Monoclonal antibodies specifically targeting fungal enolase 1, their preparation and application

Abstract

This invention provides a monoclonal antibody specifically targeting fungal enolase 1, its preparation, and its application. The monoclonal antibody of this invention comprises the gene and amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL). The monoclonal antibody or antibody fragment of this invention specifically targeting fungal enolase 1 can bind with high affinity to the fungal Eno1 protein and also bind to fungal cells, thereby inhibiting fungal adhesion, infection, and invasion of host cells. The monoclonal antibody provided by this invention has the characteristics of high specificity and good efficacy. When used alone, it can significantly treat invasive fungal infections. When used in combination with existing antifungal small molecule compound drugs, it can significantly enhance the efficacy of the small molecule compound drugs, achieving better therapeutic effects.

Description

Technical Field

[0001] This invention belongs to the fields of monoclonal antibody technology, immunology technology and biomedicine, and specifically relates to a monoclonal antibody that specifically targets fungal enolase 1 and its preparation and application. Background Technology

[0002] Systemic fungal infections are one of the major diseases threatening human health today, with Candida albicans being the most common pathogen causing invasive fungal infections. This is especially true for patients with severe burns, HIV / AIDS, or those on long-term immunosuppressants who have immunodeficiency or dysbiosis of normal flora.

[0003] Candida albicans is the most common invasive pathogenic fungus in my country. Currently, the lack of early specific diagnostic techniques and low-toxicity, effective antifungal drugs is the biggest challenge that systemic fungal infections pose to clinical diagnosis and treatment.

[0004] Currently, the main antifungal drugs used clinically include polyenes (amphotericidal B), azoles (such as fluconazole), and echinocandins (caspofungin). To provide immediate treatment and prevent missing the optimal window for intervention, clinicians are often forced to prioritize the use of large doses of antifungal drugs, with azoles (fluconazole) still being the most commonly used. However, azoles are bacteriostatic agents, and long-term clinical use has led to high levels of resistance in fungi, with sensitivity decreasing by tens of times. Furthermore, the development of novel antifungal drugs is extremely slow. Candida albicans primarily infects immunodeficient or weakened hosts, making antibody therapy crucial for enhancing passive immunity. Therefore, developing monoclonal antibodies with highly effective antifungal activity and enhanced immunity could become an important means of preventing and treating fungal infections.

[0005] Enolase (Eno), also known as 2-phospho-D-glycerate hydrolyase, is responsible for catalyzing the conversion of 2-phosphoglyceric acid (2PG) to phosphoenolpyruvate (PEP) during cytoplasmic glycolysis, and is crucial for the growth and metabolism of organisms. Enolase may be encoded by multiple genes in different species. For example, the human genome has at least four genes encoding enolase (ENO1, ENO2, ENO3, and ENO4), and the genome of *Saccharomyces cerevisiae* contains at least five: ENO1, ENO2, ERR1 (Enolase-Related Region 1), ERR2, and ERR3. However, in the *Candida albicans* genome, there is only one enolase-encoding gene, ENO1, and the encoded protein is only one *Candida albicans* Eno1 (CaEno1). CaEno1 is the most abundant protein in Candida albicans and is a multifunctional protein distributed both inside and on the cell surface. Eno1 on the cell surface is an integrated cell wall protein, considered a major cell surface antigen and virulence factor, involved in the formation of invasive Candida albicans infection. After infection, the host can produce protective antibodies in response to stimulation. Studies by Pitarch et al. found that anti-Eno1 antibodies appeared in the serum of animal models of invasive Candida infection and clinical patients, and high titers of fungal Eno1 antibodies were found in the serum of patients with candidemia and better prognosis, suggesting that Eno1-IgG in the serum of Candida patients has great potential in the diagnosis of candidemia. Other studies have found that mice immunized with CaEno1 showed significantly reduced bacterial load in their kidney tissue and significantly increased survival rate when reinfected with Candida albicans.

[0006] In summary, CaEno1 is crucial for fungal cell growth, virulence, and pathogenicity. The localization of CaEno1 differs from that in mammalian cells: CaEno1 is abundant both intracellularly and on the cell surface, while in mammalian cells, Eno1 is only found in the cytoplasm. Targeting CaEno1 to screen and design specific monoclonal antibody drugs does not affect the function of Eno1 protein in human cells, offering an advantage over small molecule compound drugs (which can cross the cell membrane and affect intracellular Eno1 protein function). Therefore, CaEno1 is an ideal target for developing antibody drugs to treat invasive Candida albicans infections.

[0007] Despite extensive research on antifungal drugs based on monoclonal antibody immunotherapy over the past 30 years, no antifungal antibody drug has yet been approved for market release. In 2018, Professor An Maomao applied for a patent for "Anti-Eno1 Antibody and Its Use" (application number: 201811471173.6), but this patent only reported the effect of its antibody in combination with a known antifungal drug (anifenidine), without verifying its antifungal effect when used alone, nor providing results on its combination with other antifungal drugs. Furthermore, it did not verify whether its antibody could bind to fungal cells. Summary of the Invention

[0008] This invention is made to solve the above-mentioned problems, and aims to provide a monoclonal antibody that specifically targets fungal enolase 1, as well as its preparation and application.

[0009] This invention provides a monoclonal antibody that specifically targets fungal enolase 1, characterized in that: the monoclonal antibody specifically targeting fungal enolase 1 comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1:

[0010] MKLWLNWIFLVTLLNGIQCEVKLVESGGGLVQPGGSLRISCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKTKGYTTQYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCARDDDGVRFAYWGQGTLVTVSA,

[0011] The heavy chain variable region contains complementarity-determining regions HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is: DYYMS, the amino acid sequence of HCDR2 is: FIRNKTKGYTTQYSASVKG, and the amino acid sequence of HCDR3 is: DDDGVRFAY.

[0012] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.

[0013] MKLPVRLLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLIHRNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPFTFGSGTKLEIK,

[0014] The light chain variable region contains complementarity-determining regions LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is: RSSQSLIHRNGNTYLH, the amino acid sequence of LCDR2 is: KVSNRFS, and the amino acid sequence of LCDR3 is: SQSTHVPFT.

[0015] This invention also provides a gene encoding the above-mentioned monoclonal antibody specifically targeting fungal enolase 1, characterized by the following: the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:3:

[0016] ATGAAGTTGTGGCTGAACTGGATTTTCCTTGTAACACTTTTAAATGGTATCCAGTGTGAGGTGAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGTTCCTGAGAATCTCCTGTGCAACTTCTGGATTCACCTTCACTGATTACTACATGAGCTGGGTCCGCCAGCCTCCAGGAAAGGCACTTGAGTGGTTGGGTTTTAT TAGAAACAAAACTAAAGGTTACACAACACAGTACAGTGCATCTGTGAAGGGTCGGTTCACTATCTCCAGAGATAACTCCCAAAGTATCCTCTATCTTCAAATGAACACCCTGAGAGCTGAGGACAGTGCCACTTATTACTGTGCAAGAGATGATGACGGCGTCCGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA,

[0017] The nucleotide sequence of HCDR1 is: GATTACATGAGC.

[0018] The nucleotide sequence of HCDR2 is as follows:

[0019] TTTATTAGAAACAAAACTAAAGGTTACACAACACAGTACAGTGCATCTGTGAAGGGT,

[0020] The nucleotide sequence of HCDR3 is as follows:

[0021] GATGATGACGGCGTCCGGTTTGCTTAC,

[0022] The nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO:4:

[0023] ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCCGATCTAGTCAGAGCCTTATACACAGAAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCA GTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAAATAAAA,

[0024] The nucleotide sequence of LCDR1 is as follows:

[0025] CGATCTAGTCAGAGCCTTATACACAGAAATGGAAACACCTATTTACAT,

[0026] The nucleotide sequence of LCDR2 is as follows:

[0027] AAAGTTTCCAACCGATTTTCT,

[0028] The nucleotide sequence of LCDR3 is as follows:

[0029] TCTCAAAGTACACATGTTCCATTCACG.

[0030] This invention also provides a method for preparing a monoclonal antibody that specifically targets fungal enolase 1, characterized by the following steps:

[0031] Step S1: The full-length Candida albicans Eno1 protein molecule was obtained by expression and purification using a prokaryotic expression system;

[0032] Step S2: Using the full-length Candida albicans Eno1 protein molecule as an antigen, BALB / c female mice are immunized to obtain immunized mice. Spleen cells from the immunized mice are then collected and hybridoma cell lines are prepared using the hybridoma method. After one and two subclonal screenings, Eno1 monoclonal antibodies specifically targeting CaEno1 and their Eno1 monoclonal antibody cell lines are obtained.

[0033] The present invention also provides a method for diagnosing fungal infections, comprising contacting a monoclonal antibody or a fragment thereof that specifically targets fungal enolase 1 with a sample from a subject.

[0034] This invention also provides the application of monoclonal antibodies that specifically target fungal enolase 1 in the preparation of antifungal drugs and antifungal drug potentiators.

[0035] Furthermore, the present invention provides the use of a monoclonal antibody or antibody fragment thereof specifically targeting fungal enolase 1 in the preparation of antifungal drugs for the prevention or treatment of invasive fungal infections (such as Candida albicans) and antifungal drug potentiators.

[0036] Furthermore, the aforementioned antifungal drug potentiators are combined with antifungal small molecule compound drugs (such as fluconazole) to form combination drugs.

[0037] The role and effect of invention

[0038] According to the present invention, a monoclonal antibody specifically targeting fungal enolase 1, and its preparation and application, the present invention provides the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence of the monoclonal antibody specifically targeting fungal enolase 1, which are completely different from the sequences in existing patents. The monoclonal antibody or its antibody fragments specifically targeting fungal enolase 1 in the present invention can not only bind with high affinity to the fungal Eno1 protein, but also bind to fungal cells, thereby inhibiting fungal adhesion, infection, and invasion of host cells.

[0039] Meanwhile, the monoclonal antibody provided by this invention has the characteristics of high specificity and good efficacy. When used alone, the monoclonal antibody can significantly treat invasive fungal infections, and when used in combination with existing antifungal small molecule compound drugs, it can also significantly enhance the efficacy of small molecule compound drugs, achieving better therapeutic effects. Attached Figure Description

[0040] Figure 1 This is the expression of the Candida albicans Eno1 recombinant protein containing a 6*His-tag fusion tag in an embodiment of the present invention;

[0041] Figure 2 This is an electrophoresis image of the purification of the recombinant Candida albicans Eno1 protein in an embodiment of the present invention;

[0042] Figure 3 This refers to the detection results of the binding reaction during the screening of mouse immunization and monoclonal antibody cell lines in the embodiments of the present invention;

[0043] Figure 4This is the detection result of the ELISA method used in the embodiments of the present invention to detect the binding of monoclonal antibody to Candida albicans Eno1 antigen;

[0044] Figure 5 This is the detection result of the ELISA method used in the embodiments of the present invention to detect the binding of monoclonal antibody ENO1-B to Candida albicans Eno1 antigen;

[0045] Figure 6 The results of flow cytometry detection of the binding of monoclonal antibodies to Candida albicans are shown in the embodiments of the present invention.

[0046] Figure 7 This is the detection result of the monoclonal antibody inhibiting the adhesion of fungus SC5314 to cells in the embodiments of the present invention;

[0047] Figure 8 These are the results of live colony count detection in the construction of the Candida system infection mouse model and the in vivo efficacy detection of monoclonal antibodies in the embodiments of the present invention;

[0048] Figure 9 The results show the mouse survival time of the Candida system infection mouse model and the in vivo efficacy detection of monoclonal antibodies in the embodiments of the present invention. Detailed Implementation

[0049] This invention discloses a method for preparing a monoclonal antibody specifically targeting fungal enolase 1, comprising the following steps: Step S1, expressing and purifying the full-length Candida albicans Eno1 (CaEno1) protein molecule using a prokaryotic expression system (E. coli system). Step S2, immunizing BALB / c female mice with the full-length Candida albicans Eno1 (CaEno1) protein molecule as an antigen, collecting spleen cells from the immunized mice, preparing hybridoma cell lines using classic hybridoma technology, and then obtaining, through primary and secondary subclonal screening, a high-affinity and biologically active Eno1 monoclonal antibody specifically targeting CaEno1 and its Eno1 monoclonal antibody cell line. Finally, identifying the Eno1 monoclonal antibody cell line by subtype and sequencing the antibody to confirm the Eno1 monoclonal antibody ENO1-B subtype (IgG2 subtype) and heavy and light chain sequence composition.

[0050] To make the technical means, creative features, objectives and effects of this invention easier to understand, the following embodiments, in conjunction with the accompanying drawings, specifically illustrate the monoclonal antibody that specifically targets fungal enolase 1 and its preparation and application.

[0051] Example 1: Expression of Candida albicans Eno1 recombinant protein containing a 6*His-tag fusion tag

[0052] Using the complete protein amino acid sequence of Candida albicans Eno1 as the target sequence, the gene sequence of the complete Candida albicans Eno1 protein was artificially synthesized by our company. The gene sequence was then cloned into a pET-21a(+) plasmid containing a 6*His-tag using restriction enzyme sites (5' Nde I and 3' Xho I). After sequencing identification, the recombinant plasmid containing the accurate target sequence was transformed into E. coli competent cells BL21(DE3) pLysS and inoculated onto LB solid medium (containing 100 μg / ml Ampicillin). On the second day, single colonies were picked from LB resistant plates and cultured in LB liquid medium (containing 100 μg / ml Ampicillin) at 37°C with shaking for 8–12 h. Inoculate the bacterial culture at a ratio of 1:100 into 100 ml of LB liquid medium (containing 100 μg / ml Ampicillin) and incubate at 37°C with shaking at 240 rpm. When the OD600nm value of the bacterial culture reaches 0.6-0.8, add IPTG to the bacterial culture to a final concentration of 0.1-0.5 mM, and induce overnight at 16°C, or 12 h at 25°C, or 4-6 h at 37°C. Collect the induced bacterial culture, centrifuge at 13000 rpm for 5-10 min at 4°C, discard the supernatant, collect the bacterial cells, and freeze at -80°C for later use. The expression of the recombinant Candida albicans Eno1 protein containing the 6*His-tag fusion tag is as follows: Figure 1 As shown.

[0053] Full-length amino acid sequence of Eno1 protein:

[0054] MSYATKIHARYVYDSRGNPTVEVDFTTDKGLFRSIVPSGASTGVHEALELRDGDKSKWLGKGVLKAVANVNDIIAPALIKAKIDVVDQAKIDEFLLSLDGTPNKSKLGANAILGVSLAAANAAAAAQGIPLYKHIANISNAKKGKFVLPVPFQNVLNGGSHAGGALAFQEFMIAPTGVSTFSEALRIGSEVYHNLKSLTKKKYGQSAGNVGDEGGVAPDIKTPKEALDLIMDAIDKAGYKGKVGIAMDVASSEFYKDGKYDLDFKNPESDPSKWLSGPQLADLYEQLISEYPIVSIEDPFAEDDWDAWVHFFERVGDKIQIVGDDLTVTNPTRIKTAIEKKAANALLLKVNQIGTLTESIQAANDSYAAGWGVMVSHRSGETEDTFIADLSVGLRSGQIKTGAPARSERLAKLNQILRIEEELGSEAIYAGKDFQKASQL

[0055] Gene sequence of 6*His-tagged Candida albicans Eno1 recombinant protein:

[0056] CAT ATGCTC GAG .

[0057] Example 2: Purification of Candida albicans Eno1 recombinant protein

[0058] The bacterial cells, frozen at -80°C, were resuspended in sterile PBS (pH=7.4). The cells were then sonicated in an ice-water bath (200W, 5s on, 10s off, total 10min) until the bacterial suspension was clear and transparent. The suspension was then centrifuged at 13000 rpm, 4°C for 30min, and the supernatant was collected. A small amount of the supernatant sample was analyzed by SDS-PAGE electrophoresis to detect recombinant protein expression. The electrophoresis result is shown below. Figure 2 As shown.

[0059] Under the premise of verifying the high-level expression of recombinant protein by SDS-PAGE electrophoresis, the supernatant was first filtered and sterilized using a 0.22 μm filter. Then, a Ni-NTA Agarose affinity column was added to the supernatant, and the mixture was stirred at room temperature for 60 min. The mixture was then allowed to stand at room temperature, allowing the Ni-NTA Agarose affinity column, now bound to the recombinant protein, to settle naturally under gravity. The supernatant was discarded, and the Ni-NTA Agarose affinity column was washed three times with filtered sterile PBS (pH=7.4) and resuspended before being added to a packing column. The column was first washed with 5 volumes of BD buffer (containing 30 mM imidazole) until the permeate solution containing Coomassie Brilliant Blue staining solution did not turn blue. Then, the target protein was eluted with 5 volumes of BB buffer (containing 150 mM imidazole), and the sample was collected. The protein purified by the Ni-NTA Agarose affinity column was further purified using a HiLoad 16 / 60 Superdex 200 prep grade column (GE Healthcare). After protein quantification, aliquot the protein into 100 μl tubes and store at -80°C until use.

[0060] Example 3: Screening, identification, and antibody sequencing of mouse immunization and monoclonal antibody cell lines.

[0061] Three BALB / c mice were immunized with recombinant Candida albicans Eno1 protein as an antigen (four immunizations plus one booster immunization). Mouse serum was analyzed by ELISA. Mice meeting quality requirements (1:4000 dilution, OD450nm value >1) were selected. Spleens were harvested under aseptic conditions, and spleen cells were fused with Sp2 / 0 myeloma cells and seeded into 96-well plates (coated with feeder cells). Hybridoma cells were successfully obtained through selective culture. The culture supernatant of the hybridoma cells was collected from the 96-well plates, and the antibody-antigen binding strength of each well was detected by ELISA (the cells were coated with Candida albicans Eno1 recombinant protein antigen one day prior to the immunization). The results are shown below. Figure 3As shown in Figure A, fusion cells from wells with strong binding reactions (i.e., high OD450nm values, indicating positive clones) were selected and subjected to subclonal selection and culture in a 96-well plate using a limiting dilution method (one-time subcloning). The antibody-antigen binding reaction strength in each well of the one-time subcloning plate was also detected by ELISA. The results are shown in Figure A. Figure 3 As shown in B, after culturing subclones with high OD450nm values, a second subclone selection was performed in 96-well plates. The antibody-antigen binding reaction strength in each well was detected by ELISA. The results are shown below. Figure 3 As shown in Figure C, based on the characteristics of hybridoma cells such as state, quality, and growth, suitable secondary subclonal hybridoma cell lines were selected for amplification culture and cryopreservation. After subtype identification of the secondary subclonal hybridoma cell lines, total RNA was extracted from the hybridoma cell lines, and antibody sequencing was performed using 5'RACE technology to confirm that the hybridoma cell lines were monoclonal antibody-producing hybridoma cell lines.

[0062] Example 4: ELISA detection of the binding of monoclonal antibody to Candida albicans Eno1 antigen

[0063] Add 100 µl of protein dilution buffer to each well of an ELISA-specific microplate (Nunc-Immuno, 96-well plate) and coat it with 0.1 µg of CaEno1 antigen. Then, place the plate with the sample added in a refrigerator at 4°C overnight. Discard the liquid in the plate, fill each well with PBST (PBS + 0.05% Tween-20), let stand for 1 min, then blot dry. Repeat five times, then invert and let stand for 5 min. Next, add 200 µl of 5% (v / w) BSA blocking solution to each well and incubate at 37°C for 1 h. Wash the plate five times with PBST, letting stand for 1 min each time, then invert and let stand for 5 min. Add different monoclonal samples (starting at the same concentration) to the first row of wells in a 96-well plate, serially diluted 2-fold from left to right, and incubate overnight at 4°C. Wash the plate five times with PBST, letting stand for 1 min each time, then invert and let stand for 5 min. Add 100 µl of anti-mouse secondary antibody (horseradish peroxidase HRP goat anti-mouse IgG) dilution (1:10000) to each well, incubate at 37°C for 1 h, wash the plate five times with PBST, letting stand for 2 min each time, then invert and let stand for 5 min. Add 100 µl of TMB substrate (Substratesolution from the ELISA kit) to each well under light-protected conditions and incubate at 37°C for 30 min. Stop the reaction by adding 50 µl of 1 M H₂SO₄ to each well. Measure the OD450nm value of each well using a microplate reader, record the raw data, and plot the data (X-axis: monoclonal antibody concentration; Y-axis: OD value). Analyze the data using Prism software.

[0064] Figure 4 This is the detection result of the ELISA method used in the embodiments of the present invention to detect the binding of monoclonal antibody to Candida albicans Eno1 antigen. Figure 5 This is the detection result of the ELISA method used in the embodiments of the present invention to detect the binding of monoclonal antibody ENO1-B to Candida albicans Eno1 antigen.

[0065] like Figure 4 and Figure 5 As shown, antigen-antibody interaction studies revealed that ENO1-B monoclonal antibody can specifically bind to the Candida albicans Eno1 antigen with high affinity.

[0066] Example 5: Flow cytometry detection of the binding of monoclonal antibodies to Candida albicans

[0067] Standard strain SC5314 was taken from -80℃, activated twice on SDA plates, and cultured in YPD liquid medium at 30℃ with shaking for 12-16 h. The overnight bacterial culture was centrifuged at 5000 rpm for 2-3 min, the supernatant was discarded, and the cells were resuspended after washing three times with sterile PBS containing 2% BSA. 500 μl of the bacterial culture was added to each 1.5 ml EP tube, and the mixture was pipetted to achieve a final bacterial concentration of 2 × 10⁻⁶. 6 Cells / ml. After incubating at 30℃ for 1 hour, wash three times with sterile PBS and discard the supernatant. Add 50 μg / ml IgG control, target antibody alone, and target antibody + 50 μg / ml FLC to the above 1.5 ml EP tubes, respectively. Incubate vertically at 4℃ overnight, centrifuge at 5000 rpm for 3 min, discard the supernatant, and wash three times with sterile PBS. Dilute FITC antibody with sterile PBS (protected from light, 1:2000), add 500 μl to each tube, incubate at 30℃ for 1 hour, centrifuge at 5000 rpm for 3 min, discard the supernatant, and wash three times with sterile PBS. Add 500 μl of FACS to each tube, mix well by pipetting, incubate for 5 min, centrifuge at 4℃, 1500 rpm for 3 min, and discard the supernatant. Add 400 μl of Fix buffer and mix thoroughly by pipetting. Incubate overnight at 4°C, protected from light. Filter through a 200-mesh sieve before flow cytometry. The detection process must be strictly performed according to the operating instructions of the flow cytometer (FACS Verse™, BD Biosciences).

[0068] Figure 6 This is the result of flow cytometry detection of the binding interaction between monoclonal antibodies and Candida albicans in an embodiment of the present invention.

[0069] like Figure 6As shown, flow cytometry results indicate that ENO1-B monoclonal antibody can bind to Candida albicans cells, and the more Candida albicans cells bind with increasing ENO1-B monoclonal antibody concentration, exhibiting a dose-dependent relationship.

[0070] Example 6: Antibodies affect the adhesion of Candida albicans to cells

[0071] Candida albicans SC5314 cultured in YPD liquid medium for 12 h was centrifuged at 3000 rpm for 5 min at 4°C, and the supernatant was discarded to collect the bacterial cells. The cells were washed three times with sterile PBS (pH=7.4) and centrifuged at 3000 rpm for 5 min at 4°C. The cells were then resuspended in DMEM medium containing 10% fetal bovine serum (FBS). The bacterial suspension was gently pipetted 60 times to disperse the cells, resulting in a uniform distribution of single bacterial cells. The cells were then counted to 2 × 10⁻⁶ cells using a hemocytometer. 6 The bacterial culture was serially diluted 10-fold to a final concentration of 200 cells / ml, and a certain concentration of monoclonal antibody was added. The mixture was then incubated. The bacterial culture was then evenly added to the bottom of the wells of a 6-well plate containing 80-90% confluence of human cells. The plate was incubated at 37°C with 5% CO2 for 1 hour to allow bacterial adhesion to the cells. The cell supernatant was carefully discarded, and the plate was washed three times with sterile PBS (pH=7.4). Wells not washed with PBS were used as positive controls. 1 ml of YPD medium (approximately 45°C) was added to each well. After the YPD medium solidified, the plate was inverted and incubated in a 30°C fungal incubator for 48 hours. The number of viable Candida albicans clones was counted, and the adhesion rate was calculated as (number of adhered clones / initial number of clones) × 100%.

[0072] Figure 7 This is the detection result of the monoclonal antibody inhibiting the adhesion of fungus SC5314 to cells in the embodiments of the present invention.

[0073] like Figure 7 As shown in the study on the interaction between Candida albicans and human cells, the results showed that the ENO1-B monoclonal antibody could significantly reduce the adhesion of Candida albicans to cells.

[0074] Example 7: Construction of a mouse model of systemic Candida infection and in vivo efficacy detection of monoclonal antibodies

[0075] After activating the standard strain SC5314 twice on SDA plates, single colonies were picked and inoculated into YPD liquid medium and cultured overnight (16 h) at 30°C with shaking (200 rpm / min). The cells were collected by centrifugation at 3000-5000 rpm for 5 min at 4°C, washed three times with sterile PBS, and resuspended in sterile PBS. The cells were then counted using a hemocytometer, and the bacterial concentration was adjusted to 5 × 10⁻⁶.6 cells / ml. Female C57BL / 6 mice aged 8-10 weeks were randomly divided into four groups (n=8 per group): model group, ENO1-B monotherapy group, FLC monotherapy group, and ENO1-B+FLC group. For the ENO1-B monotherapy group and the ENO1-B+FLC group, each mouse was injected with the monoclonal antibody ENO1-B (2 mg / kg / mouse) via the tail vein beforehand. Two hours after antibody injection, each mouse was injected with 200 μl of the above-mentioned bacterial solution via the tail vein, resulting in a bacterial load of 1 × 10⁻⁶ cells / ml per mouse. 6 CFU (Cellular Activated Cells) were administered to mice intraperitoneally 2 hours after infection. For the kidney bacterial load assay, mice were euthanized 2 days after infection. Kidneys from each group were harvested under aseptic conditions, homogenized using a vibratory homogenizer, and then serially diluted 10-fold with sterile PBS to create different concentrations. 100 μl of each homogenate was evenly spread onto SDA plates. The SDA plates were then incubated at 30°C for 48 hours. The number of Candida albicans colonies on the plates was counted, and the number of viable colonies (CFU) in the kidneys of each mouse was determined by logarithmic analysis. 10 Plot the CFU / g Kidney concentration on the y-axis. For the survival rate experiment, the survival status of mice was observed daily after drug administration. Dead mice were removed and their survival time was recorded. After 30 days of continuous observation, the remaining surviving mice were euthanized. The Kaplan-Meier method (Log-rank test) in GraphPad Prism 6.0 software was used to perform significance analysis on the mouse survival rate experiment results.

[0076] Figure 8 These are the results of live colony count detection in the construction of the Candida system infection mouse model and the in vivo efficacy detection of monoclonal antibodies in the embodiments of the present invention; Figure 9 The results show the mouse survival time of the Candida system infection mouse model and the in vivo efficacy detection of monoclonal antibodies in the embodiments of the present invention.

[0077] like Figure 8 and Figure 9 As shown, in vivo studies in mice revealed that ENO1-B monoclonal antibody not only significantly reduced the bacterial load in the kidney tissue of mice systemically infected with Candida albicans and prolonged the survival time of infected mice when used alone, but also significantly reduced the bacterial load in the kidney tissue of mice systemically infected with Candida albicans and prolonged the survival time of infected mice when used in combination with fluconazole, compared with fluconazole alone, thus significantly enhancing the in vivo efficacy of fluconazole and acting as an synergist.

[0078] The role and effect of the embodiments

[0079] Following the preparation of the ENO1-B monoclonal antibody according to Examples 1-7, the antifungal efficacy of the ENO1-B monoclonal antibody was studied in vitro and in vivo using the clinically opportunistic pathogenic fungus Candida albicans as a model organism. The in vitro results showed that the ENO1-B monoclonal antibody specifically binds to the Candida albicans antigen Eno1 with high affinity; it also binds to Candida albicans cells, and the number of bound Candida albicans cells increases with increasing ENO1-B monoclonal antibody concentration, exhibiting a dose-dependent relationship; furthermore, it significantly reduces the adhesion of Candida albicans to cells. According to the results of in vivo studies in mice, the ENO1-B monoclonal antibody obtained in the examples not only significantly reduced the bacterial load in the kidney tissue of mice systemically infected with Candida albicans and prolonged the survival time of infected mice when used alone, but also significantly reduced the bacterial load in the kidney tissue of mice systemically infected with Candida albicans and prolonged the survival time of infected mice when used in combination with fluconazole, compared with fluconazole alone, further significantly enhancing the in vivo efficacy of fluconazole and acting as an synergist.

[0080] Therefore, the monoclonal antibody that specifically targets fungal enolase 1 of the present invention has the ability to specifically bind to pathogenic fungi and can significantly inhibit the adhesion of pathogenic fungi to host cells. It has significant antifungal effects when used alone or in combination with known antifungal drugs, and the sequence of the monoclonal antibody is a novel sequence.

[0081] The above embodiments are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention.

Claims

1. A monoclonal antibody specifically targeting fungal enolase 1, characterized in that, The monoclonal antibody that specifically targets fungal enolase 1 contains a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the heavy chain variable region includes complementarity-determining regions HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is: DYYMS, The amino acid sequence of HCDR2 is: FIRNKTKGYTTQYSASVKG. The amino acid sequence of HCDR3 is: DDDGVRFAY. The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2. The light chain variable region includes complementarity-determining regions LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is: RSSQSLIHRNGNTYLH, The amino acid sequence of LCDR1 is: KVSNRFS, The amino acid sequence of LCDR1 is: SQSTHVPFT.

2. A gene encoding a monoclonal antibody specifically targeting fungal enolase 1 as described in claim 1, characterized in that, The nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO:

3. The nucleotide sequence encoding HCDR1 is: GATTACATGAGC. The nucleotide sequence encoding HCDR2 is as follows: TTTATTAGAAACAAAACTAAAGGTTACACAACACAGTACAGTGCATCTGTGAAGGGT, The nucleotide sequence encoding HCDR3 is as follows: GATGATGACGGCGTCCGGTTTGCTTAC, The nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO:

4. The nucleotide sequence encoding LCDR1 is as follows: CGATCTAGTCAGAGCCTTATACACAGAAATGGAAACACCTATTTACAT, The nucleotide sequence encoding LCDR2 is as follows: AAAGTTTCCAACCGATTTTCT, The nucleotide sequence encoding LCDR3 is as follows: TCTCAAAGTACACATGTTCCATTCACG.

3. The use of a monoclonal antibody specifically targeting fungal enolase 1 as described in claim 1 in the preparation of antifungal drugs and antifungal drug potentiators, wherein, The fungus is Candida albicans.

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