Fully human monoclonal antibody hm0699 against staphylococcus aureus alpha-hemolysin and uses thereof

Abstract

This invention discloses a fully human monoclonal antibody Hm0699 against Staphylococcus aureus α-hemolysin and its applications. The antibody 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. 5, SEQ ID NO. 6, and SEQ ID NO. 7; the amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10. This antibody can specifically bind to Staphylococcus aureus α-hemolysin and can be used for the treatment, prevention, or diagnosis of Staphylococcus aureus infections. It represents an important direction in the research field of "non-antibiotic" treatment of methicillin-resistant Staphylococcus aureus infections and control of drug resistance development.

Description

Technical Field

[0001] This invention relates to the field of antibodies, specifically to Hm0699, a fully human monoclonal antibody against Staphylococcus aureus α-hemolysin, and also to the application of Hm0699. Background Technology

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is a typical example of a "superbug," characterized by its high pathogenicity, wide transmission routes, and tendency to cause outbreaks, as well as its tendency to develop multidrug resistance. MRSA is a significant pathogen causing hospital-acquired and community-acquired infections, characterized by acute, purulent infections. Systemically, it can lead to severe infections and complications such as sepsis, acute pneumonia, endocarditis, septic arthritis, and osteomyelitis, with a mortality rate as high as 20%. Locally, it can cause persistent purulent infections of the skin and soft tissues. Furthermore, MRSA exotoxins can cause fatal systemic infections such as food poisoning, scalded skin syndrome, and toxic shock syndrome.

[0003] Alpha-hemolysin is a 33.2 kDa water-soluble protein secreted by most pathogenic Staphylococcus aureus strains, targeting almost all mammalian cells by forming stable amphiphilic transmembrane pores. It is considered a key protein regulating Staphylococcus aureus wound healing. In a rat model of Staphylococcus aureus-induced pneumonia, alpha-hemolysin has been shown to disrupt the air-blood barrier of the lungs, while Staphylococcus aureus mutants lacking alpha-hemolysin are unlikely to induce pneumonia-related death. Furthermore, alpha-hemolysin has been shown to be a key determinant of Staphylococcus aureus-induced ocular infections. Based on this evidence, alpha-hemolysin may be a potential therapeutic target for Staphylococcus aureus infections.

[0004] Currently, there are no approved vaccines or specific drug treatments for alpha-hemolysin. Current research proposes various treatment approaches, including small molecule therapeutics, vaccines targeting alpha-hemolysin, and monoclonal antibodies targeting alpha-hemolysin. Antibodies, which exert their effects immediately, are excellent partners to antibiotics and vaccines, representing the best approach for infections associated with emergency interventions and potentially the best option for immunocompromised patients who cannot be properly vaccinated. They represent a potential solution for alleviating and treating pneumonia and Staphylococcus aureus infections caused by alpha-hemolysin. Passive immunotherapy based on antibody drugs for MRSA infection shows great promise for development and application, potentially becoming a new immunoprophylactic approach for "antibiotic-free" treatment of MRSA infection and controlling the development of drug resistance. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide a fully human monoclonal antibody Hm0699 against Staphylococcus aureus α-hemolysin; a second objective of the present invention is to provide a nucleotide sequence encoding the antibody Hm0699; a third objective of the present invention is to provide a vector or host containing the nucleotide sequence; a fourth objective of the present invention is to provide a method for producing a fully human monoclonal antibody Hm0699 against Staphylococcus aureus α-hemolysin; a fifth objective of the present invention is to provide the use of the antibody Hm0699 against Staphylococcus aureus enterotoxin B in the preparation of reagents that specifically bind Staphylococcus aureus α-hemolysin; and a sixth objective of the present invention is to provide the use of the antibody Hm0699 against Staphylococcus aureus enterotoxin B in the preparation of medicaments for treating, preventing, or diagnosing Staphylococcus aureus infections.

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

[0007] 1. A fully human monoclonal antibody Hm0699 against Staphylococcus aureus α-hemolysin, wherein the antibody comprises a heavy chain and a light chain, wherein the amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the heavy chain are as shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7 or CDR variants with equivalent functions; and the amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the light chain are as shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 or CDR variants with equivalent functions.

[0008] Preferably, the constant region of the antibody Hm0699 includes any one of human IgM, IgA, or IgA constant region.

[0009] Preferably, the amino acid sequence of the heavy chain is as shown in SEQ ID NO.3 or a sequence that has at least 85% homology with SEQ ID NO.3 and specifically binds to the full-length amino acid or some amino acid of Staphylococcus aureus α-hemolysin B; the amino acid sequence of the light chain is as shown in SEQ ID NO.4 or a sequence that has at least 85% homology with SEQ ID NO.4 and specifically binds to the full-length amino acid or mutant amino acid of Staphylococcus aureus α-hemolysin B.

[0010] Preferably, the full-length amino acid composition of Staphylococcus aureus α-hemolysin is shown in SEQ ID NO.1; the mutant amino acid composition of Staphylococcus aureus α-hemolysin is shown in SEQ ID NO.2.

[0011] Preferably, in this invention, the equilibrium dissociation constant of the antibody Hm0699 binding to α-hemolysin is not higher than 1 × 10⁻⁶.-8 M.

[0012] 2. The nucleotide sequence encoding the antibody Hm0699.

[0013] 3. A vector or host containing the nucleotide sequence.

[0014] 4. A method for producing a fully human monoclonal antibody Hm0699 against Staphylococcus aureus α-hemolysin, comprising culturing host cells containing the nucleotide sequence encoding the antibody, collecting the expressed antibody, and separating and purifying it using Protein A / G.

[0015] 5. The application of the antibody Hm0699 against Staphylococcus aureus enterotoxin B in the preparation of reagents that specifically bind to Staphylococcus aureus α-hemolysin.

[0016] 6. The application of the antibody Hm0699 against Staphylococcus aureus enterotoxin B in the preparation of drugs for the treatment, prevention or diagnosis of Staphylococcus aureus infection.

[0017] The beneficial effects of this invention are as follows: This invention discloses an antibody against Staphylococcus aureus α-hemolysin, which can be used to detect and / or visualize α-hemolysin, and is therefore effective in diagnostic methods and assays. The antibody described herein can also treat sepsis caused by MRSA infection, and is of great significance for the prevention, treatment, and detection of Staphylococcus aureus. Attached Figure Description

[0018] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0019] Figure 1 This is a sorting diagram of a single plasma cell.

[0020] Figure 2 The results of SDS-PAGE analysis of the fully human anti-Staphylococcus aureus α-hemolysin monoclonal antibody are shown.

[0021] Figure 3 To analyze the binding activity of the fully human anti-Staphylococcus aureus α-hemolysin monoclonal antibody M0699 to Staphylococcus aureus α-hemolysin.

[0022] Figure 4 To analyze the results of antigen-antibody interactions using biomembrane interference technology.

[0023] Figure 5 This is a graph showing the results of Hla denatured gel Western blotting.

[0024] Figure 6Results of establishing an animal model of Staphylococcus aureus α-hemolysin lethality.

[0025] Figure 7 The results of the protective effect evaluation of the Staphylococcus aureus α-hemolysin-induced animal model.

[0026] Figure 8 Results of establishing an animal model of Staphylococcus aureus sepsis.

[0027] Figure 9 To evaluate the experimental results of an animal model of Staphylococcus aureus sepsis.

[0028] Figure 10 Results of establishing an animal model of Staphylococcus aureus pneumonia.

[0029] Figure 11 The results of the protective effect evaluation of the animal model of Staphylococcus aureus pneumonia. Detailed Implementation

[0030] 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 and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0031] This invention discloses monoclonal antibodies that bind to Staphylococcus aureus α-hemolysin, including humanized or human antibodies and single-domain antibodies. Such antibodies can be used to detect and / or visualize Staphylococcus aureus α-hemolysin, and are therefore effective in diagnostic methods and assays. The antibodies described herein can also treat sepsis caused by MRSA infection, and are therefore effective for both treatment and prevention.

[0032] Example 1: Wild-type Staphylococcus aureus α-toxin (wHla) and mutant Staphylococcus aureus α-toxin (Hla) H35L Expression and purification of )

[0033] Genomic DNA from a Staphylococcus aureus strain (ATCC accession number BAA-1556) was amplified by PCR to produce the wHla gene. The H35L variant was then generated through site-directed mutagenesis of the wild-type gene using the QuickChange II XL site-directed mutagenesis kit. After DNA sequencing confirmation, the variant was expressed in Escherichia coli and cultured overnight at 37°C in LB medium containing ampicillin. Cells were harvested by centrifugation. wHla protein and Hla were purified using Ni-NTA. H35L The amino acid sequence of the protein wHla is shown in SEQ ID NO.1. H35L The protein amino acid sequence is shown in SEQ ID NO.2.

[0034] Example 2: Isolation of peripheral blood mononuclear cells (PBMCs)

[0035] Healthy volunteers and volunteers who have recovered from severe Staphylococcus aureus infection were recruited. Venous blood samples were collected in heparinized anticoagulant tubes. PBMC cells were isolated using density centrifugation. The specific procedures were as follows: Venous blood was centrifuged at 22°C, 400×g for 15 min; the clear plasma layer at the top was collected and stored at -80°C; the supernatant was collected and thoroughly mixed with an equal volume of RPMI 1640 (Gibco), then slowly added to a sterile centrifuge tube containing lymphocyte separation medium, maintaining the intact liquid layer. The tube was centrifuged at 2000 rpm for 20 min. Mononuclear cells from the cloudy layer were collected into a sterile centrifuge tube, and at least 5 times the volume of RPMI 1640 was added. The tube was centrifuged at 1500 rpm for 10 min. The cells were washed twice, and the cells were resuspended in an appropriate amount of RPMI 1640 at a concentration of 1×10⁻⁶. 7 / pieces are frozen in liquid nitrogen for later use.

[0036] Example 3: Flow cytometry sorting of single plasma cells

[0037] Flow cytometry sorting of individual plasma cells: Using Hla protective antigen protein (HPLC purity >95%) as the antigen, serum was analyzed by ELISA to determine the antibody titer of the sample. Samples with high antibody titers were selected, and individual plasma cells were sorted by flow cytometry. Gating was performed using CD3 / CD14 / CD16 / CD235a-CD19+CD20+ / -CD38hi CD27hi to separate plasma cell populations at different time points. Serological experiments and B lymphocyte phenotype analysis ensured the acquisition of a large number of individual plasma cells from a plasma cell population >3%, from which the gene for the fully human monoclonal antibody against Hla was isolated. The sorted cells were numerous and in good condition. Figure 1 ).

[0038] Example 4: Cloning of anti-Hla antibody and expression of fully human antibody

[0039] The first strand of cDNA was synthesized using Superscript V reverse transcriptase (Invitrogen, Carlsbad, CA) and random primers. Human IgV was amplified by PCR using the Ig primer set (heavy chain constant region primer sequence: 5'-gcggccctgggctgcctggtcaag-3'; light chain constant region primer sequence: 5'-aggagagtgtcacagagcaggacag-3'). H and V K / L , will amplify V H and V K / LThe product was cloned into the TOPO TA vector and sequenced. The amplified product was then re-amplified by PCR, and the product was identified by 1.2% agarose gel electrophoresis.

[0040] Antibody gene sequencing and bioinformatics analysis: Antibody gene PCR products that were positive by gel electrophoresis and whose heavy and light chains could be matched were purified using the Qiagen PCR product purification kit, and sequenced from both the forward and reverse directions. The antibody gene family, mutation rate, subtype and CDR region were analyzed using the IMGT online server (http: / / imgt.cines.fr / ). New antibody genes were entered into the antibody gene library.

[0041] The PCR product of the antibody variable region gene, which was identified as positive by gel electrophoresis and whose heavy and light chains could be paired, was ligated into the pcDNA3.3 vector using the TA cloning method to construct a fully human anti-Hla antibody expression vector. The expression vector was then transformed into DH5α competent cells and cultured overnight at 37°C on ampicillin-containing plates. Ten single colonies were picked and PCR was performed using specific primers under the following conditions: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 100 s, 28 cycles; 72°C extension for 5 min. 5 μL of the PCR product was then detected by 1% agarose gel electrophoresis.

[0042] The results showed that transformants containing antibody heavy chain and light chain genes were identified in the positive transformants. The heavy chain gene sequence is shown in SEQ ID NO.11; the light chain gene sequence is shown in SEQ ID NO.12.

[0043] Example 5: Expression and purification of fully human anti-Hla antibody

[0044] The vector plasmid from the positive transformants obtained in Example 4 was transformed into DH5α competent cells and amplified in large quantities. After extracting a large amount of recombinant plasmid, IgV was added. H V K / L HEK293F cells were co-transfected with polyethyleneimine (PEI) and cultured in OPM medium at 37°C with 5% CO2 for 120 h. The supernatant was collected by centrifugation at 3000×g for 30 min at 4°C and purified using Protein A affinity chromatography. Antibody expression and purification were assessed by SDS-PAGE. SDS-PAGE results (see [link to SDS-PAGE results]). Figure 2The results showed that the transfected cells successfully expressed the antibody, named the fully human H1A monoclonal antibody M0699, or simply M0699 antibody or M0699 monoclonal antibody. The relative molecular weight of this antibody is approximately 160-180 kDa, with the heavy chain at approximately 55 kDa and the light chain at approximately 25 kDa. The amino acid sequence of the heavy chain is shown in SEQ ID NO.3, and the amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the heavy chain are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7. The amino acid sequence of the light chain is shown in SEQ ID NO.4, and the amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10.

[0045] Example 6: Detection of antibody binding activity

[0046] Wild-type wHla was used as the antigen, 100 μL / well was coated onto ELISA plates, incubated overnight at 4°C, and blocked with blocking buffer at 37°C for 2 h. Overlapping PCR transfection expression antibody (primary antibody) stock solution was added. Positive controls consisted of positive plasma sample stock solution (1:50 dilution) and M0699 antibody (1:1000 dilution). Negative controls consisted of negative plasma sample stock solution (1:50 dilution) and negative control-irrelevant antibody IgG1 0.5 μg / mL. Blocking buffer was added to each well for the blank control, with 3 replicates. The plates were incubated at 37°C for 1 h.

[0047] Wash the plate once with PBST buffer (4 cycles), add 100 μL of Anti-Human HRP-IgG secondary antibody diluted 1:5000 per well, and incubate at 37°C for 40 min. Wash the plate once with PBST buffer (4 cycles), protect from light, add 100 μL of TMB chromogenic buffer per well, and incubate at 37°C for 5 min in the dark. Stop the reaction by adding 50 μL of 2M stop solution per well. OD 450nm Detect the absorbance value. Calculate the mean value of the irrelevant antibody IgG1 in the negative control, and determine the threshold (3 times the mean value). Values ​​greater than the threshold are considered positive.

[0048] Experiments showed that the fully human Hla monoclonal antibody M0699 can bind to wHla ( Figure 3 The EC50 values ​​were 0.00516 μg / mL (Table 1).

[0049] Table 1. EC50 for M0699 and wHla

[0050] Ag EC50 (μg / mL) Ab wHla M0699 0.00516

[0051] Example 7: Determination of antibody-antigen affinity using biomembrane interferometry (BLI).

[0052] Biomembrane interferometry was used to measure the kinetic parameters of M0699 and wHla. 50 nM of the isolated M0699 monoclonal antibody was immobilized onto an AHC sensor. Two-fold serial dilutions of wHla (31.3 nM to 500 nM) were added according to the sample plate arrangement. The procedure was followed: baseline detection - loading detection - quenching - quenching - plate washing - baseline detection - binding detection - dissociation detection. The binding rate constant (kon), dissociation rate constant (kdis), and equilibrium dissociation constant (KD) were calculated. The results are shown in Table 2. The KD for M0699 and wHla was 7.09 × 10⁻⁶. -9 M( Figure 4 ).

[0053] Table 2. Binding kinetics data of M0699 and wHla

[0054] Ag KD(M) Kon(1 / Ms) Kdis(1 / s) wHla <![CDATA[7.09×10 -9 ]]> <![CDATA[3.84×10 3 ]]> <![CDATA[2.72×10 -5 ]]>

[0055] Example 8: Determination of the epitope type of M0699 antibody

[0056] Protein sample preparation: Add 10 μg wHla sample to 5 μL reducing loading buffer and boil in a water bath for 5 min. Install the electrophoresis tank and pre-cast gel, add electrophoresis buffer, remove the comb, and load the sample. Set the electrophoresis apparatus voltage to 120V and run the electrophoresis. Stop electrophoresis when the bromophenol blue reaches 1 cm from the bottom edge. Remove the electrophoresis gel, rinse with water, and transfer it to a semi-dry electroporator. Set the voltage to 22V and transfer for 23 min. After transfer, place the PVDF membrane in TBST solution containing 5% skim milk powder and block overnight at 4℃. Wash the membrane three times with TBST solution, 5 min each time; add 1 μg / mL M0699 solution and incubate at 37℃ for 1 h. After washing three times, add 1:5000 diluted Anti-Human HRP-IgG (secondary antibody) and incubate at 37℃ for 40 min.

[0057] Wash the membrane three times with TBST solution for 5 minutes each time; place the membrane in a clean petri dish, add about 1 mL of DAB colorimetric solution in the dark, and rinse with water to stop the reaction when the bands are clear. Figure 5 The results showed that M0699 could bind to denatured wH1a, thus indicating that the epitope of the M0699 antibody is a linear epitope.

[0058] Example 9: Establishment of an α-toxin lethal model and evaluation of treatment.

[0059] Twenty mice were randomly divided into four groups of five. Each group was injected intravenously with 100 μL of 2 μg wH1a, 3 μg wH1a, 4 μg wH1a, and 8 μg wH1a respectively. The survival time of the mice was observed, and the results are as follows: Figure 6As shown, 2 μg wHla can cause 100% mortality in mice within 72 hours, with a relatively slow mortality trend. Therefore, this dose was chosen as the lethal dose for the toxin model.

[0060] Twenty-four mice were divided into three groups of eight each. Each group received a tail vein injection of 2 μg wHla (100 μL / mouse). 0.5 h later, the two antibody groups received tail vein injections of 40 μg and 20 μg M0699 (100 μL / mouse), respectively. The control group received a tail vein injection of PBS as a negative control. Mice were observed for 72 h, and their survival was recorded. Results are as follows: Figure 7 As shown, in mice, 20 μg of M0699 antibody provided 75% protection, while 40 μg provided 100% protection. This means that within 72 hours, 40 μg of M0699 antibody could completely neutralize the toxin response induced by 2 μg of wHla. In summary, these experimental results demonstrate the crucial role of α-toxins in Staphylococcus aureus pathogenesis and provide evidence for the use of antibodies to treat diseases caused by Staphylococcus aureus α-toxins, thereby limiting the severity and even death associated with Staphylococcus aureus infection.

[0061] Example 10: Establishment and protective evaluation of a MRSA sepsis infection mortality model.

[0062] Take 25 mice and divide them into 5 groups of 5 mice each. Administer 1×10 8 CFU USA300, 1.5×10 8 CFU USA300, 2.0×10 8 CFU USA300, 2.5×10 8 CFU USA300, 3.0×10 8 Mice in each group were injected intravenously with 100 μL of CFU USA300 per tail vein. The mice were observed for 7 days, and their survival was recorded. Results are as follows: Figure 8 As shown, 2.0×10 8 Mice challenged with CFU USA300 via tail vein showed a 100% mortality rate within 7 days; therefore, this dose was chosen as the optimal dose for the MRSA sepsis model.

[0063] Forty mice were divided into four groups of ten each. Group 1 received 800 μg of M0699 antibody via tail vein, Group 2 received 400 μg of M0699 antibody via tail vein, Group 3 received 200 μg of M0699 antibody via tail vein, and Group 4 received PBS via tail vein as a negative control. The total dose was 100 μL per mouse. After 24 hours, mice were challenged via tail vein with 2.0 × 10⁻⁶ PBS. 8 CFU USA300 was used to observe the mice for 10 days, and their survival status was recorded. Results are as follows: Figure 9As shown, 800 μg of M0699 antibody protected 60% of mice, 400 μg of M0699 antibody protected 40% of mice, and 200 μg of M0699 antibody protected 30% of mice. These results indicate that M0699 antibody can resist sepsis caused by MRSA infection to a certain extent and can prevent or delay the progression of the disease to a certain extent.

[0064] Example 11: Establishment and protective evaluation of a mortality model of MRSA pneumonia infection.

[0065] Twenty-five mice were taken and divided into five groups of five. Each group received 1.0 × 10⁻⁶ mice. 8 CFU USA300, 2.0×10 8 CFU USA300, 4.0×10 8 CFU USA300, 5.0×10 8 CFU USA300, 6.0×10 8 Mice were challenged with CFU USA300 at a dose of 20 μL per mouse via endotracheal intubation. Survival status was recorded for 7 days. Results are as follows: Figure 10 As shown, 5.0×10 8 Mice challenged with CFU USA300 via endotracheal intubation showed a 100% mortality rate within 7 days; therefore, this dose was selected as the optimal dose for the MRSA pneumonia model.

[0066] Forty mice were randomly divided into four groups of ten each. Group 1 received 800 μg of M0699 antibody via tail vein, Group 2 received 400 μg of M0699 antibody via tail vein, Group 3 received 200 μg of M0699 antibody via tail vein, and Group 4 received PBS via tail vein as a negative control. The total dose volume for all groups was 100 μL per mouse. Twenty-four hours later, mice were challenged with 5.0 × 10⁻⁶ styes via endotracheal intubation. 8 CFU USA300 was used to observe the mice for 7 days, and their survival status was recorded. Results are as follows: Figure 11 As shown, 800 μg of M0699 antibody protected 80% of mice, 400 μg of M0699 antibody protected 60% of mice, and 200 μg of M0699 antibody protected 40% of mice. These results indicate that M0699 antibody can significantly resist pneumonia caused by MRSA infection and, to some extent, enhance the body's clearance of the invading substance and inhibit its systemic spread.

[0067] The above-described embodiments are merely preferred embodiments provided to fully illustrate 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 all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A fully human monoclonal antibody Hm0699 against Staphylococcus aureus α-hemolysin, characterized in that: The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.3, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.

4.

2. The nucleotide encoding the antibody Hm0699 of claim 1.

3. A vector or host containing the nucleotide of claim 2.

4. A method for producing a fully human monoclonal antibody Hm0699 against Staphylococcus aureus α-hemolysin, characterized in that: The host cells containing the nucleotides encoding claim 2 are cultured, the expressed antibodies are collected, and the antibodies are separated and purified using Protein A / G.

5. The use of the fully human monoclonal antibody Hm0699 according to claim 1 in the preparation of a medicament for treating or diagnosing Staphylococcus aureus infection.

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