Neutralizing anti-idiotypic antibody of anti-D antibody and its application

Abstract

The present invention discloses a neutralizing anti-idiotypic antibody of an anti-D antibody, comprising 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 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; and the amino acid sequence of the HCDR3 is shown in SEQ ID No. 3. This neutralizing anti-idiotypic antibody has the ability to neutralize anti-D-mediated hemagglutination of red blood cells and can be used to detect mixed samples containing multiple blood type antibodies unexpectedly containing anti-D. It can also be used for risk assessment and clinical diagnosis and treatment of Rh-HDFN.

Description

Technical Field

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

[0002] In clinical transfusion therapy, the Rh blood group system is second only to the ABO blood group system in importance. The focus on Rh-related red blood cell alloimmunity stems from the D antigen (RHD), which is the most immunogenic of all minor blood group antigens. Approximately 20–30% of RhD(-) patients develop anti-D antibodies after receiving large transfusions of RhD(+) red blood cells. Studies have shown that Rh-negative individuals may develop anti-D antibodies after receiving more than 2 ml of Rh-positive red blood cells for the first time. Recent advances in experimental technology and data sharing have revealed that anti-D antibodies are not limited to Rh-negative (D antigen-deficient) individuals. Some individuals with D variants, resulting from mutations in the RHD gene that result in incomplete expression of the D antigen, can also develop anti-D antibodies upon exposure to D antigens due to the absence of D antigen epitopes. Anti-D antibodies are primarily IgG antibodies and can cause acute or chronic hemolytic transfusion reactions and hemolytic disease of the fetus or newborn (HDFN). Therefore, accurate detection of anti-D antibodies is essential for transfusion safety and guides the prevention, diagnosis, and treatment of Rh-HDFN.

[0003] According to the principles 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 address this, the International Society of Blood Transfusion, the British Society of Hematology, and the Chinese Blood Transfusion Association have all established emergency blood use rules for Rh-negative recipients. This means that in emergency situations, Rh-negative recipients can receive a full dose of Rh-positive blood in a single transfusion. However, this requires that the Rh-negative recipient does not have anti-D antibodies. For Rh-negative recipients, accurately detecting anti-D antibodies and eliminating their effects in emergency situations presents a challenge in transfusing blood to Rh-negative recipients.

[0004] During pregnancy, if an Rh-negative or D-variant woman carries an Rh-positive fetus, she may be stimulated by D antigens through fetomaternal bleeding and develop anti-D. Current preventive measures include administering anti-D immune globulin (derived from the blood of someone with anti-D) within 28 weeks of gestation and 72 hours of delivery, unless the mother's body has already developed anti-D. This prevents the development of anti-D in Rh-negative women by clearing the antigen and inhibiting antibody production. While anti-D immune globulin can prevent the development of anti-D in some Rh-negative women, it can also fail to suppress the development of anti-D in others. Furthermore, if an Rh-negative woman has already developed anti-D, anti-D immune globulin is ineffective. In women who develop anti-D, the maternal anti-D can destroy the fetus's and newborn's red blood cells, causing alloimmune hemolytic disease of the newborn. Accurately detecting anti-D and measuring the amount of anti-D in Rh-negative or D-variant pregnant women will provide important testing basis for assessing the risk of HDFN and taking corresponding prevention and treatment measures (such as plasma exchange, neonatal transfusion or exchange blood, etc.).

[0005] Current methods for detecting and identifying RBC blood group antibodies rely on immunoserological methods. Due to the lack of access to single, purified RBC blood group antigens, these methods rely on selecting RBCs carrying multiple antigens for antibody detection and determining the specificity of the unexpected antibodies based on a specific reactivity pattern. Mixed samples that may contain multiple unexpected anti-D blood group antibodies are difficult to distinguish using conventional spectral RBC identification methods. Furthermore, the current method for detecting and identifying IgG anti-D is the indirect antiglobulin method, which uses an anti-Fc-terminal secondary antibody as an intermediary to induce agglutination of RBCs bound to IgG antibodies. This method is cumbersome and significantly impacts the operator's skill level. Furthermore, the amount of anti-D is typically expressed as a titer or titer, and detection methods employ serial dilutions of the antibody and the indirect antiglobulin method, which also presents a drawback of crude results and inability to accurately determine the amount of anti-D. Therefore, to address these issues, it is necessary to develop a "mock RhD antigen" that specifically binds to anti-D in vitro. In addition, 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 developed anti-D, the "simulated RhD antigen" must also have the function of neutralizing anti-D antibodies.

[0006] Because the RhD protein is a transmembrane protein, in addition to folding across the membrane, it also needs to interact with Rh-related proteins to form a spatial conformation to possess antigenicity. Extracting the protein from red blood cell membranes would lose its quaternary structure and RhD antigenicity. Similarly, synthesizing the protein through genetic engineering would only yield amino acid chains that lack RhD antigenicity. Therefore, generating anti-idiotypic antibodies that can mimic the antigen is a pressing issue. Summary of the Invention

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

[0008] The present invention adopts the following technical solution: a neutralizing anti-idiotypic antibody of an anti-D antibody, comprising 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, 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 have amino acid sequences that are more than 90% identical.

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

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

[0015] The amino acid sequence of the 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 substituting, 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 a heavy chain constant region of mouse IgM, IgG1, IgG2a, IgG2b or IgG3, and a κ or λ type light chain constant region.

[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 a 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 antibodies in serum or neutralizing anti-D antibodies.

[0024] The present invention also discloses the use of the neutralizing anti-idiotypic antibody of the above-mentioned 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 anti-D antibodies.

[0025] The beneficial effects of the present invention are: 1. The anti-idiotypic antibody can specifically bind to the Fab end of the anti-D antibody, acting as an antigen mimic. 2. The anti-idiotypic antibody can be used to neutralize anti-D-mediated erythrocyte agglutination, thereby enabling the development of various diagnostic reagents for detecting mixed samples containing unexpected anti-D antibodies of various blood types, and 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 immunogen prepared by pepsin digestion;

[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 lanes 1 and 3, which are non-reduced; 2 and 4 refer to lanes 2 and 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 3The figure is the SDS-PAGE protein electrophoresis diagram of the 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 fitting plot for neutralizing anti-idiotypic antibodies against anti-D antibodies.

[0033] Figure 5 The graph shows the detection of the deagglutination effect of the neutralizing anti-idiotypic antibody of anti-D antibody on red blood cells; among them: a is the deagglutination effect of the neutralizing anti-idiotypic antibody 13-2#; b is the neutralizing effect of the 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 will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] As used herein, the term "antibody" refers to immunoglobulin molecules comprising four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof, such as IgM. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region comprises one domain, CL1. The VH and VL regions are further subdivided into hypervariable regions, known as complementarity determining regions (CDRs), interspersed with conserved regions known as framework regions (FRs). Framework regions are abbreviated as FRs. From N-terminus to C-terminus, both the light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0038] The term "antigen-binding portion" of an antibody refers to the portion or segment of an intact antibody molecule 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 can be prepared from intact antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques. 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 composed of amino acid residues that mimic the hypervariable regions 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, an Fd fragment in the present invention refers to an antibody fragment consisting of the VH and CH1 domains; an Fv fragment consists of the VL and VH domains in a single arm of an antibody; and a dAb fragment consists of the VH domain.

[0039] The complementarity determining region (CDR) is the region of the variable region that has the greatest impact on the affinity and specificity of an antibody. CDRs typically include CDR1, CDR2, and CDR3. There are two common definitions for VH or VL CDR sequences: the Kabat definition and the Chothia definition. CDR sequences in VH and VL sequences can be determined based on either the Kabat or Chothia definition. In the present invention, the Kabat definition of CDR sequences is used. In the present invention, the CDR1, CDR2, and CDR3 of the heavy chain variable region are referred to as HCDR1, HCDR2, and HCDR3, respectively; the 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] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope, e.g., the ability of an antibody to bind to a specific antigen with an affinity at least twice as great as its affinity for a non-specific antigen. However, it should be understood that an antibody can specifically bind to two or more sequence-related antigens. For example, an antibody of the present invention can specifically bind to a human anti-D antibody.

[0042] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may exist in a small number of individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, so long as they exhibit the desired biological activity.

[0043] As used herein, 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-associated disease" includes diseases and / or conditions associated with anti-D antibodies. Anti-D antibody-associated diseases or conditions include one or more of hemolytic disease of the fetus or newborn.

[0044] As used herein, 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 the symptoms, complications, conditions or progression, development, severity or recurrence associated with a disease. The term "prevention" refers to the administration to a subject not suffering from 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 anti-D antibody content can be detected, or the possible role of preventing or treating anti-D antibody-related diseases can be determined.

[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 shown in SEQ ID NO:1, the HCDR2 sequence is shown in SEQ ID NO:2, and the HCDR3 sequence is 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 antibodies disclosed herein are anti-idiotypic monoclonal antibodies directed against D antibodies. Their class and subtype can be determined by any method known in the art. Generally, antibody class and subtype can be determined using antibodies specific for a particular antibody class and subtype, and ELISA assays can be used to determine the anti-D idiotypic antibody isotype.

[0064] Neutralizing anti-idiotypic antibodies may also comprise murine or human antibody constant regions. Murine antibody constant regions include murine IgM, IgG1, IgG2a, IgG2b, or IgG3 heavy chain constant regions, and kappa or lambda light chain constant regions. In some embodiments, the neutralizing anti-idiotypic antibodies of the present invention are primate anti-D antibodies. Preferably, the primate anti-D antibody is selected from a human anti-D antibody or a monkey anti-D antibody.

[0065] A method for preparing a neutralizing anti-idiotypic monoclonal antibody to an anti-D antibody may include: culturing host cells under expression conditions to express the neutralizing anti-idiotypic monoclonal antibody to the anti-D antibody; and isolating and purifying the expressed neutralizing anti-idiotypic monoclonal antibody to the anti-D antibody. The neutralizing anti-idiotypic monoclonal antibody to the anti-D antibody is purified to a substantially homogeneous substance, for example, as a single band on SDS-PAGE electrophoresis, by affinity purification of the neutralizing anti-idiotypic antibody to the anti-D antibody, native 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 neutralizing anti-idiotypic antibodies of the aforementioned anti-D antibodies, and the use of neutralizing anti-idiotypic antibodies for detecting the level of anti-D antibodies in serum or for neutralizing anti-D antibodies. Another use is in medicaments for preventing and / or treating diseases or conditions associated with anti-D antibodies. Diseases or conditions associated with anti-D antibodies include one or more of hemolytic disease of the fetus or newborn.

[0067] The term "individual" as used herein 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" are used interchangeably.

[0068] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or alleviate the target pathological condition or condition. Individuals in need of treatment include those who already have the condition, as well as those who will develop the condition or for whom the condition is to be prevented. Therefore, in the present invention, the individual to be treated has been diagnosed with the condition or is predisposed or susceptible to the condition. The anti-D antibody-associated disease is one or more of hemolytic disease of the fetus or newborn.

[0069] As a specific example, the present invention provides a neutralizing anti-idiotypic antibody of murine anti-D antibody 13-2# for detecting and neutralizing anti-D antibody, where 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 was purified from erythrocyte lysate 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 expected value, such as Figure 1 As shown in Figure A. The anti-D antibody was ultrafiltered to replace the solvent in 50mM citric acid pH 3.0 buffer, pepsinized at 37°C for 2h, and purified by Mabsure LX to remove the Fc fragment and the intact protein. The collected flow-through was passed through a 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 immunoassays.

[0072] 2. Hybridoma preparation of neutralizing anti-idiotypic antibodies against 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 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 negative control, and the optical density was detected by microplate reader at 450nm wavelength. 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. Prior to fusion, mice were intraperitoneally injected with 50 μg of anti-DF(ab')2 protein for three consecutive days. One day prior to fusion, peritoneal macrophages from normal Kunming mice were used as a trophoblast layer and seeded into 96-well plates. Spleens from immunized mice were fused with the non-secretory myeloma SP2 / 0 cell line and plated onto the trophoblast layer in 96-well plates. The fused cells were then subjected to HAT selection.

[0075] 3. After the hybridoma cells have grown in HAT medium for 12 days, collect 50 μl of the hybridoma supernatant and assay the antibody titer by ELISA. Positive, negative, and blank controls should be set up. Collect all positive clones and screen again by ELISA 2 days after replacing the HAT medium. Clones that are positive twice will proceed to the next step of testing.

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

[0077] Screening was performed using a microplate reader to measure OD620. The specific method was as follows: 75 μl of hybridoma supernatant and 5 μl of anti-D antibody were added to a 96-well microplate. The mixture was incubated at room temperature for 10 minutes. Then, 20 μl of type O red blood cells were added, maintaining the reaction volume at 100 μl. A positive control for agglutination without hybridoma supernatant and a negative control for secondary antibody were set up. The plates were gently mixed and incubated in a 37°C incubator for 0.5 hours. The red blood cells were washed with saline and centrifuged at 200 g for 5 minutes. This wash was repeated twice. 100 μl of anti-human IgG secondary antibody was added, and the plates were centrifuged at 300 g for 5 minutes. The plates were gently shaken, and red blood cell agglutination was observed visually. The optical density was measured using a microplate reader at OD620. The test results show 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 increased transmittance of the wells. The target antibody is the clone with low transmittance and high OD620 value. The wells marked in red are candidate target clones, and the candidate clones are obtained.

[0078] Candidate hybridoma cells were subcloned using the limiting dilution method. Feeder cells were pre-plated in 96-well cell culture plates to provide nutritional support for the growth of monoclonal hybridoma cells. After centrifugation of candidate hybridoma cells in the exponential growth phase, the concentration of the cell culture medium was adjusted to a cell count of 0.8 cells per well. Once hybridoma cells had formed colonies, positive clones were screened by ELISA, and subclone 13-2 was selected from clone 13. Positive clones were expanded and cryopreserved for subsequent antibody production and hybridoma variable region sequencing.

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

[0080] Antibody Preparation: After hybridoma cell expansion and culture, 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 beforehand. Each mouse received 1 ml of intraperitoneal injection. On the 10th day, ascites was extracted and the mice were sacrificed. After centrifugation of the ascites, the supernatant was filtered through a 0.45 μM filter to remove impurities. The antibody in the supernatant was purified using Protein A. SDS-PAGE analysis of the purified antibody showed that the light and heavy chains of the antibody had the correct molecular weight. Figure 3 It can be seen that the antibody purity is greater than 95%.

[0081] The ForteBio Octet red 96e molecular interaction analysis system is an advanced label-free, real-time monitoring technology primarily used for comprehensive quantitative analysis of biomolecular interactions. This example uses the ForteBio method to quantitatively detect the affinity of neutralizing anti-idiotypic antibodies to immunogens. The AMC probe is used to bind to the Fc region 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 and hardly dissociates. The dissociation curve is straight, and 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 the Fortebio method

[0083]

[0084] 5. Neutralizing anti-idiotypic antibody on red blood cell agglutination neutralization experiment:

[0085] Experiment 1: Take a 96-well plate, add 50μg of 13-2# antibody and different volumes of IgG-D antibody, among which IgG-D is selected from Shanghai Blood Biotechnology Co., Ltd., and the national device registration number is: 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℃ 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 Figure a, neither the red blood cell blank control nor the secondary antibody negative control group produced red blood cell agglutination; 5 μl of anti-D antibody can cause obvious red blood cell agglutination, and as the concentration of anti-D antibody increases, the agglutination phenomenon becomes more obvious; 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: Anti-D antibodies were obtained from two sources: IgG-D and IgM / GD. IgG-D was obtained from Shanghai Blood Biopharmaceutical Co., Ltd., with National Medical Device Registration No. 20223401104; IgM / GD was obtained from Millipore, with clones TH-28 / MS-26. A 96-well plate was prepared, and neutralizing anti-idiotypic antibody and anti-D antibody were added. The anti-D antibody was used in 5 μl or 10 μl volumes, with a final concentration of 500 μg / ml. The plate was incubated at room temperature for 10 minutes. Then, 20 μl of type O red blood cells were added, maintaining the reaction volume at 100 μl. A red blood cell blank control and a secondary antibody negative control were established. After mixing, the plate was incubated at 37°C for 0.5 hours. The red blood cells were washed with 200 μl of normal saline and centrifuged at 200 g for 5 minutes. This wash was repeated twice. Then, 100 μl of anti-IgG secondary antibody was added, and the plate was centrifuged at 300 g for 5 minutes, followed by shaking. In order to present the aggregation state of red blood cells more intuitively, the automatic scanning and photographing function of the Elisapot instrument is used to display the aggregation 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 them: "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 As shown in the results of 1A, 2A, 3A, and 4A 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] 6. Sequencing of the variable region gene of the neutralizing anti-idiotypic antibody of anti-D antibody:

[0089] Hybridoma antibody subtypes were determined using a mouse monoclonal antibody subtype identification kit (Proteintech, Cat. PK20002). 50 μl of hybridoma supernatant was diluted 1:50 with 1× PBST and added to the sample wells of the plate at 50 μl / well. 1× goat anti-mouse IgM + IgG-HRP was then added to the sample wells at 50 μl / well. Mix gently on a mixer and incubate at room temperature for 1 hour. Discard the liquid in the wells, wash the plate three times with 1× PBST, and pat dry on absorbent paper. Add the freshly prepared color development solution to the wells at 100 μl / well and develop at room temperature in the dark for 10-20 minutes. Add 100 μl / well of stop solution to each well. Read the OD450 value on a microplate reader. 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 first-strand cDNA was synthesized using reverse transcriptase (TaKaRa, Cat#2690A). PCR amplification of the light and heavy chains was performed using primers selected according to the antibody isotype of the hybridoma cells. Specific PCR-amplified bands were recovered from the gel and cloned into the TA / Blunt-Zero vector (Nanjing Novozymes Biotech Co., Ltd., Cat#C601-01). At least five colonies were sequenced for each antibody, and the sequencing results were analyzed by IgBLAST to determine the antibody CDR region sequences.

[0093] VII. Recombinant Expression and Identification of Neutralizing Anti-Idiotypic Antibodies to Anti-D Antibodies:

[0094] 7.1 Construction of mouse recombinant antibody vector:

[0095] The heavy and light chain nucleic acid variable region sequences obtained above were synthesized and cloned into a mouse IgG1 backbone eukaryotic expression vector for heavy chain expression using homologous recombination technology to obtain the corresponding heavy chain expression vector and light chain expression vector, resulting in four different light chain expression vectors with eight idiotypic antibodies. HEK293 cells (ATCC, Cat No. CRL-1573) were cultured in OPM-293CD05 serum-free medium (Ao Pu Mai, Cat No. 81075-001) at 36.5°C, 7.5% CO2, and suspension culture at 120 rpm. During transfection, the recombinant light and heavy chain plasmids were mixed in 10 mL of OPM-293CD05 medium at a weight ratio of 1:1 (total DNA volume of 100 mg). 100 μL of PEI (concentration of 3 mg / mL) was then added, vortexed rapidly, and incubated 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 minutes, and the supernatant was collected and filtered through a 0.45 μm filter. The antibody was applied to a 5 mL Protein A affinity chromatography column (GE) and eluted with 50 mM citric acid (pH 3.0) buffer at a flow rate of 5 mL / min. The entire elution peak was collected and the pH of the collected eluate was adjusted to approximately 7.0 with 1 M Tris HCl (pH 9.0) buffer. The obtained protein was detected by SDS-PAGE and Coomassie Brilliant Blue staining. 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 was used to detect the binding of neutralizing anti-idiotypic antibodies to the immunogen. An ELISA plate was coated with anti-DF(ab')2 protein at a concentration of 1 μg / ml. A 1 μg / ml concentration of mouse recombinant antibody was added to the ELISA plate, 100 μl per well. PBS served as a blank control. The secondary antibody used was HRP-conjugated goat anti-mouse IgG (Suzhou Botelon Immunotechnology Co., Ltd., Cat. No. BF03001). Optical density was measured at 450 nm using a microplate reader. The results are shown in Table 3. The prepared recombinant anti-D neutralizing anti-idiotypic antibody bound to the antigen, indicating that the variable region sequence was correct.

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

[0101]

[0102] 7.4 Identification of Recombinant Neutralizing Anti-Idiotypic Antibodies by Red Blood Cell Deagglutination

[0103] Take a 96-well plate, add 50 μg of recombinant idiotypic antibody and 5 μl of IgG-D antibody respectively, 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 in a 37°C constant temperature box for 0.5h, wash the 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 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, but 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, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences, 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; The light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences, wherein: The amino acid sequence of the LCDR1 is shown in SEQ ID No. 4; The amino acid sequence of the LCDR2 is shown in SEQ ID No. 5; The amino acid sequence of the LCDR3 is shown in SEQ ID No. 6; SEQ ID No. 5 is as follows: AAT.

2. The neutralizing anti-idiotypic antibody of anti-D antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

7.

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

8.

4. The neutralizing anti-idiotypic antibody of anti-D antibody according to claim 3, wherein 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 constant region of the light chain of the neutralizing anti-idiotypic antibody is a kappa or lambda type light chain constant region.

5. The neutralizing anti-idiotypic antibody of anti-D antibody according to claim 4, wherein The antigen-binding portion of the neutralizing anti-idiotypic antibody is a F(ab')2 fragment.

6. The neutralizing anti-idiotypic antibody of anti-D antibody according to claim 5, wherein The neutralizing anti-idiotypic antibody is a mouse antibody.

7. Use of a neutralizing anti-idiotypic antibody of an anti-D antibody according to any one of claims 1 to 6, characterized in that The neutralizing anti-idiotypic antibody is used to prepare a reagent for detecting the content of anti-D antibodies in serum or to prepare a reagent for neutralizing anti-D antibodies.

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