A monoclonal antibody against Clostridium difficile toxin B and its preparation method and application

Abstract

The present invention relates to the field of medical biological antibodies, specifically a monoclonal antibody against Clostridium difficile toxin B, the amino acid sequences of its heavy chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO:1-3, respectively, and the amino acid sequences of its light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO:4-6, respectively. The present invention also provides a preparation method and application of the monoclonal antibody. The mouse-derived neutralizing antibody screened by the present invention has good antibody activity and has the potential to be developed into a marketed product.

Description

Technical Field

[0001] The present invention relates to the field of medical biological antibodies, and in particular to a monoclonal antibody against Clostridium difficile toxin B and a preparation method and application thereof. Background Art

[0002] Clostridium difficile is the main pathogen of antibiotic-associated diarrhea and pseudomembranous colitis. The excessive reproduction of pathogenic Clostridium difficile in the intestine leads to intestinal flora imbalance, releasing a large number of virulence factors, and ultimately inducing Clostridium difficile infection (CDI).

[0003] Currently, monoclonal antibodies (McAbs) are widely used in the treatment of infectious diseases. In 2016, Bezlotoxumab, a fully human monoclonal antibody product against Clostridium difficile toxin B (TcdB), was approved for marketing by the US Food and Drug Administration (FDA), becoming the first McAb drug for the treatment of CDI patients aged ≥18 years (Gerding DN, File TM, Jr., McDonald L C. Diagnosis and Treatment of Clostridium difficile Infection (CDI) [J]. Infect Dis Clin Pract (Baltim Md), 2016, 24 (1): 3-10. Wilcox MH, Gerding DN, Poxton IR, et al. Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection [J]. N Engl J Med, 2017, 376 (4): 305-317.). Although it has shown good results in reducing the recurrence rate of CDI and improving the overall cure rate, the treatment cost is as high as US$50,000 and is generally only suitable for severe patients with a high risk of recurrence (Prabhu VS, Dubberke ER, Dorr MB, et al. Cost-effectiveness of Bezlotoxumab Compared With Placebo for the Prevention of Recurrent Clostridium difficile Infection [J]. Clin Infect Dis, 2018, 66 (3): 355-362.). In addition, the drug is prone to cause obvious side effects such as vomiting, abdominal pain, and diarrhea in patients. Therefore, it is urgent to develop therapeutic antibody drugs with a wider range of applications, lower prices, and fewer side effects to improve the overall cure rate of CDI. Summary of the invention

[0004] In order to solve the above problems, the purpose of the present invention is to provide a monoclonal antibody against Clostridium difficile toxin B and a preparation method and application thereof.

[0005] Studies have reported that Toxin of Clostridium difficile B (TcdB) plays a key role in the pathogenic process of CDI (Aktories K, Schwan C, Jank T. Clostridium difficile Toxin Biology [J]. Annu Rev Microbiol, 2017, 71: 281-307.), and the receptor binding domain (RBD) of TcdB is the key antigenic epitope for the toxin to enter the human body and cause disease (Tian JH, Fuhrmann SR, Kluepfel-Stahl S, et al. A novel fusion protein containing the receptor binding domains of C. difficile toxin A and toxin B elicits protective immunity against lethal toxin and spore challenge in preclinical efficacy models [J]. Vaccine, 2012, 30 (28): 4249-4258.). The present invention uses recombinantly expressed TcdB RBD protein as a target antigen to immunize Balb / c mice, prepare hybridoma cells, screen positive clones by limiting dilution method, determine hybridoma cell lines by forward and reverse screening and Tcd B secondary screening, verify neutralization activity by mouse animal experiments, and analyze antibody affinity by surface plasmon resonance technology (SPR).

[0006] In a first aspect of the present invention, there is provided a monoclonal antibody (2F3) against Clostridium difficile toxin B, wherein the amino acid sequences of heavy chain CDR1, CDR2 and CDR3 are:

[0007] CDR1: GYTFTSYW, as shown in SEQ ID NO: 1;

[0008] CDR2: IYPGNNNT, as shown in SEQ ID NO: 2;

[0009] CDR3: SRGDYGSSYALDY, as shown in SEQ ID NO: 3;

[0010] The amino acid sequences of light chain CDR1, CDR2, and CDR3 are:

[0011] CDR1: QDVNTA, as shown in SEQ ID NO: 4;

[0012] CDR2: SASYRYTGV, as shown in SEQ ID NO:5;

[0013] CDR3: QQRHVPPRT, as shown in SEQ ID NO:6.

[0014] Furthermore, the monoclonal antibody is selected from a fragment comprising a heavy chain CDR region and a light chain CDR region, a fragment comprising a heavy chain variable region and a light chain variable region, or a complete antibody molecule having full-length heavy and light chains.

[0015] Furthermore, in the monoclonal antibody, its heavy chain comprises 4 FR regions, and its light chain comprises 4 FR regions; the amino acid sequences of the 4 FR regions of the heavy chain are shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively;

[0016] EVKLVESGTVLARPGASVKMSCKAS(SEQ ID NO:7);

[0017] LHWVKQRPGQGLEWIGA(SEQ ID NO:8);

[0018] SYSQKFKGKAKLTAVTSTSTAYMDLSSLTNDDSAVYYC(SEQ ID NO:9);

[0019] WGQGTSVTVSSA (SEQ ID NO: 10).

[0020] The amino acid sequences of the four FR regions of the light chain are shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0021] DIVMTQAHKFMSTSVGDRVSFTCKAS(SEQ ID NO:11);

[0022] AWYQQKPGQSPKLLIY(SEQ ID NO:12);

[0023] PDRFTGSGSGTDFTFTITSVQAEDLAVYYC(SEQ ID NO:13);

[0024] FGGGTKVEIKRA (SEQ ID NO: 14).

[0025] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO:15.

[0026] In a preferred embodiment of the present invention, the amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO:16.

[0027] VH:

[0028] EVKLVESGTVLARPGASVKMSCKAS GYTFTSYW LHWVKQRPGQGLEWIGA IYPGNNNT SYSQKFKGKAKLTAVTSTSTAYMDLSSLTNDDSAVYYC SRGDYGSSYALDY WGQGTSVTVSSA (SEQ ID NO: 15)

[0029] VL:

[0030] DIVMTQAHKFMSTSVGDRVSFTCKAS QDV IAWYQQKPGQSPKLLIY SASYRYTGV PDRFTGSGSGTDFTFTITSVQAEDLAVYYC QQQRHVPPRT FGGGTKVEIKRA (SEQ ID NO: 16)

[0031] The underlined regions are the heavy chain CDR region and the light chain CDR region.

[0032] The second aspect of the present invention provides a polynucleotide comprising nucleotides encoding the above-mentioned monoclonal antibody.

[0033] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:17, and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:18.

[0034] Nucleotide sequence encoding the heavy chain variable region:

[0035] VH:GAGGTGAAGCTGGTGGAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCA GTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGTTGCACTGGGTAAAACAGAGGCCTGGACAGGGTTCTGGAATGGATTGGCGCTATTTATCCTGGAAAATAATAATACTAGTTACAGCCAGAAGTTCAAGGGCAAGG CCAAACTGACTGCAGTCACATCCACCAGTACTGCCTACATGGACCTCAGCAGCCTGACAAATGATGACTCTGCGGTCTATTACTGTTCAAGAGGGGACTACGGTAGTAGTTATGCTCTGGACTACTGGGGTCAAGGAACCTCAGTCACCGT CTCCTCAGCC(SEQID NO:17)

[0036] Nucleotide sequence encoding the light chain variable region:

[0037] VL:GATATTGTGATGACGCAGGCTCACAAATTCATGTCCACATCAGTAGGAGACAG GGTCAGCTTCACCTGCAAGGCCAGTCAGGATGTGAATACTGCTATAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGCTACACTGGAGTACCTGATCGCTTCACTGGC AGTGGATCTGGGACGGATTTCACTTTCACCATCACCAGTGTGCAGGCTGAAGACCTGGCAGTGTATTACTGTCAGCAACGTCATGTTCCTCCTCGGACGTTCGGTGGAGGCACCAAGGTGGAAATCAAACGGGCT(SEQ ID NO:18)

[0038] Furthermore, the upstream and downstream primer sequences for amplifying the heavy chain variable region are shown as SEQ ID NO:21 and SEQ ID NO:22, respectively; the upstream and downstream primer sequences for amplifying the light chain variable region are shown as SEQ ID NO:23 and SEQ ID NO:24, respectively.

[0039] The third aspect of the present invention provides a recombinant expression vector comprising the above-mentioned polynucleotide.

[0040] The fourth aspect of the present invention provides a host cell transformed with the above-mentioned recombinant expression vector.

[0041] The fifth aspect of the present invention provides the use of the above monoclonal antibody in the preparation of a drug for treating Clostridium difficile infection.

[0042] Furthermore, the drug uses the monoclonal antibody as an active ingredient.

[0043] The sixth aspect of the present invention provides a drug for treating Clostridium difficile infection, which comprises the monoclonal antibody.

[0044] The seventh aspect of the present invention provides the use of the above-mentioned monoclonal antibody in the preparation of a Clostridium difficile detection reagent or a detection kit.

[0045] Furthermore, the detection reagent includes the above monoclonal antibody which is biologically or chemically labeled;

[0046] The detection kit includes a labeled complex obtained by biologically or chemically labeling the monoclonal antibody.

[0047] The eighth aspect of the present invention provides a Clostridium difficile detection kit, which includes a labeled complex obtained by biologically or chemically labeling the above-mentioned monoclonal antibody.

[0048] The ninth aspect of the present invention provides a method for preparing the above-mentioned monoclonal antibody, comprising: culturing the above-mentioned host cell to induce the expression of the monoclonal antibody.

[0049] Further, the method specifically includes the following steps:

[0050] (A) Construction of TcdB RBD recombinant expression plasmid to induce expression of TcdB RBD protein;

[0051] (B) The purity of TcdB RBD protein after three-step column chromatography was above 90% as determined by CE-SDS;

[0052] (C) BALB / c mice were immunized with TcdB RBD recombinant protein, and spleen cells were fused with SP2 / 0 to prepare hybridoma cells;

[0053] (D) Cloning and screening of positive hybridoma cell lines by limiting dilution method, and preparing ascites animal experiment to verify the neutralizing activity of antibodies;

[0054] (E) Primers are designed to clone the heavy and light chain variable region genes of the antibody from the hybridoma cell genome, and sequencing analysis is performed to determine the candidate antibody sequences.

[0055] The advantages and beneficial effects of the present invention are:

[0056] 1. Recombinantly expressing the TcdB RBD region as an antigen to immunize animals greatly improves the efficiency of obtaining neutralizing antibodies.

[0057] 2. Antibody screening used TcdB RBD and TcdB cross-screening to further prove the reliability of the screening results.

[0058] 3. The mouse neutralizing antibody 2F3 screened out in the present invention has good antibody activity and has the potential to be developed into a marketed product. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 .Identification results of TcdB RBD gene PCR products.

[0060] Figure 2 .Identification of recombinant plasmid enzyme digestion; M: DL5000 DNA Marker; 1: PET-22b vector; 2: enzyme digested PET-22b vector; 3: recombinant plasmid Pet-TcdB RBD; 4: enzyme digested recombinant plasmid Pet-TcdB RBD.

[0061] Figure 3 .PCR identification results of TcdB RBD recombinant expression plasmid; M: DL2000 DNA Marker; 1-10: TcdBRBD recombinant expression plasmid positive clones.

[0062] Figure 4 .Homology comparison results of TcdB RBD recombinant expression plasmid.

[0063] Figure 5 .Chromatographic purification results of protein expression products.

[0064] Figure 6 .SDS-PAGE analysis of TcdB RBD recombinant protein expression and purification results; M: protein maker; 1: supernatant without induced lysis of bacteria; 2: precipitate without induced lysis of bacteria; 3: supernatant with induced lysis of bacteria; 4: precipitate with induced lysis of bacteria; 5: flow-through of Ni column chromatography; 6: elution with 20mmol / L imidazole on Ni column chromatography; 7: elution with 60mmol / L imidazole on Ni column chromatography; 8: flow-through of hydrophobic chromatography; 9: sample peak of hydrophobic chromatography; 10: sample peak of anion exchange chromatography.

[0065] Figure 7.Western Blot detection of TcdB RBD recombinant protein expression and purification results; M: Western Blotmaker; 1: supernatant without induced lysis of bacteria; 2: precipitate without induced lysis of bacteria; 3: supernatant with induced lysis of bacteria; 4: precipitate with induced lysis of bacteria; 5: flow-through of Ni column chromatography; 6: elution of Ni column chromatography with 20mmol / L imidazole; 7: elution of Ni column chromatography with 60mmol / L imidazole; 8: flow-through of hydrophobic chromatography; 9: sample peak of hydrophobic chromatography; 10: sample peak of anion exchange chromatography.

[0066] Figure 8 .CE-SDS results of TcdB RBD recombinant protein after three-step chromatography purification

[0067] Fig. 9 .Western Blot analysis of TcdB RBD recombinant protein specificity results; M: Western Blot maker; 1: TcdB RBD protein; 2: pure TcdB of Clostridium difficile.

[0068] Fig.10 .Cell fusion process.

[0069] Fig.11 . Purification of monoclonal antibodies and SDS-PAGE analysis results; M: protein maker; 1: non-reduced IgG; 2: reduced IgG.

[0070] Fig.12 . Specificity analysis results of monoclonal antibodies; M1: protein maker; M2: Western Blot maker; 1: non-reduced TcdB; 2: reduced TcdB.

[0071] Fig.13 .Results of toxicity test of Clostridium difficile toxin B.

[0072] Fig.14 .Affinity analysis results of neutralizing antibody 2F3 and TcdB.

[0073] Fig.15 .Affinity analysis results of Bezlotoxumab and TcdB.

[0074] Fig.16 .Identification results of PCR products of VH, Vκ, and Vλ chains of mouse antibodies; M: DL5000 DNA Marker; 1-3: VH; 4-5: Vκ; 6: Vλ.

[0075] Fig.17 .Antibody gene structure analysis results. DETAILED DESCRIPTION

[0076] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0077] Embodiment 1:

[0078] 1. Materials and Methods

[0079] 1.1 Test equipment and reagents

[0080] 1.1.1 Virus seeds and cells

[0081] The toxin-producing strain of Clostridium difficile LCL084 was provided by the Clostridium Laboratory of Lanzhou Institute of Biological Products. SP2 / 0 myeloma cells were provided by the Fifth Research Laboratory of Lanzhou Institute of Biological Products.

[0082] 1.1.2 Carrier

[0083] E.coil BL21 (DE3) competent cells were prepared by the Fifth Research Laboratory of Lanzhou Institute of Biological Products Co., Ltd. PET-22b vector was provided by the Fifth Research Laboratory of Lanzhou Institute of Biological Products Co., Ltd.

[0084] 1.2.3 Experimental animals

[0085] Female BALB / c mice and female Kunming mice were provided by the SPF animal room of Lanzhou Institute of Biological Products.

[0086] 1.2 Main reagents and equipment

[0087] Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; high-fidelity KOD DNA polymerase was purchased from TOYOBO, Japan; LB culture medium was purchased from Invitrogen, USA; BamHⅠ and XhoⅠ restriction endonucleases, DL2000 DNA Marker, nucleic acid dyes, IPTG, ECL ultrasensitive chemiluminescence kit, IPTG, goat anti-rabbit monoclonal antibody, and HRP-labeled goat anti-mouse IgG were purchased from Shanghai Sangon Biotechnology Co., Ltd.; Ni Sepharose TM High Performance, DEAE Sepharose FastFlow, Phenyl Sepharose Fast Flow, and Protein G filler were purchased from GE, USA; Clostridium difficile pure toxin B and anti-Clostridium difficile toxin B monoclonal antibody were purchased from Abcam, UK; gel recovery kit, PCR purification kit, and plasmid minipreparation kit were all purchased from QIAGEN, Germany; PVDF membrane, protein pre-stained Marker, and cell culture plates were purchased from Thermo, USA; newborn fetal bovine serum, RPMI 1640 culture medium, HT additive, and HAT supplement were purchased from Gibco, USA; Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from SIGMA, USA; PEG 1450 was purchased from Bioss; 96-well ELISA plate was purchased from Costar, USA; Envision microplate reader was purchased from PerkinElmer; PCR amplifiers were purchased from Bio-Rad; electrophoresis instrument was purchased from Beijing Liuyi Instrument Factory; fluorescence microscope was purchased from Olympus IX71; capillary electrophoresis instrument, high-speed centrifuge and PA 800plus SDS-MW Analysis Kit were purchased from Beckman Coulter; NanoVue microspectrophotometer, chromatography system AKTAAVANT, Biacore T200, ImageQuant gel imaging system were purchased from GE; CO2 incubator was purchased from SANYO; cell analyzer was purchased from Count star.

[0088] 1.2 Experimental methods

[0089] 1.2.1 Construction of TcdB RBD prokaryotic expression plasmid

[0090] According to the Clostridium difficile toxin B gene sequence provided by the NCBI database (sequence number: CP046327.1), the genomic DNA of the LCL084 toxin-producing strain was used as a template, BamHⅠ and XhoⅠ were selected as restriction sites, and primers were designed using Primer5.0 software to amplify the TcdB RBD gene sequence. The primer is represented by tcdB-RBD, and TcdB RBD consists of 533 amino acids, located at 1834-2366aa of the TcdB protein. The PCR primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0091] Table 1. Clostridium difficile toxin B RBD PCR primers

[0092]

[0093] The TcdB RBD gene was amplified by PCR using the genomic DNA of the toxin-producing strain LCL084 of Clostridium difficile as a template. The PCR product and the PET-22b vector were digested by BamHI and XhoⅠ, respectively, and the 1% agarose gel electrophoresis was used to identify and recover the gel. After digestion, the size of TcdBRBD was about 1600bp. The vector and sequence were connected using T4 DNA ligase, transformed into E.coil BL21 (DE3), coated on LB agar plates containing ampicillin (AMP) resistance, and the positive clones were selected for sequencing, and the strains with 100% sequence match were retained.

[0094] 1.2.2 Recombinant expression and purification of TcdB RBD protein

[0095] The recombinant bacteria activated overnight in advance were inoculated into LB (Amp) medium at a ratio of 1:100, cultured at 37°C, 220rpm until OD600 = 0.6-0.8, and a certain amount of IPTG with a final concentration of 1mM was added, and induced for 12h at 25°C, 220rpm; centrifuged at 9500rpm for 30min, dissolved the bacterial precipitate with PBS buffer, and crushed the bacteria with a pressure crusher. Centrifuged at 10000g for 15min, collected the supernatant and precipitate, and analyzed the expression of TcdB RBD protein by SDS-PAGE and Western Blot. If the expression was detected successfully, Ni Sepharose was used. TMThe supernatant of the bacteria was manually purified with High Performance medium. After washing with 20mM imidazole, the imidazole concentration was increased successively for gradient elution. The conductivity of the collected Ni affinity chromatography eluate was adjusted to 120mS / cm. The Phenyl Sepharose Fast Flow hydrophobic chromatography column was equilibrated with 0.05M PB buffer containing 1mol / L (NH4)2SO4, and linear elution was performed with 0.05M PB buffer (pH 7.0, conductivity 5mS / cm). The conductivity of the collected sample peak eluate was adjusted to 5mS / cm. The DEAE anion chromatography column was equilibrated with 0.05M PB buffer (pH 7.0, conductivity 5mS / cm), and linear elution was performed with 0.05M PB buffer (pH 7.0, conductivity 85mS / cm) containing 1mol / L NaCl, and the sample peak eluate was collected. CE-SDS and Western-Blot were used to analyze the purity and activity of TcdB RBD protein after three-step chromatography purification.

[0096] 1.2.3 Preparation of anti-TcdB monoclonal antibody

[0097] BALB / c mice were immunized with highly pure TcdB RBD recombinant protein. Seven days after the third immunization, blood was collected from the tail vein, and the serum titer was determined by ELISA. The mouse spleen cells with the highest antibody titer were selected for the preparation of hybridomas. Three days after the pure antigen booster immunization, the mouse immune spleen cells were fused with SP2 / 0 myeloma cells under the action of PEG1450, and the cells were pressure-screened using HAT selection medium. When the hybridoma cells in the 96-well cell culture plate were cloned to more than 1 / 4 of the entire well field of view, the hybridoma cells were cross-screened by ELISA using TcdB RBD recombinant protein and pure TcdB. The absorbance OD was selected according to the indirect ELISA test results. 450 Positive hybridoma cells with high readings, small number of cell clones, and good cell status are cloned and cultured by limiting dilution method. Cloning is terminated until all monoclonal cell lines are positive by indirect ELISA test results. Positive monoclonal hybridoma cells are expanded and subcultured. During the subculture, indirect ELISA test is also performed to detect the cell subculture stability, and the cells are frozen. Take BALB / c female mice, inject 0.2mL paraffin oil intraperitoneally for mouse sensitization, and inject about 1×10 6 When the abdomen of the mouse is obviously swollen and the mouse has difficulty in moving, use dissecting scissors to cut the mouse abdominal skin to expose the peritoneum, insert a sterile syringe needle into the mouse abdomen, collect the ascites and centrifuge it at 4℃ (12000rpm×30min), remove the fat and paraffin oil in the mouse abdominal cavity, and collect the middle layer of ascites for purification.

[0098] 1.2.4 Purification of mouse ascites

[0099] The ascites was first extracted by salting out with saturated ammonium sulfate precipitation method, centrifuged at 4°C (10,000 rpm × 30 min), the supernatant was discarded, the precipitate was resuspended with PBS buffer, filtered with a 0.45 μm filter membrane, and purified with Protein G agarose gel. A 5 mL Protein G pre-packed column was connected to the corresponding column position of the AKTA Avant protein purification system, and washed with purified water, 0.1 mol / L NaOH, and purified water for 5 column volumes, respectively, and then PBS was used to balance the filler for 10 column volumes. After UV 280 was zeroed, the cell culture supernatant and ascites crude pure samples were loaded at a loading of no more than 30 mg / mL, and the impurities were washed for 5 column volumes with impurity removal buffer (0.5M NaCl+10mM PB, pH 7.0-7.4), PB was rebalanced, and eluted with 0.03M NaAC (pH=3.7), and the elution peak with UV 280>50mAU was collected, and 1mol / L Tris-HCl (pH=10.2) was added to adjust the pH to about 7.0. 5 column volumes were washed with 0.1mol / L NaOH and pure water respectively, and the pre-loaded column was stored in 20% ethanol. The concentration of purified antibodies was determined by NanoVue ultra-micro spectrophotometer Protein A280 method.

[0100] 1.2.5 Specificity analysis of monoclonal antibodies

[0101] Western-Blot was used to identify the specificity of the monoclonal antibody. The pure TcdB of Clostridium difficile (positive control) and the TcdB RBD protein to be detected were separated by SDS-PAGE electrophoresis. After transfer to the membrane, the membrane was blocked for 2 hours at room temperature. The purified anti-TcdB monoclonal antibody was used as the primary antibody and incubated at room temperature for 1 hour. The membrane was washed with PBS-T for 3 to 5 times, each time for 10 minutes. The secondary antibody was HRP-labeled sheep anti-mouse monoclonal antibody, which was bound at room temperature for 1 hour. The membrane was washed with PBS-T for 3 to 5 times, each time for 10 minutes. The substrate was added for color development, exposed for 30 seconds, and the results were observed.

[0102] 1.2.6 Monoclonal Antibody Titer Detection

[0103] Coat a 96-well ELISA plate with pure Clostridium difficile toxin B, block overnight at 4°C, wash the plate, add the diluted monoclonal antibody from the first row in sequence, incubate at 37°C for 1h, wash the plate, add the peroxidase-labeled goat anti-mouse secondary antibody, incubate at 37°C for 45min, wash the plate, add substrate A solution and B solution, incubate for 15min, add the stop solution, and detect the OD with an ELISA reader 450 The positive control is the serum of mice immunized with antigen, and the negative control is the serum of mice not immunized. 450 The maximum dilution corresponding to a reading >2.1 times that of the negative control and greater than 0.2 was taken as the antibody titer.

[0104] 1.2.7 Analysis of neutralizing activity of monoclonal antibodies

[0105] 1.2.7.1 Toxicity assay of Clostridium difficile toxin B

[0106] The toxicity of TcdB was determined by referring to the 2015 edition of the Pharmacopoeia of Botulinum Toxin Type A titer determination method. The toxicity of TcdB was measured at a median lethal dose (LD50) in mice. 50 ) is the detection standard. See the literature for the LD of TcdB in CD1 mice 50 1×10 -3 mg / kg, and toxin B was serially diluted according to this dose. A total of 5 toxin dose dilutions were set, and the toxin doses were 0.25×10 -3 mg / kg, 0.5×10 -3 mg / kg, 1×10 -3 mg / kg, 2×10 -3 mg / kg and 4×10 -3 mg / kg, 2.5mL for each dilution, select female Kunming mice (SPF grade) weighing about 14-16g, and inject 4 mice for each dilution. Each mouse is injected intraperitoneally with 0.5mL, and continuously observed for 96 hours after injection, and the death of mice is recorded and the results are statistically analyzed.

[0107] LD was calculated by modified Koch method 50 , the calculation method is as follows:

[0108] LqCy 50 = maximum dose logarithm value - difference between the logarithm values ​​of two adjacent dose groups × (sum of mortality rates in each dose group - 0.5).

[0109] 1.2.7.2 Neutralization test

[0110] The mouse test was used to qualitatively analyze the neutralizing activity of monoclonal antibodies. The test set up a sample group, a toxin group, a toxin inactivation control group, and a blank group. The sample groups were quantitatively aspirated with different dilutions of the antibody solution to be tested (2F3 sample group, 4H7 sample group, 7C2 sample group, 8C10 sample group, 9C9 sample group, 9A12 sample group, 9H7 sample group, and 10F7 sample group). Then, the 5LD 50The corresponding toxin dose dilutions were mixed, the mixture was incubated at 37℃ for 1h, and immediately injected intraperitoneally into Kunming mice weighing 14-16g. Four mice were injected with each dilution, and each mouse was injected intraperitoneally with 0.5mL. The antibody solution in the sample group was divided into 3 different dilutions (low dilution B1; medium dilution B2; high dilution B3); the IgG concentrations corresponding to the three dilutions of B1, B2, and B3 were 10μg / mL, 1μg / mL, and 0.1μg / mL, respectively. In the toxin group, Kunming mice were injected with diluted toxin solution; the toxin inactivated control group was injected with toxin solution treated with heat inactivation at 100℃; the blank group Kunming mice were injected with PBS buffer; the survival of the mice was observed for 4 consecutive days after injection, and the results were statistically analyzed.

[0111] 1.2.8 Analysis of monoclonal antibody affinity by surface plasmon resonance

[0112] Coupling ligand: Mouse monoclonal antibody and the marketed anti-TcdB McAbBezlotoxumab were coated on the CM5 chip by amino coupling method. The concentration of the coupled antibody was 5μg / ml, the coating solution was 10mM sodium acetate with pH=5.0, and the running buffer was HBS-EP+. First, the chip was activated with EDC and NHS, and then the mouse antibody was bound to the chip at a flow rate of 10μL / min for 7 minutes, and then ethanolamine was used to block the unbound epitope. The other channel without antibody coupling after activation was used as the reference channel for detection.

[0113] Affinity determination: The running buffer is HBS-EP+, the flow rate is 30μL / min, and when the baseline is stable, TcdB is diluted with running buffer in a gradient manner and slowly flows over the chip surface as an analyte, and the affinity determination is performed using a single cycle method. The flow rate is 30μL / min, the binding is 120s, the dissociation is 600s, and the medium is regenerated with a pH 1.5 Gly-HCl buffer for 30s. The resulting sensorgram is fitted using its analysis software to obtain the affinity data of the antibody and antigen.

[0114] 1.2.9 Retrieval of antibody variable region sequences

[0115] The total RNA of hybridoma cells was extracted by Trizol method, reverse transcribed to synthesize the first strand of cDNA, and PCR cloned to obtain the variable region gene of hybridoma cell antibody, and the gene was cloned into T vector, and the positive clone was selected for sequencing. Primers were designed according to the principle of independently amplifying VH, Vκ and Vλ chains in mice, and the primer sequences are shown in Table 2.

[0116] Table 2. Primers for PCR amplification of VH, Vκ, and Vλ chains of mouse antibodies

[0117]

[0118]

[0119] 2. Experimental results

[0120] 2.1 Amplification of target gene and product identification results

[0121] Using the genomic DNA of the toxin-producing strain LCL084 of Clostridium difficile as a template, the PCR product of TcdB RBD was amplified. After 1% agarose gel electrophoresis analysis, a target band of about 1600 bp was observed, which was consistent with the expected size. Figure 1 shown.

[0122] 2.2 Plasmid construction results

[0123] The TcdB RBD sequence amplification product and vector PET-22b in 2.1 were double-digested with BamHⅠ and XhoⅠ, and analyzed by 1% agarose gel electrophoresis. Bands of about 1600 bp and 5500 bp were observed, respectively, and the sizes were consistent with expectations. The agarose gel electrophoresis identification results of the TcdBRBD sequence amplification product and the vector PET-22b digestion product are shown in Figure 2. Figure 2 shown.

[0124] The recombinant expression plasmid was transformed into E.coil BL21 (DE3), and positive clones were picked after overnight culture. A total of 10 single cells were cultured separately. 5 μL of the transformed single cell culture product was taken for PCR and verified by 1% agarose gel electrophoresis. A positive band appeared at 1600 bp, which was consistent with expectations. The identification results of the recombinant plasmid are shown in Figure 3 .

[0125] The positive bacteria were sent for sequencing, and the sequencing results were aligned with the master sequence by Megalign. The homology between the TcdBRBD gene sequence inserted in the plasmid and the master sequence was 100%, that is, there was no gene mutation, indicating that the recombinant plasmid was constructed correctly. Figure 4 shown.

[0126] 2.3 Expression and purification of TcdB RBD recombinant protein

[0127] The induced TcdB RBD recombinant protein was analyzed by Western Blot technology, and HRP-labeled mouse anti-His tag monoclonal antibody was used to detect whether the recombinant protein carried the tag. After detection, the His tag was positive, proving that the TcdBRBD recombinant protein was successfully expressed. The protein expression product was then separated and purified by affinity chromatography, hydrophobic chromatography, and anion exchange chromatography. The results of each chromatography purification of the protein expression product are shown in Figure 1. Figure 5 shown.

[0128] The eluates from each peak were collected separately, and after SDS-PAGE electrophoresis, staining, and decolorization, a protein band of about 60KD was visible in the eluate of the highest peak, which may be the target protein, and the protein purity increased successively. The SDS-PAGE and Western-Blot results of the purified samples of TcdB RBD recombinant protein are shown in Figure 2. Figure 6 and Figure 7 shown.

[0129] 2.4 Analysis results of properties of TcdB RBD recombinant protein

[0130] 2.4.1 CE-SDS analysis of protein purity results

[0131] The purity of TcdB RBD protein after three-step chromatography purification was tested using the non-reducing CE-SDS method, and the target protein peak was integrated using 32Karat software. Figure 8 , the results showed that the purity of TcdB RBD protein was about 91%, indicating that the purification effect was good.

[0132] 2.4.2 Results of specificity analysis of TcdB RBD recombinant protein

[0133] The specificity of TcdB RBD recombinant protein was detected by Western-Blot method. First, pure TcdB of Clostridium difficile (positive control) and TcdB RBD protein to be detected were fixed on PVDF membrane, rabbit toxin B monoclonal antibody was used as primary antibody binding protein, and HRP-labeled goat anti-rabbit monoclonal antibody was used as secondary antibody to analyze the binding of pure TcdB of Clostridium difficile and TcdB RBD recombinant protein with rabbit toxin B monoclonal antibody. It was found that both showed positive reactions, indicating that TcdB RBD recombinant protein was successfully expressed. The results are shown in Fig. 9 shown.

[0134] 2.5 Results of serum anti-TcdB antibody titers in BALB / c mice

[0135] The titer of mouse serum was detected by indirect ELISA method, and the serum of unimmunized mice was used as negative control. The results are shown in Table 3, OD 450 Mice with a serum dilution factor greater than 20,000 when the reading is about 1.0 can be used for the preparation of hybridoma cells. As shown in the table, the above five mice can all be used for the preparation of monoclonal antibodies, among which mouse No. 2 has the highest antibody titer (>160,000) and is selected to prepare anti-TcdB RBD monoclonal antibodies.

[0136] Table 3. BALB / c mouse serum anti-TcdB RBD protein antibody titer results

[0137]

[0138] 2.6 Cell fusion results

[0139] Three days after booster immunization of BALB / c mice, spleen cells were taken from the mice and fused with SP2 / 0 myeloma cells under the action of PEG1450 fusion agent. After fusion, the cell fusion was observed under an inverted microscope. The results are as follows: Fig.10 As shown, the red circle indicates that the fused cells began to divide on the second day after fusion, slowly splitting from one into two. As the fusion time prolonged, the cells gradually split into cell clusters. On the twelfth day, clear and bright cell clusters could be observed under the microscope, indicating that the cell fusion was successful.

[0140] 2.7 Screening and cloning results of positive hybridoma cells

[0141] The indirect ELISA method was used to screen positive hybridoma cells. The positive cells were first screened with TcdB RBD recombinant protein, and then screened with pure TcdB. 450 Positive hybridoma cells with high readings, small number of cell clones and good cell status were cloned and cultured. After three rounds of limiting dilution cloning screening, a total of 8 positive hybridoma cell lines were obtained, which were named as: 2F3, 4H7, 7C2, 8C10, 9C9, 9A12, 9H7, 10F7. The results are shown in Table 4. During the passage, they were tested by indirect ELISA to determine that they were all positive hybridoma cell lines that stably secreted specific monoclonal antibodies.

[0142] Table 4. Positive screening results of hybridoma cells

[0143]

[0144]

[0145] 2.8 Large-scale preparation and purification results of monoclonal antibodies

[0146] Monoclonal antibodies were prepared in large quantities using the mouse ascites method. Taking one of the antibodies as an example, the crude ascites was purified using the AKTA Avant protein chromatography system and the Protein G pre-packed column. The sample peak eluate was measured by the NanoVue ultra-micro spectrophotometer to obtain a protein concentration of 1500μg / ml. Because the monoclonal antibody has a large molecular weight (150-180KD), it was reduced with β-mercaptoethanol, and the purity of the purified monoclonal antibody was analyzed by reducing and non-reducing SDS-PAGE electrophoresis. The results are shown in Figure 2. Fig.11As shown in the figure, it can be seen that the monoclonal antibody is reduced to 55KD (heavy chain) and 25KD (light chain) by the reducing agent β-mercaptoethanol, and the molecular weight of the non-reduced monoclonal antibody is about 150KD, which is consistent with the molecular weight of IgG. And the high-purity monoclonal antibody is obtained by the Protein G medium purification method, and the antibody purity reaches more than 90%.

[0147] 2.9 Results of specificity analysis of monoclonal antibodies

[0148] The specificity of monoclonal antibodies was analyzed and identified by Western-Blot method. Taking one of the antibodies as an example, the results are as follows: Fig.12 As shown, the left figure is the SDS-PAGE electrophoresis of TcdB reduction and non-reduction, and the right figure is the Western-Blot result of monoclonal antibody binding to TcdB. The results show that the monoclonal antibody secreted by the hybridoma cell line can specifically recognize the whole TcdB toxin molecule protein (molecular weight 270KD) after reduction and non-reduction treatment.

[0149] 2.10 Monoclonal Antibody Titer Test Results

[0150] The indirect ELISA method was used to determine the titer of the purified monoclonal antibodies. Taking one of the antibodies as an example, the results are shown in Table 5. The titer of the antibody was 1:3.2×10 6 .

[0151] Table 5. Monoclonal antibody titer test results

[0152]

[0153]

[0154] 2.11 Results of monoclonal antibody neutralization activity analysis

[0155] 2.11.1 Results of toxicity test of Clostridium difficile toxin B

[0156] The results of the toxicity test of Clostridium difficile toxin B are as follows Fig.13 As shown in the table, the mortality rate of mice in the 0.25μg / kg and 0.5μg / kg toxin dose groups was below 50%, 50% of mice died in the 1μg / kg toxin dose group, and all mice died in the 2μg / kg and 4μg / kg toxin dose groups. Therefore, the toxin toxicity of TcdB is calculated to be LD 50 =1μg / kg. The experimental results are consistent with the LD of TcdB in CD1 mice referenced in the literature 50 Numeric value.

[0157] 2.11.2 Neutralization test results

[0158] The neutralization activity of monoclonal antibodies was determined by mouse test. The test set up sample group, toxin group, toxin inactivated control group and blank group respectively; the survival of mice after injection is shown in Table 6. The mice in toxin inactivated control group and blank group survived 96h after injection without symptoms. All mice in toxin group and 4H7, 7C2, 9C9, 9A12, 9H7 sample groups died at each dilution 24h after injection. Mice in 2F3, 8C10, 10F7 sample groups survived 24h after injection, but only one mouse survived in low dilution B1 in 8C10 and 10F7 sample groups, and mice in B2 and B3 dilution groups died. Since only one mouse survived in B1 group, it cannot be determined that they are neutralizing active antibodies. 24 hours after injection, mice in 2F3-B3 became significantly ill, 3, 2, and 1 mice survived in 2F3-B1, 2F3-B2, and 2F3-B3, respectively. The results show that the survival of mice also presents a certain gradient as the antibody dilution increases from low to high. After 48 hours, there are still 2 and 1 mice surviving in the 2F3-B1 and 2F3-B2 groups, respectively, and all mice in 2F3-B3 died. It is judged to be a neutralizing antibody.

[0159] Table 6. Results of monoclonal antibody neutralization activity analysis

[0160]

[0161]

[0162] 2.12 Neutralizing Antibody Affinity Analysis Results

[0163] The affinity of neutralizing antibody 2F3 and the marketed anti-TcdB McAb Bezlotoxumab to TcdB was analyzed by Biacore T200. The single-cycle method was used to determine the antibody affinity. The coupling amount of antibody 2F3 to the chip was 3413.5RU, and the coupling amount of Bezlotoxumab to the chip was 4412.6RU. TcdB was diluted two-fold in HBS-EP Buffer, with a total of 5 dilutions. Each step on the curve in the figure represents the response value of the antibody binding to TcdB of different dilutions. The change in the response value can reflect the interaction process between the antibody and TcdB. The equation was fitted with a one-to-one binding model. The affinity results are shown in Fig.14 , Fig.15 As shown in the figure, the ka and kd values ​​represent the binding rate constant and dissociation rate constant of antibody 2F3 and TcdB, respectively. By calculating the ratio of kd / ka, the equilibrium dissociation constant KD of the interaction between the two can be obtained. Generally, the smaller the KD value, the higher the affinity between the two. The equilibrium dissociation constants KD of neutralizing antibody 2F3 and Bezlotoxumab with TcdB calculated by the software are 1.188E-8M and 4.985E-9M, respectively.

[0164] 2.13 PCR amplification of VH, Vκ, and Vλ chains of mouse antibodies

[0165] Using mouse hybridoma cell cDNA as a template, the VH, Vκ, and Vλ chains of the mouse antibody were amplified and analyzed by 1% agarose gel electrophoresis. Fig.16 It can be seen that PCR amplified VH and Vκ chains, but did not amplify Vλ chains, indicating that the light chain of the mouse antibody is κ type. The VH electrophoresis band is around 360 bp, and the Vκ electrophoresis band is around 340 bp, and the band size is consistent with expectations.

[0166] 2.14 Antibody variable region sequence analysis results

[0167] The verified positive bacteria were sent for sequencing, and the sequencing results were compared with the NCBI database using BLAST software. After comparison, it was found that the amplified VH and Vκ sequences were the variable region sequences of mouse antibodies. The variable region gene sequences of mouse antibodies obtained by sequencing were further analyzed for gene structure using the V-BASE2 database to locate the complementary determining regions CDR and framework regions FR on the antibody VH and VL. The results of the antibody gene structure analysis are shown in the figure. Fig.17 shown.

[0168] 3. Discussion

[0169] Clostridium difficile LCL084 strain is a strict obligate anaerobe with harsh culture conditions. The strain has low toxin production, and the toxins TcdA and TcdB have large molecular weights and similar physical and chemical properties, which brings great challenges to the antigen preparation of the present invention. According to literature reports, TcdB RBD is a key antigenic epitope for toxins to enter the human body and cause disease. And the large-scale preparation of the protein can be achieved using a prokaryotic expression system. Therefore, the present invention chooses to clone the TcdB RBD gene into the PET-22b prokaryotic expression vector and transform Escherichia coli for recombinant expression. After three-step chromatography purification, the CE-SDS results show that the purity of the recombinant protein is more than 90%. The purity of the protein fully meets the antigen immunity requirements for the preparation of McAb by hybridoma technology, which greatly reduces the antibody screening intensity of the present invention and lays a solid foundation for the successful screening of positive McAb cell lines.

[0170] Mouse in vivo test is a common standard method for determining the neutralizing activity of anti-toxin McAb. The present invention uses animal test to analyze the antibody neutralizing activity of 8 positive hybridoma cell lines screened. The neutralized pure TcdB and McAb mixed solution is intraperitoneally injected into mice. Based on the death of mice, antibodies with inhibitory effects on toxins are preliminarily screened. The results show that only the 2F3 antibody can neutralize 5 LD 50The toxin TcdB of the mouse was detected by the PCR, while the other 7 antibodies could not produce neutralizing protection against the mice. This method successfully screened out a McAb with neutralizing activity.

[0171] The present invention uses SPR technology to evaluate the affinity of McAb through a single cycle method. Mouse-derived neutralizing antibody 2F3 and fully humanized Bezlotoxumab (anti-TcdB McAb that has been marketed) are selected to be fixed to the surface of a CM5 chip by an amino coupling method, and the pure TcdB antigen molecules after gradient dilution are slowly transferred to the surface of the sensor chip as a mobile phase through a microfluidic chuck. According to the change in the response value, the whole process of the interaction between the antibody and TcdB is reflected. Using this technology, the equilibrium dissociation constant KD of the mouse-derived neutralizing antibody 2F3 and TcdB is measured to be 1.188E-8M; while the equilibrium dissociation constant KD of Bezlotoxumab and TcdB is 4.985E-9M; there is no significant difference in the affinity data of the two, indicating that the mouse-derived neutralizing antibody 2F3 screened by the present invention has good antibody activity and has the potential to be developed into a marketed product.

[0172] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A monoclonal antibody against Clostridium difficile toxin B, characterized in that: The amino acid sequences of its heavy chain CDR1, CDR2, and CDR3 are: CDR1: GYTFTSYW, as shown in SEQ ID NO: 1; CDR2: IYPGNNNT, as shown in SEQ ID NO: 2; CDR3: SRGDYGSSYALDY, as shown in SEQ ID NO: 3; The amino acid sequences of light chain CDR1, CDR2, and CDR3 are: CDR1: QDVNTA, as shown in SEQ ID NO: 4; CDR2: SASYRYTGV, as shown in SEQ ID NO:5; CDR3: QQRHVPPRT, as shown in SEQ ID NO:

6.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody is selected from a fragment comprising a heavy chain CDR region and a light chain CDR region, a fragment comprising a heavy chain variable region and a light chain variable region, or a complete antibody molecule having full-length heavy and light chains.

3. A polynucleotide, characterized in that It comprises nucleotides encoding the monoclonal antibody according to claim 1 or 2.

4. A recombinant expression vector, characterized in that: It comprises the polynucleotide according to claim 3.

5. A host cell transformed with the recombinant expression vector according to claim 4.

6. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a medicament for treating Clostridium difficile infection.

7. A drug for treating Clostridium difficile infection, characterized in that: It comprises the monoclonal antibody according to claim 1 or 2.

8. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a Clostridium difficile detection reagent or detection kit.

9. A Clostridium difficile detection kit, characterized in that: The detection kit comprises a labeled complex obtained by biologically or chemically labeling the monoclonal antibody according to claim 1 or 2.

10. The method for preparing the monoclonal antibody according to claim 1 or 2, comprising: Cultivate the host cell according to claim 5 to induce the expression of the monoclonal antibody.

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