Humanized broad-spectrum anti-HIV-1 (Human Immunodeficiency Virus-1) monoclonal antibody and application thereof

Abstract

The invention discloses a humanized broad-spectrum anti-HIV-1 (human immunodeficiency virus-1) monoclonal neutralizing antibody, which is obtained by screening through a single B cell flow sorting-antibody gene amplification pairing expression technology, has a unique CDR (CDR region), can be specifically combined with HIV-1, and can effectively neutralize various pseudoviruses, even Tier 3 pseudoviruses. The invention also relates to a preparation method and application of the neutralizing antibody. The antibody provided by the invention can be used for emergency prevention and / or treatment of HIV-1 variant strains, has the characteristics of full human source, high expression and good stability, and is suitable for industrialization. In addition, the antibody can also be used for preparing an HIV-1 virus detection reagent, finding effective neutralizing epitopes and developing HIV-1 vaccines.

Description

Technical Field

[0001] This invention relates to HIV-1 broad-spectrum neutralizing antibodies and belongs to the field of peptide technology. Background Technology

[0002] Human immunodeficiency virus (HIV) belongs to the genus Lentivirus of the family Retroviridae. It is a single-stranded RNA virus. HIV-1 has a stronger replication capacity and pathogenicity than HIV-2 and is the leading cause of the global AIDS epidemic and infection. The HIV-1 envelope glycoprotein (Env) is a key molecule for viral invasion of host cells, composed of gp120 and gp41 generated by the cleavage of the precursor gp160. Three pairs of gp120 / gp41 heterodimers trimerize to form a functional spike complex distributed on the viral surface. gp120 is mainly responsible for receptor binding and contains five conserved regions (C1–C5) and five hypervariable regions (V1–V5). Among them, the CD4 binding site (CD4bs), the V1V2 region, and the V3 region are important targets for neutralizing antibodies. The virus first binds to the host's CD4 receptor via gp120, subsequently exposing a co-receptor binding site (CCR5 or CXCR4), triggering viral entry. Following this, gp41 undergoes a conformational change, exposing a fusion peptide and mediating viral fusion with the cell membrane. Previous studies have shown that neutralizing antibodies primarily target key epitopes of gp120 or interfere with the conformational changes of gp41, thereby effectively blocking HIV-1 infection.

[0003] As HIV-1 vaccine research deepens, inducing broadly neutralizing antibodies has gradually become a core objective of next-generation vaccine development. Through germline targeting strategies and in-depth analysis of antibody development pathways, efforts are being made to explore methods for stably inducing bNAbs. Isolating antibodies from vaccine subjects offers significant advantages compared to long-term infected individuals. A uniformly designed immunogen and vaccination protocol not only effectively reduces interference from non-target antibodies but also ensures the traceability of antibody development pathways and the standardization of sample results. These characteristics provide ideal conditions for studying the generation mechanism and functional properties of bNAbs, while significantly improving research efficiency and the reproducibility of results. Analyzing the epitopes, functions, and evolutionary characteristics of screened HIV-1-specific antibodies can not only provide key candidate molecules for the development of anti-AIDS drugs but also provide reverse guidance for the design of AIDS vaccines.

[0004] The purpose of this invention is to provide a monoclonal antibody against HIV-1 with broad-spectrum neutralizing activity, and based on this, to provide the application of the said monoclonal antibody against HIV-1 in HIV-1 vaccine development and anti-HIV-1 drugs. Summary of the Invention

[0005] Based on the above-mentioned objectives, this invention first provides a humanized broad-spectrum anti-HIV-1 monoclonal antibody. The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3, and the light chain variable regions CDR1, CDR2, and CDR3 of the antibody are shown as amino acids 26-33, 51-58, and 97-108 of SEQ ID NO.1 and amino acids 23-34, 52-54, and 91-98 of SEQ ID NO.3, respectively.

[0006] In a preferred embodiment, the amino acid sequences of the variable region of the antibody heavy chain and the variable region of the light chain are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively, and the antibody having this technical solution is named antibody "DRVI-rTV-1" in this invention.

[0007] In a more preferred embodiment, the amino acid sequence of the antibody heavy chain constant region is shown in SEQ ID NO. 5, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 7.

[0008] Secondly, the present invention also provides a polynucleotide encoding the above-mentioned humanized broad-spectrum anti-HIV-1 monoclonal antibody. The polynucleotide encoding the variable region of the antibody heavy chain and the polynucleotide encoding the variable region of the antibody light chain are shown in SEQ ID NO.2 and SEQ ID NO.4, respectively. The antibody having this technical solution is named antibody "DRVI-rTV-1" in the present invention.

[0009] In a preferred embodiment, the polynucleotide sequence encoding the antibody heavy chain constant region is shown in SEQ ID NO.6, and the polynucleotide sequence of the light chain constant region is shown in SEQ ID NO.8.

[0010] Third, the present invention also provides a vector for expressing the above-mentioned humanized broad-spectrum neutralizing anti-HIV-1 monoclonal antibody. The vector contains polynucleotides encoding the variable region of the antibody heavy chain and the variable region of the antibody light chain. The vector can be a eukaryotic expression vector commonly used in genetic engineering. In a specific embodiment of the present invention, the vector is IgH (heavy chain expression vector), Igκ (κ light chain expression vector), or Igλ (λ light chain expression vector) (see Tiller et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning, J Immunol Methods. 2008 January 1; 329(1-2): 112–124. This prior art document is incorporated herein by reference).

[0011] Fourth, the present invention provides a host cell expressing the above-mentioned humanized broad-spectrum neutralizing anti-HIV-1 virus monoclonal antibody, wherein the host cell contains the above-mentioned vector.

[0012] The host cell can be a eukaryotic host cell commonly used in genetic engineering. In one specific embodiment of the present invention, the host cell is a 293F cell.

[0013] Fifth, the present invention provides the application of the above-mentioned humanized broad-spectrum neutralizing active anti-HIV-1 monoclonal antibody in the preparation of HIV-1 treatment and / or prevention drugs. The antibody can be used to develop clinical therapeutic drugs, targeted drugs, HIV-1 recombinant proteins and subunit vaccines.

[0014] Finally, this invention provides the application of the above-mentioned humanized broad-spectrum neutralizing anti-HIV-1 monoclonal antibody in the preparation of HIV-1 virus detection reagents.

[0015] The humanized, broad-spectrum neutralizing monoclonal antibody against HIV-1 provided by this invention was obtained from HIV-1 vaccine subjects through single B-cell flow cytometry sorting-antibody gene amplification and pairing expression technology. It possesses a unique CDR region, specifically binds to HIV-1, and can effectively neutralize various pseudoviruses of different neutralizing grades. It exhibits significant broad-spectrum neutralizing ability against multiple different representative HIV-1 strains worldwide. Therefore, the antibody provided by this invention can be used to prepare HIV-1 prevention and / or therapeutic drugs, featuring full humanization, high expression, and good stability, making it suitable for industrialization. Furthermore, this antibody can also be used to prepare HIV-1 virus detection reagents for detecting viral antigens and for identifying effective neutralizing antigenic epitopes. Attached Figure Description

[0016] Figure 1 Schematic diagram of flow cytometry sorting of HIV-1 specific B cells; Figure 2 Figure 1 shows the binding affinity of antibody DRVI-rTV-1 to three gp140 variants. Figure 3 Graph showing the neutralizing ability of antibody DRVI-rTV-1 against pseudoviruses; Figure 4 Neutralization curve of antibody DRVI-rTV-1 against pseudovirus; Figure 5 Antibody-antigen affinity curve of antibody DRVI-rTV-1 detected using BLI technology. Detailed Implementation

[0017] 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 a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0018] Example 1: Screening and identification of humanized monoclonal antibodies against HIV-1 1.1 Prepare cell lysis buffer: 20 μL of cell lysis buffer per well, containing 0.5 μL RNase removal agent, 5 μL 5× First Strand buffer, 1.25 μL 0.1M DTT, 0.0625 μL Igepal, and 13.25 μL water. Cover with sealing film and store at 4°C until use.

[0019] 1.2 Sample preparation: (1) This invention is based on the previously established clinical trial cohort study of the self-developed replication-type VTT vector HIV vaccine (rTV). PBMCs cells from subjects in the IIa clinical trial cohort completed at the end of 2021 were selected from the subjects' follow-up points 4 weeks after the second dose of vaccination for resuscitation: After removing the cryopreserved cell tubes from liquid nitrogen, they were quickly placed in a 37°C water bath. When the cells thawed and ice cores were formed, they were removed and opened in a biosafety cabinet. R10 + Benzonase medium was slowly added (5 mL of R10 + Benzonase medium was used per cell line). The cells were centrifuged at 1500 rpm for 10 minutes, the supernatant was discarded, and the cells were resuspended in the residual liquid. 10 mL of R10 was added, mixed, and 50 μL was taken for cell counting. The cells were centrifuged at 1500 rpm for 10 minutes. Cell concentration was adjusted: The supernatant was discarded, the cells were resuspended in the residual liquid, and the concentration was adjusted using R10 medium. 2.5 × 10⁶ cells were added to 96-well U-shaped plates. 6 One per hole; (2) Prepare a 2 mM EDTA / PBS solution, hereinafter referred to as E-PBS; (3) Place the 96-well cell plate in a centrifuge and centrifuge at 2000 rpm for 3 minutes at 4°C, then discard the supernatant; (4) Add 50 μL of Live-Dead working solution to each well (prepare Live-Dead (PB) working solution: dilute with PBS at a ratio of 1:800 and mix well), mix well, and incubate on ice in the dark for 20 minutes. (5) Add 150 μL of E-PBS to each well and centrifuge at 2000 rpm for 3 minutes at 4℃; (6) Discard the supernatant, add 50 μL of extracellular antibody mixture (2 μL Anti-CD3-Pacific Blue, 2 μL Anti-CD8-Pacific Blue, 1.5 μL Anti-CD14-Pacific Blue, 1 μL Anti-CD19-BV510, 2 μL Anti-CD20-ECD, 2.5 μL Anti-CD27-APCCy7, 5 μL Anti-IgG-FITC, 2.5 μL Anti-IgM-PercpCy5.5, 0.5 μL Anti-IgD-PECy7, 5 μL GP140-probe-PE, 5 μL GP140-probe-APC, make up the difference with E-PBS if necessary), mix well, and incubate on ice for 60 minutes in the dark; (7) Add 150 μL of E-PBS to each well, centrifuge at 2000 rpm for 3 minutes at 4℃, and discard the supernatant; (8) Add 200 μL of E-PBS to each well, centrifuge at 2000 rpm for 3 minutes at 4℃, and discard the supernatant; (9) Add 200 μL of E-PBS to each well, mix well, filter the cell suspension of the same sample into the same branching tube, and store at 4°C in the dark for sorting. (10) Single-staining tube control: Set up one unstained tube control (add 1 drop of compensating microspheres, 200 μL E-PBS) and 11 individually stained tubes (add 1 drop of compensating microspheres to each tube, and add 2 μL Anti-CD3-Pacific Blue, 2 μL Anti-CD8-Pacific Blue, 1.5 μL Anti-CD14-Pacific Blue, 1 μL Anti-CD19-BV510, 2 μL Anti-CD20-ECD, 2.5 μL Anti-CD27-APCCy7, 5 μL Anti-IgG-FITC, 2.5 μL Anti-IgM-PercpCy5.5, 0.5 μL Anti-IgD-PECy7, 5 μL GP140-probe-PE, 5 μL GP140-probe-APC, and 50 μL UV working solution to each well), mix well, incubate on ice in the dark for 20 minutes, and then add 150 μL of UV working solution to each well. Centrifuge in E-PBS at 2000 rpm for 3 minutes at 4℃, discard the supernatant, and resuspend in 200 μL of E-PBS.

[0020] 1.3 Single B cell flow cytometry sorting: Select LIVE CD3 - CD8 - CD14 - CD19 + CD20 + CD27 + IgG + IgM - GP140-probe + The cells were sorted, resulting in a total of 8 cell types. First, single lymphocyte populations were identified, followed by CD3+ cells of viable cells. - CD8 - CD14 - To exclude T cells and macrophages, then circle CD19. + CD20 + B cells, then CD27 + Memory B cells, then circle IgG. + IgM - B cells were selected, and memory B cells that bound the probe GP140 were circled. One of these B cells was sorted into each well of a 96-well plate containing the following lysis buffer (Table 1). After sorting, the 96-well plates were immediately sealed with sealing film, solidified on dry ice, and then transferred to a -80°C freezer overnight. PCR was performed the next day. Results: The flow cytometry sorting results are as follows... Figure 1As shown.

[0021] Table 1. B cell lysis buffer system

[0022] 1.4 Amplification of the fully human antibody variable region gene using RT-PCR technology Prepare the RT-PCR reaction system as shown in Table 2, add 6 μL to each well, and perform the RT-PCR reaction.

[0023] Table 2. RT-PCR reaction system

[0024] RT-PCR reaction program settings: 42℃ for 10 min, 25℃ for 10 min, 50℃ for 50 min, 94℃ for 5 min, and store at 4℃.

[0025] (1) Two rounds of PCR reaction were performed, and the reaction system was as follows: First-round PCR reaction system (Table 3) Supplementary primer sequences (Table 4) Table 3. First-round PCR reaction system

[0026] Table 4. Primer sequences used for the first round of PCR amplification are as follows:

[0027]

[0028] The target fragments amplified by the primers used in the first round of PCR amplification are shown in Table 5.

[0029] Table 5. Amplified target fragments using primers for the first round of PCR amplification

[0030] The procedure for the first round of PCR reaction is shown in Table 6.

[0031] Table 6. First-round PCR procedure

[0032] After the first round of PCR reaction was completed, the second round of PCR reaction was carried out. The system for the second round of PCR reaction is shown in Table 7.

[0033] Table 7. Second round PCR reaction system

[0034] The primer sequences for the second round of PCR are shown in Table 8.

[0035] Table 8. Primer sequences for the second round of PCR

[0036] The target fragments amplified by the primers used in the second round of PCR amplification are shown in Table 9.

[0037] Table 9. Amplification target fragments of primers used in the second round of PCR amplification

[0038] The procedure for the second round of PCR reaction is shown in Table 10.

[0039] Table 10. Second Round PCR Procedure

[0040]

[0041] (2) Electrophoresis, sequencing, and family analysis The second round of PCR products were detected by electrophoresis, and the heavy and light chain products were directly sequenced. The sequencing results were analyzed using the antibody family gene database (http: / / www.imgt.org / IMGT_vquest / vquest), and antibody variable region sequences with restriction enzyme sites were designed and synthesized (heavy chain: 5'-Age I, 3'-Sal I; Kappa chain: 5'-Age I, 3'-BsiWI; Lambda chain: 5'-Age I, 3'-Xho I), resulting in a total of 3 pairs of heavy and light chain clones.

[0042] 1.5 Construction of monoclonal antibody expression vectors and plasmid transformation The synthesized gene was digested with the corresponding enzymes and recovered by gel electrophoresis. The variable region gene was ligated with the corresponding vectors IgH (heavy chain expression vector), Igκ (κ light chain expression vector), and Igλ (λ light chain expression vector) using T4 DNA ligase (see Tiller et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning, J Immunol Methods. 2008 January 1; 329(1-2): 112–124., which is incorporated herein by reference). The ligation was performed overnight at 16°C, followed by transformation. 10 μL of the ligation product was added to 50 μL of DH5α competent cells, shaken well, incubated on ice for 30 minutes, and then heat-shocked at 42°C for 45 seconds. After placing the centrifuge tubes on ice for 2 minutes, add 1 mL of non-resistant LB medium, incubate at 37°C with shaking at 200 rpm for 1 hour, centrifuge at 4000 rpm for 4 minutes, and spread the remaining bacterial solution onto resistant LB plates. Incubate upside down at 37°C for 14-16 hours. Pick single colonies and inoculate them into resistant LB liquid medium, incubate at 37°C with shaking at 200 rpm for 14-16 hours, and then extract plasmids (Omega PlasmidMidi Kit).

[0043] 1.6 Antibody Expression and Purification The concentration of 293F cells was adjusted to 1.2 × 10⁻⁶. 6 Cells / mL, cultured for 2 hours. Prepare solution A: Add 500 μg antibody heavy chain DNA and 500 μg antibody light chain DNA to 25 mL of Opti-MEM. Prepare solution B: Add 5 mL of PEI transfection reagent to 25 mL of Opti-MEM and let stand for 5 minutes. Mix solutions A and B, let stand for 20 minutes, and add dropwise to 1 L of 293F cells while shaking. Incubate at 37°C, 120 rpm with shaking for 5 days using 8% CO2.

[0044] Antibodies were purified using a Protein A affinity column (GE Health). Antibody concentrations were determined using a NanoDrop 2000 micro-volume spectrophotometer (Thermo Scientific), and the antibodies were incubated at 4°C until detection. This step resulted in the expression of three antibodies from three light / heavy chain pairings (antibody amount > 10 μg, antibody concentration > 10 μg / mL).

[0045] Example 2: Determination of antibody binding ability to HIV-1 gp140 protein Enzyme-linked immunosorbent assay (ELISA) evaluates the binding ability of antibodies to HIV-1 gp140 protein. The experimental procedure is briefly described below: (1) Dilute the antigen protein with PBS to 0.5 μg / mL, and coat each well with 100 μL of the antigen protein into a 96-well ELISA plate. Incubate at 4°C overnight (12-14 hours). (2) Wash the plate 5 times with 150 μL PBS-T solution (PBS containing 0.05% Tween-20), 3 minutes each time; (3) Add 150 μL of blocking solution (PBS, 2% BSA + 5% skim milk powder) to each well and block at 37°C for 2 hours; (4) Wash the plate three times with 150 μL PBS-T solution, for 3 minutes each time; (5) Dilute the monoclonal antibody to 10 μg / mL, and set up a negative control and a positive control with 10 μg / mL 2G12 antibody; (6) Take 100 μL of the diluted sample and add it to the ELISA plate. Incubate at 37°C for 1 hour. (7) Wash the plate 5 times with 150 μL PBS-T solution, 3 minutes each time; (8) Add 100 μL of horseradish enzyme-labeled goat anti-human antibody diluted 1:5000 with blocking buffer to each well and incubate at 37°C for 1 hour; (9) Wash the plate 5 times with 150 μL PBS-T solution, 3 minutes each time; (10) Add 100 μL of TMB substrate mixture to each well and develop color for 5 minutes at room temperature in the dark. (11) Add 50 μL of stop solution directly to each well to terminate the reaction. Read the absorbance values ​​at 450 nm and 630 nm wavelengths using a microplate reader. The OD value is the absorbance value at 630 nm wavelength minus the absorbance value at 450 nm wavelength. See the results below. Figure 2 The results showed that DRVI-rTV-1 has the ability to bind to GP140 of the AE, BC, and B isoforms, and binds to EC. 50 The concentrations (μg / mL) were 0.034, 0.028, and 2.266, respectively, with the strongest binding ability to GP140 of the BC subtype.

[0046] Example 3: Determination of the neutralizing activity of the antibody against HIV-1 pseudovirus 3.1 Packaging of pseudoviruses: The HIV-1 env protein gene was synthesized and inserted into the pcDNA3.1 expression plasmid to construct pcDNA-env.

[0047] 3×10 6 3 million 293T cells were seeded in T75 cell culture flasks and cultured at 37°C with 5% CO2 for 20-24 hours. 5 μg of plasmid pcDNA-env was mixed with 10 μg of pSG3. Δenv After viral infection, 30 μL of PEI transfection reagent was added, and the mixture was allowed to stand for 20 minutes. The reagent was then added dropwise to 293T cells while shaking continuously. The mixture was then incubated in a 5% CO2 incubator at 37°C. 48 hours after transfection, the culture supernatant was collected and filtered to obtain HIV-1 pseudoviruses, which were then frozen and stored at -80°C.

[0048] 3.2 Neutralization Experiment: Add 100 μL of serially diluted antibody buffer, VRC01 as a positive antibody control, and VACV-Ab as a negative antibody control to each well of a 96-well plate. Dilute the pseudovirus to 4 × 10⁻⁶ using DMEM complete medium. 3 TCID 50 Add 50 μL of the pseudovirus to each well in columns 3-11 at a concentration of 200 TCID⁻¹. 50 / well. Place the above 96-well plate in a cell culture incubator (37℃, 5% CO2) and incubate for 1 hour. When the incubation time is half an hour, remove the prepared TZM-bl cells, discard the culture medium, add PBS buffer to wash the cells, discard the PBS, add trypsin-EDTA digestion, and then add complete culture medium to resuspend the cells and count the cells. Dilute the cell suspension to 1×10⁻⁶. 5 Cells / mL. Incubate for 1 hour, then add 100 μL of cells to each well of a 96-well plate, making the cell density 1 × 10⁶ cells per well. 4 Gently shake the 96-well plate back and forth and side to side to disperse the cells evenly in the wells. Place the 96-well plate in a cell culture incubator and incubate at 37°C and 5% CO2 for 48 hours. Remove the 96-well plate from the cell culture incubator and use a multichannel pipette to aspirate 150 μL of supernatant from each sample well. Then add 100 μL of luciferase assay reagent and react at room temperature in the dark for 3 minutes. After the reaction, mix well by shaking on a plate shaker and read the luminescence value using a multi-functional plate reader. Calculate the neutralization inhibition rate: Based on the neutralization inhibition rate results, calculate the IC50 of the antibody. 50 .

[0049]

[0050] From three tested antibodies, we screened out one antibody, DRVI-rTV-1, which has neutralizing ability against multiple HIV-1 pseudoviruses and across multiple pseudovirus neutralization levels. The neutralizing abilities of antibody DRVI-rTV-1 against HIV-1 pseudoviruses MW965, ZM249M, X1632_S2_B10, CH181.12, CH120.6, BJMSM2249, 2019GX011, and 2019GX037_5 were 2.967 μg / mL, 25.45 μg / mL, 24.43 μg / mL, 15.74 μg / mL, 32.62 μg / mL, 33.07 μg / mL, 31.18 μg / mL, and 85.32 μg / mL, respectively. Detailed neutralization results can be found in [link to detailed results]. Figure 3-4 Among them, MW965, ZM249M, X1632_S2_B10, CH181.12, and CH120.6 represent pseudovirus neutralization levels (Tier 1A, 2, 2, 2, and 3), respectively. BJMSM2249, 2019GX011, and 2019GX037_5 are pseudoviruses packaged from the env protein of domestically prevalent HIV-1 strains. The eight pseudoviruses neutralized by antibody DRVI-rTV-1 cover five subtypes: C, G, CRF07_BC, CRF08_BC, and CRF01_AE.

[0051] Example 4: Antigen-antibody affinity kinetics determination Using the Octet RH16 system (Sartorius, Germany) and Protein A sensor, the affinity of antibody DRVI-rTV-1 and the VRC01 antibody (as a positive control) for AE-GP140, BG505-GP140 monomers, CN54-GP140 trimers, and RL42-GP140 trimers was detected by BLI technology. Antibody DRVI-rTV-1 was diluted to a concentration of 5 μg / mL with PBST (PBS containing 0.02% Tween 20 and 0.1% bovine serum albumin) and then immobilized on the Protein A sensor (Sartorius, Germany) for 300 seconds. After a 120-second washing step with PBST, the biosensor probe was immersed in wells containing serially diluted antigens (5000 nM, 2500 nM, 1250 nM, 625 nM, 312.5 nM, 156.25 nM, and 78.125 nM) and bound for 240 seconds, followed by a 300-second dissociation step. The kD value was calculated using a 1:1 binding model in data analysis software 9.0. The kD value of antibody DRVI-rTV-1 against AE-GP140, BG505-GP140 monomers, CN54-GP140 trimers, and RL42-GP140 trimers was found to be 3.37 × 10⁻⁶. -71.54×10- 7 <1.0×10 -12 1.50×10 -6 The results are shown in Table 11. The kD values ​​in Table 11 refer to the dissociation constant, R... 2 It is the coefficient of determination in statistics. Figure 5 The antibody DRVI-rTV-1 showed strong affinity for all four proteins, with the strongest affinity for the BC subtype CN54-GP140.

[0052] Table 11. Antibody-antigen affinity of antibodies DRVI-rTV-1 and VRC01

[0053] This invention screened a monoclonal antibody against HIV-1, DRVI-rTV-1. Sequencing of its heavy and light chain gene amplification products yielded the following gene coding sequences and amino acid sequences for the antibody's heavy and light chains: The polynucleotide sequence of the heavy chain variable region of antibody DRVI-rTV-1 is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in amino acids 26-33, 51-58, and 97-108 of SEQ ID NO.1. The polynucleotide sequence of the light chain variable region is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.3. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in amino acids 23-34, 52-54, and 91-98 of SEQ ID NO.3.

[0054] The polynucleotide sequence of the heavy chain constant region of the above antibody is shown in SEQ ID NO.6, and the polynucleotide sequence of the light chain constant region is shown in SEQ ID NO.8 (kappa chain). The amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.7.

Claims

1. A humanized broad-spectrum neutralizing monoclonal antibody against HIV-1, characterized in that, The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3, and the light chain variable regions CDR1, CDR2, and CDR3 of the humanized broad-spectrum neutralizing anti-HIV-1 monoclonal antibody are shown as amino acids 26-33, 51-58, and 97-108 of SEQ ID NO.1 and amino acids 23-34, 52-54, and 91-98 of SEQ ID NO.3, respectively.

2. The humanized broad-spectrum neutralizing anti-HIV-1 monoclonal antibody according to claim 1, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region of the humanized broad-spectrum neutralizing anti-HIV-1 monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively.

3. The monoclonal antibody according to claim 2, characterized in that, The amino acid sequence of the heavy chain constant region of the humanized broad-spectrum neutralizing active anti-HIV-1 monoclonal antibody is shown in SEQ ID NO.5; the amino acid sequence of the light chain constant region is shown in SEQ ID NO.

7.

4. A polynucleotide encoding the humanized broad-spectrum neutralizing anti-HIV-1 monoclonal antibody as described in claim 2 or 3, characterized in that, The sequences of the polynucleotides encoding the heavy chain variable region and the light chain variable region of the humanized broad-spectrum neutralizing anti-HIV-1 monoclonal antibody are shown in SEQ ID NO.2 and SEQ ID NO.4, respectively.

5. The polynucleotide encoding the humanized broad-spectrum neutralizing anti-HIV-1 monoclonal antibody according to claim 4, characterized in that, The polynucleotide sequences of the heavy chain constant region and the light chain constant region of the humanized broad-spectrum neutralizing anti-HIV-1 monoclonal antibody are shown in SEQ ID NO.6 and SEQ ID NO.8, respectively.

6. A carrier containing the polynucleotide of claim 4 or 5.

7. A host cell containing the vector of claim 6.

8. The host cell according to claim 7, characterized in that, The host cell was a 293F cell.

9. The use of the humanized broad-spectrum neutralizing anti-HIV-1 monoclonal antibody according to any one of claims 1-3 in the preparation of HIV-1 treatment and / or prevention drugs.

10. The use of the humanized broad-spectrum neutralizing anti-HIV-1 monoclonal antibody according to any one of claims 1-3 in the preparation of HIV-1 virus detection reagents.

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