Anti-adenovirus monoclonal antibodies or their antigen-binding fragments and their nucleic acid molecules and applications

Abstract

This invention discloses an anti-adenovirus monoclonal antibody or its antigen-binding fragment, its nucleic acid molecule, and its applications. The technical problem this invention aims to solve is how to inhibit adenoviruses, such as human adenovirus type 55 (HAdV55). This invention provides an anti-adenovirus monoclonal antibody or its antigen-binding fragment containing six complementarity-determining regions: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, and HCDR3 are as shown in positions 26-33, 51-58, and 97-112 of sequence 2, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as shown in positions 27-38, 56-58, and 95-103 of sequence 2, respectively. The anti-adenovirus monoclonal antibody of this invention can effectively inhibit adenovirus HAdV55 infection.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody against adenovirus or its antigen-binding fragment, its nucleic acid molecule, and its applications. Background Technology

[0002] Human adenovirus is a double-stranded DNA virus belonging to the genus Adenovirus of mammals in the family Adenoviridae. The virus has an icosahedral structure, no outer envelope, and a genome length of about 36kb. The genomic DNA and viral structural proteins combine to form the viral core, which is covered by a capsid. The capsid consists of 252 capsomeres, of which 240 are hexagonal proteins and 12 are pentagonal proteins, with a diameter of about 70-90nm.

[0003] Currently, seven subgroups (AG) and 67 different serotypes of adenovirus have been identified, of which 55 subtypes can infect humans and cause disease. Adenovirus infection in humans most commonly infects the respiratory tract, causing respiratory diseases. In addition, some viruses can also cause infections of the urinary tract and gastrointestinal system. Studies have shown that adenoviruses causing respiratory infections include groups A, C, E, and B1, with subgroup B1 being the most prevalent. Subgroup B2 can infect the human urinary system, while groups F and D can infect the human gastrointestinal system and conjunctival system, respectively. Currently, the known adenoviruses causing respiratory infections worldwide are subgroups B1: 1, 2, 3, 4, 7, 14, and 55. Among these, subtypes 3, 4, 7, 14, and 55 are the most common types causing outbreaks. Adenovirus infection can also induce a strong humoral immune response in humans, producing specific antibodies and neutralizing antibodies. These antibodies can not only neutralize the virus and clear the infection, but also maintain immunity for a considerable period of time. Studies have shown that once a person is infected with adenovirus, they generally will not be reinfected with the same type of adenovirus. This immune protection can last for more than 10 years. The neutralizing antibodies induced by the adenovirus infection process play a crucial role in this process. In addition, antibodies produced by the mother can also protect the infant from severe adenovirus infection.

[0004] Currently, there are no specific antiviral drugs for adenovirus in clinical use worldwide, and drug development is mostly limited to chemical drugs—nucleoside analogs against DNA viruses—while specific biological drugs targeting adenovirus are still lacking. Therefore, adenovirus neutralizing antibodies could serve as an effective and specific antiviral treatment, and researching adenovirus neutralizing antibodies should be an important direction for future development in the treatment of adenovirus infections. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to inhibit adenoviruses, such as human adenovirus type 55.

[0006] To address the above technical problems, the present invention first provides a monoclonal antibody against adenovirus or its antigen-binding fragment.

[0007] The monoclonal antibody against adenovirus or its antigen-binding fragment provided by the present invention includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes three complementarity-determining regions HCDR1, HCDR2 and HCDR3; the light chain variable region includes three complementarity-determining regions LCDR1, LCDR2 and LCDR3.

[0008] The amino acid sequence of HCDR1 mentioned above is positions 26-33 of sequence 2.

[0009] The amino acid sequence of HCDR2 mentioned above is positions 51-58 of sequence 2.

[0010] The amino acid sequence of HCDR3 mentioned above is positions 97-112 of sequence 2.

[0011] The amino acid sequence of LCDR1 mentioned above is positions 27-38 of sequence 4.

[0012] The amino acid sequence of LCDR2 mentioned above is positions 56-58 of sequence 4.

[0013] The amino acid sequence of the above LCDR3 is positions 95-103 of sequence 4.

[0014] The antibodies mentioned above can be full-length antibodies. The antigen-binding fragments mentioned above can be Fab fragments, Fv fragments, Fab′ fragments, F(ab′)2 fragments, single-chain antibodies (ScFv), nanobodies (single-domain antibodies), or minimal recognition units (MRUs).

[0015] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. This antigen-binding fragment retains at least some of the binding specificity of the parent antibody. Generally, when activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent antibody's binding affinity to the target. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target.

[0016] The term "Fab fragment" refers to a heterodimer composed of a heavy chain (Fd) and a complete light chain linked by disulfide bonds, containing only one antigen-binding site. The aforementioned heavy chain (Fd) refers to approximately half of the H chain portion of the Fab (containing approximately 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0017] The term "Fv fragment" refers to a vector containing VH and VL genes that can be constructed separately, co-transfected into cells to express them separately, and then assembled into a functional Fv antibody; alternatively, a stop codon can be set between VH and VL in the vector to express two small protein fragments, which can then be bound together by non-covalent bonds to form an Fv antibody (Fv fragment).

[0018] The term "Fab′ fragment" contains a portion of a light chain and a heavy chain containing the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of two Fab′ fragments to form the F(ab′)2 molecule.

[0019] The term "F(ab′)2 segment" contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab′)2 segment consists of two Fab′ segments held together by the disulfide bond between the two heavy chains.

[0020] The term "single-chain antibody (ScFv)" refers to a polypeptide formed by linking a light chain variable region and a heavy chain variable region. This polypeptide can spontaneously fold into its native conformation, maintaining the specificity and affinity of Fv.

[0021] The term "nanobody (single-domain antibody)" refers to an antibody containing only the VH fragment, obtained by expressing the V region of the antibody heavy chain through genetic engineering. The ability of single-domain antibodies to bind to antigens and their stability are essentially the same as those of complete antibodies.

[0022] The term "minimum recognition unit (MRU)" refers to a single CDR structure containing only the variable region, with a molecular weight of only about 1% of that of a complete antibody, which can bind to the corresponding antigen.

[0023] The monoclonal antibody mentioned above can be a fusion antibody of the single-chain antibody mentioned above.

[0024] In the above-mentioned monoclonal antibody or its antigen-binding fragment, the amino acid sequence of the heavy chain variable region can be sequence 6, and the amino acid sequence of the light chain variable region can be sequence 8.

[0025] In one specific embodiment of the present invention, a humanized monoclonal antibody named h55Ab9-8 is provided. The amino acid sequence of the heavy chain variable region of h55Ab9-8 is sequence 6, and the amino acid sequence of the light chain variable region is sequence 8.

[0026] In the above-mentioned monoclonal antibody or its antigen-binding fragment, the amino acid sequence of the heavy chain variable region can be sequence 2, and the amino acid sequence of the light chain variable region can be sequence 4.

[0027] In one specific embodiment of the present invention, a human-mouse chimeric monoclonal antibody named 55Ab9-8 is provided. The amino acid sequence of the heavy chain variable region of h55Ab9-8 is sequence 2, and the amino acid sequence of the light chain variable region is sequence 4.

[0028] The present invention also provides nucleic acid molecules encoding the above-mentioned monoclonal antibodies or their antigen-binding fragments.

[0029] The aforementioned nucleic acid molecules can be DNA, such as cDNA, genomic DNA, or recombinant DNA; they can also be RNA, such as mRNA or hnRNA.

[0030] The aforementioned nucleic acid molecules may be genes encoding the aforementioned monoclonal antibodies or their antigen-binding fragments.

[0031] The aforementioned genes can be DNA molecules containing the coding genes for HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the aforementioned DNA molecules can be A) or B):

[0032] A) The coding gene for HCDR1 is nucleotides 76-99 of sequence 5, the coding gene for HCDR2 is nucleotides 151-174 of sequence 5, the coding gene for HCDR3 is nucleotides 289-336 of sequence 5, the coding gene for LCDR1 is nucleotides 79-114 of sequence 7, the coding gene for LCDR2 is nucleotides 166-174 of sequence 7, and the coding gene for LCDR3 is nucleotides 283-309 of sequence 7.

[0033] B) The coding gene for HCDR1 is nucleotides 76-99 of sequence 1, the coding gene for HCDR2 is nucleotides 151-174 of sequence 1, the coding gene for HCDR3 is nucleotides 289-336 of sequence 1, the coding gene for LCDR1 is nucleotides 79-114 of sequence 3, the coding gene for LCDR2 is nucleotides 166-174 of sequence 3, and the coding gene for LCDR3 is nucleotides 283-309 of sequence 3.

[0034] The above-mentioned genes can be DNA molecules of either C) or D):

[0035] C) The gene encoding the above-mentioned heavy chain variable region and the gene encoding the above-mentioned light chain variable region, wherein the nucleotide sequence of the gene encoding the above-mentioned heavy chain variable region is sequence 5 and the nucleotide sequence of the gene encoding the above-mentioned light chain variable region is sequence 7.

[0036] D) The gene encoding the above-mentioned heavy chain variable region and the gene encoding the above-mentioned light chain variable region, wherein the nucleotide sequence of the gene encoding the above-mentioned heavy chain variable region is sequence 1 and the nucleotide sequence of the gene encoding the above-mentioned light chain variable region is sequence 3.

[0037] The DNA molecules in A) and C) above encode the h55Ab9-8 gene. The DNA molecules in B) and D) above encode the 55Ab9-8 gene.

[0038] This invention also provides biomaterials related to the aforementioned nucleic acid molecules.

[0039] The biomaterial provided by this invention may be any of the following:

[0040] B1) An expression cassette containing any of the above-mentioned nucleic acid molecules;

[0041] B2) A recombinant vector containing the expression cassette described in B1);

[0042] B3) Recombinant microorganisms containing the above-mentioned nucleic acid molecules;

[0043] B4) Recombinant microorganisms containing the expression cassette described in B1);

[0044] B5) Recombinant microorganisms containing the recombinant vector described above in B2);

[0045] B6) Transgenic animal cell lines containing the above-mentioned nucleic acid molecules;

[0046] B7) Transgenic animal cell lines containing the expression cassettes described in B1);

[0047] B8) Transgenic animal cell lines containing the above-mentioned recombinant vectors (B2).

[0048] In the aforementioned biological materials, the aforementioned expression cassette refers to DNA capable of expressing the aforementioned monoclonal antibody or its antigen-binding fragment in host cells. This DNA may include not only a promoter to initiate transcription of the aforementioned monoclonal antibody or its antigen-binding fragment gene, but also a terminator to terminate transcription of the aforementioned monoclonal antibody or its antigen-binding fragment gene. Furthermore, the aforementioned expression cassette may also include an enhancer sequence. Recombinant vectors containing the aforementioned monoclonal antibody gene expression cassette can be constructed using existing expression vectors.

[0049] Among the aforementioned biological materials, the aforementioned vectors may be plasmids, granules, bacteriophages, or viral vectors.

[0050] Among the aforementioned biological materials, the aforementioned microorganisms may be yeast, bacteria, algae, or fungi.

[0051] Among the aforementioned biological materials, the aforementioned transgenic animal cell lines may be non-reproductive materials. The aforementioned animal cell lines are not germ cells, fertilized eggs, or embryonic stem cells.

[0052] This invention protects the use of any of the above-described monoclonal antibodies or their antigen-binding fragments or any of the above-described nucleic acid molecules, the uses being (e1) and / or (e2).

[0053] (e1) The use of the above-mentioned monoclonal antibody or its antigen-binding fragment or any of the above-mentioned nucleic acid molecules in the preparation of adenovirus inhibitors.

[0054] (e2) Use of the above-described monoclonal antibody or its antigen-binding fragment or any of the above-described nucleic acid molecules in the preparation of a medicament for the prevention and / or treatment of diseases caused by adenovirus infection.

[0055] This invention also protects products. These products may be adenovirus inhibitors, or medicaments for the prevention and / or treatment of diseases caused by adenovirus infection, and may contain any of the aforementioned monoclonal antibodies or their antigen-binding fragments.

[0056] The adenovirus mentioned above can be a human adenovirus; the human adenovirus mentioned above can be human adenovirus type 55.

[0057] The aforementioned adenovirus inhibitors can neutralize the aforementioned adenoviruses. These adenovirus inhibitors can inhibit the infection of animals or humans by the aforementioned adenoviruses.

[0058] In practical applications, the monoclonal antibody or its antigen-binding fragment of the present invention can be administered directly to patients as a drug, or mixed with a suitable carrier or excipient and administered to patients to achieve the purpose of treating and / or preventing HIV infection. The carrier materials here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. Various dosage forms can be formulated using these materials, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. Suppositories can be vaginal suppositories, vaginal rings, or ointments, creams, or gels suitable for vaginal application. They can be ordinary preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into tablets. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, and ethylcellulose. Various carriers known in the art can be widely used to formulate unit dosage forms into suppositories. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.To formulate unit-dose dosage forms for injection, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. Additionally, to prepare isotonic injections, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injection formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Moreover, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation.

[0059] The above dosage forms can be used for administration via injection, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracerebrospinal injection or infusion, etc.; cavity administration, such as rectal, vaginal and sublingual; respiratory administration, such as nasal administration; and mucosal administration.

[0060] Experiments have shown that the 55Ab9-8 antibody binds to human adenovirus type 55 at an effective half-maximum concentration (EC50). 50 The value is 0.0098 nM (results are as follows). Figure 4 (as shown); the half-maximal inhibitory concentration (IC50) of 55Ab9-8 antibody against HADV55. 50 The value is 0.067 nM (results are as follows). Figure 5 (as shown); the half-maximal effective concentration (EC50) of h55Ab9-8 antibody binding to HADV55. 50 The value is 0.0702 nM (results are as follows). Figure 6 (as shown); the half-maximal inhibitory concentration (IC50) of h55Ab9-8 against HADV55 50 The value is 0.3886 nM (results are as follows). Figure 7 (As shown). The results indicate that the monoclonal antibodies 55Ab9-8 and h55Ab9-8 prepared in this invention can effectively inhibit adenovirus HADV55 infection. Attached Figure Description

[0061] Figure 1 This study aimed to detect specific antibodies against human adenovirus type 55 in mouse serum.

[0062] Figure 2 This is for SDS-PAGE electrophoresis detection of purified 55Ab9-8.

[0063] Figure 3 SDS-PAGE electrophoresis detection of purified human adenovirus type 55.

[0064] Figure 4 The binding activity of antibody 55Ab9-8 to human adenovirus type 55 was measured.

[0065] Figure 5The efficacy of antibody 55Ab9-8 against human adenovirus type 55 infection.

[0066] Figure 6 The binding activity of the humanized antibody h55Ab9-8 to human adenovirus type 55 was measured.

[0067] Figure 7 The efficacy of the humanized antibody h55Ab9-8 against human adenovirus type 55 infection. Detailed Implementation

[0068] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0070] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0071] The HAdV55 virus in the following examples is human adenovirus type 55 (hereinafter referred to as adenovirus type 55), which is described in the doctoral dissertation: Study on the interaction between human adenovirus type 55 and the host based on multi-omics, Wang Kaiying, 2021; the public may obtain this biological material from the Academy of Military Medical Sciences of the Chinese People's Liberation Army in accordance with the relevant national biosafety regulations. This biological material is only used to repeat the relevant experiments of this invention and shall not be used for other purposes.

[0072] The sequences involved in the following embodiments are shown in Table 1.

[0073] Table 1. Relevant sequences of monoclonal antibodies

[0074]

[0075]

[0076]

[0077] Example 1: Discovery of Antibodies

[0078] I. Mouse Immunization and Preparation of Polyclonal Antibodies

[0079] 1. Immunization: SPF-grade female Balb / c mice aged 4-6 weeks were randomly divided into groups of 2. Purified HAdV55 inactivated virus particles (prepared in step 1 of Example 3) were added to 1 / 10 volume of aluminum adjuvant (Bioss, C5084), and thoroughly emulsified and mixed. 100 μl (containing 25 μg of virus particles) was injected intramuscularly into each mouse. Unimmunized mice were set up as a blank control group. Immunization was carried out at 2-week intervals using the same dose and method, for a total of 4 immunizations.

[0080] 2. Preparation of adenovirus type 55 antiserum: Blood was collected two weeks after the last immunization. After being placed at 4°C overnight, the serum was centrifuged at 5,000 rpm for 30 min. The serum was then transferred to EP tubes and inactivated for complement at 56°C for 30 min to obtain adenovirus type 55 antiserum. The serum was aliquoted and frozen at -80°C for later use.

[0081] 3. Detection of antibody titers in serum:

[0082] (1) Take 200 ng of HADV55 inactivated virus prepared in Example 3, add carbonate coating buffer (pH 9.6) to 100 μL, add 100 μL to each well of the microplate (Corning, catalog number: 9018), and coat overnight at 4°C.

[0083] (2) After completing the above steps, take the enzyme-labeled plate, wash it 3 times with PBST, add PBS blocking solution containing 2% (mass percentage) BSA, and incubate at 37°C for 2 hours.

[0084] (3) After completing the above steps, take the enzyme-labeled plate, discard the blocking solution, add 100 μL of diluted mouse serum per well (mouse serum is serially diluted 4 times with 2% BSA, with dilution factors of 100, 400, 1600, 6400, 25600, 102400, 409600, and 1638400 respectively), incubate at 37°C for 90 min, and then wash the plate 3 times with PBST.

[0085] (4) After completing the above steps, take the enzyme-labeled plate, add 100 μL of 1:4000 diluted HRP-labeled anti-human IgG antibody (Zhongshan Jinqiao, catalog number ZB-2304) to each well, incubate at 37℃ for 45 min, and wash the plate 3 times with PBST.

[0086] (5) After completing the above steps, take the enzyme-labeled plate, add 50 μL of OPD substrate color development solution to each well, and incubate at room temperature for 10 minutes.

[0087] (6) After completing the above steps, take the enzyme-labeled plate and add 50 μL of 1M sulfuric acid solution to each well to terminate the enzyme-linked reaction.

[0088] (7) The optical density value was measured using a dual-wavelength microplate reader at 492nm / 630nm.

[0089] The results are as follows Figure 1 As shown in the figure, the horizontal axis represents serum dilution, and the vertical axis represents optical density. Analysis showed that, compared to pre-immunization mouse serum samples, post-immunization serum exhibited effective and specific binding activity against HAdV55, confirming the production of specific antibodies.

[0090] II. Preparation of the Immune Library

[0091] Two weeks after the final immunization, mice were sacrificed, and spleens were harvested. Spleen cells were isolated, and total RNA was extracted from the isolated spleen cells using a total RNA extraction kit (Tiangen, DP430). Using the extracted total RNA as a template, the variable regions of the heavy and light chains were synthesized separately using a first-strand cDNA synthesis kit (Thermo Scientific, K1621). Gene-specific primers were used for reverse transcription, with the primer pairing regions located in the antibody heavy and light chain constant regions, respectively. The specific sequences were PmCGR: 5'-TGCATTTGAACTCCTTGCC-3' and PmCKR: 5'-CCATCAATCTTCCACTTGA C-3'. The synthesized cDNA was immediately stored at -70°C for later use. Then, using the cDNA obtained from reverse transcription as a template, primers were synthesized according to the reference (Journal of Immunological Methods, 201(1997), 35–55), and the variable regions of the mouse antibody heavy chain and light chain were amplified by PCR. Then, single-chain antibodies (scFv) were constructed using overlap extension PCR. Finally, the prepared mouse single-chain antibody gene was cloned into the pADSCFV-S vector (see invention patent 201510097117.0) to construct an scFv library. The library capacity reached 2 × 10⁻⁶. 8 The accuracy rate was 92%.

[0092] II. Screening of mouse single-chain antibody libraries against human adenovirus type 55

[0093] Using purified HADV55 inactivated virus particles (prepared in step one of Example 3) as the antigen, the mouse single-chain antibody phage library constructed above was screened using a solid-phase screening strategy (experimental protocol referenced Phage Display: A Universal Laboratory Guide / edited by (US) Clackson, T. and (US) Lowman, HB; translated by Ma Lan et al. Chemical Industry Press, 2008.5). Three rounds of screening were performed, ultimately obtaining single-chain antibody clone 55Ab9-8 that specifically binds to HADV55 inactivated virus particles. The amino acid sequence of its heavy chain variable region is positions 1-123 from the N-terminus of Sequence 2 (amino acid residues 26-33 from the N-terminus form HCDR1, amino acid residues 51-58 form HCDR2, and amino acid residues 97-112 form HCDR3), and the corresponding coding gene nucleotide sequence is positions 1-369 from the 5' end of Sequence 1. The amino acid sequence of the light chain variable region of the 55Ab9-8 antibody is positions 1-113 from the N-terminus of sequence 4 (ammonia residues 27-38 from the N-terminus form LCDR1, amino acid residues 56-58 form LCDR2, and amino acid residues 95-103 form LCDR3), and the corresponding nucleotide sequence of the coding gene is positions 1-339 from the 5' end of sequence 3.

[0094] Example 2: Preparation of 55Ab9-8 antibody

[0095] I. Construction of Recombinant Plasmids

[0096] 1. Replace the small fragment between the HindIII and BamHI recognition sites in pcDNA3.1(+) (Invitrogen, V79020) with a DNA molecule whose nucleotide sequence is sequence 1 (heavy chain gene of 55Ab9-8 antibody), keeping the other nucleotides of the pcDNA3.1(+) vector unchanged, to obtain the recombinant expression vector pcDNA-9-8H containing the heavy chain gene (which has been verified by sequencing).

[0097] pcDNA-9-8H contains the heavy chain gene of the monoclonal antibody 55Ab9-8, whose nucleotide sequence is sequence 1. The nucleotide sequence of the variable region (VH) is nucleotides 1-369 of sequence 1, containing the coding genes for HCDR1, HCDR2, and HCDR3. The coding gene for HCDR1 is nucleotides 76-99 of sequence 1, the coding gene for HCDR2 is nucleotides 151-174 of sequence 1, and the coding gene for HCDR3 is nucleotides 289-336 of sequence 1. Nucleotides 370-666 encode CH1, nucleotides 667-711 encode Hinge, nucleotides 712-1041 encode CH2, nucleotides 1042-1359 encode CH3, and nucleotides 1360-1362 are stop codons.

[0098] The pcDNA-9-8H expression amino acid sequence is the heavy chain of the monoclonal antibody 55Ab9-8 antibody in sequence 2. The amino acid sequence of the variable region of the heavy chain is positions 1-123 of sequence 2. The variable region of the heavy chain includes three complementarity-determining regions HCDR1, HCDR2 and HCDR3. The amino acid sequence of HCDR1 is positions 26-33 of sequence 2, the amino acid sequence of HCDR2 is positions 51-58 of sequence 2, and the amino acid sequence of HCDR3 is positions 97-112 of sequence 2. Amino acid residues 124-222 form the heavy chain constant region CH1, amino acid residues 223-237 form the heavy chain hinge region Hinge, amino acid residues 238-347 form the heavy chain constant region CH2, and amino acid residues 348-453 form the heavy chain constant region CH3.

[0099] 2. Replace the small fragment between the HindIII and BamHI recognition sites in pcDNA3.1(+) (Invitrogen, V79020) with a DNA molecule whose nucleotide sequence is sequence 3 (the light chain variable region gene of the 55Ab9-8 antibody), while keeping the other nucleotides of pcDNA3.1(+) unchanged, to obtain the recombinant expression vector pcDNA-9-8K containing the light chain gene (which has been verified by sequencing).

[0100] pcDNA-9-8K contains the light chain gene of the monoclonal antibody 55Ab9-8, whose nucleotide sequence is sequence 3. The nucleotide sequence encoding the variable region (VL) of the light chain is positions 1-339 of sequence 3, containing the coding genes for LCDR1, LCDR2, and LCDR3. The coding gene for LCDR1 is nucleotides 79-114 of sequence 3, the coding gene for LCDR2 is nucleotides 166-174 of sequence 3, and the coding gene for LCDR3 is nucleotides 283-309 of sequence 3. Nucleotides 348-654 encode the constant region CL of the light chain, and nucleotides 655-657 are the stop codons.

[0101] The pcDNA-9-8K expression amino acid sequence is the light chain of the monoclonal antibody 55Ab9-8 antibody of sequence 4. The amino acid sequence of the variable region of the light chain is positions 1-113 of sequence 4. The variable region of the light chain includes three complementarity-determining regions LCDR1, LCDR2 and LCDR3. The amino acid sequence of LCDR1 is positions 27-38 of sequence 4, the amino acid sequence of LCDR2 is positions 56-58 of sequence 4, and the amino acid sequence of LCDR3 is positions 95-103 of sequence 4. The amino acid residues at positions 117-218 constitute the constant region CL of the light chain.

[0102] The CDRs mentioned above are sequences defined according to the Kabat numbering system.

[0103] II. Preparation of 55Ab9-8 antibody

[0104] 1. FreeStyle was used the day before transfection. TM HEK 293-F cells (Invitrogen, catalog number: R79007) were adjusted to a concentration of 1.0 × 10⁻⁶. 6 Cells / mL were inoculated into culture flasks and cultured at 37°C, 5% CO2, and 125 rpm for 24 hours.

[0105] 2. After completing step 1, on the day of transfection, take the culture flask, add the transfection complex to the culture flask, and culture in a cell shaker at 37°C, 5% CO2, and 125 rpm. Start monitoring cell viability after 48 hours. When the cell viability drops to 80-85%, centrifuge at 1,000 rpm for 10 minutes to collect the culture supernatant.

[0106] Transfection complex: Dilute 24 μL of FectoPRO transfection reagent in 3 mL of FreeStyle 293 medium (Gibco12338-018), mix gently, add 12 μg of pcDNA-9-8H and 12 μg of pcDNA-9-8K prepared in step one, mix well, and incubate at room temperature for 10 min.

[0107] 3. After completing step 2, filter the supernatant through a 0.45 μm filter membrane to remove impurities, and add 10×PB to adjust the ion concentration to be close to that of the binding buffer. Purify the antibody using an AKTA purification system (GE, AKTA EXPLORER). Install the HiTrap MabSelect Xtra purification column in the AKTA purification instrument, set the corresponding system parameters, equilibrate the purification column with binding buffer and load the sample, then continue equilibration, and then wash the pre-packed column with citric acid solution (pH 3.0) to elute the antibody protein. Start collecting when UV280 reaches 100 and stop collecting when UV280 drops to 100, and replace the buffer with citrate solution (pH 6.0) to obtain the 55Ab9-8 antibody solution.

[0108] 4. The antibody solution purified in step 3 was subjected to SDS-PAGE electrophoresis to detect antibody expression. The results showed that the molecular weight of the 55Ab9-8 antibody was consistent with the expectation. Figure 2 The left image shows non-reducing polyacrylamide gel electrophoresis (non-reducing SDS-PAGE), with lane 2 from the left containing a full-length monoclonal antibody with a molecular weight of approximately 150 kilodaltons. The right image shows reducing polyacrylamide gel electrophoresis (reducing SDS-PAGE), with lane 4 from the left showing two bands representing the light and heavy chains, with molecular weights of 25 kilodaltons and 50 kilodaltons, respectively. M represents the protein marker. Protein concentration was determined using a NanoDrop UV spectrophotometer (ThermoScientific) and was found to be 0.65 mg / mL.

[0109] Example 3: Detection of the binding ability of 55Ab9-8 antibody

[0110] I. Preparation of Human Adenovirus Type 55 Virus Stock Solution, Concentrated Solution and Inactivated Virus

[0111] 1. Preparation of adenovirus viral fluid

[0112] Adenovirus culture: A549 cells (Beijing Union Medical College Cell Resource Center, catalog number: 25) were cultured in standard DMEM + 10% (volume percentage) FBS medium. The A549 cells were passaged in 75 cm⁻¹ cells one day before virus inoculation. 2In the cell culture flask, ensure the cell density reaches 75%-90% by the time of virus inoculation the next day. On the day of inoculation, slowly aspirate the cell culture medium from the flask, add 5 mL of LDM to gently rinse the cells and discard the solution, then add another 3 mL of LDM. DMEM + 2% (volume percentage) FBS; use a micropipette to aspirate HADV55 virus into a cell culture flask, infect with an MOI ≈ 0.001, shake the flask several times to disperse the virus evenly, and incubate at 37°C, 5% CO2 for 2 hours, shaking the flask approximately every 30 minutes during this period; after adsorption, discard the virus culture medium, add 15 mL of fresh DMEM + 2% (volume percentage) FBS, and then incubate the cell culture flask at 37°C, 5% CO2 for further culture; observe the cytopathic effect daily (cytopathic effects will appear after viral infection and proliferation, manifested as cell shrinkage, shedding, etc.); when 75%-100% of cells show cytopathic effects, harvest the virus culture, freeze and thaw twice at -80°C, centrifuge at 4,000 rpm for 5 minutes to remove cell debris, collect the supernatant, aliquot and store at -80°C, which is the HADV55 virus stock solution.

[0113] 2. Adenovirus titer determination

[0114] One day before the experiment, A549 cells in good growth condition were harvested, digested with trypsin, and then the cell density was adjusted to 3 × 10⁻⁶ cells using DMEM + 10% (volume percentage) FBS. 5 / mL, inoculated into 96-well cell culture plates, 100μL per well, and incubated at 37℃ and 5% CO2; on the day of the experiment, remove the 96-well plates, discard the culture medium, wash once with serum-free medium, add DMEM + 2% (v / v) FBS, 100μL / well; then use serum-free medium to serially dilute the HADV55 virus solution to be tested 10-fold. -1 ~10 -8 Eight dilutions were performed. The diluted virus was added at 10 μL / well to eight wells of a prepared 96-well plate. A blank control group was also included. After the procedure, the cells were incubated at 37°C with 5% CO2. Cell death was counted after 7 days, and the TCID of the original virus solution was calculated using the formula below. 50 .

[0115] Distance ratio = (Percentage of lesions with a rate higher than 50% - 50%) / (Percentage of lesions with a rate higher than 50% - Percentage of lesions with a rate lower than 50%)

[0116] LgTCID 50 = Distance ratio × Difference between the logarithms of dilutions + Logarithm of dilutions with a lesion rate higher than 50%.

[0117] Based on testing and calculation, the viral titer of the HAdV55 virus stock solution used in this study was 4.36 × 10⁻⁶.7 TCID 50 / mL, virus titer of concentrated virus solution = 2.7 × 10⁻⁶ 9 TCID 50 / mL.

[0118] 3. Inactivation and purification of adenovirus

[0119] Transfer the HAdV55 virus stock solution to a 500mL sample vial, adjust the pH to 7.6 with sodium bicarbonate, then add β-propiolactone at a ratio of 1:2000 while stirring. After thorough mixing, continue stirring at 4℃ for inactivation. After 24 hours, adjust the pH to 7.6 again, and add β-propiolactone at a ratio of 1:2000. Continue stirring at 4℃ for another 24 hours for inactivation. Take at least 1‰ of the sample volume and hydrolyze it in a 37℃ water bath for 4 hours (adjust the pH to around 7.0 with sodium bicarbonate when the sample turns yellow). After hydrolysis, take the sample from the previous day and transfer it to a 25cm sample. 2 A549 cells from one 25cm cell culture flask were seeded at a rate of 1 mL of sample. 2 A549 cells were seeded proportionally (less than 1 mL was counted as 1 mL), and cells seeded with non-inactivated virus and untreated empty cells were set up as controls. The cells were incubated in a 37°C, 5% CO2 incubator and observed. After 7 days, the cells were blindly passaged into new A549 cells and observed again. This blind passage was repeated for 3 generations. Cells in the non-inactivated virus sample showed cytopathic effects while cells in the inactivated experimental group and the blank cells did not. The inactivation test results were considered reliable and the inactivation was complete. Otherwise, the inactivation was incomplete and the cells needed to be inactivated and tested again.

[0120] The virus stock solution, confirmed to be completely inactivated by inactivation testing, was centrifuged at 4,000 rpm for 10 minutes to remove cell debris. The Sepharose 4 Fast Flow gel column was equilibrated with PBS buffer and loaded with the sample. The sample was then eluted with PBS, and the first elution peak was the target virus peak. This elution peak was collected, and 12.5 mL of heavy-density cesium chloride solution (42.23 g cesium chloride + 57.77 mL 10 mM Tris-HCl (pH 7.9-8)) was added to an Amicon-Ultra-15 ultrafiltration tube (50 kDa). Then, 12.5 mL of light-density cesium chloride solution (22.39 g cesium chloride + 77.61 mL 10 mM Tris-HCl (pH 7.9-8)) was slowly added, followed by 15 mL of virus suspension. The solution was balanced and centrifuged in an ultracentrifuge (Beckman L100-XP) at 25,000 rpm and 4°C for 2 hours. The bands between the light and heavy density cesium chloride solutions were collected, dialyzed with PBS, and filtered to obtain HADV55 inactivated virus.

[0121] 4. Preparation of adenovirus concentrate: The inactivated HADV55 virus obtained in step 3 was transferred into an ultrafiltration tube (MILLIPORE, catalog number UFC805008) with a molecular weight cutoff of 50 kD. The tube was centrifuged at 4,000 rpm until the volume was reduced to 1 / 30 of the initial volume. The retentate was collected, aliquoted, and stored at -80℃ to obtain the HADV55 virus concentrate. Samples were taken for routine reducing SDS-PAGE analysis. The SDS-PAGE results are shown below. Figure 3 Lane 2 from the left contains the HADV55 inactivated virus obtained in step 3, and lane 3 from the left contains the HADV55 virus concentrate obtained in step 4; M stands for protein marker.

[0122] II. Analysis of the antigen-binding ability of 55Ab9-8 antibody

[0123] The experiment was repeated three times, with each repetition following the same pattern:

[0124] 1. Take 200 ng of HADV55 inactivated virus prepared in step 1, add carbonate coating buffer (pH 9.6) to 100 μL, add 100 μL to each well of the microplate (Corning, catalog number: 9018), and coat overnight at 4°C.

[0125] 2. After completing the above steps, take the ELISA plate, wash it 3 times with PBST, add PBS blocking solution containing 2% (w / w) BSA, and incubate at 37°C for 2 hours.

[0126] 3. After completing the above steps, take the ELISA plate, discard the blocking solution, add 100 μL of 55Ab9-8 antibody solution from Example 2 diluted 2 times (initial concentration 10 μg / mL, diluted with DMEM medium) to each well, set up a total of 24 gradients, with 2 wells for each gradient, incubate at 37℃ for 90 min, and then wash the plate 6 times with PBST.

[0127] 4. After completing the above steps, take the ELISA plate, add 100 μL of 1:4000 diluted HRP-labeled anti-human IgG antibody (Zhongshan Jinqiao, catalog number ZB-2304) to each well, incubate at 37℃ for 45 min, and wash the plate 3 times with PBST.

[0128] 5. After completing the above steps, take the ELISA plate, add 50 μL of OPD substrate chromogenic solution to each well, and incubate at room temperature for 10 minutes.

[0129] 6. After completing the above steps, take the ELISA plate and add 50 μL of 1M sulfuric acid solution to each well to terminate the enzyme-linked reaction.

[0130] 7. The optical density value was measured using a dual-wavelength microplate reader at 492nm / 630nm. Figure 4 OD in492-630 = Absorbance at 492nm wavelength - Absorbance at 630nm wavelength. Half-maximum effect concentration (EC50) 50 This refers to the distinct plateau phases observed at low and high antibody concentrations. The antibody concentration corresponding to half the signal value during the upper plateau phase is the measured OD. 492-630 After the value is obtained, EC can be calculated using the following formula. 50 value.

[0131]

[0132] y represents the detected OD 492-630 The values ​​are min and max, which are the observed minimum and maximum values, respectively. Hill Slope refers to the absolute value of the maximum slope of the curve (i.e., the midpoint of the curve).

[0133] The results are as follows Figure 4 As shown, the half-maximal effective concentration (EC50) of 55Ab9-8 antibody binds to human adenovirus type 55. 50 The value is 0.0098 nM.

[0134] Example 4: Efficacy against human adenovirus type 55 infection

[0135] The experiment was repeated three times, with each repetition following the same pattern:

[0136] 1. One day before the experiment, A549 cells in good growth condition were taken, digested with trypsin, and the cell density was adjusted to 3 × 10⁻⁶ cells / year using DMEM + 10% (volume percentage) FBS. 5 / mL, seeded into 96-well cell culture plates, 100μL per well, and cultured at 37℃ with 5% CO2.

[0137] 2. After completing step 1, remove the 96-well plate on the day of the experiment, discard the culture medium, wash once with serum-free culture medium, and add 100 μL of DMEM + 2% (volume percentage) FBS to each well; then perform the following operations in groups:

[0138] Experimental group (55Ab9-8): The 55Ab9-8 antibody prepared in Example 2 was diluted with serum-free medium to obtain test antibody solutions containing different concentrations (starting concentration of 100 μg / mL, serially diluted 2-fold, for a total of 22 gradients) of 55Ab9-8 antibody, with 2 wells for each gradient; the test antibody solutions were mixed with the HAdV55 virus stock solution prepared in step 1 of Example 3 (diluted with serum-free medium to a virus concentration of 2 × 10⁻⁶). 3 TCID 50 Mix the contents of the sample (100 μL / well) at a volume ratio of 1:1, incubate at 37°C for 1.5 h, then add the mixture to the wells. Continue incubating at 37°C in a 5% CO2 incubator for 1 h.

[0139] Positive antibody control group: Anti-human adenovirus type 55 mouse serum (prepared in Example 1) was diluted with serum-free medium and serially diluted 2-fold to a total of 22 dilutions. The antibody solution to be tested was mixed with the HAdV55 virus stock solution prepared in step 1 of Example 3 (diluted with serum-free medium to a virus concentration of 2×10⁻⁶). 3 TCID 50 Mix the contents of the sample (100 μL / well) at a volume ratio of 1:1, incubate at 37°C for 1.5 h, then add the mixture to the wells. Continue incubating at 37°C in a 5% CO2 incubator for 1 h.

[0140] Irrelevant antibody control group: Irrelevant antibodies (described in invention patent CN102993305B) were diluted with serum-free culture medium to obtain test antibody solutions containing different concentrations (initial concentration of 100 μg / mL, serially diluted 2-fold, for a total of 22 gradients) of Anti-EGFR antibodies; the test antibody solutions were then mixed with the HAdV55 virus stock solution prepared in step 1 of Example 2 (diluted with serum-free culture medium to a virus concentration of 2 × 10⁻⁶). 3 TCID 50 Mix the contents of the sample (100 μL / well) at a volume ratio of 1:1, incubate at 37°C for 1.5 h, then add the mixture to the wells. Continue incubating at 37°C in a 5% CO2 incubator for 1 h.

[0141] Empty cell control group (CELL): Serum-free medium was added to the wells after incubation at 37°C for 1.5 h, and the wells were incubated for another 1 h at 37°C with 5% CO2.

[0142] Virus (VIRUS): Serum-free culture medium was mixed with the HAdV55 virus stock solution prepared in step 1 of Example 3 (diluted with serum-free culture medium to a virus concentration of 2 × 10⁻⁶). 3 TCID 50 Mix the contents of the sample (100 μL / well) at a volume ratio of 1:1, incubate at 37°C for 1.5 h, then add the mixture to the wells. Continue incubating at 37°C in a 5% CO2 incubator for 1 h.

[0143] After completing step 2, take the 96-well plate, discard the supernatant, add DMEM + 2% (volume percentage) FBS medium (100 μL / well), and continue culturing at 37°C with 5% CO2 for 1 week until obvious cytopathic effects appear. After incubation at 37°C (at least 1 hour), replace with 2% FBS-DMEM and return to the incubator for continued culture, observing cell changes daily. After typical CPE appears, wash the aforementioned 96-well cell culture plate twice with sterile PBS, add DMEM medium (containing 10% FBS and 10% CCK8 reagent) at 100 μL / well, and incubate at 37°C for 1-2 hours. Measure the absorbance at 450 nm using a microplate reader. Calculate the inhibition rate (%) of each antibody against cytopathic effects = (450 nm absorbance of the antibody and virus mixed incubation group - 450 nm absorbance of the virus group) / (450 nm absorbance of the empty cell control group - 450 nm absorbance of the virus group) × 100%. The inhibition rate was calculated using the detected OD450nm data. Then, the regression equation and correlation coefficient R for each experimental concentration were obtained by plotting the logarithm of the concentration (10) on the x-axis and the probability value corresponding to the inhibition rate on the y-axis. The IC50 was calculated using the probability value corresponding to the inhibition rate of 50%. 50 value.

[0144] The results are as follows Figure 5 As shown, the half-maximal inhibitory concentration (IC50) of the 55Ab9-8 antibody against HADV55 is... 50 The value is 0.067 nM.

[0145] Example 5: Humanization and Testing of 55Ab9-8

[0146] I. Humanization of 55Ab9-8

[0147] The 55Ab9-8 antibody was humanized to obtain a humanized version of the antibody heavy chain and a humanized version of the antibody light chain. The antibody light chain and the two heavy chains were then paired to obtain the h55Ab9-8 antibody.

[0148] The amino acid sequence of the heavy chain of the h55Ab9-8 antibody is sequence 6. The amino acid sequence of the variable region of the heavy chain is positions 1-123 of sequence 6. The variable region of the heavy chain includes three complementarity-determining regions: HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is positions 26-33 of sequence 6, the amino acid sequence of HCDR2 is positions 51-58 of sequence 6, and the amino acid sequence of HCDR3 is positions 97-112 of sequence 6. Amino acid residues 124-222 form the heavy chain constant region CH1, amino acid residues 223-237 form the heavy chain hinge region Hinge, amino acid residues 238-347 form the heavy chain constant region CH2, and amino acid residues 348-453 form the heavy chain constant region CH3.

[0149] The amino acid sequence of the light chain variable region of the h55Ab9-8 antibody is sequence 8. The amino acid sequence of the light chain variable region is positions 1-113 of sequence 8. The light chain variable region includes three complementarity-determining regions (LCDR1, LCDR2, and LCDR3). The amino acid sequence of LCDR1 is positions 27-38 of sequence 8, the amino acid sequence of LCDR2 is positions 56-58 of sequence 8, and the amino acid sequence of LCDR3 is positions 95-103 of sequence 8. The amino acid residues at positions 117-218 constitute the light chain constant region (CL).

[0150] II. Construction of h55Ab9-8 recombinant plasmid

[0151] 1. Replace the small fragment between the HindIII and BamHI recognition sites in pcDNA3.1(+) (Invitrogen, V79020) with a DNA molecule whose nucleotide sequence is sequence 5 (heavy chain gene of h55Ab9-8 antibody), keeping the other nucleotides of pcDNA3.1(+) unchanged, to obtain the recombinant expression vector pcDNA-9-8H2 containing the heavy chain gene (which has been verified by sequencing).

[0152] pcDNA-9-8H2 contains the heavy chain gene of the monoclonal antibody h55Ab9-8, whose nucleotide sequence is sequence 5. The nucleotide sequence of the gene encoding the variable region (VH) is nucleotides 1-369 of sequence 5, containing the genes encoding HCDR1, HCDR2, and HCDR3. The gene encoding HCDR1 is nucleotides 76-99 of sequence 5, the gene encoding HCDR2 is nucleotides 151-174 of sequence 5, and the gene encoding HCDR3 is nucleotides 289-336 of sequence 5. Nucleotides 370-666 encode CH1, nucleotides 667-711 encode Hinge, nucleotides 712-1041 encode CH2, nucleotides 1042-1359 encode CH3, and nucleotides 1360-1362 are stop codons.

[0153] The pcDNA-9-8H2 expression is the heavy chain of the monoclonal antibody h55Ab9-8, which is the amino acid sequence of sequence 6. The amino acid sequence of the variable region of the heavy chain is positions 1-123 of sequence 6. The variable region of the heavy chain includes three complementarity-determining regions: HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is positions 26-33 of sequence 6, the amino acid sequence of HCDR2 is positions 51-58 of sequence 6, and the amino acid sequence of HCDR3 is positions 97-112 of sequence 6. The amino acid residues from positions 124-222 form the heavy chain constant region CH1, the amino acid residues from positions 223-237 form the heavy chain hinge region, the amino acid residues from positions 238-347 form the heavy chain constant region CH2, and the amino acid residues from positions 348-453 form the heavy chain constant region CH3.

[0154] 2. Replace the small fragment between the HindIII and BamHI recognition sites in pcDNA3.1(+) (Invitrogen, V79020) with a DNA molecule whose nucleotide sequence is sequence 7 (the light chain gene of the h55Ab9-8 antibody), keeping the other nucleotides of pcDNA3.1(+) unchanged, to obtain the recombinant expression vector pcDNA-9-8K2 containing the light chain gene (which has been verified by sequencing).

[0155] pcDNA-9-8K2 contains the light chain gene of the monoclonal antibody 55Ab9-8, whose nucleotide sequence is sequence 7. The nucleotide sequence encoding the variable region (VL) of the light chain is positions 1-339 of sequence 7, containing the coding genes for LCDR1, LCDR2, and LCDR3. The coding gene for LCDR1 is nucleotides 79-114 of sequence 7, the coding gene for LCDR2 is nucleotides 166-174 of sequence 7, and the coding gene for LCDR3 is nucleotides 283-309 of sequence 7. Nucleotides 348-654 encode the light chain constant region CL, and nucleotides 655-657 are stop codons.

[0156] The pcDNA-9-8K2 expression amino acid sequence is the light chain of the monoclonal antibody 55Ab9-8, which is sequence 8. The amino acid sequence of the variable region of the light chain is positions 1-113 of sequence 8. The variable region of the light chain includes three complementarity-determining regions, LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is positions 27-38 of sequence 8, the amino acid sequence of LCDR2 is positions 56-58 of sequence 8, and the amino acid sequence of LCDR3 is positions 95-103 of sequence 8. The amino acid residues at positions 117-218 constitute the constant region CL of the light chain.

[0157] The CDRs mentioned above are sequences defined according to the Kabat numbering system.

[0158] III. Preparation of h55Ab9-8 antibody

[0159] The h55Ab9-8 antibody solution was prepared according to step two of Example 2. The binding activity of h55Ab9-8 to the antigen HAdV55 was detected according to Example 3, and the results are as follows... Figure 6 As shown, the half-maximal effective concentration (EC50) of h55Ab9-8 antibody binding to HADV55 is... 50 The value is 0.0702 nM.

[0160] IV. Efficacy of h55Ab9-8 antibody against human adenovirus type 55 infection

[0161] The half-maximum inhibitory concentration (IC50) of the h55Ab9-8 antibody against HADV55 was detected according to the method in Example 4. 50 The result is as follows: Figure 7 As shown. The results indicate that the antibody h55Ab9-8 prepared in this invention can effectively inhibit HADV55 infection, and its IC50 value is [missing information]. 50 =0.3886nM.

[0162] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A monoclonal antibody against adenovirus or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises three complementarity-determining regions HCDR1, HCDR2 and HCDR3; and wherein the light chain variable region comprises three complementarity-determining regions LCDR1, LCDR2 and LCDR3. The amino acid sequence of HCDR1 is positions 26-33 of sequence 2. The amino acid sequence of HCDR2 is positions 51-58 of sequence 2. The amino acid sequence of HCDR3 is positions 97-112 of sequence 2. The amino acid sequence of LCDR1 is positions 27-38 of sequence 4. The amino acid sequence of LCDR2 is positions 56-58 of sequence 4. The amino acid sequence of LCDR3 is positions 95-103 of sequence 4.

2. The monoclonal antibody or its antigen-binding fragment as described in claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is sequence 6, and the amino acid sequence of the light chain variable region is sequence 8.

3. The monoclonal antibody or its antigen-binding fragment as described in claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is sequence 2, and the amino acid sequence of the light chain variable region is sequence 4.

4. A nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment as described in claim 1, 2 or 3.

5. The nucleic acid molecule as described in claim 4, characterized in that: The nucleic acid molecule is a gene encoding the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3.

6. The nucleic acid molecule according to claim 5, characterized in that: The gene is a DNA molecule containing the coding genes for HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the DNA molecule is A) or B): A) The coding gene for HCDR1 is nucleotides 76-99 of sequence 5, the coding gene for HCDR2 is nucleotides 151-174 of sequence 5, the coding gene for HCDR3 is nucleotides 289-336 of sequence 5, the coding gene for LCDR1 is nucleotides 79-114 of sequence 7, the coding gene for LCDR2 is nucleotides 166-174 of sequence 7, and the coding gene for LCDR3 is nucleotides 283-309 of sequence 7. B) The coding gene for HCDR1 is nucleotides 76-99 of sequence 1, the coding gene for HCDR2 is nucleotides 151-174 of sequence 1, the coding gene for HCDR3 is nucleotides 289-336 of sequence 1, the coding gene for LCDR1 is nucleotides 79-114 of sequence 3, the coding gene for LCDR2 is nucleotides 166-174 of sequence 3, and the coding gene for LCDR3 is nucleotides 283-309 of sequence 3.

7. The nucleic acid molecule according to claim 5, characterized in that: The gene is a DNA molecule of type C or D: C) The gene encoding the heavy chain variable region and the gene encoding the light chain variable region, wherein the nucleotide sequence of the gene encoding the heavy chain variable region is sequence 5 and the nucleotide sequence of the gene encoding the light chain variable region is sequence 7; D) The gene encoding the heavy chain variable region and the gene encoding the light chain variable region, wherein the nucleotide sequence of the gene encoding the heavy chain variable region is sequence 1 and the nucleotide sequence of the gene encoding the light chain variable region is sequence 3.

8. A biomaterial, wherein the biomaterial is any one of the following: B1) An expression cassette containing any of the nucleic acid molecules described in claims 4-7; B2) A recombinant vector containing the expression cassette described in B1); B3) Recombinant microorganisms containing any of the nucleic acid molecules described in claims 4-7; B4) Recombinant microorganisms containing the expression cassette described in B1); B5) Recombinant microorganisms containing the recombinant vector described in B2); B6) A transgenic animal cell line containing any of the nucleic acid molecules described in claims 4-7; B7) Transgenic animal cell lines containing the expression cassette described in B1); B11) Transgenic animal cell lines containing the recombinant vector described in B2); The animal cell line is not a germ cell, fertilized egg, or embryonic stem cell.

9. Use of the monoclonal antibody or its antigen-binding fragment according to any one of claims 1-3 or the nucleic acid molecule according to any one of claims 4-7, wherein the use is (e1) and / or (e2): (e1) Use of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-3 or the nucleic acid molecule as described in any one of claims 4-7 in the preparation of adenovirus inhibitors. (e2) Use of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-3 or the nucleic acid molecule as described in any one of claims 4-7 in the preparation of a medicament for the prevention and / or treatment of diseases caused by adenovirus infection; The adenovirus mentioned is human adenovirus type 55.

10. A product, said product being an adenovirus inhibitor or a medicine for the prevention and / or treatment of diseases caused by adenovirus infection, said product containing a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-3.

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