Antigen-binding protein neutralizing respiratory syncytial virus and use thereof

Abstract

Provided are an antigen-binding protein neutralizing respiratory syncytial virus (RSV) and a use thereof. The antigen-binding protein comprises a single-domain antibody containing CDR1, CDR2, and CDR3 antigen complementarity-determining regions, and has the function of exerting virus-neutralizing activity by recognizing and binding to RSV surface proteins. The antigen-binding protein can prevent or treat diseases caused by RSV infection.

Description

Antigen-binding proteins neutralizing respiratory syncytial virus and uses thereof

[0001] This application claims priority to Chinese application No. 202410532831.7, filed on April 29, 2024, entitled “Antigen-binding proteins neutralizing respiratory syncytial virus and uses thereof”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, in particular to an antigen-binding protein neutralizing respiratory syncytial virus and uses thereof. BACKGROUND

[0003] Respiratory syncytial virus (RSV) is one of the most common pathogens causing lower respiratory tract infection in humans, posing a serious health threat to children, the elderly and immunocompromised individuals. After a short replication in the nasal cavity and upper respiratory tract of susceptible individuals, RSV spreads to the bronchial wall and alveoli of the lower respiratory tract, causing intense inflammatory response and acute lower respiratory tract infection (LRTI).

[0004] RSV infection is globally seasonal, and almost all children under the age of 3 have been infected with RSV, and will have repeated infections for their entire lives. The incidence of RSV disease in developing countries is as high as 5.2%, much higher than the 2.4% in developed countries, accounting for 65% of global medical expenses.

[0005] In view of the above serious disease burden, the treatment drugs for RSV-related infections are very limited. So far, the only drug approved for the treatment of RSV infection is ribavirin, but due to safety issues such as teratogenicity and limited efficacy, relevant guidelines no longer recommend it. Therefore, it is of great value to develop new drugs for the treatment and prevention of RSV infection. SUMMARY

[0006] In view of the above defects, the present application provides a single-domain antibody (VHH) in the first aspect, which comprises antigenic complementarity determining regions containing CDR1, CDR2 and CDR3, and has the function of exerting virus neutralization activity by recognizing and binding to RSV surface protein.

[0007] In one or more embodiments, the binding is to RSV Pre-F protein (RSV Pre-F).

[0008] In one or more embodiments, the RSV is selected from RSV A2 strain, RSV 18537 strain, RSV 9320 strain, RSV Long strain.

[0009] The present application also provides an antigen binding protein comprising a single domain antibody (VHH) targeting a RSV surface protein, the complementarity determining regions CDR of the single domain antibody comprising a CDR1, a CDR2 and a CDR3, wherein the CDR1 comprises a sequence as set forth in SEQ ID NO: 6 or 9 or a sequence having at least 80% sequence identity thereto, and / or the CDR2 comprises a sequence as set forth in SEQ ID NO: 7 or 10 or a sequence having at least 80% sequence identity thereto, and / or the CDR3 comprises a sequence as set forth in SEQ ID NO: 8 or a sequence having at least 80% sequence identity thereto.

[0010] In one or more embodiments, the antigen binding molecule specifically binds to RSV.

[0011] In one or more embodiments, the antigen binding molecule specifically binds to a pre-fusion F protein of RSV.

[0012] In one or more embodiments, the CDR1 comprises a sequence as set forth in SEQ ID NO: 6 or a sequence having at least 80% sequence identity thereto, the CDR2 comprises a sequence as set forth in SEQ ID NO: 7 or a sequence having at least 80% sequence identity thereto, and the CDR3 comprises a sequence as set forth in SEQ ID NO: 8 or a sequence having at least 80% sequence identity thereto.

[0013] In one or more embodiments, the CDR1 comprises a sequence as set forth in SEQ ID NO: 9 or a sequence having at least 80% sequence identity thereto, the CDR2 comprises a sequence as set forth in SEQ ID NO: 10 or a sequence having at least 80% sequence identity thereto, and the CDR3 comprises a sequence as set forth in SEQ ID NO: 11 or a sequence having at least 80% sequence identity thereto.

[0014] In one or more embodiments, the CDR1 is as set forth in SEQ ID NO: 6, the CDR2 is as set forth in SEQ ID NO: 7, and the CDR3 is as set forth in SEQ ID NO: 8; or the CDR1 is as set forth in SEQ ID NO: 9, the CDR2 is as set forth in SEQ ID NO: 10, and the CDR3 is as set forth in SEQ ID NO: 11.

[0015] In one or more embodiments, the FR1 of the single domain antibody has the FR1 of the VHH as set forth in SEQ ID NO: 1 or 3. In one or more embodiments, the FR2 of the single domain antibody has the FR2 of the VHH as set forth in SEQ ID NO: 1 or 3. In one or more embodiments, the FR3 of the single domain antibody has the FR3 of the VHH as set forth in SEQ ID NO: 1 or 3. In one or more embodiments, the FR4 of the single domain antibody has the FR4 of the VHH as set forth in SEQ ID NO: 1 or 3.

[0016] In one or more embodiments, the single domain antibody has an amino acid sequence as set forth in SEQ ID NO: 1 or 3, or at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth in SEQ ID NO: 1 or 3.

[0017] In one or more embodiments, the antigen binding protein is a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody, or an antigen binding fragment thereof comprising one, two or more of the single domain antibodies.

[0018] In one or more embodiments, the multivalent single domain antibody or multispecific single domain antibody links multiple single domain antibodies via a linker. The linker consists of 1-15 amino acids selected from G and S.

[0019] In one or more embodiments, the antigen binding fragment of the heavy chain antibody is a VHH.

[0020] In one or more embodiments, the antigen binding protein is a heavy chain antibody comprising a heavy chain constant region having CH2 and CH3.

[0021] In one or more embodiments, the heavy chain antibody is a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody.

[0022] In one or more embodiments, the heavy chain constant region is a human heavy chain constant region, preferably the heavy chain constant region is a heavy chain constant region of human IgGl.

[0023] In one or more embodiments, the CH2 and CH3 of the heavy chain antibody are CH2 and CH3 of human IgGl Fc.

[0024] In one or more embodiments, the heavy chain constant region is as set forth in SEQ ID NO: 5, or at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth in SEQ ID NO: 5.

[0025] In one or more embodiments, the heavy chain antibody is as set forth in (1) SEQ ID NO: 2 or SEQ ID NO: 4, or (2) has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4.

[0026] In one or more embodiments, the antigen binding protein, single domain antibody, or heavy chain antibody is a chimeric or humanized antibody.

[0027] In one or more embodiments, the RSV is selected from the group consisting of RSV A2 strain, RSV 18537 strain, RSV 9320 strain, RSV Long strain.

[0028] The present application also provides a nucleic acid molecule encoding

[0029] (1) the antigen binding protein as described in any of the embodiments herein, and / or

[0030] (2) a complement of (1).

[0031] The present application also provides a nucleic acid construct comprising the nucleic acid molecule as described herein.

[0032] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or an integration vector, preferably, the backbone of the expression vector is pCDNA3.4.

[0033] The present application also provides a host cell selected from the group consisting of:

[0034] (1) expressing and / or secreting the antigen binding protein as described in any of the embodiments herein;

[0035] (2) comprising the nucleic acid molecule as described herein; and / or

[0036] (3) comprising the nucleic acid construct as described herein.

[0037] The present application also provides a method of producing the antigen binding protein as described in any of the embodiments herein, comprising: culturing the host cell as described herein under conditions suitable for producing the antigen binding protein, and optionally purifying the antigen binding protein from the culture.

[0038] The present application also provides a pharmaceutical composition comprising the antigen binding protein, the nucleic acid molecule, the nucleic acid construct, or the host cell as described in any of the embodiments herein, and a pharmaceutically acceptable excipient.

[0039] In one or more embodiments, the pharmaceutical composition is used for preventing or treating a disease associated with RSV infection.

[0040] In one or more embodiments, the disease is a respiratory tract infection including pharyngitis, acute lower respiratory tract infection (LRTI), bronchiolitis, pneumonia, and the like.

[0041] In one or more embodiments, the RSV is selected from the group consisting of RSV A2 strain, RSV 18537 strain, RSV 9320 strain, RSV Long strain.

[0042] The present application also provides the use of the antigen binding protein, nucleic acid molecule, nucleic acid construct or host cell of any one of the embodiments herein in the manufacture of a medicament or kit for preventing or treating a disease associated with RSV infection.

[0043] In one or more embodiments, the disease is acute lower respiratory tract infection (LRTI).

[0044] In one or more embodiments, the RSV is selected from the group consisting of RSV A2 strain, RSV 18537 strain, RSV 9320 strain, RSV Long strain.

[0045] The present application also provides a method of neutralizing RSV, comprising administering to a patient in need thereof a therapeutic dose of the antigen binding protein, nucleic acid molecule, nucleic acid construct, host cell or pharmaceutical composition of any one of the embodiments herein.

[0046] The present application also provides a kit for detecting RSV, comprising the antigen binding protein, nucleic acid molecule, nucleic acid construct or host cell of any one of the embodiments herein.

[0047] In one or more embodiments, the kit further comprises a reagent for detecting the binding of RSV to the antigen binding protein. For example, the reagent for detecting the binding is an enzyme-linked immunoassay.

[0048] In one or more embodiments, the detecting reagent is a detectable label that can be attached to the antigen binding protein, such as biotin. The detectable label is attached to the antigen binding protein or is present separately in the kit.

[0049] The present application also provides a non-diagnostic method of detecting the presence of RSV in a sample, comprising incubating the sample with the antigen binding protein of any one of the embodiments herein, and detecting the binding of RSV to the antigen binding protein, thereby determining the presence of RSV in the sample. The detecting is by enzyme-linked immunoassay.

[0050] The present application also provides the use of the antigen binding protein of any one of the embodiments herein in the manufacture of a kit for detecting RSV in a sample. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1: Result data of 3F5-Fc antigen binding protein binding to RSV Pre-F protein

[0052] Figure 2: Result of detecting the binding activity of hu3F5-Fc antigen binding protein to RSV Pre-F protein

[0053] Figure 3: Result of detecting the affinity of hu3F5-Fc antigen binding protein to RSV Pre-F

[0054] Figure 4: Result of neutralization of different subtypes of respiratory syncytial virus by hu3F5-Fc antigen binding protein

[0055] Figure 5: Detection of lung tissue virus titer of a mouse model of respiratory syncytial virus infection after injection of hu3F5-Fc antigen binding protein DETAILED DESCRIPTION

[0056] There are mainly three proteins on the surface of RSV, fusion protein (F) and attachment protein (G) and small hydrohobic protein (SH). F protein has important biological activity and can mediate the fusion process of RSV with human cell membrane, and plays an important role in the process of infection formation. The isolated antigen binding protein described in the present application can have high affinity in vitro and pharmacodynamic effect of neutralizing RSV virus activity in vitro with respiratory syncytial virus fusion pre-F protein (Pre-F).

[0057] In this paper, RSV is selected from RSV A2 strain, RSV 18537 strain, RSV 9320 strain, RSV Long strain.

[0058] Specifically, the present application uses recombinant expressed RSV Pre-F protein to immunize alpaca, and obtains high-quality single-domain antibody gene library. Then the phage display technology is used to screen the antibody gene library, so as to obtain RSV specific single-domain antibody gene. Then the gene is transferred to Expi293 cells, so as to obtain antigen binding protein with a purity of up to 95%, and then high affinity, high specificity, high functional activity single-domain antibody is identified by ELISA and BLI method.

[0059] An "antigen binding protein" herein in connection with RSV is a protein having the function of recognizing and binding to a surface protein of RSV, preferably, the function of recognizing and binding to a fusion protein F of RSV; more preferably, the function of recognizing and binding to a pre-fusion F protein (RSV Pre-F) of RSV. The antigen binding protein described herein includes, but is not limited to, an antibody, an antigen binding fragment of an antibody, a heavy chain antibody, a single domain antibody, a nanobody, a minibody, an affibody, a target binding region of a receptor, a cell adhesion molecule, a ligand, an enzyme, a cytokine, and a chemokine.

[0060] The antigen binding protein described herein can be a monovalent or multivalent or multispecific antibody comprising one, two or more single domain antibodies described herein, wherein the multispecificity can be against RSV Pre-F and another antigen, or against multiple epitopes of RSV Pre-F.

[0061] The term "antibody" herein includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments, especially antigen binding fragments, such as Fab, F(ab')2 and Fv. In some embodiments herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0062] A basic tetrameric antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites; while IgA antibodies contain 2-5 basic tetrameric units, which can combine with the J chain to form multivalent assemblies. In the case of IgG, a tetrameric unit is typically about 150,000 Daltons. Each light chain is linked to the heavy chain by a covalent disulfide bond, while two heavy chains are linked to each other by one or more disulfide bonds, the number of which depends on the isoform of the heavy chains. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at its N-terminus, followed by three (CH1, CH2, and CH3 for each α and γ chain) and four (CH1, CH2, CH3, and CH4 for the μ and ε isoforms) constant domains (CH), and a hinge region located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. The VL is aligned with the VH, while the CL is aligned with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form interfaces between the variable domains of the light and heavy chains. Pairs of VH and VL together form an antigen-binding site. For information on the structure and properties of different classes of antibodies, see Basic and Clinical Immunology, 8th Edition, edited by Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Light chains from any vertebrate species can be classified into one of two distinct types, called K and λ, based on the amino acid sequence of their constant heavy chain domains. Immunoglobulins can be classified into different classes or isotypes based on the amino acid sequence of their constant heavy chain domains (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with heavy chains called α, δ, ε, γ, and μ, respectively. Based on relatively minor differences in CH sequence and function, the γ and α classes can be further subdivided into subclasses; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0063] A "heavy chain antibody" as described herein is an antibody derived from a Camelid or a Chondrichthyan. In contrast to the 4-chain antibodies described above, heavy chain antibodies lack a light chain and a heavy chain constant region 1 (CH1), and only comprise 2 heavy chains consisting of a variable region (VHH) and other constant regions, the variable region being connected to the constant region by a hinge-like region structure. Each heavy chain of a Camelid heavy chain antibody comprises one variable region (VHH) and two constant regions (CH2 and CH3), and each heavy chain of a Chondrichthyan heavy chain antibody contains one variable region and five constant regions (CH1-CH5). Antigen binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. By fusion with the constant region of human IgG Fc, heavy chain antibodies can have CH2 and CH3 of human IgG Fc.

[0064] As used herein, the terms "single domain antibody", "heavy chain variable region domain of a heavy chain antibody", "VHH", "nanobody" are used interchangeably and all refer to a single domain antibody that specifically recognizes and binds to RSV. A single domain antibody is the variable region of a heavy chain antibody. Typically, a single domain antibody contains three CDRs and four FRs. A single domain antibody is the smallest functional antigen binding fragment. Typically, a single domain antibody is constructed by cloning the variable region of the heavy chain of an antibody naturally lacking a light chain and a heavy chain constant region 1 (CH1) after obtaining the antibody.

[0065] Binding molecules comprising two or more single domain antibodies are multivalent single domain antibodies; binding molecules comprising two or more single domain antibodies of different specificity are multispecific single domain antibodies. Multivalent single domain antibodies or multispecific single domain antibodies comprise multiple single domain antibodies connected by a linker. The linker typically consists of 1-15 amino acids selected from G and S, for example (G 4S)3.

[0066] Herein, heavy chain antibodies and antibodies are intended to distinguish different combinations of antibodies. Due to the similarity of the structures of both, the following description of the structure of antibodies applies to heavy chain antibodies as well, except where indicated otherwise.

[0067] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of an antibody. The variable domains of the heavy chain and light chain can be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding sites.

[0068] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies, and are used to confer antigen binding specificity and particular antigenic specificity of a particular antibody. However, the variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in three segments called hypervariable regions (HVRs) both in the light chain and the heavy chain variable domains, i.e., HCDR1, HCDR2, HCDR3 in the variable region of the heavy chain (which can be referred to as CDR1, CDR2, CDR3 in heavy chain antibodies) and LCDR1, LCDR2, and LCDR3 in the variable region of the light chain. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of the naturally occurring heavy and light chains each comprise four FR regions, FR1, FR2, FR3, and FR4, mostly following a -strand conformation, connected by three HVRs. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies. In general, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains do not participate directly in binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0069] The "Fc region" (fragment, crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or to the first component (Clq) of the classical complement system. In IgG antibody isotypes, the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of each of the two heavy chains of the antibody. While the boundaries of the Fc region of immunoglobulin heavy chains can vary, the human IgG heavy chain Fc region is usually defined as spanning from an amino acid residue located at C226 or P230, to the carboxy-terminus of the heavy chain, where the numbering is according to the EU index as in Kabat. As used herein, the Fc region can be a native sequence Fc or variant Fc.

[0070] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding and / or variable region of the intact antibody. Antibody fragments preferably are antigen binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragments capable of increasing the half-life of the antibody by chemical modification or by incorporation into a liposome. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, which contains the constant region of the antibody. The Fab fragment is composed of an entire light chain and the variable region of a heavy chain, and one heavy chain constant region domain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen combining site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment containing both light chains and the hinge region of the heavy chain. Fab' fragments differ from Fab fragments by having an additional few residues at the carboxy terminus of the CH1 domain, including the one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined in the Fc region, which is also the region recognized by Fc receptors (FcR) found on certain types of cells.

[0071] "Fv" is the minimum antibody fragment that contains a complete antigen- recognition and binding site. This fragment consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. From the folding of these two domains emanate the three pairs of disulfide bonds that give rise to the six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues that form the antigen binding site. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. "Single-chain Fv" also is abbreviated "sFv" or "scFv" are antibody fragments that comprise the VHand VLdomains of antibody, linked by a synthetic linker from one to another so as to form a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VHand VLdomains, which enables the sFv to form the desired structure for antigen binding. Illustrative of the antibodies described herein are the sequences shown in the following table:

[0072] Table 1

[0073] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translation modifications (e.g., isomerization, amidation) that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, which does not contain 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. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including, for example, the hybridoma method, the phage display method, recombinant DNA methods, and the technique of generating human or human-like antibodies in transgenic animals, single-cell sequencing methods.

[0074] "Monoclonal antibodies" herein also include "chimeric" antibodies in which the variable region is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0075] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain sequences derived from non-human immunoglobulin. Thus, "humanized antibodies" typically refer to non-human antibodies in which the variable domain framework regions have been exchanged for sequences found in human antibodies. Typically, in humanized antibodies, the entire antibody (except the CDRs) is encoded by human-derived polynucleotides or is identical to such antibodies except for the CDRs. The CDRs (some or all of which are encoded by nucleic acids derived from a non-human organism) are grafted into the beta-sheet framework of a human antibody variable region to create an antibody whose specificity is dictated by the grafted CDRs. Methods for creating such antibodies are well known in the art, for example, using mice with genetically engineered immune systems. In the present application, antibodies, single domain antibodies, heavy chain antibodies, etc. include humanized variants of each of the recited antibodies.

[0076] "Human antibodies" refer to antibodies having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the techniques disclosed herein for generating human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries.

[0077] In the present application, the single-domain antibody has CDR1 as shown in SEQ ID NO: 6 or 9, CDR2 as shown in SEQ ID NO: 7 or 10, and CDR3 as shown in SEQ ID NO: 8 or 11.

[0078] The FR1, FR2, FR3, and FR4 of the single-domain antibody described herein can be selected from the FR1, FR2, FR3, and FR4 of the single-domain antibody as shown in SEQ ID NO: 1 or 3. In one or more embodiments, the FR1 to FR4 of the single-domain antibody are as shown in any one of the rows of Table 1. Preferably, the amino acid sequence of the single-domain antibody described herein is as shown in SEQ ID NO: 1.

[0079] When the single-domain antibody described herein is linked to a heavy chain constant region, the heavy chain constant region can be the constant region of a camelid or human heavy chain antibody, comprising CH2 and CH3. Preferably, the antibody constant region is derived from the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, more preferably from the constant region of any one of IgGl, IgG2, IgG3, IgG4. In one or more embodiments, the heavy chain constant region is CH2 and CH3 of human IgG Fc, as shown in SEQ ID NO: 5. The heavy chain antibody is as shown in (1) SEQ ID NO: 2 or SEQ ID NO: 4, or (2) has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0080] The present application also includes derivatives and analogs of the various antibodies (e.g., single-domain antibodies, heavy chain antibodies or antigen-binding fragments thereof, multivalent single-domain antibodies, multispecific single-domain antibodies, antibodies or antigen-binding fragments thereof) described. "Derivatives" and "analog" refer to polypeptides that substantially retain the same biological function or activity of the various antibodies of the present application. Derivatives or analogs of the present application can be (i) polypeptides having substitution groups at one or more amino acid residues, or (ii) polypeptides formed by fusing the mature polypeptide to another compound, such as a compound that prolongs the half-life of the polypeptide, e.g., polyethylene glycol, or (iii) polypeptides formed by fusing additional amino acid sequences to the polypeptide sequence, such as a leader or secretion sequence, or a sequence or protein that is used to purify the polypeptide, or a proprotein sequence, or a fusion protein with a 6*His tag. These derivatives and analogs are within the scope of those skilled in the art in light of the teachings herein.

[0081] Without materially affecting the activity of the antibody, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids can be altered in the sequences of the present application by one skilled in the art to obtain variants of the sequences of the antibody or functional fragment thereof. These variants include, but are not limited to, deletion, insertion, and / or substitution of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, as well as addition of one or several (typically within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or the N-terminus. In the art, conservative substitutions with similar or identical properties are often made without altering the function of the protein. For example, substitutions of amino acids with similar properties in the FR and / or CDR regions of the variable region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues can or can not be encoded by the genetic code. For another example, addition of one or several amino acids at the C-terminus and / or the N-terminus also typically does not alter the function of the protein. All of these are considered to be within the scope of the present application.

[0082] Variants of the various antibodies described herein include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to the DNA encoding the various antibodies of the present application under conditions of high or low stringency, and polypeptides or proteins obtained using antisera against the various antibodies of the present application.

[0083] In some embodiments, the sequences of the variants of the present application can have at least 95%, 96%, 97%, 98%, or 99% identity to the sequences from which they are derived. The sequence identity of the sequences of the present application can be measured using sequence analysis software, for example, the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. The present application also includes molecules having the variable region of the heavy chain of an antibody with CDRs, as long as the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0084] The antibodies of the present application can be prepared using methods conventional in the art, such as the hybridoma technology well known in the art. The single domain antibodies and heavy chain antibodies of the present application can be prepared using methods conventional in the art, such as the phage display technology well known in the art. Alternatively, the various antibodies of the present application can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the various antibodies of the present application. Transformation can be performed using any known method, for example including packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene- mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of the DNA into nuclei, etc. Mammalian cell lines useful as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantially the RSV Pre-F binding properties.

[0085] The present application also provides polynucleotides encoding the above-mentioned antibodies or fragments thereof. The polynucleotides of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand. The present application also includes degenerate variants of the polynucleotide sequence encoding the fusion protein, i.e., nucleotide sequences that encode the same amino acid sequence but differ in nucleotide sequence.

[0086] As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be made that all encode the antibodies or antigen-binding fragments thereof of the present application. Thus, given a particular amino acid sequence, one skilled in the art can make any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. Therefore, the present application also relates to polynucleotides that hybridize to the above-described polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application specifically relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing under lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0087] The nucleotide full-length sequences of the various antibodies of the present application or fragments thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis. One possible method is to synthesize the relevant sequences by artificial synthesis, especially when the length of the fragments is relatively short. Generally, longer fragments can be obtained by first synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6*His) to form a fusion protein.

[0088] Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning them into vectors, which are then introduced into cells, and then the relevant sequences are isolated from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules that exist in isolated form. At present, it is possible to obtain the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present application by chemical synthesis.

[0089] Accordingly, the present application also relates to nucleic acid constructs comprising the appropriate DNA sequences as described above and an appropriate promoter or control sequence, such as expression and recombination vectors. These vectors can be used to transform appropriate host cells to enable them to express proteins. The vectors typically contain sequences for plasmid propagation and for cloning and expression of foreign nucleotide sequences. The sequences, which in certain embodiments are collectively referred to as "flanking sequences," typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of nucleic acids encoding antibodies to be expressed, and optional marker elements.

[0090] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples of such host cells are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Sf9 cells; and eukaryotic cells, such as CHO, COS7, 293 cells, etc.

[0091] In certain embodiments, the host cell can be various functional cells known in the art, such as various killer cells, including but not limited to cytokine-induced killer cells (CIK), dendritic cell-stimulated cytokine-induced killer cells (DC-CIK), cytotoxic T lymphocytes (CTL), gamma delta T cells, natural killer cells (NK), tumor infiltrating lymphocytes (TIL), lymphokine-activated killer cells (LAK), CD3 AK cells (anti-CD3 mAb killer cells), and CAR-T / TCR-T cells. In certain embodiments, the killer cell is a T cell or a NK cell. Exemplary NK cells include but are not limited to primary NK cells, NK cell lines (such as NK92), and NKT cells. In certain embodiments, the NK cell is a primary NK cell. Exemplary T cells include but are not limited to peripheral blood T lymphocytes, cytotoxic killer T cells (CTL), helper T cells, suppressor / regulatory T cells, gamma delta T cells, and T cells of mixed cell populations such as cytokine-induced killer cells (CIK), tumor infiltrating lymphocytes (TIL), etc. In certain embodiments, the T cell is a peripheral blood T lymphocyte and a T cell derived from TIL.

[0092] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known in the art. When the host is a prokaryote, such as E. coli, the transformation of the host cell can be effected by the use of techniques such as CaCl2treatment. Another method is the use of MgCl2. If necessary, the transformation can be performed by electroporation. When the host is a eukaryote, the transformation can be effected by the use of techniques such as calcium phosphate precipitation, conventional mechanical methods such as microinjection, electroporation, lipofection, etc.

[0093] The resulting transformant can be cultured in conventional media using conventional techniques, the polypeptide encoded by the gene of the application being expressed. The medium used in the culture will depend upon the host cell used. The culture is conducted under conditions suitable for growth of the host cell. When the host cell has reached an appropriate cell density, the selected promoter is induced by the appropriate method (e.g., temperature shift or chemical induction). The cells are then cultured for an additional period.

[0094] The polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted from the cell. If desired, 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 these methods include, but are not limited to, conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0095] All aspects of the various antibodies described herein can be used to prepare a medicament for preventing or treating a disease associated with RSV infection.

[0096] The pharmaceutical compositions herein contain the binding molecules described herein, and a pharmaceutically acceptable carrier, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives, and / or adjuvants. The carrier preferably is nontoxic to recipients at the dosages and concentrations employed. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition can contain substances that improve, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeability of the composition. Such substances are known in the art. The optimal pharmaceutical composition will be determined by one of skill in the art, taking into account the intended route of administration, mode of delivery, and desired dosage.

[0097] Pharmaceutical compositions for in vivo administration are typically provided in the form of a sterile preparation. Sterilization is accomplished by filtration through a sterile filtration membrane. This method can be used in sterilizing the composition when it is lyophilized, either prior to or following lyophilization and reconstitution. The pharmaceutical compositions of the present application can be selected for parenteral delivery. Compositions for parenteral administration can be stored in a lyophilized form or in solution. They are prepared for administration by conventional means, such as with physiological saline or aqueous dextrose and other auxiliary agents. Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. Alternatively, the compositions can be selected for delivery by inhalation or through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising the antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome vehicles, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art.

[0098] Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, or in a dehydrated or lyophilized form. The compositions can be stored in unit or multi-dose containers, for example, as a ready-to-use form or in a form to be reconstituted prior to administration (e.g., lyophilized). The present application also provides kits for producing single dose administration units. The kits of the present application can each contain a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of the present application, kits containing single and multiple chamber pre-filled syringes (e.g., liquid syringes and lyophilized syringes) are provided.

[0099] The present application also provides methods of preventing or treating a disease associated with RSV infection by administering a binding molecule according to any of the embodiments of the present application or a pharmaceutical composition thereof. As used herein, the terms "subject," "individual," "patient," and "subject" are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g., a rat, a mouse, a dog, a cat, a rabbit, etc.), and most preferably a human.

[0100] The therapeutically effective amount of a pharmaceutical composition containing a binding molecule of the present application to be employed will depend, for example, on the therapeutic effect desired and the target. Those skilled in the art will appreciate that appropriate dosage levels for treatment will vary depending on the molecule delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and / or condition (age and general health) of the patient. In certain embodiments, the clinician can titer the dosage and modify the route of administration as required by a particular patient. For example, about 10 micrograms per kilogram body weight to about 50 milligrams per kilogram body weight per day.

[0101] The frequency of dosage will depend on the pharmacokinetic parameters of the binding molecule in the formulation being used. Normally, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, or as two or more doses (which can or can not contain the same amount of the desired molecule) over time, or by an implantation device, or by a sustained-release system.

[0102] The route of administration of the pharmaceutical composition is according to known methods, for example, by oral, by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal or intralesional routes of injection; by sustained release systems or by implantation devices.

[0103] The present application also provides a test kit comprising a single-domain antibody recognizing a RSV protein, a RSV binding molecule, a nucleic acid molecule, a nucleic acid construct or a host cell, a lysis medium for lysing the sample, general reagents and buffers required for the detection, such as various buffers, detection labels, detection substrates, etc.

[0104] Particular embodiments

[0105] Item 1. An antigen binding protein comprising a single-domain antibody, the complementarity determining regions CDR of said single-domain antibody comprising a CDR1, a CDR2 and a CDR3, wherein,

[0106] the CDR1 comprises a sequence as set forth in SEQ ID NO: 6 or 9 or a sequence having at least 80% sequence identity thereto, and / or

[0107] the CDR2 comprises a sequence as set forth in SEQ ID NO: 7 or 10 or a sequence having at least 80% sequence identity thereto, and / or

[0108] the CDR3 comprises a sequence as set forth in SEQ ID NO: 8 or 11 or a sequence having at least 80% sequence identity thereto,

[0109] Preferably, the antigen binding protein targets a RSV surface protein; more preferably, the antigen binding protein targets a RSV pre-fusion F protein.

[0110] Item 2. The antigen binding protein according to item 1, characterized in that the CDR1 comprises a sequence as set forth in SEQ ID NO: 6 or a sequence having at least 80% sequence identity thereto, the CDR2 comprises a sequence as set forth in SEQ ID NO: 7 or a sequence having at least 80% sequence identity thereto, and the CDR3 comprises a sequence as set forth in SEQ ID NO: 8 or a sequence having at least 80% sequence identity thereto,

[0111] Preferably, CDR1 comprises the sequence set forth in SEQ ID NO: 9 or a sequence having at least 80% sequence identity thereto, CDR2 comprises the sequence set forth in SEQ ID NO: 10 or a sequence having at least 80% sequence identity thereto, and CDR3 comprises the sequence set forth in SEQ ID NO: 11 or a sequence having at least 80% sequence identity thereto,

[0112] Preferably, CDR1 is as set forth in SEQ ID NO: 6, CDR2 is as set forth in SEQ ID NO: 7, and CDR3 is as set forth in SEQ ID NO: 8; or CDR1 is as set forth in SEQ ID NO: 9, CDR2 is as set forth in SEQ ID NO: 10, and CDR3 is as set forth in SEQ ID NO: 11.

[0113] Item 3. The antigen binding protein of item 2, characterized in that,

[0114] FR1 of the single domain antibody has FR1 of the VHH set forth in SEQ ID NO: 1 or 3, and / or

[0115] FR2 of the single domain antibody has FR2 of the VHH set forth in SEQ ID NO: 1 or 3, and / or

[0116] FR3 of the single domain antibody has FR3 of the VHH set forth in SEQ ID NO: 1 or 3, and / or

[0117] FR4 of the single domain antibody has FR4 of the VHH set forth in SEQ ID NO: 1 or 3,

[0118] Preferably, the single domain antibody has an amino acid sequence as set forth in SEQ ID NO: 1 or 3, or has at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1 or 3.

[0119] Item 4. The antigen binding protein of item 3, characterized in that the antigen binding protein is a monovalent or multivalent single domain antibody, a multispecific single domain antibody or a heavy chain antibody comprising one, two or more of the single domain antibodies,

[0120] Preferably, the antigen binding protein has one or more features selected from the group consisting of:

[0121] the multivalent single domain antibody or multispecific single domain antibody links multiple single domain antibodies via a linker, preferably the linker consists of 1-15 amino acids selected from the group consisting of G and S,

[0122] The antigen binding protein is a heavy chain antibody comprising a heavy chain constant region having CH2 and CH3; preferably, the heavy chain constant region is a human heavy chain constant region; more preferably, the heavy chain constant region is a heavy chain constant region of human IgGl; further preferably, the CH2 and CH3 of the heavy chain antibody are CH2 and CH3 of human IgGl Fc.

[0123] Item 5. A nucleic acid molecule comprising:

[0124] (1) a coding sequence of the antigen binding protein according to any one of items 1-4, and / or

[0125] (2) a complement of (1).

[0126] Item 6. A nucleic acid construct comprising the nucleic acid molecule according to item 5,

[0127] Preferably, the nucleic acid construct is a cloning vector, an expression vector or an integration vector.

[0128] Item 7. A host cell, the host cell:

[0129] (1) expressing and / or secreting the antigen binding protein according to any one of items 1-4;

[0130] (2) comprising the nucleic acid molecule according to item 5; and / or

[0131] (3) comprising the nucleic acid construct according to item 6.

[0132] Item 8. A method of producing the antigen binding protein according to any one of items 1-4, comprising: culturing the host cell according to item 7 under conditions suitable for production of the antigen binding protein, and optionally purifying the antigen binding protein from the culture.

[0133] Item 9. A pharmaceutical composition comprising the antigen binding protein according to any one of items 1-4, the nucleic acid molecule according to item 5, the nucleic acid construct according to item 6 or the host cell according to item 7, and a pharmaceutically acceptable excipient,

[0134] Preferably, the pharmaceutical composition is for preventing or treating a disease associated with RSV infection.

[0135] Item 10. Use of the antigen binding protein according to any one of items 1-4, the nucleic acid molecule according to item 5, the nucleic acid construct according to item 6 or the host cell according to item 7 in the manufacture of a medicament or a kit for preventing or treating a disease associated with RSV infection,

[0136] Preferably, the disease comprises respiratory tract infection including pharyngitis, acute lower respiratory tract infection (LRTI), bronchiolitis, pneumonia.

[0137] Item 11. A kit for detecting RSV, comprising the antigen binding protein according to any one of items 1 to 4, the nucleic acid molecule according to item 5, the nucleic acid construct according to item 6 or the host cell according to item 7,

[0138] Preferably, the kit further comprises a detection reagent for detecting the binding of RSV to the antigen binding protein,

[0139] More preferably, the detection reagent is a detectable label capable of binding to the antigen binding protein, the detectable label being attached to the antigen binding protein or being present separately in the kit.

[0140] Item 12. A non-diagnostic method for detecting the presence of RSV in a sample, the method comprising: incubating the sample with the antigen binding protein according to any one of items 1 to 4, and detecting the binding of RSV to the RSV binding molecule, thereby determining the presence of RSV in the sample, preferably the detection is by enzyme-linked immunoassay.

[0141] Item 13. Use of the antigen binding protein according to any one of items 1 to 4 in the manufacture of a kit for detecting RSV in a sample.

[0142] The application is further illustrated by the following examples without thereby limiting the application to the examples described. The experimental methods in the following examples, where no specific conditions are indicated, are carried out according to routine methods and conditions, or according to the instructions of the commercial suppliers.

[0143] Example

[0144] Example 1: Immunization of an alpaca with recombinantly expressed RSV Pre-F protein

[0145] One alpaca was immunized with recombinantly expressed RSV Pre-F protein, the immunization interval was 21 days, and 10 days after the last immunization, peripheral blood was collected and serum was isolated. The immunization effect was detected by ELISA.

[0146] Example 2: Isolation of PBMC and construction of a phage display library displaying RSV Pre-F antigen binding proteins on the surface

[0147] PBMC were prepared from 100 ml peripheral blood collected from the immunized alpaca using EasySep TMHuman Biotin Positive Selection Kit II kit (STEMCELL) was used to isolate plasma cells in PBMC, and after isolation, the plasma cells were lysed using Trizol lysis solution, and total RNA was extracted using an RNA extraction kit (TIANGEN). After reverse transcription of the extracted total RNA using PrimeScript TM II 1st Strand eDNA Synthesis Kit kit (Takara), cDNA library preparation was performed, and further PCR technology was used to amplify the VHH antibody fragment. The pDisplay vector and the above obtained VHH PCR gel recovery product were digested with restriction endonuclease SfilI (Takara), and T4 ligase was used for ligation. The ligation product was electrotransformed into E. coli and a phage library displaying VHH antibodies was established, and the library capacity and diversity were calculated.

[0148] Example 3: Phage display library panning of RSV Pre-F antigen binding proteins

[0149] The recombinantly expressed RSV Pre-F protein was coated onto an enzyme-labeled plate for overnight coating at 4°C, then the phage display library was added to the enzyme-labeled plate coated with RSV Pre-F protein, and after 1 h of incubation at 4°C, the recombinant phage bound to RSV Pre-F protein was eluted using TEA buffer and infected with E. coli ER2738 in the logarithmic growth phase for amplification. Through 3-4 rounds of panning, the positive E. coli clones were enriched.

[0150] Example 4: Preparation of phage supernatant for screening

[0151] The positive E. coli clones obtained by panning were picked and cultured in a 96-well deep well plate to an OD 600 The value was about 0.5, helper phage M13KO7 was added, and superinfection was performed at 37°C for 15 min; after superinfection, the bacterial solution was placed in a shaker at 37°C, 225 rpm for 45 min; after incubation, the bacterial solution was centrifuged to discard the supernatant, resuspended with 2YT-AK medium, and incubated at 30°C, 210 rpm overnight. The bacterial solution was centrifuged at 4000 rpm for 10 min, and the phage supernatant for screening was obtained.

[0152] Example 5: Obtaining antigen binding protein sequences targeting RSV Pre-F using enzyme-linked immunoassay (ELISA) technology and DNA sequencing

[0153] RSV Pre-F protein (1 μg / mL, 100 μL / well) was coated to the bottom of a 96-well enzyme plate using CBS buffer, incubated at 37°C for 2 h; the enzyme plate was washed with PBST, and blocked with MPBS at 4°C overnight (an additional blank plate was blocked at the same time as a blank control); the blocking solution was discarded, and the plate was washed with 200 μL PBST for 4 times, then 50 μL PBST was added to each well, and 50 μL centrifuged phage supernatant was added to each well, and incubated at 4°C for 1 h; the supernatant was discarded, and the plate was washed with PBST for 5 times; HRP-Anti M13 secondary antibody (iCareAb) was diluted with PBST, 100 μL was added to each well, and incubated at 4°C for 45 min, then the secondary antibody was washed away, the plate was washed with PBST for 5 times, TMB was used for color development at room temperature for 10 min, and the reaction was terminated with hydrochloric acid, and the OD value was detected 450 The clones with S / N ratio greater than 4 were selected, and the corresponding bacterial solution was used for sequencing, and the variable region amino acid sequence of the antigen binding protein of 3F5 was finally obtained, and the sequence is shown as SEQ ID NO: 1.

[0154] Example 6: Expression and purification of Fc-fused antigen binding protein in Expi293 cells

[0155] The variable region coding sequence of 3F5 obtained by sequencing analysis was fused with the Fc fragment of human immunoglobulin γ1 (IgG1) and expressed in the vector pcDNA3.4. PEI was used to transfect Expi293 cells for recombinant expression, and after 7 days, the cell supernatant was collected and purified using Protein A magnetic beads, and the antigen binding protein with a purity of more than 95% was collected, and the 3F5-Fc antigen binding protein was obtained, and the sequence is shown as SEQ ID NO: 2.

[0156] Example 7: ELISA method for detecting the binding of antigen binding protein to RSV Pre-F

[0157] The RSV Pre-F protein was diluted with PBS buffer to 1 μg / ml, 100 μl per well, coated on a 96-well plate (Thermo) at 4°C overnight; the next day, the 96-well plate was removed, and the plate was washed with PBST (containing 0.5% PBS) for 1 min each time, and the residual water was completely shaken off. 200 μl of PBST containing 5% BSA was added to the sample well, and the plate was sealed at 37°C for 1 h; then the plate was washed with PBST, and the water in the well was shaken off. 100 μl of the sample to be tested (3F5-Fc antigen binding protein expressed and purified above) was added to the 96-well plate, and the plate was incubated at 4°C overnight. After the 96-well plate was removed, the plate was washed with PBST, and 100 μL of goat anti-human IgG secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, HRP, Thermo, 31413) was added to each well, and the plate was incubated at 37°C for 1 h. Then the plate was washed with PBST for 5 times, 100 μL of substrate solution (TMB-ELISA Substrate Solution, Invitrogen) was added to each well, and the plate was incubated at 37°C for 15 min; 1 mol of phosphoric acid (100 μL) was added to each well to stop the reaction, and the absorbance was detected at 450 nm wavelength on an enzyme-labeled instrument (SpectraMax190, Molecular Devices), with PBS as a negative control.

[0158] The results of the binding of the 3F5-Fc antigen binding protein to the RSV Pre-F protein are shown in FIG. 1, and the results show that the antigen binding protein has strong binding ability to the RSV Pre-F protein, and the EC 50 Data, the EC 50 value of the 3F5-Fc antigen binding protein is 3.929 ng / mL.

[0159] Example 8: Humanization of antigen binding protein

[0160] The humanized variable region amino acid sequence of hu3F5 was obtained by selecting the closest human germline sequence to the camel antibody as a template through NCBI database alignment (https: / / www.ncbi.nlm.nih.gov / igblast / ), humanizing the antigen binding protein variable region coding sequence of 3F5, and optimizing the post-translational modification sites. The sequence is shown as SEQ ID NO: 3. The coding sequence of hu3F5 was fused with the Fc fragment of human immunoglobulin γ1 (IgG1), and subcloned into the expression vector PCDNA3.4. Recombinant expression was performed by transfecting Expi293 cells with PEI. After 7 days, the cell supernatant was collected, and the antigen binding protein with a purity of more than 95% was collected by Protein A magnetic bead purification. The hu3F5-Fc antigen binding protein was obtained, and the sequence is shown as SEQ ID NO: 4.

[0161] The binding activity of the above antigen binding protein to RSV Pre-F was detected by the method of Example 7. The experimental results are shown in FIG. 2. The above antigen binding protein has strong binding ability to RSV Pre-F protein, and the EC 50 Data, the EC 50 value of hu3F5-Fc antigen binding protein is 19.13 ng / mL.

[0162] Example 9: BLI method for detecting the binding of antigen binding protein to RSV Pre-F

[0163] The kinetic equilibrium constant of the antigen binding protein and RSV Pre-F was determined by biofilm layer interference technology (BLI). First, the concentration of RSV Pre-F protein was diluted to 200 nM using PBST (containing 0.05% Tween 20), and the hu3F5-Fc antigen binding protein to be tested was diluted to 250 nM, then 2-fold dilution was performed in 7 gradients, and the affinity between the antigen binding protein and RSV Pre-F was detected at 25°C using Octet (Sartorius, OCTET-RED384). The RSV Pre-F protein was solidified to the Ni-NTA probe, the solidification time was set to 5 min, the threshold was set to 0.6 nm, and then it was balanced in PBST buffer for 60 s. Subsequently, the Ni-NTA probe with solidified RSV Pre-F protein entered the gradient-diluted antigen binding protein to be tested, and the binding threshold reached more than 0.7 nm, and then it was balanced in PBST buffer for dissociation for 600 s. After the measurement, the regeneration step was repeated 5 times for 30 s in 10 mM glycine and PBST buffer. The Octet high-throughput analysis software Octet Analysis Studio 12.1 was used to perform curve fitting analysis and calculate the binding rate constant K on , the dissociation rate constant K off , and the equilibrium dissociation constant K D .

[0164] The results of the affinity detection of hu3F5-Fc antigen binding protein and RSV Pre-F are shown in FIG. 3 and Table 2, and the results show that the above antigen binding protein has strong affinity with RSV Pre-F protein, and the K D value of hu3F5-Fc antigen binding protein is <1.0E-12 M.

[0165] Table 2

[0166] RSV virus neutralization experiments were performed by Nanjing Novozyme Biotech Co., Ltd. to evaluate the activity of the antigen binding protein in neutralizing multiple RSV strains (RSV A2 strain, RSV 18537 strain, RSV 9320 strain, and RSV Long strain): the density of digested HEP2 cells was adjusted to 2-3 × 10 5 / mL, 100 μL per well was inoculated into a 96-well plate, and it was cultured at 37°C, 5% CO2 overnight. The next day, when the confluence reached about 90%, the experiment could be started. 2 × 10 4PFU / mL RSV and antigen binding protein were mixed in a certain ratio into 96-well plates (Nest), and incubated at 37°C, 5% CO2 for 1 h. The sample was added to the cells prepared in advance, and incubated at 37°C, 5% CO2 for 2 h. The neutralization product was aspirated, 100 μL of DMEM medium containing 2% FBS was added to each well, and incubation was continued for 21-22 h. The cell supernatant was discarded, the cells were fixed with 4% paraformaldehyde for 10 min, and then the fixing solution was discarded, and the cells were washed once with PBS. 100 μL of 2% BSA diluent was added to each well, and incubated at 37°C, 5% CO2 for 30 min. Then the blocking solution was discarded, 50 μL of fluorescently labeled detection antibody was added to each well, and incubated at 37°C, 5% CO2 for 1 h; then washed with PBST for 3 times, the 96-well plate was shaken well, and then read with a fluorescent (enzyme-linked) immunospot analyzer (CTL, S6 Ultra M2).

[0167] The results of the above RSV virus neutralization experiment for detecting the neutralization of hu3F5-Fc antigen binding protein on different strains are shown in Figure 4, and the results show that hu3F5-Fc antigen binding protein has good virus neutralization effect on RSV A2 strain, RSV 18537 strain, RSV 9320 strain and RSV Long strain; the IC 50 values of hu3B4-Fc antigen binding protein for neutralizing RSV A2 strain, RSV 18537 strain, RSV 9320 strain and RSV Long strain are 0.586 ng / mL, 4.744 ng / mL, 9.146 ng / mL and 126.7 ng / mL, respectively. 50 50 50 50

[0168] Example 11: Evaluation of in vivo pharmacodynamic effect of anti-RSV infection

[0169] A BALB / c mouse infection model was established by intranasal RSV (RSV A2 strain, inoculation amount: ~10 5 CFU / 50 μl / mouse), and hu3F5-Fc antigen binding protein (2 mg / kg) was injected through the tail vein (PBS buffer as negative control); the mice were sacrificed on the 5th day after infection with respiratory syncytial virus, and the lung tissue was taken for virus titer detection.

[0170] ​​​​The results of the above experiment are shown in Figure 5. The results show that the hu3F5-Fc antigen binding protein described herein has a strong pharmacodynamic effect against respiratory syncytial virus infection. After 5 days of tail vein injection of hu3F5-Fc antigen binding protein (2 mg / kg), the lung tissue virus titer of the respiratory syncytial virus infected mouse model was lower than the detection limit (LLOQ, lowest limit of detection).

[0171] Part of the sequence

[0172] SEQ ID NO: 1_3F5 variable region amino acid sequence

[0173] SEQ ID NO: 3_hu3F5 variable region amino acid sequence

Claims

1. An antigen-binding protein comprising a single-domain antibody, wherein the complementarity-determining region (CDR) of the single-domain antibody comprises CDR1, CDR2, and CDR3, wherein, CDR1 contains the sequence shown in SEQ ID NO: 6 or 9, or a sequence having at least 80% sequence identity with it, and / or CDR2 contains the sequence shown in SEQ ID NO: 7 or 10 or a sequence having at least 80% sequence identity with it, and / or CDR3 contains the sequence shown in SEQ ID NO: 8 or 11, or a sequence having at least 80% sequence identity with it. Preferably, the antigen-binding protein targets RSV surface proteins; more preferably, the antigen-binding protein targets the RSV pre-fusion F protein.

2. The antigen-binding protein of claim 1, characterized in that CDR1 comprises the sequence shown in SEQ ID NO: 6 or a sequence having at least 80% sequence identity with it, CDR2 comprises the sequence shown in SEQ ID NO: 7 or a sequence having at least 80% sequence identity with it, and CDR3 comprises the sequence shown in SEQ ID NO: 8 or a sequence having at least 80% sequence identity with it. Preferably, CDR1 comprises the sequence shown in SEQ ID NO: 9 or a sequence having at least 80% sequence identity with it, CDR2 comprises the sequence shown in SEQ ID NO: 10 or a sequence having at least 80% sequence identity with it, and CDR3 comprises the sequence shown in SEQ ID NO: 11 or a sequence having at least 80% sequence identity with it. Preferably, CDR1 is as shown in SEQ ID NO: 6, CDR2 is as shown in SEQ ID NO: 7, and CDR3 is as shown in SEQ ID NO: 8; or CDR1 is as shown in SEQ ID NO: 9, CDR2 is as shown in SEQ ID NO: 10, and CDR3 is as shown in SEQ ID NO:

11.

3. The antigen-binding protein as described in claim 2, characterized in that, The FR1 of the single-domain antibody has the FR1 of VHH shown in SEQ ID NO: 1 or 3, and / or The FR2 of the single-domain antibody has the FR2 of VHH as shown in SEQ ID NO: 1 or 3, and / or The FR3 of the single-domain antibody has the FR3 of VHH shown in SEQ ID NO: 1 or 3, and / or The FR4 of the single-domain antibody has the FR4 of VHH shown in SEQ ID NO: 1 or 3. Preferably, the single-domain antibody has an amino acid sequence as shown in SEQ ID NO: 1 or 3, or has at least 80% sequence identity with an amino acid sequence as shown in SEQ ID NO: 1 or 3.

4. The antigen binding protein of claim 3, wherein The antigen-binding protein is a monovalent or multivalent single-domain antibody, a multispecific single-domain antibody, or a heavy chain antibody comprising one, two, or more of the aforementioned single-domain antibodies. Preferably, the antigen-binding protein has one or more of the following characteristics: said multivalent single-domain antibody or multispecific single-domain antibody connects multiple single-domain antibodies via a linker, preferably, said linker consists of 1-15 amino acids selected from G and S, said antigen-binding protein is a heavy chain antibody comprising a heavy chain constant region with CH2 and CH3; preferably, said heavy chain constant region is a human heavy chain constant region; more preferably, said heavy chain constant region is a heavy chain constant region of human IgG1; further preferably, CH2 and CH3 of said heavy chain antibody are CH2 and CH3 of human IgG1 Fc.

5. A nucleic acid molecule comprising: (1) a coding sequence of the antigen-binding protein of any one of claims 1-4, and / or (2) a complementary sequence of (1).

6. A nucleic acid construct comprising the nucleic acid molecule of claim 5, preferably, said nucleic acid construct is a cloning vector, an expression vector or an integration vector.

7. A host cell, said host cell: (1) expresses and / or secretes the antigen-binding protein of any one of claims 1-4; (2) comprises the nucleic acid molecule of claim 5; and / or (3) comprises the nucleic acid construct of claim 6.

8. A method of making an antigen binding protein according to any one of claims 1-4, comprising: culturing the host cell of claim 7 under conditions suitable for producing the antigen-binding protein, and optionally purifying the antigen-binding protein from the culture.

9. A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1-4, the nucleic acid molecule of claim 5, the nucleic acid construct of claim 6 or the host cell of claim 7, and a pharmaceutically acceptable excipient, preferably, said pharmaceutical composition is used for preventing or treating a disease associated with RSV infection.

10. Use of the antigen-binding protein of any one of claims 1-4, the nucleic acid molecule of claim 5, the nucleic acid construct of claim 6 or the host cell of claim 7 in the manufacture of a medicament or a kit for preventing or treating a disease associated with RSV infection, preferably, said disease comprises respiratory tract infection including pharyngitis, acute lower respiratory tract infection (LRTI), bronchiolitis, pneumonia.

11. A kit for detecting RSV, comprising the antigen-binding protein of any one of claims 1-4, the nucleic acid molecule of claim 5, the nucleic acid construct of claim 6 or the host cell of claim 7, preferably, said kit further comprises a detection reagent for detecting the binding of RSV to said antigen-binding protein, more preferably, said detection reagent is a detectable label capable of binding to said antigen-binding protein, said detectable label being attached to said antigen-binding protein or being present separately in the kit.

12. A non-diagnostic method of detecting the presence of RSV in a sample, the method comprising: incubating the antigen-binding protein of any one of claims 1-4 with a sample, and detecting the binding of RSV to the RSV-binding molecule, thereby determining the presence of RSV in the sample, preferably, said detecting is by enzyme-linked immunoassay.

13. Use of the antigen-binding protein of any one of claims 1-4 in the manufacture of a kit for detecting RSV in a sample.

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