Therapeutic chimeric monoclonal antibody and application thereof

Abstract

The invention discloses a therapeutic chimeric monoclonal antibody and application thereof. The monoclonal antibody M01 is obtained from a mouse of an immune polypeptide vaccine MAP-Loop2, and the MAP-Loop2 polypeptide vaccine is a polypeptide fragment designed on the basis of a neisseria gonorrhoeae MtrE protein Loop2 structure. All sequences of immunized mouse B cells BCR are obtained through single cell sequencing, and a variable region sequence with the highest occurrence frequency is screened and cloned to a human IgG1 antibody constant region for expression and purification and is named as M01. Researches prove that the M01 has preventive and therapeutic effects on neisseria gonorrhoeae infection in a mouse vaginal infection model. According to the invention, the chimeric antibody with the Loop2 structure and exposed on the surface of the neisseria gonorrhoeae MtrE protein is prepared, so that a complement-dependent antibody sterilization reaction is effectively initiated; neisseria gonorrhoeae infection is effectively treated, the problem of drug resistance enhancement caused by antibiotic treatment is avoided, and a new treatment means is provided for gonorrhoeae prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a therapeutic chimeric monoclonal antibody and its applications. Background Technology

[0002] Neisseria gonorrhoeae ( Neisseria gonorrhoeae Neisseria gonorrhoeae is a Gram-negative diplococcus belonging to the genus Neisseria. It typically consists of two connected cell bodies with flattened or slightly concave surfaces, appearing kidney-shaped or coffee-bean-like. Neisseria gonorrhoeae infection can cause a variety of diseases, including symptomatic urogenital diseases, asymptomatic mucosal infections, and rare disseminated Neisseria gonorrhoeae infections.

[0003] With the continued use of antibiotics, Neisseria gonorrhoeae has shown increasingly strong resistance. Analysis of antibiotic resistance data indicates that before the use of modern antibiotics, Neisseria gonorrhoeae did not contain antibiotic resistance elements; it is speculated that the widespread use and even abuse of modern antibiotics has driven the development of resistance in Neisseria gonorrhoeae. Recently, a large number of studies have reported that Neisseria gonorrhoeae has developed resistance to all known and currently recommended antibiotics for treatment (Zhu et al. “Ceftriaxone-Resistant Gonorrhea - China, 2022” Morbidity and Mortality Weekly Report, 2024, 73(12):255-259. doi: 10.15585 / mmwr.mm7312a2; Unemo et al. “WHO global gonococcal antimicrobial surveillance programmes, 2019-22: aretrospective observational study” Lancet Microbe, 2025:101181. doi: 10.1016 / j.lanmic.2025.101181).

[0004] Multiple transferable resistance (MtrCDE) systems expel hydrophobic substances (such as fatty acids, long-chain lipids, antimicrobial peptides, bile salts, and antibiotics) from bacteria. Competitive experiments in infection models showed that knocking out the MtrCDE efflux pump in Neisseria gonorrhoeae led to reduced bacterial adaptability. The FarA-FarB-MtrE efflux pump primarily mediates the efflux of hydrophobic substances, and point mutations in its regulatory elements can induce drug resistance changes in Neisseria gonorrhoeae strains. Multiple transferable resistance protein E (MtrE) is an outer membrane channel protein of the two efflux pump systems. It consists of an outer membrane β-barrel, a large periplasmic spatial domain, and two surface-exposed loop structures. Its surface loop structure, Loop2, is highly conserved. An analysis of 4566 Neisseria gonorrhoeae Loop2 sequences in the Public MLST Database (PubMLST) showed that only 27 Loop2 sequences had amino acid mutations, indicating that the sequence is highly conserved. Moreover, this structure is exposed on the bacterial surface and plays an important role in the survival of Neisseria gonorrhoeae (Wang et al. “Gonococcal MtrE and its surface-expressed Loop 2 are immunogenic and elicit bactericidal antibodies” Journal of Infection, 2018, 77(3):191-204. doi: 10.1016 / j.jinf.2018.06.001).

[0005] In the existing technology, there are no publicly reported applications of monoclonal antibodies targeting the Loop2 epitope of the MtrE protein of Neisseria gonorrhoeae in the treatment of gonorrhea. Summary of the Invention

[0006] To address the aforementioned shortcomings in the prior art, this invention provides a therapeutic chimeric monoclonal antibody and its applications.

[0007] This invention develops a chimeric monoclonal antibody targeting the Loop2 structure on the surface of the Neisseria gonorrhoeae MtrE protein. This monoclonal antibody, M01, was obtained from mice immunized with the MAP-Loop2 peptide vaccine, a peptide fragment designed based on the Loop2 structure of the Neisseria gonorrhoeae MtrE protein. The complete sequence of the BCR (Biocytogenetic Response Code) of immunized mouse B cells was obtained through single-cell sequencing. The most frequently occurring variable region sequence was screened, cloned into the constant region of a human IgG1 antibody, purified, and named M01. Studies have confirmed that M01 has both preventative and therapeutic effects against Neisseria gonorrhoeae infection in a mouse vaginal infection model.

[0008] This invention first provides a therapeutic chimeric monoclonal antibody that targets the Loop2 structure on the surface of the MtrE protein of Neisseria gonorrhoeae. The therapeutic chimeric monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region contains heavy chain complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region contains light chain complementarity-determining regions CDRL1, CDRL2, and CDRL3. Specifically, the CDRH1 sequence is GFSLSTSGM; the CDRH2 sequence is YWDDD; the CDRH3 sequence is CARRARGYAMDYW; the CDRL1 sequence is KSSQSLLNSGNQKNYLA; the CDRL2 sequence is GASTRES; and the CDRL3 sequence is CQNDHSYPYTF.

[0009] Preferably, the heavy chain variable region contains heavy chain constant regions HFR1, HFR2, HFR3, and HFR4, and the light chain variable region contains light chain constant regions LFR1, LFR2, LFR3, and LFR4; wherein, the HFR1 sequence is QVTLKESGPGILQPSQTLSLTCSFS; the HFR2 sequence is GVSWIRQPSGKGLEWLAHI; the HFR3 sequence is KRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYY; the HFR4 sequence is GQGTSVTVSS; the LFR1 sequence is DIVMTQSPSSLSVSAGEKVTMSC; the LFR2 sequence is WYQQKPGQPPKLLIY; the LFR3 sequence is GVPDRFTGSGSGTDFTLTISSVQAEDLAVYY; and the LFR4 sequence is GGGTKLEIK.

[0010] Preferably, the therapeutic chimeric monoclonal antibody comprises a heavy chain and a light chain, the sequence of the heavy chain being as shown in SEQ ID No. 1 and the sequence of the light chain being as shown in SEQ ID No. 2.

[0011] The present invention further provides a gene sequence encoding the therapeutic chimeric monoclonal antibody.

[0012] The present invention further provides the use of the therapeutic chimeric monoclonal antibody in the preparation of a medicament for the prevention or treatment of Neisseria gonorrhoeae infection.

[0013] The present invention further provides the application of the therapeutic chimeric monoclonal antibody in the preparation of a drug for treating Neisseria gonorrhoeae.

[0014] The present invention also provides a medicament for the prevention or treatment of Neisseria gonorrhoeae infection and / or for the treatment of Neisseria gonorrhoeae, wherein the active ingredient is the therapeutic chimeric monoclonal antibody.

[0015] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0016] The binding ability of chimeric monoclonal antibody M01 to the surface protein MtrE of Neisseria gonorrhoeae was detected by ELISA and Western blotting, respectively. The results showed a significant binding between M01 and MtrE, and the signal increased with increasing antibody concentration, indicating the specificity of this interaction. The bactericidal activity of chimeric antibody M01 against the international reference standard strain ATCC 49226 of Neisseria gonorrhoeae was evaluated by in vitro antibody bactericidal assays. The results showed that chimeric monoclonal antibody M01 had a good in vitro bactericidal effect against the standard strain of Neisseria gonorrhoeae. Subsequently, the therapeutic effect of chimeric monoclonal antibody M01 was evaluated using a mouse vaginal infection model. The results showed that chimeric monoclonal antibody M01 has a good therapeutic effect against Neisseria gonorrhoeae infection.

[0017] Compared with existing antibiotic treatment regimens, the present invention has the following beneficial effects: This invention prepares a chimeric antibody that precisely targets the Loop2 structure exposed on the surface of the MtrE protein of Neisseria gonorrhoeae, effectively triggering a complement-dependent antibody bactericidal response; it effectively treats Neisseria gonorrhoeae infection, avoids the problem of increased drug resistance caused by antibiotic treatment, and provides a new treatment method for gonorrhea prevention and control. Attached Figure Description

[0018] Figure 1 The structure of the Loop2 protein from Neisseria gonorrhoeae MtrE and the MAP-Loop2 polypeptide synthesized based on this structure are shown. Figure 1 A in the diagram represents the structure of the MtrE protein Loop2 from Neisseria gonorrhoeae. Figure 1 B in the text represents the MAP-Loop2 polypeptide.

[0019] Figure 2 The binding affinity of the chimeric monoclonal antibody M01 to the MtrE protein of Neisseria gonorrhoeae was assessed using ELISA and Western blotting, and the in vitro bactericidal activity of M01 against the international standard reference strain of Neisseria gonorrhoeae was evaluated. Figure 2 In the image, A represents the ELISA test result. Figure 2 In the image, B represents the result of the Western Blot analysis. Figure 2 In this context, C represents the in vitro bactericidal ability of M01 against the international standard reference strain of Neisseria gonorrhoeae, and mAb bactericidal activity titer represents the antibody bactericidal activity titer.

[0020] Figure 3 To evaluate the therapeutic effect of the chimeric monoclonal antibody M01 in a mouse model of vaginal gonorrhea infection. Among other things, Figure 3 In this context, A represents the change in the number of infected mice over time. Figure 3 B in the figure represents the change in bacterial load in the vagina of infected mice over time. Figure 3 In the figure, C represents the area under the curve (AUC) of the daily CFU count of vaginal bacteria in colonized mice. Detailed Implementation

[0021] Specific embodiments of the invention are described in detail in the following examples. These are illustrative of the invention and not intended to limit its scope. Unless otherwise stated, all reagents are commercially available.

[0022] Example 1 This embodiment discloses a method for preparing a precisely targeted monoclonal antibody. Specifically, it involves the synthesis and immunization process of MAP-Loop2, the preparation of single-cell suspension and B cell screening, B cell immune library sequencing, and the preparation method of chimeric monoclonal antibody, including the following steps: A1: Figure 1 This paper presents the structure of the Loop2 protein from *Neisseria gonorrhoeae* MtrE and the MAP-Loop2 polypeptide synthesized based on this structure. A quadruple Loop2 polypeptide, constructed using a lysine core and synthesized by Genscript Biotech, is named MAP-Loop2. The sequence of each Loop2 polypeptide is: Ser-Val-Glu-Leu-Gly-Gly-Leu-Phe-Lys-Ser-Gly-Thr-Gly (SVELGGLFKSGTG). The four Loop2 peptide segments are linked by C-terminal carboxyl groups (-COOH) to the α-amino (-NH2) and ε-amino (-NH2) side chains of two lysine residues, respectively, forming amide bonds (peptide bonds). These two lysine residues then form amide bonds with the α-amino and ε-amino sides of a third lysine residue through their carboxyl groups, thus constituting a branched lysine core.

[0023] A2: Immunize 4-5 week old SPF-grade female BABL / c mice with MAP-Loop2 (50 µg) mixed with CpG (20 µg) adjuvant, once every two weeks, for a total of 5 immunizations, and then wait for use.

[0024] A3: Mice were euthanized by cervical dislocation. The lower abdomen of the mice was disinfected with alcohol swabs. The abdomen was aseptically opened, and the spleen was removed from the abdominal cavity and placed in a culture dish containing Hanks' medium. Excess tissue was washed and removed, and the spleen was cut into 3 segments. After dissociation, the spleen cells were resuspended in erythrocyte lysis buffer.

[0025] A4: After screening B cells using flow cytometry, they were sent to a biotechnology company for single-cell sequencing to obtain the BCR sequences of all B cells.

[0026] A5: The most frequently occurring antibody variable region sequence was selected, and a chimeric monoclonal antibody synthesized by Genscript Biotech Co., Ltd. and fused with the human IgG1 constant region was named M01.

[0027] The sequence of the M01 antibody is as follows: SEQ ID No.1 (heavy chain sequence): QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYYCARRARGY AMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; SEQ ID No.2 (light chain sequence): DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPYTFG GGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0028] The heavy chain variable region contains heavy chain complementarity-determining regions CDRH1, CDRH2, and CDRH3, where CDRH1 sequence is GFSLSTSGM; CDRH2 sequence is YWDDD; and CDRH3 sequence is CARRARGYAMDYW. The heavy chain variable region contains heavy chain constant regions HFR1, HFR2, HFR3, and HFR4, where HFR1 sequence is QVTLKESGPGILQPSQTLSLTCSFS; HFR2 sequence is GVSWIRQPSGKGLEWLAHI; HFR3 sequence is KRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYY; and HFR4 sequence is GQGTSVTVSS.

[0029] The light chain variable region contains light chain complementarity-determining regions CDRL1, CDRL2, and CDRL3, where CDRL1 sequence is KSSQSLLNSGNQKNYLA; CDRL2 sequence is GASTRES; and CDRL3 sequence is CQNDHSYPYTF. The light chain variable region contains light chain constant regions LFR1, LFR2, LFR3, and LFR4, where LFR1 sequence is DIVMTQSPSSLSVSAGEKVTMSC; LFR2 sequence is WYQQKPGQPPKLLIY; LFR3 sequence is GVPDRFTGSGSGTDFTLTISSVQAEDLAVYY; and LFR4 sequence is GGGTKLEIK.

[0030] Example 2 Recombinant protein MtrE (Gene ID: 66753569) was expressed and purified using an *E. coli* expression system. The binding affinity of chimeric antibody M01 to MtrE protein was detected by ELISA and Western blotting. The bactericidal activity of antibody M01 against the international reference strain ATCC 49226 of *Neisseria gonorrhoeae* was monitored by in vitro bactericidal assays. The ELISA method is as follows: A1: Using purified MtrE protein as antigen, a 96-well Maxisorp microtiter ELISA plate was coated with a concentration of 150 ng / well overnight at 4°C.

[0031] A2: After washing, add 200 µL of blocking solution to each well and seal at 37°C for 1 h.

[0032] A3: After washing, add 50 µL of M01 diluted 2-fold with ELISA dilution buffer to each well as primary antibody, and incubate at 37°C for 1 h.

[0033] A4: After washing, add 50 µL of HRP-labeled goat anti-human IgG diluted 1:5000 with ELISA dilution buffer to each well as a secondary antibody, and incubate at 37°C for 1 h.

[0034] A5: After washing, add 100 µL of TMB substrate to each well, incubate in the dark for 3-5 min, then add 200 µL of ELISA stop solution to terminate the reaction. Measure the absorbance of each well at 450 nm using a microplate reader.

[0035] The Western blot method is as follows: A1: Mix the purified MtrE protein with the loading buffer and heat in boiling water for 15 min to denature the protein. Add the denatured sample to an SDS-PAGE gel and perform electrophoresis at a constant voltage of 120 V until Coomassie Brilliant Blue moves to the bottom of the gel, thus separating the proteins according to their molecular weight.

[0036] A2: The protein was then transferred from the gel to a PVDF membrane at 350 mA for 150 min. Blocking was performed at room temperature for 1 h using blocking buffer to reduce nonspecific binding.

[0037] A3: The chimeric monoclonal antibody M01 at concentrations of 10 µg / mL, 2 µg / mL, 400 ng / mL, and 80 ng / mL, respectively, was used as the primary antibody and incubated with the blocked membrane at room temperature for 1 h. After washing 3-5 times with PBST, the membrane was incubated with HRP-labeled goat anti-human IgG antibody as the secondary antibody for another 1 h at room temperature.

[0038] A4: Finally, the results were analyzed using a Western blot imaging analyzer.

[0039] The SBA method is as follows: A1: Dilute the antibody with SBA diluent, and perform serial 2-fold dilutions.

[0040] A2: Take 1 OD of overnight cultured Neisseria gonorrhoeae and dilute it to 4 × 10⁻⁶ with SBA diluent. 3 CFU, with 1:20 complement from young rabbits, was dispensed into 96-well plates at a ratio of 25 µL per well.

[0041] A3: Take 25 µL of pre-diluted serum and mix it evenly with the bacterial suspension. Set up an antibody-free control group. Incubate at 37°C in a 5% CO2 incubator for 1 h. Then, add 10 µL of the mixed suspension to a GC plate.

[0042] A4: Place the plates in a 37°C, 5% CO2 incubator and incubate for 16-18 h. Count the bacteria under a 10x microscope. The highest serum dilution that can kill more than 50% of the bacteria compared to the control group without serum is defined as the SBA titer of the serum.

[0043] like Figure 2 As shown, the results indicate that the minimum binding concentration of M01 to MtrE is 375 ng / mL. With increasing antibody concentration, the absorbance continuously increases, showing a significant difference compared to the control antibody (human IgG1 backbone antibody against H7N9 influenza virus HA protein, #HG1K) and the PBS control group (Mock group). Western blot results show that M01 can specifically bind to the MtrE protein. With increasing antibody concentration, the immunoblot gray value continuously increases, indicating that the binding of M01 to MtrE is specific. Antibody bactericidal experiments against the international standard strain of Neisseria gonorrhoeae, ATCC 49226, show that the minimum effective bactericidal concentration of the chimeric antibody M01 is 2 µg / mL.

[0044] Example 3 Evaluation of the therapeutic effect of chimeric antibody M01 on a mouse model of vaginal Neisseria gonorrhoeae ATCC 49226 infection.

[0045] A1: Select 6-7 week old SPF female BALC / c mice. After monitoring the estrous cycle, select mice in the interestrus period and administer 0.1 mg of β-estradiol subcutaneously to each mouse 2 days before infection, on the day of infection, and on the second day after infection.

[0046] A2: Neisseria gonorrhoeae ATCC 49226 was prepared to a concentration of 1×10⁻⁶ using a pre-prepared buffer solution containing 0.5 mM CaCl₂, 1 mM MgCl₂, and 1% (w / v) gelatin. 9 The bacterial culture at CFU / mL was inoculated into the mouse vagina twice, in doses of 10 µL each time.

[0047] A3: After sampling on Day 1 of infection, all mice were given either 10 µg / 10 µL of antibody or 10 µL of PBS placebo via vaginal administration daily as controls.

[0048] A4: Swab samples were taken from the vagina of each group of mice daily and inoculated onto culture plates containing selective antibiotics. The viral load of Neisseria gonorrhoeae was determined by counting the number of colonies formed at different dilutions. In this experiment, 4 colony-forming units (CFU) were set as the minimum detection limit to ensure the accuracy and reliability of the results.

[0049] The results are as follows Figure 3 As shown in the figure, the control antibody group and the PBS placebo group began to clear the infection on day 5, and completed clearance on days 9 and 11, respectively. In contrast, the chimeric monoclonal antibody M01 group showed a faster reduction in the number of infected mice, starting clearance on day 4, and by day 5, all Neisseria gonorrhoeae had been cleared from the mice in this group. The comparison of bacterial load in the vagina of infected mice yielded similar results. The bacterial load in the vagina of mice in the control antibody group and the PBS placebo group was consistently higher than that in the M01 group. The area under the curve (AUC) of daily CFU counts of colonized vaginal bacteria in mice also yielded similar results. The AUC of the control antibody group and the PBS placebo group was larger than that of the M01 group. These results indicate that the chimeric monoclonal antibody M01 has a good therapeutic effect on Neisseria gonorrhoeae infection.

Claims

1. A therapeutic chimeric monoclonal antibody, characterized in that, The therapeutic chimeric monoclonal antibody targets the Loop2 structure on the surface of the MtrE protein of Neisseria gonorrhoeae. The therapeutic chimeric monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region contains heavy chain complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region contains light chain complementarity-determining regions CDRL1, CDRL2, and CDRL3. Specifically, the CDRH1 sequence is GFSLSTSGM; the CDRH2 sequence is YWDDD; the CDRH3 sequence is CARRARGYAMDYW; the CDRL1 sequence is KSSQSLLNSGNQKNYLA; the CDRL2 sequence is GASTRES; and the CDRL3 sequence is CQNDHSYPYTF.

2. The therapeutic chimeric monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region contains heavy chain constant regions HFR1, HFR2, HFR3, and HFR4, and the light chain variable region contains light chain constant regions LFR1, LFR2, LFR3, and LFR4; wherein, the HFR1 sequence is QVTLKESGPGILQPSQTLSLTCSFS; the HFR2 sequence is GVSWIRQPSGKGLEWLAHI; the HFR3 sequence is KRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYY; the HFR4 sequence is GQGTSVTVSS; the LFR1 sequence is DIVMTQSPSSLSVSAGEKVTMSC; the LFR2 sequence is WYQQKPGQPPKLLIY; the LFR3 sequence is GVPDRFTGSGSGTDFTLTISSVQAEDLAVYY; and the LFR4 sequence is GGGTKLEIK.

3. The therapeutic chimeric monoclonal antibody according to claim 2, characterized in that, It contains a heavy chain and a light chain, the sequence of which is shown in SEQ ID No. 1 and the sequence of which is shown in SEQ ID No.

2.

4. The gene sequence encoding the therapeutic chimeric monoclonal antibody according to any one of claims 1-3.

5. The use of the therapeutic chimeric monoclonal antibody according to any one of claims 1-3 in the preparation of a medicament for the prevention or treatment of Neisseria gonorrhoeae infection.

6. The use of the therapeutic chimeric monoclonal antibody according to any one of claims 1-3 in the preparation of a medicament against Neisseria gonorrhoeae.

7. A drug for the prevention or treatment of Neisseria gonorrhoeae infection, and / or for the treatment of Neisseria gonorrhoeae infection, characterized in that, The active ingredient is the therapeutic chimeric monoclonal antibody as described in any one of claims 1-3.

8. The medicament according to claim 7, characterized in that, It also includes pharmaceutically acceptable carriers.

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