Antibodies or antigen-binding fragments thereof against hcp protein of acinetobacter baumannii and use thereof

Abstract

The application provides an antibody or antigen-binding fragment thereof against Hcp protein of Acinetobacter baumannii, which comprises an HCDR1 functional region peptide segment with an amino acid sequence of SEQ ID NO:1, an HCDR2 functional region peptide segment with an amino acid sequence of SEQ ID NO:2, and / or an HCDR3 functional region peptide segment with an amino acid sequence of SEQ ID NO:3. The application also provides use of the antibody or antigen-binding fragment thereof in diagnosis, prevention or treatment of Acinetobacter baumannii infection, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the fields of medicine and immunology, specifically to antibodies against Acinetobacter baumannii Hcp protein or their antigen-binding fragments and their applications. Background Technology

[0002] Acinetobacter baumannii is a Gram-negative bacterium widely distributed in the environment. Drug-resistant Acinetobacter baumannii has become a major epidemic pathogen in hospitals, exhibiting extremely high infectivity and pathogenicity in immunocompromised populations. With the continuous increase in the clinical isolation rate of Acinetobacter baumannii and the emergence of multidrug-resistant and pan-drug-resistant strains, infection with this bacterium has become a major public health problem both domestically and globally. In recent years, infections caused by Acinetobacter baumannii have two significant characteristics: persistently high incidence and persistently high resistance rates. To reduce the infection rate, resistance rate, and mortality rate of Acinetobacter baumannii, numerous treatment strategies have been developed in recent years both domestically and internationally. However, the development of new antimicrobial drugs takes a long time and cannot keep pace with the continuous development of drug resistance and adaptation mechanisms in Acinetobacter baumannii strains. Therefore, immunotherapy has emerged as a promising strategy to replace the use of antimicrobial drugs, shortening the development cycle and slowing down bacterial resistance.

[0003] As a core component of T6SS, the detection of hemolysin-coregulated protein (Hcp) is the most important method for verifying T6SS activity. In Acinetobacter baumannii, Hcp is not only a core protein of T6SS, but also an effector. This protein is often secreted into the supernatant of bacterial cultures, sputum of infected individuals, and even serum, indicating that T6SS is in an active state (T6SS+). T6SS plays a crucial role in the survival and pathogenicity of Gram-negative bacteria. For example, Hcp proteins are important in the biofilm formation of *Burkholderia cepacia* and *Acinetobacter baumannii*; the Hcp protein family is involved in the inflammatory response, cytoskeleton remodeling, and apoptosis of human brain microvascular endothelial cells (HBMEC) induced by E. coli K1; furthermore, studies have found that negative regulators of T6SS exist on drug resistance plasmids in *Acinetobacter baumannii*, indicating a strong association between T6SS and the bacterium's drug resistance. T6SS was also one of the earliest proteins used as a vaccine target, playing a significant role in bacterial immune evasion by non-specifically binding to the Fc fragment of antibodies to evade antibody killing. Currently, there are no antibody drugs for *Acinetobacter baumannii* used clinically. The emergence of therapeutic antibodies has significant practical and strategic implications for effectively controlling *Acinetobacter baumannii* infection and large-scale outbreaks, and for significantly reducing community infection rates and hospital infection mortality rates.

[0004] Based on the aforementioned background technology, it is both necessary and urgent to develop antibodies against Acinetobacter baumannii. Summary of the Invention

[0005] To address the aforementioned needs, this invention provides an antibody against Acinetobacter baumannii Hcp protein and the nucleotide sequence of its encoding gene; this invention also provides the application of this antibody in the preparation of reagents that specifically bind to Acinetobacter baumannii Hcp protein, and in the diagnosis of Acinetobacter baumannii infection.

[0006] This invention first provides an antibody against Acinetobacter baumannii Hcp protein or an antigen-binding fragment thereof, which includes a heavy chain variable region, said heavy chain variable region comprising:

[0007] The HCDR1 functional region peptide with the amino acid sequence of SEQ ID NO:1, the HCDR2 functional region peptide with the amino acid sequence of SEQ ID NO:2, and / or the HCDR3 functional region peptide with the amino acid sequence of SEQ ID NO:3.

[0008] In one embodiment of the invention, a light chain variable region is further included, the light chain variable region comprising:

[0009] The LCDR1 functional region peptide with the amino acid sequence of SEQ ID NO:4, the LCDR2 functional region peptide with the amino acid sequence of SEQ ID NO:5, and / or the LCDR3 functional region peptide with the amino acid sequence of SEQ ID NO:6.

[0010] In one embodiment of the invention, the amino acid sequence of the non-CDR functional region of the heavy chain variable region has 75%, 80%, 85%, 90%, 95%, or 100% similarity to the heavy chain variable region with amino acid sequence SEQ ID NO:7.

[0011] In one embodiment of the invention, the amino acid sequence of the heavy chain is SEQ ID NO:8.

[0012] In one embodiment of the invention, the amino acid sequence of the non-CDR functional region of the light chain has 75%, 80%, 85%, 90%, 95%, or 100% similarity to the variable region of the light chain with amino acid sequence SEQ ID NO:9.

[0013] In one embodiment of the invention, the amino acid sequence of the light chain is SEQ ID NO:10.

[0014] In one embodiment of the present invention, it further comprises an Fc segment, wherein the FC segment is a murine or humanized peptide segment; preferably, it comprises a heavy chain with the amino acid sequence SEQ ID NO:8 and a light chain with the amino acid sequence SEQ ID NO:10.

[0015] Another aspect of the present invention provides a gene encoding the antibody or its antigen-binding fragment described above, comprising a heavy chain encoding gene with the nucleotide sequence SEQ ID NO:11, and / or a light chain encoding gene with the nucleotide sequence SEQ ID NO:12.

[0016] Another aspect of the present invention provides the use of the above-described antibody or its antigen-binding fragment in the preparation of reagents for the diagnosis, prevention or treatment of Acinetobacter baumannii infection; preferably, the use is for the specific binding of Acinetobacter baumannii Hcp protein.

[0017] The present invention further provides a composition for diagnosing, preventing or treating Acinetobacter baumannii infection, comprising the above-described antibody or its antigen-binding fragment.

[0018] The beneficial effects of the above-described technical solution of the present invention are as follows:

[0019] The antibody or its antigen-binding fragment provided by this invention can specifically bind to the Acinetobacter baumannii Hcp protein, and can be used to prepare reagents for diagnosing Acinetobacter baumannii infection. Attached Figure Description

[0020] Figure 1 This is an image showing the SDS-PAGE detection results of the Hcp monoclonal antibody in this invention.

[0021] Figure 2 This is a graph showing the detection of Hcp monoclonal antibody and Hcp activity in this invention. Detailed Implementation

[0022] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0023] Unless otherwise specified, all reagents used in this embodiment are of analytical grade, and the progress of all chemical reactions is detected by thin-layer chromatography.

[0024] Example 1: Obtaining lymphocytes from mouse spleen and lymph nodes

[0025] Lymphocytes were obtained from mice immunized with Hcp protein. Mouse lymphocyte culture medium was prepared as follows: Fetal bovine serum (FBS) was thawed in a 37°C water bath beforehand, and 2 ml of FBS was added to 100 ml of RPMI 1640 to prepare R2 culture medium (final serum concentration 2%). The operation was performed in a fume hood. Mice were euthanized by cervical dislocation and placed in a right lateral decubitus position to expose the spleen, which was then disinfected with alcohol. The skin was grasped with forceps, a small incision was made with scissors, and the inner peritoneum was separated. The spleen was gently pulled out of the peritoneum with the forceps, and fat, connective tissue, and connecting blood vessels were removed. The separated spleen was placed in the culture medium. Culture dishes were prepared in advance, each containing 2 ml of red cleavage solution. The separated spleen was placed on a 70 μm filter screen, and the spleen was ground and broken up against the screen using the plunger of a syringe in a short time to disperse it into single cells. After grinding, an appropriate volume of R2 (approximately 3 ml) was added to thoroughly rinse out any remaining cells from the filter screen. Add the rinsed culture medium containing cells to a centrifuge tube, centrifuge (1800 rpm / 4℃ / 6 min), discard the supernatant, and resuspend an appropriate amount (depending on the cell volume, generally 1.5-3 ml) in R2.

[0026] Cut open the mouse's abdomen at the midpoint of its forehead and abdomen to expose the lateral thigh. Push the skin aside and locate the bean-shaped lymph node on the inner side of the lateral thigh. Remove the lymph node. Prepare culture dishes, adding 1 ml of R2 to each. Select a rough-surfaced glass slide, moisten it, and place the lymph node between two rough surfaces. Crush the lymph node and, after grinding, use a pipette to draw culture medium and wash the cells off the slide. Add the washed culture medium containing cells to a centrifuge tube and centrifuge (1800 rpm / 4℃ / 6 min). Discard the supernatant and resuspend the cells in fresh culture medium to obtain a cell suspension.

[0027] Example 2: Flow cytometry sorting of single Hcp antigen-specific memory B lymphocytes

[0028] Hcp protein was biotinylated and co-incubated with the aforementioned lymphocytes. CD19 was used. + IgG + SpA5 PE-CY7+ CD3 - CD14 - CD56 - Using a gating method, individual SpA5 antigen-specific memory B lymphocytes were sorted, collected in DMEM medium containing 20%, placed on ice, and used for 10× single-cell library construction within 30 minutes.

[0029] Example 3: 10×Genomics single-cell BCR sequencing

[0030] Gel beads containing barcode information were mixed with cells, enzymes, and isolated oil beads to form GEMs (GelBeads-In-Emulsion). Theoretically, each GEM encapsulates a single cell and can function as a separate reaction system. Subsequently, mRNA reverse transcription was performed. The barcode sequence in the V(D)J library (purchased from 10X Genomics, catalog number PN-1000263) was linked to a switch oligo sequence. The mRNA was first reverse transcribed into cDNA using ploy(T) primers (purchased from 10X Genomics, catalog number PN-1000263). When reverse transcription reached the 5' end, the sequence CCC was added to the end, complementary to the GGG sequence of TSO, and transcription continued, thus adding the barcode sequence to the 5' end of the cDNA sequence. Then, V(D)J region enrichment was performed. Nested PCR amplification was performed using universal primers with a 5' adapter and sequential nested primers for the BCR constant region (purchased from 10X Genomics, catalog number PN-1000253) to enrich the V(D)J region of the BCR. The reagent used was the V2 Chemistry Dual Index at the 5' end. Finally, a library was constructed. The V(D)J cDNA was randomly fragmented using enzymes. By controlling the fragment lengths, the final sequence could cover the entire V(D)J fragment cDNA sequence. End repair with A was added, and the sequencing adapter P7 was added. PCR amplification was then performed to construct the BCR V(D)J sequencing library. After the constructed library passed quality control, paired-end sequencing was performed using the Illumina platform using PE150. The nucleotide sequence of the heavy chain encoding gene is SEQ ID NO:11, and the nucleotide sequence of the light chain encoding gene is SEQ ID NO:12.

[0031] Example 4: Construction of antibody plasmid expression vector

[0032] Based on the BCR sequences (SEQ ID NO:11 and SEQ ID NO:12) obtained from single-cell sequencing, a fully murine anti-Hcp antibody expression vector was constructed by cloning NotI / XbaI into the vector pcDNA3.1(+) (synthesized by GenScript Biotech Co., Ltd.), and then transformed into TOP10 strains. The strains were inoculated into LB (Amp... + In the process, a large number of amplifications were performed, and plasmids were extracted rapidly.

[0033] Example 5: Expression and purification of whole-mouse anti-Hcp antibody

[0034] 5.1 293F cell resuscitation

[0035] (1) Take out 293F cells (purchased from Wuhan Shangen Biotechnology Co., Ltd.) from the liquid nitrogen tank, put them into EP gloves, and put them into a 37°C water bath as soon as possible to fully thaw them.

[0036] (2) Add 2 mL of OPM-293 CD05 Medium to the cells, mix well, transfer to a 10 mL centrifuge tube, centrifuge at 500×g for 5 min, remove the supernatant, add 2 mL of OPM-293 CD05 Medium, resuspend the cells, aspirate into a 250 mL culture flask, and add 48 mL of OPM-293 CD05 Medium.

[0037] (3) Shake the cells well and place them in a shaker in a 37°C constant temperature incubator for culture.

[0038] (4) Passage 1 flask of cells to 1 / 3.

[0039] 5.2 Cell Transfection

[0040] (1) Prepare two 1000mL culture flasks and adjust the concentration of 293F cells to 1×10^6 / mL. Culture them in a shaker at 37℃ for 4 hours.

[0041] (2) Shake to mix the melted plasmid, then briefly centrifuge to ensure the liquid is at the bottom of the EP tube.

[0042] (3) PEI melts at room temperature.

[0043] (4) Add 150 μg of H chain plasmid to each 50 mL centrifuge tube and 200 μg of L chain plasmid to each 50 mL centrifuge tube. Gently rotate the centrifuge tube to mix it thoroughly.

[0044] (5) After vortexing, briefly centrifuge the PEI and add 10ml-V(H)-V(L)PEI (1μg / μL) to each 50mL centrifuge tube. Quickly tighten the cap, shake to mix thoroughly, and incubate at 37℃ for 15min.

[0045] (6) Carefully add the transfection mixture along the side wall of the bottle to the culture flask containing 293F cells prepared in step (1), and incubate for 5 days in a shaker in a 37°C, 5% CO2 incubator.

[0046] 5.3 Antibody Purification

[0047] (1) Collect 293F cells that have been transfected for 5 days into a plastic bottle, centrifuge at 3000×g at room temperature for 35 min, and collect the supernatant.

[0048] (2) Add anti-human IgG Protein Abeads (purchased from Beyotime, catalog number P2015-200ml) packing material to the affinity chromatography column and wash twice with PBS.

[0049] (3) Collect the transfection supernatant after centrifugation, add all of the Protein A beads suspension to the transfection supernatant, and mix thoroughly.

[0050] (4) Wrap the food in plastic wrap and shake it overnight on a 13.0 rpm rotating shaker.

[0051] (5) Pass the suspension of transfection supernatant after Protein A beads through a column under gravity and discard the flow-through liquid.

[0052] (6) Use about 20 mL of Protein A IgG Binding buffer (purchased from Thermo Scientific, catalog number 21001) to pass through the column and elute the beads under gravity.

[0053] (7) Repeat (6) twice.

[0054] (8) Add 2 mL of 1 M Tris HCl (purchased from Solarbio, catalog number T1160) to a 50 mL ultrafiltration tube, add 10 mL of IgG Elution buffer (purchased from Thermo Scientific, catalog number 21001) to the column, and collect the eluent using an ultrafiltration tube.

[0055] (9) Gently shake the liquid in the ultrafiltration tube to mix it, centrifuge at 3000×g and 4℃ for 30min to concentrate the liquid to about 0.2mL, and discard the ultrafiltration tube to filter out the liquid.

[0056] (10) Add 10 mL of PBS to the concentrate, shake gently to mix, centrifuge at 3000×g, 4℃ for 30 min, concentrate the liquid to about 0.2 mL, discard the ultrafiltration tube and filter out the liquid.

[0057] (11) Repeat (6) once.

[0058] (12) The protein content of the ultrafiltration concentrate was detected by NanoDrop and stored at 4°C.

[0059] Example 6: SDS-PAGE Detection and Identification of Whole-Rat Anti-Hcp Antibody

[0060] (1) Prepare SDS-PAGE gel according to the instructions of the Yaxin PAGE Gel Rapid Preparation Kit.

[0061] (2) Take 2 μg of purified antibody, adjust the volume to 12 μL with ultrapure water, and add 3 μL of protein loading buffer (non-reduced). Take another 2 μg of the same purified antibody, adjust the volume to 12 μL with ultrapure water, and add 3 μL of protein loading buffer (reduced).

[0062] (3) Place the above antibodies in a metal bath and heat at 90°C for 10 minutes.

[0063] (4) Fix the SDS-PAGE gel onto the gel plate and place it in the electrophoresis tank, then add the electrophoresis solution.

[0064] (5) Take 10 μL of heated protein and add it to the corresponding well, and add 2 μL of marker to the marker well.

[0065] (6) Connect the electrodes, turn on the electrophoresis apparatus, adjust the voltage to 80V, electrophore until the sample bands separate, adjust the voltage to 120V, reach 1cm from the bottom of the PAGE gel, and turn off the power.

[0066] (7) Use tweezers to pry open the gel preparation plate, carefully remove the PAGE gel, and place it in the staining box.

[0067] (8) Add instant blue to the staining box so that the staining solution covers the PAGE gel, place it on a horizontal shaker, shake it slowly, and stain for 15 minutes at room temperature.

[0068] (9) Discard the instant blue dye, add tap water until it covers the PAGE gel, place it on a horizontal shaker, shake slowly, and decolorize at room temperature until the base color is completely removed. Then remove the PAGE gel and take a picture (e.g. Figure 1 (As shown).

[0069] The heavy chain amino acid sequence of the obtained monoclonal antibody is SEQ ID NO:8, and the heavy chain variable region amino acid sequence is SEQ ID NO:7. The amino acid sequences of the heavy chain HCDRs are HCDR1 SEQ ID NO:1, HDR2 SEQ ID NO:2, and HCDR3 SEQ ID NO:3, respectively.

[0070] The amino acid sequence of the light chain is SEQ ID NO:10, the amino acid sequence of the variable region of the light chain is SEQ ID NO:9, and the LCDRs sequences of the light chain are LCDR1 SEQ ID NO:4, LCDR2 SEQ ID NO:5, and LCDR3 SEQ ID NO:6, respectively.

[0071] Example 7 Evaluation of the binding activity of the whole-mouse anti-Hcp antibody

[0072] (1) Coating: Dilute the antigen Hcp with coating solution to 2 μg / mL and add it to the ELISA plate at a rate of 100 μL / well. Incubate overnight at 4°C.

[0073] (2) Sealing: Wash the plate once (3 cycles, 300 μL per cycle), add 300 μL of BSA blocking solution to each well, and seal at 37°C for 2 hours.

[0074] (3) Dilution of monoclonal antibodies: The purified monoclonal antibodies were diluted with PBST to 2 μg / mL; positive serum from mice was used as a positive control and diluted 100 times with PBST; and irrelevant antibodies were used as a negative control.

[0075] (4) Serial dilution: Add 100 μL of PBST to all wells except for rows 1 and 12. Add 200 μL of the monoclonal antibody diluted in (3) to row 1 of the coated ELISA plate. Transfer 100 μL from row 1 to row 2 and mix 10 times with a multi-channel pipette. Perform serial dilutions starting from row 1. Serially dilute up to row 11, discarding 100 μL of the solution.

[0076] (5) Add the diluted positive control, negative control and blank control to the corresponding wells in row 12 according to the sample loading template, 100 μL / well. Seal the ELISA plate with plastic wrap and incubate at 37°C for 1 h.

[0077] (6) Add secondary antibody: Wash the plate once (3 cycles, 300 μL per cycle), and dilute the secondary antibody with PBST at a ratio of 1:10000. Add 100 μL of the diluted secondary antibody to each well and incubate at 37°C for 45 min.

[0078] (7) Development and termination: Wash the plate once (3 cycles, 300 μL per cycle), add 100 μL of TMB development solution to each well, place at 37°C in the dark for 5 min, and terminate with 100 μL of termination solution.

[0079] (8) Detect the OD450 value using an ELISA reader (read the OD450 value within 15 minutes after termination). For example... Figure 2 As shown.

[0080] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

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Claims

1. An antibody against Acinetobacter baumannii Hcp protein or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: The HCDR1 functional region peptide with amino acid sequence SEQ ID NO:1, the HCDR2 functional region peptide with amino acid sequence SEQ ID NO:2, and the HCDR3 functional region peptide with amino acid sequence SEQ ID NO:

3. The amino acid sequence of the heavy chain variable region is SEQ ID NO:7; The light chain variable region includes: The peptide segments of the LCDR1 functional region with the amino acid sequence of SEQ ID NO:4, the peptide segments of the LCDR2 functional region with the amino acid sequence of SEQ ID NO:5, and the peptide segments of the LCDR3 functional region with the amino acid sequence of SEQ ID NO:

6. The amino acid sequence of the variable region of the light chain is SEQ ID NO:

9.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The amino acid sequence of the heavy chain is SEQ ID NO:

8.

3. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The amino acid sequence of the light chain is SEQ ID NO:

10.

4. The antibody or antigen-binding fragment thereof as described in any one of claims 1-3, characterized in that, It also includes an Fc segment, which is a murine peptide segment.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, It contains a heavy chain with the amino acid sequence SEQ ID NO:8 and a light chain with the amino acid sequence SEQ ID NO:

10.

6. The gene encoding the antibody or its antigen-binding fragment as described in any one of claims 1-5, characterized in that, It includes a heavy chain coding gene with SEQ ID NO:11 and a light chain coding gene with SEQ ID NO:

12.

7. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-5 in the preparation of a reagent for diagnosing Acinetobacter baumannii infection.

8. A composition for diagnosing Acinetobacter baumannii infection, characterized in that, It comprises an antibody or an antigen-binding fragment thereof as described in any one of claims 1-5.

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