Monoclonal antibody and application thereof in resisting acinetobacter baumannii infection

Abstract

The invention belongs to the technical field of monoclonal antibodies, and particularly relates to a monoclonal antibody and application thereof in resisting acinetobacter baumannii infection. A high-throughput monoclonal antibody is used for separation through a Beacon platform, C3 monoclone with the antibody having more excellent functions is screened out, and amino acid sequences of a complementarity determining region 1, a complementarity determining region 2 and a complementarity determining region 3 of a heavy chain of the C3 monoclone are respectively GYTITEYTMH, GINPNGGTS and GAYY; the amino acid sequences of a complementarity determining region 1, a complementarity determining region 2 and a complementarity determining region 3 of the light chain are respectively KASQDIKSYLT, YARTLAD and LQHGESPLT. The monoclonal antibody provided by the invention is beneficial to bacterial removal, reduction of release of inflammatory mediators and reduction of host damage, solves the technical problem of lack of the monoclonal antibody for efficiently resisting acinetobacter baumannii infection of targeted outer membrane protein in the prior art, and has ideal popularization and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of monoclonal antibodies, and specifically relates to a monoclonal antibody and an application thereof in resisting Acinetobacter baumannii infection. Background Art

[0002] Acinetobacter baumannii (A. baumannii) is an opportunistic pathogen commonly found in the environment that can cause serious infections in human tissues such as the lungs and blood, especially in immunocompromised intensive care unit (ICU) patients. Unlike typical Gram-negative bacteria, A. baumannii has a strong adhesion ability, promoting colonization at the site of infection and enabling biofilm formation or cell invasion. Studies have reported a mortality rate of 56.2% in infected patients, and new treatment strategies need to be urgently explored.

[0003] Antibiotics are the main treatment for Acinetobacter baumannii infections. However, A. baumannii is resistant to multiple antibiotics, which makes treatment extremely difficult. A. baumannii vaccines are safe, have few adverse reactions, and are not prone to drug resistance. However, due to several factors such as antigen sequence variation, no vaccine has passed Phase I clinical trials. Probiotic therapy and fecal microbiota transplantation (FMT) have shown some potential in the treatment of A. baumannii infections, but their mechanisms are still unclear and require further study. In recent years, monoclonal antibody (mAb) therapy has become a promising method for the treatment of A. baumannii infections. Unlike antibiotics, antibacterial monoclonal antibodies selectively target pathogens without disrupting normal bacterial colonization in the human body, thereby minimizing the risk of dysbiosis and bacterial resistance. In addition, monoclonal antibodies can be used in combination with antibiotics to reduce the use of antibiotics and alleviate related adverse reactions.

[0004] Current monoclonal antibody development technologies face various challenges. For example, hybridoma technology has the problem of reduced diversity of monoclonal antibodies due to competition between different hybridomas. Some low-yielding monoclones may show faster growth rates, surpassing high-secreting clones. In addition, low-affinity monoclonal antibodies are produced due to the random pairing of the heavy chain variable region (VH) and the light chain variable region (VL). These limitations make traditional methods inefficient in antibody development, and the antibodies obtained do not meet application requirements. Summary of the invention

[0005] The present invention is intended to provide a monoclonal antibody to solve the technical problem in the prior art of lacking a highly effective monoclonal antibody targeting outer membrane protein (OMP) against Acinetobacter baumannii infection.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A monoclonal antibody, which is monoclonal antibody C1; the amino acid sequences of the complementary determining region 1, complementary determining region 2 and complementary determining region 3 of its heavy chain are GYTITEYTMH, GINPNNGGTS, GAYY respectively; the amino acid sequences of the complementary determining region 1, complementary determining region 2 and complementary determining region 3 of its light chain are KASQDIKSYLT, YARTLAD, LQHGESPLT respectively.

[0008] Furthermore, the amino acid sequence of the variable region of the heavy chain of monoclonal antibody C1 is shown in SEQ ID NO.1; the amino acid sequence of the variable region of the light chain of monoclonal antibody C1 is shown in SEQ ID NO.5.

[0009] Furthermore, the amino acid sequence of the heavy chain of monoclonal antibody C1 is shown in SEQ ID NO.3; and the amino acid sequence of the light chain of monoclonal antibody C1 is shown in SEQ ID NO.7.

[0010] Furthermore, the monoclonal antibody C1 is used to bind to the outer membrane protein of Acinetobacter baumannii; the outer membrane protein is OMP38.

[0011] The technical solution also provides an application of a monoclonal antibody in the preparation of a drug for resisting Acinetobacter baumannii infection, wherein the monoclonal antibody is used to bind to and inhibit Acinetobacter baumannii; the monoclonal antibody is used to reduce the release of inflammatory mediators and thus reduce host damage.

[0012] The present technical scheme also provides an antibody composition comprising a monoclonal antibody, which also includes monoclonal antibody G4; the amino acid sequences of the complementary determining region 1, complementary determining region 2 and complementary determining region 3 of the heavy chain of monoclonal antibody G4 are GFTFSSYAMS, EISSGGTYTY, EGDGYVWFPY, respectively; the amino acid sequences of the complementary determining region 1, complementary determining region 2 and complementary determining region 3 of the light chain of monoclonal antibody G4 are RASGNIHNYLA, NAKTLAD, QHFLTTPRALT, respectively.

[0013] Furthermore, the dosage ratio of monoclonal antibody G4 to monoclonal antibody C3 is 1:1.

[0014] Furthermore, the amino acid sequence of the variable region of the heavy chain of monoclonal antibody G4 is shown in SEQ ID NO.9, the amino acid sequence of the variable region of the light chain of monoclonal antibody G4 is shown in SEQ ID NO.13; the amino acid sequence of the heavy chain of monoclonal antibody G4 is shown in SEQ ID NO.11; and the amino acid sequence of the light chain of monoclonal antibody G4 is shown in SEQ ID NO.15.

[0015] The technical solution also provides an application of an antibody composition in the preparation of a drug for resisting Acinetobacter baumannii infection.

[0016] Furthermore, the raw materials of the drug include a pharmaceutically acceptable carrier.

[0017] Furthermore, the protection rate of the monoclonal antibody C1 against the host infected with the highly virulent Acinetobacter baumannii LAC-4 strain was 75%. The protection rate of the monoclonal antibody C1 and G4 against the host infected with the highly virulent Acinetobacter baumannii LAC-4 strain was 100%.

[0018] The technical solution also provides an application of a monoclonal antibody in the preparation of a kit for detecting Acinetobacter baumannii.

[0019] In summary, the principle and beneficial effects of this technical solution are:

[0020] Infections caused by Acinetobacter baumannii are a global public health problem due to their high pathogenicity. Unlike antibiotics, monoclonal antibodies (mAbs) show the potential to reduce drug resistance, thereby minimizing adverse reactions. Currently, the therapeutic efficacy of mAbs against A. baumannii is unclear. This technical solution isolates mAbs against A. baumannii outer membrane proteins (OMPs) in a high-throughput manner. The binding ability of Omp38-specific mAbs to various A. baumannii strains was confirmed by enzyme-linked immunosorbent assay (ELISA). Intravenous administration of Omp38-specific mAbs provided significant protection against lethal and sublethal A. baumannii infections. In addition, analysis of the C3 binding conformation of Omp38 mAbs revealed the potential mechanism of the mAb's broad-spectrum binding activity against A. baumannii. Taken together, these findings suggest that mAbs against Omp38 facilitate bacterial clearance from the host, minimize the release of inflammatory mediators, and reduce host damage, highlighting the potential of Omp38-specific mAbs in the clinical treatment of A. baumannii infections.

[0021] More specifically, in the development of this patented technology, we used the Beacon platform to separate high-throughput monoclonal antibodies and obtained monoclonal antibodies targeting OMP of Acinetobacter baumannii. We identified 10 monoclonal antibodies targeting OMP from immunized mice, of which 6 were mAbs targeting Omp38, which can effectively and widely neutralize multiple strains of Acinetobacter baumannii and protect mice from infection. Among them, the C3 monoclonal antibody has the best protective effect against infected animals and is the first choice for further development of drugs to treat Acinetobacter baumannii infections.

[0022] Acinetobacter baumannii contains a series of potential drug targets, including porins, efflux pumps, outer membrane vesicles (OMVs), outer membrane proteins (OMPs), metal acquisition systems, secretion systems, phospholipases, and capsular polysaccharides. This technical solution selects OMPs as targets for the development of monoclonal antibodies. These proteins play a unique role in promoting bacterial adaptation to antibiotics and host-induced stress. Omp38 (also known as OmpA) is a prominent Acinetobacter baumannii OMP that regulates the adhesion, invasion, and biofilm formation of Acinetobacter baumannii and contributes to inflammation and other host responses. Omp38 is highly conserved among different strains of Acinetobacter baumannii. Overproduction of Omp38 is a risk factor for increased hospital pneumonia, bacteremia, and patient mortality. The monoclonal antibodies developed in this technical solution can effectively bind to Omp38 and effectively inhibit Acinetobacter baumannii. In addition, Omp38 is not homologous to any protein encoded by the human genome, which ensures that the administration of antibodies that inhibit it will not cause unnecessary harm to the host.

[0023] In contrast, Hamideh Barati et al. produced immune serum by subcutaneous inoculation of mice with Omp38, demonstrating the safety and efficacy of the antiserum in bacterial killing, biofilm inhibition, bacterial adhesion, and intracellular proliferation inhibition. However, they did not isolate monoclonal antibodies targeting Omp38 from the serum, while our study involved immunizing mice with OMP and identifying monoclonal antibodies targeting Omp38. At present, the mechanism of mAb-mediated protection against Acinetobacter baumannii infection is not fully understood. The monoclonal antibodies developed in this protocol can not only be applied to medical practice, but also be used for further basic research such as mechanism of action. As a basic scientific research tool, we can explore the effects of Omp38-specific monoclonal antibodies on the pathobiology of Acinetobacter baumannii. In addition, the monoclonal antibodies obtained in this protocol can effectively bind to the membrane proteins of Acinetobacter baumannii. Therefore, the monoclonal antibodies can also be used in relevant Acinetobacter baumannii detection kits to detect the target bacteria through the binding reaction of antigen and antibody.

[0024] Further, C3 and G4 were used in combination, and it was found that the combined use could obtain a 100% host protection rate against highly virulent Acinetobacter baumannii infection, which shows that C3 and G4 have a certain synergistic effect and have the potential to further develop antibody combination drugs. Antibody therapy for Acinetobacter baumannii is still in the research and development stage. The above findings have created a foundation for antibody cocktail therapy for Acinetobacter baumannii infection. Antibody cocktail therapy provides new ideas for the treatment of complex Acinetobacter baumannii infections, especially when facing bacteria that are prone to drug resistance. It shows its unique advantages. By combining the effects of multiple antibodies, better therapeutic effects can be achieved, and compared with single drugs, rationally designed antibody combinations may have lower toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The SDS-PAGE electrophoresis diagram of OMP isolated from Acinetobacter baumannii in Example 1, the mouse immunization procedure, and the polyclonal IgG antibody titer after mouse immunization.

[0026] Figure 2 The high-throughput analysis principle of Example 2 and a microscopic image of cells mounted on the device chip.

[0027] Figure 3 The high-throughput analysis results and PCR identification results of Example 2 are shown.

[0028] Figure 4 The results of identification of the monoclonal antibody variable regions of Example 2 (F4, A2, B4, C3, G1, G4, H4, G3 and E6).

[0029] Figure 5 These are the results of identification of the monoclonal antibody variable regions of Example 2 (A6, B1, D4, D6, F1, F3, H1, H3, D1, G5, E1, E4, E5, G6, H2).

[0030] Figure 6 The SDS electrophoresis results of some monoclonal antibodies in Example 2 are shown.

[0031] Figure 7 The results are the results of the study on the binding activity of the monoclonal antibody of Example 3 against recombinant Omp38 and highly virulent Acinetobacter baumannii LAC-4.

[0032] Figure 8 The results are the results of a study on the binding activity of the monoclonal antibody of Example 3 against clinical strains of Acinetobacter baumannii.

[0033] Fig. 9 The experimental results of the protective effect of the Omp38-specific monoclonal antibody of Example 4 on mice infected with a lethal dose of Acinetobacter baumannii.

[0034] Fig.10 These are the experimental results of the protective effect of the Omp38-specific monoclonal antibody of Example 4 against sublethal infection of mice with Acinetobacter baumannii.

[0035] Fig.11 This is the flow cytometry test result after the mice in Example 4 were infected with a sublethal dose of Acinetobacter baumannii.

[0036] Fig.12 These are the results of a study on the potential mechanism of the broad-spectrum binding activity of the monoclonal antibody C3 in Example 5.

[0037] Fig.13 These are the experimental study results of the monoclonal antibody C3 of Example 6 that inhibits bacterial adhesion, intracellular proliferation and biofilm formation and induces antibacterial and bactericidal effects.

[0038] Fig.14 These are the results of a study on the effects of monoclonal antibodies with IgG1 as a constant region in the LAC-4 infection model of Example 8. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods and can be carried out according to the described recombinant technology (see Molecular Cloning, Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The materials, reagents, etc. used can all be obtained from commercial sources.

[0040] Example 1: Animal Immunization

[0041] (1) Animals and bacterial strains

[0042] Specific pathogen-free (SPF) female BALB / c mice and C57BL / 6 mice aged 6-8 weeks were housed under pathogen-free conditions. The strains of Acinetobacter baumannii used in the study included ATCC17978 (standard strain) and LAC-4 (highly virulent strain).

[0043] (2) Preparation of outer membrane protein (OMP)

[0044] Acinetobacter baumannii ATCC17978 was spread on Mueller Hinton agar solid medium (24 g Mueller Hinton agar solid powder, 15 g agar powder and 1000 ml ultrapure water) by the three-line method and cultured in a 37°C incubator for 12 hours. A single colony was transferred to 20 ml Mueller Hinton agar liquid medium (24 g Mueller Hinton agar and 1000 ml high-purity water) and cultured overnight at 37°C and 220 rpm for activation. The activated bacterial culture was then expanded to 1 L and cultured at 37°C and 220 rpm for 3 hours until OD 600Reach 0.8. Acinetobacter baumannii was subsequently resuspended in 10mL of PBS at pH 7.2, and the bacteria were broken in a homogenizer at 750 bar pressure for 10 minutes. After centrifugation to remove bacterial debris and sediment, the mixture was centrifuged again to collect the supernatant, which contained bacterial membrane proteins, including inner membrane proteins and outer membrane proteins. The membrane protein was resuspended in 5mL of 2% lauroyl sarcosine and incubated at 37°C for 30 minutes to dissolve the bacterial inner membrane protein. The mixture was centrifuged at 16000rpm for 1 hour to obtain an insoluble component, i.e., outer membrane protein (OMP). The purification of OMP was carried out according to the conventional means of the prior art. After washing with 2mL of 62.5mM Tris-HCl at pH 6.8, OMP was resuspended in 2mL of 5% SDS and incubated at 4°C for 10 minutes. 6mL of methanol was added, the mixture was vortexed vigorously and centrifuged at 12000rpm for 10 seconds. Then, 2 mL of chloroform was added, the mixture was vortexed vigorously and centrifuged at 12000 rpm for 10 seconds. After separation of the liquid phase, 6 mL of ultrapure water was added, the mixture was vortexed vigorously, the mixture was centrifuged at 12000 rpm for 1 minute, and the upper phase was discarded. 6 mL of methanol was added to the lower phase, the mixture was vortexed vigorously and centrifuged at 9000 rpm for 2 minutes, the upper phase was discarded, and the resulting precipitate was OMP.

[0045] (3) Mouse immunization procedure

[0046] At 0, 14 and 21 days, 50 μg OMP and 50 μl QuickAntibody Mouse3W (Biodragon, KX0210042) were injected into the calf muscles of 6-8 week-old BALB / c mice. On the 21st day, a small amount of tail vein blood was collected for enzyme-linked immunosorbent assay (ELISA) to measure the antibody titer against OMP in the serum. The mice with the highest titer of anti-OMP antibodies were selected for further steps. Three days before the Beacon system test, the last booster in saline was injected intraperitoneally.

[0047] (4) Immune effects of OMPs

[0048] After the above process, OMPs were successfully isolated from Acinetobacter baumannii. SDS-PAGE analysis showed that the main band of OMPs was approximately 38 kDa, which was consistent with previous studies ( Figure 1 A). Compared with the non-immunized group, the OMP-immunized group had higher OMP-specific polyclonal IgG antibody titers after three OMP immunizations ( Figure 1 B and 1C). Therefore, OMPs of Acinetobacter baumannii can induce the production of high titers of specific polyclonal IgG antibodies.

[0049] Example 2: Screening of monoclonal antibodies

[0050] (1) Isolation of single OMP-specific antibody-secreting cells using the Berkeley Lights Beacon system

[0051] Loading Antibody Secreting Cells (ASC) onto OptoSelect TM 11k chip and cultured in plasmablast survival medium (Berkeley Lights, 75002051), which is designed to increase antibody secretion and maintain cell viability. Single-cell encapsulation was achieved by optically transferring B cells into nanoliter-sized chambers NanoPens using photoelectric localization (OEP) technology. Through the above operations, thousands of ASCs were distributed to NanoPens on multiple chips. Fluorescence detection was then performed to screen antibodies that bind to OMP. The mixture was then applied to the OptoSelect TM 11k chip. Biotinylated OMPs were coupled to streptavidin-coated assay beads (Berkeley Lights, 520-00053) to prepare coupled beads. These beads were incubated with fluorescently labeled anti-mouse secondary antibodies (Alexa Fluor TM 568, Thermo Fisher, a-11004) was mixed at a dilution of 1:100. Cells secreting antigen-specific antibodies were identified in NanoPens near fluorescent beads. Antigen-specific cells were selectively exported to individual wells of a 96-well RT-PCR plate containing lysis buffer (Qiagen, 1070498).

[0052] (2) Single B cell sequencing and plasmid construction

[0053] After output from Beacon system, Opto Plasma B Discovery cDNA synthesis kit (Berkeley Lights, 750-02030) is used to amplify the heavy chain variable region VH and light chain variable region VL of the antibody secreted by OMP in conjunction with B cells. RNA is purified and separated from single B cells by Agencourt RNAClean XP Beads (Beckman Coulter, A63987). According to the manufacturer's scheme (Berkeley Lights, 750-02030), first-strand cDNA synthesis and total cDNA amplification are performed. The total cDNA obtained is purified by Agencourt AMPure XP Beads (Beckman Coulter, A63881), and sequenced with Opto Plasma B Discovery Sanger Prep Kit (Berkeley Lights, 750-02041). The nucleotide sequence of amplicon is determined by reverse Sanger sequencing. Analysis of VH and VL, complementarity determining region (CDR) sequences, and percentage of somatic mutations was performed using Geneious Prime (version 2021.0.3) and the IMGT database (https: / / www.imgt.org / ). The paired heavy and light chain sequences of the selected monoclonal antibodies were codon-optimized, synthesized by Shenggong Biotechnology, cloned, and inserted into separate mammalian expression vectors containing mouse IgG2a constant regions.

[0054] (3) Expression and purification of monoclonal antibodies

[0055] ExpiCHO cells were cultured in MetaCellTM CHO-310 medium (Cellplus Bio, L1013) to approximately 8.00 × 10 6 Cells / mL to 10.00×10 6 The density of cells / mL was 2×10 cells / mL, and the cell survival rate was greater than 98%. Subsequently, the vector was co-transfected into 2×10 8 ExpiCHO cells (Celetrix, 1228). ExpiCHO cells were incubated at 37°C and 7% CO2.

[0056] On the first day after transfection (18-22 hours later), 10% MetaCellTM CHO TransFeed (CellplusBio, L1008) and 0.7% MetaCellTM Tier Enhancer (Cellplus Bio, L1009) were added to the cell culture medium. After a 5-day incubation period, the cell culture supernatant was collected and analyzed by Pierce TM Spin columns (ThermoScientific TM , 89898) for purification. The bound monoclonal antibody (mAb) was washed with a buffer containing 100mM Pro-Ac pH 3.5, and the pH was adjusted to 5.0 with 0.5M Arg. The mAb was then resuspended in PBS by centrifugation through a 50kDa MWCO membrane centrifugal filter unit (Millipore, UFC905008). The purified antibody was verified by SDS-PAGE and stored at -80°C.

[0057] (4) High-throughput analysis of single B cells secreting OMP-specific monoclonal antibodies

[0058] Biotinylated OMP (dark grey) and streptavidin-coupled polystyrene beads (light grey) were introduced into the channel above the NanoPens. Single B cells secreted antibodies (purple Y-shapes) into the NanoPens, and antibodies bound to OMP were detected by fluorescent anti-mouse IgG secondary antibodies (green with red asterisks). Figure 2 A). Therefore, the reactive antibodies diffused out of the NanoPens emit fluorescent signals ( Figure 2 B). Three days after the last boost, bone marrow and spleen cells were harvested from immunized mice. Plasma cells were enriched and loaded into the Beacon fluidics instrument in a support medium to promote cell survival. High-throughput single-cell analysis confirmed that 10,661 cells were successfully loaded into a single NanoPens on the device chip ( Figure 2 B, left). In addition, IgG beads were loaded into individual NanoPens on the chip to quantify the number of antibody-secreting plasma cells ( Figure 2 B, right). Of the 10,661 cells loaded into NanoPens, 47 were identified as producing OMP-specific antibodies ( Figure 3 A). We isolated 47 single cells from the Beacon platform and performed PCR amplification of light and heavy chain sequences ( Figure 3 B). Finally, the gene information encoding the VH and VL regions was successfully obtained from 24 candidate cells ( Figure 3 B).

[0059] Using the IMGT database, we performed CDR analysis and identified several antibodies, such as B1, F4, H3, F1, F3, D4, D6, and A6, which have highly similar sequences in the CDRs of VH and VL ( Figure 4 and Figure 5 Considering the similarity of VH and VL CDRs between different monoclonal antibodies, we selected several antibodies for further analysis, including F4, A2, B4, C3, G1, G4, H4, G3, and E6 ( Figure 4 ). Subsequently, we analyzed the variable region germline characteristics of the VH and VL of these antibodies. The VH of F4, A2, B4, and C3 belong to the VH1-18 family, while their light chains belong to the VK14-126 family ( Figure 5 ). In contrast, the VH of G1 and E6 belong to the VH1-18-26 and VH5-9-4 families, respectively, and their VL belong to the VK1-117 and VK19-93 families ( Figure 4 ). Antibody G4 has a VH of the VH5-9-4 family and a VL of the VK12-41 family ( Figure 4 ). The VH of H4 and G3 belong to the VH1-42-1 family, while their VL belongs to the VK9-124 family ( Figure 4 In addition, with the exception of G3 VH (89.93%), the VHs had more than 90% identity with their respective germline genes in the variable regions ( Figure 4 Finally, we expressed and purified mAbs containing the IgG2a constant region (IgG2a mAbs) and the variable regions of the above selected antibodies in CHO cells ( Figure 6 ).

[0060] The monoclonal antibodies with excellent effects specifically screened out in this scheme include C3 and G4, which can be used alone or in combination. The corresponding experimental data will be provided later to prove the antibody effect. The specific structural information of the two antibodies is now described. For the specific information of the two antibodies, see Table 1. For the light chain variable region or heavy chain variable region of the two antibodies, the connection order of the complementary determining region and the framework region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0061] Table 1: Sequence information of heavy chain variable region and light chain variable region of monoclonal antibodies C3 and G4

[0062]

[0063] The complementary determining regions and the framework regions are connected in the above order to form the heavy chain variable region / light chain variable region. For the above variable regions, the constant regions used in this technical solution are the mouse IgG2a constant region (heavy chain) and the mouse Kappa light chain constant region (light chain), and the constant regions are added to form complete light chains and heavy chains, as shown in Table 2.

[0064] Table 2: Summary of heavy chain and light chain (variable region) information (underline indicates constant region)

[0065]

[0066]

[0067]

[0068] The nucleotide sequences of the light chain and heavy chain are integrated into the empty expression vector pcDNA3.4 (Fenghui Biology, item number ZT179) by conventional molecular cloning methods of the prior art to obtain expression vectors: pcDNA3.4-C3-HC (mIgG2a), pcDNA3.4-G4-HC (mIgG2a), pcDNA3.4-C3-LC (mKappa), pcDNA3.4-G4-LC (mKappa). The expression vector is then transferred into CHO cells, followed by cell culture and protein expression. According to biological principles, after the heavy and light chains are synthesized in the cells, they will assemble themselves to form complete antibody molecules, thereby obtaining monoclonal antibodies C3 and G4. It is well known that the heavy / light chain variable region (especially the complementary determining region) of an antibody is the key part for the antibody to recognize an antigen, and the skeleton region in the variable region and the constant region of the antibody can be selected according to actual conditions to adapt to different application scenarios. This technical solution only uses the skeleton region and the constant region in the variable region shown in Tables 1 and 2 as representatives to study the immune effect of monoclonal antibodies. It is also in line with the concept of the present invention to select other framework region sequences and other types of constant regions to adapt to other application scenarios.

[0069] Example 3: In vitro evaluation of antibody effects

[0070] Enzyme-linked immunosorbent assay was performed to characterize the antibody effect. Recombinant Omp38 protein was synthesized for detection. The Omp38 gene was cloned and inserted into pET30a, which was then transformed into Escherichia coli BL21 (DE3). Overexpressed Omp38 (about 38 kDa) formed insoluble inclusion bodies. Omp38 was purified from the inclusion bodies by Ni-NTA affinity chromatography under denaturing conditions. Omp38 bound to the nickel column was eluted with an imidazole gradient eluent and detected by SDS-PAGE. In order to facilitate expression and purification in Escherichia coli, we added methionine to the N-terminus and 6 histidines to the C-terminus during plasmid design. Therefore, the recombinantly expressed Omp38 has 1 more methionine and 6 more histidines than the natural Omp38, and the molecular weight is 972.05 Da. The amino acid sequence of recombinant Omp38 is as follows (SEQ ID NO.17):

[0071] MGVTVTPLLLGYTFQDSQHNNGGKDGNLTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGASAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYDFDGVNRGTRGTSEEGTLGNAGVGAFWRLNDALSLRTEARATYNADEEFWNYTALAGLNV VLGGHLKPAAPVVEVAPVEPTPVTPQPQELTEDLNMELRVFFDTNKSNIKDQYKPEIAKVAEKLSEYPNATARIEGHTDNTGPRKLNERLSLARANSVKSALVNEYNVDASRLSTQGFAWDQPIADNKTKEGRAMNRRVFATITGSRTVVVQPGQEAAAPAAAQHHHHHH.

[0072] The amino acid sequence of recombinant Omp38 is as follows (SEQ ID NO.18):

[0073]

[0074] The recombinant Omp38 was added at 0.4 μg / well or 1×10 7 The amount of CFU / well of Acinetobacter baumannii strain was coated on a 96-well ELISA plate (LABSELECT, 31111) at 4°C overnight. After washing three times with PBS containing Tween 20 (0.05%) (Sigma-Aldrich, St. Louis, MO, USA), the plate was blocked with PBS containing 2% FBS (Gibco, A5669701) for 1 hour. It was then washed three times. Recombinant monoclonal antibodies or isotype control monoclonal antibodies at different dilutions were then added and incubated at 37°C for 1 hour. After washing three times, the plate was incubated with HRP-conjugated goat anti-mouse IgG (H+L) (AB Clonal, AS003) at 37°C for 1 hour. The substrate TMB (Beyotime, P0209) was added and the mixture was reacted in the dark to monitor the color development. The reaction was stopped by adding 1N hydrochloric acid and the absorbance was measured at 450nm. EC was determined based on the results. 50 .

[0075] For cytokine analysis, serum from infected mice was collected and stored at -80 °C until use. Cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α), were measured by ELISA kits (Dakewe, 1210603, 1211003 and 1217203) according to the manufacturer's instructions. The absorbance was detected at 450 nm by a microplate reader (Thermo Fisher Scientific, USA).

[0076] The statistical analysis of experimental data was performed as follows: Descriptive statistics of continuous variables were expressed as mean ± standard deviation. When comparing data from more than two groups, statistical differences between experimental and control groups were assessed by one-way analysis of variance. A significance threshold of P < 0.05 was used. All statistical analyses were performed using GraphPad Prism 8.0.

[0077] The experimental results are as follows:

[0078] (1) Binding activity of Omp38-specific monoclonal antibody to LAC-4 Acinetobacter baumannii

[0079] As one of the most abundant OMPs in Acinetobacter baumannii, Omp38 plays an important role in bacterial adhesion and invasion of host cells. We expressed and purified recombinant Omp38 for further investigation of the specificity of the above monoclonal antibodies ( Figure 7 A). ELISA confirmed the strong interaction of monoclonal antibodies (including F4, G1, B4, G4, C3 and A2) with Omp38 ( Figure 7 B). However, H4, G3, and E6 did not bind to Omp38, suggesting that they may bind to other OMPs ( Figure 7 B). Next, we tested the binding activity of the monoclonal antibodies against the highly virulent Acinetobacter baumannii LAC-4 (ST10 subtype). All of the above Omp38-specific monoclonal antibodies showed affinity for LAC-4, with EC50 values ​​ranging from 1.08 μg / ml to 37.39 μg / ml ( Figure 7 C).

[0080] (2) Binding activity of Omp38-specific monoclonal antibodies against clinical strains of Acinetobacter baumannii

[0081] To investigate the ability of the monoclonal antibodies to bind to strains other than LAC-4, we collected nine clinical strains of A. baumannii and identified their sequence types. Notably, strains 3, 4, 5, 7, and 9 belonged to ST2, while strains 1, 2, 6, and 8 were identified as ST193, ST205, ST63, and ST584, respectively ( Figure 8 A). ELISA demonstrated that monoclonal antibodies F4, A2, C3, G1, G4, and B4 could bind to various strains of Acinetobacter baumannii ( Figure 8 B).

[0082] It can be seen from the above experimental results that monoclonal antibodies such as F4, G1, B4, G4, C3 and A2 have strong interactions and binding activity with Omp38, and can be used as substances for detecting outer membrane protein Omp38 or Acinetobacter baumannii. In the development of microbial detection kits, antigen protein detection kits, etc. in the prior art, the development of antibody molecules that target and bind to target molecules is the key to successful research. The mutual binding between the monoclonal antibodies of this solution and the Omp38 antigen molecules can be used to achieve the detection of target molecules or microorganisms.

[0083] Example 4: In vivo evaluation of antibody effects

[0084] (1) Animal models

[0085] 1 C57BL / 6 mice were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital. Then, non-invasive intratracheal inoculation was performed with 1.4 × 10 7 (lethal dose) or 1×10 6(Sublethal dose) CFU of LAC-4 attack mice, then immediately intravenously inject monoclonal antibody (Mouse IgG2a Isotype Control, Invitrogen, 02-6200). Then take the infected tissue, use the conventional method of the prior art to detect the amount of microbial infection (bacterial load), and then determine the inhibitory effect of the antibody on Acinetobacter baumannii. Continuous 10 times dilutions were inoculated on tryptone soy agar (TSA), and CFU counts were performed to detect the amount of LAC-4. The bacterial load and lung pathology of the mouse lungs were measured 24 hours after sublethal stimulation. The survival rate of mice was monitored every day for 72 hours.

[0086] (2) Histopathology

[0087] Formalin-fixed and paraffin-embedded (FFPE) tissues were then cut into 5 mm thick sections and stained with hematoxylin and eosin (H&E) according to the manufacturer's instructions (Solarbio, G1120). Images were acquired by microscopy (×200, Nikon TE2000). Acute lung injury (ALI) in mouse lungs was determined by a scoring system according to published guidelines.

[0088] (3) Flow cytometry

[0089] The lungs were cut into small pieces and digested in a standard test tube containing 5 ml of digestion buffer (0.5 mg / ml collagenase and 20 mg / ml DNase diluted in RPMI medium) for 45 minutes. The cell suspension was then passed through a 70 μm pore size cell strainer and the remaining cells were lysed with ACK buffer (Invitrogen, A1049201). The cells were blocked with mouse BD FC Block (BD Pharmingen, 553141) and then stained with fluorescently labeled monoclonal antibodies as follows: CD4 fluorescein isothiocyanate (FITC) (clone GK1.5); CD45-PerCP Cyanine 5.5 (clone HI30); F4 / 80 phycoerythrin (PE)-cyanine 7 (clone BM8); LY6G allogeneic cobalt blue protein (APC) (clone 1A8-LY6G); L / D APC-CY7 (LIVE / DEAD TM Fixable near-infrared dead cell staining kit, Invitrogen); and CD8 AmCyan (clone SK1). Cells were stained with antibodies (1:200) for 30 minutes at 4°C. Data were acquired using a BD FACSCanto II cell analyzer and analyzed by FlowJo software (Tree Star, Ashland, OR).

[0090] (4) Experimental results

[0091] (4.1) Omp38-specific monoclonal antibodies protect mice from lethal Acinetobacter baumannii infection

[0092] To evaluate the protective effect of the monoclonal antibody against infection with the highly virulent A. baumannii strain LAC-4 in mice, eight animals per group were inoculated intratracheally with a lethal dose of LAC-4 and immediately treated with the Omp38-specific monoclonal antibody. Survival was monitored within 72 h after infection ( Fig. 9 A). It is noteworthy that 4 of the 6 candidate monoclonal antibodies, namely F4, A2, C3 and G4, showed protective effects against LAC-4 infection, with protection rates of 50%, 50%, 75% and 62.5%, respectively ( Fig. 9 B). For the use of monoclonal antibodies alone, this scheme directly injects the antibodies according to the dosage requirements. The protection rate of 75% for C3 and 62.5% for G4 are relatively excellent data, which are worthy of further development. In the future, the monoclonal antibodies will be studied as drugs to form dosage forms suitable for clinical applications, improve the bioavailability of antibodies and protect antibodies from the negative effects of the in vivo environment. The protective effect of monoclonal antibodies will be further improved after drug development. In addition, the sequence of the antibodies developed in this scheme (especially the CDR region) is reported for the first time, and the experiment verifies the ability of the antibodies to bind to Acinetobacter baumannii and the protective effect on infected animals. Therefore, the monoclonal antibodies in this scheme are non-obvious compared to the prior art, especially monoclonal antibodies C3 and G4.

[0093] In recent years, antibody cocktail therapy has become a more effective treatment option than single antibody therapy because it has advantages such as increased antibody diversity and complexity. It is worth noting that a double antibody mixture (C3+G4) with a final dose of 15 mg / kg per mouse, that is, 7.5 mg / kg of each antibody, can achieve a 100% survival rate, while a three-antibody mixture (C3+G4+A2; C3+G4+F4, 5 mg / kg of each antibody) or a four-antibody mixture (C3+G4+A2+F4, 3.75 mg / kg of each antibody) with a final dose of 15 mg / kg could not protect all mice from death ( Fig. 9C). It can be seen that the simultaneous use of C3 and G4 can effectively improve the immune effect to 100% survival rate when the total dose remains unchanged, which shows that C3 and G4 have a certain synergistic effect. If there is no synergistic effect, when the total dose is the same, the effect of the combined use of C3 and G4 will be between the effects of the two alone, that is, between 62.5% and 75%. There is no synergistic effect between C3 and G4 and other monoclonal antibodies. Even the simultaneous use of four antibodies will reduce the survival rate to nearly 40%, and the effect of any one antibody alone is worse. Therefore, the combined use of C3 and G4 has achieved unexpected technical effects and ensured a 100% survival rate. If the antibody needs to be used alone, the effect of C3 is the most significant. If a single-component antibody preparation needs to be prepared, C3 is preferred.

[0094] In the medical practice of fighting against Acinetobacter baumannii infection, single-component antibody therapy and cocktail therapy using a combination of multiple antibodies each have their own advantages, and medical personnel or drug researchers can choose the appropriate method according to the actual situation. The successful development of this technical solution provides a variety of options for medical personnel or drug researchers. They can use the various monoclonal antibodies developed in this study alone for treatment or for more in-depth drug development, or they can choose a suitable antibody combination. The latter can achieve better therapeutic effects by combining the effects of multiple antibodies. The former has a relatively simple production process, the drug effect is relatively controllable, and the manufacturing cost will be relatively low.

[0095] (4.1) Omp38-specific monoclonal antibodies protect mice from sublethal lung infection with Acinetobacter baumannii

[0096] We established an animal model with 5 mice per group, which showed aspiration pneumonia symptoms very similar to the typical clinical manifestations of Acinetobacter baumannii infection ( Fig.10 Importantly, at 24 hours post-infection, mice treated with C3 or G4 had a 2885-fold reduction in LAC-4 lung bacterial load, while mice treated with F4 or A2 had an 857-fold reduction compared with mice treated with an isotype control mAb ( Fig.10 B). After infection with A. baumannii, increased severity of inflammation, leukocyte infiltration, and cytokine burst were observed. To evaluate the potential anti-inflammatory effects of Omp38-specific mAbs, we investigated their effects on cytokine concentrations within the lung. Treatment with Omp38-specific mAbs resulted in a significant decrease in the levels of pro-inflammatory cytokines (IL-6, TNF) and the anti-inflammatory cytokine IL-10, which was associated with a decrease in bacterial load ( Fig.10C). Flow cytometry analysis further showed that the lungs of mAb-treated mice were infiltrated with total leukocytes, neutrophils, mononuclear macrophages / monocytes, and CD4 + The frequency of T cells was significantly reduced, indicating reduced inflammation ( Fig.10 D. Fig.11 To evaluate the potential of monoclonal antibodies in reducing lung pathology, lungs were collected 24 hours after infection for H&E staining. The results showed that Omp38-specific monoclonal antibodies reduced inflammatory cell infiltration and alveolar wall thickening, ultimately reducing damage to lung tissue structure ( Fig.10 E). At 24 h post-infection, lung injury was assessed by the acute lung injury (ALI) scoring system, and mice treated with Omp38-specific mAb scored lower than those treated with isotype control ( Fig.10 F), showing that Omp38-specific monoclonal antibodies reduced damage to lung tissue architecture.

[0097] Example 5: Omp38 monoclonal antibody C3 binding conformation analysis

[0098] To explore the antigen-antibody binding conformation, we selected mAb C3 for further analysis due to its superior in vivo efficacy and ability to bind to various A. baumannii strains. Using AlphaFold 3 and GeoBiologics, we first predicted the binding model between mAb C3 and the A. baumannii reference strain in NCBI GenBank (ATG88079.1), which suggested that C3 might bind to the extracellular domain of Omp38 ( Fig.12 A). In addition, we found three loop structures in this domain ( Fig.12 A and 12B). The residues in the three loops of various A. baumannii strains, including the reference strain LAC-4 and the nine clinical strains mentioned above, were subsequently extracted and compared. Notably, some A. baumannii strains, such as strains 2 and 6, exhibited the same loop structure as Omp38; therefore, we performed subsequent analyses on these strains ( Fig.12 C). The 11 strains were finally divided into 5 groups, and we identified 5 binding conformations based on homology modeling. To explore whether the changes in the loop affect the binding affinity of C3, we performed 10ns kinetic calculations on each complex and calculated the interaction energy between C3 and different Acinetobacter baumannii strains using the CHARMm tool in Discovery Studio 2.5. It is worth noting that C3 showed similar interaction energy for various strains, which is consistent with the ELISA results ( Figure 8 B), indicating that C3 has a broad-spectrum binding activity ( Fig.12 D).

[0099] We further analyzed five binding conformations, focusing on the interactions between the three loop regions and C3 CDRs of the various A. baumannii strains mentioned above. Since hydrogen bonds are essential for achieving high specificity and affinity of antigen-antibody interactions in most cases, we took the reference strain as an example and emphasized the key hydrogen bonds involved in the interactions between the light and heavy chains and the loop CDRs based on their 3D spatial structures ( Fig.12 A). After conformational superposition and alignment of the five bound conformations, we found that each loop contains several key residues whose side chains contribute to the formation of hydrogen bonds between the antigen and the mAb ( Fig.12 E). Among the five binding conformations, several key residues are highly conserved, such as N23 in loop1 ( Fig.12 E). Although other key residues differ in different strains, they still provide key hydrogen bonds for binding to the antibody. For example, when T122 in loop3 is mutated to Y122, hydrogen bonds are still formed between the hydroxyl group and the corresponding residue of the antibody ( Fig.12 E) The conservation of this binding mode may explain the broad-spectrum binding activity of C3.

[0100] Example 6: Protective mechanism of Omp38 monoclonal antibody C3 against Acinetobacter baumannii infection

[0101] Epithelial cells are the first line of host immune protection. A. baumannii biofilms promote bacterial adhesion and colonization on the surface of respiratory epithelial cells, leading to intracellular proliferation and host cell death. Since Omp38 has been reported to play an important role in adhesion, intracellular proliferation, and biofilm formation, we explored the effect of C3 on the pathobiology of A. baumannii. C3 inhibited biofilm formation, adhesion, and intracellular proliferation of A. baumannii compared with isotype controls ( Fig.13 A-13C).

[0102] Antibody-dependent cellular phagocytosis (ADCP) is an immune mechanism of elimination in which infected cells are targeted by mAbs to promote their clearance from the body by phagocytic immune cells such as macrophages. A previous study has shown that macrophages play an important role in the early host defense against A. baumannii infection by efficiently phagocytosing and killing A. baumannii. Therefore, we investigated the role of C3 in ADCP. In the presence of mAb C3 at MOIs of 3 and 30, macrophage uptake of the highly virulent A. baumannii LAC-4 strain was increased, but there was no difference at MOIs of 300 and 3000 ( Fig.13 D). These results confirm the role of C3 in antibacterial killing via induction of ADCP.

[0103] exist Fig.13In the figure, A is the adhesion test result of Acinetobacter baumannii ATCC17978 in the presence of mAb C3 or isotype control. A549 cells were infected with Acinetobacter baumannii at an MOI of 1:100, and mAb C3 or isotype control was added to the cell culture. After removing external non-adherent bacteria, the degree of bacterial adhesion to A549 cells was quantified by serial dilution method. B is the intracellular proliferation of Acinetobacter baumannii ATCC17978 in the presence of mAb C3 or isotype control. A549 cells were infected with Acinetobacter baumannii at an MOI of 1:100, and mAb C3 or isotype control was added to the cell culture. The intracellular proliferation rate (Ipro) was determined by calculating the number of viable intracellular bacteria present 24 hours after infection and the number of bacteria present 4 hours after infection. C is the biofilm formation of ATCC 17978 in the presence of mAb C3 or isotype control. A total of 1.0×10 7 CFU / mL Acinetobacter baumannii and mAb or isotype control were co-incubated at 37°C for 26 hours. The formed biofilm was then fixed with methanol for 20 minutes and stained with 0.1% crystal violet for 30 minutes. After washing with saline, 95% ethanol was added to loosen the biofilm. Finally, the absorbance was measured at 600nm by a microplate reader. D is the phagocytosis test of C3 and isotype control at different MOIs. Acinetobacter baumannii LAC-4 strain was incubated with RAW264.7 cells stimulated with palmitoyl-2-cysteine ​​serine lysine-4 (Pam2CSK4) at different MOIs (1:3, 1:30, 1:300 and 1:3000), and mAb C3 or isotype control was added to the cell culture. After incubation for 5 hours under gentle shaking, the bacteria in the supernatant were verified by serial dilution. Each assay was performed three times, and all data are presented as the mean ± SEM of three independent experiments. The bacterial loads between the two groups were compared by unpaired Student's t test. ns: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0104] Example 7: Safety Assessment of Omp38 Monoclonal Antibody C3

[0105] The safety of mAb C3 in vitro and in vivo was subsequently confirmed. Flow cytometry analysis showed that C3 did not affect late apoptosis (Annexin V + 7-AAD + ) cells or early apoptosis (Annexin V + 7-AAD -) cells. CCK-8 assay showed that mAb C3 did not affect the survival rate of various cells (293T cells, RAW264.7 cells or A549 cells) after treatment with 50 μg / mL for different durations or with different concentrations of C3 for 5 hours. For toxicity analysis, mice were treated with 15 mg / kg C3 or the same volume of PBS. At 72 hours after injection, serum was collected to detect serum biochemical indicators, including total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), alkaline phosphatase (ALP), albumin / globulin (A / G), direct bilirubin (TBIL), urea (urea), serum creatinine (SCR), glucose (GLU), creatine kinase (CK) and triglyceride (TG) levels. In addition, liver, kidney, intestine, lung, spine, brain, spleen and heart were collected to detect pathological changes. The results showed that there was no change in biochemical indicators and no pathological damage to organs after treatment with C3.

[0106] Example 8: Constant region type selection

[0107] Studies have shown that mouse IgG1 only binds to FcγRIIB and FcγRIII receptors, while mouse IgG2a monoclonal antibodies can bind to FcγRI, FcγRI1B, FcγRIII, and FcγRIV receptors. In addition, mouse FcγRI and FcγRIV are mainly expressed on monocytes and macrophages and show high-affinity interactions with IgG2a monoclonal antibodies, which is crucial in Acinetobacter baumannii infection. The paired VH and VL sequences of the selected monoclonal antibodies (including F4, A2, C3, and G4) were codon-optimized, synthesized by Shenggong Biotechnology, cloned, and inserted into a separate mammalian expression vector containing a constant mouse IgG1 region ( Fig.14 A). We established a lethal LAC-4 infection model under the same conditions as IgG2a-treated mice and treated mice with 15 mg / kg IgG1 monoclonal antibody ( Fig.14 B). Notably, 3 of the 4 candidate monoclonal antibodies (F4, C3, and G4) delayed mouse mortality, whereas A2 had no protective effect against LAC-4 infection. Interestingly, the double antibody cocktail (C3+G4) at a final dose of 15 mg / kg per mouse (7.5 mg / kg per antibody) resulted in a 16.7% survival rate (1 in 6 mice) ( Fig.14 C) The experimental results show that antibodies with IgG1 constant region are not effective, and antibodies with IgG2a constant region are able to work.

[0108] exist Figure 4In Figure 1, A shows: non-reducing PAGE shows that the antibody is 150 kDa; reducing PAGE shows that the antibody heavy chain is 55 kDa and the light chain is 25 kDa; IPI is used as a positive control. B shows a schematic diagram of Omp38-specific monoclonal antibody treatment of lethal LAC-4 infection model mice. Six mice were non-invasively inoculated intratracheally with a lethal dose (1.4×10 7 CFU) of LAC-4 and were immediately treated with 15 mg / kg mAbs F4, C3, G4, A2 or isotype control. Survival was monitored 72 hours after infection. C is Kaplan-Meier survival curve: survival curve of infected mice treated with single monoclonal antibodies or monoclonal antibody mixtures and isotype control group (n=6 per group). Survival curves were compared by log-rank (Mantel-Cox) test.

[0109] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A monoclonal antibody, characterized in that It is the monoclonal antibody C1; the amino acid sequences of the complementary determining region 1, complementary determining region 2 and complementary determining region 3 of its heavy chain are GYTITEYTMH, GINPNNGGTS, GAYY, respectively; the amino acid sequences of the complementary determining region 1, complementary determining region 2 and complementary determining region 3 of its light chain are KASQDIKSYLT, YARTLAD, LQHGESPLT, respectively.

2. A monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the variable region of the heavy chain of monoclonal antibody C1 is shown in SEQ ID NO.1; the amino acid sequence of the variable region of the light chain of monoclonal antibody C1 is shown in SEQ ID NO.

5.

3. A monoclonal antibody according to claim 2, characterized in that: The amino acid sequence of the heavy chain of monoclonal antibody C1 is shown in SEQ ID NO.3; the amino acid sequence of the light chain of monoclonal antibody C1 is shown in SEQ ID NO.

7.

4. A monoclonal antibody according to claim 3, characterized in that: It is used to bind to the outer membrane protein of Acinetobacter baumannii; the outer membrane protein is OMP38.

5. Use of a monoclonal antibody according to any one of claims 1 to 5 in the preparation of a drug for resisting Acinetobacter baumannii infection, characterized in that: The monoclonal antibody is used for binding to and inhibiting Acinetobacter baumannii; the monoclonal antibody is used for reducing the release of inflammatory mediators and thus reducing host damage.

6. An antibody composition comprising a monoclonal antibody according to any one of claims 1 to 5, characterized in that: It also includes monoclonal antibody G4; the amino acid sequences of the complementary determining region 1, complementary determining region 2 and complementary determining region 3 of the heavy chain of monoclonal antibody G4 are GFTFSSYAMS, EISSGGTYTY, EGDGYVWFPY, respectively; the amino acid sequences of the complementary determining region 1, complementary determining region 2 and complementary determining region 3 of the light chain of monoclonal antibody G4 are RASGNIHNYLA, NAKTLAD, QHFLTTPRALT, respectively.

7. The antibody composition according to claim 6, characterized in that The dosage ratio of monoclonal antibody G4 and monoclonal antibody C3 is 1:

1.

8. The antibody composition according to claim 7, characterized in that The amino acid sequence of the variable region of the heavy chain of monoclonal antibody G4 is shown in SEQ ID NO.9, and the amino acid sequence of the variable region of the light chain of monoclonal antibody G4 is shown in SEQ ID NO.13; the amino acid sequence of the heavy chain of monoclonal antibody G4 is shown in SEQ ID NO.11; and the amino acid sequence of the light chain of monoclonal antibody G4 is shown in SEQ ID NO.

15.

9. Use of the antibody composition according to claim 8 in the preparation of a medicament for resisting Acinetobacter baumannii infection.

10. Use of the antibody composition according to claim 9 in the preparation of a drug for resisting Acinetobacter baumannii infection, characterized in that: The raw materials of the medicine include a pharmaceutically acceptable carrier.

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