CN111606993B

CN111606993B - Fully human broad-spectrum neutralizing antibody 4F1 for resisting respiratory syncytial virus and application thereof

Info

Publication number: CN111606993B
Application number: CN201910143346.XA
Authority: CN
Inventors: 孙兵; 王宾; 凌志洋; 赵干; 伊春艳
Original assignee: Fudan University; Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Fudan University; Center for Excellence in Molecular Cell Science of CAS
Priority date: 2019-02-26
Filing date: 2019-02-26
Publication date: 2022-06-28
Anticipated expiration: 2039-02-26
Also published as: WO2020173460A1; CN111606993A

Abstract

The invention discloses a fully human neutralizing antibody for resisting respiratory syncytial virus fusion protein and application thereof. The invention particularly discloses a fully human monoclonal antibody 4F1 aiming at a respiratory syncytial virus fusion protein (F protein) and a pre-fusion F protein (preF protein), a nucleic acid sequence of a coding antibody and an antibody fragment and a preparation method thereof. In vitro and in vivo experiments prove that the 4F1 antibody can effectively prevent and control RSV infection, has lower immunogenicity for human bodies, can avoid antibody-mediated immune rejection from other species such as human anti-mouse and the like, and can be clinically used for preventing and treating respiratory syncytial virus infection.

Description

Fully human broad-spectrum neutralizing antibody 4F1 for resisting respiratory syncytial virus and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a full-human broad-spectrum neutralizing antibody 4F1 for resisting respiratory syncytial virus and application thereof.

Background

RSV causes acute upper and lower respiratory tract infections and is one of the important pathogens of respiratory tract infections in infants and young children worldwide, while RSV is also recognized as an important pathogen in certain high-risk adults, such as elderly individuals, adults with chronic lung disease, and immunocompromised adults (e.g., bone marrow transplant patients). There are 3000 million new cases of infection each year worldwide, and the number of deaths due to RSV infection is about 20 million. Among pneumonia syndrome of children under 2 years of age in China, RSV is the most common viral pathogen (17.0%). There is an urgent need worldwide for safe, effective, and inexpensive methods of prophylactic treatment of RSV viral infections.

Over the past several decades, methods of preventing and treating RSV infection have been investigated, including vaccines, antiviral compounds (ribavirin), antisense drugs, RNA interference techniques, and antibody products such as immunoglobulins or intravenous monoclonal antibodies. Palivizumab is the only antibody drug approved for RSV prevention in high-risk children. However, in china there is no vaccine for RSV or commercially available therapeutic drug, and only ribavirin is approved for the treatment of RSV infection, but there are severe side effects. The palivizumab targets a conserved region of the F protein, has a broad spectrum of action, but currently, the dosage of the palivizumab required by each administration is 15mg/kg (body weight), the palivizumab is passively immunized 5 times a year, and the defects of high dosage, multiple administration and high price exist. With the development of immunology and molecular biology, genetic engineering antibodies are rapidly developed, and the production technologies of chimeric antibodies, humanized antibodies and fully human antibodies are continuously developed, so that the HAMA reaction is minimized or even eliminated, and particularly, the fully human antibodies are the future development direction of antibody drugs.

Thus, there remains a need in the art to develop more effective fully human monoclonal antibodies capable of preventing and controlling RSV infection.

Disclosure of Invention

The invention aims to provide a fully human monoclonal antibody capable of preventing and controlling RSV infection.

In a first aspect of the present invention, there is provided a heavy chain variable region of an antibody, said heavy chain variable region comprising the following three Complementarity Determining Regions (CDRs):

CDR1 shown in SEQ ID NO. 3,

CDR2 shown in SEQ ID No. 4, and

CDR3 shown in SEQ ID NO. 5.

In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and capable of retaining the binding affinity of the respiratory syncytial virus fusion protein (preferably the pre-fusion F protein).

In another preferred embodiment, the heavy chain variable region further comprises a human FR region or a murine FR region.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 1.

In a second aspect of the invention, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region as described in the first aspect of the invention.

In another preferred embodiment, the heavy chain of said antibody further comprises a heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin.

In a third aspect of the present invention, there is provided an antibody light chain variable region comprising the following three complementarity determining regions CDR:

CDR 1' shown in SEQ ID NO. 6,

the amino acid sequence is CDR 2' of LGS, and

CDR 3' as shown in SEQ ID NO. 7.

In another preferred embodiment, the light chain variable region further comprises an FR region of human or murine origin.

In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID No. 2.

In a fourth aspect of the invention, there is provided a light chain of an antibody, said light chain having a light chain variable region as described in the third aspect of the invention.

In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin.

In a fifth aspect of the invention, there is provided an antibody having:

(1) a heavy chain variable region according to the first aspect of the invention; and/or

(2) A light chain variable region according to the third aspect of the invention;

alternatively, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.

In another preferred embodiment, said antibody is an antibody specific against respiratory syncytial virus, preferably an antibody specific against respiratory syncytial virus fusion protein, preferably the pre-fusion F protein.

In another preferred embodiment, the antibody is selected from the group consisting of: an antibody of animal origin, a chimeric antibody, a humanized antibody, or a combination thereof.

In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

In another preferred embodiment, the antibody is in the form of a drug conjugate.

In another preferred embodiment, the heavy chain variable region sequence of the antibody is as shown in SEQ ID No. 1; and the light chain variable region sequence of the antibody is shown as SEQ ID NO. 2.

In a sixth aspect of the present invention, there is provided a recombinant protein having:

(i) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

In another preferred example, the tag sequence comprises a 6His tag, a GGGS sequence and a FLAG tag.

In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

In another preferred embodiment, said recombinant protein specifically binds to a respiratory syncytial virus fusion protein, preferably to the pre-fusion F protein.

In a seventh aspect of the invention, there is provided a CAR construct wherein the scFv segment of the antigen binding region of the CAR construct is a binding region that specifically binds to an RSV fusion protein (preferably the pre-fusion F protein) and wherein the scFv has a heavy chain variable region according to the first aspect of the invention and a light chain variable region according to the third aspect of the invention.

In an eighth aspect of the invention there is provided a recombinant immune cell expressing an exogenous CAR construct according to the seventh aspect of the invention.

In another preferred embodiment, the immune cell is selected from the group consisting of: NK cells, T cells.

In another preferred embodiment, the immune cell is from a human or non-human mammal (e.g., a mouse).

In a ninth aspect of the present invention, there is provided an antibody drug conjugate comprising:

(a) an antibody portion selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, or a combination thereof; and

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, and the like.

In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or a linker.

In a tenth aspect of the invention, there is provided the use of an active ingredient selected from the group consisting of: the variable region of a heavy chain according to the first aspect of the invention, the variable region of a heavy chain according to the second aspect of the invention, the variable region of a light chain according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, or a combination thereof, and the active ingredient for use in the preparation of a medicament, a reagent, a detection panel or a kit.

In another preferred embodiment, the reagent, detection plate or kit is for detecting respiratory syncytial virus.

In another preferred embodiment, the medicament is for the treatment or prevention of respiratory syncytial virus infection.

In another preferred embodiment, the reagent comprises a chip and immune microparticles coated with antibodies.

In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) an active ingredient selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an immune cell according to the eighth aspect of the invention, an antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof; and

(ii) A pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

In another preferred embodiment, the pharmaceutical composition is an injection.

In another preferred embodiment, said pharmaceutical composition is for use in the prevention and/or treatment of respiratory syncytial virus infection.

In a twelfth aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; or

(2) A recombinant protein according to the sixth aspect of the invention;

(3) a CAR construct according to the seventh aspect of the invention.

In another preferred embodiment, the polynucleotide has the sequence shown in SEQ ID No. 8 and/or SEQ ID No. 9.

In a thirteenth aspect of the invention, there is provided a vector comprising a polynucleotide according to the twelfth aspect of the invention.

In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

In a fourteenth aspect of the invention, there is provided a genetically engineered host cell comprising a vector according to the thirteenth aspect of the invention or having a polynucleotide according to the twelfth aspect of the invention integrated into its genome.

In a fifteenth aspect of the present invention, there is provided a method of detecting respiratory syncytial virus in a sample, the method comprising the steps of:

(1) contacting the sample with an antibody according to the fifth aspect of the invention;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of respiratory syncytial virus in the sample.

In another preferred embodiment, the detection is for non-therapeutic, non-diagnostic purposes.

The invention also provides a method for detecting respiratory syncytial virus fusion protein in a sample, the method comprising the steps of:

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of respiratory syncytial virus fusion protein in the sample.

In another preferred embodiment, the respiratory syncytial virus fusion protein is the respiratory syncytial virus pre-fusion F protein.

In a sixteenth aspect of the present invention, there is provided a detector board comprising: a substrate (support plate) and a test strip comprising an antibody according to the fifth aspect of the invention or an immunoconjugate according to the ninth aspect of the invention.

In a seventeenth aspect of the present invention, there is provided a kit comprising:

(1) a first container comprising an antibody according to the fifth aspect of the invention; and/or

(2) A second container comprising a secondary antibody directed against the antibody according to the fifth aspect of the invention;

alternatively, the kit comprises a detection plate according to the sixteenth aspect of the invention.

In an eighteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) culturing a host cell according to the fourteenth aspect of the invention under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.

In a nineteenth aspect of the invention, there is provided a method of treating respiratory syncytial virus infection, the method comprising: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, an antibody-drug conjugate of said antibody, or a CAR-T cell expressing said antibody, or a combination thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Drawings

Figure 1 shows flow sorted RSV F protein-specific binding memory B cells.

FIG. 2 shows agarose gel electrophoresis patterns of the light and heavy chain genes of the matched antibodies.

FIG. 3 shows the binding activity of the 4F1 antibody to RSV A2 pre-fusion F protein (A), B9320 pre-fusion F protein (B) and A2 post-fusion F protein (C). Among them, the 4F1 antibody can bind to pre-fusion F proteins of RSV types a and B.

Figure 4 shows an antibody neutralizing activity dose fit curve for the 4F1 antibody against RSV a2(a) and B9320 (B); and antibody neutralizing activity dose-fit curves of Palivizumab (Palivizumab) against RSV a2(C) and B9320 (D).

Figure 5 shows a comparison of 4F1 and palivizumab in a mouse prevention experiment for their ability to reduce pulmonary viral load.

Figure 6 shows a comparison of pathological lung injury after passive immunization with 4F1 and palivizumab in a mouse prevention experiment.

Detailed Description

The inventor unexpectedly obtains a fully human monoclonal antibody 4F1 aiming at the respiratory syncytial virus fusion protein (F protein) and the pre-fusion F protein (preF protein) through extensive and intensive research. The antibody can be combined with pre-fusion F proteins of RSV A and B viruses in a broad spectrum, has high combination neutralization activity on respiratory syncytial viruses, has broad recognition spectrum and broad spectrum neutralization, and can inhibit or prevent the respiratory syncytial viruses from infecting susceptible cells. In vitro and in vivo experiments prove that the 4F1 antibody can effectively prevent and control RSV infection. On the basis of this, the present invention has been completed.

The invention screens a full-human broad-spectrum neutralizing antibody 4F1 against RSV virus from PBMC of a healthy volunteer by a single-cell RT-PCR technology. ELISA binding experiments and cell level micro-neutralization experiments prove that the 4F1 antibody has broad-spectrum binding activity and broad-spectrum micro-neutralization activity on RSV type A and B proteins. Compared with the currently marketed Palivizumab (Palivizumab) antibody, the 4F1 is found to be remarkably superior to the Palivizumab antibody no matter in a cell level micro-neutralization experiment or a mouse prevention experiment, and the 4F1 antibody belongs to a fully human antibody, does not contain a mouse-derived component, and has lower immunogenicity and higher safety, so that the potential clinical application value of the antibody in resisting RSV infection is indicated, and a novel candidate drug for resisting RSV infection is provided for clinic. The antibody of the invention is combined with the RSV F protein prefusion form, a great deal of research proves that the neutralizing antibody recognition site aiming at the F protein is mainly on the pre-fusion F protein, and the discovery of the 4F1 antibody epitope also provides some new ideas and references for the design of RSV vaccine.

Term(s) for

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

The three letter codes and the one letter codes for the amino acids used according to the invention are described in J.biol. chem,243, p3558 (1968).

As used herein, the term "treatment" refers to the administration of therapeutic agents, including the monoclonal antibodies to respiratory syncytial virus fusion protein, and preferably the pre-fusion F protein, of the present invention and compositions thereof, to patients having one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a particular sequence may, but need not, be 1, 2, or 3.

"sequence identity" as referred to herein means the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between a sequence described in the present invention and a sequence with which it is identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

Respiratory Syncytial Virus (RSV)

RSV belongs to the genus pneumovirus of the family Paramyxoviridae, and is a minus-strand RNA virus consisting of 15222 nucleotides. RSV is an enveloped virus, the genome of which totally encodes 11 proteins including a fusion protein F and adsorptionProtein G, small hydrophobin SH, matrix protein M, nucleoprotein N, phosphoprotein P, large polymerase protein L, small matrix protein M2 and nonstructural proteins NS1 and NS2, wherein fusion protein F, adsorption protein G and small hydrophobin SH proteins are located on the viral surface. RSV has two major surface glycoproteins, G and F. G protein mediates binding of virus to the cell surface and simultaneously mimics the chemokine CX₃The action of C chemotactic factor, interacting with the receptor thereof, and enhancing the inflammatory response after RSV infection; the F protein mediates fusion of the viral and cellular membranes and also promotes fusion of the membrane of the infected cell with the surrounding cells to form syncytia. According to serology, RSV exists in two distinct antigen groups or subtypes, a and B, distinguished primarily by differences in the G protein. Most RSV proteins are highly conserved between the two subgroups, with fusion F protein showing 91% amino acid similarity and G protein having only 53% homology between a and B. The protein F is highly conserved, and the induced neutralizing antibody can simultaneously inhibit virus infection of A and B subtypes, thereby having important significance in the aspects of anti-RSV monoclonal antibody medicaments and vaccine development. The F protein belongs to a type I transmembrane protein, and the F protein firstly synthesizes a precursor protein F0. F0 trimerizes in the endoplasmic reticulum and is treated with cellular furin-like protease at two conserved sites, producing F1, F2 and Pep27 polypeptides, eventually forming two fragments of F1 and F2 linked by disulfide bonds, F2-F1 heterodimers form trimers to assemble the mature F protein. The F1 subunit comprises heptad repeat region A (HRA), domain I/II, heptad repeat region B (HRB), transmembrane protein (TM), cytoplasmic region (CP), and the F2 subunit consists of heptad repeat region C (HRC). The hydrophobic N-terminus of F1 (137-155) plays an important role in mediating fusion. The F protein-mediated membrane fusion process is also the process of changing the structure from the pre-fusion state to the fusion state. It adopts a metastable prefusion conformation. Upon binding of the G protein to a receptor on the target cell membrane, the pre-fusion F protein (pre-fusion F protein) begins to trigger the allosteric conversion to a stable post-fusion form (post-fusion). Due to its key role in RSV invasion and high conservation, the RSV F protein is the target for neutralizing antibodies and the primary antigen for vaccine development. Numerous studies have demonstrated that neutralizing antibody recognition sites for F proteins are predominantly on pre-fusion F proteins, based on pre The immunogenicity and protective effect of the recombinant protein designed by fusion F are obviously better than those of a vaccine designed by post-fusion F protein.

As used herein, the terms "pre-fusion F protein", "pre F protein" are used interchangeably and all refer to the pre-fusion form of fusion protein F of respiratory syncytial virus.

In order to further obtain a brand-new RSV antibody medicament with better curative effect and search a new antibody recognition epitope, the invention utilizes the single-cell RT-PCR technology to separate a broad-spectrum neutralizing antibody 4F1 from PBMC of human peripheral blood. The discovery of new antibodies provides a new choice for broad-spectrum neutralizing antibody therapy application on one hand, and the discovery of new epitopes provides a new idea for the development of broad-spectrum vaccines on the other hand.

Antibodies

As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form a partially folded structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly 5 classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

As used herein, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The invention also includes monoclonal antibodies having the corresponding amino acid sequences of said monoclonal antibodies against respiratory syncytial virus fusion proteins, preferably pre-fusion F protein, monoclonal antibodies having the variable region chains of said monoclonal antibodies against respiratory syncytial virus fusion proteins, preferably pre-fusion F protein, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having light and heavy chains with hypervariable regions (complementarity determining regions, CDRs) so long as the hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous to the hypervariable regions of the light and heavy chains of the invention.

As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the anti-respiratory syncytial virus fusion protein monoclonal antibody or fragment thereof. The invention also comprises a cell surface marker or antigen combined with the monoclonal antibody or the fragment thereof for resisting the respiratory syncytial virus fusion protein.

The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of the antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab')₂A fragment comprising a bivalent fragment of two Fab fragments connected by a disulfide bridge on the chain compare region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding.

The invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab')₂A fragment; an antibody heavy chain; the light chain of the antibody.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically comprise at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation.

The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is administered at a rate of about less than 10^-7M, e.g. less than about 10^-8M、10^-9M or l0^-10M or less affinity (KD) binding.

As used herein, the term "antigenic determinant" refers to a three-dimensional spatial site on an antigen that is not contiguous and is recognized by an antibody or antigen-binding fragment of the invention.

The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art. The term "murine antibody" is in the present invention a monoclonal antibody directed against the respiratory syncytial virus fusion protein, prepared according to the knowledge and skill in the art. The term "chimeric antibody" is an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. The term "humanized antibody", also known as CDR-grafted antibody (CDR), refers to an antibody produced by grafting murine CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. The humanized antibody can overcome the heterogenous reaction induced by the chimeric antibody carrying a great deal of murine protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. To avoid reduced immunogenicity and reduced activity, the human antibody variable region framework sequences may be minimally back-mutated or back-mutated to retain activity.

In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multispecific.

As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody which primarily contributes to antigen binding. One of the most common definitions of the 6 CDRs is provided by Kabat E.A et al, (1991) Sequences of proteins of immunological interest, NIH Publication 91-3242).

In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises the following three complementarity determining regions CDRs:

CDR1:GFSFYSYS(SEQ ID NO.:3)，

CDR 2: VVYDGNHQ (SEQ ID No.:4), and

CDR3：TARSLVITLAGAGRDDY(SEQ ID NO.:5)。

in another preferred embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 1, wherein the underline marks are the amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 in this order.

EVQLVQSGGGVVRPGRSLRLSCAASGFSFYSYSVHWVRQAPGKGLEWVADVVYDGNHQHYTESVRGRFSISRDTSTNTVYLQMGSLRPEDTALYYCTARSLVITLAGAGRDDYWGQGTRVTVSS(SEQ ID NO.:1)

In another preferred embodiment, the nucleic acid coding sequence for the heavy chain variable region is as shown in SEQ ID NO. 8, wherein the nucleic acid coding sequences for the heavy chain variable region CDR1, CDR2 and CDR3 are underlined.

GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAGCTTCTATTCCTATTCTGTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGATGTTGTATATGATGGAAATCATCAACATTACACGGAGTCCGTGAGGGGCCGATTCTCCATCTCCAGAGACACCTCCACCAATACGGTGTATCTGCAAATGGGCAGCCTGAGGCCTGAAGACACGGCTCTTTATTACTGTACGGCCCG CAGTTTGGTCATAACGCTCGCGGGGGCGGGTCGAGATGACTATTGGGGCCAGGGAACTCGGGTCACCGTCTCCTCA(SEQ ID NO.:8)

In a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and a heavy chain constant region, which may be murine or human.

As used herein, the terms "light chain variable region" and "V_L"may be used interchangeably.

In a preferred embodiment of the invention, the light chain variable region of the antibody according to the invention has complementarity determining regions CDRs selected from the group consisting of:

CDR1’：QSLLHSNGYTY(SEQ ID NO.:6)，

CDR 2': LGS, and

CDR3’：VQDLQTSLT(SEQ ID NO.:7)。

in another preferred embodiment, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 2, wherein the amino acid sequences of the light chain variable region CDR1 ', CDR2 ' and CDR3 ' are indicated by double underlining.

DIVMTQSPLSLSVTPGEPASISCKSSQSLLHSNGYTYLDWYLQKPGKSPQLLIFLGSSRASGVPARFSGSGSGTDFTLEISRVEAEDVGVYYCVQDLQTSLTFGGGTKVDIK(SEQ ID NO.:2)

In another preferred embodiment, the nucleic acid encoding sequence of the light chain variable region is as shown in SEQ ID NO. 9, wherein the nucleic acid encoding sequences of the light chain variable region CDR1 ', CDR2 ' and CDR3 ' are indicated by double underlining.

GATATTGTGATGACTCAGTCTCCACTCTCCCTGTCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTCCATAGTAATGGATACACTTATTTGGATTGGTACCTGCAGAAGCCAGGGAAGTCTCCACAACTCCTGATCTTTTTGGGTTCTAGTCGGGCCTCCGGGGTCCCTGCCAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGGAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTACTACTGCGTGCAAGATCTACAAAC TTCCCTCACTTTCGGCGGAGGGACCAAAGTGGATATCAAA(SEQ ID NO.:9)

In a preferred embodiment of the invention, the light chain of the antibody comprises the light chain variable region and a light chain constant region, which may be murine or human.

The function of the antibody is determined by the gene sequence specific to the light chain and heavy chain variable regions of the antibody, and the antibody can be widely combined with pre-fusion F proteins of RSV A and B viruses and can prevent respiratory syncytial virus from infecting susceptible cells. Using the antibody variable region gene or Complementary Determining Region (CDR) gene, different forms of genetically engineered antibodies can be engineered and produced in any expression system using prokaryotic and eukaryotic cells.

In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and refer to an antibody that specifically binds to an anti-respiratory syncytial virus fusion protein, preferably the pre-fusion F protein, such as a protein or polypeptide having a heavy chain variable region (the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID No.: 8) and/or a light chain variable region (the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID No.: 9). They may or may not contain the initial methionine.

In another preferred embodiment, the antibody is a murine or human murine chimeric monoclonal antibody directed against a respiratory syncytial virus fusion protein, preferably a pre-fusion F protein, whose heavy chain constant region and/or light chain constant region may be humanized. More preferably, the humanized heavy or light chain constant region is that of human IgG1, IgG2, or the like.

In general, the antigen binding properties of an antibody can be described by 3 specific regions in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest, since at least some of them are involved in binding to an antigen. Thus, the invention includes those molecules having the light and heavy chain variable regions of a monoclonal antibody with CDRs that are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein.

The invention includes not only complete monoclonal antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies. For example, the Fc fragment is modified on the basis of the antibody of the invention, and three mutation points M252Y/S254T/T256E are introduced into a CH2 region in order to prolong the half life of the antibody.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

The antibody of the present invention refers to a polypeptide having an anti-respiratory syncytial virus fusion protein (preferably pre-fusion F protein) binding activity, comprising the above-mentioned CDR region. The term also includes variants of the polypeptides comprising the above CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids that are similar or analogous in performance do not typically alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

The invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the invention also encompasses fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 60 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region as shown in SEQ ID NO. 8 or 9 or may be a degenerate variant. As used herein, "degenerate variant" means in the present invention a nucleic acid sequence which encodes a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but differs from the coding region sequence set forth in SEQ ID No. 8 or 9.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) denaturant is added during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ℃ and the like; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. And the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID No. 1 and/or SEQ ID No. 2.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following may be usedThe DNA transfection method comprises the following steps: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.

Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.

Couplable therapeutic agents include, but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptides, gut peptide analogs, albumin, antibody fragments, cytokines, and hormones.

Therapeutic agents that may also be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. a prodrug activating enzyme; 10. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-described antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the present invention can be directly used for binding to a fusion protein (preferably pre-fusion F protein) molecule of respiratory syncytial virus, and thus can be used to prolong the half-life of the drug, and in addition, other therapeutic agents can be used simultaneously.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 10 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Detection use and kit

The antibodies of the invention are useful in detection applications, for example, for detecting a sample, thereby providing diagnostic information.

In the present invention, the specimen (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus, a biopsy as used in the present invention may comprise a tissue sample prepared, for example, by endoscopic methods or by needle or needle biopsy of an organ.

Samples for use in the present invention include fixed or preserved cell or tissue samples.

The invention also provides a kit containing the antibody (or fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

The main advantages of the invention

(1) The fully human monoclonal antibody can specifically recognize and combine with a pre-fusion F protein of the respiratory syncytial virus, has high neutralizing activity on the respiratory syncytial virus, can combine with RSV A and RSV B viruses, and effectively inhibits or prevents the respiratory syncytial virus from infecting susceptible cells.

(2) The fully human monoclonal antibody has broad-spectrum binding activity and broad-spectrum neutralizing activity on RSV A and B viruses, can effectively neutralize various respiratory syncytial viruses, and is remarkably superior to the antibodies (such as Palivizumab antibodies) sold on the market at present in cell level micro-neutralization experiments and mouse prevention experiments.

(3) The invention is the fully human monoclonal antibody 4F1, does not contain a mouse-derived part, has lower immunogenicity and higher safety for human bodies, and can avoid the human anti-mouse and other species-derived antibody-mediated immune rejection reactions.

(4) The fully human monoclonal antibody 4F1 of the invention is combined with a RSV F protein prefusion form, a large number of researches prove that a neutralizing antibody recognition site aiming at the F protein is mainly on pre-fusion F protein, and the discovery of 4F1 antibody epitope also provides some new ideas and references for the design of RSV vaccine.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Experiments in which specific conditions are not specified in the examples or test examples of the present invention are usually performed under conventional conditions or under conditions recommended by the manufacturers of raw materials/goods; reagents of specific sources are not indicated, and are conventional reagents purchased in the market.

The following describes the process of preparation of neutralizing fully human monoclonal antibody capable of neutralizing respiratory syncytial virus fusion protein and analysis of antibody characteristics according to the present invention.

Example 1 Single cell RT-PCR method for obtaining antibody genes and antibody expression

1. Acquisition of Peripheral Blood Mononuclear Cells (PBMC)

Peripheral blood was collected from healthy volunteers using conventional Ficoll-Paque (manufactured by Kabushiki Kaisha Co., Ltd.)

-H (CEDARLANE) Corp.) Density gradient centrifugation to give 10⁷The above Peripheral Blood Mononuclear Cells (PBMCs).

The Ficoll separation method comprises the following steps:

(1) blood was collected and 20ml of whole blood was collected in a 50ml centrifuge tube (containing 1ml of 4% sodium citrate beforehand), and mixed 8-10 times by inversion. (even at a final sodium citrate concentration of 0.4%);

(2) adding equal volume of RPMI1640 (containing sodium citrate), and mixing;

(3) a15 ml clear centrifuge tube was used to spread 3ml of lymphocyte separation medium, and 6ml of blood sample was carefully added thereto. Forming a separation interface (or 4ml of separation medium plus 8ml of blood sample);

(4) centrifuging at room temperature for 800g and 20min (2000rpm and 20 min);

(5) Carefully sucking the interface layer cells and transferring the interface layer cells to a new tube;

(6) RPMI1640 (containing sodium citrate) was added and the liquid density was reduced by dilution. Centrifugation at 800g/2000rpm for 10 min. Removing the supernatant;

(7) washing cells with RPMI1640 for 2-3 times for use

2. Acquisition of RSV fusion protein F-specific memory B cells

BD horizons using FITC-CD19/APC-IgG/BV421 and PE-RSV F proteins as markers^TMFixable visual Stain 780 removes dead cells, obtains specific B cells to 96-well RT-PCR plates by a flow cytometer, and obtains F protein specific memory B cells by one cell per well.

(1) The RSV A2 pre-fusion F protein is expressed by a CHO expression system of mammalian cells; reference Invitrogen ExpicCHO-S^TMExpression System handbook; the A2F protein sequence is designed according to the strategy adopted by Jason S.McLellan.science 2013 by referring to UniProtKB/Swiss-Prot: P03420.1 and the design of RSV pre-fusion F protein, and is subjected to whole gene synthesis by Shanghai Jie Ruili company and is constructed on an expression vector of invitrogen pcDNA3.1.

(2) RSV a2 pre-fusion F protein for Biotin (Biotin) labeling: No-Weigh Sulfo-NHS-LC-Biotin (purchased from PIERCE, refer to EZ-Link Sulfo-NHS-LC-Biotin Protocol from PIERCE) 10mM reagent; two further markers FITC-CD19 and APC-IgG were purchased from BD Bioscience; SA Streptavidin PE and Streptavidin BV421 (available from BD Co.) were used to detect biotin-labeled F protein;

(3) Labeling of sorted cells: grouping PBMC cells, adding a marker into the experimental group and the control group according to the number of the cells, dyeing in a dark place, marking, resuspending by PBS, and filtering by using a 40 mu m BD falcon filter membrane;

(4) sorting of specific B cells: lymphocytes were selected from PBMCs using BD FACS infilux screening according to the forward and lateral angle, and then adjusted to compensate by different controls to obtain specific memory B cells for RSV F protein, which were sorted into 96-well plates for RT-PCR (reverse transcription PCR), one cell per well, and the plates were placed on dry ice.

3. Antibody gene acquisition and vector construction

The obtained single memory B cell is subjected to RT-PCR to obtain cDNA, then an antibody gene variable region is obtained through nested-PCR, agarose nucleic acid gel is run, and a gel block which can be paired by a heavy chain and a light chain is recovered and sequenced. The antibody gene sequences were obtained by search through the IgBLAST website (https:// www.ncbi.nlm.nih.gov/projects/IgBLAST /). And then, respectively connecting the antibody genes to corresponding IgH, Ig kappa and Ig lambda expression vectors through AgeI and SalI enzyme cutting sites, AgeI and BsiwI enzyme cutting sites and AgeI and XhoI enzyme cutting sites. Fully human antibody expression vectors IgH, Ig kappa and Ig lambda (expressing antibody heavy chain, kappa chain, lambda chain respectively) were given away by Patrick Wilson laboratories and the vector sequences are given in NCBI GenBank: FJ475055, FJ475056 and FJ 517647.

4. Antibody expression and purification

CHO cells were transiently transfected for fully human antibody expression. One day before transfection (day-1), ExpicCHO-S was fractionated^TMCells, final density 3X 10⁶–4×10⁶Viable cells/mL, cells were grown overnight. The following day (day 0), viable cell density and percent viability were determined. The cell density should reach about 7X 10⁶–10×10⁶Viable cells/mL. The survival rate should be 95-99%, so that the transfection can be continued. Cells were diluted to a final density of 6X 10⁶Viable cells/mL. Expi Fectamine was prepared using a pre-chilled reagent (4 ℃ C.)^TMCHO/plasmid DNA complex. Incubation of Expi Fectamine at Room temperature^TMCHO/plasmid DNA complexes for 1-5 minutes, then the solution slowly transferred to CHO cell culture flask, in the addition process gently shaking the flask. The cells were placed on a orbital shaker (37 ℃ incubator with 8% CO)₂Under humidified air conditions). Culturing for 7-11 days, collecting supernatant when half of the cells die, and purifying antibody。

The antibody was purified using Protein G Agarose 4FF packing (from GE). The collected CHO cell suspension is firstly centrifuged at 4000rpm and 4 ℃ for 30min, and the collected supernatant is filtered by 0.45um filter for purification. Taking a gravity type centrifugal column, adding Protein G Agarose 4FF filler, stabilizing the filler by using 20% ethanol with 3 times of column volume, then balancing the column by using binding buffer with 5 times of column volume, then loading a sample, balancing the column by using the binding buffer with 10 times of column volume, finally eluting the column by using elution buffer with 3 times of column volume, and adding neutralization buffer into the eluted antibody solution to enable the pH value of the eluted sample to be about 7.5. The antibody solution was dialyzed 3 times against 5L 1 × PBS, and the antibody was concentrated and stored at-80 ℃.

The experimental results are as follows:

as shown in FIG. 1, several specific B cells of RSV-F protein were obtained, accounting for approximately 0.3% of the total memory B cell count, using FITC-CD19/APC-IgG/BV421 and the PE double positive marker as specific markers.

The single cell RT-PCR and Nested-PCR method to obtain the matching antibody heavy and light chain variable region gene, the molecular weight is about 400bp, the electrophoresis pattern is shown in figure 2. Agarose gel cutting was recovered and sequenced. The sequencing result is shown inhttp:// www.ncbi.nlm.nih.gov/igblast and http:// www.imgt.org @And (3) comparing to obtain the gene information of the antibody germ line and the high variable region information of the heavy and light chain genes of the antibody, and constructing an expression vector and performing subsequent expression and purification. Finally, by the technology, a fully human monoclonal antibody which neutralizes RSV type A and B viruses in a broad spectrum is successfully obtained and is named as 4F 1.

The heavy chain variable region gene sequence of the fully human antibody 4F1 is as follows, wherein underlined are hypervariable region sequences in the variable region of the heavy chain gene, and are the heavy chain gene CDR1, CDR2 and CDR3 sequences in this order.

The heavy chain variable region amino acid sequence of fully human antibody 4F1 is as follows, with the heavy chain amino acid CDR1, CDR2, and CDR3 sequences being in that order underlined.

The light chain variable region gene sequence of fully human antibody 4F1 is as follows, wherein the underlined hypervariable region sequences in the light chain gene variable region are the CDR1 ', CDR2 ' and CDR3 ' sequences in that order.

The light chain variable region amino acid sequence of fully human antibody 4F1 is as follows, with the light chain amino acid CDR1 ', CDR2 ' and CDR3 ' sequences being in that order underlined.

Example 2 analysis of antibody Properties

1. ELISA for detecting the antigen-binding Activity of antibodies

To investigate the ability of the 4F1 antibody to bind to RSV type a and type B viral F proteins, ELISA was used to test whether the expressed antibodies recognized RSV a2 and B9320 pre-fusion F proteins and a2 post-fusion F protein. The A2F protein sequence is referenced to UniProtKB/Swiss-Prot: P03420.1, the B9320 sequence is referenced to UniProtKB/Swiss-Prot: Q6V2E7, the RSV pre-fusion F protein is designed according to the strategy adopted by Jason S.McLellan.science 2013, and the RSV post-fusion F protein is designed according to Davide Corti. Nature 2013, and carrying out whole-gene synthesis in Shanghai Jie Co., Ltd, and constructing the gene into an expression vector of invitrogen pcDNA3.1. Expressed by a mammalian cell CHO expression system; reference Invitrogen ExpicCHO-S^TMExpression System handbook. Palivizumab (Palivizumab,

) As a positive control antibody, purchased from yapei corporation. Coated F protein ELISA plates, 0.5. mu.g/mL, 100. mu.L per well, 4 ℃ overnight. The next day the plates were washed 3 times with PBST. Blocking with 2% BSA at 200. mu.L/well, 37 ℃ for 2 h. The plates were washed again 3 times with PBST. 4F1 and control antibody were tested at 12 concentrations, 3-fold gradient dilution, 2 replicate wells, starting at a test concentration of 30. mu.g/ml. The sample was loaded at 100. mu.L/well, 37 ℃ for 2 h. PBST wash plate 3 times. Sheep Anti-Human IgG (Goat Anti-Human IgG) (Fc specific) -Peroxidase antibody (sigma), 1:5000 dilution, 100. mu.L per well, 37 ℃, 1 h. PBST wash plate 3 times. Adding 100 mu L/hole of substrate TMB to develop color, and if the color is lighter, reacting for 15min at 37 ℃ in a dark place. 2M H was added ₂SO₄The reaction was stopped at 50. mu.L per well. Determination of OD₄₅₀And performing data processing. Palivizumab (Palivizumab) PVZ was used as a positive control antibody, and a fully human antibody against an unrelated virus was used as a negative isotype control antibody (NC).

As a result, as shown in FIG. 3, the 4F1 antibody can bind to the pre-fusion F proteins of RSV type A and B viruses in a broad spectrum, and has a binding ability comparable to that of the positive control antibody. 4F1 did not bind to post-fusion F protein type A, and palivizumab was reported to bind to both forms of F protein. The results indicate that the 4F1 antibody binds to both RSV type a and type B pre-fusion F forms with a broad spectrum, and that the 4F1 antibody and palivizumab bind to two different epitopes on the F protein.

2. Virus TCID₅₀Measurement of

Using TCID₅₀Experimental verification titers of diluted virus solutions in RSV microneutralization experiments. 200. mu.l of the diluted virus solution was added to wells of a 96-well cell plate B2-D2, and 100. mu.l of the cell culture solution was added to the other wells. Sucking 100 μ l of virus solution from B2-D2 well, adding into B3-D3 well, mixing, and sequentially performing 2 times of gradient dilution, wherein the virus dilution is 18 concentration points, 3 duplicate wells. The plates were then tested at 37 ℃ and 5% CO₂Incubate in the incubator for 2 hours. HEp2 cells were seeded at a density of 25,000 cells per well in the test plate and 5% CO at 37 deg.C ₂Culturing in an incubator for 5 days.

After 5 days of culture, the supernatant was discarded and the cells in the test plate were fixed with 80% acetone. The intracellular viral content was measured using ELISA. Raw data for virus TCID₅₀Calculation (Karber method). The calculation formula is as follows:

TCID₅₀well ═ Antilog10[ (number of positive wells/3) -0.5 }. times.0.3]/2

Positive well determination criteria: test well readings > (Medium controls +3 × Medium controls SD values)

QC standard: the titer of the diluted virus solution should be 50-2000TCID₅₀A hole.

3. Virus micro-neutralization assay

To investigate the ability and broad spectrum of the 4F1 antibody to neutralize RSV virus, a gradient dilution of the 4F1 antibody was incubated with different viruses and the ability of the 4F1 antibody to neutralize the virus was examined by a micro-neutralization assay. Test strains A2 and B9320 (from ATCC) were each diluted to 4000TCID in cell culture broth₅₀And/ml. Antibody and 200TCID diluted by multiple₅₀Adding virus/well into 96-well cell culture plate at equal volume ratio, mixing well at 37 deg.C and 5% CO₂Incubate in the incubator for 2 hours. HEp2 cells were then seeded into the test plate at a density of 25,000 cells per well and 5% CO at 37 deg.C₂Culturing in an incubator for 5 days. Antibodies were tested at 9 concentrations, 3-fold gradient dilution, 3 replicate wells, starting at 4000 ng/ml.

After 5 days of culture, the supernatant was discarded and the cells in the test plate were fixed with 80% acetone. The intracellular viral content was measured using ELISA. The raw data were used for the calculation of the neutralizing activity of the antibody at different concentrations. The calculation formula is as follows:

percent activity (%) ═ test well readings-mean of virus controls)/(mean of cell controls-mean of virus controls) × 100

EC₅₀Values were calculated by Prism software, neutralizing activity curve fit methodThe method is sigmoidal dose-response (variable slope).

The results are shown in FIG. 4 and Table 1. The dose-fit curves of antibody neutralizing activity of the 4F1 antibody and the control antibody Palivizumab against RSV strains are shown in fig. 4. Palivizumab exhibits neutralizing activity against both RSV A2 and B9320, its IC₅₀The values were 384.3ng/ml and 367.2ng/ml, respectively. IC of 4F1 for RSV A2 and B9320₅₀The values were 4.368ng/ml and 23.51ng/ml, respectively. The neutralizing activity of the 4F1 antibody on RSV A2 and B9320 is better than that of palivizumab (IC)₅₀The lower is approximately 80-100 times. Negative antibody NC showed no neutralizing activity against the test virus strain at the tested concentration, IC₅₀The value was greater than the highest assay concentration 4000ng/ml (see Table 1).

TABLE 1 antibody IC₅₀Value of

4. Detection of animal prevention effect of 4F1 antibody

This example tests the ability of antibodies 4F1 and Palivizumab of the invention to prevent RSV infection in mice. Female BalB/c mice, 6-8 weeks old, were placed in advance in a biosafety secondary laboratory animal laboratory. day0 mice were injected intraperitoneally with 15mg/kg, 3mg/kg, 0.6mg/kg, 0.12mg/kg of 4F1 antibody, Palivizumab and PBS, respectively. After 24h, the mice were anesthetized with ether and challenged intranasally with RSV A2 virus (10) ⁷PFU/50 ul/mouse). Five days later the animals were sacrificed to harvest lungs. The left lung was removed from each mouse and fixed with 4% paraformaldehyde, embedded with paraffin and stained with conventional hematoxylin-eosin (HE), and the lung was observed for pathological damage and inflammatory cell infiltration with light microscopy. Homogenating right lung tissue of each mouse, extracting total RNA with EZ-press RNA Purification Kit, inverting into cDNA, performing relative quantification on RSV genome by real-time quantitative RT-PCR method, comparing gene sequences of NP protein coding RSV, selecting conserved segment for primer design, and taking beta actin of mouse as internal reference gene (see Table 2). Reference to Toyobo KOD

qPCR Mix instruction manual.

TABLE 2 real-time quantitative RT-PCR primers

The results are shown in fig. 5, and different doses of 4F1 and palivizumab were both effective in reducing RNA levels of pulmonary virus compared to the PBS group. The reduction in pulmonary viral load was more pronounced with the 4F1 antibody than with palivizumab. The inhibiting effect of the 4F1 with the administration dose of 0.12mg/kg on RSV virus is more obvious than that of the palivizumab with the administration dose of 15 mg/kg.

The results are shown in FIG. 6. Compared with the PBS control group, the lung tissue pathological results show that the 4F1 antibody can obviously reduce the pathological damage conditions of the mouse lung in the high-dose and low-dose groups, including the reduction of the infiltration conditions of inflammatory cells around the pulmonary interstitium and the bronchus, the maintenance of the normal morphological structure of the alveoli, the reduction of bleeding points and other indexes. And the palivizumab only has a relatively obvious protective effect in a high-dose group of 15 mg/kg.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Life science research institute of Chinese academy of sciences

FUDAN University

<120> full-human broad-spectrum neutralizing antibody 4F1 for resisting respiratory syncytial virus and application thereof

<130> P2018-2432

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<170> PatentIn version 3.5

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Claims

1. An anti-respiratory syncytial virus antibody, characterized in that said antibody has:

(1) a heavy chain variable region comprising the following three complementarity determining regions CDRs:

SEQ ID NO. 3 shows the CDR1,

CDR2 shown in SEQ ID No. 4, and

CDR3 shown in SEQ ID No. 5; and

(2) a light chain variable region comprising the following three complementarity determining regions CDRs:

CDR 1' shown in SEQ ID NO. 6,

the amino acid sequence is CDR 2' of LGS, and

CDR 3' as shown in SEQ ID NO. 7.

2. The antibody of claim 1, wherein said antibody has a heavy chain having a heavy chain variable region and a heavy chain constant region; and a light chain having a light chain variable region and a light chain constant region.

3. The antibody of claim 1, wherein the heavy chain variable region sequence of the antibody is as set forth in SEQ ID No. 1; and the light chain variable region sequence of the antibody is shown as SEQ ID NO. 2.

4. A recombinant protein, said recombinant protein having:

(i) the antibody of claim 1; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

5. The recombinant protein according to claim 4, wherein said recombinant protein specifically binds to a respiratory syncytial virus fusion protein.

6. A CAR construct, wherein the scFv segment of the antigen binding region of the CAR construct is a binding region that specifically binds to an RSV fusion protein and has a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following three complementarity determining regions CDRs:

SEQ ID NO. 3 shows the CDR1,

SEQ ID NO. 4 CDR2, and

a CDR3 shown in SEQ ID NO. 5; and

the light chain variable region comprises the following three Complementarity Determining Regions (CDRs):

CDR 1' shown in SEQ ID NO. 6,

the amino acid sequence is CDR 2' of LGS, and

CDR 3' as shown in SEQ ID NO. 7.

7. A recombinant immune cell expressing an exogenous CAR construct according to claim 6.

8. An immunoconjugate, wherein said immunoconjugate comprises:

(a) an antibody moiety selected from the group consisting of: the antibody of claim 1, or a combination thereof; and

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, or a combination thereof.

9. The immunoconjugate of claim 8, wherein the detectable label comprises a radionuclide, an enzyme.

10. Use of an active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 4, or a combination thereof, wherein the active ingredients are used to prepare a medicament, a test panel or a kit, wherein the test panel or kit is used to detect respiratory syncytial virus; the medicament is for use in the treatment or prevention of respiratory syncytial virus infection.

11. A pharmaceutical composition comprising:

(i) an active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 4, the immune cell of claim 7, the immunoconjugate of claim 8, or a combination thereof; and

(ii) a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of claim 11, wherein said pharmaceutical composition is for preventing or treating respiratory syncytial virus infection.

13. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) the antibody of claim 1;

(2) the recombinant protein of claim 4; and

(3) the CAR construct of claim 6.

14. The polynucleotide of claim 13, wherein the polynucleotide has the sequence shown in SEQ ID No. 8 and SEQ ID No. 9.

15. A vector comprising the polynucleotide of claim 13.

16. A genetically engineered host cell comprising the vector or genome of claim 15 having the polynucleotide of claim 13 integrated therein.

17. A method for the non-therapeutic, non-diagnostic purpose of detecting respiratory syncytial virus in a sample, which method comprises the steps of:

(1) contacting a sample with the antibody of claim 1; and

18. A method for detecting a non-therapeutic, non-diagnostic purpose respiratory syncytial virus fusion protein in a sample, the method comprising the steps of:

(1) contacting a sample with the antibody of claim 1; and

19. The method of claim 18, wherein said respiratory syncytial virus fusion protein is respiratory syncytial virus pre-fusion F protein.

20. A test board, said test board comprising: a substrate and a test strip comprising the antibody of claim 1 or the immunoconjugate of claim 8.

21. A kit comprising:

(1) a first container comprising the antibody of claim 1; and/or

(2) A second container comprising a secondary antibody against the antibody of claim 1;

alternatively, the kit comprises a test plate according to claim 20.

22. A method of producing a recombinant polypeptide, the method comprising:

(a) culturing the host cell of claim 16 under conditions suitable for expression; and

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being the antibody of claim 1 or the recombinant protein of claim 4.