Monoclonal antibodies targeting RSV F protein, their detection kits and applications

Abstract

This invention relates to monoclonal antibodies targeting the RSV F protein, related products, and applications. The monoclonal antibody (AD5) of this invention, or its antigen-binding fragment, specifically binds to the RSV F protein Site III antigenic epitope. The antibody AD5 of this invention combines the characteristics of targeting a highly conserved epitope (Site III) with high neutralizing activity, overcoming the major drawback of existing commercially available antibodies with low neutralizing activity when targeting highly conserved epitopes.

Description

Technical Field

[0001] This invention relates to the field of immunology technology, specifically to a monoclonal antibody targeting RSV F protein, its detection kit, and its applications. Background Technology

[0002] Respiratory syncytial virus (RSV) is one of the leading pathogens causing severe respiratory infections in infants, the elderly, and immunocompromised individuals, resulting in a significant disease burden and strain on healthcare resources worldwide. RSV is the leading cause of hospitalizations for acute lower respiratory tract infections in children under 5 years of age globally. Almost all children will be infected with RSV before the age of 2, with approximately half experiencing two infections during this period. RSV infection is also prevalent in adults and is often underestimated, with particularly severe impacts on the elderly, especially those ≥65 years of age.

[0003] Of the three transmembrane glycoproteins of RSV, the F protein is highly conserved, has a well-defined virus-neutralizing antibody binding site, and the ability to induce a strong neutralizing antibody response in natural infection. The F protein has two conformations: pre-F and post-F. Pre-F has six major antigenic sites (Ø, I, II, III, IV, and V), while post-F has four major antigenic sites (I, II, III, and IV).

[0004] Currently, three RSV prophylactic antibody drugs have been approved for marketing globally: nicevirumab, palizumab, and clarotrevirumab. Of these, only nicevirumab has been approved in China. Nicevirumab is expensive, and its target, Site Ø, is a "dominant non-conserved epitope" with strong neutralizing activity. However, because it is only briefly exposed before viral invasion, it faces significant evolutionary pressure and is prone to mutation to evade immune recognition. Escape mutations against nicevirumab have been detected in clinical isolates. Site Ø exhibits some variation between RSV A and B subtypes, which explains why some antibodies targeting Site Ø (such as nicevirumab's precursor D25) have insufficient neutralizing ability against certain type B strains (such as B18537). Palizumab's target, Site II, is relatively conserved, but its neutralizing activity is weaker. It requires monthly injections during RSV epidemic season, is expensive, and has a limited target population; therefore, it is not yet available in China. The exact target of another drug, clarotrevirumab, is still unclear; an NDA application has been submitted in China and accepted. Therefore, there is still a need in this field for an RSV F protein antibody that targets a conserved epitope and has high neutralizing activity. Summary of the Invention

[0005] Purpose of the invention

[0006] In view of the problems or needs existing in the prior art, the purpose of this invention is to provide a monoclonal antibody targeting RSV F protein, its detection kit and application.

[0007] Solution

[0008] To achieve the above objectives, this invention, through extensive screening, obtained a monoclonal antibody targeting the RSV F protein, particularly a monoclonal antibody or its antigen-binding fragment that specifically binds to the Site III sequence fragment in the RSV F protein. This invention provides a monoclonal antibody (AD5) targeting the Site III epitope of the RSV F protein. The Site III antigenic epitope is highly conserved in RSV A / B subtypes; amino acid position 305 is leucine (L) in RSV A subtype strains and isoleucine (I) in RSV B subtype strains, with almost no variation within subtypes. The antibody AD5 targeting the Site III antigenic epitope of this invention exhibits high neutralizing activity against both RSV A and B subtypes, regardless of viral subtype. Site III spans the core structure of the F protein, and single-point mutations are difficult to completely escape; multiple key amino acids must be altered simultaneously. Studies show that antibodies targeting this epitope develop resistance slowly under continuous viral evolutionary pressure, providing strong and durable protection and exhibiting excellent anti-escape capabilities. The antibody AD5 of this invention combines the characteristics of targeting a highly conserved epitope (Site III) and having high neutralizing activity, overcoming the main drawback of existing commercially available antibodies that have low neutralizing activity when targeting highly conserved epitopes.

[0009] Specifically, the present invention provides the following technical solution:

[0010] In a first aspect, the present invention provides a monoclonal antibody targeting the RSV F protein, comprising a heavy chain variable region and a light chain variable region, wherein,

[0011] The heavy chain variable region includes: complementarity-determining region HCDR1 as shown in SEQ ID NO:1, complementarity-determining region HCDR2 as shown in SEQ ID NO:2 and complementarity-determining region HCDR3 as shown in SEQ ID NO:3, respectively.

[0012] And / or, the light chain variable region comprises: a complementarity-determining region LCDR1 as shown in SEQ ID NO:4, a complementarity-determining region LCDR2 as shown in AAS, and a complementarity-determining region LCDR3 as shown in SEQ ID NO:5, respectively.

[0013] The monoclonal antibody of the present invention specifically targets the antigenic epitope Site III of RSV F protein, and can simultaneously target a highly conserved epitope (Site III) and have high neutralizing activity.

[0014] As a feasible embodiment, the heavy chain variable region further includes the amino acid sequences H-FR1 as shown in SEQ ID NO:6, H-FR2 as shown in SEQ ID NO:7, H-FR3 as shown in SEQ ID NO:8, and H-FR4 as shown in SEQ ID NO:9, respectively.

[0015] And / or, the light chain variable region further comprises the amino acid sequences of frame region L-FR1 as shown in SEQ ID NO:10, frame region L-FR2 as shown in SEQ ID NO:11, frame region L-FR3 as shown in SEQ ID NO:12, and frame region L-FR4 as shown in SEQ ID NO:13.

[0016] As a possible embodiment, the heavy chain variable region comprises, or is composed of, an amino acid sequence as shown in SEQ ID NO:14 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:14;

[0017] And / or, the light chain variable region comprises, or is composed of, an amino acid sequence as shown in SEQ ID NO:15 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:15.

[0018] As one possible embodiment, the monoclonal antibody comprises:

[0019] The heavy chain variable region, whose amino acid sequence is shown in SEQ ID NO:14; and,

[0020] The light chain variable region has the amino acid sequence shown in SEQ ID NO:15.

[0021] As a feasible embodiment, a signal peptide is also present at the N-terminus of the heavy chain variable region, and the signal peptide sequence of the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:27; optionally, the nucleotide sequence encoding the signal peptide is shown in SEQ ID NO:28.

[0022] And / or, at the N-terminus of the light chain variable region, there is a signal peptide, the signal peptide sequence of which has an amino acid sequence as shown in SEQ ID NO:29; optionally, the nucleotide sequence encoding the signal peptide is shown in SEQ ID NO:30.

[0023] As one possible embodiment, the monoclonal antibody comprises:

[0024] A heavy chain comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO:23 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:23; and,

[0025] Light chains comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO:25 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:25.

[0026] As a possible embodiment, it also includes an antigen-binding fragment selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies, human antibodies, chimeric antibodies, or bispecific or multispecific antibodies.

[0027] In a second aspect, a polynucleotide is provided that encodes a monoclonal antibody as described in the first aspect.

[0028] As one possible embodiment, the polynucleotide is a polynucleotide group, which includes:

[0029] (I) A first polynucleotide comprising a DNA molecule or its corresponding mRNA molecule having nucleotide sequences as shown in SEQ ID NO:16, 17, and 18, respectively; and,

[0030] (II) A second polynucleotide comprising a DNA molecule or its corresponding mRNA molecule having nucleotide sequences as shown in SEQ ID NO:19, GCCGCCAGC, and SEQ ID NO:20, respectively.

[0031] As one possible embodiment, the polynucleotide sequence includes:

[0032] (I) A first polynucleotide comprising a DNA molecule or its corresponding mRNA molecule with the nucleotide sequence shown in SEQ ID NO:21; and,

[0033] (II) A second polynucleotide comprising a DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:22;

[0034] Alternatively, the polynucleotide group may also include:

[0035] (III) A third polynucleotide comprising a DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:24; and,

[0036] (IV) A fourth polynucleotide comprising a DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:26.

[0037] Thirdly, a nucleic acid construct is provided that comprises the polynucleotides described in the second aspect.

[0038] As a possible embodiment, it also includes at least one expression regulatory element operatively linked to the polynucleotide.

[0039] Fourthly, a recombinant vector is provided, comprising a polynucleotide as described in the second aspect, or a nucleic acid construct as described in the third aspect.

[0040] Fifthly, a transformed host cell is provided, wherein the transformation comprises a polynucleotide as described in the second aspect, a nucleic acid construct as described in the third aspect, or a recombinant vector as described in the fourth aspect.

[0041] In a sixth aspect, a pharmaceutical composition is provided comprising the monoclonal antibody described in the first aspect, the polynucleotide described in the second aspect, the nucleic acid construct described in the third aspect, the recombinant vector described in the fourth aspect, or the transformed cells described in the fifth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0042] As one possible embodiment, the pharmaceutical composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation;

[0043] Alternatively, the pharmaceutical composition is a nasal spray, which is selected from aerosols, sprays, and powders;

[0044] Alternatively, the pharmaceutical composition may be the oral formulation selected from tablets, powders, pills, granules, soft / hard capsules, film-coated formulations, and ointments;

[0045] Alternatively, the pharmaceutical composition may be a sublingual tablet;

[0046] Alternatively, the pharmaceutical composition may be in the form of fine granules;

[0047] Alternatively, the pharmaceutical composition may be a powder or granule.

[0048] Alternatively, the pharmaceutical composition may be in the form of small pellets;

[0049] Alternatively, the pharmaceutical composition may be a parenteral preparation, which may be a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation.

[0050] In a seventh aspect, a reagent or kit for the specific detection of RSV virus is provided, comprising a monoclonal antibody as described in the first aspect, a polynucleotide as described in the second aspect, a nucleic acid construct as described in the third aspect, a recombinant vector as described in the fourth aspect, and / or a transformed host cell as described in the fifth aspect.

[0051] In some preferred embodiments, the kit is a detection or diagnostic kit, wherein the monoclonal antibody of the present invention contained therein further includes a detectable marker; in some preferred embodiments, the kit further includes a second antibody that specifically recognizes the monoclonal antibody of the present invention or an anti-idiotype antibody; preferably, the second antibody further includes a detectable marker; such detectable markers are well known to those skilled in the art, including but not limited to radioactive isotopes, fluorescent substances, luminescent substances, colored substances, and enzymes (e.g., horseradish peroxidase).

[0052] In a feasible implementation, the sample is a biological sample from the subject; in a specific implementation, the sample is a nasal or oral swab sample from the subject.

[0053] This method can be used for diagnostic purposes (e.g., the sample is from a patient) or for non-diagnostic purposes (e.g., the sample is a cell sample, not from a patient).

[0054] The general methods for using monoclonal antibodies to detect the presence or level of a target protein or antigen in a sample are well known to those skilled in the art. In some preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive assays, and similar methods.

[0055] Eighthly, a method for preparing a monoclonal antibody as described in the first aspect is provided, the method comprising: expressing the monoclonal antibody or its antigen-binding fragment in a host cell transformed as described in the fifth aspect under conditions suitable for expression of the monoclonal antibody or its antigen-binding fragment, and recovering the expressed monoclonal antibody or its antigen-binding fragment from a culture of the host cell.

[0056] A ninth aspect provides the use of the monoclonal antibody as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the recombinant vector as described in the fourth aspect, the transformed host cell as described in the fifth aspect, and / or the pharmaceutical composition as described in the seventh aspect in the preparation of a product for the specific detection of the presence or level of RSV virus in a sample.

[0057] Tenthly, the use of the monoclonal antibody as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the recombinant vector as described in the fourth aspect, the transformed host cell as described in the fifth aspect, and / or the pharmaceutical composition as described in the sixth aspect in the preparation of products for the prevention, relief, treatment, or adjunctive treatment of RSV virus or complications arising therefrom.

[0058] In one aspect, the present invention provides a method for detecting RSV virus, comprising administering an effective amount of the monoclonal antibody as described in the first aspect above to a patient in need.

[0059] Preferably, the RSV virus includes RSV A subtype virus and RSV B subtype virus.

[0060] The dosage of the active ingredient in the pharmaceutical composition of the present invention varies depending on the target patient, the target organ, symptoms, method of administration, etc. It can be determined based on the doctor's judgment, taking into account the type of dosage form, method of administration, patient's age and weight, patient's symptoms, etc.

[0061] Beneficial effects

[0062] This invention provides a monoclonal antibody (AD5) targeting Site III of the RSV F protein. The antigenic epitope Site III is highly conserved in RSV A / B subtypes. Amino acid position 305 is leucine (L) in RSV A subtype strains and isoleucine (I) in RSV B subtype strains, with almost no variation within subtypes. The antibody AD5 targeting the Site III antigenic epitope of this invention exhibits high neutralizing activity against both RSV A and B subtypes, regardless of viral subtype. Site III spans the core structure of the F protein, making complete escape difficult with single-point mutations; multiple key amino acids must be altered simultaneously. Studies show that antibodies targeting this epitope develop resistance slowly under continuous viral evolutionary pressure, providing strong and durable protection and demonstrating excellent anti-escape capabilities. The antibody AD5 of this invention combines targeting a highly conserved epitope (Site III) with high neutralizing activity, overcoming the major drawback of existing commercially available antibodies with low neutralizing activity when targeting highly conserved epitopes. Attached Figure Description

[0063] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0064] Figure 1 The AD5 antibody protein in Example 2 in SuperdexTM Peak chromatogram of the purified eluent from 200 Increase 10 / 300 GL.

[0065] Figure 2 The result is the SDS-PAGE assay of the purified AD5 antibody protein from Example 2.

[0066] Figure 3 The image shows the SPR detection kinetics curve for the binding of AD5 antibody to RSV antigen F protein in Example 4.

[0067] Figure 4 The results of neutralization activity assays of AD5 antibody against RSV A and RSV B subtypes in Example 5 are shown, where A represents RSV A subtype and B represents RSV B subtype.

[0068] Figure 5 The results of the detection of the melting temperature and aggregation initiation temperature of AD5 antibody in Example 6 are shown, where A is the melting temperature and B is the aggregation initiation temperature. Detailed Implementation

[0069] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0070] Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0071] To facilitate a better understanding of this invention, certain technical terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For specific definitions and terms in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular forms used herein (including the claims) include their corresponding plural forms unless otherwise expressly specified herein.

[0072] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as these variations are suitable for carrying out the disclosed methods.

[0073] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0074] As used herein, the term “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted inclusively, that is, including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when explicitly indicated by the opposite terms, such as “only one” or “exactly one” or when “consisting of” is used in the claims, will it refer to only one number or one element of the list.

[0075] The term "percentage (%) amino acid sequence identity," or simply "identity," is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to those in a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0076] The term "antibody" refers to any form of antibody that has the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camel-derived single-domain antibodies.

[0077] The term "monoclonal antibody" refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations, which may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a large number of antibodies targeting different epitopes (or specific to different epitopes). The modifier "monoclonal" indicates the characteristic of antibodies derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of antibodies through any particular method.

[0078] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0079] In this invention, the heavy chain complementarity determination region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determination region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0080] "Antigen-binding fragments" refer to antigen-binding fragments of antibodies and antibody analogs, which typically include at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. Antigen-binding fragments include those selected from Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)2, biantibodies, peptides containing CDRs, etc.

[0081] The “Fab fragment” consists of a light chain, a heavy chain, CH1, and a variable region.

[0082] The “Fc” region contains two heavy chain segments that contain the CH2 and CH3 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.

[0083] The “Fab′ fragment” contains a light chain and a heavy chain portion that includes the VH domain, the CH1 domain, and the constant region between the CH1 and CH2 domains. Interchain disulfide bonds are formed between the two heavy chains of the two Fab′ fragments to form the F(ab′)2 molecule.

[0084] The “F(ab′)2 segment” contains two light chains and two heavy chain segments containing the VH domain, the CH1 domain, and the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab′)2 segment consists of two Fab′ segments held together by disulfide bonds between the two heavy chains.

[0085] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0086] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment containing the VH and VL domains of the antibody, which are contained within a single polypeptide chain. Generally, scFv polypeptides contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding.

[0087] A "dual antibody" is a small antigen-binding fragment with two antigen-binding sites. The fragment contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH-VL or VL-VH). By using a linker so short as to prevent pairing between the two domains on the same chain, the domain pairs with a complementary domain of the other chain to form two antigen-binding sites.

[0088] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of the protein, for example, the binding reaction of the monoclonal antibody of this invention to the RSV F protein, within a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0089] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can usually be expressed by the equilibrium dissociation constant (K0). D The equilibrium dissociation constant is the ratio of the dissociation rate constant to the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay described herein. The term "non-binding" protein or cell refers to a protein or cell that does not bind to it, or does not bind to it with a high affinity, i.e., Kdis and kon bind to the protein or cell. D 1.0×10 -6 M or higher, more preferably 1.0 × 10 -5 M or higher, more preferably 1.0 × 10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0 × 10 -2 M or higher.

[0090] For IgG antibodies, the term "high affinity" refers to the affinity for the antigen's K+. D 1.0×10- 6 M or lower, preferably 5.0 × 10 -8 M or lower, more preferably 1.0 × 10 -8 M or lower, 5.0×10 -9 M or lower, more preferably 1.0 × 10-11 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding in the IgM subtype refers to K... D 10 -6 M or lower, preferably 10 -7 M or lower, preferably 10 -8 M or lower.

[0091] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides (see U.S. Patent No. 8,278,036, belonging to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0092] The preferred embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following embodiments are given only for illustrative purposes and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0093] Information on some of the sequences involved in this invention is shown in Table 1 below.

[0094] Table 1. Sequence information involved in this invention

[0095]

[0096] Note: Control antibodies were sourced from: D25 (PDB: 4JHA), 4D7 (DOI: 10.1371 / journal.pone.0164789), Palivizumab heavy chain (Genbank: AGG15976.1), Palivizumab light chain (Genbank: AGG15975.1); MPE8 (PDB: 7SEM), 101F (PDB: 3O45), and hRSV90 (PDB: 5TPN), all of which were synthesized by a commissioned company.

[0097] All antibody plasmids use pCAGGS as their vector backbone, which is commercially available.

[0098] Example 1: Screening and identification of AD5 antibodies

[0099] This invention involves isolating peripheral blood mononuclear cells (PBMCs) from a healthy individual. Using fluorescently labeled RSV F protein as a probe, the protein specifically binds to receptors on the surface of B cells. Simultaneously, surface markers such as CD19 and CD27 are stained, and RSV F antigen-specific memory B cells are sorted using flow cytometry. Subsequently, the target B cells are sorted as single cells. The sorted single B cells are directly lysed and reverse transcribed to obtain cDNA from each cell. Using this cDNA as a template, a nested PCR strategy is employed to amplify the heavy chain variable region gene (VH) and the light chain variable region gene (VL) of the antibody. Finally, the PCR products are sequenced to achieve natural pairing and sequence analysis of the light and heavy chain genes, thereby obtaining the complete AD5 gene sequence.

[0100] Example 2: Expression and purification of AD5 antibody

[0101] The heavy chain of the AD5 antibody (coding sequence of AD5-VH signal peptide (SEQ ID NO:27) + sequence encoding AD5 heavy chain (SEQ ID NO:24)) and the light chain (coding sequence of AD5-VH signal peptide (SEQ ID NO:29) + sequence encoding AD5 heavy chain (SEQ ID NO:26)) were inserted into the pCAGGS plasmid. Positive clones were screened and purified to obtain the plasmid. The AD5 antibody heavy chain and light chain plasmids were co-transfected into 293F cells at a ratio of 2:3. After suspension culture for 5 days, the expression supernatant was collected. After centrifugation and filtration to remove impurities, the supernatant was processed using a Protein A affinity chromatography column and Superdex. TM 200 Increase 10 / 300 GL molecular sieve chromatography column for antibody protein purification. Specifically:

[0102] (1) Cell preparation: Adjust the density of 293F cells to 1.8×10⁻⁶ cells one day in advance. 6 Cells / mL, cultured overnight at 37°C with shaking under 5% CO2. When the cell density increased to 2.5-3.0 × 10⁻⁶ cells / mL... 6 It can be used for plasmid transfection when the concentration is as low as cells / mL.

[0103] (2) Protein expression: Take 25 mL of 150 mM sterile NaCl solution and mix it with 1 mg of plasmid (the coding sequence of the heavy chain plasmid (SEQ ID NO:27) + the sequence encoding the AD5 heavy chain (SEQ ID NO:24) + pCAGGS plasmid) and the light chain plasmid (the coding sequence of the AD5-VH signal peptide (SEQ ID NO:29) + the sequence encoding the AD5 heavy chain (SEQ ID NO:26) + pCAGGS plasmid) in a 2:3 ratio) filtered through a 0.22 μm filter membrane and let it stand for 5 min to obtain solution A. Take 25 mL of 150 mM sterile NaCl solution and mix it with 5 mL of transfection reagent and let it stand for 5 min to obtain solution B. Slowly add solution B to solution A and mix well. After standing for 15 min, add it dropwise to the 293F cell suspension and culture at 37°C with shaking under CO2-free conditions. Add 35 mL of feed solution to the cell culture medium 24 hours after transfection, and then add feed solution every 48 hours thereafter. This system can be scaled up proportionally.

[0104] (3) Affinity chromatography purification: Five days after transfection, the cell suspension was collected and centrifuged at 8000g at 4°C to remove cell debris. Protein A affinity chromatography column and Superdex were used for purification. TM The antibody protein in the supernatant was purified using a 200 Increase 10 / 300 GL molecular sieve chromatography column. Solution A of the Protein A column was 20 mM Na3PO4 at pH 7.0; Solution B was 0.1 M glycine-hydrochloric acid at pH 3.0. A 100% solution B environment allowed the antibody protein to elute from the Protein A column. Immediately afterwards, 0.25 times the volume of solution C (1 M Tris at pH 9.0) was added to the eluent to prevent protein denaturation due to prolonged exposure to the acidic environment of solution B. SDS-PAGE was used to identify protein size. Samples containing the target protein were selected, concentrated to an appropriate volume using a 30 kDa ultrafiltration tube, and thoroughly centrifuged to remove impurities before being analyzed by Superdex. TM The antibody was further purified using a 200 Increase 10 / 300 GL molecular sieve chromatography column in PBS buffer. The purified sample was then subjected to SDS-PAGE gel electrophoresis for identification, retaining protein samples of the correct size and with single bands.

[0105] Antibody proteins in Superdex TMThe peak position in 200 Increase 10 / 300 GL molecular sieve is approximately 12 mL. Figure 1 Under reducing conditions, the disulfide bonds connecting the light and heavy chains are broken, and they coexist in SDS-PAGE as both heavy and light chains. Figure 2 Finally, the purified antibody protein was concentrated using an ultrafiltration tube and transferred to PBS buffer. After measuring the concentration and aliquoting, the protein was flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0106] Example 3: Competitive binding assay of AD5 antibody

[0107] The overlapping of epitopes between the AD5 antibody and the reference antibody in this invention was analyzed through a competitive binding assay, thereby analyzing the binding epitopes of the AD5 antibody. Monoclonal antibody D25 was used as the representative antibody targeting Site Ø, 4D7 as the representative antibody targeting Site I, Palivizumab as the representative antibody targeting Site II, MPE8 as the representative antibody targeting Site III, 101F as the representative antibody targeting Site IV, and hRSV90 as the representative antibody targeting Site V. These reference antibodies were conjugated with biotin and used as the "detection antibody" in the competitive binding assay. The AD5 antibody was used as the "blocking antibody." Specific methods included:

[0108] (1) Detection of antibody biotinylation: Using a 30kDa concentration tube, the detection antibody proteins D25, 4D7, Palivizumab, MPE8, 101F and hRSV90 were respectively replaced with PBS solution. Biotinylation reagent was added at a molar ratio of 20:1 to antibody protein. The mixture was mixed evenly and placed at room temperature. After 30 minutes, the solution was replaced at least three times using a 30kDa concentration tube to remove free biotinylation reagent.

[0109] (2) Coating: The preF7P antigen protein (for details of its preparation, please refer to Huang Q, Lang Q, Li Y, et al. Highly scalable prefusion-stabilized RSV F vaccine with enhanced immunogenicity and robust protection. Nat Commun. 2025;16(1):7805.) was diluted to 2 μg / mL with ELISA coating buffer, and 100 μL was added to each well. The mixture was then incubated overnight at 4°C.

[0110] (3) Blocking: Discard the coating solution, add 150 μL of 5% skim milk to each well, block at 37°C for 1 hour, and then discard.

[0111] (4) Binding: Add 100 μL of 200 μg / mL “blocking antibody” (AD5 antibody) to each well and incubate at 37°C for 1 h.

[0112] (5) Competitive binding: Add the above-mentioned "detection antibody" to the "blocking antibody" to a final concentration of 1 μg / mL, incubate at 37°C for 1 h, and wash three times with PBST.

[0113] (6) Incubation with secondary antibody: Add streptavidin-HRP secondary antibody diluted 1:500 (product code: 35105ES60, HRPStreptavidin, Yisheng Biotechnology), incubate at 37℃ for 1 h, and then wash five times with PBST.

[0114] (7) Color development: Add 50 μL TMB colorimetric solution to develop color, and terminate with 2M sulfuric acid. Read the value at a wavelength of 450 nm.

[0115] Using 5% skim milk instead of the "blocking antibody" served as the control group in this experiment. When the absorbance of the "blocking antibody" was less than 30% of that of the control group, it was considered that the "blocking antibody" and the "detection antibody" competed with each other; when the absorbance was greater than 70% of that of the control group, it was considered that the "blocking antibody" and the "detection antibody" did not compete; when the absorbance was between 30% and 70% of that of the control group, it was considered that the binding epitopes of the two antibodies partially competed. The results are shown in Table 2.

[0116] Table 2. Results of the competitive binding assay for AD5 antibody

[0117]

[0118] The results in Table 2 show that the AD5 antibody does not compete with the binding epitopes of D25, 4D7, Palivizumab, 101F, and hRSV90 antibodies, but competes with the binding epitope of MPE8 antibody, indicating that the AD5 antibody binds to Site Ⅲ.

[0119] Example 4: Affinity detection of AD5 antibody

[0120] Surface plasmon resonance (SPR) is a commonly used method for analyzing intermolecular interactions. It can not only detect the existence of specific bindings between molecules, but also obtain the association rate constant (K2) through data analysis software. a ), dissociation rate constant (K) d ), dissociation equilibrium constant (K)D The instrument used in this experiment, the BIAcore 8K, is based on SPR technology and is used for intermolecular interaction analysis.

[0121] We used the BIAcore 8K to detect the affinity of the AD5 antibody, using the BIAcore 8K-specific ProteinA chip. The SPR buffer was PBS containing 0.05% Tween-20, and the chip regeneration solution was 10mM Glycine-HCl at pH 1.7.

[0122] (1) Antibody capture: Dilute AD5 antibody to 5 μg / mL with SPR buffer and capture antibody: The diluted AD5 antibody flows through the chip at a flow rate of 10 μl / min for 60s. The amount of antibody captured by the chip is about 2000RU.

[0123] (2) Antigen binding: The preF7P antigen protein was serially diluted 2-fold from an appropriate initial concentration to 5 concentration gradients (1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM), and flowed sequentially from low to high concentration through a Protein A chip containing captured antibodies. The conditions were: binding flow rate 30 μl / min, duration 120 s; dissociation flow rate 30 μl / min, duration 600 s; regeneration flow rate 30 μl / min, duration 30 s; and experimental data were recorded.

[0124] (3) Data processing: Based on the curve type obtained in the previous step, use BIAcore ® The 8K Evaluation software was used to fit the curve and calculate K. a K d and K D The result is as follows Figure 3 As shown.

[0125] Figure 3 Experimental results show that the AD5 antibody has very high affinity, K D =0.059±0.001nM (reaching the picomolar level).

[0126] Example 5: Detection of neutralizing activity of AD5 antibody

[0127] The PRNT (Proteinization and Restriction of Viruses) immunostaining method utilizes the principle of specific antibody-antigen binding. Antibodies are incubated with RSV virus, and the antibody's ability to neutralize the virus is detected. Unneutralized virus infects host cells, forming plaques. Specific detection antibodies are added to label the viral antigen, and a chromogenic substrate is used to visualize the infected area. The plaque inhibition rate is calculated, and the half-maximal inhibitory concentration (IC50) of the antibody is assessed. 50 ).

[0128] (1) Neutralization assay: AD5 antibody was serially diluted 3-fold in 12 steps, with 6 replicates. The diluted antibody was diluted in DMEM medium containing 2% FBS and 1% antibiotic. The serially diluted antibody was transferred to a new 96-well plate and mixed with RSV virus (100 PFU / well) (50 μL antibody + 50 μL virus). After incubation at 37°C for 30 min, Hep2 cells were added at 100 μL per well (approximately 1.5 × 10⁻⁶ cells). 4 (Cells / well). Include virus control wells: 50 μL virus dilution + 50 μL LMEM + 100 μL cell suspension per well. Include cell control wells: 100 μL LMEM + 100 μL cell suspension per well. Incubate at 37℃ in a 5% CO2 incubator for 44–48 h.

[0129] (2) Fixation: After culture, carefully discard the culture medium in the 96-well cell culture plate and wash the plate three times with 200 μL / well of 0.05% PBST. Then add 100 μL of pre-cooled anhydrous methanol solution to each well and fix at room temperature for 10 min. Discard the anhydrous methanol solution in the 96-well cell culture plate.

[0130] (3) Blocking: The 96-well plate was washed 3 times with 200 μL / well of 0.05% PBST and then blocked at 37°C for 1 h with 100 μL / well of blocking solution.

[0131] (4) Add detection antibody: Dilute Palivizumab antibody / HRP (1.0 mg / mL) with blocking buffer to prepare a 5 μg / mL detection antibody working solution. Prepare fresh before use. Discard the blocking buffer in the 96-well plate, wash 3 times with 200 μL / well of 0.05% PBST, add 100 μL / well of detection antibody working solution, and incubate at 37°C for 1 h.

[0132] (5) Color development: Discard the detection antibody working solution in the 96-well plate, wash 5 times with 200 μL / well of 0.05% PBST, and add KPL TrueBlue at 50 μL / well. TM Apply the Peroxidase Substrate solution, gently agitate to ensure even coverage, and let stand for approximately 15 minutes, depending on the color development. After staining, discard the solution, rinse thoroughly with laboratory-grade water, and gently shake off any remaining liquid.

[0133] (6) Data Acquisition and Analysis: After the plate has dried, use an ELISA speckle analyzer to read the plate, record the number of spots in each well, and analyze and statistically analyze the data using Microsoft Office EXCEL to calculate the half-maximal inhibitory concentration (IC50) of the antibody. 50 ).

[0134] The neutralizing activity of AD5 antibody against RSV subtype A strain (ATCC: VR-26PQ™) and RSVB subtype strain (ATCC: VR-1580PQ™) was detected using the above methods, and the results are as follows: Figure 4 Experimental results showed that AD5 antibody was effective against RSV A subtype strain ( Figure 4 A) and RSV B subtype strain ( Figure 4 Both B) in the solution have good neutralizing activity, IC 50 The values ​​were 228.9 ng / mL and 91.91 ng / mL, respectively.

[0135] Example 6: Determination of the melting temperature of AD5 antibody

[0136] Label-free DSF (Nano DSF) is based on the shift in the emission spectrum of tryptophan during protein unfolding. By detecting fluorescence changes, the thermal stability of proteins can be assessed in a label-free environment. The melting temperature (Tm value) of an antibody is a key indicator of its thermal stability; a higher Tm value generally indicates better thermal stability. This study used nano DSF technology to detect the melting temperature of the AD5 antibody and also monitored its aggregation tendency (Tagg).

[0137] (1) Sample preparation: Dilute the test antibodies AD5 and MPE8 separately with the same batch of PBS buffer solution and adjust the concentration to 1 mg / mL. Then centrifuge the sample at 12000 rpm for 10 min at 4℃, take the supernatant, and remove any possible small aggregates to avoid interference with the detection results.

[0138] (2) Set instrument parameters: detect the process of antibody from natural state to complete unfolding from 25℃ to 95℃ at a heating rate of 1℃ / min.

[0139] (3) Sample loading and detection: Use a capillary tube to draw about 10 μL of sample, ensuring that no air bubbles are generated. Place the capillary tube into the sample tray of the instrument, run the program, and monitor the changes in fluorescence ratio (350 nm / 330 nm) and scattered light signal (reflecting the aggregation state).

[0140] (4) Data processing and analysis: The software will automatically calculate the fluorescence ratio (F350 / F330) with respect to the first derivative with respect to temperature. The temperature corresponding to the peak of the derivative curve is the melting temperature (Tm) of the antibody. Analyze static light scattering. The point at which the signal begins to rise sharply is the aggregation initiation temperature (Tagg).

[0141] The Tm and Tagg values ​​of AD5 and MPE8 antibodies were detected using the methods described above, and the results are shown in Table 3. Figure 5 As shown, the thermostability of the two antibodies was compared under the same conditions. Figure 5 The letter A indicates that the melting temperature of antibody AD5 in this invention is higher than that of MPE8, by approximately 2°C. Meanwhile, Figure 5 The B in the figure indicates that AD5 also has a higher Tagg level, increasing by approximately 9°C. These data suggest that the AD5 antibody exhibits better thermal stability and anti-aggregation capabilities, indicating better druggability and a longer shelf life during its development.

[0142] Table 3. Comparison of Tm and Tagg values ​​for AD5 and MPE8 antibodies.

[0143]

[0144] This invention provides a monoclonal antibody (AD5) targeting Site III of the RSV F protein. The antigenic epitope Site III is highly conserved in RSV A / B subtypes. Amino acid position 305 is leucine (L) in RSV A subtype strains and isoleucine (I) in RSV B subtype strains, with almost no variation within subtypes. The antibody AD5 targeting Site III antigen of this invention has high neutralizing activity against both RSV A and B subtypes, regardless of viral subtype. Site III spans the core structure of the F protein, and single-point mutations are difficult to completely escape; multiple key amino acids must be altered simultaneously. Studies show that antibodies targeting this epitope exhibit slow resistance development and strong durable protection under continuous viral evolutionary pressure, demonstrating excellent anti-escape capabilities. The antibody AD5 targeting Site III of this invention has higher conservation, conformational stability advantages, and the potential to reduce the risk of viral escape, making it of significant development value. Especially in addressing RSV subtype mutations, reducing the risk of viral escape, and providing long-term protection, antibodies targeting Site III may become an important supplement to existing RSV control strategies. The antibody AD5 of this invention combines the characteristics of targeting a highly conserved epitope (Site III) and having high neutralizing activity, overcoming the main drawback of existing commercially available antibodies that have low neutralizing activity when targeting highly conserved epitopes.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody targeting RSV F protein, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes: complementarity-determining region HCDR1 as shown in SEQ ID NO:1, complementarity-determining region HCDR2 as shown in SEQ ID NO:2 and complementarity-determining region HCDR3 as shown in SEQ ID NO:3, respectively. The light chain variable region includes: complementarity-determining region LCDR1 as shown in SEQ ID NO:4, complementarity-determining region LCDR2 as shown in AAS, and complementarity-determining region LCDR3 as shown in SEQ ID NO:5, respectively.

2. The monoclonal antibody of claim 1, wherein, The heavy chain variable region further includes the amino acid sequences shown as frame region H-FR1 as shown in SEQ ID NO:6, frame region H-FR2 as shown in SEQ ID NO:7, frame region H-FR3 as shown in SEQ ID NO:8, and frame region H-FR4 as shown in SEQ ID NO:9, respectively. And / or, the light chain variable region further comprises the amino acid sequences shown as frame region L-FR1 as shown in SEQ ID NO:10, frame region L-FR2 as shown in SEQ ID NO:11, frame region L-FR3 as shown in SEQ ID NO:12, and frame region L-FR4 as shown in SEQ ID NO:

13.

3. The monoclonal antibody of claim 1, wherein, The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:14; And / or, the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

15.

4. The monoclonal antibody of claim 1, wherein, The monoclonal antibody comprises: Heavy chain, comprising the amino acid sequence shown in SEQ ID NO:23; and, Light chain, which contains an amino acid sequence as shown in SEQ ID NO:

25.

5. A polynucleotide encoding a monoclonal antibody as described in any one of claims 1-4.

6. The polynucleotide of claim 5, wherein, The polynucleotide is a polynucleotide group, which includes: (I) A first polynucleotide comprising a DNA molecule or its corresponding mRNA molecule having nucleotide sequences as shown in SEQ ID NO:16, 17, and 18, respectively; and, (II) A second polynucleotide comprising a DNA molecule or its corresponding mRNA molecule having a nucleotide sequence as shown in SEQ ID NO:19, GCCGCCAGC, or SEQ ID NO:20, respectively.

7. The polynucleotide of claim 6, wherein, The polynucleotide sequence includes: (I) A first polynucleotide comprising a DNA molecule or its corresponding mRNA molecule with the nucleotide sequence shown in SEQ ID NO:21; and, (II) A second polynucleotide comprising a DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:22; Alternatively, the polynucleotide group may also include: (III) A third polynucleotide comprising a DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:24; and, (IV) A fourth polynucleotide comprising a DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:

26.

8. A nucleic acid construct comprising the polynucleotide as described in any one of claims 5-7.

9. The nucleic acid construct of claim 8, wherein, It also includes at least one expression regulatory element operatively linked to the polynucleotide.

10. A recombinant vector comprising a polynucleotide as described in any one of claims 5-7, or a nucleic acid construct as described in claim 8 or 9.

11. A transformed host cell, wherein the cell is transformed with a polynucleotide as described in any one of claims 5-7, or a nucleic acid construct as described in claim 8 or 9, or a recombinant vector as described in claim 10.

12. A pharmaceutical composition comprising a monoclonal antibody according to any one of claims 1 to 4, a polynucleotide according to any one of claims 5 to 7, a nucleic acid construct according to claim 8 or 9, or a recombinant vector according to claim 10, and a pharmaceutically acceptable carrier and / or excipient.

13. The pharmaceutical composition of claim 12, wherein, The pharmaceutical composition is in the form of a nasal spray, oral preparation, suppository, or parenteral preparation.

14. The pharmaceutical composition of claim 12, wherein, The pharmaceutical composition is a nasal spray, which is selected from aerosols, sprays, and powder sprays.

15. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutical composition is an oral preparation selected from tablets, powders, pills, granules, soft / hard capsules, film-coated preparations, and ointments.

16. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutical composition is a sublingual tablet.

17. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutical composition is a fine granule.

18. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutical composition is a powder.

19. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutical composition is in the form of small pills.

20. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutical composition is a parenteral preparation, which can be a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation.

21. A reagent or kit for the specific detection of RSV virus, comprising the monoclonal antibody as described in any one of claims 1-4.

22. A method for preparing a monoclonal antibody according to any one of claims 1-4, the method comprising: Under conditions suitable for the expression of the monoclonal antibody, the host cells transformed according to claim 11 are made to express the monoclonal antibody, and the expressed monoclonal antibody is recovered from the culture of the host cells.

23. The use of a monoclonal antibody as described in any one of claims 1-4, a polynucleotide as described in any one of claims 5-7, a nucleic acid construct as described in claim 8 or 9, a recombinant vector as described in claim 10, or a transformed host cell as described in claim 11 in the preparation of a product for the specific detection of the presence or level of RSV virus in a sample.

24. The use of a monoclonal antibody as described in any one of claims 1-4, a polynucleotide as described in any one of claims 5-7, a nucleic acid construct as described in claim 8 or 9, a recombinant vector as described in claim 10, a transformed host cell as described in claim 11, or a pharmaceutical composition as described in any one of claims 12 to 20 in the preparation of a product for the prevention, relief, treatment, or adjunctive treatment of RSV virus or complications arising therefrom.

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