Antibody or antigen binding fragment simultaneously aiming at Zika virus and encephalitis B virus, nucleic acid, recombinant vector and application
By designing humanized antibodies or antigen-binding fragments of specific heavy and light chain variable regions CDR sequences, the problem of lack of broad-spectrum antibodies in the prior art is solved, and efficient neutralization and diagnostic treatment of Zika virus and JE virus are achieved.
Patent Information
- Application Number
- CN202510395660.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-31
- Publication Date
- 2025-08-22
AI Technical Summary
There is a lack of broad-spectrum antibodies against Japanese encephalitis and Zika virus in the prior art. The existing drugs have problems such as high side effects, high treatment costs and low efficiency. Due to the differences in flavivirus species, there are differences in neutralization efficiency of the same antibody for different flaviviruses.
An antibody or antigen-binding fragment targeting both Zika virus and JE virus was designed, containing specific heavy and light chain variable region CDR sequences, and a humanized framework region design was used to reduce immunogenicity while retaining high specific binding capacity and complement activation function.
It realizes efficient neutralization ability to Zika virus and J-encephalitis in in vitro and animal models, reduces the immune rejection in the human body, and is suitable for the preparation of diagnostic and therapeutic products.
Smart Images
Figure CN120518752A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of immunology, and in particular to an antibody or antigen-binding fragment, nucleic acid, recombinant vector and application thereof that are simultaneously directed against Zika virus and Japanese encephalitis virus. Background Art
[0002] Japanese encephalitis virus and Zika virus are flaviviruses that have currently attracted public attention. Usually, these flaviviruses are transmitted by insects. Once humans are infected with flaviviruses, they can cause a variety of diseases, including congenital brain damage, infant gastroenteritis and chronic diarrhea in human immunodeficiency virus (HIV)-positive patients.
[0003] To date, there is no specific drug for flavivirus. Only some drugs have been identified as antiviral agents for flavivirus, including interferon and ribavirin, thiazolidine derivatives, polymerase inhibitors, helicase inhibitors, etc. Currently, the most effective drug for flavivirus is the combination of interferon and ribavirin. However, this therapy is only effective for some patients and has the disadvantages of large side effects, high treatment costs, and ineffectiveness against some types of flavivirus.
[0004] Given this, developing specific drugs for flaviviruses is crucial. Antibody drugs, a crucial component of the biopharmaceutical field, currently have over 100 approved for the treatment of various human diseases. However, there are currently no antibodies targeting Japanese encephalitis virus (JEV) or Zika virus. Some JEV-specific antibody drugs are under investigation, but due to the species diversity of flaviviruses, antibodies vary in their neutralizing activity and protective efficacy against flaviviruses. Consequently, most antibody drugs can only neutralize a single flavivirus. Therefore, there is an urgent need to develop a broad-spectrum flavivirus antibody. Summary of the Invention
[0005] The purpose of the present invention is to provide a broad-spectrum flavivirus antibody.
[0006] To achieve the above objectives, the first aspect of the present invention provides an antibody or antigen-binding fragment that is simultaneously directed against Zika virus and Japanese encephalitis virus, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the heavy chain variable region CDR1 shown in SEQ ID No. 2, the heavy chain variable region CDR2 shown in SEQ ID No. 3, and the heavy chain variable region CDR3 shown in SEQ ID No. 4; the light chain variable region comprising the light chain variable region CDR1 shown in SEQ ID No. 6, the light chain variable region CDR2 shown in SEQ ID No. 7, and the light chain variable region CDR3 shown in SEQ ID No. 8.
[0007] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1.
[0008] Preferably, the amino acid sequence of the light chain variable region is shown as SEQ ID No.5.
[0009] Preferably, the antibody or antigen-binding fragment further comprises a heavy chain framework region, and the heavy chain framework region is derived from human IgG.
[0010] Preferably, the antibody or antigen-binding fragment further comprises a light chain framework region, and the light chain framework region is derived from human IgG.
[0011] Preferably, the antibody or antigen-binding fragment further comprises a heavy chain constant region, and the heavy chain constant region is derived from human IgG.
[0012] Preferably, the antibody or antigen-binding fragment further comprises a light chain constant region, and the light chain constant region is of human κ type.
[0013] The antibodies or antigen-binding fragments provided by the present invention that are simultaneously directed against Zika virus and Japanese encephalitis virus are humanized antibodies, which can reduce the immunogenicity of the antibodies or antigen-binding fragments to the greatest extent. At the same time, the six CDR regions of the antibodies or antigen-binding fragments provided by the present invention have high specific binding ability to Japanese encephalitis virus and Zika virus. The present invention provides a clear antibody variable region sequence to ensure the stability and functional consistency of the antibody structure. The humanized design of the constant region further reduces immunogenicity while retaining the antibody's effector functions such as complement activation and FcR binding.
[0014] Preferably, the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 1; (b) a light chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 5; (c) The heavy chain variable region sequence as in (a) and the light chain variable region sequence as in (b).
[0015] Preferably, the antibodies and antigen-binding fragments include: 1) a heavy chain variable region sequence that has at least 95% sequence identity with the amino acid sequence shown in SEQ ID No. 1; 2) a light chain variable region sequence that has at least 95% sequence identity with the amino acid sequence shown in SEQ ID No. 5; 3) The heavy chain variable region sequence in 1) and the light chain variable region sequence in 2).
[0016] Preferably, the antigen-binding fragment provided by the present invention is selected from any one or more of Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody (e.g., scFv), human antibody, chimeric antibody or bispecific or multispecific antibody.
[0017] The second aspect of the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment of the first aspect.
[0018] The third aspect of the present invention provides a recombinant vector, which comprises the nucleic acid described in the second aspect.
[0019] A fourth aspect of the present invention provides a use of the antibody or antigen-binding fragment described in the first aspect, the use comprising: Application 1: Using the antibody or antigen-binding fragment of any one of claims 1 to 7 for the preparation of a drug for treating diseases caused by Japanese encephalitis virus and / or Zika virus; Application 2: Using the antibody or antigen-binding fragment described in any one of claims 1 to 7 in the preparation of diagnostic products for Japanese encephalitis virus and / or Zika virus infection.
[0020] Preferably, the drug for treating diseases caused by Japanese encephalitis virus and / or Zika virus and the diagnostic product for Japanese encephalitis virus and / or Zika virus infection include pharmaceutically acceptable carriers, diluents or excipients.
[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses an antibody or antigen-binding fragment that can be used to simultaneously target Zika virus and Japanese encephalitis virus. The antibody or antigen-binding fragment can simultaneously neutralize Zika virus and Japanese encephalitis virus. The CDR of the antibody or antigen-binding fragment is derived from mice, while other parts are of human origin. The immunogenicity of the antibody or antigen-binding fragment in the human body is low. At the same time, experiments have shown that the antibody or antigen-binding fragment exhibits extremely strong antigen-neutralizing ability both in vitro and in animal models. The antibody or antigen-binding fragment is suitable for the preparation of diagnostic and therapeutic products for Zika virus and Japanese encephalitis virus. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is a diagram of the SDS-PAGE electrophoresis results in Example 3 of the present invention; Figure 2 This is a diagram showing the results of an indirect immunofluorescence experiment in Example 4 of the present invention; Figure 3 This is a graph showing the results of the antibody neutralization experiment in Example 5 of the present invention; Figure 4 This is a graph showing the results of a bioprotection test of the 2A10-CDR monoclonal antibody in Example 6 of the present invention against mice infected with JEV and ZIKV; Figure 5This is a graph showing the effect of the 2A10-CDR monoclonal antibody in Example 7 of the present invention on the viral load in the serum of mice infected with JEV and ZIKV. DETAILED DESCRIPTION
[0023] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only intended to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter ranges described in the present invention. Reasonable variations derived therefrom are still within the scope of protection of the claims of the present invention.
[0024] It should be noted that the endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.
[0025] Terminology Notes: Antibody: An immunoglobulin molecule composed of two pairs of polypeptide chains, each pair consisting of a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as kappa (κ) and lambda (λ). Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon (μ), defining the five classes of antibodies: IgM, IgD, IgG, IgA, and IgE. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant region of an antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL region has seven components arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding site. The assignment of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest [Bethesda, Md. (1987 and 1991)] or the definitions of Chothia et al. (1989, Nature. 342:878-883). The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
[0026] Antigen binding fragment: refers to a polypeptide comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen to which the full-length antibody is bound, and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen binding portion thereof". Generally referring to, Fundamental Immunology, Ch. 7 [Paul, W., ed., 2nd edition, Raven Press, NY (1989)], which is incorporated herein by reference in its entirety for all purposes. Antigen binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabody, dAb) and such polypeptides, which comprise at least a portion of an antibody sufficient to confer polypeptide-specific antigen binding ability. In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), in which the VL and VH domains are paired to form a monovalent molecule via a linker that enables them to be produced as a single polypeptide chain [see, e.g., Bird et al., 1988, Science. 242:423-426. and Huston et al., 1988, Proc. Natl. Acad. Sci. USA. 85:5879-5883.]. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA. 90:6444-6448.). Other linkers useful in the present invention are described by Alfthan et al., 1995, Protein Eng. 8:725-731. Choi et al., 2001, Eur. J. Immunol. 31: 94-106.In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a bivalent antibody in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites [see, e.g., Holliger P. et al., 1993, Proc. Natl. Acad. Sci. USA .90:6444 6448. and Poljak RJ et al., 1994, Structure. 2:1121-1123. Antigen-binding fragments of antibodies (e.g., the above-mentioned antibody fragments) can be obtained from a given antibody (e.g., monoclonal antibody LZY3412 provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies. In the present invention, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.
[0027] Japanese encephalitis virus: refers to the Japanese encephalitis virus (JEV), the official classification name of the International Committee on Taxonomy of Viruses (ICTV). The two have the same meaning and can be used interchangeably.
[0028] Japanese encephalitis and Japanese encephalitis: refer to encephalitis caused by JEV infection; the two terms have the same meaning and can be used interchangeably.
[0029] Zika fever: refers to a fever-related disease caused by ZIKV infection. The two terms have the same meaning and can be used interchangeably.
[0030] As described in the background art, there are currently no specific drugs for diseases caused by flavivirus infections. The only antiviral agents identified as effective against flavivirus infections have the disadvantages of significant side effects, high treatment costs, and low efficacy. Although antibody drugs are considered to have the potential to become specific drugs for the treatment of flavivirus infections, due to the species differences of flaviviruses, the neutralization efficiency of the same antibody against different flaviviruses varies.
[0031] Based on this, a specific embodiment of the present invention provides an antibody or antigen-binding fragment that is simultaneously directed against Zika virus and Japanese encephalitis virus. The antibody or antigen-binding fragment provided in the specific embodiment of the present invention includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the heavy chain variable region CDR1 set forth in SEQ ID No. 2, the heavy chain variable region CDR2 set forth in SEQ ID No. 3, and the heavy chain variable region CDR3 set forth in SEQ ID No. 4; and the light chain variable region includes the light chain variable region CDR1 set forth in SEQ ID No. 6, the light chain variable region CDR2 set forth in SEQ ID No. 7, and the light chain variable region CDR3 set forth in SEQ ID No. 8.
[0032] More specifically, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1.
[0033] More specifically, the amino acid sequence of the light chain variable region is shown in SEQ ID No.5.
[0034] The relevant sequences provided by the present invention can ensure the stability of the antibody structure and the consistency of its functions.
[0035] More specifically, the antibody or antigen-binding fragment further comprises a heavy chain framework region, wherein the heavy chain framework region is derived from human IgG.
[0036] More specifically, the antibody or antigen-binding fragment further comprises a light chain framework region, wherein the light chain framework region is derived from human IgG.
[0037] More specifically, the antibody or antigen-binding fragment further comprises a heavy chain constant region derived from human IgG.
[0038] The present invention significantly reduces the immune rejection reaction of antibodies in the human body by replacing the human framework region.
[0039] More specifically, the antibody or antigen-binding fragment further comprises a light chain constant region, and the light chain constant region is of human kappa type.
[0040] Furthermore, a specific embodiment of the present invention also provides a nucleic acid, which can be used to encode at least the heavy chain variable region shown in SEQ ID No. 1 and the light chain variable region shown in SEQ ID No. 5.
[0041] Furthermore, the aforementioned nucleic acid should contain a CMV promoter, a signal peptide sequence and a restriction enzyme cleavage site.
[0042] More specifically, a specific embodiment of the present invention provides a recombinant vector, which at least comprises the aforementioned nucleic acid.
[0043] More specifically, a specific embodiment of the present invention provides a host cell transfected with the aforementioned vector for producing and expressing the aforementioned antibody or antigen-binding fragment that is simultaneously directed against Zika virus and Japanese encephalitis virus.
[0044] The technical solutions of the present invention are further described below by means of specific examples. Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventional meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters provided by the manufacturers.
[0045] Example 1 Antibody design Using pdb_00005szf (2A10) as the backbone, the CDR sequences of SEQ ID No. 2~4 and SEQ ID No. 6~8 were transplanted into the corresponding CDR positions of 2A10 using CDR-Grafting technology, thereby obtaining the heavy and light chains of antibodies targeting both Zika virus and Japanese encephalitis virus. The antibody was named 2A10-CDR.
[0046] After sequencing, the VH sequence of 2A10-CDR is shown as SEQ ID No. 1; the VL sequence of 2A10-CDR is shown as SEQ ID No. 5.
[0047] Example 2 Recombinant expression vector construction The nucleotide sequence was inferred based on the amino acid sequence of the 2A10-CDR antibody and after codon optimization, the nucleotide sequences encoding the heavy chain / light chain variable regions of the 2A10-CDR antibody were synthesized and cloned into the expression vector pCAGGS containing the nucleotide sequences encoding the heavy chain / light chain constant regions, thereby obtaining recombinant vectors encoding the heavy chain and light chain of the 2A10-CDR antibody, respectively.
[0048] The structure of the heavy chain expression construct is as follows, and the construct is named pCAGGS-2A10-HCDR: Heavy chain coding sequence (5'-3'): CMV promoter-EcoR I restriction site-signal peptide sequence gene-VH gene-CH gene-Xho I restriction site.
[0049] The structure of the construct for expressing the light chain is as follows, and the construct is named pCAGGS-2A10-LCDR: Light chain coding sequence (5'-3'): CMV promoter-EcoR I restriction site-signal peptide sequence gene-VL gene-CL gene-Xho I restriction site.
[0050] Among them, the signal peptide sequence encoded by the signal peptide sequence gene is shown as SEQ ID No.9; the VH sequence encoded by the VH gene is shown as SEQ ID No.1, and the CH sequence encoded by the CH gene is shown as SEQ ID No.10; the VL sequence encoded by the VL gene is shown as SEQ ID No.5, and the CL sequence encoded by the CL gene is shown as SEQ ID No.11.
[0051] Example 3 2A10-CDR antibody expression and purification The pCAGGS-2A10-HCDR and pCAGGS-2A10-LCDR prepared in Example 2 were co-transfected into 293T cells. The mass ratio of pCAGGS-2A10-HCDR to pCAGGS-2A10-LCDR used was 1.07:0.93. Each 10 cm 2 293T cells plated on a plate were transfected with 20 μg of recombinant plasmid (pCAGGS-2A10-HCDR and pCAGGS-2A10-LCDR) and 40 μg of polyethyleneimine (PEI). Cell supernatants were collected 48 and 96 hours after transfection and filtered through a 0.22 μm syringe filter. Antibodies were then purified using a Pierce protein A / G agarose column (Thermo Fisher), and monoclonal antibodies were concentrated using a 50 kDa molecular weight cutoff ultrafiltration tube (Millipore). SDS-PAGE electrophoresis (reducing and non-reducing conditions) was then performed and stained with Coomassie Brilliant Blue. The SDS-PAGE results are shown in Figure 2. Figure 1 As shown, Figure 1 In the figure, the "M" band is the marker band, the "-" band is the non-reducing SDS-PAGE electrophoresis band, and the "DTT" band is the reducing SDS-PAGE electrophoresis band. Figure 1 It can be seen that in the presence of dithiothreitol (DTT), SDS-PAGE electrophoresis showed a light chain band of about 25 kDa and a heavy chain band of about 50 kDa, which confirmed that 2A10-CDR was successfully expressed in 293T cells.
[0052] Example 4 Indirect immunofluorescence assay Place the sterile slide into the wells of a 24-well cell culture plate and plate Vero cells on each well (1*10 5cells / well) and cultured in a 37°C / 5% CO2 incubator for 24 h.
[0053] Vero cells were infected with JEV and ZIKV (MOI=1) for 48 h.
[0054] Discard the liquid in each well, add 0.5 ml of 4% paraformaldehyde to each well and fix the cells for 1 h at room temperature, discard the fixative, and wash the wells 4 times with PBS.
[0055] Add 0.5 ml of 0.5% Triton X-ray to each well and permeabilize the membrane at room temperature for 40 min. Discard the liquid in each well and wash the wells 4 times with PBS.
[0056] Add 0.5 ml of 5% bovine serum albumin to each well and block at room temperature for 1 h. Discard the liquid in each well and wash the wells 4 times with PBS.
[0057] Add 0.2 ml of 2A10-CDR monoclonal antibody (100 μg / ml) to each well and incubate at room temperature for 1 h.
[0058] The antibody in each well was discarded, the wells were washed 5 times with PBST, 0.2 ml of FITC-labeled mouse anti-human IgG monoclonal antibody (recognizing 2A10-CDR) was added to each well, and incubated at room temperature for 1 h.
[0059] Discard the antibody in each well, wash the wells 5 times with PBST, stain each well with DAPI for 10 min, and wash the wells 5 times with PBST.
[0060] The experimental results are as follows Figure 2 As shown by Figure 2 It can be seen that the 2A10-CDR monoclonal antibody does not recognize the control group cells (no green fluorescence on the cells), but can recognize the JEV-infected Vero cells and the ZIKV-infected Vero cells (green fluorescence appears in a large number of cells).
[0061] Example 5 2A10-CDR antibody neutralization experiment Vero cells were plated in 24-well plates (1*10 5 / well), and cultured in a 37℃ / 5% CO2 incubator for 24 h.
[0062] The 2A10-CDR mAb to be tested was diluted 3-fold in a 96-well plate (starting from 33 μg / ml), and the virus to be tested [JEV or ZIKV, 50 viral plaque-forming units (PFU) / well] was added to each well and incubated at 37°C for 1 h.
[0063] The Vero cell supernatant was discarded, and the virus / mAb mixture was added to the cells and infected at 37°C for 1.5 h.
[0064] The virus solution in each well was discarded, and 0.5 ml of 1% methylcellulose culture medium was added to each well to cover the cells and cultured for 3 days.
[0065] Add 1 ml of 4% paraformaldehyde to each well and fix the cells for 1 h at room temperature, then wash the plate.
[0066] Add 0.5 ml of 0.5% crystal violet staining solution to each well and stain at room temperature for 10 min, then wash the plate.
[0067] After natural drying, the number of virus plaques in each well was counted and the neutralization titer of diluted monoclonal antibody against each virus was calculated (half inhibitory concentration, PRNT 50 PRNT was calculated using GraphPad Prism 6 software. 50 value.
[0068] The experimental results are shown in Table 1 and Figure 3 As shown in Figure 2, 2A10-CDR mAb can inhibit JEV and ZIKV with extremely high neutralizing activity. The average PRNT of 2A10-CDR mAb against JEV was 50 The average PRNT for ZIKV neutralization was 79.4 ng / ml. 50 The value is 1584.9 ng / ml.
[0069] Table 1: Neutralization titer test results of 2A10-CDR monoclonal antibody against JEV and ZIKV (half inhibitory concentration, PRNT) 50 ) Virus <![CDATA[2A10 - CDR monoclonal antibody PRNT 50 Value (ng / ml)]]> JEV 79.4 ± 1.3 ZIKV 1584.9 ± 1.3 Example 6 2A10-CDR monoclonal antibody biological protection test One-day-old C57BL / 6 mice were injected subcutaneously with JEV (10 PFU) or ZIKV (1000 PFU) into the back. Two hours later, 25 μg of 2A10-CDR monoclonal antibody was injected subcutaneously into the back for a mouse survival experiment. Mouse body weight and mortality were recorded daily. Data were analyzed, statistically analyzed, and graphed using Prism 6.
[0070] The results are as follows Figure 4 As shown in the results, after JEV infection, the survival rate of mice treated with 2A10-CDR mAb was significantly higher than that of mice not treated with mAb (50% versus 9%, P=0.0175). In addition, the administration of 2A10-CDR mAb significantly reduced the weight loss of mice.
[0071] After ZIKV infection, the survival rate of mice treated with 2A10-CDR mAb was significantly higher than that of mice not treated with mAb (86.7% versus 0, P<0.0001). Furthermore, administration of 2A10-CDR mAb significantly reduced body weight loss in mice.
[0072] Example 7 Effect of 2A10-CDR antibody on JEV and ZIKV viral loads in serum One-day-old C57BL / 6 mice were injected subcutaneously with either JEV (10 PFU) or ZIKV (1000 PFU) into the back. Two hours later, 25 μg of 2A10-CDR monoclonal antibody was injected subcutaneously into the back. Three days later, the mice were sacrificed and serum viral loads were measured using a Vero cell-based plaque formation assay (PFA). Data were analyzed, statistically analyzed, and graphed using Prism 6.
[0073] The results are as follows Figure 5 As shown in the results, after JEV infection, the viral load in the serum of mice given 25 μg of 2A10-CDR mAb was significantly lower than that in the serum of mice not given mAb (251 PFU / ml versus 2238 PFU / ml, P=0.0088).
[0074] After ZIKV infection, the viral load in the serum of mice given 25 μg of 2A10-CDR mAb was significantly lower than that in the serum of mice not given mAb (239 PFU / ml versus 1584 PFU / ml, P=0.0238).
[0075] It can be seen from the above examples and related experimental data that the 2A10-CDR antibody provided in the specific embodiments of the present invention is a novel humanized antibody that can simultaneously neutralize JEV and ZIKV. The 2A10-CDR antibody has an excellent neutralizing effect on JEV and ZIKV and is an antibody with a broad-spectrum anti-flavivirus effect. This provides ideas for the subsequent development of broad-spectrum anti-flavivirus agents.
[0076] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will fall within the protection scope of the present invention.
Claims
1. An antibody or antigen-binding fragment targeting both Zika virus and Japanese encephalitis virus, characterized in that: The antibody or antigen-binding fragment includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes the heavy chain variable region CDR1 shown in SEQ ID No.2, the heavy chain variable region CDR2 shown in SEQ ID No.3, and the heavy chain variable region CDR3 shown in SEQ ID No.4; the light chain variable region includes the light chain variable region CDR1 shown in SEQ ID No.6, the light chain variable region CDR2 shown in SEQ ID No.7, and the light chain variable region CDR3 shown in SEQ ID No.
8.
2. The antibody or antigen-binding fragment according to claim 1, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.
1.
3. The antibody or antigen-binding fragment according to claim 1, wherein The amino acid sequence of the light chain variable region is shown in SEQ ID No.
5.
4. The antibody or antigen-binding fragment according to claim 1, wherein The antibody or antigen-binding fragment further comprises a heavy chain framework region, and the heavy chain framework region is derived from human IgG.
5. The antibody or antigen-binding fragment according to claim 1, wherein The antibody or antigen-binding fragment further comprises a light chain framework region, and the light chain framework region is derived from human IgG.
6. The antibody or antigen-binding fragment according to claim 1, wherein The antibody or antigen-binding fragment further comprises a heavy chain constant region, which is derived from human IgG.
7. The antibody or antigen-binding fragment according to claim 1, wherein The antibody or antigen-binding fragment further comprises a light chain constant region, and the light chain constant region is of human κ type.
8. A nucleic acid, characterized in that The nucleic acid is used to encode the antibody or antigen-binding fragment according to any one of claims 1 to 7.
9. A recombinant vector, characterized in that The recombinant vector comprises the nucleic acid according to claim 8.
10. A use of the antibody or antigen-binding fragment according to any one of claims 1 to 7, characterized in that: The applications include: Application 1: Using the antibody or antigen-binding fragment of any one of claims 1 to 7 for the preparation of a drug for treating diseases caused by Japanese encephalitis virus and / or Zika virus; Application 2: Using the antibody or antigen-binding fragment described in any one of claims 1 to 7 in the preparation of diagnostic products for Japanese encephalitis virus and / or Zika virus infection.