Anti-respiratory syncytial virus single-domain antibody and use thereof

Abstract

The application discloses an anti-respiratory syncytial virus single-domain antibody and application thereof. The anti-respiratory syncytial virus single-domain antibody or an antigen binding fragment thereof of the application can specifically recognize a pre-F protein of a respiratory syncytial virus, can be well combined with a cell expressing an RSV F protein, can neutralize the respiratory syncytial virus, and has high neutralization activity to an A2 strain and a B9320 strain of the respiratory syncytial virus, can effectively prevent and control infection of the respiratory syncytial virus, provides a new thought for detection of the respiratory syncytial virus and prevention and treatment of diseases related to the respiratory syncytial virus infection, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of molecular immunology, specifically relating to a single-domain antibody against respiratory syncytial virus or its antigen-binding fragment. Background Technology

[0002] Respiratory syncytial virus (RSV) belongs to the genus Pneumovirus in the family Paramyxoviridae. It primarily causes lower respiratory tract infections such as bronchiolitis and pneumonia in infants under 6 months of age, and upper respiratory tract infections such as rhinitis and colds in older children and adults. RSV is highly contagious, causing millions of children to be hospitalized annually due to RSV infection, with the majority of severe cases occurring in infants under 6 months of age. Elderly individuals and high-risk groups with weakened immune systems are prone to developing cerebral obstructive pulmonary disease (COPD) and associated cardiopulmonary complications after RSV infection. RSV not only threatens human health but also places a significant economic burden on healthcare systems. Currently, clinical treatment for RSV is mainly symptomatic, with relatively few specific therapeutic drugs available. Therefore, the development of RSV vaccines or antibody drugs is urgent and crucial.

[0003] RSV is a single-stranded, negative-sense RNA virus. The RSV genome is approximately 15.6 kb in length and encodes 11 proteins, including 8 structural proteins (fusion protein F, adhesion protein G, and hydrophobic proteins SH, M, M2-1, N, P, and L) and 3 non-structural proteins (NS1, NS2, and M2-2). Fusion protein F and adhesion protein G are two major envelope glycoproteins. F is a typical type I glycoprotein, which, after being cleaved by cellular proteases into F1 and F2, possesses biological activity and enables the viral envelope to fuse with the host cell membrane to form multinucleated giant cells. G protein is a type II glycoprotein that binds to host cell membrane receptors, mediating viral entry into the host cell. Compared to G protein, F protein exhibits less variation and is relatively stable, making it an important target for drug development.

[0004] Studies have shown that the F protein, which plays a crucial role in viral attack on host cells, undergoes a conformational change, specifically a transition from the pre-F protein to the post-F protein. RSV first synthesizes an inactive F0 precursor in the host cell, which, after a series of processing modifications including furin protease, eventually becomes the mature, functional trimer pre-F. When the virus infects the cell, the F protein undergoes a conformational change, transforming from pre-F to post-F, enabling viral fusion with the host cell and thus entry into the host cell. The pre-F protein has been shown to induce most highly neutralizing antibodies after natural infection or immunization, making it a preferred antigen for vaccine development.

[0005] Camels and alpacas, among other camel species, can produce naturally occurring heavy-chain antibodies that lack the light chain. These molecules contain only a single heavy-chain variable region (VHH) and two conventional CH2 and CH3 regions, yet possess complete antigen-binding function and do not aggregate as easily as artificially engineered single-chain antibody fragments (scFv). Due to their unique structural properties, single-domain antibodies combine the advantages of traditional antibodies and small-molecule drugs, overcoming the drawbacks of traditional antibodies such as long development cycles, low stability, and stringent storage conditions. They possess advantages such as high affinity, strong tissue penetration, high stability, and simple structure, and have been widely used in biopharmaceutical research and development in recent years.

[0006] Therefore, the present invention provides a single-domain antibody against respiratory syncytial virus or its antigen-binding fragment, which can specifically recognize and bind to the pre-F protein, neutralize respiratory syncytial virus, and better prevent and treat respiratory syncytial virus infection. Summary of the Invention

[0007] The single-domain antibody against respiratory syncytial virus (RSV) or its antigen-binding fragment developed in this invention can specifically recognize the RSV pre-F protein and bind well to cells expressing RSV F protein to neutralize RSV. In particular, it has highly efficient neutralizing activity against RSV A2 and B9320 strains, and can effectively prevent and control RSV infection.

[0008] This invention provides a single-domain antibody against respiratory syncytial virus (RSV) or its antigen-binding fragment thereof, wherein the single-domain antibody or its antigen-binding fragment comprises CDR1, CDR2, and CDR3, wherein...

[0009] (a) The amino acid sequence of CDR1 is as shown in SEQ ID NO: 1, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same as the amino acid sequence shown in SEQ ID NO: 1, or has one or more (preferably two or three) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in SEQ ID NO: 1.

[0010] (b) The amino acid sequence of CDR2 is as shown in SEQ ID NO: 2, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence shown in SEQ ID NO: 2, or has one or more (preferably two or three) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in SEQ ID NO: 2; and

[0011] (c) The amino acid sequence of CDR3 is as shown in SEQ ID NO:3, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same as the amino acid sequence shown in SEQ ID NO:3, or has one or more (preferably two or three) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in SEQ ID NO:3.

[0012] In some embodiments, the single-domain antibody or its antigen-binding fragment has any of the amino acid sequences shown in SEQ ID NO: 4-6, or has at least 80%, 85%, 90%, 95% or more identity with any of the amino acid sequences shown in SEQ ID NO: 4-6, or has one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any of the amino acid sequences shown in SEQ ID NO: 4-6.

[0013] The present invention provides an immunoglobulin comprising any of the single-domain antibodies or antigen-binding fragments thereof described above and an immunoglobulin Fc region.

[0014] In some embodiments, the immunoglobulin Fc region is selected from IgG or IgA.

[0015] In some embodiments, the Fc region is selected from human IgG1, IgG2, IgG3 and / or IgG4, or an Fc region amino acid sequence that has one or more amino acid mutations (preferably substitutions, insertions or deletions) with human IgG1, IgG2, IgG3, IgG4.

[0016] In some embodiments, the Fc region further includes one or more mutations, namely, a methionine mutation to leucine, and / or an asparagine mutation to serine.

[0017] In some embodiments, the Fc mutation includes mutating methionine (M) at position 430 to leucine (L), i.e., (M430L), and / or mutating asparagine (N) at position 436 to serine (S), i.e., (N436S), based on the native Fc fragment (amino acid sequence as shown in SEQ ID NO: 7), wherein the amino acid sequence is numbered with reference to the EU index of Kabat et al. In some embodiments, the mutated Fc amino acid sequence is shown in SEQ ID NO: 15.

[0018] In some embodiments, the immunoglobulin includes a chimeric antibody or an antigen-binding fragment thereof, and / or a humanized antibody or an antigen-binding fragment thereof.

[0019] The present invention provides a nucleic acid molecule that encodes a single-domain antibody against respiratory syncytial virus as described in any of the preceding claims, or an antigen-binding fragment thereof, or an immunoglobulin as described in any of the preceding claims.

[0020] The present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecules.

[0021] The present invention provides a recombinant cell comprising the above-mentioned nucleic acid molecules and / or the above-mentioned recombinant vector, and capable of expressing the above-mentioned anti-respiratory syncytial virus single-domain antibody or its antigen-binding fragment or the above-mentioned immunoglobulin.

[0022] The present invention provides an antibody conjugate comprising a single-domain antibody against respiratory syncytial virus as described in any of the preceding claims or an antigen-binding fragment thereof, or an immunoglobulin as described in any of the preceding claims and a marker, wherein the marker is selected from one or more of enzyme labeling, biotin labeling, chemiluminescent dye labeling, and radioactive labeling.

[0023] The present invention provides a pharmaceutical composition comprising an anti-respiratory syncytial virus single-domain antibody or its antigen-binding fragment as described in any of the preceding claims, or an immunoglobulin as described in any of the preceding claims, and a pharmaceutically acceptable carrier.

[0024] The present invention provides a detection kit comprising an anti-respiratory syncytial virus single-domain antibody or its antigen-binding fragment as described in any of the above claims, or an immunoglobulin as described in any of the above claims, or comprising the above antibody conjugates, or comprising the above pharmaceutical compositions.

[0025] The present invention also provides the use of the anti-respiratory syncytial virus single-domain antibody or its antigen-binding fragment as described in any of the above claims, the immunoglobulin as described in any of the above claims, the antibody-drug conjugates or pharmaceutical compositions described above in the preparation of medicaments for the prevention and / or treatment of respiratory syncytial virus infection or in the preparation of diagnostic reagents for the detection of respiratory syncytial virus.

[0026] Abbreviations and Terminology Definitions

[0027] The following abbreviations are used in this article. CDR: Complementarity-determining region in the variable region of immunoglobulins; IgG: Immunoglobulin G; IgA: Immunoglobulin A.

[0028] The term "single-domain antibody" used in this article refers to a naturally occurring antibody lacking a light chain, found in the peripheral blood of camels. This antibody contains only a single heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but unlike artificially engineered single-chain antibody fragments (cFv), it does not easily adhere to each other or even aggregate into clumps. More importantly, the VHH structure cloned and expressed individually possesses structural stability and antigen-binding activity comparable to the original heavy chain antibody, and is the smallest known unit capable of binding to target antigens. The VHH is 2.5 nm in size, 4 nm in length, and has a molecular weight of only 15 kDa, hence it is also called a nanobody (Nb).

[0029] The term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to bind to the antigen the antibody is bound to. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0030] The term "immunoglobulin" refers to a polypeptide or protein with single, dual, or multiple specificities, or with monovalent, divalent, or multivalent binding properties, including but not limited to: IgG, IgM, IgA, IgD, IgE, and all subclasses of immunoglobulins, such as the IgG subclasses IgG1, IgG2, IgG3, and IgG4 found or produced in animal cells, including human cells. "Heavy chain antibodies" formed by the fusion of a single-domain antibody (such as VHH) or its antigen-binding fragment with the Fc region of an immunoglobulin, as well as other antibodies or fragments with similar specific structures, also fall within the scope of "immunoglobulin" as defined in this application. In some embodiments, the immunoglobulin comprises a single-domain antibody or its antigen-binding fragment that specifically binds to human metapneumovirus as described above, and the Fc region of an immunoglobulin.

[0031] The term "amino acid" refers to twenty common, naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). In some embodiments, the term "amino acid" also includes non-natural amino acids. Any suitable non-natural amino acid may be used. In some embodiments, the non-natural amino acid contains a reactive moiety for conjugating the agent with MIAC.

[0032] Various methods / systems exist in this field for defining and describing CDRs. These systems and / or definitions have been developed and refined over many years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used, defining CDRs based on sequence variability; Chothia defines CDRs based on the position of structural loop regions; the IMGT system defines CDRs based on sequence variability and position within variable domain structures; AbM is defined using Oxford Molecular's AbM antibody modeling software and represents a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures and is similar to Chothia in several ways. In this invention, the numbering of amino acid positions (e.g., amino acid residues in the Fc region) and the target regions (e.g., CDRs) are performed using the Kabat system.

[0033] The term "EU index" refers to the residue numbering scheme of human IgG1 EU antibodies. Unless otherwise stated herein, references to residue numbering in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise stated herein, references to residue numbering in the constant domain of an antibody refer to residue numbering according to the EU numbering system (see, for example, U.S. Provisional Patent Application 60 / 640,323, Figure concerning EU numbering scheme).

[0034] The term "specific" means that one of the molecules involved in specific binding does not exhibit significant binding to any molecules other than one or more of its binding partner molecules. Furthermore, the term is also used when a domain containing an antibody-variable region is specific to a particular epitope among multiple epitopes in an antigen. When the epitope bound by the domain containing the antibody-variable region is contained in several different antigens, an antigen-binding molecule containing the domain containing the antibody-variable region can bind to various antigens having said epitope.

[0035] The term "chimeric antibody" is an antibody molecule (or its antigen-binding fragment) wherein (1) the constant region or a portion thereof is altered, replaced, or replaced such that the antigen-binding site (variable region) is linked to a constant region of a different or altered type, effector function, and / or kind, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug, etc.) that confers novel properties to the chimeric antibody; or (2) the variable region or a portion thereof is altered, replaced, or replaced with a variable region having a different or altered antigen specificity. For example, an antibody can be modified by replacing its constant region with a constant region derived from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its antigen-recognition specificity while exhibiting reduced antigenicity in the human body compared to the original antibody.

[0036] The term "humanized antibody" refers to a chimeric antibody containing amino acid residues derived from human antibody sequences. Humanized antibodies may contain some or all of the CDR or HVR from non-human animals or synthetic antibodies, while the frame region and constant region of the antibody contain amino acid residues derived from human antibody sequences. This overcomes the heterologous reactions induced by chimeric antibodies carrying a large number of heterologous protein components. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or from publicly available references. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reverse mutations can be performed on the variable region frame sequence of the human antibody to maintain activity.

[0037] The term "amino acid mutation" refers to a mutation or change in amino acids in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion, or substitution of one or more amino acids based on the original protein or polypeptide.

[0038] The term "identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a control polypeptide sequence after sequence alignment and, where necessary, nicking to obtain the maximum percentage sequence identity. Comparisons for determining percentage amino acid sequence identity can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, like BLAST software or the FASTA package.

[0039] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence.

[0040] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for delivering bound molecules. Carriers can be anti-adhesives, adhesives, coating agents, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, humectants, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0041] The terms "prefusion F-trimer protein" and "pre-F protein" are used interchangeably and all refer to the prefusion form of the respiratory syncytial virus fusion protein F.

[0042] The term "neutralizing activity" refers to immunoglobulins with antiviral activity that can specifically recognize viral antigens and effectively bind to and neutralize viral activity, preventing viral invasion of target cells and blocking viral replication in target cells, thus playing an important role in antiviral activity. Attached Figure Description

[0043] Figure 1 The neutralizing activity of multiple concentrations of antibody-B9320 strain live virus was demonstrated.

[0044] Figure 2 The binding activity of the humanized antibody to CHOK1 RSV F cells;

[0045] Figure 3 This study aimed to evaluate the efficacy of anti-RSV drugs based on a mouse infection model. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments. However, the scope of protection of the present invention is not limited to the following embodiments. It should also be understood that the terminology used in the embodiments of the present invention is for describing specific implementations and not for limiting the scope of protection of the present invention. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0047] Example 1: Animal Immunization

[0048] Recombinant RSV Prefusion F-trimer Protein (catalog number: DRA230) was used as the immunogen, mixed with Freund's adjuvant for immunization. The antigen and adjuvant were emulsified 1:1 to form a homogeneous mixture and stored at 4°C. After recording the camel's ear tag, the immunization experiment began. Injections were made on both sides near the lymph nodes in the camel's neck, with two injection points on each side. 0.4 mL of the mixed antigen was injected at each point. After immunization, the camel was observed for half an hour to confirm it was in good condition and showed no discomfort. Immunizations were performed on days 0, 21, 42, and 63. On day 28, 10 mL of blood was collected from the camel's neck vein; on days 49 and 70, 50 mL of blood was collected each time. A portion of the blood was taken for serum titer testing. Immunization was performed every two weeks for a total of seven immunizations. After the 6th and 7th immunizations, blood was collected 5-7 days later, with 25-30 mL of blood collected in three blood collection tubes each time. Blood samples were collected before the 4th, 5th, and 6th immunizations for immune evaluation. Blood was drawn from the jugular vein of the camel, 5 mL each time. The blood was centrifuged at 400 xg for 30 minutes on the same day after being pre-cooled to 25°C, and the supernatant serum was separated and stored. Lymphocytes were then separated by adding 3 mL of cell separation medium to a 15 mL centrifuge tube, followed by the slow addition of 3 mL of blood. Care was taken to prevent mixing between the blood and the separation medium. The centrifuge was then pre-cooled to room temperature and centrifuged at 400 g for 30 minutes. The blood separation was observed, and the cotton-like supernatant immune cells were carefully aspirated using a 200 µL pipette and transferred to a new 15 mL centrifuge tube. The supernatant serum was stored in a new centrifuge tube at -80°C. 10 mL of room-temperature PBS buffer was added to each tube, and the tubes were centrifuged at 400 g for 20 minutes at 25°C. The supernatant was removed, and 5 mL of room-temperature PBS buffer was added to each tube, and the tubes were centrifuged at 400 g for 20 minutes at 25°C. Cell counts were performed using a hemocytometer. Remove the supernatant, and use RNAiso Plus to lyse the isolated lymphocytes to obtain 10 7 / mL of solution, store at -80 ℃.

[0049] Example 2: Database Construction and Screening

[0050] PBMCs were isolated from blood collected after the seventh immunization using lymphocyte separation medium. Total RNA was extracted from the PBMCs, and the RNA was reverse-engineered into cDNA using random primers. The VHH fragment was specifically amplified, and two large E. coli libraries were constructed. Phage display technology was used, and solid-phase panning was performed with Recombinant RSV Prefusion F-trimer Protein (catalog number: DRA230). Phages bound to Recombinant RSV Prefusion F-trimer Protein on the solid-phase panning plate were recovered by infecting TG1 E. coli cells. The infected phages were amplified overnight and purified by precipitation with PEG6000 / NaCl. After three rounds of panning, single clones with good binding to Recombinant RSV Prefusion F-trimer Protein were screened by phage ELISA.

[0051] Example 3 Construction and expression of chimeric antibodies

[0052] Monoclonal antibodies that showed good binding to Recombinant RSV Prefusion F-trimer Protein (catalog number: DRA230) obtained from phage library screening were sequenced. The sequenced antibody fragments were then synthesized and constructed into a human IgG framework. Subsequently, molecular cloning technology was used to insert the antibody fragments into the PCDNA3.1 vector to construct a mammalian cell expression plasmid. The plasmid was then introduced into the host cell line CHO cells using liposome transfection. Fermentation supernatant was obtained using cell fed-batch. The fermentation supernatant was then purified through a series of steps including affinity chromatography and ion exchange chromatography to finally obtain the constructed antibody.

[0053] Example 4: Binding activity of chimeric antibody to CHOK1 RSV F cells

[0054] CHOK1 RSV F cells were added to 96-well U-shaped plates at approximately 6E4 cells / well. Chimeric antibodies were diluted to 2 µg / mL and added to each well at 50 µL / well. The plates were incubated at 4°C for 1 h. After washing twice with dilution buffer, secondary antibody (goat anti-mouse IgG, cat: HS221, 1:500 dilution) was added and the plates were incubated at 4°C for 0.5 h. After washing twice with dilution buffer, the cells were analyzed by flow cytometry. Most antibodies showed good binding activity to CHOK1 RSV F cells.

[0055] Table 1. Binding activity of chimeric antibodies to CHOK1 RSV F cells

[0056]

[0057]

[0058] Example 5: Neutralizing activity of single-concentration chimeric antibody against live A2 strain virus

[0059] Hep2 cells were seeded into 96-well cell culture plates one day in advance and cultured overnight in a cell culture incubator (37 ℃, 5% CO2). The cells were used the next day when the cell density reached 90%. The antibody dilution solution was PBS + 5% HIFBS. Each sample was diluted to twice the detection concentration according to the sample concentration (it needs to be mixed with the virus 1:1 during the detection process, and the final concentration will be diluted 2 times). The espiratory syncytial virus-A2-GFP was diluted to the appropriate concentration. The same volume of virus was added to the antibody at the above dilution concentration and mixed well (the final virus amount per well was 700 TCID50). At the same time, a cell control group (CC) and a virus control group (VC) were set up: the CC group was added with the corresponding volume of antibody dilution and virus dilution, and the VC group was added with the corresponding volume of antibody dilution and virus. The above neutralization system was placed in a cell culture incubator for 1 h. The cell supernatant of the 96-well plate prepared in advance was taken out and added to the neutralization system and placed in a cell culture incubator for 2 h. The neutralization system was aspirated and 100 μL of DMEM + 2% FBS + 1% double antibody medium was added and the culture was continued for 22-24 h before detection.

[0060] Results: The number of green fluorescent spots per cell well was determined using an AID fluorescence enzyme-linked immunospot analyzer; the virus inhibition rate (i.e., neutralization rate) was calculated using Microsoft Office.

[0061] Virus inhibition rate (%) = [1 - (number of fluorescent spots)] 实验组 -Number of fluorescent spots 细胞对照 ) / (number of fluorescent spots) 病毒对照 -Number of fluorescent spots 细胞对照 )]×100%

[0062] Table 2 Neutralizing activity of single-concentration chimeric antibodies against live A2 strain virus

[0063]

[0064]

[0065] Example 6: Neutralizing activity of chimeric antibodies against live A2 strain virus

[0066] Hep2 cells were seeded into 96-well cell culture plates one day in advance and cultured overnight in a cell culture incubator (37 ℃, 5% CO2). Cells were used the following day when the cell density reached 90%. Antibody dilution was performed using PBS + 5% HIFBS, with serially diluted antibody concentrations. Espiratory syncytial virus-A2-GFP was diluted to the appropriate concentration, and the same volume of virus was added to each diluted antibody concentration and mixed well (final virus amount per well: 700 TCID50). Cell control (CC) and virus control (VC) groups were set up: the CC group received the corresponding volumes of antibody and virus dilution, and the VC group received the corresponding volumes of antibody and virus. The neutralization system was incubated for 1 h. The cell supernatant from the prepared 96-well plates was removed and added to the neutralization system, and incubated for 2 h. The neutralization system was then aspirated, and 100 μL of DMEM + 2% FBS + 1% double antibody medium was added, followed by further incubation for 22–24 h before detection.

[0067] Results: The number of green fluorescent spots per cell well was determined using an AID fluorescence enzyme-linked immunospot analyzer; the virus inhibition rate (i.e., neutralization rate) was calculated using Microsoft Office.

[0068] Virus inhibition rate (%) = [1 - (number of fluorescent spots)] 实验组 -Number of fluorescent spots 细胞对照 ) / (number of fluorescent spots) 病毒对照 -Number of fluorescent spots 细胞对照 )]×100%

[0069] Table 3 Neutralizing activity of chimeric antibodies against live A2 strain virus

[0070]

[0071] Example 7 Neutralizing activity of multi-concentration chimeric antibodies against live B9320 strain virus

[0072] Hep2 cells were seeded into 96-well cell culture plates one day in advance and cultured overnight in a cell culture incubator (37 ℃, 5% CO2). Cells were used the following day when the cell density reached 90%. Antibody dilution was performed using PBS + 5% HIFBS, with serially diluted antibody concentrations. Respiratory syncytial virus-9320F-GFP was diluted to the appropriate concentration, and the same volume of virus was added to each diluted antibody concentration and mixed well (final virus amount per well: 700 TCID50). Cell control (CC) and virus control (VC) groups were set up: the CC group received the corresponding volumes of antibody and virus dilution, and the VC group received the corresponding volumes of antibody and virus. The neutralization system was incubated for 1 h. The cell supernatant from the prepared 96-well plates was removed and added to the neutralization system, with one replicate per sample and concentration. The plates were incubated for 2 h. The neutralization system was then aspirated, and 100 μL of DMEM + 2% FBS + 1% double antibody medium was added, followed by further incubation for 22–24 h before detection.

[0073] Results: The number of green fluorescent spots per cell well was determined using an AID fluorescence enzyme-linked immunospot analyzer; the virus inhibition rate (i.e., neutralization rate) was calculated using Microsoft Office.

[0074] Virus inhibition rate (%) = [1 - (number of fluorescent spots)] 实验组 -Number of fluorescent spots 细胞对照 ) / (number of fluorescent spots) 病毒对照 -Number of fluorescent spots 细胞对照 )]×100%

[0075] Table 4. Neutralizing activity of multi-concentration chimeric antibodies against live B9320 virus strain

[0076]

[0077] Example 8: Human-centered engineering design

[0078] The variable region of the chimeric antibody 69 was humanized, with the design principle being to avoid introducing protein modification sites such as glycosylation, deamidation, and isomerization, as well as integrin binding sites and cysteine ​​residues. Reversion mutations of key amino acids in the framework region should maintain the original physicochemical and biochemical activities. The specific methods are as follows:

[0079] The variable region of chimeric antibody 69 was aligned with the human Germline sequence using the IgBLAST tool, and the FR (fragmented finite element) was replaced with the human Germline sequence with the highest sequence similarity. Then, based on this humanization, several important amino acids affecting antibody affinity were reverse-mutated, i.e., mutated to the original camel-derived FR site. The humanization percentage was calculated as the similarity ratio between the designed sequence framework and the Germline sequence framework. The designed humanized sequence was aligned with the human Germline sequence, and sequences with a humanization percentage of over 90% were selected. The humanization results are summarized in Table 5.

[0080] Table 5. Humanization Variable Region Sequence

[0081]

[0082] Example 9 Construction and expression of humanized antibodies

[0083] The designed antibody sequence was genetically synthesized and constructed into a human IgG framework. Then, using molecular cloning technology, the antibody fragment was inserted into the PCDNA3.1 vector to construct a mammalian cell expression plasmid. The plasmid was then introduced into the host cell line CHO cells using liposome transfection. Fermentation supernatant was obtained using a cell fed-batch method. The fermentation supernatant was then purified by affinity chromatography to finally obtain the constructed humanized antibody. The Fc region sequences of humanized antibodies 1 to 3 are shown in SEQ ID NO: 7, and the variable region sequences of humanized antibodies 4 and 5 are shown in SEQ ID NO: 6. The Fc region of humanized antibodies 4 and 5 underwent amino acid mutation (the mutated Fc amino acid sequence is shown in SEQ ID NO: 15) to extend the half-life.

[0084] Table 6 Humanized Antibodies

[0085]

[0086] The expression levels in the supernatant were compared, and the purity of the purified antibody was determined by SEC-HPLC. The results are shown in Table 7.

[0087] Table 7. Expression level and purity of humanized antibodies

[0088]

[0089] Example 10 Binding activity of humanized antibody to CHOK1 RSV F cells

[0090] Approximately 6E4 CHOK1 RSV F cells were added to each well of a 96-well U-shaped plate. 50 μL of humanized antibody was added to each well, diluted to 2 μg / mL, and incubated at 4°C for 1 h. After washing twice with dilution buffer, secondary antibody (goat anti-mouse IgG, cat: HS221, 1:500 dilution) was added and incubated at 4°C for 0.5 h. Flow cytometry was used for analysis after washing twice with dilution buffer. Results are shown below. Figure 2 Humanized antibodies have excellent binding activity.

[0091] Example 11 Neutralizing activity of humanized antibodies against live viruses

[0092] The neutralizing activity of humanized antibodies against live RSV strains was detected. Hep2 cells were seeded in 96-well cell culture plates one day in advance and cultured overnight in a cell culture incubator (37℃, 5% CO2). Cells were used the following day when the cell density reached 90%. All antibodies were diluted to 20 μg / mL, and then further diluted 10-fold to 2 μg / mL as the starting concentration for antibody serial dilution. The antibody dilution buffer was 2% FBS DMEM (it needs to be mixed 1:1 with the virus during the assay, and the final antibody concentration will be diluted 2-fold). RSV-A2-GFP, RSV-Long-GFP, and RSV-9320-GFP viruses were diluted to 1500 TCID in maintenance medium. 50 50 μL of RSV-9393 virus was diluted to 100 TCID using maintenance medium. 50 50 μL of RSV-18537 virus was diluted to 100 TCID using maintenance medium. 50 / 50μL. Take an equal volume of diluted antibody and mix it with the virus suspension (the final viral load per well for RSV-A2-GFP, RSV-Long-GFP, and RSV-9320-GFP is 1500 TCID). 50 / 50μL, RSV-9393 viral load is 100 TCID 50 / 50μL, RSV-18537 viral load is 100 TCID 50 A positive control group was set up (50 μL / mL), with Nirsevimab as the positive control. The amino acid sequence of Nirsevimab consists of SEQ ID NO: 13 and SEQ ID NO: 14. Simultaneously, a cell control group (CC) and a virus control group (VC) were set up: the CC group was given the corresponding volume of antibody diluent and virus diluent (maintenance medium), and the VC group was given an equal volume of antibody diluent and virus suspension (RSV-A2-GFP, RSV-Long-GFP, RSV-9320-GFP, 1500 TCID50 viral load). 50 / hole, RSV-9393 viral load 100 TCID 50 / hole, RSV-18537 viral load is 100 TCID 50 The above neutralization system was incubated in a cell culture incubator (37℃, 5% CO2) for 1 h. The cell supernatant from the prepared 96-well plate was removed, and 50 μL of the neutralization system was added to each well. Two replicates were set up for each sample. The plates were then incubated in a cell culture incubator (37℃, 5% CO2) for 24 h before detection. The RSV-A2-GFP, RSV-Long-GFP, and RSV-9320-GFP virus neutralization systems were detected after 5 days of incubation. The number of green fluorescent spots per well was measured using an AID fluorescence enzyme-linked immunosorbent assay (ELISA) analyzer for the RSV-A2-GFP, RSV-Long-GFP, and RSV-9320-GFP virus neutralization systems. The neutralizing titer of the antibody was calculated by comparing the reduction of fluorescent spots between the experimental group and the virus control group (VC). The lesions in the RSV-18537 and RSV-9393 virus neutralization systems were observed using an inverted fluorescence microscope. The virus inhibition rate (i.e., neutralization rate) of the RSV-A2-GFP, RSV-Long-GFP, and RSV-9320-GFP virus neutralization systems was calculated using Microsoft Excel.

[0093] Virus inhibition rate (%) = [1 - (number of fluorescent spots in experimental group - number of fluorescent spots in cell control) / (number of fluorescent spots in virus control - number of fluorescent spots in cell control)] × 100%

[0094] RSV-18537 and RSV-9393 virus neutralization experiments were conducted. CPE at various antibody dilutions was observed and recorded using an inverted fluorescence microscope. NT was calculated using the Reed-Muench method. 50 .

[0095] Table 8. Neutralizing activity of humanized antibodies against live virus

[0096]

[0097] Table 9. Virus inhibition rate of humanized antibodies against RSV-A2-GFP strain

[0098]

[0099] Table 10. Virus inhibition rate of humanized antibodies against RSV-9320-GFP strain

[0100]

[0101] Table 11 Inhibition rate of humanized antibodies against RSV-Long-GFP strain

[0102]

[0103]

[0104] Table 12 Inhibition rate of humanized antibodies against RSV-9393 strain

[0105]

[0106] "-" indicates that the hole has no CPE and has a neutralizing effect; "+" indicates that the hole has CPE and has no neutralizing effect.

[0107] Table 13 Inhibition rate of humanized antibodies against RSV-18537 strain

[0108]

[0109] "-" indicates that the hole has no CPE and has a neutralizing effect; "+" indicates that the hole has CPE and has no neutralizing effect.

[0110] Example 12 Rat PK test of humanized antibodies

[0111] The experiment was divided into three groups, with each group using SD rats (one male and one female) for a rat pharmacokinetic (PK) test. The drug was administered via tail vein bolus injection at a dose of 1 MPk. Blood samples were collected from the jugular / orbital veins of the experimental animals, and the actual blood collection time was recorded. After collection, the blood samples were left at room temperature for half an hour before centrifugation (centrifuge pre-cooled at 4°C, centrifugation conditions: 4°C, 3000 rpm for 10 minutes). Serum was separated after centrifugation and transferred to labeled centrifuge tubes. Blood drug concentration was determined using ELISA.

[0112] 100 μL / well was coated with 1.0 mg / mL RSV(A) Pre F protein (source: Kaika Biotechnology) and incubated overnight at 4°C. Discard the coating solution, wash 300 μL / well with 1*PBST (0.05%) 4 times with a plate washer, block with 2% BSA (300 μL / well), and incubate at 37℃ for 1 h; discard the blocking solution, wash 300 μL / well with 1*PBST (0.05%) 4 times with a plate washer; dilute the sample with 2% BSA, add 100 μL / well to the plate, prepare a standard curve solution with 2% BSA and blank rat serum diluted accordingly, add 100 μL / well to the plate, and incubate at 37℃ for 1 h; discard the liquid, wash 300 μL / well with 1*PBST (0.05%) 4 times with a plate washer, dilute goat anti-human Fc-HRP at a ratio of 1:10000, add 100 μL / well to the plate, and incubate at 37℃ for 45 minutes. Discard the liquid. Add 300 μL of 1*PBST (0.05%) to each well and wash the plate 6 times with a plate washer. Pat the wells dry on clean paper. Add 100 μL of Solarbiotin to each well, wrap the plate with aluminum foil, and incubate at 37°C in the dark for 3 minutes. Stop the reaction by adding 100 μL of 1M hydrochloric acid to each well. Read the values ​​at 450 nm using a microplate reader and analyze the data. Use pharmacokinetic software to process the plasma drug concentration data using a non-compartmental model. Calculate the relevant pharmacokinetic parameters using the linear logarithmic trapezoidal method. The results show that humanized antibody 4 and humanized antibody 5 have longer half-lives than humanized antibody 3.

[0113] Table 14 Results of rat PK test of humanized antibodies

[0114]

[0115] Example 13 Mouse challenge test of humanized antibody

[0116] BALB / c mice were challenged with the virus via intranasal instillation on day 0 (virus: RSA A2; titer (lgTCID)). 50 ( / mL): 6.47), administered intramuscularly on day 1, and blood was collected from the eyes of mice on day 5. Lung tissue was then used for virus titer detection. A positive control group was set up, with Nirsevimab as the positive control. The amino acid sequence consists of SEQ ID NO: 13 and SEQ ID NO: 14. The specific experimental protocol is shown in Table 15.

[0117] Table 15 Test Protocol

[0118]

[0119] Challenge procedure: On Day 0, groups G1-G4 were challenged via nasal drops. Animals were anesthetized with 5% chloral hydrate (100 μL intraperitoneally) until the mice no longer exhibited pain response (no reaction to light touch on the mouse's paws). Using a 100 μL pipette, 50 μL of virus dilution was accurately drawn and slowly dripped into both nasal cavities. The mice were kept in a static, nose-up position for 3-5 minutes, and then carefully returned to their respective cages, avoiding vigorous movement.

[0120] Administration procedure: Groups G1 to G4 were administered the drug via intramuscular injection one day before the challenge. Before administration, each mouse was weighed and administered the drug via intramuscular injection according to its actual body weight.

[0121] Mice were euthanized on day 5 post-infection. They were placed on a sterile dissecting board in a biosafety cabinet, with their limbs immobilized in a supine position. The chest and abdomen were disinfected by spraying with 75% ethanol to prevent contamination. Using sterile scissors, the skin was incised along the midline of the abdomen and dissected laterally to fully expose the abdominal muscle layers. The lower end of the sternum was lifted with forceps, and the ribs were cut along both sides of the costal arch (avoiding damage to lung tissue) to create a "V"-shaped opening, exposing the heart and lungs. The trachea near the jaw was grasped with forceps and completely incised. The trachea was gently pulled upwards with forceps, and the ventral tissue connection was interrupted with scissors. The lungs were removed, and surface blood was quickly absorbed with sterile filter paper. They were weighed using an electronic balance (accurate to 0.1 mg). The left lung was collected and preserved in 1.5 mL of 4% paraformaldehyde, and the right lung was collected in 1 mL of 1×PBS. The lungs were homogenized at -20°C (65 Hz for 60 s), centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant was collected. Throughout the procedure, the sample was kept at a low temperature, and the tissue weight and grinding fluid volume were recorded for subsequent concentration calculations. Results are shown below. Figure 3 Both humanized antibody 4 and humanized antibody 5 can significantly reduce viral load in lung tissue.

[0122] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A single-domain antibody against respiratory syncytial virus (RSV) or its antigen-binding fragment, characterized in that, The single-domain antibody or its antigen-binding fragment comprises CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2 and the amino acid sequence of CDR3 is shown in SEQ ID NO:

3.

2. The anti-respiratory syncytial virus single-domain antibody or its antigen-binding fragment according to claim 1, characterized in that, The single-domain antibody or its antigen-binding fragment has any one of the amino acid sequences shown in SEQ ID NO: 4-6.

3. An immunoglobulin, characterized in that, It comprises the single-domain antibody or its antigen-binding fragment as described in claim 1 or 2 and the Fc region of an immunoglobulin.

4. The immunoglobulin according to claim 3, characterized in that, The immunoglobulin Fc region is selected from IgG or IgA.

5. The immunoglobulin according to claim 4, characterized in that, The Fc region is selected from human IgG1, IgG2, IgG3 and / or IgG4, or an Fc region amino acid sequence that has one or more amino acid mutations with human IgG1, IgG2, IgG3, IgG4.

6. The immunoglobulin according to claim 5, characterized in that, The immunoglobulins include chimeric antibodies or their antigen-binding fragments, and / or humanized antibodies or their antigen-binding fragments.

7. A nucleic acid molecule encoding a single-domain antibody against respiratory syncytial virus as described in claim 1 or 2, or an antigen-binding fragment thereof, or an immunoglobulin as described in any one of claims 3-6.

8. A recombinant vector comprising the nucleic acid molecule of claim 7.

9. A recombinant cell comprising the nucleic acid molecule of claim 7 and / or the recombinant vector of claim 8, and capable of expressing the single-domain antibody against respiratory syncytial virus or its antigen-binding fragment or immunoglobulin.

10. An antibody conjugate, characterized in that, The product comprises a single-domain antibody against respiratory syncytial virus as described in claim 1 or 2, or an antigen-binding fragment thereof, or an immunoglobulin and a marker as described in any one of claims 3-6, wherein the marker is selected from one or more of enzyme labeling, biotin labeling, chemiluminescent dye labeling, and radiolabeling.

Images