Monoclonal antibody aiming at human metapneumovirus as well as preparation method and application thereof

Abstract

The invention provides a monoclonal antibody aiming at human metapneumovirus (hMPV) or an antigen binding fragment thereof, a related product thereof, and a preparation method and application thereof. The monoclonal antibody or the antigen binding fragment of the monoclonal antibody can be specifically bound with each subtype F protein of the hMPV with higher affinity, and has higher neutralizing activity on each subtype virus of the hMPV, so that the monoclonal antibody or the antigen binding fragment of the monoclonal antibody has extremely high potential to be developed into an hMPV antigen detection kit and an hMPV broad-spectrum therapeutic antibody; the method shows a remarkable application prospect in the field of clinical detection and treatment of the hMPV, and has an extremely high application value.

Description

Technical Field

[0001] This invention relates to the fields of immunology and molecular virology, specifically to a monoclonal antibody against human metapneumovirus, its preparation method, and its application. Background Technology

[0002] Human metapneumovirus (hMPV) is a single-stranded negative-sense RNA virus belonging to the genus metapneumovirus in the family Pneumoviridae. hMPV is mainly transmitted through the respiratory tract, including droplet transmission and contact with contaminated surfaces. It can cause acute respiratory infections in infants, the elderly, and immunocompromised individuals, leading to diseases such as bronchiolitis and pneumonia.

[0003] Currently, hMPV is prevalent globally, especially during the winter and spring seasons. Statistics show that hMPV infection is the second most common cause of acute respiratory infections in infants and young children, after respiratory syncytial virus (RSV). Since the beginning of 2024, the infection rate of hMPV has further increased due to factors such as global climate change and increased population mobility.

[0004] Currently, there are no effective specific treatments for hMPV, and its clinical treatment mainly focuses on symptomatic and supportive care, such as oxygen therapy, fluid replacement, and antipyretics. This exposes infants and high-risk groups to significant health risks. Therapeutic antibodies have become a key research area in hMPV treatment. Therapeutic antibodies have advantages such as strong targeting and rapid onset of action, and have shown potential in the treatment of other viral infectious diseases. Studies have identified some antibodies with hMPV neutralizing activity, such as those targeting... Epitopes include ADI61026 targeting epitope I, DS7 targeting epitope I, M1C7s targeting epitope II, MPE8 targeting epitope III, 101F targeting epitope IV, MPV467 targeting epitope V, and MPV458 targeting the trimer. However, it is worth noting that hMPV has four subtypes: A1, A2, B1, and B2. The antigenicity of each subtype differs, and the virus is prone to mutation, which undoubtedly poses a challenge to the development of broad-spectrum neutralizing antibodies.

[0005] In conclusion, developing a therapeutic antibody that can efficiently neutralize various subtypes of hMPV while possessing high affinity and broad-spectrum activity is of paramount importance for filling the gap in clinical treatment of hMPV and reducing the health risks to infants and high-risk groups. Summary of the Invention

[0006] In response to the needs or shortcomings of existing technologies in the field of hMPV treatment, this invention provides a monoclonal antibody against hMPV that can bind to the antigen proteins of various hMPV subtypes with high affinity and can efficiently neutralize various hMPV subtypes of the virus, as well as related products, preparation methods and applications.

[0007] Specifically, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof against human metapneumovirus, comprising a heavy chain variable region and a light chain variable region, wherein,

[0009] The heavy chain variable region includes:

[0010] The amino acid sequences are HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively;

[0011] The light chain variable region includes:

[0012] The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; among them, the amino acid sequence of SEQ ID NO:5 is GAS.

[0013] In a specific implementation, the heavy chain variable region further includes four frame regions H-FR1, H-FR2, H-FR3 and H-FR4 arranged alternately with HCDR1, HCDR2 and HCDR3 in sequence, and the light chain variable region further includes four frame regions L-FR1, L-FR2, L-FR3 and L-FR4 arranged alternately with LCDR1, LCDR2 and LCDR3 in sequence;

[0014] Preferably, the amino acid sequences of H-FR1 to H-FR4 are as shown in SEQ ID NO:7 to 10, respectively; and / or, the amino acid sequences of L-FR1 to L-FR4 are as shown in SEQ ID NO:11 to 14, respectively.

[0015] Preferably, the monoclonal antibody or its antigen-binding fragment comprises:

[0016] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:15 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:15; and,

[0017] The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:16.

[0018] In a preferred embodiment, the monoclonal antibody or its antigen-binding fragment comprises:

[0019] The heavy chain variable region, whose amino acid sequence is shown in SEQ ID NO:15; and,

[0020] The light chain variable region has the amino acid sequence shown in SEQ ID NO:16.

[0021] In addition, the monoclonal antibody or its antigen-binding fragment further includes a constant region; preferably, the constant region is selected from any one of the following: the constant region of IgG, IgA or IgM antibody.

[0022] In a preferred embodiment, the monoclonal antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; preferably, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:17; and preferably, the amino acid sequence of the light chain constant region is shown in SEQ ID NO:18.

[0023] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment comprises:

[0024] A heavy chain comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO:19 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:19; and,

[0025] Light chains comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:20.

[0026] More preferably, the monoclonal antibody or its antigen-binding fragment comprises:

[0027] The heavy chain, whose amino acid sequence is shown in SEQ ID NO:19; and,

[0028] The light chain has the amino acid sequence shown in SEQ ID NO:20.

[0029] In some specific embodiments, the monoclonal antibody or its antigen-binding fragment also has a leader sequence (e.g., a signal peptide sequence) at the N-terminus of its heavy chain variable region and / or light chain variable region.

[0030] In some feasible implementations, the antigen-binding fragment of the monoclonal antibody is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies, human antibodies, chimeric antibodies, or bispecific or multispecific antibodies.

[0031] The monoclonal antibody or its antigen-binding fragment of the present invention can specifically recognize conserved epitopes of hMPV and exhibits highly efficient neutralizing activity against all subtypes of hMPV, demonstrating broad-spectrum and high-efficiency activity.

[0032] Secondly, the present invention provides a polynucleotide encoding a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above. This polynucleotide is not limited to any particular method of its production and can be obtained using genetic engineering recombination techniques or chemical synthesis methods.

[0033] In a feasible implementation, the polynucleotide is a polynucleotide group.

[0034] In some preferred embodiments, the polynucleotide group comprises:

[0035] (I) A first polynucleotide encoding the heavy chain variable regions HCDR1, HCDR2, and HCDR3 of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the first polynucleotide being a DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequences shown in SEQ ID NO:21, 22, and 23 (which respectively encode the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 as HCDR1, HCDR2, and HCDR3); and,

[0036] (II) A second polynucleotide encoding the light chain variable regions LCDR1, LCDR2, and LCDR3 of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the second polynucleotide is a DNA molecule or its corresponding mRNA molecule containing the nucleotide sequences shown in SEQ ID NO:24, 25, and 26 (which can encode the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, LCDR1, LCDR2, and LCDR3); wherein, the nucleotide sequence shown in SEQ ID NO:25 is GGCGCCAGC.

[0037] More preferably, the polynucleotide sequence comprises:

[0038] (I) A first polynucleotide encoding the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the first polynucleotide being a DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:27 (which may encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:15); and,

[0039] (II) A second polynucleotide encoding the light chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the second polynucleotide is a DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:28 (which may encode the amino acid sequence shown in SEQ ID NO:16).

[0040] In some specific embodiments, the first polynucleotide encoding the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention and / or the second polynucleotide encoding the light chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention further includes a nucleotide sequence located at the 5' end encoding a leader sequence (e.g., a signal peptide sequence).

[0041] Preferably, the polynucleotide sequence further includes:

[0042] (III) A third polynucleotide encoding the heavy chain constant region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, said third polynucleotide is a DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:29 (which encodes the heavy chain constant region of the amino acid sequence shown in SEQ ID NO:17); and,

[0043] (IV) A fourth polynucleotide encoding the light chain constant region of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the fourth polynucleotide is a DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:30 (which may encode the light chain constant region of the amino acid sequence shown in SEQ ID NO:18).

[0044] In the most preferred embodiment, the polynucleotide group comprises:

[0045] (I) A first polynucleotide encoding a heavy chain of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the first polynucleotide is a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO:31, which encodes a heavy chain with an amino acid sequence as shown in SEQ ID NO:19; and,

[0046] (II) A second polynucleotide encoding a light chain of the monoclonal antibody or its antigen-binding fragment of the present invention, preferably, the second polynucleotide is a DNA molecule or its corresponding mRNA molecule with a nucleotide sequence as shown in SEQ ID NO:32, which may encode a light chain with an amino acid sequence as shown in SEQ ID NO:20.

[0047] Thirdly, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the second aspect above, and at least one expression regulatory element operatively linked to the polynucleotide.

[0048] Fourthly, the present invention provides an expression vector comprising a polynucleotide as described in the second aspect above, or a nucleic acid construct as described in the third aspect above.

[0049] Preferably, the expression vector is a eukaryotic expression vector.

[0050] Fifthly, the present invention provides a transformed host cell comprising the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above.

[0051] The host cells include, but are not limited to: prokaryotic cells, such as Escherichia coli cells; eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cells can also be cell lines, such as the 293T cell line.

[0052] Preferably, the host cell is a eukaryotic cell, and more preferably a mammalian cell.

[0053] Sixthly, the present invention provides a method for preparing a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above, the method comprising:

[0054] (1) Under conditions suitable for the expression of the monoclonal antibody or its antigen-binding fragment, the transformed host cells as described in claim 13 are cultured to express the monoclonal antibody or its antigen-binding fragment.

[0055] (2) The expressed monoclonal antibody or its antigen-binding fragment is recovered from the culture of the host cell.

[0056] In a seventh aspect, the present invention provides a pharmaceutical conjugate comprising a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above, and an effector molecule conjugated directly or indirectly to the monoclonal antibody or the antigen-binding fragment thereof via a spacer.

[0057] Preferably, the effector molecule is a detectable label, toxin, and / or chemotherapeutic agent;

[0058] More preferably, the drug conjugate is an ADC molecule.

[0059] In an eighth aspect, the present invention provides a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a transformed host cell as described in the fifth aspect above, and / or a pharmaceutical conjugate as described in the seventh aspect above, as well as a pharmaceutically acceptable carrier and / or excipient.

[0060] In a ninth aspect, the present invention provides a kit comprising a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a transformed host cell as described in the fifth aspect above, a drug conjugate as described in the seventh aspect above, and / or a pharmaceutical composition as described in the eighth aspect above.

[0061] In a tenth aspect, the present invention provides the use of monoclonal antibodies or antigen-binding fragments thereof as described in the first aspect above, polynucleotides as described in the second aspect above, nucleic acid constructs as described in the third aspect above, expression vectors as described in the fourth aspect above, transformed host cells as described in the fifth aspect above, drug conjugates as described in the seventh aspect above, and / or pharmaceutical compositions as described in the eighth aspect above in any of the following aspects:

[0062] (1) Use in the preparation of medicaments for the prevention and / or treatment of human metapneumovirus infectious diseases;

[0063] (2) Use in the preparation of products for detecting the presence or level of human metapneumovirus in a sample and / or for diagnosing human metapneumovirus infection;

[0064] (3) Application in the preparation of products for neutralizing the virulence of human metapneumovirus in samples.

[0065] Optionally, the sample is a biological sample of the subject.

[0066] Eleventhly, the present invention provides a method for preventing and / or treating human metapneumovirus infection in a subject, the method comprising: administering to the subject a preventive and / or therapeutically effective amount of the following substances: a monoclonal antibody or its antigen-binding fragment as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a transformed host cell as described in the fifth aspect above, a drug conjugate as described in the seventh aspect above, and / or a drug composition as described in the eighth aspect above.

[0067] In a feasible implementation, the subject is a human being.

[0068] The monoclonal antibody or its antigen-binding fragment, or the pharmaceutical composition of the present invention, can be administered to a subject via any suitable route of administration, including but not limited to oral, oral, sublingual, topical, parenteral, rectal, intrathecal, or nasal routes.

[0069] The substance can be used alone or in combination, or in combination with other pharmaceutically active agents (e.g., other antiviral drugs).

[0070] The "effective dose for prevention and / or treatment" may vary depending on the recipient, the organ involved, the symptoms, the method of administration, etc. It may be determined based on the doctor's judgment, taking into account factors such as the type of dosage form, the method of administration, the patient's age and weight, and the patient's symptoms.

[0071] In a twelfth aspect, the present invention provides a method for detecting the presence or level of human metapneumovirus in a sample, the method comprising using a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a transformed host cell as described in the fifth aspect above, a drug conjugate as described in the seventh aspect above, and / or a drug composition as described in the eighth aspect above.

[0072] In some preferred embodiments of the method, the monoclonal antibody or its antigen-binding fragment further includes a detectable marker.

[0073] In some other preferred embodiments of the method, the method further includes using a second antibody carrying a detectable marker to detect the monoclonal antibody of the present invention or its antigen-binding fragment.

[0074] This method can be used for diagnostic purposes (e.g., the sample is from a patient) or for non-diagnostic purposes (e.g., the sample is a cell sample, not from a patient).

[0075] Therefore, in some specific embodiments, the present invention provides a method for diagnosing whether a subject is infected with human metapneumovirus, comprising: detecting the presence or absence of human metapneumovirus in a sample from the subject using a monoclonal antibody or antigen-binding fragment of the present invention as described in the first aspect above. In some preferred embodiments, the monoclonal antibody or antigen-binding fragment of the present invention further includes a detectable marker; in other preferred embodiments, the method further includes using a second antibody carrying a detectable marker to detect the monoclonal antibody or antigen-binding fragment of the present invention or an anti-idiotype antibody. In feasible embodiments, the subject is a human.

[0076] General methods for detecting the presence or level of a target virus or antigen in a sample using monoclonal antibodies or their antigen-binding fragments are well known to those skilled in the art. In some preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive assays, and similar methods.

[0077] Beneficial effects

[0078] The monoclonal antibody against hMPV or its antigen-binding fragment provided by this invention has the following significant advantages:

[0079] (1) Broad-spectrum binding ability: The hMPV monoclonal antibody of the present invention can specifically bind to the F protein of hMPV A / B subtype;

[0080] (2) High affinity properties: According to affinity testing, the binding affinity of the hMPV monoclonal antibody of the present invention to the target antigen can reach the nanomolar (nM) level. This high affinity property significantly enhances the stability of antibody-antigen interaction and provides a solid foundation for related applications.

[0081] (3) Strong neutralizing activity: In vitro neutralization experiments have confirmed that the hMPV monoclonal antibody of the present invention exhibits strong neutralizing activity against all subtypes of hMPV. This characteristic enables it to effectively inhibit the infection of all subtypes of hMPV virus, making it a highly promising candidate molecule for clinical anti-hMPV virus treatment.

[0082] Given the aforementioned significant advantages, the monoclonal antibody or its antigen-binding fragment of the present invention has extremely high potential to be developed into a human metapneumovirus (hMPV) antigen detection kit. This kit can achieve a lower detection limit when detecting hMPV, thus exhibiting higher detection sensitivity. Simultaneously, the monoclonal antibody or its antigen-binding fragment of the present invention demonstrates strong neutralizing activity against all hMPV subtypes, exhibiting significant advantages in broad-spectrum activity and high efficiency. Therefore, it has great potential to be developed into a broad-spectrum, therapeutic antibody against hMPV.

[0083] In summary, the monoclonal antibody or its antigen-binding fragment against hMPV provided by this invention shows significant application prospects in the clinical detection and treatment of hMPV and has extremely high application value. Attached Figure Description

[0084] 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.

[0085] Figure 1 The image shows the SDS-PAGE identification results of the monoclonal antibody RH22. The left image is the electrophoresis image of the sample without dithiothreitol treatment, and the right image is the electrophoresis image of the sample after dithiothreitol treatment.

[0086] Figure 2 Figure 1 shows the binding kinetics curves of monoclonal antibody RH22 with hMPV subtype A F protein (Figure A) and hMPV subtype B F protein (Figure B), as detected by surface plasmon resonance assay.

[0087] Figure 3 The neutralizing ability of the monoclonal antibody RH22 against hMPV is shown, where the horizontal axis represents the Log antibody concentration (ng / mL) and the vertical axis represents the neutralization inhibition rate (%). Detailed Implementation

[0088] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0089] Unless otherwise expressly stated, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0090] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0091] The term "percentage (%) sequence identity" is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing vacancies) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percentage amino acid sequence identity.

[0092] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes 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. Specifically, the antigen-binding fragment can be selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, biantibodies, etc.

[0093] The “Fab” segment consists of a light chain, a heavy chain CH1, and a variable region.

[0094] The “Fab’” fragment contains a light chain and a heavy chain portion that includes the VH domain, the CH1 domain, and the constant region between the CH1 and CH2 domains. Interchain disulfide bonds are formed between the two heavy chains of the two Fab’ fragments to form the F(ab’)2 molecule.

[0095] The “F(ab')2” segment contains two light chains and two heavy chain segments containing the VH domain, the CH1 domain, and the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 segment consists of two Fab' segments held together by disulfide bonds between the two heavy chains.

[0096] The “Fv” region contains variable regions from both the heavy and light chains, but lacks constant regions.

[0097] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment containing the VH and VL domains of the antibody, which are contained within a single polypeptide chain. Generally, scFv polypeptides contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding.

[0098] A "bispecific antibody" is a small antigen-binding fragment with two antigen-binding sites. The fragment contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH-VL or VL-VH). By using a linker short enough not to pair between the two domains on the same chain, the domain pairs with a complementary domain of the other chain to form two antigen-binding sites.

[0099] 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.

[0100] The sequence information of the monoclonal antibody RH22 described in the following examples is shown in Table 1 below.

[0101] Table 1. Sequence information of monoclonal antibody RH22

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] Example 1: Isolation of hMPV F-specific B cells, 10×Genomics sequencing, and preparation of monoclonal antibodies

[0111] Animal immunization: Balb / c mice that had been repeatedly immunized with RSV-F protein trimer and hMPV-F protein trimer were used. Lymph nodes of the immunized mice were collected, ground into single-cell suspensions, and hMPV F antigen protein (final concentration 400 nM) was added to the single-cell suspension as bait. The cells were incubated on ice for 30 min. After incubation, the cells were washed twice with PBS. Then, the following antibodies were added: Anti-mouse CD138 / BV711, Anti-mouse CD93 / APC, Anti-mouse CD38 / PE-Cy7, Anti-mouse IgD / BV510, Anti-mouse GL7 / FITC, Anti-mouse7AAD Percp, Anti-mouse B220 / BV421, and Anti-His / PE. The cells were incubated on ice in the dark for 30 min for staining. After staining, the cells were washed twice with PBS. The cells were then transferred to flow cytometry tubes.

[0112] Single-cell sorting: Single cells with 7AAD-, CD93-, CD138- / weakly+, CD38- / weakly+, IgD-, GL7+, B220+, and His+ were sorted using a BD Influx flow cytometer. These are hMPV F protein-specific germinal center B cells. The sorting process was carried out at 4°C, and the flow rate was adjusted to 8000 cells / s for optimal results. The target cells were collected in a sterile 1.5 mL EP tube.

[0113] High-throughput sequencing and antibody library construction: Using 10×Genomics single-cell sequencing technology, the sorted specific B cells were sequenced, and the BCR sequences of the B cells were compiled and summarized to construct an antibody library targeting the hMPV F protein. This part was mainly completed by Beijing Yimeitongde Technology Development Co., Ltd., and the main steps included:

[0114] 1. Cell quality control: Centrifuge germinal center B cells at 500g at 4℃, remove the supernatant slowly and gently, then add about 40μL of DPBS buffer containing 3% BSA, gently pipette and mix the cells, then take 5μL of cell solution and stain with an AO / PI double staining kit, and detect the number and viability of cells under a cell counter. Cells with a viability of more than 85% and a number of not less than 5000 can be used for the instrument.

[0115] 2. Prepare the reverse transcription reaction system: Take 10 μL of RT Enzyme Mix B, 2.0 μL of Additive A, 6.4 μL of Poly-dT RT Primer and 18.8 μL of RT Reagent B and mix them on ice. Mix thoroughly with a pipette to prepare the reverse transcription system.

[0116] 3. Preparation of water-in-oil emulsion: Mix 35 μL of B cell sample with 35 μL of the above reverse transcription system, and mix thoroughly with a pipette. Then add 70 μL of the mixture to the first row of the 10×Chromium chip, and add 50 μL of gel beads and 45 μL of partitioning oil droplets to the second and third rows, respectively. Then use a microfluidic system to bind the single cells and gel beads and encapsulate them with oil droplets to form a water-in-oil structure, called GEM (Gel Bead Inemulsion).

[0117] 4. Reverse transcription: Add 100 μL of GEM to the PCR tube and place the PCR tube into the PCR instrument to start reverse transcription.

[0118] 5. cDNA Recovery and Amplification: Add 125 μL of Recovery 2 Agent to the sample and let it stand at room temperature for 2 min. At this point, the original emulsion will turn into a pink oil phase and a clear aqueous phase. Carefully remove the oil phase from the bottom with a pipette, then add 200 μL of Dynabeads Cleanup Mix, which contains 8 μL Dynabeads MyOneSILANE, 182 μL Buffer Sample Clean Up 1, 5 μL Nuclease-free Water, and 5 μL Additive A. Mix thoroughly with a pipette and let it stand at room temperature for 10 min. Remove GeIbeads with a magnetic pole and dissolve and recover the cDNA with 35 μL of elution buffer (composed of 98 μL EB + 1 μL 10% Tween 20 + 1 μL Additive A). Amplify the cDNA in PCR.

[0119] 6. Amplifying the antibody sequence: The target sequence of the antibody is amplified using multiplex PCR primers, and then the amplification product is ligated into an Illumina sequencing adapter via Post-PCR to construct a single-cell library of specific memory B cells. After the reaction is complete, the qualified library is sequenced using Illumina, and the BCL data is converted into a FASTQ file using Illumina bcl2fastq2.20 software.

[0120] Preparation of monoclonal antibodies: The antibody light and heavy chain plasmids were co-transfected into HEK293F cells for antibody expression. The protein was then purified by protein A affinity chromatography and molecular sieve chromatography, and identified by SDS-PAGE to obtain high-purity antibody protein.

[0121] The SDS-PAGE identification results of the monoclonal antibody RH22 of the present invention obtained through efficacy screening in the following examples are as follows: Figure 1 As shown, Figure 1 In the middle, the left image shows the gel electrophoresis bands of the sample without dithiothreitol treatment, which shows a clear single band. Based on the molecular weight position of this band, it is determined to be the total antibody. The right image shows the gel electrophoresis bands of the sample treated with dithiothreitol, which shows two clear bands. Based on their molecular weight positions, these two bands are determined to be the heavy chain and light chain of the antibody, respectively.

[0122] Example 2: Determination of the binding ability of monoclonal antibody RH22 to antigenic peptide

[0123] In this embodiment, the binding affinity of the monoclonal antibody RH22 obtained in Example 1 to hMPV A isotype F protein (a complete Pre-F protein, whose amino acid sequence is shown in SEQ ID NO:33 and whose DNA coding sequence is shown in SEQ ID NO:35) and hMPV B isotype F protein (a complete Pre-F protein, whose amino acid sequence is shown in SEQ ID NO:34 and whose DNA coding sequence is shown in SEQ ID NO:36) was determined using surface plasmon resonance analysis. The specific procedure is as follows:

[0124] Surface plasmon resonance (SPR) analysis was performed using a Biacore 8K (GE Healthcare) instrument, employing a protein A chip (GE Healthcare). Based on the specific binding of protein A to the Fc fragment of the antibody, the antibody protein was immobilized on the chip, with an immobilization amount of approximately 500 RU. The antigen protein was serially diluted 2-fold with PBST solution (pH 7.4), with a total of five dilution gradients. After sequentially adding various buffer solutions and samples, the instrument was run and the response values ​​were recorded. The kinetic curves of antibody-antigen binding were analyzed using BIAevaluation software 8K (GE Healthcare).

[0125] The binding kinetics curves of monoclonal antibody RH22 with hMPV A subtype F protein and hMPV B subtype F protein are shown in the figure below. Figure 2 Figures A and B in the table, along with the kinetic constants, are shown in Table 2.

[0126] Table 2

[0127] Ka(1 / Ms) kd(1 / s) KD(M) hMPV AF-RH22 3.99e+05 3.32e-04 8.34e-10 hMPV BF-RH22 8.97e+04 2.79e-04 3.31e-09

[0128] Depend on Figure 2 As shown in Figures A and B and Table 2, RH22 exhibits an affinity of nM for both hMPV subtype A F protein and hMPV subtype B F protein, indicating that the antibody possesses extremely strong binding ability against different hMPV subtype F proteins.

[0129] Example 3: Detection of the neutralizing activity of monoclonal antibody RH22 against hMPV virus

[0130] In this embodiment, the neutralizing activity of the monoclonal antibody RH22 against the hMPV virus TN / 99 / 4-6 subtype was detected by flow cytometry. The specific steps are as follows:

[0131] (1) Cell plating: Seed VeroE6 cells in good growth condition into 24-well cell culture dishes. When the cells grow to a confluence of more than 70% the next day, they can be used for neutralization experiments.

[0132] (2) Diluting the antibody: The antibody to be tested (i.e., monoclonal antibody RH22) was serially diluted from 1 μg / mL in DMEM medium containing 1% FBS, for a total of 11 concentration gradients, with 2 replicates for each gradient.

[0133] (3) In vitro incubation of virus and test antibody: Take 200 μL of diluted test antibody and mix it with an equal volume of 200 μL of hMPV TN / 99 / 4-6 subtype virus solution, and incubate at 37℃ for 1 h. At the same time, set up a positive control (200 μL of DMEM containing 1% FBS + 200 μL of virus) and a negative control (400 μL of DMEM containing 1% FBS).

[0134] (4) Infecting cells: Discard the cell supernatant, wash the cells once with sterile PBS, add 350 μL of virus antibody mixture to the cells, culture at 37°C and 5% CO2 for 1 h, then add 150 μL of DMEM containing 1% FBS and continue culturing for 47 h.

[0135] (5) Result Interpretation: Cells were collected and transferred into 96-well round-bottom plates. After washing the cells once with PBS, 100 μL of fixative was added to each well of the 96-well plate, and the cells were fixed at 4°C in the dark for 30 min. After washing the cells twice with 10× washing buffer diluted to 1× working concentration with deionized water, the cells were stained with 5 μg / mL of MPE8 antibody labeled with FITC fluorescence at 4°C in the dark for 30 min. The cells were washed twice, and the proportion of FITC-positive cells was detected by FACS Canto. Since the MPE8 antibody can recognize and bind to the F protein of hMPV virus, the proportion of FITC-positive cells can indicate the infection status of hMPV TN / 99 / 4-6 subtype virus. Based on the change in the proportion of FITC-positive cells after incubation with different concentrations of monoclonal antibody RH22, the neutralization inhibition rate (%) and neutralization capacity (expressed as IC50) of the monoclonal antibody of this application against hMPV virus can be calculated.

[0136] Based on the proportion of positive cells at different antibody treatment concentrations, the neutralization inhibition rate (%) of monoclonal antibody RH22 against hMPV virus was calculated using the formula: Y = (1 - X / Z) × 100%, where Y is the neutralization inhibition rate (%), X is the proportion of positive cells in the antibody wells, and Z is the proportion of positive cells in the positive control wells (i.e., wells with virus-infected cells but without the antibody). The obtained data were processed using GraphPad software to generate a fitting curve and obtain the half-maximal inhibitory concentration (IC50) value. The results are shown below. Figure 3 As shown in the figure. The relevant results are statistically summarized in Table 3.

[0137] Table 3

[0138] nab IC50 (ng / ml) RH22 37.44

[0139] Figure 3 The results in Table 3 show that the RH22 antibody has a very strong neutralizing ability against hMPV virus, with an IC50 value of 37.44 ng / ml, indicating that the monoclonal antibody RH22 has extremely high application potential and value in the fight against hMPV virus.

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody or its antigen-binding fragment against human metapneumovirus, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes: The amino acid sequences are HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; The light chain variable region includes: The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; wherein the amino acid sequence shown in SEQ ID NO:5 is GAS.

2. The monoclonal antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region also includes four frame regions H-FR1, H-FR2, H-FR3 and H-FR4 arranged alternately with HCDR1, HCDR2 and HCDR3 in sequence; the light chain variable region also includes four frame regions L-FR1, L-FR2, L-FR3 and L-FR4 arranged alternately with LCDR1, LCDR2 and LCDR3 in sequence. Preferably, the amino acid sequences of H-FR1 to H-FR4 are as shown in SEQ ID NO:7 to 10, respectively; and / or, the amino acid sequences of L-FR1 to L-FR4 are as shown in SEQ ID NO:11 to 14, respectively.

3. The monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2, characterized in that, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:15 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:15; And / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:16; Preferably, the monoclonal antibody or its antigen-binding fragment comprises: The heavy chain variable region, whose amino acid sequence is shown in SEQ ID NO:15; and, The light chain variable region has the amino acid sequence shown in SEQ ID NO:

16.

4. The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3, wherein, The monoclonal antibody or its antigen-binding fragment further includes a constant region; Preferably, the constant region comprises a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:17 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:

18.

5. The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4, wherein, The monoclonal antibody or its antigen-binding fragment comprises: Heavy chains comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO:19 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:19; and, Light chains comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO:20 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:20; Preferably, the monoclonal antibody or its antigen-binding fragment comprises: The heavy chain, whose amino acid sequence is shown in SEQ ID NO:19; and, The light chain has the amino acid sequence shown in SEQ ID NO:

20.

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

7. A polynucleotide encoding a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-6.

8. The polynucleotide of claim 7, wherein, The polynucleotide is a polynucleotide group, which includes: (I) A DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:21, 22, 23; and, (II) A DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:24, 25, 26; wherein the nucleotide sequence shown in SEQ ID NO:25 is GGCGCCAGC.

9. The polynucleotide of claim 7 or 8, wherein, The polynucleotide sequence includes: (I) A DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:27; and, (II) A DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:28; Preferably, the polynucleotide sequence further includes: (III) A DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:29; and, (IV) A DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:

30.

10. The polynucleotide according to any one of claims 7-9, wherein, The polynucleotide sequence includes: (I) A DNA molecule or its corresponding mRNA molecule with the nucleotide sequence shown in SEQ ID NO:31; and, (II) DNA molecules or their corresponding mRNA molecules with nucleotide sequences as shown in SEQ ID NO:

32.

11. A nucleic acid construct comprising a polynucleotide as described in any one of claims 7-10, and at least one expression regulatory element operatively linked to said polynucleotide.

12. An expression vector comprising a polynucleotide as described in any one of claims 7-10, or a nucleic acid construct as described in claim 11.

13. A transformed host cell, wherein the transformation comprises a polynucleotide as described in any one of claims 7-10, a nucleic acid construct as described in claim 11, or an expression vector as described in claim 12.

14. A method for preparing a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-6, the method comprising: (1) Under conditions suitable for the expression of the monoclonal antibody or its antigen-binding fragment, the transformed host cells as described in claim 13 are cultured to express the monoclonal antibody or its antigen-binding fragment. (2) The expressed monoclonal antibody or its antigen-binding fragment is recovered from the culture of the host cell.

15. A drug conjugate comprising a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-6, and an effector molecule conjugated directly or indirectly via a spacer to the monoclonal antibody or the antigen-binding fragment thereof; Preferably, the effector molecule is a detectable label, toxin, and / or chemotherapeutic agent; More preferably, the drug conjugate is an ADC molecule.

16. A pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-6, a polynucleotide as described in any one of claims 7-10, a nucleic acid construct as described in claim 11, an expression vector as described in claim 12, a transformed host cell as described in claim 13, and / or a pharmaceutical conjugate as described in claim 15, and a pharmaceutically acceptable carrier and / or excipient.

17. A kit comprising a monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-6, a polynucleotide as described in any one of claims 7-10, a nucleic acid construct as described in claim 11, an expression vector as described in claim 12, a transformed host cell as described in claim 13, a pharmaceutical conjugate as described in claim 15, and / or a pharmaceutical composition as described in claim 16.

18. The use of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-6, the polynucleotide as described in any one of claims 7-10, the nucleic acid construct as described in claim 11, the expression vector as described in claim 12, the transformed host cell as described in claim 13, the drug conjugate as described in claim 15, and / or the pharmaceutical composition as described in claim 16 in any of the following aspects: (1) Use in the preparation of medicaments for the prevention and / or treatment of human metapneumovirus infectious diseases; (2) Use in the preparation of products for detecting the presence or level of human metapneumovirus in a sample and / or for diagnosing human metapneumovirus infection; (3) Application in the preparation of products for neutralizing the virulence of human metapneumovirus in samples.

19. The application as described in claim 18, characterized in that, The sample is a biological sample from the subject.

Images