Anti-neuraminidase monoclonal antibody and use thereof

Abstract

Provided are an anti-neuraminidase human monoclonal antibody obtained by means of multiple rounds of in-vitro screening and recombinant modification, and the use thereof in the preparation of a product for preventing, detecting, or treating influenza viruses. The provided monoclonal antibody exhibits significant neuraminidase inhibitory activity and broad-spectrum binding capacity to influenza virus antigens, shows excellent preventive and therapeutic effects against multiple seasonal influenza viruses, provides a new technical option for resisting influenza infection, and has great application value.

Description

Monoclonal antibodies against neuraminidase and their applications Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to monoclonal antibodies against neuraminidase and their applications. Background Technology

[0002] The surface of the influenza virus contains two glycoproteins—hemagglutinin. and neuraminidase Both work synergistically to promote the viral infection process.

[0003] Hemagglutinin (HA) binds to sialic acid (SA) receptors on the cell surface and plays a crucial role in viral adsorption and membrane fusion, making it a primary target for influenza virus infection prevention. Neuraminidase (NA), also known as sialic acid glycoprotein, is a surface glycoprotein of the influenza virus that cleaves sialic acid, promoting the release of viral particles from infected cells. Simultaneously, neuraminidase can assist the virus in penetrating the mucus layer by cleaving decoy receptors in mucin.

[0004] Neuraminidase is also an important target for antiviral therapy against influenza, and both neuraminidase inhibitors and antibodies have been found to have protective effects. Neuraminidase inhibitors are widely used in antiviral therapy, and antibodies targeting neuraminidase can inhibit neuraminidase activity by inhibiting the active site or binding to epitopes surrounding the active site. Currently reported neuraminidase antibodies 8H2, 6C6, and 3D4 primarily target the N1 subtype, while antibodies CR14011, CR14012, and CR14017 primarily target influenza B virus neuraminidase. However, although several monoclonal antibodies against human neuraminidase have been discovered in recent years, the understanding of their targeted neuraminidase epitopes is still incomplete. Conserved epitopes of the neuraminidase protein cannot be fully elucidated, especially those with broad-spectrum neuraminidase inhibitory activity. Furthermore, the number of existing specific broad-spectrum antibodies against neuraminidase is limited, and they cannot simultaneously target both influenza A and B viruses. The neutralization mechanism of neuraminidase-targeting antibodies is not yet fully understood. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a monoclonal antibody against neuraminidase and its applications.

[0006] The first objective of this invention is to provide a monoclonal antibody against neuraminidase or an antigen-binding fragment thereof.

[0007] A second object of the present invention is to provide the use of the monoclonal antibody or its antigen-binding fragment in the preparation of products for the prevention and / or treatment of influenza viruses.

[0008] A third objective of this invention is to provide a biomaterial.

[0009] A fourth object of the present invention is to provide the use of the said biological material in the preparation of monoclonal antibodies against neuraminidase or antigen-binding fragments thereof.

[0010] A fifth object of the present invention is to provide a method for preparing monoclonal antibodies against neuraminidase or antigen-binding fragments thereof.

[0011] A sixth objective of this invention is to provide a medicine for the prevention and / or treatment of influenza viruses.

[0012] To achieve the above objectives, the present invention is implemented through the following solution:

[0013]

[0014] A monoclonal antibody against neuraminidase or an antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment thereof comprising fragment (1) or fragment (2).

[0015]

[0016] Preferably, the monoclonal antibody or its antigen-binding fragment further comprises an Fc segment. The present invention does not have a particular limitation on the species origin of the Fc segment, including but not limited to human Fc segments, mouse Fc segments, rabbit Fc segments, or biologically acceptable modifications or mutations thereof.

[0017] More preferably, the Fc segment is a human Fc segment or a biologically acceptable modification or mutation thereof.

[0018] Preferably, the fragment (1) further comprises a heavy chain constant region (1) and a light chain constant region (1), the amino acid sequence of the heavy chain constant region (1) being as shown in SEQ ID NO.31, and the amino acid sequence of the light chain constant region (1) being as shown in SEQ ID NO.32.

[0019] Preferably, the fragment (2) further comprises a heavy chain constant region (12) and a light chain constant region (2), the amino acid sequence of the heavy chain constant region (2) being shown in SEQ ID NO.31 and the amino acid sequence of the light chain constant region (2) being shown in SEQ ID NO.32.

[0020] The use of any of the monoclonal antibodies or their antigen-binding fragments in the preparation of products for the prevention and / or treatment of influenza viruses should also be within the scope of protection of this invention.

[0021] A biological material, which is any one of the following (1) to (3):

[0022] (1) A nucleic acid molecule encoding any of the monoclonal antibodies or their antigen-binding fragments;

[0023] (2) A carrier containing the nucleic acid molecule described in (1);

[0024] (3) Host cells containing the vector in (2).

[0025] This invention does not have any special limitations on the source and type of host cells. Prokaryotic and eukaryotic cells that can conventionally carry the vector, such as Escherichia coli, HEK293 cells, and CHO cells, can all achieve the purpose of this invention.

[0026] Preferably, the host cell in (3) is a mammalian cell.

[0027] More preferably, the host cell mentioned in (3) is a 293T cell.

[0028] The application of the biomaterials in the preparation of monoclonal antibodies against neuraminidase or their antigen-binding fragments should also be within the scope of protection of this invention.

[0029] The application of the biomaterials in the preparation of products for the prevention and / or treatment of influenza viruses should also be within the scope of protection of this invention.

[0030] A method for preparing a monoclonal antibody against neuraminidase or an antigen-binding fragment thereof, comprising culturing the host cells in (3) of the biological material under conditions that allow expression of the monoclonal antibody or the antigen-binding fragment thereof, and recovering the monoclonal antibody or the antigen-binding fragment thereof from the cultured host cell culture.

[0031] This invention does not have any special limitations on the source and type of host cells. Prokaryotic and eukaryotic cells that can conventionally carry the vector, such as Escherichia coli, HEK293 cells, and CHO cells, can all achieve the purpose of this invention.

[0032] Preferably, the host cell is a mammalian cell.

[0033] More preferably, the host cell described herein is a 293T cell.

[0034] An medicament for the prevention and / or treatment of influenza viruses, comprising any of the said monoclonal antibodies or their antigen-binding fragments, and a pharmaceutically acceptable carrier and / or excipient.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The monoclonal antibody provided by this invention has significant neuraminidase inhibitory activity and broad-spectrum anti-influenza virus antigen binding ability. It has excellent preventive and therapeutic effects against a variety of seasonal influenza viruses, providing a new technical option for combating influenza infection and has important application value. Attached Figure Description

[0037] Figure 1 shows the SDS-PAGE detection results of F6 antibody and FF34 antibody.

[0038] Figure 2 shows the antigen binding of neuraminidase monoclonal antibodies in an ELISA experiment; A is F6 antibody; B is FF34 antibody.

[0039]

[0040]

[0041] Embodiments of the present invention

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0043] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0044] Example 1: Preparation of neuraminidase monoclonal antibody

[0045]

[0046]

[0047] Table 1 Nested PCR reaction system

[0048]

[0049] Table 2. First-round nested PCR program

[0050]

[0051] Table 3. Second round of nested PCR program

[0052]

[0053] After the second round of nested PCR, variable region fragments of the heavy and light chains of two neuraminidase monoclonal antibodies (F6 antibody and FF34 antibody) were obtained.

[0054] According to the IMGT method for defining domains, the specific domain divisions of the F6 antibody and the FF34 antibody are as follows:

[0055]

[0056] 3. Expression, purification, and identification of neuraminidase monoclonal antibodies

[0057] (1) Construction of recombinant plasmids

[0058]

[0059] (2) Antibody expression

[0060]

[0061] (3) Purification

[0062]

[0063] (4) Identification

[0064]

[0065] Example 2: Antigen binding of neuraminidase monoclonal antibody

[0066] 1. Antigen preparation and purification

[0067]

[0068] 2. ELISA experiment

[0069]

[0070]

[0071] Example 3: Enzyme activity inhibitory ability of neuraminidase monoclonal antibody

[0072] 1. Antigen preparation and purification

[0073]

[0074] 2. Experimental identification of the antibody's effect on the active site of NA enzyme

[0075]

[0076] 3. Ella assay to identify antibody binding.

[0077]

[0078]

[0079]

[0080] The above results indicate that both F6 and FF34 antibodies can inhibit the activity of neuraminidase in various types of influenza viruses.

[0081] Example 4: Protective ability of neuraminidase monoclonal antibody

[0082] 1. Antibody prevention experiment

[0083] BALB / c mice aged 6–8 weeks were used as experimental animals. One day before challenge, each mouse was intraperitoneally injected with F6 antibody, FF34 antibody, or PBS at a dose of 10 mg / kg (antibody / body weight). Twenty-four hours later, the mice were challenged with influenza virus with a 5×LD50 concentration. Mice were monitored for body weight for 14 consecutive days after challenge. Mice whose body weight fell below 75% of their initial body weight were euthanized, and the survival rate was recorded. .

[0084]

[0085] 2. Antibody therapy experiment

[0086]

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A monoclonal antibody against neuraminidase or its antigen-binding fragment, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises fragment (1) or fragment (2); The fragment (1) comprises a heavy chain variable region (1) and a light chain variable region (1); the heavy chain variable region (1) comprises CDR-F6-H1 to CDR-F6-H3, wherein the amino acid sequence of CDR-F6-H1 is shown in SEQ ID NO.2, the amino acid sequence of CDR-F6-H2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR-F6-H3 is shown in SEQ ID NO.4; the light chain variable region (1) comprises CDR-F6-L1 to CDR-F6-L3, wherein the amino acid sequence of CDR-F6-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-F6-L2 is GAS, and the amino acid sequence of CDR-F6-L3 is shown in SEQ ID NO.11; The fragment (2) comprises a heavy chain variable region (2) and a light chain variable region (2); the heavy chain variable region (2) comprises CDR-FF34-H1 to CDR-FF34-H3, wherein the amino acid sequence of CDR-FF34-H1 is shown in SEQ ID NO.17, the amino acid sequence of CDR-FF34-H2 is shown in SEQ ID NO.18, and the amino acid sequence of CDR-FF34-H3 is shown in SEQ ID NO.19; the light chain variable region (2) comprises light chain variable regions of CDR-FF34-L1 to CDR-FF34-L3, wherein the amino acid sequence of CDR-FF34-L1 is shown in SEQ ID NO.25, the amino acid sequence of CDR-FF34-L2 is AAS, and the amino acid sequence of CDR-FF34-L3 is shown in SEQ ID NO.

26.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region (1) further comprises FR-F6-H1 to FR-F6-H4, wherein the amino acid sequence of FR-F6-H1 is shown in SEQ ID NO.5, the amino acid sequence of FR-F6-H2 is shown in SEQ ID NO.6, the amino acid sequence of FR-F6-H3 is shown in SEQ ID NO.7, and the amino acid sequence of FR-F6-H4 is shown in SEQ ID NO.8; the light chain variable region (1) further comprises FR-F6-L1 to FR-F6-L4, wherein the amino acid sequence of FR-F6-L1 is shown in SEQ ID NO.12, the amino acid sequence of FR-F6-L2 is shown in SEQ ID NO.13, the amino acid sequence of FR-F6-L3 is shown in SEQ ID NO.14, and the amino acid sequence of FR-F6-L4 is shown in SEQ ID NO.

15.

3. The monoclonal antibody or its antigen-binding fragment according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region (1) is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region (1) is shown in SEQ ID NO.

9.

4. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region (2) further comprises FR-FF34-H1 to FR-FF34-H4, wherein the amino acid sequence of FR-FF34-H1 is shown in SEQ ID NO.20, the amino acid sequence of FR-FF34-H2 is shown in SEQ ID NO.21, the amino acid sequence of FR-FF34-H3 is shown in SEQ ID NO.22, and the amino acid sequence of FR-FF34-H4 is shown in SEQ ID NO.23; the light chain variable region (2) further comprises FR-FF34-L1 to FR-FF34-L4, wherein the amino acid sequence of FR-FF34-L1 is shown in SEQ ID NO.27, the amino acid sequence of FR-FF34-L2 is shown in SEQ ID NO.28, the amino acid sequence of FR-FF34-L3 is shown in SEQ ID NO.29, and the amino acid sequence of FR-FF34-L4 is shown in SEQ ID NO.

30.

5. The monoclonal antibody or its antigen-binding fragment according to claim 4, characterized in that, The amino acid sequence of the heavy chain variable region (2) is at least 95% identical to that of SEQ ID NO.16; the amino acid sequence of the light chain variable region (2) is at least 95% identical to that of SEQ ID NO.

24.

6. The use of the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 5 in the preparation of products for the prevention, detection or treatment of influenza viruses.

7. A biomaterial, characterized in that, It is any one of the following (1) to (3): (1) A nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 5; (2) A carrier containing the nucleic acid molecule described in (1); (3) Host cells containing the vector in (2).

8. The use of the biomaterial of claim 7 in the preparation of monoclonal antibodies against neuraminidase or antigen-binding fragments thereof.

9. A method for preparing a monoclonal antibody against neuraminidase or an antigen-binding fragment thereof, characterized in that, Under conditions that allow expression of the monoclonal antibody or its antigen-binding fragment, the host cell of claim 7 is cultured, and the monoclonal antibody or its antigen-binding fragment is recovered from the cultured host cell culture.

10. A drug for the prevention and / or treatment of influenza viruses, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 5, and a pharmaceutically acceptable carrier and / or excipient.

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