WO2023143629A1 - Molécule entière d'igg anti rhd de l'érythrocyte humain complètement humanisée, son procédé de préparation et son utilisation - Google Patents

Molécule entière d'igg anti rhd de l'érythrocyte humain complètement humanisée, son procédé de préparation et son utilisation Download PDF

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WO2023143629A1
WO2023143629A1 PCT/CN2023/077780 CN2023077780W WO2023143629A1 WO 2023143629 A1 WO2023143629 A1 WO 2023143629A1 CN 2023077780 W CN2023077780 W CN 2023077780W WO 2023143629 A1 WO2023143629 A1 WO 2023143629A1
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rhd
seq
antibody
erythrocyte
human
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陈玉平
冯振卿
王布强
唐奇
陈芳芳
杨婷婷
傅强
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江苏力博医药生物技术股份有限公司
南京医科大学
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    • C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention belongs to the field of immunology, and in particular relates to a fully human anti-human erythrocyte RhD full molecule IgG and a preparation method and application thereof.
  • erythrocyte blood group antigens Antigenic molecules on the surface of erythrocytes are called erythrocyte blood group antigens.
  • Erythrocyte blood group antigens and their corresponding in vivo antibodies are important indicators for clinical treatment and detection of blood transfusion, organ transplantation, and neonatal hemolysis, as well as the basis for research on blood group-related diseases.
  • 368 erythrocyte blood group antigens have been found and confirmed, which can be classified into 39 blood group systems, 5 blood group sets and 2 blood group series (low frequency antigen 700 series: 17 antigens; high-frequency antigen 90 ⁇ series: 7 antigens).
  • the most important blood group system is the ABO blood group system, whose coding gene is located on chromosome 9, and other blood group systems include MNS, P1PK, Rh, LU, KEL, LE, FY, JK, DI and other blood group systems .
  • the Rh blood group system is the most complex, and the coding gene is located on chromosome 1. Because of gene recombination and mutation, many different antigens are produced. In addition to the common D, C, c, E, and e antigens, there are also many rare antigens expressed, such as HrB, Bea, Evans and other antigens, among which RhD has the strongest antigenicity.
  • Rh blood group system antigen was first discovered on the red blood cells of rhesus monkeys (Rhesus monkeys), it was named after its initial letter Rh. Its main antigens D, C, c, E, e are coded and expressed by RhD gene and RhCE gene. RhD gene is expressed in chromosome 1 1P 34.3-36.1 , with a full length of 57295bp. The RhD gene and the RhCE gene are arranged in tandem, with opposite directions. The two genes are only 30kb apart. The RhD protein has 417 amino acids, a type IV transmembrane protein, and has 12 transmembrane transmembrane structures. It has 6 extracellular domain structures.
  • RhD antibodies are seldom produced in the natural state, and are generally produced by RhD-negative individuals who are pregnant with RhD-positive blood babies, or in cases of definite contact with RhD-positive blood by mistake. After an individual produces RhD antibody, it will lead to intravascular hemolysis and extravascular hemolysis. Due to the destruction of red blood cells, hemolytic disease of the newborn (haemolytic disease of the fetus and newborn, HDFN) may occur, which may lead to fetal hemolysis, anemia, and high biliary redness. hyperlipidemia, and even death. In order to avoid the occurrence of HDFN and delayed hemolytic transfusion reaction, prophylactic use of RhD antibody is a necessary clinical treatment. At the same time, in idiopathic thrombocytopenic purpura disease, RhD antibody also has a therapeutic effect, and it is a kind of combined drug for its treatment.
  • RhD protein antibodies Most of the anti-human RhD protein antibodies currently used in clinical testing are rhesus monkey hybridomas or polyclonal antibodies. At the same time, in the treatment of neonatal hemolytic disease, idiopathic thrombocytopenic purpura (ITP), and RhD(-) individuals with blood transfusion errors, anti-human RhD protein antibodies are often used. In order to avoid rejection caused by species differences, relevant therapeutic antibodies are often required to be of human origin during clinical application. At present, most of the therapeutic antibodies against human RhD protein used at home and abroad are isolated and purified from The peripheral blood of donors with positive expression of RhD protein is limited in quantity and cannot be produced industrially.
  • RhD antigen-negative red blood cells by repeatedly immunizing RhD antigen-negative red blood cells to produce high-titer serum to prepare polyclonal antibodies of mouse or rabbit origin, there is a risk of blood-borne transmission of viruses or other pathogens between different individuals, and the titer between different batches is unstable. Quality control costs are high.
  • the classic hybridoma monoclonal antibody preparation method requires an antigen with high immunogenicity to immunize mice.
  • RhD protein not only has a large molecular weight and complex spatial structure, but also has a large number of glycosylation site modifications in the natural RhD protein. Therefore, it is very difficult to prepare recombinant human RhD protein by gene synthesis and artificial expression.
  • mice mount an effective immune response.
  • phage surface display antibody library technology a large number of RhD-positive B cell samples are required for phage library construction, and repeated enrichment, screening, and amplification are required to screen for more suitable antibodies. The process is cumbersome and expensive.
  • the light and heavy chain variable regions of the antibody obtained by phage screening are randomly paired, and the affinity of the antibody is poor.
  • antibody engineering technology needs to be used for affinity maturation, which leads to the antibody being prone to strong antigenicity, which affects clinical application.
  • Single-cell transcriptome sequencing technology has also been applied to the development of monoclonal antibodies, which can quickly obtain high-throughput B cell transcriptome genetic information, including paired antibody variable region gene sequences.
  • B cell transcriptome genetic information including paired antibody variable region gene sequences.
  • RhD protein human erythrocyte surface antigen RhD protein.
  • the red blood cell surface antigen system is complex, and the common ABO antigen system is co-expressed with the RhD antigen system. Therefore, it is impossible to obtain B cells that can secrete RhD antibodies through cell sorting, and it is also impossible to sequence RhD antigen-positive red blood cells (red blood cells do not contain transcripts). group genetic information).
  • the invention discloses a fully human whole molecular antibody capable of specifically recognizing human erythrocyte RhD antigen, a preparation method and application thereof, and belongs to the field of biopharmaceuticals.
  • the invention discloses a fully human whole molecular antibody with specific recognition of human erythrocyte RhD antigen.
  • the nucleic acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO.1, and the nucleic acid sequence of the antibody heavy chain variable region is It is shown in SEQ ID NO.2; the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO.4; the full length of the antibody light chain
  • the nucleic acid sequence (including the constant region) is SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.7; the full-length nucleic acid sequence (including the constant region) of the antibody heavy chain is SEQ ID NO.6, and the amino acid sequence is SEQ ID NO.
  • nucleic acid sequence of the antibody light chain antigen complementary region CDR is sequentially shown in SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11; the amino acid sequence is sequentially SEQ ID NO.15, SEQ ID NO.16 , shown in SEQ ID NO.17; the nucleic acid sequence of the antibody heavy chain antigen complementary region CDR is shown in sequence as SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14; the amino acid sequence is shown in sequence as SEQ ID NO.18 , shown in SEQ ID NO.19, SEQ ID NO.20.
  • the invention discloses a preparation method of an all-human all-molecular antibody with specific recognition of human erythrocyte RhD antigen.
  • the method includes: recruiting donors who tested positive for RhD antibody to obtain peripheral blood, and separating PBMC; sorting to obtain specific B cells; screening and enriching B cells that can specifically express anti-human erythrocyte RhD antigen by using phenotypic differences. cell; Obtain paired fully human anti-RhD antibody light and heavy chain variable region gene sequences through single B cell BCR library construction PCR sequencing and bioinformatics analysis; use genetic engineering antibody technology to express fully human anti-RhD in eukaryotic cells Whole Molecular Antibody.
  • the invention also discloses an expression vector containing the DNA molecule encoding the fully human anti-human erythrocyte RhD whole molecule IgG, and cells containing the expression vector.
  • a method for preparing the fully human anti-human erythrocyte RhD full molecule IgG comprising the steps of culturing the above-mentioned cells containing the expression vector, and recovering the fully human anti-human erythrocyte RhD full molecule IgG from the cell culture.
  • composition comprising the fully human anti-human erythrocyte RhD whole molecule IgG or an antigen-binding fragment of the present invention and a pharmaceutically acceptable carrier.
  • a method of treating a pathological condition characterized by erythrocyte RhD antigen exposure in a subject comprising administering to the subject an effective amount of the composition.
  • the fully human anti-human erythrocyte RhD whole molecule antibody or its antigen-binding fragment is conjugated with a therapeutic agent.
  • the fully human anti-human erythrocyte RhD whole molecule antibody or its antigen-binding fragment is conjugated with a label.
  • said label is selected from the group consisting of radioisotopes, fluorescent dyes and enzymes.
  • a method for detecting erythrocyte RhD protein in a sample from human peripheral blood is by contacting the fully human anti-human erythrocyte RhD full molecular antibody or an antigen-binding fragment thereof with the sample and detecting the protein bound to
  • the fully human anti-human erythrocyte RhD whole molecule antibody of the erythrocyte RhD protein is carried out.
  • the fully human anti-human erythrocyte RhD whole molecule antibody or its antigen-binding fragment is used in erythrocyte agglutination analysis or ELISA analysis.
  • the fully human anti-human erythrocyte RhD full-molecule IgG described in the present invention is used for preventing neonatal hemolytic disease, treating idiopathic thrombocytopenic purpura (ITP) or preventing RhD (+) blood from being wrongly transfused into Application of drugs for sensitization to rhesus D antigen in RhD(-) individuals.
  • the advantage and innovation of the above method is that the prepared anti-RhD antibody is fully human, and there will be no rejection caused by species-specificity in clinical treatment, which meets the safety requirements of clinical medication and avoids the traditional mouse-derived antibody.
  • Sourced process development process At the same time, in the antibody gene sequence prepared and screened by this method, the heavy and light chains of the variable region of the antibody belong to the natural pairing, which has high affinity and stability. Compared with the phage display technology, the development of antibody affinity maturation is not required. step.
  • the invention discloses the application of a fully human whole molecule antibody with specific recognition of human erythrocyte RhD antigen in clinical diagnosis and clinical treatment.
  • the fully human anti-human erythrocyte RhD whole molecular antibody can effectively recognize the RhD antigen on the surface of human erythrocytes, and can be used for erythrocyte agglutination analysis or ELISA analysis related to human erythrocyte RhD antigen; it can be used for preventing neonatal hemolytic disease and treating idiopathic thrombocytopenia Infectious Purpura (ITP) or to prevent RhD(+) blood transfusion into Sensitization to rhesus monkey D antigen in RhD(-) individuals.
  • ITTP idiopathic thrombocytopenia Infectious Purpura
  • Figure 1 is a flow cytometry diagram of B cell magnetic bead sorting and enrichment. Among them: A is PBMC, B is the CD19+ B cell population in the lymphocyte population accounting for 9.57%; C is the B cell population enriched by magnetic beads, and D is the CD19+ B cell population accounting for 83.3%.
  • Fig. 2 is a flow cytometric diagram of RhD antibody-positive B cells.
  • A is lymphocyte group
  • B is CD19+/IgG+B cell group, accounting for 3.64%
  • C is CD19+/IgG+B cell group combined with red blood cells
  • IgG+/CD19+/CD235a/b+RBC/B cell group is 0,83%.
  • Figure 3 is a cluster analysis screening diagram. For antibody sequence clustering analysis, it was divided into five clusters, and one case was selected as a candidate antibody gene sequence in each cluster, namely G9, G11, D7, D3, and E.
  • Figure 4 is a UV map of antibody expression and purification. It can be seen from the UV map of protein purification that the antibody protein is adsorbed on the Protein A column in the early stage, and a high-sharp absorption peak can be seen in the later elution antibody.
  • Fig. 5 is an electrophoresis staining diagram of antibody expression and purification supernatant protein (wherein: A is Cox blue staining, B is silver staining).
  • A is Cox blue staining
  • B is silver staining.
  • Input lane antibody heavy and light chain bands and other irrelevant protein bands can be seen.
  • In the Flow through lane only irrelevant protein bands can be seen.
  • the antibody protein is purified and enriched.
  • Figure 6 is the specific detection of antibodies against erythrocyte antigens.
  • the antibody with the clone number D7 can specifically agglutinate RhD-positive red blood cells, and RhD-negative red blood cells do not agglutinate.
  • Fig. 7 is a detection diagram of antibody-induced erythrocyte agglutination titer. The results indicated that the above-mentioned fully human anti-human erythrocyte RhD antibody (D7) (0.1mg/ml) agglutinated RhD positive erythrocyte titer, the strong positive reached 1:512, and the weak positive reached 1:2048.
  • D7 fully human anti-human erythrocyte RhD antibody
  • Figure 8 is a silver staining image of antibody co-immunoprecipitated protein electrophoresis.
  • the expression of the target protein was detected by silver staining method, and a specific protein band was seen at 40kDa, which could be confirmed as human RhD protein by mass spectrometry.
  • Figure 10 is flow cytometric detection of antibody-mediated red blood cell lysis.
  • A is the result of incubation of unsensitized RhD positive red blood cells and monocytes;
  • B is the result of incubation of fully human whole molecule antibody (D7) and RhD negative red blood cells, and then incubation with monocytes;
  • C is full human The results of the incubation of the whole molecule antibody (D7) and RhD positive red blood cells, and the monocyte re-incubation, the results indicate that after the incubation of the monocytes and the RhD positive red blood cells combined with the fully human whole molecule antibody (D7), the ratio of red blood cells to significantly increased in the control group.
  • Figure 11 is the protective experimental detection of fully human anti-RhD antibody. It shows that after 9 hours of red blood cell transfusion, the human red blood cells in the mice in the experimental group are lower than those in the control group (p ⁇ 0.05), and the fully human anti-human red blood cell RhD antibody (D7) can mediate O-type RhD+ red blood cells. Cell removal.
  • Example 1 Enrichment of B cells that can secrete RhD antibody
  • Example 2 Screening for differences in erythrocyte phenotypes
  • Example 1 Enrichment of B cells that can secrete RhD antibody
  • RhD antibody titer>1:1228 Donors who tested positive for RhD antibody (serum antibody titer>1:128) were recruited, peripheral blood was obtained, and blood type identification was performed.
  • lymphocyte separation medium 2:1
  • B cell isolation Kit (Miltenyi), mix 10ul of B cells and biotin-labeled mixed antibodies Incubate for 10 minutes at 2-8°C;
  • FIG. 1 Flow cytometry results, A is PBMC, B is the CD19 + B cell population in the lymphocyte population accounts for 9.57%; C is the B cell population enriched by magnetic beads, D is the CD19 + B cell population accounts for 83.3% .
  • Example 2 Screening for differences in erythrocyte phenotypes
  • lymphocyte separation medium Take 5ml of lymphocyte separation medium in a 15ml centrifuge tube; slowly add the incubated cell mixture to the upper layer of lymphocyte separation medium (for 2ml samples, an equal volume of medium can be added);
  • Figure 2A is the lymphocyte population
  • B is the CD19 + /IgG + B cell population, accounting for 3.64%
  • C is the B cell population combined with red blood cells
  • the IgG + /CD19 + /CD235a/b + RBC/B cell population is 0, 83%.
  • Example 3 Single B cell BCR region library construction PCR sequencing and screening
  • cDNA quality detection For cDNA quality detection, perform pooling and purification on each column of cDNA enrichment products on the 96-well plate. The concentration of cDNA was detected by Qubit, and the fragment distribution of cDNA was detected by capillary electrophoresis.
  • BCR enrichment through semi-nested PCR, design two PCR Primers in the constant region (C region) of the BCR heavy chain and light chain respectively.
  • variable region clustering analysis is performed after independent BCR assembly, and the effective heavy and light chain variable region nucleic acid sequences of the antibody are obtained by screening and eliminating redundant data.
  • the antibody sequence clustering analysis was divided into five clusters, and one case was selected as the candidate antibody gene sequence in each cluster, respectively G9, G11, D7, D3, E5 ( Figure 3), candidate antibodies G9, G11,
  • the amino acid sequences of D7, D3, and E5 are as follows:
  • variable region sequence nucleic acid synthesis respectively recombined into eukaryotic expression vectors pFUSE-CHIg-hG1, pFUSE-CLIg-hl, pFUSE-CLIg-hl, containing human antibody constant region sequence
  • pFUSE-CLIg-hk both vector plasmids were purchased from Invitrogen, USA;
  • the expression vector is transformed into competent bacteria, the bacteria are shaken to amplify, and the plasmid of the expression vector is extracted;
  • the eluted protein is passed through a 30kDa ultrafiltration column and centrifuged at 4000g/min for 20min to obtain a concentrated antibody solution;
  • Figure 4 shows the UV map of protein purification.
  • the antibody protein is adsorbed on the Protein A column in the early stage, and a high-sharp absorption peak is seen in the later elution antibody.
  • Figure 5 the heavy and light chain bands of the antibody and other unrelated protein bands can be seen in the input lane, and only unrelated protein bands can be seen in the Flow through lane, and the heavy and light chain bands of the antibody can be seen clearly in the elution lane, without other protein bands, and the antibody protein is purified and enriched.
  • the blood type card is placed in a cassette centrifuge and centrifuged at 900g/min for 2min, 1200g/min for 3min;
  • the antibody whose clone number is D7 can specifically agglutinate RhD-positive red blood cells, and RhD-negative red blood cells do not agglutinate (see Figure 6), indicating that D7 has the ability to specifically recognize human red blood cells Fully human whole molecule antibody to RhD antigen.
  • the full-length light chain amino acid sequence of the fully human anti-human erythrocyte RhD antibody (D7) is SEQ ID NO.7
  • the full-length heavy chain amino acid sequence is SEQ ID NO.8
  • the light chain amino acid variable region sequence is SEQ ID NO. .3
  • the heavy chain amino acid variable region sequence is SEQ ID NO.4
  • CDR1 is SEQ ID NO.15
  • CDR2 is SEQ ID NO.16
  • CDR3 is SEQ ID NO.17.
  • CDR1 is SEQ ID NO.18
  • CDR2 is SEQ ID NO.19
  • CDR3 is SEQ ID NO.20.
  • the full-length nucleic acid sequence of the corresponding light chain in the expression plasmid of this antibody is SEQ ID NO.5, and the full-length nucleic acid sequence of the heavy chain is SEQ ID NO.6; the variable region of the light chain is SEQ ID NO.1, and the variable region of the heavy chain is SEQ ID NO.1.
  • the region is SEQ ID NO.2, wherein CDR1 in the variable region of the light chain is SEQ ID NO.9, CDR2 is SEQ ID NO.10, and CDR3 is SEQ ID NO.11; CDR1 in the variable region of the heavy chain is SEQ ID NO. .12, CDR2 is SEQ ID NO.13, and CDR3 is SEQ ID NO.14.
  • the antibody was diluted to 24 wells;
  • the blood type card is placed in a cartridge centrifuge and centrifuged at 900g/min for 2min, 1200g/min for 3min;
  • Figure 7A is the result of U-plate erythrocyte agglutination
  • Figure 7B is the result of anti-human globulin microcolumn microcolumn gel
  • the complex is subjected to protein electrophoresis
  • Mononuclear cells THP-1 were added to a 96-well plate at a cell concentration of 2 ⁇ 10 6 cells/ml, 100ul per well, and cultured overnight in an incubator to make the cells semi-adherent and activated;
  • control group was incubated with RhD negative red blood cells and fully human whole molecular antibody (D7);
  • Monocytes, phagocytic red blood cell mononuclear cells and combined red blood cell monocytes are all separated by lymphocyte separation fluid to remove unbound red blood cells;
  • Figure 9 shows that the OD value of the RhD positive erythrocyte group is significantly higher than that of the negative control group, suggesting that the fully human anti-human erythrocyte RhD antibody (D7) promotes the destruction of erythrocytes by monocytes after recognizing and binding to erythrocytes, and the content of hemoglobin in the supernatant is high , the OD value increased;
  • Figure 10A is the result of incubation of unsensitized RhD positive red blood cells and monocytes;
  • B is the result of incubation with fully human whole molecular antibody (D7) and RhD negative red blood cells, and monocytes again Figure;
  • Figure C is the result of incubation with fully human whole molecule antibody (D7) and RhD positive red blood cells, and then incubation with monocytes, suggesting that monocytes and fully human whole molecule antibody (D7) combined with RhD positive red blood cells After incubation, the proportion of red blood cells was significantly higher than that of the control group, indicating that the antibody in
  • Feed NOD-SCID mice collect their tail vein blood, centrifuge at 12000g/min for 10min;
  • mice The proportion of residual human erythrocytes in the mice was detected by flow cytometry, and the results are shown in FIG. 11 .
  • FIG 11 shows that after 9 hours of red blood cell transfusion, the human red blood cells in the mice in the experimental group were lower than those in the control group (p ⁇ 0.05), and the fully human anti-human red blood cell RhD antibody (D7) could mediate the elimination of O-type RhD+ red blood cells.

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  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne une molécule entière d'IgG anti RhD de l'érythrocyte humain complètement humanisée, et son procédé de préparation et son utilisation. La molécule entière d'IgG anti RhD de l'érythrocyte humain complètement humanisée comprend une région variable de chaîne lourde et une région variable de chaîne légère, caractérisée en ce que : la séquence d'acides aminés de la région variable de chaîne légère de l'anticorps est représentée par SEQ ID NO 3, et la séquence d'acides aminés de la région variable de chaîne lourde est représentée par SEQ ID NO 4. Le procédé consiste : à cribler et enrichir des lymphocytes B capables d'exprimer spécifiquement un antigène anti RhD de l'érythrocyte humain au moyen d'érythrocytes ayant une différence phénotypique ; à établir une bibliothèque de régions BCR de lymphocyte B unique et à réaliser une analyse de séquençage PCR au moyen d'une région BCR de lymphocyte B unique pour obtenir des séquences de gène appariées de régions variables de chaîne légère et lourde d'un anticorps anti RhD complètement humanisé ; puis à exprimer l'anticorps à molécule entière dans des cellules eucaryotes à l'aide d'une technologie d'anticorps obtenus par génie génétique. Un anticorps à molécule entière anti RhD de l'érythrocyte humain complètement humanisé soumis à une expression recombinante peut reconnaître efficacement un antigène RhD sur la surface d'érythrocytes humains, conduisant à une réaction d'agglutination d'érythrocytes RhD-positifs.
PCT/CN2023/077780 2022-01-26 2023-02-23 Molécule entière d'igg anti rhd de l'érythrocyte humain complètement humanisée, son procédé de préparation et son utilisation WO2023143629A1 (fr)

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CN114409791B (zh) * 2022-01-26 2023-01-24 南京医科大学 一种全人源抗人红细胞RhD全分子IgG及其制备方法和应用

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CN114409791A (zh) * 2022-01-26 2022-04-29 南京医科大学 一种全人源抗人红细胞RhD全分子IgG及其制备方法和应用

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CN101023101A (zh) * 2004-07-20 2007-08-22 西福根有限公司 抗－恒河猴d重组多克隆抗体和生产方法
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WO1991005800A1 (fr) * 1989-10-19 1991-05-02 Fondation Nationale De Transfusion Sanguine HETERO-ANTICORPS ANTI-RhD ET COMPOSITION PHARMACEUTIQUE LES CONTENANT
CN101178409A (zh) * 2006-11-07 2008-05-14 上海血液生物医药有限责任公司 一种单克隆抗体IgM型的RhD血型定型试剂
CN102272160A (zh) * 2008-12-31 2011-12-07 印度血清及疫苗有限公司 抗rhd单克隆抗体
AU2014200308A1 (en) * 2008-12-31 2014-02-06 Bharat Serums And Vaccines Ltd. Anti-RhD monoclonal antibodies
AR082221A1 (es) * 2011-07-14 2012-11-21 Bharat Serums & Vaccines Ltd Anticuerpos monoclonales anti-rhd (rhesus d)
CN114409791A (zh) * 2022-01-26 2022-04-29 南京医科大学 一种全人源抗人红细胞RhD全分子IgG及其制备方法和应用

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