A single-domain antibody specifically binding to human metapneumovirus

Abstract

This invention discloses a single-domain antibody that specifically binds to human metapneumovirus (HMV) and its applications. The single-domain antibody and its antigen-binding fragment provided by this invention can specifically recognize and bind to the pre-F protein of HMV, neutralizing the virus. It exhibits particularly high neutralizing activity against HMV strains A1, A2, and B1, effectively preventing and controlling HMV infection. This invention provides a new approach for the detection of HMV and the prevention and treatment of HMV-related diseases, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of molecular immunology, specifically relating to a single-domain antibody or its antigen-binding fragment that specifically binds to human metapneumovirus. Background Technology

[0002] Human metapneumovirus (HMPV) is a respiratory virus discovered in recent years, circulating globally and causing acute respiratory infections in humans. HMPV is a single-stranded, negative-sense RNA virus belonging to the Paramyxoviridae family. The structure of the HMPV virus includes a matrix protein (M) located inside the viral lipid bilayer. The viral particle contains three membrane surface glycoproteins: fusion (F) protein, glycoprotein (G), and short water-transfer protein (SH). Encapsulated within the viral envelope is a nucleoprotein complex (RNAP) composed of a helical genomic RNA-encapsulated nucleoprotein (N), viral RNA-dependent RNA polymerase (L), phosphoprotein (P), and matrix 2 protein (M2). The HMPV surface glycoprotein F mediates the fusion of the virus and the cell membrane. The F protein sequence is highly conserved across different HMPV subtypes, and antibodies induced by G and SH proteins have been reported to be non-protective. Therefore, the F protein is an attractive target for the development of neutralizing antibodies and vaccines against HMPV infection. Surface fusion (F) glycoproteins mediate viral fusion and are the primary target for neutralizing anti-HMPV antibodies. Both Pre-F and Post-F proteins of HMPV (Pre-F being the precursor conformation of the F protein before membrane fusion, and Post-F being the conformation formed after fusion) can effectively induce neutralizing antibodies. Pre-F and Post-F proteins can be detected simultaneously in the serum of HMPV-infected individuals; however, the neutralizing capacity of serum extracted with Pre-F protein is significantly reduced, indicating that the neutralizing antibodies produced in humans are mainly those targeting the Pre-F site. The Ø epitope of HMPV pre-F is glycosylated, therefore its main neutralizing epitope is the III epitope. Due to the presence of this glycosylation site, the immunogenicity of HMPV pre-F is low, requiring special immunization booster treatment.

[0003] Camels and alpacas can produce naturally occurring heavy-chain antibodies that lack the light chain. These molecules contain only a single heavy-chain variable region (VHH) and two conventional CH2 and CH3 regions, yet possess complete antigen-binding function and do not aggregate as easily as artificially engineered single-chain antibody fragments (scFv). Due to their unique structural properties, single-domain antibodies combine the advantages of traditional antibodies and small-molecule drugs, overcoming the drawbacks of traditional antibodies such as long development cycles, low stability, and stringent storage conditions. They possess advantages such as high affinity, strong tissue penetration, high stability, and simple structure, and have been widely used in biopharmaceutical research and development in recent years.

[0004] Therefore, the present invention provides a single-domain antibody or antigen-binding fragment thereof that specifically binds to human metapneumovirus, which can specifically recognize and bind to HMPV pre-F protein, neutralize human metapneumovirus, and better prevent and treat respiratory viral infections. Summary of the Invention

[0005] The single-domain antibody and its antigen-binding fragment that specifically bind to human metapneumovirus developed in this invention can specifically recognize and bind to the pre-F protein of human metapneumovirus. It can bind well to HMPV F cells and neutralize human metapneumovirus, especially showing highly efficient neutralizing activity against human metapneumovirus A1, A2 and B1 strains, and can effectively prevent and control human metapneumovirus infection.

[0006] This invention provides a single-domain antibody or antigen-binding fragment thereof that specifically binds to human metapneumovirus, wherein the single-domain antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3, wherein...

[0007] (a) The amino acid sequence of CDR1 is as shown in SEQ ID NO: 1, 8, 15 or 21, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with the amino acid sequence shown in SEQ ID NO: 1, 8, 15 or 21, or has one or more (preferably two or three) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared with the amino acid sequence shown in SEQ ID NO: 1, 8, 15 or 21.

[0008] (b) The amino acid sequence of CDR2 is as shown in SEQ ID NO: 2, 9 or 16, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence shown in SEQ ID NO: 2, 9 or 16, or has one or more (preferably two or three) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in SEQ ID NO: 2, 9 or 16; and

[0009] (c) The amino acid sequence of CDR3 is as shown in SEQ ID NO: 3 or 10 or is GDD, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with the amino acid sequence shown in SEQ ID NO: 3 or 10 or GDD, or has one or more (preferably two or three) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared with the amino acid sequence shown in SEQ ID NO: 3 or 10 or GDD.

[0010] In some embodiments, the amino acid sequences of the single-domain antibody that specifically binds to human metapneumovirus or its antigen-binding fragment, namely CDR1, CDR2 and CDR3, are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0011] In some embodiments, the amino acid sequences of the CDR1, CDR2, and CDR3 of the single-domain antibody or its antigen-binding fragment that specifically binds to human metapneumovirus are shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

[0012] In some embodiments, the amino acid sequences of the single-domain antibody or its antigen-binding fragment that specifically binds to human metapneumovirus, CDR1 and CDR2, are shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and the amino acid sequence of CDR3 is GDD.

[0013] In some embodiments, the amino acid sequences of the single-domain antibody or its antigen-binding fragment that specifically binds to human metapneumovirus, CDR1 and CDR2, are shown in SEQ ID NO: 21 and SEQ ID NO: 16, respectively, and the amino acid sequence of CDR3 is GDD.

[0014] In some embodiments, the heavy chain variable region of the single-domain antibody that specifically binds to human metapneumovirus or its antigen-binding fragment has any of the amino acid sequences shown in SEQ ID NO: 4-7, 11-14, 18-20, 22-24, or sequences having at least 80%, 85%, 90%, 95% or more identity with any of the amino acid sequences shown in SEQ ID NO: 4-7, 11-14, 18-20, 22-24, or sequences having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences shown in SEQ ID NO: 4-7, 11-14, 18-20, 22-24.

[0015] The present invention provides an immunoglobulin comprising a single-domain antibody or its antigen-binding fragment as described in any of the above claims and an immunoglobulin Fc region.

[0016] In some embodiments, the immunoglobulin Fc region is selected from IgG or IgA.

[0017] In some embodiments, the Fc region is selected from human IgG1, IgG2, IgG3 and / or IgG4 or an amino acid sequence having one or more amino acid mutations (preferably substitutions, insertions or deletions) with human IgG1, IgG2, IgG3, IgG4.

[0018] In some embodiments, the Fc region further includes one or more mutations, namely, a methionine mutation to leucine, and / or an asparagine mutation to serine.

[0019] In some embodiments, the Fc mutation includes mutating methionine (M) at position 430 to leucine (L), i.e., (M430L), and / or mutating asparagine (N) at position 436 to serine (S), i.e., (N436S), based on the native Fc fragment (amino acid sequence as shown in SEQ ID NO: 28), wherein the amino acid sequence is numbered with reference to the EU index of Kabat et al. In some embodiments, the mutated Fc amino acid sequence is shown in SEQ ID NO: 34.

[0020] In some embodiments, the immunoglobulin includes a chimeric antibody or an antigen-binding fragment thereof, and / or a humanized antibody or an antigen-binding fragment thereof.

[0021] The present invention provides a nucleic acid molecule that encodes a single-domain antibody or antigen-binding fragment thereof that specifically binds to human metapneumovirus as described in any of the preceding claims, or an immunoglobulin as described in any of the preceding claims.

[0022] The present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecules.

[0023] The present invention provides a recombinant cell comprising the above-mentioned nucleic acid molecules and / or the above-mentioned recombinant vector, and capable of expressing the single-domain antibody that specifically binds to human metapneumovirus or its antigen-binding fragment or any of the above-mentioned immunoglobulins.

[0024] The present invention provides an antibody conjugate comprising a single-domain antibody or antigen-binding fragment thereof that specifically binds to human metapneumovirus as described in any of the preceding claims, or an immunoglobulin and a marker as described in any of the preceding claims, wherein the marker is selected from one or more of enzyme labeling, biotin labeling, chemiluminescent dye labeling, and radioactive labeling.

[0025] The present invention provides a pharmaceutical composition comprising a single-domain antibody that specifically binds to human metapneumovirus as described in any of the preceding claims, or an antigen-binding fragment thereof, or an immunoglobulin as described in any of the preceding claims, and a pharmaceutically acceptable carrier.

[0026] The present invention provides a detection kit comprising a single-domain antibody that specifically binds to human metapneumovirus as described above, or an antigen-binding fragment thereof, or an immunoglobulin as described above, or comprising the antibody conjugates described above, or comprising the pharmaceutical composition described above.

[0027] The present invention also provides the use of a single-domain antibody or antigen-binding fragment thereof that specifically binds to human metapneumovirus as described in any of the above claims, an immunoglobulin, antibody-drug conjugate or pharmaceutical composition as described in any of the above claims in the preparation of a medicament for the prevention and / or treatment of human metapneumovirus infection or in the preparation of a detection reagent for the detection of human metapneumovirus.

[0028] Abbreviations and Terminology Definitions

[0029] The following abbreviations are used in this article. CDR: Complementarity-determining region in the variable region of immunoglobulins; IgG: Immunoglobulin G; IgA: Immunoglobulin A.

[0030] The term "single-domain antibody" refers to an antibody obtained through genetic engineering methods. There are three main types of methods: The first type is the heavy chain variable region obtained from camel HCAbs (Hepatocellular Cartilaginous Abs), which is a single folded unit that retains complete antigen-binding activity and is the smallest natural antibody fragment. The second type is the heavy chain variable region obtained from IgNARs of cartilaginous fish such as sharks, denoted as VNAR. The third type is the heavy or light chain variable region obtained from human or mouse monoclonal antibodies, which retains antigen-binding activity but has significantly reduced affinity and solubility. In some cases, single-domain antibodies are engineered from camel HCAbs, and their heavy chain variable domains are referred to in this paper as "VHH" (heavy chain variable domain of heavy chain antibody).

[0031] The term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to bind to the antigen the antibody is bound to. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0032] The term "immunoglobulin" refers to a polypeptide or protein with single, dual, or multiple specificities, or with monovalent, divalent, or multivalent binding properties, including but not limited to: IgG, IgM, IgA, IgD, IgE, and all subclasses of immunoglobulins, such as the IgG subclasses IgG1, IgG2, IgG3, and IgG4 found or produced in animal cells, including human cells. "Heavy chain antibodies" formed by the fusion of a single-domain antibody (such as VHH) or its antigen-binding fragment with the Fc region of an immunoglobulin, as well as other antibodies or fragments with similar specific structures, also fall within the scope of "immunoglobulin" as defined in this application. In some embodiments, the immunoglobulin comprises a single-domain antibody or its antigen-binding fragment that specifically binds to human metapneumovirus as described above, and the Fc region of an immunoglobulin.

[0033] The term "amino acid" refers to twenty common, naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). In some embodiments, the term "amino acid" also includes non-natural amino acids. Any suitable non-natural amino acid may be used. In some embodiments, the non-natural amino acid contains a reactive moiety for conjugating the agent with MIAC.

[0034] Various methods / systems exist in this field for defining and describing CDRs. These systems and / or definitions have been developed and refined over many years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used, defining CDRs based on sequence variability; Chothia defines CDRs based on the position of structural loop regions; the IMGT system defines CDRs based on sequence variability and position within variable domain structures; AbM is defined using Oxford Molecular's AbM antibody modeling software and represents a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures and is similar to Chothia in several ways. In this invention, the numbering of amino acid positions (e.g., amino acid residues in the Fc region) and the target regions (e.g., CDRs) are performed using the Kabat system.

[0035] The term "EU index" refers to the residue numbering scheme of human IgG1 EU antibodies. Unless otherwise stated herein, references to residue numbering in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise stated herein, references to residue numbering in the constant domain of an antibody refer to residue numbering according to the EU numbering system (see, for example, U.S. Provisional Patent Application 60 / 640,323, Figure concerning EU numbering scheme).

[0036] The term "specificity" refers to the ability to specifically bind to a given target antigen. A binding peptide can be monospecific and contain one or more binding sites that specifically bind to a target, or a peptide can be multispecific and contain two or more binding sites that specifically bind to the same or different targets.

[0037] The term "chimeric antibody" is an antibody molecule (or its antigen-binding fragment) wherein (1) the constant region or a portion thereof is altered, replaced, or replaced such that the antigen-binding site (variable region) is linked to a constant region of a different or altered type, effector function, and / or kind, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug, etc.) that confers novel properties to the chimeric antibody; or (2) the variable region or a portion thereof is altered, replaced, or replaced with a variable region having a different or altered antigen specificity. For example, an antibody can be modified by replacing its constant region with a constant region derived from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its antigen-recognition specificity while exhibiting reduced antigenicity in the human body compared to the original antibody.

[0038] The term "humanized antibody" refers to a chimeric antibody containing amino acid residues derived from human antibody sequences. Humanized antibodies may contain some or all of the CDR or HVR from non-human animals or synthetic antibodies, while the frame region and constant region of the antibody contain amino acid residues derived from human antibody sequences. This overcomes the heterologous reactions induced by chimeric antibodies carrying a large number of heterologous protein components. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or from publicly available references. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reverse mutations can be performed on the variable region frame sequence of the human antibody to maintain activity.

[0039] The term "amino acid mutation" refers to a mutation or change in amino acids in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion, or substitution of one or more amino acids based on the original protein or polypeptide.

[0040] The term "identity" refers to the percentage of amino acid residues in a candidate sequence that are identical to those in a control polypeptide sequence after sequence alignment and, where necessary, the introduction of gaps to obtain the maximum percentage sequence identity. Comparisons for determining percentage amino acid sequence identity can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, like BLAST software or the FASTA package.

[0041] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are effectively linked when placed in a functional relationship with another nucleic acid sequence.

[0042] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for delivering bound molecules. Carriers can be anti-adhesives, adhesives, coating agents, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, humectants, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0043] The term "pre-F protein" refers to the pre-fusion form of the human metapneumovirus fusion protein F.

[0044] The term "neutralizing activity" refers to immunoglobulins with antiviral activity that can specifically recognize viral antigens and effectively bind to and neutralize viral activity, preventing viral invasion of target cells and blocking viral replication in target cells, thus playing an important role in antiviral activity. Attached Figure Description

[0045] Figure 1 The inhibition rate of chimeric antibody 86 against the A2 strain;

[0046] Figure 2 The inhibition rate of chimeric antibody 88 against the A2 strain;

[0047] Figure 3 The inhibition rate of chimeric antibody 90 against the A2 strain;

[0048] Figure 4 The binding activity of chimeric antibody 86, humanized antibodies 4, 5, and 6 to HMPV pre-F protein was studied.

[0049] Figure 5 The binding activity of chimeric antibody 88, humanized antibodies 7 and 8 to HMPV pre-F protein;

[0050] Figure 6 The binding activity of chimeric antibody 90, humanized antibody 9, and 10 to HMPV pre-F protein was studied.

[0051] Figure 7 The humanized antibody showed neutralizing activity against three pairs of live A2 strain viruses.

[0052] Figure 8 This study evaluates the efficacy of anti-HMPV drugs based on a mouse infection model. Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments. However, the scope of protection of the present invention is not limited to the following embodiments. It should also be understood that the terminology used in the embodiments of the present invention is for describing specific implementations and not for limiting the scope of protection of the present invention. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0054] Example 1: Expression and purification of antigen

[0055] The pcDNA3.1(+) vector was used as the antigen expression vector. The nucleotide sequence of the HMPV pre-F protein was synthesized. The vector and target fragment were double-digested with HindIII and XhoI, recovered, and ligated using DNA ligase. The resulting fragment was transformed into *E. coli* competent cells DH5α. Positive clones were selected, and plasmid extraction and enzyme digestion verification were performed to obtain a recombinant plasmid containing the HMPV pre-F protein nucleotide sequence, named pcDNA3.1(+)-V3B-Δ12-3P-DS454. Expi-CHO cells were cultured at 37℃, 8% CO2, and 100 rpm until a cell density of 6 × 10⁶ cells / year was reached. 6 Cells / mL, the constructed vector pcDNA3.1(+)-V3B-Δ12-3P-DS454 was transfected into the above cells using liposomes at a plasmid concentration of 1 μg / mL. The liposome concentration was determined according to the ExpiCHO™ Expression System kit. Cells were cultured at 32℃, 5% CO2, and 100 rpm for 7–10 days. Feeding was performed once 18–22 h after transfection and again between 5–8 days. The culture product was centrifuged at 4000 rpm, filtered through a 0.22 μm filter, and the supernatant was collected. The expressed HMPV pre-F protein was purified using a nickel column (the amino acid sequence of the pre-F protein is shown in SEQ ID NO: 25).

[0056] Example 2 Animal Immunization

[0057] Using the self-made HMPV pre-F protein as an immunogen, Freund's adjuvant was added for immunization. The antigen and adjuvant were emulsified 1:1 to form a homogeneous mixture and stored at 4°C. Camel ear tags were recorded, and then the immunization experiment began. Injections were made on both sides near the lymph nodes in the camel's neck, with two injection points on each side. 0.4 mL of the mixed antigen was injected at each point. After immunization, the camel was observed for half an hour to confirm it was in good condition and showed no discomfort. Immunizations were performed on days 0, 21, 42, and 63. On day 28, 10 mL of blood was collected from the camel's neck vein; on day 49 and day 70, 50 mL of blood was collected each time. A portion of the blood was taken for serum titer testing. Immunization was performed every two weeks, for a total of seven immunizations. After the 6th and 7th immunizations, blood was collected 5-7 days later, with 25-30 mL of blood collected each time in three blood collection tubes. Blood samples were collected before the 4th, 5th, and 6th immunizations for immune evaluation. Blood was drawn from the jugular vein of the camel, 5 mL each time. The blood was centrifuged at 400 xg for 30 minutes on the same day after being pre-cooled to 25°C, and the supernatant serum was separated and preserved. Lymphocytes were then separated by adding 3 mL of cell separation medium to a 15 mL centrifuge tube, followed by the slow addition of 3 mL of blood. Care was taken to prevent mixing between the blood and the separation medium. The centrifuge was then pre-cooled to room temperature and centrifuged at 400 g for 30 minutes. The blood separation was observed, and the cotton-like supernatant immune cells were carefully aspirated using a 200 μL pipette and transferred to a new 15 mL centrifuge tube. The supernatant serum was preserved in a new centrifuge tube at -80°C. 10 mL of room-temperature PBS buffer was added to each tube, and the tubes were centrifuged at 400 g for 20 minutes at 25°C. The supernatant was removed, and 5 mL of room-temperature PBS buffer was added to each tube, and the tubes were centrifuged at 400 g for 20 minutes at 25°C. Cell counts were performed using a hemocytometer. Remove the supernatant, and use RNAiso Plus to lyse the isolated lymphocytes to obtain 10 7 / mL of solution, store at -80℃.

[0058] Example 3: Database Construction and Screening

[0059] PBMCs were isolated from blood collected after the fifth immunization using lymphocyte separation medium. Total RNA was extracted from the PBMCs, and the RNA was reverse-engineered into cDNA using random primers. The VHH fragment was specifically amplified, and two large E. coli libraries were constructed. Phage display technology was used, and solid-phase panning was performed with HMPV pre-F protein. Phages bound to the recombinant HMPV pre-F protein on the solid-phase panning plate were recovered by infecting TG1 E. coli cells. The infected phages were amplified overnight and purified by precipitation with PEG6000 / NaCl. After three rounds of panning, single clones with good binding to HMPV pre-F protein were screened by phage ELISA.

[0060] Example 4 Construction and expression of chimeric antibodies

[0061] Monoclonal antibodies that showed good binding to the HMPV pre-F protein, obtained from phage library screening, were sequenced. The sequenced antibody fragments were then synthesized and constructed into a human IgG framework. Molecular cloning technology was then used to insert the antibody fragments into the PCDNA3.1 vector to construct a mammalian cell expression plasmid. The plasmid was then introduced into the host cell line CHO cells using liposome transfection. Fermentation supernatant was obtained using a cell fed-batch method. The fermentation supernatant was then purified through a series of steps, including affinity chromatography and ion exchange chromatography, to finally obtain the constructed antibody.

[0062] Example 5: Binding activity of chimeric antibody to 293 HMPV F cells

[0063] 293 HMPV F cells were added to 96-well U-shaped plates at a concentration of 1E5 / well. Chimeric antibodies were diluted to 2 µg / mL and added to each well at a concentration of 50 µL / well. The plates were incubated at 4°C for 1 h. After washing twice with dilution buffer (PBS + 2% FBS), secondary antibody (goat antimouse IgG, cat: HS221, 1:500 dilution) was added and the plates were incubated at 4°C for 0.5 h. After washing twice with dilution buffer, the cells were analyzed by flow cytometry. A positive control (SEQ ID NO: 26 and SEQ ID NO: 27) was included in this experiment. The results showed that most chimeric antibodies could bind to 293 HMPV F cells.

[0064] Table 1. Binding activity of chimeric antibodies to 293 HMPV F cells

[0065]

[0066] Example 6: ELISA method for detecting protein binding activity

[0067] The binding activity of the antibody to HMPV pre-F protein was determined by ELISA. Plates coated with HMPV pre-F were incubated at 37°C for 1 h, blocked with 2% PBS-BSA, and washed three times with PBST. A candidate antibody was used as the primary antibody (diluted to 0.1 µg / mL with PBS), and goat anti-mouse IgG-FC-HRP (source: Beijing TransGen Biotech, catalog number: HS201-01) was used as the secondary antibody. After TMB staining, the absorbance of each well was read at 450 nm. The binding of the antibody to HMPV pre-F was detected, and the results are shown in Table 2. The results showed that most chimeric antibodies bound to the HMPV pre-F protein.

[0068] Table 2. Binding activity of chimeric antibodies to HMPV pre-F protein

[0069]

[0070]

[0071] Example 7: Neutralizing activity of chimeric antibody against live A1 strain virus

[0072] Vero E6 cells were seeded into 96-well cell culture plates one day in advance and cultured overnight in a cell culture incubator (37℃, 5% CO2). Cells were used the following day when the cell density reached 90%. All antibodies were diluted to 10 μg / mL using PBS as the antibody dilution buffer. Humanmetapneumovirus A1 NL / 1 / 00 was diluted to the appropriate concentration, and the same volume of virus was added to each diluted antibody well (final virus amount per well: 200 TCID50). The neutralization system was incubated at room temperature for 1 h. The prepared cell supernatant from the 96-well plate was then added to the neutralization system and incubated in a cell culture incubator (37℃, 5% CO2) for 1 h. 50 mL of culture medium was added, and the plate was incubated again in a cell culture incubator (37℃, 5% CO2) for 72 h before detection. The number of green fluorescent wells at each concentration gradient was read using an inverted fluorescence microscope. The results are shown in Tables 3-4. The results show that nearly half of the chimeric antibodies have neutralizing activity against Human metapneumovirus A1 NL / 1 / 00 at concentrations of 10 μg / mL or 2.5 μg / mL.

[0073] Table 3 Neutralizing activity of chimeric antibodies against A1 strain

[0074]

[0075]

[0076] Note: + indicates a positive result at a concentration of 10 μg / mL or 2.5 μg / mL, indicating neutralizing activity; - indicates a negative result at a concentration of 10 μg / mL or 2.5 μg / mL, indicating no neutralizing activity.

[0077] The antibodies with neutralizing activity described above were serially diluted, and the neutralizing activity of each antibody at different concentrations against the live Humanmetapneumovirus A1 NL / 1 / 00 was detected. A positive control 1 was included in this experiment; the amino acid sequence of positive control 1 consists of SEQ ID NO: 26 and SEQ ID NO: 27.

[0078] Table 4 Neutralizing activity of chimeric antibody gradient concentrations against A1 strain

[0079]

[0080] Table 5 Neutralizing activity of chimeric antibody gradient concentrations against A1 strain

[0081]

[0082]

[0083] Example 8: Neutralizing activity of chimeric antibody against live A2 strain virus

[0084] LLC-MK2 cells were seeded into 96-well cell culture plates one day in advance and cultured overnight in a cell incubator (37℃, 5% CO2). Cells were used the following day when the cell density reached 90%. All antibodies were serially diluted with 2% FBS 2.5 μg / mL TPCK trypsin-opti-MEM. Human metapneumovirus A2-GFP was diluted to the appropriate concentration, and the same volume of virus was added to each diluted antibody and mixed well (final virus amount per well was 100 TCID50). The neutralization system was incubated at room temperature for 1 h. The cell supernatant from the prepared 96-well plates was then added to the neutralization system, and the cells were incubated for another 7 days (33℃, 5% CO2) before detection. The number of green fluorescent spots per cell well was determined using an AID fluorescence ELISA analyzer. The virus inhibition rate (i.e., neutralization rate) was calculated using Microsoft Office.

[0085] Virus inhibition rate (%) = [1 - (number of fluorescent spots)] 实验组 -Number of fluorescent spots 细胞对照 ) / (number of fluorescent spots) 病毒对照 -Number of fluorescent spots 细胞对照 )]×100%

[0086] The results are shown in Tables 6 and 7. Figure 1-3Chimeric antibodies 86, 88, and 90 exhibit excellent virus-neutralizing activity against the A2 strain.

[0087] Table 6 Spot Count Results

[0088]

[0089] Table 7. Inhibition rate of chimeric antibodies against A2 strain

[0090]

[0091] Example 9: Neutralizing activity of multi-concentration chimeric antibodies against live B1 strain virus

[0092] Vero E6 cells were seeded into 96-well cell culture plates one day in advance and cultured overnight in a cell culture incubator (37℃, 5% CO2). Cells were used the following day when the cell density reached 90%. All antibodies were serially diluted with PBS. Humanmetapneumovirus B1 NL / 1 / 99 was diluted to the appropriate concentration, and the same volume of virus was added to each diluted antibody and mixed well (final virus amount per well was 200 TCID50). The neutralization system was incubated at room temperature for 1 h. The cell supernatant from the prepared 96-well plate was then added to the neutralization system and incubated in a cell culture incubator (37℃, 5% CO2) for 1 h. 50 mL of culture medium was added, and the plate was incubated again in a cell culture incubator (37℃, 5% CO2) for 72 h before detection. The number of green fluorescent wells at each concentration was read using an inverted fluorescence microscope. The neutralizing activity of the chimeric antibody against the B1 strain is shown in Table 8.

[0093] Table 8 Neutralizing activity of chimeric antibodies against strain B1

[0094]

[0095] Example 10: Human-centered engineering design

[0096] The variable regions of camel-derived single-domain antibodies were humanized, with the design principle being to avoid introducing protein modification sites such as glycosylation, deamidation, and isomerization, as well as integrin binding sites and cysteine ​​residues. Reversion mutations of key amino acids in the framework region should maintain the original physicochemical and biochemical activities. Using IgBLAST, the variable regions of chimeric antibodies 3, 86, 88, and 90 were aligned with human Germline sequences, and the FR (fractional ligation) sites were replaced with the human Germline sequence with the highest sequence similarity. Then, based on this humanization, several key amino acids affecting antibody affinity were reverse-mutated, i.e., mutated to the original camel-derived FR sites. The humanization percentage was defined as the similarity ratio between the designed sequence framework and the Germline sequence framework. The designed humanized sequences were aligned with human Germline sequences, and sequences with a humanization percentage of 85% or higher were selected. Finally, humanization was carried out on chimeric antibody 3 to obtain humanized antibodies 1, 2, and 3; humanization was carried out on chimeric antibody 86 to obtain humanized antibodies 4, 5, and 6; humanization was carried out on chimeric antibody 88 to obtain humanized antibodies 7 and 8; and humanization was carried out on chimeric antibody 90 to obtain humanized antibodies 9 and 10. The antibody sequences are shown in Table 9.

[0097] Table 9 Antibody Sequences

[0098]

[0099] Example 11 Construction and Expression of Humanized Antibodies

[0100] The designed antibody sequence was genetically synthesized, and then the antibody fragment was inserted into the PCDNA3.1 vector using molecular cloning technology to construct a mammalian cell expression plasmid. The plasmid was then introduced into the host cell line CHO cells using liposome transfection. The fermentation supernatant was obtained using cell Fed-batch, and the fermentation supernatant was purified by affinity chromatography. Finally, the constructed humanized antibody was obtained.

[0101] The expression levels of the supernatant were compared, and the purity of the purified antibody was determined by SEC-HPLC. The results are shown in Table 10.

[0102] Table 10. Expression level and purity of humanized antibodies

[0103]

[0104] Example 12 Binding activity of humanized antibody to HMPV pre-F protein

[0105] The binding activity of the antibody to HMPV pre-F protein was determined by ELISA. Plates coated with HMPV pre-F were incubated at 37°C for 1 h, blocked with 2% PBS-BSA, and washed three times with PBST. Candidate antibodies were used as primary antibodies, and as shown in Table 11, they were diluted with PBS to 0.1, 0.02, and 0.004 µg / mL. As shown in Table 12, starting at a concentration of 1 µg / mL, 3-fold serial dilutions were performed to obtain 11 or 12 concentration gradients (chimeric antibody 86, humanized antibody 4, humanized antibody 5, and humanized antibody 6 were diluted to 11 concentration gradients, and the remaining antibodies were diluted to 12 concentration gradients). Goat anti-mouse IgG-FC-HRP (source: Beijing TransGen Biotech, catalog number: HS201-01) was used as the secondary antibody. After TMB colorimetry, the absorbance of each well was read at a wavelength of 450 nm. The binding of the antibody to HMPV pre-F was detected, and the results are shown in Tables 11 and 12. Figure 4-6 The results showed that most humanized antibodies bound to the HMPV pre-F protein.

[0106] Table 11 Binding activities of humanized antibodies 1, 2, and 3 with HMPV pre-F protein

[0107]

[0108] Table 12. Binding activity of antibody to HMPV pre-F protein

[0109]

[0110] Example 13 Neutralizing activity of humanized antibody against live A1 strain virus

[0111] Vero E6 cells were seeded into 96-well cell culture plates one day in advance and cultured overnight in a cell culture incubator (37℃, 5% CO2). Cells were used the following day when the cell density reached 90%. All antibodies were diluted to 10 μg / mL using PBS as the antibody dilution buffer. Humanmetapneumovirus A1 NL / 1 / 00 was diluted to the appropriate concentration, and the same volume of virus was added to each diluted antibody well (final virus amount per well was 200 TCID50). The neutralization system was incubated at room temperature for 1 h. The prepared cell supernatant from the 96-well plate was then added to the neutralization system and incubated in a cell culture incubator (37℃, 5% CO2) for 1 h. 50 mL of culture medium was added, and the plate was incubated again in a cell culture incubator (37℃, 5% CO2) for 72 h before detection. The number of green fluorescent wells at each concentration gradient was read using an inverted fluorescence microscope. The results are shown in Table 13. The results showed that humanized antibody 2 and humanized antibody 3 had significant neutralizing activity and were comparable, while humanized antibody 1 had weaker neutralizing activity.

[0112] Table 13 Neutralizing activity of humanized antibodies against live A1 strain virus

[0113]

[0114] Example 14 Neutralizing activity of humanized antibody against live A2 strain virus

[0115] LLC-MK2 cells were seeded into 96-well cell culture plates one day in advance and cultured overnight in a cell incubator (37℃, 5% CO2). Cells were used the following day when the cell density reached 90%. All antibodies were serially diluted with 2% FBS 2.5 μg / mL TPCK trypsin-opti-MEM. Human metapneumovirus A2-GFP was diluted to the appropriate concentration, and the same volume of virus was added to each diluted antibody and mixed well (final virus amount per well was 100 TCID50). The neutralization system was incubated at room temperature for 1 h. The cell supernatant from the prepared 96-well plates was then added to the neutralization system, and the cells were incubated for another 7 days (33℃, 5% CO2) before detection. The number of green fluorescent spots per cell well was determined using an AID fluorescence ELISA analyzer. The virus inhibition rate (i.e., neutralization rate) was calculated using Microsoft Office.

[0116] Virus inhibition rate (%) = [1 - (number of fluorescent spots)] 实验组 -Number of fluorescent spots 细胞对照 ) / (number of fluorescent spots) 病毒对照 -Number of fluorescent spots 细胞对照 )]×100%

[0117] See results Figure 7 Tables 14 and 15.

[0118] Table 14 Spot Count Results

[0119]

[0120] Table 15 Inhibition rate of humanized antibodies against A2 strain

[0121]

[0122] Example 15 Neutralizing activity of humanized antibody against live B1 strain virus

[0123] Vero E6 cells were seeded into 96-well cell culture plates one day in advance and cultured overnight in a cell culture incubator (37℃, 5% CO2). Cells were used the following day when the cell density reached 90%. All antibodies were serially diluted with PBS. Humanmetapneumovirus B1 NL / 1 / 99 was diluted to the appropriate concentration, and the same volume of virus was added to each diluted antibody well (final virus amount per well: 200 TCID50). The neutralization system was incubated at room temperature for 1 h. The cell supernatant from the prepared 96-well plate was then added to the neutralization system and incubated in a cell culture incubator (37℃, 5% CO2) for 1 h. 50 mL of culture medium was added, and the plate was incubated again in a cell culture incubator (37℃, 5% CO2) for 72 h before detection. The number of green fluorescent wells at each concentration gradient was read using an inverted fluorescence microscope. The results are shown in Table 16.

[0124] Table 16 Neutralizing activity of humanized antibodies against B1 strain

[0125]

[0126] Example 16 Construction and Expression of Humanized Antibodies

[0127] The heavy chain variable region of humanized antibody 3 was linked to the N-terminus of humanized antibody 3 using a linker, and the Fc region was mutated to extend its half-life (the mutated Fc amino acid sequence is shown in SEQ ID NO: 34), resulting in humanized antibody 13; the Fc region of humanized antibody 10 was mutated to extend its half-life (the mutated Fc amino acid sequence is shown in SEQ ID NO: 34), resulting in humanized antibody 11; the heavy chain variable region of humanized antibody 10 was linked to the N-terminus of humanized antibody 10 using a linker, and the Fc region was mutated to extend its half-life (the mutated Fc amino acid sequence is shown in SEQ ID NO: 34), resulting in humanized antibody 12. The designed antibody sequence was synthesized, and then molecular cloning technology was used to insert the antibody fragment into the PCDNA3.1 / PVAC vector to construct a mammalian cell expression plasmid. This plasmid was then introduced into the host cell line CHO cells using liposome transfection. Fermentation supernatant was obtained using a cell feed-batch method, and the supernatant was purified by affinity chromatography to finally obtain the constructed antibody. The antibody sequence is shown in Table 17.

[0128] Table 17 Antibody Sequences

[0129]

[0130] Example 17 Neutralizing activity of humanized antibodies against live viruses

[0131] The neutralizing activity of humanized antibody 11-13 against HMPV live virus strains was tested. The detection method for strain A1 was the same as in Example 7, the detection method for strain A2 was the same as in Example 8, and the detection method for strain B1 was the same as in Example 9. The results are shown in Tables 18 and 19. Humanized antibody 11-13 has broad-spectrum neutralizing activity against live virus.

[0132] Table 18 Neutralizing activity of humanized antibodies against A2 strain

[0133]

[0134] Table 19 Neutralizing activity of humanized antibodies against A1 and B1 strains

[0135]

[0136] Note: "+" indicates neutralizing activity, and "-" indicates no neutralizing activity.

[0137] Example 18 Rat PK test of humanized antibodies

[0138] SD rats (3 males and 3 females) were used for a rat pharmacokinetic (PK) test of the humanized antibody. The drug was administered via tail vein bolus injection at a dose of 1 mpk. Blood samples were collected from the jugular / orbital veins of the experimental animals, and the actual blood collection time was recorded. After collection, the blood samples were left at room temperature for half an hour before centrifugation (centrifuge pre-cooled at 4°C; centrifugation conditions: 4°C, 3000 rpm, 10 minutes). Serum was separated after centrifugation and transferred to labeled centrifuge tubes. Blood drug concentration was determined using ELISA.

[0139] 100 μL / well was coated with 1.0 mg / mL HMPV pre-F protein and incubated overnight at 4°C. Discard the coating solution, wash 300 μL / well with 1*PBST (0.05%) 4 times with a plate washer, block with 2% BSA (300 μL / well), and incubate at 37℃ for 1 h; discard the blocking solution, wash 300 μL / well with 1*PBST (0.05%) 4 times with a plate washer; dilute the antibody with 2% BSA, add 100 μL / well to the plate, prepare a standard curve solution with 2% BSA and blank rat serum with the corresponding dilution factor, add 100 μL / well to the plate, and incubate at 37℃ for 1 h; discard the liquid, wash 300 μL / well with 1*PBST (0.05%) 4 times with a plate washer, dilute goat anti-human Fc-HRP at a ratio of 1:10000, add 100 μL / well to the plate, and incubate at 37℃ for 45 minutes. Discard the liquid. Add 300 μL of 1*PBST (0.05%) to each well and wash the plate 6 times with a plate washer. Pat the wells dry on clean paper. Add 100 μL of Solarbiotin to each well, wrap the plate with aluminum foil, and incubate at 37°C in the dark for 3 minutes. Stop the reaction by adding 100 μL of 1M hydrochloric acid to each well. Read the values ​​at 450 nm using a microplate reader and analyze the data. Use pharmacokinetic software to process the plasma drug concentration data using a non-compartmental model. Calculate the relevant pharmacokinetic parameters using the linear logarithmic trapezoidal method. The results are shown in Table 20. The results indicate that the humanized antibody exhibits superior metabolic stability in vivo and a longer duration of action.

[0140] Table 20 Results of rat PK test of humanized antibodies

[0141]

[0142] Example 19 Mouse challenge test of humanized antibody

[0143] BALB / c mice were challenged with the virus (HMPV-A2; titer (lgTCID50 / mL): 5.5) via intranasal instillation on day 0, and administered intramuscularly on day 1. Blood was collected from the eyes of the mice on day 5, and lung tissue was harvested for virus titer detection. A positive control 2 was included, with its amino acid sequence consisting of SEQ ID NO: 32 and SEQ ID NO: 33. The detailed experimental protocol is shown in Table 21.

[0144] Table 21 Test Protocol

[0145]

[0146] Challenge procedure: On day 0, G1-G4 groups were challenged via nasal drops. Animals were anesthetized with 5% chloral hydrate (100 μL intraperitoneally) to confirm that the mice no longer had a pain response (no response to light touch on the mouse's paw). Using a 100 μL pipette, 50 μL of virus dilution was accurately drawn and slowly dripped into both nasal cavities. The mice were kept in a static, nose-up position for 3-5 minutes, and then carefully returned to their cages, avoiding vigorous movement.

[0147] Administration procedure: Groups G1 to G4 were administered the drug via intramuscular injection one day before the challenge. Before administration, each mouse was weighed and administered the drug via intramuscular injection according to its actual body weight.

[0148] Mice were euthanized on day 5 post-infection. They were placed on a sterile dissecting board in a biosafety cabinet, with their limbs immobilized in a supine position. The chest and abdomen were disinfected by spraying with 75% ethanol to prevent contamination. Using sterile scissors, the skin was incised along the midline of the abdomen and dissected laterally to fully expose the abdominal muscle layers. The lower end of the sternum was lifted with forceps, and the ribs were cut along both sides of the costal arch (avoiding damage to lung tissue) to create a "V"-shaped opening, exposing the heart and lungs. The trachea near the jaw was grasped with forceps and completely incised. The trachea was gently pulled upwards with forceps, and the ventral tissue connection was interrupted with scissors. The lungs were removed, and surface blood was quickly absorbed with sterile filter paper. The lungs were weighed using an electronic balance (accurate to 0.1 mg). The left lung was collected and preserved in 1.5 mL of 4% paraformaldehyde, and the right lung was collected in 1 mL of 1×PBS. The lungs were homogenized at -20°C (65 Hz for 60 s), centrifuged at 4°C and 8000 rpm for 10 min, and the supernatant was collected. Throughout the procedure, keep the sample in a low-temperature environment as much as possible, and record the tissue weight and the volume of the grinding fluid for subsequent concentration calculation.

[0149] See results Figure 8Regarding viral load, the G1 model group detected the highest viral copy number compared to the same period last year, while the viral load in the G4 group was significantly lower than that in the model group (P=0.0152), confirming the validity of the infection model. Among them, the copy numbers of humanized antibody 11 and humanized antibody 12 were significantly lower than those in the G1 model group, and there was no significant difference in viral load between them and the G4 group.

[0150] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A single-domain antibody or antigen-binding fragment thereof that specifically binds to human metapneumovirus, characterized in that, The single-domain antibody or its antigen-binding fragment comprises CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is as shown in SEQ ID NO: 1, 8, 15 or 21, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 2, 9 or 16, and the amino acid sequence of CDR3 is as shown in SEQ ID NO: 3 or 10 or is GDD.

2. The single-domain antibody or antigen-binding fragment thereof that specifically binds to human metapneumovirus according to claim 1, characterized in that, in, (1) The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; (2) The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively; (3) The amino acid sequences of CDR1 and CDR2 are shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and the amino acid sequence of CDR3 is GDD; or (4) The amino acid sequences of CDR1 and CDR2 are shown as SEQ ID NO: 21 and SEQ ID NO: 16, respectively, and the amino acid sequence of CDR3 is GDD.

3. The single-domain antibody or antigen-binding fragment thereof that specifically binds to human metapneumovirus according to claim 1 or 2, characterized in that, The heavy chain variable region of the single-domain antibody or its antigen-binding fragment has any one of the amino acid sequences shown in SEQ ID NO: 4-7, 11-14, 18-20, 22-24.

4. An immunoglobulin, characterized in that, It comprises the single-domain antibody or its antigen-binding fragment as described in any one of claims 1-3 and the Fc region of an immunoglobulin.

5. The immunoglobulin according to claim 4, characterized in that, The immunoglobulin Fc region is selected from IgG or IgA.

6. The immunoglobulin according to claim 5, characterized in that, The Fc region is selected from human IgG1, IgG2, IgG3 and / or IgG4, or an amino acid sequence that has one or more amino acid mutations with human IgG1, IgG2, IgG3, IgG4.

7. The immunoglobulin according to claim 6, characterized in that, The immunoglobulins include chimeric antibodies or their antigen-binding fragments, and / or humanized antibodies or their antigen-binding fragments.

8. A nucleic acid molecule encoding a single-domain antibody or antigen-binding fragment thereof that specifically binds to human metapneumovirus as described in any one of claims 1-3, or an immunoglobulin as described in any one of claims 4-7.

9. A recombinant vector comprising the nucleic acid molecule of claim 8.

10. A recombinant cell comprising the nucleic acid molecule of claim 8 and / or the recombinant vector of claim 9, and capable of expressing the single-domain antibody or antigen-binding fragment thereof that specifically binds to human metapneumovirus or an immunoglobulin.

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