An antibody against human metapneumovirus and uses thereof

Abstract

This invention relates to an antibody against human metapneumovirus and its uses. The antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1-3, respectively; and the light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4-6, respectively. The antibody of this invention exhibits good broad-spectrum binding ability to hMPV A / B strain F protein and highly efficient neutralizing activity against human metapneumovirus.

Description

Technical Field

[0001] This invention relates to the fields of immunology and molecular biology, and more particularly to an antibody against human metapneumovirus and its uses. Background Technology

[0002] Human metapneumovirus (hMPV) is one of the main pathogens causing severe lower respiratory tract infections in infants, the elderly, and immunocompromised individuals. hMPV infection can lead to symptoms such as cough, wheezing, and difficulty breathing, and in severe cases, it can even be life-threatening.

[0003] hMPV is a single-stranded negative-sense RNA virus belonging to the family Pneumoviridae and the genus Metapneumovir. Its genome is approximately 13.3 kb in length and encodes nine proteins: nucleocapsid protein N, phosphoprotein P, matrix protein M, small hydrophobic protein SH, adhesion protein G, fusion protein F, M2-1, M2-2, and polymerase subunit protein L.

[0004] Although several hMPV vaccine and neutralizing antibody projects are underway, no vaccine or neutralizing antibody has yet been successfully approved for market release. Currently, some hMPV combination vaccines that simultaneously target respiratory syncytial virus (RSV) and / or human parainfluenza virus type 3 (HPIV3) have entered clinical trials. For example, Vicebio Australia's VXB-241 vaccine and AstraZeneca's IVX-A12 vaccine have entered Phase I and Phase III clinical trials, respectively.

[0005] The global disease burden caused by hMPV is very heavy each year. hMPV infection is particularly common in infants and young children, and can lead to serious illnesses such as bronchiolitis and pneumonia. In the elderly and immunocompromised individuals, hMPV infection can also cause severe respiratory illnesses and even death. To better combat hMPV infection, there is an urgent need to develop more effective hMPV monoclonal antibodies. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide an antibody against human metapneumovirus and its use therein, which has highly efficient neutralizing activity against human metapneumovirus.

[0007] To achieve the above objectives, a first aspect of the present invention provides an antibody against human metapneumovirus, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 6.

[0008] In this application, HCDR represents a complementary determinant region (CDR) in the heavy chain variable region, for example, HCDR1 refers to CDR1 in the heavy chain variable region; LCDR represents a complementary determinant region (CDR) in the light chain variable region, for example, LCDR1 refers to CDR1 in the light chain variable region.

[0009] In a preferred embodiment of the present invention, the heavy chain variable region further comprises HFR1 with the amino acid sequence shown in SEQ ID NO: 7, HFR2 with the amino acid sequence shown in SEQ ID NO: 8, HFR3 with the amino acid sequence shown in SEQ ID NO: 9, and HFR4 with the amino acid sequence shown in SEQ ID NO: 10; and The light chain variable region further includes LFR1 with an amino acid sequence as shown in SEQ ID NO: 11, LFR2 with an amino acid sequence as shown in SEQ ID NO: 12, LFR3 with an amino acid sequence as shown in SEQ ID NO: 13, and LFR4 with an amino acid sequence as shown in SEQ ID NO: 14.

[0010] In this application, HFR represents a frame region (FR) in the Heavy Chain Variable Region, for example, HFR1 refers to FR1 in the Heavy Chain Variable Region; LFR represents a frame region (FR) in the Light Chain Variable Region, for example, LFR1 refers to FR1 in the Light Chain Variable Region.

[0011] In this application, each heavy chain variable region and light chain variable region typically contains 3 CDRs and up to 4 FRs, wherein the CDRs and FRs are arranged from the amino terminus to the carboxyl terminus in, for example, the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In the heavy chain variable region of the antibody of this application, the CDRs and FRs are arranged from the amino terminus to the carboxyl terminus in the following order: HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, HFR4; and in the light chain variable region of the antibody of this application, the CDRs and FRs are arranged from the amino terminus to the carboxyl terminus in the following order: LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4.

[0012] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.

[0013] In a preferred embodiment of the present invention, the antibody is a fully humanized monoclonal antibody.

[0014] A second aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the aforementioned antibody.

[0015] In a preferred embodiment of the invention, the nucleotide sequence encoding the antibody comprises: a nucleotide sequence encoding the heavy chain variable region, as shown in SEQ ID NO: 17; and a nucleotide sequence encoding the light chain variable region, as shown in SEQ ID NO: 18.

[0016] A third aspect of the present invention provides a carrier comprising the above-described nucleic acid molecules.

[0017] A fourth aspect of the present invention provides a cell comprising the above-described nucleic acid molecule or the above-described carrier.

[0018] A fifth aspect of the present invention provides a pharmaceutical composition comprising the antibody, the nucleic acid molecule, the carrier or the cell, and a pharmaceutically acceptable carrier.

[0019] The sixth aspect of the present invention provides the use of the above-described antibody, nucleic acid molecule, carrier, cell, or pharmaceutical composition in the preparation of a drug for the prevention and / or treatment of human metapneumovirus or in the preparation of a human metapneumovirus detection reagent.

[0020] The beneficial effects of this invention are as follows: The antibody against human metapneumovirus (hMPV) of this invention possesses a unique CDR region, exhibiting excellent broad-spectrum binding ability to the F protein of hMPV A / B strains; and it can effectively neutralize hMPV, particularly showing highly efficient neutralizing activity against hMPV A2 and B1 strains, with an IC50 value of [missing information]. 50 The concentrations were 33.66 ng / mL and 20.85 ng / mL, respectively. Therefore, the antibodies of the present invention can be used to prepare drugs for the prevention and / or treatment of human metapneumovirus. Furthermore, the antibodies of the present invention can be used to prepare human metapneumovirus detection reagents for the detection of viral antigens and for the identification of effective neutralizing antigenic epitopes. Attached Figure Description

[0021] Figure 1 Flowchart for antibody screening using phage display technology.

[0022] Figure 2 : Binding ability curves of RMFP2E03 to the F protein (pre-F / post-F) of hMPV A2 and B2 strains; (A) Binding ability curve of antibody to hMPV A2 pre-F; (B) Binding ability curve of antibody to hMPV B2 pre-F; (C) Binding ability curve of antibody to hMPV A2 post-F.

[0023] Figure 3 : Binding kinetics curves of RMFP2E03 interacting with the F protein (pre-F / post-F) of hMPV A2 and B2 strains; (A) Binding kinetics curve of antibody to hMPV A2 pre-F; (B) Binding kinetics curve of antibody to hMPV B2 pre-F; (C) Binding kinetics curve of antibody to hMPV A2 post-F.

[0024] Figure 4 (A) In vitro neutralization ability curve of RMFP2E03 against hMPV; (B) Neutralization ability curve of antibody against hMPV A2 strain;

[0025] Figure 5 Epitope competition diagram between RMFP2E03 and other hMPV neutralizing antibodies. Detailed Implementation

[0026] It should be noted that, unless otherwise defined, the technical or scientific terms used in this application shall have the ordinary meaning as understood by one of ordinary skill in the art.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all medicinal materials and reagents used in the following examples are commercially available products.

[0028] When used herein and in the appended claims, the singular forms “a,” “an,” “another,” and “the” include the plural referents unless the context clearly indicates otherwise.

[0029] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0030] In this application, the term "antibody" has its conventional meaning in the art, referring to an immunoglobulin molecule composed of four polypeptide chains, namely two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Analysis of the amino acid sequences of different antibody heavy and light chains revealed that the amino acid sequences near the N-terminus of both heavy and light chains exhibit significant variation, while the amino acid sequences in other parts remain relatively constant. Therefore, the region of significant variation in the amino acid sequence near the N-terminus in the antibody light and heavy chains is termed the variable region (V), and the region of relatively stable amino acid sequence near the C-terminus is termed the constant region (C). The V regions of the heavy and light chains are abbreviated as VH and VL, respectively, and the C regions of the heavy and light chains are abbreviated as CH and CL, respectively. Within the variable region of an antibody, a small subset of amino acid residues exhibits particularly strong variations. These regions, where the composition and sequence of amino acid residues are more prone to variation, are called hypervariable regions (HVRs). There are three hypervariable regions each in the V region of the L and H chains. Because these regions can form precise complementarity with antigenic determinants in their spatial structure, they are also called complementarity determining regions (CDRs). Common CDR classification rules for antibodies include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well-known to those skilled in the art. When using websites that apply these rules, simply inputting the VH and VL sequences and selecting the corresponding rule will yield CDR sequences based on different rules. Those skilled in the art should understand that the scope of protection of this application covers combinations of CDR sequences obtained through analysis using different rules. The six CDR regions of an antibody collectively determine the antibody's recognition ability and specificity for the corresponding antigen. Those skilled in the art should understand that when this application defines the amino acid sequences of the six CDR regions, the antibody's recognition ability and specificity for the corresponding antigen are predictable.

[0031] In this application, the term "variable region" refers to a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen. The variable regions (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures and can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) interspersed within more conserved regions (called frame regions (FRs)).

[0032] In this application, the term “complementarity-determining region” (CDR, such as CDR1, CDR2, and CDR3) refers to certain amino acid residues in an antibody variable region that are essential for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2, and CDR3. Each complementarity-determining region may contain amino acid residues from “complementarity-determining regions” as defined by Kabat (Kabat et al., Sequences of Proteins of Immulological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. 1991)) and / or residues from “hypervariant rings” (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)).

[0033] In this application, the complementarity-determining region (CDR) and frame region (FR) of a given antibody may be identified using the Kabat system (Kabat et al.: Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991).

[0034] In this application, the term "fully humanized antibody" refers to the process of transferring the entire human antibody-encoding gene into a genetically engineered animal lacking the antibody gene through transgenic or transchromosomal techniques, thereby enabling the animal to express human antibodies and achieving the goal of fully humanizing the antibody. The aim of "fully humanization" is to eliminate the immunogenicity of non-human antibodies in the human body while preserving affinity to the greatest extent possible. It is advantageous to select a human skeleton sequence most similar to the non-human antibody skeleton sequence as a template for humanization. In some cases, it may be necessary to replace one or more amino acids in the human skeleton sequence with corresponding residues from the non-human skeleton to avoid loss of affinity.

[0035] In this application, "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, i.e., the group comprising a single antibody is identical except for the possibility of mutations (e.g., natural mutations) that may be present in very small amounts. Therefore, the term "monoclonal" indicates the nature of the antibody, i.e., it is not a mixture of unrelated antibodies. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Besides their specificity, monoclonal antibody formulations have the advantage that they are generally not contaminated by other antibodies. The term "monoclonal" should not be construed as requiring the antibody to be produced by any specific method. The term monoclonal antibody specifically includes chimeric antibodies, humanized antibodies, and human antibodies. In this application, the term "neutralizing antibody" refers to an antibody with neutralizing activity. The term "neutralizing activity" refers to an immunoglobulin with antiviral activity that can specifically recognize viral self-antigens and effectively bind to and neutralize viral activity, preventing viral invasion of target cells and blocking viral replication in target cells to play an important role in antiviral activity.

[0036] In this application, the binding affinity between the antibody and the antigen can be determined by methods commonly used in the art, such as ELISA, typically using ECG. 50 and IC 50 EC 50 The half-maximal effective concentration (IC50) is the antibody concentration that achieves 50% of the maximum biological effect. 50 The half-maximal inhibitory concentration (MCI) is the concentration of antibody required to inhibit a specific biological process (such as the binding reaction of the F protein of antigen hMPV to the antibody) by half. 50 and IC 50 The smaller the value, the stronger the binding ability of the antibody to the antigen.

[0037] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein is inserted, thereby enabling the protein to be expressed. Vectors can transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cell. Examples of vectors include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site. Vectors may also include components that facilitate their entry into the cell, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0038] In this application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to a patient, which may contain the antibodies, nucleic acid molecules, carriers or cells described in this application, and may also contain one or more pharmaceutically acceptable excipients, such as: carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.

[0039] As described in the background section, the technical problem to be solved by the present invention is to provide an antibody with highly efficient neutralizing activity against human metapneumovirus. To solve this technical problem, the present invention uses phage display technology to screen and obtain a fully humanized monoclonal antibody against human metapneumovirus, RMFP2E03.

[0040] The fusion protein F and adhesion protein G of human metapneumovirus (HMV) are crucial for viral invasion of the human body. Because the G protein is highly prone to mutation, while the F protein is relatively conserved, it has become a good target for scientists developing neutralizing antibodies against HMV. The F glycoprotein exists in two forms: the pre-fusion conformation (Pre-F) and the post-fusion conformation (post-F). Previous studies have shown that targeting specific epitopes in the Pre-F conformation is more effective in neutralizing HMV, making Pre-F the preferred antigen for HMV vaccines. The antibody RMFP2E03 screened in this invention is a neutralizing antibody with a strong binding affinity to the Pre-F glycoprotein.

[0041] In addition, the antibody RMFP2E03 obtained by screening in this invention is a fully humanized antibody. Humanized antibodies can eliminate the immunogenicity of non-human antibodies in the human body, while retaining affinity to the greatest extent possible.

[0042] Examples 3-6 of the present invention demonstrate that: RMFP2E03 exhibits good broad-spectrum binding ability to hMPV A / B strain F protein.

[0043] RMFP2E03 exhibits highly effective neutralizing activity against human metapneumovirus (hMPV). Its IC50 against hMPV A2 strain... 50 The concentration was 33.66 ng / mL; the IC50 against hMPV B1 strain was... 50 It was 20.85 ng / mL.

[0044] RMFP2E03 competes with antibodies DS7 and 101F, indicating that antibody RMFP2E03 binds to site I and site IV epitopes.

[0045] This demonstrates that the antibody possesses a unique CDR region, enabling it to specifically bind to pre-F and post-F glycoproteins and effectively neutralize human metapneumovirus.

[0046] In summary, the antibody RMFP2E03 obtained by screening in this invention is a fully humanized monoclonal antibody with highly efficient neutralizing activity.

[0047] 1. RMFP2E03 antibody The RMFP2E03 antibody contains both heavy chain variable regions and light chain variable regions.

[0048] 1.1 Heavy Chain Variable Region of RMFP2E03 Antibody The heavy chain variable region of the RMFP2E03 antibody contains three complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and four frame regions (HFR1, HFR2, HFR3, and HFR4), as shown below: HFR1: EVRLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO: 7) HCDR1: GASISSDDYY (SEQ ID NO: 1) HFR2: WTWIRQHPGKALEWIGY (SEQ ID NO: 8) HCDR2: IHYSGNT (SEQ ID NO: 2) HFR3: YHNPSLKSRVTLSVGASKNQFSLSLSSVTAADTAVYFC (SEQ ID NO: 9) HCDR3: ARGPTGLMDV (SEQ ID NO: 3) HFR4: WGQGTMVTVSS (SEQ ID NO: 10) The amino acid sequence of the heavy chain variable region is as follows: EVRLQESGPGLVKPSQTLSLTCTVSGASISSDDYYWTWIRQHPGKALEWIGYIHYSGNTYHNPSLKSRVTLSVGASKNQFSLSLSSVTAADTAVYFCARGPTGGLMDVWGQGTMVTVSS (SEQ ID NO: 15) The nucleotide sequence encoding the heavy chain variable region is as follows: gaggtgcggctgcaggagtccggcccaggactggtgaagccttcacagacgctgtcactcacctgcactgtctctggtgcctccatcagcagtgatgattactactggacctggatccgccagcacccagggaaggccctggagtggattgggtacatccattatagtgggaacacct accacaacccgtccctcaagagtcgagttaccctatcagtgggcgcctctaagaaccagttctccctgagtctgagctctgtgacggccgcggacacggccgtgtatttctgtgcgagggggcctacgggtggcctcatggacgtctggggccaagggacaatggtcaccgtctcttca (SEQ ID NO: 17) 1.2 Light Chain Variable Region of RMFP2E03 Antibody The light chain variable region of the RMFP2E03 antibody contains three complementarity-determining regions (LCDR1, LCDR2, and LCDR3) and four frame regions (LFR1, LFR2, LFR3, and LFR4), as shown below: LFR1: AIRMTQSPSSSLSASVGDRVTITCRAS (SEQ ID NO: 11) LCDR1: QDISTW (SEQ ID NO: 4) LFR2: VNWYQYKVGKAPQLLIY (SEQ ID NO: 12) LCDR2: GAF (SEQ ID NO: 5) LFR3: YLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 13) LCDR3: QQAKSFPLT (SEQ ID NO: 6) LFR4: FGGGTKLEI (SEQ ID NO: 14) The amino acid sequence of the light chain variable region is as follows: AIRMTQSPSSSLSASVGDRVTITCRASQDISTWVNWYQYKVGKAPQLLIYGAFYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPLTFGGGTKLEI (SEQ ID NO: 16) The nucleotide sequence encoding the light chain variable region is as follows: gccatccggatgacccagtctccatcttccctgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcaggatattagcacctgggtaaactggtatcagtacaaagtagggaaagccccccagctcctgatctatggtgcattctat ttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgccacttactattgtcaacaggctaagagtttccctctcactttcggcggagggaccaagctggagatc (SEQ ID NO: 18) The present invention also relates to nucleic acid molecules comprising a nucleotide sequence encoding an antibody of the present invention. The nucleotide sequence encoding the antibody comprises: a nucleotide sequence encoding the heavy chain variable region, as shown in SEQ ID NO: 17; and a nucleotide sequence encoding the light chain variable region, as shown in SEQ ID NO: 18.

[0049] The present invention also provides a vector comprising one or more nucleic acid molecules of the present invention, such as a plasmid, phage, bacteriophage, or viral vector, wherein a nucleic acid molecule encoding an antibody of the present invention is inserted into the vector.

[0050] The antibodies described herein can be prepared by recombinantly expressing nucleotide sequences encoding light and heavy chains or portions thereof in cells, such as host cells. To express antibodies recombinantly, host cells can be transfected with one or more recombinant expression vectors carrying nucleotide sequences encoding light and / or heavy chains or portions thereof, so that the light and heavy chains are expressed in the host cells. Standard recombinant DNA methodologies used to prepare and / or obtain nucleic acids encoding heavy and light chains, incorporate these nucleic acids into recombinant expression vectors, and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds.), *Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989)*, Ausubel, FM et al. (eds.), *Current Protocols in Molecular Biology*, Greene Publishing Associates, (1989), and Boss et al. U.S. Patent No. 4,816,397.

[0051] Furthermore, the nucleotide sequences encoding the variable regions of the heavy and / or light chains can be converted into nucleotide sequences encoding, for example, full-length antibody chains, Fab fragments, or ScFv: for example, DNA fragments encoding the variable regions of the light or heavy chains can be operatively ligated (so that the amino acid sequences encoded by both DNA fragments are within the frame) to another DNA fragment encoding, for example, antibody constant regions or flexible linkers. Sequences of the human heavy and light chain constant regions are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIHP Publication No. 91-3242), and DNA fragments including these regions can be obtained by standard PCR amplification.

[0052] Therefore, embodiments of the present invention also include a host cell containing the carrier or nucleic acid molecule, wherein the host cell may be a higher eukaryotic host cell such as a mammalian cell, a lower eukaryotic host cell such as a yeast cell, or a prokaryotic cell such as a bacterial cell.

[0053] The antibodies of the present invention can be combined with at least one other reagent (e.g., a stabilizing compound) to prepare a pharmaceutical composition comprising the antibodies of the present invention and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0054] The antibody provided by this invention can be used to prepare drugs for the prevention and / or treatment of human metapneumovirus (HMV), and features full humanization, high expression, and good stability, making it suitable for industrialization. Furthermore, this antibody can also be used to prepare HMV detection reagents for detecting viral antigens and for identifying effective neutralizing antigenic epitopes.

[0055] The technical solution provided by the present invention will be further described below with reference to specific embodiments. The following embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention.

[0056] Example 1: Screening of fully human monoclonal antibodies against hMPV 1.1 Establishment of a phage display antibody library (1) Resuscitation of peripheral blood mononuclear cells (PBMCs) from recovered patients with acute respiratory infection: After the frozen cells were taken out, they were placed in a water bath at 37°C. After thawing, they were transferred to centrifuge tubes containing 1640 medium and centrifuged at 300 g for 5 min. The supernatant was discarded, and 500 μL of Trizol reagent was added. After thorough mixing, the mixture was added to Eppendorf tubes and allowed to stand at room temperature for 5 min.

[0057] (2) Add 200 μL of chloroform to each tube, mix thoroughly for 15 s, let stand at room temperature for 3 min, and centrifuge at 12000g for 15 min at 4℃.

[0058] (3) Take the upper aqueous phase into a new Eppendorf tube, add an equal volume of isopropanol, mix thoroughly, let stand at room temperature for 10 min, centrifuge at 12000 g for 10 min at 4℃, and discard the supernatant.

[0059] (4) Add 1 mL of 75% ethanol, mix by inverting for 10 s, centrifuge at 12000 g for 5 min at 4℃, discard the supernatant, and dry at room temperature.

[0060] (5) Add 20 μL of sterile, enzyme-free water to each Eppendorf tube to dissolve the precipitate.

[0061] (6) Measure RNA concentration.

[0062] (7) The extracted RNA was used to construct a phage display antibody library by Chengdu Renyu Biotechnology Co., Ltd.

[0063] 1.2 Screening of hMPV neutralizing antibodies First round of screening (1) Antigen coating: 1.5 μg each of the pre-fusion F glycoprotein (Sinochem 40993-V08H1) and post-fusion F glycoprotein (Sinochem 40993-V08H3) of hMPV A2 strain were mixed and diluted with antigen coating solution (an aqueous solution containing 1.59 g / L Na2CO3 and 2.94 g / L NaHCO3) to a protein concentration of 10 μg / mL. The solution was added to a 96-well ELISA plate (Corning, catalog number 9018) at 100 μL / well and coated overnight at 4°C.

[0064] (2) Blocking: On the second day, discard the coating solution in the ELISA plate, pat dry on paper, wash once with PBS solution (phosphate buffer), then add 350 μL of 3% BSA (bovine serum albumin) / PBS blocking solution and block at 37℃ for 2 h.

[0065] (3) Background subtraction: Take 200 μL of antibody library (titer 2×10⁻⁶) 11 Mix 800 μL of 3% BSA / PBS blocking buffer (pfu / mL) and incubate at room temperature for 1 h.

[0066] (4) Binding: Discard the blocking solution, pat dry on paper, wash 3 times with PBS solution, add the antibody library with background removed to the antigen-coated ELISA plate, 100 μL / well, and incubate at 37℃ with slow shaking at 100 rpm for 1 h.

[0067] (5) Washing: Wash once with PBS solution, then wash 10 times with 0.1% PBST solution (phosphate buffer containing 0.1% Tween-20), 1 min each time.

[0068] (6) Elution: Add 100 μL of elution buffer (glycine solution at pH 3.0) to each well and incubate at room temperature with slow shaking at 100 rpm for 10 min. Use a pipette tip to remove the elution buffer and add 10 μL of neutralization buffer (Tris-HCl at pH 8.0). Mix well and then measure the titer.

[0069] (7) Titer determination: The eluted antibody library was serially diluted 10-fold (10... -1 ~10 -8 Take 100 μL of 10 -4 ~10 -8The diluted antibody was added to 200 μL of TG1 strain in the logarithmic growth phase, incubated at 37°C for 30 min, and then spread on 2YTAG medium (2YT solid medium containing 0.1% ampicillin (Amp), 0.1% glucose and 1.5% agar) plates and incubated overnight at 37°C. The colony count was calculated the next day.

[0070] (8) Add the obtained antibody library to 20 mL OD 600 In a TG1 bacterial culture with a concentration of 0.6-0.8, incubate at 37°C for 60 minutes.

[0071] (9) Add 4 μL of ampicillin and incubate at 37℃ and 180 rpm for 60 min to achieve a TG1 concentration of 1×10⁻⁶. 9 pfu.

[0072] (10) Add 2×10 to the culture 10 PBS solution of pfu M13KO7 helper phage (Chengdu Renyu Biotechnology Co., Ltd., catalog number A001) was incubated at 37°C for 30 min.

[0073] (11) Add 30 mL of 2YT medium and 6 μL of Amp, and incubate at 37℃ and 180 rpm for 60 min.

[0074] (12) Centrifuge at 5000 rpm for 10 min at room temperature, discard the supernatant, collect the bacterial pellet, add 50 mL of new 2YTAK medium (2YT medium containing 100 μg / mL Amp and 50 μg / mL kanamycin) to reselect the pellet, and incubate in a 200 mL Erlenmeyer flask at 37°C and 220 rpm for 16 h.

[0075] Second / Third Round Screening According to the screening scheme, with other conditions remaining unchanged, the second round of screening (washing 15 times with 0.3% PBST solution in step (5)) and the third round of screening (washing 20 times with 0.5% PBST solution in step (5)) were carried out by increasing the elution pressure in turn for screening and titration.

[0076] 1.3 Purification of phage antibody library (1) After three rounds of screening, the culture cultured for 16 h was transferred to a 100 mL centrifuge tube and centrifuged at 4℃ and 12000 rpm for 20 min. The supernatant was then transferred to a new centrifuge tube.

[0077] (2) Add 1 / 4 volume of phage precipitate (a solution containing 20% ​​PEG / 2.5 M NaCl) to the supernatant and incubate on ice for 4 h.

[0078] (3) Centrifuge at 4℃ and 12000 rpm for 20 min, discard the supernatant, and centrifuge briefly again to remove the supernatant.

[0079] (4) The precipitate was resuspended in 1 mL of PBS solution and centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was then transferred to a new centrifuge tube.

[0080] (5) Titer determination: The supernatant (antibody library) from (4) above is serially diluted 10-fold (10... -1 ~10 -10 Take 100 μL of 10 -6 ~10 -10 Antibodies of varying dilutions were added to 200 μL of TG1 strain in the logarithmic growth phase, incubated at 37°C for 30 min, spread on 2YTAG medium plates, and incubated overnight at 37°C. The colony count was calculated the next day.

[0081] 1.4 Preparation of monoclonal phage supernatant (1) After three rounds of phage antibody library screening, single colonies were picked from the phage antibody library plates that were eluted and purified in the last round of screening and inoculated into 96-well deep-well plates containing 300 μL of 2YTAG medium and cultured at 37°C and 700 rpm until the logarithmic growth phase.

[0082] (2) Take 100 μL of bacterial culture from each well into a sterile 96-well plate and store at 4 °C.

[0083] (3) Add M13KO7 helper phage diluted with 2YT medium (titer 2×10⁻⁶). 10 100 μL of pfu / mL (approximately 10 times the bacterial concentration) was incubated at 37°C for 30 min, followed by incubation at 37°C and 600 rpm for 30 min.

[0084] (4) Centrifuge the 96-well deep plate at 4000 rpm for 10 min, discard the supernatant, add 300 μL of 2YTAK medium to each well to resuspend the bacterial cells, and incubate overnight at 700 rpm and 37 ℃.

[0085] 1.5 Monoclonal phage ELISA (1) Coating target proteins: The pre-fusion conformation F glycoprotein of hMPV A2 strain (Essential Shenzhou 40993-V08H1) and the post-fusion conformation F glycoprotein (Essential Shenzhou 40993-V08H3) and the pre-fusion conformation F glycoprotein of hMPV B2 strain (Essential Shenzhou 41082-V08H) were coated in a 384-well ELISA plate (Corning, catalog number 3702) at 25 ng / 25 μL antigen coating solution per well and incubated overnight at 4°C.

[0086] (2) Washing: Discard the coating supernatant, pat dry, and wash 3 times with 0.3% PBST solution for 1 min each time.

[0087] (3) Blocking: Add 3% skim milk powder / PBS solution, 100 μL / well, and block at 37℃ for 1 h.

[0088] (4) Washing: Discard the blocking solution, pat dry, and wash 5 times with 0.3% PBST solution for 1 min each time.

[0089] (5) Phage supernatant binding: 50 μL of 1% skim milk powder / PBS solution and 50 μL of monoclonal phage supernatant obtained in 1.4 were mixed at a ratio of 1:1 and added to the target protein-coated ELISA plate. The plate was then incubated at 37°C with slow shaking at 100 rpm for 1 h.

[0090] (6) Washing: Discard the liquid, pat dry, and wash 5 times with 0.3% PBST solution for 1 min each time.

[0091] (7) Secondary antibody binding: Anti-M13-HRP (Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number 11973-MM05T-H) antibody was diluted 1:5000 in 1% skim milk / PBS solution, 100 μL / well was added to the ELISA plate and incubated at 37℃ with slow shaking at 100 rpm for 1 h.

[0092] (8) Washing: Discard the liquid, pat dry, and wash 5 times with 0.3% PBST solution for 1 min each time.

[0093] (9) Add 50 μL of TMB chromogenic solution, incubate at 37℃ for 10 min, add 25 μL of ELISA stop solution, and read the OD value using a microplate reader. 450 Numerical value.

[0094] (10) Take the positive clone bacterial culture stored at 4℃ in 1.4(2) and entrust the company to sequence multiple antibodies. According to ELISA OD 450Based on numerical values ​​and the non-repetitive amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the antibody BCR (B cell antigen receptor) heavy and light chains, an antibody RMFP2E03 with strong binding ability to hMPV F protein was finally screened.

[0095] Example 2: Antibody Expression and Purification 2.1 Cloning of the heavy and light chains of antibody BCR The BCR heavy chain and light chain variable region sequences of the antibody RMFP2E03, which were correctly sequenced, were cloned into the heavy chain expression vector IgGvec-Hb (Addgene, catalog number 80795) and the light chain expression vector IgGvec-L (Addgene, catalog number 183703) respectively, using conventional molecular cloning methods.

[0096] 2.2 Antibody Expression and Purification The concentration of Expi 293 cells was adjusted to 3 × 10⁻⁶. 6 / mL, the transfection reagent is prepared as follows: Prepare solution A: in 3mL of Gibco... TM Add 50 μg of recombinant antibody plasmid (heavy chain plasmid: light chain plasmid = 1:2) to Opti-MEM. For solution B: Add 180 μL of Expi Fectamine transfection reagent to 3 mL of Opti-MEM and vortex thoroughly to mix. Mix solutions A and B thoroughly and incubate at room temperature for 20 min. Add the mixture to 50 mL of Expi 293 cells and incubate at 37°C (8% CO2) for 5 days at 120 rpm.

[0097] After 5 days of culture, the cell supernatant was collected and the antibody was purified using Protein A affinity magnetic beads. The antibody concentration was measured using a NanoDrop2000 micro spectrophotometer, and the procedure was performed according to the manufacturer's instructions.

[0098] Example 3: Determination of antibody binding activity against hMPV A / B F protein in different conformations (1) Dilute the pre-fusion conformation F glycoprotein of hMPV A2 strain (Essence 40993-V08H1) and the post-fusion conformation F glycoprotein (Essence 40993-V08H3) and the pre-fusion conformation F glycoprotein of hMPV B2 strain (Essence 41082-V08H) with antigen coating buffer, respectively, and add them to a 384-well ELISA plate, 25 ng / 25 μL / well, and coat overnight at 4℃.

[0099] (2) The next day, discard the supernatant, add 100 μL of 3% BSA / PBS blocking solution to each well, incubate at 37 ℃ for 2 h, discard the blocking solution, and wash once with PBST solution (0.3% Tween-20).

[0100] (3) The prepared test antibody was diluted with PBS solution containing 0.3% BSA to a concentration of 10 μg / mL, and serially diluted 3-fold with PBS solution. A total of 8 gradients were prepared and added to the blocked ELISA plate, 25 μL per well, and incubated in a 37 ℃ incubator for 1 h.

[0101] (4) Discard the supernatant solution, wash 5 times with PBST solution (0.3% Tween-20), and pat dry on absorbent paper.

[0102] (5) Add 25 μL of anti-human IgG-Fc-HRP antibody (Sigma-Aldrich, catalog number A1070) diluted 1:10000 with 0.3% BSA / PBS solution to each well and incubate at 37 °C for 1 h.

[0103] (6) Discard the supernatant solution, wash 3 times with PBST solution (0.3% Tween-20), and pat dry on absorbent paper.

[0104] (7) Add 50 μL of TMB chromogenic solution (Solepro, PR1210) to each well, incubate at room temperature for 10 min, and then add 25 μL of ELISA stop solution (Solepro, C1058) to each well. Read the absorbance (OD) at 450 nm using a microplate reader. 450 (as shown in Table 1 and) Figure 2 (As shown).

[0105] (8) Obtain the OD 450 The values ​​were fitted using a four-parameter Logistic model in GraphPad Prism 11.0 software via nonlinear regression, and EC was calculated. 50 value.

[0106] Table 1. Antibody efficacy against different conformations of hMPV FA / B isoform F protein 50 Based on the results (Table 1 and...) Figure 2 As can be seen, the antibody of the present invention has a good ability to bind to the F protein of hMPV A / B strains.

[0107] Example 4: Binding kinetics of antibody Fab fragment to hMPV A / B F protein in different conformations 4.1 Preparation and purification of antibody Fab fragments (1) Preparation of cutting conditions: Prepare Fab antibody cutting buffer: Premixed solution: 1.418 g disodium hydrogen phosphate + 400 mL ddH2O + 10 mL 0.5 M EDTA, fully dissolved, and adjusted to 500 mL; When using: 35 mg L-cysteine ​​hydrochloride + 10 mL premixed solution, fully dissolved, and temporarily stored at 4℃. Add 65 μL of immobilized papain (coupled to 4% agarose microspheres) (Thermo Fisher Scientific, catalog number 20341) to a centrifuge column equipped with a 0.45 μm sieve plate, and centrifuge at 1000 g for 2 min at room temperature to remove the storage solution. Then add 500 μL of pre-cooled Fab antibody cutting buffer, centrifuge at 1000 g for 2 min at room temperature, and repeat the washing twice. Next, the complete IgG antibody solution prepared in Example 2 was transferred to a 50 kDa ultrafiltration tube and centrifuged at 12000 g for 10 min at 4 °C. The antibody preservation solution was replaced with Fab antibody cleavage buffer, and the antibody concentration was adjusted to 1 mg / mL.

[0108] (2) Antibody cutting: Install the plunger of the centrifuge column, mix 300 μg of IgG antibody with pretreated papain resin, add Fab antibody cutting buffer to a final volume of 250 μL, and cut by rotation at 37°C for 5 h.

[0109] (3) Recovery and identification of Fab antibodies: After cleavage, remove the plunger and centrifuge at 1000 g for 2 min at room temperature to collect the flow-through. Add 125 μL PBS to the centrifuge column and centrifuge at 1000 g for 2 min at room temperature to collect and combine the flow-through. Add 400 μL Protein A agarose beads to the combined flow-through and incubate by rotation at room temperature for 15 min, then centrifuge at 1000 g for 2 min and retain the flow-through. Wash the magnetic beads twice with 200 μL PBS and combine the flow-through. Ultrafilter the combined flow-through using a 10 kDa ultrafiltration tube and replace the Fab antibody cleavage solution with PBS. The purity of the obtained antibody Fab fragment is identified by SDS-PAGE.

[0110] 4.2 Antibody Fab Segment Biomembrane Interference Technology To detect the binding of hMPV F protein to the RMFP2E03 Fab fragment, the Octet BLI molecular interaction analysis system and the HIS1K (Anti-Penta-HIS) probe (Sartorius, catalog number 18-5122) were used. First, 200 μL of hMPV F protein at a concentration of 5 μg / mL (Octet® sample diluent, Sartorius, catalog number 18-1104) was immobilized on the sensor surface per well. The Fab fragment molecule was serially diluted using Octet® sample diluent to final concentrations of 100 nM, 40 nM, 16 nM, 6.4 nM, 2.56 nM, and 1.02 nM, and 200 μL of diluent was transferred to the sample plate.

[0111] Detection process: Baseline 1 (60 seconds) → Sample loading (60 seconds) → Baseline 2 (60 seconds) → Binding (100 seconds) → Dissociation (300 seconds) → Regeneration (5 seconds) → Neutralization (5 seconds) → Regeneration (5 seconds) → Neutralization (5 seconds) → Regeneration (5 seconds) → Neutralization (5 seconds)

[0112] Table 2. Binding kinetics of antibody Fab fragment to hMPV FA / B F protein in different conformations The results are shown in Table 2 and... Figure 3 As shown, the dissociation constant K of the antibody for three different conformations of F protein is... D All reached 10 -11 The M-level indicates that the antibody has a very high affinity for the three different conformations of F protein. Its binding constant Ka and dissociation rate constant Kdis also indicate that the antibody has the characteristics of strong binding and slow dissociation.

[0113] Example 5: Determination of the neutralizing activity of the antibody against hMPV A2 / B1 (1) One day in advance, add 100 μL of LLC-MK2 cell (ATCC, CCL-7) culture medium (concentration of 2×10⁻⁶) to each well of a 96-well cell culture plate. 5 ( / mL), and cultured in a 37℃ 5% CO2 cell culture incubator for 16 h.

[0114] (2) Add 50 μL of serially diluted test antibody to each well of a 96-well plate, and then add 50 μL of hMPV virus A2 (CAN97-83 strain, ViraTree, number M121) or B1 (NL / 1 / 99 strain, Zhejiang Difulunsi Biotechnology Co., Ltd.), both containing recombinant GFP (Green Fluorescent Protein), 0.1 MOL per well. Mix thoroughly and incubate at 37℃ for 1 h.

[0115] (3) Discard the culture medium in the 96-well cell culture plate, add 100 μL of antibody-virus mixture to each well, and incubate in a 37℃ cell culture incubator for 2 h.

[0116] (4) After removing the supernatant, wash the cells with PBS and add 100 μL of Opti-MEM medium containing 2% fetal bovine serum (FBS). Incubate in a 5% CO2, 37°C incubator for 72 h.

[0117] (5) Discard the cell supernatant, wash the cells with PBS solution, add 4% paraformaldehyde, fix at room temperature for 30 minutes, discard the supernatant, wash the cells with PBS solution again, discard the supernatant, and then place them in a microplate fluorescent spot counter to read the data. The obtained data were analyzed by nonlinear regression using a four-parameter logistic model in GraphPad Prism 11.0 software to calculate the IC50 of the antibody against different hMPV strains. 50 .

[0118] Table 3. IC50 of antibody against hMPV FA / B isotype 50 Based on the results (Table 3 and...) Figure 4 It can be seen that RMFP2E03 has highly efficient neutralizing activity against human metapneumovirus. Its IC50 against hMPVA2 strain... 50 The concentration was 33.66 ng / mL, which was not significantly different from MPE8; the IC50 for hMPV B1 strain was... 50 The concentration was 20.85 ng / mL, which was significantly better than MPE8.

[0119] Example 6: Antibody Binding Epitope Analysis and Specific Recognition Study Based on Biomembrane Interference Technology To determine the specific antigen-binding epitope of RMFP2E03, the Octet BLI molecular interaction analysis system and HIS1K (Anti-Penta-HIS) probe (Sartorius, catalog number 18-5122) were used. Following the procedure in Example 4, hMPV F protein (5 μg / mL) was immobilized on the sensor surface. IgG antibodies against each target epitope were diluted to 100 nM with Octet® sample dilution buffer (Sartorius, catalog number 18-1104), 200 μL per well, and detection was performed according to the following procedure.

[0120] Detection process: Baseline 1 (60 seconds) → Sample loading (60 seconds) → Baseline 2 (60 seconds) → Binding (100 seconds) → Dissociation (100 seconds) → Regeneration (5 seconds) → Neutralization (5 seconds) → Regeneration (5 seconds) → Neutralization (5 seconds) → Regeneration (5 seconds) → Neutralization (5 seconds)

[0121] This study used reference antibodies representing different antigen-binding epitopes, including: ADI-61026 (site 0, Sinopharm 68253-H001A), DS7 (site I, Antibody Systems RVV22902), MPV467 (site II, Sinopharm 68273-H001A), MPE8 (site III, Sinopharm 68239-H001A), 101F (site IV, Bipsy RSF-M309a), and SAN27-14 (site V, Sinopharm 68250-H001A). These reference antibodies served as competitive antibodies, competing with RMFP2E03 for hMPV A2 pre-F, hMPV B2 pre-F, and hMPV A2 post-F binding assays, respectively.

[0122] The results show (e.g.) Figure 5 The antibody RMFP2E03 competes with antibodies DS7 and 101F, indicating that antibody RMFP2E03 binds to site I and site IV epitopes.

Claims

1. An antibody against human metapneumovirus, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:

6.

2. The antibody according to claim 1, wherein: The heavy chain variable region further comprises HFR1 with the amino acid sequence shown in SEQ ID NO: 7, HFR2 with the amino acid sequence shown in SEQ ID NO: 8, HFR3 with the amino acid sequence shown in SEQ ID NO: 9, and HFR4 with the amino acid sequence shown in SEQ ID NO: 10; and The light chain variable region further includes LFR1 with an amino acid sequence as shown in SEQ ID NO: 11, LFR2 with an amino acid sequence as shown in SEQ ID NO: 12, LFR3 with an amino acid sequence as shown in SEQ ID NO: 13, and LFR4 with an amino acid sequence as shown in SEQ ID NO:

14.

3. The antibody according to claim 1 or 2, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

4. The antibody according to any one of claims 1-3, wherein, The antibody is a fully humanized monoclonal antibody.

5. A nucleic acid molecule comprising a nucleotide sequence encoding an antibody according to any one of claims 1-4.

6. The nucleic acid molecule according to claim 5, wherein, The nucleotide sequence encoding the antibody comprises: a nucleotide sequence encoding the heavy chain variable region, as shown in SEQ ID NO: 17; and a nucleotide sequence encoding the light chain variable region, as shown in SEQ ID NO:

18.

7. A vector comprising the nucleic acid molecule of claim 5 or 6.

8. A cell comprising the nucleic acid molecule of claim 5 or 6 or the vector of claim 7.

9. A pharmaceutical composition comprising an antibody according to any one of claims 1-4, a nucleic acid molecule according to claim 5 or 6, a carrier according to claim 7 or a cell according to claim 8, and a pharmaceutically acceptable carrier.

10. Use of the antibody of any one of claims 1-4, the nucleic acid molecule of claim 5 or 6, the vector of claim 7, the cell of claim 8, or the pharmaceutical composition of claim 9 in the preparation of a drug for the prevention and / or treatment of human metapneumovirus or in the preparation of a human metapneumovirus detection reagent.

Images