Monoclonal antibodies against hla-g molecules and uses thereof

Abstract

The application discloses an antibody (YHWG-1) against HLA-G molecules and application, the antibody (YHWG-1) is produced by hybridoma with the preservation number of CCTCC NO:202120, using all currently known 7 HLA-G isomer molecules (HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6 and HLA-G7) shared, the antigen peptide (QTDRMNLQTLRGYYNQSEAS) of the 72~91 amino acid sequence in the HLA-G molecule heavy chain alpha 1 domain is immunogen.The application provides nucleotide and the amino acid sequence coded by the YHWG-1 antibody of the application, and the application also provides the application of the YHWG-1 antibody for HLA-G isomer molecule immunohistochemical, immunoblotting and flow cytometry detection.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the following aspects of an anti-HLA-G molecule antibody (YWHG-1): using an antigenic peptide common to all seven currently known HLA-G isoforms (HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, and HLA-G7), located at amino acid sequence 72-91 (QTDRMNLQTLRGYYNQSEAS) of the α1 domain of the HLA-G molecule heavy chain, as an immunogen, to prepare a monoclonal antibody (YWHG-1) against the HLA-G molecule; the nucleotide encoding the YWHG-1 antibody of this invention and its encoded amino acid sequence, and the use of the antibody (YWHG-1) for immunohistochemistry, Western blotting, and flow cytometry detection, etc. Background Technology

[0002] The human leukocyte antigen-G (HLA-G) gene, 6.0 kb in length, is located on the distal side of the short arm of human chromosome 6, at 6p21.3. During protein translation, exon 1 of the HLA-G mRNA encodes a signal peptide; exons 2, 3, and 4 encode the extracellular α1, α2, and α3 domains, respectively; exon 5 encodes the transmembrane region; exon 6 encodes the intracellular segment of the HLA-G molecule, containing only 6 amino acid residues; exon 7 is not transcribed due to the stop codon in exon 6; and exon 8 corresponds to the 3′UTR of the HLA-G gene. The initial HLA-G transcript undergoes alternative splicing to produce seven mature mRNAs, each encoding a different molecular weight isoform (HLA-G1, -G2, -G3, -G4, -G5, -G6, and HLA-G7). HLA-G1, HLA-G2, HLA-G3, and HLA-G4 contain transmembrane regions and are membrane-bound isomers; HLA-G5, HLA-G6, and HLA-G7 lack transmembrane structures and are soluble isomers. The molecular weights of the HLA-G1 to -G7 isomers are 39 kD, 31 kD, 22 kD, 30 kD, 34 kD, 23 kD, and 16 kD, respectively.

[0003] HLA-G1 is encoded by full-length HLA-G mRNA and consists of extracellular α1, α2, and α3 domains, a transmembrane region, and an intracellular domain. HLA-G2 lacks the α2 domain and consists of extracellular α1 and α3 domains, a transmembrane region, and an intracellular domain. HLA-G3 lacks both α2 and α3 domains and consists of an extracellular α1 domain, a transmembrane region, and an intracellular domain. HLA-G4 lacks the α3 domain and consists of extracellular α1 and α2 domains, a transmembrane region, and an intracellular domain. The extracellular domains are the same as those of HLA-G1 and HLA-G2, but they are encoded by HLA-G mRNA containing intron 4. Because intron 4 contains a stop codon, the encoded protein molecule lacks the transmembrane region encoded by exon 5, forming a soluble HLA-G molecule. The mRNA encoding HLA-G7 contains a stop codon in intron 2, and its extracellular region consists only of an α1 domain and two amino acid residues encoded by intron 2. Figure 1 ).

[0004] Under normal physiological conditions, HLA-G molecules are expressed only on extravillous trophoblast cells at the maternal-fetal interface, maintaining maternal-fetal immune tolerance during pregnancy. Under pathological conditions, HLA-G molecules can be induced to express in pathological tissues such as tumor cells and viral infections, and are closely related to the occurrence and progression of diseases. HLA-G molecules are an important immune tolerance molecule in the body and also an important immune checkpoint molecule. Their immunosuppressive function is mainly achieved by binding to immunosuppressive receptors immunoglobulin-like transcript-2 (ILT2 / LILRB1 / CD85j) and immunoglobulin-like transcript-4 (ILT4 / LILRB2 / CD85d), transmitting inhibitory signals, and inducing immune tolerance. Specific mechanisms of action include: ① Binding to ILT-2 expressed on T cells, NK cells, and B cells, inhibiting the immune killing activity of T cells and NK cells, and inhibiting B cell proliferation and antibody secretion. ② It binds to ILT-2 and ILT-4 expressed on dendritic cells (DCs), inhibiting DC maturation and differentiation, and inducing the production of tolerant DCs. ③ It binds to ILT-2 and ILT-4 expressed on bone marrow-derived suppressor cells (MDSCs) and macrophages (Mф), inducing the differentiation of pro-inflammatory and anti-tumor M1 macrophages into immune-resistant M2 macrophages. Therefore, HLA-G plays an important role in the development and progression of diseases such as tumors. Several tumor immunotherapy targeted by HLA-G have entered Phase I clinical trials in the United States and other countries.

[0005] HLA-G binds to receptors ILT-2 and ILT-4 with molecular structure specificity. The binding site for receptors ILT-2 and ILT-4 is the α3 domain of the extracellular region of HLA-G. ILT-2 binds only to the HLA-G / β2m complex, while ILT-4 can bind not only to HLA-G / β2m but also to free HLA-G molecules without β2m. Due to differences in expression mechanisms and molecular structures among HLA-G1, -G2, -G3, -G4, -G5, -G6, and HLA-G7 isoforms, ILT-2 can bind to both HLA-G1 and HLA-G, while ILT-4 can bind to all HLA-G1, HLA-G2, HLA-G5, and HLA-G6 isoforms. Since the extracellular regions of HLA-G3, -G4, and HLA-G7 isoforms lack the α3 domain, they cannot bind to ILT-2 and ILT-4. Different HLA-G isoforms can exert specific immunological effects during pathophysiological processes. Under different pathological conditions, especially in tumor tissues and cells, HLA-G isoform expression exhibits wide heterogeneity, and the expression of different HLA-G molecular isoforms has specific clinical significance. Therefore, analyzing the expression of specific HLA-G isoforms and their expression profiles is of great importance for elucidating the biological functions and clinical significance of specific HLA-G isoforms.

[0006] Currently, the antibodies used domestically and internationally for HLA-G molecular immunohistochemistry and immunoblotting include 4H84, MEM-G1, and MEM-G2. Antibody 4H84 recognizes the extracellular α1 domain, a domain present in all HLA-G molecules, and can detect the seven known HLA-G isoforms containing the α1 domain (HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, and HLA-G7). Antibodies MEM-G1 and MEM-G2 are obtained by immunizing mice with the full-length HLA-G heavy chain; their specific recognition sites cannot be predicted. Theoretically, these two antibodies are similar to antibody 4H84 and can recognize the aforementioned HLA-G isoforms. However, the detection specificity of antibodies 4H84, MEM-G1, and MEM-G2 is limited, and cross-reactivity exists in immunohistochemistry and immunoblotting, leading to false positives. Furthermore, these three antibodies cannot be used for flow cytometry detection.

[0007] Therefore, in the context of targeted precision medicine, existing antibodies for detecting HLA-G molecules are not satisfactory, and there is an urgent need to develop more monoclonal antibodies against HLA-G molecules with better specificity and affinity. Summary of the Invention

[0008] In view of the above-mentioned shortcomings of existing HLA-G antibodies, the purpose of this invention is to provide a specific monoclonal antibody against HLA-G molecules (YWHG-1), the nucleotide sequence encoding the antibody (YWHG-1) of this invention and the amino acid sequence it encodes; and the use of the YWHG-1 antibody for immunohistochemistry, immunoblotting and flow cytometry detection, etc.

[0009] This invention uses an antigenic peptide located in the α1 domain of the HLA-G molecule heavy chain, common to all seven known HLA-G isoforms (HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, and HLA-G7), as the immunogen. Its amino acid sequence is shown in SEQ ID No. 19 (QTDRMNLQTLRGYYNQSEAS). The QTDRMNLQTLRGYYNQSEAS peptide is located in the α1 domain of the HLA-G molecule heavy chain (…). Figure 1 The area shown in the dashed box was used to prepare a specific monoclonal antibody against HLA-G molecules (YWHG-1).

[0010] Based on the inventor's research findings, this invention provides a monoclonal antibody (YWHG-1) against HLA-G molecules, comprising at least one or more of the light chain hypervariable regions CDR1, CDR2, and CDR3, and / or one or more of the heavy chain hypervariable regions CDR1, CDR2, and CDR3;

[0011] The amino acid sequence of the monoclonal antibody (YWHG-1) light chain is as shown in SEQ ID No. 1, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 1 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of the hypervariable region CDR1 of the antibody light chain is the sequence QSFVHSNGNIY shown in SEQ ID No. 2, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 2 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of the hypervariable region CDR2 of the light chain is the sequence KVS shown in SEQ ID No. 3, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 3 formed by substitution, deletion, or addition of one or more amino acids; and the amino acid sequence of the hypervariable region CDR3 of the light chain is the sequence FQGSHVPPT shown in SEQ ID No. 4, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 4 formed by substitution, deletion, or addition of one or more amino acids.

[0012] The amino acid sequence of the heavy chain of the monoclonal antibody (YWHG-1) is as shown in SEQ ID No. 5, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 5 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of the hypervariable region CDR1 of the antibody heavy chain is the sequence GYIFTSYW shown in SEQ ID No. 6, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 6 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of the hypervariable region CDR2 of the heavy chain is the sequence IYPSDSYT shown in SEQ ID No. 7, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 7 formed by substitution, deletion, or addition of one or more amino acids; and the amino acid sequence of the hypervariable region CDR3 of the heavy chain is the sequence TRFGYPFDY shown in SEQ ID No. 8, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 8 formed by substitution, deletion, or addition of one or more amino acids.

[0013] Furthermore, the monoclonal antibody (YWHG-1) further includes a light chain framework region (FR) and a heavy chain framework region; wherein, the light chain framework region includes one or more of light chains FR1, FR2, FR3, and FR4; the amino acid sequence of light chain FR1 is the sequence DIVITQDELSLTVSLGDQASISCRTS shown in SEQ ID No. 9, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 9 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of light chain FR2 is the sequence LEWFLQKPGQSPKLLIY shown in SEQ ID No. 10, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 10 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of light chain FR3 is the sequence SRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYC shown in SEQ ID No. 11, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 11 formed by substitution, deletion, or addition of one or more amino acids. The sequence shown has an equivalent amino acid sequence; the amino acid sequence of the light chain FR4 is the sequence FGGGTKLEIK shown in SEQ ID No. 12 or an amino acid sequence with the same function as the sequence shown in SEQ ID No. 12 formed by substitution, deletion, or addition of one or more amino acids; the heavy chain frame region includes one or more of heavy chains FR1, FR2, FR3, and FR4, wherein: the amino acid sequence of the heavy chain FR1 is the sequence QLQESGTVLVRPGASVKLSCKAS shown in SEQ ID No. 13 or an amino acid sequence with the same function as the sequence shown in SEQ ID No. 13 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of the heavy chain FR2 is the sequence INWVKQRPGQGLEWIGN shown in SEQ ID No. 14 or an amino acid sequence with the same function as the sequence shown in SEQ ID No. 14 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of the heavy chain FR3 is SEQ ID... The amino acid sequence shown in SEQ ID No. 15 is NFNQKFEDKATLTVDTSSSTAYMQFSSPTSEDSAVYYC, or an amino acid sequence with the same function as the sequence shown in SEQ ID No. 15 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of the heavy chain FR4 is the sequence WGQGTTLTVSS of SEQ ID No. 16, or an amino acid sequence with the same function as the sequence shown in SEQ ID No. 16 formed by substitution, deletion, or addition of one or more amino acids;

[0014] Furthermore, the nucleotide sequence encoding the light chain in the monoclonal antibody (YWHG-1) is the sequence shown in SEQ ID No. 17, or a nucleotide sequence with the same function as the sequence shown in SEQ ID No. 17 formed by replacing, deleting, or adding one or more nucleotides to the sequence; and the nucleotide sequence encoding the heavy chain in the monoclonal antibody (YWHG-1) is the sequence shown in SEQ ID No. 18, or a nucleotide sequence with the same function as the sequence shown in SEQ ID No. 18 formed by replacing, deleting, or adding one or more nucleotides to the sequence.

[0015] According to one aspect of the present invention, a preferred monoclonal antibody against the HLA-G molecule (YWHG-1) is provided, said monoclonal antibody (YWHG-1) is produced from a hybridoma with accession number CCTCC NO: C202120, deposited at the China Center for Type Culture Collection on August 17, 2021. The address of the China Center for Type Culture Collection is Wuhan University, Wuhan, Hubei Province, China, 430072, China.

[0016] The present invention also provides the use of the anti-HLA-G molecular antibody (YWHG-1) for HLA-G molecular immunohistochemistry, immunoblotting and flow cytometry detection, which has the characteristics of high specificity and strong affinity.

[0017] To provide a clearer understanding of the inventive concept and technical solutions of this application, the following will further explain this application through specific embodiments and accompanying drawings. The technical solutions described in the embodiments are merely preferred embodiments and should not be construed as limiting the scope of this application. For those skilled in the art, several improvements and adjustments can be made without departing from the technical principles of this application, and these improvements and adjustments should also be considered to fall within the protection scope of this application.

[0018] Unless otherwise defined, all technical terms used herein shall be deemed to have the same meaning as understood by one of ordinary skill in the art. The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to the 20 commonly used amino acids.

[0019] The hypervariable region of light chain or hypervariable region of heavy chain mentioned in this invention is also called the complementarity determining region (CDR).

[0020] The term "sequence" as used in this invention can refer to amino acid sequences containing certain biologically equivalent amino acids or "conserved substitutions," while "other sequences" can refer to amino acids with non-equivalent functions or "non-conserved substitutions," which are genetically engineered to improve the properties of CDR or CDR-containing antibodies. Without substantially affecting antibody activity, those skilled in the art can manipulate the sequences in this application, i.e., replace, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain variants of the antibody or its functional fragment sequence. These should be considered to be included within the scope of protection of this application. For example, amino acids with similar properties can be substituted in the variable region. The sequences of the variants mentioned in this invention can have at least 95%, 96%, 97%, 98%, or 99% sequence identity with their source sequences. Sequence identity can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. Various amino acid sequences described herein are detailed in the sequence listing. Attached Figure Description

[0021] Figure 1 These are schematic diagrams of the molecular structures of seven different HLA-G isomers and the locations of immune antigen peptides.

[0022] Figure 2 This involves the identification of the heavy and light chain subclasses of the antibody (YWHG-1).

[0023] Figure 3 The antibody (YWHG-1) was tested for purity using SDS-PAGE.

[0024] Figure 4 The antibody affinity constant of the (YWHG-1) antibody was determined by ELISA.

[0025] Figure 5 The antibody (YWHG-1) is used for immunoblotting to detect HLA-G1~HLA-G7 molecules.

[0026] Figure 6 The antibody (YWHG-1) was used to detect intracellular HLA-G1~HLA-G7 molecules by flow cytometry.

[0027] Figure 7 The antibody (YWHG-1) was used to detect HLA-G molecule expression in gastric cancer tumor tissue by immunoblotting.

[0028] Figure 8 The antibody (YWHG-1) was used for immunohistochemical detection of HLA-G molecule expression in gastric cancer tissue. Detailed Implementation

[0029] 1. Preparation of anti-HLA-G monoclonal antibody (YWHG-1)

[0030] ① Antigenic peptide synthesis

[0031] An antigenic peptide located in the α1 domain of the HLA-G molecule heavy chain, amino acid sequence 72-91, was synthesized, SEQ No. 19QTDRMNLQTLRGYYNQSEAS.

[0032] ② Mouse immunization

[0033] Four SPF-grade female BALB / c mice were initially immunized with 60 μg / mouse. A first booster immunization was performed with 30 μg / mouse. A second booster immunization was performed with 30 μg / mouse. A third booster immunization was performed with 30 μg / mouse. Blood was collected from the orbital sinus for serum titer determination. The immunized mice were coated with “SEQ No. 19 QTDRMNLQTLRGYYNQSEAS” and ELISA was used to determine the titer. The mice were incubated overnight at 4°C with “SEQ No. 19 QTDRMNLQTLRGYYNQSEAS” at 2 μg / ml; then blocked at 37°C for 2 hours with 2% milk. Serum was serially diluted 2-fold starting from 200-fold. The blank control was PBS, and the negative control was a 200-fold dilution of negative serum. Before fusion, mice were immunized with a shock immunization using 50 μg of the “SEQ No. 19 QTDRMNLQTLRGYYNQSEAS” immunogen. In the fusion experiment, mouse spleen cells and SP2 / 0 cells were fused using the PEG method. After fusion, the cells were screened and cultured in a semi-solid medium (containing HAT).

[0034] ③ Monoclonal cell screening

[0035] Select 10 plates × 93 cell clones and culture them in 96-well cell culture plates (pre-coated with thymocytes, 100 μL / well). Coat the plates with "SEQ No. 19 QTDRMNLQTLRGYYNQSEAS", dilute "SEQ No. 19 QTDRMNLQTLRGYYNQSEAS" with coating buffer to a final concentration of 2 μg / ml, 100 μL / well, incubate overnight at 4°C; then wash 3 times with washing buffer. Block with 2% milk blocking buffer, 200 μL / well, incubate at 37°C for 2 h; then wash 3 times with washing buffer. Add primary antibody (cell culture supernatant), negative control (SP2 / 0 culture supernatant), blank control (PBS), and positive control (positive serum PBS diluted 1000 times), 100 μL / well each, incubate at 37°C for 1 h; then wash 3 times with washing buffer. Secondary antibody diluted 20,000 times with PBS, 100 μL / well, incubated at 37°C for 1 h; washed 3 times with washing buffer after incubation. For color development, 100 μL / well of development solution, development time approximately 5 min. Stop the reaction by adding 50 μL of stop solution to each well. Measure absorbance at dual wavelengths (450, 630). Selected clones were screened using ELISA for the first time to obtain positive hybridoma cell lines. The positive cell lines were then coated again with “SEQ No. 19 QTDRMNLQTLRGYYNQSEAS” and screened again using ELISA for the second time to obtain positive hybridoma cell lines. After multiple screenings, a monoclonal antibody against the fragment SEQ No. 19 QTDRMNLQTLRGYYNQSEAS was obtained and named YWHG-1 (accession number CCTCC NO: C202120).

[0036] ④ Identification of subclasses of anti-HLA-G monoclonal antibody (YWHG-1)

[0037] Ten positive cell lines were selected for subclass identification. The coating antibody was diluted to 0.5 μg / ml with 100 mM PBS (pH 7.4), and 0.1 ml was added to each well. The cells were incubated overnight at 4°C. After washing twice with PBS-T, 200 μl of blocking buffer was added to each well, and the cells were incubated at 37°C for 2 h. After washing three times with PBS-T, 100 μl of hybridoma supernatant was added to each well, and the cells were incubated at 37°C for 1 h. After washing three times with PBS-T, 0.1 ml of HRP-labeled antibody diluted 1:10000 (κ,λ) or 1:20000 (other) blocking buffer was added to each well, and the cells were incubated at 37°C for 1 h. After washing three times with PBS-T, 50 μl of substrate solution was added to each well, and the absorbance was measured at dual wavelengths (450, 630) within 10–20 min. The antibody subtype was again confirmed using Thermo's Pierce Rapid Isotyping Kits – Mouse, Catalog #26178, to be the monoclonal antibody subtype produced by this cell line as IgG1 for the heavy chain and kappa(κ) for the light chain. Figure 2 ).

[0038] ⑤ Antibody purification

[0039] Sample pretreatment: Dilute 1:3 with the appropriate conjugation buffer, centrifuge at 12000 rpm for 10 min at 4℃, and filter through a 0.22 μm filter to remove fat, cell debris, and small particulate matter. Equilibration: Equilibrate the column with 10 column volumes of the appropriate conjugation buffer at a flow rate of 1 ml / min. Sample loading: Inject the sample into the top inlet of the column and collect the eluent at a flow rate of 1 ml / min. Washing: Pass the sample through the column with 5 column volumes of conjugation buffer at a flow rate of 1 ml / min. Elution: Elute the antibody with 5 column volumes of elution buffer and collect it in the EP tube at a flow rate of 1 ml / min. Immediately adjust the pH to 7.0 with 1 M pH 9.0 Tris-HCl buffer. Equilibration: Equilibrate the column back to pH 7.0 with 10 column volumes of conjugation buffer at a flow rate of 1 ml / min. Dialysis: Dialyze the antibody overnight using 0.01 M PBS buffer, changing the buffer 3 times.

[0040] ⑥ SDS-PAGE assay for antibody purity

[0041] Prepare SDS-PAGE gels, with a separating gel concentration of 12%. Sample preparation: After adding sample buffer, boil the sample in water for 10 min. Sample loading: 10 μL per well. Gel running: Stacking gel 80 V, 30 min; separating gel 120 V, 60 min. Stop 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 gently shake on a shaker for at least 30 min (staining time needs to be adjusted according to gel thickness). Remove the gel and rinse several times in water, then add Coomassie Brilliant Blue destaining solution and shake. Destain the gel until the bands are roughly visible (1 hour); for complete destaining, change the destaining solution 2-3 times and shake for at least 24 hours. After destaining, the gel can be scanned and recorded using an ECL gel imaging system. The purified antibody YWHG-1 has a purity >90% ( Figure 3 ).

[0042] ⑦ Determination of the affinity constant of antibodies for ELISA detection

[0043] Dilute the antigen (SEQ No. 19 QTDRMNLQTLRGYYNQSEAS) with coating buffer to a final concentration of 2 μg / ml, 100 μl / well, incubate overnight at 4°C; then wash twice with washing buffer. Block with blocking buffer, 200 μl / well, incubate at 37°C for 2 h; then wash once with washing buffer. Serially dilute the purified antibody 2-fold (PBS) starting at 200-fold, with a blank control in PBS, 100 μl / well for both, incubate at 37°C for 1 h; then wash three times with washing buffer. Add secondary antibody diluted 20000-fold in PBS, 100 μl / well, incubate at 37°C for 1 h; then wash three times with washing buffer. Develop colorimetric solution, 100 μl / well, for 5-15 min. Add 50 μl of stop solution to each well to stop the reaction. Measure absorbance at dual wavelengths (450, 630) and plot for analysis. According to the Logistic regression equation, the affinity constant ≈ 150000 × A / antibody concentration (A is the antibody dilution factor corresponding to 1 / 2 OD value; dilution factor = 25600; YWHG-1 antibody concentration = 1.9 mg / mL), and the affinity constant of antibody YWHG-1 is 2.02 × 10⁹ L / mol. Figure 4 ).

[0044] ⑧ Immunoblotting to detect the HLA-G isoform recognition specificity of antibodies.

[0045] HLA-G isoform standard proteins HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7, and HLA-G molecules lacking the α1 domain were electroporated onto membranes, blocked with 5% skim milk powder at room temperature for 4 h, and washed with 0.2% TBS (Teween-20 PBS). The antibody (YWHG-1, 1.0 ug / ml) was added, and its recognition specificity was detected. The membrane was incubated overnight at 4°C, and then washed. HRP-labeled rabbit anti-mouse IgG antibody was added, and the membrane was incubated at room temperature for 30 min. After washing, the membrane was incubated for 1–3 min using a Dako REAL™ EnVision™ detection system (DAKO). The results showed that the antibody (YWHG-1) specifically recognized the standard protein and did not cross-react with other HLA-G isoforms, demonstrating good specificity for HLA-G isoforms containing the α1 domain (HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7). Figure 5 ).

[0046] II. Application Examples

[0047] Example 2.1. Flow cytometry detection of intracellular HLA-G1~HLA-G7 molecules using the antibody (YWHG-1).

[0048] Cultures of K562-expressing cells in the logarithmic growth phase (blank control), K562-HLA-G1, K562-HLA-G2, K562-HLA-G3, K562-HLA-G4, K562-HLA-G5, K562-HLA-G6, and K562-HLA-G7 were collected. Intracellular flow cytometry analysis was performed.

[0049] Intracellular molecular expression detection by flow cytometry: K562, K562-HLA-G1, K562-HLA-G2, K562-HLA-G3, K562-HLA-G4, K562-HLA-G5, K562-HLA-G6, and K562-HLA-G7 cell cultures were collected in flow cytometry tubes, washed twice by centrifugation with 2% BSA / PBS (250g), and 250 μL of cell permeabilizing agent (BD Cytofix / Cytoperm™) was added. The mixture was thoroughly mixed and incubated at 4°C for 20 minutes. Then, 1 mL of BD Perm / Wash™ was added, and the cells were washed twice by centrifugation (250g). After washing, K562, K562-HLA-G1, K562-HLA-G2, K562-HLA-G3, K562-HLA-G4, K562-HLA-G5, K562-HLA-G6, and K562-HLA-G7 cells were resuspended in 100 μL of BD Perm / Wash™. A FITC-labeled purified antibody (YWHG-1) containing 1 μL (1.0 mg / mL) was added, and the cells were incubated at 4°C for 30 minutes. The cells were then washed three times (250 g) by centrifugation with 1 mL of 1× BD Perm / Wash™. The cells were then resuspended in 1× BD Perm / Wash™ to a 300 μL cell suspension and analyzed by flow cytometry. The results showed that the antibody (YWHG-1) could specifically recognize intracellularly expressed HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, and HLA-G7 molecules. Figure 6 ).

[0050] Example 2.2. Detection of HLA-G molecule expression in gastric cancer tumor tissue by immunoblotting of the antibody (YWHG-1).

[0051] Preparation of single-cell suspensions from 62 frozen gastric cancer tumor tissues: Mechanical separation was used first, followed by enzymatic digestion. Samples were removed, equilibrated to room temperature, washed three times with HBSS buffer, and then cut into 5-6 mm tissue fragments. After washing the tissue fragments three times with HBSS buffer, they were digested with type IV collagenase (1 mg / ml) and hyaluronidase (10 ng / ml) at 37°C in 2% fetal bovine serum (FBS) HBSS buffer for 4 hours. Following digestion, the cells were filtered through a 70 µm cell filter to obtain a cell suspension. The cell suspension was centrifuged and lysed with tissue cell lysis buffer. After lysis at 4°C for 2 hours, the cells were centrifuged at 12,000 rpm for 30 minutes, and the lysate was collected for later use.

[0052] The supernatant from the lysate of 62 gastric cancer tumor tissues was subjected to denaturing PAGE electrophoresis. After semi-dry electroporation, the membrane was blocked with 2% albumin / PBS at room temperature for 4 h, and washed with 0.2% TBS (Teween-20 PBS). The antibody (YWHG-1) was added to detect HLA-G molecule expression. The membrane was incubated overnight at 4°C, washed, and then incubated with HRP-labeled rabbit anti-mouse IgG antibody at room temperature for 30 min. After washing, the membrane was incubated for 1-3 min using a Dako REAL™ EnVision™ detection system (DAKO). The results showed that the antibody (YWHG-1) could specifically detect the expression of HLA-G molecules in colorectal cancer tumor tissues. Figure 7 ).

[0053] Example 2.3. The antibody (YWHG-1) was used for immunohistochemical detection of HLA-G molecule expression in gastric cancer tissue.

[0054] Gastric cancer tissue was fixed in 10%-12% neutral formalin and embedded in paraffin. Tissue sections underwent standard slide preparation procedures including baking, dewaxing, hydration, and antigen retrieval. A suitable amount of 1% BSA was added to the tissue, covering the tissue and its edges by 2 mm, and incubated at room temperature for 10 min for blocking. The antibody (YWHG-1) (1 mg / mL, 1:500 dilution) was added, and the slide was incubated overnight (16-20 h) in a humidified chamber at 4°C. After washing with TBS buffer, secondary antibody (TBS diluted antibody at a goat anti-mouse ratio of 1:300) was added, and the slide was incubated at 37°C for 30 min. After washing with TBS buffer again, DAB working solution was added as the chromogenic agent. Once the tissue was fully stained, the slide was rinsed in running water for 5 min and then soaked in distilled water for 5 min. After HE counterstaining, dehydration, clearing, and mounting, the tissue sections were observed in each field of view under an optical microscope. Brownish-brown staining indicates positive HLA-G expression in gastric cancer tissue cells. The intensity of HLA-G expression is determined by the depth of the brownish-brown staining. Figure 8 HLA-G molecules were highly expressed in gastric cancer tissue samples (A), (B), (C), and (D). Figure 8 ). sequence list <110> Taizhou Enze Medical Center (Group) <120> A monoclonal antibody against HLA-G molecules and its uses <160> 19 <170> SIPOSequenceListing 1.0 <210> 1 <211> 112 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 1 天冬氨酸-异亮氨酸-缬氨酸-异亮氨酸-苏氨酸-谷氨酰胺-天冬氨酸-谷氨酸-亮氨酸-丝氨酸-亮氨酸-苏氨酸-缬氨酸-丝氨酸-亮氨酸-甘氨酸 1 5 10 15 天冬氨酸-谷氨酰胺-丙氨酸-丝氨酸-异亮氨酸-丝氨酸-半胱氨酸-精氨酸-苏氨酸-丝氨酸-谷氨酰胺-丝氨酸-苯丙氨酸-缬氨酸-组氨酸-丝氨酸 20 25 30 天冬酰胺-甘氨酸-天冬酰胺-异亮氨酸-酪氨酸-亮氨酸-谷氨酸-色氨酸-苯丙氨酸-亮氨酸-谷氨酰胺-赖氨酸-脯氨酸-甘氨酸-谷氨酰胺-丝氨酸 35 40 45 脯氨酸-赖氨酸-亮氨酸-亮氨酸-异亮氨酸-酪氨酸-赖氨酸-缬氨酸-丝氨酸-丝氨酸-精氨酸-苯丙氨酸-丝氨酸-甘氨酸-缬氨酸-脯氨酸 50 55 60 天冬氨酸-精氨酸-苯丙氨酸-丝氨酸-甘氨酸-丝氨酸-甘氨酸-丝氨酸-甘氨酸-苏氨酸-天冬氨酸-苯丙氨酸-苏氨酸-亮氨酸-赖氨酸-异亮氨酸 65 70 75 80 丝氨酸-精氨酸-缬氨酸-谷氨酸-丙氨酸-谷氨酸-天冬氨酸-亮氨酸-甘氨酸-异亮氨酸-酪氨酸-酪氨酸-半胱氨酸-苯丙氨酸-谷氨酰胺-甘氨酸 85 90 95 丝氨酸-组氨酸-缬氨酸-脯氨酸-脯氨酸-苏氨酸-苯丙氨酸-甘氨酸-甘氨酸-甘氨酸-苏氨酸-赖氨酸-亮氨酸-谷氨酸-异亮氨酸-赖氨酸 100 105 110 <210> 2 <211> 11 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 2 谷氨酰胺-丝氨酸-苯丙氨酸-缬氨酸-组氨酸-丝氨酸-天冬酰胺-甘氨酸-天冬酰胺-异亮氨酸-酪氨酸 1 5 10 <211> 3 <212> PRT ​<213> Hybridoma (YWHG-1 Hybridoma) <400> 3 Lys Val Ser 1 <210> 4 <211> 9 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 4 Phe Gln Gly Ser His Val Pro Pro Thr 1 5 <210> 5 <211> 114 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 5 Gln Leu Gln Glu Ser Gly Thr Val Leu Val Arg Pro Gly Ala Ser Val 1 5 10 15 Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr Trp Ile 20 25 30 Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Asn 35 40 45 Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Phe Asn Gln Lys Phe Glu Asp 50 55 60 Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln 65 70 75 80 Phe Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg 85 90 95 Phe Gly Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser <210> 6 <211> 8 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 6 Gly Tyr Ile Phe Thr Ser Tyr Trp 1 5 <210> 7 <211> 8 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 7 Ile Tyr Pro Ser Asp Ser Tyr Thr 1 5 <210> 8 <211> 9 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 8 Thr Arg Phe Gly Tyr Pro Phe Asp Tyr 1 5 <210> 9 <211> 26 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 9 Asp Ile Val Ile Thr Gln Asp Glu Leu Ser Leu Thr Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Thr Ser 20 25 <210> 10 <211> 17 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 10 Leu Glu Trp Phe Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 11 <211> 36 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 11 Ser Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly 20 25 30 Ile Tyr Tyr Cys 35 <210> 12 <211> 10 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 12 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 13 <211> twenty three <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 13 Gln Leu Gln Glu Ser Gly Thr Val Leu Val Arg Pro Gly Ala Ser Val 1 5 10 15 Lys Leu Ser Cys Lys Ala Ser 20 <210> 14 <211> 17 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 14 Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Asn <210> 15 <211> 38 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 15 Asn Phe Asn Gln Lys Phe Glu Asp Lys Ala Thr Leu Thr Val Asp Thr 1 5 10 15 Ser Ser Ser Thr Ala Tyr Met Gln Phe Ser Ser Pro Thr Ser Glu Asp 20 25 30 Ser Ala Val Tyr Tyr Cys 35 <210> 16 <211> 11 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 16 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1 5 10 <210> 17 <211> 334 <212> DNA <213> Hybridoma (YWHG-1 Hybridoma) <400> 17 gatatcgtga taacccaaga tgaactctcc ctgactgtca gtcttggaga tcaggcctcc 60 atctcttgca gaactagtca gagttttgta catagtaatg gaaacatcta tttagaatgg 120 ttcctgcgaa accaggccag tctccaaaac tcctgatcta caaagtttcc agccgatttt 180 ctggggtccc agacaggttc agtggcagtg gatcagggac agatttcaca ctcaagatca 240 gcagagtgga ggctaggatc tgggaattta ttactgcttt caaggttcac atgttcctcc 300 gacgttcggt ggaggcacca agctggaaat caaa 334 <210> 18 <211> 340 <212> DNA <213> Hybridoma (YWHG-1 Hybridoma) <400> 18 cagctgcagg agtctgggac tgttctggtg aggcctgggg cttcagtgaa gctgtcctgc 60 aaggcttctg gctacatttt caccagctac tggataaact gggtgaagca gaggcctgga 120 caaggcctga gtggatcgga aatatttatc cttctgatag ttatactaac ttcaatcaaa 180 agttcgagga caaggccaca ttgactgtag acacatcctc cagcacagcc tacatgcagt 240 tcagcagccc gacactgagg actctgcggt ctattattgt acaagatttg gttacccctt 300 tgactactgg ggccaaggca caactctcac agtctcctca 340 <210> 19 <211> 20 <212> PRT <213> Hybridoma (YWHG-1 Hybridoma) <400> 19 Gln Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr Asn Gln 1 5 10 15 Ser Glu Ala Ser 20

Claims

1. A monoclonal antibody YWHG-1 against HLA-G molecule, wherein the monoclonal antibody YWHG-1 is produced by a hybridoma with a preservation number of CCTCC NO: C202120.

2. Anti-HLA-G molecule monoclonal antibody YWHG-1, characterized in that, The monoclonal antibody YWHG-1 comprises light chain hypervariable regions CDR1, CDR2 and CDR3, and heavy chain hypervariable regions CDR1, CDR2 and CDR3. The amino acid sequence of the antibody light chain variable region of the monoclonal antibody YWHG-1 is shown in SEQ ID No. 1; the amino acid sequence of the antibody light chain hypervariable region CDR1 is QSFVHSNGNIY shown in SEQ ID No. 2; the amino acid sequence of the light chain hypervariable region CDR2 is KVS shown in SEQ ID No. 3, and the amino acid sequence of the light chain hypervariable region CDR3 is FQGSHVPPT shown in SEQ ID No.

4. The amino acid sequence of the antibody heavy chain variable region of the monoclonal antibody YWHG-1 is shown in SEQ ID No. 5; the amino acid sequence of the antibody heavy chain hypervariable region CDR1 is GYIFTSYW shown in SEQ ID No. 6; the amino acid sequence of the heavy chain hypervariable region CDR2 is IYPSDSYT shown in SEQ ID No. 7, and the amino acid sequence of the heavy chain hypervariable region CDR3 is TRFGYPFDY shown in SEQ ID No. 8.

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