Humanized anti-beta2-gp1 monoclonal antibodies, methods of making and uses thereof

Abstract

The present application relates to the technical field of antibodies, in particular to humanized anti-beta2-GP1 monoclonal antibody, preparation method and application thereof. The present application constructs a natural full human source library, adopts phage display technology, screens out a high affinity antigen binding fragment, assembles a variable region sequence with an IgG / IgM / IgA constant region to obtain a full-length IgG / IgM / IgA type antibody sequence. The anti-beta2-GPI antibody is expressed by transient transfection, prepared by protein A / L affinity purification method and subjected to binding activity determination. The results show that the specific beta2-GPI antibody obtained by the above method has strong antigen binding activity, strong specificity and good affinity.

Description

TECHNICAL FIELD

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

[0002] Anti-phospholipid antibodies (aPLs) are a group of autoantibodies targeting phospholipids and / or phospholipid-binding proteins. 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 found in malignant tumors, infectious diseases, after the use of certain drugs, and even in some healthy people. Among them, lupus anticoagulant (LA), anticardiolipin antibody (aCL), and anti-β2-glycoprotein I (β2-GPI) antibody are laboratory indicators in the classification criteria for APS, and are widely used in clinical practice. They have also become one of the most common autoantibody detection projects in clinical laboratories. The standardization of aPLs detection is crucial to its clinical application. The standardization of the clinical application of LA, aCL, and anti-β2-GPI antibody detection has 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, which is mainly detected based on the ability of LA to prolong the coagulation test time of different pathways dependent on phospholipids in vitro; (2) Anti-negative charge 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-thrombin antibodies, anti-protein C antibodies, anti-protein S antibodies, anti-annexin A2 antibodies, anti-annexin A5 antibodies, etc. Among them, LA, aCL-IgG antibodies, aCL-IgM antibodies, anti-β2-GPI-IgG antibodies, and anti-β2-GPI-IgM antibodies have been used as laboratory indicators in the 2006 revised APS classification criteria of the International Society on Thrombosis and Hemostasis (ISTH).

[0004] The aCL antibody and anti-β2-GPI antibody detection generally includes IgG, IgM and IgA subtypes, and it is recommended to detect aCL-IgG antibody and aCL-IgM antibody, anti-β2-GPI-IgG antibody and anti-β2-GPI-IgM antibody. The aCL-IgA antibody and anti-β2-GPI-IgA antibody are not included in the APS classification standard at present, and the high-titer positive of the aCL-IgA antibody and anti-β2-GPI-IgA antibody alone is less clinically appeared, and the exact clinical significance still needs to be further studied. If the IgG and IgM types of the aCL antibody and anti-β2-GPI antibody are negative, but the clinical APS is suspected, it is recommended to detect the aCL antibody and anti-β2-GPI-IgA antibody.

[0005] ELISA is a conventional detection method for aCL and anti-β2-GPI antibody, and is widely used in clinical practice at present. The ELISA detection method mainly relies on the comparison analysis and positive discrimination of the serum sample to be detected by the calibration and control in the kit. At present, these calibration and control are mixed and prepared into a certain concentration gradient by using the positive serum of patients and the serum of healthy people. Therefore, it is necessary to have a stable serum source, and the serum raw material should be representative. Considering the biological safety problem, the serum sample is strictly managed in each hospital, resulting in the shortage of serum raw material in the production process of the ELISA kit. On the other hand, the serum raw material collected in the clinic has serious individual differences, and often causes the fluctuation of the detection data of the kit and the inconsistency of the result interpretation in the use process. This is a problem that cannot be avoided in the application process of the ELISA kit produced by using the clinical serum. At the same time, the detection results of the ELISA kits provided by different manufacturers are quite different, and the false positive rate is relatively high. The causes of the false positive may come from the interference of the non-specific antibody and the preparation of the calibration and control in the kit. SUMMARY

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

[0007] The present application provides an anti-β2-GP1 monoclonal antibody,

[0008] The amino acid sequences of the three CDR regions of the heavy chain are respectively shown as SEQ ID NO: 1, 2 and 3, and the amino acid sequences of the three CDR regions of the light chain are respectively shown as SEQ ID NO: 4, 5 and 6.

[0009] In some embodiments, the present application provides an anti-β2-GP1 monoclonal antibody,

[0010] The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 7;

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

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

[0013] Antibodies are traditionally prepared by immunizing animals to obtain polyclonal serum, but the preparation cycle is long and the amount of antibodies obtained is limited. The present application uses phage display technology to screen and obtain the anti-β2-GP1 monoclonal antibody described in the present application, which has high specificity, high sensitivity and good affinity.

[0014] The present application provides a biological material, 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) a host cell transformed or transfected with the expression vector;

[0018] 4) a culture of the host cell, wherein the culture contains the anti-β2-GP1 monoclonal antibody.

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

[0020] In some embodiments, the expression module further comprises a single or multiple nucleic acids described in the present application combined in series, fusion expression or other feasible ways to form an expression module, which is not limited by the present application.

[0021] In some embodiments, the present application further provides a transcription unit, which refers to a DNA sequence from the start of the promoter to the end of the terminator. The promoter and the terminator can further comprise a regulatory fragment on both sides or between them, which can 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 linked to the nucleic acid sequence, such as an enhancer of the promoter, an ITR sequence, a polyA, a MIS signal peptide, etc.

[0022] In some embodiments, the host cell described in the present application is derived from plants, animals, microorganisms or viruses, which is not limited by the present application. The host cell is transformed or transfected by a vector constructed using recombinant DNA technology, so that the transformed host cell has the ability to replicate the vector encoding the protein or express the desired protein.

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

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

[0025] In some embodiments, the label comprises a chemical label and a biological label.

[0026] The chemical label comprises an isotope and / or a chemical drug.

[0027] The biological label comprises biotin, avidin or an enzyme; the enzyme comprises horseradish peroxidase or alkaline phosphatase.

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

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

[0030] In some embodiments, the conjugation medium is selected from colloidal gold, polystyrene flat plate or beads.

[0031] The present application provides the use of at least one of i-iii below in the preparation of a product for detecting anti-phospholipid 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 application provides a product for detecting anti-phospholipid syndrome, comprising at least one of the following ①-④:

[0037] i. the anti-β2-GP1 monoclonal antibody;

[0038] ii. the biological material;

[0039] iii. the labeled antibody;

[0040] iv. the conjugate.

[0041] The application acquires whole blood of APS patients, especially beta2-GPI antibody positive patients, and acquires lymphocytes thereof through lymphocyte separation, constructs a natural whole human source library, screens high-affinity antigen binding fragments by using phage display technology, assembles variable region sequences with IgG / IgM / IgA constant region to obtain full-length IgG / IgM / IgA type antibody sequences. The anti-beta2-GPI antibody is expressed by transient transfection into HEK 293T cells, and is prepared by using protein L affinity purification method and subjected to binding activity and cell activity determination. The results show that the specific beta2-GPI antibody is obtained by the above method, and the antibody has strong antigen binding activity, high specificity, high sensitivity and good affinity. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Figure 2 shows a reduced SDS-PAGE map of beta2-GPI-IgG, beta2-GPI-IgM and beta2-GPI-IgA antibodies, wherein five lanes from left to right are Marker, GP144-IgA, GP144-IgG, GP144-IgG, GP144-IgM. DETAILED DESCRIPTION

[0043] The application provides a humanized anti-beta2-GP1 monoclonal antibody, a preparation method and applications thereof, and those skilled in the art can refer to the content herein, and appropriately improve process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as included in the application. The method and applications of the application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and applications herein without departing from the content, spirit and scope of the application, to realize and apply the technology of the application.

[0044] The test materials used in the application are all ordinary commercially available products, which can be purchased in the market. The application is further described below in combination with examples.

[0045] Example 1 Production of Anti-beta2-GPI Antibody

[0046] The phage display technology is used to screen specific antibodies of human beta2-GPI protein from a natural whole human source library. For this purpose, 400 mL of 2xYT / ampicillin medium is inoculated with glycerol bacteria of phage display whole human single-chain antibody natural library, so that the cell density reaches OD600=0.1, and the culture is shaken at 37°C and 200 rpm until the cell density reaches OD600=0.5. 10 mL of M13KO7 helper phage is added, and the culture is shaken at 37°C and 200 rpm for 30 min, and then the culture is centrifuged at 4000 rpm for 20 min. The supernatant is collected, and 1 / 10 volume of 20% PEG8000 / 2.5M NaCl is added, and the mixture is shaken at 4°C overnight. The precipitate is collected by centrifugation at 4000 rpm for 20 min, and the precipitate is dissolved in 10 mL of TBS, and the solution is centrifuged at 4000 rpm for 20 min. The supernatant is collected, and the concentration of the solution is adjusted to 1x108 pfu / mL. 12M13KO7 helper phage (purchased from Invitrogen) was infected and incubated for 30 minutes. After 50 mg / L kanamycin was added, the culture was incubated at 37°C and 200 rpm for 30 minutes, then the precipitate was separated by centrifugation, resuspended in 400 mL 2xYT / ampicillin / kanamycin medium, and incubated at 37°C and 200 rpm for 16 hours. Finally, the cells were separated by centrifugation (20 minutes, 5000xg, 4°C) and discarded, and the supernatant was filtered with a 0.45 μm filter membrane, then 1 / 4 volume of 20% (w / v) PEG8000, 2.5 M NaCl solution was added and incubated in an ice bath for 1 hour to precipitate the phage particles. Then the precipitate was centrifuged (20 minutes, 8000xg, 4°C), the supernatant was discarded, and the phage was resuspended in 25 mL pre-cooled PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4), and centrifuged (5 minutes, 20000xg, 4°C). To the supernatant, 1 / 4 volume of 20% (w / v) PEG8000, 2.5 M NaCl solution was added, and the phage particles were precipitated again in an ice bath for 30 minutes. The precipitate was centrifuged (30 minutes, 20000xg, 4°C), and the phage precipitate was resuspended again in 2 mL pre-cooled PBS, kept on ice for 30 minutes and centrifuged (30 minutes, 17000xg, 4°C). The supernatant was mixed with PBS containing 4% (w / v) BSA at a ratio of 1:1, incubated at room temperature for 30 minutes on a rotary mixer, and then directly used for screening.

[0047] Using the above phage antibody library, four rounds of directed screening were carried out against human β2-GPI antigen coated magnetic beads (Antibody). The screening scheme is as follows: the phage antibody library was incubated with human β2-GPI antigen coated magnetic beads (Antibody) at room temperature for 2 hours. Then the magnetic beads were washed with PBST (containing 0.1% Tween-20) buffer to remove non-specific binding or weakly binding phage. The strongly binding phage was eluted from the magnetic beads with glycine-hydrochloric acid (pH 2.2), neutralized with Tris neutralization solution (pH 9.1), and used to infect E. coli ER2738 in the middle of the logarithmic growth phase, and used for the next round of screening. In the four rounds of screening, the amount of magnetic beads was 50 μL, 20 μL, 10 μL and 10 μL, and the number of PBST washes was 10 times, 10 times, 15 times and 20 times, respectively.

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

[0049] For single phage ELISA, the above phage supernatant was detected by an anti-β2-GPI antibody detection kit (Antibody, magnetic microparticle chemiluminescence method). The HRP-labeled anti-M13 antibody (naked antibody purchased from Yiqiao Shenzhou) diluted with PBST 1 / 5k was used to replace the enzyme conjugate component in the kit, and the automatic chemiluminescence detector was used for detection. The results of clone number and signal value detection are shown in Table 1 below, in which the signal values of clone numbers GP58, GP108 and GP144 are relatively strong, and the reactivity of GP144 is the strongest. The antibody sequence information of GP58, GP108 and GP144 is shown in Table 2 below

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

[0051]

[0052]

[0053]

[0054] Table 2 Antibody sequence information of preferred construction

[0055]

[0056]

[0057] Example 2, Expression of Anti-β2-GPI Fully Human Antibody

[0058] The three antibodies were constructed into human IgG / IgM / IgA complete antibodies, respectively, and the DNA encoding the antibodies was obtained in a total synthesis manner according to the anti-β2-GPI fully human antibody of the present application, and the DNA was connected into a eukaryotic expression plasmid pcDNA 3.1; the plasmid was added into a serum-free culture medium, mixed uniformly, then LipofectamineTM 2000 was added, mixed, and placed at room temperature for standby as a transfection liquid.

[0059] The logarithmic phase HEK 293T cells were taken, and the cell density was adjusted to 10 6 / mL, then inoculated into a culture dish, and cultured at 37°C in a 5% CO2 cell incubator overnight, and replaced with a serum-free DMEM culture medium before transfection. The aforementioned transfection liquid was added into the cell culture dish, and cultured overnight, then replaced with a 10% FBS-containing DMEM culture medium with antibiotics for continuous culture, and the supernatant was collected after the culture was completed, and stored at 4°C for standby.

[0060] Example 3, purification of the anti-β2-GPI fully human antibody

[0061] Protein A / L purification columns (Protein A for IgG recombinant antibodies, and Protein L for IgA / IgM recombinant antibodies) were used, and a PBS solution was added for column equilibration. After equilibration, the cell supernatant obtained in Example 2 was loaded, and the operation was performed at 4°C. After loading was completed, the column was continuously equilibrated with a PBS solution. After baseline equilibration, elution was performed with a 0.1 mol / L glycine solution (pH 2.5). When a peak was displayed on the ultraviolet absorbance photometer, the protein was collected, and collected into an EP tube containing a Tris-HCl buffer (pH 9.0) in advance, so as to reduce the time of the protein under acidic conditions and avoid protein aggregation. After elution was completed, the column was regenerated, eluted with 10 times the column volume of a 0.1 mol / L glycine solution (pH 2.5), then washed with a Tris-HCl buffer (pH 9.0), and finally washed with 5 times the column volume of 20% ethanol. The collected eluted protein sample was dialyzed, filtered to remove bacteria, and stored at -20°C.

[0062] The collected protein was detected by SDS-PAGE, and the detection result of β2-GP144 is shown in Figure 1 , indicating that the antibody protein was successfully purified, and the eukaryotic expression antibody structure was complete.

[0063] Example 4, determination of the reactivity of the anti-β2-GPI antibody

[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 (Antibody Biological, magnetic particle anti-β2-GPI-IgG / β2-GPI-IgM / β2-GPI-IgA antibody detection kit) was used to automatically detect the above antibody diluents by a full-automatic chemiluminescence detector. Meanwhile, the EBVVCA-IgA kit was used to detect the corresponding IgA antibodies of the three antibodies to verify the specificity of the antibodies.

[0065] Table 3: β2-GPI antibody detection results on a magnetic particle platform antibody detection kit

[0066] Packet IgG IgM IgA EBV VCA-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] The three antibodies were all negative on the EBV VCA-IgA kit, and were β2-GPI specific antibodies. The β2-GPI 44 different subtype modified antibodies had strong detection results on the three corresponding kits, and could be preferably used for the preparation of β2-GPI-IgG / IgM / IgA kit quality control.

[0068] The above is only a preferred embodiment of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. An anti-β2-GP1 monoclonal antibody, characterized in that, the three CDR regions of the heavy chain thereof comprise: the amino acid sequence of CDR1 as shown in SEQ ID NO: 1, the amino acid sequence of CDR2 as shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 as shown in SEQ ID NO: 3; the three CDR regions of the light chain thereof comprise: the amino acid sequence of CDR1 as shown in SEQ ID NO: 4, the amino acid sequence of CDR2 as shown in SEQ ID NO: 5, and the amino acid sequence of CDR3 as shown in SEQ ID NO:

6.

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

8.

3. The anti-β2-GPl monoclonal antibody as claimed in 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, comprising at least one of: 1) a nucleic acid encoding the anti-β2-GP1 monoclonal antibody according to any one of claims 1-3; 2) an expression vector comprising the nucleic acid; 3) a host cell transformed or transfected with the expression vector; 4) culturing the host cell to obtain a culture containing the anti-β2-GP1 monoclonal antibody.

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

6. The labeled antibody of claim 5, wherein, The label comprises a chemical label and a biological label; The chemical label comprises an isotope and / or a chemical drug; The biological label comprises biotin, avidin or an enzyme.

7. Conjugate characterized in that, comprising a coupling medium and the anti-β2-GP1 monoclonal antibody according to any one of claims 1-3; The coupling medium is selected from colloidal gold, polystyrene flat plates or beads.

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

9. A product for the detection of antiphospholipid syndrome, characterized in that, comprising at least one of the following ①-④: ① the anti-β2-GP1 monoclonal antibody according to any one of claims 1-3; 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.

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