Monoclonal antibody specifically binding to EsxB and therapeutic reagent thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a monoclonal antibody specifically combined with EsxB 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 complementary determining regions VLCDR1, VLCDR2 and VLCDR3, the heavy chain variable region comprises three complementary determining regions VHCDR1, VHCDR2 and VHCDR3, the light chain variable region is an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical with SEQ.ID.NO.1, and the heavy chain variable region is an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical with SEQ.ID.NO.2; the therapeutic reagent comprises a monoclonal antibody, a single-chain antibody, a human-mouse chimeric antibody or a humanized antibody; the monoclonal antibody with high specificity, high neutralization activity and high protection force for EsxB provided by the invention has the characteristic of mediating complement-dependent cytotoxic effect, has excellent in-vivo antibacterial activity, and can support the construction of an anti-EsxB chimeric or humanized genetic engineering antibody with neutralization activity.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a monoclonal antibody specifically binding to EsxB and a therapeutic agent thereof. Background Art

[0002] Staphylococcus aureus (S. aureus) can cause a range of diverse human infections, ranging from relatively minor skin and wound infections to more severe, life-threatening conditions such as pneumonia, sepsis, and deep tissue infections. S. aureus can persist in infected hosts in both planktonic and sessile (biofilm-based) forms. This potent, β-hemolytic, Gram-positive, and halotolerant pathogen readily colonizes the skin, various mucosal surfaces, soft tissues, bone, and indwelling medical devices. Approximately 30% of humans are asymptomatic carriers of S. aureus strains that harbor genes for antibiotic resistance, S. aureus enterotoxins, and other virulence factors. Extracellular secretory protein B (EsxB) is a factor associated with abscess formation secreted by the Early Secretory Antigenic Target-6 (ESAT-6) secretion system of Staphylococcus aureus. It is present in most Staphylococcus aureus substrains and can promote the persistence and spread of Staphylococcus aureus in 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 major pathogen causing both community- and hospital-acquired infections and a common cause of hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP). Although a multivalent MRSA vaccine has entered Phase III clinical trials, its effectiveness is delayed and its efficacy in emergency treatment of infected patients is poor. Therefore, there is an urgent need to explore new approaches to control MRSA infections, such as antimicrobial nanomaterials, small molecule antimicrobial substances, antimicrobial antibodies, and antibody-antibiotic conjugates (AACs).

[0004] Antimicrobial antibodies, as the core effector molecules of the immune system against bacterial infections, are essentially specific immunoglobulins produced by B lymphocytes after being stimulated by bacterial antigens. They mediate antimicrobial effects through multi-dimensional mechanisms by recognizing bacterial surface antigen epitopes (such as capsular polysaccharides, flagellin and other antigenic determinants): first, they can specifically neutralize bacterial exotoxins through antigen binding sites, blocking the binding effect of toxins with host cell receptors; second, they can bind to Fc receptors on the surface of macrophages through the Fc segment, mediate opsonized phagocytosis, and significantly enhance the efficiency of phagocytes in taking up bacteria; third, they can activate the complement system through the classical pathway, triggering bacterial lytic death. Antimicrobial antibodies play a key role in the immune defense against bacterial infections due to their high specificity in antigen recognition and the diversity of their effector mechanisms, and are one of the important research areas 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) connected by interchain disulfide bonds. The variable region (V region) at the N-terminus of the H and L chains consists of the hypervariable region / complementarity determining region (HVR / CDR) and the framework 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 complements the antigenic determinant. Monoclonal antibodies (mAbs) are classified as human or mouse based on their species of origin. Mouse antibodies are immunogenic when used in humans and are prone to eliciting immune responses. These immune responses can lead to clearance of the mouse antibody and immune complex-mediated hypersensitivity reactions. In the 1980s, recombinant DNA technology for producing chimeric antibodies containing human constant region and mouse variable region genes emerged. The resulting genetically engineered antibodies greatly reduced the side effects of mouse-derived antibodies. In addition to chimeric antibodies, single-chain antibodies or humanized antibodies can also be constructed based on the variable region sequences of mouse-derived mAbs to further reduce the side effects of mouse-derived mAbs. In the genetic modification process, the most important thing is to first obtain a mouse-derived parent mAb with high specificity, high affinity, and neutralizing activity, and then clone its light chain and heavy chain variable region genes before starting to construct genetically engineered antibodies.

[0006] Although antibacterial antibodies are an important research direction for combating antibiotic resistance, their clinical application is limited by the lack of effective antigenic targets. Existing research on specific antibodies targeting Staphylococcus aureus EsxB is significantly limited: the lack of mAbs with high affinity, high neutralizing activity, and strong bacteriolytic effects makes it difficult to effectively mediate complement-dependent cytotoxicity and opsonophagocytosis, resulting in limited therapeutic efficacy against S. aureus infections. Furthermore, the construction of genetically engineered antibodies targeting EsxB lacks a high-quality mouse parental antibody as a foundation, severely restricting the development of therapeutic antibodies against this target.

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

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

[0009] In order to overcome the shortcomings of the above-mentioned prior art, the object of the present invention is to provide a monoclonal antibody that specifically binds to EsxB and a therapeutic agent thereof. The monoclonal antibody has a high affinity for EsxB and can specifically recognize EsxB 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 accurate recognition and efficient neutralization of EsxB, thereby solving the problems of low affinity, insufficient lytic activity and lack of high-quality templates for the construction of genetically engineered antibodies in the prior art of anti-EsxB antibodies.

[0010] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0011] In a first aspect, a monoclonal antibody that specifically binds to EsxB, comprising 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, and 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;

[0012] 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.

[0013] Furthermore, the amino acid sequences of the three complementarity determining regions VLCDR1, VLCDR2 and VLCDR3 of the light chain variable region (VL) are:

[0014] VLCDR1:Ser-Ala-Arg-Ser-Ser-Ile-Ser-Tyr-Ile-His;

[0015] VLCDR2:Asp-Ala-Ser-Lys-Leu-Ala-Ser;

[0016] VLCDR3: Phe-Gln-Gly-Ser-Gly-Tyr-Pro-Leu-Thr.

[0017] Furthermore, the amino acid sequences of the three complementarity determining regions VHCDR1, VHCDR2 and VHCDR3 of the heavy chain variable region (VH) are:

[0018] VHCDR1: Gly-Tyr-Tyr-Ile-His;

[0019] VHCDR2:

[0020] Arg-Phe-Asn-Pro-Tyr-Asn-Gly-Ala-Ser-Asp-Tyr-Asn-Gln-Asn-Phe-Arg-Asp-Lys-Ala-Ser;

[0021] VHCDR3: Thr-His-Thr-Ser-Gly-Tyr-Val-Trp-Ala-Met-Asp-Tyr.

[0022] In a second aspect, a single-chain antibody against EsxB comprises VLCDR1, VLCDR2, VLCDR3 or / and VHCDR1, VHCDR2, VHCDR3 located in the monoclonal antibody.

[0023] In a third aspect, a human-mouse chimeric antibody against EsxB comprises VLCDR1, VLCDR2, VLCDR3 or / and VHCDR1, VHCDR2 and VHCDR3 located in the monoclonal antibody.

[0024] In a fourth aspect, a humanized antibody against EsxB comprises VLCDR1, VLCDR2, VLCDR3 or / and VHCDR1, VHCDR2, VHCDR3 located in the monoclonal antibody.

[0025] In a fifth aspect, a therapeutic agent targeting EsxB comprises the monoclonal antibody, single-chain antibody, human-mouse chimeric antibody or humanized antibody.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The monoclonal antibody provided by the present invention has a high affinity for EsxB and can specifically recognize EsxB through the light chain and heavy chain variable regions.

[0028] 2. The light chain and heavy chain variable region genes and amino acid sequences of the monoclonal antibodies provided by the present invention are unique.

[0029] 3. The monoclonal antibodies provided by the present invention have high neutralizing activity, can mediate complement-dependent cytotoxicity, and achieve specific lysis of Staphylococcus aureus, and are expected to provide new strategies and means for the treatment of Staphylococcus aureus infectious diseases.

[0030] 4. The monoclonal antibody provided by the present invention has high protective power. In the in vivo antibacterial activity experiment, when the dose reaches 8 mg / kg, it can produce a 100% protection rate in the mouse model of the lethal Staphylococcus aureus challenge experiment. It is of great significance for the effective treatment and improvement of prognosis of Staphylococcus aureus infectious diseases, especially Staphylococcus aureus infectious diseases with multiple antibiotic resistance.

[0031] In summary, the present invention provides a monoclonal antibody against EsxB with high specificity, high neutralizing activity, and high protective power, which has the characteristics of mediating complement-dependent cytotoxic effects and excellent in vivo antibacterial activity. It can provide support for the construction of anti-EsxB chimeric or humanized genetically engineered antibodies with neutralizing activity, and is of great significance for the effective treatment and improvement of prognosis of Staphylococcus aureus infectious diseases, especially Staphylococcus aureus infectious diseases with multiple antibiotic resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the SDS-PAGE result of EsxB recombinant protein.

[0033] Figure 2 This is the SDS-PAGE result of FL-EsxB No.1 mAb.

[0034] Figure 3 This is the result of gene homology sequence detection of the light chain variable region of FL-EsxB No.1 mAb.

[0035] Figure 4 This is the result of homology sequence detection of the heavy chain variable region gene of FL-EsxB No.1mAb.

[0036] Figure 5 This is the result of amino acid sequence homology detection of the light chain variable region of FL-EsxB No.1 mAb.

[0037] Figure 6 This is the result of amino acid sequence homology detection of the heavy chain variable region of FL-EsxB No.1 mAb.

[0038] Figure 7 This is the result of in vitro bacteriolysis experiment mediated by FL-EsxB No.1mAb.

[0039] Figure 8 These are the results of the in vivo antibacterial activity experiment of FL-EsxB No.1 mAb.

[0040] In the accompanying drawings, 1. molecular weight standard (Marker), 2. bovine serum albumin (BSA), 3. mouse ascites, 4. permeate, 5. purified mAb. DETAILED DESCRIPTION

[0041] The following is combined with Figure 1 To the attached Figure 8 The present invention is described in further detail.

[0042] In the early stage, the extracellular secretory protein B (essextracellular B, EsxB) secreted by the ESAT-6 secretion system of Staphylococcus aureus was selected as the target protein of the vaccine to prepare a Staphylococcus aureus vaccine. The vaccine produced high levels of neutralizing antibodies in mice, and the antibody had a good effect in the complement-mediated lysis test, with a lysis rate of up to 91.6%, providing new strategies and means for the treatment of Staphylococcus aureus infection.

[0043] Based on this antibody, the present invention provides a monoclonal antibody that specifically binds to EsxB and a therapeutic agent thereof. The light and heavy chain variable regions (including their amino acid and nucleotide sequences) of this monoclonal antibody can support the construction of high-affinity anti-EsxB chimeric or humanized antibodies. This EsxB-specific antibody exhibits complement-mediated bacteriolytic activity in vitro and is effective in treating Staphylococcus aureus infections in vivo.

[0044] In a first aspect, a monoclonal antibody that specifically binds to EsxB, comprising 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, and 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;

[0045] 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.

[0046] Furthermore, the amino acid sequences of the three complementarity determining regions VLCDR1, VLCDR2 and VLCDR3 of the light chain variable region (VL) are:

[0047] VLCDR1:Ser-Ala-Arg-Ser-Ser-Ile-Ser-Tyr-Ile-His;

[0048] VLCDR2:Asp-Ala-Ser-Lys-Leu-Ala-Ser;

[0049] VLCDR3: Phe-Gln-Gly-Ser-Gly-Tyr-Pro-Leu-Thr.

[0050] Furthermore, the amino acid sequences of the three complementarity determining regions VHCDR1, VHCDR2 and VHCDR3 of the heavy chain variable region (VH) are:

[0051] VHCDR1: Gly-Tyr-Tyr-Ile-His;

[0052] VHCDR2:

[0053] Arg-Phe-Asn-Pro-Tyr-Asn-Gly-Ala-Ser-Asp-Tyr-Asn-Gln-Asn-Phe-Arg-Asp-Lys-Ala-Ser;

[0054] VHCDR3: Thr-His-Thr-Ser-Gly-Tyr-Val-Trp-Ala-Met-Asp-Tyr.

[0055] In a second aspect, a single-chain antibody against EsxB comprises VLCDR1, VLCDR2, VLCDR3 or / and VHCDR1, VHCDR2, VHCDR3 located in the monoclonal antibody.

[0056] In a third aspect, a human-mouse chimeric antibody against EsxB comprises VLCDR1, VLCDR2, VLCDR3 or / and VHCDR1, VHCDR2 and VHCDR3 located in the monoclonal antibody.

[0057] In a fourth aspect, a humanized antibody against EsxB comprises VLCDR1, VLCDR2, VLCDR3 or / and VHCDR1, VHCDR2, VHCDR3 located in the monoclonal antibody.

[0058] In a fifth aspect, a therapeutic agent targeting EsxB can be used to treat Staphylococcus aureus infection, comprising the monoclonal antibody, single-chain antibody, human-mouse chimeric antibody or humanized antibody.

[0059] Example 1

[0060] The mAb prepared in Example 1 was named FL-EsxB No. 1. The three complementarity determining regions of its light chain variable region (VL) had an amino acid sequence that was 100% identical to SEQ.ID.NO.3. The specific amino acid sequences were:

[0061] VLCDR1:Ser-Ala-Arg-Ser-Ser-Ile-Ser-Tyr-Ile-His;

[0062] VLCDR2:Asp-Ala-Ser-Lys-Leu-Ala-Ser;

[0063] VLCDR3: Phe-Gln-Gly-Ser-Gly-Tyr-Pro-Leu-Thr.

[0064] The three complementary determining regions of the heavy chain variable region (VH) of FL-EsxB No. 1 mAb prepared in Example 1 have 100% identical amino acid sequences to SEQ.ID.NO.4. The specific amino acid sequences are:

[0065] VHCDR1: Gly-Tyr-Tyr-Ile-His;

[0066] VHCDR2:

[0067] Arg-Phe-Asn-Pro-Tyr-Asn-Gly-Ala-Ser-Asp-Tyr-Asn-Gln-Asn-Phe-Arg-Asp-Lys-Ala-Ser;

[0068] VHCDR3: Thr-His-Thr-Ser-Gly-Tyr-Val-Trp-Ala-Met-Asp-Tyr.

[0069] The gene sequence encoding the light chain variable region (VL) of the anti-EsxB FL-EsxB-No.1 mAb 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.

[0070] Unless otherwise specified, the experimental methods used in the following experimental procedures are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following experimental procedures can be obtained from commercial channels.

[0071] 1. Expression and purification of Staphylococcus aureus extracellular secretory protein B (EsxB)

[0072] Place the EsxB prokaryotic expression plasmid containing a 6×His tag on ice and add 100 μL of deionized water to resuspend the plasmid. Pipette 1 μL of the plasmid solution and carefully add it to the BL21 (DE3) competent cells. After gently flicking to mix, let it stand on ice for 5 minutes. Then flick again to mix, and repeat 6 times. Then transfer it to a 42°C constant temperature water bath and let it stand for 90 seconds. Then place the competent cells on ice for 5 minutes. Add 900 μL of LB liquid culture medium to the competent cells in the bacterial operation table, and then transfer it to a 37°C constant temperature oscillator and incubate at 180 rpm for 45 minutes. After the incubation is completed, pipette 100 μL of liquid and evenly spread it on the LB solid culture medium. After the bacterial solution is completely absorbed, place it in a 37°C bacterial incubator and incubate overnight. Single colonies with plump, rounded surfaces were selected and transferred to LB liquid medium containing Kana. The cells were incubated at 180 rpm in a 37°C shaker to allow bacterial growth and monitor the absorbance (OD) of the culture. When the OD reached 0.5–0.6, IPTG was added to a final concentration of 0.1 mmol / L. The shaker temperature was adjusted to 20°C, and the cells were incubated for 23 h to induce protein expression. After induction, the cells were harvested by centrifugation (4200 rpm for 10 min at 4°C), resuspended in deionized water, and the pellet was disrupted by sonication in an ice-water bath (amplitude 6, 15% power, 180 min, ultrasound on for 1 s, off for 2 s). The supernatant was collected by centrifugation (18,000 × g for 10 min at 4°C). Use deionized water to wet the pre-packed column, and then add 5mL of nickel column filler. After the liquid in the nickel column flows out naturally, add about 30mL of deionized water to clean the nickel column. Then add 40mL of equilibrium buffer to equilibrate the nickel column. After the equilibrium is completed, mix the supernatant of the bacterial lysate with the nickel column filler, and use HularMixer to vertically mix the mixture for 2h in a 4℃ constant temperature incubator. After the mixing is completed, the mixture is loaded back into the pre-packed column. After the liquid in the mixture flows out naturally, elute the nickel column with 40mL of phosphate buffer saline (PBS), 5mmol / L elution buffer, 100mmol / L elution buffer and 500mmol / L elution buffer in sequence, and collect the eluate. The collected eluate was dialyzed overnight with dialysate at 4℃ and washed with EtEraser TM HP endotoxin removal kit was used to remove endotoxins from the recombinant protein solution; SDS-PAGE was used to determine the molecular weight and purity of the recombinant protein. Figure 1 As shown, the recombinantly expressed EsxB component was 11KD and had a purity of 90%, which met the requirements of the next experiment; the concentration of the recombinant protein was determined by the BCA method.

[0073] 2. Preparation and Purification of Mouse Anti-EsxB High-Affinity mAb

[0074] 2.1 Immunization regimen

[0075] For the first immunization, 100 μg of EsxB recombinant 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 at 30,000 rpm for 15 minutes to form a stable oil-in-water structure. The 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 with 100 μg of EsxB recombinant protein dissolved in 2.5 ml of PBS and mixed with an equal volume of Freund's incomplete adjuvant. The treatment and injection procedures were the same as before. Three weeks later, a third immunization was performed with 100 μg of EsxB recombinant protein dissolved in 2.5 ml of PBS and injected intraperitoneally at a dose of 0.5 ml per mouse. Ten days after the third immunization, the titer of immune sera was determined by indirect ELISA.

[0076] 2.2 Immune serum titer determination

[0077] Dilute EsxB recombinant protein to 5 μg / ml in coating buffer, add 100 μl per well of the ELISA plate, and refrigerate at 4°C overnight. After washing with 0.05% Tween 20-PBS (PBST), add immune mouse serum serially diluted in 0.1% BSA-PBS and incubate in a 37°C water bath for 1 hour. After washing with PBST, add a working concentration of HRP-labeled goat anti-mouse Ig (H+L) and incubate in a 37°C water bath for 1 hour. After washing with PBST, add ABTS colorimetric solution and develop at room temperature for 30 minutes. OD410 values ​​are then measured for determination. Cell fusion can be performed when the serum titer reaches 1:64,000.

[0078] 2.3 Cell fusion

[0079] Three days before cell fusion, 10 μg of EsxB recombinant protein, resuspended in 1 ml of PBS, was injected intraperitoneally into mice for booster immunization. One day before fusion, peritoneal macrophages from female BALB / c mice were sterilely rinsed, washed once with incomplete RPMI 1640, and resuspended in 40 ml of RPMI 1640 supplemented with 20% fetal calf serum. 100 μl / well of the solution was plated into a 96-well cell culture plate and incubated at 37°C in a 5% CO2, saturated relative humidity incubator until ready to use.

[0080] SP2 / 0 cells in the logarithmic growth phase were collected by centrifugation, with a total number of 5 to 6 × 10 7Discard the supernatant and wash once with incomplete RPMI 1640. Take logarithmically growing Sp2 / 0 myeloma cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in incomplete culture medium, count the cells, take the desired number of cells, and wash twice with incomplete culture medium. Simultaneously, prepare a suspension of immune spleen cells and wash twice with incomplete culture medium. Mix myeloma cells and spleen cells at a ratio of 1:10 or 1:5, wash twice with incomplete culture medium in a 50ml plastic centrifuge tube, centrifuge at 1200 rpm for 8 minutes, discard the supernatant, aspirate any remaining liquid with a pipette, and gently tap the bottom of the centrifuge tube to loosen the cell pellet. Allow to fusate at room temperature. Add 1ml of 45% PEG preheated at 37°C over 30 seconds with stirring. After 90 seconds, add incomplete culture medium preheated at 37°C to terminate the PEG treatment. Continue adding 1ml, 2ml, 3ml, 4ml, 5ml, and 10ml of the incomplete culture medium every 2 minutes. Centrifuge at 800 rpm for 6 min, discard the supernatant, gently resuspend with 40 ml of 20% calf serum RPMI 1640, add the fused cell suspension to a 96-well plate containing feeder cells, 100 μl / well, and culture in a 37°C, 5% CO2, and relative saturated humidity incubator.

[0081] 24 hours after fusion, add 50 μl of 5×HAT selection medium to each well. After 3 days, change the medium once and use 1×HAT / HT selection medium. After 3 days, change the medium again and use 1×HT medium. After about 2 to 3 days, collect the supernatant in the well and detect positive clones by indirect ELISA.

[0082] EsxB-positive clones were selected for cloning. Spleens were aseptically removed from BALB / c mice, and a splenocyte suspension was prepared. The cells were resuspended in 80 ml of RPMI 1640 supplemented with 20% calf serum and 2×HT, and 100 μl / well was plated onto a 96-well cell culture plate. The plate was then incubated at 37°C, 5% CO2, and a relative humidity of 50°C until ready for use. Hybridoma cells were adjusted to a density of 10 cells / ml and 100 μl / well was plated onto a 96-well cell culture plate containing the splenocyte suspension. The plate was cultured for 6-7 days in a 37°C, 5% CO2, and a relative humidity of 50°C. After colonies emerged, they were retested and cloned repeatedly until 100% positive results were obtained twice in a row. A hybridoma cell line secreting a highly specific mAb for EsxB was obtained, designated FL-EsxB No. 1. The culture supernatant was collected and ascites fluid was prepared.

[0083] 2.4 Determination of subclasses of FL-EsxB No.1

[0084] Coat goat anti-mouse Ig (H+L) on an ELISA plate, coating six wells per antibody strain, and incubate overnight at 4°C. Wash three times with 0.15M PBS-Tween 20. Add hybridoma cell culture supernatant to each of the six wells and incubate at 37°C for 1 hour. Wash three times with 0.15M PBS-Tween 20, then add HRP-labeled rat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA, or IgM antibodies. Incubate at 37°C for 1 hour. Wash three times with 0.15M PBS-Tween 20, then add ABTS colorimetric solution and develop at room temperature for 30 minutes. Measure OD410 values ​​and determine the results (IgG1 subclass, κ light chain).

[0085] Purification of FL-EsxB No.1 antibody using 2.5Q Sepharose Fast Flow column chromatography

[0086] Centrifuge the ascites at 4°C, 3000rpm, for 5 minutes. Add saturated ammonium sulfate solution while stirring in an ice bath within 30 minutes to make the final concentration of ammonium sulfate 40%. Let it stand at 4°C for 1 hour. Centrifuge at 4°C, 12000rpm for 10 minutes and discard the supernatant. Resuspend the precipitate with an appropriate amount of 45% saturated ammonium sulfate, centrifuge at 4°C, 12000rpm for 10 minutes, discard the supernatant, and repeat the above steps once. Dissolve the precipitate with 0.02M Tris-HCl buffer, remove the salt by gel filtration, and then load the column according to the instructions provided by the Q Sepharose Fast Flow manufacturer. Load the sample at a flow rate of 5ml / min. After loading the sample, wash away the non-bound proteins with buffer solution starting from 2 column volumes, and elute with increasing concentrations of 1M NaCl as the eluent at a flow rate of 5ml / min. Collect the target protein according to the changes in the protein curve. Take a small amount of sample and dilute it appropriately. Detect the protein content by UV spectrophotometer and identify the purity of the antibody by SDS-PAGE. Figure 2 As shown, the antibody purity is 90%, which meets the requirements of the next experiment.

[0087] 3. Sequencing of the light and heavy chain variable region genes of FL-EsxB No.1 mAb

[0088] 3.1 FL-EsxB No.1 mAb hybridoma cell culture and total RNA extraction

[0089] Hybridoma cells secreting FL-EsxB No. 1 mAb were revived according to routine laboratory procedures and cultured in RPMI 1640 medium supplemented with 20% calf serum in an incubator at 37°C in 5% CO2 until the logarithmic growth phase. Total RNA was extracted using TRIZOL Reagent according to the manufacturer's instructions.

[0090] 3.2 The sequencing of the light chain and heavy chain variable region genes and the determination of the CDR regions of FL-EsxB No.1 mAb were commissioned to Beijing Anbiqi Biotechnology Co., Ltd.

[0091] 4. Nucleotide sequence homology analysis of the light and heavy chain variable regions of FL-EsxB No.1 mAb

[0092] 4.1 Nucleotide sequence homology analysis of the light chain variable region of FL-EsxB No.1 mAb

[0093] Nucleotide sequence homology analysis (Blastn) was performed in the GenBank database. The FL-EsxB No.1 mAb light chain variable region gene sequence had the highest homology with the light chain variable region gene of Sequence ID: MF467040.1, clone number 376C7L, at 304 / 317 (96%). Figure 3 Although the nucleotide sequence encoding the light chain variable region of FL-EsxB No. 1 mAb has certain homology with the light chain variable region sequences of other antibodies, no light chain variable region gene sequence identical to that of the present invention was found, indicating that the nucleotide sequence of the light chain variable region of FL-EsxB No. 1 mAb disclosed in the present invention is unique in terms of gene sequence.

[0094] 4.2 Nucleotide Sequence and Homology Analysis of the Heavy Chain Variable Region of FL-EsxB No.1 mAb

[0095] Nucleotide sequence homology analysis (Blastn) was performed in the GenBank database. The FL-EsxB No.1 mAb heavy chain variable region gene sequence had the highest homology with the heavy chain variable region gene of clone number 2-2-B of Sequence ID: AY639151.1, with a homology of 279 / 294 (95%). Figure 4 Although the nucleotide sequence encoding the heavy chain variable region of FL-EsxB No. 1 mAb has certain homology with the heavy chain variable region sequences of other antibodies, no heavy chain variable region gene sequence identical to that of the present invention was found, indicating that the nucleotide sequence of the heavy chain variable region of FL-EsxB No. 1 mAb disclosed in the present invention is unique in terms of gene sequence.

[0096] 5. Amino Acid Sequence Homology Analysis of the Light and Heavy Chain Variable Regions of FL-EsxB No.1 mAb

[0097] 5.1Amino acid sequence homology analysis of the light chain variable region of FL-EsxB No.1 mAb

[0098] The nucleotide sequence of the light chain variable region of FL-EsxB 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-EsxB No. 1 mAb is shown in SEQ ID NO. 3. Amino acid sequence homology analysis (Blastp) was performed against the Genbank protein database. The search results showed that the amino acid sequence of the light chain variable region of FL-EsxB No. 1 mAb shared the highest homology with the amino acid sequence of the mouse light chain variable region protein with Sequence ID: ATI98468.1, at 100 / 106 (94%). Figure 5 Homology analysis showed that although the amino acid sequence of the light chain variable region of FL-EsxB No. 1 mAb shared some homology with the amino acid sequences of light chains of other mouse-derived antibodies, no identical light chain amino acid sequence was found with the antibody of the present invention, indicating that the light chain of FL-EsxB No. 1 mAb disclosed in the present invention is also unique in amino acid sequence.

[0099] 5.2 Amino Acid Sequence Homology Analysis of the Heavy Chain Variable Region of FL-EsxB No.1 mAb

[0100] The nucleotide sequence of the heavy chain variable region of FL-EsxB No. 1 mAb was translated into an amino acid sequence, and amino acid sequence homology analysis was performed. The amino acid sequence of the heavy chain variable region of FL-EsxB No. 1 mAb is shown in SEQ ID NO. 4. Amino acid sequence homology analysis (Blastp) was performed against the Genbank protein database. The search results showed that the amino acid sequence of the heavy chain of FL-EsxB No. 1 mAb had the highest homology with the amino acid sequence of the mouse heavy chain variable region protein with Sequence ID: ABK59912.1, with a homology of 100 / 121 (83%). Figure 6 Homology analysis showed that although the amino acid sequence of the variable region of the heavy chain of FL-EsxB No. 1 mAb showed some homology with the amino acid sequences of heavy chains of other mouse-derived antibodies, no identical heavy chain amino acid sequence was found with the antibody of the present invention, indicating that the heavy chain of FL-EsxB No. 1 mAb disclosed in the present invention is also unique in amino acid sequence.

[0101] 6. In vitro bacteriolytic activity of FL-EsxB No.1 mAb

[0102] Complement-dependent cytotoxicity (CDC) refers to the cytotoxic effect of complement. Specifically, specific antibodies bind to pathogens or corresponding antigens on the cell membrane, forming antigen-antibody complexes. Activating C1q, which in turn activates the classical complement pathway, results in a membrane-attacking complex that lyses the corresponding pathogen or target cell. This is the primary mechanism by which antibodies kill Staphylococcus aureus.

[0103] Staphylococcal protein A (SpA), located on the cell wall of Staphylococcus aureus, binds nonspecifically to the Fc region of IgG in the serum of various mammals, a key mechanism of immune escape. When designing complement-dependent lysis experiments mediated by the FL-EsxB No.1 mAb, it is necessary to block the SpA binding site with a nonspecific antibody. Because SpA has a higher affinity for rabbit IgG than for mouse IgG1, blocking the SpA binding site with rabbit serum is ideal.

[0104] MRSA strain USA300 was streaked onto LB agar plates, and single clones were picked for amplification. When the bacteria amplified to an OD600 of 0.5, the bacteria were collected and diluted 1:10 with normal saline. 0.2 ml was added to each test tube. At the same time, 0.2 ml of inactivated rabbit serum (56°C, 30 min) diluted 1:10 with normal saline was added to block the SpA binding site.

[0105] 6.1 Experimental Grouping:

[0106] (1) Experimental group 1: 32 μg of FL-EsxB No. 1 mAb, 0.2 ml of USA300, 0.2 ml of 1:10 diluted inactivated rabbit serum, 0.2 ml of 1:10 diluted fresh guinea pig serum (complement), and 0.2 ml of 1:10 diluted fresh guinea pig serum (complement). The total volume was made up to 2 ml with normal saline. The final concentration of FL-EsxB No. 1 mAb was 16 μg / ml.

[0107] (2) Experimental group 2: 16 μg of FL-EsxB No. 1 mAb, 0.2 ml of USA300, 0.2 ml of 1:10 diluted inactivated rabbit serum, 0.2 ml of 1:10 diluted fresh guinea pig serum (complement), and 0.2 ml of 1:10 diluted fresh guinea pig serum (complement). The total volume was made up to 2 ml with normal saline. The final concentration of FL-EsxB No. 1 mAb was 8 μg / ml.

[0108] (3) Experimental group 3: 8 μg of FL-EsxB No. 1 mAb, 0.2 ml of USA300, 0.2 ml of 1:10 diluted inactivated rabbit serum, 0.2 ml of 1:10 diluted fresh guinea pig serum (complement), and 0.2 ml of 1:10 diluted fresh guinea pig serum (complement). The total volume was made up to 2 ml with normal saline. The final concentration of FL-EsxB No. 1 mAb was 4 μg / ml.

[0109] (4) Experimental group 4: 4 μg of FL-EsxB No.1 mAb, 0.2 ml of USA300, 0.2 ml of 1:10 diluted inactivated rabbit serum, 0.2 ml of 1:10 diluted fresh guinea pig serum (complement), and 0.2 ml of 1:10 diluted fresh guinea pig serum (complement). The total volume was made up to 2 ml with normal saline. The final concentration of FL-EsxB No.1 mAb was 16 μg / ml.

[0110] (5) Control group 1: 32 μg of irrelevant antibody (mouse anti-human CD147 antibody), 0.2 ml of USA300, 0.2 ml of inactivated rabbit serum diluted 1:10, 0.2 ml of fresh guinea pig serum (complement) diluted 1:10, and the total volume was made up to 2 ml with normal saline;

[0111] (6) Control group 2: 32 μg of FL-EsxB No. 1 mAb, 0.2 ml of USA300, 0.2 ml of inactivated rabbit serum diluted 1:10, and the total volume was made up to 2 ml with normal saline;

[0112] (7) Control group 3: 0.2 ml of USA300, 0.2 ml of inactivated rabbit serum diluted 1:10, 0.2 ml of fresh guinea pig serum (complement) diluted 1:10, and the total volume was made up to 2 ml with normal saline;

[0113] (8) Control group 4: 0.2 ml of USA300, 0.2 ml of inactivated rabbit serum diluted 1:10, and the total volume was made up to 2 ml with normal saline.

[0114] 6.2 Experimental steps:

[0115] Place the labeled test tubes on an ice bath, with 6 tubes in each 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 shake once every 5 to 10 minutes. After the incubation, perform a gradient dilution of the reaction solution, take 100 μl and spread it on an LB agar plate, and incubate in a 37°C incubator for 16 hours. Count the number of bacterial colonies, calculate the lysis rate, and draw a bar graph.

[0116] 6.3 Experimental Results

[0117] No obvious lysis occurred in the three control groups. In the four experimental groups, the lysis rate increased with the increase of FL-EsxBNo.1mAb concentration. Figure 7As shown, when the concentrations of FL-EsxB No. 1 mAb were 2 μg / ml, 4 μg / ml, 8 μg / ml, and 16 μg / ml, the lysis rates were 12.6%, 32.3%, 56.9%, and 93.1%, respectively. The data were analyzed using GraphPad Prism (v.8.0.2) software to compare the significant differences between the groups. The results of this example demonstrate that FL-EsxB No. 1 mAb has the ability to mediate complement-dependent cytotoxicity and is expected to provide new strategies and approaches for the treatment of MRSA.

[0118] 7. In vivo antibacterial activity of FL-EsxB No.1 mAb

[0119] 7.1 Determination of the Absolute Lethal Dose in Mice

[0120] Staphylococcus aureus USA300 stored at -80°C was streaked onto an LB agar plate and incubated in a 37°C incubator for 16 h. A single colony was picked, 5 mL of LB liquid medium was added, and the plate was transferred to a constant temperature oscillator at 180 rpm at 37°C for bacterial amplification. When the OD600 value of the bacterial solution increased to approximately 0.4-0.5, the plate was expanded to a 200 ml system. When the OD600 value of the bacterial solution increased to approximately 0.4-0.5, the bacterial solution was graded diluted and evenly spread on LB solid medium. The plate was incubated in a 37°C bacterial incubator overnight. The number of colonies on each LB solid medium was counted the next day, and the colony-forming units (CFU) were calculated.

[0121] Different concentrations of bacterial solution were injected into blank mice via the tail vein in a volume of 100 μL. The physical condition and survival rate of mice in each group were continuously observed for 14 days. The bacterial dose that caused all 10 mice to die within 14 days was taken as the absolute lethal dose (LD). 100 LD determined in this experiment 100 2.56×10 8 CFU / piece.

[0122] 7.2 Protective effect of FL-EsxB No.1 mAb in lethal challenge experiment

[0123] Different doses of FL-EsxB No.1 mAb were injected intraperitoneally, and 24 h later, the LD was determined to be 7.1. 100 A lethal challenge test model was established in mice, with 12 mice in each group, to investigate the in vivo antibacterial activity of FL-EsxB No.1 mAb.

[0124] (1) Experimental groups:

[0125] A. FL-EsxB No. 1 mAb dose 8 mg / kg;

[0126] B. FL-EsxB No. 1 mAb dose 4 mg / kg;

[0127] C. FL-EsxB No. 1 mAb dose 2 mg / kg;

[0128] D. irrelevant antibody control group (mouse anti-human CD147 antibody) 8 mg / kg;

[0129] E. Blank control group, intraperitoneal injection of 0.5 ml PBS.

[0130] (2) Experimental results

[0131] To investigate the protective effect of FL-EsxB No.1mAb in the lethal challenge experiment, different doses of FL-EsxB No.1mAb were injected intraperitoneally. After 24 hours, the LD 100 A lethal challenge test model was established in mice, with 12 mice per group, to investigate the in vivo antibacterial activity of FL-EsxB No.1 mAb. GraphPad Prism (v.8.0.2) software was used for statistical analysis. Survival Analyses was used to compare significant differences between the two groups. 100 Dose (2.56×10 8 After the inoculation of Staphylococcus aureus USA300 (CFU), the survival of mice in each group was observed for 14 days. Figure 8 As shown, all mice in the irrelevant antibody control group (mouse anti-human CD147 antibody, 8 mg / kg) and the blank control group (PBS) died within 3 days after the challenge, indicating that the irrelevant antibody did not provide effective protection for the mice. The survival rate of mice in the 2 mg / kg FL-EsxB No.1mAb group was 30%, which was significantly better than the irrelevant antibody group and the PBS control group (p<0.01). The survival rate of mice in the 4 mg / kg FL-EsxB No.1mAb group was 70%, which was significantly better than the 2 mg / kg FL-EsxB No.1mAb group, the irrelevant antibody group, and the PBS control group (p<0.05). When the 8 mg / kg FL-EsxB No.1mAb dose was used, it provided sufficient protection for mice at the current challenge dose, with a survival rate of 100% in all groups of mice, demonstrating excellent in vivo antibacterial activity and providing new hope for the treatment of clinical MRSA infections.

[0132] 8. Based on the light and heavy chain variable region sequences of FL-EsxB No.1 mAb, the following biological products were designed and constructed

[0133] 8.1 Construction of single-chain antibodies

[0134] 8.1.1 Single-chain antibody molecule design

[0135] Single-chain Fv (scFv) is a recombinant protein formed by linking the light chain variable region (VL) and heavy chain variable region (VH) of an antibody via a linker. In this study, a flexible and stable (Gly4Ser)3 linker was selected for the VL and VH gene sequences of the FL-EsxB No.1 mAb. This linker ensures proper spatial folding of the VL and VH regions, maintaining the antibody's antigen-binding activity.

[0136] 8.1.2 Vector Construction

[0137] The designed VL-linker-VH or VH-linker-VL gene sequence is inserted into the pCDNA3.1 eukaryotic expression vector. This vector contains the promoter (CMV promoter), terminator, selection marker (neomycin resistance gene) and other elements required for efficient expression in eukaryotic cells.

[0138] When constructing the vector, the VL and VH gene fragments were first amplified from a plasmid containing the light and heavy chain variable region genes of the FL-EsxBNo.1 mAb using PCR technology. The VL (or VH) and linker genes, and the linker gene and VH (or VL) were then sequentially linked using overlap extension PCR (SOE-PCR) to obtain the complete single-chain antibody gene sequence. This sequence was digested with restriction endonucleases and ligated with an expression vector treated with the same enzymes. The resulting sequence was then transformed into competent E. coli DH5α cells. Positive clones were screened for resistance to ampicillin or kanamycin, and the plasmids were extracted and sequenced to ensure the correct ligation sequence.

[0139] 8.1.3 Host System Transformation and Expression

[0140] Transfect the eukaryotic expression vector into human embryonic kidney (HEK293) cells using lipofectamine. Before transfection, culture the cells until the logarithmic growth phase and perform the transfection reagent according to the manufacturer's instructions. After transfection, plate the cells in a culture medium containing a selection marker, such as neomycin (G418), and perform clonal screening to obtain cell lines stably expressing the single-chain antibody.

[0141] 8.1.4 Purification and identification of expression products

[0142] Eukaryotically expressed single-chain antibodies are typically secreted into the cell culture medium, the supernatant collected by centrifugation, and purified using antigen-reverse affinity chromatography. The purified single-chain antibodies are characterized by molecular weight and specificity using methods such as SDS-PAGE electrophoresis and Western blot, and their binding activity to the EsxB antigen is tested using indirect ELISA to ensure that the prepared single-chain antibodies have the correct structure and good antigen-binding ability.

[0143] 8.1.5 Advantages of Single-Chain Antibodies

[0144] (1) 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 (about 25 kDa) have a small molecular weight and can penetrate the biofilm matrix, interfering with bacterial adhesion or toxin activity.

[0145] (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 a small molecular weight and low immunogenicity. Humanization can further reduce immunogenicity and improve drug safety. Alternatively, PEGylation can be used to extend the half-life and reduce the risk of immune reactions with repeated administration.

[0146] (3) Application and multifunctional transformation of single-chain antibodies:

[0147] 1) Diagnostic Applications: Single-chain antibodies can be used to rapidly detect staphylococcal toxins (such as enterotoxins) in clinical samples (e.g., blood, secretions) and can be further developed into portable test strips to reduce testing time.

[0148] 2) Therapeutic applications: Single-chain antibodies can be fused or cross-linked with toxin proteins (such as ricin toxin) to construct immunotoxins that specifically kill Staphylococcus aureus expressing the toxin; or single-chain 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).

[0149] 8.2 Construction of Human-Mouse Chimeric Antibodies

[0150] 8.2.1 Vector Design and Construction

[0151] Human-mouse chimeric antibodies are chimeric gene expression products formed by combining the light and heavy chain variable region genes of mouse 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) were used. These vectors have pre-cloned human antibody light chain (kappa type) or heavy chain constant region (IgG1 subclass) genes and contain regulatory elements suitable for eukaryotic cell expression, such as promoters, enhancers, and terminators.

[0152] The specific steps are as follows: Amplify the VL and VH gene fragments from plasmids containing the light and heavy chain variable region genes of the FL-EsxBNo.1 mAb, respectively. These fragments are then inserted into the AbVEC1.1-IGKC or AbVEC2.0-IGHG1 expression vectors using restriction endonucleases, ligating them to the human antibody constant region genes to form complete chimeric antibody genes. Transform competent E. coli DH5α cells, screen positive clones for resistance to ampicillin or kanamycin, and extract plasmids for sequencing verification to ensure the correct ligation sequence.

[0153] 8.2.2 Eukaryotic Cell Transfection and Screening

[0154] Human embryonic kidney (HEK293) cells were co-transfected with the constructed vectors containing the antibody light and heavy chain chimeric genes using lipofectamine. Prior to transfection, the cells were cultured to the logarithmic growth phase and the transfection reagent instructions were followed. Following transfection, the cells were plated in a culture medium containing neomycin (G418) for clonal screening to obtain cell lines stably expressing the single-chain antibody.

[0155] To improve transfection efficiency and screening results, positive clones can be subcloned using limiting dilution to ensure a single, high-expressing cell line is obtained. The expression level of the chimeric antibody in the cell culture supernatant is detected by ELISA, and high-expressing cell lines are selected for subsequent culture.

[0156] 8.2.3 Expression and purification of chimeric antibodies

[0157] The screened high-expressing cell lines are cultured on a large scale in serum-free medium. Batch, fed-batch, or perfusion methods can be used to increase antibody production. During the culture process, control culture conditions such as temperature, pH, and dissolved oxygen concentration to ensure optimal cell growth and antibody expression.

[0158] By utilizing the fact that chimeric antibodies contain human antibody constant regions, high-purity chimeric antibodies can be obtained through purification using Protein A or Protein G affinity chromatography. The purified antibodies are then structurally identified using methods such as immunoblotting and mass spectrometry to confirm the correct connection between the mouse variable region and the human constant region.

[0159] 8.2.4 Advantages of Chimeric Antibodies

[0160] (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. The immunogenicity of chimeric antibodies is significantly reduced compared to mouse-derived antibodies, significantly improving drug safety. Alternatively, the half-life of chimeric antibodies can be extended through PEGylation, reducing the risk of immune reactions with repeated administration.

[0161] (2) Application and multifunctional transformation of chimeric antibodies:

[0162] 1) Diagnostic applications: Chimeric antibodies can be used to rapidly detect staphylococcal toxins (such as enterotoxins) in clinical samples (e.g., blood, secretions) and can be further developed into portable test strips to reduce testing time;

[0163] 2) Therapeutic applications: Chimeric antibodies can be fused or cross-linked with toxin proteins (such as ricin toxin) to construct immunotoxins, which 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).

[0164] 8.3 Construction of Humanized Antibodies

[0165] 8.3.1 CDR Region Transplantation Design

[0166] The construction of humanized antibodies uses the complementary determining region (CDR) transplantation technology, which transplants the CDR regions (i.e., hypervariable regions, including CDR1, CDR2, and CDR3) in the light and heavy chain variable regions of mouse antibodies into the framework region (FR) of the human antibody variable region. First, a human antibody FR sequence that is highly similar to the human IgG variable region sequence is selected from the database. Then, the mouse 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 the CDR conformation and antigen binding activity need to be analyzed and optimized, and site-directed mutagenesis is performed if necessary to maintain the specificity and affinity of the antibody.

[0167] 8.3.2 Gene Synthesis and Vector Construction

[0168] Based on the designed humanized antibody light and heavy chain variable region gene sequences (including mouse CDR regions and human FR regions), 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, and correctly linked to the antibody constant region genes to form a complete humanized antibody gene. E. coli DH5α competent cells were transformed, and positive clones were screened for ampicillin or kanamycin resistance. Plasmids were extracted and sequenced to ensure the correct ligation sequence.

[0169] 8.3.3 Cell transfection and expression optimization

[0170] Human embryonic kidney (HEK293) cells were co-transfected with the constructed vectors containing the humanized antibody light and heavy chain genes using lipofectamine. Prior to transfection, cells were cultured to the logarithmic growth phase and the transfection reagent instructions were followed. Following transfection, cells were plated in a culture medium containing neomycin (G418) for clonal screening to obtain cell lines stably expressing the single-chain antibody.

[0171] To improve transfection efficiency and screening results, positive clones can be subcloned using limiting dilution to ensure a single, high-expressing cell line is obtained. The expression level of the chimeric antibody in the cell culture supernatant is detected by ELISA, and high-expressing cell lines are selected for subsequent culture.

[0172] 8.3.4 Purification and Identification of Humanized Antibodies

[0173] The screened high-expressing cell lines are cultured on a large scale in serum-free medium. Batch, fed-batch, or perfusion methods can be used to increase antibody production. During the culture process, control culture conditions such as temperature, pH, and dissolved oxygen concentration to ensure optimal cell growth and antibody expression.

[0174] By utilizing the characteristic of chimeric antibodies containing human antibody constant regions, high-purity chimeric antibodies can be obtained through purification using Protein A or Protein G affinity chromatography. The purified antibodies are then structurally characterized by immunoblotting, mass spectrometry, and other methods to confirm the correct connection between the CDR and FR regions and the human constant region.

[0175] 8.3.5 Advantages of Humanized Antibodies

[0176] (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. The immunogenicity of humanized antibodies is significantly reduced compared to mouse-derived antibodies and chimeric antibodies, and drug safety is greatly improved. Humanized antibodies should have lower immunogenicity and better biocompatibility; or the half-life of chimeric antibodies can be extended through PEGylation to reduce the risk of immune reactions with repeated administration.

[0177] (2) Application and multifunctional transformation of chimeric antibodies:

[0178] 1) Diagnostic Applications: Humanized antibodies can be used to rapidly detect staphylococcal toxins (such as enterotoxins) in clinical samples (e.g., blood, secretions) and can be further developed into portable test strips to reduce testing time.

[0179] 2) Therapeutic applications: Humanized antibodies can be fused or cross-linked with toxin proteins (such as ricin toxin) to construct immunotoxins, which 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).

[0180] Nucleotide and amino acid sequence table SEQ.ID.NO.1

[0181]

[0182] SEQ.ID.NO.2

[0183]

[0184] SEQ.ID.NO.3

[0185]

[0186]

[0187] SEQ.ID.NO.4

[0188]

[0189] The present invention immunizes BALB / c mice with recombinant EsxB protein, obtains hybridoma cells through cell fusion and cloning screening, and produces and purifies the FL-EsxB No.1 monoclonal antibody. This monoclonal antibody specifically binds to EsxB through its VL and VH variable regions, mediating complement-dependent cytotoxicity and bacterial lysis. In vivo, an 8 mg / kg dose achieved a 100% protection rate against lethally challenged mice. This antibody exhibits high specificity, high neutralizing activity, and high protective efficacy, demonstrating its significance for the effective treatment and improved prognosis of multi-antibiotic-resistant Staphylococcus aureus infections.

Claims

1. A monoclonal antibody that specifically binds to EsxB, comprising a light chain variable region (VL) and a heavy chain variable region (VH), characterized in that: The light chain variable region (VL) comprises three complementarity determining regions VLCDR1, VLCDR2 and VLCDR3, the heavy chain variable region (VH) comprises three complementarity determining regions VHCDR1, VHCDR2 and VHCDR3, 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; 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.

2. The monoclonal antibody according to claim 1, wherein The amino acid sequences of the three complementarity determining regions VLCDR1, VLCDR2 and VLCDR3 of the light chain variable region (VL) are: VLCDR1:Ser-Ala-Arg-Ser-Ser-Ile-Ser-Tyr-Ile-His; VLCDR2:Asp-Ala-Ser-Lys-Leu-Ala-Ser; VLCDR3: Phe-Gln-Gly-Ser-Gly-Tyr-Pro-Leu-Thr.

3. The monoclonal antibody according to claim 1, wherein The amino acid sequences of the three complementarity determining regions VHCDR1, VHCDR2 and VHCDR3 of the heavy chain variable region (VH) are: VHCDR1: Gly-Tyr-Tyr-Ile-His; VHCDR2: Arg-Phe-Asn-Pro-Tyr-Asn-Gly-Ala-Ser-Asp-Tyr-Asn-Gln-Asn-Phe-Arg-Asp-Lys-Ala-Ser; VHCDR3: Thr-His-Thr-Ser-Gly-Tyr-Val-Trp-Ala-Met-Asp-Tyr.

4. A single-chain antibody against EsxB, characterized in that It comprises VLCDR1, VLCDR2, VLCDR3 or / and VHCDR1, VHCDR2, VHCDR3 located in the monoclonal antibody.

5. A human-mouse chimeric antibody against EsxB, characterized in that: It comprises VLCDR1, VLCDR2, VLCDR3 or / and VHCDR1, VHCDR2 and VHCDR3 located in the monoclonal antibody.

6. A humanized antibody against EsxB, characterized in that It comprises VLCDR1, VLCDR2, VLCDR3 or / and VHCDR1, VHCDR2, VHCDR3 located in the monoclonal antibody.

7. A therapeutic agent targeting EsxB, characterized in that It comprises the monoclonal antibody, single-chain antibody, human-mouse chimeric antibody or humanized antibody.

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