CN109651510B - anti-Eno 1 antibodies and uses thereof - Google Patents
anti-Eno 1 antibodies and uses thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56961—Plant cells or fungi
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
- G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida
Abstract
The invention discloses an anti-Eno 1 antibody and application thereof, relating to the technical field of antibodies. An antibody or fragment thereof that specifically binds 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 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 antibodies provided by the invention have the ability to bind to the pathogenic fungal Eno1 protein and inhibit fungal attack on host cells, and can be used alone or in combination with existing antifungal chemicals to treat invasive fungal infections.
Description
Technical Field
The invention relates to the technical field of antibodies, and particularly relates to an anti-Eno 1 antibody.
Background
Fungus enolase 1 (enolase, eno 1) is also called 2-phosphate-D-glycerolate hydrolase and consists of 440 amino acids, and the enolase has the basic function of participating in glycolysis reaction and catalyzing the interconversion of 2-phosphoglycerate (2-PGE) and phosphoenolacetone. In recent years, it has been found that invasive fungal enolases are distributed not only in the cytoplasm but also on the surface of the invasive fungal cell wall. The Eno1 molecule can infect a host by binding to plasminogen to activate the fibrinolytic system. Plasminogen is a precursor of plasmin, is present in most vertebrates and is a key component of the fibrinolytic system. Plasminogen is cleaved at the N-terminal part by plasminogen activator (plasminogen activator) and converted into plasmin (plasmin) with serine protease activity, which converts collagenase, and therewith degrades fibrin and other extracellular matrices such as laminin and fibronectin. After the enolase on the surface of the invasive fungus is combined with plasminogen, the enolase can be accelerated to be activated into plasmin and initiate a series of downstream reactions, so that the invasive fungus can degrade the tissue barrier of the invasive fungus by using a host fibrinolytic system to carry out tissue infection or migration and diffusion. The inhibition of the activity of Eno1 molecules on the surfaces of fungal cells can effectively inhibit the invasion of fungi to hosts. The literature reports that rabbit serum for immunizing candida albicans Eno1 protein can inhibit the adhesion of candida albicans to human intestinal epithelial cells, and the Eno1 protein plays an important role in the adhesion of candida albicans to host epithelial cells. When the candida albicans Eno1 protein is immunized by the mice to infect the candida albicans again, the bacteria carrying amount of the kidney, brain tissues, lung and spleen of the mice is obviously reduced. This phenomenon may be associated with Th 1-mediated adaptive immune responses. In conclusion, the fungus Eno1 protein is highly conserved in pathogenic fungus cells, and the biological function is important (closely related to the virulence, stress response and the like of the fungus); the positioning of the fungus cells is different from that of the mammal cells (the mammal cell Eno1 is positioned in cytoplasm, the fungus cell Eno1 is positioned on the surface of cell wall), the design of antibody drugs based on the Eno1 protein on the surface of the fungus cell wall can not influence the Eno1 protein of the human cells, and can not be replaced by small molecular compound drugs (the small molecular compound can penetrate through the cell membrane to influence the function of the Eno1 protein of the human cells); the fungal Eno1 protein structure can induce protective antibody production in infected patients. Therefore, the Eno1 protein is an ideal action target of the antifungal infection antibody medicine.
In recent years, the incidence of invasive fungal infections has increased year by year with the development of medical treatment technologies, such as hematopoietic stem cell transplantation, solid organ transplantation, high-strength immunosuppressive agents, broad-spectrum antibiotics, the widespread use of various catheterization techniques, and the increase in patients in intensive care, elderly, and aids. Invasive fungal infections are located at positions 4-6 in China, the United states and Europe. Because invasive fungi have high adaptability, the invasive fungi are highly resistant after being converted into hypha and form a biofilm in vivo, and the research and development of novel antifungal medicaments and related early diagnosis methods are slow, so that the fatality rate of invasive fungal infection reaches up to 40 percent, and the method poses serious threat to human health. The mortality rate of candidemia is as high as 40%, and the mortality rate of invasive aspergillus infection which cannot be diagnosed in time is as high as more than 90%, so that the candidemia becomes an infectious disease seriously threatening the health of human beings.
At present, clinically available antifungal drugs are very limited, azole antifungal drugs such as fluconazole, itraconazole, voriconazole and the like are still applied more frequently, and the drug resistance phenomenon of fungi is increasingly serious along with the long-term clinical application; some fungi such as candida krusei, aspergillus fumigatus and the like have inherent drug resistance; the fungus has the characteristic of high adaptability, can be highly resistant to drugs after biofilm is formed in vivo and converted into hypha, even the prevalence of 'super-resistant' fungus appears, and the drug resistance also becomes one of the main reasons for the failure of invasive fungal infection treatment. Therefore, the research of efficient novel antifungal drugs is a problem to be solved urgently in clinic. At present, no industrialized monoclonal antibody developed aiming at the Eno1 protein of the targeted fungi exists.
Disclosure of Invention
The invention aims to: overcomes the defects of the prior art, provides an antibody or a functional fragment thereof which specifically binds to fungal Eno1 protein, and provides application thereof based on the antibody or the functional fragment thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
an antibody or fragment thereof that specifically binds 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 combination of CDRs: VH-CDR1, VH-CDR2 and VH-CDR3;
the light chain variable region (VL) comprises a CDR combination of: VL-CDR1, VL-CDR2 and VL-CDR3;
the heavy chain variable region is:
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 method for preparing the ArYEGNYVSY,
the light chain variable region is:
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.
Further, the heavy chain variable region (VH) comprises a sequence selected from: and SEQ ID NO:1, an amino acid sequence having at least 75% identity to the amino acid sequence set forth in seq id no; preferably, the at least 75% identity is any percentage identity greater than or equal to 75%, such as at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity.
And/or, the light chain variable region (VL) comprises a sequence selected from: and SEQ ID NO:2, or a pharmaceutically acceptable salt thereof, and an amino acid sequence having at least 75% identity to the amino acid sequence set forth in figure 2. Preferably, the at least 75% identity is any percentage identity greater than or equal to 75%, such as at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity.
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 group consisting of:
as shown in SEQ ID NO:1 or an amino acid sequence substantially identical to SEQ ID NO:1, a heavy chain variable region having an amino acid sequence with at least 75% identity to the amino acid sequence set forth in seq id no;
and to SEQ ID NO:2, and a light chain variable region having an amino acid sequence with at least 75% identity to the amino acid sequence set forth in seq id No. 2.
Further, 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; preferably, the antibody is IgA, igD, igE, igG or IgM, more preferably IgG1.
Said fragment is selected from the group consisting of scFv, fab, F (ab') 2 Or an Fv fragment.
In another aspect, the invention also provides a nucleic acid molecule encoding a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, a heavy chain or a light chain as in the aforementioned antibody or fragment thereof.
In another aspect, the present invention also provides a conjugate comprising the aforementioned antibody or a fragment thereof.
In another aspect, the present invention also provides a vector comprising the aforementioned nucleic acid molecule. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome or a phage vector and the like.
In another aspect, the present invention also provides a pharmaceutical composition comprising the aforementioned antibody or fragment thereof, or nucleic acid molecule, or conjugate, or carrier; and optionally pharmaceutically acceptable adjuvants.
In another aspect, the present invention also provides a use of the aforementioned antibody or fragment thereof, or the aforementioned nucleic acid molecule, or the aforementioned conjugate, or the aforementioned vector, or the aforementioned pharmaceutical composition for the preparation of a medicament for preventing or treating fungal infection.
In another aspect, the present invention also provides a method of diagnosing a fungal infection, said method comprising contacting the aforementioned antibody or fragment thereof, or the aforementioned nucleic acid molecule, or the aforementioned conjugate, or the aforementioned vector, or the aforementioned pharmaceutical composition, with a sample from a subject.
The fungal infection is a skin and soft tissue infection, a deep fungal infection, or an invasive fungal infection.
Preferably, the fungus is a pathogenic fungus capable of causing an infection in a mammal, such as a human, preferably a fungus of the genera candida, cryptococcus and mycete, such as candida albicans.
Preferably, the combination drug can also be made in combination with other antifungal drugs such as azole antifungal drugs (e.g., fluconazole, itraconazole, voriconazole, posaconazole, isaconazole, etc.), echinocandin antifungal drugs (e.g., caspofungin, anidulafungin, micafungin, etc.), polyene antifungal drugs (e.g., amphotericin, etc.), allylamine antifungal drugs (e.g., terbinafine, etc.), or pyrimidine antifungal drugs (e.g., 5-fluorocytosine, etc.).
Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages and positive effects as examples: the antibodies have specific binding affinity for fungal Eno1 protein and thus have the ability to bind to pathogenic fungal Eno1 protein and inhibit fungal attack on host cells, and can be used to treat invasive fungal infections, either alone or in combination with existing antifungal chemicals.
Invasive fungal infection is mostly a patient with low immune function, and the monoclonal antibody drug for treating fungal infection has unique advantages in terms of immune regulation. Meanwhile, eno1 is mainly located on the fungal cell wall, mammalian cells have no cell wall structure, homologous proteins do not exist on the surface of the cell wall, the Eno1 monoclonal antibody medicine is mainly targeted on fungal cell wall antigens, the off-target possibility is extremely low, and the potential toxic and side effects are small.
Drawings
FIG. 1 shows the recombinant expression of the full-length sequence of the His-tag fused Candida albicans Eno1 protein provided by the embodiment of the present invention.
FIG. 2 shows the purification of recombinant Candida albicans Eno1 protein provided by the embodiment of the present invention.
FIG. 3 is a graph showing analysis of the results of detecting the binding of an antibody to Eno1 protein by ELISA method according to the embodiment of the present invention.
FIG. 4 shows the results of experiments on the activity of the Eno1 antibody of Candida albicans Eno1 protein.
FIG. 5 shows the results of the animal model replication of candidemia and the detection of the therapeutic effect of monoclonal antibody provided by the embodiment of the invention.
Detailed Description
The anti-Eno 1 antibodies disclosed herein and their uses are described in further detail below with reference to the accompanying drawings and specific examples. It should be noted that technical features or combinations of technical features described in the following embodiments should not be considered in isolation, and they may be combined with each other to achieve better technical effects. In the drawings of the embodiments described below, the same reference numerals appearing in the respective drawings denote the same features or components, and may be applied to different embodiments. Thus, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.
It is noted that techniques, methods and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail, but are intended to be part of the specification where appropriate. In all examples shown and discussed herein, any particular value should be construed as merely illustrative, and not limiting. Thus, other examples of the exemplary embodiments may have different values.
Example 1: recombinant expression of His-tag fused Candida albicans Eno1 protein C-terminal sequence
Taking C-terminal amino acid of Candida albicans Eno1 protein as a target sequence, artificially synthesizing a corresponding base sequence, and cloning the corresponding base sequence into a Pet-21a plasmid containing a His tag by utilizing enzyme cutting sites NdeI and XhoI. The recombinant plasmid was transformed into competent cells BL21 (DE 3) pLysS, and a single colony was picked the next day and inoculated into LB liquid medium containing 100. Mu.g/ml ampicillin, followed by shaking culture at 37 ℃ overnight. Inoculating overnight culture solution at a ratio of 1: 100 into LB liquid culture medium containing 100. Mu.g/ml ampicillin, and performing shaking culture at 200rpm at 37 deg.C until OD 600 About 0.6-0.8, IPTG was added to the bacterial suspension to a final concentration of 0.5mM, and induction was carried out at 37 ℃ for 4.5 hours. And (4) centrifuging the induced bacterial liquid at 8,000 rpm for 3min, collecting thalli, and storing at-80 ℃. Recombinant expression of the full-length sequence of the His-tag fused Candida albicans Eno1 protein is shown in FIG. 1.
Full-length amino acid sequence of Eno1 protein:
expressing the full-length base sequence of the Candida albicans Eno1 protein:
example 2: purification of recombinant Candida albicans Eno1 protein
Crushing escherichia coli for inducing expression of recombinant candida albicans Eno1 protein by using an ultrasonic crusher, working at 180W for 3s, and pausing for 3s for 7-9min;13 Centrifuging at 000rpm for 30min, collecting supernatant, and filtering with 0.22 μmL filter for sterilization; mixing the Ni column and the supernatant on a rotary mixer for 1h at room temperature, and loading the Ni column into a filler column; eluting with BD solution (containing imidazole at concentration of 30 mM) with 5 times of bed volume and Ni column nonspecific binding protein until the protein color developing solution does not change color, and eluting with BB solution (containing imidazole at concentration of 300 mM) with 5 times of bed volume to obtain target protein; the eluent containing the target protein is concentrated by a 10KD concentration tube to replace the solvent into PBS buffer. The electrophoretogram is shown in FIG. 2.
Example 3: balb/c mice immunized by recombinant candida albicans Eno1 protein
Referring to Antibodies a Laboratory Manual, second Edition (Edward A. Greenfield 2012), 8-week-old Balb/c mice were immunized in a total of 42 days at 14-day intervals. The immunogenic candida albicans Eno1 protein was emulsified in complete or incomplete freund's adjuvant and injected in unilateral fashion into the subcutaneous tissue and peritoneal cavity of the mouse at the dorsum cervicales, caudate root, groin 3. On the 35 th day of immunization, tail vein blood was collected, and spleen cells of immunized mice were fused with myeloma cells after antibody titer detection by ELISA.
Example 4: screening and identification of anti-Eno 1 protein monoclonal antibody hybridoma cell strain and determination of antibody sequence
Taking recombinant candida albicans Eno1 protein immune Balb/c mouse spleen cells and myeloma cells P3X63Ag8.653 to fuse by using a PEG or electrofusion method, inoculating the fused hybridoma cells into a 96-well plate, adding a culture medium containing HAT culture medium and HT after 24 hours, and screening the hybridoma cells; after culturing in a 96-well plate for 10-14 days, cell supernatants were taken for ELISA experiments to screen hybridoma clones capable of secreting anti-Eno 1 antibody.
Adding hybridoma mother clones secreting anti-Eno 1 antibodies into a 96-well plate paved with feeder cells by a limiting dilution method, observing and marking monoclonal cells under a microscope after 2-3 days, and screening monoclonal hybridoma cells capable of secreting anti-Eno 1 monoclonal antibodies by an ELISA (enzyme-linked immunosorbent assay) experiment after 7 days.
After the monoclonal hybridoma secreting the anti-Eno 1 monoclonal antibody is subjected to expanded culture, extracting total RNA of the cell according to the steps of an RNAfast200 kit (Shanghai Feijie Biotechnology Co., ltd.); reverse transcribing hybridoma cell total RNA to cDNA using 5 XPrimeScript RT Master Mix (Takara); amplification of antibody light chain variable region IgVL (kappa) and heavy chain variable region IgVL (kappa) Using degenerate primers (Anke Krebber. 1997) and Extaq PCR reagents (Takara)Variable region V H A sequence; purifying the PCR amplification product by using a PCR clean-up Gel extraction kit (Macherey-Nagel Co.); connecting the amplified PCR product to a T vector and transforming escherichia coli competent cells according to the specification of a pClone007Simplevector Kit (Scutellaria Biotech limited), and performing DNA sequencing after strain amplification and plasmid extraction to obtain a monoclonal antibody variable region sequence.
Example 5: ELISA method for detecting binding of antibody and Eno1 protein
Fungal Eno1 protein was diluted to 1. Mu.g/ml with PBS buffer and 100. Mu.l per well coated in 96-well plates (Microwell 96F 167008, thermo) and incubated overnight at 4 ℃; the next day the 96-well plates were removed and washed with PBST (0.5% PBS) and the residual water was spun off thoroughly after 1min of each soak. 200. Mu.l of BSA-containing PBST (5% assay) was added to each well and blocked at 37 ℃ for 1 hour; the plates were then washed with PBST and the wells were spin dried. 100. Mu.l of each sample to be tested was added to the 96-well plate and incubated overnight at 4 ℃. After the 96-well plate was removed and washed with PBST, 100. Mu.l of a secondary antibody was added to each well, and incubated at 37 ℃ for 1 hour. Washing with PBST for 5 times, adding 100 μ l of Substrate Solution (Invitrogen) per well, and incubating at 37 deg.C for 10min; after the reaction was terminated by adding 50. Mu.l of 2N sulfuric acid to each well, absorbance was measured at a wavelength of 450nm using a microplate reader (Multiskcin FC, thermo).
The results of the measurements are shown in FIG. 3.
Example 6: experiment for inhibiting activity of candida albicans Eno1 protein by Eno1 antibody
Mix 1ug Eno1 protein with 9ug and incubate for 10min at 37 ℃ incubator. Separately add kit reagents (Sigma company); enolase Substrate Mix 2. Mu.l, enolase Converter 2. Mu.l, enolase Developer 2. Mu.l Peroxidase Substrate 2. Mu.l the reaction system was made up to 50. Mu.l with Enolase Assay Buffer. And (3) putting the mixed sample into a 37 ℃ incubator for 2h, and reading the absorbance light value of 570nm by using a multifunctional microplate reader.
The results of the experiment are shown in FIG. 4.
Example 7: the candida blood animal model replication and monoclonal antibody treatment effect detection is to culture candida in a logarithmic growth phase for 12 hours, wash the candida in a PBS buffer solution for 3 times, then suspend the candida in the PBS buffer solution, and adjust the concentration of the bacteria solution. C57BL/6 mice were infected by tail vein injection. Randomly dividing the model mice into a model group, an antifungal small molecule drug treatment group, a monoclonal antibody drug treatment group, an antifungal small molecule drug + monoclonal antibody drug treatment group according to the body weight; 2 hours after Candida infection, each group of animals was given the corresponding drug treatment via the tail vein, and the model group was given an equivalent volume of PBS buffer. The survival time of experimental animals is recorded by observation every day, or the kidney of the mice is killed 48 hours after infection, weighed, homogenized and coated on SDA solid culture medium to detect the bacterial load of the tissues.
The results of the test are shown in FIG. 5.
Other embodiments
In the foregoing description, the disclosure of the present invention is not intended to limit itself to these aspects. Rather, the various components may be selectively and operatively combined in any number within the intended scope of the disclosure. In addition, terms like "comprising" and "having" should be interpreted as inclusive or open-ended, rather than exclusive or closed-ended, by default, unless explicitly defined to the contrary. All technical, scientific, or other terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs unless defined otherwise. Common terms found in dictionaries should not be interpreted too ideally or too realistically in the context of related art documents unless the present disclosure expressly limits them to that. Any changes and modifications of the present invention based on the above disclosure may be made by those of ordinary skill in the art and shall fall within the scope of the appended claims.
Sequence listing
<110> Shanghai Kopara-technology of medicine Co., ltd
<120> anti-Eno 1 antibodies and uses thereof
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130 135 140
Gly Cys Thr Thr Thr Gly Gly Ala Ala Thr Thr Gly Ala Gly Ala Gly
145 150 155 160
Ala Thr Gly Gly Thr Gly Ala Cys Ala Ala Ala Thr Cys Cys Ala Ala
165 170 175
Ala Thr Gly Gly Thr Thr Ala Gly Gly Thr Ala Ala Ala Gly Gly Thr
180 185 190
Gly Thr Thr Thr Thr Gly Ala Ala Ala Gly Cys Cys Gly Thr Thr Gly
195 200 205
Cys Cys Ala Ala Thr Gly Thr Thr Ala Ala Thr Gly Ala Cys Ala Thr
210 215 220
Cys Ala Thr Thr Gly Cys Cys Cys Cys Ala Gly Cys Thr Thr Thr Ala
225 230 235 240
Ala Thr Ala Ala Ala Ala Gly Cys Cys Ala Ala Gly Ala Thr Cys Gly
245 250 255
Ala Thr Gly Thr Thr Gly Thr Cys Gly Ala Cys Cys Ala Ala Gly Cys
260 265 270
Thr Ala Ala Gly Ala Thr Thr Gly Ala Thr Gly Ala Ala Thr Thr Cys
275 280 285
Thr Thr Gly Thr Thr Gly Thr Cys Cys Thr Thr Gly Gly Ala Cys Gly
290 295 300
Gly Thr Ala Cys Thr Cys Cys Ala Ala Ala Cys Ala Ala Ala Thr Cys
305 310 315 320
Cys Ala Ala Ala Thr Thr Gly Gly Gly Thr Gly Cys Cys Ala Ala Thr
325 330 335
Gly Cys Thr Ala Thr Cys Thr Thr Gly Gly Gly Thr Gly Thr Thr Thr
340 345 350
Cys Thr Thr Thr Gly Gly Cys Thr Gly Cys Thr Gly Cys Cys Ala Ala
355 360 365
Thr Gly Cys Thr Gly Cys Cys Gly Cys Thr Gly Cys Thr Gly Cys Thr
370 375 380
Cys Ala Ala Gly Gly Cys Ala Thr Thr Cys Cys Ala Thr Thr Gly Thr
385 390 395 400
Ala Cys Ala Ala Ala Cys Ala Cys Ala Thr Thr Gly Cys Cys Ala Ala
405 410 415
Cys Ala Thr Thr Thr Cys Cys Ala Ala Thr Gly Cys Cys Ala Ala Gly
420 425 430
Ala Ala Ala Gly Gly Thr Ala Ala Ala Thr Thr Cys Gly Thr Thr Thr
435 440 445
Thr Gly Cys Cys Ala Gly Thr Thr Cys Cys Ala Thr Thr Cys Cys Ala
450 455 460
Ala Ala Ala Cys Gly Thr Thr Thr Thr Gly Ala Ala Cys Gly Gly Thr
465 470 475 480
Gly Gly Thr Thr Cys Cys Cys Ala Thr Gly Cys Thr Gly Gly Thr Gly
485 490 495
Gly Thr Gly Cys Thr Thr Thr Ala Gly Cys Thr Thr Thr Cys Cys Ala
500 505 510
Ala Gly Ala Ala Thr Thr Thr Ala Thr Gly Ala Thr Thr Gly Cys Cys
515 520 525
Cys Cys Ala Ala Cys Thr Gly Gly Thr Gly Thr Cys Thr Cys Cys Ala
530 535 540
Cys Thr Thr Thr Cys Thr Cys Thr Gly Ala Ala Gly Cys Thr Thr Thr
545 550 555 560
Gly Ala Gly Ala Ala Thr Thr Gly Gly Thr Thr Cys Ala Gly Ala Ala
565 570 575
Gly Thr Thr Thr Ala Cys Cys Ala Cys Ala Ala Cys Thr Thr Gly Ala
580 585 590
Ala Ala Thr Cys Thr Thr Thr Gly Ala Cys Cys Ala Ala Gly Ala Ala
595 600 605
Gly Ala Ala Ala Thr Ala Cys Gly Gly Thr Cys Ala Ala Thr Cys Cys
610 615 620
Gly Cys Thr Gly Gly Thr Ala Ala Cys Gly Thr Cys Gly Gly Thr Gly
625 630 635 640
Ala Cys Gly Ala Ala Gly Gly Thr Gly Gly Thr Gly Thr Thr Gly Cys
645 650 655
Thr Cys Cys Ala Gly Ala Thr Ala Thr Cys Ala Ala Ala Ala Cys Thr
660 665 670
Cys Cys Ala Ala Ala Gly Gly Ala Ala Gly Cys Thr Thr Thr Gly Gly
675 680 685
Ala Cys Thr Thr Gly Ala Thr Cys Ala Thr Gly Gly Ala Thr Gly Cys
690 695 700
Cys Ala Thr Thr Gly Ala Cys Ala Ala Ala Gly Cys Cys Gly Gly Thr
705 710 715 720
Thr Ala Cys Ala Ala Ala Gly Gly Thr Ala Ala Gly Gly Thr Thr Gly
725 730 735
Gly Thr Ala Thr Thr Gly Cys Cys Ala Thr Gly Gly Ala Thr Gly Thr
740 745 750
Thr Gly Cys Thr Thr Cys Ala Thr Cys Thr Gly Ala Ala Thr Thr Cys
755 760 765
Thr Ala Cys Ala Ala Gly Gly Ala Cys Gly Gly Thr Ala Ala Ala Thr
770 775 780
Ala Cys Gly Ala Cys Thr Thr Gly Gly Ala Cys Thr Thr Thr Ala Ala
785 790 795 800
Ala Ala Ala Cys Cys Cys Ala Gly Ala Ala Thr Cys Cys Gly Ala Cys
805 810 815
Cys Cys Ala Thr Cys Thr Ala Ala Ala Thr Gly Gly Thr Thr Gly Thr
820 825 830
Cys Thr Gly Gly Cys Cys Cys Ala Cys Ala Ala Thr Thr Gly Gly Cys
835 840 845
Thr Gly Ala Cys Thr Thr Ala Thr Ala Thr Gly Ala Ala Cys Ala Ala
850 855 860
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
885 890 895
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
915 920 925
Gly Gly Gly Thr Cys Cys Ala Cys Thr Thr Cys Thr Thr Thr Gly Ala
930 935 940
Ala Ala Gly Ala Gly Thr Thr Gly Gly Thr Gly Ala Cys Ala Ala Gly
945 950 955 960
Ala Thr Cys Cys Ala Ala Ala Thr Thr Gly Thr Cys Gly Gly Thr Gly
965 970 975
Ala Thr Gly Ala Thr Thr Thr Gly Ala Cys Thr Gly Thr Cys Ala Cys
980 985 990
Thr Ala Ala Cys Cys Cys Thr Ala Cys Cys Ala Gly Ala Ala Thr Cys
995 1000 1005
Ala Ala Gly Ala Cys Thr Gly Cys Cys Ala Thr Thr Gly Ala Ala Ala
1010 1015 1020
Ala Gly Ala Ala Ala Gly Cys Cys Gly Cys Thr Ala Ala Thr Gly Cys
1025 1030 1035 1040
Thr Thr Thr Gly Thr Thr Gly 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
1060 1065 1070
Thr Gly Ala Cys Thr Gly Ala Ala Thr Cys Thr Ala Thr Ala Cys Ala
1075 1080 1085
Ala Gly Cys Thr Gly Cys Thr Ala Ala Cys Gly Ala Thr Thr Cys Thr
1090 1095 1100
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 Ala
1125 1130 1135
Cys Ala Gly Ala Thr 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 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 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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| BR112022019853A2 (en) * | 2020-05-11 | 2022-11-22 | Hunilife Biotechnology Inc | IMMUNOCONJUGATE THAT SPECIFICALLY BINDS ENO-1, COMPOSITION FOR DIAGNOSING OR IMAGING CELLS OR TISSUE EXPRESSING ENO-1, PHARMACEUTICAL COMPOSITION FOR USE IN THE TREATMENT OF AN INFLAMMATORY DISEASE, METHOD FOR TREATMENT OF AN INFLAMMATORY DISEASE, AND USE OF THE IMMUNOCONJUGATE |
| CN112111462B (en) * | 2020-09-14 | 2022-08-23 | 兰州大学 | Enolase ENO1 monoclonal antibody and application thereof |
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