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WO2023184734A1 - 一种抗hla-g1，-g2，-g5及hla-g6异构体分子的单克隆抗体及其用途 - Google Patents

一种抗hla-g1，-g2，-g5及hla-g6异构体分子的单克隆抗体及其用途 Download PDF

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WO2023184734A1
WO2023184734A1 PCT/CN2022/100233 CN2022100233W WO2023184734A1 WO 2023184734 A1 WO2023184734 A1 WO 2023184734A1 CN 2022100233 W CN2022100233 W CN 2022100233W WO 2023184734 A1 WO2023184734 A1 WO 2023184734A1
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hla
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amino acid
acid sequence
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颜卫华
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台州恩泽医疗中心(集团)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the field of biomedicine and relates to the following aspects of anti-HLA-G isomer molecules (HLA-G1, HLA-G2, HLA-G5 and HLA-G6) antibodies (YWHG-3): using HLA-G isomers
  • the antigenic peptide common to molecules (HLA-G1, HLA-G2, HHLA-G5 and HLA-G6) and located in the 209-226th amino acid sequence (KTHVTTHHPVFDYEATLRC) of the heavy chain ⁇ 3 domain of the HLA-G molecule is the immunogen to prepare anti-HLA - Monoclonal antibodies (YWHG-3) of G isoform molecules (HLA-G1, HLA-G2, HLA-G5 and HLA-G6); nucleotides encoding the YWHG-3 antibody of the present invention and their encoded
  • the amino acid sequence, and the antibody (YWHG-3) are used for immunohistochemistry, western blotting and flow cytometry detection.
  • Human leukocyte antigen-G human leukocyte antigen-G (human leukocyte antigen-G, HLA-G) gene, with a full length of 6.0kb, is located at 6p21.3, the distal short arm of human chromosome 6.
  • exon 1 of HLA-G mRNA encodes the signal peptide
  • exons 2, 3 and 4 encode the ⁇ 1, ⁇ 2 and ⁇ 3 domains of the extracellular region respectively
  • exon 5 encodes Transmembrane region
  • exon 6 encodes the intracellular segment of HLA-G molecule containing only 6 amino acid residues; because exon 6 contains a stop codon, exon 7 is not transcribed
  • the initial transcript of HLA-G can be alternatively spliced to produce 7 types of mature mRNA, encoding 7 isoform molecules with different molecular weights (HLA-G1, -G2, -G3, -G4, -G5, -G6 and HLA -G7).
  • HLA-G1, HLA-G2, HLA-G3 and HLA-G4 contain trans-cell membrane regions and are membrane-bound isomers; HLA-G5, HLA-G6 and HLA-G7 lack trans-cell membrane structures and are soluble isomers.
  • the molecular weights of HLA-G1 ⁇ -G7 isomer molecules are 39kD, 31kD, 22kD, 30kD, 34kD, 23kD and 16kD respectively.
  • HLA-G1 is encoded by full-length HLA-G mRNA and consists of extracellular ⁇ 1, ⁇ 2 and ⁇ 3 domains, a transmembrane region and an intracellular domain.
  • HLA-G2 lacks ⁇ 2 domain and consists of extracellular ⁇ 1 and ⁇ 3 domains, transmembrane region and intracellular domain;
  • HLA-G3 lacks ⁇ 2 and ⁇ 3 domains and consists of extracellular ⁇ 1 domain, transmembrane region and Composition of intracellular domain;
  • HLA-G4 lacks ⁇ 3 domain and consists of extracellular ⁇ 1 and ⁇ 2 domains, transmembrane region and intracellular domain; extracellular domain is the same as HLA-G1 and HLA-G2 respectively.
  • HLA-G mRNA containing intron 4 contains a stop codon, the encoded protein molecules lack the transmembrane region encoded by exon 5, forming soluble HLA-G molecules. . Since the mRNA encoding HLA-G7 contains a stop codon in intron 2, the extracellular region only contains the ⁇ 1 domain and is connected by the two amino acid residues encoded by intron 2 ( Figure 1).
  • HLA-G molecules Under normal physiological conditions, HLA-G molecules are only expressed in extravillous trophoblast cells at the maternal-fetal interface, maintaining maternal-fetal immune tolerance during pregnancy. Under pathological conditions, HLA-G molecules can be inducibly expressed in tumor cells and virus-infected cells, and are closely related to the occurrence and progression of the disease. HLA-G molecule is an important immune resistance-causing molecule in the body and an important immune checkpoint molecule. Its immunosuppressive function is mainly through binding to the immunosuppressive receptor immunoglobulin-like transcript-2 (immunoglobulin-like transcript- 2, ILT2/LILRB1/CD85j) and immunoglobulin-like transcript-4 (ILT4/LILRB2/CD85d), transmit inhibitory signals and induce immune tolerance.
  • immunoglobulin-like transcript-2 immunoglobulin-like transcript- 2, ILT2/LILRB1/CD85j
  • ILT4/LILRB2/CD85d immunoglobulin-like transcript-4
  • HLA-G plays an important role in the occurrence and development of tumors and other diseases.
  • a number of tumor immune targeted therapies based on HLA-G as targets have entered phase I clinical trials in the United States and other countries.
  • HLA-G binds to receptors ILT-2 and ILT-4 and has molecular structure specificity.
  • the ⁇ 3 domain of HLA-G extracellular region is the site where receptors ILT-2 and ILT-4 bind to HLA-G.
  • ILT-2 only binds to the HLA-G/ ⁇ 2 m complex, while ILT-4 can not only bind to HLA-G/ ⁇ 2 m, but also bind to free HLA-G molecules that do not contain ⁇ 2 m. Due to differences in expression mechanisms and molecular structures of HLA-G1, -G2, -G3, -G4, -G5, -G6 and HLA-G7 isoform molecules.
  • ILT-2 can bind to HLA-G1 and HLA-G
  • ILT-4 can bind to HLA-G1, HLA-G2, HLA-G5 and HLA-G6 isoform molecules.
  • the extracellular regions of HLA-G3, -G4, and HLA-G7 isoforms do not contain ⁇ 3 domains and cannot bind to ILT-2 and ILT-4.
  • Different HLA-G isoform molecules can exert specific immunological effects in pathophysiological processes. Under different pathological conditions, especially in tumor tissue cells, there is extensive heterogeneity in the expression of HLA-G isoforms, and the expression of different HLA-G molecular isoforms has specific clinical significance. Therefore, analyzing the molecular expression of specific HLA-G isoforms and the molecular expression profiles of different HLA-G isoforms is of great significance for elucidating the biological functions and clinical significance of specific HLA-G isoform molecules.
  • the antibodies used for HLA-G immunohistochemistry and western blotting at home and abroad mainly include 4H84, MEM-G1, MEM-G2, 2A12 and 5A6G7.
  • Antibody 4H84 whose recognition site is located in the extracellular ⁇ 1 domain of all seven HLA-G isoform molecules, can detect the seven currently known HLA-G isoform molecules containing the ⁇ 1 domain (HLA- G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6 and HLA-G7), but the expression of specific HLA-G isoform molecules cannot be distinguished in immunohistochemistry.
  • Antibodies MEM-G1 and MEM-G2 were obtained from mice immunized with the full-length HLA-G heavy chain. The specific recognition sites cannot be predicted. Theoretically, these two antibodies are similar to antibody 4H84 and can recognize 7 HLA-G isomers. molecules (HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6 and HLA-G7), but it is also unable to distinguish the expression of specific HLA-G isoform molecules.
  • Antibodies 2A12 and 5A6G7 are obtained by immunizing mice with 22 amino acid residues located at the carboxyl terminus of HLA-G5 and HLA-G6 molecules, and can recognize HLA-G5 and HLA-G6 molecules. However, antibodies 2A12 and 5A6G7 can also recognize other unknown non-HLA-G5 and HLA-G6 molecules with molecular weights in the range of 36-175kDa. In the application of immunohistochemistry, there may be cross-reactions, false positives, and inability to correct HLA -G5 and HLA-G6 molecular expression.
  • the monoclonal antibodies used to detect HLA-G molecules by flow cytometry include 87G and MEM-G/9, but they can only detect HLA-G1 and HLA-G5 molecules.
  • the purpose of the present invention is to provide an anti- Monoclonal antibodies (YWHG-3) specific for the HLA-G isoform molecules (HLA-G1, HLA-G2, HLA-G5 and HLA-G6) of the domain, encoding the antibody (YWHG-3) of the invention Nucleotide sequence and its encoded amino acid sequence; and the YWHG-3 antibody is used for immunohistochemistry, immunoblotting, flow detection and other purposes.
  • the present invention adopts the antigenic peptide shared by HLA-G isomeric molecules (HLA-G1, HLA-G2, HLA-G5 and HLA-G6) and located at the 209th to 226th amino acid sequence of the heavy chain ⁇ 3 domain of the HLA-G molecule.
  • Immunogen its amino acid sequence is the amino acid sequence shown in SEQ ID No. 19 (KTHVTTHHPVFDYEATLRC).
  • the KTHVTHHPVFDYEATLRC peptide is located in the ⁇ 3 domain of the heavy chain of the HLA-G molecule (the area shown in the dashed box in Figure 1), and based on this, anti-HLA-G isoform molecules (HLA-G1, HLA-G2, HLA-G5 and HLA -G6) specific monoclonal antibody (YWHG-3).
  • the present invention provides an anti-HLA-G molecule monoclonal antibody (YWHG-3), which at least includes one or more of the light chain hypervariable regions CDR1, CDR2 and CDR3, or/ and one or more of the heavy chain hypervariable regions CDR1, CDR2 and CDR3;
  • the amino acid sequence of the monoclonal antibody (YWHG-3) antibody light chain is as shown in SEQ ID No. 1 or has the same function as the sequence shown in SEQ ID No. 1 formed by replacing, deleting or adding one or more amino acids.
  • Amino acid sequence; the amino acid sequence of the antibody light chain hypervariable region CDR1 is the sequence QSLVHSTGNTF shown in SEQ ID No. 2 or the sequence shown in SEQ ID No. 2 formed by replacing, deleting or adding one or more amino acids.
  • Amino acid sequences with equivalent functions; the amino acid sequence of the light chain hypervariable region CDR2 is the sequence KVS shown in SEQ ID No. 3 or the sequence KVS shown in SEQ ID No. 3 by replacing, deleting or adding one or more amino acids.
  • the sequence shown has an amino acid sequence with equivalent functions, and the amino acid sequence of the light chain hypervariable region CDR3 is the sequence SQSTHVPWT shown in SEQ ID No. 4 or the sequence SQSTHVPWT formed by replacing, deleting or adding one or more amino acids and SEQ ID
  • the sequence shown in No. 4 has an amino acid sequence with equivalent functions;
  • the amino acid sequence of the heavy chain of the monoclonal antibody (YWHG-3) antibody is as shown in SEQ ID No. 5 or has the same function as the sequence shown in SEQ ID No. 5 formed by replacing, deleting or adding one or more amino acids.
  • the amino acid sequence of the antibody heavy chain hypervariable region CDR1 is the sequence GTFFSDYN shown in SEQ ID No.6 or the sequence GTFFSDYN shown in SEQ ID No.6 by replacing, deleting or adding one or more amino acids.
  • the sequence has an amino acid sequence with equivalent functions.
  • the amino acid sequence of the heavy chain hypervariable region CDR2 is the sequence ISDGGRST shown in SEQ ID No. 7 or the same sequence as SEQ ID No. 7 formed by replacing, deleting or adding one or more amino acids.
  • the sequence shown has an amino acid sequence with equivalent functions
  • the amino acid sequence of the heavy chain hypervariable region CDR3 is the sequence VRDDY shown in SEQ ID No. 8 or the sequence VRDDY formed by replacing, deleting or adding one or more amino acids and SEQ ID
  • the sequence shown in No. 8 has an amino acid sequence with equivalent functions.
  • the monoclonal antibody also includes a light chain framework region (FR) and a heavy chain framework region; wherein, the light chain framework region includes light chain FR1, FR2, FR3 and FR4.
  • the light chain framework region includes light chain FR1, FR2, FR3 and FR4.
  • One or more of the light chain FR1, the amino acid sequence of the light chain FR1 is the sequence DIMMTQTPLSLPVSLGDQASISCRSS shown in SEQ ID No. 9 or the sequence formed by replacing, deleting or adding one or more amino acids and the sequence shown in SEQ ID No. 9
  • the sequence has an amino acid sequence with equivalent functions;
  • the amino acid sequence of the light chain FR2 is the sequence LNWYLQKPGQSPKLLIY shown in SEQ ID No. 10 or the sequence shown in SEQ ID No.
  • amino acid sequence of the light chain FR3 is the sequence shown in SEQ ID No. 11 NRFSGVSDRFSGSGSGTDFTLKIRRVEAEDLGVYFC or is formed by replacing, deleting or adding one or more amino acids and having the same sequence as the sequence shown in SEQ ID No. 11 Amino acid sequence with equivalent functions; the amino acid sequence of the light chain FR4 is the sequence FGGGTKLEIK shown in SEQ ID No. 12 or is equivalent to the sequence shown in SEQ ID No. 12 by replacing, deleting or adding one or more amino acids.
  • the heavy chain framework region includes one or more of heavy chain FR1, FR2, FR3 and FR4, wherein: the amino acid sequence of the heavy chain FR1 is the sequence LVQLQQSGGGLVKPGGSLKLSCAAS shown in SEQ ID No. 13 Or an amino acid sequence having the same function as the sequence shown in SEQ ID No. 13 formed by replacing, deleting or adding one or more amino acids; the amino acid sequence of the heavy chain FR2 is the sequence MYWVRQTPEKRLEWVAT shown in SEQ ID No. 14 or An amino acid sequence having the same function as the sequence shown in SEQ ID No.
  • the amino acid sequence of the heavy chain FR3 is the sequence FYLDSVKGRFTISRDNAKNNLYLQINNLKSEDTAIYYC or the sequence shown in SEQ ID No. 15.
  • the amino acid sequence of the heavy chain FR4 is the sequence WGQGTTLTVSS of SEQ ID No. 16 or has been replaced or deleted Or add one or more amino acids to form an amino acid sequence with equivalent functions to the sequence shown in SEQ ID No. 16;
  • nucleotide sequence encoding the light chain in the monoclonal antibody is the sequence shown in SEQ ID No. 17 or the sequence is formed by replacing, deleting or adding one or more nucleotides.
  • the nucleotide sequence encoding the heavy chain in the monoclonal antibody (YWHG-3) is the sequence shown in SEQ ID No. 18 or this sequence.
  • the invention provides a preferred monoclonal antibody (YWHG-3) against HLA-G isoform molecules (HLA-G1, HLA-G2, HLA-G5 and HLA-G6),
  • the monoclonal antibody (YWHG-3) is produced by a hybridoma with the deposit number CCTCC NO: 202121.
  • the deposit institution is: China Type Culture Collection Center. The preservation time is August 11, 2021. China Type Culture Collection Center The address is Wuhan University, Wuhan City, Hubei province, China, postal code 430072.
  • the invention also provides the anti-HLA-G isomer molecule (HLA-G1, HLA-G2, HLA-G5 and HLA-G6) antibody (YWHG-3) for use in HLA-G isomer molecules (HLA- G1, HLA-G2, HLA-G5 and HLA-G6) immunohistochemistry, western blotting and flow cytometry, etc. It has the characteristics of high specificity and strong affinity.
  • the "hypervariable region” is also called a complementarity determining region (CDR).
  • CDR complementarity determining region
  • sequence mentioned in the present invention may refer to amino acid sequences containing certain biologically equivalent amino acids or "conservative substitutions", and “other sequences” may include functionally non-equivalent amino acids or "non-conservative substitutions", which are genetically modified.
  • sequence of the variant mentioned in the present invention may be at least 95%, 96%, 97%, 98% or 99% identical to its source sequence. Sequence identity can be measured using sequence analysis software. For example, the computer program BLAST uses default parameters, especially BLASTP or TBLASTN. The various amino acid sequences described herein are detailed in the Sequence Listing.
  • Figure 1 is a diagram of the molecular structures of seven different HLA-G isomers and the positions of immune antigen peptides.
  • Figure 2 is the identification of heavy chain and light chain subclasses of the antibody (YWHG-3).
  • Figure 3 is the SDS-PAGE detection of the antibody purity of the antibody (YWHG-3).
  • Figure 4 is the measurement of the antibody affinity constant of the antibody (YWHG-3) by ELISA.
  • Figure 5 is the detection of HLA-G5 molecule concentration by ELISA method of the antibody (YWHG-3).
  • Figure 6 shows the immunoblotting of the antibody (YWHG-3) to detect HLA-G1, -G2, -G5 and HLA-G6 molecules.
  • Figure 7 shows the flow cytometric detection of intracellular HLA-G1, -G2, -G5 and HLA-G6 molecules using the antibody (YWHG-3).
  • Figure 8 shows the application of the antibody (YWHG-3) to immunohistochemical detection of the expression of HLA-G1, -G2, -G5 and HLA-G6 molecules in gastric cancer tissue.
  • the antigenic peptide located in the 209th to 226th amino acid sequence of the ⁇ 3 domain of the HLA-G molecule heavy chain was synthesized, SEQ No. 19 KTHVTHHPVFDYEATLRC.
  • mice For the first time, 4 SPF grade BALB/c female mice were immunized, 60ug/mouse. For the first booster immunization of mice, 30ug/mouse. Second booster immunization of mice, 30ug/mouse. The third booster dose for immunized mice is 30ug/mouse. Blood was taken from the orbit to measure the serum titer. Use "SEQ No.19 KTHVTHHPVFDYEATLRC" to wrap the plate and measure the titer of immunized mice by ELISA. Use "SEQ No.
  • the positive cell lines were plated again with "SEQ No. 19 KTHVTHHPVFDYEATLRC", and the ELISA method was used for a second screening to obtain positive hybridoma cell lines. After multiple screenings, a monoclonal antibody against the SEQ No. 19 KTHVTHPVFDYEATLRC fragment was obtained, named YWHG-3 (deposit number CCTCC NO: 202121).
  • the 10 screened positive cell lines were identified for subtypes, dilute the coating antibody to 0.5ug/ml with 100mM PBS (pH7.4), add 0.1ml to each well, and incubate at 4°C overnight. Wash twice with PBS-T, add 200ul blocking solution to each well, and incubate at 37 0 C for 2 hours. Wash 3 times with PBS-T; add 100ul hybridoma supernatant to each well and incubate at 37 0 C for 1 hour.
  • Sample pretreatment dilute 1:3 with the corresponding coupling buffer, centrifuge at 12,000 rpm for 10 minutes at 4°C, and filter with a 0.22 ⁇ m filter to remove fat, cell debris, and small particulate matter.
  • Equilibrate Equilibrate the column with 10 times the column volume of the corresponding coupling buffer, keeping the flow rate at 1ml/min.
  • Loading the sample Inject the sample into the upper end of the column, collect the effluent, and keep the flow rate at 1ml/min.
  • Clean impurities Use 5 times the column volume of coupling buffer to pass through the column, keeping the flow rate at 1 ml/min.
  • Elution Use 5 times the column volume of elution buffer to elute the antibody, collect it in the above-mentioned EP tube, and keep the flow rate at 1ml/min. Immediately adjust the pH to 7.0 with 1M Tris-Hcl buffer pH 9.0. Equilibrate: Equilibrate the column back to pH 7.0 with 10 column volumes of coupling buffer, maintaining the flow rate at 1 ml/min. Dialysis: Use 0.01M PBS buffer to dialyze the antibody overnight and change the medium three times.
  • Sample preparation Add sample buffer and boil in boiling water for 10 minutes. Sample loading: 10ul per well.
  • Gel running 80V for stacking gel, 30min; 120V, 60min for separating gel. Stop the electrophoresis when the bromophenol blue front reaches the bottom of the glass plate and remove the gel.
  • Staining and destaining Immerse the gel in Coomassie Brilliant Blue staining solution and shake slowly on a shaker for more than 30 minutes (the staining time needs to be adjusted appropriately according to the thickness of the gel). Take out the gel and rinse it in water several times, then add Coomassie Brilliant Blue destaining solution and shake.
  • Dilute the antigen with coating solution SEQ No. 19 KTHVTHPVFDYEATLRC, to a final concentration of 2ug/ml, 100ul/well, 4°C overnight; then wash twice with washing solution.
  • Block with blocking solution 200ul/well, incubate at 37°C for 2 hours; then wash once with washing solution.
  • the purified antibody was diluted 2 times in gradient (PBS) starting from 200 times, and the blank control (blank) was PBS, both 100ul/well, incubated at 37°C for 1 hour; and then washed 3 times with washing solution.
  • HLA-G isoform standard proteins HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6 and HLA-G7 isoform molecular standard proteins use 5% to remove The cells were blocked with fat milk powder for 4 hours at room temperature and washed with 0.2% TBS (Tewen-20 PBS).
  • TBS Tewen-20 PBS
  • the results show: The results indicate that the antibody (YWHG-3) can specifically recognize HLA-G1, -G2, -G5 and HLA-G6 standard proteins, and has no cross-reaction with other HLA-G isomeric molecules, and has good
  • the specificity of HLA-G isoform molecules (HLA-G1, -G2, -G5 and HLA-G6) containing ⁇ 3 domain was determined (Figure 5).
  • Example 2.1 Detection of HLA-G5 molecule concentration by ELISA method of the antibody (YWHG-3)
  • the final concentrations are 4ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml, and 0.25ug/ml. 0.125ug/ml and 0.0625ug/ml, with the coating solution as the blank control.
  • biotin-labeled antibody (YWHG-3) with a final concentration of 1.0ug/ml, 100ul/well, incubate at 37°C for 1 hour; then wash 3 times with washing solution.
  • PBS to dilute 1.0ug/ml of peroxidase-conjugated Streptavidin, 100ul/well, incubate at 37°C for 1 hour; remove and wash 3 times with washing solution.
  • TMB chromogenic solution 100ul/well, color development time is 5-15min.
  • Add 50ul of stop solution to each well to stop. Measure the absorbance value at wavelength (450) and draw a graph for analysis.
  • the OD 450 of 4ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml, 0.25ug/ml, 0.125ug/ml, 0.0625ug/ml and coating solution blank control are 1.856, 1.490, 1.347, 1.092, respectively. 0.664, 0.388, 0.263 and 0.045.
  • Example 2.2 Flow cytometric detection of intracellular HLA-G1, -G2, -G5 and HLA-G6 molecules using the antibody (YWHG-3)
  • K562 (blank control), K562-HLA-G1, K562-HLA-G2, K562-HLA-G5 and K562-HLA-G6 were taken in the logarithmic growth phase. Detected by intracellular flow cytometry.
  • Flow cytometry detection of intracellular molecule expression Collect K562, K562-HLA-G1, K562-HLA-G2, K562-HLA-G5 and K562-HLA-G6 cell cultures in flow tubes, centrifuge and wash twice with 2% BSA/PBS (250g), add 250ul of cell membrane-breaking agent (BD Cytofix/Cytoperm TM ), mix thoroughly, and place in a 4°C refrigerator for 20 minutes. Add 1ml BD Perm/Wash TM and centrifuge twice (250g).
  • BSA/PBS 250g
  • BD Cytofix/Cytoperm TM cell membrane-breaking agent
  • the antibody (YWHG-3) is used for immunohistochemistry to detect the expression of HLA-G molecules (HLA-G1, HLA-G2, HLA-G5 and HLA-G6) in gastric cancer tissue.
  • the gastric cancer tissues were fixed in 10%-12% neutral formalin and embedded in paraffin. Tissue sections undergo conventional production processes such as baking, dewaxing, hydration and antigen retrieval. Drop an appropriate amount of 1% BSA onto the tissue, covering the tissue and tissue edges by 2 mm, and incubate at room temperature for 10 minutes for sealing.
  • the antibody (YWHG-3) (1 mg/mL, 1:500 dilution) was added dropwise and kept in a humidified box at 4°C overnight (16-20 h). Wash with TBS buffer, dropwise add secondary antibody (TBS diluted antibody goat anti-mouse ratio 1:300), and incubate in a 37°C incubator for 30 minutes.
  • HLA-G isoform molecules HLA-G1, HLA-G2, HLA-G5 and HLA-G6
  • HLA-G isoforms can be identified based on the depth of tan coloring of the cells. Expression intensity of molecules (HLA-G1, HLA-G2, HLA-G5 and HLA-G6).
  • Figure 8 shows that HLA-G molecules are highly expressed in (A), (B), (C) and (D) gastric cancer tissue samples ( Figure 8).

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Abstract

本发明公开了一种抗HLA-G1，-G2，-G5及HLA-G6异构体分子的单克隆抗体(YWHG-3)及用途，所述抗体(YWHG-3)由保藏编号为CCTCC NO:202121的杂交瘤产生，采用HLA-G异构体分子(HLA-G1，HLA-G2，HHLA-G5及HLA-G6)共有的，位于HLA-G分子重链α3结构域第209～226位氨基酸序列抗原肽(KTHVTHHPVFDYEATLRC)为免疫原。本发明提供了编码本发明所述YWHG-3抗体的核苷酸及其编码氨基酸序列,本发明还提供了所述YWHG-3抗体用于HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G5及HLA-G6)免疫组化、免疫印迹及流式细胞术等检测的用途。

Description

一种抗HLA-G1，-G2，-G5及HLA-G6异构体分子的单克隆抗体及其用途 技术领域
本发明属于生物医药领域，涉及抗HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G5及HLA-G6)抗体(YWHG-3)的以下方面：采用HLA-G异构体分子(HLA-G1，HLA-G2，HHLA-G5及HLA-G6)共有的，位于HLA-G分子重链α3结构域第209～226氨基酸序列(KTHVTHHPVFDYEATLRC)的抗原肽为免疫原，制备抗HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G5及HLA-G6)的单克隆抗体(YWHG-3)；编码本发明所述YWHG-3抗体的核苷酸及其编码的氨基酸序列，以及所述抗体(YWHG-3)用于免疫组化、免疫印迹及流式细胞术检测等用途。
背景技术
人类白细胞抗原-G(human leukocyte antigen-G,HLA-G)基因，全长6.0kb，位于人类第6号染色体短臂远侧6p21.3。在蛋白质翻译过程中，HLA-G mRNA第1外显子编码信号肽，第2、3和第4外显子分别编码细胞外区的α1、α2和α3结构域，第5位外显子编码跨膜区；第6外显子编码仅含6个氨基酸残基的HLA-G分子胞内段；由于外显子6内含有终止密码子，第7外显子不被转录；第8外显子对应于HLA-G基因的3′UTR。HLA-G初始转录产物经选择性剪接，可产生7种成熟mRNA，分别编码7种不同分子量的异构体分子(HLA-G1、-G2、-G3、-G4、-G5、-G6及HLA-G7)。其中HLA-G1、HLA-G2、HLA-G3及HLA-G4含有跨细胞膜区，为膜结合型异构体；HLA-G5、HLA-G6及HLA-G7缺乏跨细胞膜结构，为可溶性异构体。HLA-G1～-G7异构体分子的分子量分别为39kD,31kD,22kD,30kD,34kD,23kD及16kD。
HLA-G1是由全长HLA-G mRNA编码，由胞外区α1、α2及α3结构域，跨膜区及胞内结构域组成。HLA-G2缺乏α2结构域，由胞外区α1及α3结构域，跨膜区及胞内结构域组成；HLA-G3缺乏α2和α3结构域，由胞外区α1结构域，跨膜区及胞内结构域组成；HLA-G4缺乏α3结构域，由胞外区α1及α2结构域，跨膜区及胞内结构域组成；胞外区结构域分别与HLA-G1及HLA-G2相同，但它们由含有内含子4的HLA-G mRNA编码，因内含子4中含有终止密码子，所编码的蛋白分子缺乏由外显子5所编码的跨膜区，形成可溶性HLA-G分子。编码HLA-G7的mRNA由于内含子2中含有终止密码子，胞外区仅含α1结构域及由内含子2所编码的2个氨基酸残基相连(图1)。
在正常生理情况下，HLA-G分子仅表达在母胎界面的绒毛外滋养层细胞，维持妊娠过程中的母胎免疫耐受。病理情况下，HLA-G分子能在肿瘤细胞及病毒感染等病理组织细胞中诱导性表达，与疾病的发生及进展密切相关。HLA-G分子是体内一个重要的免疫致耐分子，也是一种重要的免疫检查点分子，其免疫抑制功能主要通过结合免疫抑制性受体免疫球蛋白样转录物-2(immunoglobulin-like transcript-2,ILT2/LILRB1/CD85j)、免疫球蛋白样转录物-4(ILT4/LILRB2/CD85d),传递抑制信号，诱导免疫耐受。具体作用机制有：①与表达在T细胞、NK细胞和B细胞上的ILT-2结合，抑制T细胞和NK细胞免疫杀伤活性，抑制B细胞增殖和抗体分泌等功能。②与树突状细胞(DC)上表达的ILT-2、ILT-4结合，抑制DC细胞的成熟和分化，诱导产生耐受性DC细胞。③与骨髓来源抑制性细胞(MDSC)和巨噬细胞(Mф)上表达的ILT-2、ILT-4结合，诱导促炎抗肿瘤的M1型巨噬细胞向免疫致耐性M2型巨噬细胞分化等。因此，HLA-G在肿瘤等疾病的发生发展中起到重要作用。基于HLA-G为靶点的多项肿瘤免疫靶向治疗已在美国等进入I期临床试验。
HLA-G与受体ILT-2、ILT-4结合，具有分子结构特异性。受体ILT-2、ILT-4与HLA-G结合的位点HLA-G胞外区α3结构域。ILT-2仅结合HLA-G/β 2m复合物，而ILT-4不仅可以与HLA-G/β 2m结合，同时可以与不含β 2m的游离HLA-G分子结合。由于HLA-G1、-G2、-G3、-G4、-G5、-G6及HLA-G7异构体分子在表达机制及分子结构上存在差异。ILT-2能与HLA-G1和HLA-G结合，而ILT-4能与HLA-G1，HLA-G2，HLA-G5及HLA-G6异构体分子结合。如HLA-G3、-G4、及HLA-G7异构体胞外区不含α3结构域，不能与ILT-2和ILT-4结合。不同HLA-G异构体分子可在病理生理过程中发挥特定的免疫学效应。在不同病理状态下，尤其在肿瘤组织细胞中，HLA-G异构体表达存在广泛的异质性，不同HLA-G分子异构体的表达具有特定的临床意义。因此，分析特定HLA-G异构体分子表达及不同HLA-G异构体分子表达谱，对于阐述特定HLA-G异构体分子的生物学功能及其临床意义具有重要意义。
目前国内外用于HLA-G免疫组化和免疫印迹的抗体主要有4H84，MEM-G1，MEM-G2，2A12及5A6G7。抗体4H84，其识别位点位于7种HLA-G异构体分子均具有的胞外区α1结构域，能够检测目前已知的含有α1结构域的7种HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G3，HLA-G4，HLA-G5，HLA-G6及HLA-G7)，但在免疫组化中不能区分具体特定HLA-G异构体分子的表达。抗体MEM-G1及MEM-G2由全长HLA-G重链免疫小鼠得到，具体识别位点无法预测，这两种抗体理论上，与抗体4H84相似，能够识别7种HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G3，HLA-G4，HLA-G5，HLA-G6及HLA-G7)，但同样不能区分具体特定HLA-G异构体分子的表达。抗体2A12及5A6G7，由位于HLA-G5和HLA-G6分子羧基端22个氨基 酸残基免疫小鼠获得，能够识别HLA-G5和HLA-G6分子。但抗体2A12及5A6G7同时还可以识别其他分子量在36-175kDa范围内的未知的其他非HLA-G5和HLA-G6分子，在免疫组化的应用上可能存在交叉反应，出现假阳性，不能正确HLA-G5和HLA-G6分子表达。用于流式细胞术检测HLA-G分子的单抗有87G及MEM-G/9，但只能检测HLA-G1及HLA-G5分子。
目前尚无检测HLA-G1，HLA-G2，HLA-G5及HLA-G6异构体分子的抗体。
因此，在靶向免疫治疗及精准医疗的背景下，现有的检测HLA-G分子的抗体还不尽如人意，迫切需要开发更多针对不同HLA-G异构体分子的单克隆抗体。
发明内容
鉴于现有HLA-G抗体存在上述不足，及目前尚无检测HLA-G1，HLA-G2，HLA-G5及HLA-G6异构体分子的抗体，本发明的目的是提供一种抗含有α3结构域的HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G5及HLA-G6)的特异性单克隆抗体(YWHG-3)，编码本发明所述抗体(YWHG-3)的核苷酸序列及其编码的氨基酸序列；以及所述YWHG-3抗体用于免疫组化、免疫印迹及流式检测等用途。
本发明采用HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G5及HLA-G6)共有的，位于HLA-G分子重链α3结构域第209～226氨基酸序列的抗原肽为免疫原，其氨基酸序列为SEQ ID No.19所示氨基酸序列(KTHVTHHPVFDYEATLRC)。KTHVTHHPVFDYEATLRC肽段位于HLA-G分子重链α3结构域(图1虚框内所示区域)，并基于此制备抗HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G5及HLA-G6)的特异性单克隆抗体(YWHG-3)。
根据发明人的研究成果，本发明提供了一种抗HLA-G分子的单克隆抗体(YWHG-3)，至少包括轻链高变区CDR1、CDR2和CDR3中的一种或多种，或/和重链高变区CDR1、CDR2和CDR3中的一种或多种；
所述单克隆抗体(YWHG-3)抗体轻链的氨基酸序列如SEQ ID No.1所示或经替换、缺失或添加一个或多个氨基酸形成的与SEQID No.1所示序列具有同等功能的氨基酸序列；所述抗体轻链高变区CDR1的氨基酸序列为SEQ ID No.2所示的序列QSLVHSTGNTF或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.2所示的序列具有同等功能的氨基酸序列；所述轻链高变区CDR2的氨基酸序列为SEQ ID No.3所示的序列KVS或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.3所示的序列具有同等功能的氨基酸序列，和所述轻链高变区CDR3的氨基酸序列为SEQ ID No.4所示的序列SQSTHVPWT或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.4所示的序列具有同等功能的氨基酸序列；
所述单克隆抗体(YWHG-3)抗体重链的氨基酸序列如SEQ ID No.5所示或经替换、缺 失或添加一个或多个氨基酸形成的与SEQ ID No.5所示序列具有同等功能的氨基酸序列；所述抗体重链高变区CDR1的氨基酸序列为SEQ ID No.6所示的序列GFTFSDYN或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.6所示的序列具有同等功能的氨基酸序列，所述重链高变区CDR2的氨基酸序列为SEQ ID No.7所示的序列ISDGGRST或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.7所示序列具有同等功能的氨基酸序列，和所述重链高变区CDR3的氨基酸序列为SEQ ID No.8所示的序列VRDDY或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.8所示的序列具有同等功能的氨基酸序列。
进一步地，所述单克隆抗体(YWHG-3)还包括轻链框架区(Framework region，FR)和重链框架区；其中，所述轻链框架区包括轻链FR1、FR2、FR3和FR4中的一种或多种，所述轻链FR1的氨基酸序列为SEQ ID No.9所示的序列DIMMTQTPLSLPVSLGDQASISCRSS或该序列经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.9所示序列具有同等功能的氨基酸序列；所述轻链FR2的氨基酸序列为SEQ ID No.10所示的序列LNWYLQKPGQSPKLLIY或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.10所示序列具有同等功能的氨基酸序列；所述轻链FR3的氨基酸序列为SEQ ID No.11所示的序列NRFSGVSDRFSGSGSGTDFTLKIRRVEAEDLGVYFC或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.11所示序列具有同等功能的氨基酸序列；所述轻链FR4的氨基酸序列为SEQ ID No.12所示的序列FGGGTKLEIK或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.12所示序列具有同等功能的氨基酸序列；所述重链框架区包括重链FR1、FR2、FR3和FR4中的一种或多种，其中：所述重链FR1的氨基酸序列为SEQ ID No.13所示的序列LVQLQQSGGGLVKPGGSLKLSCAAS或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.13所示序列具有同等功能的氨基酸序列；所述重链FR2的氨基酸序列为SEQ ID No.14所示的序列MYWVRQTPEKRLEWVAT或经替换、缺失或添加一个或多个氨基酸形成的具有与SEQ ID No.14所示序列同等功能的氨基酸序列；所述重链FR3的氨基酸序列为SEQ ID No.15所示的序列FYLDSVKGRFTISRDNAKNNLYLQINNLKSEDTAIYYC或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.15所示序列具有同等功能的氨基酸序列；所述重链FR4的氨基酸序列为SEQ ID No.16的序列WGQGTTLTVSS或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.16所示序列具有同等功能的氨基酸序列；
进一步地，所述单克隆抗体(YWHG-3)中编码轻链的核苷酸序列为SEQ ID No.17所示的序列或该序列经替换、缺失或添加一个或多个核苷酸形成的与SEQ ID No.17所示序列具有同等功能的核苷酸序列，所述单克隆抗体(YWHG-3)中编码重链的核苷酸序列为SEQ ID  No.18所示的序列或该序列经替换、缺失或添加一个或多个核苷酸形成的与SEQ ID No.18所示序列具有同等功能的核苷酸序列。
根据本发明的一个方面，本发明提供了一种优选的抗HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G5及HLA-G6)的单克隆抗体(YWHG-3)，所述单克隆抗体(YWHG-3)由保藏编号为CCTCC NO:202121的杂交瘤产生，保藏机构为：中国典型培养物保藏中心，保藏时间为2021年8月11日，中国典型培养物保藏中心的地址是中国湖北省武汉市武汉大学内，邮编430072。
本发明还提供了所述抗HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G5及HLA-G6)抗体(YWHG-3)用于HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G5及HLA-G6)免疫组化、免疫印迹及流式细胞术等检测的用途，具有特异性高，亲和力强等特点。
为更清楚了解本申请的发明构思和技术方案，下文将通过具体实施例和附图进一步解释本申请，其中关于实施方式中的技术方案仅是优选的实施方式，不应被解释为限制本申请的范围。对于本领域技术人员来说，在不脱离本申请的技术原理的前提下，还可以做出若干改进和调整，这些改进和调整也应视为落入本申请的保护范围之中。
除非另有定义，本文使用的所有科技术语应视为具有本领域普通技术人员所理解的相同含义。氨基酸残基的缩写是本领域中所用的指代20个常用氨基酸的标准3字母和/或1字母代码。
本发明中提及的轻链高变区或重链高变区，其中“高变区”又被称之为互补决定区(complementarity determining region，CDR)。
本发明中提及的“序列”可以指包含某些生物学功能等同的氨基酸序列或“保守性替代”，“其它序列”可以包含功能非等同的氨基酸或“非保守性替代”，其经基因工程改造以改进CDR或含CDR抗体的特性。在不实质性地影响抗体活性的前提下，本领域技术人员可以对本申请中的序列进行操作，即替换、添加和/或缺失一个或多个(例如1、2、3、4、5、6、7、8、9或10个或多个)氨基酸，以获得所述抗体或其功能性片段序列的变体。它们应被视为包括在本申请保护的范围内。例如，在可变区将具有类似性质的氨基酸进行替换。本发明中提及的变体的序列可以与其来源序列有至少有95％、96％、97％、98％或99％的一致性。序列一致性可以使用序列分析软件测量。例如使用缺省参数的计算机程序BLAST，尤其是BLASTP或TBLASTN。本文所述的多种氨基酸序列详见序列表。
附图说明
图1是7种不同HLA-G异构体分子结构模式图及免疫抗原肽段位置。
图2是所述抗体(YWHG-3)重链和轻链亚类鉴定。
图3是所述抗体(YWHG-3)SDS-PAGE检测抗体纯度检测。
图4是所述抗体(YWHG-3)ELISA法抗体亲和常数测定。
图5是所述抗体(YWHG-3)ELISA法HLA-G5分子浓度检测。
图6是所述抗体(YWHG-3)免疫印迹检测HLA-G1，-G2，-G5及HLA-G6分子。
图7是所述抗体(YWHG-3)流式检测胞内HLA-G1，-G2，-G5及HLA-G6分子。
图8是所述抗体(YWHG-3)应用于免疫组化检测胃癌组织中HLA-G1，-G2，-G5及HLA-G6分子的表达。
具体实施方式
1.抗HLA-G单克隆抗体(YWHG-3)制备
①抗原肽合成
合成位于HLA-G分子重链α3结构域第209～226氨基酸序的抗原肽，SEQ No.19 KTHVTHHPVFDYEATLRC。
②小鼠免疫
初次免疫4只SPF级BALB/c雌性小鼠，60ug/只。第一次加强免疫小鼠，30ug/只。第二次加强免疫小鼠，30ug/只。第三次加强免疫小鼠，30ug/只。眼眶取血，测血清效价。用“SEQ No.19 KTHVTHHPVFDYEATLRC”包板，ELISA测定免疫小鼠效价。用“SEQ No.19 KTHVTHHPVFDYEATLRC”，2ug/ml，4℃包被过夜；2％牛奶，37℃封闭2h；血清从200倍开始2倍梯度稀释，空白对照(blank)为PBS，阴性对照(negative)为阴性血清200倍稀释。融合前，用“SEQ No.19 KTHVTHHPVFDYEATLRC”免疫原50ug，冲击免疫小鼠。融合实验，取小鼠脾细胞与SP2/0细胞，采用PEG法进行融合，融合完细胞用半固体培养基(含HAT)进行筛选培养。
③单克隆细胞筛选
挑10板×93个细胞单克隆，培养于96孔细胞培养板(事先用胸腺细胞铺板，100ul/孔)。用“SEQ No.19 KTHVTHHPVFDYEATLRC”包板，用包被液稀释“SEQ No.19 KTHVTHHPVFDYEATLRC”，终浓度为2ug/ml，100ul/孔，4℃，过夜；后用洗液洗涤3次。2％牛奶封闭液封闭，200ul/孔，37℃孵箱，2h；后用洗液洗涤3次。加入一抗(细胞培养上清)、阴性对照(SP2/0培养上清)、空白对照(PBS)、阳性对照(阳性血清PBS 1000倍稀释)，均为100ul/孔，37℃孵箱，1h；后用洗液洗涤3次。加入PBS稀释20000倍的二抗，100ul/孔，37℃孵箱，1h；取出后用洗液洗涤3次。显色，显色液100ul/孔，显色时间为 5min左右。每孔加入50ul终止液终止。双波长(450，630)测吸光值。对挑选的克隆采用ELISA方法，做第一次筛选，得到阳性杂交瘤细胞株。将阳性的细胞株，用“SEQ No.19 KTHVTHHPVFDYEATLRC”再次包板，采用ELISA方法，做第二次筛选，得到阳性杂交瘤细胞株。经多次筛选后得到一株抗SEQ No.19 KTHVTHHPVFDYEATLRC片段的单克隆抗体，命名为YWHG-3(保藏编号为CCTCC NO:202121)。
④抗HLA-G单克隆抗体(YWHG-3)亚类鉴定
将筛选出来的10株阳性细胞株进行亚类鉴定，用100mM PBS(pH7.4)稀释包被抗体至0.5ug/ml,每孔加0.1ml,4℃，过夜。PBS-T洗2次,每孔加入200ul封闭液，37 0C孵育2h。PBS-T洗3次；每孔加入100ul杂交瘤上清，37 0C孵育1h。PBS-T洗3次；用封闭液1：10000(κ,λ)或1：20000(其它的)稀释的HRP标记的抗体0.1ml每孔，分别加入适当的孔中，370C孵育1h。PBS-T洗3次；每孔加50ul底物溶液，10-20min内于双波长(450，630)测吸光值。抗体亚型再次用Thermo公司的小鼠抗体亚型快速鉴定法(Pierce Rapid Isotyping Kits–Mouse，Catalog”#26178)确定该细胞株产生的单抗亚型为重链为IgG1型，轻链为kappa(κ)型(图2)。
⑤抗体纯化
样本预处理：用相应的偶联缓冲液以1:3稀释，12000rpm 4℃离心10min，0.22μm滤膜过滤，除去脂肪、细胞残渣及小颗粒物质。平衡：用10倍柱体积的相应偶联缓冲液平衡柱子，保持流速为1ml/min。上样：把样品注入柱子上端接口，收集流出液，保持流速为1ml/min。洗杂：用5倍柱体积的偶联缓冲液过柱，保持流速为1ml/min。洗脱：用5倍柱体积洗脱缓冲液洗脱抗体，收集于上述EP管中，保持流速为1ml/min。立即用1M pH9.0的Tris-Hcl缓冲液调整pH值至7.0。平衡：用10柱体积的偶联缓冲液平衡柱子回pH7.0，保持流速为1ml/min。透析：使用0.01M PBS缓冲液将抗体透析过夜，换液3次。
⑥SDS-PAGE检测抗体纯度
配置SDS-PAGE胶，分离胶的浓度为12％。样品制备：样品加上样缓冲液后沸水煮浴10min。上样：每孔10ul。跑胶：浓缩胶80V，30min；分离胶120V，60min。溴酚蓝前沿到达玻璃板底部时停止电泳，取出凝胶。染色与脱色：将凝胶浸入考马斯亮蓝染色液中，置摇床上缓慢震荡30min以上(染色时间需根据凝胶厚度适当调整)。取出凝胶在水中漂洗数次，再加入考马斯亮蓝脱色液、震荡。凝胶脱色至大致看清条带1h，完全脱色则需更换脱色液2～3次，震荡达24h以上。凝胶脱色后可通过ECL凝胶成像系统扫描记录。纯化后得到的抗体YWHG-3纯度>90％(图3)。
⑦ELISA检测抗体的亲和常数测定
用包被液稀释抗原，SEQ No.19 KTHVTHHPVFDYEATLRC，终浓度为2ug/ml，100ul/孔，4℃，过夜；后用洗液洗涤2次。封闭液封闭，200ul/孔，37℃孵箱，2h；后用洗液洗涤1次。纯化抗体从200倍开始2倍梯度稀释(PBS)，空白对照(blank)为PBS，均为100ul/孔，37℃孵箱，1h；后用洗液洗涤3次。加入PBS稀释20000倍的二抗，100ul/孔，37℃孵箱，1h；取出后用洗液洗涤3次。显色，显色液100ul/孔，显色时间为5-15min。每孔加入50ul终止液终止。双波长(450，630)测吸光值，作图分析。根据Logistic拟合方程，亲和常数≈150000×A/抗体浓度(A为1/2OD值所对应的抗体稀释倍数；稀释倍数＝204800；YWHG-3抗体浓度＝2.8mg/mL)，抗体YWHG-3的亲和常数为1.10×10 10L/mol(图4)。
⑧免疫印迹检测抗体的HLA-G异构体识别特异性。
将HLA-G异构体标准蛋白HLA-G1，HLA-G2，HLA-G3，HLA-G4，HLA-G5，HLA-G6及HLA-G7异构体分子标准蛋白电转膜后，用5％去脂奶粉室温封闭4h，0.2％TBS(Teween-20 PBS)洗涤。加入所述抗体(YWHG-3，1.0ug/ml)，检测其识别特异性，4℃孵育过夜，洗涤；加入HRP标记兔抗鼠IgG抗体,室温孵育30min，洗涤后，用Dako REAL TMEnVision TM检测系统(DAKO)孵育1-3min。结果显示：结果说明所述抗体(YWHG-3)能特异性识别HLA-G1，-G2，-G5及HLA-G6标准蛋白，与其他HLA-G异构体分子不存在交叉反应,具有很好设别含有α3结构域HLA-G异构体分子(HLA-G1，-G2，-G5及HLA-G6)特异性(图5)。
二、应用实施例
实施例2.1.所述抗体(YWHG-3)ELISA法HLA-G5分子浓度检测
用包被液(0.1M,pH＝9.6NaHCO3)2倍梯度稀释HLA-G5蛋白，配置终浓度为分别为4ug/ml，2ug/ml，1ug/ml，0.5ug/ml,0.25ug/ml，0.125ug/ml及0.0625ug/ml，以包被液为空白对照。100ul/孔包板，4℃，过夜；后用洗液洗涤2次。200ul/孔1％BSA封闭，37℃孵箱，2h；后用洗液洗涤3次。
洗涤后，加终浓度为1.0ug/ml的生物素标记抗体(YWHG-3)，100ul/孔，37℃孵箱，1h；后用洗液洗涤3次。加入PBS稀释1.0ug/ml的过氧化物酶标记链酶亲和素(Peroxidase-conjugated Streptavidin)，100ul/孔，37℃孵箱，1h；取出后用洗液洗涤3次。显色，TMB显色液100ul/孔，显色时间为5-15min。每孔加入50ul终止液终止。波长(450)测吸光值，作图分析。4ug/ml，2ug/ml，1ug/ml，0.5ug/ml,0.25ug/ml，0.125ug/ml，0.0625ug/ml及包被液空白对照的OD 450分别为1.856,1.490，1.347，1.092,0.664，0.388,0.263及0.045。结果显示，抗体(YWHG-3)特异性识别HLA-G5，且与HLA-G5浓度具 有显著相关性[HLA-G5(ug/ml)＝1.7355x(OD) 2-1.2268x(OD)；R 2＝0.9504](图6)。
实施例2.2.所述抗体(YWHG-3)流式检测胞内HLA-G1，-G2，-G5及HLA-G6分子检测
分别取对数生长期表达K562(空白对照)，K562-HLA-G1，K562-HLA-G2，K562-HLA-G5及K562-HLA-G6细胞培养物。通过细胞内流式细胞术检测。
细胞内分子表达流式检测：流式管收集K562，K562-HLA-G1，K562-HLA-G2，K562-HLA-G5及K562-HLA-G6细胞培养物，2％BSA/PBS离心洗涤2次(250g)，加入250ul细胞破膜剂(BD Cytofix/Cytoperm TM)，充分混匀，4℃冰箱放置20分钟。加入1ml BD Perm/Wash TM离心洗涤两遍(250g)。洗涤后，100ul BD Perm/Wash TM重悬K562，K562-HLA-G1，K562-HLA-G2，K562-HLA-G5及K562-HLA-G6细胞，加入含有加入1ul(1.0mg/ml)的FITC标记的纯化抗体(YWHG-3),4℃孵育30分钟，用1ml 1×BD Perm/Wash TM离心洗涤3遍(250g)；将细胞用1×BD Perm/Wash TM重悬成300ul细胞悬液，流式细胞检测。结果显示：所述抗体(YWHG-3)能特异性识别胞内表达的K562-HLA-G1，K562-HLA-G2，K562-HLA-G5及K562-HLA-G6分子(图7)。
实施例2.3.
所述抗体(YWHG-3)应用于免疫组化检测胃癌组织中HLA-G分子(HLA-G1，HLA-G2，HLA-G5及HLA-G6)表达。
所取胃癌组织于10％-12％中性福尔马林固定，石蜡包埋。组织切片经烤片，脱蜡，水化及抗原修复等常规制片流程。组织上滴加适量1％BSA，其覆盖组织及组织边缘2mm，室温孵育10min进行封闭。滴加所述抗体(YWHG-3)(1mg/mL，1:500稀释)，4℃冰箱湿盒过夜(16-20h)。TBS缓冲液冲洗，滴加二抗(TBS稀释抗体羊抗鼠比例1：300)，37℃恒温箱孵育30min。TBS缓冲液冲洗，滴加显色剂DAB工作液，待组织显色完全后，将玻片置于流水中冲洗5min，蒸馏水浸泡5min。HE复染、脱水、透明、封片后，光学显微镜观察组织切片各视野情况。棕褐色着色的细胞为胃癌组织细胞HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G5及HLA-G6)阳性表达，根据细胞棕褐色着色深浅，判别HLA-G异构体分子(HLA-G1，HLA-G2，HLA-G5及HLA-G6)的表达强度。其中图8为HLA-G分子在(A),(B),(C)及(D)胃癌组织样本中高表达(图8)。

Claims (5)

  1. 一种抗HLA-G1，-G2，-G5及HLA-G6异构体分子的单克隆抗体(YWHG-3)，所述单克隆抗体(YWHG-3)由保藏编号为CCTCC NO:202121的杂交瘤产生，保藏机构为：中国典型培养物保藏中心，保藏时间为2021年8月11日。
  2. 一种抗HLA-G1，-G2，-G5及HLA-G6异构体分子的单克隆抗体(YWHG-3)，其特征在于，所述单克隆抗体(YWHG-3)至少包括轻链高变区CDR1、CDR2和CDR3中的一种或多种，或/和重链高变区CDR1、CDR2和CDR3中的一种或多种；
    所述单克隆抗体(YWHG-3)抗体轻链氨基酸序列如SEQ ID No.1所示或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.1所示序列具有同等功能的氨基酸序列；所述抗体轻链高变区CDR1氨基酸序列为SEQ ID No.2所示的序列QSLVHSTGNTF或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.2所示的序列具有同等功能的氨基酸序列；所述轻链高变区CDR2的氨基酸序列为SEQ ID No.3所示的序列KVS或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.3所示的序列具有同等功能的氨基酸序列，和所述轻链高变区CDR3氨基酸序列为SEQ ID No.4所示序列SQSTHVPWT或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.4所示的序列具有同等功能的氨基酸序列；
    所述单克隆抗体(YWHG-3)抗体重链核苷酸序列如SEQ ID No.5所示或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.5所示序列具有同等功能的氨基酸序列；所述抗体重链高变区CDR1氨基酸序列为SEQ ID No.6所示的序列GFTFSDYN或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.6所示的序列具有同等功能的氨基酸序列，所述重链高变区CDR2氨基酸序列为SEQ ID No.7所示的序列ISDGGRST或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.7所示序列具有同等功能的氨基酸序列，和所述重链高变区CDR3的氨基酸序列为SEQ ID No.8所示的序列VRDDY或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.8所示的序列具有同等功能的氨基酸序列。
  3. 根据权利要求2所述的一种抗HLA-G1，-G2，-G5及HLA-G6异构体分子的单克隆抗体(YWHG-3)，其特征在于，所述单克隆抗体(YWHG-3)还包括轻链框架区(Framework region，FR)和重链框架区；其中，所述轻链框架区包括轻链FR1、FR2、FR3和FR4中的一种或多种，所述轻链FR1的氨基酸序列为SEQ ID No.9所示的序列DIMMTQTPLSLPVSLGDQASISCRSS或该序列经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.9所示序列具有同等功能的氨基酸序列；所述轻链FR2的氨基酸序列为SEQ ID No.10所示的序列LNWYLQKPGQSPKLLIY或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.10所示序列具有同等功能 的氨基酸序列；所述轻链FR3的氨基酸序列为SEQ ID No.11所示的序列NRFSGVSDRFSGSGSGTDFTLKIRRVEAEDLGVYFC或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.11所示序列具有同等功能的氨基酸序列；所述轻链FR4的氨基酸序列为SEQ ID No.12所示的序列FGGGTKLEIK或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.12所示序列具有同等功能的氨基酸序列；
    所述重链框架区包括重链FR1、FR2、FR3和FR4中的一种或多种，其中：所述重链FR1的氨基酸序列为SEQ ID No.13所示的序列LVQLQQSGGGLVKPGGSLKLSCAAS或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.13所示序列具有同等功能的氨基酸序列；所述重链FR2的氨基酸序列为SEQ ID No.14所示的序列MYWVRQTPEKRLEWVAT或经替换、缺失或添加一个或多个氨基酸形成的具有与SEQ ID No.14所示序列同等功能的氨基酸序列；所述重链FR3的氨基酸序列为SEQ ID No.15的序列FYLDSVKGRFTISRDNAKNNLYLQINNLKSEDTAIYYC或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.15所示序列具有同等功能的氨基酸序列；所述重链FR4的氨基酸序列为SEQ ID No.16的序列WGQGTTLTVSS或经替换、缺失或添加一个或多个氨基酸形成的与SEQ ID No.16所示序列具有同等功能的氨基酸序列；
  4. 根据权利要求2所述的一种抗HLA-G1，-G2，-G5及HLA-G6异构体分子的单克隆抗体(YWHG-3)，其特征在于，所述单克隆抗体(YWHG-3)中编码轻链可变区的核苷酸序列为SEQ ID No.17所示的序列或该序列经替换、缺失或添加一个或多个核苷酸形成的与SEQ ID No.17所示序列具有同等功能的核苷酸序列，所述单克隆抗体(YWHG-3)中编码重链可变区的核苷酸序列为SEQ ID No.18所示的序列或该序列经替换、缺失或添加一个或多个核苷酸形成的与SEQ ID No.18所示序列具有同等功能的核苷酸序列。
  5. 权利要求1-4中任一项所述的抗HLA-G1，-G2，-G5及HLA-G6异构体分子的单克隆抗体(YWHG-3)用于免疫组化、免疫印迹及胞内流式细胞术的用途。
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