Humanized monoclonal antibody h3D8 for inhibiting HTNV infection and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a humanized monoclonal antibody h3D8 for inhibiting HTNV infection and application of the humanized monoclonal antibody h3D8. The humanized monoclonal antibody h3D8 comprises a heavy chain and a light chain; the amino acid sequence of the heavy chain is as shown in SEQ ID NO. 1; the amino acid sequence of the light chain is as shown in SEQ ID NO. 2. The invention provides a humanized anti-HTNV virus infection antibody h3D8. The antibody is combined with a virus to block the combination of the virus and a cell receptor so as to avoid infection of a host. The h3D8 antibody provided by the invention is a safe and effective anti-HTNV humanized antibody, has huge application potential, is expected to enter a clinical test to evaluate the antibody drug, and has huge application transformation potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a humanized monoclonal antibody h3D8 for inhibiting HTNV infection and its uses. Background Art

[0002] Hemorrhagic fever with renal syndrome (HFRS) caused by Hantaan virus (HTNV) infection poses a hazard to public health, and clinically features high fever, bleeding, and hypotension. So far, there is no specific therapeutic drug for treating HFRS.

[0003] Monoclonal antibody drugs have characteristics such as strong targeting and low toxicity and side effects, and are a class of potential antiviral drugs. Currently, monoclonal antibodies developed for HFRS caused by HTNV virus infection include murine m3D8 and other antibodies screened using the hybridoma technique. Although these murine monoclonal antibodies have significant protective effects, they have a problem of strong immunogenicity when used. Summary of the Invention

[0004] To solve the problem of strong immunogenicity of murine monoclonal antibodies developed for HFRS caused by HTNV virus infection in the prior art, the present invention provides a humanized monoclonal antibody h3D8 for inhibiting HTNV infection and its uses.

[0005] The present invention provides a humanized monoclonal antibody h3D8 for inhibiting HTNV infection, and the humanized monoclonal antibody h3D8 includes a heavy chain and a light chain.

[0006] The amino acid sequence of the heavy chain is as shown in SEQ ID NO.1. Among them, the heavy chain includes a heavy chain variable region; the heavy chain variable region includes heavy chain complementarity-determining regions (CDRs).

[0007] The amino acid sequence of the light chain is as shown in SEQ ID NO.2. Among them, the light chain includes a light chain variable region; the light chain variable region includes light chain complementarity-determining regions.

[0008] The object of the present invention is to transform the antibody sequence of murine m3D8 into humanized form, improve the affinity of the antibody, and reduce immunogenicity. The humanized monoclonal antibody h3D8 provided by the present invention includes a heavy chain and a light chain. The humanized monoclonal antibody provided by the present invention has the advantages of strong affinity and low immunogenicity. By transplanting the CDR region sequence of murine m3D8 into the framework gene of the human variable region, the problems of strong immunogenicity and short half-life in the prior art are solved.

[0009] Among them, the heavy chain and light chain of the humanized antibody retain the CDR sequences of murine m3D8, reducing immunogenicity.

[0010] Preferably, the heavy chain comprises a heavy chain variable region. The heavy chain variable region comprises variable region 1 with the amino acid sequence as shown in SEQ ID NO.3, or the heavy chain variable region comprises variant variable region 1 having 1 to 3 amino acid differences from variable region 1 with the amino acid sequence as shown in SEQ ID NO.3.

[0011] The light chain comprises a light chain variable region. The light chain variable region comprises variable region 2 with the amino acid sequence as shown in SEQ ID NO.4, or the light chain variable region comprises variant variable region 2 having 1 to 3 amino acid differences from variable region 2 with the amino acid sequence as shown in SEQ ID NO.4.

[0012] Preferably, the heavy chain variable region comprises heavy chain antigen - complementary determining regions. The heavy chain antigen - complementary determining regions comprise at least one of HCDR1, HCDR2, and HCDR3, or the heavy chain antigen - complementary determining regions comprise at least one of an HCDR variant of HCDR1 having 1 to 3 amino acid differences, an HCDR variant of HCDR2 having 1 to 3 amino acid differences, and an HCDR variant of HCDR3 having 1 to 3 amino acid differences.

[0013] The amino acid sequence of HCDR1 is as shown in SEQ ID NO.5 or SEQ ID NO.6; the amino acid sequence of HCDR2 is as shown in SEQ ID NO.7 or SEQ ID NO.8; the amino acid sequence of HCDR3 is as shown in SEQ ID NO.9 or SEQ ID NO.10.

[0014] The light chain variable region comprises light chain antigen - complementary determining regions. The light chain antigen - complementary determining regions comprise at least one of LCDR1, LCDR2, and LCDR3, or the light chain antigen - complementary determining regions comprise at least one of an LCDR variant of LCDR1 having 1 to 3 amino acid differences, an LCDR variant of LCDR2 having 1 to 3 amino acid differences, and an LCDR variant of LCDR3 having 1 to 3 amino acid differences.

[0015] The amino acid sequence of LCDR1 is as shown in SEQ ID NO.11 or SEQ ID NO.12; the amino acid sequence of LCDR2 is ATS or as shown in SEQ ID NO.13; the amino acid sequence of LCDR3 is as shown in SEQ ID NO.14 or SEQ ID NO.15.

[0016] The heavy-chain antigen complementary determining regions and light-chain antigen complementary determining regions of the humanized antibody provided by the present invention retain the CDR sequences of murine m3D8, reducing the immunogenicity.

[0017] The present invention also provides the coding nucleic acid of the humanized monoclonal antibody h3D8.

[0018] Furthermore, the coding nucleic acid includes one or more nucleic acid molecules optimized by the heavy-chain or light-chain CDRs. The above heavy-chain and light-chain CDR sequences are transplanted into the framework genes of human variable regions to form a complete sequence encoding the humanized monoclonal antibody h3D8.

[0019] The present invention also provides an expression vector containing the coding nucleic acid.

[0020] Furthermore, the expression vector includes, but is not limited to, plasmids, viruses, and phages.

[0021] The present invention also provides a recombinant cell containing the coding nucleic acid or containing the expression vector.

[0022] The present invention also provides a method for preparing the humanized monoclonal antibody or its variant, and the preparation method is as follows: culturing the recombinant cell under conditions that enable the expression of the monoclonal antibody h3D8 or its variant.

[0023] Specifically, the recombinant cell includes, but is not limited to, microorganisms, plant cells, or animal cells. The vector of the present invention can be introduced into the recombinant cell by methods known to those skilled in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0024] The present invention also provides the use of the humanized monoclonal antibody h3D8, the coding nucleic acid, the expression vector, or the recombinant cell in the preparation of a drug for preventing and / or treating diseases caused by HTNV virus infection.

[0025] The diseases include, but are not limited to, hemorrhagic fever with renal syndrome.

[0026] The present invention also provides a pharmaceutical composition for preventing and / or treating diseases caused by HTNV virus infection.

[0027] The pharmaceutical composition includes the humanized monoclonal antibody h3D8, the coding nucleic acid, the expression vector, or the recombinant cell.

[0028] The diseases include, but are not limited to, hemorrhagic fever with renal syndrome.

[0029] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable adjuvant.

[0030] Preferably, the pharmaceutically acceptable adjuvant includes any one or more of a diluent, an excipient, a filler, a wetting agent, a disintegrant, a flavoring agent, and a binder.

[0031] The present invention also provides a method for preventing or treating HTNV infection, the method comprising administering to a subject in need thereof the humanized monoclonal antibody h3D8 or its variant, the encoding nucleic acid, the expression vector, the recombinant cell, and / or the pharmaceutical composition, kit, and / or device.

[0032] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a humanized monoclonal antibody h3D8 that inhibits HTNV infection. This humanized monoclonal antibody has the advantages of strong affinity and low immunogenicity. By transplanting the CDR region sequence of murine m3D8 into the framework gene of the human variable region, the problems of strong immunogenicity and short half-life in the prior art are solved.

[0033] 2. The variable region of an antibody is divided into a hypervariable region and a framework region. The hypervariable region is the binding site of the antibody and the antigen, called the complementarity determinant region (CDR). The other parts of the variable region serve as the framework region, mainly playing a role in supporting the CDR. In the process of further exploring to improve antibody humanization, it is found that the affinity of specific binding between the antigen and the antibody mainly depends on the 6 CDRs on the antibody. Therefore, if the CDR region of the human antibody is replaced with the CDR region of the murine antibody, the highest degree of humanization of the recombinant antibody can reach 97%. This can not only reduce the heterogenicity of the antibody but also maintain the affinity of the murine antibody. Such a recombinant antibody is called a humanized monoclonal antibody, also known as a CDR-grafted antibody. Compared with chimeric antibodies, since the murine part in the antibody is further reduced, the human anti-mouse antibody reaction is greatly reduced. The monoclonal antibody that inhibits HTNV infection provided by the present invention is a humanized monoclonal antibody, which can improve the affinity of the antibody and reduce the immunogenicity.

[0034] 3. The humanized monoclonal antibody h3D8 that inhibits HTNV infection provided by the present invention is a high-expression sequence, with high expression level and few impurities, easy to purify and collect, reducing the purification cost, and being beneficial to large-scale production and application.

[0035] 4. The present invention provides a humanized monoclonal antibody h3D8 that inhibits HTNV infection. This humanized monoclonal antibody h3D8 that inhibits HTNV infection binds to the virus to block the binding of the virus to the cell receptor, thereby avoiding infection of the host.

[0036] 5. The present invention provides a humanized monoclonal antibody h3D8 that is safe and effective in inhibiting HTNV infection, has great application potential, is expected to enter clinical trials to evaluate this antibody drug, and has great potential for application transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the SDS-PAGE purity identification diagram of the human-derived h3D8 antibody in the present invention.

[0038] Figure 2 It is the evaluation diagram of the neutralizing effect of human-derived h3D8 antibody and murine m3D8 antibody against HTNV at the cellular level; among them, Figure 2 Figure A in it is the evaluation diagram of the neutralizing effect of human-derived h3D8 antibody against HTNV; Figure 2 Figure B in it is the evaluation diagram of the neutralizing effect of murine m3D8 antibody against HTNV; among them, the human-derived h3D8 antibody is the humanized monoclonal antibody h3D8 that inhibits HTNV infection.

[0039] Figure 3 It is the evaluation diagram of the litter mouse protection effect of human-derived h3D8 antibody and murine m3D8 antibody at the animal level; among them, the human-derived h3D8 antibody is the humanized monoclonal antibody h3D8 that inhibits HTNV infection. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0041] The humanized monoclonal antibody h3D8 provided by the present invention includes a heavy chain and a light chain.

[0042] The amino acid sequence of the heavy chain is shown as SEQ ID NO.1.

[0043] The amino acid sequence of the light chain is shown as SEQ ID NO.2.

[0044] Furthermore, the heavy chain includes a heavy chain variable region; the light chain includes a light chain variable region.

[0045] That is, the humanized monoclonal antibody h3D8 provided by the present invention includes a heavy chain variable region and a light chain variable region.

[0046] The heavy chain variable region includes variable region 1 with an amino acid sequence shown as SEQ ID NO.3, or the heavy chain variable region includes a variable region variant 1 having 1 to 3 amino acid differences from variable region 1 with an amino acid sequence shown as SEQ ID NO.3.

[0047] The light chain variable region includes variable region two with an amino acid sequence as shown in SEQ ID NO.4, or the light chain variable region includes variable region variant two with 1 to 3 amino acid differences from variable region two with an amino acid sequence as shown in SEQ ID NO.4.

[0048] Among them, the sequence as shown in SEQ ID NO.3 is as follows: MDWTWRILFLVAAATGAHSQVQLQESGPGLVKPSETLSLTCTASGYTFTSYVIHWVRQTPGKGLEWIGYINPFNDGTKYNEKFKGRATLSSDRSKNQASLKLKSVTAADTAIYYCARGDYVAWFGYWGRGTLVTVSS.

[0049] The sequence as shown in SEQ ID NO.4 is as follows: DIVMTQSPSSLSASLGERVSLTCRASQDIGISLNWLQQEPDGTIKRLIYATSNLDSSVPTRFSGSRSGSDYSLTISSLESGDFVDYYCLQYASFPFTFGSGTKLEMK.

[0050] Furthermore, the heavy chain variable region includes heavy chain antigen - complementary determining regions; the light chain variable region includes light chain antigen - complementary determining regions.

[0051] That is, the humanized monoclonal antibody h3D8 provided by the present invention includes heavy chain antigen - complementary determining regions and light chain antigen - complementary determining regions.

[0052] The heavy chain antigen - complementary determining region includes at least one of HCDR1, HCDR2, and HCDR3, or the heavy chain antigen - complementary determining region includes at least one of an HCDR variant of HCDR1 with 1 to 3 amino acid differences, an HCDR variant of HCDR2 with 1 to 3 amino acid differences, and an HCDR variant of HCDR3 with 1 to 3 amino acid differences.

[0053] The amino acid sequence of HCDR1 is as shown in SEQ ID NO.5 (GYTFTSYV) or SEQ ID NO.6 (SYVIH); the amino acid sequence of HCDR2 is as shown in SEQ ID NO.7 (INPFNDGT) or SEQ ID NO.8 (YINPFNDGTKYNEKFKG); the amino acid sequence of HCDR3 is as shown in SEQ ID NO.9 (ARGDYVAWFGY) or SEQ ID NO.10 (GDYVAWFGY).

[0054] The light chain complementarity determining region includes at least one of LCDR1, LCDR2, and LCDR3, or the light chain complementarity determining region includes at least one of an LCDR variant of LCDR1 with 1 to 3 amino acid differences, an LCDR variant of LCDR2 with 1 to 3 amino acid differences, and an LCDR variant of LCDR3 with 1 to 3 amino acid differences.

[0055] The amino acid sequence of the LCDR1 is shown as SEQ ID NO.11 (QDIGIS) or SEQ ID NO.12 (RASQDIGISLN); the amino acid sequence of the LCDR2 is ATS or is shown as SEQ ID NO.13 (ATSNLDS); the amino acid sequence of the LCDR3 is shown as SEQ ID NO.14 (LQYASFPFT) or SEQ ID NO.15 (LQYASFPFT).

[0056] Example 1 1. Preparation of Monoclonal Antibody - Heavy Chain Sequence Clone the sequence of the h3D8 antibody into an IgG expression vector. Use an endotoxin - free plasmid large - scale extraction kit to extract the plasmid. Culture Expi - 293 cells in SMM - 293 - TII medium at 37 °C under 8% CO2. When the cell density reaches 1×10 6 cells / mL, transfect the h3D8 plasmid. The plasmid transfection concentration is 1 μg / mL. According to the ratio of the mass of the plasmid: the volume of the liposome of 1:3, use blank DMEM medium to dilute the plasmid and the liposome respectively, let them stand at room temperature for 5 min, and then add the liposome - blank DMEM medium incubation mixture to the plasmid - DMEM incubation mixture and mix and let stand at room temperature for 20 min. Then add the plasmid - liposome incubation mixture to the culture flask, mixing while adding. Place it in a shaker for culture, and the culture conditions are 37 °C, 125 rpm, 8% CO2. After transfection for 6 h, centrifuge (500 rpm, 5 min) to collect the cells and replace the fresh medium. After transfection for 5 d, collect the supernatant to obtain the cell culture solution containing the h3D8 antibody.

[0057] Among them, the h3D8 antibody is the humanized monoclonal antibody h3D8 that inhibits HTNV infection, also known as the human - derived h3D8 antibody.

[0058] The IgG expression vector is from the vector currently stored in this laboratory.

[0059] The endotoxin - free plasmid large - scale extraction kit is from Omega brand, and the product number is: D6926 - 04.

[0060] The main components of SMM-293-TII medium are glutamine, HEPES, sodium bicarbonate, amino acids, vitamins, phenol red, trace elements and PluronicÒ F-68.

[0061] Expi-293 cells were obtained from Thermo Fisher, with the product number: A14527.

[0062] The h3D8 plasmid was constructed by the Department of Microbiology and Pathogenic Biology of the Air Force Medical University.

[0063] Liposome transfection reagent was purchased from Diyi, catalog number: DY20001.

[0064] 2. Purification and detection of monoclonal antibodies (1) Affinity chromatography: After flushing the system with dd H2O, a PBS system with pH 7.4 and a pre-packed column for protein A purification were used. After transiently transfecting the h3D8 plasmid into Expi 293 cells for expression for 5 days, the supernatant was harvested, centrifuged, and filtered using a 0.22 µm filter membrane. The supernatant was purified using an AKATA puro protein purifier, and the sample injection flow rate was set to 1 mL / min. The sample was eluted with glycine at pH 2.7, and the pH value of the antibody was neutralized and adjusted with 1M Tris-Hcl (pH 9.0) to obtain the h3D8 antibody.

[0065] Among them, the h3D8 antibody is the humanized monoclonal antibody h3D8 that inhibits HTNV infection, also known as the human h3D8 antibody.

[0066] Protein A protein purification prepacked column: GenScript, catalog number: L00680.

[0067] The concentration of PBS was 0.1 M and it was purchased from Beijing Coolbo with the catalog number PM5090.

[0068] (2) Testing: The purity of the antibody was determined by SDS-PAGE with a separation gel mass ratio of 10%. Figure 1 shown.

[0069] Depend on Figure 1 It can be seen that the size of the heavy chain and light chain (indicated by red arrows) of the h3D8 antibody is consistent with expectations, and the target band is single without any impurities, and the purity is greater than 95%, indicating that high-purity h3D8 antibody was successfully purified.

[0070] The h3D8 antibody includes a heavy chain and a light chain; The amino acid sequence of the heavy chain is shown in SEQ ID NO.1: AGCFISMLGAREVCTGGTCQASSCDRESPGSGACRPSHSFTRRQGEALLRVVVVQSLMHHGA*EDVPLLPPALVHGELAVEEEGAVGVQHGRRGLVVVLRLPIALPLHGDVAGIEAFDQAGQADLVLGQLIPGWGQGVHLWFSGLPCRDRGWYSGHSQGRGWAEPASVHVALAPHGSHLWLWRWFSRWGLGGLCWRPCTCTPCHSASPGAGR*GR*PHGTCCCTAPPAALSWHYAPPRRPRTS*T*PQGLRGSRPPPRM*PQGSGRS*GCPWVLGGRGRLTVPPGVQVLRKRWRWTCQHPAGACPWMQATLGHLSRLELEGEAWAGLPGHGGHV*VLSQDLGSAERRLGVTRIWEERRCPS*GSGEFGLWGGLRSGAGSSRIWLLAGLSPAPAPLCAGPGLGAPSLCLPRAWGKSQKTLSLSMSGAGRGLRVKRQTGPALLPWTGAA*PGCVQAGALSLASSRHPPSLCWPLTNFLVHLGVAGLVIHVADVGLGAQAAGGHGHHAAEGVES*GL*DSREGVHAAGQGA*VPRHRHRFGEVVLDQAAQGRCAPRGALGGGCQGEDRWALGGGAAHGHQGAAAPVAEPGHVVAAGAVVDGGVGGGHALQLQAGLVLAAVAAQGGAALELLVVLGAVVEGVDVADPLQALAGGLAHPVDHVAGEGVAAGGAGQAQGLAGLHQAGAALLQLHLGVSSCGCCHQEEDPPGPVH。

[0071] The nucleotide sequence of the nucleic acid encoding the above heavy chain is shown in SEQ ID NO.16:

[0072] The amino acid sequence of the light chain is shown in SEQ ID NO.2: MVLQTQVFISLLLWISGAYGDIQMTQTPSSLSASVGDRVTITCRASQDIGISLNWLQQKPGEAIKRLIYATSNLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASFPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0073] The nucleotide sequence of the nucleic acid encoding the above light chain is shown in SEQ ID NO.17: ATGGTGTTGCAGACCCAGGTCTTCATAAGCTTGTTGCTCTGGATCTCTGGTGCCTACGGGGACATCCAGATGACCCAGACCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCGGCATCAGCCTGAACTGGCTGCAGCAGAAGCCCGGCGAGGCCATCAAGCGCCTGATCTACGCCACCAGCAACCTGGACAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCCGCAGCGGCAGCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGTACGCCAGCTTCCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT。

[0074] 3. Activity evaluation of monoclonal antibodies (1)Neutralization activity evaluation: Vero E6 cells were inoculated in a 96-well plate at a cell density of 1.5×10 4 cells / well. After diluting the h3D8 antibody and murine m3D8 antibody at 100 μg / mL to 11 concentrations by 3-fold dilution, they were respectively mixed with HTNV virus to obtain antibody-virus mixtures.

[0075] Place the antibody-virus mixture at 4 °C overnight. On the second day, add the antibody-virus mixture to a 96-well plate to infect Vero E6 cells. After 4 hours of infection, discard the supernatant and add 2% carboxymethyl cellulose covering solution (mass / volume). Place it in a cell culture incubator at 37 °C with 5% CO2 (volume concentration) for 5 days. Then discard the covering solution, wash it 3 times with PBS, add 4% paraformaldehyde (mass / volume), fix it at room temperature for 2 hours, and then discard the 4% paraformaldehyde (mass / volume). After washing 3 times with PBS, add 0.5% Triton-X100 (mass / volume), place it at room temperature for 12 minutes, discard the 0.5% Triton-X100 (mass / volume), and wash it 3 times with PBS. Then add 3% BSA and block it at room temperature for 1 hour, discard the 3% BSA (mass / volume), and wash it 3 times with PBS. Add the primary antibody (1A8 antibody, diluted 1:1000) and incubate it at 4 °C overnight. On the second day, recover the primary antibody, wash it 3 times with PBS, add HRPlabled Goat anti mouse (diluted 1:1000), place it at room temperature for 4 hours, wash it 3 times with PBS, add the single-component TMB chromogenic solution in the dark, place it at 37 °C for 10 minutes, wash it once with PBS and spin dry, then scan the 96-well plate with an Elisport counting plate, count the number of spots in each group, and calculate the half maximal inhibitory concentration. The results are as Figure 2 shown.

[0076] Among them, the English name of the half maximal inhibitory concentration is half maximal inhibitory concentration, IC50.

[0077] It can be Figure 2 seen that high concentrations of h3D8 antibody and murine m3D8 antibody can significantly inhibit HTNV infection, and the half maximal inhibitory concentration (IC50) is 0.07 µg / mL and 2.58 µg / mL respectively, indicating that the neutralizing activity of h3D8 antibody is superior to that of m3D8 antibody.

[0078] Among them, the murine m3D8 antibody was prepared by the Department of Microbiology and Pathogen Biology, Air Force Medical University, and its amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.18: QVQLQESGPDLVKPGASVKMSCKASGYTFTSYVIHWVQQKPGQGLEWIGYINPFNDGTKYNEKFKGKAALTSDRSSSTAYMEFSSLTSEHSAVYYCARGDYVAWFGYWGQGTTVTVSS.

[0079] Its amino acid sequence of the light chain variable region is as shown in SEQ ID NO.19: DIQLTQDQLSLSVSLGDQASISCRSRQSLVHINGNTYLHWYLQRPGQSPKLLIYRVSNRFSGVADRFSGSGSGTDFTLKISRVEAEDLGVYFCSQGTHVPLTFGAGTKLEI。

[0080] Vero E6 cells were preserved by the Department of Microbiology and Pathogen Biology, Air Force Medical University.

[0081] HTNV virus was preserved by the Department of Microbiology and Pathogen Biology, Air Force Medical University.

[0082] Overnight means the time ≥ 12 h.

[0083] Paraformaldehyde: PFA.

[0084] Triton-X100 was sourced from MP Biomedical, and the product number is: N0.194854.

[0085] BSA was purchased from Yeasen Biotech, and the product number is: 36101ES76.

[0086] HRP labled Goat anti mouse was purchased from Sangon Biotech, and the product number is: D110087-0001.

[0087] (2)Animal protection experiment: Newborn Kunming litter mice at 3 days old were intraperitoneally injected with 25 mg / kg of humanized h3D8 antibody and murine m3D8 antibody respectively. After 24 h, 3600 PFU of HTNV was inoculated intraperitoneally, and the survival days of the mice were continuously observed and counted every day.

[0088] Among them, the Kunming litter mice were sourced from the Animal Experiment Center of Air Force Medical University.

[0089] As Figure 3 is known, the animal protection rates of humanized h3D8 antibody and murine m3D8 antibody at a dose of 25 mg / kg are 100% and 70% respectively.

[0090] From the above results, it can be seen that the effect of h3D8 antibody is better than that of murine m3D8 antibody.

[0091] In summary, the monoclonal antibody (humanized monoclonal antibody h3D8 that inhibits HTNV infection) prepared in the present invention has a good effect against HTNV infection.

[0092] It should be noted that when the present invention involves numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, preferred embodiments of the present invention are described.

[0093] Although the preferred embodiments of the present invention have been described, once those skilled in the art learn the basic creative concept, additional changes and modifications can be made to these embodiments, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A humanized monoclonal antibody h3D8 that inhibits HTNV infection, characterized in that, The humanized monoclonal antibody h3D8 comprises a heavy chain and a light chain; The amino acid sequence of the heavy chain is as shown in SEQ ID NO.1; The amino acid sequence of the light chain is as shown in SEQ ID NO.

2.

2. The humanized monoclonal antibody h3D8 according to claim 1, wherein The heavy chain comprises a heavy chain variable region; the heavy chain variable region comprises variable region 1 with an amino acid sequence as shown in SEQ ID NO.3, or the heavy chain variable region comprises variant variable region 1 having 1 to 3 amino acid differences from variable region 1; The light chain comprises a light chain variable region; the light chain variable region comprises variable region 2 with an amino acid sequence as shown in SEQ ID NO.4, or the light chain variable region comprises variant variable region 2 having 1 to 3 amino acid differences from variable region 2.

3. The humanized monoclonal anti-antibody h3D8 according to claim 2, characterized in that, The heavy chain variable region comprises a heavy chain antigen - complementary determining region; the heavy chain antigen - complementary determining region comprises at least one of HCDR1, HCDR2 and HCDR3, or the heavy chain antigen - complementary determining region comprises at least one of an HCDR variant of HCDR1 having 1 to 3 amino acid differences, an HCDR variant of HCDR2 having 1 to 3 amino acid differences and an HCDR variant of HCDR3 having 1 to 3 amino acid differences; The amino acid sequence of HCDR1 is as shown in SEQ ID NO.5 or SEQ ID NO.6; the amino acid sequence of HCDR2 is as shown in SEQ ID NO.7 or SEQ ID NO.8; the amino acid sequence of HCDR3 is as shown in SEQ ID NO.9 or SEQ ID NO.10; The light chain variable region comprises a light chain antigen - complementary determining region; the light chain antigen - complementary determining region comprises at least one of LCDR1, LCDR2 and LCDR3, or the light chain antigen - complementary determining region comprises at least one of an LCDR variant of LCDR1 having 1 to 3 amino acid differences, an LCDR variant of LCDR2 having 1 to 3 amino acid differences and an LCDR variant of LCDR3 having 1 to 3 amino acid differences; The amino acid sequence of LCDR1 is as shown in SEQ ID NO.11 or SEQ ID NO.12; the amino acid sequence of LCDR2 is ATS or as shown in SEQ ID NO.13; the amino acid sequence of LCDR3 is as shown in SEQ ID NO.14 or SEQ ID NO.

15.

4. The coding nucleic acid of the humanized monoclonal antibody h3D8 according to claim 1.

5. An expression vector containing the coding nucleic acid according to claim 4.

6. A recombinant cell containing the coding nucleic acid according to claim 4, or containing the expression vector according to claim 5.

7. Use of the humanized monoclonal antibody h3D8 according to claim 1, the coding nucleic acid according to claim 4, the expression vector according to claim 5 or the recombinant cell according to claim 6 in the preparation of a medicament for preventing and / or treating diseases caused by HTNV virus infection.

8. A pharmaceutical composition for preventing and / or treating diseases caused by HTNV virus infection, characterized in that, The pharmaceutical composition comprises the humanized monoclonal antibody h3D8 as claimed in claim 1, the coding nucleic acid as claimed in claim 4, the expression vector as claimed in claim 5 or the recombinant cell as claimed in claim 6.

9. The pharmaceutical composition according to claim 8, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

10. The pharmaceutical composition according to claim 9, wherein The pharmaceutically acceptable adjuvant comprises any one or more of a diluent, an excipient, a filler, a wetting agent, a disintegrant, a flavoring agent and a binder.

Images