WO2024011774A1 - Anticorps monoclonal dirigé contre les molécules hla-g2 et hla-g6 et son utilisation - Google Patents
Anticorps monoclonal dirigé contre les molécules hla-g2 et hla-g6 et son utilisation Download PDFInfo
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- 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
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
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- 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
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- 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
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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- C07K2317/00—Immunoglobulins specific features
- 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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- C—CHEMISTRY; METALLURGY
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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Definitions
- the invention belongs to the field of biomedicine and relates to the following aspects of anti-HLA-G2 and HLA-G6 isomer molecular antibodies (YWHG-26): using the consensus sequence of HLA-G2 and HLA-G6 isomers, located in the ⁇ 1 and ⁇ 3 connecting regions
- the antigen peptide (RGYYNQSEAKPPKTHVTHHPV) is used as an immunogen to prepare a monoclonal antibody (YWHG-26) against HLA-G molecules; the nucleotide encoding the YWHG-26 antibody of the present invention and its encoded amino acid sequence, and the antibody (YWHG-26) is used for molecular immunoblotting and immunohistochemistry detection of HLA-G2 and HLA-G6 isoforms.
- 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.
- targeted therapies for tumors related to HLA-G and ILT have been carried out one after another.
- the expression of HLA-G isoforms is widely heterogeneous, 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.
- antibody 4H84 has a recognition site located in the extracellular ⁇ 1 domain of all HLA-G molecules, and can detect seven HLA-G isoform molecules currently known to contain ⁇ 1 domains (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, and the specific recognition sites cannot be predicted. Antibodies MEM-G1 and MEM-G2 are similar in theory to antibody 4H84 and can recognize the above-mentioned HLA-G isoform molecules, but they are also unable to distinguish the expression of specific HLA-G isoform molecules.
- the purpose of the present invention is to provide a specific monoclonal antibody against HLA-G2 and HLA-G6 isoform molecules ( YWHG-26), the nucleotide sequence encoding the antibody (YWHG-26) of the present invention and its encoded amino acid sequence; and the use of the YWHG-26 antibody for detection such as immunoblotting and immunohistochemistry.
- the present invention adopts the consensus sequence of HLA-G2 and HLA-G6 isomers.
- the antigen peptide (RGYYNQSEAKPPKTHVTHHPV) located in the connecting region of ⁇ 1 and ⁇ 3 is used as the immunogen.
- Its amino acid sequence is the amino acid sequence shown in SEQ ID No. 19 (RGYYNQSEAKPPKTHVTHHPV) ( The area shown in the dotted box in Figure 1), and based on this, specific monoclonal antibodies (YWHG-26) against HLA-G2 and HLA-G6 isoform molecules were prepared.
- the present invention provides a monoclonal antibody (YWHG-26) against HLA-G2 and HLA-G6 isoform molecules, including at least one of the light chain hypervariable regions CDR1, CDR2 and CDR3.
- YWHG-26 a monoclonal antibody against HLA-G2 and HLA-G6 isoform molecules, including at least one of the light chain hypervariable regions CDR1, CDR2 and CDR3.
- the amino acid sequence of the monoclonal antibody (YWHG-26) 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 QSIVHSNGNTY 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 FQGSHVPLT shown in SEQ ID No. 4 or the sequence 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-26) 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 GYAFSTYW shown in SEQ ID No.6 or the sequence GYAFSTYW 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 IYPGDGDT shown in SEQ ID No.
- 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 amino acid sequence of the light chain FR1 is the sequence DIMLTQTPLSLPVSLGDQASISCRSS shown in SEQ ID No. 9 or the sequence is formed by replacing, deleting or adding one or more amino acids and is 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 LEWYLQKPGQSPKLLIY 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 NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC or has the same sequence as the sequence shown in SEQ ID No. 11 by replacing, deleting or adding one or more amino acids.
- Amino acid sequence with equivalent functions; the amino acid sequence of the light chain FR4 is the sequence FGAGTKLELK 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 shown in SEQ ID No. 13 LGQLQESGAELVRPGSSVKISCKAS 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 MNWVKQRPGQGLEWIGQ 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 RYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFC or the sequence shown in SEQ ID No. 15.
- the amino acid sequence formed by replacing, deleting or adding one or more amino acids has the same function as the sequence shown in SEQ ID No. 15;
- the amino acid sequence of the heavy chain FR4 is the sequence WGQGTLLTVSA 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-26) is the sequence shown in SEQ ID No. 18 or this sequence.
- the present invention provides a preferred monoclonal antibody (YWHG-26) against HLA-G2 and HLA-G6 isoform molecules.
- the monoclonal antibody (YWHG-26) is deposited under the deposit no. It was produced from the hybridoma of CCTCC NO:C202240.
- the depository institution is: China Type Culture Collection Center. The preservation date is March 8, 2022. The address of China Type Culture Collection Center is Wuhan University, Wuhan City, Hubei province, China, Postal Code 430072 .
- the invention also provides the use of the anti-HLA-G molecule antibody (YWHG-26) for HLA-G molecule immunoblotting and immunohistochemistry detection, and has the characteristics of high specificity and strong affinity.
- YWHG-26 anti-HLA-G molecule antibody
- the "hypervariable region” is also called a complementarity determining region (CDR).
- CDR complementarity determining region
- sequences 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 modified by genes.
- 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-26).
- Figure 3 is the SDS-PAGE detection of the antibody purity of the antibody (YWHG-26).
- Figure 4 is the determination of the antibody affinity constant of the antibody (YWHG-26) by ELISA
- Figure 5 is the immunoblot detection of HLA-G2 and HLA-G6 molecules using the antibody (YWHG-26).
- Figure 6 shows the application of the antibody (YWHG-26) to immunohistochemical detection of HLA-G2 and HLA-G6 molecule expression in colorectal cancer tissue.
- the antigen peptide (SEQ No. 19 RGYYNQSEAKPPKTHVTHHPV) located in the consensus sequence of HLA-G2 and HLA-G6 isomers and located in the connecting region of ⁇ 1 and ⁇ 3 is synthesized as the immunogen.
- 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. Wrap the plate with "SEQ No.19 RGYYNQSEAKPPKTHVTHHPV" and measure the titer of immunized mice by ELISA.
- the selected clones were screened for the first time using ELISA method to obtain positive hybridoma cell lines.
- the positive cell lines were plated again with "SEQ No. 19 RGYYNQSEAKPPKTHVTHHPV", and the ELISA method was used for a second screening to obtain positive hybridoma cell lines.
- YWHG-26 monoclonal antibody against the SEQ No. 19 RGYYNQSEAKPPKTHVTHHPV fragment was obtained, named YWHG-26 (deposit number: CCTCC NO: C202240).
- 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, maintaining a flow rate of 1 ml/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.
- Example 2.1 Detection of HLA-G isoform recognition specificity of antibodies by immunoblotting.
- HLA-G isoform standard protein HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, and HLA-G7 molecules were electrotransferred to the membrane, they were blocked with 5% skim milk powder at room temperature for 4 hours. , washed with 0.2% TBS (Tewen-20PBS).
- TBS Tewen-20PBS
- Example 2.2 The antibody (YWHG-26) is used for immunohistochemical detection of HLA-G2 and HLA-G6 molecular expression in colorectal cancer tissue.
- the colorectal 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-26) (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.
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Abstract
La présente invention concerne un anticorps monoclonal dirigé contre les molécules HLA-G2 et HLA-G6 et son utilisation. L'anticorps (YWHG-26) est produit à partir d'un hybridome ayant le numéro de dépôt de CCTCC NO : C202240, la séquence commune d'isomères HLA-G2 et HLA-G6, à savoir, le peptide antigénique situé dans la région de connexion α1 et α3 (RGYYNQSEAKPPKTHVTHHPV) étant utilisé en tant qu'immunogène. La présente invention concerne un nucléotide codant pour l'anticorps YWHG-26 selon la présente invention et une séquence d'acides aminés codée par celui-ci. La présente invention concerne en outre l'utilisation de l'anticorps YWHG -26 pour la détection, tel que l'immunotransfert et l'immunohistochimie de molécules isomères HLA-G2 et HLA-G6.
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CN114605543A (zh) * | 2021-12-17 | 2022-06-10 | 台州恩泽医疗中心(集团) | 一种抗hla-g异构体分子hla-g5及hla-g6的单克隆抗体及其用途 |
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CN113045656A (zh) * | 2020-07-27 | 2021-06-29 | 台州恩泽医疗中心(集团) | 抗hla-g异构体分子hla-g5及hla-g6的单克隆抗体及其用途 |
CN114605543A (zh) * | 2021-12-17 | 2022-06-10 | 台州恩泽医疗中心(集团) | 一种抗hla-g异构体分子hla-g5及hla-g6的单克隆抗体及其用途 |
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