CN118126165A - Antibodies and uses binding to respiratory syncytial virus F protein - Google Patents

Abstract

The application discloses an antibody binding to respiratory syncytial virus F protein and application thereof. In a first aspect of the application there is provided an antibody or antigen binding fragment thereof that binds to respiratory syncytial virus F protein, comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of a certain amino acid sequence, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of a certain amino acid sequence. The antibody or antigen binding fragment thereof according to the embodiments of the present application has at least the following beneficial effects: the antibody or the antigen binding fragment thereof provided by the embodiment of the application can be combined with preF and postF proteins with high affinity, so that the antibody or the antigen binding fragment thereof can be applied to detection of respiratory syncytial virus or respiratory syncytial virus F protein.

Description

Antibodies binding to respiratory syncytial virus F protein and their applications

Technical Field

The present application relates to the field of antibody technology, and in particular to antibodies binding to respiratory syncytial virus F protein and their applications.

Background technique

Respiratory syncytial virus (RSV) is a filamentous enveloped, negative-sense, single-stranded RNA virus belonging to the order Mononegavirales, family Pneumoviridae, genus Orthopneumovirus. It is mainly transmitted through aerosolized droplets and close contact. After replicating in the epithelial cells of the nasopharynx and upper respiratory tract for a short period of time, RSV infection may spread to the small bronchioles or alveoli of the lower respiratory tract. The host immune response to RSV infection increases mucus production and inflammation, leading to airway narrowing and is the most common cause of bronchiolitis. In addition, RSV is also one of the most important pathogens causing viral respiratory infections in infants, the elderly, and immunocompromised people.

The genome of this virus is 15.2kb in length, contains 10 genes, and encodes 11 proteins. RSV virus particles contain a lipid bilayer, expressing fusion protein (F), attachment protein (G) and small molecule hydrophobic protein (SH). Among them, F and G proteins are more abundant than SH protein. The F protein plays a key role in mediating the fusion of RSV virus and host cell membrane and in the pathogenic process, and the amino acid sequence homology of the extracellular region of the F protein of different RSV strains is as high as 95%, which is highly conservative. Therefore, it is necessary to provide specific antibodies against the F protein of RSV to meet the needs of diagnosing RSV.

Summary of the invention

The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes an antibody that binds to the F protein of respiratory syncytial virus and its application.

In a first aspect of the present application, an antibody or an antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus is provided, comprising a heavy chain variable region and a light chain variable region;

A1) the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO.10, HCDR2 as shown in SEQ ID NO.11 and HCDR3 as shown in SEQ ID NO.12, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO.18, LCDR2 as shown in SEQ ID NO.19 and LCDR3 as shown in SEQ ID NO.20; or,

A2) the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO.13, HCDR2 as shown in SEQ ID NO.11 and HCDR3 as shown in SEQ ID NO.14, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO.21, LCDR2 as shown in SEQ ID NO.22 and LCDR3 as shown in SEQ ID NO.23; or,

A3) The heavy chain variable region includes HCDR1 as shown in SEQ ID NO.15, HCDR2 as shown in SEQ ID NO.16, and HCDR3 as shown in SEQ ID NO.17, and the light chain variable region includes LCDR1 as shown in SEQ ID NO.24, LCDR2 as shown in SEQ ID NO.22, and LCDR3 as shown in SEQ ID NO.25.

The antibodies or antigen-binding fragments thereof according to the embodiments of the present application have at least the following beneficial effects:

The antibody or antigen-binding fragment thereof provided in the embodiments of the present application can bind to preF and postF proteins with high affinity, and thus can be applied to the detection of respiratory syncytial virus or respiratory syncytial virus F protein.

In some embodiments of the present application, A1) the heavy chain variable region has an amino acid sequence with at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO.2, and the light chain variable region has an amino acid sequence with at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO.3; or,

A2) the heavy chain variable region has an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO.4, and the light chain variable region has an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO.5; or

A3) The heavy chain variable region has an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO.6, and the light chain variable region has an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO.7.

In some embodiments of the present application, the antibody or antigen-binding fragment thereof is selected from at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, Fc, Fd, and a domain antibody.

In some embodiments of the present application, it also includes a heavy chain constant region and a light chain constant region, the heavy chain constant region has an amino acid sequence that has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.8; and/or the light chain constant region has an amino acid sequence that has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.9.

In some embodiments of the present application, the antibody or antigen-binding fragment thereof is any one of a human antibody, a humanized antibody and a chimeric antibody.

In a second aspect of the present application, a biomaterial is provided, the biomaterial comprising any one of B1) to B4);

B1) a nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment thereof;

B2) an expression cassette containing the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule B1) or the expression cassette B2);

B4) A recombinant cell containing B1) a nucleic acid molecule or B2) an expression cassette or B3) a recombinant vector.

The third aspect of the present application provides a pharmaceutical composition, comprising C1) and C2):

C1) an effective dose of the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned biological material;

C2) at least one of a pharmaceutically acceptable carrier, a diluent, a buffer and an excipient.

In a fourth aspect of the present application, a kit is provided, comprising the aforementioned antibody or antigen-binding fragment thereof.

The fifth aspect of the present application provides a use of the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned pharmaceutical composition, or the aforementioned kit in the preparation of a diagnostic, preventive or therapeutic product for respiratory syncytial virus.

In a sixth aspect of the present application, a method for detecting respiratory syncytial virus in a sample is provided, comprising contacting the sample with the aforementioned antibody or antigen-binding fragment thereof.

Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is the result of Example 1, (a) is the gel column chromatography and SDS-PAGE diagram of RSV pre-fusion conformation F protein, (b) is the gel column chromatography and SDS-PAGE diagram of RSV post-fusion conformation F protein. Wherein, the lane M of the SDS-PAGE diagram is Marker, the lane 1 is the +DTT reducing condition, and the lane 2 is the -DTT non-reducing condition.

Figure 2 is an SDS-PAGE image of RD5, RD6 and RD7 purified in Example 4. Wherein, lane _M1 is a marker of SDS-PAGE, lane 1 is RD5 antibody under +DTT reducing conditions, lane 2 is RD5 antibody under -DTT non-reducing conditions, lane 3 is RD6 antibody under +DTT reducing conditions, lane 4 is RD6 antibody under -DTT non-reducing conditions, lane 5 is RD7 antibody under +DTT reducing conditions, lane 6 is RD7 antibody under -DTT non-reducing conditions, and lane 7 is bovine serum albumin.

FIG3 is the result of Octet Red96 detection of the affinity of RD5, RD6, RD7 and preF/postF in Example 5.

FIG. 4 is the result of the antigen binding epitope analysis of RD5, RD6 and RD7 by Octet Red96 in Example 6.

Detailed ways

The following will clearly and completely describe the concept of the present application and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of them. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present application.

The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

In the description of this application, "several" means more than one, "more" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples" or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

The term "antibody" refers to an immunoglobulin that specifically binds to an antigen. Accordingly, "antigen" refers to a target antigen to which an antibody can selectively bind, and the specific types of target antigens include polypeptides, proteins, nucleic acids, lipids, carbohydrates, haptens or other natural or synthetic compounds. Specifically, in the present application, an antigen refers to respiratory syncytial virus F protein, and an immunoglobulin that specifically binds to respiratory syncytial virus F protein refers to an immunoglobulin that binds to respiratory syncytial virus F protein with a _KD of 1nM, 500pM, 200pM, 100pM, 50pM, 20pM, 10pM, 5pM, 2pM, 1pM or less. In addition, for antigens other than respiratory syncytial virus F protein, immunoglobulins as antibodies will not specifically bind to them, but this does not mean that antibodies cannot bind to other antigens, for example, immune cross-reactions are also known in the art.

Wherein, _KD is the equilibrium dissociation constant of antibody and antigen, which is the ratio of _Koff to _Kon , and the unit is recorded as molar concentration (M). _KD can be used to measure the binding affinity between antibody and its antigen, and the smaller the _KD value, the stronger the affinity. The _KD value of antibody can be carried out by any method known in the art, including but not limited to the method of surface plasmon resonance (SPR), for example, SPR chip or related analysis system such as Biacore T200 can be used.

The types of antibodies specifically include monoclonal antibodies, polyclonal antibodies, multispecific antibodies, multivalent antibodies, etc. In addition, antibodies may be modified in a variety of ways, such as PEGylation, glycosylation, formation of antibody molecule conjugates (such as ADC), addition of purification tags, or fusion with other proteins.

Taking a full-length antibody as an example, it contains two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds or a polymer formed thereof. According to the conservative differences in the amino acid sequences, the heavy chain and the light chain can be divided into a variable region (V) and a constant region (C). Among them, each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (composed of domains CH1, hinge, CH2 and CH3), and each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The variable region includes three complementary determining regions (CDRs) and is separated by a framework region (FR), which is arranged from the amino terminus to the carboxyl terminus as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, so VH contains three CDRs: HCDR1, HCDR2, HCDR3, and VL contains three CDRs: LCDR1, LCDR2 and LCDR3.

The term "complementarity determining region" refers to the high-frequency mutation area in the variable region. The CDRs of a heavy chain and a light chain interact with each other to form the antigen binding site of the full-length antibody, which is complementary to the three-dimensional structure of the antigen epitope. The high heterogeneity of CDR determines the diversity of antibodies, thereby recognizing different antigen epitopes.

Among them, the light chain of the antibody is divided into κ chain and λ chain. The heavy chain of the antibody can usually be divided into μ chain, δ chain, γ chain, α chain and ε chain, and antibodies are divided into five types: IgM, IgD, IgG, IgA and IgE. IgG and IgA can be further divided into different subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, whereby the individual antibodies constituting the antibody population are substantially identical and bind to the same antigenic epitope, except for trace amounts of antibody variants resulting from mutations that may be present. It is understood that monoclonal antibodies are monospecific or polyspecific. Specifically, monoclonal antibodies typically bind to one antigenic epitope, but may also have polyspecificity. Accordingly, a "polyclonal antibody" refers to a collection of different antibodies directed against the same antigen, such as a collection of different antibodies produced by B lymphocytes, including mixed antibodies directed against different antigenic epitopes.

The term "multispecific" refers to an antibody that can bind to two or more different antigenic epitopes of the same antigen, or can bind to two or more different antigens or a combination thereof. Multispecific antibodies may have cross-reactivity to other related antigens, for example, to the same antigen from other species (homologous), or may bind to an epitope shared between two or more different antigens.

The term "epitope" refers to the part of an antigen that can specifically bind to an antibody, usually composed of amino acids or chemically active groups on the side chains, such as polar or non-polar or hydrophobic groups, and optionally has at least one of a specific three-dimensional structural feature and a specific charge feature.

Depending on the origin of the antibody, the antibody can be a human antibody, a humanized antibody or a chimeric antibody.

The term "human antibody" refers to an antibody whose variable region is derived from a human immunoglobulin sequence and, if the antibody has a constant region, the constant region is also derived from a human immunoglobulin sequence, wherein the human immunoglobulin sequence may be a human germline sequence or a mutant form thereof, such as a mutant form produced by random mutation or site-directed mutation.

The term "humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species antibody (donor antibody) and at least one FR is derived from a human antibody (recipient antibody). Among them, non-human species include other mammals, such as animals of the order Carnivora, Perissodactyla, Artiodactyla, Rodentia, Lagomorpha, and other animals of the order Primates, specifically non-human species such as dogs, cats, pigs, sheep, cattle, horses, rabbits, mice, monkeys, orangutans, gorillas, chimpanzees, etc.

The term "chimeric antibody" refers to antibodies in which different parts are derived from different animal species, such as antibodies whose variable regions are derived from one species and whose constant regions are derived from another species, specifically, the variable regions are derived from mouse antibodies and the constant regions are derived from human antibodies or vice versa. Chimeric antibodies are mainly used to reduce immunogenicity in applications and to increase the yield during preparation, for example, where mouse antibodies have a higher hybridoma yield but higher human immunogenicity, so that human/mouse chimeric antibodies are used. It is understood that chimeric antibodies also include situations in which different parts of an antibody are derived from different animal individuals.

In the case of known antibody complementary determining region sequences, those skilled in the art can easily replace part of the antibody sequence (e.g., constant region or framework region) with a sequence from another species by techniques such as DNA recombination to form a chimeric antibody or a humanized antibody, and substantially retain the binding specificity of the source antibody. For example, when the source antibody is a mouse antibody, its constant region or framework region can be replaced with a human antibody constant region, or conversely, when the source antibody is a human antibody, its constant region or framework region can be replaced with a mouse antibody constant region or framework region to reduce the immunogenicity of the antibody when used in different species or to utilize its specific functions, such as ADCC-related activity.

In order to further reduce the immunogenicity of the antibody or for other purposes, other sequences in the antibody molecule except the CDR sequence can be replaced with the corresponding sequence of another antibody molecule (from the same or different species) (which may include several amino acid mutations as appropriate) while substantially retaining the binding specificity of the source antibody.

The term "antigen-binding fragment" refers to an antibody fragment in an immunoglobulin that retains the ability to specifically bind to the antigen bound by the full-length antibody, which can be a synthetic, enzymatically obtained or genetically engineered polypeptide. Antibody binding fragments include at least one or more CDR fragments, and specific fragment types include Fab, Fab', F(ab')2, Fv, scFv, Fc, Fd, domain antibodies, etc. Among them, the Fab fragment contains the variable region and CH1 of a heavy chain and a light chain, for example, it can be a papain cleavage product of an antibody. The Fab' fragment contains the variable region and CH1 of a heavy chain and the regional fragment between CH1 and CH2 and a light chain. The F(ab')2 fragment is composed of two Fab' fragments connected by a disulfide bond between the heavy chains, for example, it can be a pepsin cleavage product of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and the hydrophobic interaction of the CH3 domain. The Fv fragment is composed of a heavy chain variable region and a light chain variable region. scFv is composed of a heavy chain variable region and a light chain variable region and a linker connecting the heavy chain variable region and the light chain variable region. Through correct folding, the heavy chain variable region and the light chain variable region interact through non-covalent bonds to form an Fv segment, so that scFv can better retain its affinity activity for the antigen. Fc is composed of two heavy chain fragments including CH1 and CH2. Fd is composed of a heavy chain variable region and CH1. Domain antibodies are composed of heavy chain variable regions or light chain variable regions. In some cases, the heavy chain variable regions can be connected by a linker to form a bivalent domain antibody. The heavy chain variable regions of the bivalent domain antibodies can target the same or different antigens. Domain antibodies were first discovered in camels. They naturally lack light chains, but still have antigen binding ability. Compared with ordinary antibody molecules, domain antibodies have some advantages, such as better stability against pH, heat, and proteases, and can target and bind to antigen epitopes on antigen molecules that are difficult for ordinary antibody molecules to access.

Methods for preparing antibodies are known in the art, including but not limited to separating cells expressing specific antibodies after immunizing animals with antigens (e.g., preparing antibodies through hybridoma technology), screening antibody technology using phage antibody libraries, transforming cells through genetic engineering technology and making them express antibody molecules, etc. In one specific example, the embodiments herein provide a method for preparing antibody molecules by introducing recombinant expression vectors encoding antibody heavy chains and light chains into 293F cells.

The term "RSV F protein" refers to the type I integral membrane fusion protein of RSV, which plays a key role in the pathogenesis of RSV by mediating fusion between the virus and host cell membranes. The F protein has two different conformations, one is a lollipop-shaped prefusion conformation (preF), which exists on the virus surface before the virus interacts with the host cell; and the other is a crutch-shaped postfusion conformation (postF), which is usually obtained after the fusion of the virus and host cell membranes, but there is also spontaneous rearrangement from the highly metastable preF to the energy-preferred postF. The preF and postF conformations are different as antigens, although preF, which can induce most high- and medium-titer neutralizing antibodies, has become the first choice for vaccine development, but both are potential candidate antigens. Due to its high conservation, the RSV F protein can be an F protein derived from any one of RSV A subtype or B subtype, such as RSV A2, RSV A2cpts-530, RSV A2 cpts-530/1009, RSV Long, RSV18537, RSV rA2cp248/404, RSV 2-20, RSV 3-12, RSV 58-104, RSV 9320, RSVS B WV/14617/85, RSV L19, etc.

The term "sequence identity" refers to the amount of consistency between two amino acid sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Typically, before calculating the percentage of consistency between two amino acid sequences, a sequence alignment is performed and gaps are introduced if any. If the amino acid residues in the two sequences are the same at a certain alignment position, the two sequences are considered to be consistent or matched at that position; if the amino acid residues in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence consistency. In other algorithms, the number of gaps and/or the length of the gaps are also taken into account. The specific alignment method can use the publicly available alignment software BLAST (available on the webpage ncbi.nlm.nih.gov) to obtain the best sequence alignment and calculate the sequence consistency between the two amino acid sequences using the default settings.

The term "nucleic acid molecule" refers to at least one of DNA and RNA of genome, mRNA, cDNA or synthetic origin. It is understood that those skilled in the art can mutate or modify the nucleotide sequence of the nucleic acid molecule by known methods, such as directed evolution and point mutation, as long as the antibody or antigen-binding fragment thereof encoded by the mutated or modified nucleic acid molecule has the same function, they are all derived from the nucleotide sequence of the embodiment of the present application and belong to the nucleic acid molecule of the embodiment of the present application.

The term "expression cassette" refers to a construct containing the necessary regulatory elements for the expression of the contained nucleic acid molecule in a recombinant cell. Specific regulatory elements include promoters, polyadenylation sites, etc.

The term "recombinant vector" refers to a molecule containing all elements for replication, integration, amplification and/or expression of the nucleic acid molecules contained in the recombinant cell. All elements include, but are not limited to, promoters, polyadenylation sites, terminators, replication initiation sites, transcription initiation sequences, enhancers, selection elements, reporter genes, etc. For example, antibiotic resistance genes or selection marker genes (such as ampicillin resistance gene AmpR, thymidine kinase gene TK, etc.) for screening and target protein coding sequences can be inserted into the multiple cloning site (MCS). Optional recombinant vector types include plasmids, bacteriophages, lentiviruses, adenoviruses, adeno-associated viruses, etc.

The term "recombinant cell" refers to a cell in which a nucleic acid molecule is replicated, integrated, amplified and/or expressed. Recombinant cells include, but are not limited to, bacteria, fungi, insect cells, plant cells, and animal cells. Among them, bacteria, for example, can be Escherichia coli or Bacillus subtilis, fungi, for example, can be yeast or Aspergillus, insect cells, for example, can be S2 Drosophila cells or Sf9 cells, plant cells, for example, can be cells of Arabidopsis thaliana or tobacco, and animal cells, for example, can be mammalian cells, such as cells of mice, rats, pigs, sheep, monkeys, orangutans, or human beings, such as 293T cells, 293F cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, etc. It is understood that these recombinant cells can all be non-reproductive materials.

The first aspect of the embodiment of the present application relates to an antibody or an antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three CDRs, namely HCDR1, HCDR2 and HCDR3; and the light chain variable region comprises three CDRs, namely LCDR1, LCDR2 and LCDR3. Among them, HCDR1 to HCDR3 and LCDR1 to LCDR3 are respectively selected from the following groups:

A1) HCDR1 with the amino acid sequence shown in SEQ ID NO.10: GFSLNTRKMG;

HCDR2 with the amino acid sequence shown in SEQ ID NO.11: IDWDDDK;

HCDR3 having an amino acid sequence as shown in SEQ ID NO.12: GRTDYYDGNGYYLGYFDN;

LCDR1 having an amino acid sequence as shown in SEQ ID NO. 18: QSIATY;

LCDR2 having an amino acid sequence as shown in SEQ ID NO. 19: ATS;

LCDR3 having an amino acid sequence as shown in SEQ ID NO. 20: QQSYNNPWT;

A2) HCDR1 with the amino acid sequence shown in SEQ ID NO.13: GFSLNDRKMC;

HCDR2 with the amino acid sequence shown in SEQ ID NO.11: IDWDDDK;

HCDR3 having an amino acid sequence as shown in SEQ ID NO. 14: ARTEYYDRNGYYLGYLDY;

LCDR1 having an amino acid sequence as shown in SEQ ID NO.21: QIIASY;

LCDR2 having an amino acid sequence as shown in SEQ ID NO.22: AAS;

LCDR3 having an amino acid sequence as shown in SEQ ID NO. 23: QQSYSNPWT;

A3) HCDR1 with the amino acid sequence shown in SEQ ID NO.15: GFTVSGEY;

HCDR2 with the amino acid sequence shown in SEQ ID NO.16: IYSGGGT;

HCDR3 having an amino acid sequence as shown in SEQ ID NO. 17: ARNTPHCRSTSPLCYFYMDV;

LCDR1 having an amino acid sequence as shown in SEQ ID NO.24: QNIDNY;

LCDR2 having an amino acid sequence as shown in SEQ ID NO.22: AAS;

The amino acid sequence of LCDR3 is shown in SEQ ID NO.25: QRSYSTPPEGT.

Those skilled in the art can also understand that, based on the specific antibody sequences provided herein, a small number of amino acids can be replaced, deleted, added, and the binding ability or biological activity of the resulting products to the corresponding antigen (F protein) can be verified or screened, thereby obtaining the corresponding variants of the antibodies binding to the F protein provided in the embodiments of the present application, and these variants should also be included in the scope of the embodiments of the present application.

Therefore, in some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof corresponding to the CDR combination of A1) has an amino acid sequence with at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity to the amino acid sequence as shown in SEQ ID NO.2, and the light chain variable region has an amino acid sequence with at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity to the amino acid sequence as shown in SEQ ID NO.3; the heavy chain variable region of the antibody or antigen-binding fragment thereof corresponding to the CDR combination of A2) has an amino acid sequence with at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity to the amino acid sequence as shown in SEQ ID NO. The light chain variable region has an amino acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identical to the amino acid sequence shown in SEQ ID NO.4, and the light chain variable region has an amino acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identical to the amino acid sequence shown in SEQ ID NO.5; the heavy chain variable region of the antibody or antigen-binding fragment thereof corresponding to the CDR combination of A3) has an amino acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identical to the amino acid sequence shown in SEQ ID NO. The light chain variable region has an amino acid sequence that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity to the amino acid sequence shown in SEQ ID NO.6, and the light chain variable region has an amino acid sequence that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity to the amino acid sequence shown in SEQ ID NO.7.

In some embodiments, the antibody or antigen-binding fragment thereof is selected from at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, Fc, Fd, and a domain antibody. In some embodiments, the antibody or antigen-binding fragment thereof further comprises at least one of a heavy chain constant region and a light chain constant region. It is understood that the heavy chain constant region and the light chain constant region may be full-length or partial fragments thereof, such as CH1, CH2, etc. In some specific embodiments, the heavy chain constant region has an amino acid sequence having a sequence identity of at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) with the amino acid sequence shown in SEQ ID NO.8. In some specific embodiments, the light chain constant region has an amino acid sequence with at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity to the amino acid sequence shown in SEQ ID NO. 9. In some embodiments, the antibody or antigen-binding fragment thereof is any one of a human antibody, a humanized antibody, and a chimeric antibody.

In some specific embodiments, the antibody or antigen-binding fragment thereof further comprises a tag sequence at the N-terminus and/or C-terminus, including a tag sequence that facilitates dissolution and/or purification. Specifically, the tag sequence comprises at least one of a His tag, a GGGS sequence, a FLAG tag, a strep tag, a nus tag, a maltose binding protein, and a GST tag. It is understood that one or more tag sequences may be included; multiple tag sequences may include a combination of multiple identical tag sequences, or a combination of multiple different tag sequences.

The second aspect of the embodiments of the present application relates to a biomaterial, which may be a nucleic acid molecule encoding the aforementioned antibody or its antigen-binding fragment; an expression cassette containing the nucleic acid molecule; a recombinant vector containing the nucleic acid molecule or the expression cassette; a recombinant cell containing the nucleic acid molecule or the expression cassette or the recombinant vector.

The third aspect of the embodiment of the present application relates to a pharmaceutical composition, including an effective dose of an antibody or an antigen-binding fragment thereof or a biomaterial. In some embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier. Among them, the pharmaceutically acceptable carrier includes solid or liquid diluents, fillers, antioxidants, stabilizers and other substances that can be safely administered, which are suitable for human and/or animal administration without excessive adverse side effects, and are suitable for maintaining the vitality of the drug or active agent located therein. In some embodiments, the pharmaceutical composition also includes other substances that have preventive or therapeutic effects on respiratory syncytial virus or its related diseases or symptoms or complications. For example, after respiratory syncytial virus infection, the clinical manifestations are lower respiratory tract infection, cough, fever, runny nose, wheezing, dyspnea, sore throat, headache, muscle pain, fatigue and bacterial infection, so these other substances can be substances with antitussive, antipyretic, analgesic, antibacterial and other effects, or other antibodies to respiratory syncytial virus, such as nesvir monoclonal antibody and palivizumab. It is understood that the ingredients in the pharmaceutical composition can be administered to the subject simultaneously or sequentially. Among them, the effective dose refers to the amount of active compound (such as antibody) sufficient to cause the biological or medical response desired by the clinician in the subject. Specifically, the effective dose can be determined by those skilled in the art based on factors such as the route of administration, the subject's weight, age, and condition. For example, a typical daily dose range can be 0.01 mg to 100 mg of active ingredient per kg body weight, such as 0.01 mg, 0.02 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.5 mg, 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, and 100 mg.

A fourth aspect of the embodiments of the present application relates to a kit, which includes the aforementioned antibody or antigen-binding fragment thereof.

In some of the embodiments, the kit can be any one of a detection plate, a detection chip, a detection test strip, etc. for respiratory syncytial virus or respiratory syncytial virus F protein.

In some embodiments, the antibody or antigen-binding fragment thereof can be used to form a kit in the form of a solid (such as a lyophilized powder), a liquid (such as a solution), or directly coated on a substrate (such as a test paper or a chip).

In some embodiments, the kit detects respiratory syncytial virus or respiratory syncytial virus F protein by any one of immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, electrochemiluminescence, etc. Enzyme-linked immunosorbent assay includes but is not limited to any one of direct method, indirect method, sandwich method and competitive method. Immunochromatography includes but is not limited to any one of fluorescent microsphere immunochromatography, colloidal gold immunochromatography, latex microsphere immunochromatography, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography and quantum dot immunochromatography.

In some embodiments, the antibody or antigen-binding fragment thereof can be used as at least one of a coating antibody and a detection antibody in the kit.

In some embodiments, the antibody or antigen-binding fragment thereof is bound to a solid substrate such as magnetic beads, microspheres, microplates, nitrocellulose membranes, microfluidic chips, etc.

In some embodiments, a detectable marker is coupled to the antibody or its antigen-binding fragment, or a separate detectable marker that can be coupled to the antibody or its antigen-binding fragment is also included in the kit. Specifically, the detectable marker can be at least one of a radionuclide, a fluorescent substance, a chemiluminescent substance, a colored substance, an aggregation-induced luminescent substance, a micro-nanoparticle, an enzyme, and the like.

The fifth aspect of the embodiments of the present application relates to the use of an antibody or an antigen-binding fragment thereof, or a pharmaceutical composition, or a kit in the preparation of a diagnostic, preventive or therapeutic product for respiratory syncytial virus.

Specifically, the antibody or its antigen-binding fragment can be used to prepare a diagnostic, preventive or therapeutic product for respiratory syncytial virus, the pharmaceutical composition can be used to prepare a preventive or therapeutic product for respiratory syncytial virus, and the kit can be used to prepare a diagnostic product for respiratory syncytial virus.

The embodiments of the present application also relate to the use of an antibody or an antigen-binding fragment thereof, or a pharmaceutical composition, or a kit in the diagnosis, prevention or treatment of respiratory syncytial virus infection. Specifically, the antibody or its antigen-binding fragment can be used to diagnose, prevent or treat respiratory syncytial virus infection, the pharmaceutical composition can be used to prevent or treat respiratory syncytial virus infection, and the kit can be used to diagnose respiratory syncytial virus infection.

The embodiments of the present application also relate to a method for diagnosing, preventing or treating respiratory syncytial virus infection, comprising contacting an antibody or an antigen-binding fragment thereof, or a pharmaceutical composition, or a kit with a subject. Specifically, a method for diagnosing, preventing or treating respiratory syncytial virus infection comprises contacting an antibody or an antigen-binding fragment thereof with a subject; a method for preventing or treating respiratory syncytial virus infection comprises administering a pharmaceutical composition to a subject; a method for diagnosing respiratory syncytial virus infection comprises administering a kit to a subject.

A sixth aspect of the embodiments of the present application relates to a method for detecting respiratory syncytial virus in a sample, the method comprising contacting the sample with the aforementioned antibody or antigen-binding fragment thereof.

In some embodiments of the present application, the method for detecting respiratory syncytial virus in a sample is a method for non-disease diagnosis or treatment purposes, such as environmental virus monitoring and analysis, etc. Since the transmission routes of respiratory syncytial virus include respiratory droplets and close contact transmission, indirect contact transmission through virus-containing secretions or pollutants, and aerosol transmission under specific conditions, the virus can be monitored by testing environmental samples including water, air, aerosols, etc.

In some embodiments of the present application, the sample includes at least one of an environmental sample, a biological sample, etc. In some embodiments, the environmental sample includes but is not limited to at least one of water (e.g., domestic water, industrial water, medical water, agricultural water, and other types of sewage and wastewater, or rivers, ocean water, etc.), air, soil, compost, sludge (e.g., river sludge, wastewater sedimentation tank sludge, etc.), volcanic ash, frozen soil, and food (e.g., solid food, fluid food, beverage, etc.). In some embodiments, the biological sample includes but is not limited to at least one of body fluids (e.g., blood, tissue fluid, lymph, cerebrospinal fluid, urine, sweat, sputum, saliva, gastric juice, intestinal juice, pancreatic juice, bile, prostatic fluid, vaginal secretions, semen, serous cavity effusion, joint cavity effusion, bronchoalveolar lavage fluid, amniotic fluid, etc.), skin, feces, intestinal contents, and histological samples (e.g., samples obtained by surgery, endoscopy, or percutaneous puncture biopsy).

In the examples of the present application, the RSV pre-fusion conformation F protein DS-Cav1 (preF) was used as bait, and three human monoclonal antibodies were isolated from healthy adult peripheral blood lymphocytes (PBMCs) using single-cell sequencing technology, which were named RD5, RD6 and RD7. All three strains can bind to RSV F protein with high affinity, binding to both pre-fusion F protein and post-fusion F protein. These three monoclonal antibodies can be used for RSV diagnosis and antigen quantification. In addition, the three monoclonal antibodies bind to regions outside the φ, II, and IV epitopes, and can be used for the development of antibody conjugates targeting F protein.

The antibody or its antigen-binding fragment provided in the embodiment of the present application can specifically bind to the respiratory syncytial virus F protein (pre-fusion conformation and/or post-fusion conformation), that is, the respiratory syncytial virus F protein is used as the target antigen of the antibody. Due to the antigen binding specificity of the antibody provided in the embodiment of the present application, the antibody can be used to detect whether there is RSV virus in a sample (such as blood) from a subject. Accordingly, the embodiment of the present application provides an RSV detection kit that can include the antibody or its antigen-binding fragment provided in the present embodiment.

The present invention is further described below through specific examples.

Example 1: Expression and purification of RSV virus preF and postF

(1) The pre-fusion F protein preF encoding the RSV A2 strain was constructed with a Thrombin restriction site, 6 histidine sequences, 1 GS [glycine-serine] linker sequence, 1 sequence encoding a Strep II tag, and 1 stop codon at the C-terminus (the final amino acid sequence is shown in SEQ ID NO.1) and inserted into the EcoR I and XhoI restriction sites of the mammalian expression vector pCAGGS. The ligation product was transformed into DH5α Escherichia coli competent cells. Then, a single clone was picked and inoculated into 4 mL of LB medium and cultured for 6 to 8 hours; then it was inoculated into 300 mL of LB medium and cultured for 12 to 16 hours, the bacteria were collected, and the plasmid was extracted using an endotoxin-free plasmid extraction kit (TIANGEN) to obtain pCAGGS-RSV-preF.

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNTKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQ SYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLGSGYIPEAPRDGQAYVRKDGEWVLLSTFLLVPRGSHHHHHHGSWSHPQFGK (SEQ ID NO. 1).

(2) The post-fusion F protein postF encoding RSV A2 strain was constructed to have amino acids 1 to 513 (three mutations P102A, I379V and M447V were used to enhance expression, and the amino acid sequence of fusion peptide residues 137 to 146 was deleted), and the C-terminus was provided with an HRV 3C restriction site sequence, an 8×His tag and a Streptag II tag (the amino acid sequence is shown in SEQ ID NO. 26). The plasmid pCAGGS-RSV-postF was finally obtained according to the method in (1).

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKL MSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLASLEVLFQGPSSHHHHHHHH (SEQ ID NO. 26).

(3) After expressing the above two plasmids in suspension mammalian cells 293F for 5 to 7 days, the supernatant was collected and filtered with a 0.22 μm filter membrane. The protein was purified using an affinity column HisTrap HP (5 mL, GE healthcare) and a gel filtration column Hiload16/60Superdex 200 pg (GE healthcare). After elution from the His column, the elution peak containing the target protein was collected, concentrated, and subjected to molecular sieve chromatography. The size and purity of the target protein were determined based on the protein peak position and the results of SDS-PAGE.

The results are shown in Figure 1. The molecular sieve results show that the protein peak position is 10.9mL, which is consistent with the theoretical molecular weight of the F protein trimer of 180kDa. The SDS-PAGE results show that under reducing (+DTT, reducing) and non-reducing (-DTT, non-reducing) conditions, the F protein is around ~60kDa, suggesting that the F protein subunits form trimers through non-covalent bonding.

Example 2: Isolation of RSV preF protein-specific memory B cells

With the informed consent of the patients who were confirmed to be infected with RSV virus and discharged from the hospital after recovery, 3-10 mL of blood was collected to separate PBMCs. The separated PBMCs (10 ⁷ /mL) were incubated with 200 nM preF protein in Example 1 on ice for half an hour; then washed twice with PBS, and then mixed with the following antibodies (all purchased from BD): anti-human CD3/PE-Cy5, anti-humanCD16/PE-Cy5, anti-human CD235a/PE-Cy5, anti-human CD19/APC-Cy7, anti-human CD27/Pacific Blue, anti-human CD38/APC, anti-human IgG/FITC and anti-His/PE. After incubation on ice for half an hour, the cells were washed twice with PBS. Subsequently, PBMCs were sorted using FACSAria III to collect PE-Cy5-APC-APC-Cy7+Pacific Blue+FITC+PE+ cells (i.e., memory B cells) and directly collected into a 96-well plate at 1 cell/well.

Example 3: Single B cell PCR and cloning of human monoclonal antibody IgG1

The cells obtained in Example 2 were subjected to PCR in a 96-well plate, and 50U of Superscript III reverse transcriptase and a final concentration of 0.5μM human IgG, IgM, IgD, IgA1, IgA2, Igκ and Igλ constant region primers were added, and the mixture was reacted at 37°C for 1h. After cDNA amplification, two rounds of PCR reactions were performed in a 96-well PCR plate to obtain VH, Vκ and Vλ genes. After two rounds of PCR, the PCR products were recovered by 1.2% agarose gel electrophoresis and delivered to a sequencing company for reverse sequencing. The variable region sequences of the heavy and light chains were compared using the IGMT information database, and the variable region sequences of the heavy and light chains of the target antibody were obtained after pairing analysis.

The analysis results are as follows, and a total of three paired antibodies were obtained: RD5, RD6 and RD7. According to the sequencing results, the amino acid sequence of the heavy chain variable region of RD5 is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.3; the amino acid sequence of the heavy chain variable region of RD6 is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5; the amino acid sequence of the heavy chain variable region of RD7 is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7.

MDILCSTLLLLTVPSWVLSQVTLRESGPALVKPTQTLTLTCSFSGFSLNTRKMGVTWIR QSPGKALEWLARIDWDDDKFYSTSLKARLTISKDTSKNQVVLKMTNVDPADTATYFCGRTDYYDGNGYYLGYFDNWGQGILVTVSS (SEQ ID NO. 2);

MDMRVPAQLLGLLLLWLRGARCDIQLTQSPSSLSASVGDRVIITCRASQSIATYLNWYQ QKPGKAPNLLIYATSMSPGGVPLRFSGSGSGTDFTLTISSMQPEDFATYYCQQSYNNPWTLG QGTKVEMR (SEQ ID NO. 3);

MDILCSTLLLLTVPSWVLSQVTLRESGPALVKPTQTLTLTCSFSGFSLNDRKMCVSWLR QPPGKALEWLARIDWDDDKFYSTSLKTRLTISKDTSKNQVVLQMTNVDPVDTATYYCARTEYYDRNGYYLGYLDYWGQGILVTVSS (SEQ ID NO. 4);

MDMRVPAQLLGLLLLWLRGARCDIQMTQSPASLSASVGDRVTITCRSSQIIASYLNWYQQKPGKAPTLLIYAASTLQSGVPARFSGTGSGTDFTLTISGLQLEDFATYYCQQSYSNPWTLGQGTKVEIR (SEQ ID NO. 5);

QVTLRESGPALVKPTQTLTLTCSFSGFSLSSRGMCVSWIRQPPGKALEWLARIDWDDD KHYSTSLKTRLTISKDTSRNQVVLTMADVDPLDTATYYCARTALYDSDSFYLFYFDFWGQTGVTVSS (SEQ ID NO. 6);

MDMRVPAQLLGLLLLWLRGARCGIQMTQSPSSLSASVGDRVTITCRASQNIDNYLNW YQQQPGKAPRLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQRSYSTPPEGTFGQGTKVEIK (SEQ ID NO. 7).

Among them, the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 in the heavy chain variable region of RD5 are shown in SEQ ID NO.10, 11, 12, 18, 19 and 20, respectively; the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 in the heavy chain variable region of RD6 are shown in SEQ ID NO.13, 11, 14, 21, 22 and 23, respectively; the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 in the heavy chain variable region of RD7 are shown in SEQ ID NO.15, 16, 17, 24, 22 and 25, respectively.

The light chain sequencing results of the three antibodies were compared with the germline genes, and it was determined that RD5, RD6 and RD7 all used CLκ.

The correct variable region sequence was analyzed and connected with the corresponding constant regions of heavy chain CH and light chain CLκ by bridge PCR, and cloned into the expression vector pCAGGS to obtain a recombinant plasmid containing the light and heavy chain coding genes of the specific antibody. The amino acid sequence of the heavy chain constant region CH is shown in SEQ ID NO.8, and the amino acid sequence of the light chain constant region CLκ is shown in SEQ ID NO.9.

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO. 8);

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECS (SEQ ID NO. 9).

Example 4: Expression and purification of monoclonal antibodies

The pCAGGS plasmid containing the heavy chain and light chain coding sequences was co-transfected into 293F cells. 24 hours after transfection, a working concentration of 2.2 mM VPA was added and cultured for 5 days. The cells were harvested, the supernatant was collected by centrifugation, filtered with a 0.22 μm filter membrane, and passed through a HiTrap Protein AHP (5 ml, GE Healthcare) column at a speed of 2 mL/min.

According to the operating instructions of the affinity column, the initial elution purification of the antibody was completed. The antibody was further purified by gel filtration chromatography column (superdex200PG, GE Healthcare) to obtain high-purity antibodies. As shown in Figure 2, the bands of RD5, RD6, and RD7 under non-reducing conditions were displayed at 150 kDa, while the heavy chain of 55 kDa and the light chain of 25 kDa were obtained under reducing conditions. After purification by gel filtration chromatography column, it was finally concentrated and stored at -80°C for future use.

Example 5: Antibody Binding Performance Detection

The Octet Red96 system was used to detect the interaction between purified antibodies and pre-F/postF by biomembrane interferometry (BLI). The three antibodies were immobilized on the ProteinA probe at a concentration of 10 μg/ml, and then the real-time response values of the probes in preF or postF wells at different dilution concentrations (12.5-200 nM) were detected. All proteins used in the experiment were exchanged into a buffer consisting of 10 mM HEPES, pH 7.4, 150 mM NaCl and 0.005% v/v Tween-20 to measure the binding. The antibodies bound to the probe were regenerated with 10 mM glycine (pH 1.7). The binding constant ( _KD value) of each preF/postF interaction with the antibody was calculated using the instrument's own software, and the results are shown in Figure 3.

As can be seen from the figure, RD5, RD6, and RD7 can bind to both RSV pre-fusion F protein and RSV post-fusion F protein. The affinity value K _D of RD5 binding to preF and postF is <1pM; the affinity K _D of RD6 binding to preF is 527pM, and the affinity K _D of binding to postF is 22.5pM; the affinity K _D of RD7 binding to preF is 199pM, and the affinity K _D of binding to postF is <1pM. Therefore, these three monoclonal antibodies show extremely high affinity for both preF and postF.

Example 6: Antibody epitope competition experiment

The binding competition experiments between antibodies and preF and postF proteins were determined by real-time, label-free biolayer interferometry on Octet RED96 biosensors (PallForteBio). The entire experiment was performed in PBST buffer containing 0.005% Tween-20 at 30°C, and the 96-well plate was shaken at 1000 rpm. The Ni-NTA biosensor was first loaded with 20 μg/mL pre-F protein for 300 s and then bound to the first mAb for 900 s to obtain saturation. Then, the biosensor was immersed in the second mAb to be detected in the presence of the first antibody, and the irrelevant antibody Z30 was used as a negative control. All sensors were regenerated with 10 mM glycine HCl (pH 1.7) and re-saturated with 10 mM NiCl _2. The binding reaction was monitored in real time during the experiment and measured at the end of each step. The response of the two mAbs to preF and postF was compared, and the data were processed using BIA evaluation software. Palivizumab (PDB number: 2HWZ_H and 2HWZ_L) that binds to epitope II of the F protein, D25 (PDB number: 4JHW_H and 4JHW_L) that binds to epitope φ of the F protein, and RV11 (PDB number: 8WSQ_H and 8WSQ_L) that binds to epitope IV/V were used as positive control antibodies in the test.

The results are shown in Figure 4. There is no competitive binding between RD5, RD6 and RD7 and Palivizumab, D25 and RV11, respectively, which indicates that the antigenic epitopes bound by RD5, RD6 and RD7 may be in the region other than epitopes II, φ and IV/V. In addition, there is competitive binding between RD5, RD6 and RD7, which indicates that the three antibodies may bind to similar antigenic epitopes.

Example 7

This embodiment provides an RSV diagnostic kit, including RSV standards of different concentration gradients, a coating plate, RSV primary antibody, enzyme-labeled RSV secondary antibody, BSA blocking solution, color developer A solution, color developer B solution, stop solution, etc.

The preparation process of the pre-coated plate is as follows: the purified RSV primary antibody is diluted with a carbonate buffer solution of pH 9.6 to obtain a mixed solution with a concentration of 5 μg/mL, and the mixed solution is coated on a 96-well plate, 0.1 mL per well, incubated at 4°C for 21 hours, the liquid in the well is discarded, and the wells are washed and patted dry; then BSA blocking solution is added to each well, 0.1 mL per well, incubated at 37°C for 2 hours, the liquid in the well is discarded, and the wells are washed and patted dry to obtain a pre-coated plate.

The primary RSV antibody was a mouse anti-RSV monoclonal antibody.

The enzyme-labeled RSV secondary antibody was labeled with HRP by sodium periodate oxidation method, and the RSV secondary antibody was RD5.

The stop solution was 2M sulfuric acid.

The method of using the kit is as follows:

Add 75 μL of sample or RSV standard to each well, set up negative control and blank control, incubate at 37℃ for 1h, discard the liquid in the well, wash and pat dry. Then add 25 μL of enzyme-labeled RSV secondary antibody to each well, incubate at 37℃ for 1h, discard the liquid in the well, wash and pat dry. Then add 100 μL of color developer A solution and color developer B solution to each well, color at 37℃ for 10min, and add 50 μL of stop solution to each well. At a wavelength of 450nm, zero the blank well and read the OD value of each well.

The quantitative method is as follows: a standard curve is drawn according to the OD values of RSV A2 standards of different concentrations and the linear range is determined. The OD value of the sample is substituted into the standard curve to obtain the RSV concentration in the sample.

The qualitative method is as follows: twice the OD value of the negative control is set as the critical OD value. When the OD value of the sample is greater than the critical OD value, it is determined to be RSV antigen positive, otherwise it is negative.

Example 8

This embodiment provides a RSV diagnostic kit, which is different from Example 7 in that the RSV secondary antibody is RD6.

Example 9

This embodiment provides a RSV diagnostic kit, which is different from Example 7 in that the RSV secondary antibody is RD7.

In Examples 7 to 9, the detection limit LOD=3SD/b was calculated according to the standard curve, where SD is the standard deviation of the blank control and b is the slope of the standard curve. The results show that all three have a lower detection limit. At the same time, clinical samples identified as negative and positive for nucleic acid were tested to determine the false positive and false negative of the kit. In addition, the test was performed to determine whether there was a cross reaction to viral samples such as influenza A virus, influenza B virus, and parainfluenza virus. The results show that the RSV diagnostic kits of Examples 7 to 9 have good specificity and sensitivity.

Example 10

This embodiment provides a RSV colloidal gold test strip, comprising a sample pad, a colloidal gold pad, a nitrocellulose membrane, a water-absorbing paper, and a PVC substrate, which are overlapped in pairs. The sample pad is provided with a spotting hole.

Among them, the colloidal gold pad is coated with colloidal gold-labeled mouse anti-RSV monoclonal antibody, and the nitrocellulose membrane is provided with a quality control line (C line) and a detection line (T line). The quality control line is coated with sheep anti-mouse IgG antibody, and the detection line is coated with RD5 antibody.

During the test, add 200 μL of sample to the spotting well, react for 15 minutes and observe the results; if both C line and T line have bands, it is positive; if only C line has bands, it is negative; if there is no C line band, the result is invalid.

Embodiment 11

This embodiment provides a RSV colloidal gold test strip, which is different from Embodiment 10 in that the detection line is coated with RD6 antibody.

Example 12

This embodiment provides a RSV colloidal gold test strip, which is different from Embodiment 10 in that the detection line is coated with RD7 antibody.

The RSV colloidal gold test strips of Examples 10 to 12 were tested with RSV A2 standards of different concentrations, and the lowest concentration of the standard when the T line appeared was determined to be the detection sensitivity of the test strip. At the same time, clinical samples identified as negative and positive for nucleic acid were tested to determine the false positive and false negative of the test strip. In addition, samples such as influenza A virus, influenza B virus, and parainfluenza virus were tested for cross reactions. The results showed that the test strips of Examples 10 to 12 all had high sensitivity, specificity, and sensitivity.

The present application is described in detail above in conjunction with the embodiments, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims (10)

1. An antibody or antigen-binding fragment thereof that binds respiratory syncytial virus F protein, comprising a heavy chain variable region and a light chain variable region;

A1 The heavy chain variable region comprises HCDR1 with an amino acid sequence shown as SEQ ID NO.10, HCDR2 with an amino acid sequence shown as SEQ ID NO.11 and HCDR3 with an amino acid sequence shown as SEQ ID NO.12, and the light chain variable region comprises LCDR1 with an amino acid sequence shown as SEQ ID NO.18, LCDR2 with an amino acid sequence shown as SEQ ID NO.19 and LCDR3 with an amino acid sequence shown as SEQ ID NO. 20; or alternatively, the first and second heat exchangers may be,

A2 The heavy chain variable region comprises HCDR1 with an amino acid sequence shown as SEQ ID NO.13, HCDR2 with an amino acid sequence shown as SEQ ID NO.11 and HCDR3 with an amino acid sequence shown as SEQ ID NO.14, and the light chain variable region comprises LCDR1 with an amino acid sequence shown as SEQ ID NO.21, LCDR2 with an amino acid sequence shown as SEQ ID NO.22 and LCDR3 with an amino acid sequence shown as SEQ ID NO. 23; or alternatively, the first and second heat exchangers may be,

A3 The heavy chain variable region comprises HCDR1 with an amino acid sequence shown as SEQ ID NO.15, HCDR2 with an amino acid sequence shown as SEQ ID NO.16 and HCDR3 with an amino acid sequence shown as SEQ ID NO.17, and the light chain variable region comprises LCDR1 with an amino acid sequence shown as SEQ ID NO.24, LCDR2 with an amino acid sequence shown as SEQ ID NO.22 and LCDR3 with an amino acid sequence shown as SEQ ID NO. 25.

2. The antibody or antigen-binding fragment thereof according to claim 1,

A1 The heavy chain variable region has an amino acid sequence that is at least 85% sequence identical to the amino acid sequence shown as SEQ ID NO.2, and the light chain variable region has an amino acid sequence that is at least 85% sequence identical to the amino acid sequence shown as SEQ ID NO. 3; or alternatively, the first and second heat exchangers may be,

A2 The heavy chain variable region has an amino acid sequence that is at least 85% sequence identical to the amino acid sequence shown as SEQ ID NO.4, and the light chain variable region has an amino acid sequence that is at least 85% sequence identical to the amino acid sequence shown as SEQ ID NO. 5; or (b)

A3 The heavy chain variable region has an amino acid sequence that is at least 85% sequence identical to the amino acid sequence set forth in SEQ ID NO.6, and the light chain variable region has an amino acid sequence that is at least 85% sequence identical to the amino acid sequence set forth in SEQ ID NO. 7.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is selected from at least one of a full-length antibody, fab ', F (ab') 2, fv, scFv, fc, fd, domain antibody.

4. The antibody or antigen-binding fragment thereof of claim 1, further comprising at least one of a heavy chain constant region, a light chain constant region, the heavy chain constant region having an amino acid sequence that is at least 85% sequence identical to the amino acid sequence set forth in SEQ ID No. 8; and/or the light chain constant region has an amino acid sequence that has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID No. 9.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is any one of a human antibody, a humanized antibody, and a chimeric antibody.

6. A biomaterial, characterized in that the biomaterial comprises any one of B1) to B4);

B1 A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 5;

B2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising B1) said nucleic acid molecule or B2) said expression cassette;

B4 A recombinant cell comprising B1) said nucleic acid molecule or B2) said expression cassette or B3) said recombinant vector.

7. A pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, or the biomaterial of claim 6.

8. Kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

9. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 7, or a kit according to claim 8, for the preparation of a diagnostic, prophylactic or therapeutic product of respiratory syncytial virus.

10. A method of detecting respiratory syncytial virus in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1-5.