A fully human SEB monoclonal antibody against Staphylococcus aureus enterotoxin B and its application

Abstract

The present invention discloses a fully human SEB monoclonal antibody against Staphylococcus aureus enterotoxin B and its applications. The amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the heavy chain of the antibody are shown as SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7 respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the light chain are shown as SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11 respectively; the antibody can specifically bind to Staphylococcus aureus enterotoxin B and can be used for the treatment, prevention, or diagnosis of Staphylococcus aureus infections and toxic shock syndrome caused by enterotoxin B, which will become an important direction in the research field of "non-antibiotic" treatment of drug-resistant Staphylococcus aureus infections and control of the development of drug resistance.

Description

Technical Field

[0001] The present invention relates to the field of immunology, and specifically relates to a fully human SEB monoclonal antibody against Staphylococcus aureus enterotoxin B, and also relates to the application of the antibody. Background Art

[0002] Staphylococcus aureus is an important pathogenic microorganism that is widely prevalent and causes hospital and community infections, showing multidrug resistance and highly drug-resistant spread. Its infections are mainly acute and suppurative, and systemic infections can lead to various severe infections and complications such as acute pneumonia, endocarditis, septic arthritis, osteomyelitis, and sepsis. The long-term abuse of antibiotics has increasingly exacerbated and highlighted the problem of S. aureus drug resistance. As a typical representative of "superbugs", Methicillin-resistant Staphylococcus aureus (MRSA) can secrete multiple toxins to destroy normal humoral immunity and cellular immunity and reshape the host immune environment, thereby achieving immune escape and causing serious damage to the human body.

[0003] Staphylococcus aureus enterotoxin B is one of the most common and potent superantigens of Staphylococcus aureus. It can bind to the α-chain of the major histocompatibility complex (MHC) class II molecule and the specific Vβ region of the T cell receptor (TCR) in humans to form a ternary complex, resulting in the massive proliferation and activation of monocytes / macrophages and T lymphocytes, and inducing high levels of pro-inflammatory cytokines and chemokines. Systemic inflammatory factors may lead to potentially fatal toxic shock syndrome (TSS) in patients. In addition, SEB is closely related to food poisoning and can cause symptoms such as vomiting, abdominal pain, and diarrhea. Since SEB is easy to produce in large quantities using genetic engineering techniques, has aerosol stability, and can be used as a lethal and disabling agent, it is an ideal biological toxin and is classified as a category B select biological warfare agent by the Centers for Disease Control and Prevention (CDC). At the same time, we still lack effective medical countermeasures for large-scale treatment of affected populations, and the concerns about SEB are even more complex.

[0004] Currently, there are no approved vaccines or specific antibody therapies for SEB. Previous studies have identified a variety of potential treatment methods, including small molecule therapeutics, vaccines, and monoclonal antibodies. Among them, monoclonal antibodies that can act immediately are an effective immunotherapy, representing the best method for emergency intervention related to infections, and may also be the best choice for immunodeficient patients who cannot be vaccinated correctly, representing the possibility of alleviating and treating toxic shock syndrome caused by SEB and Staphylococcus aureus infections. For the severe situation caused by Staphylococcus aureus resistance and the huge threat of Staphylococcus aureus enterotoxin B, the antibodies against Staphylococcus aureus enterotoxin B can help reduce the severity of Staphylococcus aureus infections, treat the biological toxicity caused by enterotoxin B, and these antibodies can also be used to detect the typing of Staphylococcus aureus enterotoxin. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide a fully human SEB monoclonal antibody against Staphylococcus aureus enterotoxin B; a second objective of the present invention is to provide a nucleic acid molecule encoding the fully human SEB monoclonal antibody; a third objective of the present invention is to provide a recombinant expression vector containing the nucleic acid molecule; a fourth objective of the present invention is to provide a host cell containing the recombinant expression vector of the nucleic acid molecule; a fifth objective of the present invention is to provide the application of the fully human SEB monoclonal antibody in the preparation of a reagent specifically binding to Staphylococcus aureus enterotoxin B; a sixth objective of the present invention is to provide the application of the fully human SEB monoclonal antibody in the preparation of a drug for treating or assisting in the treatment of Staphylococcus aureus infections.

[0006] To achieve the above objectives, the present invention provides the following technical solutions:

[0007] 1. A fully human SEB monoclonal antibody against Staphylococcus aureus enterotoxin B, characterized in that: the fully human SEB monoclonal antibody comprises a heavy chain and a light chain, and the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the heavy chain are respectively as shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7; the amino acid sequences of CDR1 and CDR3 in the variable region of the light chain are respectively as shown in SEQ ID NO.9 and SEQ ID NO.10, and the amino acid sequence of CDR2 in the variable region of the light chain is Asp Thr Lys.

[0008] Preferably, the amino acid sequence of the heavy chain of the present invention is as shown in SEQ ID NO.4; the amino acid sequence of the light chain is as shown in SEQ ID NO.8.

[0009] Preferably, the constant region of the antibody includes any one of human IgM, IgA or IgG constant regions, and the antibody is a monoclonal antibody, a human antibody, a human antibody or a single-domain antibody. The heavy chain of this monoclonal antibody can be selected from isotypes IgM, IgA, or IgG, preferably IgG.

[0010] In the present invention, the light chain of the monoclonal antibody of the present invention can be of the κ type or the λ type. In a preferred example, the light chain is of the λ type.

[0011] Preferably, the fully human SEB monoclonal antibody specifically binds to the full-length amino acid or partial amino acid sequence of Staphylococcus aureus enterotoxin B, and the full-length amino acid of Staphylococcus aureus enterotoxin B is shown as SEQ ID NO.1.

[0012] Preferably, the fully human SEB monoclonal antibody binds to Staphylococcus aureus enterotoxin B with an equilibrium dissociation constant KD of no higher than 5×10 -8 , such as: dissociating from Staphylococcus aureus enterotoxin B with a KD of 1×10 -9 M, 1×10 -10 M, 1×10 -11 M or less. Among them, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, indicating the degree of dissociation of the antibody and antigen at equilibrium. The smaller the KD, the smaller the dissociation, representing a stronger affinity between the antibody and the antigen.

[0013] In the present invention, the monoclonal antibody is generated from blood lymphocytes infected with Staphylococcus aureus, and such generated antibodies are natural, refined and selected with high affinity to achieve effective protection against infection by neutralization.

[0014] 2. A nucleic acid molecule encoding the fully human SEB monoclonal antibody.

[0015] 3. A recombinant expression vector containing the nucleic acid molecule.

[0016] 4. A host cell containing the recombinant expression vector with the nucleic acid molecule. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells. Particularly preferred is a human production cell line.

[0017] 5. Use of the fully human SEB monoclonal antibody in the preparation of a reagent specifically binding to Staphylococcus aureus enterotoxin B.

[0018] 6. Use of the fully human SEB monoclonal antibody in the preparation of a drug for treating or adjuvantly treating Staphylococcus aureus infection.

[0019] The beneficial effects of the present invention are as follows: By sorting single plasma cells, the fully human monoclonal antibody secreting anti-SEB separated is obtained. Therefore, the gene sequence of the fully human monoclonal antibody against SEB can be isolated from the separated plasma cells. The isolated gene sequence is used to construct an expression vector, and the monoclonal antibody is produced by expression in a host cell. The generated monoclonal antibody is secreted into the supernatant and purified by chromatography technology. The obtained monoclonal antibody can specifically bind to Staphylococcus aureus enterotoxin B and can be used as an antibody for detecting Staphylococcus aureus. The obtained antibody can also treat sepsis caused by MRSA infection and is of great significance in the prevention and treatment of Staphylococcus aureus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention provides the following drawings for description:

[0021] Figure 1 It is a diagram of sorting single plasma cells in the present invention.

[0022] Figure 2 It is the SDS-PAGE detection result of the SEB monoclonal antibody Hm0487 in the present invention.

[0023] Figure 3 It is the detection of the binding activity between the SEB monoclonal antibody Hm0487 and SEB in the present invention.

[0024] Figure 4 It is the result of analyzing the interaction between SEB and Hm0487 using the biolayer interferometry technology in the present invention.

[0025] Figure 5 It is the SEB denaturing gel Western Blot result diagram in the present invention.

[0026] Figure 6 It is the evaluation result diagram of the Hm0487 inhibition of the SEB lethal animal model in the present invention.

[0027] Figure 7 It is the evaluation result diagram of the Hm0487 on the MRSA sepsis animal model in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present invention will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0029] Example 1: Expression and purification of Staphylococcus aureus enterotoxin B (SEB) and mutant Staphylococcus aureus enterotoxin B (mSEB)

[0030] The genomic DNA from Staphylococcus aureus strain (ATCC accession number BAA - 1556) was used to amplify the SEB gene by PCR. Then, the L45R, Y89A, Y94A variants were generated by site - directed mutagenesis of the wild - type gene using the QuickChange II XL Site - Directed Mutagenesis Kit. After DNA sequencing confirmation, it was expressed in Escherichia coli, cultured overnight at 37°C in LB medium containing ampicillin, and the cells were harvested by centrifugation. The SEB protein and mSEB (L45R, Y89A, Y94A) protein were obtained by Ni - NTA purification. The amino acid sequence of the SEB protein is shown in SEQ ID NO.1.

[0031] Example 2: Isolation of peripheral blood mononuclear cells (PBMCs)

[0032] Healthy volunteers and volunteers who had recovered after severe Staphylococcus aureus infection were recruited. Venous blood samples were collected into anticoagulant tubes containing heparin, and PBMC cells were isolated by density centrifugation as follows: The venous blood was centrifuged at 22°C, 400×g for 15 min; the upper clear plasma layer was aspirated and stored at - 80°C; after aspirating the supernatant, it was thoroughly mixed with an equal volume of RPMI1640 (Gibco), and slowly added along the inclined tube wall to the upper layer of an equal volume of lymphocyte separation medium, and centrifuged at 2000 rpm for 20 min. The mononuclear cells in the cloudy layer were aspirated into a sterile centrifuge tube, 5 times or more volume of RPMI1640 was added, and centrifuged at 1000 rpm for 5 min. The cells were washed twice, and resuspended in an appropriate amount of RPMI1640 at 1×10 7 / tube and stored in liquid nitrogen for later use.

[0033] Example 3: Sorting of single plasma cells by flow cytometry

[0034] Sorting of single plasma cells by flow cytometry: Using the SEB protective antigen protein (HPLC purity > 95%) as an antigen to detect the antibody titer of the serum by ELISA, selecting the samples with high antibody titers, sorting single plasma cells by flow cytometry, and sorting by gating with CD3 / CD14 / CD16 / CD235a-CD19+CD20+ / -CD38hi CD27hi to isolate the plasma cell populations at different time points. Through serological experiments and analysis of B lymphocyte phenotypes, it can be ensured that we can obtain a large number of single plasma cells from a plasma cell population of > 3%, and isolate the gene sequences of fully human monoclonal antibodies against SEB. The heavy chain nucleotide sequence is shown as SEQ ID NO.11, and the light chain nucleotide sequence is shown as SEQ ID NO.12. The sorted cells are in a state with a relatively large number and good condition ( Figure 1 ).

[0035] Example 4. Cloning of anti-SEB antibody and expression of fully human antibody

[0036] Synthesize the first strand of cDNA using Superscript V reverse transcriptase (Invitrogen, Carlsbad, CA) and random primers. Use the Ig primer set (constant region primer sequence of the heavy chain: 5’-gcggccctgggctgcctggtcaag-3’ (SEQ ID NO.2); constant region primer sequence of the light chain: 5’-aggagagtgtcacagagcaggacag-3’ (SEQ ID NO.3)) to amplify human Ig V H and V K / L , clone the amplified V H and V K / L products into the TOPO TA vector and perform sequencing. Then re-amplify the above amplified products by PCR, and identify the products by agarose gel electrophoresis with a mass fraction of 1.2%.

[0037] Antibody gene sequence determination and bioinformatics analysis: Purify the antibody gene PCR products identified as positive by gel electrophoresis and with matching heavy and light chains in pairs using the Qiagen PCR product purification kit, and perform sequence determination from both the forward and reverse directions. Use the IMGT online server (http: / / imgt.cines.fr / ) to analyze the antibody gene family, mutation rate, subtype, and CDR region, and enter the new antibody genes into the antibody gene library.

[0038] The PCR products of the antibody variable region genes identified as positive by gel electrophoresis and with the heavy and light chains capable of matching into pairs were ligated to the pcDNA3.3 vector using TA cloning method to construct an expression vector for the fully human anti-SEB antibody. Then, the expression vector was transformed into DH5α competent bacteria and cultured overnight at 37 °C on a plate containing ampicillin. Ten single colonies were picked and subjected to PCR using specific primers. The reaction conditions were: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 100 s, for 28 cycles; extension at 72 °C for 5 min. 5 μL of the PCR product was detected by 1% agarose gel electrophoresis.

[0039] The results showed that transformants containing the antibody heavy and light chain genes were identified among the positive transformants.

[0040] Example 5: Expression and purification of the fully human anti-SEB antibody

[0041] The vector plasmid in the positive transformants obtained in Example 4 was transformed into DH5α for large-scale amplification. After rapid extraction of the recombinant plasmid, Ig VH, VK / L were co-transfected with the transfection reagent PolyFect into HEK293 cells. 5 - 6 hours after transfection, HEK293 basal medium (OPM) was supplemented, and the cells were cultured in a 37 °C, 5% CO2 incubator for 96 hours. The transfection supernatant was collected by centrifugation at 3000×g for 30 minutes and purified using protein A affinity chromatography; the expression and purification of the antibody were examined by SDS-PAGE. The SDS-PAGE detection results (see Figure 2 ) showed that the transfected cells successfully expressed the antibody, named the fully human SEB monoclonal antibody Hm0487, abbreviated as Hm0487 antibody or Hm0487 monoclonal antibody. The relative molecular weight of this antibody was approximately 160 - 180 KD, the heavy chain was approximately 55 KD, and the light chain was approximately 25 KD; the amino acid sequence of the heavy chain was as shown in SEQ ID NO.4, and the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region were as shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7 respectively; the amino acid sequence of the light chain was as shown in SEQ ID NO.8, and the amino acid sequences of CDR1 and CDR3 in the light chain variable region were as shown in SEQ ID NO.9 and SEQ ID NO.10 respectively; the CDR2 amino acid was Asp Thr Lys.

[0042] Example 6: Detection of the binding activity of the expressed antibody

[0043] The ELISA plates were coated with 100 μL / well of recombinantly expressed SEB protein (L45R, Y89A, Y94A) and SEB respectively as antigens, and incubated overnight at 4°C. Then they were blocked at room temperature for 2 h with the blocking solution. The Hm0487 antibody (diluted 1:1000), the negative control was an irrelevant antibody IgG at 10.5 μg / mL, and the blank was added with the blocking solution, 100 μL per well. Three replicates were set up and incubated at 37°C for 1 h.

[0044] The plates were washed once with PBST buffer (3 cycles), then 100 μL / well of Anti-Human HRP-IgG (secondary antibody) diluted 1:5000 was added and incubated at 37°C for 45 min. The plates were washed once with PBST buffer (5 cycles), then TMB at 100 μL / well was added in the dark and the color was developed at 37°C for 5 min in the dark. Then 50 μL of ELISA stop solution was added to terminate the reaction, and the absorbance was measured at 450 nm. The mean value of the irrelevant antibody IgG1 in the negative control was calculated, and the threshold (3 times the mean value) was calculated. An antibody was considered positive if its value was greater than the threshold.

[0045] The experiment showed that the fully human SEB monoclonal antibody Hm0487 could bind to SEB and mSEB (L45R, Y89A, Y94A) ( Figure 3 ), and the EC50 values were 0.00913 μg / mL and 0.00702 μg / mL respectively (Table 1).

[0046] Table 1. EC50 of Hm0487 and SEB with mSEB (L45R, Y89A, Y94A)

[0047]

[0048] Example 7. Determination of the affinity of an antibody for an antigen by biolayer interferometry (BLI)

[0049] The kinetic parameters of Hm0487 binding to SEB were measured using biolayer interferometry. The isolated Hm0487 monoclonal antibody at 200 nM was immobilized on the AHC sensor. Serial two-fold dilutions of SEB (12.5 nM to 200 nM) were added according to the sample plate arrangement, and the operation was carried out according to the program settings of "baseline detection - loading detection - quenching - quenching - plate washing - baseline detection - binding detection - dissociation detection". The association rate constant (kon), dissociation rate constant (kdis), and equilibrium dissociation constant (KD) were calculated. The results are shown in Table 2, and the KD of Hm0487 and SEB was 4.661×10 -8 M( Figure 4 ).

[0050] Table 2. Binding affinity data of Hm0487 and SEB

[0051] Ag KD(M) Kon(1 / Ms) Kdis(1 / s) SEB <![CDATA[4.461×10 -8 > <![CDATA[1.21×10 5 > <![CDATA[5.41×10 -3 >

[0052] Example 8. Determination of the epitope type of the Hm0487 antibody

[0053] Protein sample preparation: 10 μg of SEB and 10 μL of mSEB (L45R, Y89A, Y94A) were boiled in a metal bath at 100 °C for 5 min. The electrophoresis tank and electrophoresis gel were installed, electrophoresis buffer was added, and the sample was loaded after removing the comb. The voltage of the electrophoresis instrument was set to 100 V to run the electrophoresis. After the sample ran into the separation gel, the voltage was adjusted to 200 V, and the electrophoresis was terminated when the bromophenol blue ran to 1 cm from the lower edge. After rinsing the electrophoresis gel with water, it was transferred to a semi-dry electrotransfer apparatus for membrane transfer, and the voltage was set to 200 V for 90 min; the transferred PVDF membrane was placed in a TBST solution containing 5% skim milk powder and blocked at room temperature for 1 h. The membrane was washed 3 times with TBST solution, 5 min each time; 1 μg / mL of Hm0487 solution was added and incubated overnight at 4 °C. After washing the membrane 3 times, Anti-Human HRP-IgG (secondary antibody) was diluted 1:5000 with TBST, and the membrane was placed in the above solution and incubated at 37 °C for 45 min.

[0054] The membrane was washed 3 times with TBST solution, 5 min each time; the membrane was placed in a clean petri dish, and about 1 mL of DAB chromogenic solution was added dropwise to each membrane in the dark. When the bands were obvious, the reaction was terminated by rinsing with water. Figure 5 It was shown that Hm0487 could bind to denatured SEB and mSEB (L45R, Y89A, Y94A). Thus, it was determined that the Hm0487 antibody could recognize the linear epitope of SEB.

[0055] Example 9. Evaluation experiment of the enterotoxin B animal model

[0056] Twenty mice were divided into 2 groups, 10 mice in each group. 100 μg of Hm0487, 10 μg of Hm0487, and PBS were injected into the caudal vein of each group of mice, with a volume of 100 μL. After 24 h, 20 μg of SEB and 10 mg of D-galactosamine hydrochloride were injected into the caudal vein of all mice, with a volume of 100 μL. The mice were observed for 7 d and the survival status of the mice was recorded ( Figure 6 ). The neutralization and protection evaluation experiment showed that 100 μg of the Hm0487 antibody could protect 80% of the mice. This result indicated that Hm0487 could protect against enterotoxin B challenge. In summary, the experimental results illustrated the role of Staphylococcus aureus enterotoxin B in pathogenic bacteria and provided evidence for the use of an antibody that inhibits the function of Staphylococcus aureus enterotoxin B to limit the severity of diseases associated with Staphylococcus aureus infection and even death.

[0057] Example 10. Establishment and protective evaluation of a MRSA sepsis infection lethality model

[0058] Ten mice were selected and divided into two groups of five mice each. 600 μg of Hm0487 and PBS were injected into the caudal vein of each group of mice, with a volume of 100 μL. After 24 h, 1.5×108 CFU of MRSA 252 was injected into the caudal vein of all mice, with a volume of 100 μL. The mice were observed for 7 d and the survival status of the mice was recorded ( Figure 7 ). The protective evaluation experiment of MRSA sepsis showed that the 600 μg Hm0487 antibody could protect 80% of the mice. This result indicated that Hm0487 could protect against MRSA sepsis. It was shown that the fully human anti-SEB antibody Hm0487 could inhibit disease progression, enhance clearance and also inhibit the systemic spread of invasive organisms.

[0059] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

1. A fully human SEB monoclonal antibody against Staphylococcus aureus enterotoxin B, characterized in that: The fully human SEB monoclonal antibody comprises a heavy chain and a light chain. The amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the heavy chain are shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 respectively; the amino acid sequences of CDR1 and CDR3 in the variable region of the light chain are shown as SEQ ID NO.9 and SEQ ID NO.10 respectively, and the amino acid sequence of CDR2 in the variable region of the light chain is Asp Thr Lys.

2. The fully human SEB monoclonal antibody against Staphylococcus aureus enterotoxin B according to claim 1, characterized in that: The amino acid sequence of the heavy chain is shown as SEQ ID NO.4; the amino acid sequence of the light chain is shown as SEQ ID NO.

8.

3. The fully human SEB monoclonal antibody against Staphylococcus aureus enterotoxin B according to claim 1, characterized in that: The constant region of the antibody comprises any one of human IgM, IgA or IgG constant regions.

4. The fully human SEB monoclonal antibody against Staphylococcus aureus enterotoxin B according to claim 1, characterized in that: The fully human SEB monoclonal antibody specifically binds to the full-length amino acid or partial amino acid sequence of Staphylococcus aureus enterotoxin B, and the full-length amino acid of Staphylococcus aureus enterotoxin B is shown as SEQ ID NO.

1.

5. The fully human SEB monoclonal antibody against Staphylococcus aureus enterotoxin B according to any one of claims 1-4, characterized in that: The fully human SEB monoclonal antibody binds to staphylococcal enterotoxin B with an equilibrium dissociation constant KD of no higher than 5×10 -8 .

6. A nucleic acid molecule encoding the fully human SEB monoclonal antibody according to any one of claims 1-5.

7. A recombinant expression vector containing the nucleic acid molecule according to claim 6.

8. A host cell containing the recombinant expression vector according to claim 7.

9. Use of the fully human SEB monoclonal antibody according to any one of claims 1-5 in the preparation of a reagent specifically binding to Staphylococcus aureus enterotoxin B.

10. Use of the fully human SEB monoclonal antibody according to any one of claims 1-5 in the preparation of a drug for treating or adjuvantly treating Staphylococcus aureus infection.

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