Monoclonal antibody against human cd209 and cd209l, reagent and application thereof

Abstract

The application discloses a kind of anti-human CD209 and CD209L monoclonal antibody, reagent and its application, it is related to biomedical technology field.The application provides a new prevention and treatment scheme for the pathogen infection of cell infection by receptor CD209 and CD209L, and the anti-human CD209 and CD209L monoclonal antibody can specifically recognize CD209 and CD209L, and is combined with CD209 and CD209L, so as to close human cell surface receptor CD209 and CD209L, so that the protein of pathogen cannot be combined with human cell surface receptor CD209 and CD209L, and the infection of host cell by this kind of pathogen through CD209 and CD209L receptor is blocked.In addition, the monoclonal antibody provided by the application can be used for the detection of human CD209 and CD209L protein.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a monoclonal antibody against human CD209 and CD209L, a reagent thereof, and their applications. Background Technology

[0002] The ongoing COVID-19 pandemic poses a serious threat to global public health and causes severe damage to the global socio-economic system. Viruses usually enter cells by binding to receptors on the surface of host cells, leading to viral infection, and SARS-CoV-2 is no exception. Angiotensin-converting enzyme 2 (ACE2) was the first major receptor identified for SARS-CoV-2 to infect host cells. SARS-CoV-2 enters host cells by binding to ACE2 through the receptor-binding domain (RBD) of its surface glycoprotein Spike[1]. However, studies have found that susceptibility to the novel coronavirus exists in immune and non-immune cells lacking ACE2 expression, suggesting that the spike protein may use other receptors for infection.

[0003] As research continues, more and more SARS-CoV-2 receptors have been identified, including two molecules from the C-type lectin receptor family, CD209 / DC-SIGN and CD209L / L-SIGN [2,3]. CD209 and CD209L share 77% amino acid sequence identity. CD209 is mainly expressed in dendritic cells, while CD209L is mainly expressed in human hepatic sinusoidal endothelial cells (LSEC), lymphoendothelial cells, and lung endothelial cells. Both CD209 and CD209L are type II transmembrane glycoprotein receptors. Their extracellular domains consist of a neck region and a carbohydrate recognition domain (CRD). The neck region is a 23-amino acid repeat sequence that mainly participates in protein dimerization or oligomerization, while the CRD is crucial for CD209 and CD209L to recognize the mannose structure present on specific glycoproteins on pathogens. The N-glycosylation of the SARS-CoV-2 spike protein is mainly oligomannose-type glycan, which may be the reason why the SARS-CoV-2 spike protein binds strongly to CD209 and CD209L[4]. As the most common pathogen recognition receptor members in the human genome, in addition to SARS-CoV-2, CD209 and CD209L can also act as receptors for SARS-CoV, Ebola virus, dengue virus, HIV, influenza virus, hepatitis C virus, etc., mediating cis or trans viral infection of cells[5]. Therefore, CD209 and CD209L are promising therapeutic targets in antiviral drug research.

[0004] Since the advent of the first murine monoclonal antibody drug, Muromonab OKT3, in 1986, an increasing number of monoclonal antibody drugs have been approved for the treatment of various diseases, including cancer, autoimmune diseases, infectious diseases, and transplant rejection. Meanwhile, numerous monoclonal antibodies are in different stages of clinical trials. Therefore, monoclonal antibody drugs have become an important component of biomedicine. Currently, there are no specific drugs for SARS-CoV-2 on the market, and the effectiveness of existing SARS-CoV-2 vaccines and spike protein-blocking antibodies is gradually being limited by the continuous emergence of viral variants. Since the novel coronavirus infects cells by binding to host cell surface receptors (e.g., ACE2, CD209, CD209L) via the Spike protein, receptor-blocking monoclonal antibodies can also block SARS-CoV-2 infection. Furthermore, compared to viral vaccines and Spike protein-blocking antibodies, receptor-blocking antibodies are much less likely to develop resistance to emerging viral variants. In addition, receptor-blocking antibodies can not only block SARS-CoV-2 infection of host cells but also block other viruses that infect host cells via the same receptor.

[0005] We and another research group confirmed these two points by finding that blocking antibodies against the receptor ACE2 have a blocking effect on SARS-COV and SARS-COV-2 as well as multiple variants of SARS-COV-2 [6-8].

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a monoclonal antibody against human CD209 and CD209L, a reagent thereof, and their applications. This invention provides a new prevention and treatment strategy for pathogens that infect cells via receptors CD209 and CD209L. The monoclonal antibody against human CD209 and CD209L can specifically recognize and bind to CD209 and CD209L, thereby blocking the human cell surface receptors CD209 and CD209L. This prevents pathogen proteins (e.g., envelope proteins) from binding to the human cell surface receptors CD209 and CD209L, thus blocking the infection of host cells by pathogens that infect the host via CD209 and CD209L receptors. This invention has significant application value.

[0008] This invention is implemented as follows:

[0009] In a first aspect, the present invention provides a monoclonal antibody against human CD209 and CD209L, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises 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: CDR-VL1, CDR-VL2, and CDR-VL3. The amino acid sequences of CDR-VH1, CDR-VH2, and CDR-VH3 are shown in SEQ ID NO: 1-3, and the amino acid sequences of CDR-VL1, CDR-VL2, and CDR-VL3 are shown in SEQ ID NO: 4-6.

[0010] The sequence of CDR-VH1, SEQ ID NO: 1 is:

[0011] GFTFRDYA;

[0012] The sequence of CDR-VH2, SEQ ID NO: 2 is:

[0013] ISGGGSFT;

[0014] The sequence of CDR-VH3, SEQ ID NO: 3 is:

[0015] ARFATSTAMDY;

[0016] The sequence of CDR-VL1, SEQ ID NO: 4 is:

[0017] ESVDKFGFSF;

[0018] The sequence of CDR-VL2, SEQ ID NO: 5, is: GAS;

[0019] The sequence of CDR-VL3, SEQ ID NO: 6 is:

[0020] QQSKEVPRT.

[0021] This invention involves gene synthesis and vector construction to express and purify recombinant human CD209L protein. Mice are immunized with this recombinant protein as an antigen to prepare monoclonal antibody hybridoma cell lines, obtaining mouse anti-human CD209 and CD209L monoclonal antibodies. These antibodies are then screened to obtain anti-human CD209 and CD209L monoclonal antibodies capable of neutralizing and blocking these proteins. Sequencing revealed that these antibodies possess the aforementioned heavy chain complementarity-determining regions (CMRs) and light chain complementarity-determining regions. These monoclonal antibodies can be used for the detection of CD209 and CD209L proteins, as well as for the diagnosis or auxiliary diagnosis of diseases using CD209 and CD209L as biomarkers.

[0022] Considering the degeneracy of codons, the gene sequence encoding the above-mentioned (e.g., Fab segment) antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody; alternatively, the gene can be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0023] In a preferred embodiment of the present invention, the antibody is selected from any one of chimeric antibodies, F(ab')2, Fab', Fab, Fv, scFv, and bispecific antibodies.

[0024] The functional fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned functional fragments.

[0025] The functional fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by, for example, automated peptide synthesizers sold by Applied BioSystems.

[0026] In one optional embodiment, the antibody against human CD209 and CD209L is a human-mouse chimeric monoclonal antibody against human CD209 and CD209L or an scFv. In another optional embodiment, the light chain variable region and the heavy chain variable region of the above antibody are recombined to obtain a single-chain antibody (scFv) with a smaller molecular weight, which can also specifically recognize human CD209 and CD209L.

[0027] In one alternative embodiment, the antibody includes light chain backbone regions FR-L1, FR-L2, FR-L3, and FR-L4, and / or heavy chain backbone 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, and the sequences of FR-H1, FR-H2, FR-H3, and FR-H4 are shown in SEQ ID NO: 11-14, respectively.

[0028] FR-L1, SEQ ID NO:7:DIAVTQSPASLAVSLGQRATISCRAS.

[0029] FR-L2, SEQ ID NO:8:MNWFQQKPGQPPKLLMY.

[0030] FR-L3, SEQ ID NO: 9:

[0031] KPGSGVPARFSGSGSGTDFSLNIHPMEEDDIAMYFC.

[0032] FR-L4, SEQ ID NO: 10: FGGGTKLEIK.

[0033] FR-H1, SEQ ID NO: 11: EVHLVESGGGLVKPGGSLKLSCGAS.

[0034] FR-H2, SEQ ID NO: 12: MSWVRQSPEKRLEWVAE.

[0035] FR-H3, SEQ ID NO: 13:

[0036] FYADTVTGRFTVSRDNAKNTLYLEMSSLRSEDTAMYYC.

[0037] FR-H4, SEQ ID NO: 14: WGQGASVTVSS.

[0038] It should be noted that, in other embodiments, the amino acid sequences of each backbone region of the monoclonal antibody provided by the present 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 with the corresponding backbone regions described above.

[0039] In one alternative implementation, the antibody further includes an antibody constant region.

[0040] In one alternative implementation, the antibody constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0041] In one alternative implementation, the species source of the antibody constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0042] In one alternative implementation, the species source of the antibody constant region is human.

[0043] In one alternative implementation, the antibody constant region is selected from human IgG4.

[0044] This invention comprises a human-mouse chimeric monoclonal antibody with clone number 7-H7-B1, composed of the variable region of mouse antibody and the constant region of human antibody IgG4. It has been verified that the antibody has effective neutralizing activity against novel coronavirus and coronavirus, and can block infection of the human body by SARS-CoV-2, coronavirus (SARS-CoV), and all viruses that enter cells through ACE2.

[0045] Secondly, the present invention also provides the application of antibodies against human CD209 and CD209L in the preparation of a drug for the prevention or treatment of diseases caused by pathogen infection that enters cells through CD209 and / or CD209L receptors.

[0046] In a preferred embodiment of the present invention, the pathogen is selected from viruses, bacteria, parasites, or fungi.

[0047] In one alternative embodiment, the pathogen is selected from filoviruses, lymphocytic chorioencephalitis 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 pathogens may also be selected from other enveloped viruses.

[0048] In one alternative implementation, HIV is either HIV-1 or HIV-2.

[0049] Thirdly, the present invention also provides a medicament comprising the aforementioned antibodies against human CD209 and CD209L, the medicament being used to prevent or treat diseases caused by pathogen infection that enters cells via CD209 and / or CD209L receptors.

[0050] The aforementioned diseases include, but are not limited to, viral infections such as pneumonia, enteritis, encephalitis, chronic hepatitis B, loss of taste and smell, nausea, vomiting, diarrhea, skin discoloration, body aches, internal bleeding, external bleeding, and fever.

[0051] The aforementioned drugs also include pharmaceutically acceptable carriers, including but not limited to fillers, lubricants, disintegrants, binders, and flow aids.

[0052] In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier includes, but is not limited to, one or a combination of polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropylcellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, dicalcium phosphate, calcium stearate, sodium stearoyl fumarate, silicon dioxide, titanium dioxide, talc, and indigo.

[0053] Fourthly, the present invention also provides the use of antibodies against human CD209 and CD209L in the preparation of reagents or kits for detecting CD209 or CD209L.

[0054] Fifthly, the present invention also provides a reagent or kit for detecting CD209 or CD209L, comprising the aforementioned antibodies against human CD209 and CD209L.

[0055] Detection reagents include antibody reagents against human CD209 and CD209L, and antibody reagents labeled with easily detectable markers. The monoclonal antibody provided by this invention can specifically bind to CD209 and CD209L and can be used for the diagnosis or auxiliary diagnosis of diseases related to human CD209 and CD209L as markers. These diseases include, but are not limited to, human pneumonia, enteritis, encephalitis, chronic hepatitis B, loss of taste and smell, nausea, vomiting, diarrhea, skin color changes, general aches and pains, internal bleeding, external bleeding, fever, etc.

[0056] The antibodies described above are labeled with detectable markers.

[0057] Detectable markers refer to substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc. These properties enable qualitative or quantitative detection of the corresponding target.

[0058] In optional embodiments, the detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0059] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0060] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0061] In optional embodiments, the enzymes that catalyze the color development of the substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0062] In optional embodiments, the radioactive isotope includes, but is not limited to, those mentioned above. 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.

[0063] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0064] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0065] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0066] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0067] In a sixth aspect, the present invention also provides an expression cassette or vector containing a gene encoding the aforementioned antibodies against human CD209 and CD209L.

[0068] In a seventh aspect, the present invention also provides a host cell containing the aforementioned carrier.

[0069] Eighthly, the present invention also provides a method for producing the above-mentioned antibodies against human CD209 and CD209L, comprising:

[0070] The host cells described above are cultured, and antibodies against human CD209 and CD209L are isolated and purified from the culture medium or from the cultured host cells.

[0071] Based on the amino acid sequence of the antibody or its functional fragment disclosed in this invention, those skilled in the art will readily conceive of using genetic engineering or other techniques (chemical synthesis, hybridoma cells) to prepare the antibody or its functional fragment. For example, the antibody or its functional fragment can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing monoclonal antibodies against human CD209 and CD209L as described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody or its functional fragment of this invention, it falls within the protection scope of this invention.

[0072] The present invention has the following beneficial effects:

[0073] This invention provides a novel prevention and treatment strategy for pathogens that infect cells via receptors CD209 and CD209L. Monoclonal antibodies against human CD209 and CD209L specifically recognize and bind to CD209 and CD209L, thereby blocking the human cell surface receptors CD209 and CD209L. This prevents pathogen proteins (e.g., envelope proteins) from binding to these receptors, thus blocking the infection of host cells by pathogens that infect the host via CD209 and CD209L receptors. This invention has significant application value.

[0074] This invention provides monoclonal antibodies that can be used for the detection of human CD209 and CD209L proteins, as well as for the diagnosis or auxiliary diagnosis of related diseases using human CD209 and CD209L as biomarkers. Attached Figure Description

[0075] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0076] Figure 1 Statistical results of human-mouse chimeric monoclonal antibodies against human CD209L and CD209 inhibiting SARS-CoV-2 infection of 293T-CD209L and 293T-CD209 cells.

[0077] Figure 2 Statistical results of human-mouse chimeric monoclonal antibodies against human CD209L and CD209 inhibiting coronavirus (SARS-CoV) infection of 293T-CD209L and 293T-CD209 cells.

[0078] Figure 3 Statistical results of human-mouse chimeric monoclonal antibodies against human CD209L and CD209 inhibiting Ebola virus (Sudan subtype) infection of 293T-CD209L and 293T-CD209 cells. Detailed Implementation

[0079] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0080] Unless otherwise defined, 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 pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0081] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0082] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0083] Example 1

[0084] Preparation of recombinant human CD209L protein and mouse anti-human CD209 and CD209L monoclonal antibodies.

[0085] 1. Preparation of recombinant human CD209L protein

[0086] Human CD209L gene expression vector was constructed using genetic engineering techniques. Human CD209L cDNA was incorporated into a pATX2 (EcoRI / NotI restriction site) expression vector, transformed, cultured, and endotoxin-free plasmids were extracted. The plasmid (500 μg) was transfected into 500 ml of 293F cells. When cell viability decreased to approximately 50%, the cell culture supernatant was collected, centrifuged at 1000 rpm for 5 minutes to remove cells and cell debris, and then centrifuged again at 10000 rpm for 5 minutes. The cell culture supernatant was then filtered through a 0.45 μM filter. The human CD209L-Fc protein was purified by Protein A affinity chromatography.

[0087] 2. Preparation of mouse anti-human CD209L and CD209 monoclonal antibodies

[0088] (1) Immunizing mice with recombinant human CD209L-Fc protein

[0089] Four Balb / c mice were immunized with the prepared human CD209L-Fc recombinant protein. Immunization was performed by subcutaneous injection of 1 mg / 0.025 ml antigen into the axilla, paw, and groin. A primary immunization was administered, followed by 2-4 booster immunizations, each 14 days apart. Seven days after the final booster immunization, serum titers were measured. If the titer met the standard, a pulse immunization was performed by intraperitoneal injection of 0.5 ml of 200 ug / ml antigen solution. Three days later, spleen cells were harvested and fused with myeloma cells.

[0090] (2) Cell fusion

[0091] Mice that underwent shock immunization and whose serum titers met the requirements were selected. Blood was collected from the eyeballs for use as a positive control for hybridoma screening. The mice were then euthanized by cervical dislocation and disinfected in 75% alcohol for at least 30 seconds. The spleens of the mice were collected to prepare a suspension of spleen cells and counted. SP2 / 0 myeloma cells were also collected and counted. The spleen cells and myeloma cells were mixed in a 5:1 ratio in a 50ml centrifuge tube and centrifuged at 1000rpm for 5min. The supernatant was discarded, and the centrifuge tube wall was gently tapped to loosen the cell pellet. The centrifuge tube was placed in a 37℃ water bath, and preheated PEG was added to the cell pellet at a uniform rate (1ml over 1min). During the addition of PEG, the centrifuge tube was rotated while gently stirring with the tip of a pipette. The mixture was allowed to stand for 90s. Add preheated 1640 basal medium at a constant rate (first addition), 1 ml over 1 minute, while gently stirring. Add preheated 1640 basal medium at a constant rate (second addition), 2 ml over 1 minute, while gently stirring. Add preheated 1640 basal medium at a constant rate (third addition), 9 ml over 3 minutes, while gently stirring. Add preheated 1640 basal medium at a constant rate, while gently stirring until 40 ml remains. Place the centrifuge tube in a 37°C water bath and let it stand for 3 minutes. Centrifuge the confluent cell suspension at 800 rpm for 5 minutes, discard the supernatant, gently tap the centrifuge tube wall to loosen the cell pellet, add an appropriate amount of HAT medium according to the number of spleen cells, mix well by pipetting, and seed into a 96-well cell culture plate. Maintain in HAT selection medium for 7–10 days, then replace with HT medium. During the selection culture period, when hybridoma cells cover 1 / 10 of the bottom area of ​​the well, collect the cell culture supernatant to start detecting specific antibodies, and screen out the wells containing positive antibodies.

[0092] (3) ELISA detection of antibodies

[0093] Coat 96-well microplates with 100 μl (2 μg / ml) of CD209L and CD209 recombinant protein antigen solution and incubate overnight at 4°C. The next day, remove the coating solution, add 300 μl / well of 5% skim milk, and block at room temperature for 1 hour. Remove the blocking solution, wash three times with PBST, and pat dry on absorbent paper. Add 100 μl / well of primary antibody and incubate at 37°C for 60 minutes. Remove the primary antibody, wash three times with PBST, and pat dry on absorbent paper. Add 100 μl / well of secondary antibody and incubate at 37°C for 30 minutes. Remove the secondary antibody, wash three times with PBST, and pat dry on absorbent paper. Add 100 μl / well of TMB chromogenic buffer and incubate at 37°C for 5-20 minutes, observing the color development. Add 50 μl / well of 2M hydrochloric acid as stop solution, and read the absorbance using a microplate reader at a wavelength of 450-620 nm.

[0094] (4) Hybridoma subcloning

[0095] Subcloning of hybridoma cells in positive wells was performed using the limiting dilution method. The hybridoma cells to be cloned were resuspended from the culture wells and counted to a concentration of 10 cells / ml. 100 μl of diluted cells was added to each well, and the wells were incubated at 37°C with 8% CO2 for 8-9 days. The culture supernatant was collected to detect antibody activity. Positive wells with well-grown monoclonal hybridomas were selected and transferred to 24-well plates for further subcloning or expansion culture.

[0096] (5) Production of mouse anti-human CD209L and CD209 monoclonal antibodies

[0097] Mice were intraperitoneally injected with 0.5 ml of Freund's incomplete adjuvant 7-21 days prior to the event, and hybridoma cells were collected and their density adjusted to 2 × 10⁻⁶. 6 2 × 10⁶ hybridoma cells / ml were injected intraperitoneally into mice that had previously been injected with incomplete adjuvant. 6 / 0.5ml / mouse. Ascites fluid was collected from mice 7-12 days after cell injection. The ascites fluid was purified by Protein G affinity chromatography, eluted with 0.1M glycine solution (pH 2.5), and neutralized with 1M Tris-HCl solution (pH 9.0). The pH of the eluent was tested with pH paper to confirm its neutrality. The solution was then filtered through a 50KD ultrafiltration column. The antibody was replaced in PBS, and the antibody concentration was determined. At the same time, 2ug of antibody was taken for gel staining to verify antibody purity.

[0098] (6) Screening for mouse anti-human CD209L and CD209 monoclonal antibodies that can block infection of SARS-CoV-2, SARS-CoV and Ebola pseudovirus.

[0099] 293T cells expressing human CD209L and CD209 were infected with SARS-CoV-2 pseudovirus, along with control antibodies and mouse anti-human CD209L and CD209 monoclonal antibodies. Cells were incubated at 37°C for 24 hours and then collected. The SARS-CoV-2 pseudovirus infection status was detected by luciferase activity assay. Similarly, 293T cells expressing human CD209L and CD209 were infected with Ebola pseudovirus, along with control antibodies and mouse anti-human CD209L and CD209 monoclonal antibodies. Cells were incubated at 37°C for 24 hours and then collected. The Ebola pseudovirus infection status was detected by luciferase activity assay.

[0100] Through cell infection experiments, we screened out a mouse anti-human CD209L and CD209 monoclonal antibody that can simultaneously block SARS-CoV-2, SARS-CoV and Ebola pseudovirus infection: clone number 7H7B1.

[0101] 3. Hybridoma cell sequencing / variable region gene sequencing

[0102] Hybridoma cells were collected after reaching the logarithmic growth phase (clone number 7H7B1). Total RNA was extracted from the hybridoma cells, and first-strand cDNA complementary to the full-length mRNA was obtained by RT-PCR using 3'RACE and 5'RACE techniques. Using the synthesized cDNA as a template, the antibody heavy and light chain variable region genes were amplified by PCR. The antibody variable region genes were ligated into a T vector, transformed, and positive clones were selected for sequencing. The sequencing results were analyzed by bioinformatics to obtain the antibody variable region gene sequence, as shown in SEQ ID NO: 1-6.

[0103] Example 2

[0104] Preparation of CD209L and CD209 recombinant antibodies

[0105] 1. Construction of recombinant antibody plasmid

[0106] The constant regions of the heavy and light chains of the human IgG4 antibody were cloned into the pCAGGS vector. Then, the variable regions of the heavy and light chains of the obtained mouse anti-human CD209L and CD209 antibodies were inserted into the pCAGGS expression vector containing the constant region of the human IgG4 antibody, constructing a recombinant antibody expression vector. This vector was transfected into 293F mammalian cells for recombinant antibody expression (500ug / 500ml). The human-mouse chimeric monoclonal antibody with clone number 7H7B1 consists of the variable region of the mouse antibody and the constant region of the human IgG4 antibody.

[0107] 2. Expression and purification of recombinant antibodies

[0108] After transfecting 293F cells, continue culturing for 5-6 days, collect the cell culture supernatant, and purify the anti-CD209L and CD209 recombinant human-mouse chimeric antibodies in the supernatant using a Protein G affinity chromatography column.

[0109] Example 3

[0110] Characterization of anti-CD209L and CD209 recombinant human-mouse chimeric antibodies

[0111] 1. Experiments using human-mouse chimeric antibodies against CD209L and CD209 to block SARS-CoV-2 pseudovirus infection in cells expressing human CD209L and CD209:

[0112] 293T cells expressing human CD209L and CD209 were infected with SARS-CoV-2 pseudovirus. Human-mouse chimeric monoclonal antibodies against CD209L and CD209 (7H7B1, both 10ug / ml) were added, and the cells were incubated at 37 degrees Celsius for 24 hours. The viral infection status was detected by luciferase activity assay using the Promega Luciferase Assay System (Cat.#E1501).

[0113] Results reference Figure 1 As shown, the results indicated that the addition of CD209L and CD209 human-mouse chimeric monoclonal antibodies significantly inhibited viral infection of human CD209L(A) and CD209(B) 293T cells, blocking SARS-CoV-2 pseudovirus infection. Furthermore, the effect was concentration-dependent; higher concentrations of CD209L and CD209 human-mouse chimeric monoclonal antibodies resulted in better blocking efficacy.

[0114] 2. Experiments using human-mouse chimeric antibodies against C209L and CD209 to block SARS-CoV infection in cells expressing human CD209L and CD209:

[0115] 293T cells expressing human CD209L and CD209 were infected with SARS-CoV pseudovirus. Human-mouse chimeric monoclonal antibodies against CD209L and CD2092 (7H7B1, both 10ug / ml) were added, and the cells were incubated at 37 degrees Celsius for 24 hours. The viral infection status was detected by luciferase activity assay using the Promega Luciferase Assay System (Cat.#E1501).

[0116] Results reference Figure 2 As shown, the results indicated that the addition of CD209L and CD209 human-mouse chimeric monoclonal antibodies significantly inhibited coronavirus infection of human CD209L(C) and CD209(D) 293T cells, thus blocking coronavirus infection. Furthermore, the effect was concentration-dependent; the higher the concentration of CD209L and CD209 human-mouse chimeric monoclonal antibodies, the better the blocking effect.

[0117] 3. Experiments using human-mouse chimeric antibodies against C209L and CD209 to block Ebola pseudovirus infection in cells expressing human CD209L and CD209:

[0118] Ebola pseudovirus was used to infect 293T cells expressing human CD209L and CD209. Human-mouse chimeric monoclonal antibodies against CD209L and CD2092 (7H7B1, both 10ug / ml) were added, and the cells were incubated at 37 degrees Celsius for 24 hours. The viral infection status was detected by luciferase activity assay using the Promega Luciferase Assay System (Cat.#E1501).

[0119] Results reference Figure 3 As shown, the results indicated that the addition of CD209L and CD209 human-mouse chimeric monoclonal antibodies significantly inhibited Ebola pseudovirus infection of human CD209L(E) and CD209(F) 293T cells, thus blocking Ebola pseudovirus infection. Furthermore, the effect was concentration-dependent; the higher the concentration of CD209L and CD209 human-mouse chimeric monoclonal antibodies, the better the blocking effect.

[0120] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0121] References:

[0122] 1. Jun Lan, et al. Structure of the SARS-CoV-2spike receptor-bindingdomain bound to the ACE2 receptor. Nature. 2020May; 581(7807):215-220.

[0123] 2. Michel Thépaut, et al. DC / L-SIGN recognition of spike glycoproteinpromotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimeticantagonist. PLoS Pathog. 2021May20; 17(5):e1009576.

[0124] 3.Yuji Kondo,et al.L-SIGN is a receptor on liver sinusoidalendothelial cells for SARS-CoV-2virus.JCI Insight.2021Jul22;6(14):e148999.

[0125] 4.Yasunori Watanabe,et al.Site-specific glycan analysis of the SARS-CoV-2spike.Science.2020Jul 17;369(6501):330-333.

[0126] 5.Nader Rahimi.C-type Lectin CD209L / L-SIGN and CD209 / DC-SIGN:CellAdhesion Molecules Turned to Pathogen Recognition Receptors.Biology(Basel).2020Dec 22;10(1):1.

[0127] 6.Yanyun Du,et al.A broadly neutralizing humanizedACE2-targetingantibody against SARS-CoV-2 variants.Nat Commun.2021 Aug 17;12(1):5000.

[0128] 7.Yuning Chen,et al.ACE2-targeting monoclonal antibody aspotent andbroad-spectrum coronavirus blocker.Signal Transduct TargetTher.2021 Aug 25;6(1):315.

[0129] 8.Jianxia Ou,et al.ACE2-Targeting antibody suppressesSARS-CoV-2Omicron and Delta variants.Signal Transduct Target Ther.2022 Feb 9;7(1):43.

Claims

1. A monoclonal antibody against human CD209 and CD209L, comprising a heavy chain variable region and a light chain variable region, characterized in that, The heavy chain variable region includes the following three heavy chain complementarity-determining regions: CDR-VH1, CDR-VH2, and CDR-VH3. The light chain variable region includes the following three light chain complementarity-determining regions: CDR-VL1, CDR-VL2, and CDR-VL3. The amino acid sequences of 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 shown in SEQ ID NO: 4-6, and the amino acid sequence of SEQ ID NO: 5 is GAS.

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, and scFv.

3. The antibody against human CD209 and CD209L according to claim 1, characterized in that, The antibodies against human CD209 and CD209L are human-mouse chimeric monoclonal antibodies against human CD209 and CD209L or scFv.

4. The antibody against human CD209 and CD209L according to claim 1, characterized in that, The antibody includes light chain backbone regions FR-L1, FR-L2, FR-L3 and FR-L4, and / or heavy chain backbone regions FR-H1, FR-H2, FR-H3 and FR-H4.

5. The antibody against human CD209 and CD209L according to claim 1, characterized in that, The antibody also includes an antibody constant region.

6. The antibody against human CD209 and CD209L according to claim 5, characterized in that, The antibody constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

7. The antibody against human CD209 and CD209L according to claim 5, characterized in that, The species source of the antibody constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

8. The antibody against human CD209 and CD209L according to claim 7, characterized in that, The species origin of the antibody constant region is human.

9. The antibody against human CD209 and CD209L according to claim 5, characterized in that, The constant region of the antibody is selected from human IgG4.

10. The use of an antibody against human CD209 and CD209L as described in any one of claims 1-9 in the preparation of a pharmaceutical product, characterized in that, The drug is used to prevent diseases caused by infection with the novel coronavirus, SARS-CoV coronavirus, or Ebola virus.

11. A drug, characterized in that, The drug comprises the antibody against human CD209 and CD209L as described in any one of claims 1-9, and the drug is used to prevent disease caused by infection with the novel coronavirus, SARS-CoV coronavirus, or Ebola virus.

12. The use of an antibody against human CD209 and CD209L as described in any one of claims 1-9 in the preparation of a reagent or kit for detecting CD209 or CD209L.

13. A reagent or kit for detecting CD209 or CD209L, characterized in that, It includes the antibodies against human CD209 and CD209L as described in any one of claims 1-9.

14. An expression box or carrier, characterized in that, It contains a gene encoding the antibody against human CD209 and CD209L as described in any one of claims 1-9.

15. A host cell, characterized in that, It contains the carrier as described in claim 14.

16. A method for producing antibodies against human CD209 and CD209L as described in any one of claims 1 to 9, characterized in that, It includes: The host cells of claim 15 are cultured, and the antibodies against human CD209 and CD209L are isolated and purified from the culture medium or from the cultured host cells.

Images