Antigen-binding protein targeting the alpha-toxin of Staphylococcus aureus and its applications

Abstract

The present application relates to the biomedical field and discloses isolated antigen-binding proteins and derived proteins thereof, which are capable of specifically binding to Staphylococcus aureus alpha-toxin and neutralizing its biological activity. The present application also discloses the application of said isolated antigen-binding proteins and derived proteins thereof in the field of treatment and / or prevention of Staphylococcus aureus infections.

Description

[Technical Field]

[0001] This application relates to the biopharmaceutical field, specifically to an antigen-binding protein that targets the α-toxin of Staphylococcus aureus. [Background technology]

[0002] Staphylococcus aureus, belonging to the genus Staphylococcus, is an important Gram-positive pathogen that causes community-acquired respiratory infections, skin and soft tissue infections, and can also cause hospital-acquired ventilator-associated pneumonia, and various infections after surgery and various invasive procedures (e.g., tracheostomy, intravenous catheter insertion). According to data from the China Antimicrobial Resistance Surveillance Network (http: / / www.carss.cn / ) in 2019, the clinical detection rate of Staphylococcus aureus ranked 4th among all pathogens. + It ranks first in the detection rate of pathogenic bacteria. Due to the continuous development and widespread application of antimicrobial agents, Staphylococcus aureus is constantly mutating, and the phenomenon of drug resistance is becoming increasingly serious.

[0003] Alpha-toxins are the most important exotoxins released by Staphylococcus aureus, and of these, alpha-hemolysis (HLA) plays the most crucial role in the pathogenesis. Alpha-toxin is a secretory toxin protein encoded by the HLA gene of Staphylococcus aureus, expressed in almost all strains, and is the most important toxic factor influencing the pathogenicity of Staphylococcus aureus. Alpha-toxin binds to cholesterol and sphingomyelin in the host cell membrane to form heptamers, which then fold to form β-barrel transmembrane structures with a diameter of approximately 1.5 nm, rapidly lysing host red blood cells and other tissue cells. At the same time, alpha-toxin can also destroy white blood cells in infected tissue, preventing the host from eliminating the infecting Staphylococcus aureus. With the action of the host's immune system and antibiotics, Staphylococcus aureus at the site of infection releases large amounts of alpha-toxin, which, upon entering the bloodstream, activate the host's immune system, releasing excessive inflammatory factors and leading to sepsis. [Overview of the project] [Problems that the invention aims to solve]

[0004] Currently, the increasing resistance of Staphylococcus aureus to antibiotics has complicated the problem of Staphylococcus aureus infections. In the clinical treatment of Staphylococcus aureus infections, small molecule antiinfective drugs are ineffective in treating toxin-induced bodily damage and sepsis. Therefore, there is an urgent need to develop effective alternative methods for diagnosing and treating Staphylococcus aureus infections. [Means for solving the problem]

[0005] Summary of the Invention This application provides an isolated antigen-binding protein targeting Staphylococcus aureus α-toxin, possessing one or more properties selected from among those that: 1) specifically bind to Staphylococcus aureus α-toxin; 2) neutralize the biological activity of Staphylococcus aureus α-toxin; 3) have pharmacodynamic effects against diseases and symptoms caused by Staphylococcus aureus infection; and 4) have superior biological function compared to currently disclosed Staphylococcus aureus antibody molecules. This application further provides nucleic acid molecules encoding the isolated antigen-binding protein, expression vectors, host cells, immune complexes containing the antigen-binding protein, pharmaceutical compositions, methods for preparing the isolated antigen-binding protein, and applications of the isolated antigen-binding protein described in this application.

[0006] On the other hand, the present invention relates to an isolated antigen-binding protein comprising HCDR3, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 14, or SEQ ID NO: 26.

[0007] In some embodiments, the isolated antigen-binding protein comprises HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 27.

[0008] In some embodiments, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 15.

[0009] In some embodiments, the isolated antigen-binding protein comprises HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 28.

[0010] In some embodiments, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 16.

[0011] In some embodiments, the isolated antigen-binding protein comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 28, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 27, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 14, or SEQ ID NO: 26.

[0012] In some embodiments, the isolated antigen-binding protein comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 16, and SEQ ID NO: 28; HCDR2 comprises an amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 15, and SEQ ID NO: 27; and HCDR3 comprises an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 14, and SEQ ID NO: 26.

[0013] In some embodiments, the isolated antigen-binding protein comprises HCDR1, HCDR2, and HCDR3 selected from any one of the following groups: 1) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1; 2) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14; and 3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 28, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 27, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 26.

[0014] In some embodiments, the isolated antigen-binding protein comprises H-FR1, the C-terminus of which is directly or indirectly bound to the N-terminus of which is which is which, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 54.

[0015] In some embodiments, the H-FR1 comprises an amino acid sequence represented by one of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, and SEQ ID NO: 33.

[0016] In some embodiments, the isolated antigen-binding protein comprises H-FR2, which is located between HCDR1 and HCDR2, and which comprises the amino acid sequence shown in SEQ ID NO: 55 or SEQ ID NO: 56.

[0017] In some embodiments, the H-FR2 comprises an amino acid sequence represented by one of SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 30, and SEQ ID NO: 34.

[0018] In some embodiments, the isolated antigen-binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 57 or SEQ ID NO: 58.

[0019] In some embodiments, the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 31, and SEQ ID NO: 35.

[0020] In some embodiments, the isolated antigen-binding protein comprises H-FR4, the N-terminus of the H-FR4 binds to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 59.

[0021] In some embodiments, the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 32, and SEQ ID NO: 36.

[0022] In some embodiments, the isolated antigen-binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 54, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 55 or SEQ ID NO: 56, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 57 or SEQ ID NO: 58, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 59.

[0023] In some embodiments, the isolated antigen-binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein the H-FR1 comprises an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, and SEQ ID NO: 33, the H-FR2 comprises an amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 30, and SEQ ID NO: 34, the H-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 31, and SEQ ID NO: 35, and the H-FR4 comprises an amino acid sequence shown in any one of SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 32, and SEQ ID NO: 36.

[0024] In some embodiments, the isolated antigen-binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4 selected from any one of the following groups: 1) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7; 2) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 9, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 10, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 11; 3) H-FR1 includes the amino acid sequence shown in SEQ ID NO: 17, H-FR2 includes the amino acid sequence shown in SEQ ID NO: 18, H-FR3 includes the amino acid sequence shown in SEQ ID NO: 19, and H-FR4 includes the amino acid sequence shown in SEQ ID NO: 20; 4) H-FR1 includes the amino acid sequence shown in SEQ ID NO: 8, H-FR2 includes the amino acid sequence shown in SEQ ID NO: 21, H-FR3 includes the amino acid sequence shown in SEQ ID NO: 22, and H-FR4 includes the amino acid sequence shown in SEQ ID NO: 23; 5) H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 29, H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 30, H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 31, and H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 32; and 6) H-FR1 includes the amino acid sequence shown in SEQ ID NO: 33, H-FR2 includes the amino acid sequence shown in SEQ ID NO: 30, H-FR3 includes the amino acid sequence shown in SEQ ID NO: 35, and H-FR4 includes the amino acid sequence shown in SEQ ID NO: 36.

[0025] In some embodiments, the isolated antigen-binding protein comprises VH, which comprises the amino acid sequence shown in SEQ ID NO: 60 or SEQ ID NO: 61.

[0026] In some embodiments, the VH includes an amino acid sequence represented by one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 37, and SEQ ID NO: 38.

[0027] In some embodiments, the isolated antigen-binding protein contains VHH. In some embodiments, the VHH comprises the amino acid sequence shown in SEQ ID NO: 60 or SEQ ID NO: 61.

[0028] In some embodiments, the VHH comprises an amino acid sequence represented by one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 37, and SEQ ID NO: 38.

[0029] In some embodiments, the isolated antigen-binding protein includes a constant region of the antibody heavy chain.

[0030] In some embodiments, the antibody heavy chain constant region is derived from the human IgG constant region. In some embodiments, the antibody heavy chain constant region is derived from the human IgG1 constant region.

[0031] In some embodiments, the antibody heavy chain constant region includes the amino acid sequence shown in SEQ ID NO: 39.

[0032] In some embodiments, the isolated antigen-binding protein contains an amino acid sequence represented by one of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70.

[0033] In some embodiments, the isolated antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0034] In some embodiments, the antigen-binding fragment comprises Fab, Fab', Fv fragment, Bs-Fv, F(ab')2, F(ab)2, scFv, di-scFv and / or dAb.

[0035] In some embodiments, the antibody is one or more selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully humanized antibodies.

[0036] In some embodiments, the isolated antigen-binding protein can specifically bind to Staphylococcus aureus α-toxin.

[0037] In some embodiments, the isolated antigen-binding protein can neutralize the biological activity of Staphylococcus aureus α-toxin.

[0038] In some embodiments, the isolated antigen-binding protein can prevent and / or treat diseases and / or symptoms and their complications.

[0039] In some embodiments, the disease and / or symptoms and their complications are caused or mediated by Staphylococcus aureus α-toxin.

[0040] In some embodiments, the disease and / or symptoms and their complications include bacteremia and / or sepsis.

[0041] On the other hand, this application further provides a polypeptide molecule comprising the isolated antigen-binding protein.

[0042] In some embodiments, the polypeptide molecule includes a fusion protein. In some embodiments, the polypeptide molecule comprises a fusion protein formed with two or more isolated antigen-binding proteins and antibody heavy chain constant regions.

[0043] In some embodiments, the polypeptide molecule comprises an amino acid sequence represented by one of SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0044] On the other hand, this application further provides an immune complex comprising the isolated antigen-binding protein.

[0045] On the other hand, this application further provides nucleic acid molecules encoding the isolated antigen-binding protein or the polypeptide molecule.

[0046] On the other hand, this application further provides a vector containing the nucleic acid molecule. On the other hand, this application further provides cells containing the nucleic acid molecule or the vector.

[0047] On the other hand, the present application further provides a pharmaceutical composition comprising the isolated antigen-binding protein, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector and / or the cell, and an optional pharmaceutically acceptable carrier.

[0048] On the other hand, the present application further provides a method for preparing the isolated antigen-binding protein, comprising culturing the cells under conditions that express the antigen-binding protein.

[0049] On the other hand, this application further provides applications of the isolated antigen-binding protein, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cells, and / or the pharmaceutical composition in the manufacture of drugs for preventing and / or treating diseases and / or symptoms and their complications.

[0050] In some embodiments, the disease and / or symptoms and their complications are caused or mediated by Staphylococcus aureus α-toxin.

[0051] In some embodiments, the disease and / or symptoms and their complications include sepsis and / or bacteremia.

[0052] On the other hand, the antigen-binding protein, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition described in this application may be used alone or in combination with other molecules.

[0053] On the other hand, the present application further provides a method for detecting Staphylococcus aureus α-toxin in a sample, comprising using the isolated antigen-binding protein, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cells, and / or the pharmaceutical composition.

[0054] On the other hand, the present application further provides a reagent or reagent kit for detecting Staphylococcus aureus α-toxin in a sample, comprising the isolated antigen-binding protein, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cells, and / or the pharmaceutical composition.

[0055] On the other hand, this application further provides applications in the manufacture of reagent kits for detecting the presence and / or content of Staphylococcus aureus α-toxin in a sample of the isolated antigen-binding protein, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cells, and / or the pharmaceutical composition.

[0056] Those skilled in the art will readily recognize other aspects and advantages of this application from the following detailed description. The following detailed description shows and describes only exemplary embodiments of this application. As will be understood by those skilled in the art, the content of this application allows them to modify the specific embodiments disclosed without departing from the spirit and scope of the invention to which this application relates. Accordingly, the drawings and description of this application are illustrative and not limiting.

[0057] Brief explanation of the drawing The specific features of the invention relating to this application are described in the appended claims. The features and advantages of the invention relating to this application can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail below. A brief description of the drawings is as follows. [Brief explanation of the drawing]

[0058] [Figure 1] Figure 1 shows the detection of the antigen-binding ability of the antigen-binding protein described in this application to Staphylococcus aureus α-toxin protein by ELISA measurement. [Figure 2] Figure 2 shows the neutralizing effect of the antigen-binding protein described in this application on the hemolytic activity of recombinant Staphylococcus aureus α-toxin. [Figure 3] Figure 3 shows the therapeutic effect of the antigen-binding protein described in this application on mouse sepsis caused by Staphylococcus aureus α-toxin. [Figure 4] Figure 4 shows the therapeutic effect of the antigen-binding protein described in this application on bacteremia in mice caused by Staphylococcus aureus α-toxin. [Figure 5] Figure 5 shows the detection of the antigen-binding ability of the antigen-binding protein described in this application to Staphylococcus aureus α-toxin protein by ELISA measurement. [Figure 6] Figure 6 shows the neutralizing effect of the antigen-binding protein described in this application on the hemolytic activity of recombinant Staphylococcus aureus α-toxin. [Figure 7] Figure 7 shows the therapeutic effect of the antigen-binding protein described in this application on mouse sepsis caused by Staphylococcus aureus α-toxin. [Figure 8] Figure 8 shows the therapeutic effect of the antigen-binding protein described in this application on bacteremia in mice caused by Staphylococcus aureus α-toxin. [Figure 9] Figure 9 shows the neutralizing effect of the tetravalent antigen-binding protein described in this application on the hemolytic activity of Staphylococcus aureus α-toxin. [Figure 10] Figure 10 shows the neutralizing effect of the tetravalent antigen-binding protein described in this application on the hemolytic activity of Staphylococcus aureus α-toxin. [Figure 11] Figure 11 shows the neutralizing effect of the tetravalent antigen-binding protein described in this application on the hemolytic activity of Staphylococcus aureus α-toxin. [Modes for carrying out the invention]

[0059] Modes for carrying out the invention The embodiments of this application will be described below based on specific examples. Those skilled in the art will readily understand the advantages and effects of the present invention from the information disclosed herein.

[0060] Term definition In this application, the term "Staphylococcus aureus" is interchangeable with "Staphylococcus aureus" and "S. aureus," which generally belong to the Gram-positive bacterium. In this application, the term encompasses all types and forms of Staphylococcus aureus and its variants, homologs, and functionally active fragments.

[0061] In this application, the term "Staphylococcus aureus α-toxin" is also referred to as "α-toxin," "α-hemolysin," "hemolysin A," "Hla," and "hemolysin A toxin," and generally refers to the pore-forming protein hemolysin produced by Staphylococcus aureus. In this application, the α-toxin of Staphylococcus aureus may include forms such as wild-type Staphylococcus aureus α-toxin and its variants, derivatives, homologs, recombinant proteins, monomers, and oligomers. The oligomer form may include forms different from the monomer of the α-toxin, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., or multimer forms such as α-toxin in heptamer-prepore form.

[0062] In this application, the term “isolated” generally means obtained in its natural state by artificial means. When a particular “isolated” substance or component appears in nature, it may be because the natural environment in which it exists has been altered, or the substance has been isolated from the natural environment, or both. For example, if an unisolated polynucleotide or polypeptide exists naturally in a living animal, a highly pure version of the same polynucleotide or polypeptide isolated in this naturally occurring state is called isolated. The term “isolated” does not exclude the presence of mixtures of artificial or synthetic substances, or other impurities that do not affect the activity of the substance.

[0063] In this application, the term “isolated antigen-binding protein” typically refers to an antigen-binding protein that has been deviated from its naturally occurring state. Such “isolated antigen-binding protein” may include an antigen-binding moiety and, optionally, a framework or framework moiety that enables the antigen-binding moiety to adopt a three-dimensional structure that facilitates binding to the antigen. Antigen-binding proteins may include, for example, antibody-derived protein framework regions (FRs), or alternative protein framework regions or artificial framework regions having transplanted CDRs or CDR derivatives. Such frameworks include, but are not limited to, antibody-derived framework regions into which mutations have been introduced to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthesized framework regions, for example, those containing biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Examples of antigen-binding proteins include, but are not limited to, human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, single-chain antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, Fab, Fab', Fv fragments, Bs-Fv, F(ab')2, F(ab)2, scFv, di-scFv, dAb, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies and their fragments.

[0064] In this application, the term "CDR" is also called "complementarity-determining region" and typically refers to a region within the variable domain of an antibody whose sequence is highly variable and / or forms a structurally defined loop. Typically, an antibody contains six CDRs, three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In some embodiments, naturally occurring camel antibodies consisting only of heavy chains may function normally and stably even in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996). Antibody CDRs can be determined by various coding systems, including CCG, Kabat, AbM, Chothia, IMGT, and a comprehensive consideration of Kabat / Chothia. These coding systems are well-known in this field; see http: / / www.bioinf.org.uk / abs / index.html#kabatnum for details. For example, the numbering of the amino acid sequence of the antigen-binding protein can be determined using the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system@imgt.cines.fr; http: / / imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27: 209-212; Ruiz et al., 2000 Nucleic Acids Res. 28: 219-221; Lefranc et al., 2001, Nucleic Acids Res. 29: 207-209; Lefranc et al., 2003, Nucleic Acids Res. 31: 307-310; Lefranc et al., 2005, DevComp Immunol 29: 185-203).For example, the CDR of the antigen-binding protein can be determined by the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY AcadSci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0065] In this application, the term "FR" generally refers to a more highly conserved portion of the antibody variable domain, known as the framework region. Typically, the variable domains of the natural heavy and light chains each contain four FR regions, namely four in the VH (H-FR1, H-FR2, H-FR3, and H-FR4) and four in the VL (L-FR1, L-FR2, L-FR3, and L-FR4).

[0066] In this application, the terms “variable domain” and “variable region” may be used interchangeably and typically refer to a portion of the antibody heavy chain and / or light chain. The variable domains of the heavy chain and light chain are respectively referred to as “V H " and "V L These domains can be referred to as "VH" and "VL" (or "VL" respectively). These domains are typically the most variable parts of the antibody (compared to other antibodies of the same type) and contain the antigen-binding site.

[0067] In this application, the term “variable” generally refers to the possibility that specific segments of the variable domain may differ substantially in sequence between antibodies. The variable domain mediates antigen binding, determining the specificity of a particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domain. It is usually concentrated in three segments called hypervariable regions (CDRs or HVRs) of the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The variable domains of the natural heavy and light chains each contain four FR regions, which typically employ a β-pleated sheet structure, forming a cyclic linkage and sometimes forming part of the β-pleated sheet structure. The CDRs in each chain are held at very close proximity to the FR regions and, together with the CDRs in the other chain, promote the formation of the antibody's antigen-binding site (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0068] In this application, the term “antibody” typically refers to an immunoglobulin or a fragment thereof or a derivative thereof, encompassing any polypeptide containing an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and transplanted antibodies. For the purposes of this invention, the term “antibody” also includes, for example, antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function (e.g., specifically binding to Staphylococcus aureus α-toxin). Such fragments typically include an antigen-binding domain. A basic quadruplex antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetrameric units and other polypeptides called J chains, and contain 10 antigen-binding sites, while IgA antibodies contain 2-5 basic quadrivalent units that can bind to and polymerize with the J chains to form multivalent aggregates. In the case of IgG, the quadrivalent units are typically around 150,000 daltons. Each light chain is attached to a heavy chain by a disulfide covalent bond, and two heavy chains are linked to each other by one or more disulfide bonds depending on the isotype of the heavy chain. Each heavy and light chain further has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for α and γ chains, and four CH domains for μ and ε isotypes. Each light chain has a variable domain (VL) at the N-terminus and a constant domain at the other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are thought to form an interface between the light chain and the heavy chain variable domain. VH and VL pair up to form a single antigen-binding site.For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The light chain (L) from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on the amino acid sequence of its constant domain. Immunoglobulins can be assigned to different classes or isotypes based on the amino acid sequence of their heavy chain (CH) constant domain. Currently, there are five types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each possessing a heavy chain called α, δ, ε, γ, and μ, respectively.

[0069] In this application, the term “antigen-binding fragment” usually refers to one or more fragments having the ability to specifically bind to an antigen (e.g., Staphylococcus aureus α-toxin). In this application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0070] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, intact antibodies can be digested with papain. After the antibody is digested with papain, two identical antigen-binding fragments are produced: the "Fab" fragment and the remaining "Fc" fragment (i.e., the Fc region as described above). The Fab fragment may consist of the complete light chain, the variable region of the heavy chain, and the first constant region (CH1) of the heavy chain (VH).

[0071] In this application, the term "Fab' fragment" usually refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which is slightly larger than a Fab fragment. For example, a Fab' fragment may include all light chains, all variable domains of heavy chains, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment may include some or all of the 220–330 amino acid residues of the heavy chain.

[0072] In this application, the term "F(ab')2" typically refers to an antibody fragment produced by pepsin digestion of an intact antibody. The F(ab')2 fragment includes two Fab fragments linked by disulfide bonds and a portion of a hinge region. The F(ab')2 fragment has divalent antigen-binding activity and can crosslink antigens.

[0073] In this application, the term "Fv fragment" typically refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or some of the heavy chain variable regions and light chain variable regions, and lacking the heavy chain constant regions and light chain constant regions. The heavy chain variable regions and light chain variable regions include, for example, the CDR. For example, an Fv fragment contains all or some of the amino-terminal variable regions of about 110 amino acids of the heavy chain and light chain.

[0074] In this application, the term "scFv" typically refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are adjacent (e.g., via a synthesized linker such as a short flexible polypeptide linker), can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, the scFv may have the VL and VH variable regions in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise a VL-linker-VH or a VH-linker-VL.

[0075] In this application, the term "dAb" usually refers to an antigen-binding fragment having a VH domain, a VL domain, or having either a VH domain or a VL domain, e.g., Ward et al. (Nature, 1989 Oct 12;341(6242):544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and other disclosed patent applications such as WO 06 / 030220, WO 06 / 003388 and Domantis Ltd. The term "dAb" usually includes sdAb. The term "sdAb" usually refers to a single-domain antibody. A single-domain antibody usually refers to an antibody fragment consisting only of the antibody heavy chain variable region (VH domain) or the antibody light chain variable region (VL).

[0076] In this application, the term "VHH" usually refers to variable antigen-binding domains derived from heavy chain antibodies of camelids (camels, dromedaries, llamas, alpacas, etc.) (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and overview; Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH is also referred to as nanobody (Nb).

[0077] In this application, the term "monoclonal antibody" generally refers to a preparation of an antibody molecule with a single molecular composition. Generally, monoclonal antibodies have a high degree of specificity for a single antigen site. Furthermore, unlike conventional polyclonal antibody preparations (which usually have different antibodies corresponding to different determinants), each monoclonal antibody corresponds to a single determinant in the antigen. In addition to this specificity, the advantages of monoclonal antibodies are that they can be synthesized by culturing hybridomas and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method. For example, the monoclonal antibodies used in this application can be produced in hybridoma cells or by recombinant DNA methods.

[0078] In this application, the term "chimeric antibody" generally refers to an antibody whose variable region originates from one species and whose constant region originates from another species. Typically, the variable region originates from antibodies of experimental animals such as rodents ("parental antibodies"), and the constant region originates from human antibodies. Therefore, the resulting chimeric antibody has a lower likelihood of inducing a harmful immune response in human individuals compared to a parental antibody (e.g., one derived from an alpaca).

[0079] In this application, the term “humanized antibody” typically refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., alpaca antibody) are replaced with corresponding amino acids derived from human immunoglobulin. In the CDR region, small additions, deletions, insertions, substitutions, or modifications of amino acids are also permitted, as long as the antibody retains its ability to bind to a specific antigen. Humanized antibodies optionally include at least a portion of the constant region of human immunoglobulin. “Humanized antibodies” retain antigen specificity similar to the original antibody. A “humanized” form of a non-human (e.g., alpaca) antibody may be a chimeric antibody containing minimal sequences derived from non-human immunoglobulin. In some cases, CDR region residues of human immunoglobulin (receptor antibody) may be replaced with CDR region residues of a non-human species (donor antibody) (e.g., alpaca, mouse, rat, rabbit, or non-human primate) having desirable properties, affinity, and / or capabilities. In some cases, FR region residues of human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications not present in receptor or donor antibodies. These modifications can be made to further improve antibody performance, such as binding affinity.

[0080] The term "fully humanized antibody" typically refers to an antibody containing only human immunoglobulin sequences. When produced in mice, mouse cells, or hybridomas derived from mouse cells, fully humanized antibodies may contain mouse glycans. Similarly, "mouse antibody" or "rat antibody" refers to an antibody containing only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans, in transgenic animals possessing human immunoglobulin germline sequences, or by phage display or other molecular biological methods. Exemplary techniques that can be used to produce antibodies are described in U.S. Patents 6,150,584, 6,458,592, and 6,420,140. Other techniques, such as the use of libraries, are well known in the art.

[0081] In this application, the terms “polypeptide molecule,” “polypeptide,” and “peptide” are interchangeable and generally refer to polymers of amino acid residues. The term “fusion protein” generally refers to a polypeptide having at least two covalently linked parts. Each part may be a polypeptide with different properties. These properties may be biological properties such as in vitro or in vivo activity. These properties may also be simple chemical or physical properties such as binding to a target molecule or catalysis of a reaction. These two parts can be directly linked by a single peptide bond or a peptide linker.

[0082] In this application, the term “nucleic acid molecule” usually refers to a nucleotide, deoxyribonucleotide, or ribonucleotide of any length in an isolated form, or an analogue isolated from the natural environment or artificially synthesized.

[0083] In this application, the term “vector” typically refers to a nucleic acid delivery tool that can insert a polynucleotide encoding a protein and thereby express that protein. A vector can express the genetic material elements it carries within a host cell by transforming, transfecting, or transfecting the host cell. Examples of vectors include plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phages or M13 phages; and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). A vector may contain multiple elements that control expression, including a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, and a reporter gene. A vector may also contain a replication start site. The vector may, but is not limited to, further contain components that assist in cell entry, such as viral particles, liposomes, or protein membranes.

[0084] In this application, the term “cell” generally refers to a single cell, cell line, or cell culture that may or may have been a recipient of a subject’s plasmid or vector containing the nucleic acid molecules or vectors described in this invention. A cell may include offspring of a single cell. Due to spontaneous, accidental, or intentional mutations, the offspring may not necessarily be completely identical to the original parent cell (either in terms of whole DNA complementation or genome). A cell may include a cell transfected in vitro with the vector of this application. A cell may be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cells such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, or NS0 cells.

[0085] In this application, the term "immune complex" generally refers to a complex formed by the conjugation (for example, covalently by a binding molecule) of the other reagents (e.g., chemotherapeutic agents, radioactive elements, cell proliferation inhibitors, and cytotoxic agents) with the antibody or its antigen-binding fragment, and the complex can deliver the other reagents to target cells (e.g., tumor cells) by specifically binding the antibody or its antigen-binding fragment to an antigen on the target cell.

[0086] In this application, the term “pharmaceutical composition” generally refers to a composition for preventing / treating a disease or symptom. The pharmaceutical composition may comprise an isolated antigen-binding protein, a nucleic acid molecule, a vector and / or a cell as described in this application, and an optional pharmaceutically acceptable adjuvant. The pharmaceutical composition may also further comprise one or more suitable formulations of pharmaceutically effective carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Preferably, the acceptable components of the composition are non-toxic to the recipient at the dose and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, freeze-dried compositions, and lyophilized compositions.

[0087] In this application, the term “pharmaceutically acceptable carrier” generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cells or mammals being exposed at the doses and concentrations used. Physiologically acceptable carriers include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium and / or nonionic surfactants.

[0088] In this application, the terms “specifically binding” or “specific” generally refer to measurable and reproducible interactions, such as the binding of a target to an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) may bind to that target more readily and / or for a longer period of time with higher affinity and binding strength than an antibody that binds to other targets. In some embodiments, an antibody specifically binds to an epitope on a protein that is conserved across different species of proteins. In some embodiments, specific binding may include, but is not required, exclusive binding.

[0089] In this application, the term “subject” generally refers to a human or a non-human animal, including but not limited to a cat, dog, horse, pig, cattle, sheep, rabbit, mouse, rat, or monkey.

[0090] In this application, the protein, polypeptide and / or amino acid sequence referred to herein should be understood to include at least variants or homologs having the same or similar function as the protein or polypeptide.

[0091] In this application, the variant may be, for example, a protein or polypeptide obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence of the protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to Staphylococcus aureus α-toxin). For example, the functional variant may include a protein or polypeptide having amino acid modifications by substitution, deletion, and / or insertion of at least 1, for example, 1-30, 1-20, or 1-10, and further, for example, 1, 2, 3, 4, or 5 amino acids. The functional variant can substantially retain the biological properties of the protein or polypeptide before modification (e.g., substitution, deletion, or addition). For example, the functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding ability) of the protein or polypeptide before modification. For example, the substitution may be a conservative substitution.

[0092] In this application, the homologue may be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even higher) sequence homology to the amino acid sequence of the protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to Staphylococcus aureus α-toxin).

[0093] In this application, homology usually refers to the approximation, similarity, or association between two or more sequences. The "sequence homology percentage" is calculated by comparing two sequences aligned within a comparison window and determining the number of positions in both sequences where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is present. The number of matching positions is then divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to calculate the sequence homology percentage. Alignment for determining the sequence homology percentage can be performed in various ways known in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve the greatest alignment over the entire length of the sequences being compared or within the region of the target sequence. The homology may be measured by FASTA and BLAST. For a description of the FASTA algorithm, see WRPearson and D.J.Lipman, “An Improved Tool for Biological Sequence Comparison,” Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.), 85:2444-2448, 1988; and D.J.Lipman and WRPearson, “Rapid and Highly Sensitive Protein Similarity Search,” Science, 227:1435-1441, 1989. For a description of the BLAST algorithm, see S. Altschul, W. Gish, W. Miller, EWMyers and D. Lipman, “A Basic Local Alignment Search Tool,” Journal of Molecular Biology 215:403-410, 1990.

[0094] In this application, the term "includes" usually means "to have," "to contain," or "to encompass." In some cases, it may mean "to be" or "to consist of."

[0095] In this application, the term "approximately" generally means that the value varies within a range of 0.5% to 10% above or below the specified value, for example, within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0096] Detailed description of the invention Isolated antigen-binding protein The antibody CDR, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region may come into contact with the antigen or its epitope. Antibody CDRs can be determined by various coding systems, including CCG, Kabat, Chothia, IMGT, AbM, and a comprehensive consideration of Kabat / Chothia. These coding systems are well-known in the art; see http: / / www.bioinf.org.uk / abs / index.html#kabatnum for specific examples. Those skilled in the art can determine the CDR region using different coding systems based on the antibody sequence and structure. The CDR region may differ depending on the coding system. In this application, the CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants of the CDR amino acid sequence in which one or more amino acids are substituted, deleted, and / or added. For example, amino acids 1-30, 1-20, or 1-10, and further, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9, are substituted, deleted, and / or inserted; further including homologs thereof, the homologs may be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even higher) sequence homology to the amino acid sequence of the CDR. In some embodiments, the isolated antigen-binding protein described in this application is defined by a Chothia coding system.

[0097] On the other hand, this application provides an isolated antigen-binding protein comprising HCDR3. In some embodiments, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26.

[0098] In this application, the isolated antigen-binding protein may further comprise HCDR2. In some embodiments, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 52.

[0099] In some embodiments, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 28.

[0100] In this application, the isolated antigen-binding protein may further comprise HCDR1. In some embodiments, HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 53.

[0101] In some embodiments, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 28.

[0102] In this application, the isolated antigen-binding protein may further comprise HCDR1, HCDR2, and HCDR3. For example, HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 28, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 27, and HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 14, or SEQ ID NO: 26.

[0103] In this application, the isolated antigen-binding protein comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 16, and SEQ ID NO: 28, HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 15, and SEQ ID NO: 27, and HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 14, and SEQ ID NO: 26.

[0104] For example, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 3, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 1.

[0105] For example, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 16, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 15, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 14.

[0106] For example, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 28, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 26.

[0107] In this application, the isolated antigen-binding protein may include at least one CDR selected from the amino acid sequences shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 37, and SEQ ID NO: 38, wherein the CDR may include, and is not limited to, CDRs obtained by any method of splitting, as shown in the table below. Any CDR split by any method is protected by the claims of this application as long as its sequence is identical to that of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 37, or SEQ ID NO: 38.

[0108] For example, by splitting the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13 using the method shown in Table 1, the antigen-binding protein isolated according to this application may contain the CDR shown in the table below.

[0109] [Table 1]

[0110] For example, by splitting the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 25 using the method shown in Table 2, the isolated antigen-binding protein according to this application may contain the CDR shown in the table below.

[0111] [Table 2]

[0112] For example, by splitting the amino acid sequence shown in SEQ ID NO: 37 or SEQ ID NO: 38 using the method shown in Table 3, the isolated antigen-binding protein according to this application may contain the CDR shown in the table below.

[0113] [Table 3]

[0114] In this application, the isolated antigen-binding protein may include VHH. In some embodiments, the CDR3 of VHH may include the amino acid sequence shown in SEQ ID NO: 1, the CDR2 of VHH may include the amino acid sequence shown in SEQ ID NO: 2, and the CDR1 of VHH may include the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the CDR3 of VHH may include the amino acid sequence shown in SEQ ID NO: 14, the CDR2 of VHH may include the amino acid sequence shown in SEQ ID NO: 15, and the CDR1 of VHH may include the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the CDR3 of the VHH may include the amino acid sequence shown in SEQ ID NO: 26, the CDR2 of the VHH may include the amino acid sequence shown in SEQ ID NO: 27, and the CDR1 of the VHH may include the amino acid sequence shown in SEQ ID NO: 28.

[0115] In this application, the isolated antigen-binding protein may include H-FR1, the C-terminus of H-FR1 may be directly or indirectly bound to the N-terminus of HCDR1. In some embodiments, H-FR1 may include the amino acid sequence shown in SEQ ID NO: 54.

[0116] In some embodiments, H-FR1 may include the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, H-FR1 may include the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, H-FR1 may include the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, H-FR1 may include the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, H-FR1 may include the amino acid sequence shown in SEQ ID NO: 33.

[0117] In this application, the isolated antigen-binding protein may include H-FR2, which may be located between HCDR1 and HCDR2. In some embodiments, H-FR2 may include the amino acid sequence shown in SEQ ID NO: 55. In some embodiments, H-FR2 may include the amino acid sequence shown in SEQ ID NO: 56.

[0118] In some embodiments, the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 34.

[0119] In this application, the isolated antigen-binding protein may contain H-FR3, and the H-FR3 may be located between the HCDR2 and the HCDR3. In some embodiments, the H-FR3 may contain the amino acid sequence shown in SEQ ID NO: 57. In some embodiments, the H-FR3 may contain the amino acid sequence shown in SEQ ID NO: 58.

[0120] In some embodiments, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 19. In some embodiments, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 22. In some embodiments, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 31. In some embodiments, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 35.

[0121] In this application, the isolated antigen-binding protein may include H-FR4, the N-terminus of which can bind to the C-terminus of HCDR3. In some embodiments, H-FR4 may include the amino acid sequence shown in SEQ ID NO: 59.

[0122] In some embodiments, the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 23. In some embodiments, the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 36.

[0123] In this application, the isolated antigen-binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4. For example, H-FR1 may include the amino acid sequence shown in SEQ ID NO: 54, H-FR2 may include the amino acid sequence shown in SEQ ID NO: 55 or SEQ ID NO: 56, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 57 or SEQ ID NO: 58, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 59.

[0124] In some embodiments, the isolated antigen-binding protein may comprise H-FR1, H-FR2, H-FR3, and H-FR4, where H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, and SEQ ID NO: 33; H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 30, and SEQ ID NO: 34; H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 31, and SEQ ID NO: 35; and H-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NO: 7, SEQ It may contain the amino acid sequence shown in any one of ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 32, and SEQ ID NO: 36.

[0125] In this application, the isolated antigen-binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4. For example, the isolated antigen-binding protein H-FR1, H-FR2, H-FR3, and H-FR4 may each contain the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively. For example, the isolated antigen-binding protein H-FR1, H-FR2, H-FR3, and H-FR4 may each contain the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. For example, the isolated antigen-binding proteins H-FR1, H-FR2, H-FR3, and H-FR4 may each contain the amino acid sequences shown sequentially at SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively. For example, the isolated antigen-binding proteins H-FR1, H-FR2, H-FR3, and H-FR4 may each contain the amino acid sequences shown sequentially at SEQ ID NO: 8, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively. For example, the isolated antigen-binding proteins H-FR1, H-FR2, H-FR3, and H-FR4 may each contain the amino acid sequences shown sequentially at SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively. For example, the isolated antigen-binding proteins H-FR1, H-FR2, H-FR3, and H-FR4 may each contain the amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively.

[0126] In this application, the isolated antigen-binding protein may include VH. In some embodiments, VH may include the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, VH may include the amino acid sequence shown in SEQ ID NO: 61.

[0127] In some embodiments, the VH may include the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the VH may include the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the VH may include the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the VH may include the amino acid sequence shown in SEQ ID NO: 25. In some embodiments, the VH may include the amino acid sequence shown in SEQ ID NO: 37. In some embodiments, the VH may include the amino acid sequence shown in SEQ ID NO: 38.

[0128] In this application, the isolated antigen-binding protein may include VHH. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 61.

[0129] In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 25. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 37. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 38.

[0130] In this application, the isolated antigen-binding protein may include an antibody heavy chain constant region. In some embodiments, the antibody heavy chain constant region may be derived from the heavy chain constant region of any immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. For example, the antibody heavy chain constant region may be derived from the human IgG heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region may include its variants. In some embodiments, the antibody heavy chain constant region may be derived from the heavy chain constant region of any of human IgG1-4. In some embodiments, the isolated antigen-binding protein may be derived from the human IgG1 heavy chain constant region. For example, the human IgG1 heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 39. For example, the isolated antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 62. For example, the isolated antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 63. For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO: 64. For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO: 68. For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO: 69. For example, the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO: 70.

[0131] In this application, the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof.

[0132] In some embodiments, the antigen-binding fragment may include Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, bs-Fv, di-scFv and / or dAb.

[0133] In this application, the isolated antigen-binding protein may include VHH. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 61.

[0134] In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 25. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 37. In some embodiments, the VHH may include the amino acid sequence shown in SEQ ID NO: 38.

[0135] In this application, the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully humanized antibody.

[0136] For example, the VH of the humanized antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 13. For example, the VH of the humanized antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 25. For example, the VH of the humanized antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 38.

[0137] In this application, the isolated antigen-binding protein may include a tetravalent antigen-binding protein and / or a tetravalent antigen-binding protein Fc fusion protein. For example, two sequences encoding the variable region of an anti-α-toxin antigen-binding protein (e.g., two huA3 molecules or two huG1 molecules as described in this application, or one huA3 molecule and one huG1 molecule; e.g., two A3 molecules or two G1 molecules or one A3 molecule and one G1 molecule as described in this application) can be linked in series and fused with a human IgG1 Fc fragment. For example, a bivalent Fc fusion protein can be obtained by connecting the C-terminus of one VHH to the N-terminus of a second VHH and the C-terminus of the second VHH to the N-terminus of an Fc fragment, and the bivalent Fc fusion protein can be subcloned into a plasmid and transfected into cells for recombinant expression to obtain the tetravalent antigen-binding protein as described in this application. For example, the isolated antigen-binding protein may include the huA3-huG1-Fc antigen-binding protein. For example, the isolated antigen-binding protein may include the huA3-huA3-Fc antigen-binding protein. For example, the isolated antigen-binding protein may include the huG1-huG1-Fc antigen-binding protein. For example, the isolated antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 65. For example, the isolated antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 66. For example, the isolated antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 67.

[0138] The isolated antigen-binding protein described in this application may contain heavy and / or light chain sequences having one or more conservative sequence modifications. “Conservative sequence modifications” refer to amino acid modifications that do not significantly affect or alter the antibody binding properties. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications may be introduced into the antibody of the present invention by known articulate techniques, for example, site-directed mutations or PCR-mediated induction mutations. Conservative amino acid substitutions are substitutions that replace an amino acid residue with an amino acid residue having a similar side chain. In this art, families of amino acid residues having similar side chains are already well known. These families include amino acids with alkaline side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR region of the isolated antigen-binding protein described in this application can be replaced with amino acid residues from other amino acid families of the same side chain family. As is well known to those skilled in the art, some conservative sequence modifications do not disrupt antigen binding.For example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem.32:6862-35; Adib-Conquy et al., (1998) Int. Immunol.10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0139] The Staphylococcus aureus α-toxin antigen-binding proteins described herein can be identified, screened, or characterized by various detection methods known in the art.

[0140] For example, the antigen-binding activity of the antigen-binding protein or fusion protein of this application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS), immunohistochemistry, and immunofluorescence.

[0141] In this application, the isolated antigen-binding protein can specifically bind to Staphylococcus aureus α-toxin. In some embodiments, the binding of the isolated antigen-binding protein to Staphylococcus aureus α-toxin can be detected by ELISA. For example, the antigen-binding protein described in this application has EC levels of approximately 0.010 μg / mL or less, approximately 0.009 μg / mL or less, approximately 0.008 μg / mL or less, approximately 0.007 μg / mL or less, approximately 0.006 μg / mL or less, approximately 0.005 μg / mL or less, approximately 0.004 μg / mL or less, approximately 0.003 μg / mL or less, approximately 0.002 μg / mL or less, and approximately 0.001 μg / mL or less. 50 The value allows it to bind to Staphylococcus aureus α-toxin.

[0142] In this application, the isolated antigen-binding protein can neutralize the biological activity of Staphylococcus aureus α-toxin. For example, rabbit red blood cells can be added to Staphylococcus aureus α-toxin and the antigen-binding protein, and the antihemolytic activity can be measured using a microplate reader. For example, the antigen-binding protein described in this application can completely neutralize 18.75 ng of recombinant Staphylococcus aureus α-toxin in amounts of approximately 200 ng or less, approximately 100 ng or less, approximately 75 ng or less, approximately 60 ng or less, approximately 50 ng or less, approximately 45 ng or less, approximately 40 ng or less, and approximately 35 ng or less.

[0143] In this application, the isolated antigen-binding protein can prevent and / or treat a disease and / or symptoms and its complications. In some embodiments, the disease and / or symptoms and its complications can be caused by or mediated by Staphylococcus aureus α-toxin. In some embodiments, the disease and / or symptoms and its complications may include sepsis and / or bacteremia.

[0144] Polypeptide molecules, nucleic acid molecules, vectors, cells, immune complexes, and pharmaceutical compositions On the other hand, this application provides a polypeptide molecule that may contain the isolated antigen-binding protein described herein.

[0145] In some embodiments, the polypeptide molecule may include a fusion protein.

[0146] In some embodiments, the polypeptide molecule comprises an isolated antigen-binding protein described in any one of two or more claims of this application, and a fusion protein comprising an isolated antigen-binding protein described in any one of two or more claims of this application and an antibody heavy chain constant region.

[0147] For example, the polypeptide molecule may comprise two antigen-binding domains and an antibody heavy chain constant region. Of these, the first antigen-binding domain may comprise an isolated antigen-binding protein described in any one of the paragraphs of this application, and the second antigen-binding domain may comprise an isolated antigen-binding protein described in any one of the paragraphs of this application.

[0148] In some embodiments, the polypeptide molecule may contain an amino acid sequence represented by any one of SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0149] On the other hand, this application provides an isolated nucleic acid molecule capable of encoding the isolated antigen-binding protein described herein. For example, it may be (i) amplified in vitro, for example by polymerase chain reaction (PCR); (ii) produced by genetic recombination, for example by cloning; (iii) purified, for example by enzymatic cleavage and gel electrophoresis; or (iv) produced or synthesized by synthesis, for example by chemical synthesis.

[0150] On the other hand, this application provides a vector which may contain the nucleic acid molecule described herein. The vector may also contain other genes, such as a marker gene that enables selection of the vector under appropriate conditions in a suitable host cell. Furthermore, the vector may also contain expression regulatory elements that enable appropriate expression of the coding region in a suitable host. Such regulatory elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other regulatory elements that regulate gene transcription or mRNA translation. The vector can express the genetic material elements it carries in a host cell by transforming, transducing, or transfecting the host cell. The vector may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. The vector may further contain, but is not limited to, components that assist in cell entry, such as, for example, viral particles, liposomes, or protein membranes.

[0151] On the other hand, this application provides cells that may contain nucleic acid molecules or vectors described herein. In some embodiments, various or each host cell may contain one or one type of nucleic acid molecule or vector described herein. In some embodiments, various or each host cell may contain multiple (e.g., two or more) or multiple types (e.g., two or more) of nucleic acid molecules or vectors described herein. For example, a vector described herein can be introduced into a host cell, such as a eukaryotic cell, such as a plant-derived cell, fungal or yeast cell. In some embodiments, the cell may be a bacterial cell (e.g., Escherichia coli), a yeast cell, or another eukaryotic cell, such as a COS cell, Chinese hamster ovary (CHO) cell, CHO-K1 cell, LNCAP cell, HeLa cell, 293T cell, COS-1 cell, SP2 / 0 cell, NS0 cell, or myeloma cell. A vector described herein can be introduced into a host cell by a method well known in this field, such as electroporation, lipofectine transfect, or lipofectamin transfect.

[0152] On the other hand, this application provides an immune complex that may contain the isolated antigen-binding protein described herein.

[0153] In some embodiments, isolated antigen-binding proteins or fragments thereof described in this application can be conjugated to other agents such as chemotherapeutic agents, toxins, immunotherapeutic agents, imaging probes, and spectroscopic probes. Such conjugation can be mediated by one or more covalent or non-covalent interactions and may include chelation. Various linkers known in the art can be used to form immune complexes. Furthermore, immune complexes can be provided in the form of fusion proteins that can be expressed by polynucleotides encoding the immune complex. The immune complex may further include, for example, antibody-drug conjugates (ADCs). Suitable drugs may include cytotoxins, alkylating agents, DNA subgroove linking molecules, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In ADCs, the antibody and therapeutic agent can be crosslinked by a cleavable linker such as a peptide linker, disulfide linker, or hydrazone linker.

[0154] On the other hand, the present application further provides a pharmaceutical composition which may comprise an isolated antigen-binding protein as described in the present application, a polypeptide molecule as described in the present application, an immune complex as described in the present application, a nucleic acid molecule as described in the present application, a vector and / or a cell as described in the present application, and an optional pharmaceutically acceptable carrier.

[0155] In some embodiments, the pharmaceutical composition may further comprise one or more suitable formulations of pharmaceutically effective adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Preferably, the acceptable components of the composition are nontoxic to the recipient at the dose and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, freeze-dried compositions, and lyophilized compositions.

[0156] In some embodiments, the pharmaceutical composition may contain two or more active compounds, which are generally complementary compounds that do not adversely affect each other. The type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.

[0157] In some embodiments, the pharmaceutically acceptable carrier includes any solvent, dispersion medium, coating, isotonic agent, and absorption retarder suitable for drug administration, and is generally safe and non-toxic.

[0158] In some embodiments, the pharmaceutical composition may be administered parenterally, transdermally, intracavitarially, intraarterially, intrathecally, and / or intranasally, or by direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the administration of the pharmaceutical composition may be carried out in a variety of ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. In some embodiments, the pharmaceutical composition may be administered without interruption. Uninterrupted (or continuous) administration may be achieved by a small pump system attached to the patient, as described, for example, in WO2015 / 036583, which can measure the amount of therapeutic agent flowing into the patient's body.

[0159] Preparation method On the other hand, this application provides a method for preparing the isolated antigen-binding protein. The method includes culturing the host cells described in this application under conditions that express the antigen-binding protein. For example, methods such as suitable culture media, suitable temperature and suitable incubation time, which are well known to those skilled in the art, can be used.

[0160] Any method suitable for producing monoclonal antibodies can be used to produce the antigen-binding proteins of this application. For example, animals can be immunized using conjugated or naturally occurring Staphylococcus aureus α-toxin or fragments thereof. Appropriate immunization methods, including adjuvants, immunostimulants, and repeated inoculations for immune enhancement, can be used, and one or more methods can be used. In some embodiments, nucleic acid fragments from immunized alpaca peripheral blood lymphocytes are extracted and cloned into vectors, and isolated antigen-binding proteins against Staphylococcus aureus α-toxin are screened and enriched using a phage surface display system.

[0161] Any appropriate form of Staphylococcus aureus α-toxin can also be used as an immunogen (antigen) to produce non-human antibodies specific to Staphylococcus aureus α-toxin and to screen the biological activity of the antibodies. For example, the induced immunogen may be a natural homodimer or a full-length Staphylococcus aureus α-toxin containing single / multiple epitope-containing peptides. The immunogen may be used alone or in combination with one or more immunogenicity enhancers known in the art.

[0162] Methods and Applications On the other hand, this application provides applications of the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immune complex, and / or the pharmaceutical composition in the manufacture of drugs for preventing and / or treating diseases and / or symptoms and their complications.

[0163] On the other hand, the present application further provides a method for preventing and / or treating diseases and / or symptoms and their complications, comprising administering to a subject in need of the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cells, the immune complex and / or the pharmaceutical composition described in the present application.

[0164] In this application, the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immune complex, and / or the pharmaceutical composition may be used alone or in combination with other drugs to prevent and / or treat a disease and / or symptoms. In some embodiments, the other drug may be any currently known drug having antibacterial activity. The disease and / or symptoms may be related diseases caused by Staphylococcus aureus infection or other infections.

[0165] In this application, the administration can be carried out in various ways, such as intravenously, intratumorally, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally.

[0166] On the other hand, the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immune complex, and / or the pharmaceutical composition described in this application can be used for the prevention and / or treatment of diseases and / or symptoms.

[0167] In this application, the disease and / or symptoms can be caused or transmitted by Staphylococcus aureus α-toxin.

[0168] In this application, the disease and / or symptoms may be complications of a disease and / or symptoms caused or mediated by Staphylococcus aureus.

[0169] In this application, the disease and / or symptoms and their complications include bacterial infections.

[0170] In this application, the disease and / or symptoms and their complications include bacteremia and / or sepsis.

[0171] In some embodiments, the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immune complex, and / or the pharmaceutical composition described in this application can be used alone or in combination with other drugs.

[0172] On the other hand, the present application further provides a method for detecting Staphylococcus aureus α-toxin in a sample, comprising administering the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cells, the immune complex and / or the pharmaceutical composition.

[0173] In some embodiments, the method for detecting Staphylococcus aureus α-toxin in the sample may be an in vitro method. For example, the isolated antigen-binding protein described in this application may be brought into contact with the ex vivo sample to detect the presence and / or content of Staphylococcus aureus α-toxin in the sample. In some embodiments, the method for detecting Staphylococcus aureus α-toxin in the sample is not for therapeutic purposes. In some embodiments, the method for detecting Staphylococcus aureus α-toxin in the sample is not a diagnostic method.

[0174] On the other hand, the present application further provides a reagent or reagent kit for detecting Staphylococcus aureus α-toxin in a sample, comprising the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immune complex and / or the pharmaceutical composition.

[0175] On the other hand, this application further provides applications in the manufacture of reagent kits for detecting the presence and / or content of Staphylococcus aureus α-toxin in a sample of the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immune complex, and / or the pharmaceutical composition.

[0176] The following examples are not limited to theory, but are merely intended to illustrate the antigen-binding protein of this application, its preparation method, and applications, and do not limit the scope of the invention of this application. [Examples]

[0177] Example 1: Recombinant expression of Staphylococcus aureus α-toxin for immunizing alpacas Alpacas were subcutaneously immunized four times with the prepared Staphylococcus aureus α-toxin. The immunization process is shown in Table 4.

[0178] [Table 4]

[0179] Example 2: Construction and screening of a library of Staphylococcus aureus α-toxin antigen-binding proteins. 100 ml of peripheral blood lymphocytes from immunized alpacas were collected and Qi a Total RNA from cells was extracted using a reagent kit from gen. Reverse transcription kit (Qi a RNA was reverse transcribed into cDNA using (gen). The variable region nucleic acid fragment encoding the antibody was amplified by PCR. The PCR product and the cloning vector pComb3Xss were enzymatically cleaved with the restriction enzyme SfilI (Takara), and the cloning vector was ligated into the phage vector pComb3Xss using T4 ligase. The product was then electrotransformed into E. coli electroporation-competent cells Top10.

[0180] Example 3: Panning against Staphylococcus aureus α-toxin antigen-binding protein 96-well plates were coated with α-toxin fusion protein and stored overnight at 4°C. The following day, they were blocked with 5% BSA and 100 μL of phage was added. After allowing to react at room temperature for 1 hour, unbound phages were removed by panning with PBST. Finally, triethylamine was used. n The phages specifically bound to α-toxin were dissociated and used to infect E. coli TG1 in the logarithmic growth phase. The phages were then generated and purified for the next screening. The same screening process was repeated 3-4 times. This enriched the positive clones, achieving the objective of screening α-toxin-specific antibodies in the antibody library using phage display technology.

[0181] Example 4 Screening of specific single positive clones of phages by enzyme-linked immunosorbent assay (ELISA) The obtained Staphylococcus aureus α-toxin-positive phages were used to infect Escherichia coli and spread onto plates. Then, 210 single colonies were selected and cultured to generate and purify phages. 96-well plates were coated with α-toxin fusion protein and stored overnight at 4°C. The obtained sample phages (blank phages for the control group) were added and reacted at room temperature for 1 hour. After washing, mouse anti-HA tag antibody was added and reacted at room temperature for 1 hour. After washing, goat anti-mouse HRP antibody (Beijing Dingguo Biotechnology Co., Ltd.) was added and reacted at room temperature for 1 hour. After washing, TMB chromogenic solution was added and reacted at wavelength 4 50 Absorbance was measured in nm. Wells with an OD value more than three times that of the control well were marked as positive clone wells. Plasmids were extracted, and bacteria from the positive clone wells were cultured in LB solution containing 100 μL of ampicillin for sequencing. Finally, the antigen-binding protein sequence was obtained.

[0182] The sequences of the antigen-binding proteins that were measured are as follows: A3 SEQ ID NO: 12 QVQLVESGGGLVQAGGSLTLSCVAS GFTFShr GMTWVRQAPGKELDYVGEI NIPGDD VDYDISVKGRFTISRDNAKNTVYLHMTNLKVEDTATYWCAQ AGENMRLVT RRQGTQVTVST G1 SEQ ID NO: 24 QVQLVESGGDLVQPGGSLTLACEAS GFTISST GMTWVRQAPGKELDWVGDI NIPGTD TDYDISVKGRFTISKDHAKNTVYLQMTSLRPEDTAIYYCAK QGANLRQGV QSPGTLVTVSS F6 SEQ ID NO: 37 QVQLVESGGGLAQPGGSLRLSCEAS GSTLDSY TVAWFRQAPGKEREGVSCA SRSGAS TNYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAA YRNFGNFCVMGRYEYDE WGQGTQVTVSS However, the underlined portions indicate CDR regions that have been divided using the Chothia numbering scheme.

[0183] Example 5: Expression and purification of Fc fusion antigen-binding protein in Expi293 cells The variable region coding sequences of antigen-binding proteins A3, G1, and F6, obtained by sequencing and analysis, were fused with the Fc fragment of human immunoglobulin γ1 (IgG1) and subcloned into the expression vector PCDNA3.4. Expi293 cells were transfected with PEI and recombinantly expressed. After 7 days, the cell supernatant was collected and purified using Protein A magnetic beads. Antigen-binding proteins with a purity of 95% or higher were collected, yielding A3-Fc antigen-binding protein, G1-Fc antigen-binding protein, and F6-Fc antigen-binding protein. Of these, the amino acid sequence of A3-Fc antigen-binding protein is shown in SEQ ID NO: 62, the amino acid sequence of G1-Fc antigen-binding protein is shown in SEQ ID NO: 63, and the amino acid sequence of F6-Fc antigen-binding protein is shown in SEQ ID NO: 64.

[0184] Example 6 Detection of antigen-binding protein binding to Staphylococcus aureus α-toxin by ELISA Staphylococcus aureus α-toxin protein was diluted to 1 μg / mL in PBS buffer, and 100 μL per well was used to coat a 96-well plate (Thermo). The plate was incubated overnight at 4°C. The next day, the 96-well plate was removed, washed with PBST (containing 0.5% PBS), immersed for 1 minute each time, and the remaining water was completely dried. 200 μL of PBST containing 5% BSA was added to the sample wells and blocked at 37°C for 1 hour. The plate was then washed with PBST and the water in the wells was dried. 100 μL of each test sample was added to the 96-well plate and incubated overnight at 4°C. The 96-well plate was removed, washed with PBST, and then 100 μL of goat anti-human IgG secondary antibody (thermo, 31413, Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, HRP) was added to each well and incubated at 37°C for 1 hour. The plates were then washed five times with PBST, 100 μL of substrate solution (Invitrogen) was added to each well, and the mixture was incubated at 37°C for 10 minutes. After stopping the reaction by adding 50 μL of 1 mol of sulfuric acid to each well, the absorbance at a wavelength of 450 nm was detected using a microplate reader (Multiskcin FC, Thermo). Human IgG was used as a negative control.

[0185] Figure 1 shows the results of the binding detection of A3-Fc antigen-binding protein, G1-Fc antigen-binding protein, and F6-Fc antigen-binding protein to Staphylococcus aureus α-toxin. The results show that the above three types of antigen-binding proteins have a strong affinity for Staphylococcus aureus α-toxin, and among them, A3-Fc antigen-binding protein showed the highest EC 50 The value was 0.009 μg / mL, and the EC of the G1-Fc antigen-binding protein 50 The value was 0.007 μg / mL, and the EC of F6-Fc antigen-binding protein 50 The value is 0.003 μg / mL.

[0186] Example 7 Evaluation of the neutralization of hemolytic activity of Staphylococcus aureus α-toxin by antigen-binding protein AR-301 is a fully human antibody drug (Salvecin, tosatoxumab) targeting alpha-toxin developed by Alidis Pharmaceuticals. The neutralizing effect of AR301 on the hemolytic activity of Staphylococcus aureus alpha-toxin was evaluated using a rabbit erythrocyte hemolysis model induced by alpha-toxin. 18.75 ng of recombinant Staphylococcus aureus alpha-toxin protein was mixed with antigen-binding protein or AR-301 in a fixed ratio, added to a 96-well plate (Thermo), incubated at 37°C for 10 minutes, and then 5% rabbit erythrocytes were added and incubated at 37°C for 1 hour. The sample was centrifuged at 3000 rpm for 5 minutes, and the absorbance at a wavelength of 405 nm was detected using a microplate reader (Multiskcin FC, Thermo) to measure the anti-hemolytic activity.

[0187] Figure 2 shows the results of detecting the neutralizing effects of A3-Fc antigen-binding protein, G1-Fc antigen-binding protein, and F6-Fc antigen-binding protein against Staphylococcus aureus α-toxin using the rabbit erythrocyte hemolysis model with the above-mentioned α-toxin. The results show that 50 ng of A3-Fc antigen-binding protein (A), G1-Fc antigen-binding protein (B), and F6-Fc antigen-binding protein (C) had complete neutralizing effect against 18.75 ng of recombinant α-toxin; 50 ng of the control antibody AR-301 could not completely neutralize 18.75 ng of recombinant Staphylococcus aureus α-toxin (D). The neutralizing effect of the A3-Fc antigen-binding protein, G1-Fc antigen-binding protein, and F6-Fc antigen-binding protein disclosed in this invention against Staphylococcus aureus α-toxin is significantly superior to that of the control antibody AR-301.

[0188] Example 8: Reproduction of a mouse sepsis model induced by Staphylococcus aureus α-toxin and evaluation of the pharmacodynamic effects of antigen-binding proteins. C57BL / 6 mice were pre-injected with different doses of antigen-binding protein, and 30 minutes later, 2.5 μg of recombinant Staphylococcus aureus α-toxin protein was injected into the tail vein. Human IgG was used as a control. The survival time of the mice was observed.

[0189] Figure 3 shows the results of testing the therapeutic effects of A3-Fc antigen-binding protein, G1-Fc antigen-binding protein, and F6-Fc antigen-binding protein using a mouse sepsis model induced by Staphylococcus aureus α-toxin. The results show that the three antigen-binding proteins described in this application—A3-Fc antigen-binding protein (187.5 μg / kg, A), G1-Fc antigen-binding protein (125 μg / kg, B), and F6-Fc antigen-binding protein (250 μg / kg, C)—have a complete protective effect.

[0190] Example 9: Reproduction of a mouse bacteremia model induced by Staphylococcus aureus infection and evaluation of the therapeutic effect of antigen-binding protein. Staphylococcus aureus USA300 was activated from a -80°C refrigerator, activated for two generations in TSA medium, inoculated into 2 ml of TSB and cultured for 12 hours. After washing three times with PBS, 6 × 10⁶ cells were collected. 7 CFU mice were infected in the tail vein. Two hours after infection, an antigen-binding protein was injected, and the survival of the mice was observed using human IgG as a control.

[0191] Figure 4 shows the results of evaluating the antiinfective pharmacodynamic effects of A3-Fc antigen-binding protein and G1-Fc antigen-binding protein using a mouse bacteremia model caused by Staphylococcus aureus. The results show that A3-Fc antigen-binding protein and G1-Fc antigen-binding protein have a significant protective effect against the mouse bacteremia model caused by Staphylococcus aureus, and their pharmacodynamic effects are superior to those of the control antibody AR-301.

[0192] Example 10 Humanization of Staphylococcus aureus α-toxin antigen-binding protein By aligning the NCBI database (https: / / www.ncbi.nlm.nih.gov / igblast / ), we selected the human germline sequence most closely resembling the camel antibody as a template. We then humanized A3, G1, and F6, and optimized the post-translational modification sites of the antigen-binding proteins.

[0193] The humanized sequences of the obtained antigen-binding proteins are as follows: huA3 SEQ ID NO: 13 QVQLVESGGGLVQPGGSLRLSCAAS GFTFShr GMTWVRQAPGKGLEYVGEI NIPGDD VDYDISVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAR AGENMRLVT RGQGTLVTVSS huG1 SEQ ID NO: 25 QVQLVESGGGLVQPGGSLRLSCAAS GFTISST GMTWVRQAPGKGLEWVGDI NIPGTD TDYDISVKGRFTISKDHAKNTVYLQMNSLRAEDTAVYYCAK QGANLRQGV QGQGTLVTVSS huF6 SEQ ID NO: 38 EVQLVESGGGLVQPGGSLRLSCAAS GSTLDSY TVAWVRQAPGKGLEWVSCA SRSGAS TNYANSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAA YRNFGNFCVMGRYEYDE WGQGTLVTVSS However, the underlined portions indicate CDR regions that have been divided using the Chothia numbering scheme.

[0194] The variable region coding sequences of the antigen-binding proteins huA3, huG1, and huF6 were fused with the Fc fragments of human immunoglobulin γ1 (IgG1), respectively, and subcloned into the expression vector PCDNA3.4. Expi293 cells were transfected with PEI and recombinantly expressed. After 7 days, the cell supernatant was collected and purified using Protein A magnetic beads. Antigen-binding proteins with a purity of 95% or higher were collected, yielding huA3-Fc antigen-binding protein, huG1-Fc antigen-binding protein, and huF6-Fc antigen-binding protein. The humanized sequences of each of the above antigen-binding proteins are as follows: huA3-Fc SEQ ID NO: 68 QVQLVESGGGLVQPGGSLRLSCAAS GFTFShr GMTWVRQAPGKGLEYVGEI NIPGDD VDYDISVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARAGENMRLVTRGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK huG1-Fc SEQ ID NO: 69 QVQLVESGGGLVQPGGSLRLSCAASGFTISSTGMTWVRQAPGKGLEWVGDINIPGTDTDYDISVKGRFTISKDHAKNTVYLQMNSLRAEDTAVYYCAKQGANLRQGVQGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK huF6-Fc SEQ ID NO: 70 EVQLVESGGGLVQPGGSLRLSCAASGSTLDSYTVAWVRQAPGKGLEWVSCASRSGASTNYANSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAYRNFGNFCVMGRYEYDEWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The binding activity of the above antigen-binding protein and Staphylococcus aureus α-toxin was detected by the method of Example 6. The experimental results are shown in Figure 5, and it was found that the above three types of antigen-binding proteins have strong affinity for the α-toxin of Staphylococcus aureus. However, the EC 50 value of the huA3-Fc antigen-binding protein is 0.005970 μg / mL, and the EC 50 value of the huG1-Fc antigen-binding protein is 0.006353 μg / mL, and the EC 50 value of the huF6-Fc antigen-binding protein is 0.002721 μg / mL.

[0195] Using the rabbit red blood cell hemolysis model with α-toxin of Example 7, the neutralizing effect of the above antigen-binding protein on the hemolytic activity of Staphylococcus aureus α-toxin was evaluated. The experimental results are shown in Figure 6, and the neutralizing effects of huA3-Fc, huG1-Fc, and huF6-Fc on Staphylococcus aureus α-toxin are all significantly superior to that of the control antibody AR-301.

[0196] The pharmacodynamic effects of antigen-binding proteins were evaluated using a mouse sepsis model induced by Staphylococcus aureus α-toxin. C57BL / 6 mice were pre-injected with different doses of antigen-binding proteins, and 30 minutes later, 2.5 μg of recombinant Staphylococcus aureus α-toxin protein was injected into the tail vein, with human IgG used as a control. The survival time of the mice was observed. Figure 7 shows the results of testing the therapeutic effects of huA3-Fc antigen-binding protein and huG1-Fc antigen-binding protein using a mouse sepsis model induced by Staphylococcus aureus α-toxin. The results show that the protective effects of all three antigen-binding proteins described in this application—A3-Fc antigen-binding protein, G1-Fc antigen-binding protein, and huG1-Fc antigen-binding protein—are superior to those of AR301.

[0197] The therapeutic effect of antigen-binding proteins was evaluated using a mouse bacteremia model induced by Staphylococcus aureus infection. Staphylococcus aureus USA300 was activated from a -80°C refrigerator, activated for two generations in TSA medium, inoculated into 2 ml of TSB, and cultured for 12 hours. After washing three times with PBS, 6 × 10⁶ cells were collected. 7 CFU mice were infected in the tail vein. Two hours after infection, the antigen-binding protein was injected, and the survival of the mice was observed using human IgG as a control. The anti-infective pharmacodynamic effects of huA3-Fc antigen-binding protein and huG1-Fc antigen-binding protein were evaluated using a mouse bacteremia model caused by Staphylococcus aureus, and the results are shown in Figure 8. The results show that huA3-Fc antigen-binding protein and huG1-Fc antigen-binding protein have a significant protective effect against the mouse bacteremia model caused by Staphylococcus aureus, and their pharmacodynamic effects are superior to those of the control antibody AR-301.

[0198] Example 11: Preparation of Fc fusion protein of tetravalent Staphylococcus aureus α-toxin antigen-binding protein Two anti-Staphylococcus aureus α-toxin antigen-binding protein sequences (e.g., two huA3 molecules, two huG1 molecules, or one huA3 molecule and one huG1 molecule) were linked in series with a nucleic acid fragment encoding human immunoglobulin G1 (IgG1)Fc, and the resulting molecules were subcloned into a pcDNA3.4 plasmid and expressed and purified using the method described in Example 5. The molecule obtained by recombinant expression has four binding domains to Staphylococcus aureus α-toxin and is therefore called a tetravalent anti-Staphylococcus aureus α-toxin Fc antigen-binding protein. Of these, the amino acid sequence of the huA3-huA3-Fc antigen-binding protein is shown in SEQ ID NO: 65, the amino acid sequence of the huG1-huG1-Fc fusion protein is shown in SEQ ID NO: 66, and the amino acid sequence of the huA3-huG1-Fc antigen-binding protein is shown in SEQ ID NO: 67.

[0199] Using the rabbit erythrocyte hemolysis model induced by Staphylococcus aureus α-toxin in Experimental Example 7, the neutralizing effects of tetravalent huA3-huG1-Fc antigen-binding protein, huA3-Fc antigen-binding protein, and huG1-Fc antigen-binding protein on Staphylococcus aureus α-toxin were tested, and the results are shown in Figure 9. The results show that, against 18.75 ng of recombinant Staphylococcus aureus α-toxin, the neutralizing effect of the same dose of tetravalent huA3-huG1-Fc fusion protein was superior to that of divalent huA3-Fc antigen-binding protein and huG1-Fc antigen-binding protein.

[0200] Using the rabbit erythrocyte hemolysis model induced by Staphylococcus aureus α-toxin from Experimental Example 7, the neutralizing effects of tetravalent huA3-huA3-Fc antigen-binding protein and huA3-Fc antigen-binding protein on Staphylococcus aureus α-toxin were tested, and the results are shown in Figure 10. The results show that, for 18.75 ng of recombinant Staphylococcus aureus α-toxin, the neutralizing effect of the same dose of tetravalent huA3-huA3-Fc fusion protein was superior to that of divalent huA3-Fc antigen-binding protein.

[0201] Using the rabbit erythrocyte hemolysis model induced by Staphylococcus aureus α-toxin from Experimental Example 7, the neutralizing effects of tetravalent huG1-huG1-Fc antigen-binding protein and huG1-Fc antigen-binding protein on Staphylococcus aureus α-toxin were tested, and the results are shown in Figure 11. The results show that, against 18.75 ng of recombinant Staphylococcus aureus α-toxin, the neutralizing effect of the same dose of tetravalent huG1-huG1-Fc fusion protein was superior to that of huG1-Fc antigen-binding protein.

Claims

1. comprising HCDR1, HCDR2, and HCDR3 selected from any one of the following groups: 1) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1; 2) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14; and 3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 28, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 27, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

26. An isolated antigen-binding protein that targets the alpha-toxin of Staphylococcus aureus.

2. comprising H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1 comprises an amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29 and SEQ ID NO: 33, H-FR2 comprises an amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 30 and SEQ ID NO: 34, and H-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 22, SEQ The H-FR4 comprises an amino acid sequence shown in either ID NO: 31 or SEQ ID NO: 35, and the H-FR4 comprises an amino acid sequence shown in either SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 32, or SEQ ID NO:

36. An isolated antigen-binding protein that targets the α-toxin of Staphylococcus aureus as described in claim 1.

3. comprising H-FR1, H-FR2, H-FR3 and H-FR4 selected from any one of the following groups: 1) H-FR1 includes the amino acid sequence shown in SEQ ID NO: 4, H-FR2 includes the amino acid sequence shown in SEQ ID NO: 5, H-FR3 includes the amino acid sequence shown in SEQ ID NO: 6, and H-FR4 includes the amino acid sequence shown in SEQ ID NO: 7; 2) H-FR1 includes the amino acid sequence shown in SEQ ID NO: 8, H-FR2 includes the amino acid sequence shown in SEQ ID NO: 9, H-FR3 includes the amino acid sequence shown in SEQ ID NO: 10, and H-FR4 includes the amino acid sequence shown in SEQ ID NO: 11; 3) H-FR1 includes the amino acid sequence shown in SEQ ID NO: 17, H-FR2 includes the amino acid sequence shown in SEQ ID NO: 18, H-FR3 includes the amino acid sequence shown in SEQ ID NO: 19, and H-FR4 includes the amino acid sequence shown in SEQ ID NO: 20; 4) H-FR1 includes the amino acid sequence shown in SEQ ID NO: 8, H-FR2 includes the amino acid sequence shown in SEQ ID NO: 21, H-FR3 includes the amino acid sequence shown in SEQ ID NO: 22, and H-FR4 includes the amino acid sequence shown in SEQ ID NO: 23; 5) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 29, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 30, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 31, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 32; and 6) H-FR1 includes the amino acid sequence shown in SEQ ID NO: 33, H-FR2 includes the amino acid sequence shown in SEQ ID NO: 34, H-FR3 includes the amino acid sequence shown in SEQ ID NO: 35, and H-FR4 includes the amino acid sequence shown in SEQ ID NO:

36. An isolated antigen-binding protein that targets the α-toxin of Staphylococcus aureus as described in claim 1.

4. An isolated antigen-binding protein that targets the α-toxin of Staphylococcus aureus according to Claim 1, comprising VH, wherein VH comprises an amino acid sequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 37, and SEQ ID NO:

38.

5. An isolated antigen-binding protein that targets the α-toxin of Staphylococcus aureus according to Claim 1, comprising VHH, wherein VHH comprises an amino acid sequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 37, and SEQ ID NO:

38.

6. An isolated antigen-binding protein that targets the α-toxin of Staphylococcus aureus as described in Claim 1, comprising an antibody heavy chain constant region.

7. An isolated antigen-binding protein that targets the α-toxin of Staphylococcus aureus according to Claim 1, comprising the amino acid sequence shown in any one of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO:

70.

8. An isolated antigen-binding protein that targets the α-toxin of Staphylococcus aureus according to claim 1, comprising an antibody or an antigen-binding fragment thereof.

9. A polypeptide molecule comprising an isolated antigen-binding protein that targets the α-toxin of Staphylococcus aureus as described in Claim 1.

10. The polypeptide molecule according to claim 9, which is a fusion protein comprising two or more isolated antigen-binding proteins and an antibody heavy chain constant region, targeting the α-toxin of Staphylococcus aureus.

11. The polypeptide molecule according to claim 9, comprising the amino acid sequence shown in any one of SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO:

67.

12. An immune complex comprising an isolated antigen-binding protein that targets the α-toxin of Staphylococcus aureus as described in claim 1.

13. A nucleic acid molecule encoding an isolated antigen-binding protein that targets the α-toxin of Staphylococcus aureus as described in claim 1, or a polypeptide molecule containing the isolated antigen-binding protein that targets the α-toxin of Staphylococcus aureus.

14. A cell comprising the nucleic acid molecule described in claim 13 or a vector containing the nucleic acid molecule.

15. A pharmaceutical composition comprising: an isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus as described in claim 1; a polypeptide molecule containing the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus; an immune complex containing the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus; a nucleic acid molecule encoding the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus; a vector containing the nucleic acid molecule and / or a cell containing the nucleic acid molecule or a vector containing the nucleic acid molecule; and an optional pharmaceutically acceptable carrier.

16. Use in the manufacture of a drug for the prevention and / or treatment of diseases and / or symptoms and complications of humans and / or animals, of a pharmaceutical composition comprising: an isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus as described in claim 1; a polypeptide molecule containing the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus; an immune complex containing the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus; a nucleic acid molecule encoding the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus; a vector containing the nucleic acid molecule; a cell containing the nucleic acid molecule or the vector, and / or the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus.

17. The use according to claim 16, wherein the disease and / or symptoms and their complications are caused or transmitted by Staphylococcus aureus.

18. A method for detecting Staphylococcus aureus α-toxin in a sample, comprising using an isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus as described in Claim 1, a polypeptide molecule containing the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus, an immune complex containing the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus, a nucleic acid molecule encoding the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus, a vector containing the nucleic acid molecule, a cell containing the nucleic acid molecule or the vector, and / or a pharmaceutical composition containing the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus.

19. A reagent or reagent kit for detecting Staphylococcus aureus α-toxin in a sample, comprising: an isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus as described in Claim 1; a polypeptide molecule containing the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus; an immune complex containing the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus; a nucleic acid molecule encoding the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus; a vector containing the nucleic acid molecule; and a pharmaceutical composition containing the nucleic acid molecule or the vector, and / or the isolated antigen-binding protein targeting the α-toxin of Staphylococcus aureus.

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