An R4C7 monoclonal antibody and its uses

Abstract

This invention discloses an R4C7 monoclonal antibody and its uses. This R4C7 monoclonal antibody specifically binds to the extracellular domain of lymphocyte activation gene 3, which includes the V region of the heavy chain variable region. H CDR1-3 and V of the light chain variable region L CDR1-3, V H CDR1, V H CDR2 and V H The amino acid sequences of CDR3 are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5, respectively. L CDR1, V L CDR2 and V L The amino acid sequences of CDR3 are shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6, respectively. The R4C7 monoclonal antibody of this invention can bind to LAG3 with high affinity and promote the activation of various HIV latent cells, providing an important research foundation for the development of novel HIV latent activators targeting LAG3.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an R4C7 monoclonal antibody and its uses. Background Technology

[0002] Lymphocyte activation gene 3 (LAG3) is an immunosuppressive T-cell receptor. In cancer or chronic infection, persistent antigen stimulation leads to high expression of LAG3, thereby promoting T-cell exhaustion. Studies have found that LAG3 is highly expressed in HIV latent cell pools, and the existence of these pools has always been a challenge in AIDS treatment. Currently available latent activation agents all have varying degrees of drawbacks. Therefore, developing novel latent activation agents targeting the third-generation immune checkpoint molecule LAG3 may be one direction for achieving functional cure in HIV patients. Currently, most drugs targeting LAG3 are used in cancer treatment, while the application of LAG3 antibody drugs in HIV latent activation is still relatively limited. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention aims to provide an R4C7 monoclonal antibody and its uses. This invention is the first to discover a LAG3 antibody activator—the R4C7 monoclonal antibody—that reverses HIV latent cell activity in “Shock” therapy. This offers hope for immune checkpoint inhibitors in achieving the goal of shrinking the HIV latent cell reservoir through a “Shock and Kill” treatment strategy.

[0004] The specific technical solution of this invention is as follows:

[0005] The first aspect of this invention provides an R4C7 monoclonal antibody or an antibody fragment thereof, wherein the R4C7 monoclonal antibody is used to specifically bind to the extracellular domain of the immune checkpoint protein LAG3, and comprises a V-shaped heavy chain variable region. H CDR1-3 and V of the light chain variable region L CDR1-3, wherein V H CDR1, V H CDR2 and V H The amino acid sequences of CDR3 are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5, respectively. The V L CDR1, V L CDR2 and V L The amino acid sequences of CDR3 are shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6, respectively;

[0006] The antibody fragment is selected from Fab, single-chain antibody, single-domain antibody, and V. H and V L One or more of the Fv regions formed by non-covalent interactions.

[0007] Furthermore, the immune checkpoint protein LAG3 is a human-derived LAG3 protein.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region of the R4C7 monoclonal antibody is shown in SEQ ID NO.7.

[0009] And / or, the amino acid sequence of the light chain variable region of the R4C7 monoclonal antibody is shown in SEQ ID NO.8.

[0010] A second aspect of the present invention provides a polynucleotide sequence encoding the R4C7 monoclonal antibody or an antibody fragment thereof.

[0011] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the R4C7 monoclonal antibody is shown in SEQ ID NO. 9;

[0012] And / or, the nucleotide sequence encoding the light chain variable region of the R4C7 monoclonal antibody of claim 1 is shown in SEQ ID NO. 10.

[0013] A third aspect of the present invention provides a biological material expressing the R4C7 monoclonal antibody or an antibody fragment of the monoclonal antibody, or containing the polynucleotide sequence described above, wherein the biological material comprises at least one of an expression cassette, a vector, a recombinant microorganism, and a cell line.

[0014] The fourth aspect of the present invention provides the use of the R4C7 monoclonal antibody and / or an antibody fragment thereof in any of the following:

[0015] (1) Prepare reagents for detecting HIV latent reservoir; where HIV latent reservoir refers to cells or tissues where HIV is hidden under the pressure of immune response and antiviral treatment, and its existence is the key reason why AIDS cannot be cured.

[0016] (2) Preparation of reagents that specifically bind to LAG3 protein;

[0017] (3) Prepare drugs for activating the HIV virus reservoir;

[0018] (4) Prepare drugs for the prevention and / or treatment of immune diseases related to LAG3;

[0019] (5) To prepare drugs for the prevention and / or treatment of symptoms caused by immune diseases related to LAG3;

[0020] (6) To prepare a pharmaceutical composition for use in combination with other anti-HIV drugs to treat AIDS;

[0021] (7) Preparation of detection reagents for LAG3 protein.

[0022] A fifth aspect of the present invention provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the R4C7 monoclonal antibody and / or an antibody fragment thereof.

[0023] Furthermore, the pharmaceutical composition also includes an anti-HIV drug.

[0024] Furthermore, the pharmaceutical composition also includes other drugs that activate latent HIV-infected cells.

[0025] The beneficial effects of this invention are:

[0026] This invention provides an R4C7 monoclonal antibody and demonstrates its application in binding to the third-generation immune checkpoint antigen LAG3. Furthermore, this invention provides the application of the R4C7 monoclonal antibody in an HIV latent cell repository and demonstrates its ability to activate various HIV latent cell models. This invention screened and obtained a hybridoma cell line that efficiently and stably secretes and expresses a LAG3 monoclonal antibody, as well as the LAG3 monoclonal antibody secreted by it. The monoclonal antibody of this invention has high neutralizing activity; a concentration of 2 μM is sufficient to activate approximately 75% of HIV latent cells, representing the first reported LAG3 monoclonal antibody capable of activating HIV latent cells. The monoclonal antibody produced and expressed by the monoclonal cell line of this invention exhibits high titer and good specificity. Furthermore, the monoclonal cell line of this invention is produced using mouse ascites fluid, and the monoclonal antibody acquisition process is simple to operate, can be artificially intervened, and has relatively low cost. It is easy to express, collect, and purify, and the purity can reach more than 90%. The hybridoma cell line or LAG3 monoclonal antibody provided by this invention can specifically react with the extracellular domain of LAG3 protein, and has important application value in serum cytology detection of LAG3 protein expression and in the preparation of drugs that activate HIV latent reservoirs.

[0027] The development of the R4C7 monoclonal antibody in this invention provides experimental evidence for clinical research on drugs that reduce or eliminate the latent reservoir of HIV in patients. Specifically:

[0028] This invention first evaluated the affinity of the R4C7 monoclonal antibody for the LAG3 antigen. Furthermore, this invention selected the third-generation immune checkpoint LAG3 protein, and enzyme-linked immunosorbent assay (ELISA) showed that the R4C7 monoclonal antibody binds to the LAG3 protein via ECG. 50The concentration was 75 ng / mL. Surface plasmon resonance experiments showed that the R4C7 monoclonal antibody bound to the LAG3 protein at a concentration of K. D The value was 14.6 nM. Furthermore, Western blotting and ELISA experiments demonstrated that the R4C7 monoclonal antibody can bind to both linear and spatial epitopes of LAG3, making it a suitable antibody for detecting LAG3 protein. These experiments indicate that the R4C7 monoclonal antibody has good affinity for LAG3.

[0029] In cell viability assays, the R4C7 monoclonal antibody exhibited strong activation activity against HIV latent cells, significantly activating J-Lat-10.6, J-Lat-A2, and J-Lat-NIB latent cells. At a concentration of 2 μM, the activation rates for J-Lat-10.6, J-Lat-A2, and J-Lat-NIB latent cells were approximately 76%, 50%, and 38%, respectively. This indicates that the R4C7 antibody has a good activation effect on HIV latent cells and can be considered a candidate drug for developing HIV latent cell activation antibody therapies.

[0030] In further cell experiments, the activation effect of the R4C7 monoclonal antibody on the J-Lat-10.6 latent cell model was weakened after in vitro incubation with LAG3. Furthermore, knocking down the LAG3 gene in the J-Lat-10.6 latent cell model also reduced the activation effect of the antibody on latent cells. This indicates that the R4C7 monoclonal antibody does indeed exert its activation effect on HIV latent cells by binding to LAG3 in latent cells, and it has good binding specificity to the LAG3 protein. Attached Figure Description

[0031] Figure 1 This is an SDS-PAGE electrophoresis image of the R4C7 monoclonal antibody.

[0032] Figure 2 A graph showing the binding titer of R4C7 monoclonal antibody to LAG3 antigen for enzyme-linked immunosorbent assay (ELISA).

[0033] Figure 3 The graph shows the binding and dissociation curves of R4C7 monoclonal antibody and LAG3 protein as displayed by surface plasmon resonance experiments.

[0034] Figure 4 The graph shows the expression level of LAG3 protein in J-Lat-10.6 latent cells under different treatments, using R4C7 monoclonal antibody as the primary antibody.

[0035] Figure 5The images show the activation results of R4C7 monoclonal antibody on J-Lat-10.6, J-Lat-A2, and J-Lat-NIB latent cells. The groups represent the PBS group, the Prostratin positive control group, and the 2μM R4C7 antibody group, respectively.

[0036] Figure 6 The activation effect of R4C7 monoclonal antibody on J-Lat-10.6 cells after in vitro incubation with LAG3.

[0037] Figure 7 The activation effect of R4C7 monoclonal antibody on LAG3 knockdown in J-Lat-10.6 cells. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention.

[0039] Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0040] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0041] Example 1: Preparation of R4C7 monoclonal antibody

[0042] 1. R4C7 monoclonal antibody

[0043] Purified recombinant LAG3 protein with its extracellular domain was used as an immunogen. Eight- to 12-week-old BALB / c mice were immunized subcutaneously and intraperitoneally at multiple sites, with booster immunizations every two weeks for a total of three immunizations. Blood was collected from immunized mice, and antibody titers were screened by ELISA. Mice with the highest antibody titers were selected for fusion. Seven days before fusion, a booster immunization was performed. Spleen cells from these mice were fused with myeloma cells SP2 / 0. After multiple clonal screenings using limiting dilution, a monoclonal cell line was obtained, and the R4C7 monoclonal antibody, an IgG subtype, was identified. The R4C7 monoclonal antibody specifically binds to the LAG3 extracellular domain. The R4C7 monoclonal antibody consists of a heavy chain (H chain) and a light chain (L chain), each including a heavy chain variable region (V region). H ) and light chain variable region (V L ), heavy chain variable region (V H This includes the heavy chain complementarity determinant region (V). H CDR1, V H CD R2 and V H CDR3), light chain variable region (V LThis includes the light chain complementarity determinant region (V). L CDR1, V L CDR2 and V L CD R3). Among them, V H CDR1, V H CDR2 and V H The amino acid sequences of CDR3 are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5, respectively. L CDR1, V L CDR2 and V L The amino acid sequences of CDR3 are shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6, respectively.

[0044] The amino acid sequence of the heavy chain complementarity-determining region CDR1 of the R4C7 monoclonal antibody, SEQ ID NO.1:

[0045] GYTFTDYP

[0046] The amino acid sequence of the complementarity-determining region CDR1 of the light chain of the R4C7 monoclonal antibody, SEQ ID NO.2:

[0047] KSVSTSGYSY

[0048] The amino acid sequence of the heavy chain complementarity-determining region CDR2 of the R4C7 monoclonal antibody is SEQ ID NO.3:

[0049] INTETGEP

[0050] The amino acid sequence of the complementarity-determining region CDR2 of the light chain of the R4C7 monoclonal antibody is SEQ ID NO.4:

[0051] LVS

[0052] The amino acid sequence of the heavy chain complementarity-determining region CDR3 of the R4C7 monoclonal antibody is SEQ ID NO. 5:

[0053] TRGGLLDAMDY

[0054] The amino acid sequence of the complementarity-determining region CDR3 of the light chain of the R4C7 monoclonal antibody is SEQ ID NO. 6:

[0055] HHSRELPLT

[0056] The amino acid sequence of the variable region of the heavy chain of the R4C7 monoclonal antibody is SEQ ID NO.7:

[0057] The amino acid sequence of the light chain variable region of the monoclonal antibody is SEQ ID NO. 8:

[0058] ETTVTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNL ESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCHHSRELPLTFGGGTKLEIK

[0059] The nucleic acid sequence encoding the variable region of the heavy chain of the R4C7 monoclonal antibody is SEQ ID NO.9:

[0060] CAGATCCAGCTGCTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATAACCTTCACAGACTATCCAATACACTGGGTGAAGCAGGCTCCCGGAAAGGAGTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAACCGACA TATGCAGATAACTTCAAGGGACGGTTTGCCTTCTCTTTGGACACCTCTGCCAGCACTGCCTTTTTGCTGATCAACAACCTCGAAAATGAGGACACGGCTCCATTTTTCTGTACTAGAGGGGGGTTACTAGATGCTATGGACTACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA

[0061] The nucleic acid sequence encoding the variable region of the light chain of the R4C7 monoclonal antibody is SEQ ID NO.10:

[0062] GAAACAACTGTGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATC CAACCTGGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCATCACAGTAGGGAGCTTCCGCTCACGTTCGGAGGGGGGACAAAGCTGGAAATAAAAC

[0063] 2. Preparation and purification of R4C7 monoclonal antibody

[0064] In this embodiment, the ascites solution was prepared by intraperitoneal injection of 0.5 mL / mouse of Freund's incomplete adjuvant in 8-week-old male BALB / c mice. Seven days later, the mice were intraperitoneally injected with 5 × 10⁻⁶ PBS diluted with PBS. 5 0.5 mL of R4C7 monoclonal hybridoma cell suspension was collected. Mice were continuously observed. After about 10 days, when the mice's abdomens became significantly enlarged and they showed signs of slow movement, the mice were euthanized by cervical dislocation. The ascites was removed, centrifuged to remove insoluble matter, and the supernatant was stored at -80°C for later use.

[0065] R4C7 monoclonal antibody purification:

[0066] In this study, the purification of murine antibodies was performed using Protein G affinity chromatography. After thawing the ascites fluid, floating flocculent insoluble matter was scraped off with a pipette tip, and 5 volumes of Protein G binding buffer (25 mM NaH2PO3 pH 7.5, 150 mL NaCl) were added. Then, 5 mL of Protein G resin equilibrated with the binding buffer was added, and the mixture was incubated on ice with a shaker for 2 hours. After incubation, the mixture was transferred to a gravity chromatography column, the permeate was discarded, and at least 10 column volumes of binding buffer were added to wash away non-specifically bound packing material. Finally, 5 column volumes of 0.1 M glycine pH 3.0 elution buffer were added, and a 1 / 10 elution volume of 1 M Tris-HCl pH 8.5 neutralization buffer was added to the collection tube beforehand. Samples were taken for SDS-PAGE electrophoresis, and Coomassie brilliant blue staining was used to observe the purity of the purified antibody. Ultrafiltration tubes concentrate mAb and replace the buffer with sterile PBS, A 280 Protein concentration can be determined by this method, and the sample should be stored at -80℃ for later use.

[0067] Figure 1 This is a Coomassie brilliant blue staining image of the purified R4C7 monoclonal antibody. The molecular weights of the heavy chain (HC) and light chain (LC) of the R4C7 monoclonal antibody are 53 kDa and 27 kDa, respectively.

[0068] Example 2: Determination of R4C7 monoclonal antibody titer and evaluation of affinity with LAG3 protein

[0069] This embodiment uses enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) assay, and Western blotting to analyze the recognition of LAG3 protein by the R4C7 monoclonal antibody. The specific steps include the following:

[0070] ① Affinity titer determination with LAG3 protein (ELISA): 10 μg of LAG3 protein was dissolved in 10 mL of 0.05 M carbonate coating buffer at pH 9.6, and added to a 96-well plate at 200 μL / well. The plate was coated overnight at 4°C. The plate was washed three times with PBST, and blocked with 200 μL / well of PBS containing 5% skim milk blocking buffer at 37°C for 1 h. After washing three times with PBST, 100 μL of serially diluted R4C7 monoclonal antibody (adjusted to 2500 ng / mL with PBST, and down-diluted 10 times in a 2-fold gradient) was added to each well, and the plate was incubated at 37°C for 1 h. After washing the plate three times with PBST, add 100 μL of horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody to each well and incubate at 37°C for 1 h. After washing three times with PBST, perform TMB color development at 100 μL per well and incubate at room temperature in the dark for 15 min. Terminate the reaction with 100 μL of 2M H₂SO₄ per well and measure the absorbance at 450 nm. Calculate the EC... 50 The value is used to determine the binding affinity of the antibody to the LAG3 protein.

[0071] ② Identification of the binding dissociation constant (SPR) of LAG3 protein: LAG3 protein was spotted at a concentration of 1 mg / mL in a volume of 0.3 μL onto the activated 3D optical cross-linked chip. Three spots were applied for each protein concentration, with two concentrations typically used. After spotting, the chip was washed with PBS for 10 min. Immediately afterwards, the chip was immersed in 1M ethanolamine aqueous solution for 30 min in the dark. After blocking, the chip was washed with PBS for 10 min each time, for a total of three washes to ensure complete removal of the ethanolamine aqueous solution. The chip was then subjected to nitrogen blowing to remove excess moisture, followed by covering and fixation. The covered and fixed chip was mounted on a PlexArray HT100 instrument. After washing with PBS at a flow rate of 2 μL / s, antibody samples were placed. Five concentration gradients of antibody were prepared (250 nM, 125 nM, 62.5 nM, 31.3 nM, 15.6 nM). The program was set to: 300 s baseline, 300 s binding, 300 s dissociation. Run the configured program, and simultaneously configure the regeneration buffer (10mM Glycin-HCl pH 2.0) as required. After the program finishes running, configure PlexArray in Plexra. TM Preliminary data processing and data copying were performed on the Plexera analysis software configured on the HT instrument. The final copied data were processed on GraphPad Prism 8.0, and curves were plotted based on the correlation between time and corresponding intensity. Furthermore, the affinity constant (K0) was determined using the instrument's accompanying application, BIAEVAL. D ) calculation.

[0072] ③ Binding to the linear epitope of LAG3 protein: 3 mL of each of J-Lat 10.6, J-Lat A2, and J-Lat NIB cells (treated with or untreated with 1 μM Prostratin activator) were centrifuged at 600 g for 10 min. The supernatant was collected, and 1 mL of PBS solution was added for resuspending. The cells were centrifuged at 600 g for 5 min to thoroughly wash away the culture medium. The PBS solution was discarded after centrifugation, and the cells were pre-chilled on ice using a pipette. RIPA lysis buffer (containing 1% PMSF, 1× phosphatase inhibitor, and 1× protease inhibitor) was prepared. 50 μL of the lysis buffer was added to each tube of cells, and lysis was performed on ice. During lysis, the cells were vortexed for 10 s every 10 min, and immediately placed back on ice after each vortex. This process was repeated three times. The lysed cells were then centrifuged at 15000 g for 20 min in a low-temperature centrifuge at 4℃. After centrifugation, the extracted total cellular protein was transferred to a new EP tube. Total cellular protein extracted was quantified using the BCA protein quantification kit. For Western blotting experiments, the loading volume was fixed at 20 μg. Antibody R4C7 was used as the primary antibody against LAG3 protein at a concentration of 1 mg / mL, diluted 500-fold with TBST containing 2.5% BSA. The internal control was β-actin, and the secondary antibody was Goat anti-mouse IgG, diluted 4000-fold with TBST containing 2.5% BSA. Finally, LAG3 protein was detected using HRP substrate, and images were acquired using the Bio-Rad imaging system.

[0073] Experimental results are as follows Figure 2 , 3 As shown in Figure 4. Figure 2 To obtain the binding rate curve of R4C7 monoclonal antibody to LAG3 protein for enzyme-linked immunosorbent assay (ELISA), the EC50 of LAG3 protein was calculated through fitting. 50 It was 75 ng / mL. Figure 3 To detect the binding and dissociation curves of R4C7 monoclonal antibody to LAG3 protein using surface plasmon resonance (SPR) assays, the binding and dissociation constant K with LAG3 protein was calculated through fitting. D It is 14.6 nM. Figure 4 The application of R4C7 as a primary antibody for detecting LAG3 in Western blotting (WB) experiments was demonstrated. The WB results showed that using R4C7 monoclonal antibody as the primary antibody for LAG3 detection yielded a clear and single band. These binding affinity experiments indicated that R4C7 monoclonal antibody has a strong affinity for LAG3 protein in both spatial and linear epitopes.

[0074] Example 3: Activity identification of R4C7 monoclonal antibody in an activated HIV latent cell model

[0075] This embodiment uses flow cytometry to analyze the activation of latent HIV cells by R4C7 monoclonal antibody. J-Lat 10.6, J-Lat A2, and J-Lat NIB cells in good condition and in the logarithmic growth phase were cultured at 2 × 10⁶ cells / year. 5 Cells were seeded at a density of 1 / mL in 48-well plates. 2 μM of LAG3 antibody (R4C7) was added to each well. 1 μM Prostratin was added to the control wells, and the same volume of PBS as the 2 μM R4C7 antibody was added to the blank wells. The R4C7 antibody had been pre-diluted to PBS, passed through a membrane (0.22 μM), and quantified. After adding the antibody, the cells were gently shaken to mix, and then incubated at 37°C with 5% CO2 for 48 hours. The cells were then collected, centrifuged at 600g for 3 minutes, the supernatant was discarded, and the cells were washed once with 1 mL of PBS. Approximately 200 μL was reserved for resuspending the cells in flow cytometry tubes. The GFP-positive signal detected by flow cytometry represents the proportion of cells activated by the R4C7 monoclonal antibody in these cell types.

[0076] Experimental results are as follows Figure 5 As shown. Figure 5 The image shows the results of R4C7 monoclonal antibody activation of three HIV latent cell models: J-Lat 10.6, J-Lat A2, and J-Lat NIB. 2 μM of R4C7 monoclonal antibody activated all three HIV latent cell models.

[0077] Example 4: Evaluation of the activation effect of R4C7 monoclonal antibody on J-Lat 10.6 latent cells after incubation with LAG3.

[0078] J-Lat 10.6 cells in good condition and in logarithmic growth phase were used at a dose of 2×10⁶. 5 Cells were seeded at a density of 1 μM / mL in 48-well plates. 1 μM R4C7, 1 μM R4C7 incubated with 0.5 μM LAG3 protein for 1 h, and 1 μM R4C7 incubated with 1 μM LAG3 protein for 1 h were added to the wells. Blank wells contained an equal volume of PBS containing 1 μM LAG3 antibody. After adding the antibody, the cells were gently shaken to mix. The plates were then incubated at 37°C with 5% CO2 for 48 h. The cells were collected, centrifuged at 600g for 3 min, the supernatant was discarded, and the cells were washed once with 1 mL of PBS. Approximately 200 μL was reserved for resuspending the cells in flow cytometry tubes for flow cytometry analysis. The percentage of activated cells in each group was calculated based on the GFP-positive signal detected by flow cytometry.

[0079] Experimental results are as follows Figure 6 As shown. Figure 6The image shows the activation effect of R4C7 monoclonal antibody on J-Lat 10.6 latent cells after incubation with LAG3. Incubation with 1 μM LAG3 reduced the activation effect of the 1 μM monoclonal antibody on J-Lat 10.6 latent cells by approximately 26%.

[0080] Example 5: Evaluation of the activation effect of R4C7 monoclonal antibody on J-Lat 10.6 latent cells after LAG3 knockdown.

[0081] This embodiment uses CRISPR / Cas9 and Western blotting to construct a J-Lat 10.6 cell line with LAG3 knockdown. Flow cytometry is used to detect the activation effect of R4C7 monoclonal antibody on J-Lat 10.6 latent cells after LAG3 knockdown. The specific steps include the following:

[0082] First, five gRNA sequences targeting the LAG3 gene were designed and constructed into the lenti CRISPR v2 vector. Recombinant plasmids lentil CRISPR v2-gRNA-1, lentil CRISPR v2-gRNA-2, lentil CRISPR v2-gRNA-3, lentil CRISPR v2-gRNA-4, lentil CRISPR v2-gRNA-5, and the empty vector lentilCRISPR v2 were mixed with lentiviral packaging plasmids pVSVG and psPAX2 at a ratio of 2:1:1 and diluted with blank medium. Transfection reagent was used at three times the volume of DNA and also diluted with appropriate blank medium. Finally, lentiviruses carrying the corresponding gRNA sequences were packaged. 72 hours after transfection, the supernatant lentiviral particles were collected at 3000g for 10 minutes. J-Lat 10.6 cells with good infection status were infected with Puromycin. After multiple screenings using Puromycin, the J-Lat 10.6 latent cell lines with successful knockdown of LAG3 protein were finally screened by Western blotting.

[0083] J-Lat 10.6 and J-Lat 10.6 cells in good condition and in the logarithmic growth phase were compared. -LAG3 Cells at 2×10 5 Cells were seeded at a density of 1 / mL in 48-well plates, and 2 μM of LAG3 antibody (R4C7) was added to each well. After incubation at 37°C and 5% CO2 for 48 h, the cells were harvested, centrifuged at 600g for 3 min, the supernatant was discarded, and the cells were washed once with 1 mL of PBS. Approximately 200 μL was reserved for resuspending the cells in flow cytometry tubes. Flow cytometry analysis was performed to detect the effect of antibody R4C7 on J-Lat 10.6 and J-Lat 10.6. -LAG3Cell activation.

[0084] Experimental results are as follows Figure 7 As shown. R4C7 monoclonal antibody against J-Lat 10.6 -LAG3 The cell activation rate decreased from 80% to 38%, indicating that the R4C7 monoclonal antibody does indeed activate the J-Lat 10.6 latent cell line by binding to LAG3. This antibody could be further used to study novel HIV latent activators.

[0085] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any equivalent modifications made by those skilled in the art to the technical solutions of the present invention by reading the present invention specification are covered by the claims of the present invention.

Claims

1. An R4C7 monoclonal antibody or an antibody fragment thereof, characterized in that, The R4C7 monoclonal antibody specifically binds to the extracellular domain of the immune checkpoint protein LAG3, which includes the V region of the heavy chain variable region. H CDR1-3 and V of the light chain variable region L CDR1-3, wherein V H CDR1, V H CDR2 and V H The amino acid sequences of CDR3 are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5, respectively. The V L CDR1, V L CDR2 and V L The amino acid sequences of CDR3 are shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6, respectively; The antibody fragment is selected from Fab, single-chain antibody, single-domain antibody, and V. H and V L One or more of the Fv regions formed by non-covalent interactions.

2. The R4C7 monoclonal antibody or an antibody fragment thereof according to claim 1, characterized in that, The immune checkpoint protein LAG3 is a human-derived LAG3 protein.

3. The R4C7 monoclonal antibody or an antibody fragment thereof according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the R4C7 monoclonal antibody is shown in SEQ ID NO.7; And / or, the amino acid sequence of the light chain variable region of the R4C7 monoclonal antibody is shown in SEQ ID NO.

8.

4. A polynucleotide sequence, characterized in that, The polynucleotide sequence encodes the R4C7 monoclonal antibody or an antibody fragment of the monoclonal antibody as described in any one of claims 1-3.

5. The polynucleotide sequence according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the R4C7 monoclonal antibody of claim 1 is shown in SEQ ID NO. 9; And / or, the nucleotide sequence encoding the light chain variable region of the R4C7 monoclonal antibody of claim 1 is shown in SEQ ID NO.

10.

6. A biomaterial, characterized in that, The biological material expresses the R4C7 monoclonal antibody or an antibody fragment of the monoclonal antibody as described in any one of claims 1-3, or contains the polynucleotide sequence as described in claim 4 or 5, and the biological material includes at least one of an expression cassette, a vector, a recombinant microorganism, and a cell line.

7. Use of the R4C7 monoclonal antibody according to any one of claims 1-3 and / or an antibody fragment thereof in any of the following: (1) Preparation of reagents for detecting HIV virus latent reservoir; (2) Preparation of reagents that specifically bind to LAG3 protein; (3) Prepare drugs for activating the HIV virus reservoir; (4) Prepare drugs for the prevention and / or treatment of immune diseases related to LAG3; (5) To prepare drugs for the prevention and / or treatment of symptoms caused by immune diseases related to LAG3; (6) To prepare a pharmaceutical composition for use in combination with other anti-HIV drugs to treat AIDS; (7) Preparation of detection reagents for LAG3 protein.

8. A pharmaceutical composition, characterized in that, The active ingredient of the pharmaceutical composition includes the R4C7 monoclonal antibody as described in any one of claims 1-3 and / or an antibody fragment of the monoclonal antibody.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition also includes an anti-HIV drug.

10. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition also includes other drugs that activate latent HIV-infected cells.

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