A monoclonal antibody and its application in resisting acinetobacter baumannii infection

Abstract

The application belongs to the technical field of monoclonal antibodies, and particularly relates to a monoclonal antibody and application thereof in resisting infection of Acinetobacter baumannii. The antibody is separated by using a high-throughput monoclonal antibody separation platform of a Beacon platform, and C3 monoclonal antibody with more excellent functions is screened out, the amino acid sequences of the complementarity determining region 1, the complementarity determining region 2 and the complementarity determining region 3 of the heavy chain of the C3 monoclonal antibody are GYTITEYTMH, GINPNNGGTS and GAYY respectively, and the amino acid sequences of the complementarity determining region 1, the complementarity determining region 2 and the complementarity determining region 3 of the light chain of the C3 monoclonal antibody are KASQDIKSYLT, YARTLAD and LQHGESPLT respectively. The monoclonal antibody of the scheme is helpful for bacterial clearance, reduction of release of inflammatory mediators and reduction of host damage, solves the technical problem of lacking of the monoclonal antibody for resisting infection of Acinetobacter baumannii in the prior art, and has an ideal popularization and application prospect.

Description

Technical Field

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

[0002] Acinetobacter baumannii (A. baumannii) is an opportunistic pathogen commonly found in the environment, capable of causing severe infections in human tissues such as the lungs and blood, particularly in immunocompromised patients admitted to the intensive care unit (ICU). Unlike typical Gram-negative bacteria, A. baumannii possesses robust adhesion, promoting colonization at the site of infection and enabling biofilm formation or cellular invasion. Studies have reported a 56.2% mortality rate in infected patients, necessitating the urgent exploration of new treatment strategies.

[0003] Antibiotics are the mainstay of treatment for Acinetobacter baumannii infections. However, A. baumannii is resistant to multiple antibiotics, making treatment extremely difficult. Vaccines for A. baumannii are safe, have few adverse reactions, and are less likely to induce drug resistance. However, due to several factors, including antigenic sequence variation, no vaccine has yet passed Phase I clinical trials. Probiotic therapy and fecal microbiota transplantation (FMT) have shown promise in treating A. baumannii infections, but their mechanisms remain unclear and require further investigation. In recent years, monoclonal antibody (mAb) therapy has emerged as a promising approach for treating 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. Furthermore, monoclonal antibodies can be combined with antibiotics to reduce antibiotic usage and mitigate associated adverse reactions.

[0004] Current monoclonal antibody development technologies face various challenges. For example, hybridoma technology suffers from reduced monoclonal antibody diversity due to competition between different hybridomas. Some low-producing monoclonal clones may exhibit faster growth rates, surpassing high-secreting clones. In addition, due to the random pairing of the heavy chain variable region (VH) and light chain variable region (VL), low-affinity monoclonal antibodies are produced. 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 aims 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 solutions:

[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, and 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, and 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; 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 present technical solution also provides a use of a monoclonal antibody in the preparation of a drug for treating 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 thereby reduce host damage.

[0012] The present technical solution 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 and 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, 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.

[0015] This 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 pharmaceutically acceptable carriers.

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

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

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

[0020] Infections caused by Acinetobacter baumannii are a global public health concern due to their high pathogenicity. Unlike antibiotics, monoclonal antibodies (mAbs) have shown promise in reducing drug resistance and thus minimizing adverse reactions. Currently, the therapeutic efficacy of mAbs targeting A. baumannii remains unclear. This technical proposal isolates mAbs targeting 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. Furthermore, analysis of the C3 binding conformation of the Omp38 mAb revealed the underlying mechanism of the mAb's broad-spectrum binding activity against A. baumannii. Taken together, these findings suggest that mAbs targeting 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 developing this patented technology, we used the Beacon platform to isolate high-throughput monoclonal antibodies and obtained monoclonal antibodies targeting the OMP of Acinetobacter baumannii. Ten OMP-targeting monoclonal antibodies were identified from immunized mice, six of which targeted Omp38. These mAbs effectively and broadly neutralized multiple strains of A. baumannii, protecting mice from infection. Among these, the C3 monoclonal antibody demonstrated the most robust protective effect against infected animals, making it a promising candidate for further development as a treatment for A. baumannii infection.

[0022] Acinetobacter baumannii contains a range 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 A. baumannii OMP that regulates adhesion, invasion, and biofilm formation in A. baumannii and contributes to inflammation and other host responses. Omp38 is highly conserved among different A. baumannii strains. Overproduction of Omp38 is a risk factor for nosocomial pneumonia, bacteremia, and increased patient mortality. The monoclonal antibodies developed in this technical solution can effectively bind to Omp38 and effectively inhibit A. 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. generated immune serum by subcutaneously inoculating mice with Omp38 and demonstrated the safety and efficacy of the antiserum in terms of bacterial killing, biofilm inhibition, bacterial adhesion, and inhibition of intracellular proliferation. However, they did not isolate a monoclonal antibody targeting Omp38 from the serum. Our study, however, involved immunizing mice with Omp38 and identifying a monoclonal antibody targeting Omp38. Currently, the mechanism of mAb-mediated protection against A. baumannii infection is not fully understood. The monoclonal antibodies developed in this protocol have potential applications not only in medical practice but also in further basic research, including mechanism of action. They serve as basic research tools to explore the impact of Omp38-specific monoclonal antibodies on the pathobiology of A. baumannii. Furthermore, the monoclonal antibodies generated in this protocol can effectively bind to membrane proteins of A. baumannii. Therefore, the monoclonal antibodies can also be used in relevant A. baumannii detection kits to detect the target bacteria through antigen-antibody binding reactions.

[0024] Further combining C3 and G4 revealed that their combined use could achieve a 100% host protection rate against highly virulent Acinetobacter baumannii infection, indicating 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, and the above findings have laid the foundation for antibody cocktail therapy for Acinetobacter baumannii infection. Antibody cocktail therapy provides a new approach to the treatment of complex Acinetobacter baumannii infections, especially when faced with bacteria that are prone to drug resistance. By combining the effects of multiple antibodies, better therapeutic effects can be achieved, and compared to 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 This is 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 These are 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 show the binding activity of the monoclonal antibody of Example 3 against recombinant Omp38 and highly virulent Acinetobacter baumannii LAC-4.

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

[0033] Figure 9 These are the experimental results of the protective effect of the Omp38-specific monoclonal antibody of Example 4 against lethal doses of Acinetobacter baumannii infection in mice.

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

[0035] Figure 11 These are the flow cytometry test results after the mice in Example 4 were infected with a sublethal dose of Acinetobacter baumannii.

[0036] Figure 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] Figure 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] Figure 14 These are the results of a study on the effects of monoclonal antibodies with IgG1 as the constant region in the LAC-4 infection model of Example 8. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below with reference to the following 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 recombinant techniques described (see Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). All materials and reagents used are commercially available.

[0040] Example 1: Animal Immunization

[0041] (1) Animals and bacterial strains

[0042] Specific pathogen-free (SPF) female BALB / c and C57BL / 6 mice, 6-8 weeks old, 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 the OD 600Reach 0.8. Subsequently, Acinetobacter baumannii was resuspended in 10mL of PBS with a pH of 7.2 and the bacteria were broken in a homogenizer at a pressure of 750 bar 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 proteins were resuspended in 5mL of 2% lauroyl sarcosine and incubated at 37°C for 30 minutes to dissolve the bacterial inner membrane proteins. 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 a pH of 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 vigorously vortexed, and centrifuged at 12,000 rpm for 10 seconds. After separation of the liquid phase, 6 mL of ultrapure water was added, the mixture was vigorously vortexed, and centrifuged at 12,000 rpm for 1 minute, and the upper phase was discarded. 6 mL of methanol was added to the lower phase, the mixture was vigorously vortexed, and centrifuged at 9,000 rpm for 2 minutes, and the upper phase was discarded. The resulting precipitate was OMP.

[0045] (3) Mouse immunization program

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

[0047] (4) Immune effects of OMP

[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. Using photoelectric positioning (OEP) technology, single cell encapsulation is achieved by optically transferring B cells into nanoliter-sized chambers NanoPens. Through the above operations, thousands of ASCs are distributed to NanoPens on multiple chips. Fluorescence detection is then performed to screen antibodies that bind to OMP. The mixture is then applied to the OptoSelect TM 11k chip. Biotinylated OMP was coupled to streptavidin-coated assay beads (Berkeley Lights, 520-00053) to prepare coupled beads. These beads were incubated with fluorescently labeled anti-mouse secondary antibody (Alexa Fluor TM 568, Thermo Fisher, a-11004) 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 the 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 isolated from single B cells by Agencourt RNAClean XP Beads (Beckman Coulter, A63987). According to the manufacturer's protocol (Beckman Coulter, 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 the amplicon is determined by reverse Sanger sequencing. Analysis of VH and VL, complementarity-determining region (CDR) sequences, and somatic mutation percentages was performed using Geneious Prime (version 2021.0.3) and the IMGT database (https: / / www.imgt.org / ). 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 MetaCell™ CHO-310 medium (Cellplus Bio, L1013) to a size of approximately 8.00 × 10 6 cells / mL to 10.00×10 6 The density of cells / mL was 100 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) were cultured 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 column (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-shaped) into the NanoPens, and the antibodies bound to OMP were detected using a fluorescent anti-mouse IgG secondary antibody (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 final booster immunization, bone marrow and spleen cells were harvested from immunized mice. Plasma cells were enriched and loaded into a support medium in the Beacon fluidics instrument 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 panel.) 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 panel). 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 among 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 VH from the VH5-9-4 family and VL from the VK12-41 family ( Figure 4 The VH of H4 and G3 belong to the VH1-42-1 family, while their VL belong to the VK9-124 family ( Figure 4 In addition, with the exception of G3 VH (89.93%), the VHs shared greater 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 (IgG2amAbs) and the variable regions of the above-selected antibodies in CHO cells ( Figure 6 ).

[0060] The monoclonal antibodies C3 and G4, which have shown excellent efficacy, were selected for this study. Both can be used alone or in combination. Subsequent experimental data will be provided to demonstrate the effectiveness of these antibodies. The detailed structural information for these two antibodies is described below. See Table 1 for details. For both light and heavy chain variable regions, the order of the complementarity-determining regions and framework regions is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

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

[0062]

[0063] The complementarity-determining regions and framework regions are linked in the order described above to form the heavy chain variable region and the light chain variable region. For these variable regions, this technical solution uses the constant regions of the mouse IgG2a constant region (heavy chain) and the mouse kappa light chain constant region (light chain). After adding the constant regions, the complete light and heavy chains are formed, as detailed in Table 2.

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

[0065]

[0066]

[0067]

[0068] The nucleotide sequences of the light chain and heavy chain were integrated into the empty expression vector pcDNA3.4 (Fenghui Biology, Product No. ZT179) through conventional molecular cloning methods in 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 was 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 on their own 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 the antibody is the key part for the antibody to recognize the 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 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] An 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. The nickel column-bound Omp38 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 histidines than the natural Omp38, and the molecular weight is 972.05 Da larger. 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. Subsequently, it was washed three times. Recombinant monoclonal antibodies or isotype control monoclonal antibodies of 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 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. Absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA).

[0076] Statistical analysis of experimental data was performed as follows: Descriptive statistics for continuous variables are presented as mean ± standard deviation. When comparing data from more than two groups, statistical differences between experimental and control groups were assessed using 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 antibodies 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-mentioned 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 might bind to other OMPs Figure 7 B). Next, we tested the binding activity of the monoclonal antibodies to the highly virulent A. baumannii LAC-4 (ST10 subtype). All of the above Omp38-specific monoclonal antibodies showed affinity to 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 to A. baumannii clinical strains

[0081] To investigate the ability of the monoclonal antibodies to bind to strains other than the LAC-4 strain, we collected 9 A. baumannii clinical strains 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 the monoclonal antibodies F4, A2, C3, G1, G4 and B4 could bind to various A. baumannii strains Figure 8 B).

[0082] From the above experimental results, it can be seen that the monoclonal antibodies F4, G1, B4, G4, C3 and A2 have strong interaction and binding activity with Omp38 and can be used as a substance for detecting outer membrane protein Omp38 or A. baumannii. In the development of existing microbial detection kits, antigen protein detection kits, etc., developing an antibody molecule that targets the binding of the target molecule is the key to success. The mutual binding between the monoclonal antibody of the present application and the Omp38 antigen molecule can be used to achieve the detection of the target molecule or microorganism.

[0083] Example 4: In vivo experimental evaluation of the effect of the antibody

[0084] (1) Animal model

[0085] 1 C57BL / 6 mice were anesthetized by intraperitoneal injection of 50 mg / kg of sodium pentobarbital. Subsequently, 1.4 x 10 7 (mortal dose) or 1 x 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, adopt the amount (bacterial load) of the detection microbial infection of prior art conventional method, and then judge the inhibitory effect of antibody for Acinetobacter baumannii. Continuous 10 times of dilutions are inoculated on tryptone soy agar (TSA), and the amount of CFU is counted to detect LAC-4. After sublethal stimulation, the bacterial load and lung pathology of mouse lungs are measured 24 hours. The survival rate of mice is 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 using a microscope (×200, Nikon TE2000). Acute lung injury (ALI) in mouse lungs was determined using a scoring system based on 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 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. Cytometry was performed using a BD FACSCanto II cell analyzer, and data were analyzed using 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 monoclonal antibodies against infection with the highly virulent Acinetobacter baumannii strain LAC-4 in mice, eight animals per group were inoculated intratracheally with a lethal dose of LAC-4 and immediately treated with Omp38-specific monoclonal antibodies. Survival was monitored within 72 hours after infection ( Figure 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 ( Figure 9 B). For the use of monoclonal antibodies alone, this protocol directly injects the antibodies according to the dosage requirements. The 75% protection rate of C3 and the 62.5% protection rate of G4 are relatively excellent data, which are worthy of further development. Subsequently, the monoclonal antibodies will be studied as drugs to form dosage forms suitable for clinical applications, improve the bioavailability of the antibodies and protect the antibodies from the negative effects of the in vivo environment. After the drugs are developed, the protective effect of the monoclonal antibodies will be further enhanced. In addition, the sequence of the antibodies developed in this protocol (especially the CDR region) is reported for the first time, and the ability of the antibodies to bind to Acinetobacter baumannii and the protective effect on infected animals have been verified experimentally. Therefore, the monoclonal antibodies of this protocol are non-obvious compared to the prior art, especially the 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, achieved 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 failed to protect all mice from death ( Figure 9C). Thus, the simultaneous use of C3 and G4, while maintaining the same total dose, effectively boosts the immune response to 100% survival, demonstrating a certain synergistic effect between C3 and G4. Without this synergistic effect, at the same total dose, the combined effect of C3 and G4 would be somewhere between the effects of either monoclonal antibody alone, i.e., between 62.5% and 75%. There is no synergistic effect between C3, G4, and other monoclonal antibodies. In fact, using all four antibodies simultaneously reduces the survival rate to nearly 40%, even lower than the effect of any single antibody alone. Therefore, the combined use of C3 and G4 achieved an unexpected technical benefit, guaranteeing a 100% survival rate. If a single antibody is required, C3 is the most effective, and C3 is preferred for preparing a single-component antibody formulation.

[0094] In the medical practice of treating Acinetobacter baumannii infections, single-component antibody therapy and cocktail therapy using a combination of multiple antibodies each have their own advantages. Medical personnel or drug researchers can choose the appropriate method based on actual conditions. The successful development of this technical solution provides medical personnel or drug researchers with a variety of options. 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, relatively controllable drug effects, and relatively low manufacturing costs.

[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 symptoms of aspiration pneumonia very similar to the typical clinical manifestations of Acinetobacter baumannii infection ( Figure 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 to mice treated with an isotype control mAb ( Figure 10 B). Following infection with Acinetobacter baumannii, increased severity of inflammation, leukocyte infiltration, and cytokine burst were observed. To evaluate the potential anti-inflammatory effects of Omp38-specific monoclonal antibodies, we investigated their effects on cytokine concentrations in the lungs. Treatment with Omp38-specific monoclonal antibodies 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 ( Figure 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 ( Figure 10 D. Figure 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 ( Figure 10 E). 24 hours after infection, lung injury was assessed by the acute lung injury (ALI) scoring system, and mice treated with Omp38-specific monoclonal antibodies scored lower than those treated with isotype controls ( Figure 10 F), showing that Omp38-specific monoclonal antibodies reduced damage to lung tissue structure.

[0097] Example 5: Conformational Analysis of Omp38 Monoclonal Antibody C3 Binding

[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 ( Figure 12 A). In addition, we found three loop structures in this domain ( Figure 12 A and 12B). Residues within 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 subsequently analyzed these strains ( Figure 12 C). The 11 strains were ultimately divided into five groups, and we identified five binding conformations based on homology modeling. To explore whether changes in the loops affect the binding affinity of C3, we performed 10 ns kinetic calculations on each complex and calculated the interaction energy between C3 and different A. baumannii strains using the CHARMm tool in Discovery Studio 2.5. Notably, C3 exhibited similar interaction energies for various strains, which is consistent with the ELISA results ( Figure 8 B), indicating that C3 has broad-spectrum binding activity ( Figure 12 D).

[0099] We further analyzed five binding conformations, focusing on the interactions between the three loop regions and the C3 CDRs of the various A. baumannii strains mentioned above. Since hydrogen bonds are crucial for achieving high specificity and affinity in antigen-antibody interactions in most cases, we used the reference strain as an example and, based on its 3D spatial structure, highlighted the key hydrogen bonds involved in the interactions between the light and heavy chains and the loop CDRs ( Figure 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 ( Figure 12 E). Several key residues are highly conserved in the five binding conformations, such as N23 in loop 1 ( Figure 12 E). Although other key residues vary in different strains, they still provide key hydrogen bonds for binding to the antibody. For example, when T122 in loop 3 is mutated to Y122, hydrogen bonds are still formed between the hydroxyl group and the corresponding residue of the antibody ( Figure 12 E) The conservation of this binding mode may explain the broad 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 defense for 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 investigated the effect of C3 on the pathobiology of A. baumannii. Compared with the isotype control, C3 inhibited biofilm formation, adhesion, and intracellular proliferation of A. baumannii ( Figure 13 A-13C).

[0102] Antibody-dependent cellular phagocytosis (ADCP) is an immune mechanism of elimination that targets infected cells with mAbs, promoting their removal from the body by phagocytic immune cells such as macrophages. A previous study showed that macrophages play an important role in the host's early defense against A. baumannii infection by effectively 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 ( Figure 13 D). These results confirm the role of C3 in antibacterial killing through induction of ADCP.

[0103] exist Figure 13In the figure, A shows the adhesion test results 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. B shows 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 shows 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 600 nm using a microplate reader. D is the phagocytosis assay 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 with gentle shaking, 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 was subsequently confirmed in vitro and in vivo. 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 assays showed that mAb C3 did not affect the viability 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. 72 hours after injection, serum was collected to measure serum biochemical parameters, 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 after treatment with C3, there were no changes in biochemical parameters and no pathological damage to organs.

[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 ( Figure 14 A). We established a lethal LAC-4 infection model under the same conditions as those for IgG2a-treated mice and treated mice with 15 mg / kg IgG1 monoclonal antibody ( Figure 14 B). Notably, three of the four candidate monoclonal antibodies (F4, C3, and G4) delayed mouse mortality, while A2 had no protective effect against LAC-4 infection. Interestingly, the dual 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) ( Figure 14 C) The experimental results showed that antibodies with an IgG1 constant region were not very effective, while antibodies with an IgG2a constant region were more effective.

[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 the treatment of lethal LAC-4 infection model mice with Omp38-specific monoclonal antibodies. Six mice were non-invasively intratracheally inoculated 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 an isotype control. Survival was monitored 72 hours after infection. (C) Kaplan-Meier survival curves for infected mice treated with a single monoclonal antibody or a mixture of monoclonal antibodies compared with an isotype control group (n = 6 per group). Survival curves were compared using the log-rank (Mantel-Cox) test.

[0109] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are 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 variations and improvements can be made, which should also be regarded as the scope of protection 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 description can be used to interpret the content of the claims.

Claims

1. A monoclonal antibody, characterized in that It is the monoclonal antibody C3; The amino acid sequence of the variable region of the heavy chain of monoclonal antibody C3 is shown in SEQ ID NO.1; the amino acid sequence of the variable region of the light chain of monoclonal antibody C3 is shown in SEQ ID NO.

5.

2. The monoclonal antibody according to claim 1, wherein The amino acid sequence of the heavy chain of monoclonal antibody C3 is shown in SEQ ID NO. 3; the amino acid sequence of the light chain of monoclonal antibody C3 is shown in SEQ ID NO.

7.

3. Use of a monoclonal antibody according to claim 1 or 2 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.

4. An antibody composition comprising a monoclonal antibody according to claim 1 or 2, characterized in that: Also included is monoclonal antibody G4; 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.

5. The antibody composition according to claim 4, characterized in that The dosage ratio of monoclonal antibody G4 and monoclonal antibody C3 is 1:

1.

6. The antibody composition according to claim 5, characterized in that The amino acid sequence of the heavy chain of monoclonal antibody G4 is shown in SEQ ID NO.11; the amino acid sequence of the light chain of monoclonal antibody G4 is shown in SEQ ID NO.

15.

7. Use of the antibody composition according to claim 6 in the preparation of a medicament for resisting Acinetobacter baumannii infection.

8. Use of the antibody composition according to claim 7 in the preparation of a drug for treating Acinetobacter baumannii infection, characterized in that: The raw materials of the medicine include pharmaceutically acceptable carriers.

Images