Neutralizing anti-idiotype antibodies against D antibodies and uses thereof

Abstract

The invention discloses a neutralizing anti-idiotype antibody of an anti-D antibody, the neutralizing anti-idiotype antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises at least one of HCDR1, HCDR2 and HCDR3 sequences, the amino acid sequence of the HCDR1 is as shown in SEQ ID No.1, the amino acid sequence of the light chain variable region is as shown in SEQ ID No.2, the amino acid sequence of the light chain variable region is as shown in SEQ ID No.3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No.4. The amino acid sequence of the HCDR2 is as shown in SEQ ID No. 2; the amino acid sequence of the HCDR3 is as shown in SEQ ID No. 3. The neutralizing anti-idiotype antibody has the effect of neutralizing anti-D-mediated erythrocyte agglutination, can be used for detecting a mixed sample containing various anti-D blood group accidental antibodies, and can also be used for risk assessment and clinical diagnosis and treatment by using Rh-HDFN.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a neutralizing anti-idiotypic antibody of an anti-D antibody and an application thereof. Background Art

[0002] In clinical transfusion therapy, the importance of the Rh blood group system is second only to the ABO blood group system. The concern about red blood cell alloimmunity related to the Rh blood group system stems from the D antigen (RHD), which is the most immunogenic of all minor blood group antigens. About 20-30% of RhD(-) patients can produce anti-D after a large amount of RhD(+) red blood cells are transfused. Studies have found that Rh-negative individuals may produce anti-D antibodies after the first transfusion of more than 2 ml of Rh-positive red blood cells. In recent years, with the advancement of experimental technology and the sharing of data, it has been found that not only Rh-negative (D antigen-deficient) individuals will produce anti-D, but also some D variant individuals with incomplete expression of D antigen due to RHD gene mutations will also produce anti-D under the stimulation of D antigen due to the lack of D antigen epitopes. Anti-D is mainly IgG type immune antibody, which can cause acute or chronic hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN). Therefore, accurate detection of anti-D is the basic guarantee of blood transfusion safety and the basis for guiding the prevention and diagnosis of Rh-HDFN.

[0003] According to the principle of blood transfusion safety, Rh-negative recipients should receive Rh-negative red blood cells. Rh-negative blood is relatively rare and may be in short supply in emergency situations. To deal with this situation, the International Society of Blood Transfusion, the British Society of Hematology and the Chinese Blood Transfusion Association have all formulated emergency blood use rules for Rh-negative recipients, that is, in an emergency, Rh-negative recipients can receive a sufficient amount of Rh-positive blood at one time. But the prerequisite is that the Rh-negative recipient cannot have anti-D antibodies in his body. For Rh-negative recipients, how to accurately detect anti-D antibodies and eliminate the influence of anti-D antibodies in emergency situations has become a challenge facing the blood transfusion problem of Rh-negative recipients.

[0004] During pregnancy, if the fetus of an Rh-negative or D-variant pregnant woman is Rh-positive, she may be stimulated by D antigen through fetal-maternal bleeding and produce anti-D. In the current preventive measures, if the pregnant woman has not yet produced anti-D in her body, anti-D immunoglobulin preparations (anti-D extracted from the blood of people with anti-D) are injected within 28 weeks of pregnancy and 72 hours of delivery to prevent Rh-negative pregnant women from producing anti-D through the principle of antigen clearance and inhibition of antibody production; the use of anti-D immunoglobulin preparations can prevent some Rh-negative pregnant women from producing anti-D, but some Rh-negative pregnant women fail to suppress and still produce anti-D; and if Rh-negative pregnant women have already produced anti-D, the use of anti-D immunoglobulin preparations is ineffective. For pregnant women who produce anti-D, the anti-D from the mother will destroy the fetal and newborn red blood cells, causing neonatal alloimmune hemolytic disease. Accurately detecting anti-D and measuring the amount of anti-D in Rh-negative pregnant women or D-variant pregnant women will provide an important test basis for assessing the risk of HDFN and taking corresponding prevention and treatment measures (such as plasma exchange, neonatal blood transfusion or exchange blood, etc.).

[0005] At present, the detection and identification method of red blood cell blood type antibodies is an immunoserological method. Since it is impossible to obtain a single, purified red blood cell blood type antigen, its principle is to select red blood cells carrying multiple antigens for antibody detection and determine the specificity of unexpected antibodies according to a certain reaction pattern. For mixed samples of multiple blood type unexpected antibodies that may contain anti-D, it is difficult to distinguish them through the commonly used spectrum red blood cell identification method. In addition, the current method for detecting and identifying IgG type anti-D is the indirect anti-human globulin method, which uses an anti-Fc-terminal secondary antibody as an intermediary to cause agglutination of red blood cells bound to IgG antibodies. The operation is cumbersome and is greatly affected by the technical level of the operator. In addition, the amount of anti-D is usually expressed in titer or titer. The detection method is to dilute the antibody in multiple ratios and detect it with the indirect anti-human globulin method. There is also the disadvantage of rough results and the amount of anti-D cannot be accurately determined. Therefore, in order to solve the above problems, it is necessary to obtain a "simulated RhD antigen" that can specifically bind to anti-D in vitro. In order to solve the difficulties in identifying complex anti-D antibodies and the problems of blood transfusion and pregnancy in Rh-negative and D-mutated individuals who have already developed anti-D, the "simulated RhD antigen" must also have the function of neutralizing anti-D antibodies.

[0006] Since RhD protein is a transmembrane protein, in addition to folding through the membrane, it also needs to interact with Rh-related proteins to form a spatial conformation to be antigenic. If the protein is obtained by extracting it from the red blood cell membrane, the quaternary structure of the RhD protein will be lost, and the RhD antigenicity will also disappear; similarly, by synthesizing proteins through genetic engineering, only amino acid chains without RhD antigenicity can be obtained. Therefore, how to obtain anti-idiotypic antibodies to simulate antigens is a problem that needs to be solved at present. Summary of the invention

[0007] The purpose of the present invention is to provide a neutralizing anti-idiotypic antibody of an anti-D antibody and its application. The neutralizing anti-idiotypic antibody has the effect of neutralizing anti-D-mediated erythrocyte agglutination, can be used to detect mixed samples of unexpected antibodies of various blood types containing anti-D, and can also be used for risk assessment and clinical diagnosis and treatment of Rh-HDFN.

[0008] The present invention adopts the following technical scheme: a neutralizing anti-idiotypic antibody of an anti-D antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises at least one of HCDR1, HCDR2 and HCDR3 sequences, wherein:

[0009] The amino acid sequence of the HCDR1 is shown in SEQ ID No. 1;

[0010] The amino acid sequence of the HCDR2 is shown in SEQ ID No. 2;

[0011] The amino acid sequence of the HCDR3 is shown in SEQ ID No. 3;

[0012] or the amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, ... 、 SEQ ID No. 2 and SEQ ID No. 3 have amino acid sequences with more than 90% identity.

[0013] Further, the light chain variable region comprises at least one of LCDR1, LCDR2 and LCDR3 sequences, wherein:

[0014] The amino acid sequence of LCDR1 is shown in SEQ ID No.4;

[0015] The amino acid sequence of LCDR2 is shown in SEQ ID No.5;

[0016] The amino acid sequence of the LCDR3 is shown in SEQ ID No.6;

[0017] Or an amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, or an amino acid sequence having more than 90% identity with SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6.

[0018] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.7.

[0019] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID No.8.

[0020] Furthermore, the constant region of the heavy chain of the neutralizing anti-idiotypic antibody is the heavy chain constant region of mouse IgM, IgG1, IgG2a, IgG2b or IgG3, and the light chain constant region of κ or λ type.

[0021] Furthermore, the antigen-binding portion of the neutralizing anti-idiotypic antibody is a F(ab')2 fragment.

[0022] Furthermore, the neutralizing anti-idiotypic antibody is a mouse antibody.

[0023] The present invention also discloses the use of the neutralizing anti-idiotypic antibody of the anti-D antibody, and the use of the neutralizing anti-idiotypic antibody in detecting the content of anti-D antibody in serum or neutralizing anti-D antibody.

[0024] The present invention also discloses the use of the neutralizing anti-idiotypic antibody of the anti-D antibody, and the use of the neutralizing anti-idiotypic antibody in the preparation of a drug for treating diseases or conditions related to the anti-D antibody.

[0025] The beneficial effects of the present invention are as follows: 1. The anti-idiotypic antibody can specifically bind to the Fab end of the anti-D antibody and can play a role in simulating antigens. 2. The anti-idiotypic antibody can be used to neutralize the anti-D-mediated red blood cell agglutination, and then can be used to develop various diagnostic reagents, which can be used to detect mixed samples of various blood type unexpected antibodies containing anti-D, and can also be used for risk assessment and clinical diagnosis and treatment of Rh-HDFN. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The electrophoresis diagram of the preparation of human anti-D antibody and the preparation of immunogen by pepsin digestion; among which:

[0027] A is the SDS-PAGE electrophoresis diagram of the crude protein of anti-D antibody red blood cell lysate before and after purification;

[0028] B is the SDS-PAGE electrophoresis of the products before and after pepsin cleavage of anti-D antibody.

[0029] In the figure: M refers to lane M, which is a protein pre-stained marker; 1 and 3 refer to lane 1 and lane 3, which are non-reduced; 2 and 4 refer to lane 2 and lane 4, which are reduced.

[0030] Figure 2 This is a graph showing the determination of serum antibody titers in mice immunized with anti-DF(ab')2 protein.

[0031] Figure 3It is the SDS-PAGE protein electrophoresis diagram of neutralizing anti-idiotypic antibody; wherein: M refers to lane M, which is the pre-stained protein Marker, 1 refers to lane 1, which is non-reduced, and 2 refers to lane 2, which is reduced.

[0032] Figure 4 Figure 1 is the Fortebio assay fit for neutralizing anti-idiotypic antibodies against anti-D antibodies.

[0033] Figure 5 This is a test diagram of the neutralizing anti-idiotypic antibody of anti-D antibody on the deagglutination of red blood cells; among which: a is the deagglutination effect of 13-2# neutralizing anti-idiotypic antibody; b is the neutralizing effect of neutralizing anti-idiotypic antibody on red blood cell agglutination mediated by different anti-D antibodies. The anti-D antibodies selected were Shanghai anti-D antibody and Millipore anti-D antibody.

[0034] Figure 6 This is the SDS-PAGE electrophoresis of the neutralizing anti-idiotypic antibody of the recombinant anti-D antibody.

[0035] Figure 7 This is a diagram of the red blood cell deagglutination experiment using recombinant anti-idiotypic antibodies. DETAILED DESCRIPTION

[0036] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] In the present invention, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, namely two heavy chains H and two light chains L interconnected by disulfide bonds, and multimers thereof, such as IgM. Each heavy chain comprises a heavy chain variable region and a heavy chain constant region, the heavy chain variable region is abbreviated as VH, the heavy chain constant region is abbreviated as CH, and the heavy chain constant region comprises three domains, namely CH1, CH2 and CH3. Each light chain comprises a light chain variable region and a light chain constant region, the light chain variable region is abbreviated as VL, and the light chain constant region is abbreviated as CL. The light chain constant region comprises one domain CL1. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions, written as CDRs, interspersed with conserved regions called framework regions FRs. The framework regions are written as FRs. From the N-terminus to the C-terminus, both the light chain and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0038] The term "antigen binding portion" of an antibody refers to a portion or segment of a complete antibody molecule that is responsible for binding to an antigen. The antigen binding portion may comprise a heavy chain variable region, a light chain variable region, or both. The antigen binding portion of an antibody may be prepared from a complete antibody molecule using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, etc. Non-limiting examples of antigen binding portions include: Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, single-domain antibodies, dAb fragments, and minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody, such as isolated CDRs. The term "antigen binding portion" also includes other engineered molecules, such as diabodies, triabodies, tetrabodies, and minibodies. For example, the Fd fragment in the present invention refers to an antibody fragment consisting of a VH and CH1 domain; the Fv fragment consists of a VL and VH domain in a single arm of an antibody; and the dAb fragment consists of a VH domain.

[0039] The complementary determining region is the region in the variable region that has the greatest impact on the affinity and specificity of the antibody. CDRs usually include CDR1, CDR2 and CDR3; there are two common ways to define the CDR sequence of VH or VL, namely the Kabat definition and the Chothia definition, and the CDR region sequence in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In the present invention, the Kabat definition of CDR sequences is used. In the present invention, CDR1, CDR2 and CDR3 of the heavy chain variable region are referred to as HCDR1, HCDR2 and HCDR3, respectively; CDR1, CDR2 and CDR3 of the light chain variable region are referred to as LCDR1, LCDR2 and LCDR3, respectively.

[0040] For a given antibody variable region sequence, the CDR region sequence in the variable region sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis.

[0041] In the present invention, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope, such as the ability of an antibody to bind to a specific antigen with an affinity at least twice greater than its affinity for a non-specific antigen. However, it should be understood that an antibody can specifically bind to two or more antigens related to its sequence. For example, an antibody in the present invention can specifically bind to a human anti-D antibody.

[0042] In the present invention, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies constituting the population are identical except for the presence of naturally occurring mutations in a small number of individuals. The monoclonal antibodies described herein particularly include "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, and the remainder of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, as long as they can exhibit the desired biological activity.

[0043] In the present invention, the term "murine antibody" refers to any antibody in which all domain sequences are mouse sequences. Such antibodies can be produced by hybridomas. The term "anti-D antibody-related disease" includes diseases and / or conditions associated with anti-D antibodies. Anti-D antibody-related diseases or conditions include one or more of fetal newborn hemolytic disease.

[0044] In the present invention, the term "treatment" refers to any type of intervention or method performed on a subject or the administration of an active agent thereto, wherein the purpose is to reverse, alleviate, ameliorate, inhibit or relieve or prevent symptoms, complications, conditions or progression, development, severity or recurrence associated with a disease. The term "prevention" refers to administration to a subject who does not have a disease in order to prevent the disease from occurring or, if present, to minimize its effects.

[0045] In the present invention, hybridoma cells are screened and secreted antibodies that can bind to anti-D antibody proteins. The content of anti-D antibodies can be detected, or the possible role of preventing or treating anti-D antibody-related diseases can be used.

[0046] The 13-2# mouse antibody that specifically binds to the anti-D antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises at least one of HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1 sequence is as shown in SEQ ID NO:1, the HCDR2 sequence is as shown in SEQ ID NO:2, and the HCDR3 sequence is as shown in SEQ ID NO:3.

[0047] The light chain variable region comprises at least one of LCDR1, LCDR2 and LCDR3 sequences, wherein the LCDR1 sequence is shown in SEQ ID NO:4, the LCDR2 sequence is shown in SEQ ID NO:5, and the LCDR3 sequence is shown in SEQ ID NO:6.

[0048] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0049] as follows:

[0050] SEQ ID NO: 1: GHNFISFW.

[0051] SEQ ID No. 2: IYPGTGYS.

[0052] SEQ ID NO:3: ARGITTATYWYVDV.

[0053] SEQ ID NO:4: ENIYSN.

[0054] SEQ ID NO:5: AAT.

[0055] SEQ ID No. 6: QHFWGTPPT.

[0056] SEQ ID NO:7:

[0057] QVQLQQPGAELVKPGTSVKLSCKASGHNFISFWINWVKLRPGKGLEWIG DIYPGTGYSNYNKKFESKATLVDTSSSTAYMQLSSLASEDSGLYYCARGITTA TYWYVDVWGAGTTVTVSS.

[0058] SEQ ID NO:8:

[0059] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYA ATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYFCQHFWGTPPTFGGGTN LEIK.

[0060] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(107aa).

[0061] The antigen-binding portion of the neutralizing anti-idiotypic antibody is a F(ab')2 fragment.

[0062] The neutralizing anti-idiotypic antibody is a mouse antibody.

[0063] The neutralizing anti-idiotypic antibody disclosed in the present invention is an anti-idiotypic monoclonal antibody of an anti-D antibody. Its type and subtype can be determined by any method known in the art. Generally, the antibody type and subtype can be determined using antibodies specific for a particular antibody type and subtype, and the anti-D idiotypic antibody isotype can be determined using an ELISA assay.

[0064] The neutralizing anti-idiotypic antibody may also comprise a mouse or human antibody constant region. The mouse antibody constant region comprises a mouse IgM, a heavy chain constant region of IgG1, IgG2a, IgG2b or IgG3, and a κ or λ type light chain constant region. In some embodiments, the neutralizing anti-idiotypic antibody of the present invention is a primate anti-D antibody. Preferably, the primate anti-D antibody is selected from a human anti-D antibody or a monkey anti-D antibody.

[0065] The preparation method of the neutralizing anti-idiotypic monoclonal antibody of the anti-D antibody may include: culturing host cells under expression conditions to express the neutralizing anti-idiotypic monoclonal antibody of the anti-D antibody; isolating and purifying the expressed neutralizing anti-idiotypic monoclonal antibody of the anti-D antibody. The neutralizing anti-idiotypic monoclonal antibody of the anti-D antibody is purified into a substantially homogeneous substance, for example, a single band on SDS-PAGE electrophoresis, based on affinity purification of the neutralizing anti-idiotypic antibody of the anti-D antibody, non-denaturing gel purification, HPLC or RP-HPLC, size exclusion, purification on a protein A column, or any combination of these techniques.

[0066] The present invention also discloses the use of the neutralizing anti-idiotypic antibody of the anti-D antibody, the use of the neutralizing anti-idiotypic antibody in detecting the content of anti-D antibody in serum or neutralizing anti-D antibody. Another use is in a drug for preventing and / or treating diseases or conditions related to anti-D antibodies. Diseases or conditions related to anti-D antibodies are one or more of fetal neonatal hemolytic disease.

[0067] The term "individual" used refers to mammals, including but not limited to primates, cattle, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or a human. The terms "individual" and "subject" used can be used interchangeably.

[0068] "Treatment" refers to both therapeutic treatment and preventive or preventive measures, the purpose of which is to prevent or alleviate the target pathological state or disease. Individuals in need of treatment include those who already have the disease, as well as those who will develop the disease or want to prevent their disease. Therefore, the individual to be treated in the present invention has been diagnosed with the disease or is prone to or susceptible to the disease. The anti-D antibody-related disease is one or more of the fetal neonatal hemolytic disease.

[0069] As a specific embodiment, the present invention provides a neutralizing anti-idiotypic antibody of 13-2# mouse-derived anti-D antibody for detecting and neutralizing anti-D antibody, 13-2# is an artificial number. The details are as follows:

[0070] 1. Anti-D antibody purification:

[0071] The crude product of anti-D antibody red blood cell lysate was purified by Protein A affinity purification and eluted to obtain anti-D antibody. The harvested antibody molecules were identified by SDS-PAGE electrophoresis, and the molecular weight was consistent with the expectation, such as Figure 1 As shown in A. The anti-D antibody was ultrafiltered to replace the solvent into 50mM citric acid pH 3.0 buffer, pepsinized at 37℃ for 2h, purified by Mabsure LX to remove the Fc fragment and the intact protein that was not cleaved, and the collected flow was passed through the Capto L purification column to remove pepsin and other antibody fragments, and the F(ab')2 protein was eluted. The harvested protein was identified by SDS-PAGE electrophoresis. Figure 1 As shown in Figure B, the reduced molecular weight of the F(ab')2 segment of the target protein is 25 kD, and the non-reduced molecular weight is about 100 kD, which is in line with expectations and can be used for subsequent immune experiments.

[0072] 2. Preparation of hybridomas of neutralizing anti-idiotypic antibodies of anti-D antibodies:

[0073] 1. Take 6-8 week old female Balb / c mice and inject them subcutaneously with anti-DF(ab')2 protein fully emulsified with immune adjuvant, 50μg / dose / mouse, repeated three times. After three subcutaneous immunizations, collect blood from the eye sockets at 150 μ l After centrifugation, serum was collected. Anti-DF(ab')2 protein 1μg / ml concentration was coated on the enzyme-linked plate, and the mouse serum antibody titer was detected by ELISA method. The mouse serum dilution ratio started from 1:4000, and was diluted 1:3 for 5 concentrations. PBS was used as a negative control, and the optical density was detected by a microplate reader at a wavelength of 450nm. Figure 2 It can be seen that the immunization effects of the three mice are comparable, and the abundance of serum antibody titers from high to low are 1#, 2#, and 3#.

[0074] 2. Before fusion, mice were intraperitoneally injected with 50 μg of anti-DF(ab')2 protein for 3 consecutive days. One day before fusion, intraperitoneal macrophages of common Kunming mice were taken as trophoblasts and inoculated into 96-well plates. The spleen of the immunized mice was fused with the non-secretory myeloma SP2 / 0 cell line, and the cells were added to the 96-well plate covered with the trophoblasts, and the fused cells were subjected to HAT selection.

[0075] 3. After the hybridoma cells have grown in HAT medium for 12 days, take 50 μl of the hybridoma supernatant and detect the antibody titer in the supernatant by ELISA. Set up positive, negative and blank controls. Collect all positive clones, replace the HAT medium for 2 days and screen again by ELISA. The clones that are positive twice will enter the next step of testing.

[0076] 3. Neutralizing anti-idiotypic antibody screening:

[0077] Screening was performed by measuring OD620 using an ELISA instrument. The specific method is as follows: take a 96-well ELISA plate, add 75 μl of hybridoma supernatant and 5 μl of anti-D antibody, mix, incubate at room temperature for 10 min, then add 20 μl of O-type red blood cells, keep the reaction system at 100 μl, set up a positive control for agglutination without hybridoma supernatant and a negative control for secondary antibody, mix gently and incubate in a 37°C incubator for 0.5 h. Wash the red blood cells with saline, centrifuge at 200g for 5 min, and repeat the wash twice. Add 100 μl of anti-human IgG secondary antibody, centrifuge at 300g for 5 min, shake gently, visually observe the state of red blood cell agglutination, and measure the optical density value with an ELISA instrument OD620. From the test results, we can see that the secondary antibody cannot cause O-type red blood cell agglutination, and the red blood cells are evenly distributed in the plate wells; in the positive agglutination wells, the red blood cells aggregate on the side walls of the wells, resulting in an increase in the transmittance of the wells. The target antibody is a clone with low transmittance and a high OD620 value. The wells marked in red are candidate target clones, and candidate clones are obtained.

[0078] The candidate hybridoma cells were subcloned by limiting dilution method, and feeder cells were pre-plated in 96-well cell culture plates to provide nutritional support for the growth of monoclonal hybridoma cells. After the candidate hybridoma cells in the exponential growth phase were centrifuged, the concentration of the cell culture medium was adjusted to 0.8 cells per well. After the hybridoma cells formed clones, the ELISA method was used to screen positive clones, and the 13# clone selected the 13-2# subclone. The positive clones were amplified and frozen for subsequent antibody preparation and hybridoma variable region sequencing.

[0079] 4. Determination of affinity between neutralizing anti-idiotypic antibodies and antigens:

[0080] Antibody preparation: After hybridoma cells were expanded and cultured, the cell concentration was adjusted to 2e6 / ml with PBS and injected into the peritoneal cavity of male 6-8 week old Balb / c mice that had been prepared in advance. Each mouse was injected with 1ml. The ascites was extracted on the 10th day and the mice were killed. After centrifugation of the ascites, the supernatant was filtered with a 0.45μM filter to remove impurities. The antibodies in the supernatant were purified by Protein A. The purified antibody SDS-PAGE identification showed that the molecular weight of the antibody light and heavy chains was correct. Figure 3 It can be seen that the antibody purity is greater than 95%.

[0081] ForteBio Octet red 96e molecular interaction analysis system is a non-labeled, real-time monitoring advanced technology, mainly used for comprehensive quantitative analysis of biomolecular interactions. This example uses the Fortebio method to detect the affinity of neutralizing anti-idiotypic antibodies and immunogens. The AMC probe is used to bind to the Fc segment of the idiotypic antibody to detect its affinity and dissociation with the immunogen anti-DF(ab')2. Figure 4It can be seen that the neutralizing anti-idiotypic antibody can bind to the anti-DF(ab')2 molecule, and has a strong binding affinity with the anti-D antibody, almost no dissociation, and a straight dissociation curve. The equilibrium dissociation constant KD obtained by fitting calculation is less than 1.0E-12. The smaller the KD, the stronger the affinity, as shown in Table 1.

[0082] Table 1. Data on affinity between idiotypic antibodies and immunogens detected by Fortebio method

[0083]

[0084] 5. Experiment on the neutralization effect of neutralizing anti-idiotypic antibodies on red blood cell agglutination:

[0085] Experiment 1: Take a 96-well plate, add 50μg of 13-2# antibody and different volumes of IgG-D antibody, where IgG-D is selected from Shanghai Blood Biomedicine Co., Ltd., National Medical Device Registration No.: 20223401104. Incubate at room temperature for 10 minutes, then add 20μl of O-type red blood cells, keep the reaction system at 100μl, set up a red blood cell blank control and a secondary antibody negative control, mix gently and incubate at 37°C incubator for 0.5h, wash the red blood cells with 200μl of normal saline, centrifuge at 200g for 5min, repeat the wash twice, add 100μl of anti-IgG secondary antibody, centrifuge at 300g for 5min, shake gently, observe and record the red blood cell agglutination state under a microscope. Figure 5 As shown in a, there was no red blood cell agglutination in the red blood cell blank control and the secondary antibody negative control group; 5 μl of anti-D antibody could cause obvious red blood cell agglutination, and the agglutination became more obvious as the concentration of anti-D antibody increased; after premixing with 50 μg of anti-D idiotypic antibody 13-2#, compared with the corresponding anti-D antibody group, it can be seen that 13-2# antibody has a significant deagglutination effect.

[0086] Experiment 2: Two sources of anti-D antibodies were selected: IgG-D and IgM / GD. Among them, IgG-D was selected from Shanghai Blood Biomedicine Co., Ltd., with a national medical device registration number of 20223401104; IgM / GD was selected from Millipore, with clones of TH-28 / MS-26. Take a 96-well plate, add neutralizing anti-idiotypic antibody and anti-D antibody, the amount of anti-D antibody used is 5μl or 10μl, and the final concentration of neutralizing anti-idiotypic antibody is 500μg / ml. Incubate at room temperature for 10min, then add 20μl of O-type red blood cells, keep the reaction system at 100μl, set up a red blood cell blank control, a secondary antibody negative control, mix well, incubate at 37℃ incubator for 0.5h, wash the red blood cells with 200μl of normal saline, centrifuge at 200g for 5min, repeat the wash twice, add 100μl of anti-IgG secondary antibody, centrifuge at 300g for 5min, and shake. In order to present the agglutination state of red blood cells more intuitively, the automatic scanning and photographing function of the Elisapot instrument is used to display the agglutination state of red blood cells. Figure 5 As can be seen in b, compared with the secondary antibodies 5C and 5D, 5μl or 10μl of anti-D antibody can cause significant agglutination effect of red blood cells 5A, 5B and 5G, 5H; among which: "5" represents the fifth column in the figure; A, B, C, D, G and H represent the row numbers respectively. Taking 5A as an example, it represents the picture at the intersection of the "5th" column and the Ath row.

[0087] according to Figure 5 From the results of 1A, 2A, 3A, and 4A shown in b, it can be seen that the neutralizing anti-idiotypic antibody has a neutralizing effect on Shanghai IgG-D; according to the results of 6A and 7A, it is shown that there is no antagonistic effect on Millipore's IgM / GD, indicating that the anti-idiotypic antibody prepared by the present invention has selective neutralizing activity.

[0088] VI. Gene sequencing of the variable region of neutralizing anti-idiotypic antibodies of anti-D antibodies:

[0089] The hybridoma antibody subtype was determined using a mouse monoclonal antibody subtype identification kit (Proteintech, Cat. PK20002). Take 50 μl of the hybridoma supernatant and dilute it with 1×PBST 1:50, and add the sample to be tested to the sample well of the strip, 50 μL / well. Then add 1× sheep anti-mouse IgM+IgG-HRP to the sample well, 50 μL / well. Mix gently on a mixer and incubate at room temperature for 1 hour. Discard the liquid in the well, wash the plate 3 times with 1×PBST, and pat dry on absorbent paper. Add the freshly prepared color development solution to the well, 100 μL / well, and color at room temperature in the dark for 10-20 minutes. Add the stop solution to each well, 100 μL / well. The microplate reader reads OD450, and the well with the darkest color or the highest OD value corresponds to the corresponding subtype. The results are shown in Table 2.

[0090] Table 2. Identification of neutralizing anti-idiotypic antibody subtypes of mouse anti-D antibodies

[0091]

[0092] The experimental steps for sequencing the variable region genes of neutralizing anti-idiotypic antibodies of anti-D antibodies are briefly described as follows: Total RNA was extracted from hybridoma cells, and the first chain of cDNA was synthesized using reverse transcriptase (TaKaRa, Cat#2690A). According to the antibody subtype of the hybridoma cells, the corresponding primers were selected for PCR amplification of the light / heavy chain. The specific bands amplified by PCR were cut and recovered, and cloned into TA / Blunt-Zero (Nanjing Novozyme Biotechnology Co., Ltd., Cat No.C601-01), vectors. At least 5 colonies were sequenced for each antibody, and the sequencing results were analyzed by IgBLAST to determine the antibody CDR region sequence.

[0093] VII. Recombinant expression and identification of neutralizing anti-idiotypic antibodies of anti-D antibodies:

[0094] 7.1 Construction of mouse recombinant antibody vector:

[0095] The heavy chain and light chain nucleic acid variable region sequences obtained above were synthesized, and the sequences were cloned into the mouse IgG1 backbone eukaryotic expression heavy chain vector using homologous recombination technology to obtain the corresponding heavy chain expression vector and light chain expression vector, and four idiotypic antibody light chain expression vectors were obtained. HEK293 cells (ATCC, Cat No. CRL-1573) were cultured in OPM-293CD05 serum-free medium (OPM, Cat No. 81075-001), 36.5°C, 7.5% CO 2 , 120rpm suspension culture. During transfection, the recombinant light-paired heavy chain plasmid was mixed in 10mL OPM-293CD05 culture medium at a weight ratio of 1:1 (total DNA amount was 100mg), followed by the addition of 100μL PEI (concentration was 3mg / mL), rapid vortex mixing, and static incubation at room temperature for 15 minutes. The mixture was then added to the above cell culture. After 7 days of cell culture, the antibodies in the supernatant were harvested.

[0096] 7.2 Purification of recombinant antibodies:

[0097] The harvested cell culture was centrifuged at 3000×g for 20 min, and the supernatant was collected and filtered with a 0.45 μm filter. The antibody was eluted with 5 mL of Protein A affinity chromatography column (GE) with 50 mM citric acid (pH 3.0) buffer at a flow rate of 5 mL / min, and the complete elution peak was collected. At the same time, the pH of the collected eluate was adjusted to about 7.0 with 1 M Tris HCl (pH 9.0) buffer. The obtained protein was detected by SDS-PAGE and Coomassie Brilliant Blue staining, as shown in FIG. Figure 6 As shown, the reduced molecular weight of the target protein is 50KD and 25kD, indicating that the target protein is of IgG type.

[0098] 7.3 ELISA Identification of Recombinant Neutralizing Anti-Idiotypic Antibodies:

[0099] ELISA detects the binding of neutralizing anti-idiotypic antibodies to immunogens. The ELISA plate was coated with anti-DF(ab')2 protein at a concentration of 1μg / ml, and the mouse recombinant antibody concentration was 1μg / ml, added to the ELISA plate, 100μl per well, and PBS solution was used as a blank control. The secondary antibody selected was HRP-labeled sheep anti-mouse IgG ((Suzhou Botelon Immunotechnology Co., Ltd., Cat No. BF03001), and the optical density was detected by the ELISA reader at a wavelength of 450nm. The results are shown in Table 3. It can be seen that the prepared recombinant anti-D neutralizing anti-idiotypic antibody binds to the antigen, indicating that the variable region sequence is correct.

[0100] Table 3 ELISA identification of neutralizing anti-idiotypic antibodies

[0101]

[0102] 7.4 Identification of erythrocyte deagglutination by recombinant neutralizing anti-idiotypic antibodies:

[0103] Take a 96-well plate, add 50 μg of recombinant idiotypic antibody and 5 μl of IgG-D antibody, mix, incubate at room temperature for 10 minutes, then add 20 μl of O-type red blood cells, keep the reaction system at 100 μl, set up red blood cell blank control and secondary antibody negative control, mix gently and incubate at 37°C incubator for 0.5 h, wash red blood cells with 200 μl of normal saline, centrifuge at 200g for 5 minutes, repeat washing twice, add 100 μl of anti-IgG secondary antibody, centrifuge at 300g for 5 minutes, shake gently, observe and record the red blood cell agglutination state under a microscope, and record the agglutination state of red blood cells. Figure 7 It can be seen that neither the red blood cell blank control nor the secondary antibody negative control group produced red blood cell agglutination, and 5μl anti-D antibody could cause obvious red blood cell agglutination; after premixing 50μg of anti-D idiotypic antibody, compared with the corresponding anti-D antibody group, the recombinant idiotypic antibody had a significant red blood cell deagglutination effect.

Claims

1. A neutralizing anti-idiotypic antibody of an anti-D antibody, characterized in that comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising at least one of a HCDR1, HCDR2 and HCDR3 sequence, wherein: The amino acid sequence of the HCDR1 is shown in SEQ ID No. 1; The amino acid sequence of the HCDR2 is shown in SEQ ID No. 2; The amino acid sequence of the HCDR3 is shown in SEQ ID No. 3; or the amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, ... 、 SEQ ID No. 2 and SEQ ID No. 3 have amino acid sequences with more than 90% identity.

2. The neutralizing anti-idiotypic antibody of anti-D antibody according to claim 1, characterized in that The light chain variable region comprises at least one of LCDR1, LCDR2 and LCDR3 sequences, wherein: The amino acid sequence of LCDR1 is shown in SEQ ID No.4; The amino acid sequence of LCDR2 is shown in SEQ ID No.5; The amino acid sequence of the LCDR3 is shown in SEQ ID No.6; Or an amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, or an amino acid sequence having more than 90% identity with SEQ ID No.4, SEQ ID No.5 and SEQ ID No.

6.

3. The neutralizing anti-idiotypic antibody of anti-D antibody according to claim 2, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

7.

4. The neutralizing anti-idiotypic antibody of anti-D antibody according to claim 3, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID No.

8.

5. The neutralizing anti-idiotypic antibody of anti-D antibody according to claim 4, characterized in that The constant region of the heavy chain of the neutralizing anti-idiotypic antibody is the heavy chain constant region of mouse IgM, IgG1, IgG2a, IgG2b or IgG3, and the light chain constant region of κ or λ type.

6. The neutralizing anti-idiotypic antibody of anti-D antibody according to claim 5, characterized in that The antigen-binding portion of the neutralizing anti-idiotypic antibody is a F(ab')2 fragment.

7. The neutralizing anti-idiotypic antibody of anti-D antibody according to claim 6, characterized in that The neutralizing anti-idiotypic antibody is a mouse antibody.

8. Use of a neutralizing anti-idiotypic antibody of an anti-D antibody according to any one of claims 1 to 7, characterized in that: The neutralizing anti-idiotypic antibody is used for detecting the content of anti-D antibodies in serum or for neutralizing anti-D antibodies.

9. Use of a neutralizing anti-idiotypic antibody of an anti-D antibody according to any one of claims 1 to 7, characterized in that: Use of the neutralizing anti-idiotypic antibody in preparing a drug for treating diseases or disorders associated with anti-D antibodies.

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