Antibody Hm0283 against Staphylococcus aureus enterotoxin B and its application

Abstract

The present invention discloses the anti-Staphylococcus aureus enterotoxin B antibody Hm0283 and its applications. Also provided are nucleic acid molecules, expression vectors, and host cells encoding the antibody or its functional fragment, as well as processes for producing the antibody or its functional fragment. The present invention also provides immunoconjugates and pharmaceutical compositions comprising the antibody or its functional fragment, which can be used to treat, prevent, or diagnose Staphylococcus aureus infections and are expected to become important research areas in the field of Staphylococcus aureus infections and the control of drug resistance.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, in particular to an anti-Staphylococcus aureus enterotoxin B antibody Hm0283, and also to applications of the antibody. Background Art

[0002] Staphylococcal enterotoxin B (SEB), produced by the human pathogen Staphylococcus aureus, is a potent superantigen toxin that causes food poisoning and systemic intoxication, leading to a severe, life-threatening illness known as toxic shock syndrome (TSS). The SEB superantigen primarily interacts with MHC class II and TCR molecules, leading to the activation of monocytes / macrophages and T lymphocytes and inducing high levels of proinflammatory cytokines and chemokines. The high levels of inflammatory cytokines, including IL-2, TNF, and IFN-γ, can lead to respiratory failure, vascular damage, multi-organ system collapse, and death.

[0003] Currently, there is no specific drug treatment for SEB. Research has proposed a variety of treatment approaches, including small molecule drugs, vaccines, and monoclonal or polyclonal antibodies targeting SEB. The antibodies of the present invention were evaluated for their protective effects in a lethal model of Staphylococcus aureus sepsis infection. The experimental results showed that 600 μg of the antibody protected 60% of mice. This result demonstrates that fully human anti-SEB antibodies can inhibit disease progression, enhance clearance, and suppress the systemic spread of invading organisms, effectively protecting BALB / C mice from MRSA-induced sepsis. Therefore, passive immunotherapy based on antibody drugs has great potential for development and application in MRSA infection, and could become a new immunotherapy approach for the non-antibiotic treatment of MRSA infection and the control of drug resistance. Summary of the Invention

[0004] In view of this, one of the objects of the present invention is to provide an anti-Staphylococcus aureus enterotoxin B antibody Hm0283; a second object of the present invention is to provide a nucleotide sequence encoding the antibody Hm0283; a third object of the present invention is to provide a vector or host containing the nucleotide sequence; a fourth object of the present invention is to provide a method for producing the anti-Staphylococcus aureus enterotoxin B antibody Hm0283; a fifth object of the present invention is to provide the use of the anti-Staphylococcus aureus enterotoxin B antibody Hm0283 in the preparation of a reagent that specifically binds to Staphylococcus aureus enterotoxin B; a sixth object of the present invention is to provide the use of the anti-Staphylococcus aureus enterotoxin B antibody Hm0283 in the preparation of a drug for treating, preventing or diagnosing Staphylococcus aureus infection.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. Anti-Staphylococcus aureus enterotoxin B antibody Hm0283, the antibody Hm0283 comprises a heavy chain and a light chain, the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the heavy chain are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively; the amino acid sequences of the variable regions CDR1 and CDR3 of the light chain are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and the amino acid sequence of CDR2 is AAS.

[0007] Preferably, the constant region of the antibody Hm0283 comprises any one of the human IgA, IgD, IgE, IgG, or IgM constant regions; preferably IgG. The light chain of the antibody of the present invention may be κ or λ, preferably κ. Functional fragments include, but are not limited to, antibodies consisting of a VL / VH region pair and an antibody constant domain, such as scFv, Fab, F(ab')2, or Fv-specific antibodies.

[0008] Preferably, the amino acid sequence of the heavy chain is shown in SEQ ID NO.9; and the amino acid sequence of the light chain is shown in SEQ ID NO.10.

[0009] Preferably, the antibody Hm0283 of the present invention specifically binds to Staphylococcus aureus enterotoxin B or a Staphylococcus aureus enterotoxin B mutant protein.

[0010] Preferably, the equilibrium dissociation constant of antibody Hm0283 binding to SEB is no higher than 2×10 -8 M.

[0011] 2. The nucleotide sequence encoding the antibody Hm0283.

[0012] A vector or host containing the nucleotide sequence, wherein the vector is a pGEX series vector, a pET series vector, or a pcDNA3.1(+) series vector. The preferred technical solution of the expression vector is pcDNA3.1(+). The host can be a prokaryotic cell, such as Escherichia coli, Bacillus subtilis, etc.; or a lower eukaryotic cell, such as yeast cells; or a higher eukaryotic cell, such as sf9 insect cells, CHO animal cells, or 293 cells. Particularly preferred are human production cell lines, preferably 293F cells.

[0013] 4. A method for producing an anti-Staphylococcus aureus enterotoxin B antibody Hm0283, comprising culturing host cells containing the nucleotide sequence encoding the antibody, collecting the expressed antibody, and separating and purifying the antibody using Protein A / G, or using MabSelect or MabSelect PrismA.

[0014] 5. Use of the anti-Staphylococcus aureus enterotoxin B antibody Hm0283 in the preparation of a reagent that specifically binds to Staphylococcus aureus enterotoxin B.

[0015] 6. Use of the anti-Staphylococcus aureus enterotoxin B antibody Hm0283 in the preparation of a medicament for treating, preventing or diagnosing Staphylococcus aureus infection.

[0016] The beneficial effects of the present invention are as follows: the present invention provides an anti-Staphylococcus aureus enterotoxin B antibody Hm0283, which is produced by culturing host cells into which an expression vector having a signal sequence is introduced to generate a monoclonal antibody, the generated monoclonal antibody is secreted into the supernatant, and can be separated and purified by applying conventional chromatography techniques, preferably using Protein A / G for separation and purification, the prepared antibody can be used for diagnosis or determination of Staphylococcus aureus enterotoxin B, and is effective, and the antibody can also prevent or treat sepsis and pneumonia caused by MRSA infection, and is of great significance for the prevention, treatment and detection of Staphylococcus aureus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0018] Figure 1 The reduced (R) and non-reduced (N) SDS-PAGE electrophoresis images of the purified antibody Hm0283.

[0019] Figure 2 The SDS-PAGE electrophoresis diagram of wSEB and mSEB after purification.

[0020] Figure 3 This is a graph showing the results of the antibody Hm0283 and antigen binding activity test.

[0021] Figure 4 This is the result of measuring antigen-antibody affinity using biomembrane interferometry technology.

[0022] Figure 5 This figure shows the protective evaluation results of the lethal sepsis infection model. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0024] The strains and reagents are as follows:

[0025] Escherichia coli strain DH5α was purchased from Shanghai Bioengineering Co., Ltd.; plasmid extraction kit was purchased from Biomed Biotechnology Co., Ltd.; Staphylococcus aureus MRSA-252 international standard strain was purchased from ATCC, USA.

[0026] Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, NaOH, Tween-20, Na2CO3, NaHCO3, and glycine were purchased from Sinopharm Chemical Reagent Co., Ltd.; protein loading buffer and protein marker were purchased from Beyotime Biotechnology Co., Ltd.; PBS was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; bovine serum albumin V was purchased from BIOSHARP; colorimetric solution (TMB) was purchased from Tiangen Biotechnology Co., Ltd.; sulfuric acid was purchased from Chengdu Kelon Chemical Reagent Factory; AHC sensor was purchased from Forte Bio; Tryptone and yeast extract were purchased from OXOID, UK; Ampicillin was purchased from Taiji Group Southwest Pharmaceutical; Polyethylenimine was purchased from Polysciences; and Protein A affinity filler was purchased from GE, USA.

[0027] Example 1. Expression and purification of Staphylococcus aureus enterotoxin B (wSEB) and mutant Staphylococcus aureus enterotoxin B (mSEB)

[0028] The SEB gene was amplified by PCR from genomic DNA of Staphylococcus aureus (ATCC accession number BAA-1556). The wild-type gene was then subjected to site-directed mutagenesis using the QuickChange II XL Site-Directed Mutagenesis Kit to generate the L45R, Y89A, Y94A variant. After confirmation by DNA sequencing, the variant was expressed in Escherichia coli, cultured overnight at 37°C in LB medium containing ampicillin, and the cells were harvested by centrifugation. wSEB and mSEB (L45R, Y89A, Y94A) proteins were purified using Ni-NTA. The amino acid sequence of the wSEB protein is shown in SEQ ID NO. 6.

[0029] Example 2: Isolation of peripheral blood mononuclear cells (PBMC)

[0030] Healthy volunteers and volunteers who recovered from severe Staphylococcus aureus infection were recruited. Venous blood samples were collected in anticoagulant tubes containing heparin, and PBMC cells were isolated by density centrifugation. The specific operation was as follows: venous blood was collected and centrifuged at 22°C, 400×g, for 15 minutes; the upper transparent plasma layer was aspirated and frozen at -80°C; after aspirating the supernatant, it was thoroughly mixed with an equal amount of RPMI1640 (Gibco) and slowly added to the upper layer of an equal amount of lymphocyte separation solution along the inclined tube wall, centrifuged at 2000rpm for 20 minutes, and the mononuclear cells in the cloud layer were aspirated into a sterile centrifuge tube. More than 5 times the volume of RPMI1640 was added, and the cells were centrifuged at 1000rpm for 5 minutes. The cells were washed twice and resuspended in an appropriate amount of RPMI1640 at a concentration of 1×10 7 Store frozen in liquid nitrogen until use.

[0031] Example 3: Flow cytometry sorting of single plasma cells

[0032] Flow cytometry sorting of single plasma cells: ELISA testing of serum using SEB protective antigen protein (HPLC purity >95%) as antigen to determine the antibody titer of the sample. Samples with high antibody titer were selected and single plasma cells were sorted by flow cytometry. Plasma cell populations at different time points were separated by gating based on CD3 / CD14 / CD16 / CD235a-CD19+CD20+ / -CD38hiCD27hi. Through serological experiments and B lymphocyte phenotypic analysis, we can ensure that we can obtain a large number of single plasma cells from >3% of the plasma cell population, and isolate the gene sequence of the fully human monoclonal antibody against SEB from them. The heavy chain nucleotide sequence is shown in SEQ ID NO.13, and the encoded amino acid sequence is shown in SEQ ID NO.9; the variable region sequence is shown in SEQ ID NO.7, and the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3; the light chain nucleotide sequence is shown in SEQ ID NO.14, and the encoded amino acid sequence is shown in SEQ ID NO.10; the variable region sequence is shown in SEQ ID NO.8, the amino acid sequences of CDR1 and CDR3 are shown in SEQ ID NO.4 and SEQ ID NO.5, and the amino acid sequence of CDR2 is AAS.

[0033] Example 4. Expression and purification of antibodies

[0034] 1. Synthesis of Recombinant Plasmid

[0035] The DNA sequences encoding the amino acid coding sequences of the antibody heavy chain and light chain were optimized respectively. The optimized sequences are shown in SEQ ID NO.11 and SEQ ID NO.12. The optimized sequences were sent to Wuhan Jinkairui for synthesis and then ligated into the eukaryotic expression vector pcDNA3.1(+) plasmid.

[0036] 2. Transformation and Extraction of Recombinant Plasmid

[0037] Remove two tubes of E. coli DH5α competent cells from a -80°C freezer and add 1 μL of the synthesized pcDNA3.1(+)-HC and pcDNA3.1(+)-LC plasmids, respectively. Incubate on ice for 50 minutes, heat shock in a 42°C metal bath for 90 seconds, and then quickly incubate on ice for 2 minutes. Add 600 μL of LB medium, mix thoroughly, and shake at 220 rpm at 37°C for 1 hour. Spread 100 μL of the culture onto a plate containing ampicillin (Amp)-resistant LB solid medium. Place the plate upside down in a 37°C incubator. Pick a single colony and inoculate it into 10 mL of LB medium for 5 hours. Then, inoculate it into 1 L of LB liquid medium containing Amp-resistant LB medium and incubate at 37°C with shaking overnight. Collect the precipitate by centrifugation at 5000 g. Extract the plasmid using the Biomed Biotech endotoxin-free, high-purity plasmid rapid extraction kit instructions, sterilize with a 0.22 μm filter, and store at -20°C.

[0038] Preparation of Ampicillin Solution: Dissolve 500 mg of Ampicillin in 5 mL of Grade I water and filter through a 0.22 μm sterile filter.

[0039] Preparation of Amp-resistant LB solid medium: Take 1g of Tryptone, 0.5g of Yeast extract, 1g of NaCl, and 1.5g of agar powder, add 100mL of Grade I water to dissolve completely, adjust the pH to 7.4, sterilize by high pressure, cool to about 40℃, add 0.1mL of Ampicillin solution, and pour about 10mL into a sterile culture dish. After cooling, store at 4℃.

[0040] Preparation of Amp-resistant LB liquid medium: Take 1g of Tryptone, 0.5g of Yeast extract, and 1g of NaCl and dissolve them completely in 100mL of Grade I water. Adjust the pH to 7.4, sterilize by autoclave, cool to about 40℃, and add 0.1mL of Ampicillin solution.

[0041] 3.293F cell transfection and culture

[0042] The above plasmids were added to 10 ml of cell culture medium at a ratio of 1:1 between heavy chain and light chain, totaling 300 μg. 700 μl of Polyethylenimine (PEI25K) (1 mg / ml) liposomes were added to 10 ml of cell culture medium. The plasmids were slowly added to the PEI liposomes and vortexed to mix. The cells were incubated at 37°C for 15 min. 293F suspension cells were cultured to prepare 1.2×10 6 / ml, a total of 280ml; 20ml of the plasmid mixture was slowly added to 293F cells for transfection, and mixed while adding. Expression was carried out at 37°C, 5% CO2, and 125rpm. After 6 days, the supernatant was collected by centrifugation at 3000g.

[0043] 4. Antibody Preparation

[0044] Wash 5 mL of Protein A or G affinity medium three times with 20 mM PBS, pH 7.5 (20 mM PBS: Dissolve 6.02 g of Na₂HPO₄·12H₂O, 0.49 g of NaH₂PO₄·2H₂O, and 8.77 g of NaCl in 900 mL of Grade I water until completely dissolved, adjust the pH to 7.5, and bring the volume to 1 L with Grade I water). Mix the prepared centrifugation supernatant with the affinity medium and allow to bind vertically for 2 hours at room temperature. Remove unbound supernatant by air gravity column and wash three times with 20 mM PBS. To the cleaned affinity filler, add 10 mL of 100 mM glycine-HCl, pH 2.7 (solution preparation: 7.51 g of glycine, add 980 mL of grade I water to dissolve completely, adjust the pH to 2.7 with HCl, and add grade I water to 1 L) eluent, collect the flow-through, repeat the elution once with the above eluent, and combine with the above flow-through to obtain the antibody stock solution Hm0283. The purified sample was tested by 12% SDS-PAGE electrophoresis. When the purity reached more than 95%, it was sterilized by 0.22 μm filtration and stored at -80°C ( Figure 1 Then, based on the desired application, the molecular weight, isoelectric point, hydrophobicity and other physical and chemical properties are predicted through the amino acid sequence, and further purification is carried out using protein purification technologies such as ion exchange, hydrophobic or molecular sieve chromatography.

[0045] Example 5. Antibody Hm0283 and antigen binding activity detection

[0046] 1. Liquid Preparation

[0047] (1) Preparation of coating solution: Weigh 1.6 g of Na2CO3 and 2.9 g of NaHCO3, dissolve in 1 L of ddH2O, and adjust the pH to 9.6 using a pH meter;

[0048] (2) Preparation of blocking solution: 1 g bovine serum V was dissolved in 100 mL antibody diluent (1:100);

[0049] (3) Preparation of antibody diluent: Dissolve PBS in 1 L ddH2O, add 500 μL Tween-20, and adjust the pH to 7.4 using a pH meter;

[0050] (4) Preparation of PBST washing solution: same as antibody dilution solution;

[0051] (5) Preparation of stop solution (2M H2SO4): Pour 22.2 mL of concentrated sulfuric acid into 177.8 mL of ddH2O.

[0052] 2. Antigen-antibody binding activity detection

[0053] The recombinantly expressed mutant SEB protein (L45R, Y89A, Y94A) and wild-type SEB protein ( Figure 2 ) were diluted to 5 μg / mL with coating buffer, and 100 μL / well of the ELISA plate was coated overnight at 4°C. The plate was then blocked with blocking buffer for 2 hours at room temperature. Antibody Hm0283 (1:1000 dilution) was used as a negative control. An irrelevant antibody, IgG1, at 0.5 μg / mL was used as a blank. 100 μL of blocking buffer was added to each well, and triplicate wells were set up for incubation at 37°C for 1 hour.

[0054] Wash the plate once with PBST (3 cycles), add 100 μL / well of a 1:5000 dilution of Anti-Human HRP-IgG (secondary antibody), and incubate at 37°C for 45 min. Wash the plate once with PBST (5 cycles), protect from light, add 100 μL / well of TMB, and develop at 37°C for 5 min in the dark. Terminate the reaction with 50 μL of ELISA stop buffer, and measure absorbance at 450 nm. Calculate the mean of the negative control, an unrelated antibody, IgG1, and a threshold value (3 times the mean). A value greater than the threshold value is considered positive.

[0055] The experimental results showed that the fully human SEB antibody Hm0283 can bind to wSEB and mSEB ( Figure 3 ), with EC50 of 0.00612 μg / mL and 0.00615 μg / mL, respectively (Table 1).

[0056] Table 1 EC50 of antibodies Hm0283, wSEB and mSEB

[0057]

[0058] Example 6: Determination of Antigen-Antibody Affinity by Biofilm Interferometry (BLI)

[0059] Biomembrane interferometry was used to measure the affinity of the antibody for wSEB. First, 50 nM of the antibody was immobilized on the AHC sensor. Two-fold serial dilutions of wSEB (3.13 nM to 50 nM) were loaded according to the sample plate arrangement. The procedure was performed according to the "baseline test - loading test - quenching - plate washing - baseline test - binding test - dissociation test" setting. The association rate constant (Kon), dissociation rate constant (Kdis), and equilibrium dissociation constant (KD) were calculated. The results are shown in Table 2. The KD of antibody Hm0283 for wSEB is 1.245×10 -8 M( Figure 4 ).

[0060] Table 2. Binding affinity of antibody Hm0283 and wSEB

[0061]

[0062] Example 7: Evaluation of the protective effect of antibody Hm0283 on a lethal MRSA sepsis infection model

[0063] 20 mice were divided into 2 groups, 10 in each group. 600 μg of antibody Hm0283 or PBS control group were injected into the tail vein of each group of mice in a volume of 100 μL. 24 hours later, 1.5×10 8 All mice were injected with CFU MRSA 252 via tail vein in a volume of 100 μL. The mice were observed for 7 days and their survival was recorded ( Table 3 and Figure 5 MRSA sepsis protection experiments showed that 600 μg of Hm0283 antibody protected 60% of mice. This result demonstrates that Hm0283 can protect against MRSA sepsis. This suggests that the fully human anti-SEB antibody Hm0283 can inhibit disease progression, enhance clearance, and suppress the systemic spread of invading organisms.

[0064] Table 3. Evaluation of the protective effect of antibody Hm0283 on the lethal sepsis infection model

[0065]

[0066] Those skilled in the art can use the antibodies or functional fragments thereof of the present invention for any other applicable purposes.

[0067] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. Anti-Staphylococcus aureus enterotoxin B antibody Hm0283, characterized by: The antibody Hm0283 includes a heavy chain and a light chain, and the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the heavy chain are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively; the amino acid sequences of the variable regions CDR1 and CDR3 of the light chain are shown as SEQ ID NO.4 and SEQID NO.5, respectively, and the amino acid sequence of CDR2 is AAS.

2. The anti-Staphylococcus aureus enterotoxin B antibody Hm0283 according to claim 1, characterized in that: The constant region of the antibody Hm0283 includes any one of human IgA, IgD, IgE, IgG or IgM constant regions.

3. The anti-Staphylococcus aureus enterotoxin B antibody Hm0283 according to claim 1, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO.9; the amino acid sequence of the light chain is shown in SEQ ID NO.

10.

4. A nucleotide encoding the antibody Hm0283 according to any one of claims 1 to 3.

5. A vector or host containing the nucleotide sequence of claim 4.

6. A method for producing an anti-Staphylococcus aureus enterotoxin B antibody Hm0283, characterized in that: The host cells containing the nucleotide encoding the antibody according to claim 4 are cultured, the expressed antibodies are collected, and the antibodies are separated and purified by Protein A / G.

7. Use of the anti-Staphylococcus aureus enterotoxin B antibody Hm0283 according to any one of claims 1 to 3 in the preparation of a medicament for treating or diagnosing Staphylococcus aureus infection.

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