A monoclonal antibody specifically binding to esxa and therapeutic agents thereof

Abstract

The application relates to the field of biomedical technology, in particular to a monoclonal antibody specifically combined with EsxA and a therapeutic reagent; the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises three complementarity determining regions VLCDR1, VLCDR2 and VLCDR3, the heavy chain variable region comprises three complementarity determining regions VHCDR1, VHCDR2 and VHCDR3, the light chain variable region is an amino acid sequence identical to SEQ.ID.NO.1 at least 90%, 95%, 96%, 97%, 98%, 99% or 100%, and the heavy chain variable region is an amino acid sequence identical to SEQ.ID.NO.2 at least 90%, 95%, 96%, 97%, 98%, 99% or 100%; the therapeutic reagent comprises the monoclonal antibody, a single-chain antibody, a human-mouse chimeric antibody or a humanized antibody; the monoclonal antibody provided by the application is high in specificity, neutralization activity and protective power and is directed to EsxA, has the characteristics of mediating complement-dependent cytotoxicity, is excellent in in-vivo antibacterial activity and can support the construction of an anti-EsxA chimeric or humanized genetically engineered antibody with neutralization activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a monoclonal antibody that specifically binds to EsxA and its therapeutic agent. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus, S. aureus Staphylococcus aureus can cause a range of different human infections, from relatively mild skin and wound infections to more serious, life-threatening illnesses such as pneumonia, sepsis, and deep tissue infections. Staphylococcus aureus can exist in infected hosts in both planktonic and attached (biofilm-based) forms. This potent, β-hemolytic, Gram-positive, and salt-tolerant pathogen readily colonizes the skin, various mucosal surfaces, soft tissues, bones, and indwelling medical devices. Approximately 30% of people are asymptomatic carriers of Staphylococcus aureus strains that possess genes for antibiotic resistance, Staphylococcus aureus enterotoxin, and other virulence factors. Ess extracellular protein A (EsxA), a factor secreted by the Early Secretory Antigenic Target-6 (ESAT-6) secretion system of Staphylococcus aureus and associated with abscess formation, is present in most Staphylococcus aureus substrains and promotes the persistence and spread of Staphylococcus aureus within the infected host.

[0003] Antimicrobial resistance is one of the major challenges facing humanity in the 21st century. Methicillin-resistant Staphylococcus aureus (MRSA) has become a significant pathogen causing community-acquired infections and hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP). Although multivalent MRSA vaccines have entered Phase III clinical trials, their onset of action is relatively long, resulting in poor emergency treatment efficacy for already infected patients. Therefore, there is an urgent need to explore new methods to control MRSA infection, such as antimicrobial nanomaterials, small-molecule antimicrobial substances, antimicrobial antibodies, and antibody-antibiotic conjugates (AACs).

[0004] Antimicrobial antibodies, as core effector molecules in the immune system's defense against bacterial infection, are essentially specific immunoglobulins produced by B lymphocytes in response to bacterial antigens. They mediate antimicrobial effects through multidimensional mechanisms by recognizing bacterial surface antigenic epitopes (such as capsular polysaccharides and flagellin antigens): firstly, they can specifically neutralize bacterial exotoxins through antigen-binding sites, blocking the binding of toxins to host cell receptors; secondly, they can mediate opsonization by binding to Fc receptors on macrophages via their Fc fragments, significantly enhancing the uptake efficiency of bacteria by phagocytes; and thirdly, they can activate the complement system through the classical pathway, inducing lytic death of bacteria. Due to their high antigen recognition specificity and diverse effector mechanisms, antimicrobial antibodies play a crucial role in the immune defense against bacterial infection and are an important research area in addressing antibiotic resistance.

[0005] An antibody molecule is a tetrapeptide chain structure composed of two identical heavy chains (H chains) and two identical light chains (L chains) linked by interchain disulfide bonds. The variable region (V region) at the N-terminus of the H and L chains consists of a hypervariable / complementarity-determining region (HVR / CDR) and a backbone region (FR); the constant region (C region) is located near the C-terminus. The spatial structure formed by the heavy chain variable region (VH) and the light chain variable region (VL) is the antigen-binding site, where the CDR / HVR is the site where the antibody binds complementary to the antigenic determinant. Monoclonal antibodies (mAbs) can be classified into human and murine origins, etc., depending on their species origin. Murine antibodies are immunogenic when used in humans, easily inducing an immune response. These immune responses can lead to the clearance of murine antibodies and immune complex-mediated hypersensitivity reactions. In the 1980s, recombinant DNA technology emerged for producing chimeric antibodies containing human constant regions and mouse variable regions. The resulting genetically engineered antibodies significantly reduced the side effects of murine antibodies. Besides chimeric antibodies, single-chain antibodies or humanized antibodies can be constructed based on the variable region sequences of murine mAbs, further reducing the side effects of murine mAbs. In the genetic modification process, the most crucial step is to first obtain a murine parental mAb with high specificity, high affinity, and neutralizing activity, and then clone its light and heavy chain variable region genes before constructing genetically engineered antibodies.

[0006] Although antimicrobial antibodies are an important research direction in combating antibiotic resistance, their clinical application is limited by the lack of effective antigenic targets. Current research on specific antibodies against Staphylococcus aureus EsxA has significant shortcomings: there is a lack of mAbs with high affinity, high neutralizing activity, and strong bacteriolytic effects, making it difficult to effectively mediate complement-dependent cytotoxicity and opsonize phagocytosis, thus limiting the therapeutic efficacy against Staphylococcus aureus infections. Furthermore, the construction of genetically engineered antibodies targeting EsxA lacks high-quality murine parental antibodies as a foundation, severely restricting the development of therapeutic antibodies against this target.

[0007] Therefore, screening for murine EsxA-specific mAbs with high affinity and high lytic activity, cloning the light and heavy chain variable region genes from hybridoma cells, and preparing high-affinity and high-specificity antibodies against EsxA are of great significance for the effective treatment and improved prognosis of Staphylococcus aureus infections, especially those with multi-antibiotic resistance.

[0008] In the prior art, patent publication number "CN118480121A" entitled "An antibody specifically binding to Staphylococcus aureus protein A and its therapeutic agent" discloses an antibody specifically binding to Staphylococcus aureus protein A (SpA) and its therapeutic agent. This antibody can specifically bind to Staphylococcus aureus protein A via its Fab fragment, and the anti-SpA antibody can opsonize and kill Staphylococcus aureus. By screening for highly sensitive and specific antibodies against Staphylococcus aureus, treatment of disseminated Staphylococcus aureus infection, pneumonia, and systemic infection can be achieved. However, its efficacy is unsatisfactory; in a systemic Staphylococcus aureus infection model, a dosage of 100 mg / kg of SpA was used. The protection rate of 16 monoclonal antibodies is only 65%. Summary of the Invention

[0009] To overcome the shortcomings of the prior art, the present invention aims to provide a monoclonal antibody that specifically binds to EsxA and its therapeutic agent. This monoclonal antibody has a high affinity for EsxA and can specifically recognize EsxA through the light chain variable region (VL) and heavy chain variable region (VH). The monoclonal antibody also has high neutralizing activity and can mediate complement-dependent cytotoxicity, ultimately achieving precise recognition and efficient neutralization of EsxA. This solves the problems of low affinity of anti-EsxA antibodies, insufficient lysing activity, and lack of high-quality templates for the construction of genetically engineered antibodies in the prior art.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, a monoclonal antibody that specifically binds to EsxA includes a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region (VL) includes three complementarity-determining regions VLCDR1, VLCDR2 and VLCDR3, and the heavy chain variable region (VH) includes three complementarity-determining regions VHCDR1, VHCDR2 and VHCDR3.

[0012] Furthermore, the amino acid sequences of the three complementarity-determining regions VLCDR1, VLCDR2, and VLCDR3 of the light chain variable region (VL) are as follows:

[0013] VLCDR1: Gln-Asp-Ile-Asn-Phe-Asn;

[0014] VLCDR2: His-Gly-Lys;

[0015] VLCDR3: Val-Gln-Tyr-Asp-Gln-Phe-Pro-Trp-Thr.

[0016] Furthermore, the amino acid sequences of the three complementarity-determining regions VHCDR1, VHCDR2, and VHCDR3 of the heavy chain variable region (VH) are as follows:

[0017] VHCDR1: Gly-Tyr-Ser-Phe-Thr-Asp-Tyr-Ala;

[0018] VHCDR2: Ile-Asn-Pro-Tyr-Asn-Gly-Gly-Ser;

[0019] VHCDR3: Ala-Arg-Gly-Asp-Asp-Phe-Asp-Gly-Phe-Ala-Tyr.

[0020] Secondly, a single-chain antibody against EsxA, comprising VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3 located in the monoclonal antibody.

[0021] Thirdly, a human-mouse chimeric antibody against EsxA, comprising VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2, and VHCDR3 located in the monoclonal antibody.

[0022] Fourthly, a humanized antibody against EsxA, comprising VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3 located in the monoclonal antibody.

[0023] Fifthly, a therapeutic agent for EsxA, said therapeutic agent comprising the monoclonal antibody, single-chain antibody, human-mouse chimeric antibody, or humanized antibody.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. The monoclonal antibody provided by this invention has a high affinity for EsxA and can specifically recognize EsxA through the variable regions of the light and heavy chains.

[0026] 2. The light chain, heavy chain variable region genes and amino acid sequences of the monoclonal antibody provided by this invention are unique.

[0027] 3. The monoclonal antibody provided by this invention has high neutralizing activity and can mediate complement-dependent cytotoxicity, achieving specific lysis of Staphylococcus aureus, which is expected to provide a new strategy and means for the treatment of Staphylococcus aureus infectious diseases.

[0028] 4. The monoclonal antibody provided by this invention has high protective efficacy. In in vivo antibacterial activity experiments, when the dose reaches 20 mg / kg, it can produce a 100% protection rate against Staphylococcus aureus in mouse models of lethal challenge experiments. It is of great significance for the effective treatment and improved prognosis of Staphylococcus aureus infectious diseases, especially Staphylococcus aureus infectious diseases resistant to multiple antibiotics.

[0029] In summary, this invention provides a highly specific, highly neutralizing, and highly protective monoclonal antibody against EsxA, which mediates complement-dependent cytotoxic effects and exhibits excellent in vivo antibacterial activity. This provides support for the construction of chimeric or humanized anti-EsxA genetically engineered antibodies with neutralizing activity, and is of great significance for the effective treatment and improved prognosis of Staphylococcus aureus infections, especially those with multi-antibiotic resistance. Attached Figure Description

[0030] Figure 1 This is the SDS-PAGE result of the EsxA recombinant protein.

[0031] Figure 2 This is the SDS-PAGE result of FL-EsxA No.1 mAb.

[0032] Figure 3 This is the result of gene homology sequence detection for the FL-EsxA No.1 mAb light chain variable region.

[0033] Figure 4 This is the result of gene homology sequence detection for the FL-EsxA No.1 mAb heavy chain variable region.

[0034] Figure 5 This is the result of amino acid homology sequence detection of the light chain variable region of FL-EsxA No.1 mAb.

[0035] Figure 6 This is the result of amino acid homology sequence detection in the variable region of the heavy chain of FL-EsxA No.1 mAb.

[0036] Figure 7 This is the result of an in vitro lysis assay mediated by FL-EsxA No.1 mAb.

[0037] Figure 8 These are the results of the in vivo antibacterial activity assay for FL-EsxA No.1 mAb.

[0038] In the attached figures, 1. Molecular weight standard (Marker), 2. Bovine serum albumin (BSA), 3. Mouse ascites fluid, 4. Permeate, and 5. Purified mAb. Detailed Implementation

[0039] The following is in conjunction with the appendix Figure 1 To be continued Figure 8 The present invention will be described in further detail below.

[0040] In the early stages, an extracellular secretory protein A (EsxA) secreted by the ESAT-6 secretion system of Staphylococcus aureus was selected as the target protein for the preparation of a Staphylococcus aureus vaccine. This vaccine produced high levels of neutralizing antibodies in mice, and the antibodies showed good efficacy in complement-mediated lysis assays, with a lysis rate as high as 91.6%, providing a new strategy and approach for the treatment of Staphylococcus aureus infection.

[0041] Based on this antibody, this invention provides a monoclonal antibody that specifically binds to EsxA and its therapeutic agent. The variable regions of the light and heavy chains (including their amino acid and nucleotide sequences) in this monoclonal antibody can support the construction of high-affinity anti-EsxA chimeric antibodies or humanized antibodies. This EsxA-specific antibody possesses complement-mediated in vitro lysis activity and demonstrates therapeutic efficacy against Staphylococcus aureus infection in in vivo experiments.

[0042] In a first aspect, a monoclonal antibody that specifically binds to EsxA comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region (VL) comprises three complementarity-determining regions VLCDR1, VLCDR2, and VLCDR3, and the heavy chain variable region (VH) comprises three complementarity-determining regions VHCDR1, VHCDR2, and VHCDR3, wherein the light chain variable region (VL) is an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ.ID.NO.3;

[0043] The heavy chain variable region (VH) is an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ.ID.NO.4.

[0044] Furthermore, the amino acid sequences of the three complementarity-determining regions VLCDR1, VLCDR2, and VLCDR3 of the light chain variable region (VL) are as follows:

[0045] VLCDR1: Gln-Asp-Ile-Asn-Phe-Asn;

[0046] VLCDR2: His-Gly-Lys;

[0047] VLCDR3: Val-Gln-Tyr-Asp-Gln-Phe-Pro-Trp-Thr.

[0048] Furthermore, the amino acid sequences of the three complementarity-determining regions VHCDR1, VHCDR2, and VHCDR3 of the heavy chain variable region (VH) are as follows:

[0049] VHCDR1: Gly-Tyr-Ser-Phe-Thr-Asp-Tyr-Ala;

[0050] VHCDR2: Ile-Asn-Pro-Tyr-Asn-Gly-Gly-Ser;

[0051] VHCDR3: Ala-Arg-Gly-Asp-Asp-Phe-Asp-Gly-Phe-Ala-Tyr.

[0052] Secondly, a single-chain antibody against EsxA, comprising VLCDR1, VLCDR2, VLCDR3 and / or VHCDR1, VHCDR2, VHCDR3 located in the monoclonal antibody.

[0053] Thirdly, a human-mouse chimeric antibody against EsxA, comprising VLCDR1, VLCDR2, VLCDR3 and / or VHCDR1, VHCDR2 and VHCDR3 located in the monoclonal antibody.

[0054] Fourthly, a humanized antibody against EsxA, comprising VLCDR1, VLCDR2, VLCDR3 and / or VHCDR1, VHCDR2, VHCDR3 located in the monoclonal antibody.

[0055] Fifthly, a therapeutic agent targeting EsxA, for the treatment of Staphylococcus aureus infection, comprising the monoclonal antibody, single-chain antibody, human-mouse chimeric antibody, or humanized antibody.

[0056] Example 1

[0057] The mAb prepared in Example 1 was named FL-EsxA No.1. Its three complementarity-determining regions (VL) of the light chain variable region have 100% amino acid sequence identity with SEQ ID NO.3. The specific amino acid sequences are as follows:

[0058] VLCDR1: Gln-Asp-Ile-Asn-Phe-Asn;

[0059] VLCDR2: His-Gly-Lys;

[0060] VLCDR3: Val-Gln-Tyr-Asp-Gln-Phe-Pro-Trp-Thr.

[0061] The three complementarity-determining regions (MDRs) of the FL-EsxA No.1 mAb heavy chain variable region (VH) prepared in Example 1 have amino acid sequences that are 100% identical to those of SEQ.ID.NO.4. The specific amino acid sequences are as follows:

[0062] VHCDR1: Gly-Tyr-Ser-Phe-Thr-Asp-Tyr-Ala;

[0063] VHCDR2: Ile-Asn-Pro-Tyr-Asn-Gly-Gly-Ser;

[0064] VHCDR3: Ala-Arg-Gly-Asp-Asp-Phe-Asp-Gly-Phe-Ala-Tyr.

[0065] The gene sequence encoding the FL-EsxA-No.1 mAb light chain variable region (VL) of anti-EsxA is shown in SEQ.ID.NO.1, and the gene sequence encoding the heavy chain variable region (VH) is shown in SEQ.ID.NO.2.

[0066] Unless otherwise specified, the experimental methods used in the following experiments are all conventional methods, and the materials and reagents used in the following experiments are all commercially available unless otherwise specified.

[0067] 1. Expression and purification of Staphylococcus aureus extracellular protein A (EsxA)

[0068] Will contain 6 The His-tagged EsxA prokaryotic expression plasmid was placed on ice, and 100 μL of deionized water was added to resuspend the plasmid. 1 μL of the plasmid solution was carefully added to BL21(DE3) competent cells, gently tapped to mix, and incubated on ice for 5 min. This process was repeated 6 times. The cells were then transferred to a 42°C water bath and incubated for 90 s, followed by another 5 min on ice. 900 μL of LB liquid medium was added to the competent cells in a bacterial culture medium container, and the cells were then transferred to a 37°C shaker and incubated at 180 rpm for 45 min. After incubation, 100 μL of the liquid was evenly spread onto LB solid medium, and after complete absorption, the cells were incubated overnight at 37°C. Select plump, rounded single colonies and transfer them to LB broth containing Kana. Incubate the culture at 180 rpm in a 37°C shaker to amplify the bacteria and monitor the optical density (OD) value. When the OD value reaches 0.5–0.6, add IPTG solution to a final concentration of 0.1 mmol / L, adjust the shaker temperature to 20°C, and incubate for 23 h to induce protein expression. After induction, collect the bacterial cells by centrifugation (centrifugation conditions: 4200 rpm; 10 min; 4°C), then resuspend the cells in deionized water and sonicate the precipitate in an ice-water bath (centrifugation conditions: 6-gauge amplitude transformer; 15% power; 180 min; sonication on for 1 s and off for 2 s). After disruption, collect the supernatant by centrifugation (centrifugation conditions: 18000 × g; 10 min; 4°C). Wet the pre-packed column with deionized water, then add 5 mL of nickel column packing material. After the liquid in the nickel column has drained naturally, add approximately 30 mL of deionized water to wash the nickel column. Then add 40 mL of equilibration buffer to equilibrate the nickel column. After equilibration, mix the supernatant of the bacterial lysate with the nickel column packing material and incubate the mixture vertically for 2 h at 4°C using a Hular Mixer. After mixing, repack the mixture into the pre-packed column. After the liquid in the mixture has drained naturally, elute the nickel column sequentially with 40 mL of phosphate buffer saline (PBS), 5 mmol / L elution buffer, 100 mmol / L elution buffer, and 500 mmol / L elution buffer, collecting the eluent. Dialyze the collected eluent overnight at 4°C using dialysis buffer, and then use EtEraser. TM The HP endotoxin removal kit removes endotoxins from recombinant protein solutions; SDS-PAGE determines the molecular weight and purity of the recombinant protein, such as... Figure 1 As shown, the recombinant EsxA component was 11 KD with a purity of 90%, which met the requirements for the next experimental step; the concentration of the recombinant protein was determined by the BCA method.

[0069] 2. Preparation and purification of mouse anti-EsxA high-affinity mAb

[0070] 2.1 Immunization regimen

[0071] For the first immunization, 100 μg of recombinant EsxA protein was dissolved in 2.5 ml of PBS and mixed with an equal volume of Freund's complete adjuvant. The mixture was placed on ice and homogenized using a tissue homogenizer (30,000 rpm) for 15 min to form a stable water-in-oil structure. This mixture was then injected into five 8-week-old female BALB / c mice using a glass syringe. Four weeks later, a second immunization was performed. 100 μg of recombinant EsxA protein was dissolved in 2.5 ml of PBS and mixed with an equal volume of Freund's incomplete adjuvant. The treatment and injection methods were the same as before. Three weeks later, a third immunization was performed. 100 μg of recombinant EsxA protein was dissolved in 2.5 ml of PBS and injected intraperitoneally, 0.5 ml per mouse. Ten days after the third immunization, the serum titer was measured using an indirect ELISA method.

[0072] 2.2 Determination of Immune Serum Titer

[0073] The EsxA recombinant protein was diluted to 5 μg / ml with coating buffer, and 100 μl was added to each well of the ELISA plate and incubated overnight at 4°C. After washing with 0.05% Tween 20-PBS (PBST), immunized mouse serum serially diluted with 0.1% BSA-PBS was added, and the plate was incubated at 37°C for 1 h. After washing with PBST, HRP-labeled goat anti-mouse Ig (H+L) at the working concentration was added, and the plate was incubated at 37°C for 1 h. After washing with PBST, ABTS chromogenic solution was added, and the plate was incubated at room temperature for 30 min. The OD410 value was measured to determine the result. Cell fusion was performed when the serum titer reached 1:64000.

[0074] 2.3 Cell Fusion

[0075] Three days prior to cell fusion, mice were intraperitoneally injected with 10 μg of recombinant EsxA protein in 1 ml of PBS to enhance immunization. One day prior to fusion, peritoneal macrophages from female BALB / c mice were aseptically washed, washed once with incomplete RPMI 1640, resuspended in 40 ml of RPMI 1640 containing 20% ​​fetal bovine serum, and seeded at 100 μl / well in 96-well cell culture plates. The plates were then incubated at 37°C in a 5% CO2 incubator with relative humidity.

[0076] SP2 / 0 cells in the logarithmic growth phase were collected by centrifugation, with a total count of 5-6 × 10⁻⁶. 7Collect the myeloma cells, discard the supernatant, and wash once with incomplete RPMI 1640. Take logarithmically growing myeloma cells (Sp2 / 0), centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in incomplete culture medium, count the cells, and take the required number of cells. Wash twice with incomplete culture medium. Simultaneously prepare an immunosporinated spleen cell suspension and wash twice with incomplete culture medium. Mix the myeloma cells and spleen cells at a ratio of 1:10 or 1:5, wash twice with incomplete culture medium in a 50 ml plastic centrifuge tube, centrifuge at 1200 rpm for 8 min, discard the supernatant, remove the residual liquid with a dropper, and gently tap the bottom of the centrifuge tube to slightly loosen the cell pellet. Incubate at room temperature, add 1 ml of 45% PEG preheated at 37°C within 30 s while stirring, then incubate for 90 s. Add PEG preheated at 37°C to terminate the PEG reaction. Add 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, and 10 ml every 2 min. Centrifuge at 800 rpm for 6 min, discard the supernatant, gently resuspend the cells in 40 ml of 20% fetal bovine serum RPMI 1640, and add the fused cell suspension to a 96-well plate containing feeder cells at 100 μl / well. Incubate at 37°C in a 5% CO2, relative humidity incubator.

[0077] 24 hours after fusion, 50 μl of 5×HAT selection medium was added to each well. The medium was changed once after 3 days to 1×HAT / HT selection medium. The medium was changed again after 3 days to 1×HT medium. After about 2-3 days, the supernatant in the well was collected and positive clones were detected by indirect ELISA.

[0078] Cloning was performed on EsxA-positive clones. Spleens from BALB / c mice were aseptically harvested, and spleen cell suspensions were prepared. These suspensions were resuspended in 80 ml of RPMI 1640 containing 20% ​​fetal bovine serum and 2×HT. 100 μl / well was seeded into 96-well cell culture plates and incubated at 37°C with 5% CO2 and relative humidity. Hybridoma cells were adjusted to a density of 10 cells / ml, and 100 μl / well was seeded into 96-well cell culture plates containing the spleen cell suspension. These plates were incubated at 37°C with 5% CO2 and relative humidity for 6-7 days until clones emerged. Cloning was repeated until two consecutive positive results were obtained. A hybridoma cell line secreting a highly specific mAb for EsxA was obtained and named FL-EsxA No.1. The culture supernatant was collected and ascites fluid was prepared.

[0079] 2.4 Subclass determination of FL-EsxA No.1

[0080] Goat anti-mouse Ig (H+L) was coated onto an ELISA plate, with each antibody line covering 6 wells, and incubated overnight at 4°C. After washing three times with 0.15M PBS-Tween 20, the supernatant of hybridoma cell culture was added to each of the 6 wells, and incubated at 37°C for 1 h. After washing three times with 0.15M PBS-Tween 20, HRP-labeled rat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA, or IgM antibodies were added, and incubated at 37°C for 1 h. After washing three times with 0.15M PBS-Tween 20, ABTS chromogenic buffer was added, and the plate was incubated at room temperature for 30 min. The OD410 value was measured, and the result was interpreted (result indicating IgG1 subclass, κ light chain).

[0081] 2.5 Q Sepharose Fast Flow column chromatography purification of FL-EsxA No.1 antibody

[0082] Centrifuge the ascites fluid at 4°C, 3000 rpm, for 5 min. While stirring, add saturated ammonium sulfate solution over 30 min in an ice bath to achieve a final ammonium sulfate concentration of 40%. Let stand at 4°C for 1 h. Centrifuge at 4°C, 12000 rpm, for 10 min, and discard the supernatant. Resuspend the precipitate in an appropriate amount of 45% saturated ammonium sulfate, centrifuge at 4°C, 12000 rpm, for 10 min, and discard the supernatant. Repeat the above steps once. Dissolve the precipitate in 0.02 M Tris-HCl buffer, remove salt by gel filtration, and pack the column according to the manufacturer's instructions for Q Sepharose Fast Flow. Load the sample at a flow rate of 5 ml / min. After loading, wash away unbound proteins with buffer starting at 2 column volumes, using incrementally increasing concentrations of 1 M NaCl as the elution buffer at a flow rate of 5 ml / min. Collect the target protein based on the protein curve changes. Take a small sample, dilute appropriately, and detect the protein content using a UV spectrophotometer. Identify antibody purity using SDS-PAGE. Figure 2 As shown, the antibody purity is 90%, which meets the requirements for the next experimental step.

[0083] 3. Sequencing of the light and heavy chain variable region genes of FL-EsxA No.1 mAb

[0084] 3.1 Culture of FL-EsxA No.1 mAb hybridoma cells and extraction of total RNA

[0085] Hybridoma cells secreting FL-EsxA No. 1 mAb were resuscitated using standard laboratory methods and cultured in RPMI 1640 culture medium containing 20% ​​fetal bovine serum at 37°C in a 5% CO2 incubator until the logarithmic growth phase. Total RNA was extracted using TRIZOL Reagent, following the manufacturer's instructions.

[0086] 3.2 Sequencing of the light and heavy chain variable regions of FL-EsxA No.1 mAb and determination of the CDR region were commissioned to Beijing Anbiqi Biotechnology Co., Ltd.

[0087] 4. Nucleotide sequence homology analysis of the light and heavy chain variable regions of FL-EsxA No.1 mAb

[0088] 4.1 Nucleotide sequence homology analysis of the light chain variable region of FL-EsxA No.1 mAb

[0089] Nucleotide sequence homology analysis (Blastn) was performed in the GenBank database. The light chain variable region gene sequence of FL-EsxA No.1 mAb showed the highest homology (301 / 321, 94%) with the light chain variable region gene of clone No. ms6_0011_IGKV14-100_IGKJ1_IGKC with Sequence ID: PP321858.1. Figure 3 As shown. Although the nucleotide sequence encoding the light chain variable region of FL-EsxA No.1 mAb has some homology with the light chain variable region sequences of other antibodies, no light chain variable region gene sequence completely identical to that of this invention has been found, indicating that the nucleotide sequence of the light chain variable region of FL-EsxA No.1 mAb disclosed in this invention is unique in terms of gene sequence.

[0090] 4.2 Nucleotide sequence and homology analysis of the variable region of the heavy chain in FL-EsxA No.1 mAb

[0091] Nucleotide sequence homology analysis (Blastn) was performed in the GenBank database. The FL-EsxA No.1 mAb heavy chain variable region gene sequence showed the highest homology (330 / 355, 93%) with the heavy chain variable region gene of clone number 2E4 (Sequence ID: DQ913736.1). Figure 4 As shown. Although the nucleotide sequence encoding the heavy chain variable region of FL-EsxA No.1 mAb has some homology with the heavy chain variable region sequences of other antibodies, no heavy chain variable region gene sequence completely identical to that of this invention has been found, indicating that the nucleotide sequence of the heavy chain variable region of FL-EsxA No.1 mAb disclosed in this invention is unique in terms of gene sequence.

[0092] 5. Amino acid sequence homology analysis of the variable regions of the light and heavy chains in FL-EsxA No.1 mAb

[0093] 5.1 Amino acid sequence homology analysis of the light chain variable region of FL-EsxA No.1 mAb

[0094] The nucleotide sequence of the light chain variable region of FL-EsxA No.1 mAb was translated into an amino acid sequence, and amino acid sequence homology analysis was performed. The amino acid sequence of the light chain variable region of FL-EsxA No.1 mAb is shown in SEQ ID NO.3. Amino acid sequence homology analysis (Blastp) was performed in the Genbank protein database. The search results showed that the amino acid sequence of the light chain of FL-EsxA No.1 mAb had the highest homology (95 / 107, 89%) with the amino acid sequence of the mouse light chain variable region protein with clone number anti-PCB mab-4444 (Sequence ID: ACV89992.1). Figure 5 As shown. Homology analysis showed that although the amino acid sequence of the variable region of the FL-EsxA No.1 mAb light chain had some homology with the amino acid sequences of other mouse-derived antibody light chains, no completely identical antibody light chain amino acid sequence was found, indicating that the FL-EsxA No.1 mAb light chain disclosed in this invention is also unique in amino acid sequence.

[0095] 5.2 Amino acid sequence homology analysis of the variable region of the heavy chain in FL-EsxA No.1 mAb

[0096] The nucleotide sequence of the variable region of the heavy chain of FL-EsxA No.1 mAb was translated into an amino acid sequence, and amino acid sequence homology analysis was performed. The amino acid sequence of the variable region of the heavy chain of FL-EsxA No.1 mAb is shown in SEQ ID NO.4. Amino acid sequence homology analysis (Blastp) was performed in the Genbank protein database. The search results showed that the amino acid sequence of the heavy chain of FL-EsxA No.1 mAb had the highest homology (101 / 118, 86%) with the amino acid sequence of the mouse heavy chain variable region protein of anti-SARS-CoV-2 spike protein with Sequence ID: WGF13045.1. Figure 6 As shown. Homology analysis showed that although the amino acid sequence of the variable region of the FL-EsxA No.1 mAb heavy chain had some homology with the amino acid sequences of other mouse-derived antibody heavy chains, no amino acid sequence of the antibody heavy chain completely identical to that of the present invention was found, indicating that the FL-EsxA No.1 mAb heavy chain disclosed in the present invention is also unique in amino acid sequence.

[0097] 6. In vitro lysing activity of FL-EsxA No.1 mAb

[0098] Complement-dependent cytotoxicity (CDC) refers to cytotoxic effects involving complement. Specifically, specific antibodies bind to pathogens or corresponding antigens on the cell membrane surface, forming antigen-antibody complexes. These complexes activate C1q, thereby activating the classical complement pathway. The resulting membrane-attack complex exerts a lytic effect on the corresponding pathogens or target cells. This is the primary killing mechanism of antibodies against Staphylococcus aureus.

[0099] Staphylococcal Protein A (SpA) on the cell wall surface of Staphylococcus aureus can nonspecifically bind to the Fc fragment of IgG in the serum of various mammals, which is an important mechanism for Staphylococcus aureus immune evasion. When designing complement-dependent FL-EsxA No.1 mAb-mediated lysis assays, it is necessary to block the SpA binding site with a nonspecific antibody. Since SpA has a higher affinity for rabbit IgG than mouse-derived IgG1, using rabbit serum to block the SpA binding site is a more effective method.

[0100] MRSA strain USA300 was streaked onto LB agar plates, and single clones were picked for amplification. When the bacteria reached an OD600 of 0.5, they were collected and diluted 1:10 with physiological saline. 0.2 ml of the solution was added to each test tube, along with 0.2 ml of inactivated rabbit serum diluted 1:10 with physiological saline (56℃, 30 min) to block the SpA binding site.

[0101] 6.1 Experimental Grouping:

[0102] (1) Experimental group 1: FL-EsxA No.1 mAb 80μg, USA300 0.2ml, 1:10 diluted inactivated rabbit serum 0.2ml, 1:10 diluted fresh guinea pig serum (complement) 0.2ml, and physiological saline to make up to a total volume of 2ml. The final concentration of FL-EsxA No.1 mAb was 40μg / ml.

[0103] (2) Experimental group 2: FL-EsxA No.1 mAb 40μg, USA300 0.2ml, 1:10 diluted inactivated rabbit serum 0.2ml, 1:10 diluted fresh guinea pig serum (complement) 0.2ml, and physiological saline to make up to a total volume of 2ml. The final concentration of FL-EsxA No.1 mAb was 20μg / ml.

[0104] (3) Experimental group 3: FL-EsxA No.1 mAb 20μg, USA300 0.2ml, 1:10 diluted inactivated rabbit serum 0.2ml, 1:10 diluted fresh guinea pig serum (complement) 0.2ml, and physiological saline to make up to a total volume of 2ml. The final concentration of FL-EsxA No.1 mAb was 10μg / ml.

[0105] (4) Experimental group 4: FL-EsxA No.1 mAb 10µg, USA300 0.2ml, 1:10 diluted inactivated rabbit serum 0.2ml, 1:10 diluted fresh guinea pig serum (complement) 0.2ml, and added to physiological saline to a total volume of 2ml. The final concentration of FL-EsxA No.1 mAb was 5μg / ml.

[0106] (5) Control group 1: irrelevant antibody (mouse anti-human CD147 antibody) 80 μg, USA300 0.2 ml, 1:10 diluted inactivated rabbit serum 0.2 ml, 1:10 diluted fresh guinea pig serum (complement) 0.2 ml, and physiological saline to make up to a total volume of 2 ml;

[0107] (6) Control group 2: FL-EsxA No.1 mAb 80μg, USA300 0.2ml, 1:10 diluted inactivated rabbit serum 0.2ml, and physiological saline to make up to a total volume of 2ml;

[0108] (7) Control group 3: USA300 0.2ml, 1:10 diluted inactivated rabbit serum 0.2ml, 1:10 diluted fresh guinea pig serum (complement) 0.2ml, and physiological saline to make up to a total volume of 2ml;

[0109] (8) Control group 4: USA300 0.2ml, 1:10 diluted inactivated rabbit serum 0.2ml, and physiological saline to make up to a total volume of 2ml.

[0110] 6.2 Experimental Procedure:

[0111] Place the labeled test tubes on an ice bath, with 6 tubes per group. Add each component to the test tubes according to the above grouping, mix well, and incubate in a 37°C water bath for 30 minutes, gently shaking once every 5-10 minutes. After incubation, perform serial dilutions of the reaction solution, take 100µl and spread it on an LB agar plate, incubate at 37°C for 16 hours, count the number of bacterial clones, calculate the lysis rate, and draw a bar chart.

[0112] 6.3 Experimental Results:

[0113] No significant lysis was observed in the three control groups. In the four experimental groups, the lysis rate increased with increasing concentration of FL-EsxA No.1 mAb. Figure 7 As shown, when the concentrations of FL-EsxA No.1 mAb were 5 µg / ml, 10 µg / ml, 20 µg / ml, and 40 µg / ml, the lysis rates were 11.9%, 23.3%, 49.9%, and 90.1%, respectively. The data were compared for significant differences between groups using GraphPad Prism (v.8.0.2) software. The results of this embodiment demonstrate that FL-EsxA No.1 mAb has the property of mediating complement-dependent cytotoxic effects, and it is expected to provide new strategies and methods for the treatment of MRSA.

[0114] 7. In vivo antibacterial activity of FL-EsxA No.1 mAb

[0115] 7.1 Determination of the absolute lethal dose in mice

[0116] Staphylococcus aureus USA300, stored at -80℃, was streaked onto LB agar plates and incubated at 37℃ for 16 hours. Single colonies were picked, and 5 mL of LB liquid medium was added. The culture was then transferred to a constant-temperature shaker and amplified at 37℃ at 180 rpm. When the OD600 value of the bacterial suspension increased to approximately 0.4-0.5, the culture was expanded to a 200 mL system. When the OD600 value of the bacterial suspension increased to approximately 0.4-0.5, the bacterial suspension was serially diluted and evenly spread on LB solid medium. The culture was incubated overnight at 37℃. The colony count on each LB solid medium was counted the next day, and the colony-forming units (CFU) were calculated.

[0117] Different concentrations of bacterial suspension were injected into blank mice via the tail vein at a volume of 100 μL. The physical condition and survival rate of each group of mice were continuously observed for 14 days. The bacterial dose that caused all 10 mice to die within 14 days was defined as the absolute lethal dose (LD). 100 The LD determined in this experiment 100 2.56×10 8 CFU / each.

[0118] 7.2 Protective effect of FL-EsxA No.1 mAb in lethal challenge experiments

[0119] Different doses of FL-EsxA No.1 mAb were administered intraperitoneally, and the LD50 was determined at 7.1 after 24 hours. 100 A lethal challenge mouse model was established, with 10 mice in each group, to investigate the in vivo antibacterial activity of FL-EsxA No.1 mAb.

[0120] (1) Experimental groups:

[0121] A. FL-EsxA No.1 mAb dosage 20 mg / kg;

[0122] B. FL-EsxA No.1 mAb dosage 10 mg / kg;

[0123] C. FL-EsxA No.1 mAb dose 5 mg / kg;

[0124] D. Irrelevant antibody control group (mouse anti-human CD147 antibody) 20 mg / kg;

[0125] E. Blank control group, intraperitoneal injection of 0.5 ml PBS.

[0126] (2) Experimental results

[0127] The protective effect of FL-EsxA No.1 mAb in a lethal challenge experiment was investigated. Different doses of FL-EsxA No.1 mAb were administered intraperitoneally. The LD50 was determined at 7.1 after 24 hours. 100 A lethal challenge mouse model was established, with 12 mice in each group, to investigate the in vivo antibacterial activity of FL-EsxA No.1 mAb. Data were statistically analyzed using GraphPad Prism (v.8.0.2) software. Survival Analyses were used to compare significant differences between the two groups. LD50 was administered via tail vein injection. 100 Dosage (2.56×10) 8 After inoculating mice with Staphylococcus aureus USA300 (CFU), the survival of mice in each group was continuously observed for 14 days. Figure 8 As shown, all mice in the irrelevant antibody control group (mouse anti-human CD147 antibody, 20 mg / kg) and the blank control group (PBS) died within 3 days after challenge, indicating that the irrelevant antibody did not provide effective protection for mice. The survival rate of mice in the FL-EsxA No.1 mAb group at 5 mg / kg was 30%, significantly better than that of the irrelevant antibody group and the PBS control group. p <0.01). The survival rate of mice in the FL-EsxA No.1 mAb group at a dose of 10 mg / kg was 70%, which was significantly better than that in the FL-EsxA No.1 mAb group at a dose of 5 mg / kg, the irrelevant antibody group, and the PBS control group. p <0.05). When the dose of FL-EsxA No.1 mAb was 20 mg / kg, it provided sufficient protection to mice at the current challenge dose, with a survival rate of 100% in all groups, demonstrating excellent in vivo antibacterial activity and providing new hope for the clinical treatment of MRSA infection.

[0128] 8. Based on the light and heavy chain variable region sequences of FL-EsxA No.1 mAb, design and construct the following bioproducts.

[0129] 8.1 Construction of single-chain antibodies

[0130] 8.1.1 Single-chain antibody molecule design

[0131] Single-chain antibodies (scFv) are recombinant proteins formed by linking the light chain variable region (VL) and heavy chain variable region (VH) of an antibody using a linker. In this invention, a flexible and stable (Gly4Ser)3 linker peptide was selected for the VL and VH gene sequences of FL-EsxANo.1mAb. This linker peptide ensures that VL and VH are correctly folded in space, maintaining the antigen-binding activity of the antibody.

[0132] 8.1.2 Carrier Construction

[0133] The designed VL-linker-VH or VH-linker-VL gene sequence was inserted into the pCDNA3.1 eukaryotic expression vector. This vector contains elements required for efficient expression in eukaryotic cells, such as the promoter (CMV promoter), terminator, and selection marker (neomycin resistance gene).

[0134] In constructing the vector, the VL and VH gene fragments were first amplified from a plasmid containing the light and heavy chain variable regions of FL-EsxANo.1mAb using PCR. Then, overlap extension PCR (SOE-PCR) was used to sequentially ligate VL (or VH) to the linker gene and the linker gene to VH (or VL) to obtain the complete single-chain antibody gene sequence. This sequence was then digested with restriction endonucleases and ligated into an expression vector that had undergone the same digestion process. The ligation was then performed on E. coli DH5α competent cells. Positive clones were selected by ampicillin or kanamycin resistance screening, and the plasmid was extracted and sequenced to verify the correctness of the ligation sequence.

[0135] 8.1.3 Host System Transformation and Expression

[0136] The eukaryotic expression vector was transfected into human embryonic kidney cells (HEK293 cells) using liposome transfection. Before transfection, the cells were cultured to the logarithmic growth phase, following the instructions of the transfection reagent. After transfection, the cells were seeded in a culture medium containing selection markers, such as neomycin (G418), for clonal selection to obtain cell lines stably expressing single-chain antibodies.

[0137] 8.1.4 Purification and Identification of Expression Products

[0138] Eukaryotically expressed single-chain antibodies are typically secreted into cell culture medium. The supernatant is collected by centrifugation and purified using reverse affinity chromatography. The purified single-chain antibodies are then characterized for molecular weight and specificity using SDS-PAGE electrophoresis and Western blot. Their binding activity to the EsxA antigen is detected using indirect ELISA to ensure that the prepared single-chain antibodies have the correct structure and good antigen-binding ability.

[0139] 8.1.5 Advantages of Single-Chain Antibodies

[0140] (1) Highly efficient penetration of biofilms and infection foci: Staphylococcus aureus easily forms biofilms (such as catheter-related infections and periprosthetic infections), which are difficult for traditional antibodies to penetrate. Single-chain antibodies (approximately 25 kDa) have a small molecular weight and can penetrate the biofilm matrix, interfering with bacterial adhesion or toxin activity.

[0141] (2) Low immunogenicity and potential for long-term modification: Staphylococcus aureus infection is often accompanied by recurrent inflammation (such as osteomyelitis and sepsis), requiring long-term intervention. Single-chain antibodies have small molecular weight and low immunogenicity. They can be further reduced in immunogenicity and improved in drug safety through humanization; or the half-life can be extended through PEGylation to reduce the risk of immune reactions from repeated administration.

[0142] (3) Applications and multifunctional modifications of single-chain antibodies:

[0143] 1) Diagnostic applications: Single-chain antibodies can be used for rapid detection of staphylococcal toxins (such as enterotoxins) in clinical samples (such as blood and secretions), and can be further developed into portable test strips to reduce testing time;

[0144] 2) Therapeutic applications: Immunotoxins can be constructed by fusing or cross-linking single-chain antibodies with toxin proteins (such as ricin) to specifically kill Staphylococcus aureus; or single-chain antibodies can be fusing or cross-linked with antimicrobial peptides, antibiotics and other small molecule antimicrobial drugs to enhance the clearance of intracellular bacteria (such as Staphylococcus in macrophages).

[0145] 8.2 Construction of human-mouse chimeric antibodies

[0146] 8.2.1 Carrier Design and Construction

[0147] Human-mouse chimeric antibodies are chimeric gene expression products formed by combining the variable region genes of the light and heavy chains of murine antibodies with the constant region genes of human antibodies. In this invention, the AbVec light chain (Kappa type, clone number AbVEC1.1-IGKC) and heavy chain (IgG1 subclass, clone number AbVEC2.0-IGHG1) expression vectors from AddGene (https: / / www.addgene.org) are used. These vectors have been pre-cloned with either the human antibody light chain (Kappa type) or the heavy chain constant region (IgG1 subclass) gene and contain regulatory elements suitable for eukaryotic cell expression, such as promoters, enhancers, and terminators.

[0148] The specific steps are as follows: VL and VH gene fragments are amplified from plasmids containing the light and heavy chain variable regions of FL-EsxANo.1mAb, respectively. These fragments are then inserted into the AbVEC1.1-IGKC or AbVEC2.0-IGHG1 expression vectors using restriction endonucleases, ensuring correct ligation with the human antibody constant region gene to form a complete chimeric antibody gene. The cells are then transformed into *E. coli* DH5α competent cells. Positive clones are selected using ampicillin or kanamycin resistance screening. The plasmids are extracted and sequenced to verify the correct ligation sequence.

[0149] 8.2.2 Eukaryotic cell transfection and screening

[0150] Human embryonic kidney cells (HEK293 cells) were co-transfected with vectors containing the light and heavy chain chimeric genes of antibodies using liposome transfection. Before transfection, cells were cultured to the logarithmic growth phase, and the transfection reagent was used according to the manufacturer's instructions. After transfection, cells were seeded in a medium containing neomycin (G418) for clonal selection to obtain cell lines stably expressing single-chain antibodies.

[0151] To improve transfection efficiency and screening effectiveness, limiting dilution can be used to subclone positive clones, ensuring the acquisition of single, high-expressing cell lines. The expression level of chimeric antibodies in the cell culture supernatant is then detected by ELISA to screen for high-expressing cell lines for subsequent culture.

[0152] 8.2.3 Expression and purification of chimeric antibodies

[0153] The selected high-expression cell lines are then cultured on a large scale in serum-free medium, using methods such as batch culture, fed-batch culture, or perfusion culture, to increase antibody yield. During the culture process, culture conditions such as temperature, pH, and dissolved oxygen concentration are controlled to ensure that cell growth and antibody expression are at their optimal levels.

[0154] Taking advantage of the fact that chimeric antibodies contain the constant region of human antibodies, high-purity chimeric antibodies can be obtained by purification using Protein A or Protein G affinity chromatography. The purified antibodies are then analyzed by methods such as Western blotting and mass spectrometry to confirm the correct linkage between the murine variable region and the human constant region.

[0155] 8.2.4 Advantages of chimeric antibodies

[0156] (1) Low immunogenicity and long-term modification potential: Staphylococcus aureus infection is often accompanied by recurrent inflammation (such as osteomyelitis and sepsis), requiring long-term intervention. Chimeric antibodies have significantly lower immunogenicity than murine antibodies, greatly improving drug safety; or the half-life of chimeric antibodies can be extended by PEGylation, reducing the risk of immune reactions from repeated administration.

[0157] (2) Applications and multifunctional modification of chimeric antibodies:

[0158] 1) Diagnostic applications: Chimeric antibodies can be used for rapid detection of staphylococcal toxins (such as enterotoxins) in clinical samples (such as blood and secretions), and can be further developed into portable test strips to reduce testing time;

[0159] 2) Therapeutic applications: Chimeric antibodies can be fused or cross-linked with toxin proteins (such as ricin) to construct immunotoxins that can directly kill Staphylococcus aureus; or chimeric antibodies can be fused or cross-linked with antimicrobial peptides, antibiotics and other small molecule antimicrobial drugs to enhance the clearance of intracellular bacteria (such as Staphylococcus aureus in macrophages).

[0160] 8.3 Construction of humanized antibodies

[0161] 8.3.1 CDR Region Transfer Design

[0162] Humanized antibodies are constructed using complementarity-determining region (CDR) transplantation technology, which involves transplanting the CDR regions (i.e., hypervariable regions, including CDR1, CDR2, and CDR3) from the light and heavy chain variable regions of murine antibodies into the backbone region (FR) of the variable region of human antibodies. First, human antibody FR sequences highly similar to the variable region sequences of human IgG are selected from a database. Then, the murine CDR region sequence is transplanted into the human FR sequence to form a CDR-grafted antibody, i.e., a humanized antibody. During the transplantation process, key amino acid residues in the FR region that may affect CDR conformation and antigen-binding activity need to be analyzed and optimized, and site-directed mutagenesis may be performed if necessary to maintain the antibody's specificity and affinity.

[0163] 8.3.2 Gene Synthesis and Vector Construction

[0164] Based on the designed humanized antibody light and heavy chain variable region gene sequences (including mouse CDR and human FR regions), the target gene fragments were prepared using gene synthesis technology. The synthesized light and heavy chain variable region genes were inserted into the AbVEC1.1-IGKC or AbVEC2.0-IGHG1 expression vectors, respectively, to ensure correct ligation with the antibody constant region gene, forming a complete humanized antibody gene. The gene was transformed into *E. coli* DH5α competent cells, and positive clones were selected by ampicillin or kanamycin resistance. Plasmids were extracted and sequenced to verify the correct ligation sequence.

[0165] 8.3.3 Cell Transfection and Expression Optimization

[0166] Human embryonic kidney cells (HEK293 cells) were co-transfected with vectors containing the light and heavy chain humanized antibody genes, respectively, using liposome transfection. Before transfection, cells were cultured to the logarithmic growth phase, following the instructions of the transfection reagent. After transfection, cells were seeded in a medium containing neomycin (G418) for clonal selection to obtain cell lines stably expressing single-chain antibodies.

[0167] To improve transfection efficiency and screening effectiveness, limiting dilution can be used to subclone positive clones, ensuring the acquisition of single, high-expressing cell lines. The expression level of chimeric antibodies in the cell culture supernatant is then detected by ELISA to screen for high-expressing cell lines for subsequent culture.

[0168] 8.3.4 Purification and Identification of Humanized Antibodies

[0169] The selected high-expression cell lines are then cultured on a large scale in serum-free medium, using methods such as batch culture, fed-batch culture, or perfusion culture, to increase antibody yield. During the culture process, culture conditions such as temperature, pH, and dissolved oxygen concentration are controlled to ensure that cell growth and antibody expression are at their optimal levels.

[0170] Taking advantage of the fact that chimeric antibodies contain the constant region of human antibodies, high-purity chimeric antibodies can be obtained by purification using Protein A or Protein G affinity chromatography. The purified antibodies are then analyzed by immunoblotting, mass spectrometry, and other methods to confirm the correct linkage between the CDR region, FR region, and human constant region.

[0171] 8.3.5 Advantages of Humanized Antibodies

[0172] (1) Low immunogenicity and long-term modification potential: Staphylococcus aureus infection is often accompanied by recurrent inflammation (such as osteomyelitis and sepsis), requiring long-term intervention. Humanized antibodies have significantly lower immunogenicity than murine antibodies and chimeric antibodies, and the drug safety is greatly improved. Humanized antibodies should have lower immunogenicity and better biocompatibility; or the half-life of chimeric antibodies can be extended by PEGylation to reduce the risk of immune reactions from repeated administration.

[0173] (2) Applications and multifunctional modification of chimeric antibodies:

[0174] 1) Diagnostic applications: Humanized antibodies can be used for rapid detection of staphylococcal toxins (such as enterotoxins) in clinical samples (such as blood and secretions), and can be further developed into portable test strips to reduce testing time;

[0175] 2) Therapeutic applications: Immunotoxins can be constructed by fusing or cross-linking humanized antibodies with toxin proteins (such as ricin) to directly kill Staphylococcus aureus; or chimeric antibodies can be fused or cross-linked with antimicrobial peptides, antibiotics and other small molecule antimicrobial drugs to enhance the clearance of intracellular bacteria (such as Staphylococcus aureus in macrophages).

[0176] This invention involves immunizing BALB / c mice with recombinant EsxA protein, obtaining hybridoma cells through cell fusion and clonal screening, and preparing and purifying the FL-EsxA No.1 monoclonal antibody. This monoclonal antibody can specifically bind to EsxA through the VL and VH variable regions, mediating complement-dependent cytotoxicity to lyse bacteria. In in vivo experiments, a dose of 20 mg / kg achieved 100% protection against lethal challenge in mice, exhibiting high specificity, high neutralizing activity, and high protective efficacy. It is of great significance for the effective treatment and improved prognosis of Staphylococcus aureus infections resistant to multiple antibiotics.

Claims

1. A monoclonal antibody that specifically binds to EsxA, comprising a light chain variable region and a heavy chain variable region, characterized in that, The light chain variable region contains three complementarity-determining regions, VLCDR1, VLCDR2, and VLCDR3, and the heavy chain variable region contains three complementarity-determining regions, VHCDR1, VHCDR2, and VHCDR3. The amino acid sequences of the three complementarity-determining regions VLCDR1, VLCDR2, and VLCDR3 of the light chain variable region (VL) are as follows: VLCDR1:Gln-Asp-Ile-Asn-Phe-Asn, VLCDR2: His-Gly-Lys, VLCDR3: Val-Gln-Tyr-Asp-Gln-Phe-Pro-Trp-Thr; The amino acid sequences of the three complementarity-determining regions (VHCDR1, VHCDR2, and VHCDR3) of the heavy chain variable region (VH) are as follows: VHCDR1: Gly-Tyr-Ser-Phe-Thr-Asp-Tyr-Ala, VHCDR2: Ile-Asn-Pro-Tyr-Asn-Gly-Gly-Ser, VHCDR3: Ala-Arg-Gly-Asp-Asp-Phe-Asp-Gly-Phe-Ala-Tyr.

2. A single-chain antibody targeting EsxA, characterized in that, It includes VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3 located in the monoclonal antibody as described in claim 1.

3. A human-mouse chimeric antibody against EsxA, characterized in that, It includes VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3 located in the monoclonal antibody as described in claim 1.

4. A humanized antibody against EsxA, characterized in that, It includes VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3 located in the monoclonal antibody as described in claim 1.

5. A therapeutic agent targeting EsxA, characterized in that, It includes monoclonal antibodies, single-chain antibodies, human-mouse chimeric antibodies or humanized antibodies as described in any one of claims 1 to 4.

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