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

Abstract

The present application relates to a human monoclonal neutralizing antibody targeting metapneumovirus F protein and application thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: a complementarity determining region HCDR1 having an amino acid sequence 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; and / or the light chain variable region comprises: a complementarity determining region LCDR1 having an amino acid sequence as shown in SEQ ID NO:4, a complementarity determining region LCDR2 as shown in AAS, and a complementarity determining region LCDR3 as shown in SEQ ID NO:5. The metapneumovirus monoclonal neutralizing antibody MV61 of the present application can specifically bind to metapneumovirus F antigen, has high affinity (about 0.18nM), high neutralization activity to different mutant strains of metapneumovirus, and excellent broad spectrum.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and antibody engineering technology, specifically to a human monoclonal neutralizing antibody (MV61) targeting the F protein of metapneumovirus and its applications. Background Technology

[0002] Human metapneumovirus (hMPV) is a novel, highly pathogenic respiratory virus classified under the genus metapneumovirus in the family Pneumoviridae. It has a single-stranded, negative-sense RNA structure. Susceptible populations are primarily infants, the elderly, and those with weakened immune systems. Infection can cause a range of respiratory illnesses, from mild cases like the common cold and acute bronchitis to severe cases like severe pneumonia and bronchiolitis. In severe cases, it can even lead to respiratory failure, posing a significant health threat to susceptible individuals.

[0003] Serological epidemiological data indicate that hMPV is widespread globally, with significant regional and seasonal variations in its prevalence—peak seasons occur in temperate regions during winter and spring, while infection rates rise during the rainy season in tropical regions. Notably, almost all children experience at least one hMPV infection before the age of 5, and due to the virus's single-stranded negative-sense RNA structure and its antigenic susceptibility to mutation, reinfection is common across all age groups, making it one of the leading causes of hospitalization for acute respiratory infections in children worldwide.

[0004] In the field of viral disease prevention and treatment, neutralizing antibodies, as a highly efficient passive immunization method, have broad application prospects. For hMPV, its prefusion F protein is a key protein mediating the fusion of the virus with host cells and completing the invasion process, and it is also an important target for neutralizing antibodies. Although many studies in recent years have reported monoclonal antibodies targeting the hMPV F protein, and although some antibodies have been shown to bind to different antigenic epitopes on the hMPV F protein, these antibodies generally have many limitations and cannot meet the needs of clinical translation and application: most antibodies have low neutralizing activity and insufficient broad-spectrum neutralization, and cannot effectively cover multiple genotypes of hMPV such as A1, B1, A2a, and A2b; some antibodies, although showing certain neutralizing activity in in vitro experiments, have failed to show ideal protective effects in in vivo animal models, or due to insufficient humanization, they pose a high risk of immunogenicity in human applications, limiting their clinical translation and application.

[0005] Currently, there are no approved vaccines or specific treatments for hMPV, and clinical treatment mainly focuses on symptomatic support. Therefore, the development of highly effective hMPV neutralizing antibodies is of paramount importance and value, possessing significant clinical and practical significance. 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 effects. This would provide a novel and effective solution for the prevention and treatment of hMPV infection. Summary of the Invention

[0006] Object 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., MV61) targeting the F protein of metapneumovirus and its applications.

[0008] The monoclonal neutralizing antibody against metapneumovirus (e.g., MV61) of this invention can specifically bind to the metapneumovirus F antigen with high affinity (approximately 0.18 nM). It exhibits high neutralizing activity and excellent broad-spectrum neutralization against different mutant strains of metapneumovirus, especially against representative strains such as hMPV A1, hMPV B1, and A2b. It also has certain in vivo protective and preventive effects while maintaining low immunogenicity. It can be used for the prevention and treatment of human metapneumovirus infection and has good prospects for clinical application.

[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., MV61) 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 complementation-determining region HCDR1 (GASISTGGHY) as shown in SEQ ID NO:1, a complementation-determining region HCDR2 (SYGSGIT) as shown in SEQ ID NO:2, and a complementation-determining region HCDR3 (ATSRAMIRGGNDAFDI) as shown in SEQ ID NO:3. And / or, the light chain variable region comprises: an amino acid sequence having a complementarity-determining region LCDR1 (QGISTW) as shown in SEQ ID NO:4, a complementarity-determining region LCDR2 as shown in AAS, and a complementarity-determining region LCDR3 (QQADSFPWT) as shown in SEQ ID NO:5.

[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-FR1~4 are derived from human common framework sequences or human germline sequences, and the amino acid sequences of H-FR1~4 are as shown in SEQ ID NO:6~9, 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-FR1~4 are derived from human common framework sequences or human germline sequences, and the amino acid sequences of framework regions L-FR1~4 are as shown in SEQ ID NO:10~13, 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. The amino acid sequence shown in SEQ ID NO:14 is as follows: EVQLVESGPGLVNPSQTLSLTCSVSGASISTGGHYWTWIRQPAGKGLEWIGHSYGSGITQYNPPLKSRVTISADTSKNQFSLRLNSVTAADTAVYYCATSRAMIRGGNDAFDIWGQGTMVTVSS.

[0012] 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.

[0013] The amino acid sequence shown in SEQ ID NO:15 is as follows: EIVLTQSPSSVSASVGDRVTITCRASQGISTWLAWYQQKPGKAPSLLIYAASSLHSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQADSFPWTFGQGTKVDLK.

[0014] As a feasible embodiment, a signal peptide is also present at the N-terminus of the heavy chain variable region.

[0015] 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 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 being a portion or all of the constant region of human IgG1; optionally the 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, The light chain comprises a light chain variable region and a light chain constant region. Optionally, the light chain constant region is derived from at least a portion or all of the sequence of a human antibody, a primate antibody, or a mutant thereof. Optionally, the light chain constant region is selected from a λ-type or κ-type light chain constant region. Optionally, the light chain constant region includes a portion or all of the CL domain sequence. Optionally, the light chain comprises, or consists of, an amino acid sequence having, or being composed of, 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.

[0016] As one possible embodiment, the antigen-binding fragment is selected from single-chain antibodies, human antibodies, Fab, Fab', F(ab')2, Fab'-SH, Fd, scFv, Fv, dAb, complementarity-determining region fragments, chimeric antibodies, or bispecific or multispecific antibodies.

[0017] 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.

[0018] 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).

[0019] 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).

[0020] 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 is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation.

[0021] Fifthly, a reagent or kit is provided for the specific detection of metapneumovirus F protein or metapneumovirus, 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.

[0022] 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 F protein or metapneumovirus for non-diagnostic purposes. Optionally, the metapneumovirus includes at least one of the hMPV A1 strain, the hMPV B1 strain, and the hMPV A2b strain.

[0023] 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).

[0024] In some feasible embodiments, the kit provided by the present invention can be used as a detection kit or diagnostic kit, which contains the monoclonal antibody or its antigen-binding fragment of the present invention, and may also be further linked with various detectable markers to improve detection sensitivity.

[0025] In other feasible embodiments, the kit may further include a second antibody that can specifically bind to the monoclonal antibody of the present invention, its antigen-binding fragment, or a corresponding anti-idiotype antibody; preferably, the second antibody may also be conjugated with a detectable marker to assist in the detection process.

[0026] The detectable markers used are all of types commonly known to those skilled in the art, including but not limited to: radioactive isotopes, fluorescent compounds, chemiluminescent substances, chromogenic substances, and enzyme markers (such as horseradish peroxidase).

[0027] In some feasible implementations, the sample used for testing is a biological sample derived from the subject; in some specific implementations, the sample used for testing is a nasopharyngeal swab or oropharyngeal swab sample taken from the subject.

[0028] The detection method described in this invention has both diagnostic and non-diagnostic applications. For example, when the sample is a clinical sample derived from a patient, it can be used for disease diagnosis; when the sample is a cell sample cultured in vitro (not taken from a patient), it can be used for non-diagnostic experimental research.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] There is no fixed standard for the dosage of the active ingredient in the pharmaceutical composition of this invention, and it can vary depending on factors such as the patient, target organ, severity of disease, and route of administration. In actual clinical application, the dosage can be determined by the 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.

[0033] The active ingredients in this invention may include antibodies or their antigen-binding fragments, and can be administered to subjects via any suitable route of administration known in the art, including but not limited to oral, oral, sublingual, local, parenteral, rectal, intrathecal, or nasal administration, etc., and the optimal administration method can be selected according to clinical needs.

[0034] In some feasible implementations, the subjects are humans. It should be noted that the "effective dose for prevention and / or treatment" is not a fixed value and may vary depending on the recipient, organ of action, disease presentation, route of administration, etc. It 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 disease condition.

[0035] The substance can be used alone or in combination, or in combination with other antibodies (e.g., other antibodies).

[0036] Optionally, metapneumovirus includes at least one of metapneumovirus A1 strain, metapneumovirus B1 strain, and metapneumovirus A2b strain.

[0037] Advantages The monoclonal neutralizing antibody MV61 for metapneumovirus of this invention can specifically bind to the metapneumovirus F antigen with high affinity (approximately 0.18 nM). It exhibits high neutralizing activity and excellent broad-spectrum neutralization against different mutant strains of metapneumovirus, especially against representative strains such as hMPV A1, B1, and A2b. It also has certain in vivo protective and preventive effects while maintaining low immunogenicity. It can be used for the prevention and treatment of human metapneumovirus infection and has good application prospects.

[0038] The MV61 antibody targeting metapneumovirus provided by this invention has a sequence different from previously reported antibody sequences, providing new possibilities for the detection and neutralization of metapneumovirus and offering a feasible path for developing metapneumovirus products that can achieve protective or preventive effects in vivo. It is expected to be used to treat and prevent infection by various metapneumovirus strains. Attached Figure Description

[0039] 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.

[0040] Figure 1 The results of the purification molecular sieve and SDS-PAGE of hMPV F protein (DS-CavEs2 protein) in Example 1 of this invention are shown.

[0041] Figure 2 The results of molecular sieve purification and SDS-PAGE electrophoresis of the purified MV61 antibody prepared in Example 1 of this invention are shown, where A is the molecular sieve purification result and B is the SDS-PAGE electrophoresis image.

[0042] Figure 3 The results show the kinetic curves of the binding between the MV61 antibody and the DS-CavEs2 protein in Example 2 of this invention.

[0043] Figure 4 The results show the neutralization curve of the MV61 antibody against the hMPV-B1-EGFP virus in Example 3.

[0044] Figure 5 The neutralization curve results of the MV61 antibody and hMPV-A1-F-EGFP virus in Example 3 are shown.

[0045] Figure 6 The neutralization curve results of the MV61 antibody and the hMPV clinical strain A2b virus in Example 3 are shown. Detailed Implementation

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] The term “and / or” should be understood as any one of the listed options, or any combination of two or more of the listed options.

[0051] When the term "approximately" is used in conjunction with a numerical value, it means that the specified value has a certain reasonable range of fluctuation, specifically including all values ​​that are not lower than 5% of the lower limit of the value and not higher than 5% of the upper limit of the value. The range of fluctuation of the numerical value includes, but is not limited to, ±5%, ±2%, ±1% and ±0.1%, etc. All of the above deviations apply to the relevant methods disclosed herein.

[0052] The term "percentage of amino acid sequence identity (%)" is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to a reference amino acid sequence, provided that the amino acid sequences are aligned (and vacancies are introduced where necessary) to obtain the maximum percentage of sequence identity, and no conserved substitutions are considered as sequence identity. Various conventional alignment methods can be used in the art to determine the percentage of amino acid sequence identity.

[0053] 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.

[0054] 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.

[0055] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, which contain one or more, or even all, of the major amino acid residues that play a key role in the binding of an antibody or its antigen-binding fragment to a corresponding antigen or epitope. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0056] In this invention, the heavy chain complementarity determination region is represented by HCDR, which may specifically include HCDR1, HCDR2 and HCDR3; the light chain complementarity determination region is represented by LCDR, which may specifically include LCDR1, LCDR2 and LCDR3.

[0057] "Antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically contains 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, but are not limited to, those selected from Fab, Fab′, F(ab′)2, Fab′-SH, Fv, scFv, biantibodies, and peptides containing CDRs.

[0058] The “Fab fragment” consists of a light chain, a heavy chain, a CH1 domain, and a variable region.

[0059] The “Fab′ fragment” contains a light chain and a heavy chain fragment containing a VH domain, a CH1 domain, and a partial constant region sequence 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.

[0060] The “Fc” region contains two heavy chain segments, each consisting of an antibody CH2 domain and a CH3 domain. These two heavy chain segments are stabilized by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain.

[0061] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0062] "Single-chain Fv antibody (scFv antibody)" refers to a class of antigen-binding fragments containing both VH and VL domains of the antibody, with the VH and VL domains contained within a single polypeptide chain. Typically, scFv polypeptides also contain a polypeptide linker between the VH and VL domains, which allows the scFv to fold into a spatial structure suitable for antigen binding.

[0063] A "dual antibody" is a small antigen-binding fragment with two antigen-binding sites. This fragment is a polypeptide (VH) with a heavy chain variable region (VH) and a light chain variable region (VL) linked together on the same polypeptide chain. VL or VL VH). By using a connector that is so short that it cannot pair between two domains on the same chain, the domain is paired with a complementary domain of another chain to form two antigen-binding sites.

[0064] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific binding" refers to the ability to specifically recognize and bind to a target molecule in a complex mixture 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 the defined binding conditions, specific ligands / antigens preferentially bind to specific receptors / antibodies, while exhibiting minimal non-specific binding to other proteins present in the sample.

[0065] "Affinity" or "binding affinity" is used to characterize the strength of the interaction between members of a binding pair, reflecting its intrinsic binding properties. 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 onThe ratio of ) to ). Affinity can be determined by conventional experimental methods known in the art. A 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, or binds only with low 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.

[0066] 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. -6 M or lower, preferably 10 -7 M or lower, preferably 10 -8 M or lower.

[0067] 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 explicitly stated, the term also 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).

[0068] 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.

[0069] Information on some of the sequences involved in this invention is shown in Table 1 below.

[0070] Table 1. Sequence information involved in this invention

[0071] Example 1: Preparation and purification of monoclonal antibodies against metapneumovirus Expression and purification of Metapneumovirus pre-fusion stabilized F protein (DS-CavEs2 protein) The preparation method of metapneumovirus F protein (DS-CavEs2 protein) is described in the reference "Structure-based design of prefusion-stabilized human metapneumovirus fusion proteins | Nature Communications". Specifically, 500 μg of the recombinant vector expressing metapneumovirus F protein (DS-CavEs2 protein) was mixed with transfection reagent (purchased from Yiqiao Shenzhou) at a volume ratio of 1:5 and transfected into 500 mL of 293F cells for protein expression. After culturing for 4 days, the cell supernatant was collected by centrifugation (8000 rpm, 30 min). The His-tagged DS-CavEs2 protein was enriched by His-Trap affinity column. The protein elution buffer was a buffer containing 300 mM imidazole (20 mM Tris, 150 mM NaCl, pH=8.0). After elution, the protein was concentrated to 2 mL using a 30 kDa concentrator and then further purified and detected by gel filtration chromatography using a Superdex 200 Increase 10 / 300GL (GE Healthcare).

[0072] Purified molecular sieve and SDS-PAGE detection results are as follows: Figure 1 As shown, the results indicate that when eluting metapneumovirus F protein (DS-CavEs2 protein) by gel filtration chromatography, a UV 280 nM absorption peak can be detected at approximately 16 mL. SDS-PAGE electrophoresis shows that the molecular weight of the protein monomer band of metapneumovirus F protein (DS-CavEs2 protein) is approximately 60 kDa, and the protein purity is high.

[0073] Sorting of specific B cells binding to Metapneumovirus F protein (DS-CavEs2 protein) Referring to 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 purified DS-CavEs2 protein was biotinylated and then sorted into B cells.

[0074] Monocytes isolated from human peripheral blood were stained, and B cells that were positive for binding to IgG and metapneumovirus F protein (DS-CavEs2 protein) were selected. The variable region gene sequence of the antibody was obtained by reverse transcription and PCR amplification, followed by sequencing. The metapneumovirus monoclonal antibody MV61 was obtained through screening. The amino acid sequences of its heavy and light chains, and the variable regions CDR1, CDR2, and CDR3 of the heavy and light chains are shown in Table 1 above. Furthermore, the heavy and light chain variable regions are different from previously reported sequences, demonstrating specificity.

[0075] Expression and purification of MV61 antibody MV61 Antibody cloning and construction: The heavy chain variable region gene (VH) encoding the MV61 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 MV61 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) to obtain the recombinant vector, named pCAGGS-Light chain-fulllength.

[0076] MV61 Antibody expression and purification: 7.5 mL of the constructed plasmid solution containing both light and heavy chain plasmids was mixed with 9 mL of STF02 transfection reagent and co-transfected into 300 mL of 293F cells. The cells were cultured in a shaker at 37°C. After 5 days, the supernatant was collected by centrifugation (8000 rpm, 90 min). The supernatant was passed through a Protein A affinity column and eluted with 0.1 M Glycine (pH 3.0) using an AKTA Purifier to obtain the target antibody. The 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.

[0077] The plasmid solution consisted of 180 μg of light chain plasmid and 120 μg of heavy chain plasmid dissolved in 7.5 mL of 150 mM NaCl and allowed to stand for 5 min. The transfection reagent STF02 solution was prepared by dissolving 1.5 mL of STF02 in 7.5 mL of 150 mM NaCl and allowing it to stand for 5 min.

[0078] The results of SDS-PAGE analysis of purified MV61 antibody are as follows: Figure 2 As shown, the results indicate that the purified MV61 antibody has high purity, and the size of the heavy chain after melting of the MV61 antibody is approximately 50 kDa, while the size of the light chain is approximately 25 kDa.

[0079] Example 2: Detection of the ability of MV61 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: Using a GE Healthcare SA chip, streptavidin (SA) was used to bind to the biotinylated metapneumovirus F protein antigen (DS-CavEs2 protein), immobilizing the antigen on the chip (the antigen immobilization amount was controlled to be approximately 500 RU). The antibody protein Fab was serially diluted with pH 7.4 PBST solution to prepare samples with concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM. Each concentration of sample was sequentially passed through the chip surface, and the changes in response values ​​were recorded. The Fab protein was obtained by co-transfecting MV61 VH (SEQ ID NO:21) and MV61 light chain (SEQ ID NO:26) expression plasmids with a C-terminal 6xhis tag into 293FT cells. After 5 days, the supernatant was collected, enriched using a His-Trap column, and purified using a Superdex 75 size exclusion column. The kinetic curves of antibody-antigen binding were plotted and the kinetic constants were analyzed using GE Healthcare's BIAevaluation software 8K.

[0080] The results of the kinetic curves are as follows Figure 3 As shown, the results indicate that the affinity of the MV61 antibody Fab protein for the antigen hMPV F protein is 0.18 nM.

[0081] Example 3: Metapneumovirus neutralization assay to detect the neutralizing effect of MV61 antibody Neutralization experiments with Metapneumovirus with fluorescent reporter: 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, 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.

[0082] The MV61 antibody purified in Example 1 was serially diluted 3-fold at an initial concentration of 10 μg / mL, resulting in a total of 9 concentration gradients. Diluted hMPV-B1-EGFP or hMPV-A1-F-EGFP viral solution was added to each well (the viral load per well was controlled at approximately 200 μg / mL). (FFU); Transfer the mixture in the wells to a 37°C incubator and incubate for 1 hour to allow the antibody to fully interact with the virus; After incubation, add the mixture to the culture wells containing VeroE6-TMPRSS2 cells and incubate at 37°C with 5% CO2 for 24 hours; After incubation, add 4% paraformaldehyde fixative to each well and fix for at least 2 hours; Then discard the paraformaldehyde fixative in the wells, wash each well 2-3 times with PBS buffer, and use a high-content cell imaging analyzer to detect and record the green fluorescence produced after metapneumovirus infection. Based on the proportion of green fluorescent positive cells, calculate the neutralizing activity value of the antibody—the half-maximum inhibitory concentration (IC50).

[0083] The results are as follows Figure 4 , 5As shown, the results indicate that the MV61 antibody can efficiently neutralize human metapneumovirus A1 (hMPV A1) and human metapneumovirus B1 (hMPV B1) strains, and can effectively inhibit the infection of cells by the two strains of the virus at half-maximal inhibitory concentrations of 292 ng / ml and 569 ng / ml, respectively.

[0084] Neutralization experiments with Metapneumovirus clinical strains : The MV61 antibody purified in Example 1 was serially diluted 3-fold at an initial concentration of 10 μg / mL, resulting in 9 different concentration gradients. Diluted hMPV clinical strain A2b virus solution (approximately 500 TCID50 / well) was added to each well. The mixture was transferred to a 37°C incubator and incubated for 1 hour to allow for sufficient interaction between the antibody and virus. After incubation, the antibody-virus mixture was added to cells and cultured for 24-48 hours at 37°C with 5% CO2. 4% paraformaldehyde fixative was added to each well for at least 2 hours. The paraformaldehyde fixative was then discarded, and each well was washed 2-3 times with PBS buffer. A high-content cell imaging analyzer was used to detect and record the green fluorescence produced after metapneumovirus infection. The neutralizing activity value of the antibody—the half-maximal inhibitory concentration (IC50)—was calculated based on the proportion of green fluorescent positive cells.

[0085] The results are as follows Figure 6 As shown, the results indicate that the MV61 antibody can efficiently neutralize the human metapneumovirus A2b strain (hMPV clinical strain A2b) and effectively inhibit hMPV A2b virus infection (IC50 = 298 ng / ml).

[0086] The antibody MV61 of this invention can significantly reduce the viral load in the lungs of mice infected with metapneumovirus, demonstrating good preventive and protective effects.

[0087] This invention provides a monoclonal antibody MV61 targeting the metapneumovirus F protein, which can specifically bind to the metapneumovirus F antigen with high affinity (approximately 0.18 nM). It exhibits high neutralizing activity and excellent broad-spectrum activity against different mutant strains of metapneumovirus, especially against representative strains such as hMPV A1, B1, and A2b. It also has certain in vivo protective and preventive effects while maintaining low immunogenicity. It can be used for the prevention and treatment of human metapneumovirus infection and has good application prospects.

[0088] The MV61 antibody targeting metapneumovirus provided by this invention has a sequence different from previously reported antibody sequences, providing new possibilities for the detection and neutralization of metapneumovirus and offering a feasible path for developing metapneumovirus products that can achieve protective or preventive effects in vivo. It is expected to be used to treat and prevent infection by various metapneumovirus strains.

[0089] 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 AAS, 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 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-FR1~4 are derived from human common framework sequences or human germline sequences. Optionally, the amino acid sequences of H-FR1~4 are shown in SEQ ID NO:6~9, 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-FR1~4 are derived from human common framework sequences or human germline sequences, and the amino acid sequences of framework regions L-FR1~4 are as shown in SEQ ID NO:10~13, 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 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 as described in any one of claims 1 to 3, wherein, The antigen-binding fragment is selected from single-chain antibodies, human antibodies, Fab, Fab', F(ab')2, Fab'-SH, Fd, Fv, scFv, dAb, complementarity-determining region fragments, chimeric antibodies, or bispecific or multispecific antibodies.

5. The monoclonal neutralizing antibody or its antigen-binding fragment as described in any one of claims 1 to 4, 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 derived from a portion or all of the sequence of at least one 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 being a portion or all of the constant region of human IgG1; optionally the 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, The light chain comprises a light chain variable region and a light chain constant region. Optionally, the light chain constant region is derived from at least a portion or all of the sequence of a human antibody, a primate antibody, or a mutant thereof. Optionally, the light chain constant region is selected from a λ-type or κ-type light chain constant region. Optionally, the light chain constant region includes a portion or all of the CL domain sequence. Optionally, the light chain comprises, or consists of, an amino acid sequence having, or being composed of, 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 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 having a nucleotide sequence as shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively; optionally comprising a DNA molecule or its corresponding mRNA molecule having a nucleotide sequence as shown in SEQ ID NO:21; optionally comprising a DNA molecule or its corresponding mRNA molecule having a nucleotide sequence 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 is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation.

10. A reagent or kit for the specific detection of metapneumovirus F protein or 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 detecting metapneumovirus F protein or metapneumovirus for non-diagnostic purposes, 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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