Human monoclonal neutralizing antibody targeting f protein of metapneumovirus and application thereof

Abstract

This invention relates to a human monoclonal neutralizing antibody targeting the metapneumovirus F protein and its applications. It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes complementary determinant regions HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and / or, the light chain variable region includes complementary determinant regions LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, AAS, and SEQ ID NO:5, respectively. The metapneumovirus monoclonal neutralizing antibody MV32 of this invention can specifically bind to the metapneumovirus F antigen (approximately 0.6 nM) and exhibits good broad-spectrum neutralizing activity against representative strains such as hMPV A1, hMPV B1, and hMPV A2b. It also possesses certain in vivo protective and preventative effects and can be used for the prevention and treatment of human metapneumovirus infection, showing great application potential.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and antibody engineering technology, specifically to a human monoclonal neutralizing antibody (MV32) targeting the F protein of metapneumovirus and its applications. Background Technology

[0002] Human metapneumovirus (hMPV) is a novel respiratory pathogenic virus first isolated and identified in 2001 by Dutch researchers from the nasopharyngeal secretions of patients with acute respiratory infections. It belongs to the genus metapneumovirus in the family Pneumoviridae and is a single-stranded negative-sense RNA virus. 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.

[0003] Serological epidemiological surveys show that hMPV has a widespread infection prevalence globally, with almost all children experiencing at least one hMPV infection before the age of 5. Due to the variability of viral antigens, reinfection is very common. Statistics show that hMPV infection has become one of the leading causes of hospitalization for acute respiratory infections in children worldwide. During the autumn and winter respiratory disease season, its detection rate is second only to RSV, placing a heavy burden on children's health and public health security.

[0004] Neutralizing antibodies, as a passive immunization strategy, have demonstrated significant advantages and great application potential in the prevention and treatment of viral diseases due to their unique mechanism of action—specifically recognizing and binding to viral antigens and blocking viral invasion of host cells. The prefusion F protein of hMPV is a key envelope glycoprotein mediating viral invasion of host cells and is also the most important target for neutralizing antibodies. Although some studies have reported monoclonal antibodies targeting the hMPV F protein in recent years, these antibodies generally have many limitations and cannot meet the needs of clinical applications: most antibodies have limited neutralizing activity, poor broad-spectrum neutralization, or the epitopes they target are not strongly conserved, allowing viruses to easily escape their inhibitory effects through mutation. Furthermore, while some antibodies show acceptable in vitro activity, their in vivo protective effects are poor, or insufficient humanization leads to a high risk of immunogenicity, limiting their clinical translation and application.

[0005] Therefore, the development of novel hMPV neutralizing antibodies has significant clinical value and practical implications. An ideal hMPV neutralizing antibody should possess the following core characteristics: high affinity for the antigen, high neutralizing activity against the virus, excellent broad-spectrum neutralization (effectively neutralizing different hMPV genotypes), and in vivo protective activity. This would provide a novel 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., MV32) targeting the F protein of metapneumovirus and its applications.

[0008] The monoclonal neutralizing antibody against metapneumovirus of the present invention (e.g., MV32) can specifically bind to metapneumovirus F antigen (approximately 0.6 nM) and has good broad-spectrum neutralizing activity against representative strains such as hMPV A1, hMPV B1, and hMPV A2b. It also has certain in vivo protective and preventive effects and can be used to prevent and treat human metapneumovirus infection, showing great application potential.

[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 (e.g., MV32) 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 complementarity-determining regions HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; And / or, the light chain variable region comprises: an amino acid sequence having complementary determinant regions LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, AAS and SEQ ID NO:5, respectively.

[0010] Furthermore, the heavy chain variable region also includes framework regions H-FR1, H-FR2, H-FR3 and H-FR4, which are arranged alternately with complementarity-determining regions HCDR1, HCDR2 and HCDR3 in sequence. Optionally, framework regions H-FR1 to H-FR4 are derived from human common framework sequences or human germline sequences. Optionally, the amino acid sequences of H-FR1 to H-FR4 are shown in SEQ ID NO:6 to 9, respectively. And / or, the light chain variable region further includes framework regions L-FR1, L-FR2, L-FR3 and L-FR4, which are arranged alternately with complementarity-determining regions LCDR1, LCDR2 and LCDR3 in sequence. Optionally, framework regions L-FR1 to L-FR4 are derived from human common framework sequences or human germline sequences. Optionally, the amino acid sequences of framework regions L-FR1 to L-FR4 are as shown in SEQ ID NO:10 to 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%, 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 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, or a humanized modified amino acid sequence thereof.

[0012] As a feasible 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 comprising a sequence 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 derived from a sequence of at least one of human antibodies, primate antibodies, or mutants thereof, optionally the heavy chain constant region is a sequence of a sequence of 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%, 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 λ-type or κ-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 sequence; optionally the light chain comprises 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, or is composed of the above.

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

[0015] In a preferred embodiment, the monoclonal antibody or its antigen-binding fragment comprises: A heavy chain comprising, or consisting of, an amino acid sequence having, or having at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO:23; and, A light chain comprising, or consisting 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:25. 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, GCTGCATCC, 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, wherein the transformation is performed with polynucleotides as described in the first aspect, or nucleic acid constructs as described in A), or recombinant vectors as described in B), and the host cells may be mammalian cells (e.g., 293F cells).

[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 nasal spray is selected from aerosols, sprays, and powders; Optionally, the oral formulation is selected from tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated tablets, pellets, sublingual tablets, or ointments; Optionally, the parenteral preparation 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, wherein optionally, the metapneumovirus includes at least one of the hMPV A1 strain, the hMPV B1 strain, and the hMPVA2b 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 some preferred embodiments, the kit provided by the present invention is a detection kit or a diagnostic kit, wherein the monoclonal antibody or its antigen-binding fragment contained herein may be further linked to a detectable marker.

[0023] In some other preferred embodiments, the kit further comprises a second antibody capable of specifically binding to the monoclonal antibody of the present invention, its antigen-binding fragment, or a corresponding anti-idiotype antibody; preferably, the second antibody is also conjugated with a detectable label.

[0024] When a detectable label is used, it is well known to those skilled in the art, including but not limited to: radioactive isotopes, fluorescent compounds, chemiluminescent substances, chromogenic substances, and enzyme labels (such as horseradish peroxidase).

[0025] In some feasible implementations, the sample is a biological sample derived from the subject; in some specific implementations, the sample is a nasopharyngeal swab or oropharyngeal swab sample taken from the subject.

[0026] The above detection method can be used for diagnostic purposes (e.g., the sample is a clinical sample derived from a patient) or for non-diagnostic purposes (e.g., the sample is a cell sample cultured in vitro, rather than a sample taken from a patient).

[0027] The methods for detecting the presence and content level of target proteins or antigens in samples using the monoclonal antibodies or antigen-binding fragments of the present invention are all conventional and known techniques in the art.

[0028] In some preferred embodiments, the detection methods include, but are not limited to: enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, and competitive binding assay.

[0029] 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.

[0030] The dosage of the active ingredient in the pharmaceutical composition of this invention can vary depending on factors such as the patient, target organ, severity of disease, and route of administration. The actual dosage can be determined by a physician based on the actual clinical situation, taking into account factors such as the type of formulation, route of administration, age and weight of the patient, and clinical symptoms.

[0031] The active ingredients of the present invention, such as antibodies or antigen-binding fragments thereof, can be administered to subjects via any suitable route of administration, including but not limited to oral, oral, sublingual, local, parenteral, rectal, intrathecal, or nasal administration.

[0032] In some feasible implementations, the subject is a human. The "effective dose for prevention and / or treatment" may vary depending on the recipient, organ of action, symptoms, route of administration, etc., and may be determined by a physician or veterinarian based on professional judgment, taking into account factors such as dosage form, route of administration, subject's age, weight, and condition.

[0033] The substance can be used alone or in combination, or in combination with other vaccines (such as other serum vaccines).

[0034] Alternatively, the metapneumovirus includes at least one of the hMPV A1 strain, hMPV B1 strain, and hMPV A2b strain.

[0035] Beneficial effects The monoclonal neutralizing antibody MV32 for metapneumovirus of the present invention can specifically bind to metapneumovirus F antigen (approximately 0.6 nM) and has good broad-spectrum neutralizing activity against representative strains such as hMPV A1, hMPV B1 and hMPV A2b. It also has certain in vivo protective and preventive effects and can be used to prevent and treat human metapneumovirus infection, showing great application potential.

[0036] This invention provides a monoclonal antibody MV32 targeting the hMPV F protein. It exhibits strong binding affinity to hMPV and broad-spectrum neutralization of representative hMPV strains A1, B1, and A2. It also demonstrates high efficiency in vitro neutralization of hMPV, high affinity for the hMPV F protein antigen (approximately 0.6 nM), and in vivo preventative and protective effects. The MV32 antibody targeting hMPV of this invention differs in sequence from previously reported antibodies, providing a novel product for the detection and neutralization of hMPV. It also offers the possibility of providing a product with in vivo protective or preventative effects against hMPV, and has the potential to treat and prevent infection by various hMPV strains. Attached Figure Description

[0037] 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.

[0038] Figure 1The results of SDS-PAGE of the purified molecular sieve for protein F (DS-CavEs2) in Example 1 are shown.

[0039] Figure 2 The results of SDS-PAGE analysis of the purified MV32 antibody prepared in Example 1 are shown in Figure A, where A is the molecular sieve purification result and B is the SDS-PAGE electrophoresis image.

[0040] Figure 3 The results of the kinetic curves for the binding of the MV32 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.

[0041] Figure 4 The results show the neutralization curve of the MV32 antibody against the hMPV-B1-EGFP virus in Example 3.

[0042] Figure 5 The neutralization curve results of the MV32 antibody and hMPV-A1-F-EGFP virus in Example 3 are shown.

[0043] Figure 6 The neutralization curve results of the MV32 antibody and the hMPV clinical strain A2b virus in Example 3 are shown. Detailed Implementation

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0050] 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.

[0051] The term "antibody" is used in its broadest sense herein to refer to any form of antibody that retains the desired antigen-binding activity or related biological activity. This 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.

[0052] The term "monoclonal antibody" refers to an antibody derived from a largely homogeneous population of antibodies, in which the individual antibody molecules constituting the population are essentially identical, except for a small number of possible natural mutations. Monoclonal antibodies are highly specific, typically targeting only a single antigenic epitope; in contrast, conventional polyclonal antibody preparations usually contain a mixture of antibodies targeting multiple different antigenic epitopes. The modifier "monoclonal" in this document is used only to characterize the essentially homogeneous population of antibodies and should not be construed as requiring the antibody to be prepared using a specific method.

[0053] 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.

[0054] 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.

[0055] "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.

[0056] The “Fab fragment” consists of a light chain, a heavy chain, CH1, and a variable region.

[0057] The “Fab′ fragment” contains a light chain and a partial sequence including the VH domain, the CH1 domain, and a 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.

[0058] 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.

[0059] 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.

[0060] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0061] "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.

[0062] 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.

[0063] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific binding" means the ability to specifically recognize and bind to a heterogeneous population containing proteins and / or other biological reagents, such as the binding between the monoclonal antibody of this invention and the human metapneumovirus F protein. Therefore, under specified binding conditions, a particular ligand / antigen preferentially binds to a specific receptor / antibody, without generating significant amounts of nonspecific binding to other proteins present in the sample.

[0064] "Affinity" or "binding affinity" refers to the strength of the interaction between members of a binding pair, reflecting their inherent binding capacity. The affinity of molecule X for its binding partner Y is usually determined by the equilibrium dissociation constant (K0). ) represents the dissociation rate constant (k o s ) and binding rate constant (k on The ratio of ) to ). Affinity can be measured by conventional methods known in the art. One specific method for measuring affinity is the surface plasmon resonance technique described herein. The term "non-binding" protein or cell means that the protein or cell does not bind to it, or does not bind to it with 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.

[0065] For IgG antibodies, the term "high affinity" refers to a KD (kinematic density) of 1.0 × 10⁻⁶ M or lower for the antigen, preferably 5.0 × 10⁻⁶ M. -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 for the IgM subtype refers to a KD of 10. -6M or lower, preferably 10 -7 M or lower, preferably 10 -8 M or lower.

[0066] The term "nucleic acid" or "polynucleotide" refers to a single-stranded or double-stranded polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Unless otherwise expressly defined, the term includes nucleic acids containing natural nucleotide analogs that have similar binding properties to natural nucleotides and are metabolized in a similar manner (see, for example, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for their synthesis, and their use for in vivo delivery of therapeutic proteins).

[0067] 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.

[0068] Information on some of the sequences involved in this invention is shown in Table 1 below.

[0069] Table 1. Sequence information involved in this invention

[0070] Example 1: Preparation and purification of metapneumovirus monoclonal antibodies Expression and purification of the conformationally stable F protein (DS-CavEs2 protein) before metapneumovirus fusion. The preparation method of metapneumovirus F protein (DS-CavEs2) is described in the reference (Structure-based design of prefusion-stabilized human metapneumovirus fusion proteins | Nature Communications): 500 μg of the recombinant vector expressing metapneumovirus F protein (DS-CavEs2) and transfection reagent (purchased from Yiqiao Shenzhou) were transfected into 500 mL of 293F cells at a volume ratio of 1:5 to express the His-tagged DS-CavEs2 protein. After culturing for 4 days, the cell supernatant was collected by centrifugation (8000 rpm, 30 min). The His-tagged DS-CavEs2 protein was enriched using a His-Trap affinity column. The enriched protein was eluted with a buffer containing 300 mM imidazole (20 mM Tris, 150 mM NaCl, pH=8.0), concentrated to 2 mL using a 30 kDa concentrator, and further purified by gel filtration chromatography using Superdex 200 Increase 10 / 300GL (GE). Healthcare) performs purification and testing.

[0071] SDS-PAGE analysis results of purified molecular sieves are as follows: Figure 1 As shown, when metapneumovirus F protein (DS-CavEs2 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 corresponding to the protein monomer band in SDS-PAGE electrophoresis is about 60 kDa, and the protein purity is high.

[0072] and Metapneumovirus F protein ( Sorting of specific B cells by DS-CavEs2 protein binding The purified DS-CavEs2 protein was biotinylated and then used for B cell sorting. The sorting method was based on the literature "Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus" by Wang Qihui et al. | Science Translational Medicine.

[0073] Human peripheral blood mononuclear cells were isolated and stained to identify B cells that were positive for IgG and DS-CavEs2 protein binding. The variable region gene sequence of the antibody was obtained through reverse transcription and PCR amplification. The metapneumovirus monoclonal antibody MV32 was obtained, and its heavy chain, light chain, and variable regions CDR1, CDR2, and CDR3 amino acid sequences are shown in Table 1 above. Furthermore, the heavy chain and light chain variable regions are different from previously reported sequences, demonstrating specificity.

[0074] Expression and purification of MV32 antibody MV32 Antibody cloning and construction: The heavy chain variable region gene (VH) encoding the MV32 antibody (SEQ ID NO:21) was fused with the IgG1 constant region gene (CH) and constructed into the pCAGGS vector (named pCAGGS-Heavy chain-fulllength) (containing the sequence SEQ ID NO:24). The light chain variable region gene (VL) encoding the MV32 antibody (SEQ ID NO:22) was fused with the constant region gene (CL) and constructed into the pCAGGS vector (containing the sequence SEQ ID NO:26), obtaining the recombinant vector, named pCAGGS-Light chain-fulllength.

[0075] MV32 Antibody expression and purification: 7.5 mL of plasmid solution (plasmid dissolved in 150 mM NaCl) containing 180 μg of light chain plasmid and 120 μg of heavy chain plasmid, constructed above, was co-transfected into 300 mL of 293F cells. The plasmid solution was mixed with 9 mL of transfection reagent STF02 solution (1.5 mL STF02 dissolved in 7.5 mL 150 mM NaCl), allowed to stand for 10 min, and then added to 293F cells. The cells were incubated at 37°C in a shaker. After 5 days, the cells were centrifuged (8000 rpm, 90 min) to collect the supernatant. The supernatant was passed through a Protein A affinity column and eluted with 0.1 M Glycine at pH 3.0 using an AKTA Purifier to obtain the target antibody. The antibody was then concentrated and the solution was changed to PBS. After further purification using a Superdex 200 molecular sieve, the purified antibody was detected by SDS-PAGE.

[0076] The SDS-PAGE results of the purified MV32 antibody are as follows: Figure 2 As shown, the results indicate that the size of the heavy chain after antibody melting is approximately 50 kDa, the size of the light chain is approximately 25 kDa, and the purified MV32 antibody has high purity.

[0077] Example 2: Detection of the ability of MV32 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 antigen (DS-CavEs2 protein), immobilizing the DS-CavEs2 antigen on the chip (approximately 500 RU of antigen immobilization). The Fab region of the antibody protein (the Fab protein was obtained by co-transfecting MV32 VH (SEQ ID NO:21) and MV32 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) at 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 of antibody-antigen binding were plotted and the kinetic constants were analyzed using BIAevaluation software 8K (GE Healthcare).

[0078] The results of the kinetic curves are as follows Figure 3 As shown, the results indicate that the affinity of the MV32 antibody Fab protein for the antigen hMPV F protein is 0.6 nM.

[0079] Example 3: Metapneumovirus neutralization assay to detect the neutralizing effect of MV32 antibody Neutralization assay with metapneumovirus carrying a 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 obtained by replacing the F gene in hMPV-B1-EGFP virus with the F gene sequence of A1 (NL / 1 / 00) and then rescuing it using the same procedure.

[0080] The purified human MV32 antibody from Example 1 was serially diluted 3-fold starting at 10 μg / mL, resulting in 9 dilution gradients. Approximately 200 FFU / well of diluted hMPV-B1-EGFP or hMPV-A1-EGFP viral solution was added to each well. The mixture was incubated at 37°C for 1 hour, then added to VeroE6-TMPRSS2 cells and cultured for another 24 hours (37°C, 5% CO2). 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. Metapneumovirus infection was detected using a high-content cell imaging analyzer. The neutralizing activity (half-maximal inhibitory concentration, IC50) of the antibody was calculated based on the proportion of positive green fluorescence.

[0081] The results are as follows Figure 4 , 5 As shown, the results indicate that the MV32 antibody can efficiently neutralize viral infections of hMPV A1 strain (IC50 = 346 ng / ml) and hMPV B1 strain (IC50 = 1238 ng / ml).

[0082] Neutralization assay with clinical strains of metapneumovirus : The purified MV32 antibody from Example 1 was serially diluted 3-fold starting at 10 μg / mL, resulting in 9 dilution gradients. Approximately 500 TCID50 / well of diluted hMPV clinical strain A2b 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 cells, and the cells were cultured for another 24-48 hours (37°C, 5% CO2). 4% paraformaldehyde fixative was added to each well, and the cells were fixed 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 (37°C, 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) (37°C, 1 hour). After washing with PBS, the infection status of metapneumovirus 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.

[0083] The results are as follows Figure 6 As shown, the results indicate that the MV32 antibody can effectively neutralize infection by the clinical strain of hMPV A2b virus (IC50 = 665 ng / ml).

[0084] The antibody MV32 of this invention can significantly reduce the viral load of metapneumovirus in the lungs of mice, demonstrating good preventive and protective effects.

[0085] This invention provides a monoclonal antibody MV32 targeting the hMPV F protein. It exhibits strong binding affinity to hMPV and broad-spectrum neutralization of representative hMPV strains A1, B1, and A2. It also demonstrates high efficiency in vitro neutralization of hMPV, high affinity for the hMPV F protein antigen (approximately 0.6 nM), and in vivo preventative and protective effects. The MV32 antibody targeting hMPV of this invention differs in sequence from previously reported antibodies, providing a novel product for the detection and neutralization of hMPV. It also offers the possibility of providing a product with in vivo protective or preventative effects against hMPV, and has the potential to treat and prevent infection by various hMPV strains.

[0086] 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 complementarity-determining regions HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; And / or, the light chain variable region comprises: an amino acid sequence having complementary determinant regions LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, AAS and SEQ ID NO:5, respectively.

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

3. The monoclonal neutralizing antibody or its antigen-binding fragment according to claim 1, characterized in that, 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%, 95%, 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 such amino acid sequences. 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%, 95%, 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 according to any one of claims 1 to 3, 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 comprising a sequence of 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 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 is a sequence of a portion or all of the constant region of human IgG1; or constitutes thereof; and, A light chain comprising a light chain variable region and a light chain constant region, wherein the light chain constant region is optionally selected from a λ-type or κ-type light chain constant region, wherein the light chain constant region is optionally derived from at least one of the sequences of a human antibody, a primate antibody or a mutant thereof, and wherein the light chain constant region optionally includes or comprises a portion or all of the CL domain; or is composed of thereof. Optionally, the antigen-binding fragment is selected from single-chain antibodies, human antibodies, Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, chimeric antibodies, or bispecific or multispecific antibodies.

5. The monoclonal neutralizing antibody or its antigen-binding fragment according to any one of claims 1-4, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises: A heavy chain comprising, or consisting of, an amino acid sequence having, or having at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO:23; and, Light chains comprising, or consisting 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:

25.

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

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, GCTGCATCC, 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) A transformed host cell, 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), and the host cell may be a mammalian cell.

9. A pharmaceutical composition comprising the nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 6, the polynucleotide as described in claim 7, the 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 nasal spray is selected from aerosols, sprays, and powders; Optionally, the oral formulation is selected from tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated tablets, pellets, sublingual tablets, or ointments; Optionally, the parenteral preparation 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, the metapneumovirus includes at least one of the hMPV A1 strain, the hMPV B1 strain, and the hMPV 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, the metapneumovirus includes at least one of the hMPV A1 strain, hMPV B1 strain, and hMPV A2b strain.

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