An antibody targeting hr1 and a preparation method and application thereof

Abstract

The application discloses an antibody targeting HR1 and a preparation method and application thereof, wherein the amino acid sequence of HCDR1 of the antibody is shown as SEQ ID NO: 4, the amino acid sequence of HCDR2 is shown as SEQ ID NO: 6, the amino acid sequence of HCDR3 is shown as SEQ ID NO: 8, the amino acid sequence of LCDR1 is shown as SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown as SEQ ID NO: 14, and the amino acid sequence of LCDR3 is shown as SEQ ID NO: 16. The antibody has high affinity to RNA viruses, has broad-spectrum anti-RNA virus activity, and can be used as a tool for scientific research, basic medical research, diagnosis and clinical medical research.

Description

[0001] This application claims priority to Chinese patent application 2025105093316, filed on April 22, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedical technology, specifically to an antibody targeting HR1, its preparation method, and its application. Background Technology

[0003] Coronaviruses (CoV) are a large family of naturally occurring, enveloped RNA viruses with a positive-sense single-stranded genome. Nidovirales Mewa Coronaviridae Coronaviruses are named for their distinctive crown-like appearance observed under an electron microscope. Currently, seven coronaviruses are known to widely infect humans: HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 cause mild to moderate upper respiratory tract illness, while SARS-CoV, MERS-CoV, and SARS-CoV-2 cause severe infectious diseases. During its spread, SARS-CoV-2 has produced numerous mutant strains that have evaded most neutralizing antibodies, including some previously granted emergency authorization. Therefore, addressing immune-evading SARS-CoV-2 and other potential future coronaviruses presents a key challenge in developing neutralizing antibodies. Antibody development for SARS-CoV-2 primarily focuses on the mechanism by which SARS-CoV-2 enters host cells, including the binding of the S protein to angiotensin-converting enzyme 2 (ACE2) and subsequent S protein-mediated membrane fusion, a mechanism common to other RNA viruses. Viral membrane fusion with host cells is an essential pathway for viral infection, and blocking this pathway is an effective treatment for viral infections. The heptapeptide repeat sequence HR1 domain involved in the fusion exhibits fewer mutations and is relatively conserved in viruses. Antibodies possess advantages such as high affinity and target specificity, preventing viral invasion of host cells by specifically binding to the conserved HR1 sequence of the virus, thus showing great potential for treating viral infections. However, a broad-spectrum drug for the treatment and detection of multiple RNA viruses, including SARS-CoV-2, is still lacking. Summary of the Invention

[0004] To address the current technical problem of lacking a broad-spectrum drug for the treatment and detection of various RNA viruses, including SARS-CoV-2, this invention provides an antibody targeting HR1, its preparation method, and its application. This antibody has the ability to neutralize RNA viruses across a broad spectrum.

[0005] In order to solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0006] A first aspect of the present invention provides an antibody targeting HR1, the neutralizing antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequence of HCDR1 being shown in SEQ ID NO: 4, the amino acid sequence of HCDR2 being shown in SEQ ID NO: 6, the amino acid sequence of HCDR3 being shown in SEQ ID NO: 8, the amino acid sequence of LCDR1 being shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 being shown in SEQ ID NO: 14, and the amino acid sequence of LCDR3 being shown in SEQ ID NO: 16, the antibody or its antigen-binding fragment being capable of binding to an RNA virus.

[0007] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 2 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 2.

[0008] In some embodiments of the present invention, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 10 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 10.

[0009] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10.

[0010] In some embodiments of the present invention, the coronavirus is preferably selected from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV-1, batCoV-BM48-31, BANAL-20-247, RpYN06, Anser fabalis NCN2, Canada goose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1. More preferably, the coronavirus is selected from one or more of SARS-CoV-2 Alpha, Beta, Gamma, and Delta variants.

[0011] In some embodiments of the present invention, the antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv, wherein the Fv is preferably scFv; when the antibody is a full-length antibody, the heavy chain constant region of the antibody is preferably derived from the heavy chain of a human antibody or a variant thereof, and the light chain constant region is preferably derived from the κ chain or λ chain of a human antibody or a variant thereof.

[0012] In some specific embodiments of the present invention, the antibody is scFv.

[0013] A second aspect of the present invention provides an antigenic epitope peptide that binds to an antibody as described in the first aspect of the present invention; the amino acid sequence of the antigenic epitope peptide is shown in SEQ ID NO: 19 or SEQ ID NO: 47, wherein X1 and X4 represent any amino acid residues, X2 and X3 independently represent amino acid residues having smaller hydrophobic side chains, such as V, I, L, M or A, and X5 represents amino acid residue N or Q.

[0014] In some embodiments of the present invention, the amino acid sequence of the antigenic epitope peptide is as shown in SEQ ID NO: 18, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 47.

[0015] In some embodiments of the present invention, the amino acid sequence of the antigenic epitope peptide is as shown in SEQ ID NO: 18, 22 or 28.

[0016] A third aspect of the present invention provides an isolated nucleic acid that encodes an antibody as described in the first aspect of the present invention.

[0017] In some embodiments of the present invention, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 3, and / or the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 11.

[0018] A fourth aspect of the present invention provides a recombinant expression vector comprising isolated nucleic acids as described in the third aspect of the present invention.

[0019] In some embodiments of the present invention, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector, and the viral vector is preferably a retroviral vector, lentiviral vector, adenovirus vector, or adeno-associated virus vector.

[0020] A fifth aspect of the present invention provides a transformant comprising isolated nucleic acid as described in the third aspect of the present invention or a recombinant expression vector as described in the fourth aspect of the present invention, wherein the host cell of the transformant is a eukaryotic cell or a prokaryotic cell.

[0021] In some embodiments of the present invention, the eukaryotic cells are mammalian cells, such as HEK293 or CHO cells.

[0022] A sixth aspect of the present invention provides a method for preparing an antibody, the method comprising culturing a transformant as described in the fifth aspect of the present invention to prepare the antibody.

[0023] A seventh aspect of the present invention provides a pharmaceutical composition for combating RNA viruses, or for preventing or treating diseases caused by RNA virus infection, said pharmaceutical composition comprising an antibody as described in the first aspect of the present invention, and optionally a pharmaceutically acceptable carrier.

[0024] In some embodiments of the present invention, the RNA virus is selected from one or more of coronaviruses, MARV viruses, HERV viruses, and HIV viruses.

[0025] In some embodiments of the present invention, the coronavirus is selected from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV, batCoV-BM48-31, BANAL-20-247, RpYN06, Anser fabalis NCN2, Canada goose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1.

[0026] In some embodiments of the present invention, the coronavirus is selected from one or more of the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2.

[0027] An eighth aspect of the present invention provides a kit comprising an antibody as described in the first aspect of the present invention or a pharmaceutical composition as described in the seventh aspect of the present invention.

[0028] In some embodiments of the present invention, the kit further includes (1) an apparatus for administering the antibody or the pharmaceutical composition; and / or (2) instructions for use.

[0029] In some embodiments of the present invention, the kit is used to detect RNA viruses; the RNA virus is preferably one or more of coronaviruses, MARV viruses, HERV viruses, and HIV viruses; the coronavirus is selected, for example, from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV, batCoV-BM48-31, BANAL-20-247, RpYN06, Anser fabalis NCN2, Canada goose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1; more preferably, the coronavirus is selected from one or more of SARS-CoV-2 Alpha, Beta, Gamma, and Delta variants.

[0030] A ninth aspect of the present invention provides a pillbox set, the pillbox set comprising pillbox A and pillbox B, wherein: The medicine box A contains an antibody as described in the first aspect of the present invention or a pharmaceutical composition as described in the seventh aspect of the present invention; Kit B contains other antibodies against RNA viruses or pharmaceutical compositions containing said other antibodies against RNA viruses, and / or other anti-RNA virus drugs. In this invention, "other antibodies against RNA viruses" does not include antibodies targeting HR1.

[0031] In some embodiments of the present invention, the RNA virus is selected from one or more of coronaviruses, MARV virus, HERV virus, and HIV virus. The coronavirus is preferably selected from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV, batCoV-BM48-31, BANAL-20-247, RpYN06, Anserfabalis NCN2, Canada goose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1. More preferably, the coronavirus is selected from one or more of SARS-CoV-2 Alpha, Beta, Gamma, and Delta variants.

[0032] The tenth aspect of the present invention provides the use of antibodies as described in the first aspect of the present invention, isolated nucleic acids as described in the third aspect of the present invention, recombinant vectors as described in the fourth aspect of the present invention, transformants as described in the fifth aspect of the present invention, pharmaceutical compositions as described in the seventh aspect of the present invention, kits as described in the eighth aspect of the present invention, or kits as described in the ninth aspect of the present invention in the preparation of medicaments for detecting RNA viruses, inhibiting RNA viruses, preventing RNA virus infection-related diseases, or treating RNA virus infection-related diseases.

[0033] In some embodiments of the present invention, the RNA virus is selected from one or more of coronaviruses, MARV viruses, HERV viruses, and HIV viruses.

[0034] In some embodiments of the present invention, the coronavirus is selected from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV, batCoV-BM48-31, BANAL-20-247, RpYN06, Anser fabalis NCN2, Canada goose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1.

[0035] In some embodiments of the present invention, the coronavirus is selected from one or more of the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2.

[0036] The eleventh aspect of the present invention provides a drug delivery device comprising an antibody as described in the first aspect of the present invention, a pharmaceutical composition as described in the seventh aspect of the present invention, a reagent kit as described in the eighth aspect of the present invention, or a kit as described in the ninth aspect of the present invention.

[0037] In some embodiments of the invention, the drug delivery device further includes components for administering the antibody, the pharmaceutical composition, the kit, or the kit to a subject, such as a syringe or infusion device.

[0038] The twelfth aspect of the present invention provides a method for detecting RNA viruses, the method comprising contacting a sample to be tested with an antibody as described in the first aspect of the present invention, a pharmaceutical composition as described in the seventh aspect of the present invention, or a kit as described in the eighth aspect of the present invention.

[0039] In some embodiments of the present invention, the RNA virus is selected from one or more of human coronaviruses, MARV virus, HERV virus, and HIV virus; the human coronavirus is preferably selected from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV, batCoV-BM48-31, BANAL-20-247, RpYN06, Anser fabalis NCN2, Canada goose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1; more preferably, the coronavirus is selected from one or more of the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2.

[0040] In some embodiments of the present invention, the method is for non-diagnostic purposes.

[0041] The thirteenth aspect of the present invention provides a method for treating RNA viruses, or for preventing or treating diseases caused by RNA virus infection, the method comprising administering to a patient in need a therapeutically effective amount of an antibody as described in the first aspect of the present invention, a pharmaceutical composition as described in the seventh aspect of the present invention, or a kit as described in the eighth aspect of the present invention, or treating a patient in need using a drug delivery device as described in claim 12.

[0042] In some embodiments of the present invention, the RNA virus is selected from one or more of coronaviruses, MARV virus, HERV virus, and HIV virus; the coronavirus is preferably selected from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV, batCoV-BM48-31, BANAL-20-247, RpYN06, Anserfabalis NCN2, Canada goose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1; more preferably, the coronavirus is selected from one or more of SARS-CoV-2 Alpha, Beta, Gamma, and Delta variants.

[0043] The fourteenth aspect of the present invention provides antibodies as described in the first aspect of the present invention, isolated nucleic acids as described in the third aspect of the present invention, recombinant vectors as described in the fourth aspect of the present invention, transformants as described in the fifth aspect of the present invention, pharmaceutical compositions as described in the seventh aspect of the present invention, kits as described in the eighth aspect of the present invention, or kits as described in the ninth aspect of the present invention for preparing drugs for detecting RNA viruses, inhibiting RNA viruses, preventing RNA virus infection-related diseases, or treating RNA virus infection-related diseases.

[0044] In some embodiments of the present invention, the RNA virus is selected from one or more of coronaviruses, MARV virus, HERV virus, and HIV virus; the coronavirus is preferably selected from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV, batCoV-BM48-31, BANAL-20-247, RpYN06, Anserfabalis NCN2, Canada goose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1; more preferably, the coronavirus is selected from one or more of SARS-CoV-2 Alpha, Beta, Gamma, and Delta variants.

[0045] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0046] The reagents and raw materials used in this invention are all commercially available.

[0047] The positive and progressive effects of this invention are as follows: The antibody targeting HR1 provided by this invention has high affinity for RNA viruses and broad-spectrum anti-RNA virus activity, and can be used as a tool for scientific research, basic medical research, diagnostic and clinical medical research. Attached Figure Description

[0048] Figure 1 The epitopes and binding modes of the HR1 peptide that binds to SARS-CoV-2 are shown.

[0049] Figure 2A and Figure 2B To detect the affinity of 3D1 for various coronavirus HR1 peptides in the BLI experiment, the antibody was used to characterize the affinity of 3D1 for these peptides using two to three concentration gradients. The corresponding concentrations are displayed above each curve. The actual measured curves are shown in black, while the curves generated according to the fitting model are shown in corresponding colors. The final calculated affinity is displayed at the top of the curve.

[0050] Figure 3 The study demonstrated the broad-spectrum inhibitory effect of 3D1 against pseudoviruses (PsV) of various coronaviruses.

[0051] Figure 4 The study demonstrated the inhibitory effect of 3D1 on the pseudovirus of MARV.

[0052] Figure 5 The inhibitory effect of 3D1 on live SARS-CoV-2 and its derived mutants was demonstrated. Detailed Implementation

[0053] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below: In this invention, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0054] In this invention, the amino acid sequences of the listed complementarity-determining regions (CDRs) are all as defined according to the IMGT numbering rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody sequence variability, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), Kabat, and North CDR definitions based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementary determination region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementary determination region defined by any of the above-described known schemes as described in this invention.

[0055] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of protection claimed by this invention is based on the sequence defined according to the IMGT numbering rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0056] In this invention, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this invention) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this invention) and a light chain constant region (abbreviated as CL in this invention). The light chain constant region consists of one domain: CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins IgA, IgD, IgE, IgG, and IgM. A complete antibody forms a "Y" shape. The stem of the Y is formed by the second and third constant regions of the two heavy chains (and, for IgE and IgM, a fourth constant region) linked together, and disulfide bonds (interchain) are formed in the hinge. The heavy chains γ, α, and δ have constant regions consisting of three tandem (in a row) Ig domains and hinge regions for increased flexibility; the heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain," respectively. Each arm of the Y chain includes a variable region of a single heavy chain and a first constant region that binds to a variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0057] In this invention, a "Fab fragment" consists of a light chain and a heavy chain, comprising the CH1 domain and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain. A "Fab' fragment" contains a portion of a light chain and a heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of the two Fab' fragments to form the F(ab')2 molecule. An "F(ab')2 fragment" contains two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody, but lacking the constant region.

[0058] In this invention, scFv refers to a single-chain antibody fragment, which includes a heavy chain variable region, a light chain variable region, and a linker peptide of 15-20 amino acids. The VL and VH domains enable the linker peptides to pair and form monovalent molecules as single polypeptide chains [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have a general structure: NH2-VL-linker peptide-VH-COOH or NH2-VH-linker peptide-VL-COOH.

[0059] In this invention, "nucleic acid" refers to a nucleotide chain of any length and includes DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or its analogues, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.

[0060] In this invention, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of this invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of this invention can contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors can contain naturally occurring or non-naturally occurring nucleotide linkages, or both. In an exemplary aspect, modified nucleotides or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector.

[0061] The recombinant expression vector of the present invention can be any suitable recombinant expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0062] In this invention, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it may be a prokaryotic cell, such as a bacterium or protozoan.

[0063] In this invention, the pharmaceutical composition may comprise a suitable pharmaceutically acceptable carrier, such as pharmaceutical excipients, including buffers, as known in the art. "Pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions comprising the invention can be prepared by mixing antibodies of the invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Preferably, the composition is in the form of a lyophilized formulation or an aqueous solution.

[0064] The pharmaceutical compositions of the present invention may also comprise more than one active ingredient required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, antiviral agents, small molecule drugs, or immunomodulators. The active ingredients are suitably combined in amounts effective for the intended use. Sustained-release formulations can be prepared, suitable examples of which include a semi-permeable matrix of a solid hydrophobic polymer containing the antibody of the present invention, said matrix being a shaped article, such as a film or microcapsule.

[0065] In this invention, the application scenarios for "non-diagnostic purposes" include, but are not limited to: for example, detecting the presence of antigen HR1 / RNA virus in vitro in the laboratory; or using it as a positive antibody to screen other antibodies targeting HR1; or competing with other antibodies targeting HR1 to detect whether there is competition between the antibodies, i.e., whether the antigen epitopes are the same or similar.

[0066] In this invention, "RNA virus infection-related diseases" refers to a series of infectious diseases caused by RNA viruses.

[0067] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0068] Example 1: Antibody sequence, full-length antigen sequence and its epitope sequence

[0069] Sangon Biotech (Shanghai) Co., Ltd. synthesized the SARS-CoV-2 HR1 FC (FusionCore) peptide (residue range: 924-955), with the amino acid sequence HHHHHHANQFNSAIGKIQDSLSSTASALGKLQDVVNQN (SEQ ID NO: 1). The N-short of this peptide was biotinylated, and the biotinylated peptide was used as the antigen for phage screening. After multiple rounds of phage screening, an antibody 3D1 (scFv form) binding to this peptide was obtained. Its sequence information is shown in Table 1 below. The variable region sequence was defined according to the IMGT numbering system.

[0070] Table 1 Sequence information of antibody 3D1

[0071] A mixture of 15 mg / mL 3D1 (scFv) and the SARS-CoV-2 HR1 C-terminal peptide (amino acid sequence Ac-SLSSTASALGKLQDVVNQN-NH2) assembled at a 2:1 molar ratio was screened for crystallization using 576 conditions from Hampton Research and Qiagen on Mosquito (TTP LabTech). A crystallization assay was established using vapor diffusion in droplets containing 0.1 μL of protein and 0.1 μL of storage solution. The optimized storage solution conditions for crystal growth of the 3D1 scFv-DVVNQN peptide (SARS-CoV-2) complex were as follows: 0.1 M BIS-TRIS buffer (containing 25% (w / v) polyethylene glycol 3350), pH 5.5, and growth temperature of 16 °C.

[0072] Crystals were harvested by immersion in a stock solution replenished with 20% (v / v) glycerol or methanol, followed by rapid cooling and storage in liquid nitrogen until data collection. Diffraction data were collected at BL19U1 beamline at 0.97853 Å at cryogenic temperature (100 K) on the Shanghai Synchrotron Radiation Facility (SSRL). The dataset was indexed, integrated, and scaled using the HKL-2000 software package. Molecular substitution was performed in PHENIX using PHASER, with the homology models SWISS-MODEL and PIGSPro used as search models for apo 3D1 scFv or 3D1 Fab-pepDVVNQN to resolve the structure. Iterative model building and refinement were performed in COOT and PHENIX, combined with additional manual model building processes. After several rounds of refinement, a clear Fo-Fc density was observed at the 3D1 binding sites on the peptide. The peptides were manually constructed into differential density Fo-Fc maps, followed by further refinement of the complex in PHENIX.refine and manual construction cycles in COOT. BSA and para-ionic residues and their interactions were identified using PISA from the European Institute of Bioinformatics (http: / / www.ebi.ac.uk / pdbe / prot_int / pistart.html). These results are presented in [the table / link / ]. Figure 1 middle.

[0073] The co-crystal structure of the 3D1-scFv complex with the SARS-CoV-2 HR1 peptide was resolved to 1.08 Å. The structural results showed that only the electron cloud density of the six C-terminal amino acids of HR1 could be determined, which was [missing value]. 950 DVVNQN 955Furthermore, the figure shows that the DVVNQN (SEQ ID NO: 18) peptide interacts with the complementarity-determining region (CDR) of the 3D1 heavy and light chains in an extended chair-like structure (β-turn), rather than the α-helical secondary structure present in the pre-fusion or post-fusion 6-HB. This indicates that the minimal epitope of 3D1 is a hexapeptide containing a β-turn. The unique interaction pattern between Q954 and 3D1 is crucial and indispensable for 3D1's recognition of the antigen. Combining alanine scanning and peptide affinity assays (using the Biomembrane Interference (BLI) method with PBS buffer plus 0.1% Tween 20), the results showed that Q954 mutations prevent recognition, while the N955A mutation reduces the affinity of 3D1 for antigen recognition by an order of magnitude (see affinity data in Table 2 below). Therefore, the pattern of antigen sequences recognized by 3D1 is X1X2X3X4QX5 (SEQ ID NO: 19), where X1 and X4 represent any amino acid residue, X2 and X3 independently represent amino acid residues with small hydrophobic side chains such as V, I, L, M and A, and X5 represents amino acid residue N or Q. Exemplary sequences are DVVNQQ (SEQ ID NO: 20), QVVNQQ (SEQ ID NO: 21), AVVNQN (SEQ ID NO: 22), SVVNQQ (SEQ ID NO: 23), DVVNQN, DVINQN (SEQ ID NO: 24), TVVNQQ (SEQ ID NO: 25), AVVLQN (SEQ ID NO: 26), GIVQQQ (SEQ ID NO: 27) or DVVAQN (SEQ ID NO: 28).

[0074] Table 2 Affinity Data

[0075] Example 2: Verification of antibody binding activity

[0076] Based on the protein complex structures resolved by X-ray crystal diffraction, we screened a series of peptides with 3D1 binding characteristics and used the biofilm layer interferometry (BLI) method to determine the affinity of 3D1 for the HR1 peptide (sequences shown in Table 3) of various coronaviruses (including but not limited to human coronaviruses such as Alpha, Beta, Delta, and Gamma viruses, such as 229E, NL63, SARS-CoV-2, SARS-CoV, etc., as well as MARV (Marburg virus), HERV (Human Endogenous Retroviruses), and HIV (Human Immunodeficiency Virus)). A Satorious Octect RED96 instrument was used; the specific operation method is as follows: Table 3. Peptide sequences and corresponding virus names used for affinity detection in the experiment.

[0077] The peptides were dissolved and diluted to 0.05 mg / mL in Octet buffer: PBS (Sigma, #C3041) with 0.1% Tween-20 (Yeasen, #60305ES76). The peptides were captured using an activated Ni-NTA biosensor for 5–10 seconds, followed by transfer to Octet buffer for 60 seconds to remove unbound peptides and establish a baseline. After establishing the baseline, the sensor was immersed in antibody solutions of different concentrations, and binding signals were recorded for 300 seconds, followed by dissociation in Octet buffer for 300 seconds. The generated data were analyzed using FortéBio / Sartorius analysis software (version 12.0) for correction, curve fitting, and calculations. K D The value is plotted using GraphPad Prism version 9.1.1.

[0078] Depend on Figure 2A and Figure 2B It can be seen that 3D1 has an affinity of nM for the peptides corresponding to all the above viruses.

[0079] Example 3: Experimental Verification of Inhibition of Coronavirus

[0080] Pseudovirus Packaging: HEK293T cells were transfected using PEI (Polysciences, #24765). Following the PEI instructions, pcDNA3.1 plasmid containing the spike protein (encoding the spike protein of different viruses) and NL4-3-luciferase-mecherry plasmid with red fluorescence and luciferase were co-transfected. Six hours after transfection, the supernatant was discarded and replaced with fresh culture medium. Forty-eight hours later, the supernatant containing pseudovirus particles (PsV) was collected by filtration through a 0.45 μm filter membrane (Sartorius, #16533-K). The virus stock solution was concentrated using Lenti-X concentrator (Takara, #631231) according to the reagent instructions and stored at -80°C for use.

[0081] Sham virus neutralization assay: The sham viruses used in the sham virus neutralization assay were concentrated viruses prepared using the method described above. First, target cells containing different viruses were prepared at a density of 1 × 10⁶ cells per well. 4 The target cells were seeded at a density in 96-well white opaque plates (Jing'an Biotechnology, #J09601) and cultured overnight. The antibody was diluted to different concentrations (dilution factor: 3.16-fold) with medium containing 10% serum (Gibco, 10091148) and incubated with PsV at 37°C for 30 minutes using an equal volume. The supernatant of the target cells was discarded, and 100 μL of the antibody-virus mixture was transferred to a 96-well plate containing the target cells. After 16 hours, the medium was replaced with 100 μL of fresh medium per well, and the plate was cultured for another 48 hours. 50 μL of luciferase substrate (Beyotime, #RG051M) was added to each well, and after 5 minutes of reaction, the lumi chemiluminescence was measured using a microplate reader (Enspire, PekinElmer). Nonlinear regression analysis (four-parameter fitting) was performed using GraphPad Prism 9.1.1 to obtain the IC50 values ​​of the antibody against different viral pseudoviruses. 50 value.

[0082] Pseudoviruses containing various Alpha / Beta / Delta coronaviruses, including SARS-CoV, SARS-CoV-2, HCoV-NL63, SL-Rs3367, SL-WIV1, BANAL-20-52, and PCoV-GX / P2V / 2017, were packaged using NL4-3 with 293T- h ACE2 is the target cell for HCoV-229E and Hu-PDCoV at 293T- h APN was used as the target cell, MERS-CoV used huh-7 as the target cell, and CCoV-HuPn-2018 used 293T- fAPN was successfully used as the target cell to construct a pseudovirus-based infection system. The pseudovirus results showed that 3D1 can neutralize coronaviruses of the Alpha / Beta / Delta genus (see...). Figure 3 ).

[0083] Example 4: Experimental verification of inhibiting other RNA viruses

[0084] MARV (Marburg virus), a filovirus, requires its GP2 protein to form 6-HB to infect host cells. Comparison revealed a binding site for 3D1 in MARV, specifically MARV "LIKNQN" (SEQ ID NO: 47). A pseudovirus of MARV was packaged using the NL4-3 system, targeting Vero-E6 cells (see Example 3 for details). Experimental results on antibody neutralization of the MARV pseudovirus showed that 3D1 can inhibit MARV pseudovirus infection (see Example 3). Figure 4 ).

[0085] Example 5 Experimental verification of inhibition of live SARS-CoV-2 virus (Plaque Reduction Neutralization Test (PRNT))

[0086] Live virus was developed at the CL3 facility at Oxford University, using a capping layer containing 1.5% carboxymethyl cellulose to prevent satellite focus formation. Vero-TMPRSS2 cells (Vero cells stably expressing TMPRSS2) were cultured at 2 × 10⁻⁶ cells / year. 4 Cells / well were seeded into 96-well plates with black walls and clear bottoms and cultured overnight at 37°C. The next day, antibodies were diluted 9.5-fold using DMEM + 10% FBS medium. Different SARS-CoV-2 strains were diluted in the infection medium to a final MOI of 0.01 PFU / cell and added to the antibody dilution buffer, incubated at 37°C for 30 minutes. The original cell culture medium was removed, antibody-virus complexes were added, and incubated at 37°C for 18 hours. Cells were fixed with 4% PFA, permeabilized with Triton X-100, and stained with an antibody against the viral nucleocapsid protein, followed by staining with the nuclear dye Hoechst 33342 and goat anti-rabbit Alexa Fluor 647 antibody. Cells were imaged using an automated multimode plate reader (Biotek, Cytation 5). Infection foci were then counted using an ELISPOT reader. Nonlinear regression analysis (four-parameter fitting) was performed using GraphPad Prism 9.1.1.

[0087] The live virus experiments were conducted in collaboration with Nicole Zitzman's laboratory at the University of Oxford to investigate the neutralizing effect of the 3D1 antibody against wild-type and five variants of concern (VOCs): Alpha, Beta, Gamma, Delta, and Omicron. The target cells used in the experiments were Vero-TMPRSS2 cells. Figure 5 As shown, 3D1 showed excellent neutralizing effects against the British wild strain Eng2 / 20, Alpha, and Delta. The neutralizing effects against Beta and Gamma decreased but were still effective. Similar to the results of the pseudovirus experiment, 3D1 lost its neutralizing effect against Omicron.

[0088] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An antibody targeting HR1, the neutralizing antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3, characterized in that, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 6, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 8, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

16. The antibody or its antigen-binding fragment can bind to RNA viruses.

2. The antibody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:2 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

2. And / or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 10 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 10; And / or, the RNA virus is selected from one or more of coronaviruses, MARV virus, HERV virus and HIV virus, wherein the coronavirus is preferably selected from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV-1, batCoV-BM48-31, BANAL-20-247, RpYN06, Anser fabalis NCN2, Canadagoose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367 and SL-WIV1, and more preferably, the coronavirus is selected from one or more of SARS-CoV-2 Alpha, Beta, Gamma and Delta variants; And / or, the antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv, wherein the Fv is preferably scFv; when the antibody is a full-length antibody, the heavy chain constant region of the antibody is preferably derived from the heavy chain of a human antibody or a variant thereof, and the light chain constant region is preferably derived from the κ chain or λ chain of a human antibody or a variant thereof.

3. An antigenic epitope peptide, characterized in that, The antigenic epitope peptide binds to the antibody according to claim 1 or 2; the amino acid sequence of the antigenic epitope peptide is shown in SEQ ID NO: 19 or SEQ ID NO: 47, wherein X1 and X4 represent any amino acid residues, X2 and X3 independently represent amino acid residues with smaller hydrophobic side chains, such as V, I, L, M or A, and X5 represents amino acid residue N or Q. Preferably, the amino acid sequence of the antigenic epitope peptide is as shown in SEQ ID NO: 18, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 47; More preferably, the amino acid sequence of the antigenic epitope peptide is as shown in SEQ ID NO: 18, 22 or 28.

4. An isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the antibody as described in claim 1 or 2; preferably, the nucleotide sequence encoding the heavy chain variable region is as shown in SEQ ID NO: 3, and / or the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO:

11.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 4; preferably, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector, and the viral vector is preferably a retroviral vector, lentiviral vector, adenovirus vector, or adeno-associated virus vector.

6. A transformant, characterized in that, The transformant comprises the isolated nucleic acid as described in claim 4 or the recombinant expression vector as described in claim 5, and the host cell of the transformant is a eukaryotic cell or a prokaryotic cell; Preferably, the eukaryotic cells are mammalian cells, such as HEK293 or CHO cells.

7. A method for preparing an antibody, characterized in that, The method comprises culturing the transformant as described in claim 6 to prepare the antibody.

8. A pharmaceutical composition for combating RNA viruses, or for preventing or treating diseases caused by RNA virus infection, characterized in that, The pharmaceutical composition includes the antibody as described in claim 1 or 2, and optionally a pharmaceutically acceptable carrier; Preferably, the RNA virus is selected from one or more of coronaviruses, MARV virus, HERV virus, and HIV virus; the coronavirus is preferably selected from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV, batCoV-BM48-31, BANAL-20-247, RpYN06, Anser fabalis NCN2, Canadagoose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1; more preferably, the coronavirus is selected from one or more of the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2.

9. A reagent kit, characterized in that, The kit contains the antibody as described in claim 1 or 2 or the pharmaceutical composition as described in claim 8; Preferably, the kit further includes (1) an apparatus for administering the antibody or the pharmaceutical composition; and / or (2) instructions for use; And / or, the kit is used to detect RNA viruses; the RNA virus is preferably one or more of coronaviruses, MARV viruses, HERV viruses, and HIV viruses; the coronavirus is selected, for example, from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV, batCoV-BM48-31, BANAL-20-247, RpYN06, Anser fabalis NCN2, Canada goose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1; more preferably, the coronavirus is selected from one or more of SARS-CoV-2 Alpha, Beta, Gamma, and Delta variants.

10. A medicine box set, characterized in that, The pillbox set includes pillbox A and pillbox B, wherein: The medicine box A contains the antibody as described in claim 1 or 2 or the pharmaceutical composition as described in claim 8; The kit B contains other antibodies against RNA viruses or a pharmaceutical composition containing said other antibodies against RNA viruses, and / or other anti-RNA virus drugs; Preferably, the RNA virus is selected from one or more of coronaviruses, MARV virus, HERV virus, and HIV virus, and the coronavirus is preferably selected from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV, batCoV-BM48-31, BANAL-20-247, RpYN06, Anser fabalis NCN2, Canadagoose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1; more preferably, the coronavirus is selected from one or more of SARS-CoV-2 Alpha, Beta, Gamma, and Delta variants.

11. The use of the antibody as described in claim 1 or 2, the isolated nucleic acid as described in claim 4, the recombinant vector as described in claim 5, the transformant as described in claim 6, the pharmaceutical composition as described in claim 8, the kit as described in claim 9, or the kit as described in claim 10 in the preparation of medicaments for detecting RNA viruses, inhibiting RNA viruses, preventing RNA virus infection-related diseases, or treating RNA virus infection-related diseases; Preferably, the RNA virus is selected from one or more of coronaviruses, MARV virus, HERV virus, and HIV virus; the coronavirus is preferably selected from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV, batCoV-BM48-31, BANAL-20-247, RpYN06, Anser fabalis NCN2, Canadagoose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1; more preferably, the coronavirus is selected from one or more of the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2.

12. A drug delivery device, characterized in that, The drug delivery device comprises the antibody as described in claim 1 or 2, the pharmaceutical composition as described in claim 8, the kit as described in claim 9, or the kit as described in claim 10; Preferably, the drug delivery device further includes components for administering the antibody, the pharmaceutical composition, the kit, or the package to the subject, such as a syringe or infusion device.

13. A method for detecting RNA viruses, characterized in that, The method includes contacting the sample to be tested with an antibody as described in claim 1 or 2, a pharmaceutical composition as described in claim 8, or a kit as described in claim 9. Preferably, the RNA virus is selected from one or more of human coronaviruses, MARV virus, HERV virus, and HIV virus; the human coronavirus is preferably selected from one or more of HCoV-229E, HCoV-NL63, BatCoV-HKU2 / SADS-CoV, FCoV, FIPV, SARS-CoV-2, SARS-CoV, batCoV-BM48-31, BANAL-20-247, RpYN06, Anser fabalis NCN2, Canada goose CoV, Hu-PDCoV, Munia coronavirus HKU13, MtCoV, PCoV-GX / P2V / 2017, BANAL-20-52, MERS-CoV, SL-Rs3367, and SL-WIV1; more preferably, the coronavirus is selected from one or more of the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2. And / or, the method is not for diagnostic purposes.

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