The above three antibodies were constructed into human IgG / IgM / IgA complete antibodies respectively. According to the present invention, the anti-β2-GPI fully human antibody was synthesized to obtain the DNA encoding the antibody, and the DNA was connected to the eukaryotic expression plasmid pcDNA 3.1; the plasmid was added to the serum-free culture medium, mixed evenly, and LipofectamineTM 2000 was added. After mixing, it was allowed to stand at room temperature and used as the transfection liquid for later use.

[0059] Take HEK 293T cells in the logarithmic phase and adjust the cell density to 10 6 / mL, inoculate into a culture dish, culture overnight in a 37°C, 5% CO2 cell culture incubator, and change to serum-free DMEM medium before transfection. Add the aforementioned transfection liquid to the cell culture dish, culture overnight, and then change to DMEM containing 10% FBS with antibiotics to continue culturing. After the culture is completed, collect the supernatant and store it at 4°C for future use.

[0060] Example 3: Purification of fully human anti-β2-GPI antibodies

[0061] A ProteinA / L purification column (IgG recombinant antibody uses ProteinA, and IgA / IgM recombinant antibody uses Protein L) is used, and a PBS solution is added to balance the column. After balancing, the cell supernatant obtained in Example 2 is loaded, and the operation is performed at 4°C. After loading, the column is continuously balanced with a PBS solution. After baseline balancing, 0.1 mol / L glycine solution (pH 2.5) is used for elution. When the ultraviolet absorption spectrophotometer shows a peak, the protein is collected and collected into an EP tube pre-containing Tris-HCl buffer (pH 9.0) to reduce the time that the protein is in acidic conditions and avoid protein aggregation. After elution is completed, the column is regenerated, eluted with 10 column volumes of 0.1 mol / L glycine solution (pH 2.5), then rinsed with Tris-HCl buffer (pH 9.0), and finally washed with 5 column volumes of 20% ethanol. The collected eluted protein sample is dialyzed, sterilized by filtration, and stored at -20°C.

[0062] The collected proteins were detected by SDS-PAGE, and the detection results of β2-GP144 were as follows: Figure 1 As shown, it indicates that the antibody protein was successfully purified and the eukaryotic expressed antibody structure was intact.

[0063] Example 4. Determination of anti-β2-GPI antibody reactivity

[0064] The above β2-GPI-IgG / IgM / IgA antibodies were diluted with PBS, and PBS buffer was used as a negative control. The corresponding magnetic particle complete kit (Antu Bio, magnetic particle anti-β2-GPI-IgG / β2-GPI-IgM / β2-GPI-IgA antibody detection kit) was used to detect the above antibody dilutions on the fully automatic chemiluminescence analyzer. At the same time, the EBVVCA-IgA kit was used to detect the corresponding IgA antibodies of the three antibodies to verify the antibody specificity.

[0065] Table 3 β2-GPI antibody detection results using magnetic particle platform antibody detection kit

[0066] Grouping IgG IgM IgA EBVVCA-IgA PBS 2,640 1,973 2,647 2,860 β2-GP58 19,174 9,626 342,078 2,738 β2-GP108 2,048,140 1,592 1,325,439 3,862 β2-GP144 16,726,865 2,712,247 4,433,735 3,177

[0067] All three antibodies were negative in the EBV VCA-IgA kit, so they were all β2-GPI-specific antibodies. Among them, the modified antibodies of different subtypes of β2-GP144 showed strong detection results in the three corresponding kits and can be preferably used for the preparation of quality control products for β2-GPI-IgG / IgM / IgA kits.

[0068] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An anti-β2-GP1 monoclonal antibody, characterized in that: The amino acid sequences of the three CDR regions of its heavy chain are shown in SEQ ID NOs: 1, 2 and 3, respectively, and the amino acid sequences of the three CDR regions of its light chain are shown in SEQ ID NOs: 4, 5 and 6, respectively.

2. The anti-β2-GP1 monoclonal antibody according to claim 1, characterized in that Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7; The light chain variable region thereof has the amino acid sequence shown in SEQ ID NO:

8.

3. The anti-β2-GP1 monoclonal antibody according to claim 1 or 2, characterized in that The constant region of the heavy chain is any one of IgG, IgA and IgM subtypes, and the constant region of the light chain is κ type.

4. Biomaterial, characterized in that Include at least one of the following: 1) A nucleic acid encoding the anti-β2-GP1 monoclonal antibody according to any one of claims 1 to 3; 2) an expression vector comprising the nucleic acid; 3) transforming or transfecting host cells with the expression vector; 4) culturing the host cells to obtain a culture containing the anti-β2-GP1 monoclonal antibody.

5. A labeled antibody, characterized in that The invention comprises a marker and the anti-β2-GP1 monoclonal antibody according to any one of claims 1 to 3.

6. The labeled antibody according to claim 5, characterized in that The markers include chemical markers and biological markers; The chemical markers include isotopes and / or chemical drugs; The biomarkers include biotin, avidin or enzymes.

7. A conjugate, characterized in that The invention comprises a coupling medium and the anti-β2-GP1 monoclonal antibody according to any one of claims 1 to 3.

8. The conjugate according to claim 7, characterized in that The coupling medium includes a solid medium or a semi-solid medium.

9. Use of at least one of the following i-iii in the preparation of a product for detecting antiphospholipid syndrome: i. The anti-β2-GP1 monoclonal antibody according to any one of claims 1 to 3; ii. The biomaterial according to claim 4; iii. The labeled antibody according to claim 5 or 6; iv. The conjugate according to claim 7 or 8.

10. A product for detecting antiphospholipid syndrome, characterized in that: Include at least one of the following ①~④: ①. The anti-β2-GP1 monoclonal antibody according to any one of claims 1 to 3; ② The biological material according to claim 4; ③. The labeled antibody according to claim 5 or 6; ④. The conjugate according to claim 7 or 8.

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