CN116514973A - Monoclonal antibody and reagent for resisting human CD209 and CD209L and application thereof - Google Patents

Monoclonal antibody and reagent for resisting human CD209 and CD209L and application thereof Download PDF

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CN116514973A
CN116514973A CN202211482773.9A CN202211482773A CN116514973A CN 116514973 A CN116514973 A CN 116514973A CN 202211482773 A CN202211482773 A CN 202211482773A CN 116514973 A CN116514973 A CN 116514973A
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cd209l
human
antibody
cdr
antibodies
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王晨辉
杜艳芸
赵紫艳
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Sichuan Academy Of Medical Sciences Sichuan Provincial People's Hospital
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Sichuan Academy Of Medical Sciences Sichuan Provincial People's Hospital
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Abstract

The invention discloses a monoclonal antibody and a reagent for resisting human CD209 and CD209L and application thereof, and relates to the technical field of biomedicine. The invention provides a new preventive and therapeutic scheme for pathogen infection of cells infected by receptors CD209 and CD209L, and monoclonal antibodies against human CD209 and CD209L can specifically recognize CD209 and CD209L and bind to CD209 and CD209L, thereby blocking human cell surface receptors CD209 and CD209L, so that proteins of the pathogen cannot bind to human cell surface receptors CD209 and CD209L, and blocking the class of pathogen infection of host cells infected by the receptors CD209 and CD209L. In addition, the monoclonal antibody provided by the invention can be used for detecting human CD209 and CD209L proteins.

Description

Monoclonal antibody and reagent for resisting human CD209 and CD209L and application thereof
Technical Field
The invention relates to the technical field of biomedicine, in particular to a monoclonal antibody for resisting human CD209 and CD209L, a reagent and application thereof.
Background
The persistent pandemic of the new coronavirus (SARS-CoV-2) poses a serious threat to public health worldwide and causes serious damage to social economy worldwide. Viruses enter the cell, usually by binding to a receptor on the surface of the host cell, which in turn leads to the onset of viral infection, as is no exception for SARS-CoV-2. Angiotensin converting enzyme 2 (ACE 2) is the primary receptor of the first identified SARS-CoV-2-infected host cell, and SARS-CoV-2 mediates viral entry into host cell through its Receptor Binding Domain (RBD) of surface glycoprotein Spike, with ACE2 [1]. However, studies have found that there is a susceptibility to new coronaviruses in immune and non-immune cells that lack ACE2 expression, suggesting that spike proteins may utilize other receptors for infection.
With the continued depth of research, more and more new coronavirus receptors have been identified, including two molecules of the C-type lectin receptor family, CD209/DC-SIGN and CD209L/L-SIGN [2,3]. CD209 has 77% amino acid sequence identity with CD209L, where CD209 is expressed predominantly in dendritic cells and CD209L is expressed predominantly in human Liver Sinusoidal Endothelial Cells (LSEC), lymphatic endothelial cells and lung endothelial cells. CD209 and CD209L are both type II transmembrane glycoprotein receptors, whose extracellular domain consists of a neck region, which is a repeat sequence of 23 amino acids, primarily involved in protein dimerization or oligomerization, and a Carbohydrate Recognition Domain (CRD), which is critical for CD209 and CD209L to recognize the mannose structure present on a specific glycoprotein on a pathogen. N-glycosylation of SARS-CoV-2 spike protein is predominantly an oligomannose-type glycan, which may be responsible for the strong binding of SARS-CoV-2 spike protein to CD209 and CD209L [4]. As the most common pathogen recognition receptor members in the human genome, CD209 and CD209L can also be used as receptor-mediated viruses for SARS-CoV, ebola virus, dengue virus, HIV, influenza virus, hepatitis C virus, etc. in addition to SARS-CoV-2 to infect cells in cis or trans [5]. Thus, CD209 and CD209L are promising therapeutic targets in antiviral drug research.
Since the advent of the first murine monoclonal antibody drug murominab OKT3 in 1986, more and more monoclonal antibodies have been approved successively for the treatment of various diseases such as tumors, autoimmune diseases, infectious diseases, and graft rejection, and many monoclonal antibodies are in different stages of clinical research. Therefore, monoclonal antibody drugs have become an important component of biological medicine. There is no specific drug against SARS-CoV-2 on the market, and the effectiveness of existing vaccines against SARS-CoV-2 and blocking antibodies targeting spike proteins is gradually limited as viral variants continue to appear. Since the new coronavirus infected cells need to bind to host cell surface receptors (e.g., ACE2, CD209L, etc.) via Spike proteins, blocking monoclonal antibodies to the receptors can also block infection by the new coronavirus, and the possibility of resistance of blocking antibodies to viral receptors to constantly occurring viral variants is greatly reduced relative to viral vaccines and blocking antibodies to Spike proteins. In addition, blocking antibodies to the receptor can block not only new coronaviruses from infecting host cells, but also other viruses that infect host cells through the receptor.
We and another group of studies have found that blocking antibodies against the receptor ACE2 have blocking effects on both SARS-COV and various variants of SARS-COV-2 and SARS-COV-2, confirming both of these [6-8].
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a monoclonal antibody against human CD209 and CD209L, a reagent and application thereof, and provides a novel prevention and treatment scheme for pathogen infection of cells infected by the receptors CD209 and CD209L, and the monoclonal antibody against human CD209 and CD209L can specifically recognize the CD209 and CD209L and bind with the CD209 and CD209L, thereby blocking the human cell surface receptors CD209 and CD209L, so that the protein (such as envelope protein) of the pathogen cannot bind with the human cell surface receptors CD209 and CD209L, thereby blocking the infection of host cells by the pathogen of the host infected by the receptors CD209 and CD209L. The invention has important application value.
The invention is realized in the following way:
in a first aspect, the invention provides a monoclonal antibody directed against human CD209 and CD209L comprising a heavy chain variable region comprising three heavy chain complementarity determining regions: CDR-VH1, CDR-VH2 and CDR-VH3, the light chain variable region comprises three light chain complementarity determining regions: the amino acid sequences of CDR-VL1, CDR-VL2 and CDR-VL3, CDR-VH1, CDR-VH2 and CDR-VH3 are shown in SEQ ID NO:1-3, the amino acid sequences of CDR-VL1, CDR-VL2 and CDR-VL3 are as shown in SEQ ID NO: 4-6.
CDR-VH1, SEQ ID NO:1 is as follows:
GFTFRDYA；
CDR-VH2, SEQ ID NO:2 is as follows:
ISGGGSFT；
CDR-VH3, SEQ ID NO:3 is as follows:
ARFATSTAMDY；
CDR-VL1, SEQ ID NO:4 is as follows:
ESVDKFGFSF；
CDR-VL2, SEQ ID NO:5 is as follows: GAS;
CDR-VL3, SEQ ID NO:6 is as follows:
QQSKEVPRT。
the invention prepares monoclonal antibody hybridoma cell strain by gene synthesis and vector construction, expresses and purifies human CD209L recombinant protein, uses the recombinant protein as antigen to immunize mice, obtains mouse anti-human CD209 and CD209L monoclonal antibodies, and screens to obtain anti-human CD209 and CD209L monoclonal antibodies capable of neutralizing and blocking human CD209 and CD209L. The antibody was found to have the heavy chain complementarity determining region and the light chain complementarity determining region described above, by sequencing. The monoclonal antibody can be used for detecting CD209 and CD209L proteins and diagnosing or assisting in diagnosing related diseases by taking CD209 and CD209L as markers.
Taking into account the degeneracy of the codons, the sequence of the genes encoding the above (e.g., fab fragments) antibodies may be modified in their coding regions without altering the amino acid sequence to obtain genes encoding the same antibodies; the modified genes can also be artificially synthesized according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.
In a preferred embodiment of the present invention, the antibody is any one selected from the group consisting of chimeric antibodies, F (ab ') 2, fab', fab, fv, scFv and bispecific antibodies.
The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.
Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
In an alternative embodiment, the antibodies against human CD209 and CD209L are human murine chimeric monoclonal antibodies or scFv against human CD209 and CD209L. In an alternative embodiment, the light chain variable region and the heavy chain variable region of the above antibodies are recombined to obtain a smaller molecular weight single chain antibody (scFv) which is also capable of specifically recognizing human CD209 and CD209L.
In an alternative embodiment, the antibody comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4. The sequences of FR-L1, FR-L2, FR-L3 and FR-L4 are shown in SEQ ID NO:7-10, the sequences of FR-H1, FR-H2, FR-H3 and FR-H4 are shown in SEQ ID NO: 11-14.
FR-L1，SEQ ID NO：7：DIAVTQSPASLAVSLGQRATISCRAS。
FR-L2，SEQ ID NO：8：MNWFQQKPGQPPKLLMY。
FR-L3，SEQ ID NO：9：
KPGSGVPARFSGSGSGTDFSLNIHPMEEDDIAMYFC。
FR-L4，SEQ ID NO：10：FGGGTKLEIK。
FR-H1，SEQ ID NO：11：EVHLVESGGGLVKPGGSLKLSCGAS。
FR-H2，SEQ ID NO：12：MSWVRQSPEKRLEWVAE。
FR-H3，SEQ ID NO：13：
FYADTVTGRFTVSRDNAKNTLYLEMSSLRSEDTAMYYC。
FR-H4，SEQ ID NO：14：WGQGASVTVSS。
In other embodiments, each framework region amino acid sequence of a monoclonal antibody provided by the invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the corresponding framework region.
In an alternative embodiment, the antibody further comprises an antibody constant region.
In an alternative embodiment, the antibody constant region is selected from the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD.
In an alternative embodiment, the species source of the antibody constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.
In an alternative embodiment, the species source of the antibody constant region is human.
In an alternative embodiment, the antibody constant region is selected from human IgG4.
The invention uses the mouse antibody variable region and human antibody IgG4 constant region to form the human mouse chimeric monoclonal antibody with clone number of 7-H7-B1, and verifies that the human mouse chimeric monoclonal antibody has the infection activity of effectively neutralizing novel coronaviruses and coronaviruses, and can block the infection of human bodies by the novel coronaviruses (SARS-Cov-2), coronaviruses (SRAS-Cov) and all viruses entering cells through ACE 2.
In a second aspect, the invention also provides the use of an antibody against human CD209 and CD209L in the manufacture of a medicament for the prevention or treatment of a disease caused by infection by a pathogen that enters cells via CD209 and/or CD209L receptors.
In a preferred embodiment of the use of the invention, the pathogen is selected from the group consisting of a virus, a bacterium, a parasite or a fungus.
In an alternative embodiment, the pathogen is selected from the group consisting of a filovirus, lymphocytic choriocauloencephalitis virus, SARS-CoV-2, SARS-CoV, ebola virus, zika virus, dengue virus, HIV, influenza virus, hepatitis C virus, hepatitis B virus, or simian immunodeficiency virus. In other embodiments, the pathogen may be selected from other enveloped viruses.
In an alternative embodiment, HIV is HIV-1 or HIV-2.
In a third aspect, the invention also provides a medicament comprising an antibody as described above against human CD209 and CD209L for use in the prevention or treatment of a disease caused by infection by a pathogen entering cells via CD209 and/or CD209L receptors.
Such diseases include, but are not limited to, viral-induced human pneumonia, enteritis, encephalitis, chronic hepatitis b, loss of taste and smell, nausea, vomiting, diarrhea, altered skin tone, general soreness, internal bleeding, external bleeding, fever, and the like.
The above drugs also include pharmaceutically acceptable carriers including, but not limited to, fillers, lubricants, disintegrants, binders, glidants, and the like.
In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier includes, but is not limited to, one of polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, dibasic calcium phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo, or a combination thereof.
In a fourth aspect, the invention also provides the use of an antibody against human CD209 and CD209L in the preparation of a reagent or kit for detecting CD209 or CD209L.
In a fifth aspect, the invention also provides a reagent or kit for detecting CD209 or CD209L, comprising the above-mentioned antibodies against human CD209 and CD209L.
The monoclonal antibody provided by the invention can be specifically combined with the CD209 and the CD209L, and can be used for diagnosing or assisting in diagnosing related diseases taking the human CD209 and the CD209L as markers, including but not limited to human pneumonia, enteritis, encephalitis, chronic hepatitis B, gustatory smell loss, nausea, vomiting, diarrhea, skin color change, general soreness, in-vivo bleeding, in-vitro bleeding, fever and the like.
The antibody is labeled with a detectable label.
A detectable label refers to a substance of a type having properties such as luminescence, color development, radioactivity, etc., that can be directly observed by the naked eye or detected by an instrument, by which a qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, the detectable label includes, but is not limited to, fluorescent dyes, enzymes that catalyze the development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (chlorophyll), and the like).
In alternative embodiments, the enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
In alternative embodiments, the radioisotope includes, but is not limited to 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
in alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.
In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In a sixth aspect, the invention also provides an expression cassette or vector comprising genes encoding antibodies against human CD209 and CD209L as described above.
In a seventh aspect, the invention also provides a host cell comprising the vector described above.
In an eighth aspect, the present invention also provides a method for producing the above-described antibodies against human CD209 and CD209L, comprising:
culturing the above-mentioned host cells, and isolating and purifying the antibodies against human CD209 and CD209L from the culture medium or from the cultured host cells.
On the basis of the present invention, it is easy for a person skilled in the art to prepare the antibody or its functional fragment by genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells), for example, by isolating and purifying the antibody or its functional fragment from a culture product of recombinant cells capable of recombinantly expressing the monoclonal antibodies against human CD209 and CD209L as described in any of the above, and on the basis of this, it is within the scope of the present invention to prepare the antibody or its functional fragment by whatever technique is used.
The invention has the following beneficial effects:
the present invention provides a novel prophylactic and therapeutic regimen for pathogen infection of cells infected by the receptors CD209 and CD209L, whereby monoclonal antibodies against human CD209 and CD209L specifically recognize CD209 and CD209L and bind to CD209 and CD209L, thereby blocking the human cell surface receptors CD209 and CD209L, such that the pathogen proteins (e.g., envelope proteins) cannot bind to the human cell surface receptors CD209 and CD209L, thereby blocking the infection of host cells by this class of pathogens that infect hosts via CD209 and CD209L receptors. The invention has important application value.
The invention provides monoclonal antibodies useful for the detection of human CD209 and CD209L proteins, and diagnosis or diagnosis-aid of related diseases using human CD209 and CD209L as markers.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the statistical results of human murine chimeric monoclonal antibodies against human CD209L and CD209 inhibiting the infection of 293T-CD209L and 293T-CD209 cells with novel coronavirus (SARS-Cov-2).
FIG. 2 is a graph showing the statistical results of human murine chimeric monoclonal antibodies against human CD209L and CD209 against coronavirus (SARS-Cov) infection of 293T-CD209L and 293T-CD209 cells.
FIG. 3 is a graph of the statistical results of human murine chimeric monoclonal antibodies against human CD209L and CD209 against the inhibition of ebola virus (Sudan subtype) infection of 293T-CD209L and 293T-CD209 cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: ALaboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
Preparation of human CD209L recombinant protein and mouse anti-human CD209 and CD209L monoclonal antibodies.
1. Preparation of human CD209L recombinant protein
The human CD209L gene expression vector is constructed by utilizing the genetic engineering technology. We constructed human CD209L cDNA into pATX2 (restriction enzyme sites EcoRI/NotI) expression vector, transformed, shaken, and extracted endotoxin-free plasmid. Plasmid (500. Mu.g) was transfected into 500ml of 293F cells, and after the cell viability had fallen to about 50%, the cell culture supernatant was collected, centrifuged at 1000rpm for 5 minutes to remove cells and cell debris, and centrifuged at 10000rpm for 5 minutes, and the cell culture supernatant was filtered with a 0.45uM filter. The cell culture supernatant was subjected to protein A affinity chromatography to purify the human CD209L-Fc protein.
2. Preparation of mouse anti-human CD209L and CD209 monoclonal antibodies
(1) Human CD209L-Fc recombinant protein immunized mice
4 Balb/c mice were immunized with the prepared human CD209L-Fc recombinant protein and 1mg/0.025ml of antigen was subcutaneously injected in the armpit, sole and groin of the mice for immunization. The method comprises the steps of performing primary immunization and then performing 2-4 times of booster immunization, wherein the immunization interval time is 14 days, taking the serum measurement titer of the mice on the seventh day after the last booster immunization, performing intraperitoneal injection of 0.5ml 200ug/ml antigen solution for impact immunization when the measurement titer meets the standard, and taking spleen cells and myeloma cells for cell fusion after three days.
(2) Cell fusion
Selecting a mouse which is subjected to impact immunization and has consistent serum titer, removing eyeball blood for positive control of hybridoma screening, killing the mouse by cervical dislocation, sterilizing in 75% alcohol for at least 30 seconds, preparing spleen cell suspension from spleen of the mouse, counting, further taking SP2/0 myeloma cells, counting, mixing the spleen cells and the myeloma cells in a 50ml centrifuge tube according to the ratio of 5:1, centrifuging at 1000rpm for 5min, discarding supernatant, flicking the wall of the centrifuge tube, loosening cell sediment, placing the centrifuge tube in a water bath at 37 ℃, adding preheated PEG (1 ml is added in 1 min) into the cell sediment at constant speed, stirring gently by the tip of the gun head while rotating the centrifuge tube in the PEG adding process, and standing for 90s. Adding the preheated 1640 basic culture medium at a constant speed (first time), adding 1ml in 1min, and stirring gently while adding; adding the preheated 1640 basic culture medium (second time) at a constant speed, adding 2ml in 1min, and stirring gently while adding; adding the preheated 1640 basic culture medium (third time) at a constant speed, adding 9ml in 3min, and stirring gently while adding; the preheated 1640 minimal medium was added at a constant speed, stirred gently while adding up to 40ml, and the centrifuge tube was placed in a 37℃water bath for 3min. Centrifuging the fused cell suspension at 800rpm for 5min, removing the supernatant, flicking the wall of the centrifugal tube, loosening the cell sediment, adding a proper amount of HAT culture medium according to the number of spleen cells, blowing and uniformly mixing, inoculating to a 96-well cell culture plate, and changing the HT culture medium after the HAT selection culture medium is maintained for 7-10 days. During the selection culture, when hybridoma cells are distributed to 1/10 area of the bottom of the well, the cell culture supernatant is collected to start detection of specific antibodies, and wells containing positive antibodies are screened.
(3) ELISA detection antibody
100 μl (2 ug/ml) of CD209L and CD209 recombinant protein antigen solution was used to coat the 96-well ELISA plate and incubated overnight at 4deg.C. The next day the coating solution was removed, the wells were blocked for 1 hour at room temperature with 300 ul/well of 5% skim milk, the blocking solution was removed, washed 3 times with PBST, and dried on a water absorbing paper towel. The primary antibody was added to 100 ul/well for 60 minutes at 37℃and removed, washed 3 times with PBST, and dried on a water-absorbing paper towel. The secondary antibody was added to 100 ul/well and incubated at 37℃for 30 minutes, removed, washed 3 times with PBST and dried on a water-absorbing paper towel. Adding TMB color development liquid 100 ul/hole 37 ℃ for incubation for 5-20min, observing color development condition, adding stop liquid 2M hydrochloric acid 50 ul/hole, selecting wavelength of 450-620nm, and reading light absorption value by an enzyme label instrument.
(4) Hybridoma subcloning
Positive Kong Zajiao tumor cells were subcloned by limiting dilution. Hybridoma cells to be cloned were resuspended from the culture well and counted to 10 cells/ml, 100. Mu.l of diluted cells were added to each well, and the mixture was placed at 37℃in 8% CO 2 Culturing in incubator for 8-9 days, collecting supernatant of culture solution, detecting antibody activity, selecting positive well growing of monoclonal hybridoma, transferring to 24-well plate, and subcloning or enlarging cultureAnd (5) nourishing.
(5) Production of mouse anti-human CD209L and CD209 monoclonal antibodies
Mice were intraperitoneally injected with 0.5ml of Freund's incomplete adjuvant 7-21 days in advance, hybridoma cells were collected and the density was adjusted to 2X 10 6 2X 10 hybridoma cells were intraperitoneally injected into mice injected with incomplete adjuvant at a concentration of 2X 10 cells per ml 6 And (2) collecting ascites of the mice 7-12 days after cell injection, purifying the ascites of the mice by Protein G affinity chromatography, eluting by 0.1M glycine solution (pH 2.5), neutralizing by 1M Tris-HCl (pH 9.0) solution, testing whether the eluent is neutral by PH test paper, then, replacing the antibodies by an ultrafiltration column of 50KD in PBS, measuring the concentration of the antibodies, and simultaneously taking 2ug of the antibodies to run the gel to verify the purity of the antibodies.
(6) Screening of mouse anti-human CD209L and CD209 monoclonal antibodies blocking SARS-Cov-2, SARS-Cov and Ebola pseudovirus infection
New coronavirus (SARS-Cov-2) is infected with 293T cells expressing human CD209L and CD209, a control antibody and a mouse anti-human CD209L and CD209 monoclonal antibody are added simultaneously, the cells are collected by incubation for 24 hours in a 37-degree incubator, and the SARS-Cov-2 pseudovirus infection is detected by luciferase activity assay. Coronavirus (SARS-Cov) was infected with 293T cells expressing human CD209L and CD209, control antibodies and mouse anti-human CD209L and CD209 monoclonal antibodies were added simultaneously, and cells were collected by incubation in a 37 degree incubator for 24 hours, and the SARS-Cov pseudovirus infection was detected by luciferase activity assay. The ebola pseudovirus was infected with 293T cells expressing human CD209L and CD209, control antibodies and mouse anti-human CD209L and CD209 monoclonal antibodies were added simultaneously, cells were collected by incubation in a 37 degree incubator for 24 hours, and ebola pseudovirus infection was detected by luciferase activity assay.
Through a cell infection experiment, 1 mouse anti-human CD209L and CD209 monoclonal antibodies capable of simultaneously blocking SARS-Cov-2, SARS-Cov and Ebola pseudovirus infection are screened out: clone No. 7H7B1.
3. Hybridoma cell sequencing/variable region Gene sequencing
Collecting cells (clone number is 7H7B 1) when the hybridoma cells grow to a logarithmic growth phase, extracting total RNA of the hybridoma cells, obtaining first-strand cDNA complementary to full-length mRNA by RT-PCR (reverse transcription-polymerase chain reaction) through a 3'RACE technology and a 5' RACE technology, obtaining antibody heavy chain and light chain variable region genes through PCR amplification by taking the synthesized cDNA as a template, connecting the antibody variable region genes to a T vector, transforming and selecting positive clones for sequencing, and obtaining an antibody variable region gene sequence as shown in SEQ ID NO: 1-6.
Example 2
Preparation of CD209L and CD209 recombinant antibodies
1. Construction of plasmid expressing recombinant antibody
The constant regions of the heavy and light chains of the human IgG4 antibody were cloned into the pCAGGS vector, and the obtained heavy and light chain variable regions of the mouse anti-human CD209L and CD209 murine antibodies were inserted into the pCAGGS expression vector containing the constant regions of the human IgG4 antibody to construct a recombinant antibody expression vector, and 293F mammalian cells were transfected for recombinant antibody expression (500 ug/500 ml). The human mouse chimeric monoclonal antibody with clone number 7H7B1 consists of a mouse antibody variable region and a human antibody IgG4 constant region.
2. Expression and purification of recombinant antibodies
Culturing is continued for 5-6 days after transfection of 293F cells, cell culture supernatant is collected, and anti-CD 209L and CD209 recombinant human-mouse chimeric antibody in the supernatant is purified by using a Protein G affinity chromatography purification column.
Example 3
Characterization of recombinant human murine chimeric antibodies against CD209L and CD209
1. Cell experiments blocking infection of human CD209L and CD209 expressing cells with a novel coronavirus pseudovirus (SARS-Cov-2) were performed using anti-CD 209L and CD209 human murine chimeric antibodies:
new coronavirus pseudovirus (SARS-Cov-2) was used to infect 293T cells expressing human CD209L and CD209, CD209L and CD209 human murine chimeric monoclonal antibodies (7H 7B1, 10ug/ml each) were added, cells were collected by incubation in a 37℃incubator for 24 hours, and virus infection was detected by luciferase activity assay by Promega company Luciferase Assay System (Cat.#E1501).
The results, as shown in FIG. 1, demonstrate that addition of CD209L and CD209 human murine chimeric monoclonal antibodies significantly inhibited viral infection of 293T cells of human CD209L (A) and CD209 (B), blocking new coronavirus pseudovirus (SARS-Cov-2) infection. And has a certain concentration dependency, and the higher the concentration of the CD209L and the CD209 human mouse chimeric monoclonal antibody is, the better the blocking effect is.
2. Cell experiments to block coronavirus (SARS-Cov) infection of human CD209L and CD209 expressing cells were performed using anti-C209L and CD209 human murine chimeric antibodies:
coronavirus (SARS-Cov) pseudovirus was used to infect 293T cells expressing human CD209L and CD209, human mouse chimeric monoclonal antibodies (7H 7B1, 10 ug/ml) were added to CD209L and CD2092, and cells were collected by incubation in a 37℃incubator for 24 hours, and luciferase activity assay was performed by Promega company Luciferase Assay System (Cat.#E1501).
The results are shown in FIG. 2, which shows that addition of CD209L and CD209 human murine chimeric monoclonal antibodies significantly inhibited coronavirus infection of 293T cells of human CD209L (C) and CD209 (D), blocking coronavirus infection. And has a certain concentration dependency, and the higher the concentration of the CD209L and the CD209 human mouse chimeric monoclonal antibody is, the better the blocking effect is.
3. Cell experiments blocking ebola pseudovirus infection with human CD209L and CD209 were performed using anti-C209L and CD209 human murine chimeric antibodies:
ebola pseudovirus was used to infect 293T cells expressing human CD209L and CD209, CD209L and CD2092 human murine chimeric monoclonal antibodies (7H 7B1, both 10 ug/ml) were added, cells were collected by incubation in 37 degree incubator for 24 hours, and virus infection was detected by luciferase activity assay performed by Promega company Luciferase Assay System (cat.#e1501).
The results are shown with reference to fig. 3, which shows that addition of CD209L and CD209 human murine chimeric monoclonal antibodies significantly inhibited ebola false virus infection of 293T cells to human CD209L (E) and CD209 (F), blocking ebola false virus infection. And has a certain concentration dependency, and the higher the concentration of the CD209L and the CD209 human mouse chimeric monoclonal antibody is, the better the blocking effect is.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Reference is made to:
1.Jun Lan,et al.Structure of the SARS-CoV-2spike receptor-binding domain bound to the ACE2 receptor.Nature.2020May；581(7807):215-220.
2.Michel Thépaut,et al.DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2trans-infection and can be inhibited by a glycomimetic antagonist.PLoS Pathog.2021May20；17(5):e1009576.
3.Yuji Kondo,et al.L-SIGN is a receptor on liver sinusoidal endothelial cells for SARS-CoV-2virus.JCI Insight.2021Jul22；6(14):e148999.
4.Yasunori Watanabe,et al.Site-specific glycan analysis of the SARS-CoV-2spike.Science.2020Jul 17；369(6501):330-333.
5.Nader Rahimi.C-type Lectin CD209L/L-SIGN and CD209/DC-SIGN:Cell Adhesion Molecules Turned to Pathogen Recognition Receptors.Biology(Basel).2020Dec 22；10(1):1.
6.Yanyun Du,et al.A broadly neutralizing humanizedACE2-targeting antibody against SARS-CoV-2 variants.Nat Commun.2021 Aug 17；12(1):5000.
7.Yuning Chen,et al.ACE2-targeting monoclonal antibody aspotent and broad-spectrum coronavirus blocker.Signal Transduct TargetTher.2021 Aug 25；6(1):315.
8.Jianxia Ou,et al.ACE2-Targeting antibody suppressesSARS-CoV-2 Omicron and Delta variants.Signal Transduct Target Ther.2022 Feb 9；7(1):43.

Claims (10)

1. a monoclonal antibody directed against human CD209 and CD209L comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity determining regions: CDR-VH1, CDR-VH2 and CDR-VH3, the light chain variable region comprising three light chain complementarity determining regions: the amino acid sequences of CDR-VL1, CDR-VL2 and CDR-VL3, CDR-VH1, CDR-VH2 and CDR-VH3 are shown in SEQ ID NO:1-3, the amino acid sequences of CDR-VL1, CDR-VL2 and CDR-VL3 are as shown in SEQ ID NO: 4-6.
2. The antibody against human CD209 and CD209L according to claim 1, characterized in that the antibody is selected from any one of chimeric antibodies, F (ab ') 2, fab', fab, fv, scFv and bispecific antibodies;
preferably, the antibodies against human CD209 and CD209L are human murine chimeric monoclonal antibodies or scFv against human CD209 and CD 209L;
preferably, the antibody comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4;
preferably, the antibody further comprises an antibody constant region;
preferably, the antibody constant region is selected from the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD;
preferably, the species source of the antibody constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human;
preferably, the species source of the antibody constant region is human;
preferably, the antibody constant region is selected from human IgG4.
3. Use of an antibody against human CD209 and CD209L according to any of claims 1-2 for the preparation of a medicament for the prevention or treatment of a disease caused by infection by a pathogen entering cells via CD209 and/or CD209L receptors.
4. Use according to claim 3, wherein the pathogen is selected from the group consisting of viruses, bacteria, parasites or fungi;
preferably, the pathogen is selected from the group consisting of filoviruses, lymphocytic choriocaitosis virus, SARS-CoV-2, SARS-CoV, ebola virus, dengue virus, zika virus, HIV, influenza virus, hepatitis C virus, hepatitis B virus or simian immunodeficiency virus:
preferably, the HIV is HIV-1 or HIV-2.
5. A medicament comprising an antibody according to any one of claims 1-2 against human CD209 and CD209L for use in the prevention or treatment of a disease caused by infection by a pathogen entering cells via CD209 and/or CD209L receptors.
6. Use of an antibody according to any one of claims 1-2 against human CD209 and CD209L for the preparation of a reagent or kit for detecting CD209 or CD209L.
7. A reagent or kit for detecting CD209 or CD209L, comprising an antibody according to any one of claims 1-2 against human CD209 and CD209L.
8. An expression cassette or vector comprising genes encoding antibodies against human CD209 and CD209L according to any one of claims 1-2.
9. A host cell comprising the vector of claim 8.
10. A method of producing an antibody of any one of claims 1-2 against human CD209 and CD209L, comprising:
culturing the host cell of claim 9, and isolating and purifying the anti-human CD209 and CD209L antibodies from the culture medium or from the cultured host cell.
CN202211482773.9A 2022-11-24 2022-11-24 Monoclonal antibody and reagent for resisting human CD209 and CD209L and application thereof Pending CN116514973A (en)

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