CN109651510B - anti-Eno 1 antibodies and uses thereof - Google Patents

Abstract

The invention discloses an anti-Eno1 antibody and its application, and relates to the technical field of antibodies. An antibody or fragment thereof specifically binding to fungal Eno1 protein, said antibody or fragment thereof having specific binding affinity to fungal Eno1 protein; said antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL) wherein, the heavy chain variable region (VH) comprises the following CDR combinations: VH-CDR1, VH-CDR2 and VH-CDR3; the light chain variable region (VL) comprises the following CDR combinations: VL-CDR1 , VL‑CDR2 and VL‑CDR3. The antibody provided by the invention has the ability to bind to the Eno1 protein of pathogenic fungi and inhibit fungal invasion of host cells, and can be used alone or in combination with existing antifungal chemicals to treat invasive fungal infections.

Description

Anti-Eno1 antibody and use thereof

technical field

The invention relates to the technical field of antibodies, in particular to an anti-Eno1 antibody.

Background technique

Fungal enolase 1 (enolase, Eno1), also known as 2-phospho-D-glycerol hydrolase, consists of 440 amino acids. The basic function of enolase is to participate in the glycolysis reaction and can catalyze 2-phosphoglycerate ( 2-PGE) interconverts with phosphoenolacetone. In recent years, studies have found that invasive fungal enolase is not only distributed in the cytoplasm, but also distributed on the surface of the invasive fungal cell wall. Eno1 molecules can infect hosts by binding to plasminogen to activate the fibrinolytic system. Plasminogen, the precursor of fibrinolytic enzymes, exists in most vertebrates and is a key component of the fibrinolytic system. Under the action of plasminogen activator (plasminogen activator), blood plasminogen will cut off the N-terminal partial sequence, and convert it into blood plasmin (plasmin) with serine protease activity, which can convert collagenase into Collagenase, together with it, then degrades fibrin and other extracellular matrices such as laminin and fibronectin. After enolase on the surface of invasive fungi binds to plasminogen, it will accelerate its activation into plasmin and trigger a series of downstream reactions, so that invasive fungi can use the host fibrinolytic system to degrade their tissue barriers for further tissue infection or migration. Inhibiting the activity of Eno1 molecules on the surface of fungal cells can effectively inhibit the fungal invasion of the host. It has been reported in the literature that rabbit serum immune to Candida albicans Eno1 protein can inhibit the adhesion of Candida albicans to human intestinal epithelial cells, suggesting that Eno1 protein plays an important role in the adhesion of Candida albicans to host epithelial cells. When mice were immunized with Candida albicans Eno1 protein and re-infected with Candida albicans, the amount of bacteria in the kidney, brain tissue, lung, and spleen of the mice decreased significantly. This phenomenon may be related to Th1-mediated adaptive immune response. In summary, the fungal Eno1 protein is highly conserved in pathogenic fungal cells and has important biological functions (closely related to fungal virulence, stress response, etc.); the localization in fungal cells is different from that in mammalian cells (Eno1 in mammalian cells is located in In the cytoplasm; fungal cell Eno1 is located on the surface of the cell wall), antibody drugs designed based on the Eno1 protein on the surface of the fungal cell wall will not affect the Eno1 protein of human cells, and will not be replaced by small molecule compounds (small molecule compounds can affect the human body through the cell membrane) Cellular Eno1 protein function); fungal Eno1 protein structure can induce protective antibody production in infected patients. Therefore, the Eno1 protein is an ideal target for anti-fungal infection antibody drugs.

In recent years, with the development of medical treatment technologies, such as hematopoietic stem cell transplantation, solid organ transplantation, high-strength immunosuppressants, broad-spectrum antibiotics, the wide use of various catheterization techniques, and the increase in intensive care patients, elderly patients, and AIDS patients The incidence of invasive fungal infection is increasing year by year. Invasive fungal infection ranks 4-6 among nosocomial infections in China, the United States and Europe. Due to the high adaptability of invasive fungi, they will also be highly resistant to drugs after they transform into hyphae and biofilms in the body. In addition, the research and development of new antifungal drugs and related early diagnostic methods are progressing slowly, making the fatality rate of invasive fungal infections As high as 40%, it poses a serious threat to human health. The fatality rate of candidemia is as high as 40%, and the fatality rate of invasive aspergillus infection is as high as 90% without timely diagnosis and treatment. It has become an infectious disease that seriously threatens human health.

At present, the available antifungal drugs in clinical practice are very limited, and azole antifungal drugs such as fluconazole, itraconazole, voriconazole, etc. are still widely used. With the long-term clinical application, the phenomenon of fungal drug resistance It is becoming more and more serious; some fungi such as Candida krusei and Aspergillus fumigatus are inherently resistant to drugs; fungi have high adaptability, and they will also be highly resistant to drugs after forming biofilms in the body and transforming into hyphae, and even "super drug resistance" "The prevalence of fungi and drug resistance have also become one of the main reasons for treatment failure of invasive fungal infections. Therefore, it is an urgent clinical problem to study new and efficient antifungal drugs. At present, there is no industrialized monoclonal antibody developed against fungal Eno1 protein.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, provide an antibody or its functional fragment specifically binding to the fungal Eno1 protein, and provide its use based on the antibody or its functional fragment.

In order to achieve the above object, the present invention provides the following technical solutions:

An antibody or fragment thereof that specifically binds to fungal Eno1 protein, said antibody or fragment thereof having specific binding affinity for fungal Eno1 protein;

The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL);

Wherein, the heavy chain variable region (VH) comprises the following CDR combinations: VH-CDR1, VH-CDR2 and VH-CDR3;

The light chain variable region (VL) comprises the following CDR combinations: VL-CDR1, VL-CDR2 and VL-CDR3;

The heavy chain variable region is:

VH-CDR1 amino acids: GFNIKDYY,

VH-CDR2 amino acids: IDPENGNN,

VH-CDR3 amino acid: ARYEGNYVSY,

The light chain variable region is:

VL-CDR1 amino acids: QSIVHSNGYTY,

VL-CDR2 amino acids: KVS,

VL-CDR3 amino acid: FQSSHVPYT.

Further, the heavy chain variable region (VH) comprises a sequence selected from the following: an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 1; preferably, the at least 75% identity For example at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity etc. Any percent identity > 75%.

And/or, the light chain variable region (VL) comprises a sequence selected from the group consisting of an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO:2. Preferably, said at least 75% identity is for example at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or even 99% identity, etc. Any percent identity > 75%.

Further, the antibody or fragment thereof comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from:

The heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 1;

A light chain variable region having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO:2.

Further, the antibody is any one of monoclonal antibody, single chain antibody, single domain antibody, fully or partially humanized antibody or chimeric antibody; preferably, the antibody is IgA, IgD, IgE, IgG or IgM, more preferably IgG1.

Said fragment is selected from scFv, Fab, F(ab') ₂ or Fv fragments of said antibody.

In another aspect, the present invention also provides a nucleic acid molecule encoding the heavy chain CDR, light chain CDR, heavy chain variable region, light chain variable region, heavy chain or light chain in the aforementioned antibody or fragment thereof.

In another aspect, the present invention also provides a conjugate comprising the aforementioned antibody or fragment thereof.

In another aspect, the present invention also provides a vector comprising the aforementioned nucleic acid molecule. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome or a phage vector, etc.

On the other hand, the present invention also provides a pharmaceutical composition, which comprises the aforementioned antibody or its fragment, or nucleic acid molecule, or conjugate, or carrier; and optional pharmaceutically acceptable excipients.

In another aspect, the present invention also provides the aforementioned antibody or fragment thereof, or the aforementioned nucleic acid molecule, or the aforementioned conjugate, or the aforementioned carrier, or the aforementioned pharmaceutical composition in preparation for preventing or treating fungal infection Uses in medicine.

In another aspect, the present invention also provides a method for diagnosing fungal infection, the method comprising making the aforementioned antibody or fragment thereof, or the aforementioned nucleic acid molecule, or the aforementioned conjugate, or the aforementioned carrier, or the aforementioned The pharmaceutical composition is contacted with a sample from a subject.

The fungal infection is skin and soft tissue infection, deep fungal infection or invasive fungal infection.

Preferably, the fungus is a pathogenic fungus capable of infecting mammals such as humans, preferably Candida, Cryptococcus and Mycete, such as Candida albicans.

Preferably, other antifungal drugs such as azole antifungal drugs (such as fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole, etc.), echinocandin antifungal drugs ( Such as caspofungin, anidifungin, micafungin, etc.), polyene antifungals (such as amphotericin, etc.), allylamine antifungals (such as terbinafine, etc.) or pyrimidine antifungals Fungal drugs (such as 5-fluorocytosine, etc.) to make combination drugs.

Because the present invention adopts the above technical scheme, compared with the prior art, as an example, it has the following advantages and positive effects: the antibody has a specific binding affinity to the fungal Eno1 protein, so it has the ability to bind and inhibit the Eno1 protein of the pathogenic fungus The ability of fungi to invade host cells can be used alone or in combination with existing antifungal chemicals to treat invasive fungal infections.

Most of the invasive fungal infections are immunocompromised patients. As a monoclonal antibody drug, the present invention has unique advantages in treating fungal infections from the perspective of immune regulation. At the same time, Eno1 is mainly located on the fungal cell wall, while mammalian cells have no cell wall structure, and there is no homologous protein on the surface of the cell membrane. The Eno1 monoclonal antibody drug mainly targets the fungal cell wall antigen, and the possibility of off-target is extremely low, and the potential side effects are small.

Description of drawings

Figure 1 shows the recombinant expression of the full-length sequence of the Candida albicans Eno1 protein fused to the His tag provided in the example of the present invention.

Fig. 2 is the purification of the recombinant Candida albicans Eno1 protein provided by the example of the present invention.

Fig. 3 is an analysis diagram of the results of detecting the binding of the antibody to the Eno1 protein by the ELISA method provided by the embodiment of the present invention.

Fig. 4 is the experimental result of Eno1 antibody inhibiting the activity of Candida albicans Eno1 protein provided by the embodiment of the present invention.

Fig. 5 is the replication of the animal model of candidemia provided by the embodiment of the present invention and the results of detection of the therapeutic effect of the monoclonal antibody.

Detailed ways

The anti-Eno1 antibody and its application disclosed in the present invention will be further described in detail below with reference to the accompanying drawings and specific examples. It should be noted that the technical features or combinations of technical features described in the following embodiments should not be regarded as isolated, and they can be combined with each other to achieve better technical effects. In the drawings of the following embodiments, the same reference numerals appearing in each drawing represent the same features or components, which can be applied in different embodiments. Therefore, once an item is defined in one figure, it does not require further discussion in subsequent figures.

It should be noted that the technologies, methods and devices known to those of ordinary skill in the relevant fields may not be discussed in detail, but under appropriate circumstances, the described technologies, methods and devices should be regarded as part of the authorized specification. In all examples shown and discussed herein, any specific values should be construed as illustrative only, and not as limiting. Therefore, other examples of the exemplary embodiment may have different values.

Example 1: Recombinant expression of the C-terminal sequence of the Candida albicans Eno1 protein fused to the His tag

Taking the C-terminal amino acid of Candida albicans Eno1 protein as the target sequence, the corresponding base sequence was artificially synthesized, and cloned into the Pet-21a plasmid containing His tag by using restriction sites NdeI and XhoI. The recombinant plasmid was transformed into competent cells BL21(DE3)pLysS, and a single colony was picked and inoculated into LB liquid medium containing 100 μg/ml ampicillin the next day, and cultured overnight at 37°C with shaking. The overnight culture solution was inoculated into LB liquid medium containing 100 μg/ml ampicillin at a ratio of 1:100, cultured with shaking at 200 rpm at 37°C until the OD ₆₀₀ was about 0.6-0.8, and IPTG was added to the solution to a final concentration of 0.5mM , induced at 37°C for 4.5h. Take the induced bacterial liquid, centrifuge at 8,000rpm for 3min to collect the bacterial cells, and store at -80°C. See Figure 1 for the recombinant expression of the full-length sequence of the Candida albicans Eno1 protein fused to the His tag.

The full-length amino acid sequence of Eno1 protein:

Express the base sequence of the full-length Eno1 protein of Candida albicans:

Embodiment 2: Purification of recombinant Candida albicans Eno1 protein

The Escherichia coli induced to express the recombinant Candida albicans Eno1 protein was crushed with an ultrasonic breaker, worked at 180W for 3s and intermittently for 3s, and the time was 7-9min; centrifuged at 13,000rpm for 30min, collected the supernatant, and sterilized it with a 0.22μmL filter; at room temperature Mix the Ni column and the supernatant on a rotary mixer for 1 hour, and load the Ni column into the packing column; use 5 times of the column bed volume to elute the non-specific binding protein with the Ni column (containing imidazole concentration 30mM), wash Until the protein chromogenic solution does not change color, then use 5 times the column bed volume of BB solution (containing imidazole concentration 300mM) to elute the target protein; use a 10KD concentrator tube to concentrate the eluate containing the target protein and replace the solvent with PBS buffer. The electropherogram is shown in Figure 2.

Embodiment 3: Recombinant Candida albicans Eno1 protein immunizes Balb/c mice

Referring to Antibodies a Laboratory Manual, Second Edition (Edward A. Greenfield 2012), 8-week-old Balb/c mice were immunized for a total of 42 days at intervals of 14 days. The immunogenic Candida albicans Eno1 protein was emulsified in complete or incomplete Freund's adjuvant, and it was injected unilaterally into the subcutaneous tissue of the back of the neck, the root of the tail, and the groin of the mouse, and into the peritoneal cavity. Blood was collected from the tail vein on the 35th day of immunization, and after the antibody titer was detected by ELISA, spleen cells of immunized mice were fused with myeloma cells.

Example 4: Screening, identification and antibody sequence determination of anti-Eno1 protein monoclonal antibody hybridoma cell lines

The recombinant Candida albicans Eno1 protein was used to immunize the spleen cells of Balb/c mice and myeloma cells P3X63Ag8.653 were fused by PEG or electrofusion, and the fused hybridoma cells were inoculated in 96-well plates, and 24 hours later, adding HAT medium and HT medium were used to screen hybridoma cells; after culturing in 96-well plates for 10-14 days, the cell supernatant was taken for ELISA experiments to screen hybridoma mother clones capable of secreting anti-Eno1 antibodies.

The hybridoma mother clones secreting anti-Eno1 antibody were added to a 96-well plate with feeder cells by the limiting dilution method. After 2-3 days, the monoclonal cells were observed under a microscope and labeled, and after the 7th day, they were screened by ELISA to secrete anti-Eno1 Monoclonal hybridoma cells for monoclonal antibodies.

After the monoclonal hybridoma cells secreting anti-Eno1 monoclonal antibody were expanded and cultured, the total RNA was extracted according to the instructions of the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.); the hybridoma cells were mixed with 5 × PrimeScript RTMaster Mix (Takara) Total RNA was reverse-transcribed into cDNA; using degenerate primers (AnkeKrebber.1997) and Extaq PCR reagent (Takara) to amplify antibody light chain variable region IgVL (κ) and heavy chain variable region _VH sequences; using PCR clean- Up Gel extraction kit (Macherey-Nagel Company) was used to purify the PCR amplification products; according to the instructions of the pClone007SimpleVector Kit kit (Qingke Biotechnology Co., Ltd.), the amplified PCR products were connected to the T vector and transformed into Escherichia coli competent cells, and the strain was expanded. After amplification and extraction of the plasmid, DNA sequencing was performed to obtain the sequence of the variable region of the monoclonal antibody.

Embodiment 5: ELISA method detects the combination of antibody and Eno1 protein

The fungal Eno1 protein was diluted to 1 μg/ml with PBS buffer, and 100 μl per well was coated on a 96-well plate (Microwell96F 167008, Thermo) and incubated overnight at 4°C; the next day, the 96-well plate was taken out and washed with PBST (containing 0.5% PBS) Plates, thoroughly shake off residual moisture after soaking for 1 min each time. Add 200 μl of PBST containing 5% BSA to the sample wells, place at 37°C for blocking for 1 hour; then wash the plate with PBST, and spin dry the water in the wells. Add 100 μl of the sample to be tested to the 96-well plate and incubate overnight at 4°C. After removing the 96-well plate, wash the plate with PBST, add 100 μl of secondary antibody to each well, and incubate at 37°C for 1 h. After washing 5 times with PBST, add 100 μl Substrate Solution (Invitrogen) to each well, and incubate at 37°C for 10 min; add 50 μl 2N sulfuric acid to each well to terminate the reaction, and then detect the absorbance at a wavelength of 450 nm in a microplate reader (Multiskcin FC, Thermo).

The test results are shown in Figure 3.

Example 6: Eno1 antibody inhibits Eno1 protein activity of Candida albicans

Mix 1ug of Eno1 protein with 9ug and incubate for 10 minutes in a 37 degree incubator. Add kit reagents (Sigma Company); Enolase Substrate Mix 2 μl, Enolase Converter 2 μl, Enolase Developer 2 μl Peroxidase Substrate 2 μl, and Enolase Assay Buffer to make up the reaction system to 50 μl. The mixed samples were incubated at 37°C for 2 h, and the absorbance at 570 nm was read with a multi-functional microplate reader.

The experimental results are shown in Figure 4.

Example 7: Candidemia Animal Model Replication and Monoclonal Antibody Therapeutic Effect Detection Candida in the logarithmic growth phase was cultured for 12 hours, washed 3 times with PBS buffer solution, resuspended in PBS buffer solution, and adjusted bacterial solution concentration. C57BL/6 mice were infected by tail vein injection. The model mice were randomly divided into model group, antifungal small molecule drug treatment group, monoclonal antibody drug treatment group, antifungal small molecule drug + monoclonal antibody drug treatment group according to body weight; 2 hours after Candida infection, animals in each group Corresponding drug treatment was given through the tail vein, and the model group was given the same volume of PBS buffer. The experimental animals were either observed and recorded the survival time every day, or the mice were sacrificed 48 hours after infection, and the kidneys were taken, weighed, homogenized, and spread on SDA solid medium to detect the tissue load.

The test results are shown in Figure 5.

Other implementations

In the above description, the disclosure of the present invention is not intended to limit itself in these respects. Rather, the various components may be selectively and operatively combined in any number within the intended scope of the present disclosure. In addition, terms like "comprising" and "having" should be interpreted as inclusive or open by default, rather than exclusive or closed, unless they are clearly defined to the contrary. All technical, technological or other terms have the meanings understood by those skilled in the art unless defined to the contrary. Common terms found in dictionaries should not be interpreted too ideally or too impractically in the context of the relevant technical document, unless the disclosure expressly defines them as such. Any changes and modifications made by those of ordinary skill in the field of the present invention based on the above disclosures shall fall within the scope of protection of the claims.

sequence listing

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<120> Anti-Eno1 antibody and use thereof

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Ala Cys Thr Thr Gly Ala Thr Cys Ala Thr Gly Gly Ala Thr Gly Cys

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Cys Ala Thr Thr Thr Gly Ala Cys Ala Ala Ala Ala Gly Cys Cys Cys Gly Gly Thr

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Thr Ala Cys Ala Ala Ala Gly Gly Thr Ala Ala Gly Gly Thr Thr Gly

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Thr Gly Cys Thr Thr Cys Ala Thr Cys Thr Gly Ala Ala Thr Thr Cys

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Ala Cys Gly Ala Cys Thr Thr Gly Gly Ala Cys Thr Thr Thr Ala Ala

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Ala Ala Ala Cys Cys Cys Cys Ala Gly Ala Ala Thr Cys Cys Gly Ala Cys

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Cys Cys Ala Thr Cys Thr Ala Ala Ala Thr Gly Gly Thr Thr Gly Thr

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Cys Thr Gly Gly Cys Cys Cys Cys Ala Cys Ala Ala Thr Thr Gly Gly Gly Cys

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Thr Gly Ala Cys Thr Thr Ala Thr Ala Thr Gly Ala Ala Cys Ala Ala

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Thr Thr Gly Ala Thr Thr Thr Cys Cys Gly Ala Ala Thr Ala Cys Cys

865 870 875 880

Cys Ala Ala Thr Thr Gly Thr Thr Thr Cys Thr Ala Thr Thr Gly Ala

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Ala Gly Ala Thr Cys Cys Ala Thr Thr Cys Gly Cys Thr Gly Ala Ala

900 905 910

Gly Ala Thr Gly Ala Cys Thr Gly Gly Gly Ala Thr Gly Cys Thr Thr

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Gly Gly Gly Thr Cys Cys Ala Cys Thr Thr Cys Thr Thr Thr Gly Ala

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Ala Ala Gly Ala Gly Thr Thr Gly Gly Thr Gly Ala Cys Ala Ala Gly

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Ala Thr Cys Cys Ala Ala Ala Thr Thr Gly Thr Cys Gly Gly Thr Gly

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Ala Thr Gly Ala Thr Thr Thr Gly Ala Cys Thr Gly Thr Cys Ala Cys

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Thr Ala Ala Cys Cys Cys Thr Ala Cys Cys Ala Gly Ala Ala Thr Cys

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Ala Ala Gly Ala Cys Thr Gly Cys Cys Ala Thr Thr Gly Ala Ala Ala

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Ala Gly Ala Ala Ala Gly Cys Cys Gly Cys Thr Ala Ala Thr Gly Cys

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Thr Thr Thr Gly Thr Thr Gly Thr Thr Thr Gly Ala Ala Gly Gly Thr Thr

1045 1050 1055

Ala Ala Cys Cys Ala Ala Ala Thr Thr Gly Gly Thr Ala Cys Thr Thr

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Thr Gly Ala Cys Thr Gly Ala Ala Thr Cys Thr Ala Thr Ala Cys Ala

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Ala Gly Cys Thr Gly Cys Thr Ala Ala Cys Gly Ala Thr Thr Cys Thr

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Thr Ala Cys Gly Cys Thr Gly Cys Thr Gly Gly Thr Thr Gly Gly Gly

1105 1110 1115 1120

Gly Thr Gly Thr Cys Ala Thr Gly Gly Thr Thr Thr Cys Cys Cys Cys Ala

1125 1130 1135

Cys Ala Gly Ala Thr Cys Cys Cys Gly Gly Thr Gly Ala Ala Ala Cys Cys

1140 1145 1150

Gly Ala Ala Gly Ala Thr Ala Cys Thr Thr Thr Cys Ala Thr Thr Gly

1155 1160 1165

Cys Thr Gly Ala Cys Thr Thr Gly Thr Cys Ala Gly Thr Thr Gly Gly

1170 1175 1180

Thr Thr Thr Ala Ala Gly Ala Thr Cys Thr Gly Gly Thr Cys Ala Ala

1185 1190 1195 1200

Ala Thr Cys Ala Ala Gly Ala Cys Thr Gly Gly Thr Gly Cys Thr Cys

1205 1210 1215

Cys Ala Gly Cys Thr Ala Gly Ala Thr Cys Thr Gly Ala Ala Ala Gly

1220 1225 1230

Ala Thr Thr Gly Gly Cys Cys Ala Ala Ala Thr Thr Gly Ala Ala Cys

1235 1240 1245

Cys Ala Ala Ala Thr Cys Thr Thr Thr Gly Ala Gly Ala Ala Thr Cys Gly

1250 1255 1260

Ala Ala Gly Ala Ala Gly Ala Ala Thr Thr Ala Gly Gly Thr Thr Cys

1265 1270 1275 1280

Thr Gly Ala Ala Gly Cys Thr Ala Thr Cys Thr Ala Cys Gly Cys Thr

1285 1290 1295

Gly Gly Thr Ala Ala Ala Gly Ala Thr Thr Thr Cys Cys Cys Ala Ala Ala

1300 1305 1310

Ala Gly Gly Cys Thr Thr Cys Thr Cys Ala Ala Thr Thr Gly Cys Thr

1315 1320 1325

Cys Gly Ala Gly

1330

Claims (9)

1. An antibody that specifically binds fungal Eno1 protein, said antibody having specific binding affinity for fungal Eno1 protein;

the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL);

wherein the heavy chain variable region (VH) comprises the following combination of CDRs: VH-CDR1, VH-CDR2 and VH-CDR3;

the light chain variable region (VL) comprises the following CDR combinations: VL-CDR1, VL-CDR2 and VL-CDR3;

the heavy chain variable region is:

EVQLQQSGAELVRPGALVKLSCKASGFNIKDYYMNWVKQRPEQGLEWIGWIDPENGNNIYDPKFQGKASITADKSSNTAYLQLSSLTSEDTAVYYCARYEGNYVSYWAQGTLVTVSA（SEQ ID NO: 1），

VH-CDR1 amino acids: the concentration of the glass fiber is GFNIKDYY,

VH-CDR2 amino acids: the number of IDPENGNN is equal to the number of IDPENGNN,

VH-CDR3 amino acids: the process of making an ArYEGNYVSY,

the light chain variable region is:

DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGYTYLEWYLQKPGQSPKLLMYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSSHVPYTFGGGTKLEIK（SEQ ID NO: 2），

VL-CDR1 amino acids: the number of the QSIVHSNGYTY is as follows,

VL-CDR2 amino acids: the process of the KVS is carried out,

VL-CDR3 amino acids: FQSSHVPYT.

2. The antibody of claim 1, wherein: the heavy chain variable region (VH) comprises a sequence selected from: an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 1;

and/or, the light chain variable region (VL) comprises a sequence selected from: an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO. 2.

3. The antibody of claim 1, wherein: the antibody comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:

a heavy chain variable region having the amino acid sequence shown as SEQ ID NO. 1 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID NO. 1;

and a light chain variable region having an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO. 2.

4. The antibody of any one of claims 1 to 3, characterized in that: the antibody is any one of a monoclonal antibody, a single-chain antibody, a single-domain antibody, a fully or partially humanized antibody or a chimeric antibody.

5. A nucleic acid molecule encoding the antibody of any one of claims 1 to 4.

6. A conjugate comprising the antibody of any one of claims 1 to 4.

7. A vector comprising the nucleic acid molecule of claim 5;

the vector is a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome or a phage vector.

8. A pharmaceutical composition comprising the antibody of any one of claims 1 to 4, or comprising the nucleic acid molecule of claim 5, or comprising the conjugate of claim 6, or comprising the vector of claim 7;

and optionally pharmaceutically acceptable adjuvants.

9. Use of the antibody of any one of claims 1 to 4, or the nucleic acid molecule of claim 5, or the conjugate of claim 6, or the vector of claim 7, or the pharmaceutical composition of claim 8, in the manufacture of a medicament for the prevention or treatment of a candida albicans fungal infection.

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