An anti-bkv antibody and use thereof

Abstract

The application discloses an anti-BKV antibody and application thereof, and the anti-BKV antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. The application successfully obtains a specific anti-BKV monoclonal antibody, and fills a market blank.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of screening and discovery of monoclonal cell strains, and is suitable for the preparation of monoclonal antibodies. In particular, it relates to an anti-BKV antibody and its application. BACKGROUND

[0002] BK polyomavirus (BKV) infection is an infection that usually affects kidney transplant recipients. This infection usually occurs in childhood and remains dormant in healthy adults. However, the immunosuppressive drug treatment regimen for kidney transplantation can activate the infection, causing serious complications such as hemorrhagic cystitis, BKV-associated nephropathy, and ureteral stenosis. BKV infection is worldwide, and primary infection with BKV is in the low age group. According to statistics in different regions of the world, the adult infection rate of BKV is 75% to 100%, and after primary infection, it can cause viremia, and the virus reaches the kidney and becomes latent. During periods of decreased immunity or pregnancy, the virus can be reactivated and replicated, excreted through urine, contaminate the surrounding environment, and be inhaled in the form of aerosol to cause infection.

[0003] The dissemination and pathogenicity of BKV in the body are similar to those of JCV.

[0004] 1. Primary infection with BKV can invade the human body through the respiratory tract. It first proliferates in the respiratory tract and then enters the bloodstream, causing primary viremia. The virus reaches the kidney, spleen, and lung through the bloodstream, and is then excreted through the respiratory tract and urine. Generally, no clinical symptoms appear. However, in some patients, the virus does not completely disappear, but becomes latent in the kidney. After primary infection, the body's humoral immunity can be induced, and after infection, the corresponding antibodies can be detected in the body, and the continuous replication of the virus can maintain a high titer of antibodies.

[0005] 2. Latent infection and reactivation. After primary infection, the latent virus can be reactivated during periods of decreased immunity or pregnancy, proliferate in the urinary tract, contaminate the surrounding environment with viral urine, be inhaled in the form of aerosol by susceptible individuals to cause new infections, or cause hemorrhagic cystitis when proliferating in the urinary tract.

[0006] The incidence of the disease is not high, and the onset time is different. Ureteral stenosis can occur 2-10 months after transplantation. BKV infection can cause the disease alone or mixed with JCV. JCV and BKV belong to the polyomavirus family, human polyomavirus branch, have close genetic relationship and similar genetic structure and biological characteristics, but there are significant differences in infection epidemiology, virus-host interaction. BKV has been confirmed to be associated with polyomavirus-associated nephropathy (PVAN) after kidney transplantation. 5%-10% of patients with BKV urinary symptoms will develop BKVAN, and 50%-80% of them will continue to develop renal dysfunction after transplantation. This is related to the severity of inflammation, the degree of tubular cell damage and the degree of renal fibrosis. Some scholars believe that there is a mutual promotion between JCV and BKV. There have been reports of JCV and BKV recombinant fragments found in the urine of kidney transplant recipients, and reports of PVAN development due to co-infection of BKV and JCV. Some studies believe that there is mutual inhibition between JCV and BKV. In the state of immunosuppression, JCV and BKV are co-activated at a low rate, and the BKV antibody titer of the donor and the recipient hinders the development of JCV urinary symptoms in the recipient in a dose-dependent manner.

[0007] The treatment of BK virus infection after kidney transplantation may include immunosuppressive therapy, antiviral drug therapy and supportive therapy, but the above treatment methods have the problems of long treatment time and large side effects. And it cannot be distinguished whether it is a specific treatment for BKV or a treatment for JCV. Monoclonal antibody therapy uses monoclonal antibody drugs to specifically bind to specific foreign substances, stimulate immune cells such as macrophages or other immune cells to clear specific pathogens. Monoclonal antibodies are usually some targeted drugs with very strong selectivity, and basically do not kill lymphocytes expressing other antibodies. Because it kills normal cells less, the side effects are relatively small, and the damage to the human body is smaller.

[0008] There is no specific anti-BKV monoclonal antibody or corresponding product in the prior art. SUMMARY

[0009] To solve the technical problem that there is no specific anti-BKV monoclonal antibody or corresponding product in the prior art, the present application provides a monoclonal antibody capable of specifically binding to BKV. The present application is based on the discovery and preparation method of monoclonal cell strains, and uses time-resolved fluorescence analysis to screen cell strains coated with BKV protein at the same time, and to screen cell strains coated with JCV protein at the same time. When positive detection is presented with BKV protein coating and negative with JCV protein coating, a cell strain that specifically secretes anti-BKV antibody can be screened, thereby obtaining an anti-BKV monoclonal antibody.

[0010] The present application provides an anti-BKV antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3; the light chain variable region comprising LCDR1, LCDR2 and LCDR3; wherein:

[0011] the amino acid sequence of the HCDR1 is shown as SEQ ID NO: 9, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 10, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 11; the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 12, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 13, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 14.

[0012] In the present application, the amino acid sequences of the above-listed CDRs are shown according to the Kabat definition (the sequences shown in the claims of the present application are also according to the Kabat definition). However, it is well known to those skilled in the art that the CDRs of an antibody can be defined in various ways in the art, such as based on the Kabat definition and the Chothia definition based on the location of structural loop regions (see J Mol Biol 273:927-48, 1997). In the present application, the amino acid residues in the variable domain sequence can also be determined using the Combined definition rule which combines the Kabat definition and the Chothia definition, i.e. a larger range is taken based on the combination of the two. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" given to an antibody or a region thereof (e.g. a variable region) are to be understood as encompassing complementarity determining regions defined by any of the above-mentioned known schemes as described in the present application. Although the scope of the present application is requested based on the sequences shown according to the Kabat definition, the amino acid sequences corresponding to the CDRs according to other definitions of the CDRs should also fall within the scope of the present application.

[0013] In some embodiments, the framework region of the heavy chain variable region and / or the light chain variable region is a murine framework region.

[0014] In some preferred embodiments, the heavy chain variable region comprises an amino acid sequence shown as SEQ ID NO: 3 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3; and / or, the light chain variable region comprises an amino acid sequence shown as SEQ ID NO: 7 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 7.

[0015] In some embodiments, the anti-BKV antibody further comprises a heavy chain constant region and a light chain constant region.

[0016] In some preferred embodiments, the heavy chain constant region has an amino acid sequence as set forth in SEQ ID NO: 4 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 4; and / or, the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 8 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8.

[0017] In some embodiments, the anti-BKV antibody is a full-length antibody, Fab, Fab', scFab, F(ab')2, or Fv; the Fv is preferably scFv.

[0018] Examples of antigen-binding fragments of the application include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single (heavy) chain antibodies, nanobodies, and domain antibodies, etc. Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0019] The second aspect of the application provides an isolated nucleic acid encoding the anti-BKV antibody according to the first aspect of the application.

[0020] The third aspect of the application provides a recombinant expression vector comprising the isolated nucleic acid according to the second aspect of the application.

[0021] The term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits expression of an mRNA, protein, polypeptide, or peptide by a host cell when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide and the vector is contacted with a cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed within the cell. The vectors of the present disclosure are not naturally occurring in general. However, portions of the vectors can be naturally occurring. The recombinant expression vectors of the present invention can comprise any type of nucleotide, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthetic or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. Suitable vectors include those designed for propagation and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or bacteriophage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0022] The fourth aspect of the present invention provides a transformant comprising the isolated nucleic acid of the second aspect of the present invention or the recombinant expression vector of the third aspect of the present invention.

[0023] In some preferred embodiments, the host cell of the transformant is a eukaryotic cell or a prokaryotic cell.

[0024] As used herein, the term "host cell" refers to any type of cell that can contain the nucleic acids or vectors described herein. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or alga; or the host cell can be a prokaryotic cell, such as a bacterium or protozoan.

[0025] The expression vector can be transfected or introduced into a suitable host cell. Various techniques can achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, transduction of retroviruses, viral transfection, gene editing (CRISPR-Cas system, ZFN system or TALEN system), transposon (Sleeping Beauty or PiggyBAC), gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are incubated in culture medium and suitable activity is screened. Methods and conditions for culturing the produced transfected cells and for recovering the produced antibody molecules are known to those skilled in the art and can be varied or optimized based on the methods known in the art and the specific expression vector and host cell used according to the present specification. In addition, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker can provide, for example, prototrophy to an auxotrophic host, biocidal resistance (e.g., to antibiotics) or heavy metal (such as copper) resistance, etc. The selectable gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements can also be required for optimal mRNA synthesis. These elements can include splicing signals, as well as transcription promoters, enhancers and termination signals.

[0026] The fifth aspect of the present application provides a hybridoma cell strain capable of secreting the anti-BKV antibody according to the first aspect of the present application.

[0027] The sixth aspect of the present application provides a pharmaceutical composition comprising the anti-BKV antibody according to the first aspect of the present application, and a pharmaceutically acceptable carrier.

[0028] The seventh aspect of the present application provides a method for preparing an anti-BKV antibody, comprising the steps of:

[0029] culturing the transformant according to the fourth aspect of the present application or culturing the hybridoma cell strain according to the fifth aspect of the present application to obtain the anti-BKV antibody from the culture; and / or synthesizing according to the amino acid sequence shown as SEQ ID NO: 2 and SEQ ID NO: 6 by chemical methods.

[0030] The eighth aspect of the present application provides a method for preparing the hybridoma cell strain according to the fifth aspect of the present application, characterized in that the method comprises the steps of:

[0031] 1) immunize Balb / c mice with a BKV protein solution as an antigen, after 3 immunizations, perform retro-orbital blood collection on the mice, obtain mouse serum after centrifugation, and detect the mouse serum by time-resolved fluorescence analysis;

[0032] 2) fusing the SP2 / 0 myeloma cells with the spleen cells of the mice which have the immune effect after the detection in the selective medium to screen the hybridoma cells;

[0033] Preferably, the selective medium is 20% FBS (HAT) medium.

[0034] 3) subcloning the positive hybridoma cells screened in step 2) in the medium containing 10% FBS for 3 times to obtain the hybridoma cell strain stably secreting the monoclonal antibody against BKV.

[0035] The ninth aspect of the present application provides a kit for immunological detection, which comprises one or more of the anti-BKV antibody according to the first aspect of the present application, the isolated nucleic acid according to the second aspect of the present application, the recombinant expression vector according to the third aspect of the present application, the transformant according to the fourth aspect of the present application, the hybridoma cell strain according to the fifth aspect of the present application and / or the pharmaceutical composition according to the sixth aspect of the present application.

[0036] The tenth aspect of the present application provides a method for detecting BKV for non-diagnostic purposes, which comprises contacting one or more of the anti-BKV antibody according to the first aspect of the present application, the isolated nucleic acid according to the second aspect of the present application, the recombinant expression vector according to the third aspect of the present application, the transformant according to the fourth aspect of the present application, the hybridoma cell strain according to the fifth aspect of the present application and / or the pharmaceutical composition according to the sixth aspect of the present application with the sample to be detected to detect BKV.

[0037] The eleventh aspect of the present application provides the use of the anti-BKV antibody according to the first aspect of the present application, the isolated nucleic acid according to the second aspect of the present application, the recombinant expression vector according to the third aspect of the present application, the transformant according to the fourth aspect of the present application, the hybridoma cell strain according to the fifth aspect of the present application and / or the pharmaceutical composition according to the sixth aspect of the present application in the preparation of a reagent for detecting BKV.

[0038] As used herein, the term "comprising" or "including" is intended to mean that the compositions and methods include the recited elements, but not excluding others, but according to the understanding of the context, it also includes the case of "consisting of".

[0039] On the basis of the common knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily, i.e. to obtain each preferred example of the present application.

[0040] The reagents and raw materials used in the present application are commercially available.

[0041] The positive progress effect of the present application is that:

[0042] The prepared anti-BKV monoclonal antibody has good specificity and titer, and is suitable for preparing different BKV diagnostic reagents. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Time-resolved fluorescence immunoassay detection process.

[0044] Figure 2 Photo of mice after the first immunization.

[0045] Figure 3 Schematic diagram of serum collection operation of mice. DETAILED DESCRIPTION

[0046] The application will be further described below by way of examples, but the application is not limited in the scope of the examples. The experimental methods in the following examples without specific conditions are selected according to conventional methods and conditions, or according to the product instructions.

[0047] Example 1 solution preparation

[0048] 1, 1×PBS buffer

[0049] Table 1 1×PBS buffer formula

[0050]

[0051] Preparation: take a clean preparation container, respectively take Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, add purified water of about 80% of the total preparation amount, stir until fully dissolved, adjust the pH to 7.5±0.1 with 1M HCl or 1M NaOH, add purified water to the total preparation amount, mix well. Store at room temperature, the effective period is 3 months.

[0052] 2, CB coating solution

[0053] Table 2 CB coating solution formula

[0054]

[0055] Preparation: take a clean preparation container, respectively take Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, add purified water of about 80% of the total preparation amount, stir until fully dissolved, adjust the pH to 7.5±0.1 with 1M HCl or 1M NaOH, add purified water to the total preparation amount, mix well. Store at room temperature, the effective period is 3 months.

[0056] 3, 20×PBST

[0057] Table 3 20×PBST formula

[0058]

[0059] Preparation: Take a clean preparation container, weigh Na2HPO4-12H2O, NaH2PO4-2H2O, NaCl, measure Tween-20 into about 80% of the total amount of purified water, stir until fully dissolved, then add purified water to the total amount, mix well. Store at room temperature, the effective period is 3 months.

[0060] 4, Sealing liquid

[0061] Table 4 Sealing liquid formula

[0062]

[0063] Preparation: Take a clean preparation container, weigh sucrose and fish gelatin respectively, add about 80% of the total amount of 1×PBS buffer, stir until fully dissolved, then add 1×PBS buffer to the total amount, mix well. Store at 2-8℃, the effective period is 3 months.

[0064] 5, Gelatin diluent

[0065] Table 5 Gelatin diluent formula

[0066]

[0067] Preparation: Take a clean preparation container, weigh fish gelatin, measure Tween-20 into about 100% of the total amount of 1×PBS buffer, stir until fully dissolved, then mix well. Store at 2-8℃, the effective period is 3 months.

[0068] 6, Buffer A

[0069] Table 6 Buffer A formula

[0070]

[0071] Preparation: Take a clean preparation container, weigh fish gelatin and EDTA-2K, measure Tween-20 into about 100% of the total amount of 1×PBS buffer, stir until fully dissolved, then mix well. Store at 2-8℃, the effective period is 3 months.

[0072] 7, Enhancer

[0073] Table 7 Enhancer formula

[0074]

[0075] Preparation: Take a clean preparation container, weigh β-NTA and TOPO, dissolve in 1 mL of anhydrous ethanol; weigh potassium hydrogen phthalate, measure glacial acetic acid, add purified water to about 80% of the total preparation volume, stir until completely dissolved, then add purified water to the total preparation volume, add 1 mL of ethanol containing β-NTA and TOPO. Store at 2-8°C, with a shelf life of 6 months.

[0076] 8. 20% FBS (HAT) medium

[0077] Table 8. 20% FBS (HAT) medium formula

[0078]

[0079] Preparation: Take a sterilized container, and in a biological safety cabinet, measure 1640 medium, fetal bovine serum, and penicillin-streptomycin solution into the container and mix well. Dissolve HAT powder in part of the solution and add to the container. Store at 2-8°C, with a shelf life of 3 months.

[0080] 9. 15% FBS (HT) medium

[0081] Table 9. 15% FBS (HT) medium formula

[0082]

[0083] Preparation: Take a sterilized container, and in a biological safety cabinet, measure 1640 medium, fetal bovine serum, and penicillin-streptomycin solution into the container and mix well. Dissolve HT powder in part of the solution and add to the container. Store at 2-8°C, with a shelf life of 3 months.

[0084] 10. 10% FBS medium

[0085] Table 10. 10% FBS medium formula

[0086]

[0087] Preparation: Take a sterilized container, and in a biological safety cabinet, measure 1640 medium, fetal bovine serum, and penicillin-streptomycin solution into the container and mix well. Store at 2-8°C, with a shelf life of 3 months.

[0088] 11. Preparation of immunization needle

[0089] The volume ratio of the BKV protein solution to the adjuvant is 1:1. The immunization scheme is shown in Table 11. For the first immunization, 450 μL of a 2 mg / mL (diluted with 1x PBS) BKV protein solution was mixed with 450 μL of Freund's complete adjuvant. For the subsequent immunizations, 450 μL of a 2 mg / mL (diluted with 1x PBS) BKV protein solution was mixed with 450 μL of Freund's incomplete adjuvant. The mixture was then placed on a silver gun blender and shaken for 60 s, and then rapidly cooled on ice for 60 s. The immunization emulsion was obtained after repeating the shaking and cooling for 6 times, and was then absorbed into a 1 mL syringe and stored at 2-8 °C.

[0090] Table 11 Immunization scheme

[0091] Number of immunizations BKV protein solution (2 mg / mL) Adjuvant First time 450 μL Freund's complete adjuvant 450 μL Second time 450 μL Freund's incomplete adjuvant 450 μL Third time 450 μL Freund's incomplete adjuvant 450 μL

[0092] Example 2 Immunization and blood collection of mice

[0093] (1) Immunization of mice

[0094] Before the experiment officially started, the immunization and blood collection plan of the mice was first established, and finally 6 female Blab / c mice of 6 weeks old were selected. The immunization method was subcutaneous injection of BKV antigen at multiple points on the back of the mice, the immunization dose was 150 μL per mouse, and the effective amount was 150 μg. The immunization interval was 14 days. One week after the third immunization, the mice were subjected to retro-orbital blood collection (see Table 12).

[0095] In order to obtain better immunization effect, the immunization method was subcutaneous immunization, and the hair on the back of the mice was shaved before immunization to expose the skin. Then, the skin was gently lifted with the tip of the syringe, and the immunization emulsion was injected. The emulsion amount was 150 μL, which was divided into 5 immunization points, and each point was about 30 μL. The photo of the mice after the first immunization is shown in Figure 2 .

[0096] Table 12 Immunization schedule

[0097] Days Operation Adjuvant Inoculation site 0 First immunization Freund's complete adjuvant Subcutaneous 14 Second immunization Freund's incomplete adjuvant Subcutaneous 28 Third immunization Freund's incomplete adjuvant Subcutaneous

[0098] (2) Serum collection of mice

[0099] When collecting blood, the thumb and index finger of one hand pressed the both sides of the neck of the mouse, and the neck of the mouse was held tightly from the back to make the retro-orbital venous plexus congest. When the mouse was held in the hand, the mouse body was kept in a straight posture, the right hand capillary was at an angle of 45 ° with the mouse face, and the capillary was pierced from the inner corner of the eye. The capillary was rotated by the finger to pierce the vein to collect blood, as shown in Figure 3 . After blood collection, the bleeding was immediately stopped to avoid excessive bleeding or eyeball necrosis. The collected blood was quickly blown into a 1.5 mL EP tube, and was placed at room temperature for 1 hour, and then at 4 °C for 1 hour. The serum was obtained by centrifugation at 6000 rpm for 5 min, and was stored at 4 °C.

[0100] Serum detection of Example 3

[0101] 1. Detection plate coating: the BVK protein solution was diluted to 2 μg / mL with CB coating solution, 50 μL / well, added to the 96-well TRFIA microplate, and incubated at 37°C for 2 h and then placed at 4°C for 16-36 h. The coating solution was discarded, 20x PBST was diluted with purified water to 1x PBST, and the plate was washed twice. 200 μL / well of blocking solution was added, incubated at 37°C for 2 h, the blocking solution was discarded, and the plate was washed twice with 1x PBST. The coating of the detection plate was completed, and it was stored at -20°C.

[0102] 2. Titer detection: the mouse serum was diluted from the initial 1 / 1000 (gelatin diluent) and then diluted by 3-fold gradient, with the gradient being 1 / 1000, 1 / 3000, 1 / 9000, 1 / 27000, 1 / 81000, 1 / 243000, 1 / 729000, and the gelatin diluent as a BLANK control. 50 μL / well of each gradient of the diluted serum and the BLANK was added to the detection plate, and incubated at 37°C for 30 min. The supernatant was discarded, the plate was washed once with 1x PBST, 50 μL / well of Bio-YKS (Bio: biotin, B2643-100 mg, Sigma; YKS: goat anti-mouse antibody, DCY0260A, Jackson) diluted to 1 μg / mL with the gelatin diluent was added, and incubated at 37°C for 30 min. The supernatant was discarded, the plate was washed twice with 1x PBST, 50 μL / well of SA-Eu (SA: streptavidin, Z7041, Promega; Eu: DTTA-Eu, DTTA-EuNa, Shanghai Weini) diluted to 0.5 μg / mL with Buffer A was added, and incubated at 37°C for 30 min. The supernatant was discarded, the plate was washed 6 times with 1x PBST, shaken and dried, 100 μL / well of enhancer was added, and shaken for 10 min. It was placed in a time-resolved fluorescence immunoassay instrument for reading, and the results are shown in Table 13. Mouse 5# had the highest titer, and was selected for cell fusion.

[0103] Table 13. Results of mouse serum titer detection

[0104]

[0105] Example 4. Cell fusion

[0106] Anatomize mouse 5#, take its spleen and grind to collect spleen cells, meanwhile collect SP2 / 0 cells which were cultured in advance for one week. Adjust cell number SP2 / 0: spleen cell = 1:6, adjust fusion parameters of electrofusion instrument (see Table 14), and carry out cell electrofusion. Collect hybridoma cells after fusion, mix with 20% FBS (HAT) medium, 200 μL / well plate in 96-well cell culture plate. After one week of culture in 37°C, 5% CO2 cell incubator, carry out fusion medium replacement. Discard supernatant, add 200 μL / well 15% FBS (HT) medium.

[0107] Table 14 Fusion parameters of electrofusion instrument

[0108]

[0109]

[0110] Example 5 Fusion primary screening

[0111] Take 50 μL cell supernatant in 96-well cell plate and add to BKV detection plate coated in advance (coating method, refer to Example 6), and incubate at 37°C for 30 min. Subsequent detection is the same as Example 6 "discard supernatant, wash plate with 1×PBST for 2 times, add Buffer A to dilute SA-Eu to working concentration, 50 μL / well, incubate at 37°C for 30 min. Discard supernatant, wash plate with 1×PBST for 6 times, shake dry and pat dry, add enhancer 100 μL / well, shake for 10 min. Put into time-resolved fluorescence immunoassay instrument for reading (detection process, refer to Example 6), and the results are shown in Table 15. Select 12 positive strains with higher reading for the first time subcloning. Figure 1

[0112] Table 15 Fluorescence immunoassay results of fusion primary screening

[0113] #Plate number

[0114] Negative control

[0115] Positive control

[0116] Positive cell strain

[0117]

[0118]

[0119]

[0120] Example 6 Subcloning

[0121] 1. First time subcloning and detection

[0122] ​(1) First subcloning: 96-well cell culture plates were added with 10% FBS medium, 200 μL / well, and the positive strain cells were transferred to cell plate row A, from row A to row H, and the cell solution was diluted at a gradient of 1:5. After incubation at 37°C in a 5% CO2 cell incubator for one week, the first subcloning detection was performed.

[0123] (2) First subcloning detection: The detection plates coated with BKV and JCV in advance (coating method, refer to Example 6) were selected. Eight cell strains were selected from each subclone, and 100 μL / well of supernatant was added to the BKV detection plate and the JCV detection plate, 50 μL was added to the BKV detection plate, and 50 μL was added to the JCV detection plate, and incubated at 37°C for 30 min. The subsequent detection was the same as Example 6 “discard the supernatant, wash the plate twice with 1×PBST, add Buffer A to dilute the SA-Eu to the working concentration, 50 μL / well, 37°C incubation for 30 min. Discard the supernatant, wash the plate 6 times with 1×PBST, shake dry and pat dry, add 100 μL / well of enhancer, shake for 10 min. Put into the time-resolved fluorescence immunoassay instrument for reading”, the results are shown in Table 16. Select the positive strain with BKV reading > JCV reading and BKV reading > 50W (4 strains) for second subcloning. The method of second subcloning is the same as that of first subcloning, and the data below has an underline, indicating that the cell strain has become a monoclonal cell strain (6F9 No. 8).

[0124] Table 16 First subcloning detection results

[0125] Cell strain name

[0126] Positive cell strain

[0127]

[0128]

[0129] 2. Second subcloning and detection

[0130] (1) Second subcloning: the method is the same as the first subcloning, and the data below has an underline, indicating that the cell strain has become a monoclonal cell strain.

[0131] (2) Second subcloning detection:

[0132] Method: First subcloning detection, results are shown in Table 17, data below has an underline (underlined cell strain name represents that the cell strain has become a single cell mass after multiple subcloning from a multi-cell mass; that is, a cell mass cloned from a single cell, indicating that all genetic materials in the cell mass are consistent), indicating that the cell strain has become a monoclonal cell strain. Considering the higher BKV reading, lower JCV / BKV, better cell state under microscope and priority of single clone, two cell strains were selected from each cell strain for third subcloning, i.e. a total of 8 cell strains.

[0133] Table 17 Second subcloning detection results

[0134] Cell strain name

[0135] Positive cell strain

[0136]

[0137]

[0138] 3. Third subcloning and detection

[0139] (1) Third subcloning: the method is the same as the first subcloning.

[0140] (2) The method is the same as the first subcloning detection, and the results are shown in Table 18. The data below has an underline, indicating that the cell strain is a monoclonal cell strain. Considering the higher BKV reading, lower JCV / BKV, better cell state under microscope and priority of monoclonal cells, the cell strain 6F9.2.6.5 is finally selected. After subculture, the cells are collected, a part is frozen in liquid nitrogen, and a part is used for sequencing.

[0141] Table 18 Third subcloning detection results

[0142] Cell strain name

[0143] Positive monoclonal cell strain

[0144]

[0145] Example 7 Sequencing

[0146] After DNA extraction and PCR amplification, the cells are sent for sequencing, and the sequencing return results are as follows:

[0147] BKV heavy chain full-length nucleotide sequence (SEQ ID NO: 1):

[0148]

[0149] BKV heavy chain full-length amino acid sequence (SEQ ID NO: 2):

[0150] MGWSWIFLFLLSGTAGVLSEVQLKQSGPELVKPGASVKISCKTSGYTFTEYTMYWVKQSHGKSLEWIGGINPNNGDTTYKQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASFYPIYVMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPSGELQEHSAHHGHRWLLLRLQQAQCAEEQLGGRKYFHLLCVT

[0151] BKV heavy chain variable region sequence (SEQ ID NO: 3):

[0152] MGWSWIFLFLLSGTAGVLSEVQLKQSGPELVKPGASVKISCKTSGYTFTEYTMYWVKQSHGKSLEWIGGINPNNGDTTYKQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASFYPIYVMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVT

[0153] BKV heavy chain constant region sequence (SEQ ID NO: 4):

[0154] VTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPSGELQEHSAHHGHRWLLLRLQQAQCAEEQLGGRKYFHLLCVT

[0155] Kappa light chain sequence is as follows:

[0156] BKV Kappa light chain full-length nucleotide sequence (SEQ ID NO: 5):

[0157] ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGCCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGATTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTTAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG

[0158] BKV Kappa light chain full-length amino acid sequence (SEQ ID NO: 6):

[0159] MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQDSHVPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGALVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0160] BKV Kappa light chain variable region sequence (SEQ ID NO: 7):

[0161] MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQDSHVPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGALVVCFLNNFY

[0162] BKV Kappa light chain constant region sequence (SEQ ID NO: 8):

[0163] PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0164] Table 19 Light and Heavy Chain CDR Amino Acid Sequences

[0165] Number Name Sequence SEQ ID NO: 9 HCDR1 EYTMY SEQ ID NO: 10 HCDR2 GINPNNGDTTYKQKFKG SEQ ID NO: 11 HCDR3 FYPIYVMDY SEQ ID NO: 12 LCDR1 RSSQSIVHSNGNTYLE SEQ ID NO: 13 LCDR2 KVSNRFS SEQ ID NO: 14 LCDR3 FQDSHVPWT

[0166] The CDR sequences for the light and heavy chains of the anti-BKV viral antibody are shown in Table 19, with the numbering scheme being Kabat.

Claims

1. An anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof, characterized in that, The anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a HCDR1, a HCDR2 and a HCDR3; and a light chain variable region comprising a LCDR1, a LCDR2 and a LCDR3; wherein: the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 11; the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 12, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 13, and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO:

14.

2. The anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof of claim 1, wherein, The framework region of the heavy chain variable region and / or the light chain variable region is a murine framework region.

3. The anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof of claim 2, wherein, The heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3; and / or, the light chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:

7.

4. The anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof of claim 3, wherein, The heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 3; and / or, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:

7.

5. The anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region.

6. The anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof of claim 5, wherein, The heavy chain constant region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 4; and / or, the light chain constant region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:

8.

7. The anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof of claim 6, wherein, The heavy chain constant region comprises an amino acid sequence set forth in SEQ ID NO: 4; and / or, the light chain constant region comprises an amino acid sequence set forth in SEQ ID NO:

8.

8. The anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein, The antigen-binding fragment is a Fab, a Fab', a scFab, a F(ab')2, a Fv or a scFv.

9. An isolated nucleic acid, comprising, The isolated nucleic acid encodes the anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof of any one of claims 1-7.

10. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid of claim 9.

11. A transformant characterized in that, The transformant comprises the isolated nucleic acid of claim 9 or the recombinant expression vector of claim 10; and the host cell of the transformant is an animal cell or a prokaryotic cell.

12. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof of any one of claims 1-7, and a pharmaceutically acceptable carrier.

13. A method of making an anti-BK polyomavirus (BKV) antibody, characterized in that, The method comprises the following steps: culturing the transformant of claim 11 to obtain the anti-BK polyomavirus (BKV) antibody from the culture; and / or, synthesizing by chemical method according to the amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO:

6.

14. A kit for immunological detection, characterized in that The kit comprises one or more of the anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof of any one of claims 1-7, the isolated nucleic acid of claim 9, the recombinant expression vector of claim 10, the transformant of claim 11, and the pharmaceutical composition of claim 12.

15. A method of detecting BK polyomavirus (BKV) for non-diagnostic purposes, characterized in that, The method comprises contacting a sample to be tested with one or more of the anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof of any one of claims 1-7 and the pharmaceutical composition of claim 12 to detect BK polyomavirus (BKV).

16. Use of the anti-BK polyomavirus (BKV) antibody or antigen-binding fragment thereof of any one of claims 1-7, the isolated nucleic acid of claim 9, the recombinant expression vector of claim 10, the transformant of claim 11, and / or the pharmaceutical composition of claim 12 in the manufacture of a reagent for detecting BK polyomavirus (BKV).

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