A human monoclonal neutralizing antibody targeting metapneumovirus F protein and its application

Abstract

This invention relates to a human monoclonal neutralizing antibody targeting the F protein of metapneumovirus and its applications. It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes an amino acid sequence having a complementarity-determining region (CDR1) as shown in SEQ ID NO:1, a CDR2 as shown in SEQ ID NO:2, and a CDR3 as shown in SEQ ID NO:3, respectively; and / or, the light chain variable region includes an amino acid sequence having a CDR1 as shown in SEQ ID NO:4, a CDR2 as shown in KAS, and a CDR3 as shown in SEQ ID NO:5, respectively. The metapneumovirus monoclonal neutralizing antibody MV31 obtained by this invention exhibits strong binding affinity to metapneumovirus, good in vitro neutralization, high affinity, and in vivo protective activity; it provides a product for the detection and neutralization of metapneumovirus, and makes it possible to provide metapneumovirus products with in vivo protective activity, possessing the potential for treating and preventing infections of various metapneumovirus strains.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and antibody engineering technology, specifically to a human monoclonal neutralizing antibody (MV31) targeting the F protein of metapneumovirus and its applications. Background Technology

[0002] Human metapneumovirus (hMPV) is an important respiratory pathogen first discovered in 2001, belonging to the genus metapneumovirus in the family Pneumoviridae. It primarily infects infants, the elderly, and immunocompromised individuals, causing a range of respiratory illnesses from the common cold and bronchitis to severe pneumonia and bronchiolitis, with clinical symptoms similar to respiratory syncytial virus (RSV) infection. Serological studies indicate that hMPV infection is widespread in humans; almost everyone is infected with hMPV before the age of 5, and reinfection is possible. It is one of the leading causes of hospitalization for acute respiratory infections in children worldwide.

[0003] Currently, there are no specific antiviral drugs or vaccines approved for the market against hMPV infection. Clinical treatment mainly focuses on supportive therapy and symptomatic management, such as fluid replacement and oxygen therapy, which have limited effectiveness for severely ill patients. Therefore, developing effective prevention and treatment methods is an urgent unmet clinical need.

[0004] Neutralizing antibodies, as a passive immunization strategy, have shown great potential in the prevention and treatment of viral infections. The prefusion F protein of hMPV is a key protein mediating viral invasion of host cells and is also the most important target for neutralizing antibodies. Although several monoclonal antibodies targeting the hMPV F protein have been reported, most of them have limited neutralizing activity, poor broad-spectrum neutralization, or poorly conserved epitopes, allowing the virus to easily evade their inhibitory effects through mutation. Furthermore, while some antibodies show decent in vitro activity, their in vivo protective efficacy is poor, or due to insufficient humanization, their immunogenicity risk is high, limiting their clinical translational applications.

[0005] Therefore, it is very important and necessary to develop one or more novel neutralizing antibodies against human metapneumovirus (hMPV). These antibodies should have high affinity for the antigen, high neutralizing activity against the virus, excellent broad-spectrum neutralization (effectively neutralizing different hMPV genotypes), and in vivo protective effects, thereby providing a new and effective solution for the prevention and treatment of hMPV infection. Summary of the Invention

[0006] Purpose of the invention

[0007] In response to the problems or needs existing in the prior art, the present invention provides a human monoclonal neutralizing antibody (e.g., MV31) targeting the F protein of metapneumovirus and its application.

[0008] In summary, the monoclonal neutralizing antibody (e.g., MV31) of the present invention has high affinity for the antigen and high neutralizing activity against metapneumovirus strains, and can effectively neutralize different hMPV genotypes, exhibiting excellent broad-spectrum neutralization.

[0009] Solution To address the problems or needs existing in the prior art, in a first aspect, the present invention provides a monoclonal neutralizing antibody or its antigen-binding fragment (optionally a human monoclonal neutralizing antibody or its antigen-binding fragment) targeting the metapneumovirus F protein, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes an amino acid sequence having a complementarity-determining region HCDR1 as shown in SEQ ID NO:1, a complementarity-determining region HCDR2 as shown in SEQ ID NO:2, and a complementarity-determining region HCDR3 as shown in SEQ ID NO:3, respectively. And / or, the light chain variable region comprises: an amino acid sequence having a complementarity-determining region LCDR1 as shown in SEQ ID NO:4, a complementarity-determining region LCDR2 as shown in KAS, and a complementarity-determining region LCDR3 as shown in SEQ ID NO:5, respectively.

[0010] Furthermore, the heavy chain variable region also includes frame regions H-FR1, H-FR2, H-FR3, and H-FR4 arranged in sequence interleaved with the complementarity-determining regions HCDR1, HCDR2, and HCDR3. Optionally, frame regions H-FR1, H-FR2, H-FR3, and H-FR4 are derived from human common frame sequences or human germline sequences. Optionally, the amino acid sequences of H-FR1, H-FR2, H-FR3, and H-FR4 are shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively. And / or, the light chain variable region further includes frame regions L-FR1, L-FR2, L-FR3 and L-FR4 arranged in sequence interleaved with complementarity-determining regions LCDR1, LCDR2 and LCDR3. Optionally, frame regions L-FR1, L-FR2, L-FR3 and L-FR4 are derived from human common frame sequences or human germline sequences. Optionally, the amino acid sequences of frame regions L-FR1, L-FR2, L-FR3 and L-FR4 are shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively.

[0011] As a possible embodiment, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:14 or a humanized modified amino acid sequence thereof, or is composed of the same. And / or, the light chain variable region comprises, or consists of, an amino acid sequence as shown in SEQ ID NO:15, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:15, or a humanized modified amino acid sequence thereof.

[0012] As a possible embodiment, a signal peptide is also present at the N-terminus of the heavy chain variable region.

[0013] As one possible embodiment, the monoclonal antibody or its antigen-binding fragment comprises: A heavy chain comprising a heavy chain variable region and a heavy chain constant region, optionally the heavy chain constant region being derived from a portion or all of the sequence of at least one of a human antibody, a primate antibody, or a mutant thereof; optionally the heavy chain constant region comprising a portion or all of the constant region of at least one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a mutant thereof; optionally the heavy chain constant region is a portion or all of the constant region of IgG1; optionally the heavy chain constant region is a portion or all of the constant region of human IgG1; optionally the heavy chain comprises, or consists of, an amino acid sequence having, or 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, The light chain comprises a light chain variable region and a light chain constant region. Optionally, the light chain constant region is selected from the κ-type or λ-type light chain constant region. Optionally, the light chain constant region is derived from at least one of the sequences of a human antibody, a primate antibody, or a mutant thereof. Optionally, the light chain constant region includes part or all of the CL domain. Optionally, the light chain comprises or consists of an amino acid sequence having the amino acid sequence shown in SEQ ID NO:25 or 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.

[0014] As a possible embodiment, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dAb, complementarity-determining region fragment, single-chain antibody, human antibody, chimeric antibody, or bispecific or multispecific antibody.

[0015] In a second aspect, a polynucleotide is provided that encodes a monoclonal neutralizing antibody or an antigen-binding fragment thereof as described in the first aspect; Optionally, the polynucleotide is a polynucleotide group, which includes: (I) A first polynucleotide comprising a DNA molecule or its corresponding mRNA molecule with nucleotide sequences as shown in SEQ ID NO:16, 17, and 18, respectively; optionally, comprising a DNA molecule or its corresponding mRNA molecule with nucleotide sequences as shown in SEQ ID NO:21; optionally, comprising a DNA molecule or its corresponding mRNA molecule with nucleotide sequences as shown in SEQ ID NO:24; and, (II) A second polynucleotide comprising a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO:19, AAGGCGTCT, or SEQ ID NO:20, respectively; optionally, comprising a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO:22; or optionally, comprising a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO:26.

[0016] Thirdly, a biomaterial comprising the polynucleotides described in the first aspect is provided, optionally comprising: A) A nucleic acid construct comprising a polynucleotide as described in the first aspect, and optionally, further comprising at least one expression regulatory element operatively linked to the polynucleotide; B) A recombinant vector comprising the polynucleotides as described in the first aspect, or the nucleic acid constructs as described in A).

[0017] C) Transformed host cells (optionally mammalian cells, such as 293F cells) wherein the transformation involves polynucleotides as described in the first aspect, or nucleic acid constructs as described in A), or recombinant vectors as described in B).

[0018] Fourthly, a pharmaceutical composition is provided comprising the nanobody or antigen-binding fragment described in the first aspect, the polynucleotide described in the second aspect, the biomaterial described in the third aspect, and a pharmaceutically acceptable carrier and / or excipient; Optionally, the pharmaceutical composition is in the form of a nasal spray, an oral formulation, a suppository, or a parenteral formulation; Optionally, the pharmaceutical composition is a nasal spray, wherein the nasal spray is selected from aerosols, sprays, and powders; Optionally, the pharmaceutical composition is the oral formulation selected from tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated tablets, pellets, sublingual tablets, or ointments; Optionally, the pharmaceutical composition is a sublingual tablet; Optionally, the pharmaceutical composition is in the form of fine granules; Optionally, the pharmaceutical composition is a powder or granule. Optionally, the pharmaceutical composition is in the form of pellets; Optionally, the pharmaceutical composition is a parenteral preparation, which may be a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation.

[0019] Fifthly, a reagent or kit for the specific detection of metapneumovirus is provided, comprising a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect, a polynucleotide as described in the second aspect, or a biological material as described in the third aspect.

[0020] In a sixth aspect, the invention provides the use of the monoclonal antibody or antigen-binding fragment thereof described in the first aspect, the polynucleotide described in the second aspect, or the biological material described in the third aspect in the preparation of a product for the detection of metapneumovirus or metapneumovirus F protein for non-diagnostic purposes. Optionally, the metapneumovirus includes at least one of the metapneumovirus A1 strain, the metapneumovirus B1 strain, and the metapneumovirus A2b strain.

[0021] In some preferred embodiments, the kit is a detection or diagnostic kit, wherein the monoclonal antibody or its antigen-binding fragment of the present invention contained herein further includes a detectable label; in some preferred embodiments, the kit further includes a second antibody that specifically recognizes the monoclonal antibody or its antigen-binding fragment of the present invention or an anti-idiotype antibody; preferably, the second antibody further includes a detectable label; such detectable labels 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).

[0022] 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.

[0023] 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).

[0024] General methods for detecting the presence or level of a target protein or antigen in a sample using monoclonal antibodies or their antigen-binding fragments 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.

[0025] 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 blood sample, not from a patient).

[0026] In a seventh aspect, the use of a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect, a polynucleotide as described in the second aspect, a biological material as described in the third aspect, or a pharmaceutical composition as described in the fourth aspect in the preparation of a product for the prevention, relief, treatment, or adjunctive treatment of metapneumovirus or complications arising therefrom; Eighthly, the present invention provides a method for preventing, alleviating, treating or adjunctive treating metapneumovirus or infection with its complications, comprising administering to a patient in need an effective amount of a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect, a biological material as described in the third aspect, or a pharmaceutical composition as described in the fourth aspect.

[0027] 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.

[0028] The antibodies or antigen-binding fragments of the present invention may be administered to subjects via any suitable route of administration, including but not limited to oral, oral, sublingual, topical, parenteral, rectal, intrathecal, or nasal routes.

[0029] In a feasible implementation, the subject is a human being. The "effective dose for prevention and / or treatment" may vary depending on the recipient, the organ involved, the symptoms, the method of administration, etc., and may be determined based on the veterinarian's judgment, taking into account the type of dosage form, method of administration, the subject's age, weight, symptoms, etc.

[0030] The substance can be used alone or in combination, or in combination with other vaccines (such as other serum vaccines).

[0031] Optionally, metapneumovirus includes at least one of metapneumovirus A1 strain, metapneumovirus B1 strain, and metapneumovirus A2b strain.

[0032] Beneficial effects The monoclonal neutralizing antibody MV31 for metapneumovirus of the present invention can specifically bind to the metapneumovirus F antigen and has good broad-spectrum neutralizing activity and certain in vivo protective effect against representative strains of metapneumovirus A1, B1 and A2b. It can be used to prevent and treat human metapneumovirus infection and has great application prospects.

[0033] The monoclonal neutralizing antibody MV31 obtained by this invention has strong binding ability to metapneumovirus, good in vitro neutralization, high affinity, and in vivo protective effect. It provides a product for the detection and neutralization of metapneumovirus, and makes it possible to provide metapneumovirus products with in vivo protective effect. It also has the potential to treat and prevent infection by various metapneumovirus strains. Attached Figure Description

[0034] 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.

[0035] Figure 1 The results of SDS-PAGE of the purified molecular sieve for protein F (DS-CavEs2) in Example 1 are shown.

[0036] Figure 2 The results of SDS-PAGE analysis of the purified MV31 antibody prepared in Example 1 are shown in Figure A, where A represents the molecular sieve purification result and B represents the SDS-PAGE electrophoresis image.

[0037] Figure 3 The results of the kinetic curves for the binding of the MV31 antibody to the DS-CavEs2 protein in Example 2 are shown, where the horizontal axis represents time (seconds) and the vertical axis represents the response value.

[0038] Figure 4 The results show the neutralization curves of the MV31 antibody and hMPV-B1-EGFP virus in Example 3.

[0039] Figure 5 The neutralization curve results of the MV31 antibody and hMPV-A1-F-EGFP virus in Example 3 are shown.

[0040] Figure 6 The neutralization curve results of the MV31 antibody and the clinical strain A2b of hMPV in Example 3 are shown.

[0041] Figure 7The MV31 antibody of Example 4 is shown to resist hMPV-B1-EGFP and hMPV A2b virus infection in vivo. The horizontal axis shows the names of the antibody and the control, and the vertical axis shows the viral load in mouse lungs. The left figure shows hMPV-B1-EGFP virus, and the right figure shows hMPV A2b virus. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0048] The term "percentage (%) sequence identity" is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing vacancies) 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 percentage amino acid sequence identity.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] "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.

[0054] The “Fab fragment” consists of a light chain, a heavy chain, CH1, and a variable region.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0059] "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.

[0060] 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.

[0061] 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 metapneumovirus 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.

[0062] “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 generally be represented by the equilibrium dissociation constant (KD), which 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 means that the protein or cell does not bind, or does not bind with a high affinity, i.e., the KD of the bound protein or cell is 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.

[0063] For IgG antibodies, the term "high affinity" refers to a KD of 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, and more preferably 1.0 × 10⁻¹¹ M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, for IgM subtypes, "high affinity" binding refers to a KD of 10⁻⁶ M or lower, preferably 10⁻⁷ M or lower, and more preferably 10⁻⁸ M or lower.

[0064] 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)).

[0065] 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.

[0066] Information on some of the sequences involved in this invention is shown in Table 1 below.

[0067] Table 1. Sequence information involved in this invention

[0068] Example 1: Preparation and purification of metapneumovirus monoclonal antibodies Expression and purification of the conformationally stable F protein (DS-CavEs2 protein) before metapneumovirus fusion. Metapneumovirus F protein (DS-CavEs2) source reference (Structure-based design of prefusion-stabilized human metapneumovirus fusion proteins | Nature Communications): 500 μg of metapneumovirus F protein (DS-CavEs2) expression plasmid was transfected into 500 mL of 293F cells using Yiqiao Shenzhou transfection reagent at a ratio of 1:5. After 4 days of expression, the cell supernatant was collected by centrifugation at 8000 rpm for 30 minutes and His-Trap affinity column was used to enrich His-tagged DS-CavEs2 protein. The enriched protein was eluted with buffer containing 300 mM imidazole (20 mM Tris, 150 mM NaCl, pH=8.0), concentrated to 2 mL in a 30 kDa concentrator, and further purified by gel filtration chromatography using Superdex 200 Increase 10 / 300 GL (GE Healthcare).

[0069] The results of SDS-PAGE purification of molecular sieves are as follows: Figure 1 As shown, the results indicate that when the protein flows through the gel filtration chromatography column, a UV 280 nM absorption peak can be detected at the 16 mL position. The molecular weight of the protein monomer of metapneumovirus F protein (DS-CavEs2 protein) in SDS-PAGE electrophoresis is about 60 kDa, and the obtained protein has high purity.

[0070] Sorting of specific B cells that bind to the DS-CavEs2 protein The purified DS-CavEs2 protein was biotinylated and then used for B cell sorting.

[0071] Human peripheral blood mononuclear cells were isolated and stained to separate B cells that were positive for IgG and DS-CavEs2 protein binding (referring to the sorting method in the literature "Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus | Science Translational Medicine" by Wang Qihui et al.). The variable region gene sequence of the antibody was obtained through reverse transcription and PCR amplification. The metapneumovirus monoclonal antibody MV31 was obtained, and its amino acid sequences of the light and heavy chains, as well as the CDR1, CDR2, and CDR3 sequences of the light and heavy chains, are shown in Table 1 above. The light and heavy chain variable regions differ from existing sequences, demonstrating specificity.

[0072] Expression and purification of MV31 antibody Antibody cloning and construction: The heavy chain variable region gene (VH) (SEQ ID NO:21) of the MV31 antibody was fused with the IgG1 constant region gene (CH) and constructed into the pCAGGS vector (named pCAGGS-Heavy chain-full length) (containing the sequence SEQ ID NO:24). The light chain variable region gene (VL) (SEQ ID NO:22) of the antibody was fused with the constant region gene (CL) and constructed into the pCAGGS vector (named pCAGGS-Light chain-full length) (containing the sequence SEQ ID NO:26).

[0073] Antibody expression and purification: The constructed light and heavy chain plasmids were co-transfected into 300 mL of 293F cells. The plasmid solution was mixed with the transfection reagent solution, allowed to stand for 10 min, and then added to the 293F cells. The cells were incubated at 37°C in a shaker. After 5 days, the supernatant was harvested after centrifugation at 8000 rpm for 90 min. The supernatant was passed through an affinity column (Protein A) and eluted with 0.1 M Glycine (pH 3.0) using an AKTA Purifier to obtain the target antibody. The eluted antibody was then concentrated and the solution was changed to PBS. Further purification was performed using a Superdex 200 molecular sieve, and the purified antibody was detected by SDS-PAGE.

[0074] The preparation of the plasmid solution included: dissolving 120 μg of heavy chain plasmid and 180 μg of light chain plasmid in 7.5 mL of 150 mM NaCl and allowing it to stand for 5 min. The preparation of the transfection reagent solution included: dissolving 1.5 mL of transfection reagent (STF02) in 7.5 mL of 150 mM NaCl and allowing it to stand for 5 min.

[0075] The SDS-PAGE results of the purified MV31 antibody are as follows: Figure 2 As shown, the results indicate that the purified MV31 antibody has high purity, with the heavy chain size after dissociation being approximately 50 kDa and the light chain size being approximately 25 kDa.

[0076] Example 2: Detection of the ability of MV31 antibody to bind to antigen using surface plasmon resonance technology. Surface plasmon resonance analysis was performed using a Biacore 8K (GE Healthcare). The specific steps were as follows: An SA chip (GE Healthcare) was used. The SA chip bound to a biotinylated hMPV F antigen protein (DS-CavEs2 protein), immobilizing the antigen protein on the chip. The immobilized antigen amount was approximately 500 RU. The antibody protein Fab (Fab protein was obtained by co-transfecting MV31 VH (SEQ ID NO:21) and MV31 light chain (SEQ ID NO:26) expression plasmids with a C-terminus 6x his tag into 293FT cells, collecting the supernatant after 5 days, enriching it on a His-Trap column, and then purifying it using a Superdex 75 size exclusion column) was serially diluted with PBST solution (pH 7.4) to concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM. The sample was loaded onto the chip surface, and the changes in response values ​​were recorded. The kinetic curves and kinetic constants of antibody-antigen binding were analyzed using BIAevaluation software 8K (GE Healthcare).

[0077] The results are as follows Figure 3 As shown in the figure. The results show that the affinity of the MV31 antibody Fab protein for the hMPV F protein is 1.69 nM.

[0078] Example 3: Metapneumovirus neutralization assay to detect the neutralizing effect of MV31 antibody Neutralization assay of metapneumovirus with fluorescent reporter gene: The B1 metapneumovirus (hMPV-B1-EGFP) carrying the green fluorescent reporter gene was rescued using the reference (Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes). Specifically, the EGFP gene was inserted into the P and M genes of hMPV B1 cDNA (NL / 1 / 99) via homologous recombination to construct a full-length hMPV cDNA gene sequence with the EGFP insertion. This sequence was then co-transfected into BSRT7 cells with four helper plasmids expressing NP, L, M, and P proteins for hMPV-B1-EGFP rescue. The rescued virus was amplified using VeroE6 cells stably expressing TMPRSS2 (i.e., VeroE6-TMPRSS2 cells, which can be constructed by packaging TMPRSS2-IRES-mCherry into lentivirus and infecting VeroE6 cells, then sorting red fluorescent positive cells by flow cytometry). The hMPV-A1-F-EGFP virus was rescued by replacing the F gene in hMPV-B1-EGFP virus with the F gene sequence of A1 (NL / 1 / 00) using the same procedure.

[0079] The purified human MV31 antibody from Example 1 was serially diluted 3-fold starting at 10 μg / mL, for a total of 9 dilutions. Approximately 200 FFU / well of diluted hMPV-B1-EGFP or hMPV-A1-F-EGFP virus solution was added to each well. The mixture was incubated at 37°C for 1 hour. The incubated antibody-virus mixture was then added to VeroE6-TMPRSS2 cells and cultured at 37°C with 5% CO2 for 24 hours. 4% paraformaldehyde fixative was added to each well for at least 2 hours. The paraformaldehyde was discarded, and the cells were washed 2-3 times with PBS. Viral infection was detected using a high-content cell imaging analyzer. The neutralizing activity value (half-maximal inhibitory concentration, IC50) of the antibody was calculated based on the proportion of positive green fluorescence.

[0080] The results are as follows Figure 4 , 5 As shown, the MV31 antibody can effectively neutralize hMPV A1 and B1 viral infections at half-maximal inhibitory concentrations of 20.15 ng / ml and 79.93 ng / ml.

[0081] Neutralization of clinical strains of metapneumovirus : The purified MV31 antibody from Example 1 was serially diluted 3-fold starting at 10 μg / mL, for a total of 9 dilutions. The diluted hMPV clinical strain A2b virus solution (approximately 500 TCID50 / well) was added to each well. The mixture was incubated at 37°C for 1 hour. The incubated antibody-virus mixture was then added to cells and incubated at 37°C in a 5% CO2 incubator for 24-48 hours. 4% paraformaldehyde fixative was added to each well for at least 2 hours. The paraformaldehyde was discarded, and the cells were washed 2-3 times with PBS and stained with hMPV antibody MPE8 at 37°C for 2 hours. After washing 2-3 times with PBS, the cells were stained with FITC-conjugated anti-human IgG secondary antibody (BioLegend, catalog number #410706) at 37°C for 1 hour. After washing with PBS, viral infection was detected using a high-content cell imaging analyzer. The neutralizing activity value (half-maximal inhibitory concentration, IC50) of the antibody was calculated based on the proportion of green fluorescent positive cells.

[0082] The results are as follows Figure 6 As shown, the MV31 antibody can effectively neutralize the infection of the clinical strain A2b of hMPV with an IC50 activity of 18.58 ng / ml.

[0083] Example 4: The effect of MV31 antibody in preventing and protecting against metapneumovirus infection Taking the MV31 antibody (purified in Example 1 above), which exhibited the best neutralizing activity, as an example, the in vivo protective effect of the antibody was evaluated using a BALB / c mouse model. Mice were challenged with hMPV-B1-EGFP and hMPV A2b viruses via intranasal administration. MV31 antibody was administered intranasally 24 hours before challenge, at doses of 10 mg / kg and 1 mg / kg, respectively. Control mice were given 10 mg / kg of an unrelated antibody (influenza virus antibody PNSIA28, prepared according to the reference "Structural basis for a humanbroadly neutralizing influenza A hemagglutinin stem-specific antibody including H17 / 18 subtypes"). Mice that lost more than 20% of their body weight were euthanized. Results are as follows: Figure 7 As shown, the antibody can significantly reduce the viral load in the lungs of mice, demonstrating a better protective effect.

[0084] This invention provides a monoclonal antibody targeting the F protein of metapneumovirus (MPV), which exhibits strong binding affinity to MPV, good in vitro neutralization, high affinity, and in vivo protective activity. It also broadly neutralizes representative MPV strains A1, B1, and A2b. The MPV-targeting MV31 antibody of this invention differs in sequence from previously reported antibodies, providing a novel product for MPV detection and neutralization, and opening up possibilities for providing MPV prophylactic products with in vivo protective activity. It also possesses the potential to treat and prevent infection with multiple MPV strains.

[0085] 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 present invention.

Claims

1. A monoclonal neutralizing antibody or its antigen-binding fragment targeting the metapneumovirus F protein, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes an amino acid sequence having a complementarity-determining region HCDR1 as shown in SEQ ID NO:1, a complementarity-determining region HCDR2 as shown in SEQ ID NO:2, and a complementarity-determining region HCDR3 as shown in SEQ ID NO:3, respectively. And / or, the light chain variable region comprises: an amino acid sequence having a complementarity-determining region LCDR1 as shown in SEQ ID NO:4, a complementarity-determining region LCDR2 as shown in KAS, and a complementarity-determining region LCDR3 as shown in SEQ ID NO:5, respectively.

2. The monoclonal neutralizing antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region further includes frame regions H-FR1, H-FR2, H-FR3, and H-FR4, which are sequentially interleaved with the complementarity-determining regions HCDR1, HCDR2, and HCDR3. Optionally, frame regions H-FR1, H-FR2, H-FR3, and H-FR4 are derived from human common frame sequences or human germline sequences. Optionally, the amino acid sequences of H-FR1, H-FR2, H-FR3, and H-FR4 are shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively. And / or, the light chain variable region further includes frame regions L-FR1, L-FR2, L-FR3 and L-FR4 arranged in sequence interleaved with complementarity-determining regions LCDR1, LCDR2 and LCDR3. Optionally, frame regions L-FR1, L-FR2, L-FR3 and L-FR4 are derived from human common frame sequences or human germline sequences. Optionally, the amino acid sequences of frame regions L-FR1, L-FR2, L-FR3 and L-FR4 are shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively.

3. The monoclonal neutralizing antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region comprises, or consists of, an amino acid sequence as shown in SEQ ID NO:14, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:14, or a humanized modified amino acid sequence thereof. And / or, the light chain variable region comprises, or consists of, an amino acid sequence as shown in SEQ ID NO:15, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:15, or a humanized modified amino acid sequence thereof.

4. The monoclonal neutralizing antibody or its antigen-binding fragment as described in claim 1, characterized in that, The N-terminus of the heavy chain variable region also has a signal peptide.

5. The monoclonal neutralizing antibody or its antigen-binding fragment as described in claim 1, wherein, The monoclonal antibody or its antigen-binding fragment comprises: A heavy chain comprising a heavy chain variable region and a heavy chain constant region, optionally the heavy chain constant region being a partial or complete sequence derived from at least one of a human antibody, a primate antibody, or a mutant thereof; optionally the heavy chain constant region comprising a partial or complete constant region of at least one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a mutant thereof; optionally the heavy chain constant region is a partial or complete constant region of IgG1; optionally the heavy chain constant region is a partial or complete constant region of human IgG1; optionally the heavy chain comprises, or consists of, an amino acid sequence having, or being composed of, the amino acid sequence shown in SEQ ID NO:23 or having, or being composed of, 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, The light chain comprises a light chain variable region and a light chain constant region. Optionally, the light chain constant region is selected from the κ-type or λ-type light chain constant region. Optionally, the light chain constant region is derived from at least one of the sequences of a human antibody, a primate antibody, or a mutant thereof. Optionally, the light chain constant region includes part or all of the CL domain. Optionally, the light chain comprises or consists of an amino acid sequence having the amino acid sequence shown in SEQ ID NO:25 or 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.

6. The monoclonal neutralizing antibody or its antigen-binding fragment as described in any one of claims 1-5, wherein, The antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dAb, complementarity-determining region fragment, single-chain antibody, human antibody, chimeric antibody, or bispecific or multispecific antibody.

7. A polynucleotide encoding a monoclonal neutralizing antibody or an antigen-binding fragment thereof as described in any one of claims 1-6; Optionally, the polynucleotide is a polynucleotide group, which includes: (I) A first polynucleotide comprising a DNA molecule or its corresponding mRNA molecule with nucleotide sequences as shown in SEQ ID NO:16, 17, and 18, respectively; optionally, comprising a DNA molecule or its corresponding mRNA molecule with nucleotide sequences as shown in SEQ ID NO:21; optionally, comprising a DNA molecule or its corresponding mRNA molecule with nucleotide sequences as shown in SEQ ID NO:24; and, (II) A second polynucleotide comprising a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO:19, AAGGCGTCT, or SEQ ID NO:20, respectively; optionally, comprising a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO:22; or optionally, comprising a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO:

26.

8. A biomaterial comprising the polynucleotide as described in claim 7, optionally comprising: A) A nucleic acid construct comprising the polynucleotide as described in claim 7, optionally further comprising at least one expression regulatory element operatively linked to the polynucleotide; B) A recombinant vector comprising the polynucleotide as described in claim 7, or a nucleic acid construct as described in A); C) Transformed host cells, wherein the transformation contains the polynucleotide as described in claim 7, or the nucleic acid construct as described in A), or the recombinant vector as described in B).

9. A pharmaceutical composition comprising a nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 6, a polynucleotide as described in claim 7, a biomaterial as described in claim 8, and a pharmaceutically acceptable carrier and / or excipient; Optionally, the pharmaceutical composition is in the form of a nasal spray, an oral formulation, a suppository, or a parenteral formulation; Optionally, the pharmaceutical composition is a nasal spray, wherein the nasal spray is selected from aerosols, sprays, and powders; Optionally, the pharmaceutical composition is the oral formulation selected from tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated tablets, pellets, sublingual tablets, or ointments; Optionally, the pharmaceutical composition is a sublingual tablet; Optionally, the pharmaceutical composition is in the form of fine granules; Optionally, the pharmaceutical composition is a powder or granule. Optionally, the pharmaceutical composition is in the form of pellets; Optionally, the pharmaceutical composition is a parenteral preparation, which may be a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation.

10. A reagent or kit for the specific detection of metapneumovirus, comprising a monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-6, a polynucleotide as described in claim 7, and a biological material as described in claim 8.

11. The use of a monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-6, a polynucleotide as described in claim 7, a biomaterial as described in claim 8, or a pharmaceutical composition as described in claim 9 in the preparation of a product for non-diagnostic detection of metapneumovirus or metapneumovirus F protein, wherein optionally, metapneumovirus includes at least one of metapneumovirus A1 strain, metapneumovirus B1 strain, and metapneumovirus A2b strain.

12. The use of a monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-6, a polynucleotide as described in claim 7, a biomaterial as described in claim 8, or a pharmaceutical composition as described in claim 9 in the preparation of a product for the prevention, relief, treatment, or adjunctive treatment of metapneumovirus or complications arising therefrom, wherein optionally, metapneumovirus includes at least one of metapneumovirus A1 strain, metapneumovirus B1 strain, and metapneumovirus A2b strain.

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