Monoclonal antibody for resisting human B-type adenovirus and application of monoclonal antibody

Abstract

The invention belongs to the technical field of biology, and discloses an anti-human B-type adenovirus monoclonal antibody and application thereof. The monoclonal antibodies 10G12, 12F12, 2E8 and 2F5 provided by the invention can be effectively combined with the type 3 or 55 human adenovirus, and have very strong neutralizing activity on the type 3 or 55 human adenovirus. The clinical application value of preventing and treating adenovirus infection is realized; the compound can be used for detecting adenovirus, diagnosing diseases related to adenovirus infection, and preventing and / or treating adenovirus infection and / or diseases related to adenovirus infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a monoclonal antibody against human B-type adenovirus and application thereof. BACKGROUND

[0002] Human adenovirus (HAdV), a non-enveloped icosahedral double-stranded DNA virus, has tissue tropism, and B group adenoviruses such as types 3, 7, 11, 14 and 55 mainly cause respiratory tract infections. Recent data shows that the incidence of adenovirus infection is about 9.8%, which is significantly higher than the level before the COVID-19 pandemic, and is more common in preschool children aged four to six. HAdV-3 and HAdV-7 are common types of community adenovirus pneumonia outbreaks. Although most HAdV infections have mild clinical symptoms, some serotypes, such as HAdV-3, HAdV-4, HAdV-55, etc., can cause severe infections in children, the elderly and immunodeficient patients, and even cause death. The mortality rate of severe adenovirus pneumonia can reach 50%. Adenovirus infection has a variety of clinical signs and symptoms, including pneumonia, bronchitis, muscle pain, cough and high fever, and can also cause conjunctival and gastrointestinal infections. It is worth noting that the early clinical symptoms of severe adenovirus pneumonia are usually atypical and difficult to diagnose by clinical symptoms, leading to rapid disease progression and sequelae.

[0003] Infection of HAdV not only affects immunodeficient patients, but also affects healthy people, and its impact on public health cannot be ignored. At present, there is still a lack of specific and effective adenovirus treatment. However, neutralizing monoclonal antibodies can target free viruses and virus-infected cells through a series of mechanisms and exert antiviral effects in vivo, including direct neutralization of virus invasion of cells, antibody-mediated cytotoxicity, or indirect blocking of virus entry by cross-linking virus bodies. Therefore, it is urgent to develop highly efficient, safe and targeted anti-HAdV neutralizing monoclonal antibodies, which is also an important research direction in the current field of adenovirus infection treatment. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art, and develop human adenovirus neutralizing antibodies that recognize different antigen targets. To this end, the present application provides an adenovirus monoclonal antibody that can specifically target type 3 human adenovirus particles, type 55 human adenovirus particles and their hexon proteins, respectively, and shows high efficiency in neutralizing viruses. Therefore, the antibody of the present application is particularly suitable for diagnosing, preventing and treating type 3 or type 55 human adenovirus infection or diseases related to human adenovirus infection.

[0005] The first aspect of the present application aims to provide a monoclonal antibody against human B-type adenovirus.

[0006] The second aspect of the present application aims to provide an antibody.

[0007] The third aspect of the present application aims to provide a biological material of the monoclonal antibody of the first aspect of the present application or the antibody of the second aspect of the present application.

[0008] The fourth aspect of the present application aims to provide a conjugate.

[0009] The fifth aspect of the present application aims to provide an application of the monoclonal antibody of the first aspect of the present application, the antibody of the second aspect of the present application, the biological material of the third aspect of the present application or the conjugate of the fourth aspect of the present application.

[0010] The sixth aspect of the present application aims to provide a product.

[0011] The seventh aspect of the present application aims to provide a method for detecting an adenovirus.

[0012] In order to achieve the above-mentioned purposes, the technical scheme adopted by the present application is as follows:

[0013] The first aspect of the present application provides a monoclonal antibody against human adenovirus type B, characterized in that the monoclonal antibody is 10G12, 12F12, 2F5 or 2E8, and the monoclonal antibody comprises a light chain variable region and a heavy chain variable region.

[0014] The light chain variable region of the 10G12 comprises CDR1, CDR2 and CDR3, and the amino acid sequences thereof are shown in SEQ ID NO: 34-36 in sequence; the heavy chain variable region of the 10G12 comprises CDR1, CDR2 and CDR3, and the amino acid sequences thereof are shown in SEQ ID NO: 37-39 in sequence.

[0015] The light chain variable region of the 12F12 comprises CDR1, CDR2 and CDR3, and the amino acid sequences thereof are shown in SEQ ID NO: 44-46 in sequence; the heavy chain variable region of the 12F12 comprises CDR1, CDR2 and CDR3, and the amino acid sequences thereof are shown in SEQ ID NO: 47-49 in sequence.

[0016] The light chain variable region of the 2F5 comprises CDR1, CDR2 and CDR3, and the amino acid sequences thereof are shown in SEQ ID NO: 54-56 in sequence; the heavy chain variable region of the 2F5 comprises CDR1, CDR2 and CDR3, and the amino acid sequences thereof are shown in SEQ ID NO: 57-59 in sequence.

[0017] The light chain variable region of the 2E8 comprises CDR1, CDR2, CDR3, the amino acid sequences of which are shown in SEQ ID NOs: 54-56 in order; the heavy chain variable region of the 2E8 comprises CDR1, CDR2, CDR3, the amino acid sequences of which are shown in SEQ ID NOs: 64-66 in order.

[0018] In some embodiments of the present application, the amino acid sequence of the light chain variable region of the 10G12 is: A1) SEQ ID NO: 40; or A2) an amino acid sequence having at least 90% (such as 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, or 90%) homology with SEQ ID NO: 40 and being functionally identical or similar; or A3) an amino acid sequence modified from the amino acid sequence shown in SEQ ID NO: 40 by substitution, deletion, or addition of one or more amino acids and being functionally identical or similar.

[0019] In some embodiments of the present application, the amino acid sequence of the heavy chain variable region of the 10G12 is: B1) SEQ ID NO: 42; or B2) an amino acid sequence having at least 90% (such as 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, or 90%) homology with SEQ ID NO: 42 and being functionally identical or similar; or B3) an amino acid sequence modified from the amino acid sequence shown in SEQ ID NO: 42 by substitution, deletion, or addition of one or more amino acids and being functionally identical or similar.

[0020] In some embodiments of the present application, the amino acid sequence of the light chain variable region of the 12F12 is: C1) SEQ ID NO: 50; or C2) an amino acid sequence having at least 90% (such as 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, or 90%) homology with SEQ ID NO: 50 and being functionally identical or similar; or C3) an amino acid sequence modified from the amino acid sequence shown in SEQ ID NO: 50 by substitution, deletion, or addition of one or more amino acids and being functionally identical or similar.

[0021] In some embodiments of the present application, the amino acid sequence of the heavy chain variable region of the 12F12 is: D1) SEQ ID NO: 52; or D2) an amino acid sequence having at least 90% (such as 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, or 90%) homology with SEQ ID NO: 52 and being functionally identical or similar; or D3) an amino acid sequence modified from the amino acid sequence shown in SEQ ID NO: 52 by substitution, deletion, or addition of one or more amino acids and being functionally identical or similar.

[0022] In some embodiments of the present application, the amino acid sequence of the light chain variable region of the 2F5 is: E1) SEQ ID NO: 60; or E2) an amino acid sequence having at least 90% (such as 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, or 90%) homology to SEQ ID NO: 60 and functionally identical or similar; or E3) an amino acid sequence modified from the amino acid sequence set forth in SEQ ID NO: 60 by substitution, deletion, or addition of one or more amino acids and functionally identical or similar.

[0023] In some embodiments of the present application, the amino acid sequence of the heavy chain variable region of the 2F5 is: F1) SEQ ID NO: 62; or F2) an amino acid sequence having at least 90% (such as 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, or 90%) homology to SEQ ID NO: 62 and functionally identical or similar; or F3) an amino acid sequence modified from the amino acid sequence set forth in SEQ ID NO: 62 by substitution, deletion, or addition of one or more amino acids and functionally identical or similar.

[0024] In some embodiments of the present application, the amino acid sequence of the light chain variable region of the 2E8 is: G1) SEQ ID NO: 67; or G2) an amino acid sequence having at least 90% (such as 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, or 90%) homology to SEQ ID NO: 67 and functionally identical or similar; or G3) an amino acid sequence modified from the amino acid sequence set forth in SEQ ID NO: 67 by substitution, deletion, or addition of one or more amino acids and functionally identical or similar.

[0025] In some embodiments of the present application, the amino acid sequence of the heavy chain variable region of the 2E8 is: H1) SEQ ID NO: 69; or H2) an amino acid sequence having at least 90% (such as 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, or 90%) homology to SEQ ID NO: 69 and functionally identical or similar; or H3) an amino acid sequence modified from the amino acid sequence set forth in SEQ ID NO: 69 by substitution, deletion, or addition of one or more amino acids and functionally identical or similar.

[0026] In some embodiments of the present application, the monoclonal antibody comprises at least one of a full-length antibody, a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, a bispecific antibody, a multispecific antibody.

[0027] The present application provides a monoclonal antibody capable of specifically binding to human adenovirus. According to some examples of the present application, the monoclonal antibody is capable of targeting human adenovirus type 3, human adenovirus type 55, and the hexon protein thereof. The monoclonal antibody is capable of inhibiting the receptor binding domain-mediated receptor binding and / or membrane fusion process, and inhibiting the infection of the virus to the cell.

[0028] In some embodiments of the present application, the monoclonal antibody can be used to detect the presence or level of human adenovirus type 3 or 55 in a sample.

[0029] In some embodiments of the present application, the monoclonal antibody has neutralizing ability (e.g., capable of neutralizing human adenovirus type 3 or 55).

[0030] In some embodiments of the present application, the monoclonal antibody is capable of inhibiting the infection or entry of human adenovirus type 3 or 55 into a host cell, capable of neutralizing human adenovirus type 3 or 55, and thereby preventing and treating the infection of human adenovirus type 3.

[0031] In a second aspect of the present application, an antibody is provided, which is any one of M1) to M4): M1) a single-chain antibody obtained by linking the heavy chain variable region and the light chain variable region of the first aspect of the present application; M2) a fusion antibody containing the single-chain antibody of M1); M3) a Fab containing the heavy chain variable region and the light chain variable region of the first aspect of the present application; and M4) a whole antibody containing the heavy chain variable region and the light chain variable region of the first aspect of the present application.

[0032] In some embodiments of the present application, the fusion antibody has a first domain and a second domain, the first domain being the single-chain antibody of M1), and the second domain having the effect of prolonging the half-life in vivo and / or having the effect of binding to effector cells.

[0033] In some embodiments of the present application, the second domain includes (but is not limited to): an immunoglobulin Fc region (such as a human immunoglobulin Fc region); and / or serum albumin (such as human-derived HSA) or a fragment thereof, a domain binding to serum albumin (such as an anti-serum albumin antibody, including a nanobody), polyethylene glycol, a polyethylene glycol-liposome complex, or a combination thereof; and / or a molecule having affinity to a T cell surface molecule and / or capable of binding to a surface molecule present on a T cell (such as CD3).

[0034] In some embodiments of the present application, the human immunoglobulin Fc region comprises a mutation for altering Fc-mediated effector functions, including one or more of a combination of CDC activity, ADCC activity, and ADCP activity. In some embodiments of the present application, the immunoglobulin is one or more of a combination selected from the group consisting of IgG, IgAl, IgA2, IgD, IgE, and IgM. In some embodiments of the present application, the IgG is one or more of a combination selected from the group consisting of IgGl, IgG2, IgG3, and IgG4 subtypes.

[0035] In some embodiments of the present application, the first domain is linked to the N-terminus or C-terminus of the second domain.

[0036] In some embodiments of the present application, the first domain and the second domain are linked by a hinge or a linking peptide.

[0037] In some embodiments of the present application, the linking peptide is a flexible linker. In some embodiments of the present application, the amino acid sequence of the flexible linker includes, but is not limited to, GSAS, (GGCAGCGCCAGC) n , (GGCGGCGGCAGC) n , (GGCGGCGGCGGCAGC) n , YAPVDV, (GGGS) n , (GGSG) n , (GGGGS) n , (G) n , wherein 1≤n≤5, and n is an integer.

[0038] In a third aspect of the present application, a biological material related to the monoclonal antibody of the first aspect of the present application or the antibody of the second aspect of the present application is provided; the biological material is any one of a1) to a12): a1) a nucleic acid molecule encoding the monoclonal antibody of the first aspect of the present application or the antibody of the second aspect of the present application; a2) an expression cassette comprising the nucleic acid molecule of a1); a3) a recombinant vector comprising the nucleic acid molecule of a1); a4) a recombinant vector comprising the expression cassette of a2); a5) a recombinant microorganism comprising the nucleic acid molecule of a1); a6) a recombinant microorganism comprising the expression cassette of a2); a7) a recombinant microorganism comprising the recombinant vector of a3); a8) a recombinant microorganism comprising the recombinant vector of a4); a9) a transgenic cell line comprising the nucleic acid molecule of a1); a10) a transgenic cell line comprising the expression cassette of a2); a11) a transgenic cell line comprising the recombinant vector of a3); and a12) a transgenic cell line comprising the recombinant vector of a4).

[0039] In some embodiments of the application, the nucleic acid molecule in a1) can be a nucleic acid molecule encoding the monoclonal antibody.

[0040] In some embodiments of the application, the nucleic acid molecule encoding the variable region of the light chain of 10G12 is set forth in SEQ ID NO: 41, or a nucleotide sequence having at least 90% homology to SEQ ID NO: 41 and functionally identical or similar.

[0041] In some embodiments of the application, the nucleic acid molecule encoding the variable region of the heavy chain of 10G12 is set forth in SEQ ID NO: 43, or a nucleotide sequence having at least 90% homology to SEQ ID NO: 43 and functionally identical or similar.

[0042] In some embodiments of the application, the nucleic acid molecule encoding the variable region of the light chain of 12F12 is set forth in SEQ ID NO: 51, or a nucleotide sequence having at least 90% homology to SEQ ID NO: 51 and functionally identical or similar.

[0043] In some embodiments of the application, the nucleic acid molecule encoding the variable region of the heavy chain of 12F12 is set forth in SEQ ID NO: 53, or a nucleotide sequence having at least 90% homology to SEQ ID NO: 53 and functionally identical or similar.

[0044] In some embodiments of the application, the nucleic acid molecule encoding the variable region of the light chain of 2F5 is set forth in SEQ ID NO: 61, or a nucleotide sequence having at least 90% homology to SEQ ID NO: 61 and functionally identical or similar.

[0045] In some embodiments of the application, the nucleic acid molecule encoding the variable region of the heavy chain of 2F5 is set forth in SEQ ID NO: 63, or a nucleotide sequence having at least 90% homology to SEQ ID NO: 63 and functionally identical or similar.

[0046] In some embodiments of the application, the nucleic acid molecule encoding the variable region of the light chain of 2E8 is set forth in SEQ ID NO: 68, or a nucleotide sequence having at least 90% homology to SEQ ID NO: 68 and functionally identical or similar.

[0047] In some embodiments of the application, the nucleic acid molecule encoding the variable region of the heavy chain of 2E8 is set forth in SEQ ID NO: 70, or a nucleotide sequence having at least 90% homology to SEQ ID NO: 70 and functionally identical or similar.

[0048] In some embodiments of the present application, the expression cassette in a2) refers to DNA capable of expressing the monoclonal antibody in a host cell, which can include not only a promoter initiating transcription of the coding gene of the monoclonal antibody, but also a terminator terminating transcription of the coding gene of the monoclonal antibody. Further, the expression cassette can also include a replication initiation site, a transcription initiation sequence, an enhancer sequence, a selection element or a reporter gene.

[0049] In some embodiments of the present application, the vector in a3) to a4) can be a plasmid vector, a phagemid, a viral vector, a cell vector, a phage, a cosmid, a fosmid or an artificial chromosome. The vector can be a cloning vector or an expression vector.

[0050] In some embodiments of the present application, the plasmid vector can be an optional plasmid, the viral vector can be an optional virus, and the cell vector does not include reproductive material.

[0051] In some embodiments of the present application, the cell in a5) to a8) can be a bacterium (such as Escherichia coli or Bacillus subtilis, etc.), an alga, a fungus (such as yeast or Aspergillus, etc.), an insect cell (such as S2 fruit fly cell or Sf9 cell, etc.) or an animal cell (such as CHO cell, COS cell, NSO cell, HeLa cell, BHK cell or HEK293 cell, etc.). The cell does not include reproductive material.

[0052] In some embodiments of the present application, the cell can particularly be a hybridoma cell. The hybridoma cell is obtained by fusing a spleen cell with a SP2 / 0 cell.

[0053] In a fourth aspect of the present application, a conjugate is provided, comprising: the monoclonal antibody of the first aspect of the present application or the antibody of the second aspect of the present application; and a conjugating moiety.

[0054] In some embodiments of the present application, the conjugating moiety comprises at least one of a detectable label (such as a fluorescent or luminescent label), a drug, a toxin, an electron-dense marker, biotin / avidin, a spin label, an antibody (including a Fc fragment, an antibody scFv fragment), a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle and a virus coat protein.

[0055] In some embodiments of the present application, the drug is another drug for preventing and / or treating an adenovirus infection and / or a disease associated with an adenovirus infection.

[0056] In some embodiments of the present application, the adenovirus includes a human adenovirus, preferably a type 3 or type 55 human adenovirus.

[0057] In a fifth aspect of the present application, the use of the monoclonal antibody of the first aspect of the present application, the antibody of the second aspect of the present application, the biological material of the third aspect of the present application or the conjugate of the fourth aspect of the present application in at least one of b1) to b8): b1) preparing a product for preventing or treating a disease associated with an adenovirus infection; b2) detecting an adenovirus; b3) preparing a product for detecting an adenovirus; b4) inhibiting an adenovirus activity; b5) preparing a product for inhibiting an adenovirus activity; b6) preparing a product for diagnosing an adenovirus infection; b7) neutralizing a virulence of an adenovirus; and b8) preparing a product for neutralizing a virulence of an adenovirus.

[0058] In some embodiments of the present application, the adenovirus comprises a human adenovirus, preferably a human adenovirus of type 3 or type 55.

[0059] In some embodiments of the present application, the product comprises at least one of a vaccine, a drug, a reagent, a detection plate, a kit, a detection chip.

[0060] In some embodiments of the present application, the disease comprises a respiratory disease, such as pneumonia, bronchitis.

[0061] In a sixth aspect of the present application, a product is provided, the product comprising the monoclonal antibody of the first aspect of the present application, the antibody of the second aspect of the present application, the biological material of the third aspect of the present application or the conjugate of the fourth aspect of the present application.

[0062] In some embodiments of the present application, the product comprises at least one of a vaccine, a drug, a reagent, a detection plate, a kit, a detection chip.

[0063] In some embodiments of the present application, the product has at least one of c1) to c5): c1) preventing or treating a disease associated with an adenovirus infection; c2) detecting an adenovirus; c3) inhibiting an adenovirus activity; c4) diagnosing an adenovirus infection; and c5) neutralizing a virulence of an adenovirus.

[0064] In some embodiments of the present application, the product further comprises a pharmaceutically acceptable excipient.

[0065] In some embodiments of the present application, the excipient comprises at least one of a filler, a disintegrant, a diluent, a lubricant, a binder, a humectant, a flavoring agent, a suspending agent, a solvent, a sustained release agent, an emulsifying agent, an absorption enhancer, a surfactant, a preservative, a pigment, an essence and a solvent.

[0066] In some embodiments of the present application, the drug further comprises another drug for preventing and / or treating an adenovirus infection and / or a disease associated with an adenovirus infection.

[0067] In some embodiments of the application, the medicament can administer the monoclonal antibody or antigen-binding fragment thereof of the application to a subject by any appropriate route of administration. Such routes of administration include, but are not limited to, oral, buccal, sublingual, topical, parenteral, rectal, intrathecal or intranasal routes.

[0068] In some embodiments of the application, the subject is a mammal, such as a human.

[0069] In a seventh aspect of the application, there is provided a method for detecting an adenovirus, comprising contacting a sample containing or suspected of containing an adenovirus with the monoclonal antibody of the first aspect of the application, the antibody of the second aspect of the application, the conjugate of the fourth aspect of the application, the product of the sixth aspect of the application, and detecting the formation of a complex of the monoclonal antibody or antibody and the adenovirus, the method being for non-diagnostic purposes.

[0070] The application has the following advantages:

[0071] The monoclonal antibodies 10G12, 12F12, 2E8 and 2F5 provided by the application can effectively bind to human adenovirus type 3 or 55, and have strong neutralizing activity against human adenovirus type 3 or 55. Moreover, they have clinical application value for preventing and treating adenovirus infection, and can be used for detecting adenovirus, diagnosing diseases related to adenovirus infection, preventing and / or treating adenovirus infection and / or diseases related to adenovirus infection.

[0072] Specifically, the monoclonal antibodies 10G12 and 12F12 of the application can effectively bind to inactivated HAdV-3 virus particles, with half effective concentrations (EC 50 ) of 70.43 and 100.9 ng / mL respectively, but do not show binding to the hexon protein of HAdV-3, and can effectively neutralize live HAdV-3 virus, with half inhibitory concentrations (IC 50 ) of 5.33 and 13.41 μg / mL respectively; the monoclonal antibodies 2E8 and 2F5 can not only effectively bind to HAdV-55 virus particles, with half effective concentrations (EC 50 ) of 16.512 and 0.4046 μg / mL respectively, but also show binding activity to the hexon protein of HAdV-55, with half effective concentrations (EC 50 ) of 8.854 and 1.646 μg / mL respectively, and both can effectively neutralize live HAdV-55 virus, with half inhibitory concentrations (IC 50) were 3.162 and 26.572 μg / mL, respectively. Therefore, the monoclonal antibodies 10G12, 12F12, 2E8 and 2F5 provided by the present application not only have good binding activity and can effectively bind to HAdV-3 or HAdV-55 virus particles, but also can inhibit the infection of cells by type 3 or type 55 human adenovirus, and have excellent neutralization activity. The development of new drugs and universal vaccines with neutralization activity against type 3 or type 55 human adenovirus has important significance in clinical practice. BRIEF DESCRIPTION OF DRAWINGS

[0073] The present application will be further described below in conjunction with the drawings and examples, in which:

[0074] Figure 1 SDS-PAGE detection results of the monoclonal antibodies 10G12, 12F12, 2E8 and 2F5.

[0075] Figure 2 Detection results of the binding activity of the monoclonal antibodies 10G12 and 12F12 to inactivated HAdV-3 virus particles (A) and hexon protein of HAdV-3 (C), respectively, and detection results of the binding activity of the monoclonal antibodies 2E8 and 2F5 to inactivated HAdV-55 virus particles (B) and hexon protein of HAdV-55 (D), respectively.

[0076] Figure 3 Detection results of the binding activity of different concentrations of the monoclonal antibodies 10G12, 12F12, 2E8 and 2F5 to the fiber protein of HAdV-7.

[0077] Figure 4 Neutralization activity of different concentrations of the 10G12 and 12F12 antibodies against live HAdV-3 (A) and HAdV-55 (C), and neutralization activity of different concentrations of the 2E8 and 2F5 antibodies against live HAdV-55 (B) and HAdV-3 (D). DETAILED DESCRIPTION

[0078] The concept and technical effects of the present application will be described below in conjunction with examples to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0079] The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are conventional products that can be obtained by market purchase.

[0080] The molecular biological experimental methods and immunoassay methods used in the present application are basically performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995, unless otherwise specified. The examples are performed under the conventional conditions or the conditions recommended by the manufacturers, unless otherwise specified. The reagents or instruments used are all conventional products available on the market, unless otherwise specified.

[0081] "Room temperature" in the present application means 25°C ± 5°C, unless otherwise specified.

[0082] The features and properties of the present application are further described in detail below with reference to the examples.

[0083] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally comprised of two pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be assigned to a class, kappa and lambda. Heavy chains can be assigned to a class, mu, delta, gamma, alpha, or epsilon, and define a specificity of an antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The fragment, crystallizable (Fc fragment) corresponds to the CH2 and CH3 domains of an antibody, forms a crystal, has no antigen binding activity, but has functions such as fixing complement, binding to Fc receptors, etc. The constant region of an antibody can mediate 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 (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair, VH and VL, form the antibody binding site. Assignment of amino acids to each region or domain follows the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0084] As used herein, the term "monoclonal antibody" refers to an antibody or a fragment of an antibody from a population of antibody molecules of extreme homogeneity, i.e., a population of antibody molecules that are identical except for naturally occurring mutations that can arise during production. Monoclonal antibodies have high specificity for a single epitope on an antigen. An antibody fragment comprising the variable region of a heavy chain and the variable region of a light chain. Typically, a single-chain antibody also comprises a connecting polypeptide between the variable region of the heavy chain and the variable region of the light chain that enables the single-chain antibody to form the desired structure for antigen binding. Polyclonal antibodies are in contrast to monoclonal antibodies and typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can typically be obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, e.g., Journal of virological methods, 2009, 158(1-2): 171-179).

[0085] As used herein, the term "single-chain antibody" refers to a single-chain fragment variable (scFv), which is a genetically engineered fusion protein composed of the variable regions of the heavy chain and the variable regions of the light chain connected by a short linker. As used herein, the term "fusion antibody" refers to a product obtained by genetically engineering a single-chain antibody with a functional protein molecule having biological activity. The fusion antibody does not affect the binding ability of the single-chain antibody to the antigen, nor does it affect the biological activity of the functional protein molecule fused thereto. As used herein, the term "Fab" refers to an antibody fragment formed by an intact light chain (a variable region of a light chain (VL) and a constant region of a light chain (CL)) and a Fd fragment (a variable region of a heavy chain (VH) and a first constant domain of a heavy chain (CH1)) through an interchain disulfide bond.

[0086] As used herein, the terms "human adenovirus" and "HAdV" refer to a nonenveloped, linear, double-stranded DNA virus comprising a viral capsid and internal nucleic acid, the surface of each adenoviral particle primarily comprising three capsid proteins: 240 hexon trimer proteins, 12 penton trimer proteins, and 12 fiber trimer proteins. There are also four minor proteins within the capsid, namely IIIa, VI, VII, and IX, and core proteins including V, VII, Mu, TP, IVa2, and Protease. Both have the same meaning and can be used interchangeably.

[0087] As used herein, the term "myeloma cell" refers to a cell that is a fusion of a myeloma cell and a B lymphocyte. Hybridoma cells are generally prepared by fusion of tumor cells. The technique of preparing hybridoma cells refers to the phenomenon of the fusion of two or more cells to form a single cell; it allows the expression of functions of two different cells in the same cell. By fusing two cells, the major characteristics of both are maintained, i.e., a mouse spleen cell immunized with an antigen and a mouse myeloma cell. The major characteristic of the B lymphocyte is its antibody-secreting function and the ability to grow in selective medium; the mouse myeloma cell, on the other hand, can divide, proliferate under culture conditions indefinitely, i.e., so-called immortality. Only the hybrid cell, which is the fusion of the B lymphocyte and the myeloma cell, has the ability to proliferate continuously under the influence of the selective medium, forming a cell that has both the antibody-secreting function and the characteristic of cell immortality.

[0088] As used herein, "neutralizing antibody" refers to an antibody or antibody fragment that is capable of clearing or significantly reducing the virulence of a target pathogen.

[0089] As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment that binds to an antigenic protein on a virus, thereby preventing the virus from infecting a cell and / or the maturation of viral progeny and / or the release of viral progeny, an antibody or antibody fragment having neutralizing activity can prevent the amplification of the virus, thereby inhibiting or eliminating infection by the virus.

[0090] As used herein, the term "specifically binds" refers to a nonrandom binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.

[0091] In the present application, amino acids are generally represented by their commonly known single-letter and three-letter abbreviations. For example, alanine can be represented by A or Ala.

[0092] As used herein, the term "CPE" refers to the cytopathic effect, which can be observed under a microscope after a certain period of time by culturing and inoculating a cell with a cytopathogenic virus in an in vitro experiment. The cytopathic effect can be used for virus quantification.

[0093] As used herein, the term "Da" is a commonly used unit of molecular weight, Da stands for Dalton, which is the algebraic sum of the atomic weights of all atoms in a molecule. Proteins are macromolecules, so kDa (kilodalton) is often used to represent them. Dalton is the unit of atomic mass, and D or KD is often used in biochemistry, molecular biology, and proteomics.

[0094] As used herein, the term "passive immunity" refers to the specific immunity acquired by the body passively receiving antibodies, sensitized lymphocytes, or products thereof.

[0095] As used herein, the term "homology" refers to the degree of similarity between nucleotide sequences of two nucleic acid molecules or between amino acid sequences of two protein molecules. It reflects the relationship between branches derived from the same ancestor in the evolutionary process, and the essential similarity in common origin in evolution or ontogeny, but the functions are not necessarily the same.

[0096] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of mediating the expression of the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector are expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage, and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site.

[0097] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as E. coli or B. subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells, etc.

[0098] As used herein, the term "TCID50" refers to the median tissue culture infective dose, also known as the 50% tissue culture infective dose. It refers to the amount of virus required to cause cytopathic effect (CPE) in half of the cells in a culture plate well or test tube, and is used to characterize the titer of the virus.

[0099] As used in this article, the term "ELISA" stands for Enzyme-Linked Immunosorbent Assay, which is an immunoassay method that uses the specific binding of antigens or antibodies to the surface of a solid-phase carrier and the colorimetric reaction catalyzed by enzymes labeled on the antibody or antigen to detect the target analyte.

[0100] As used in this article, the term "IC" 50 "EC" refers to the half-maximal inhibitory concentration (WMC) of the antagonist being measured. In this document, it represents the antibody concentration at which the antibody neutralizes the virus to 50% of its maximum effect in an in vitro neutralization assay. As used herein, the term "EC" is... 50 "" refers to the half-maximal effect concentration. In this article, it represents the antibody concentration at which antigen-antibody binding reaches 50% of the maximum effect in an ELISA experiment.

[0101] To obtain neutralizing antibodies with protective effects, the inventors of this application first used isolated human adenovirus as an antigen. Using mouse monoclonal antibody preparation technology, high-titer mice were screened from immunized mice for cell fusion. The resulting monoclonal antibody cell lines with neutralizing activity were then expanded and cultured. Antibodies were then purified in large quantities from mouse ascites fluid, yielding antibodies 10G12, 12F12, 2E8, and 2F5. A series of functional tests were performed on these antibodies. The results showed that antibodies 10G12 and 12F12 effectively bound to human adenovirus type 3 (HAV3), inhibiting HAV3 infection of cells and exhibiting neutralizing activity against HAV3 infection. Antibodies 2E8 and 2F5 effectively bound to human adenovirus type 55 (HAV55), inhibiting HAV55 infection of cells and exhibiting neutralizing activity against HAV55 infection.

[0102] Example 1: Establishment of hybridoma cell lines and preparation of monoclonal antibodies

[0103] (1) The isolated type 3 or type 55 human adenovirus was used as an antigen to repeatedly immunize 6-8 week old female BALB / c mice with 1×10 5 TCID 50 Mice were initially immunized by subcutaneous injection of 250 μL of antigen mixed with an equal volume of complete Freund's adjuvant at multiple sites (500 μL / mice). Subsequent immunizations were performed using an equal volume mixture of incomplete Freund's adjuvant and antigen (1 × 10⁶ mice). 5 TCID 50 Mice were immunized five times (including the initial immunization), with each immunization spaced two weeks apart. On day 12 after the fourth immunization, blood was collected from the orbital cavity of mice, and serum titer was detected using an indirect ELISA method. Serum obtained from orbital cavity blood collection before the initial immunization served as a negative control. When the immunogenicity reached 100,000 or higher, mice with the highest titer were selected three days before cell fusion and immunized at a dose of 1×10⁻⁶. 5TCID 50 250 μL of antigen per mouse was emulsified with an equal volume of incomplete Freund's adjuvant (500 μL per mouse) and administered intraperitoneally as a pulse immunization to mice. Three days after the pulse immunization, spleen cells were collected from the mice and fused with SP2 / 0 cells to obtain fusion cells (hybridoma cells). The specific steps of the indirect ELISA method are as follows: 1) HADV-3 or HADV-55 was inactivated (treated in a metal bath at 56°C for 1 hour; the inactivation effect can be judged by observing whether CPE occurs after 96 hours when seeded into HEP2 cells). 8 × 10⁸ cells were coated onto a 96-well polystyrene microplate. 3 TCID 50 1) Add 100 μL of inactivated HADV-3 or HADV-3 virus particles to each well and incubate at 37°C for 2 hours. Then, block the 96-well plate with blocking buffer. 2) Inactivate mouse serum by treating it in a metal bath at 56°C for 30 minutes and use it as the antibody sample. Dilute the inactivated serum 10,000 times in the first well, and use this as the initial concentration for eight 2-fold serial dilutions. Add 100 μL of the diluted monoclonal antibody to each well of the above-mentioned ELISA plate and incubate at 37°C for 30 minutes. Wash the microplate five times with ELISA wash buffer (PBST). Add 100 μL of HRP-goat anti-mouse IgG (diluted 1:5000 with 5% skim milk powder) to each well and incubate at 37°C for 30 min. Then wash the microplate five times with ELISA wash buffer (PBST). Add 100 μL of ELISA reagent to each well and incubate at 37°C in the dark for 15 min. Finally, add 50 μL of H2SO2 solution to stop the reaction. Read the OD values ​​on a microplate reader within 15 min. 450 Absorbance value in nm. Sample well OD 450 OD of negative control well 450 A ratio > 2.1 is considered positive.

[0104] (2) Take 50 μL of cell supernatant obtained from the culture of hybridoma cells as the test sample, and screen hybridoma cells according to the indirect ELISA method established above. The hybridoma cell lines identified as positive were subcloned three times using the limiting dilution method, and finally selected strong positive single cell lines against human adenovirus type 3 or 55 for expansion culture.

[0105] (3) Take 80 μL of cell supernatant obtained from the culture of hybridoma cells as the test sample, and screen hybridoma cells according to the micro-neutralization assay. First, take 80 μL of cell supernatant into the first well, and then take 40 μL for serial dilution by 2-fold serial dilution, and dilute 8 times; prepare 200 TCID2. 50 / 35μL of HAdV-3 or HAdV-55, mixed with the diluted cell supernatant at 40 μL / well, and incubated in a 37°C incubator for 1 h; after the incubation was completed, the HEP2 cell plate was washed, 35 μL / well of the mixed solution was taken to the cell plate, and negative and positive controls were added at the same time, and incubated in a 37°C, 5% CO2 incubator for 1 h; 200 μL / well of maintenance culture medium was added after 1 h, and incubated in a 37°C, 5% CO2 incubator for 96 h, and the cytopathic effect (CPE) was observed. Finally, the single cell strain that can effectively inhibit the cytopathic effect of type 3 or type 55 human adenovirus in the cell supernatant was selected for large-scale culture.

[0106] (4) One week in advance, 500 μL / injection of incomplete Freund's adjuvant (or paraffin oil) was injected intraperitoneally into the mice, and 5×10 5 ~1×10 6 hybridoma cells (the injection volume was not higher than 500 μL) were injected into each mouse, and then the ascites produced was collected, and the ascites produced by in vivo culture of the mice was diluted and impurities were removed, and purified by affinity chromatography (Protein A agarose purification resin). The monoclonal antibodies secreted by the hybridoma cells were labeled as 10G12, 12F12, 2E8 or 2F5, 20 μL of the antibodies was added to 5 μL of SDS-PAGE Loading Buffer (5×) (product number: G3422-2), mixed, and then placed in a metal bath at 100°C for 10 min, and the antibodies were denatured, and the heavy chains and light chains were dissociated. Amy KD-PAGE gel rapid preparation kit (product number: ATG0046) was used to prepare the gel, and the sample loading amount was 20 μL, and the gel was run at 100 V for 120 min.

[0107] The SDS-PAGE detection results are shown in Figure 1 .

[0108] The heavy chains and light chains of the 10G12, 12F12, 2E8 or 2F5 monoclonal antibodies were dissociated, and two bands were presented, with the heavy chain being about 55 kD and the light chain being about 25 kD.

[0109] The hybridoma cells prepared according to the above method can continuously and efficiently secrete monoclonal antibodies, and the method for preparing the hybridoma cells is simple and easy to operate. Compared with in vitro culture, in vivo culture of the hybridoma cells in mice can effectively reduce the influence of adverse external environmental factors on the proliferation and expression of monoclonal antibodies by the hybridoma cells, and can provide a relatively stable living environment for the survival of the hybridoma cells, and is not easy to be contaminated, and has high production efficiency.

[0110] Example 2: Amplification of mouse hybridoma antibody genes

[0111] The hybridoma cells prepared above were cultured in a T75 cell culture flask to a density of 70% to 90%, centrifuged to collect the cells, washed with 10 mL of 1xPBS, and repeated twice, and the hybridoma cells were left after centrifugation for use. After extracting the RNA of the hybridoma cells using the AG SteadyPure Universal RNA Extraction Kit II (item number AG21022), the extracted RNA was reverse transcribed into cDNA using the Takara PrimeScript™ II 1st Strand cDNA Synthesis Kit (item number: 6210A) (which can be stored at -20°C or -80°C for later use). The light chain and heavy chain of the monoclonal antibodies 10G12, 12F12, 2E8 and 2F5 were respectively amplified by PCR using the Takara Taq™ Version 2.0 plus dye premix enzyme (item number: RR901A), and the PCR products obtained by amplification were analyzed by 1% to 2% agarose gel electrophoresis. The bands with a size of 300 bp to 500 bp were sent for sequencing. The sequencing results were analyzed for gene sequence on the VBASE2 website, and the heavy and light chain variable region sequences of the monoclonal antibodies 10G12, 12F12, 2E8 and 2F5 were obtained (Table 2). The PCR amplification conditions were as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 40 s, 58°C annealing for 40 s, 72°C extension for 1 min, a total of 35 cycles; and 72°C for 10 min. The primers used for PCR amplification are shown in Table 1. The heavy chain downstream primer was combined with 15 heavy chain upstream primers for sequencing, and the light chain downstream primer was combined with 16 light chain upstream primers for sequencing.

[0112] The heavy and light chain variable region sequences of the monoclonal antibodies 10G12, 12F12, 2E8 and 2F5 are shown in Table 2.

[0113] Table 1 Primer sequences

[0114]

[0115]

[0116] Table 2 Sequence information of monoclonal antibodies 10G12, 12F12, 2E8 and 2F5

[0117]

[0118]

[0119]

[0120] Example 3: Evaluation of the binding affinity of monoclonal antibodies 10G12, 12F12, 2E8, and 2F5 to inactivated HADV-3 or HADV-55 viral particles, HADV-3 or HADV-55 hexagonal proteins, and HADV-7 ciliary proteins.

[0121] In a 96-well polystyrene microplate, 8×10⁸ μL of coating was used. 3 TCID 50 100 μL of inactivated HADV-3 or HADV-55 viral particles and 100 ng / well of HADV-3 or HADV-55 hexagonal protein were added to each well. The 96-well plate was then blocked with blocking buffer. The monoclonal antibody to be tested (10G12, 12F12, 2E8, or 2F5) was diluted to 10 μg / mL, and this was used as the initial concentration for 16 serial dilutions of 2-fold. 100 μL of the diluted antibody was added to each well of the ELISA plate, and the plates were incubated at 37°C for 30 min. The plates were washed five times with ELISA wash buffer (PBST), followed by the addition of 100 μL of diluted horseradish peroxidase-labeled secondary antibody, and incubated at 37°C for 30 min. After washing the plates five times with PBST, the chromogenic reagent was added, and the plates were incubated for 15 min. The OD values ​​were then read using a microplate reader. 450 The absorbance value in nm.

[0122] The results are as follows Figure 2 As shown, monoclonal antibodies 10G12 and 12F12 can effectively bind to inactivated HADV-3 viral particles, with a half-maximal effective concentration (EC50) of 10G12. 50 The concentrations were 70.43 and 100.9 ng / mL, respectively, but no binding to the hexagonal protein of HADV-3 was observed; monoclonal antibodies 2E8 and 2F5 not only effectively bound HADV-55 viral particles, but also achieved a half-maximal effective concentration (EC50) of 100.9 ng / mL. 50 The concentrations were 16.512 and 0.4046 μg / mL, respectively, and it also showed binding activity with the hexagonal protein of HAdV-55, with a half-maximal effective concentration (EC50) of 16.512 and 0.4046 μg / mL. 50 The concentrations were 8.854 and 1.646 μg / mL, respectively.

[0123] The results are as follows Figure 3 As shown, monoclonal antibodies 10G12, 12F12, 2E8 and 2F5 did not show effective binding to HADV-7 ciliary protein.

[0124] Example 4: Evaluation of the ability of monoclonal antibodies 10G12, 12F12, 2E8, and 2F5 to neutralize live HADV-3 or HADV-55 viruses in vitro.

[0125] The purified monoclonal antibodies 10G12, 12F12, 2E8 and 2F5 were respectively diluted by 2 times from 100 μg / mL to the 8th gradient, and then mixed with 100 TCID 50 / 35 μL of HAdV-3 or HAdV-55 virus at 37℃ for 1h. After the incubation, 35 μL of the virus-antibody mixture was added to a 96-well plate pre-seeded with HEP2 cells, and incubated at 37℃ for 1h. Then 200 μL / well of maintenance medium was supplemented, and the plate was cultured at 37℃ in a 5% CO2 incubator for 4 days, and the cytopathic effect (CPE) was observed, and the neutralization titers of the monoclonal antibodies 10G12, 12F12, 2E8 and 2F5 were calculated.

[0126] The results are shown in Table 1. Figure 4 The neutralization activities of the monoclonal antibodies 10G12 and 12F12 against HAdV-3 and HAdV-55 live viruses at different concentrations showed that both of them could effectively neutralize HAdV-3 live virus, and the half inhibitory concentrations (IC 50 ) were 5.33 and 13.41 μg / mL respectively, but no neutralization activity against HAdV-55 live virus was shown; the neutralization activities of the monoclonal antibodies 2E8 and 2F5 against HAdV-3 and HAdV-55 live viruses at different concentrations showed that both of them could effectively neutralize HAdV-55 live virus, and the half inhibitory concentrations (IC 50 ) were 3.162 and 26.572 μg / mL respectively, but no neutralization activity against HAdV-3 live virus was shown; it was indicated that the monoclonal antibodies 10G12 and 12F12 targeting HAdV-3 virus particles and the monoclonal antibodies 2E8 and 2F5 targeting HAdV-55 virus particles could not show cross-protection.

[0127] In summary, the monoclonal antibodies 10G12, 12F12, 2E8 and 2F5 not only have good binding activity and can effectively bind to HAdV-3 or HAdV-55 virus particles, but also can inhibit the infection of type 3 or type 55 human adenovirus to cells, and have excellent neutralization activity. It is of great significance for the development of new drugs and universal vaccines for the neutralization of type 3 or type 55 human adenovirus in clinic.

[0128] The embodiments of the application are described in detail above with reference to the drawings, but the application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the purpose of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A monoclonal antibody against human adenovirus type B, characterized in that the monoclonal antibody is 10G12, 12F12, 2F5 or 2E8, and the monoclonal antibody includes a light chain variable region and a heavy chain variable region; The light chain variable region of the 10G12 includes CDR1, CDR2, and CDR3, whose amino acid sequences are shown in SEQ ID NO:34-36. The heavy chain variable region of the 10G12 includes CDR1, CDR2, and CDR3, whose amino acid sequences are shown in SEQ ID NO:37-39. The light chain variable region of 12F12 includes CDR1, CDR2, and CDR3, and their amino acid sequences are shown in SEQ ID NO:44-46; the heavy chain variable region of 12F12 includes CDR1, CDR2, and CDR3, and their amino acid sequences are shown in SEQ ID NO:47-49; the light chain variable region of 2F5 includes CDR1, CDR2, and CDR3, and their amino acid sequences are shown in SEQ ID NO:54-56; the heavy chain variable region of 2F5 includes CDR1, CDR2, and CDR3, and their amino acid sequences are shown in SEQ ID NO:57-59; the light chain variable region of 2E8 includes CDR1, CDR2, and CDR3, and their amino acid sequences are shown in SEQ ID NO:54-56; the heavy chain variable region of 2E8 includes CDR1, CDR2, and CDR3, and their amino acid sequences are shown in SEQ ID NO:64-66.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the 10G12 is as follows: A1)SEQ ID NO:40; or A2) An amino acid sequence that has at least 90% homology with SEQ ID NO:40 and is functionally identical or similar; or A3) An amino acid sequence that has been modified by substitution, deletion or addition of one or more amino acids as shown in SEQ ID NO:40 and is functionally identical or similar. Preferably, the amino acid sequence of the heavy chain variable region of the 10G12 is as follows: B1)SEQ ID NO:42; or B2) An amino acid sequence that has at least 90% homology with SEQ ID NO:42 and is functionally identical or similar; or B3) An amino acid sequence that has been modified by substitution, deletion or addition of one or more amino acids as shown in SEQ ID NO:42 and is functionally identical or similar. Preferably, the amino acid sequence of the light chain variable region of the 12F12 is as follows: C1)SEQ ID NO:50; or C2) An amino acid sequence that has at least 90% homology with SEQ ID NO:50 and is functionally identical or similar; or C3) An amino acid sequence that has been modified by substitution, deletion or addition of one or more amino acids as shown in SEQ ID NO:50 and is functionally identical or similar. Preferably, the amino acid sequence of the heavy chain variable region of the 12F12 is as follows: D1)SEQ ID NO:52; or D2) An amino acid sequence that has at least 90% homology with SEQ ID NO:52 and is functionally identical or similar; or D3) An amino acid sequence that has been modified by substitution, deletion or addition of one or more amino acids as shown in SEQ ID NO:52 and is functionally identical or similar.

3. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of 2F5 is: E1)SEQ ID NO:60; or E2) An amino acid sequence that has at least 90% homology with SEQ ID NO:60 and is functionally identical or similar; or E3) An amino acid sequence that has been modified by substitution, deletion or addition of one or more amino acids as shown in SEQ ID NO:60 and is functionally identical or similar. Preferably, the amino acid sequence of the heavy chain variable region of 2F5 is as follows: F1)SEQ ID NO:62; or F2) An amino acid sequence that has at least 90% homology with SEQ ID NO:62 and is functionally identical or similar; or F3) An amino acid sequence that has been modified by substitution, deletion or addition of one or more amino acids as shown in SEQ ID NO:62 and is functionally identical or similar. Preferably, the amino acid sequence of the light chain variable region of 2E8 is as follows: G1)SEQ ID NO:67; or G2) An amino acid sequence that has at least 90% homology with SEQ ID NO:67 and is functionally identical or similar; or G3) An amino acid sequence that has been modified by substitution, deletion or addition of one or more amino acids as shown in SEQ ID NO:67 and is functionally identical or similar. Preferably, the amino acid sequence of the heavy chain variable region of 2E8 is as follows: H1)SEQ ID NO:69; or H2) An amino acid sequence that has at least 90% homology with SEQ ID NO:69 and is functionally identical or similar; or H3) An amino acid sequence that has been modified by substitution, deletion or addition of one or more amino acids as shown in SEQ ID NO:69 and is functionally identical or similar.

4. An antibody, characterized in that, The antibody is any one of M1 to M4. M1) A single-chain antibody obtained by linking the heavy chain variable region and the light chain variable region as described in any one of claims 1 to 3; M2) is a fusion antibody containing the single-chain antibody described in M1; M3) contains the Fab of any one of claims 1 to 3, which includes the heavy chain variable region and the light chain variable region; M4) is a complete antibody containing the heavy chain variable region and the light chain variable region as described in any one of claims 1 to 3.

5. Biological material relating to the monoclonal antibody of any one of claims 1 to 3 or the antibody of claim 4; said biological material being any one of a1) to a12): a1) A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1 to 3 or the antibody of claim 4; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecules described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4); a9) Transgenic cell lines containing the nucleic acid molecules described in a1); a10) Transgenic cell lines containing the expression cassette described in a2); a11) Transgenic cell lines containing the recombinant vector described in a3); a12) Transgenic cell lines containing the recombinant vector described in a4).

6. A coupling comprising: The monoclonal antibody according to any one of claims 1 to 3 or the antibody according to claim 4; And the coupling part; Preferably, the coupling portion comprises at least one of the following: a detectable marker, a drug, a toxin, an electron-dense marker, a biotin / avidin protein, a spin marker, an antibody, a radionuclide, an enzyme, gold nanoparticles / nanorods, magnetic nanoparticles, and a viral capsid protein.

7. The use of the monoclonal antibody of any one of claims 1 to 3, the antibody of claim 4, the biological material of claim 5, or the conjugate of claim 6 in at least one of b1) to b8): b1) Prepare products for the prevention or treatment of adenovirus infection-related diseases; b2) Detection of adenovirus; b3) Prepare products for detecting adenovirus; b4) Inhibits adenovirus activity; b5) Prepare products that inhibit adenovirus activity; b6) Prepare products for diagnosing adenovirus infection; b7) Neutralizes the virulence of adenovirus; b8) Prepare products for neutralizing the virulence of adenoviruses.

8. The application according to claim 7, characterized in that, The product includes at least one of the following: vaccine, drug, reagent, test plate, reagent kit, and test chip.

9. A product characterized in that, The product includes any one of the monoclonal antibodies of claims 1 to 3, the antibody of claim 4, the biomaterial of claim 5, or the conjugate of claim 6; Preferably, the product has at least one function selected from c1) to c5): c1) Prevention or treatment of adenovirus infection-related diseases; c2) Detection of adenovirus; c3) Inhibits adenovirus activity; c4) Diagnosis of adenovirus infection; c5) neutralizes the virulence of adenovirus; Preferably, the product comprises at least one of a vaccine, a drug, a reagent, a test plate, a test kit, and a test chip.

10. A method for detecting adenovirus, comprising contacting a sample containing or suspected of containing adenovirus with any one of claims 1 to 3, the antibody of claim 4, the conjugate of claim 6, or the product of claim 9, and detecting the formation of an adenovirus monoclonal antibody or a complex of antibody and adenovirus, said method for non-disease diagnostic purposes.

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