Fully human anti-BK polyoma virus IgG1 monoclonal antibody BKAb03 and preparation method thereof

Abstract

The invention is applicable to the technical field of biological medicines, and provides a fully human anti-BK polyoma virus IgG1 monoclonal antibody BKAb03 and a preparation method thereof, and the antibody BKAb03 is specifically bound with a BK polyoma virus capsid protein VLP or a capsid protein monomer VP1 protein thereof; the amino acid sequences of the HCDR1, the HCDR2 and the heavy chain variable region of the HCDR3 are as shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and the light chain variable region comprises the LCDR1 as shown in SEQ ID NO: 5, the LCDR2 as shown in SEQ ID NO: 6 and the LCDR3 as shown in SEQ ID NO: 7. The antibody prepared by the invention can be used for preparing medicines for treating or preventing BK polyoma virus infection and / or BK polyoma virus related complications.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03 and its preparation method. Background Technology

[0002] BK polyomavirus, or BK virus for short, belongs to the double-stranded DNA polyomavirus family. Its genome is approximately 5000 base pairs long. It lacks an outer envelope, consisting only of a single capsid protein layer. The viral capsid is approximately 40-50 nm in diameter and serves to maintain viral particle stability. The proteins constituting the BK polyomavirus capsid include VP1, VP2, and VP3. The most important member is the VP1 protein, composed of 362 amino acids with a molecular weight of approximately 42.5 kDa. Five VP1 protein molecules are tightly linked together to form a VP1 pentamer, and 72 VP1 pentamers are tightly linked together to form the basic framework of the BK virus capsid protein. The VP1 protein is the only protein target responsible for the contact between BK polyomavirus and gangliosides on the surface of the host cell membrane, thus promoting viral invasion of the host cell. It plays a crucial role in the entire process of viral infection of the host cell and is also an important antibody binding target. The VP2 and VP3 proteins share the same C-terminal sequence and can both bind to the N-terminus of the VP1 protein, embedding themselves inside the BK polyomavirus capsid protein. In addition, the VP2 and VP3 proteins also share a nuclear localization sequence (NLS) on their C-terminal sequence, which can promote the localization of the BK polyomavirus genome to the host cell nucleus, but there is currently little research on these two proteins.

[0003] In individuals with normal immune function, BK polyomavirus remains dormant. However, in immunocompromised patients, such as kidney transplant recipients, hematopoietic stem cell transplant recipients, and HIV-infected individuals, BK polyomavirus can reactivate, causing "reinfection" and triggering serious complications that threaten the patient's life and health. In addition, HIV-infected individuals are also highly susceptible to BK polyomavirus infection due to their impaired immune function, which can lead to serious complications such as HIV-associated salivary gland disease (HIVSGD) and acute renal failure.

[0004] Current clinical treatments for combating BK polyomavirus infection include reducing immunosuppressant dosage, administering antiviral chemotherapy, intravenous immunoglobulin infusion, and adoptive T-cell therapy. However, most of these treatments have limited efficacy and are prone to unpredictable side effects, hindering their widespread application. The publicly disclosed specific anti-BK polyomavirus monoclonal antibody (MAU868) has been proven to possess excellent antiviral efficacy and good tolerability, showing great promise for application. However, monoclonal antibody resources available for clinical treatment are extremely scarce. Therefore, the development and research of specific anti-BK polyomavirus human monoclonal antibodies is urgently needed. Summary of the Invention

[0005] The purpose of this invention is to provide a fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03, which aims to solve the problems mentioned in the background art.

[0006] The present invention is implemented as follows: a fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03, wherein the antibody BKAb03 binds to the BK polyomavirus capsid protein VLP or its capsid protein monomer VP1 protein.

[0007] Its amino acid sequence includes the heavy chain variable regions of HCDR1 (CDR-complementarity-determining region) as shown in SEQ ID NO: 2, HCDR2 as shown in SEQ ID NO: 3, and HCDR3 as shown in SEQ ID NO: 4, as well as the light chain variable regions of LCDR1 as shown in SEQ ID NO: 5, LCDR2 as shown in SEQ ID NO: 6, and LCDR3 as shown in SEQ ID NO: 7.

[0008] Another objective of this invention is to provide a method for preparing a fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03, comprising the following steps:

[0009] Step S1: Construct a prokaryotic expression vector containing the BK VP1 protein gene sequence and transform it into a prokaryotic expression system. Construct and screen stable expression strains and induce expression in vitro at low temperature. The BK VP1 protein gene sequence is shown in SEQ ID NO: 1. BK VLP antigen solution is obtained through in vitro expression, two-step purification and two-step dialysis self-assembly.

[0010] Step S2: Use BK VLP antigen solution to periodically immunize fully human CA mice to construct a BK virus immunization animal model;

[0011] Step S3: In vitro sorting of target B cell populations from the spleen of fully human CA mice immunized with BK virus;

[0012] Step S4: Using 10× Genomics dual-omics combined sequencing analysis, high-throughput screening was conducted to obtain the sequence information of the fully human anti-BK polyomavirus IgG1 monoclonal antibody;

[0013] Step S5: Insert the sequence information of the fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03 into a eukaryotic expression vector and transform it into a eukaryotic expression system, wherein the eukaryotic expression system is a mammalian cell, to produce the target human IgG1 monoclonal antibody, and then obtain the pure target antibody protein solution through separation and purification.

[0014] Another objective of this invention is to provide the use of a fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03 in the preparation of drugs for the prevention and / or treatment of BK polyomavirus infection.

[0015] In this embodiment of the invention, BK virus-like particles (BK VLPs) are self-assembled in vitro to periodically immunize CA fully human mice, thereby constructing a fully human immunized animal model. Single B cell clusters that can secrete IgG1 antibodies that specifically bind to BK virus are sorted from their spleens. Then, single-cell transcriptomics sequencing and single-cell immune repertoire sequencing are combined to screen for fully human monoclonal antibody sequences with potential specificity and high affinity for BK virus IgG1.

[0016] Meanwhile, by leveraging the advantages of mammalian cell expression systems such as correct glycosylation, natural folding, and low immunogenicity, the recombinant antibodies produced are consistent with natural human antibodies. Stable target antibody expression cell lines are constructed using mammalian cell expression systems, and the produced complete antibodies can be directly used for various detections such as BK polyomavirus infection prevention detection and BK polyomavirus resistance detection, providing accurate feedback on the titer level of fully human IgG1 monoclonal antibodies.

[0017] The antibody development and preparation process can obtain highly specific human IgG1 monoclonal antibodies in a high-throughput and rapid manner. Furthermore, the antibody or its antigen-binding fragment provided in the embodiments of the present invention can be used to prepare drugs for the treatment or prevention of BK polyomavirus infection and / or BK polyomavirus-related diseases. Attached Figure Description

[0018] Figure 1 This is a design diagram of the pET30a(+) gene sequence containing the BK VP1 protein provided in an embodiment of the present invention;

[0019] Figure 2 This is an SDS-PAGE denaturing and reducing gel electrophoresis image of BK VP1 protein induced by low temperature in vitro according to an embodiment of the present invention.

[0020] Figure 3 This is a Western blot diagram of the in vitro low-temperature induced expression of BK VP1 protein provided in an embodiment of the present invention.

[0021] Figure 4 The two-step purification results provided in this embodiment of the invention are detected by SDS-PAGE denaturing-reduction gel electrophoresis.

[0022] Figure 5 The images shown are transmission electron microscopy (TEM) images of BK VLP assembly provided in this embodiment of the invention, where A, B, C, and D are different TEM images.

[0023] Figure 6 The results of the initial ELISA method for detecting serum titers in CA mice provided in this embodiment of the invention;

[0024] Figure 7 The results of detecting serum titers in CA mice using a secondary ELISA method provided in this embodiment of the invention;

[0025] Figure 8 A bar chart showing the distribution of CA mouse cell subpopulations provided in this embodiment of the invention;

[0026] Figure 9 A bar chart showing the percentage of different cell subpopulations in CA mice provided in this embodiment of the invention;

[0027] Figure 10 CellPhone image for single-cell transcriptomics analysis of CA mice provided in this embodiment of the invention;

[0028] Figure 11 The pBudCE4.1 expression vector plasmid map of the nucleotide sequence of the VP1 protein gene containing the capsid of BK-Ia serotype virus provided in this embodiment of the invention;

[0029] Figure 12 The pBudCE4.1 expression vector plasmid map of the nucleotide sequence of the VP1 protein gene containing the capsid of the BK-IVc2 serotype virus provided in this embodiment of the invention;

[0030] Figure 13 The pBudCE4.1 expression vector plasmid map of the nucleotide sequence of the VP2 protein gene in the capsid of BK-IVc2 serotype virus provided in this embodiment of the invention;

[0031] Figure 14 The pBudCE4.1 expression vector plasmid map of the nucleotide sequence of the VP3 protein gene in the capsid of BK-IVc2 serotype virus provided in this embodiment of the invention;

[0032] Figure 15 The direct ELISA method provided in this embodiment of the invention determines EC50 The value was used to assess the ability of the BKAb03 antibody to bind to the capsid protein of two serotypes of BKV, where A is the BKV-Ia serotype and B is the BKV-IVc2 serotype.

[0033] Figure 16 for Figure 15 Statistical results;

[0034] Figure 17 The ELISA method for detecting IC provided in the embodiments of the present invention 50 The efficacy of BKAb03 antibody against two serotypes of BKV was evaluated, where A is serotype BKV-Ia and B is serotype BKV-IVc2.

[0035] Figure 18 for Figure 17 The statistical results. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0037] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1: Construction and screening of stable expression strains and in vitro low-temperature induction of expression:

[0039] The obtained BK VP1 protein gene sequence was codon-optimized, and the optimized gene sequence (as shown in SEQ ID NO: 1) was cloned into a kanamycin-resistant pET30a(+) plasmid vector suitable for prokaryotic cell expression. The complete plasmid map is available in [link to full image]. Figure 1 As shown, the plasmid was transformed into BL21 <de3>Prokaryotic host cells of *E. coli* were used for antibiotic resistance screening. Working strains expressing BK VP1 protein were obtained through two rounds of resistance screening using both solid and liquid resistant culture media. After induction at 18°C, 180 rpm, and 1 mM IPTG for 12–14 h, strains quantitatively expressing BK VP1 protein were obtained. The successful expression of BK VP1 was verified by SDS-PAGE denaturing-reduction gel electrophoresis and Western blot analysis. See the results figure for details. Figure 2 , Figure 3 As shown.

[0040] Example 2: Two-step purification and two-step dialysis self-assembly:

[0041] The bacterial strain precipitated after in vitro low-temperature expression was subjected to high-pressure disruption, centrifuged at 4°C and 12000 rpm for 15 min, and the supernatant was collected. The supernatant was first purified by Capto Q anion exchange chromatography, followed by Ni... + -NTA affinity chromatography purification, followed by a two-step combined purification process, yielded a pure BK VP1 protein solution. The purification status of each step could be verified by SDS-PAGE denaturing-reduction gel electrophoresis. See details for the results. Figure 4 As shown, a pure BK VP1 protein solution was finally obtained;

[0042] By altering the salt ion strength in two different buffer systems, the BK VP1 protein first self-assembles into a BKVP1 protein pentamer. 72 BK VP1 protein pentamers are tightly packed together to form BK VP1 protein particles. The solution is as follows: The pure BK VP1 protein solution is placed in a dialysis bag with a molecular weight cutoff of 12-14 kDa. It is first dialyzed in assembly buffer A [500 mM (NH4)2SO4 + 20 mM Tris-HCl (pH 7.4) + 5% glycerol + 1 mM CaCl2] for 17 h, then dialyzed in assembly buffer B [200 mM NaCl + 20 mM Tri-HCl (pH 7.4) + 5% glycerol + 1 mM CaCl2] for 24 h, followed by another 24 h of dialysis.

[0043] The success of protein assembly was assessed by transmission electron microscopy (TEM). A 20-fold diluted solution of BK VLP protein particles was added to a 200-mesh copper grid, negatively stained with 2% phosphotungstic acid, and observed under a TEM. The TEM results are shown below. Figure 5 As shown, the obtained pure BK VLP protein particle solution has high stability and high immunogenicity, but is not infectious, and is a high-quality immune antigen.

[0044] Example 3: Construction of a fully human CA mouse BK VLP immune model:

[0045] The purified BK VLP protein particle solution was fused with an immune adjuvant to prepare an immune emulsion, which was then used to periodically immunize fully human CA mice according to Table 1.

[0046] Table 1. Timeline for constructing the fully human CA mouse BK VLP immune model

[0047] Immunization time Experimental Operation Composition of immunomodulatory agents Day -3 Blood was collected from the posterior ocular venous plexus of CA mice, and serum was obtained by centrifugation (negative control serum). Day 0 The first immunization uses the intraperitoneal multi-target method. Contains an appropriate dose of BK VLP purified protein particles (100 μg per CA mouse), aluminum hydroxide nanogel, CpG oligonucleotide adjuvant, and Freund's complete adjuvant. Day 14 The first booster immunization was administered using a multi-target intraperitoneal approach. Contains an appropriate dose of BK VLP purified protein particles (100 μg per CA mouse), aluminum hydroxide nanogel, CpG oligonucleotide adjuvant, and Freund's incomplete adjuvant. Day 21 Blood was collected from the posterior ocular venous plexus of CA mice and serum was obtained by centrifugation for the initial ELISA assay to detect serum titer in CA mice. Day 28 Second booster immunization, using intraperitoneal multi-target method Contains an appropriate dose of BK VLP purified protein particles (100 μg per CA mouse), aluminum hydroxide nanogel, CpG oligonucleotide adjuvant, and Freund's incomplete adjuvant. Day 42 Three booster shots were administered using the intraperitoneal multi-target method. Contains an appropriate dose of BK VLP purified protein particles (100 μg per CA mouse), aluminum hydroxide nanogel, CpG oligonucleotide adjuvant, and Freund's incomplete adjuvant. Day 49 The final bolus immunization uses a multi-target intraperitoneal approach. BK VLP purified protein particles (100μg dose per CA mouse) and naked antigen solution Day 56 Blood was collected from the posterior ocular venous plexus of CA mice, and serum was obtained by centrifugation. Serum titers of CA mice were then determined by a second ELISA assay.

[0048] Specifically, the two ELISA tests for CA mouse serum titers described in Table 1 include:

[0049] 50 ng / well BK VLP was coated onto an ELISA plate and incubated overnight at 4°C. The plate was then sealed with PBST containing 5% BSA at 37°C for 2 hours. Separated CA mouse serum was serially diluted 2-fold in PBST containing 5% BSA, allowing the serum dilution to bind to the antigen-coated ELISA plate at 37°C for 2 hours. The plate was washed three times with PBST, and then incubated with a secondary antibody (HRP-conjugated goat anti-mouse IgG) diluted 1:5000 in PBST containing 0.5% BSA at 37°C for 1 hour. The plate was washed three times with PBST, and the reaction was performed using tetramethylbenzidine (TMB) microporous peroxidase substrate, incubated at 37°C for 30 minutes. Finally, 2% oxalic acid aqueous solution was added to each well to stop the color development. The absorbance (OD value) was measured at 450 nm using a microplate reader. Figure 6 and Figure 7 The results of two ELISA serum titers show that the BK VLP antigen has successfully activated strong humoral immunity in CA mice, and can be used for subsequent target antibody sorting experiments.

[0050] Example 4: FACS flow cytometry sorting of target B cell populations in CA mice:

[0051] After the periodic immunization of CA mice, the mouse spleen was isolated in vitro, and mouse spleen B cell populations were collected using a magnetic bead negative enrichment kit. APC-goat anti-human IgG fluorescent antibody, Super Flour 488-BK VLP specific fluorescent antigen, and 7-AAD (7-aminoactinomycin D) reagent were added sequentially to the isolated mouse spleen B cell populations and incubated at low temperature in the dark for 30 min. After washing the cell incubation mixture twice with DPBS solution, specific fluorescent flow cytometry sorting was performed on the BD Melody flow cytometer to screen for target B cell populations: B cell populations that are double positive for APC and Super Flour 488 and negative for 7-ADD.

[0052] Example 5: Screening of target antibody sequences using dual-omics combined analysis:

[0053] By combining 10× Genomics single-cell transcriptomics (scRNA-seq) and 10× Genomics single-cell immune repertoire (scVDJ-seq) dual-omics sequencing analysis technologies, a systematic single-cell analysis was performed on the sorted target B cell population, and target antibody sequences were screened. The specific details are as follows:

[0054] (1) Bar chart showing the distribution of cell subsets in single-cell transcriptomics ( Figure 8 ) and a bar chart showing the percentage of each cell subpopulation ( Figure 9 The results showed that the blank control group CA mice had a high proportion of immature B cells and a low proportion of activated B cells, while the immune group CA mice had a significantly increased proportion of activated B cells and functional B cells and a decreased proportion of immature B cells, which preliminarily revealed the changes in immune activation at the cellular level in the immune group CA mice.

[0055] (2) Single-cell transcriptomics analysis CellPhone diagram ( Figure 10 The results showed that the interaction between different cell subsets in the immunized CA mice was significantly enhanced, which provided evidence of immune activation in the immunized CA mice at the single-cell level.

[0056] (3) According to the screening criteria, antibodies that meet the criteria are selected from the list of all antibody information obtained from the single-cell immune repertoire analysis. The partial information of the selected antibodies is shown in Table 2.

[0057] Specifically, the screening criteria described in the embodiments include:

[0058] i. The B cells that secrete antibodies must be activated B cells (such as memory B cells, plasma cells, etc.).

[0059] ii. Clonal expansion of antibody-secreting B cells ≥2;

[0060] iii. Select and retain only IgG1 subtype antibodies from all antibody types;

[0061] iv. Somatic hypermutation rate (SHR) of the antibody > 2%;

[0062] Table 2. Statistical table of antibody information obtained from standardized screening

[0063]

[0064] Example 6: In vitro synthesis of the target antibody:

[0065] From the antibody information list 2 obtained from standardized screening, the nucleotide sequence of the fully human IgG1 antibody gene with the highest SHR value, sequence number 03 (see SEQ ID NO: 8-13), was selected and directly cloned into a vector suitable for expression in mammalian cells, such as pcDNA3.1, pCMV, pEF-1α, or pCDH plasmid. A signal peptide sequence was added to the vector to ensure that the recombinant antibody protein could be correctly secreted into the extracellular environment. The constructed recombinant expression vector was transfected into HEK293 cells. After culturing the cells for 5-7 days, the cell supernatant was collected, filtered, and purified by Protein A affinity chromatography to obtain a pure antibody, which was named BKAb03.

[0066] Example 7: Preparation of BK pseudovirus particles:

[0067] The VP1 protein gene sequences of the two most prevalent BK virus genotypes, BKV-Ia (BK-D genotype) and BKV-IVc2 (A-66H genotype), along with the VP2 and VP3 protein gene sequences of BKV-IVc2 (A-66H genotype), were cloned into the pBudCE4.1 eukaryotic expression vector, respectively. The specific expression vector plasmid map is shown below. Figures 11-14 As shown, the virus was co-transfected into HEK293TT cells. The cells were lysed 48 h after transfection, and the supernatant containing BK pseudovirus particles (BK-PsV) was collected. After purification by gradient density ultracentrifugation and ultrafiltration, a pure BK pseudovirus particle (BK-PsV) solution was obtained.

[0068] Example 8, Evaluation of the neutralizing capacity of BKAb03 antibody:

[0069] 50 ng / well of BK pseudovirus particles (BK-PsV) were coated onto an ELISA plate and incubated overnight at 4°C. The plate was then sealed with PBST containing 5% BSA at 37°C for 2 hours. BKAb03 antibody and positive control antibody (MAU868) were serially diluted 4-fold in PBST containing 5% BSA, with an initial antibody concentration of 10 μg / mL (~69 nM). The antibody dilution was allowed to bind to the antigen-coated ELISA plate at 37°C for 2 hours. The plate was washed three times with PBST solution, and then incubated at 37°C for 1 hour with a secondary antibody (HRP-conjugated goat anti-mouse IgG) diluted 1:5000 in PBST containing 0.5% BSA. The plate was then washed three times with PBST and reacted with tetramethylbenzidine (TMB) microporous peroxidase substrate, incubated at 37°C for 30 min, and finally, 2% oxalic acid aqueous solution was added to each well to stop the color development. The absorbance (OD value) was measured at 450 nm using a microplate reader. Figure 15 , 16 The results of ELISA serum titer assay and the corresponding EC2 values ​​obtained after calculation can be seen in the image. 50 The results showed that the BKAb03 antibody molecule had an EC50 of approximately 0.1345. 50 The value binds to BKV-Ia serum capsid protein, and its EC50 value... 50 The EC50 value was lower than that of the positive control group MAU868 antibody. 50 Value; the BKAb03 antibody molecule has an EC50 of approximately 0.3159. 50 The value binds to BKV-IVc2 serum capsid protein, and its EC50 value... 50 The EC50 value was slightly higher than that of the positive control group MAU868 antibody. 50 The results indicate that the BKAb03 antibody molecule possesses a certain degree of potency in binding to the BK virus capsid protein, and its ability to bind to the BKV-Ia serological capsid protein is stronger and superior to that of the positive control group MAU868 antibody.

[0070] Example 9: Efficacy of BKAb03 antibody against two serotypes of BK virus infection:

[0071] Approximately 1×10 4 293FT cells were seeded in 96-well plates and an appropriate volume of DMEM medium containing 5% FBS and penicillin-streptomycin was added. The cells were incubated overnight for adherence. Different concentrations of serially diluted BKAb03 antibody and positive control antibody (MAU868) were mixed with a fixed amount of BK pseudovirus particles. The mixture was then incubated at 37°C for 1 hour and added to each well of the 96-well plate. The 96-well plate was then incubated at 37°C and 5% CO2 for 48-72 hours to ensure complete infection of 293FT cells with BK pseudovirus.

[0072] After discarding the cell supernatant, the cells were gently washed with DPBS, fixed, and permeabilized with 293FT cells. An appropriate amount of immunostaining blocking solution was added for incubation. The cells were then incubated overnight at 4°C with Anti-SV40 VP1 primary antibody (ab53977) diluted solution. After gently washing the 293FT cells with DPBS, an appropriate amount of HRP-goat anti-rabbit secondary antibody solution was added to fully cover the cells. The cells were incubated in a humidified chamber at room temperature for 1 hour. The absorbance of each well was measured at 450 nm using a microplate reader. The IC50 readings were... 50 The values ​​were determined by GraphPad Prism using nonlinear regression (curve fitting) and the formula "log(antibody) versus standardized response (variable slope)" (GraphPad software), as shown in the results. Figure 17 , 18 As shown.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03, characterized in that, The antibody BKAb03 specifically binds to the BK polyomavirus capsid protein VLP or its capsid protein monomer VP1 protein. Its amino acid sequence includes the heavy chain variable regions of HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:4, and the light chain variable regions of LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6, and LCDR3 as shown in SEQ ID NO:

7.

2. The fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03 according to claim 1, characterized in that, Its nucleotide sequence includes heavy chain variable regions of HCDR1 as shown in SEQ ID NO: 8, HCDR2 as shown in SEQ ID NO: 9, and HCDR3 as shown in SEQ ID NO: 10, as well as light chain variable regions of LCDR1 as shown in SEQ ID NO: 11, LCDR2 as shown in SEQ ID NO: 12, and LCDR3 as shown in SEQ ID NO:

13.

3. A method for preparing the fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03 as described in claim 1 or 2, characterized in that, Includes the following steps: Step S1: Construct a prokaryotic expression vector containing the BK VP1 protein gene sequence and transform it into a prokaryotic expression system. Construct and screen stable expression strains and induce expression in vitro at low temperature. The BK VP1 protein gene sequence is shown in SEQ ID NO:

1. BK VLP antigen solution is obtained through in vitro expression, two-step purification and two-step dialysis self-assembly. Step S2: Use BK VLP antigen solution to periodically immunize fully human CA mice to construct a BK virus immunization animal model; Step S3: In vitro sorting of target B cell populations from the spleen of fully human CA mice immunized with BK virus; Step S4: Using 10× Genomics dual-omics combined sequencing analysis, high-throughput screening was conducted to obtain the sequence information of the fully human anti-BK polyomavirus IgG1 monoclonal antibody; Step S5: Insert the sequence information of the fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03 into a eukaryotic expression vector and transform it into a eukaryotic expression system, wherein the eukaryotic expression system is a mammalian cell, to produce the target human IgG1 monoclonal antibody, and then obtain the pure target antibody protein solution through separation and purification.

4. The method for preparing the fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03 according to claim 3, characterized in that, In step S5, the lactating cells are HEK293 cells.

5. The method for preparing the fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03 according to claim 3, characterized in that, In step S5, the separation and purification employs at least one of salting out, chromatographic chromatography, affinity chromatography, and membrane separation.

6. The use of the fully human anti-BK polyomavirus IgG1 monoclonal antibody BKAb03 as described in claim 1 or 2 in the preparation of medicaments for the prevention and / or treatment of BK polyomavirus infection.

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