Anti-BKV antibody and application thereof

Abstract

The invention discloses an anti-BKV antibody and application thereof, the anti-BKV antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. According to the invention, the specific anti-BKV monoclonal antibody is successfully obtained, and the market vacancy is made up.

Description

Technical Field

[0001] The present invention belongs to the field of screening and discovery of monoclonal cell lines, and is applicable to the preparation of monoclonal antibodies, and specifically relates to an anti-BKV antibody and its application. Background Art

[0002] BK polyomavirus (BKV) infection is an infection that commonly affects kidney transplant recipients. This infection usually occurs in childhood and remains dormant in healthy adults. The immunosuppressive drug treatment regimen associated with kidney transplantation can activate the infection, causing serious complications such as hemorrhagic cystitis, BKV-related nephropathy, and ureteral stenosis. BKV infection is worldwide, and the primary infection of BKV is in the younger age group. According to statistics from different regions around the world, the adult infection rate of BKV is 75% to 100%. After primary infection, it can cause viremia. The virus reaches the kidneys and lurks. When the body's immunity is reduced or during pregnancy, the virus can be reactivated and replicated, excreted through urine, contaminate the surrounding area, and be inhaled in the form of aerosols to cause infection.

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

[0004] 1. Primary infection BKV may invade the human body through the respiratory tract. First, it proliferates in the respiratory tract, then enters the bloodstream, causing primary viremia. The virus flows with the blood to the kidneys, spleen, and lungs, and is then excreted through the respiratory tract and urine. Generally, there are no clinical symptoms. However, the virus is not completely eliminated in some patients, but lurks in the kidneys. Primary infection can induce the body's humoral immunity. After infection, corresponding antibodies can be detected in the body, and continuous viral replication can maintain high titer antibodies.

[0005] 2. Latent infection and reactivation. The virus that has been latent after primary infection can be reactivated and proliferate in the urinary tract when the body's immune function declines or during pregnancy, resulting in viral urine that contaminates the surrounding environment and can be inhaled by susceptible people in the form of aerosols, causing new infections or causing hemorrhagic cystitis when proliferating in the urinary tract.

[0006] The incidence of this disease is not high, and the onset time varies. Ureteral stenosis can occur 2 to 10 months after transplantation. BKV infection can cause this disease alone or in combination with JCV. JCV and BKV belong to the same family of polyomaviruses, the human polyomavirus branch, and are closely related. They have similar genetic structures and biological characteristics, but there are significant differences in infection epidemiology and virus-host interactions. BKV has been confirmed to be associated with polyomavirus-associated nephropathy (PVAN) after renal transplantation. 5% to 10% of BKVuria patients will develop BKVAN, of which 50% to 80% will continue to develop into transplanted kidney failure, which is related to the severity of inflammation, the degree of tubular cell destruction and the degree of renal fibrosis. Some scholars believe that there is a mutually reinforcing effect between JCV and BKV. There have been reports of JCV and BKV recombinant fragments found in the urine of renal transplant recipients, as well as reports of PVAN development due to co-infection of BKV and JCV. Other studies believe that there is a mutual inhibitory effect between JCV and BKV. Under immunosuppressive conditions, JCV and BKV are co-activated at a low rate, and the BKV antibody titers of donors and recipients inhibit the development of JCVuria in recipients in a dose-dependent manner.

[0007] Treatment for BK virus infection after kidney transplantation may include immunosuppressive therapy, antiviral drug therapy, and supportive therapy, but all of the above treatments have problems such as long treatment time and large side effects. And it is impossible to distinguish whether it is a treatment specifically for BKV or for JCV. Monoclonal antibody therapy uses the specificity of monoclonal antibody drugs to combine with human recognition of specific foreign substances to stimulate macrophage immune cells or other immune systems to eliminate specific pathogens. Monoclonal antibodies are usually targeted drugs with very strong selectivity. They basically do not kill lymphocytes that express other antibodies. During the use of the drug, because it kills fewer normal cells and has relatively fewer side effects, it will cause less damage to the human body.

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

[0009] In order to solve the technical problem that there is no specific anti-BKV monoclonal antibody or its corresponding product in the prior art, the present invention provides a monoclonal antibody that can specifically bind to BKV. The present invention is based on the discovery and preparation method of monoclonal cell lines, and uses time-resolved fluorescence analysis to screen cell lines with BKV protein coating and JCV protein coating at the same time. When the BKV protein coating test is positive and the JCV protein coating is negative, the cell line that specifically secretes anti-BKV antibodies can be screened, thereby obtaining anti-BKV monoclonal antibodies.

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

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

[0012] In the present invention, the amino acid sequences of the CDRs listed above are all shown according to the Kabat definition rules (the claims of the present invention are also the sequences shown according to the Kabat definition rules). However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as the Chothia definition rules based on the Kabat definition rules and the structural loop region position (see JMol Biol 273: 927-48, 1997). In the present invention, the Combined definition rules including the Kabat definition and the Chothia definition can also be used to determine the amino acid residues in the variable domain sequence. The Combined definition rule combines the scope of the Kabat definition and the Chothia definition, and takes a larger scope based on this. It should be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or its region (e.g., variable region) should be understood to cover the complementary determining region defined by any of the above-mentioned known schemes described in the present invention. Although the scope of protection claimed in the present invention is based on the sequence shown in the Kabat definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of protection of the present invention.

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

[0014] In some preferred embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:3; and / or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 85% sequence identity with 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 shown in SEQ ID NO:4 or includes an amino acid sequence that has at least 85% sequence identity with SEQ ID NO:4; and / or, the light chain constant region includes an amino acid sequence as shown in SEQ ID NO:8 or includes an amino acid sequence that has at least 85% sequence identity with 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 a scFv.

[0018] Examples of antigen-binding fragments of the present invention 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 present invention provides an isolated nucleic acid, wherein the isolated nucleic acid encodes the anti-BKV antibody according to the first aspect of the present invention.

[0020] The third aspect of the present invention provides a recombinant expression vector, which comprises the isolated nucleic acid as described in the second aspect of the present invention.

[0021] The term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits the expression of mRNA, protein, polypeptide or peptide by a host cell when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide or peptide to be expressed in the cell. The vectors disclosed herein are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may contain any type of nucleotides, including but not limited to DNA and RNA: they may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and they may contain natural, non-natural or altered nucleotides. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0022] The fourth aspect of the present invention provides a transformant, which comprises the isolated nucleic acid as described in the second aspect of the present invention or the recombinant expression vector as described in 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 a nucleic acid or vector described herein. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or algae; or the host cell can be a prokaryotic cell, such as a bacterium or a protozoan.

[0025] The expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, 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 cell is cultivated in a culture medium and screened for suitable activity. The method and conditions for cultivating the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on the present specification and the methods known in the prior art, according to the specific expression vector used and the host cell change or optimization. In addition, one or more markers of the host cell transfected can be selected by introducing, and the cell that has been stably incorporated into its chromosome by DNA can be selected. The marker can, for example, provide prototrophy, biocidal resistance (for example, antibiotics) or heavy metal (such as copper) resistance to auxotrophic hosts. Selectable marker genes can be directly connected to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

[0026] The fifth aspect of the present invention provides a hybridoma cell line, which can secrete the anti-BKV antibody as described in the first aspect of the present invention.

[0027] The sixth aspect of the present invention provides a pharmaceutical composition, which comprises the anti-BKV antibody as described in the first aspect of the present invention, and a pharmaceutically acceptable carrier.

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

[0029] Cultivate the transformant as described in the fourth aspect of the present invention or cultivate the hybridoma cell line as described in the fifth aspect of the present invention, and obtain anti-BKV antibodies from the culture; and / or, synthesize by chemical methods according to the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:6.

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

[0031] 1) Balb / c mice were immunized with BKV protein solution as an antigen. After three immunizations, blood was collected from the retroorbital area of ​​the mice and centrifuged to obtain mouse serum, which was then tested using time-resolved fluorescence analysis.

[0032] 2) fusing SP2 / 0 mouse myeloma cells with spleen cells of mice having immune effects detected in 1) and screening hybridoma cells in a selective medium;

[0033] Preferably, the selection medium is 20% FBS (HAT) medium;

[0034] 3) The positive hybridoma cells screened in step 2) were subcloned three times in a culture medium containing 10% FBS to obtain a hybridoma cell line that stably secretes anti-BKV monoclonal antibodies.

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

[0036] The tenth aspect of the present invention provides a method for detecting BKV for non-diagnostic purposes, the method comprising contacting a sample to be tested with one or more of the anti-BKV antibody as described in the first aspect of the present invention, the isolated nucleic acid as described in the second aspect of the present invention, the recombinant expression vector as described in the third aspect of the present invention, the transformant as described in the fourth aspect of the present invention, the hybridoma cell line as described in the fifth aspect of the present invention and / or the pharmaceutical composition as described in the sixth aspect of the present invention to detect BKV.

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

[0038] As used herein, the terms “comprise” or “comprising” are intended to indicate that compositions and methods include the stated elements but do not exclude other elements, but according to the context, also include the case of “consisting of…”

[0039] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

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

[0041] The positive and progressive effects of the present invention are:

[0042] The anti-BKV monoclonal antibody prepared by the invention has good specificity and potency and is suitable for preparing different BKV diagnostic reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the time-resolved fluorescence immunoassay detection process.

[0044] Figure 2 These are photos of mice after the first immunization.

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

[0046] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

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

[0052] 2. CB coating solution

[0053] Table 2 CB coating solution formula

[0054]

[0055] Preparation: Take a clean preparation container, weigh Na2CO3 and NaHCO3 respectively, add about 80% of the total amount of purified water, stir until fully dissolved, adjust the pH to 9.6±0.1 with 1M HCl or 1M NaOH, add purified water to the total amount of preparation, mix well. Store at room temperature, the validity 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 and add about 80% of the total amount of purified water, stir until fully dissolved, add purified water to the total amount of preparation, mix well. Store at room temperature, the validity period is 3 months.

[0060] 4. Blocking solution

[0061] Table 4 Blocking solution formula

[0062]

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

[0064] 5. Gelatin diluent

[0065] Table 5 Gelatin diluent formula

[0066]

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

[0068] 6. Buffer A

[0069] Table 6 Buffer A formula

[0070]

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

[0072] 7. Enhancement fluid

[0073] Table 7 Enhancement liquid formula

[0074]

[0075] Preparation: Take a clean preparation container, weigh β-NTA and TOPO, dissolve in 1mL of anhydrous ethanol; weigh potassium hydrogen phthalate, measure glacial acetic acid, add about 80% of the total preparation amount of purified water, stir until fully dissolved, add purified water to the total preparation amount, add 1mL of ethanol dissolved with β-NTA and TOPO and mix well. Store at 2-8℃, the validity period is 6 months.

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

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

[0078]

[0079] Preparation: Take the sterilized container and measure 1640 culture medium, fetal bovine serum, and penicillin-streptomycin solution in a biosafety cabinet and mix them. Use part of the solution to dissolve HAT powder and add it to the container. Store at 2-8℃. The shelf life is 3 months.

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

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

[0082]

[0083] Preparation: Take the sterilized container and measure 1640 culture medium, fetal bovine serum and penicillin-streptomycin solution in a biosafety cabinet and mix them. Use part of the solution to dissolve the HT powder and add it to the container. Store at 2-8℃. The shelf life is 3 months.

[0084] 10. 10% FBS medium

[0085] Table 10 10% FBS culture medium formula

[0086]

[0087] Preparation: Take the sterilized container and measure 1640 culture medium, fetal bovine serum and penicillin-streptomycin solution in a biosafety cabinet and mix well. Store at 2-8℃. The shelf life is 3 months.

[0088] 11. Preparation of Immunization Injections

[0089] The volume ratio of BKV protein solution to adjuvant is 1:1. The immunization scheme is shown in Table 11. For the first immunization, take 450μL of 2mg / mL (1×PBS dilution) BKV protein solution and mix it with 450μL of Freund's complete adjuvant. For subsequent immunizations, take 450μL of 2mg / mL (1×PBS dilution) BKV protein solution and mix it with 450μL of Freund's incomplete adjuvant. Put it on the Yingong mixer, shake for 60S, and then quickly put it on ice to cool for 60S. Repeat the shaking and cooling 6 times to obtain the immune emulsifier, and draw the immune emulsifier into a 1mL syringe and set aside at 2-8℃.

[0090] Table 11 Immunization schedule

[0091] Immunization times BKV protein solution (2 mg / mL) Adjuvant first 450μL Freund's complete adjuvant 450 μL Second time 450μL Freund's incomplete adjuvant 450 μL The third time 450μL 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 mouse immunization and blood collection plan was first formulated, and finally 6 female Blab / c mice of 6 weeks old were selected. The immunization method was to inject BKV antigen subcutaneously at multiple points on the back, the immunization dose was 150μL / mouse, the effective amount was 150μg, and the immunization interval was 14 days. One week after the third immunization, blood was collected from the mouse's posterior orbit (see Table 12).

[0095] In order to obtain a better immunization effect, the immunization method is subcutaneous. Before immunization, the hair on the back of the mouse to be immunized is shaved to expose the back skin. Then, the skin is gently lifted with the sharp needle at the front of the syringe, and the immune emulsifier is injected. 150μL of emulsified volume is divided into 5 immunization points, and each point is about 30μL. The photo of the mouse after the first immunization is shown in Figure 2 .

[0096] Table 12 Immunization schedule

[0097] Number of days operate Adjuvant Inoculation site 0 First immunization Freund's complete adjuvant Subcutaneous 14 Second immunization Freund's incomplete adjuvant Subcutaneous 28 The third immunization Freund's incomplete adjuvant Subcutaneous

[0098] (2) Serum collection from mice

[0099] When collecting blood, press the sides of the mouse's neck with the thumb and index finger of one hand, and hold the mouse's neck tightly from the back to make the retro-orbital venous plexus congested. When the mouse is held in the hand, make sure the mouse body remains in a straight position. The capillary in the right hand is at an angle of 45° to the mouse's face and inserted from the inner corner of the eye. The capillary is rotated with the finger to puncture the vein for blood collection. Figure 3 After blood collection, press to stop bleeding immediately to avoid excessive bleeding or eyeball necrosis. The collected blood was quickly blown into a 1.5mL EP tube and placed at room temperature for 1 hour. After 1 hour at 4℃, centrifuge at 6000rmp for 5 minutes, take the upper serum and store at 4℃ for later use.

[0100] Example 3 Serum detection

[0101] 1. Plate coating: Dilute the BKV protein solution to 2 μg / mL with CB coating solution, 50 μL / well, add to a 96-well TRFIA microplate, incubate at 37°C for 2 hours, and then incubate at 4°C for 16-36 hours. Discard the coating solution, dilute 20×PBST to 1×PBST with purified water, and wash the plate twice. Add 200 μL / well of blocking solution, incubate at 37°C for 2 hours, discard the blocking solution, and wash the plate twice with 1×PBST. Complete the coating of the test plate and store at -20°C for later use.

[0102] 2. Titer detection: After the mouse serum was diluted from the initial 1 / 1000 (gelatin diluent), it was diluted 3 times in a gradient of 1 / 1000, 1 / 3000, 1 / 9000, 1 / 27000, 1 / 81000, 1 / 243000, and 1 / 729000. The gelatin diluent was used as a BLANK control. 50 μl / well of each gradient of diluted serum and BLANK were added to the test plate and incubated at 37°C for 30 minutes. The supernatant was discarded, and the plate was washed once with 1×PBST. Bio-YKS (Bio: biotin, B2643-100 mg, Sigma; YKS: goat anti-mouse antibody, DCY0260A, Jackson) diluted to 1 μg / mL with gelatin diluent was added, 50 μL / well, and incubated at 37°C for 30 minutes. Discard the supernatant, wash the plate twice with 1×PBST, add Buffer A diluted to 0.5μg / mL SA-Eu (SA: streptavidin, Z7041, Promega; Eu: DTTA-Eu, DTTA-EuNa, Shanghai Wei Ni), 50μL / well, incubate at 37℃ for 30min. Discard the supernatant, wash the plate 6 times with 1×PBST, spin dry, add 100μL / well of enhancement solution, and shake for 10min. Put it into the time-resolved fluorescence immunoassay analyzer for reading. The results are shown in Table 13. Mouse 5# has the highest titer and is selected for cell fusion.

[0103] Table 13 Mouse serum titer test results

[0104]

[0105] Example 4 Cell Fusion

[0106] Mouse #5 was dissected, and its spleen was taken and grinded to collect spleen cells. SP2 / 0 cells that had been revived and cultured one week in advance were also collected. The cell number SP2 / 0: spleen cells was adjusted to 1:6, and the fusion parameters of the electrofusion instrument were adjusted (see Table 14) to perform cell electrofusion. The fused hybridoma cells were collected, mixed with 20% FBS (HAT) medium, and plated on a 96-well cell culture plate at 200 μL / well. After one week of culture in a cell culture incubator at 37°C and 5% CO2, the fusion medium was changed. The supernatant was discarded and 200 μL / well of 15% FBS (HT) medium was added.

[0107] Table 14 Fusion parameters of electrofusion instrument

[0108]

[0109]

[0110] Example 5: Fusion screening

[0111] Take 50 μL of the cell supernatant in each well of the 96-well cell plate and add it to the pre-coated BKV detection plate (the coating method is described in Example 6), and incubate at 37°C for 30 min. Subsequent detection is the same as in Example 6: "Discard the supernatant, wash the plate twice with 1×PBST, add Buffer A diluted to the working concentration of SA-Eu, 50 μL / well, and incubate at 37°C for 30 min. Discard the supernatant, wash the plate 6 times with 1×PBST, spin dry, pat dry, add 100 μL / well of enhancement solution, and shake for 10 min. Put it into the time-resolved fluorescence immunoassay analyzer for reading (see the detection process). Figure 1 ), the results are shown in Table 15, and the positive strains with higher readings (12 strains) were selected for the first subcloning.

[0112] Table 15 Results of fusion primary screening fluorescent immunoassay

[0113] #Board number

[0114] Negative control

[0115] Positive Control

[0116] Positive cell lines

[0117]

[0118]

[0119]

[0120] Example 6 Subcloning

[0121] 1. First subcloning and detection

[0122] (1) First subcloning: Add 10% FBS medium to a 96-well cell culture plate at 200 μL / well, transfer the cells of the positive strain to row A of the cell plate, and dilute the cell solution in a 1:5 gradient from row A to row H. After culturing in a cell culture incubator at 37°C and 5% CO2 for one week, perform the first subcloning test.

[0123] (2) First subclone detection: BKV and JCV detection plates were coated in advance (the coating method refers to Example 6, 8 cell lines were selected for each subclone, and 100 μL / well of supernatant was taken. 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. Subsequent detection was the same as in Example 6 "discard the supernatant, wash the plate twice with 1×PBST, add 50 μL / well of SA-Eu diluted to the working concentration in Buffer A, and incubate at 37°C for 30 min. Discard the supernatant, wash the plate 6 times with 1×PBST, spin dry, pat dry, add 100 μL / well of enhancement solution, and shake for 10 min. Put it into the time-resolved fluorescence immunoassay analyzer for reading", the results are shown in Table 16, and the positive strains (4 strains) with BKV readings > JCV readings and BKV readings > 50W were selected for the second subcloning. The method for the second subcloning was the same as the first subcloning, and the data was underlined below, indicating that the cell line had become a monoclonal cell line (6F9 No. 8).

[0124] Table 16 Results of the first subclone detection

[0125] Cell line name

[0126] Positive cell lines

[0127]

[0128]

[0129] 2. Second subcloning and detection

[0130] (1) Second subcloning: The method is the same as the first subcloning. The data is underlined, indicating that the cell line has become a monoclonal cell line.

[0131] (2) Second subclone detection:

[0132] Methods The first subclone test, the results are shown in Table 17, and there are underlines below the data (the underlined cell line name represents that the cell line has become a single cell cluster after multiple subclones from a multi-cell cluster; that is, a cell cluster cloned from a cell, indicating that all genetic materials in the cell cluster are consistent), indicating that the cell line has become a monoclonal cell line. Considering the high BKV reading, low JCV / BKV, good cell state under the microscope and monoclonal priority as much as possible, 2 cell lines were selected for each cell line again, that is, a total of 8 cell lines were subcloned for the third time.

[0133] Table 17 Second subclone detection results

[0134] Cell line name

[0135] Positive cell lines

[0136]

[0137]

[0138] 3. The third subcloning and detection

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

[0140] (2) The method is the same as the first subclone test. The results are shown in Table 18. The data is underlined, indicating that the cell line has become a monoclonal cell line. Taking into account the high BKV reading value, low JCV / BKV, good cell status under the microscope and the priority of monoclonality, the cell line 6F9.2.6.5 was finally selected for establishment. After subculture, the cells were collected, some of which were frozen in liquid nitrogen and some were used for sequencing.

[0141] Table 18 The third subclone detection results

[0142] Cell line name

[0143] Positive monoclonal cell line

[0144]

[0145] Example 7 Sample Submission for Sequencing

[0146] After DNA extraction and PCR amplification, the cells were sent for sequencing, and the results returned by sequencing were as follows:

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

[0148]

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

[0150] MGWSWIFLFLLSGTAGVLSEVQLKQSGPELVKPGASVKISCKTSGYTFTEYTMYWVKQSHGKSLEWIGGINPNNGDTTYKQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASFYPIYVMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPSGELQEHSAHHGHRWLLLRLQQAQCAEEQLGGRKYFHLLCVT

[0151] Variable region sequence of the BKV heavy chain (SEQ ID NO: 3):

[0152] MGWSWIFLFLLSGTAGVLSEVQLKQSGPELVKPGASVKISCKTSGYTFTEYTMYWVKQSHGKSLEWIGGINPNNGDTTYKQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASFYPIYVMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVT

[0153] Constant region sequence of the BKV heavy chain (SEQ ID NO: 4):

[0154] VTWNGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQ INSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPSGELQEHSAHHGHRWLLLRLQQAQCAEEQLGGRKYFHLLCVT

[0155] The kappa light chain sequence is as follows:

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

[0157] ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGCCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGATTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTTAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG

[0158] Full-length amino acid sequence of BKV kappa light chain (SEQ ID NO: 6):

[0159] MKLPVRLLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQDSHVP WTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGALVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

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

[0161] MKLPVRLLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQSI VHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQDSHVPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGALVVCFLNNFY

[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] serial 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 of the light and heavy chains of the anti-BKV virus antibodies are shown in Table 19, and the numbering scheme is Kabat.

Claims

1. An anti-BKV antibody, characterized in that 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; the light chain variable region comprises LCDR1, LCDR2 and LCDR3; wherein: The amino acid sequence of the HCDR1 is shown in SEQ ID NO:9, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:10, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:11; the amino acid sequence of the LCDR1 is shown in SEQ ID NO:12, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:13, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:

14.

2. The anti-BKV antibody according to claim 1, characterized in that The framework region of the heavy chain variable region and / or the light chain variable region is a murine framework region; Preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:3; and / or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

7.

3. The anti-BKV antibody according to claim 1 or 2, characterized in that The anti-BKV antibody further comprises a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:4; and / or, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

8.

4. The anti-BKV antibody according to claim 1 or 2, characterized in that The anti-BKV antibody is a full-length antibody, Fab, Fab', scFab, F(ab')2 or Fv; the Fv is preferably scFv.

5. An isolated nucleic acid, characterized in that The isolated nucleic acid encodes the anti-BKV antibody according to any one of claims 1-4.

6. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid of claim 5.

7. A transformant, characterized in that: The transformant comprises the isolated nucleic acid of claim 5 or the recombinant expression vector of claim 6; Preferably, the host cell of the transformant is a eukaryotic cell or a prokaryotic cell.

8. A hybridoma cell line, characterized in that: The hybridoma cell line can secrete the anti-BKV antibody according to any one of claims 1 to 4.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-BKV antibody according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

10. A method for preparing an anti-BKV antibody, characterized in that: The method comprises the following steps: Cultivating the transformant as claimed in claim 7 or culturing the hybridoma cell line as claimed in claim 8, and obtaining anti-BKV antibodies from the culture; and / or, synthesizing by chemical methods according to the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO:

6.

11. A kit for immunological detection, characterized in that: The kit comprises one or more of the anti-BKV antibody according to any one of claims 1 to 4, the isolated nucleic acid according to claim 5, the recombinant expression vector according to claim 6, the transformant according to claim 7, the hybridoma cell line according to claim 8, and the pharmaceutical composition according to claim 9.

12. A method for detecting BKV for non-diagnostic purposes, characterized in that: The method comprises contacting one or more of the anti-BKV antibody of any one of claims 1 to 4, the isolated nucleic acid of claim 5, the recombinant expression vector of claim 6, the transformant of claim 7, the hybridoma cell line of claim 8, and the pharmaceutical composition of claim 9 with the sample to be tested to detect BKV.

13. Use of the anti-BKV antibody according to any one of claims 1 to 4, the isolated nucleic acid according to claim 5, the recombinant expression vector according to claim 6, the transformant according to claim 7, the hybridoma cell line according to claim 8 and / or the pharmaceutical composition according to claim 9 in the preparation of a reagent for detecting BKV.

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